GR I1111111 111111I 1111

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2
19 May, 1999
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Linda S. Kahl, Ph.D.
Regulatory Policy Branch, HFS-206
Center for Food Safety and Applied Nutrition
Food and Drug Administration
200 C Street, S.W.
Washington,D.C.20204

Dear Dr. Kahl,

We areherebysubmitting, in triplicate,agenerallyrecognizedas
safe (GRAS) notification, in accordance with proposed 21 C.F.R. 5
170.36, for Novo Nordisks a-amylase enzyme preparation,
produced by Bacillus licheniformis carrying a modified gene coding
0 fora-amylase from Bacillus licheniformis. Thea-amylaseenzyme
preparation is to be used in the food industry as a processing aid in
the liquefactionof starch.

Please contact me by direct telephone at 919 494-3152or direct fax

at 919494-3420 if youhaveanyquestions or requireadditional
information.

Sincerely,

Scott H. Shore, Ph.D.

Senior Regulatory Specialist

Enclosures (3 binders)
ry
c
 14 May, 1999

RE: GRAS Notification - Exemption Claim

Dear Sir or Madam:

Pursuant to the proposed 21C.F.R. §170.36(~)(1)NovoNordiskBioChemNorth

AmericaInc.hereby claims that a-amylase enzymepreparationsproducedby
submerged fermentation of Bacillus licheniformis carrying the gene coding for a
modified a-amylase from Bacillus licheniformis are Generally Recognized as Safe;
therefore, theyare exempt from statuatory premarket approval requirements.

The following informationis provided in accordance with the proposed regulation:

Proposed §170.36(c)(l)(i) The name and address offhe notifier.

Novo Nordisk BioChemNorth America Inc.
77 Perry Chapel ChurchRd., Box 576
Franklinton, NC 27525

Proposed §170.36(c)(l)(ii) The common or usual name of notified substance.

a-Amylaseenzyme preparation from Bacillus licheniformis carryingthegene
coding fora modified a-amylase from Bacillus licheniformis.

Proposed §170.36(c)(l)(iii) Applicable conditions of use.

@ Theabovedescribed a-amylase preparation is to be usedinthefoodindustry as a
processing aid in the liquefaction of starch.Theenzymepreparationisusedat
minimum levels necessary to achieve the desired effect
andaccording to
requirements fornormal production following Good ManufacturingPractices.

Proposed §170.36(c)(l)(iv) Basis for GRAS determination.

This GRAS determination is based on scientific procedures.

Proposed §170.36(c)(1)(v) Availability of information.

A notification package providing a summary of the information which supports this
GRAS determination is enclosedwiththisletter.Thepackageincludes a safety
evaluation of the production strain, the enzyme, and the manufacturing process, as
well as an evaluation of dietary exposure. Complete data and information that are
thebasisfor this GRASdeterminationare available to theFoodandDrug
Administration for review and copying upon request. *
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Date Job$ Carroll (
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Termamyl@LC

an a-amylase preparation

produced by Bacillus licheniformis expressing

a gene encoding a modified

a-amylase from Bacillus lichenformis

Annette Gnrtzsche-Larsen, Enzyme Regulatory Affairs, NNNS, Denmark

Scott Shore, Enzyme Regulatory Affairs, NNBNA, USA
Lori Gregg, Enzyme Regulatory Affairs, NNBNA, USA
 TABLE OF CONTENTS 0
PAGE z
0
1. GENERAL INTRODUCTION 3 3
0
2. PRODUCTION MICROORGANISM 4 z
2.1 Production Strain 4
2.2 Recipient Organism 5
2.3 Plasmid K

2.4 Stability of the Transformed Plasmid Genetic Sequences

2.5 Absence of rDNA Sequences in the Enzyme Preparation 6
2.6 Absence of Production Organismin the Product 6
2.7 Antibiotic Resistance Gene 6

3. ENZYME IDENTITY AND SUBSTANTIAL EQUIVALENCE 7

3.1 Enzyme Identity 7
3.2 Amino Acid Sequence 8
3.3 Sequence Comparison to other8.lichenifomis a-amylases 9
3.4 Enzymatic Activity 10

4. MANUFACTURING PROCESS 10
4.1 Raw Materials 10

a
4.2 Fermentation Process 11
4.3 Recovery Process 12
4.4 Quality Control of Finished Product 13
-
5. COMPOSITION AND SPECIFICATIONS 13
5.1 Formulation 13
5.2 Specifications 13

6. APPLICATION 14
6.1 Mode of Action 14
6.2 Application 14
6.3 Use Levels 14
6.4 Enzyme Residues in the Final Food 14

7. SAFETY EVALUATION 15
7.1 Safety of the Production Strain 15
7.2 Safety of thea-amylase enzyme 16
7.3 Safety of the Manufacturing Process 18
7.4 Safety Studies 18
7.5 Estimates of Human Consumption and Safety Margin 18
7.6 Results and Conclusion 20

8. LIST OF APPENDICES 21

9. LIST OF REFERENCES 22

Novo Nordisk I Termamyl LC a-amylase information

 1. GENERAL INTRODUCTION 0

-
2
Termamyl@LC is the NovoNordisk N S trade name used for an 0
3
a-amylase preparation produced submerged
by fermentation of 0
Bacillus licheniformis carrying a modified gene coding for a-amylase a 2
from Bacillus licheniformis.

Theenzyme preparation is to beusedin the foodindustryas a

processing aidin the liquefaction of starch.

The enzyme is a heat-stable a-amylase, which can operate at lower

pHand
lower calcium levels
than
conventional
thermostable
a-amylases.Theenzyme is anendo-amylase,whichhydrolyses
1,4-a-glucosidiclinkages in amyloseandamylopectin.Starch is
thereby broken downto soluble dextrins and oligosaccharides.

The following sections provide information on the enzyme and the

production organism which are the basis for our determination of the
general recognition of safety of an a-amylase enzyme preparation
(commercialproduct
trade
name,Termamyl LC) produced by
8.lichenifomis expressing a gene encoding a modified a-amylase
from B. lichenifomis. Our safety evaluation in Section 7 includes an
@ evaluation of the publishedliteraturesupporting the safety of the
productionstrain, the enzyme,andthemanufacturingprocess as
well as an evaluation of dietary exposure tothe enzyme preparation.

The
safety of the production organism
must be the prime
consideration in assessingtheprobable degree of safety of an
enzymepreparationintendedforuse in food’. Theproduction
organism for Termamyl LC, B. lichenifomis, is discussed in Sections
2 and 7. Carbohydraseandproteaseenzymepreparations from
€3. lichenifomis have been affirmed by FDA asGRAS2.
Anessentialaspect of the safetyevaluation of foodcomponents
derived from genetically modified organisms is the identification and
characterization of the insertedgeneticmaterial 3-8. Thegenetic
modificationsused to developtheproductionmicroorganismand
a-amylase enzyme are well defined and are described in Section 2.
Data
showing this modified
a-amylase to be substantially
equivalent6”’ to naturally
occurring a-amylases is provided in
Section 3. Thesafetystudiesdescribed in Section 7 show no
evidence to indicate that anyoftheincorporateda-amylase or

e
plasmid vector DNA sequences encode or expressa harmful or toxic
substance.

Novo Nordisk I Termamyl LC a-amylase information 3

 I
Y

’a
v)
5
L
Thesafety ofthe processing aidsandothersubstancesused in the
z
0

-
manufacturing
process is included in Section 4. Finally, an
0
evaluation of potential dietary exposure and a calculation of a safety >
margin is included
7. in Section 0
z
It should be noted that within some of the attachments, the terms
“Novozym935”,“SP935”,and“LowCaAmylase”areused to
describeTermamylLC.Thesethreenamesare internal names
used before a decision concerning the trade name was made.

2. PRODUCTION MICROORGANISM

2.1 Production
Strain

The microbial production strain for Termamyl LC,

designated
LiH1159, (production code Ad-I-O/B) is
derivative
a of the
6. licheniformis natural isolate,AmericanTypeCultureCollection
Strain(ATCC)9789.ThisATCC9789strainis the ancestor of
6. lichenifomis strainsthathavebeenusedsafely for industrial
production of enzymes since 1972 including an enzyme preparation
that was the subject of the GRAS affirmation petition12 submitted by
Novo Nordisk and affirmed by FDA in T983l3.Theclassification of
thisstrainandtheproductionmicroorganismisbased on generally
accepted taxonomic characteristic^'^.

Strain
LiH
1159 was constructed by
common transformation
procedures using well-known plasmid vector^'^"^ and
well-
characterized B. licheniformis a-amylase DNA sequences. The
recombinant plasmid DNAwasintegrated into the 6. licheniformis
host
strain
chromosome by
homologous recombination. The
6. lichenifomis a-amylase gene (amyL) was modified by well-known
techniques designed to introducespecific desired modifications in
the coding s e q ~ e n c e ’ ~ ~
The
* ~ .identity and location of these changes
were verified by DNA sequencing in both orientations several times.
The genetic construction was evaluated at every step to assess the
incorporation of the desiredfunctionalgeneticinformationand to
ensure that no unintendedsequenceswereincorporated in the
production strain.

Thisgeneticallymodifiedproductionorganismcomplieswith the
OECD (Organization for Economic Co-operation and Development)
criteria
for GILSP (Good Industrial Large ScalePractice)
, - microorganisms21. The genetically modified strain meets the criteria

0-
..- -:3_.
r/l “-7-

forasafeproductionmicroorganismasdescribedbyParizaand
Foster
and
several
expert
groupsM.
These
criteria
include the

Novo Nordisk / Termamyl LC a-amylase information 4

0 identification and characterization of the host strain, plasmid vector, 0
2

-
andinsertedgeneticsequencesgiveninSections2.2and2.3below.
0
2.2 Recipient
Organism
L
The
recipient
microorganism, designated SJI707, used in the
construction of the a-amylaseproductionstrainisasporulation
deficientand alkaline proteasenegative derivative of a natural
isolate of B. lichenifomis, ATCC 9789.

Plasmid
2.3

The4.39kbamylaseexpressionplasmid, pLiHl108, used in the

construction of theproductionstrain,enabledincorporationand
amplification of the modified B. lichenifomis a-amylase gene in the
recipient
organism.Plasmid pLiHl108 contains strictly
defined
bacterial chromosomal DNA fragments,DNA from well-characterized
bacterialcloningvectors,andspecificsyntheticoligonucleotide
sequences.The
particular
DNA
sequencesinclude:
coding
sequencefor the modifieda-amylaseenzyme; 5’ and3’flanking

e
fragmentsfrom the wild-type B. lichenifomis a-amylasecoding
sequence, a non-functional origin of replication from plasmid pEI94,
and
well-knownsequences from Bacillus cloningvector
plasmid
pUB1O I . Plasmids pE194 and pUBl10 are certified plasmid vectors
under the NIH Guidelinesz.

Plasmid pLiHl108 contains

the
following
genetic
material
(see
Appendix A):

1.77 kb DNA from theBacillus cloning vector, pUBl10

0.25 kb DNA frompEI94 containing nonfunctional replication origin
0.63 kb DNA from the5’ region of theB. lichenifomis a-amylase gene
0.19 kb DNAfrom the3’ region of theB. lichenifomis a-amylase gene
1.55kbDNAfrom the modified B. lichenifomis a-amylasecoding
sequence

2.4 Stability of the Transformed Plasmid Genetic Sequences

The presence and configuration of the introduced DNA sequences

weredetermined by Southernhybridization to assess the stability
and potential for transfer of genetic material as a component of the
safety evaluation of the production rnicroorgani~m~~.The
transforming plasmid DNA sequences are stably integrated into the
B. lichenifomis chromosomeand do notcontainanysequences
1) known to promote gene transfer.Theintroducedgeneticmaterial is

Novo Nordisk I Termamyl LC a-amylase information 5

@
poorly
mobilizable for genetic
transfer to other
organisms
and
is 0
mitotically z0
2.5 Absence of rDNA Sequences in the Enzyme Preparation

Samples of the a-amylase enzyme preparation have been analyzed

bydotblot hybridization for thepresence of recombinantDNA
sequences used in the production strain
construction as an
additionalcomponent of thesafetyevaluation of the production
micr~organism~~~. NorecombinantDNAsequencesweredetected.
Thedetection limit is 200 pgplasmid DNNg sample.Therefore,
there is no potential for transfer of recombinant DNA sequences to
other organisms which ingest foods processed with the a-amylase
enzyme preparation.

2.6 Absence of Production Organism in the Product

The
absence of the production
organism is an
established
specification for the commercialproduct,
TermamylLC.
The
production organism does not end up in food and therefore the first
step in the safety assessment as described by IFBC4is satisfactorily
addressed.

2.7 Antibiotic
Resistance
Gene

Plasmid pLiH 1108 contains a kanamycin resistance gene, encoding

an aminoglycoside 3’-phosphotransferase II protein, APH(3’) I I . FDA
has evaluated the safety of this gene and gene productfor use in the
development of geneticallyengineeredtomato,oilseedrape,and
TheFDA’s
evaluation of
issues
and the agency’s
conclusions are also applicable for assessment of the kanamycin
resistance gene present in the Termamyl LC production strain. As
discussed by FDA in its decision23 and further elaboratedin the draft
guidance and report on the use of antibiotic resistance marker genes
in transgenic plants24,the evaluation of the safety of the use of an
antibiotic resistance marker gene should include an assessment of
the safety of the protein encoded by the gene and consideration of
the potential for horizontal transfer of the antibiotic resistance gene.

