Optimization and partial characterization of culture conditions for the production of alkaline protease from Bacillus licheniformis P003
© Sarker et al.; licensee Springer. 2013
Received: 20 May 2013
Accepted: 18 September 2013
Published: 4 October 2013
Proteolytic enzymes have occupied a pivotal position for their practical applications. The present study was carried out under shake flask conditions for the production of alkaline protease from Bacillus licheniformis P003 in basal medium containing glucose, peptone, K2HPO4, MgSO4 and Na2CO3 at pH 10. The effect of culture conditions and medium components for maximum production of alkaline protease was investigated using one factor constant at a time method along with its characterization. Maximum level of enzyme production was obtained after 48h of incubation with 2% inoculum size at 42°C, under continuous agitation at 150 rpm, in growth medium of pH 9. Highest enzyme production was obtained using 1% rice flour as carbon source and 0.8% beef extract as organic nitrogen source. Results indicated that single organic nitrogen source alone was more suitable than using in combinations and there was no significant positive effect of adding inorganic nitrogen sources in basal medium. After optimization of the parameters, enzyme production was increased about 20 fold than that of in basal medium. The crude enzyme was highly active at pH 10 and stable from pH 7–11. The enzyme showed highest activity (100%) at 50°C, and retained 78% relative activity at 70°C. Stability studies showed that the enzyme retained 75% of its initial activity after heating at 60°C for 1h. The enzyme retained about 66% and 46% of its initial activity after 28 days of storage at 4°C and room temperature (25°C) respectively. Mn2+ and Mg2+ increased the residual activity of the enzyme, whereas Fe2+ moderately inhibited its residual activity. When pre-incubated with Tween-20, Tween-80, SDS and H2O2, each at 0.5% concentration, the enzyme showed increased residual activity. These characteristics may make the enzyme suitable for several industrial applications, especially in leather industries.
Proteases execute a large variety of functions and have numerous applications in detergent, food, pharmaceutical and leather industries (Gupta et al. 2002). Alkaline proteases hold a major share of the enzyme market with two third shares in detergent industry alone (Anwar and Saleemuddin 2000, Haki and Rakshit 2003). Although there are many microbial sources available for protease production, only a few are considered as commercial producers (Beg et al. 2002). Of these, species of Bacillus dominate in the industry (Gupta et al. 2002). Due to enhancement of such demand of proteases for specific properties, scientists are looking for newer sources of proteases. In addition, for effective use in industries, alkaline proteases need to be stable and active at high temperature and pH and in the presence of surfactants, oxidizing agents, and organic solvents (Johnvesly and Naik 2001, Fu et al. 2003, Rahman et al. 2006, Bhunia et al. 2011). Culture condition is also another important parameter to consider that can influence the cost and rate of enzyme production (Beg et al. 2002). About 30-40% of the cost of industrial enzymes depends on the cost of the growth medium (Joo et al. 2003). In addition, extracellular protease production from Bacillus species is significantly influenced by medium composition and some physical factors, such as fermentation period, aeration, inoculum density, incubation temperature and pH of growth medium (Puri et al. 2002; Genckal and Tari 2006; Nadeem et al. 2006). In this study, strategies were applied to optimize the production of alkaline protease from Bacillus licheniforms P003. The enzyme was also partially characterized in this study.
Results and discussion
The demand of eco-friendly technology is increasing day by day to reduce the pollution at industrial level. Being an eco-friendly compound, enzymes got application at different industrial sectors. There is no general medium for protease production by different microbial strains (Pandey et al. 2000). Every microorganism evidences its own idiosyncratic physicochemical and nutritional requirements for growth and enzyme secretion (Reddy et al. 2008). Enzyme production was carried out under shake flask culture at 37°C and 150 rpm for the production of alkaline protease from B. licheniformis P003 in basal medium containing Glucose 10.0 g/l, Peptone 10.0 g/l, K2HPO4 1.0 g/l, MgSO4 0.2 g/l, Na2CO3 5.0 g/l at initial pH 10. After 48 h fermentation, enzyme activity of 46.10 U/ml was obtained. To increase the production of enzyme, optimization of culture conditions and medium components were then studied. Initially, the traditional one-fact-at-a-time method was employed for selection of appropriate medium ingredients and their apparent concentrations of the enzyme production.
