Optimization of alkaline protease production from a locally isolated Bacillus sp. ZR-5: Potential application as a detergent additive

Document Type : Original Research Papers


1 Department of Pharmaceutical Biotechnology, Isfahan Pharmaceutical Sciences Research Center, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran.

2 Chemical Engineering Department, School of Chemical and Petroleum Engineering, Shiraz University, Shiraz 71345, Iran

3 Department of Biochemistry, Isfahan Pharmaceutical Sciences Research Center, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran.

4 Department of Biology, School of Basic Sciences, University of Isfahan, Isfahan, Iran


The aim of this study was to optimize protease production using a novel Bacillus sp. ZR-5 strain
isolated from the soil, and evaluate its application in detergent industry. The proteolytic activity
of the strain was demonstrated using gelatin hydrolysis screening test. Protease production
optimization was carried out using a two-step approach: a conventional method in order to
identify the best carbon and nitrogen sources followed by the application of response surface
methodology (RSM) to optimize the factors, which include temperature, pH and incubation
time. Glucose or fructose (5 g/L), wheat bran (5 g/L), temperatures of 25 and 55°C, pH 10.0 and
an approximate incubation time of 44 h, were determined as the optimal conditions according to
optimization processes. Validation tests were carried out under these conditions and the results
were in good agreement with RSM predicted data. The in-gel activity (zymogram) test showed
two hydrolytic zones with 66.2 and 36.5 kDa molecular weight on the casein containing
polyacrylamide gel. The high compatibility in the presence of detergent powder and washing
performance test suggested that the crude enzyme could be an appropriate choice as a detergent
additive in detergent industries.