Asstatedabove in Section2.5,norecombinantDNAsequences
were detected in the Termamyl LC enzyme product and as stated in
Section 2.6, the absence of
theproduction
organism is an

~a
established specification for the Termamyl LC enzymeproduct.
Consequently, the kanamycin antibiotic resistance gene would not
be present in either the Termamyl LC enzyme product or in foods
derived
from starch processed with
the
TermamylLC
enzyme

Novo Nordisk / Termamyl LC a-amylase information 6

 Y
=
VI
.I

product.Therefore, the use of theTermamylLCenzymeproductas 8

-
aprocessing aid inthe liquefaction of starchdoesnotposeasafety 2
0
concern due to any potential for transfer of the kanamycin resistance >
gene to other organisms. 0

FDA’ssafety evaluation andconclusions regarding the potential

direct effect of ingestion of the APH(3’) II protein and the potential
effect of the biological activity of APH(3’) II protein on the therapeutic
efficacy of orally administered antibiotic^^^ is also applicable to the
use of the kanamycin resistancegene in the Termamyl LC
production strain. The kanamycin antibiotic resistance gene used in
theTermamylLCproductionstrainisthesameas the kanamycin
resistance gene evaluated by FDA 23 and in each case encodes the
same protein, APH(3’) II. High temperature, in excess of 100” C, as
well as otherconditionsandstepsused during industrialstarch
processing (low pH, filtration, ion e x ~ h a n g e )would
~ ~ . ~be
~ expected
to inactivate and remove APH(3’) II protein. It is unlikely there would
beanyAPH(3’) II protein in anyfinalfoodproductderivedfrom
starch
processed with the Termamyl LCenzyme product. In
addition,the biological activity of APH(3’) II is destroyed during
digestion.Therefore, the use of thekanamycinresistancegene
encodingAPH(3”) II in the TermamylLCproductionstrainwillnot

0 compromise the therapeutic use of orally administered kanamycinor

neomycin.

3. ENZYME IDENTITY AND SUBSTANTIAL EQUIVALENCE

3.1
Enzyme Identity

Key enzyme andprotein chemical characteristicsof the

Termamyl LC a-amylaseare given below:

Classification: a-amylase (generic name)

IUB EC nomenclature: a-amylase
IUB No.: 3.2.1.1
CAS No.: 9000-90-2
EINECS No.: 232-565-6
Specificity: 1,4-a-glucosidic linkages in amylose
and amylopectin.
Molecular weight: 55 kDa
Amino acid sequence: The total nucleotide and amino acid
sequences have been determined

Novo Nordisk/ Termamyl LC a-amylase information 7

0
3.2 Amino Acid Sequence (see Appendix B, Figure 1)

-
The
Termamyl LC B. lichenifomis a-amylaseenzymehas
been >
developed to improveenzymeperformancebyincreasingstabilityat 0
lowpH, low calcium concentration,andhightemperature.The 2
starting point for the Termamyl LC enzyme was the B. lichenifomis
a-amylase gene used in the. production of Novo Nordisk’s product
Termamyl(subject of GRASP 3G002612, affirmedbyFDA in 198313
as 21 CFR $184.1027). The Termamyl LC enzyme was constructed
bysplicing in the NH,-terminalregion of the B. amyloliquefaciens
a-amylasesequenceandchangingthe DNA codingsequence for
other specific amino acid residues.

The Termamyl a-amylase enzyme requires calcium ions for stability.

In industrial applications such as starch processing, up to 40 ppm
calcium is added. The addition of calcium is costly and the calcium
must be removed after liquefaction to avoid fouling of evaporators
and inactivation of glucose isomerase. This is done by ion exchange
whichresults in large volumes of wastewaterwith high levels of
salts.

a
Severalconceptswereapplied in selectingtheappropriateamino
acidresidues to achieve the desiredeffect.The identification of
specific
residues to be changed was based on information from
publishedscientific literature onthe
structure
and function of
a-amylases,
analysis of publicly
accessible
protein
sequence
databases, and internal Novo Nordisk research.

The general structure of a-amylases is characterized by interactions

of three common domains and several calcium ion binding site~~~,~~.
The primary approach used to engineer an improved a-amylase was
to change amino
acid
residues
that
would stabilize the
domainldomain andcalciumion
interactions.
In
addition,
other
amino acid substitutions were incorporatedthat have been shown to
increase thermostabilityof B. lichenifomis a - a m y l a ~ e ~ ~ . ~ ~ .

Finally,
Novo
Nordisk
constructed
hybrid
a B. lichenifomis /
B. amyloliquefaciens a-amylasethatdisplayedimprovedstability.
6. lichenifomis and B. amyloliquefaciens a-amylase genes are highly
hom01ogo~~~’. There are nine differences in the first 35 NH,-terminal
amino
acid
residues
between
mature B. lichenifomis and
B. amyloliquefaciens a-amylase enzymes. The B. amyloliquefaciens

e
matureenzymeisalso two aminoacidresiduesshorteratthe
NH,-terminai end than the matureB. lichenifomis enzyme.

Novo Nordisk/ Termamyl LC a-amylase information 8

 z
ur
.I

‘EI
5
0 Thecompleteamino acid
sequence for
the
TermamylLC
mature
z0

-
enzyme is given as the top sequence of the
alignment
shown
in
Appendix B, Figure 1. The amino acid residue designated 1 in these
figures is thefirstalanineinthematureTermamyla-amylaseenzyme
sequence.AdifferencebetweentheTermamylenzymeandthe a zB
Termamyl LC enzyme is indicated above the Termamyl LC sequence
by an either an asterisk * or an arrow 4.The asterisks indicate amino
acidresiduedifferencesduetotheincorporationoftheNH2-terminal
region of the B. amyloliguefaciens sequence.Thearrowsindicate
changes that were made to increase stability.

Thecodingsequence for the NH,-terminal region of themature

Termamyl LC gene corresponds to the first thirty-five amino acids of
the mature B. amyloliquefaciens a-amylase enzyme. The splicing of
the NH2-terminal region of the B. amyloliquefaciens sequence results
in the differences indicated in AppendixB, Figure 1 by the locations of
the asterisks.

Fiveadditionalaminoacidchangesweremadeasindicated in
Appendix B, Figure 1 by the locations of the arrows. These changes
provide increased stability at low pH, low calcium concentration, and
high temperature.

. @ 3.3 Sequence
Comparison to other B. licheniformis a-amylases
(see Appendix B, Figure2)

The Termamyl LC B. licheniformis a-amylase amino acid sequence

has been compared to other B. licheniformis a-amylase amino acid
sequences (AppendixB,
Figure 2): NovoNordisk’s TermamyI3’,
GenBank protein
accessionsequence
M385703, and
GenBank
nucleotideaccessionsequenceE0115834.Homologyamongthese
B. licheniformis a-amylases is indicated in the table below:

Termamyl
Termamyl
LC M38570 E01158
100 LC Termamyl
0 Termamyl
M38570 100
E01 158 I00

TermamylLCis96.7%identical to Termamyl,96.3%identical to
M38570 and 93.0% identical to E01 158. This is within the range of
similarity of thethreeother B. lichenifomis a-amylaseswitheach
other: Termamyl is 99.2% identical to M38570 and 96.0% identical to
@ E01158;M38570is95.6%identicalto EO1158.

Novo Nordisk/ Termamyl LC a-amylase information 9

 Y
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v
1 1

None of thedifferencesamongthesefoura-amylasesarefound in
z8

-
conserved amino acid residues identified as structurally or functionally
0
important for a-amylasesin genera127~28.
B
3.4 Activity 2

Termamyl LC a-amylase is functionally equivalent to B. licheniformis

a-amylase (Termamyl) that is affirmed as GRAS as a carbohydrase in
= 21CFR$184.1027. Three experiments(AppendicesC, D, E) were
carried out to verify that Termamyl LC a-amylase exhibited catalytic
propertiesand functional effects in starchhydrolysissubstantially
I similar to those of Termamyla-amylase.Theexperimentsconducted
were:monitoringchanges in carbohydratespectraduringstarch
hydrolysis by HPLC (Appendix C), monitoring the release of reducing
sugars(Appendix D), andanalysisofglucoseyieldandoligo-
saccharidecompositionbyHPLC(AppendixE).Theseexperiments
demonstrate that Termamyl LC is functionally equivalentto Termamyl
with respect to the starch hydrolytic process and products.

a
4. MANUFACTURING PROCESS

This section describes the manufacturing process for Termamyl LC.

Thequalitymanagementsystemused in themanufacturingprocess
forTermamylLC complies withtherequirements of IS0 9002.
Termamyl LC is also manufactured in accordance with current good
manufacturing practices.

4.1 Raw Materials

Raw materialsused for fermentationandforrecoveryconform to

FoodChemicalsCodexspecificationsexceptthoserawmaterials
whichdonotappear in the FCC.Forthosenotappearing in the
FCC, internalspecificationshavebeenmade in line withFCC
requirements. On arrival at Novo Nordisk N S , the raw materials are
sampledby the Quality ControlDepartmentandsubjected to the
appropriate analysesto ensure their conformanceto specifications.

Theantifoamsand flocculants used in fermentationandrecovery

and listed below in Sections 4.2.1 and 4.3.1 are used in accordance
withtheEnzyme Technical Associationsubmission to FDA on
antifoams and flocculants dated April 10, 1998. The maximum use
level of these antifoams and flocculants in the Termamyl LC product
is less than 1%.

Novo Nordisk/ Termarnyl LC a-amylase information 10

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m

4.2 0
0 2

-
TermamylLC is manufactured by
submerged fed-batch pure culture 0
fermentation of the geneticallymodifiedstrain of B. licheniformis
described in Section 2. Aflowsheet of the fermentationprocessis 2
shown in Appendix F, Figure 1. All equipment is carefully designed,
constructed,operated,cleaned,andmaintained
contamination by foreign microorganisms. During
so as to prevent
all steps of
&pj
f
fermentation, physical and chemical control measures are taken and
microbiological analyses are conductedto ensure absence offoreign
microorganisms andto confirm strain identity.

4.2.1 Raw Materials for the Fermentation Process

The raw materials used in the fermentation process arelisted below.
The list includes both the raw materials used for the seed fermentor
as well as those used for the main fermentor.
Starch and/or starch hydrolysates*, Glucose*
1 Potato Protein
Corn Steep Liquor, Soy Meal
Minerals (phosphates, sulfates)*
H Alkali and acid (ammonia, sodium hydroxide, sodium carbonate
and phosphoric acid)* for pH adjustments

0
Polymeric antifoam compounds- P2000 (polypropylene glycol)*
and Pluronic PE6100 (polyoxypropylene-polyoxyethylene
copolymer),
H Potablewater
*Conform to FCC specifications

4.2.2 Production Organism

Each batch of the fermentation process is initiated with a lyophilized
stock culture of the production organism B. licheniformis, production
code Ad-I-OIB, described in Section 2. Each new batch of the stock
cultureisthoroughlycontrolledforidentity,absence of foreign
microorganisms, and enzyme-generating ability before use.
4.2.3 Criteria for the Rejection of Fermentation Batches
Growth
characteristics
during
fermentation
are
observed both
macroscopically and microscopically. Samples are taken from both
theseedfermentorand the mainfermentorbeforeinoculation, at
regularintervals during cultivation,andbefore transfedharvest.
Thesesamplesaretestedformicrobiologicalcontaminationby
microscopy and by plating on a nutrient agar followed by a 24-48
hour incubation period.

Novo Nordisk I Termamyl LC a-amylase information I1

e The fermentation is declared "contaminated" if one of the following
conditions
are
fulfilled:
I.Infection is observed in 2 or more samples by microscopy
2. Infection is observed in two successive agar plates at a
minimum interval of 6 hours
Any contaminated fermentationis rejected.
4.3
Recovery
Process
The
recoveryprocess
multistep
ais operation
which
starts
immediately after the fermentation process and consists of both the
purificationand the formulationprocesses.Aflowsheet of the
recovery process is shown in Appendix F, Figure 2.
4.3.1 Raw Materials in the Recovery Process
rn Alkali and acidsfor pH adjustment (sodium hydroxide,
potassium hydroxide, acetic acid, phosphoric acid)*
Precipitation/flocculatingagent [calcium chloride*, sodium
aluminate, Superfloc A I 30, (polyacrylamide acrylate
copolymer) and C521 (Dimethylamine epichlorohydrin

e
copolymer)]
rn Filter aids (diatomaceous earth/Celite, Dicalite, Perlite or
similar)*
Stabilizers andformulation aids (sodium chloride, sucrose)*
rn Water
*Conform to FCC specification

4.3.2Purification Process

Theenzymeisrecoveredfromtheculturebrothbythefollowing
series of operations:

1. Pretreatment- flocculationlpH adjustment

2. Primary Separation - drum filtration

3. Concentration - evaporation

4. Pasteurization

5. Pre- and Germ Filtration - for removal of residual production

strain organisms and as a general
precaution against microbial
degradation

Novo Nordisk / Termamyl LC a-amylase information

 4.3.3 Formulation and Standardization Processes 0
2
The enzyme concentrate is blended with water, sodium chloride, and 0
sucroseand, if necessary,adjusted to thedesiredpH.Theproduct is B
2
standardized according to the product specification.

4.4 Quality Control of FinishedProduct

The final productsare analyzed according tothe specifications given I

in Section 5.

5. COMPOSITION AND SPECIFICATIONS

5.1 Formulation

Termamyl LC will be available in a liquid formulation with an activity

of 120 KNU(L)/g and with the following typical composition:

Enzyme solids (E-TOS) approx.

3%
Sodium Chloride approx. 14%
Sucrose approx. 24%
Water approx. 59%

Further information on the product is given in the enclosed Product

Sheet (AppendixG).