Effect of culture conditions for production of extracellular protease from B. licheniformis P003 in shake-flask cultivation
Protease activity (U/ml)
Relative activity (%)
Protease activity (U/mg)
Total soluble protein (mg/ml)
Incubation temperature (°C)
Incubation period (hr)
Inoculums volume (%, ml)
B. licheniformis P003 was cultivated in basal medium at pH 10.0 for different incubation period ranging from 24 to 120 hours at a temperature of 37°C and 150 rpm and enzyme assay was carried out every 24 hours interval. The time course data revealed that maximum level of alkaline protease was produced (48.10 U/ml enzyme activity and 1.178 mg/ml protein concentration) after 48h of cultivation period (Table 1). Xiong et al. (2010) found maximum protease production by B. licheniformis after 48 h of incubation. Olajuyigbe and Ajele (2008) also found maximum protease production of 18.4 U/ml after 48 h incubation by B. licheniformis LBBL-11. Whereas, Akcan (2012) found highest alkaline protease production after 24 h by B. licheniformis ATCC 12759. Inoculum concentration was also studied for maximum alkaline protease production. According to data presented in Table 1, optimum concentration of inoculum for protease production was found at 2.0%. Protease production was sharply decreased at a concentration of 2.5%. Total protein content was also highest at 2.0% inoculum concentration.
Depicting the protease activity and total soluble protein of the culture supernatant of B. licheniformis P003 grown on different carbon source in presence of 1% peptone
Protease activity (U/ml)
Protease activity (U/mg)
Total protein content (mg/ml)
Depicting the protease activity and total soluble protein of the culture supernatant of B. licheniformis P003 grown on different nitrogen source in presence of 1% glucose
Nitrogen sources (%)
Protease activity (U/ml)
Protease activity (U/mg)
Total protein content (mg/ml)
Control (Peptone, 1%)
Yeast Extract (1%)
Beef Extract (1%)
P (0.5%) + T (0.5%)
P (0.5%) + YE (0.5%)
P (0.5%) + BE (0.5%)
T (0.5%) + YE (0.5%)
T (0.5%) + BE (0.5%)
BE (0.5%) + YE (0.5%)
Alkaline protease production was significantly increased about 20 fold (930.02 U/ml) using the simulated media after optimization of different parameters in comparison to the basal medium (46.10 U/ml).
Effect of surfactants and oxidants on protease stability
Surfactants/ oxidizing agent
Residual activity (%)
The results in this study indicated that optimization of culture conditions played a central role for improving yield through the shake flask fermentation process. Maximum protease activity was obtained after optimization of the nutritional components and fermentation processes. Along with production optimization, this work also describes the partial characterization of alkaline protease from B. licheniformis P003. These characteristics indicated that alkaline protease produced by B. licheniformis P003 might be an excellent candidate for use as detergent additive in laundry industry and as a dehairing and bating agent in tanneries. Further studies on protease produced by B. licheniformis P003 would make it feasible for commercialization of the enzyme.
Materials and methods
The bacterial culture B. licheniformis P-003 was obtained from the Microbial Biotechnology Division, National Institute of Biotechnology, Ganakbari, Savar, Dhaka. Stock culture of the organism was maintained on nutrient agar medium at 4°C in refrigerator for routine laboratory use and 15% glycerol broth at −20°C for long term preservation.
Preparation of seed culture
Vegetative inoculums were used in the present studies. 50 ml of inoculums medium containing nutrient broth 13 g/l, pH 7.4±0.2 was transferred to a 100 ml conical flask and cotton plugged. It was sterilized in an autoclave at 15 lbs/inch2 pressure at 121°C for 20 min. After cooling to room temperature, a loopful of freshly grown culture was aseptically transferred to it. The flask was incubated overnight at 37°C and 150 rpm in a rotary shaking incubator.
