African Journal of Biotechnology Vol. 11(8), pp.

1977-1980, 26 January, 2012

Available online at [Link]
DOI: 10.5897/AJB11.2529
ISSN 16845315 2012 Academic Journals

Full Length Research Paper

Isolation and partial purification of lysozyme from

saliva of Bali cattle (Bos sondaicus) using an aqueous
mixture of polyethylene glycol (PEG) with sodium
sulfate
Sulaiman Ngongu Depamede1,3*, Niken Asri2, Nur Indah Julisaniah2,
Bambang F. Suryadi2 and Djoko Kisworo1
1
Microbiology and Biotechnology Laboratory, Faculty of Animal Sciences, University of Mataram, Jalan Majapahit No.
62, Mataram 83125, Indonesia.
2
Faculty of Mathematics and Life Sciences, Mataram University, Jl. Majapahit No. 62 Mataram 83125, Indonesia.
3
Immunobiology Laboratory, Faculty of Medicine, University of Mataram, Mataram, Indonesia.
Accepted 31 October, 2011

Lysozyme from saliva of Bali cattle (Bos sondaicus) was successfully isolated through an aqueous
mixture system of polyethylene glycol (PEG 4000) and salt (Na2SO4). About 100 g/ml lysozyme of 14.2
kDa, determined through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), per
4800 g/ml saliva was obtained. Based on turbidity assay of Staphylococcus aureus, it was revealed
that inhibition activities of 40 g/ml isolated lysozyme were comparable to 800 g/ml of a commercial
hen egg white lysozyme.

Key words: Lysozyme, Bali cattle, Bos sondaicus, PEG, aqueous mixture system.

INTRODUCTION

Lysozyme (EC [Link], N-acetylmuramic-hydrolase) with saliva (Tenovuo, 2002a) in relation to oral health, as well
a molecular weight around 14.3 kDa is one of the as the overall body health, considering the mouth as one
components of the animal innate immune systems of the entrance of various pathogens. In line with this,
(Janeway et al., 2005; Cegielska-Radziejewska et al., lysozyme has also been used not only for the treatment
2008). It is usually present in the body tissues such as in of dry-mouth (xerostomia) for instance, but also for
the lungs (Travis et al., 1999) and body fluids such as gastrointestinal infections (Tenovuo, 2002b). Moreover
tears, breast milk and in saliva (Hankiewicz and some researchers are interested in revealing the links
Swierczek, 1974). In human saliva, lysozyme was found between levels of salivary lysozyme and several diseases
to be about 20 to 80 g/ml, and has the ability as an such as odd hypertension (Qvarnstrom et al., 2008).
innate defense system (Tenovuo, 1989). As a natural It is now evident that, aside from the lysozyme
immune system, lysozyme also has the ability to bacteriolytic action, a dimeric form of lysozyme exhibits
hydrolyze peptidoglycan of bacterial cell walls (Araki et therapeutic, antiviral and anti-inflammatory properties
al., 2003; Islam et al., 2006). Studies on lysozyme in (Cegielska-Radziejewska et al., 2008). Increasing
saliva have been done extensively, especially in human demand for the availability of lysozyme from various
natural sources, both for industry and for research
purposes has opened up opportunities to explore of new
sources of lysozyme. One of these sources is the saliva
*Corresponding author. E-mail: sulaiman_n@[Link]. of Bali cattle (Bos sondaicus). This paper presents partial
purification and characterization of lysozyme isolated
Abbreviations: PEG, polyethylene glycol; ddW, double from the saliva of Bali cattle using polyethylene glycol
distillated water; SDS-PAGE, sodium dodecyl sulfate (PEG)/Na2SO4 aqueous mixture. Successes in isolating
polyacrylamide gel electrophoresis. lysozyme from saliva of Bali cattle do not only open up
1978 Afr. J. Biotechnol.

Estimation of total protein

Total protein in saliva, as well as the isolated lysozyme, was

estimated by measurement of the absorbance at 280 nm in a UV-
Vis spectrophotometer (Shimadzu UV-160, Japan) using a BSA
standard curve.

Determination of molecular weight of the enzyme

Molecular weight of the enzyme was determined by 15% sodium

dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
using a commercial lysozyme as standard. The gel was stained
using coomassie brilliant blue staining system (Volker et al., 1985).