5.2 Specifications

Termamyl LC complieswiththepuritycriteriarecommended for

enzymepreparationsasdescribed in FoodChemicalsCodex, 4'h
edition 199632. In addition,Terrnamyl LC also conforms to the
GeneralSpecifications for EnzymePreparationsUsed in Food
Processing as proposed by the Joint FAONVHO Expert Committee
on Food Additivesin Compendium of Food Additive specification^^^.

The following specifications have been establishedfor Termamyl LC:

a-amylase activity, KNU(L)/g according to declaration
Heavy metals not more than 30 ppm
Lead not more than5 ppm
Arsenic not more than 3 ppm
Total viable count/g not more than5 x I O 4
Total coliforms/g not more than30
Enteropathogenic E. co/i/25 g negative by test

0 Salmonella/25 g
Antibiotic
activity
Production organism
negative by test
negative bytest
negative by test

Novo Nordisk / Termamyl LC a-amylase information 13

 6. APPLICATION 8
2

-
6.1 Mode of Action 0
B
Termamyl LC is a heat-stable a-amylase, which can operate at lower e
pHandlower calcium than conventionalheat-stable a-amylases. h#
Theenzyme hydrolyses 1,4-a-glucosidiclinkages in amylose and 1
%
amylopectin, forming soluble dextrins and oligosaccharides.

Application
6.2

In the starch industry, Termamyl LC is an a-amylase used for the

enzymatic liquefaction of starch in the production of syrups25*26,37~38.

Inthealcohol industry, TermamylLCisana-amylaseused for

thinning of starch in distilling mashes3'.

6.3 Use
Levels

Theenzyme preparation is usedatminimumlevelsnecessary to

achieve the desired effect and according to requirements for normal
production following Good Manufacturing Practices (GMP).

@ Therecommended dosage for TermamylLC is 0.45 kgper ton of

starch. The optimum dosage depends on the desired effect and the
specific circumstancesin the processing plant.

6.4 Enzyme Residues in theFinalFood

Termamyl LC is used as a processing aid in starch hydrolysis and is

notadded directly to food.Furthermore,thea-amylaseenzyme
proteinisinactivatedandremovedduringtheconditionsandsteps
used for industrial starch processing(hightemperature(>IOO"C),
low pH (<5), filtration, carbon treatment, and ion e ~ c h a n g e ) ~This
~.~~.
hasbeenconfirmedbyimmunochemical (ELISA) analysis for the
presence of residualenzymeproteininsyrupsamplestakenat
several stages in the normal starch hydrolysis process. There was no
detectable enzyme protein in the final syrup sample. The detection
limit for the experimental assay 70 is pg/ml.

Novo Nordisk / Termamyl LC a-amylase information 14

 z
v)
v
.I

7. SAFETY EVALUATION
z8

-
Asstatedmost recently by FDA4’n4’, issuesrelevanttoa safety >
0
evaluation of an enzymepreparationarethesafety of theenzyme 0
source,
enzyme
component,
manufacturing
process,
and
a a m
97
consideration of dietary exposure.Eachoftheseisaddressed
below.
/ I
Safety 7.1 of the Strain
Production

Thesafety of the productionorganism

mustbe
the prime
consideration in assessingtheprobabledegree of safety of an
enzymepreparationintendedforuse in food’. If the organism is
nontoxigenicandnonpathogenic,thenitisassumedthatfoodor
foodingredients produced fromtheorganism,usingcurrentGood
ManufacturingPractices, is safetoconsume4.ParizaandFoster
(1983)defineanontoxigenicorganismas“onewhich does not
produce injurious
substances at levels that are detectableor
demonstrably harmful under ordinary conditions of use or exposure’’
andanonpathogenicorganismas“one that isveryunlikely to
produce disease under ordinary circumstances”. B. licheniformis is
notahuman pathogen and it isnot t o x i g e n i ~ ’ ~ * ~Carbohydrase
*-~~.

@ andproteaseenzymemixtures

published literature establishing

from B. licheniformis havebeen
affirmedby the FDAasGeneralllyRecognized
that
as Safe2 based on
B. licheniformis is widely
recognizedasaharmlesscontaminant of foodandunpublished
studies which corroborate that food uses of enzyme products from
B. licheniformis are safe13.

Because thegenetic
modifications
are
well
characterizedand
specific (see Section 2),and the incorporated plasmid DNA does not
encodeandexpressanyknownharmful or toxicsubstances, the
a-amylase enzyme preparation derived from the genetically modified
6. licheniformis is considered safe4vg. Section 7.3 describesthe
safetystudies@ that wereperformedon the enzymepreparation
made from the genetically modified B. licheniformis production strain
described in Section 2.

An evaluation of the genetically modified production microorganism

forTermamylLC,according to theconceptsinitially outlined by
Pariza and Foster, 1983 and further developed by IFBC in 1990, the
EU SCF in 1991, the OECD in 1992, lLSl Europe Novel Food Task
Force in 1996, and FAONVHO in 1996, demonstrates the safety of
this
genetically
modified
production
microorganismstrain.
The

@ components of this evaluation: the identity of thehoststrain,a

description of the plasmid used, the sourcesandfunctions of the

Novo Nordisk / Termamyl LC a-amylase information 15

 Y
v)
m
.I

5
@ introducedgeneticmaterial,anoutline
the
production
strain,
andsomecharacteristics
strain and the enzyme derived
of thegeneticconstruction of
of the
production
from it are given in Sections 2 and 3.
Z
0

of Safety7.2 the a-Amylase Enzyme a z

Enzymeproteinsthemselves
concern^^^‘"*^^. Asshown
do notgenerally
in Section
3,
Termamyl
raise safety
LC is an r l
wY?
a-amylase, IUB EC 3.2.1 .I, catalyzing the degradation of starch by D
hydrolysis of 1,4-a-glucosidic linkages in amylose and amylopectin.

7.2.1
a-Amylases

Thereisalonghistory of safeuseofa-amylasesfromseveral
microbial source organisms, including B. lichenifomis’. In particular,
bacterial a-amylases have been used in the starch industry sincethe
1 9 5 0 ~and,
~more
~recently,
thermostablea-amylases from
B. licheniformisand B. stearothermophilushavebeen ~ ~ e d In~ ~ , ~ ~ .
1973,GRAS petition 3G0026I2,whichproposedtheaffirmation of
the GRAS status of carbohydrase and protease preparations from
B. licheniformis,
was
accepted
filing
for
by the FDA”.

0 Carbohydrases from €3.licheniformis have been marketed in the US

as GRAS since that time.In1983,
carbohydraseand
protease
enzyme product
derived
the FDAaffirmed that mixed
from
B. licheniformisis GRAS whenused to hydrolyzestarchesand
proteins in the production of certain foods13.Species of Bacillus
have a long history of safe use in food prod~ction’~~. Therefore, the
transfer of an a-amylase gene from a nonpathogenic, nontoxigenic
source, B. lichenifomis, to thesamesafehost, B licheniformis, is
regarded as a safe system for enzyme prod~ction’-~.

7.2.2 SubstantialEquivalence

Severalexpertgroups,aswell as FDAandFDAscientists have

discussedtheconcept of substantialequivalencerelative to food
safety a s ~ e s s m e n t ~. ~Essentially
~ ” ’ ~ ~ all these groups conclude that
if afoodingredient is substantiallyequivalent to anexisting food
ingredient known to be safe, then no further safety considerations
other than thosefor the existing ingredient are necessary.

In addition, FDA has applied this concept in the determination that

several
enzyme preparationsare
safe for use in food10.47.4a . In
particular, differences in glycosylation between enzyme proteins was

I
~
0 considered.

safely
FDA has

consumed but having

also
stated that enzyme

differences in specific
proteins
demonstrated to besubstantiallyequivalent to enzymesknown to be
properties
due to

Novo Nordisk / Termamyl LC a-amylase information 16

 *
In
.I

a changes in the enzymeaminoacidsequencebynaturalselection, 8

z

-
chemicalmodification, or site-directedmutagenesiswouldnot raise
0
safety concernsg~”. >
0
z
m
,
TheTermamylLC a-amylase enzymefromthegeneticallymodified e
B. lichenifomis is substantiallyequivalent to a-amylase from an B;f
unmodified B. licheniformis. TermamylLCa-amylase is functionally
equivalent and within the range of natural variation of B. licheniformis
a-amylases (See section 3).

As noted by Kessler et al (1992), there is variation, due to genetic

polymorphism, in DNA and amino acid sequence, protein structure,
and optimal conditions of use among enzymes that have the same
fundamentalcatalyticactivity.a-Amylasesfromdifferentstrains of
B. lichenifomis exhibit different p r o p e r t i e ~
and
~ ~have
~ ~ ~nearly, but not
exactly,
identical
amino
acid
sequences (see
Section 3 and
Appendix B, Figure 2).

Termamyl LC has been modified to have increased stability at lower

calciumconcentrations,highertemperatures,andlowerpHthan
naturallyoccurring B. lichenifomis a-amylases(seeSection 3).

0 However, the properties and amino acid sequence

a-amylase are
within
the
range
of
naturally
occurring
biological
of Termamyl LC

diversity of B. lichenifomis a-amylases (see Section3). Termamyl LC

hasthesamemolecularweight,96.7%aminoacididentity,and
significantly similar structure as other B. lichenifomis a-amylases. In
addition,Termamyl LC hasthesamefunctionalactivityas other
Bacillus a-amylases (see Section3).

Novo Nordisk and other enzyme manufacturers have produced and

marketedsignificantamounts of modifiedenzymes for non-food
technical applications such as use in laundry detergents for several
years, the first one developed in 199125~4931*Append’x . These modified
enzymes are substantially equivalent
to
traditional
microbially
derived
proteases, amylases
and
lipases.
NovoNordisk has
communicated with the US Environmental Protection
Agency
regarding the manufacture and use of these enzymes. These and
other
manufacturer’s modified
products
have
been
safely
manufactured and used.

The Termamyl LC a-amylaseissubstantiallyequivalent to other

B.lichenifomis a-amylases andis safe for usein food.

Novo Nordisk / Termamyl LC a-amylase information 17

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m

0 7.3 Safety of the Manufacturing

Process
0
Termamyl LC meets the general
enzymepreparationsasoutlined
andadditional
requirements
in the monograph on Enzyme
for
e
5
Preparations. in the Food Chemicals Codex. As described in 8
Section 4, the a-amylase preparation is producedin accordance with
currentgoodmanufacturingpractices,usingingredients that are I ’y”B
‘I
acceptablefor general use in foods,andunderconditions that I
ensureacontrolledfermentation.Thesemethodsarebased on
generally available and accepted methods usedfor the production of
microbial en~yrnes~’”~.

7.4
Safety
Studies

Thissection describes thestudiesandanalysesperformed to

evaluate the safetyof theuse of Termamyl LC.

7.4.1 Description of Test Material

Thesafetystudiesdescribedbelowwereallconducted on a test
batch of the a-amylase preparationwhichwasobtainedby the

0 mixing of four sub-batches, each of which were produced according

tothedescription given in Section4,except that standardizationwas
omitted.

7.4.2 Studies

The following studies were performed:

1) Gene mutation (Ames test with S.typhimuriurn).

2)Chromosomalaberrations (in vitro cytogeneticswithhuman
lymphocytes).
3) Subchronic toxicity (Rats- 13 weeks)

The results of these tests are summarized in Appendix I.

7.5 Estimates of HumanConsumptionandSafetyMargin

Asstated in Section 6.4, Novo Nordisk has experimentally verified

that the Termamyl LC a-amylase enzyme protein is inactivated and
removed during the conditions and steps used for industrial starch
processing. Therefore, the potential human exposure to the enzyme
from the consumptionof food is negligible.

Novo Nordisk / Termamyl LC a-amylase information 18

 2
ul
m
.I

In addition,
dietary
exposure to anenzyme preparation that is 6
@ ;e

-
derived
from
a controlled fermentation ofnonpathogenic,
a
nontoxigenicmicroorganismthatdoesnotproduceantibiotics,and >
0
that is processed using substancesthatareacceptablefor general 0
use in foods,would not ordinarilypresentabasisforsafety
concern”. Termamyl LC meets these criteria.

However,inorder to demonstratethehypothetical“worstcase”
situation, the following calculations are made assuming that all sugar
andsweetenersareproducedfromstarch in processes using
Termamyl LC as a processing aid. Furthermore, it is assumed that
all enzyme protein is retained in the sugar and sweeteners.

Per caDita consumDtion of suaar and sweeteners

The average human intake of sugar and sweeteners is estimated by

usingwellestablishedstatisticsfromvarious c o ~ n t r i e s ~ ~The
’~~.
consumption of refined sugar and high fructose corn syrupin the US
is 143 g per person per day. This is the highest of the daily intake
figures and is usedin thecalculations below.

Fiaures used in the calculation

0 Consumption of sugarandsweeteners:143g/day
Maximum
Termamyl LC dosage: 0.05%
Yield sweetener/starch: 1 kg sweetener / 1 kg starch
E-TOS: 3%
average
Weight
person: 60 kg
“Worstcase”dailvintake Der person of TermamvlLCE-TOSfrom
suaar and sweeteners:
143 gx 0.0005 x 0.03 = 2 x 1O3 g E-TOS per day

For an averaqe rlerson weiqhinq60 kq this corresoonds to:

3.3 x 10-5g E-TOS/kg body weightlday

It
must,however, be emphasizedthatthesefiguresareover-
estimated because:
- Notallsugar is fromstarch.
- Not all high-fructose corn syrup is from glucose (starch).
- Theenzymeproteinisinactivatedandremovedduringstarch
processing.