Fermentation and separation of culture filtrates
The seed culture (1 ml) was transferred to 100 ml of basal medium in a 250-ml Erlenmeyer flask. Basal medium contained Glucose 10.0 g/l, Peptone 10.0 g/l, K2HPO4 1.0 g/l, MgSO4 0.2 g/l, Na2CO3 5.0 g/l (initial pH 10.0). The inoculated flasks were placed in a thermostated orbital shaker for 48 hours, at 37°C and 150 rpm. Samples were withdrawn at regular intervals and centrifuged in a refrigerated centrifuge machine at 10000 rpm for 15 minutes at 4°C. The cell free supernatant was preserved at 4°C and used for enzyme assay and protein estimation.
Soluble protein estimation
Extracellular soluble protein in culture filtrate was estimated by Lowry’s method using bovine serum albumin (BSA) used as Standard (Lowry et al. 1951). 2 ml of analytical reagent was added to 0.2 ml suitably diluted test samples (enzyme solution). The mixture was mixed well and allowed to stand for 10 min at 50°C. Then 0.2 ml of the folin-ciocalteau reagent was added and shaken to mix well and incubated at room temperature for about 30 min. Optical density of the reaction mixture was measured at 600 nm, against a blank prepared with 0.2 ml buffer. A standard curve was constructed with each experiment using bovine serum albumin as a known protein. The amount of the soluble protein was calculated from the standard curve as mg protein per ml of test samples.
Determination of enzyme activity
Protease activity was determined by Anson method (Anson, 1938; Bhunia et al., 2010) using 1% casein as substrate. 0.2 ml of enzyme solution was added to 0.8 ml of substrate solution (1% V/V, casein with 50 mM Glycine-NaOH buffer, pH 10.0) and incubated at 50°C for 20 min independently with respective controls. The reaction was stopped by adding 1 ml of 10% TCA followed by holding 10 min at room temperature and then subsequently followed by centrifugation at 8000 rpm for 15 min at 4°C. After that 1 ml of supernatant was added to 3ml of 0.4M Na2CO3 solution. Then 0.5 ml of Folin reagent was immediately added to each tube, vortexed and left for 30 min at room temperature. This provides coloration (measured at OD660 nm) equivalent to 1 μmol of tyrosine, in the presence of the Folin-Ciocalteau reagent by using a tyrosine standard curve (Folin and Ciocalteu 1927). The protease activity was expressed as the difference of absorbance at 660 nm between the control and the test sample. One unit of protease activity was defined as the amount of enzyme liberating 1 μg of tyrosine/min under assay conditions. Enzyme units were measured using tyrosine (0–100 mg) as standard.
Optimization of fermentation parameters
The fermentation condition for protease production by B. licheniformis P003 was studied. The experiments were carried out systematically in such a way that the parameter optimized in one experiment was maintained at its optimum level in the subsequent experiments. Various process parameters that enhance the yield of protease under submerged fermentation were investigated by taking one factor at a time. The impact of initial pH (8.0-11.0), temperature (32-45°C), incubation time (24–120 h), inoculums concentration (0.5-3.5%) and medium composition (carbon, organic and inorganic nitrogen) were evaluated. All the experiments were conducted in triplicate and then the mean values were considered.
Data analysis was performed using SPSS software version 10 (Chicago, USA). The results were presented as mean ± SE.
