Characterization of alkaline protease from plant growth promoting rhizobacteria and their potential applications
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Date
2015
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YSPU
Abstract
Rhizosphere of apple trees of Trans Himalayas is great niche of proteolytic bacteria which may have
potential for use in industries. A total of 76 proteolytic bacteria were obtained from rhizosphere soil and root
endosphere of apple trees from different sites of four locations viz., Chamba, Shimla, Kinnaur and Kullu of
Himachal Pradesh, India, which were screened for plant growth promoting traits including phosphate
solubilization, IAA, hydrogen cyanide production and production of antifungal metabolite. Among them, 17
isolates that exhibited maximum alkaline protease activity were selected. The diversity elucidation and
differentiation of isolates was achieved through their biochemical characterization and amplified ribosomal
DNA restriction analysis with three tetra-cutter restriction enzymes (Hpa II, Hinf I and TaqI), the representative cluster groups were identified by 16S rDNA sequencing. This brings to light the predominance of Bacillus sp. and presence of remarkable intraspecies functional and genotypic diversity in apple rhizosphere. Proteolytic acitivity among selected isolates ranged between 50 to 1610 μg/ml/min at pH 8.0 and maximum was observed for Bacillus amyloliquefaciens SP1 (1610 μg/ml/min). Maximum enzyme production for B. amyloliquefaciens SP1 was observed, when fermentation medium, supplemented with 1.0% gelatin at pH 8.0 was incubated at 35°C for 48 h with 1.0% inoculun size and maltose as carbon source. Further optimization by Plackett Burman design and Response surface methodology resulted 2.08-fold increase in alkaline protease production (3350 μg/ml/min). The enzyme was purified by gel permeation and cation exchange chromatography and had a molecular mass of 43 kDa. Protease activity was optimum at pH 8.0 and 60°C. The half-life of the enzyme at 50, 60 and 65⁰C was 77, 19.8 and 13.33 min. The protease had Km, V max and Ea values of 0.125 mg/ml, 12820 μg min-1 and 34.52 kJ/mol in casein hydrolysis, respectively. All metal ions except Hg2+ and Cu2+ were well tolerated. The deduced internal amino acid sequence of B. amyloliquefaciens SP1 protease by MALDI-TOF MS resembled the sequence with D-alanyl-D-alanine carboxypeptidase of Bacillus subtilis strain 168. An alkaline protease gene was amplified from genomic DNA, cloned and successfully expressed in competent cells of E. coli DH5 . Most reliable three dimensional structure for protease was predicted by using Phyre2 server. This study reported the shortest time of 1:30 min at 3630 μg/ml/min and 4:30 min at74 μg/ml/min of enzyme activity for hydrolysis of gelatin layers of used X-ray film. It has revealed strain removal property and dehairing activity for animal hide without chemical assistance and without hydrolyzing fibrous proteins. This study also reported
significant role of protease in antagonistic activity against C. michiganensis, R. solanacearum and F.
oxysporium.
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Keywords
enzymes, alkalinity, productivity, bacteria, proteins, animal husbandry, polysaccharides, poultry equipment, amino acids, inorganic acid salts