Novo Nordisk / Termamyl LC a-amylase information I9

a Exposure from Production of Alcohol

The enzyme is used in the early stages of alcohol production. It is

added to the starch, which is subjected to further processing steps,
including hydrolysis to fermentable sugars. The sugars are fermented
to produce alcohol and the alcohol is distilled. During these steps the
enzyme is inactivated. In addition, the inactivated enzyme protein is
not transferred withthe distillate, and thusit is not possible for enzyme
or enzyme residuesto be present in the final alcohol.

Safetv Marain

The safety margin is calculated as the dose level with no observed

adverseeffect(NOAEL) in ratsdividedbytheestimatedhuman
consumption.

of 3.6%
The test batch used for the 13 weeks rat study has a content
TOS.The
batch
hasan
a-amylase
activity of 216KNU(T)/g,
corresponding to 147 KNU(L)/g.

The dose level in the 13 weeks oral toxicity study in rats, which could
be given without any toxic effects, was equivalentto 0.58 g E-TOSlkg
body weighffday.

The safety margin in a “worst case situation”, can thus be calculated

to be:
0.58 g E-TOS/3.3 x lo5 g E-TOS = 1.8 x I O 4
7.6 ResultsandConclusion

Theresults of the tests described in section7.4.2show that the

Termamyl LC a-amylase enzyme preparation does not exhibit any
mutagenicactivity,clastogenicactivity or toxiceffectunder the
conditions of eachspecifictest. On the basis of theevaluation
contained in Sections 7.1 - 7.5, a review of the published literature,
the history of use of B. licheniformis,and the limited and well defined
nature of the geneticmodifications,theTermamylLC a-amylase
enzymepreparationcan be safelymanufacturedandusedasa
processing aid in the starch and alcohol industry as well as in other
food or non-food applications.

Novo Nordisk / Termamyl LC a-amylase information 20

 Y
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8. LIST OFAPPENDICES 8
2
@ A. pLiHllO8 plasmid map 0
>
0
B. Amino acid sequence comparisons
Figure I Termamyl LC I Termamyl / BAN
Figure 2 Termamyl LC / 3 B. lichenifomis a-amylases

C. Experiment Report- “Characterization of NOVOZYM 935 -

starch hydrolysis”; Reference No. 1998-05771-01

D. Experiment Report- “Characterization of NOVOZYM 935 -

reducing sugar formation during liquefaction”;
Reference No. 1998-05788-01

E. Experiment Report- “Saccharification of starch liquefied with

NOVOZYM 935”; Reference No. 1998-05791-02

F. Manufacturing Process Flow Diagrams

Figure 1 Fermentation
Figure 2 Recovery

a
G. Novo Nordisk Product Sheet for Termamyl LC

H. Novo Nordisk Detergent Industry Product Range Sheet

I. Summary of Toxicity Data, Low Ca Amylase,

Batch No. PPY 5977, July 3, 1998, Ref.: AMPa 1998-05943-01.

Novo Nordisk / Termamyl LC a-amylase information 21

9. LIST OF REFERENCES

1. Pariza, M.W. and Foster, E.M.. Determining the Safety of 0

5
Enzymes Used in Food Processing.Journal of FoodProtection, b
46:5:453-468, 1983. 2
2. Food and Drug Administration. 21 CFR g184.1027 Mixed
carbohydrase and protease enzyme product.

3. Berkowitz, D. and Maryanski, J.. Implications of biotechnology

on international food standards and codesof practice. Joint
FAONVHO Food Standards Programme, Codex Alimentarius
Commission, Eighteenth Session, Geneva, July 3-1 2, 1989.

4. IFBC (International Food Biotechnology Council). Chapter 4:

Safety Evaluation of Foods and Food Ingredients Derived from
Microorganisms in Biotechnologies and Food: Assuringthe
Safety of Foods Produced by Genetic Modification. Regulatory
Toxicology and Pharmacology 12:Sl-SI 96, 1990.

5. EU Scientific Committee for Food. Guidelinesfor the presentation

of data on food enzymes. Reportsof the Scientific Committee for
Food, 27th series, 1991.

6. Organisation for Economic Cooperation and Development,

Safety Evaluation of Foods Derived by Modern Biotechnology,
1993.

7. FAONVHO. Biotechnology and Food Safety, Report of a Joint

FAONVHO Consultation. FA0 Food and Nutrition Paper 61.
Rome, Italy. 1996.

8. Jonas, D.A., Antignac, E., Antoine, J.M., Classen, H.G., Huggett,

A., Knudsen, I., Mahler, J., Ockhuizen, T., Smith, M., Teuber, M.,
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Foods, Guidelines prepared bylLSl Europe Novel Food Task
Force. Food Chemical Toxicology 34:931-940, 1996.

9. Kessler, D.A., Taylor, M.R., Maryanski, J.H., Flamm, E.L., and

Kahl, L.S. The Safety of Foods Developedby Biotechnology.
Science 256:1747-I 749, and 1832, 1992.

10.
Food
and
Drug
Administration.
Enzyme
preparations
from 000830
animal and plant sources; Affirmation of GRAS status as direct
food ingredients. Fed. Regist. 60:32904-32912, 1995.

Novo Nordisk / Termamyl LC a-amylase information 22

 Y
tn
L

1I.
Food and Drug Administration. Substances generally recognized 0
as safe. Proposed Rule. Fed. Regist. 62:18938-18964, 1997. 2

-
0
>
12.Novo Nordisk N S . GRAS Petition 3G0026 proposing that mixed 0
carbohydrase and protease enzyme product derived from
Bacillus lichenifomis be affirmed as GRAS. Notice of filing. Fed. gfl 2
Regist. 38:26214, 1973.
rf
13. Food and Drug Administration. Direct food substances affirmed
as GRAS: Mixed carbohydrase and protease enzyme product.
Fed. Regist. 48:239-240, 1983.

14. Priest, F.G. and Alexander, B.J. A frequency matrix for

probabilistic identification of some bacilli. Gen. Micro. 134:301I-
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vector for Bacillus subtilis. Plasmid, 30:312-315, 1993.

lG.Villafane, R., Bechhofer, D., Narayanan, C.S., and Dabnau, D.

Replication control of genes of plasmid pE194. Journal of
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@ 17.McKenzie, T., Hoshino, T., Tanaka, T.,andSueoka,N. The

nucleotide sequence of the pUBl10: Some salient features in
relation to replication and its regulation. Plasmid 1593-103,
1986.

18. Bolivar, F.,Rodriquez, R.L., Greene, P.J., Betlach, M.C.,

Heyneker, H.L., and Boyer, H.W.. Construction and
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vitro preparation and specific mutagenesis of DNA fragments:
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20. Horton, R.M., Hunt, H.D., Ho, S.N., Pullen, J.K., and Pease, L.R.
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gene splicing by overlap extension. Gene 77:61-68, 1989.
...
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Safety Considerations for Biotechnology, 1992.

Novo Nordisk / Termamyl LC a-amylase information 23

 Y
z
In

22. Department of Health andHumanServices,NationalInstitutes of z2

-
Health
(NIH),
Guidelines
for
Research
Involving
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DNA FR 34496, 1994 as >
0
amended,
FR
59 40170,60 FR 20726,61 FR 1482,61 FR 0
2
8%
10004,62 FR 4782.

23. Food and Drug Adminstration. Secondary Direct Food Additives

Permitted in Food for Human Comsumption;
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Permitted in Feed and
Drinking
Water
Animals;
of
Aminoglycoside 3'-phosphotransferase II. Fed. Regist.
59:26700-26711, 1994.

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of Antibiotic Resistance Marker Genes in Transgenic Plants;
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63147505-47506, 1998.

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Enzymology, 2nd ed., Eds. Godfrey,T., and West, S.,
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a
26. Uhlig,H., Industrial Enzymes and Their Applications, John Wiley
and Sons, Inc., 1998.

27.Machius, M., Wiegand, G., and Huber, R.. Crystal structure of

calcium-depleted Bacillus licheniformis a-amylase at 2.2 A
resolution. J. Mol. Biol. 246545-559, 1995.

28. Machius, M., Declerck, N., Huber, R., and Wiegand, G..
Activation of Bacillus licheniformis a-amylase through a
disorder-+order transition of the substrate-binding site mediated
by a calcium-sodium-calcium metal triad. Structure 6:281-292,
1998.

29.Declerck, N., Joyet, P., Trosset, J., Garnier, J., and Gaillardin, C.
Hyperthermostable mutants ofBacillus lichenifomis a-amylase:
multiple amino acid replacements and molecular modelling.
Protein Engineering 8:10:1029-1037, 1995.

30. Joyet, P., Declerck, N., and Gaillardin, C.. Hyperthermostable

variants of a highly thermostable alpha-amylase. Biotechnology
1011579-1583,1992.

Novo Nordisk/ Termamyl LC a-amylase information 24

31.Yuuki, T., andUdaka, S.. Complete nucleotide
sequence
gene codingfor heat and pH-stablea-amylase of Bacillus
of a

licheniformis: Comparison of the amino acid sequences of three

-
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VI
I-

8
2
0
>
bacterial liquefying a-amylases
deduced
from the DNA 0
sequences. Journal of Biochem.
98:1147-1156,
1985. .a 2

32. Novo Nordisk Patent 6.licheniformis sequence W096/23874

33. Udaka, S., Tsukagoshi, N., and Yamagata, H.. Bacterial amylase
genes and their expression. J. Jpn. SOC. Starch. Sci. 33:112-
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34. Gurei, G.E..Patent JPI 98708389-A2, 17-04-1 987.

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Sciences, Food and Nutrition Board, Committeeon Food
Chemicals Codex, National Academy Press, Washington, D.C.,
p. 133, 1996.

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Processing, Joint FAONVHO Expert Committeeon Food
Additives, Compendium of Food Additive Specifications,Vol. 1,
Annex 1, FAO, 1992.

37. James, J. and Simpson, B.K.. Applicationof Enzymes in Food

Processing. CRC Reviews in Food Science and Nutrition.
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38.Crabb1 W.D. and Mitchinson, C.. Enzymes involvedin the

processing of starch to sugars. TIBTECH. 15349-352, 1997.

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ed., Eds. Godfrey, T., and West, S., Chapter 2.1, pp 13 - 47,
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From Rhizopus niveus: Affirmation of GRAS status as aDirect
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Enzyme Preparations Derived FromBacillus subtilis or Bacillus
amyloliquefaciens; Affirmation of GRAS Status as Direct Food
Ingredients. Fed. Regist. 64:19887-19895, 1999.

Novo Nordisk / Termamyl LC a-amylase information 25

 *VI
5
Environmental
42. Protection Agency.
Microbial
Products of 8
Biotechnology;
Proposed
Rule.
Fed.
Regist.
59:45526-45585, z

-
1994.
Microbial
Products of Biotechnology; Final Rule.
Fed. >
0
Regist. 62:17909-I 7958.
Final
Decision
Document:
TSCA 0
z
Section 5(H)(4) Exemption for Bacillus licheniformis. Item No.
3168. 8-
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industrial useof Bacillus licheniformis: a review. Appl. Microbiol.
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44. Paarup, A.M.. Summary of Toxicity Data, Low Ca Amylase,

Batch No. PPY 5977, Ref.: AMPa 1998-05943-01, July 3, 1998.

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Derived From New Plant Varieties. Fed. Regist. 57:22984-
23005,1992.

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enzyme preparations: Affirmation of the GRAS status. Fed.
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48. Food and Drug Administration. Secondary Direct Food Additives

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49. Morgan F.J. and Priest, F.G.. Characterization of a thermostable

a-amylase from Bacillus licheniformis NClB 6346. Journ. of Appl.
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50.Vihinen, M., and Mantsala, P.. Microbial Amylolytic €nzymes in

Critical Reviews in Biochemistry and Molecular Biology, ed.
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51. Rubingh, D.N.. Protein engineering from a bioindustrial point of

view. Current Opinion in Biotech., 8:417-422, 1997.

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4thed.,
Ed.
Kroschwitz,
J.I.,
Volume
pp.
567-620,
9, 1994. 000894

Novo Nordisk I Terrnamyl LC a-amylase information 26

 Y

'a
VI
'El
8
53.Aunstrup1K., Andersen, O., Falch, E.A., and Nielsen, T.K. 2

-
0
Production of Microbial Enzymes in Microbial Technology, 2"d >
ed.,
Vol. I, Eds. Peppler, H.J.
and Perlman, D., Chapter 9, 0
z
+y
pp.282-309, 1979.

5#.Aunstrup, K.. Production, Isolation, and Economics of

Extracellular Enzymes in Applied Biochemistry and If
Bioengineering, Volume 2, Enzyme Technology, Eds. Wingard,
L.B., Katchalski-Katzir, E. And Goldstein, L, pp. 28-68, 1979.

55.Chemical Economics Handbook. Source: Panorama of EU

Industry, 1994, European Commission, 1994.

56. Department of Agriculture, Economic Research Service, "Sugar

and Sweeteners-Situation and outlook Report", June 1991.

