Research Article | Volume: 3, Issue: 4, July-August, 2015

Isolation and optimization of alkaline protease producing Bacteria from undisturbed soil of NE-region of India falling under Indo-Burma biodiversity hotspots

Onkar Nath Tiwari Thiyam Bidyababy Devi Kangjam Sarabati Devi Gunapati Oinam Thingujam Indrama Keithellakpam Ojit Oinam Avijeet Lakreiphy Ningshen   

Open Access   

Published:  Aug 24, 2015

DOI: 10.7324/JABB.2015.3406

A total of 28,000 bacterial isolates were encountered during 2008-2011 from north eastern region of India, which is falling under Indo-Burma biodiversity hot-spots and were preserved in glycerol stock solution at -80°C in deep freezer. 19,834 isolates were biochemically characterized and screened for protease activity in culture conditions further 1,579 isolates were found to be good amount of protease producing bacteria where, the zone of inhibition was observed in the range between 20-45 mm on skim milk agar medium at different pH range i.e. 4.0, 7.0 and 10.0. Out of 1,579 protease producing bacteria, 425 isolates produced protease activity at acidic pH-4.0, 547 bacteria at alkaline pH-10.0 and 607 bacteria at neutral pH-7.0. Compared to acidic and neutral pH, the highest proteolytic activity was exhibited by the isolates which were grown in alkaline medium. In this study, 5 isolates forming larger zone as a result of casein hydrolysis were further studied for quantitative production of extracellular alkaline protease activity. Isolate no. B-2 was observed as a highest potential protease producer bacterium. Upon 16S rRNA analysis, it displayed maximum similarity with Bacillus sp. and the sequences were deposited in GenBank database. Different cultural parameters like effect of pH, temperature, time and inoculum sizes were optimized for maximal enzyme production. Maximum yield of enzyme was obtained at a pH of 10.0 with 1 ml of inoculum in the medium after 48 hours of incubation and maintained at a temperature of 37ºC. The present investigation indicates the potential use of these microorganisms as biotechnological tools for various industrial activities.

Keyword:     Alkaline proteases Bacillus sp. 16S rRNA gene sequence proteolytic activity optimization.


Onkar Nath Tiwari, Thiyam Bidyababy Devi, Kangjam Sarabati Devi, Gunapati Oinam, Thingujam Indrama, Keithellakpam Ojit, Oinam Avijeet, Lakreiphy Ningshen. Isolation and optimization of alkaline protease producing Bacteria from undisturbed soil of NE-region of India falling under Indo-Burma biodiversity hotspots. J App Biol Biotech. 2015; 3 (04): 025-031.

Copyright: Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license.

HTML Full Text


1. Verma OP, Prashansa K, Shruti S, Abha S. Production of Alkaline Protease by Bacillus subtilis (MTCC7312) using Submerged Fermentation and Optimization of Process Parameters. European Journal of Experimental Biology. 2011; 1: 124-129.

2. Chu WH. Optimization of extracellular alkaline protease production from species of Bacillus. Journal of Industrial Microbiology and Biotechnology. 2007; 34: 241-245.

3. Jellouli K, Bougatef A, Manni L, Agrebi R, Siala R, Younes I, Nasri M. Molecular and biochemical characterization of an extracellular serine-protease from Vibrio metschnikovii J1. Journal of Industrial Microbiology and Biotechnology. 2009; 36: 939-948.

4. Saeki K, Ozaki K, Kobayashi T, Ito S. Detergent alkaline proteases: enzymatic properties, genes, and crystal structures. Journal of Biosciences and Bioengineering. 2007; 103: 501-508.

5. Maurer KH. Detergent proteases. Current Opinion in Biotechnology. 2004; 15: 330-334.

6. Haddar A, Agrebi R, Bougatef A, Hmidet N, Sellami-Kamoun A, Nasri M. Two detergent stable alkaline serine-proteases from Bacillus mojavensis A21: purification, characterization and potential application as a laundry detergent additive. Bioresource Technology. 2009; 100: 3366-3373.

