TRÌNH BÀY CÁC BƯỚC SẢN XUẤT ENZYME PROTEASE TÁI TỔ HỢP TỪ CHỦNG BACILLLUS SP Ở VIỆT NAM. BIẾT TRÊN GENEBANK CÓ MỘT SỐ TRÌNH TỰ MÃ HÓA PROTEASE CỦA MỘT SỐ LOÀI TRONG CHI BACILLUS?
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Pak. J. Bot., 40(5): 2161-2169, 2008.
INFLUENCE OF CULTURE CONDITIONS ON PRODUCTION
AND ACTIVITY OF PROTEASE FROM
MUSSARAT SHAHEEN, AAMER ALI SHAH, ABDUL HAMEED, FARIHA HASAN
Department of Microbiology,
Quaid-i-Azam University, Islamabad, Pakistan.
Abstract
Bacillus subtilis BS1 was used in the present study for the production of protease. For protease production optimum pH and temperature were found to be 11 and 50˚C, respectively. Soybean (197PU/mg) and casein (168 PU/mg) proved as the best substrates for the production of enzyme.Maximum production of protease (126 and 121 PU/mg) was shown in 1.5 and 4.5% of Sodium chloride (NaCl) concentration, respectively. Maximum activity was observed at pH 9 at 90ºC, in crude extract, after 20 minutes of incubation. EDTA slightly enhanced proteolytic activity, whereas, Na, K, Ca, Li, Mg, Cu and Fe inhibited the activity of protease. Due to maximum production of protease in the presence of cheaper, low concentrations of substrate and stability at alkaline pH, high temperature and salt-tolerance, these characteristics makes the strain and its enzymes usefull in different industries.
Introduction
Proteases represent the class of enzymes that occupy a pivotal position with respect
to their physiological roles as well as their commercial applications. More than 75% of industrial enzymes are hydrolases. Protein-degrading enzymes constitute about 40% of all enzymes sales (Leisola et al., 2001). They perform both degradative and synthetic functions. A number of eukaryotic and prokaryotic organisms are reported to produce proteolytic enzymes (Sakka et al., 1986). Many different types of extracellular hydrolytic enzymes as well as some non-hydrolytic enzymes (Lipases, α and β-amylases, cellulases, xylanase, penicillinase, invertase, β-glycosidase, cytase, phosphatases, deaminases, urease, asperginase) are produced by microorganisms e.g., Actinoplanes sp., Arthrobacter sp., Aspergillus sp., Bacillus cereus, Bacillus coaglulans, Bacillus subtilis, Candida sp.,Clostridium sp., Lactobacillus sp., Penicillium sp., Pseudomonas sp., Rhizopus sp., Streptococcus sp., Streptomyces sp., (James & David, 1986). The biosynthesis of proteolytic enzymes by microorganisms is not only of scientific but also of great practical importance. Bacillus subtilis produces both neutral and alkaline protease (Dhandapani & Vijayaragavan, 1994). Proteases produced from Bacillus subtilis have wider specificity than that of trypsin and chymotrypsin of animal origin. They are present in a large variety of commercially available enzymes differing in biological source, activity, purity, physical form and characteristics such as pH and temperature optima (Cheetham, 1995). Enzymes are vulnerable to various environmental factors. Their activitymay be significantly diminished or destroyed by a variety of physical or chemical agen resulting in a loss of the functions performed by the enzymes (Pelczar et al., 1986). The present study was aimed to optimize pH, temperature, substrate and salt concentration for maximum production of protease from Bacillus subtilis BS1 and its stability.
MUSSARAT SHAHEEN ET AL., 2162
Materials and Methods
Microorganisms: Bacillus subtilis BS1, used in the present study, was isolated from
sugar scum from Crescent Sugar Mills Ltd., Faisalabad. Cultures were maintained on
Nutrient agar slants. Qualitative test for protease: Proteolytic activity of Bacillus subtilis BS1 was detected on the basis of appearance of clear zones around the bacterial colonies. Luria casein agar (1%) plates were used for this purpose.