- Akcan N: Production of extracellular protease in submerged fermentation by Bacillus licheniformis ATCC 12759. Afr J Biotechnol 2012, 11(7):1729-1735.Google Scholar
- Anson ML: The estimation of pepsin, trypsin, papain and cathepsin with hemoglobin. J Gen Physiol 1938, 22: 79-89. 10.1085/jgp.22.1.79View ArticleGoogle Scholar
- Anwar A, Saleemuddin M: Alkaline protease from Spilosoma obliqua: potential applications in bio-formulations. Biotechnol Appl Biochem 2000, 3192: 85-89.View ArticleGoogle Scholar
- Beg QK, Saxena RK, Gupta R: De-repression and subsequent induction of protease synthesis by Bacillus mojavensis under fed-batch operations. Process Biochem 2002, 37(10):1103-1109. 10.1016/S0032-9592(01)00320-XView ArticleGoogle Scholar
- Bhunia B, Dutta D, Chaudhuri S: Selection of suitable carbon, nitrogen and sulphate source for the production of alkaline protease by Bacillus licheniformis NCIM-2042. Not Sci Biol 2010, 2(2):56-59.Google Scholar
- Bhunia B, Dutta D, Chaudhuri S: Extracellular alkaline protease from Bacillus licheniformis NCIM-2042: improving enzyme activity assay and characterization. Engineering in Life Sciences 2011, 11(2):207-215. 10.1002/elsc.201000020View ArticleGoogle Scholar
- Deng A, Wu J, Zhang Y, Zhang G, Wen T: Purification and characterization of a surfactant-stable high-alkaline protease from Bacillus sp. B001. Bioresour Technol 2010, 101: 7100-7116. 10.1016/j.biortech.2010.03.130View ArticleGoogle Scholar
- Feng YY, Yang WB, Ong SL, Hu JY, Ng WJ: Fermentation of starch for enhanced alkaline protease production by constructing an alkalophilic Bacillus pumilus strain. Appl Microbiol Biotechnol 2001, 57: 153-160. 10.1007/s002530100765View ArticleGoogle Scholar
- Folin O, Ciocalteu V: On tyrosine and tryptophan determination in proteins. J Biol Chem 1927, 73: 627-650.Google Scholar
- Fu Z, Hamid SBA, Razak CNA, Basri M, Bakar Salleh A, Rahman RNZA: Secretory expression in Escherichia coli and single-step purification of a heat-stable alkaline protease. Protein Expr Purif 2003, 28(1):63-68. 10.1016/S1046-5928(02)00637-XView ArticleGoogle Scholar
- Genckal H, Tari C: Alkaline protease production from alkalophilic Bacillus sp. isolated from natural habitats. Enzyme Microb Technol 2006, 39: 703-710. 10.1016/j.enzmictec.2005.12.004View ArticleGoogle Scholar
- Ghorbel B, Sellami-Kamoun A, Nasri M: Stability studies of protease from Bacillus cereus BG1. Enzyme Microb Technol 2003, 32: 513-518. 10.1016/S0141-0229(03)00004-8View ArticleGoogle Scholar
- Gupta R, Beg QK, Khan S, Chauhan B: An overview on fermentation, downstream processing and properties of microbial alkaline proteases. Appl Microbiol Biotechnol 2002, 60(4):381-395. 10.1007/s00253-002-1142-1View ArticleGoogle Scholar
- Haki GD, Rakshit SK: Developments in industrially important thermostable enzymes: a review. Bioresour Technol 2003, 89(1):17-34. 10.1016/S0960-8524(03)00033-6View ArticleGoogle Scholar
- Hossain MS, Azad AK, Sayem SMA, Mostafa G, Hoq MM: Production and partial characterization of feather degrading keratinolytic serine protease from Bacillus licheniformis MZK-3. J Biol Sci 2007, 7(4):599-606.View ArticleGoogle Scholar
- Johnvesly B, Naik GR: Studies on production of thermostable alkaline protease from thermophilic and alkaliphilic Bacillus sp. JB-99 in a chemically defined medium. Process Biochem 2001, 37(2):139-144. 10.1016/S0032-9592(01)00191-1View ArticleGoogle Scholar
- Joo HS, Kumar CG, Park GC, Paik SR, Chang CS: Oxidant and SDS-stable alkaline protease from Bacillus clausii I-52: production and some properties. J Appl Microbiol 2003, 95: 267-272. 10.1046/j.1365-2672.2003.01982.xView ArticleGoogle Scholar