Novo Nordisk I Termamyl LC a-amylase information 27

 pE 194 origin
I

8.licheniformis

pLiHl108

B. licheniformis

Termamyl LC
cc -amylase

pLiHl108 plasmid map

Amino Acid Sequence Comparison
Figure 1

1 * * * ** * *** 50
TermamylLC ..VNGTLMQY FEWYTPNDGQ HWKRLQNDAE HLSDIGITAV WIPPAYKGTS
Termamyl ANLNGTLMQY FEWYMPNDGQ HWRRLQNDSA YLAEHGITAV WIPPAYKGTS
BAN ..VNGTLMQY FEWYTPNDGQ HWKRLQNDAE HLSDIGITAV WIPPAYKGLS

51 , < 100
TermamylLC QADVGYGAYD LYDLGEFHQK GTVRTKYGTK GELQSAIKSL HSRDINVYGD
Termamyl QADVGYGAYD LYDLGEFHQK GTVRTKYGTK GELQSAIKSL HSRDINVYGD
BAN QSDNGYGPYD LYDLGEFQQK GTVRTKYGTK SELQDAIGSL HSRNVQVYGD

101 150
TermamylLC WINHKGGAD ATEDVTAVEV DPADRNRVIS GEHLIKAWTH FHFPGRGSTY
Termamyl WINHKGGAD ATEDVTAVEV DPADRNRVIS GEHLIKAWTH FHFPGRGSTY
BAN WLNHKAGAD ATEDVTAVEV NPANRNQETS EEYQIKAWTD FRFPGRGNTY

151 -1 J -1 198
TermamylLC SDFKWYWYHF DGTDWDESRK LNRIYKF..Q GKTWDWEVSN EFGNYDYLMY
Termamyl SDFKWHWYHF DGTDWDESRK LNRIYKF..Q GKAWDWEVSN ENGNYDYLMY
BAN SDFKWHWYHF DGADWDESRK ISRIFKFRGE GKAWDWEVSS ENGNYDYLMY

199 4, 248
TermamylLC ADIDYDHPDV VAEIKRWGTW YANELQLDGF RLDAVKHIKF SFLRDWVNHV
Termamyl ADIDYDHPDV AAEIKRWGTW YANELQLDGF RLDAVKHIKF SFLRDWVNHV
BAN ADVDYDHPDV VAETKKWGIW YANELSLDGF RIDAAKHIKF SFLRDWVQAV

249 J 298
TermamylLC REKTGKEMFT VAEYWSNDLG ALENYLNKTN FNHSVFDVPL HYQFHAASTQ
Termamyl REKTGKEMFT VAEYWQNDLG ALENYLNKTN FNHSVFDVPL HYQFHAASTQ
BAN RQATGKEMFT VAEYWQNNAG KLENYLNKTS FNQSVFDVPL HFNLQAASSQ

299 348
TermamylLC GGGYDMRKLL NGTWSKHPL KSVTFVDNHD TQPGQSLEST VQTWFKPLAY
Termamyl GGGYDMRKLL NGTWSKHPL KSVTFVDNHD TQPGQSLEST VQTWFKPLAY
BAN GGGYDMRRLL DGTWSRHPE KAVTFVENHD TQPGQSLEST VQTWFKPLAY

349 398
TermamylLC AFILTRESGY PQVFYGDMYG TKGDSQREIP ALKHKIEPIL KARKQYAYGA
Termamyl AFILTRESGY PQVFYGDMYG TKGDSQREIP ALKHKIEPIL KARKQYAYGA
BAN AFILTRESGY PQVFYGDMYG TKGTSPKEIP SLKDNIEPIL KARKEYAYGP

399 448
TermamylLC QHDYFDHHDI VGWTREGDSS VANSGLAALI TDGPGGAKRMWGRQNAGET
Termamyl QHDYFDHHDI VGWTREGDSS VANSGLAALI TDGPGGAKRMWGRQNAGET
BAN QHDYIDHPDV IGWTREGDSS AAKSGLAALI TDGPGGSKRM YAGLKNAGET

449 483
TermamylLC WHDITGNRSE PWINSEGWG EFHVNGGSVS I W QR.
Termamyl WHDITGNRSE PWINSEGWG EFHVNGGSVS IYVQR.
BAN WYDITGNRSD TVKIGSDGWG EFHVNDGSVS IYVQK.
@ Amino Acid Sequence Comparison
Figure 2

1 * * * ** * *** 50
TermamylLC ..VNGTLMQY FEWYTPNDGQ HWKRLQNDAE HLSDIGITAV WIPPAYKGTS
Termamyl ANLNGTLMQY FEWYMPNDGQ HWRRLQNDSA YLAEHGITAV WIPPAYKGTS
M38570 ANLNGTLMQY FEWYMPNDGQ HWKRLQNDSA YLAEHGITAV WIPPAYKGTS
E01158 ANLNGTLMQY FEWYMPNDGQ HWKRLQNDSA YLAEHGITAV WIPPAYKGTS

51 , \ 100
TermamylLC QADVGYGAYD LYDLGEFHQK GTVRTKYGTK GELQSAIKSL HSRDINVYGD
Termamyl QADVGYGAYD LYDLGEFHQK GTVRTKYGTK GELQSAIKSL HSRDINVYGD
M38570 QADVGYGAYD LYDLGEFHQK GTVRTKYGTK GELQSAIKSL HSRDINVYGD
E01158 QADVGYGAYD LYDLGEFHQK GTVRTKYGTK GELQSAIKSL HSRDINVYGD

101 150
TermamylLC VVINHKGGAD ATEDVTAVEVDPADRNRVIS GEHLIKAWTH FHFPGRGSTY
T e 1 VVINHKGGAD ATEDVTAVEV DPADRNRVIS GEHLIKAWTH
rmamy FHFPGRGSTY
M38570 VVINHKGGAD ATEDVTAVEVDPADRNRVIS GEHRIKAWTH FHFPGRGSTY
E01158 VVINHKGGAD ATEDVTAVEVDPADRNRVIS GEHLIKAWTH FHFPGRGSTY

151 -1 -1 -1 198
TermamylLC SDFKWYWYHF DGTDWDESRK LNRIYKF..Q GKTWDWEVSN EFGNYDYLMY
Termamyl SDFKWHWYHF DGTDWDESRK LNRIYKF..Q GKAWDWEVSN ENGNYDYLMY
M38570 SDFKWHWYHF DGTDWDESRK LNRIYKF..Q GKAWDWEVSN ENGNYDYLMY
E01158 SDFKWHWYHF DGTDWDESRK LNRIYKF Q GKAWDWEVSN ENGNYDYLML

199 .1 248
TermamylLC ADIDYDHPDV VAEIKRWGTW YANELQLDGF RLDAVKHIKF SFLRDWVNHV
Termamyl ADIDYDHPDV AAEIKRWGTW YANELQLDGF RLDAVKHIKF SFLRDWVNHV
M38570 ADIDYDHPDV AAEIKRWGTW YANELQLDGF RLDAVKHIKF SFLRDWVNHV
E01158 ADIDYDHPDV PAEIKRWGTW YANELQLDGF RLDAVKHIKF SFLRDWVNHV

249 -1 2 98
TermamylLC REKTGKEMFT VAEYWSNDLG ALENYLNKTNFNHSVFDVPL HYQFHAASTQ
T e REKTGKEMFT VAEYWQNDLG ALENYLNKTNFNHSVFDVPL
rmamyl HYQFHAASTQ
M38570 REKTGKEMFT VAEYWQNDLG ALENYLNKTNFNHSVFDVPL HYQFHAASTQ
E01158 RAKTGKEMFT VAEYWQNDLG ALENYLNKTN FNHSVFDVPL HYQFHAASTQ

299 348
TermamylLC GGGYDMRKLL NGTVVSKHPL KSVTFVDNHD TQPGQSLEST VQTWFKPLAY
Termamyl GGGYDMRKLL NGTVVSKHPL KSVTFVDNHD TQPGQSLEST VQTWFKPLAY
M38570 GGGYDMRKLL NSTVVSKHPL KAVTFVDNHD TQPGQSLEST VQTWFKPLAY
E01158 GGGYDMRKLL NGTVVSKHPL KSVTFVDNHD TQPGQSLEST VQTWFKPLAY

349 398
TermamylLC AFILTRESGY PQVFYGDMYG TKGDSQREIP ALKHKIEPIL KARKQYAYGA
Termamyl AFILTRESGY PQVFYGDMYG TKGDSQREIP ALKHKIEPIL KARKQYAYGA
M38570 AFILTRESGY PQVFYGDMYG TKGDSQREIP ALKHKIEPIL KARKQYAYGA
E01158 AFILTRESGY PQVFYGDMYG TKGDSQREIP ALKHKIEPIL KARKQYAYGA

399 448
TermamylLC QHDYFDHHDI VGWTREGDSS VANSGLAALI TDGPGGAKRM YVGRQNAGET
Termamyl QHDYFDHHDI VGWTREGDSS VANSGLAALI TDGPGGAKRM YVGRQNAGET
M38570 QHDYFDHHDI VGWTREGDSS VANSGLAALI TDGPGGAKRM YVGRQNAGET
E01158 QHDYFDHHDI VGWTREGDTS VANSGLAALI TDGPGGQSE. CMSAGKTRET
449 483
TermamylLC WHDITGNRSE PWINSEGWG EFHVNGGSVS IYVQR.
Termamyl WHDITGNRSE PVVINSEGWG EFHVNGGSVS IYVQR.
M38570 WHDITGNRSE PVVINSEGWG EFHVNGGSVSIYVQR.
E01158 WHDITGNRSE PWINSEGWE SFTVNGGSVS IYVQR
 e0

-
Application TechnologyI
Starch Degrading Enzymes
I9986577161
1998-07-02
z
BEN'LoSc 95!l
If
Characterisation of NOVOZYM 935 - starch hydrolysis

Purpose

To verify that the catalytic properties (action pattern on starch) of Novozym 935 were
identical to thoseof the native enzyme, starch hydrolysis experiments were carried out
as described below,and changes in the carbohydrate spectra monitoredby HPLC.
For comparison, thermostable a-amylases from Bacillus licheniformis (Termamyl 120L
type L) and Bacillus stearothermophilus (Termamyl 120L type S ) were included in the
trials.

Background

Thermostable a-amylases havebeen used for many years in the Starch processing
industry for the manufacture of glucoseand fructose syrups. They are employed in
the first stageof the process - liquefaction. Duringliquefaction, a starch suspension
(typically 35% dry solids (DS) is heated to 105-110°C in a jet cooker in the presence
of a thermostable endo-acting a-amylase, which partially hydrolyses the starch,
thereby reducing viscosity.

The liquefaction process is completed before significant starch hydrolysishas taken

place, but if the reaction
is allowed to proceed, differences in the action pattern of the
amylases on starch can be observed. For example, thermostable a-amylases from
Bacillus licheniformis generate significant amounts of maltopentaose, maltotriose and
maltose, whereas the thermostable a-amylase from Bacillus stearothermophilus pro-
duces significantly more maltohexaose.

Novozym 935is a protein engineered variant of the commercially available enzyme

Termamyl12OL type L derived from Bacillus licheniformis. The enzyme has been
and it is more stable than
modified to make it more robust under industrial conditions
the conventional enzyme athigher temperatures and lower pH values. What is more
important, Novozym 935 does not require additional calciumfor stabilisation.

Work carried out

15.0 g Waxy corn starch (Cerestar044201 WE 5676 5/97) were suspended in 300 ml
0.01M pH 6.0 acetate buffer and heated to 100 "Cand cooked for two minutes under
constant stirring.After cooling, 209 aliquots were weighedout into 6 x 50 ml Blue-cap
flasks with magnetic stirrers. The flasks were cappedand allowed to equilibrate in a
water bath, at60"C,for 30 minutes.

~ "
.

1R3 106
 1998-05771-01

The following enzyme samples were tested.

Termamyl 120L
type L (B.licheniformis) batch AXR 04059
105.6
KNUlg'

Termamyl120Ltype S (B Aearothermophilus) batch AEP 3011105.3KNUlg

Novozyme
935 (B.licheniformis) batch
980415 HGO 172.5
KNU/g

The enzymes were dilutedin Milli-Q waterso that the final concentration (KNU/g
starch) wasas follows: * '

Enzyme Termamyl type L Termamyl type S Novozyme

935
starch KNUlg 130 115 166

1 ml of diluted enzyme was added each to flask. 2 ml samples were taken at regular
intervals and the reaction stopped by addingthe 2 ml sampleto a test-tube containing
2 ml deionised waterto which had been added 2 drops of 1M HCI. After thorough
mixing, the tube was sealedwith a glass stopper andthen placed in a boiling water
bath for 15 minutesto inactivate the enzyme.(l,2 and 3)

After cooling,the sample wasfiltered through a 0.2 pm before being submittedfor

HPLC analysis(4).

Results

From the attached HPLC chromatograms,it can be seen that,on prolonged hydroly-
sis, the major oligosaccharides produced by Novozyme 935 and Termamyl 120L
type L are maltopentaose, maltotriose and maltose. Termamyl 120L typeS has a
slightly different action pattern in that significant amountsof maltohexaose are formed
initially.

Conclusion

and Termamyl 120L typeL on waxy corn starch

The action patterns of Novozym 935
are almost identical.

References

1) Experimental Plan: LoSc-016-98, 060598

2) Laboratory Journal: LoSc 6914, pages069-070,060598
3) Standard Procedure: ABF-SP-5008.01/01Hydrolysis of Amylopectin
4) Standard Method: ABF-SM-5122.02/01Determination ofsugars (DPI-
DP10+) in Hydrolysed Starch syrups
5) Standard
Method:
EAL-SM-0009.01/02

KNU: Kilo Novo Units analysed according to Novo Nordisk standard method EAL-SM-
0009.01/02(5)
2
088%h03
a ,
 1998-05771 -01

Carbohydrate profile of starch hydrolysed with Terrnamyl 120L type L (B.licheniformis)"

1 hour 2 hours 4 hours

U
n

"

n
n

6 hours 24 hours 48 hours

..DP = degree of polymerisation. DP I = mono-saccharide, DP2 = di-saccharide etc

3
 1998-05771-01

Carbohydrate profile of starch hydrolysed with Termamyl 120L type S (B .stearothermophilus)

1 hour

6 hours 24 hours 48 hours

8%

I
 1998-05771-01

Carbohydrate profile of starch hydrolysed with Novozym 935 (B.Iicheniformis)

1 hour 2 hours 4 hours

i i , \

i L-

24 hours

5
 0
>
0
Application TechnologyI 1998-05788-01
2
Starch Degrading Enzymes 1998-07-02
BEN/LoSc 8%
-
Characterisation of NOVOZYM 935 reducing sugar formation rf
during liquefaction

Purpose

To demonstrate that the catalytic properties (starch hydrolysis) of Novozym 935 are
similar to those of the thermostable a-amylases from Bacillus licheniformis (Termamyl
120L type L) and Bacillus stearothermophilus (Termamyl 120L type S), starch hy-
drolysis experiments were carried out as described belowand the release of reducing
sugars was monitoredby the Neocuproine method(3).