Quantitative test for protease: Liquid medium (250ml) containing (g/l): Gelatin, 15;
casein hydrolysate, 0-5; glycerol, 3ml, with pH 8, was used to screen the bacterial strain
for the production of protease. About 250ml medium was poured in two 1000ml
Erlenmeyer flasks, and pH was adjusted by using 0.1N NaOH and 0.1N CH3COOH. The
medium was autoclaved at 121°C, 15psi for 20 minutes. Oven sterilized, 3ml of 20%
glycerol solution was added to the medium aseptically. Bacillus subtilis BS1 was
inoculated in the medium and the flasks were kept in shaking incubator at 37°C with
150rpm. Samples were collected after 24, 48, 72 and 96 hours and centrifuged at 10,
000rpm for 30 minutes at 4°C. Cell free supernatant was used as the crude enzyme for theestimation of enzyme activity.
To check the activity of enzyme in culture supernatant, method of Kunitz (1965) was
used with casein as a substrate. One unit of enzyme activity is defined as that amount of
enzyme which releases a micro mole tyrosine under standard conditions of assay, 45°C,
pH 8.5 and reaction time one hour.
Optimization of culture conditions for production of protease: Different culture
conditions, like pH (5-11), temperature (40, 50, 60 and 70˚C), different substrates
(gelatin, casein, soybean) and salt concentration (0, 1.5, 3.0, 4.5 and 6%), were optimized
for production of protease from Bacillus subtilis BS1. Samples were collected after 24,
48 and 72hrs and centrifuged at 1000rpm for 30 minutes at 4˚C. Supernatant was used as
crude extract. Proteolytic activity was measured under standard assay conditions.
Stability of enzyme in crude enzyme extract: Protease was produced from Bacillus
subtilis BS1 under optimized conditions. After 48 hours of incubation, cell free
supernatant was taken as crude enzymes extract and used for the characterization. The
effect of pH (4-11), temperature (40, 50, 60, 70, 80, 90, 100˚C) and metal ions (20mM
solution of NaCl, KCl, CaCl2, LiCl2, MgSO4, CuSO4, FeCl2, BaCl2 and HgCl2 and
chelating agent EDTA) on the activity of crude enzyme was studied. The crude enzyme
was incubated for 20 minutes and enzyme activity was measured before and after the
treatment under standard assay conditions. Buffers (0.02M) of different pH values were
used: acetate buffer (pH 4, 5), phosphate buffer (6, 7), Tris (pH 8) and glycine-NaOH
buffer (pH 9, 10, 11) for assay.
Results and Discussion
Qualitative and quantitative analysis: Bacillus subtilis BS1, was found to produce
protease on the basis of clear zones around the bacterial colonies on 1% casein agar
PRODUCTION AND ACTIVITY OF PROTEASE FROM BACILLUS SUBTILIS
plates at 37˚C after 24 hours. These clear zones were due to hydrolysis of different
substrates (Khire & Pant, 1992). Maximum production of protease by Bacillus subtilis
BS1 was 131 U/ml after 48hrs, at 37˚C, pH 8 and 150rpm, in Gelatin casein medium. The
production was 129, 123 and 109 U/ml after 24, 72 and 96 hrs, respectively.
Optimization of culture conditions for protease production
Effect of pH:
Production of protease was observed at various pH values ranging from 4 to
11. The maximum production of protease was 523 PU/mg, at pH 11 after 48hrs (Fig. 1).
Kobayashi (1995) reported optimal activity of bacterial protease at pH 11. Cheetham
(1995), Muderrizade et al., (2001) and Kumar (2002) also reported the production of an
alkaline protease at 11.5 from Bacillus sp. The study reveals that the enzyme secretion is
greatly influenced by the change in initial pH of the environment (Sarkar et al., 1998).
Enzymes possess many ionizable groups so that pH changes may alter the conformation of
the enzyme (Cheetham, 1995).
Effect of temperature:
Only few microbial strains are able to grow at elevated temperatures (Cheetham, 1995). Effect of various temperatures, 40, 50, 60 and 70˚C on protease production was studied. It was observed that enzyme production was maximum (301 PU/mg) at 50˚C (Fig. 2). Sookkeo et al., (2000) and Kobayashi (1995) reported production of protease at such high temperatures. Mao & Freedman (1992) found that maximum protease production could be achieved by controlled pH and temperature.