- Kumar CG, Takagi H: Microbial alkaline proteases: from a bioindustrial viewpoint. Biotechnol Advances 1999, 17: 561-594. 10.1016/S0734-9750(99)00027-0View ArticleGoogle Scholar
- Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem 1951, 193(1):265-75.Google Scholar
- Mabrouk SS, Hashem AM, El-Shayeb NMA, Ismail AS, Abdel-Fattah AF: Optimization of alkaline protease productivity by Bacillus licheniformis ATCC 21415. Bioresour Technol 1999, 69: 155-159. 10.1016/S0960-8524(98)00165-5View ArticleGoogle Scholar
- Nadeem M, Shahjahan B, Syed QA: Microbial production of alkaline proteases by locally isolated Bacillus subtilis PCSIR-5. Pak J Zool 2006, 38: 109-118.Google Scholar
- Nadeem M, Qazi JI, Baig S, Syed Q: Effect of medium composition on commercially important alkaline protease production by Bacillus licheniformis N-2. Food Technol Biotechnol 2008, 46(4):388-394.Google Scholar
- Naidu KSB, Devi KL: Optimization of thermostable alkaline protease production from species of Bacillus using rice bran. Afr J Biotechnol 2005, 4(7):724-726.View ArticleGoogle Scholar
- Nejad ZG, Yaghmaei S, Hosseini RH: Production of extracellular protease and determination of optimal condition by Bacillus licheniformis BBRC 100053. IJE Transactions B: Applications. 2009, 22(3):221-228.Google Scholar
- Olajuyigbe FM, Ajele JO: Some properties of extracellular protease from Bacillus licheniformis lbbl-11 isolated from “iru”, a traditionally fermented African locust bean condiment. Global J Biotechno Biochem 2008, 3(1):42-46.Google Scholar
- Pandey A, Nigam P, Soccol CR, Soccol VT, Singh D, Mohan R: Advances in microbial amylases. Biotechnol Appl Biochem 2000, 1: 135-152.View ArticleGoogle Scholar
- Paul D, Rahman A, Ilias M, Hoq M: Production and characterization of keratinolytic protease of Bacillus licheniformis MZK-03 grown on feather mill. Bangladesh J Microbiol 2007, 24(1):57-61.Google Scholar
- Puri S, Beg QK, Gupta R: Optimization of alkaline protease production from Bacillus sp. by response surface methodology. Cur Micobiol 2002, 44: 286-290.Google Scholar
- Rahman RNZA, Geok LP, Basri M, Salleh AB: An organic solvent-stable alkaline protease from Pseudomonas aeruginosa strain K: enzyme purification and characterization. Enzyme Microb Technol 2006, 39(7):1484-1491. 10.1016/j.enzmictec.2006.03.038View ArticleGoogle Scholar
- Reddy LVA, Wee YJ, Yun JS, Ryu HW: Optimization of alkaline protease production by batch culture of Bacillus sp. RKY3 through plackett–burman and response surface methodological approaches. Bioresour Technol 2008, 99: 2242-2249. 10.1016/j.biortech.2007.05.006View ArticleGoogle Scholar
- Saeki KS, Katsuya O, Tohru K, Susumu I: Detergent alkaline proteases: Enzymatic properties, genes, and crystal structures. J Biosci Bioeng 2007, 103: 501-508. 10.1263/jbb.103.501View ArticleGoogle Scholar
- Sayem SMA, Alam MJ, Hoq MM: Effect of temperature, pH and metal ions on the activity and stability of alkaline protease from novel Bacillus licheniformis MKZ03. Proc Pakistan Acad Sci 2006, 43(4):257-262.Google Scholar
- Srividya S, Mala M: Influence of process parameters on the production of detergent compatible alkaline protease by a newly isolated Bacillus sp . Y. Turk J Biol 2011, 35: 177-182.Google Scholar
- Xiong Y, Wang Y, Yu Y, Li Q, Wang H, Chen R, He N: Production and characterization of a novel bioflocculant from Bacillus licheniformis . Appl Environ Microbiol 2010, 76(9):2778-2782. 10.1128/AEM.02558-09View ArticleGoogle Scholar
- Yu J, Jin H, Choi W, Yoon M: Production and characterization of an alkaline protease from Bacillus licheniformis MH31. Agric Chem Biotechnol 2006, 49: 135-139.Google Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.