Volume 9, Issue 4 (2018)                   JMBS 2018, 9(4): 507-515 | Back to browse issues page

XML Persian Abstract Print

Download citation:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:

Borhani M, Etemadifar Z, Emtiazi G, Jorjani E. Molecular Detection and Optimization of Extracellular Alkaline Protease Production of Bacillus pseudofirmus MSB22 Using Response Surface Methodology. JMBS 2018; 9 (4) :507-515
URL: http://biot.modares.ac.ir/article-22-24438-en.html
1- Biology Department, Sciences Faculty, University of Isfahan, Isfahan, Iran
2- Biology Department, Sciences Faculty, University of Isfahan, Isfahan, Iran, University of Isfahan, Hezarjarib Street, Isfahan, Iran. Postal Code: 8174673441 , z_etemadifar@yahoo.com
3- Biology Department, Sciences Faculty, Gonbad Kavvous University, Golestan, Iran
Abstract:   (9100 Views)
Aims: Alkaline protease is one of the most important groups of industrial enzymes with many applications. The aim of this study was to determine the physicochemical parameters affecting the production of alkaline protease enzyme produced by Bacillus pseudofirmus MSB22 by one-factor-at-a-time (OFAT) method and optimize the production of this enzyme by the response surface methodology (RSM) in the form of a rotatable central composite design.
Materials and Methods: In the present experimental study, the isolation of microorganism producing alkaline protease from wastewater from sausage and lunch meat factories in Isfahan was carried out. The morphological and biochemical characteristics of the strain were performed according to the Bergey's book and amplification of 16S rRNA gene sequences. Detection of metalloproteinase gene and alkaline serine protease was done by polymerase chain reaction (PCR) reaction and enzyme activity measurement was performed by Folin reagent. Screening of variables effective in enzyme production was done, using one-factor-at-a-time method and optimization was performed by response surface methodology. MEGA 6 software was used for phylogenetic analyses. To analyze the data, the Design Expert 7 software and the one-way analysis of variance were used.
Findings: The maximum protease production, which was 1.85 times higher than that of OFAT method and 3.45 times higher than unoptimized conditions was obtained, using 1% w/v xylose, 3% w/v beef extract, 4% v/v inoculation size, pH 10, and 30°C. The established quadratic model had a great ability to predict responses to new observations due to a high value of the predicted determination coefficient.
Conclusion: OFAT and RSM strategies are useful screening and optimization methods, respectively and sub I and sub II genes (alkaline serine protease genes) are detected in Bacillus pseudofirmus MSB22.
Full-Text [PDF 933 kb]   (2234 Downloads)    
Subject: Agricultural Biotechnology
Received: 2016/08/30 | Accepted: 2018/09/6 | Published: 2018/12/21

1. Rai SK, Roy JK, Mukherjee AK. Characterisation of a detergent-stable alkaline protease from a novel thermophilic strain Paenibacillus tezpurensis sp. nov. AS-S24-II. Appl microbiol biotechnol. 2010;85(5):1437-50. [Link] [DOI:10.1007/s00253-009-2145-y]
2. Jisha VN, Smitha RB, Pradeep S, Sreevedi S, Unni KN, Sajith S, et al. Versatility of microbial proteases. Adv Enzym Res. 2013;1(3):39-51. [Link] [DOI:10.4236/aer.2013.13005]
3. Ferracini-Santos L, H Sato H. Production of alkaline protease from Cellulosimicrobium cellulans. Braz J Microbiol. 2009;40(1):54-60. [Link] [DOI:10.1590/S1517-83822009000100008]
4. Anbu P, Annadurai G, Hur BK. Production of alkaline protease from a newly isolated Exiguobacterium profundum BK-P23 evaluated using the response surface methodology. Biologia. 2013;68(2):186-93. [Link] [DOI:10.2478/s11756-013-0159-5]
5. Singh SK, Singh SK, Tripathi VR, Khare SK, Garg SK. Comparative one-factor-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. 2011;10:114. [Link] [DOI:10.1186/1475-2859-10-114]
6. Vaishnav D, Suthar J, Oza T, Dave G, Sheth N. A statistical approach for the enhanced production of thermostable alkaline protease showing detergent compatibility activity from Bacillus circulans. Biocatal Biotransform. 2014;32(3):151-60. [Link] [DOI:10.3109/10242422.2014.913579]
7. Bach HJ, Hartmann A, Schloter M, Munch JC. PCR primers and functional probes for amplification and detection of bacterial genes for extracellular peptidases in single strains and in soil. J Microbiol Methods. 2001;44(2):173-82. [Link] [DOI:10.1016/S0167-7012(00)00239-6]
8. Cupp-Enyard C. Sigma's non-specific protease activity assay-casein as a substrate. J Vis Exp. 2008;(19):e899. [Link] [DOI:10.3791/899]
9. Gupta R, Beg QK, Khan S, Chauhan B. An overview on fermentation, downstream processing and properties of microbial alkaline proteases. Appl Microbiol Biotechnol. 2000;60(4):381-95. [Link]
10. Bhunia B, Basak B, Dey A. A review on production of serine alkaline protease by Bacillus spp. J Biochem Technol. 2012;3(4):448-57. [https://www.researchgate.net/publication/237006217]
11. Chi Z, Ma C, Wang P, Li HF. Optimization of medium and cultivation conditions for alkaline protease production by the marine yeast Aureobasidium pullulans. Bioresour Technol. 2007;98(3):534-8. [Link] [DOI:10.1016/j.biortech.2006.02.006]
12. Patel R, Dodia M, Singh SP. Extracellular alkaline protease from a newly isolated haloalkaliphilic Bacillus sp.: Production and optimization. Process Biochem. 2005;40(11):3569-75. [Link] [DOI:10.1016/j.procbio.2005.03.049]
13. Naidu K.S.B., Devi K.L. Optimization of thermostable alkaline protease production from species of Bacillus using rice bran. Afr J Biotechnol. 2005;4(7):724-6. [Link] [DOI:10.5897/AJB2005.000-3132]
14. Sen Sh, Dasu Veeranki V, Mandal B. Effect of physical parameters, carbon and nitrogen sources on the production of alkaline protease from a newly isolated Bacillus pseudofirmus SVB1. Ann Microbiol. 2009;59(3):531-8. [Link] [DOI:10.1007/BF03175142]
15. Shafee N, Norarati Aris S, Abd Rahman RNZ, Basir M, Salleh AB. Optimization of environmental and nutritional conditions for the production of alkaline protease by a newly isolated bacterium Bacillus cereus strain 146. J Appl Sci Res. 2005;1(1):1-8. [Link]
16. Patel RK, Dodia MS, Joshi RH, Singh SP. Production of extracellular halo-alkaline protease from a newly isolated haloalkaliphilic Bacillus sp. isolated from seawater in Western India. World J Microbiol Biotechnol. 2006;22(4):375-82. [Link] [DOI:10.1007/s11274-005-9044-x]

Add your comments about this article : Your username or Email:

Send email to the article author

Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.