7. Anwar A, Saleemuddin M. Alkaline proteases. A Review. Bioresource Technology. 1998; 6: 175-183.

8. Gupta R, Beg QK, Lorenz P. Bacterial alkaline proteases: molecular approaches and industrial applications. Applied Microbiology and Biotechnology. 2002; 59: 15-32.

9. Takagi H, Kondou M, Hisatsuka T, Nakamuris K, Tsai YC, Yamasaki M. Effect of an alkaline elastase from an alkalophillic Bacillus strain on the tenderization of beef meat. Journal of Agricultural and Food Chemistry. 1992; 46: 2364-2368.

10. Wilson SA, Young OA, Coolbear T, Daniel RM. The use of protease from extreme thermophilic for meat tenderization. Meat Science Journal. 1992; 32: 93-103.

11. George S, Raju V, Krishnan MRV, Subramanian TV, Jayaraman K. Production of protease by Bacillus amyloliquefaciens in solid state fermentation and its application in the unhairing of hides and skins. Process Biochemistry. 1995; 30: 457-462.

12. Usharani B, Muthuraj M. Production and characterization of protease enzyme from Bacillus laterosporus. African Journal Microbiological Research. 2010; 4: 1057-1063.

13. Denizci AA, Kazan D, Abeln ECA, Erarslan A. Newly isolated Bacillus clausii GMBAE 42: an alkaline protease producer capable to grow under highly alkaline conditions. Journal of Applied Microbiology. 2004; 96: 320-327.

14. Borsosi AK, Micsinai A, Rusznyak A, Vladar P, Kovacs G, Toth EM, Marialigeti K. Diversity of alkaliphilic and alkalitolerant bacteria cultivated from decomposing reed rhizomes in Hungarian Soda Lake. Microbial Ecology. 2005; 50: 9-18.

15. Gupta R, Beg QK, Lorenz P. Bacterial alkaline proteases: molecular approaches and industrial applications. Applied Microbiology and Biotechnology. 2002; 59: 15-32.

16. Sidra A, Samia A, Sadia S, Shika AR. Screening, Isolation and Characterization of Alkaline Protease Producing Bacteria from Soil. Pakistan Journal of Biological Sciences. 2006; 9: 2122-2126.

17. Rao MM, Tanksale Ghatge MS, Deshpande VV. Molecular and biotechnological aspects of microbial proteases. Microbiology and Molecular Biology Reviews. 1998; 62: 597-635.

18. Newman SM, Boynton JE, Gillham NW, Randolph- Anderson BL, Johnson AM, Harris EH. Transformation of Chloroplast Ribosomal RNA Genes in Chlamydomonas: molecular and genetic characterization of integration events. Genetic Society of America. 1990; 126: 875-888.

19. Nubel U, Garcia-Pichel F, Muyzer G. PCR primers to amplify 16S rRNA gene from cyanobacteria. Applied Enviromental Microbiology. 1997; 63: 3327-3332.

20. Tunga R, Shrivastava B, Banerjee R. Purification and characterization of a protease from solid-state cultures of Aspergillus parasiticus. Process Biochemistry. 2003; 38: 1553-1558.

21. Cheng K, Lu FP, Li M, Liu LL, Liang XM. Purification and biochemical characterization of a serine alkaline protease TC4 from a new isolated Bacillus alcalophilus TCCC11004 in detergent formulations. African Journal of Biotechnology. 2010; 9: 4942-4953.

22. Kumar CG, Takagi H. Microbial alkaline proteases from a bioindustrial viewpoint. Biotechnology Advance. 1999; 17: 561-594.

23. Krishnaveni K, Kumar DJM, Balakumaran MD, Ramesh S, Kalaichelvan PT. Production and optimization of extracellular alkaline protease from Bacillus subtilis isolated from dairy effluent. Der Pharmacia Lettre. 2012; 4: 98-109.

24. Porwal S, Lal S, Cheema S, Kalia VC. Phylogeny in aid of the present and novel microbial lineages: Diversity in Bacillus. PLoS One. 2009; 4: 11-27.

