BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF AN EGYPTIAN MARINE ISOLATE "Alcaligenes faecalis" PRODUCING THERMOSTABLE PROTEASES
Abstract
An Egyptian marine bacterium, isolated from Hamam Pheroon, South Sinai region was able to produce thermostable proteases, the isolate was identified morphologically, biochemically, and confirmed molecularly by 16S rRNA sequencing with 99% similarity to Alcaligenes faecalis. It exhibited optimum activity of 328.3 U/mg after ten min, incubation at 65C and pH 7. Both ammonium sulphate and sephadex G-100 purification methods enhanced the yield of Alcaligenes faecalis strain HFW-9081 to 125 and 121% as well as the specific activity to 458.9 and 590 U/mg, respectively, compared to cell free supernatant. However, relative protease activity was reduced to 35.8% when H2O2 was added. On the other hand, the activities increased 7.5 folds when Tween-80 was used as a surfactant. Genetic background of the protease genes in Alcaligenes faecalis was analyzed using bioinformatics database for the proteases amino acids sequences in the desired bacteria; and it specified that Alcaligenes faecalis has four different protease genes; these genes encode for various peptidases family groups. The variation in the peptidase family groups provides the protease enzymes with many features making them able to remain active under various environmental stresses. The overall results showed promising thermostable proteases isolated from local marine Egyptian bacterium; that can be used potentially in many industrial applications.References
Akel, H., F. Al-Quadan and T. K. Yousef (2009). Characterization of a purified thermostable protease from hyperthermophilic Bacillus strain HUTBS71. Eur. J. Sci. Res., 31: 280-288.
Al-Saman, M. A., Safinaz Farfour, A. A. Tayel and N. Rizk (2015). Bioactivity of lectin from Egyptian Jatropha curcas seeds and its potentiality as antifungal agent. Global Advanced Research J. Microbiol., 4: 087-097.
Amund, O. O., O. Omidiji and O. Ilori (1990). Purification and properties of a neutral protease produced by Lactobacillus brevis. Journal of biotechnology, 13: 361-365.
Asker, M. M., M. G. Mahmoud, K. El- Shebwy and M. S. A. El-Aziz (2013). Purification and characterization of two thermostable protease fractions from Bacillus megaterium. Journal of Genetic Engineering and Biotechnology, 11: 103-109.
Bahobil, A. S. (2011). Production, purification and characterization of alkaline and thermostable protease by Shewanella putrefaciens- EGKSA21 isolated from El- Khorma governorate KSA. Life Science Journal-Acta Zhengzhou University Overseas Edition, 8: 580-590.
Barberis, S., E. Quiroga, S. Morcelle, N. Priolo and J. M. Luco (2006). Study of phytoproteases activity in aqueous-organic biphasic systems using linear free energy relationships. Journal of Molecular Catalysis B: Enzymatic, 38: 95-103.
Basu, B. R., A. K. Banik and M. Das (2008). Production and characterization of extracellular protease of mutant Aspergillus niger AB100 grown on fish scale. World Journal of Microbiology and Biotechnology, 24: 449-455.
Beena, A. K., P. I. Geevarghese and K. K. Jayavadana (2012). Detergent potential of a spoilage protease enzyme liberated by a psychrotrophic spore former isolated from sterilized skim milk. American J. of food technol., 7: 89-95.
Bommarius, A. S. (2015). Biocatalysis: A status report. Annual review of chemical and biomolecular engineering, 6: 319-345.
Brenner, D. J., N. R. Krieg and J. T. Staley (2005). Bergey’s Manual of Systematic Bacteriology Second Edition. Vol. (2), The Proteobacteria part B, The Gammaproteobacteria. Springer Science and Business Media, Inc., New York, USA.
El-Eskafy H. M. (2015). Biochemical and molecular studies of thermostable protease from hyperthermophilic microorganism. M. Sc. Thesis, Department of microbial biotechnology, Genetic Engineering and Biotechnology Research Institute, University of Sadat City, Egypt.
Evans, E. C. and A. Abdullahi (2012). Effect of surfactant inclusions on the yield and characteristics of protease from Bacillus subtilis. In Proc. Rom. Acad. Series B (2: 108-112.
Ghorbel-Frikha, B., A. Sellami-Kamoun, N. Fakhfakh, A. Haddar, L. Manni and M. Nasri (2005). Production and purification of a calciumdependent protease from Bacillus cereus BG1. Journal of Industrial Microbiology and Biotechnology, 32: 186-194.
`Guangrong, H., Y. Tiejing, H. Po and J. Jiaxing (2006). Purification and characterization of a protease from Thermophilic Bacillus strain HS08. African Journal of Biotechnology, 5(24).
Gupta, A. and S. K. Khare (2006). A protease stable in organic solvents from solvent tolerant strain of Pseudomonas aeruginosa. Bioresource technology, 97: 1788- 1793.
Gupta, A., B. Joseph, A. Mani and G. Thomas (2008). Biosynthesis and properties of an extracellular thermostable serine alkaline protease from Virgibacillus pantothenticus. World journal of Microbiology and Biotechnology, 24: 237-243.