Inhibition of lysozyme on Staphylococcus aureus

A reduction on turbidity was used for determining the lysozyme

inhibition on S. aureus as modified from Jenzano et al. (1986) and
Kumar et al. (2001). S. aureus was cultured in Luria-Bertani broth
media, pH 7 at 37C. Isolated lysozyme (40 g/ml) was added into
the culture and as a comparison, a commercial hen egg white
lysozyme (800 g/ml) was also included. S. aureus cultured with LB
medium alone was considered as control. Optical density (Abs) at
600 nm was observed at zero hour of incubation, then the optical
density was observed at 2 and 6 h. Finally, percent inhibition was
Figure 1. Lane 1, Commercial hen egg white lysozyme; lane 2,
determined as percentage reduction in turbidity compared to
molecular weight marker; lane 3, representation of SDS-PAGE
control, calculated by the formula;
profile of lysozyme isolated from Bali cattle (Bos sondaicus) saliva
by aqueous mixture of PEG4000/Na2SO4.
Abs of control S. aureus Abs of treated S. aureus
Inhibition (%) = 100%
Abs of control S. aureus

opportunities to study the role of lysozyme in the

diversification of species, but also open up additional
RESULTS AND DISCUSSION
sources of lysozyme.
As a natural antimicrobial, lysozyme has been used both
MATERIALS AND METHODS in food and cosmetics industry. This has led to the
increase in the demand for lysozyme from year to year.
Collection of sample So far lysozymes derived from hen egg white are the
most widely used sources of natural lysozyme
A total of 35 ml saliva was collected from oral cavity of B. sondaicus (Cegielska-Radziejewska et al., 2008). Exploration of
using a 20 ml syringe without needle. The saliva was collected from
three bulls and two cows, age range between 1.5 and 2.5 years old,
natural lysozyme from various species has been done
with good health condition based on exterior examination. The extensively, but rarely from saliva of domestic cattle such
cattle were raised traditionally on a farm, fed with native grass and as B. sondaicus. In this study we succeeded in isolating
local roughage with water ad libitum in West Lombok District, the lysozyme from saliva of Bali cattle with aqueous
Lombok Island, Indonesia. The saliva was centrifuged (3,000 g) mixture of PEG 4000/Na2SO4, a method modified from
for 5 min at 4C. Supernatant was collected, added with 0.2% (v/v) Su and Chiang (2006).
of NaN3, aliquoted and kept at 4C until used within 2 weeks.
Following mixing and centrifugation of the aqueous
phases, three layers were observed designated as F1 on
Isolation and purification of lysozyme top, F2 in the middle and F3 at the bottom part. With this
method, lysozyme was obtained in the F1 phase. The
The method adopted for isolation of the lysozyme was from Su and concentration of isolated lysozyme was about 100 g/ml
Chiang (2006). This method was based on PEG/sodium sulfate from 4800 g/ml total protein of post-dialysis of original
aqueous two-phase system. The stock solution of PEG 4000 (50%,
w/w) and sodium sulfate (30%, w/w) were prepared in double saliva. The lysozyme concentration was comparable to
distillated water (ddW). To avoid protein precipitation, polymers, those of human saliva lysozyme; 20 to 80 g/ml
buffer, salt and ddW were mixed before adding saliva. The pH of (Tenovuo, 1989). The purity and molecular weight
the system was nine and the total weight was 10 g. The phases specifications of this lysozyme (42 kDa) were verified on
were mixed gently for 60 min at room temperature followed by the results of SDS-PAGE analysis (Figure 1). In addition,
centrifugation at 1500 g for 20 min. The formed fractions were
dialyzed separately against phosphate buffered saline (PBS) pH 7,
the inhibitory activities of the salivary lysozyme are
and then analyzed for the protein concentration and the lysozyme presented in Figure 2. In this study, 40 and 800 g/ml of
activity. isolated saliva lysozyme and commercial hen egg white
 Depamede et al. 1979

Figure 2. BCL, Percentage inhibitions of lysozyme isolated from

Bali cattle (Bos sondaicus); CWEL, commercial hen egg white
lysozyme; PBS, phosphate buffered saline without lysozyme as
control, based on turbidimetric assay of S. aureus during 2 and 6 h
cultures. Data obtained from the two independent experiments were
carried out in duplicate.

lysozyme were used respectively, for turbidimetric assay AKNOWLEDGEMENTS

of S. aureus.
The results of this study indicate that lysozyme isolated We thank the farmers of Kapek Gunung Sari, West
from Bali cattle saliva has a potent ability to lyse the cell Lombok, Indonesia for providing their Bali cattle used as
wall of S. aureus even though S. aureus with their source of saliva in this study. The authors are also
peptidoglycan layer has been reported resistant to the thankful to the Deans of Faculty of Animal Science and
hydrolysis effects of lysozyme (Bera et al., 2005). Faculty of Mathematics and Life Sciences, Mataram
Interestingly, Ibrahim et al. (1994) as well as Cegielska- University, for providing the facilities to carry out this work
Radziejewska et al. (2008) revealed that the modified throughout 2010. Thank you also to Mr. Suparman for his
lysozyme enhanced its hydrolysis effects both against technical assistance.
gram-negative bacteria (Escherichia coli K12) and gram-
positive bacteria (S. aureus). Although the present works
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