25. Johnvesly B, Naik GR. Study on production of thermostable alkaline protease from thermophilic and alkaliphilic Bacillus spp. JB-99 in a chemically defined medium. Process Biochemistry. 2001; 37: 139-144.

26. Aoyama M, Yasuda M, Nakachi K, Kobamoto N, Oku H, Kato F. Soybean-milk-coagulating activity of Bacillus pumilus derives from a serine proteinase. Applied Microbiology and Biotechnology. 2000; 53: 390-395.

27. Feng YY, Yang WB, Ong SL, Hu JY, Nig WJ. Fermentation of starch for enhanced alkaline protease production by constructing an alkalophilic Bacillus pumilus strain. Applied Microbiology and Biotechnology. 2001; 57: 153-160.

28. Adinarayana K, Ellaiah P, Prasad DS. Purification and partial characterization of thermostable serine alkaline protease from a newly isolated Bacillus subtilis PE-11. Journal of Pharmaceutical Science and Technology. 2003; 4: 56-64.

29. Al-Shehri L, Abdul-Rahman M, Yasser S. Production and some properties of protease produced by Bacillus licheniformis isolated from Tihamet aseer, Saudi Arabia. Pakistan Journal of Biological Sciences. 2004; 7:1631-1635.

30. Schallmey M, Singh A, Ward OP. Developments in the use of Bacillus species for industrial production. Canadian Journal of Microbiology. 2004; 50: 1-17.

31. Tsujibo H, Miyamoto K, Hasegana T, Inamori Y. Purification, properties, and partial aminoacid sequences of thermostable xylanases from Streptomyces thermoviolaceus OPC-520. Journal of Applied Bacteriology. 1990; 69: 520-529.

32. Dunaevsky TE, Pavyukova EB, Gruban TN, Belyakova GA, Belozershy MA. An extracellular protease of the micromycete Alternaria alternate. Process Biochemistry. 1996; 61: 1350-1354.

33. Mukesh KDJ, Krishnaveni K, Balakumaran MD, Ramesh S, Kalaichelvan PT. Scholars Research Library. Der Pharmacia Lettre. 2012; 4: 98-109.

34. Usharani B, Muthuraj M. Production and characterization of protease enzyme from Bacillus laterosporus. African Journal of Microbiological Research. 2010; 4: 1057-1063.

35. VijayAnand S, Hemapriya J, Selvin J, Kiran S. Production and Optimization of Haloalkaliphilic Protease by an Extremophile Halobacterium Sp. Js1, Isolated from Thalassohaline Environment. Glob. Journal of Biotechnology and Biochemistry. 2010; 5: 44-49.

36. Fulzele R, DeSa E, Yadav A, Shouche Y, Bhadekar R. Characterization of novel extracellular protease produced by marine bacterial isolate from the Indian Ocean. Braz. Journal of Microbiology. 2011; 42: 1364-1373.

37. El-Safey EM, Abdul-Raouf UM. Production, purification and characterization of protease enzyme from Bacillus subtilis. International Conferences For Development and The Environment In The Arab World, Assiut University, Egypt; 2004, p. 14.

38. Rahman RNZR, Geok LP, Basri M, Salleh AB. Physical factors affecting the production of organic solvent-tolerant protease by Pseudomonas aeruginosa strain K. Bioresource Technology. 2005; 96: 429-436.

39. Kaur M, Dhillon S, Chaudhray K, Singh R. Production and characterization of a Termostable Alkaline Protease from Bacillus polymyxa. Indian Journal of Microbiology. 1998; 38: 63-67.

40. Dawson PSS, Kurz WGW. Continuous phased culture -A technique for growing, analyzing and using Microbial cells. Biotechnology and Bioengineering. 1969; 11: 843-851.

41. Olajuiyigbe FM, Ajele TO. Production dynamics of extracellular protease from Bacillus species. African Journal of Biotechnology. 2005; 4: 776-779.

Article Metrics

313 Absract views 174 PDF Downloads 487 Total views

Related Search

By author names

Citiaion Alert By Google Scholar

Name Required
Email Required Invalid Email Address

Comment required