Gupta, R., Q. Beg and P. Lorenz (2002). Bacterial alkaline proteases: molecular approaches and industrial applications. Applied Microbiology and Biotechnology, 59: 15-32.
Habib, S. A., A. N. M. Fakhruddin, S. Begum and M. M. Ahmed (2011). Production and characterization of thermo-alkaline extracellular protease from Halobacterium sp. AF1. Asian J. Biotechnol., 3: 345-356.
Hinderhofer, M., C. A. Walker, A. Friemel, C. A. Stuermer, H. M. Möller and A. Reuter (2009). Evolution of prokaryotic SPFH proteins. BMC Evolutionary Biology, DOI:10.1186/1471-2148-9-10. http://www.ncbi.nlm.nih.gov/nuccore/NZ_LQAS01000005.1?from=4624an dto=5619 http://www.ncbi.nlm.nih.gov/nuccore/NZ_LQAS01000005.1?from=5638an dto=6540 http://www.ncbi.nlm.nih.gov/nuccore/NZ_LSUO01000024.1?from=207504 andto=208430 http://www.ncbi.nlm.nih.gov/protein/976160005?report=genbankandlog$=pr ottopandblast_rank=1andRID=V6 72V508011
Lowry, O. H., N. J. Rosebrough, A. L. Farr and R. J. Randall (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265-275.
Merheb-Dini, C., H. Cabral, R. S. Leite, L. M. Zanphorlin, D. N. Okamoto, G. O. B. Rodriguez, L. Juliano, E. C. Arantes, E. Gomes and R. da Silva (2009). Biochemical and functional characterization of a metalloprotease from the thermophilic fungus Thermoascus aurantiacus. Journal of agricultural and food chemistry, 57: 9210-9217.
Mohamed, A., M. Abd El-Mongy, H. Mahrous, A. I. El-Batal, H. A. Hamza (2013). Partial characterization of a novel bacteriocin substance produced by Lactobacillus spp. Egyptain J. of Botany, Special Issue, 187-200.
Nascimento, W. C. A. D. and M. L. L. Martins (2004). Production and properties of an extracellular protease from thermophilic Bacillus sp. Brazilian Journal of Microbiology, 35: 91-96.
Patel, R., M. Dodia and S. P. Singh (2005). Extracellular alkaline protease from a newly isolated haloalkaliphilic Bacillus sp.: Production and optimization. Process Biochemistry, 40: 3569-3575.
Phylip, F. (1989). Phylogeny Inference Package. (Version 3.3). Cladistics, 5: 164-166. Phylip, F. (2000). Phylogeny Inference Package (Version 3.3). Cladistics; 5:164-6.
Rai, S. K., J. K. Roy and A. K. Mukherjee (2010). Characterisation of a detergent- stable alkaline protease from a novel thermophilic strain Paenibacillus tezpurensis sp. nov. AS-S24-II. Applied microbiology and biotechnology, 85: 1437-1450.
Rao, M. B., A. M. Tanksale, M. S. Ghatge and V. V. Deshpande (1998). Molecular and biotechnological aspects of microbial proteases. Microbiology and Molecular Biology Reviews, 62: 597-635.
Rawlings, N. D. and A. J. Barrett (1993). Evolutionary families of peptidases. Biochemical Journal, 290: 205- 218.
Rawlings, N. D., D. P. Tolle and A. J. Barrett (2004). Evolutionary families of peptidase inhibitors. Biochemical Journal, 378: 705-716. Ray, A. (2012). Protease enzymepotential industrial scope. Int. J. Tech., 2: 1-4.
Regar, R. K., V. K. Gaur, G. Mishra, S. Jadhao, M. Kamthan and N. Manickam (2016). Draft genome sequence of Alcaligenes faecalis strain IITR89, an indole-oxidizing bacterium. Genome Announcements, 4: e00067-16.
Scandurra, R., V. Consalvi, R. Chiaraluce, L. Politi and P. C. Engel (2000). Protein activity in extremophilic archaea. Front BioSci., 5: D787- D795. Sevinc, N. and E. Demirkan (2011). Production of Protease by Bacillus sp. N-40 isolated from soil and its enzymatic properties. Journal of Biological and Environmental Sciences, 5: 95-103.
Srinivasan, T. R., S. Das, V. B. R. Philip and N. Kannan (2009). Isolation and characterization of thermostable protease producing bacteria from tannery industry effluent. Recent Research in Science and Technology, 1: 63-66.
Tari, C., H. Genckal and F. Tokatli (2006). Optimization of a growth medium using a statistical approach for the production of an alkaline protease from a newly isolated Bacillus sp. L21. Process Biochemistry, 41: 659-665.
Weng, M., X. Deng, J. Wu and G. Zou (2014). Thermoactivity of subtilisin nattokinase obtained by site-directed mutagenesis. Wuhan University Journal of Natural Sciences, 19: 229-234.