Background

Thermostable a-amylases areused in the starch processing industryfor the liquefac-

tion of starch.The liquefaction process is completedbefore significant starch hydroly-
sis has taken place, but if the reaction is allowedto proceed, the enzyme continuesto
break downthe starch, generating reducing sugars which can be measured andex-
pressed as DE' .

Work carried out

Corn starch slurries were prepared by suspending 1.8 kg Cerestar C'Pharm GL

03406 (89 % starch) in deionised water and makingup to 30 kg. The pH was adjusted
to 6.0 at ambient temperature,after the addition of4.40 g CaCI2.2H20.

The following enzymes were used:

Termarnyll2OL
type L (6.licheniforrnis)
batch AXR 04059 105.6
KNUlg"

Termarnyl120Ltype S (B .stearothermophilus)batch AEP 3011105.3KNUlg

Novozym
935 (B.licheniformis)
batch
980415 HGO 172.5
KNUlg

The following amountsof a-amylase were added:

1) Termarnyll2OL type L 63 NUlg DS"

2) Terrnarnyll2OL type S 52 NUlg DS
3) Novozym935 71 NUlg DS

..DE = Dextrose equivalent. % reducing sugars expressedas D-glucose

KNU: Kilo Novo Units analysed accordingto Novo Nordisk standard method EAL-SM-
0009.01102 (4)
a. .

DS = Dry solids (% weightlweight)

a.
 1998-05788-01

pH adjustment, the conductivity of the starch slurries

After the addition of enzyme and
was adjusted to350pS using NaCI. The standard conditions were as follows:

Substrate concentration 35 % w/w (initial)

31.6-31.9% w/w (final)
Temperature 105"C, 5 min (Primary liquefaction)
95"c, 120 min (Secondary liquefaction)
pH (initial) 6.0

level Calcium 40 mgkg

After jet-cooking (Primary liquefaction), the liquefied starch was collectedand trans-
ported in sealed thermos-flasks from the pilot plant to the laboratory, wheresecondary
liquefaction was continued at 95 "C. Samples weretaken at regular intervalsand the
reaction stopped. by lowering thepH with HCI. After dilution, reducing sugars were
determined using the Neocuproine method (3).

Results
Time DE
(mins) 935
Novozyrne Termamyl
type L Termamyl
type S
2.2 15 2.8 3.9
30 5.2 4.3 5.7
45 6.7 (6.4) 8.1
60 8.2 6.4 10.0
90 11.4 9.9 14.5
120 14.7 12.0 16.6

Reducing sugar formation

16 --

14 -~

12 -.
10 "

w
n
8 --

6 --

4 "

2 "

0 20 40 60 80 100 120
Time (mins.)

I Novozym 935 Termamyl type L A Termamyl type S 1

2
 1998-05788-01

Conclusion

Reducing sugar formationfollows a similar coursefor all three enzymes.

References

1) Experimental Plan: LoSc-018-98, 060598

2) Laboratory Journal: LoSc 6914, pages 080-083, 190598
3) Standard Method: ABF- SM-5160.01/01Determination of Dextrose
Equivalent (Neocuproine Method)
4) Standard
Method:
EAL-SM-0009.01/02

3
 L
0
z

-
5!
0
Application TechnologyI
Starch Degrading Enzymes
199805791-02
z

Saccharification of starch liquefied with NOVONM 935

Purpose

In order to verifythat starch liquefied with Novozym935 was essentiallythe same as

starch liquefied with other commercially available enzymes,liquefaction trials, followed
by saccharification, wereset up as follows.

Background

The conversion of starch to glucose comprises two hydrolytic stages: liquefaction

and saccharification. During liquefaction,a starch slurry containing 30-40%dry sol-
ids, is pumped through a "jet-cooker" where the temperature is raised to typically
105°C by direct steam injection. To reduce the viscosity of the cooked starch, a ther-
mostable a-amylaseis added tothe starch slurry during liquefaction. The slurry is
maintained at this temperaturefor 5 minutes, after which it is flash-cooled to about
95°C. The enzyme, which is still active,is allowed to continue the hydrolysisfor up to
two hours. After this treatment, the starch willhave a (DE)*of 10-15.

After liquefaction, the partially hydrolysed starchis converted to D-glucose

(saccharification) by the action of the enzyme glucoamylase. The processis carried
out at 60"C,pH 4.5 for a period of 48-96 hours. The yield of D-glucose is dependent
on the liquefaction process. There might be losses due to by-product formation orto
incomplete hydrolysis resulting from the choice liquefying
of enzyme.

Work carried out

Liquefaction

Corn starch slurries were prepared by suspending11.8 kg Cerestar C*PharmGL

03406 (89 YO starch) in deionised waterand making up to 30 kg. The pH was adjusted
to 6.0 at ambient temperature,after the addition of 4.40 g CaCI2. 2Hz0.

The following enzymes were used:

Termamyl
120L type(B.licheniformis)
L batch AXR 04059 105.6
KNUIg"

Termamyl120L type S (B .stearothermophilus) batch AEP 3011105.3KNUIg

Novozym 935 (B.licheniformis) batch

980415 HGO 172.5
KNUlg

LDE (dextrose equivalent): reducing sugar (%) expressed as D-glucose

KNU: Kilo Novo Units analysed accordingto Novo Nordisk standard method EAL-SM-
0009.01/02(5)

R
183106
 1998-05791-02

The following amounts of a-amylase were added:

1) Termamyll20Ltype L 63 NUlg DS-

2) Termamyl 120L type S 52 NUlg DS
3) Novozyme 935 71 NUlg DS

After the addition of enzyme and pH adjustment, the conductivity of the starch slurries
was adjusted to350pS using NaCI. The standard conditions wereas follows (1,2):

Substrate concentration 35 % w h (initial)

31.6-31.9YOw/w (final)
Temperature 105"C,5 min (Primary liquefaction)
95"c, 90 min (Secondary liquefaction)
pH (initial) 6.0

40 level Calcium mgkg

After jet-cooking(Primary liquefaction), the liquefied starchwas collected and

transported in sealed thermos-flasks-from the pilot plant to the laboratory, where
secondary liquefaction was continued at95 "C for 90 minutes. After 90 minutes, the pH
was lowered to4.0 and held there for 10 minutes to inactivate the enzymes.

Saccharification

4009 of substrate (equivalent to 124 g DS liquefied starch) were transferredto 500 ml

blue cap flasks which were then placed 60°C.The saccharification
in a water bath at
experiments were carried out in these 500ml blue-cap flasks, which were fitted with
magnetic stirrers, according to ABF-SP-5004.01 (3).The pH's were adjustedat the
start of saccharification, with the pH electrode calibrated
at the saccharification
temperature. No pH adjustment was made during the course of saccharification. The
following saccharification enzyme (glucoamylase) was used:

A M G (AMR 2530C) 437 AGUlg-

(1,2):
The standard conditions used for saccharification were as follows

Substrate
concentration 31 %
(initial)
w/w
34 % w/w (final)
Temperature 60°C
(initial) pH
0.24 dosage Enzyme AGU/g

-....
DS = Dry solids (% weighheight)
AMG = Amyloglucosidase unit (6)
2
 1998-05791 -02

Samples were takenat set intervalsand heated in a boiling water bathfor 15 minutes
to inactivate the enzymes.After cooling, the samples were diluted1:2 with deionised
water and then treated with a mixedbed ion-exchange resin (Bio-Rad AG 501K8 (D))
for 30 minutes to remove ash and solubleN. Before being analysed by HPLC (ABF-
SM-5121.02) (4), the samples were filtered (Sartorius MlNlSART NML 0.2 micron).
The results aregiven in the tables below.

Results

Liquefaction: Novozym 935 (B.licheniformis) batch 980415 HGO

Start DE: 11

hours PH DP3
DPq- DP2 DP4+
24 4.1 0.4
93.0 2.0 4.6
48 4.0 0.3
95.6 2.7 1.4
95.7
72 3.9 0.3 3.3 0.7
95.5
95 3.8 3.7 0.4 0.4

Liquefaction: Termamyl 12OL type L (B.licheniformis) batchAXR 04059

Start DE: 10

hours PH DP3
DP1 DPz DP4+
24 4.1 0.4
92.9 2.0 4.8
48
95.5 4.0 0.3 2.7 1.5
95.6
72 3.9 3.3 0.3 0.7
95
95.5 3.8 0.4 3.7 0.4

Liquefaction: Termamyl 120Ltype S (B stearothermophilus) batch AEP 3011

Start DE: 15

hours PH DP3DPl DP2 DP4+

92.6
24 4.2 1.9 0.4 5.1
48
95.4 4.1 0.3 2.7 1.6
72
95.7 3.9 0.3 3.3 0.7
95
95.5 3.8 0.4 3.7 0.5

Conclusion

There is no detectable difference in the glucose yield or carbohydrate composition

after saccharification of starch, liquefied with Novozym935 or Termarnyl type L orS

References

1) Experimental Plan: LoSc-018-98, 060598

2) Laboratory Journal: LoSc 6914, pages080-085,060598
3) Standard Procedure: ABF-SP-5004.01/01
Saccharification
4) Standard Method: ABF-SM-5121.02/01Determination of sugars
(DP1-
DP4+) in Hydrolysed Starch syrups.
5 ) Standard method EAL-SM-0009.01/02
6) Standard method EAL-SM-0022.01/02

... DP: Degree of polymerisation. DP1= monosaccharides, DP2= disaccharidesetc. OOBbal4l

3
 Termamyl LC

Fermentation

FEED A CAI D
NTIFOAM

RAW
MATERIALS
G A SE X H A U S T

4 STERILE
F1 LTRATIO N

FIGURE 1
V

S
0
 Enzyme Business
g=-

Termamyl" LC
Description Termamyl LC is a thermostable alpha-amylase used for the liquefac-
tion of starch. Termamyl LC is a protein engineered alpha-amylase
produced by a genetically modified strain of Bacillus lichenformis.
Termamyl LC is a remarkable new liquefaction enzyme which can
operate at lower pH and calcium levels than conventional thermostable
alpha-amylases. This brings several advantages to its applications
which can all be translated into reduced operating costs.

Specification Appearance
Termamyl LC is a brown liquid with a density of 1.20-1.25 d m l .

Activity
Termamyl LC is available with a standard strength of 120 KNU(L)/g.
A detailed description ofNovo Nordisk's method of analysisis avail-
able on request.

Food-grade status
Termamyl LC complies with FAONHO JECFA and FCC recom-
mended purity specifications.

Packing
Termamyl LC is available in 30 kg jerrycans, 250 kg steel drums,
1200 kg Schiitz containers and in bulk by the truck load.

Application Termamyl LC has been developed for the starch industry. The product
is like other Termamyl types used forthe continuous liquefaction of
starch in steam jetcookers or similar equipment operating attempera-
tures around 105"C, thereby taking advantage of the extreme heat sta-
bility of the enzyme.
For the liquefaction of starch, a dosageof 0.45 kg Termamyl LC per
ton of starch at pH 5.4 and 5 ppm Ca is recommended as a starting
point.
Detailed recommendations concerning the application of Termamyl LC
are given in an Application Sheet, "How to Use TermamylLC for the
Liquefaction of Starch", available on request.

Safety Enzymes are proteins and inhalation of dust or aerosols may induce
sensitization and may cause allergic reactions in sensitized individuals.
Some enzymes may irritate the skin, eyes and mucous membranes
upon prolonged contact.
 . ,
, < . ? . . - The product may createeasily inhaled aerosols if splashed o r vigor-
, a o u s l sti6ed.
i . . . dry out and,create dust.
, .
Spilled
product'may
" /

- . , , < _, " I '. x

.. Spilledmaterialshould beflushed away with water. Avoid splashing.

Material Safety Data Sheets and separate materialdescribing how to
handle the product safely are available upon request.

Storage Enzymesgradually lose activity overtime

depending on
storage tem-
perature. Cool conditions are recommended.
When stored at 25"C, Termamyl LC will maintain its declared activity
for 3 months.
When stored at5"C, Termamyl LC will maintain its declared activity
for 12 months.
Extended storageand/or adverse conditions, including higher tem-
perature, may lead to higher dosage requirements.

-
Y
VI

Enzyme Business and mgulabonsLaws, th~rdparty nghts may prevent

customers horn mportmg, pmcesung, and/or
applyrng
F0
reselling ceiiam products in a gwen manner. I t IS the 2
Novo Nordisk AIS Tel. +45 4444 8888 responub!lky of the customers that thew spmfic use of
0
5
Novo Alle Fax +45 4444 1021 praduck fmrn NOMNordsk does not infnnge reievant
laws andregulabons and, fufihemre, does not mfnnge
2880 Bagsvaerd Telex 37560
2
paw
patents orolherlhm nghls
Denmark
The contenls of thts document am subject to change -,
w,lhout fufihernolice ,.