Effect of substrates:
The substrates used in industrial enzyme fermentations are normally common agricultural products like soybean, casein, starch and ordinary sugar (Cheetham, 1995). Enzyme formation is largely dependent on the condition of growth of the culture and composition of nutrient medium (Fujiwara & Masui, 1993). In the present study, casein, gelatin and soybean flour with glycerol and casein hydrolysate were used as substrates for the production of protease from Bacillus subtilis BS1, at pH 11. Maximum production of protease (197 PU/mg) was observed in case of soybean after 48 hrs of incubation while in case of casein and gelatin, the production of protease was found as 171 PU/mg and 163 PU/mg, respectively after 72 hrs of incubation. Sun (1997) reported maximum production of protease from B. sphaericus C3-41 in the presence of soybean after 48 hrs of incubation. Geoffrey & Hodges (1981) found that protease synthesis occurred at exponential phase of bacterial growth, which is associated with sporulation of B. subtilis, but according to Dercova et al., (1992), the secretions of enzyme occurs mostly between the end of the exponential phase to an early stationary phase, maximum production occurs after cell population reached its peak. High levels of proteases were produced on casein, soybean flour and starch (Mahmood et al., 2000; Sampath &Chandrakasan, 1998). Gelatin and soybean act as organic nitrogen sources and enhance the bacterial growth, while casein hydrolysate is a source of readymade amino
acids and encourage the foam formation to remove spores and cellular debris from the
culture medium (Feng et al., 2001). It also enhances the extracellular alkaline protease
synthesis. The above results showed that enzyme production was mostly decreased after
48hrs., this might be due to the depletion of nitrogen and other sources present in
medium, which were utilized by the organisms, or due to inactivation of enzyme by
acidification of the medium (Dervoca et al., 1992).
Effect of NaCl: In the present study, various salt concentrations from (0-6%) were used
to study the effect of NaCl on the production of protease from Bacillus subtilis BS1.
Maximum enzyme (126 PU/mg) was produced in the presence of 1.5% NaCl (Fig. 4),
followed by 4.5 and 3%. Tolerance of enzyme up to 5 M of NaCl over 24hrs without
losing original activity was reported by Jana et al., (1997).
Stability of protease in crude enzyme extract: Both low and high levels of temperature
and pH affect the enzyme activity and stability.
Effect of pH: The enzyme activity of the protease produced by Bacillus subtilis BS1 was
determined after incubating the crude extract for 20 minutes at 90˚C at different pH
values. The enzyme showed maximum activity (100%) at pH 9. It was shown that the
enzyme was 96 % stable at pH 6 and 89% stable at pH 10 and 11 (Fig. 5). Protease
activities at alkaline pH (8-11) were studied by Kim et al., (2001) and Feng et al., (2001).
Effect of temperature:
The effect of temperature on the activity of enzyme was studied. Maximum activity was shown at 90˚C and 50˚C, 100% and 97%, respectively (Fig. 6). Activity of protease at 45˚C for 30min., and 80˚C for 10min., and 60min., respectively was reported by Feng et al., (2001). The decrease in enzyme activity at higher temperatures might be due to the destruction of an enzyme at certain temperatures.
Effect of metal ions:
Metal ions often act as salt or ion bridges between two adjacent amino acid residues. Effects of various metal ions are shown in Fig. 7. K, Li+2, Mg+2, Cu+2, Na inhibited the activity of protease, but EDTA played important role in stimulation of enzyme activity and increased the activity up to 101%. Observations aboutMUSSARAT SHAHEEN ET AL., 2168
the stability of enzyme in the presence of EDTA are also reported by Muderrizade et al.,
(2001). Inhibition by EDTA, Hg, Fe, Cu, Fe, Mg and Li was also reported by Sun et al.,
(1997) and Sookkeo et al., (2000). In the present work, strong inhibition or stimulation by
metal ions in case of protease activity, was not observed. Alkaline proteases are generally
neither inhibited by metal chelating reagents nor activated by metal ions or reducing
agents (Kim et al., 2001).