Main Subjects

1. Gupta, R., Beg, Q.K. and Lorenz, P. (2002). Bacterial alkaline proteases: molecular approaches
and industrial applications. Appl. Microbiol. Biot 59, 15-32.
2. Masui, A., Yasuda, M., Fujiwara, N. and Ishikawa, H. (2008). Enzymatic hydrolysis of gelatin
layers on used lith film using thermostable alkaline protease for recovery of silver and PET film.
Biotechnol. Progr, 20, 1267-1269.
3. Demain, A.L. and Adrio, J.L. (2008). In Natural Compounds as Drugs Volume I. Springer, in
press., pp. 251-289.
4. Rao, M.B., Tanksale, A.M., Ghatge, M.S. and Deshpande, V.V. (1998). Molecular and
biotechnological aspects of microbial proteases. Microbiol. Mol. Biol. R 62, 597-635.
5. Joo, H.S., Kumar, C.G., Park, G.C., Kim, K.T., Paik, S.R. and Chang, C.S. (2002). Optimization
of the production of an extracellular alkaline protease from Bacillus horikoshii. Process.
Biochem, 38, 155-159.
6. Kumar, C.G. and Takagi, H. (1999). Microbial alkaline proteases: from a bioindustrial viewpoint.
Biotechnol. Adv, 17, 561-594.
7. Wang, Q., Hou, Y., Xu, Z., Miao, J. and Li, G. (2008). Optimization of cold-active protease
production by the psychrophilic bacterium Colwellia sp. NJ341 with response surface
methodology. Bioresource. Technol, 99, 1926-1931.
8. Armstrong, N.A. (2006). Pharmaceutical experimental design and interpretation. CRC Press,
9. Singh, S.K., Singh, S.K., Tripathi, V.R., Khare, S.K. and Garg, S.K. (2011). Comparative onefactor-
at-a-time, response surface (statistical) and bench-scale bioreactor level optimization of
thermoalkaline protease production from a psychrotrophic Pseudomonas putida SKG-1 isolate.
Microb. Cell. Fact, 10, 114.
10. Liu, S., Fang, Y., Lv, M., Wang, S. and Chen, L. (2010). Optimization of the production of
organic solvent-stable protease by Bacillus sphaericus DS11 with response surface methodology.
Bioresource. Technol, 101, 7924-7929.
11. Oskouie, S.F.G., Tabandeh, F., Yakhchali, B. and Eftekhar, F. (2008). Response surface
optimization of medium composition for alkaline protease production by Bacillus clausii.
Biochem. Eng. J, 39, 37-42.
12. Pillai, P., Mandge, S. and Archana, G. (2011). Statistical optimization of production and tannery
applications of a keratinolytic serine protease from Bacillus subtilis P13. Process. Biochem, 46,
13. Rao, Y.K., Lu, S.-C., Liu, B.-L. and Tzeng, Y.-M. (2006). Enhanced production of an
extracellular protease from Beauveria bassiana by optimization of cultivation processes.
Biochem. Eng. J 28, 57-66.
14. Reddy, L., Wee, Y.-J., Yun, J.-S. and Ryu, H.-W. (2008). Optimization of alkaline protease
production by batch culture of Bacillus sp. RKY3 through Plackett–Burman and response surface
methodological approaches. Bioresource. Technol, 99, 2242-2249.
15. Thys, R., Guzzon, S.O., Cladera-Olivera, F. and Brandelli, A. (2006). Optimization of protease
production by Microbacterium sp. in feather meal using response surface methodology. Process.
Biochem, 41, 67-73.
16. Fakhfakh-Zouari, N., Haddar, A., Hmidet, N., Frikha, F. and Nasri, M. (2010). Application of
statistical experimental design for optimization of keratinases production by Bacillus pumilus A1
grown on chicken feather and some biochemical properties. Process. Biochem, 45, 617-626.
17. Haddar, A., Fakhfakh-Zouari, N., Hmidet, N., Frikha, F., Nasri, M. and Kamoun, A.S. (2010).
Low-cost fermentation medium for alkaline protease production by Bacillus mojavensis A21
using hulled grain of wheat and sardinella peptone. J. Biosci. Bioeng 110, 288-294.
18. Rabbani, M., Bagherinejad, M.R., Sadeghi, H.M., Shariat, Z.S., Etemadifar, Z., Moazen, F.,
Rahbari, M., Mafakher, L. and Zaghian, S. (2013). Isolation and characterization of novel
thermophilic lipase-secreting bacteria. Braz. J. Microbiol 44, 1113-1119.
19. Mukherjee. A. K and K., R.S. (2011). A statistical approach for the enhanced production of
alkaline protease showing fibrinolytic activity from a newly isolated Gram-negative Bacillus sp.
strain AS-S20-I. New. Biotechnol, 28, 182-189.
20. Garciacarreno, F., Dimes, L. and Haard, N. (1993). Substrate-gel electrophoresis for composition
and molecular weight of proteinases or proteinaceous proteinase inhibitors. Anal. Biochem, 214,
21. Arulmani, M., Aparanjini, K., Vasanthi, K., Arumugam, P., Arivuchelvi, M. and Kalaichelvan,
P.T. (2007). Purification and partial characterization of serine protease from thermostable
alkalophilic Bacillus laterosporus-AK1. World. J. Microb. Biot 23, 475-481.
22. Hübner, U., Bock, U. and Schügerl, K. (1993). Production of alkaline serine protease subtilisin
Carlsberg by Bacillus licheniformis on complex medium in a stirred tank reactor. Appl.
Microbiol. Biotechnol., 40, 182-188.
23. Maurer, K.-H. (2004). Detergent proteases. Curr. Opin. Biotech 15, 330-334.
24. Rai, S.K. and Mukherjee, A.K. (2011). Optimization of production of an oxidant and detergentstable
alkaline β-keratinase from Brevibacillus sp. strain AS-S10-II: Application of enzyme in
laundry detergent formulations and in leather industry. Biochem. Eng. J 54, 47-56.
25. Kumar, C. (2002). Purification and characterization of a thermostable alkaline protease from
alkalophilic Bacillus pumilus. Lett. Appl. Microbiol 34, 13-17.
26. Huang, Q., Peng, Y., Li, X., Wang, H. and Zhang, Y. (2003). Purification and characterization of
an extracellular alkaline serine protease with dehairing function from Bacillus pumilus. Curr.
Microbiol, 46, 0169-0173.
27. Miyaji, T., Otta, Y., Nakagawa, T., Watanabe, T., Niimura, Y. and Tomizuka, N. (2006).
Purification and molecular characterization of subtilisin‐like alkaline protease BPP‐A from
Bacillus pumilus strain MS‐1. Lett. Appl. Microbiol, 42, 242-247.