8 1131s-GB 200 Aug. 9998 0 Nova Nadlak U S B

i .
"9 Enzyme Business

Detergent Industry

Pro teases Amylases

Alcalase' Terrnamyl'
A bacterlal protease effectlve under neutral and A bactertal amylase whlch is able to work at rela-
mildly alkaline condittons (pH 7-10) Sultable for tlvely high (alkalme) pH values (up to pH 11) and
soaking preparations and llquld as well as powder at hlgh temperatures (up to 100°C).
detergents Alcalase is also characterized by being
non aggresslve towards wool
BAN
A traditlonal bactertal amylase whrch is effective at
Savinase' neutral and mlldly alkaline conditions and at low
A bacterial protease whlch is effectlve under alka- and moderate wash temperatures (Only avatlable
line condltions (pH 8-1 1) Savinase IS the most as a co-granulate with Savinase )
commonly used protease and works well under
most washing conditlons.
Duramyl'
A protein-englneered variant of Termamyl charac-
Everlase" tertzed by having an excellent storage stability in
A protein-engrneered varlant of Savmase charac- bleach-contalning detergents as well as superior
terized by havtng an excellent storage stability in performance at medlum temperatures compared
-
,
.
"
bleach-contaming detergenls to Termamyl.
- .

Esperase"
A bacterial protease which IS effective under Cellulases
strongly alkaline conditions (up to pH 12 approxi-
mately) even at very hlgh temperatures
Celluzyrnem
A fungal enzyme product contatnrng a cellulase
complex active in the neutral to moderately alka-
f ipases line region Celluzyme is well-suited for washing
temperatures from 15°C to 65°C and It works on
garments made from cellulosic fibres, as for ex-
Lipolase' ample cotton and cotton blends
A fungal lipase Lipolase is effective under alkaline
condttlons (up to pH 12 approxmately) and a t a
broad temperature range Carezyme'
A monocomponent cellulase showing remarkable
colour brlghtenlng benefits that works on cotton
Lipolase Ultra and cotton-containlng blends Carezyme is effect-
A protein-engineered variant of Lipolase In gen- ive under neutral to moderately alkalme condi-
eral, the washlng performance of Lipolase Ultra IS tions Besides it is well-sulted for washmg tem-
superior to that of Lipolase at high Ionic strength, peratures rangmg from 15°C to 65°C
low temperatures and htgh pH, and various com-
blnatlons of these conditlons

LipoPrime"
A protein-engineered variant of Lipolase
LlpoPrlme shows superlor fat-removing efficacy in
comparison to Llpolase and Lipolase Ultra tn most
detergent formulatrons Product Sheets avatlable on request
 - .
, . ,
Single Enzyme Products
I. Declared
Product * activity* *

Proteases
..
iI

Alcalase 2.0 T 2 0 AU/g

Alcalase 3.0 T 3.0 AUtg
Alcalase 2.5 L. Type DX 2.5 AU/g

Esperase 4.0 T 4.0 KNPUig

Esperase 6 0 T 6:O KNPU/g
Esperase 8.0 L, Type A 8.0 KNPU/g

Everlase 6.0 T 6.0 EPU/g

Everlase 8 0 T 8 0 EPU/g
Everlase 16 L, TypeEX 16 0 EPU/g

Savinase 6.0 T, Type W 6.0 KNPU/g

.-- , .
Savlnase 8.0 T, Type W . . , 8.0 KNPU/g
Savinase 12 T, Type W 12.0 KNPUlg
Savlnase 16 L, TypeEX 16.0 KNPU/g

Amylases
BAN See list of combined products next page

Termarnyl 60 T 60 KNU/g
Termamyl 120 T 120 KNU/g
Termarnyl 300 L, Type DX 300 KNU/g

Durarnyl60 T 60 KNU/g
Duramyl 120 T 120 KNU/g
Duramyl 300 L 300 KNU/g
~~

Cellulases
Carezyrne 900 T 900 S-CEVU/g
Carezyrne 4500 T 4500 S-CEVU/g
Carezyrne 1000 L 1000 S-CEVU/g
Carezyrne 4500 L 4500 S-CEVU/g

Celluzyme 0.7 T 700 DCU/g

Lipases , _

Lipolase 100 T 100 KLU/g

Lipolase 100 L, TypeEX 100 KLU/g
".
Lipolase Ultra 50 T 50 KULU/g

LipoPrlrne 50 T 50 KLU(P)/g

Coloured Products 0
Declared
Product* activity* *

Savlnase 6 0 T, Type W Green 6.0 KNPU/g

Savinase 6.0 T,Type W Blue 6.0 KNPU/g.
I I
 r .

, .
, I
. .
Dimensions
~approx.cm

Jolume Net Tare Height Diam-eter Granu- Liquid

weight approx, length lates
Type kg kg x width

b EWC One trlp 1000 I 1000 82 116 -120 x 100 ' 0

container 600 58 98 120 x 80
(Schutz)

c' Steel drum 215 I 200 20 88 59 0

wlth epoxy . .
lining
I .

d Polyethylene drum 220 I 200 8.5 94 58

e:Jerrycan 27.5 I 25 1.4 47 29 x 26 0

~~

j. IBC (BigBag) 1000 I 1000 4 115 105x110 ' 0

Intermediate (on pallet)
Bulk Contarner. 91 x 91
on a 105 x 105 pallet (bottom of bag)
(See separate Novo
Nordisk leaflet B 377) i

m Flbre drum 50 I 40 25 57 36 0
lined wrth
polyethylene bag

.-

m
 E0
2

0
2
Enzyme Business Toxicology Date : 1998-07-03
Ref.: AMPa
File : 1998-05943-01

SUMMARY OF TOXICITY DATA

LOW CALCIUM AMYLASE

Batch No. PPY 5977

Author :
Anne-Mette Paarup

lssued by :
Novo Nordisk A/S
Novo All6
2880 Bagsvzrd
Denmark

183 1 0 6
 CONTENTS

PAGE

1. Abstract ......................................................................................................................................... 3

2. Test substance ............................................................................................................................. 3

2.1 Characterisation..................................................................................................................... 3

2.2 Production organism ............................................................................................................. 3

3. Toxicity Data .................................................................................................................................. 4

3.1 General toxicity ...................................................................................................................... 4

3.1.1 Toxicity studies by oral (gavage) administrationto rats for 13 weeks .......................... 4
. . ...........................................................................................................................
3.2 GenotoxlcQ 5

3.2.1 Testing for mutagenic activity in Salmonalla typhimurium and Escherichia coli .......... 5

3.2.2 Cytogenetic study in cultured human peripheral lymphocytes..................................... 6

4. References .................................................................................................................................. 7

0 Last page ................................................ .......................................................................... .................. a

2
Summary of Toxiclty Data (Low Calcium Amylase)

sl..
1. ABSTRACT

Low Calcium Amylase is an Alpha-amylase withhigh activity at low Calcium levels. The production
organism is a Bacillus licheniformis strain, and the enzymatic activity is expressed in KNU-Units
The toxicological programme was carried out to fulfil the toxicological requirements forregistration
of Low Calcium Amylase for use in starch industry (food grade).

All studies were carried out in accordance with the current EU and OECD guidelines andin compli-
ance with the OECD principles of Good Laboratory Practice (GLP).

The main conclusions of thesafety studies can besummarised as below

At repeated oral dosing of rats for 13 weeks threerats of the middose group and two ratsof the
highdose group died during the study (in the period between Week 9 and Week 12). The rats died
soon after dosing, and on basis of macroscopical findings and histopathological examination, the
deaths appeared to be related to the dosing procedure rather than the test substance. The NOAEL
(No Observed Adverse Effect Level) of Low Calcium Amylase wasplaced at the high-dose level, 15
mllkg bwlday (equivalent to 0.589 TOSlkg bwlday).

Low Calcium Amylasedid not induce gene-mutations in bacteria.

In an in vifro chromosome aberration test using humanelymphocytes, Low Calcium Amylase

showed no clastogenic activity.

2. TEST SUBSTANCE

2.1 Characterisation

Test Substance Low Calcium Amylase

Batch Number PPY 5977

Appearance Brown fluid

Analysis:
Enzyme Activity 216 KNU(T)Ig

Water (KF) 90.5%

Ash (600’) 5.9%

Total Organic Solids (TOS)’ 3.6%

Specific gravity (glml) 1.072

’ %TOS = 100%- % water- % ash- % dlluents

2.2 Production 0001kZ9r
The production organism is a Bacillus licheniformis strain, Ad-1-OlB (LiH 1159).
The batch of Low Calcium Amylase, Batch No. PPY 5977, used in the present toxtcological
programme is a mixture of four sub-batches.
UwUmb
3
Summary of Toxicity Data (Low Calcium Amylase)
e
3. TOXICITY DATA

3.1 General toxicity

3.1.1 Subchronic (13-wk) Oral Toxicity Study with Low Calcium Amylase in Rats.
The study was carried out in accordance with the current guidelines of OECD(408,
1981) and EU (Commission Directive 87/302/EEC).

Procedures
LOWCalcium Amylase, Batch PPY 5977 was administered to groups of ten male and
ten female Wlstar rats by oral gavage at dosages of 1, 5 or 15 mWkg bwlday. The dose
levels were chosen on basis of a preliminary dose range finding study and corre-
sponded to 232, 1158 or 3473 KNU(T)lkg bwlday and to 0.04.0.19 or 0.58 g TOSlkg
bwlday, respectively. A similarly constituted group received the vehicle (tap water) only
and served as controls. All rats received a constant dose volume of 15 mllkg bwlday.
The animals were dosed daily over a period of 13 weeks.

Discussion of results
Three rats of the middose group (females) and two rats of thehighdose group (one
male and one female) died during the study(in the period between Week 9 and Week
12). The rats died very soon after dosing with a possible exception of one rat, which
was found dead in the afternoon following dosing in the morning. Signs indicative ofa
dosing error were evident in all these rats by macroscopic examination and the follow-
ing spectrum of findings were seen: foamylbloody foamy contents in trachea, haemo-
thoraxlhydrothorax, dark red, uncollapsed lungs, or, in one case, perforation of the oe-
sophagus. This theory was supported by the histopathologicalexamination; all five
animals demonstrated lungs with haemorrhages andlor hyperaemia, and two females
exhibited haemorrhages and infiltrates of mononuclear inflammatory cellsin the wall of
trachea. In addition, a mixed inflammatory cell infiltrate with haemorrhage was ob-
served in the wall of the oesophagus in one female. These observations suggest that
the dosing material has directly come into contact with the lungs and trachea, either
due to incorrect dosing via the trachea or via perforation of the oesophagus. The other
gross and histopathological changes observed in these animals werenot remarkable
with the exception of a cortical adenoma in the adrenals of one highdose animal, be-
cause such tumour is generally found spontaneously in animals of older age. However,
no related toxic or preneoplastic lesions were found in the adrenals of any other
treated rats. Therefore, the cortical adenoma is considered to be an isolated, fortuitous
finding. For these reasons, the death ofthe rats is considered to be relatedto the dos-
ing procedure rather than the test substance,although intercurrent deaths occurred
only in the mid- and highdose group.

General condition and behaviour were not adversely affected by treatment in any of
the groups Body weight, food consumption and food conversion efficiency were not
affected by treatment. Ophthalmoscopic examination did not reveal any treatment-
related changes.

Haematology investigations in Week 13 did not show any treatment-relatedchanges.

The clinical chemistry data revealed that alanine aminotransferase activities were sig-
nificantly decreased in males and females of the high dose group, and phospholipids
concentration was decreased in the males of this group too. The biological significance
of a decrease in these parameters is not clear, and the values were well within the
range of historical control data. Therefore their toxicological significance is doubtful

The renal concentration test showed a statistically significant increase in urinary vol-
ume, associated with a decreased urinary density in both males and females of the

0 00130
lowdose group. However, these findingswere not confirmed at higher dose levels and
are therefore considered to be fortuitous. There were no differences amongthegroups
in semiquantitative urinary observation or in microscopy of the urinary sediment
4
Summary of Toxicity Data (Low Calcium Amylase)
 The absolute and the relative weight of the kidneys was significantly increased in the
females ofthe highdose group. The organ weight analysis revealed no otherstatisti-
cally significant differences. The increase in the weight of the kidneys wasnot accom-
panied by histopathological renal changes. The high NaCl content of the test sub-
stance (approx. 5%; providing an additional NaCl intake of 750 mglkg bwlday in the
highdose group) is probably the cause of the increased kidney weights. Increased
kidney weight has been reported in rats fed diets with a high NaCl content.Therefore
the increased kidney weights in the females of the highdose group are most likely the
reflection of a physiological response of the kidneys to increased work load resulting
fromenhanced diuresis. .

At the macroscopic posf modem examination at the terminationof the study no treat-
ment-related findings were demonstrated. A dilated oesophagus and a impacted stom-
ach were observed in one male of the highdose group. These findings are uncommon
in rats of this age and strain. Although thechanges were observed in an animal of the
high dose group, therelationship with the test material is not likely because the other
19 highdose animals did not exhibit these findings.

The histopathological examination of the surviving animals revealed a zona glomeru-

losa reduction in the adrenals of three highdose females, and a focal submucosal in-
flammatory cell infiltrate in the oesophagus wall of one highdose male. The incidence
of the zona glomerulosareduction was not significantlydifferent from the controls.
The high NaCl content in the test substance may be responsible for this finding since
aldosteron secretion is attenuated by sodium loading. Since zona glomerulosa reduc-
tion occurred only in three rats and its incidence was not statistically significantly dif-
ferent from the controls, no significance is attached to this finding. The inflammatory
cell infiltrate in the oesophagus wall resembles the inflammation observed in one of the
animals thatdied during the study. The inflammation suggests that damageto the oe-
sophagus may have occurred in these rats, and that this damage maybe treatment-
related rather than test substance-related.

Conclusion
Five rats in the mid- orhighdose group died during the study soon after dosing. On
the basis of macroscopic findings and histopathologicalexamination, the death ap-
peared to be related to the dosing procedurerather than the test substance.
Since the changesobserved at the highdose level were either ofdoubtful toxicological
significance or ascribed to the high NaCl content of the test substance, the NOAEL (No
Observed Adverse Effect Level) of Low Calcium Amylase, Batch 5977, wasplaced at
the highdose level, 15ml/kg bwlday (equivalent to 0.58 g TOSlkg bwlday)

3.2 Genotoxicity

3.2.1 Testing for Mutagenic Activity with Strains of Salmonella fyphimurium in a Liq-
uid Culture Assay andEscherichia coli in a Direct Plate Incorporation Assay.
The study was conducted in accordance with OECD Guideline for Testing of Chemi-
cals No. 471 (1997). andthe requirements of the Annex to EuropeanCommission Di-
rective 92/69/EEC. However, the exposure of the testbacteria in liquid culture ("treat
and plate"), as applied in this study with strains of Salmonella, is not specifically de-
scribed in any guidelines.

Procedures
The test material Low Calcium Amylase, Batch No. PPY 5977, was examined for
mutagenic activity in four histidinedependent strains of Salmonella fyphimurium,
strains TA 98, TA 100, TA 1535 and'TA 1537, using the"treat-and-plate" procedure,
and in the tryptophan-dependent strain Escherichia coli WP2uvrAusingthe direct OOBPi3%
plate incorporation method.
5
Summary of Toxlcity Data (Low Calcium Amylase)

dl..
 The bacterlawere exposed to six doses of thetest material separated with bi-sections.
The highest dose level applied was 5mg per ml (Salmonella strains) and 5mg per plate
(E.coli strain). The Salmonella strains were exposed to the test material in phosphate-
buffered nutrientbroth for three hours. After incubation, the exposed cells were sepa-
rated from thetest material by centrifugation prior to plating. The Escherichia coli was
exposed to the test material in a direct plate incorporation assay. The study was con-
ducted in both the presence and absence ofan activating system derived from rat liver
(S-9 mix).All tests included solvent (purified water) and positive controls with and
without S-9 mix. Each test with each strain was conducted in two complete and inde-
pendent experiments.

Results
No dose-related and reproducible increases in revertants to prototrophy were obtained
with any of the bacterial strains exposed to Low Calcium Amylase, Batch PPY 5977,
either in the presence or absence of S-9 mix.

The sensitivity of theindividual bacterial strains and the metabolising potential of the S-
9 mix was confirmed by significant increases in number of revertant colonies induced
by diagnostic mutagens under similar conditions.

Conclusion
It was concluded that Low Calcium Amylase, Batch PPY 5977 did not induce gene
mutations in bacteria in either the absence or presence of S-9 mix, when tested under
the conditions employed in the study.

3.2.2 Chromosomal Aberrations Assay with Human Peripheral Lymphocyte Cultures

in vitro.
The procedures and experimental design employed in this test complied with therec-
ommendations of therelevant toxicity testing guidelines of the OECD (Guideline473,
1983), and with the requirements of the Annex to European Commission Directive
92169lEEC.

The objective of the study wasto determine the potential of the test material to induce
chromosomal aberrations in human peripheral blood lymphocyte cultures in vitro.

Procedures
The study was conducted incorporating two independent tests. The tests were con-
ducted with andwithout the inclusion of a rat liver derived metabolic activating system
(S-9 mix). Cultures were exposed for 5 h in the presence of S-9 mix and for 25 h in the
absence of S-9 mix. The cells were harvested 29 h (Test 1 and 2) and 53 h (Test 2)
after treatment.

Low Calcium Amylase, Batch No. PPY 5977, was tested at concentrations ranging
from 0.078 to 10%. Treatment was established by addition of the test solution to 48- .

-
hour lymphocyte cultures established from whole, human blood. Three hours before
the cells were harvested cell division was arrested by the addition of Colcemid. which
resulted in accumulation of cells in metaphase. Slides were then prepared for micro-
scopic analyses.

Based on toxicity (Le. mitotic indices and slidelculture observatlons) and osmolality, 3
concentration levels were selected for assessment of chromosomal aberrations. A
dose level was considered to be toxic if the mitotic indices were less than 60% of the
mean vehiclecontrol values. If however, mitotic indices are greater than 60%, then
dose levels can beconsidered toxic if there are consistent changes to cell morphology
on the slides Chromosome aberration were scored by examination of 100 metaphase

0 0,0132
cells per culture,and the frequencies of cells with one or more aberrations werecal-
culated bothincluding and excluding gap-type aberrations. As culturesharvestedat
6
Summary of Toxiclty Data (Low Calclum Amylase)

a
 both culture times were negative with regards to structural aberrations, a further as-
sessment of polyploidy was made (53 h harvest).

The tests also incorporated vehicle (RPMI 1640) and positive (cyclophosphamide and
mitomycin C) control cultures. All control and test exposures were established in dupli-
cate cultures.

Results
Low Calcium Amylase was toxic in both the presence and absence of S-9 mix. In the
presence of S-9 mix, LowCalcium Amylase was judged to be toxic at 0 313% and
above (Test 1) and 1.25% and above (Test 2). In the absence of S-9 mix, Low Calcium
Amylase was judged toxic at 0.156% and above (Test 1) and 1.25% and above (Test
2)

In general all cultures treated with Low Calcium Amylase were within the confidence
limits for a negative response. The exceptions were one culture with a suspicious fre-
quency of lesions per cell (Test 2, absence of S-9 mix, 29h harvest) and one with a
positive level of lesions per cell (Test 2, presence of S-9 mix, 53h harvest). The latter
was due to a single multiply damaged c e l l . As these responses were not observed in
the duplicate cultures or were dose related, they wereconsidered to be sporadic.

An extra polyploidy assessment was carried out on cultures harvested at 53 h. In the

presence of S-9 mix, one of the cultures treated with 7.5 % and one treated with 10%
Low Calcium Amylase gave suspicious responses. In the absence of S-9 mix, oneof
the cultures treated with 0.31 3%gave a suspicious response, and one culture treated
with 10% had a positive response. As these responses were not reproduced in dupli-
cate cultures they were considered to be sporadic.

All vehicle control and untreated cultures had levels of structural aberrations within the
95% confidence limits of the historical negative control data. The knownclastogens,
cyclophosphamide in the presence of S-9 mix and mitomycin C in the absence of S-9
mix, induced positive frequencies of structural aberrations in at least one of the dose
levels assessed. These results demonstrated the sensitivity of the test system

Conclusion
It was concluded that Low Calcium Amylase, Batch No.PPY 5977, under the condi-
tions of test, did not show any evidence of clastogenic activity.

4. REFERENCES

Study Reports
Low Calcium Amylase, SP 935, Batch No.PPY 5977. Preliminary toxicity study by oral (gavage)
administration to rats for two weeks.
Novo Nordisk Study Summary,Novo Nordisk Study No.978091. November20, 1997.

Sub-chronic (13-wk) Oral Toxicity Study with Low Calcium Amylase SP 935 in Rats
TNO reportV98.539. Novo Nordisk Study No.976021. June 25, 1998.

Low Calcium Amylase, Chromosomal Aberrations Assay with Human Peripheral LymphocyteCul-
tures in vitro.
lnveresk Report No.16159. Novo Nordisk Study No.976022. June 5 , 1998.

e
Low Calcium Amylase(Batch No. PPY 5977): Test for Mutagenic Activity with Strains of Salmonella
typhimuriurn and Escherichia coli.
Novo Nordisk Study No.988008. July1, 1998.

7
Summaly of Toxtclty Data (Low Calclurn Amylase)

97,.
 Guidelines
-
OECD Guidelines for testing of Chemicals (420, 1992).

OECD Guidelines fortesting of Chemicals (408, 1981).

OECD Guidelines for testing of Chemicals (471, 1997).

OECD Guidelines fortesting of Chemicals (473, 1983).

EU. Annex to the European Communities Council. Directive 87/302/EEC.

EU. Annex to the European Communities Council. Directive 92/69/EEC Section 6.14.

EU. Annex to the European Communities Council. Directive 92/69/EEC Section B.10.

Summary of Toxlclty Data (Low Calcium Amylase)

I
 NOU-29-1999 16:25 NNBNR ERR P.01/02 3420 919 494

-.
. IlllllllIIIIIII Ill1 AM y
ul
Novo Nordisk
BioChem
2
__- - -- -

FAX TRANSMITTAL
"~
.I
~

North America,
0 Inc.

a,
Z
0 State Road 1003
PAGE 1 O F L P A G E S >
0
P. 0 . Box 576
Frankliton

-
2 North Carolina
27525
Regulatory Affairs
29 November, 1999 Tel. 919 494 3152
Fax 919 4.94 34.20

TO: Dr. Mary Ditto FAX: 202 4183131

FDNCFSAN

FROM: Scott Shore

SUBJECT: 1973 FR notice

0
Please find attached a copy of the 1973 FR notice on the filing of GRASP 3G0026,Novo's
B. lichenifomis carbohydrase and protease enzyme preparations.

Attachment
NOU-29-1999
16:25 NNBNR ERR 919 494 3420 P.02/02

. NOTICES

NOUe ls glvm purswnl to Ule Fedcrd

AvinUorr Act of 10.58. as wncndcd. that .
the Dubllc hearing !n Lllc abovc-cnLlllcd
Droccedlng now schedulcd Lo commence
on Sc~lcrnber18. 1073. (3E FR 22909) Is
hcrcby posLpolaed until furtllcr noLicc.
Dated nt Wad~bgLon.D.C.. Seylenl-
ber 6, 1973.
lcurl &VIS W. Goarso~.
AUmfrdslrativc I a w Judgc.
[PR Poc.73-19304 FlJcd PIl-?J:8:4G am)

CIVIL SERVICE COMMISSION

”

.DEPARTMENT OF COMMERCE
Notlcc of Grant of Authority To Mahe
N o m r c e r Executive As!&nmcnt
Under nrltlrorlLy or f 9.20 of Civll Scrv-
l e t Ruk N (5 CF’R 9.20). tllc Civil Scrv-
lco Colnmlsvlon w l l ~ o r i ~ . ctlrc
s Drlmrt-
mcrrt of Commcm t o nll by nom-arccr
exccuuve ac4gruncnt In l h c exccvtcd
3
scrvicc the ;)aslllon of C2cucr.Q Cou~\r.Cl.
Omce of G c l ~ ~ nCou~ucl. l Nnllorial
 I1111111111111 1I 1111
To : Mary Ditto@[email protected]
From:
Certify: N AM
Priority: Norma1
Subject : Acronym info
Date : Mon Nov 29 15:59:42 1999
Attached: None

Dear Dr. Ditto,

for EINECS, ISO, and the FR notices.

Please find below the explanation

Unlike the US that keeps just 1 chemical inventory list,

the TSCA Inventory,
Europe has 2 lists.

EINECS (European INventoryof Existing Chemical Substances)

This is the EU chemical inventory listingof about 100,000 substances,
including morethan 300 enzymes, nominated by industry as being on the
former EEC marketbetween 1/1/71 and 9/18/81. EINECSis a closed, published
list with entries byCAS and the assigned EINECS numbers.In accordance with
the EU Directiveson chemicals, these substances can legally be marketedfor
technical use withinthe EU.

ELINCS (European LIstof Notified Chemical Substances)

The open and regularly published, updated listof notified new substances in
the EU. Substances are assigned ELINCS numbers and legalized as above.
Substances not classified as dangerous substances may not be identifiable in
the published list (code or trade name), however, Member State authorities
will inform upon request if a given substance, on notEINECS, is notified.

IS0 has a website at http://www.iso.ch/

Although I thought it stood

for International Standards Organization, it
does not as they point
out on their website and excerpted below.

The International Organization for Standardization (ISO) is a worldwide

federation of national standards bodiesfrom some 130 countries, one from
each country. IS0 is a non-governmental organization establishedin 1947.
The mission of IS0 is to promote the development of standardization and
related activitiesin the world with a view to facilitating the
international exchange of goods and services, and to developing cooperation
in the spheres of intellectual, scientific, technological and economic
activity. ISO's work results in international agreements which are
published as International Standards.
ISO's name
Many people will have noticed a seeming lack of correspondence
between the officialtitle when used in full, International Organization for
Standardization, and the short form, ISO. Shouldn'tthe acronym be "IOS"?
Yes, if it werean acronym - which it is not.
In fact, "ISO" is a word, derivedfrom the Greek isos, meaning
"equal", which is the root of the prefix "iso-" that occurs in a host of
terms, such as "isometric" (of equal measure or dimensions) and "isonomy"
(equality of laws, or of people before the law).
From "equal" to "standard", the lineof thinking that led tothe
choice of "ISO" as the name of the organization is easy to follow.In
addition, the nameIS0 is used around the world to denote the organization,
thus avoiding the plethora of acronyms resulting from the translation of
"International Organizationfor Standardization" into the different national
languages of members, e.g.10s in English, OIN in French(from Organization
internationale de normalisation). Whatever the country, the short form of
the Organization's name is always ISO.

Lastly, the copy of the 1973 FR notice we haveis hard to read butI will
try faxing it to you anyway and will ask my colleagues in Denmark
if they
have a cleaner copy. It does look like it should be 25214 not 26214.

0Please
Hope this helps. We are looking forward to the Agency Response Letters.
contact
information.
meif you have any additional questionsor need more ooor414rll
Page 1
 Sincerely, Scott

Scott H. Shore, Ph.D.

Senior Regulatory Specialist
Novo Nordisk BioChem North America, Inc.
77 Perry Chapel ChurchRoad, Box 5 1 6
Franklinton,-NC 2 7 5 2 5
0 Dhone: 919 494-3152
fax : 919 494-3420
email: [email protected]