Please use this identifier to cite or link to this item: http://krishikosh.egranth.ac.in/handle/1/5810133556
Authors: ASH, KRISHNA
Advisor: Sushma, Dr. (Mrs.)
Title: Partial purification and industrial applications of alkaline protease from selected bacterial strains
Publisher: DEPARTMENT OF BIOCHEMISTRY AND BIOCHEMICAL ENGINEERING JACOB INSTITUTE OF BIOTECHNOLOGY AND BIOENGINEERING SAM-HIGGINBOTTOM UNIVERSITY OF AGRICULTURE, TECHNOLOGY AND SCIENCES PRAYAGRAJ (ALLAHABAD) -211007
Language: en
Type: Thesis
Pages: 131p.
Agrotags: null
Keywords: Key words: Alkaline protease, 16S rDNA, Purification, Characterization, Detergent compatibility, washing performance, feather degradation, keratin, Pseudomonas aeruginosa MH298778, Acinetobacter variabilis MG650110, soil samples
Abstract: Protease enzymes have immense commercial value and play a pivotal role in application of various industrial sectors. Microbial proteases are one of the important groups of industrially and commercially produced enzymes which have several applications. In this study 148 bacterial strains were isolated from 50 different soil samples of slaughter house, fish market and sewage wastes of Lucknow, Uttar Pradesh, India. Out of which ninety four strains competent of secreting extracellular alkaline protease. In preliminary screening the isolates SSB1 and SSB2 showed highest ability to hydrolyze casein and skimmed milk which was done on skim milk agar media. Based on biochemical tests the isolate showed positive for casein, starch, catalase and negative for gram staining, indole, methyl red, voges proskauer, gelatin, urea, oxidase, hemolysis and triple sugar iron test and found to be non motile. Strain SSB1 and SSB2 with the maximum yield alkaline protease was identified as Pseudomonas aeruginosa MH298778 and Acinetobacter variabilis MG650110 based on nucleotide homology and phylogenetic analysis (16S rDNA sequencing). Protease production was enhanced by optimizing the culture conditions. Many physical and chemical parameters were studied to optimize the maximum yield of alkaline protease. The maximum enzyme activity were observed with optimum incubation time, temperature, pH, carbon, nitrogen sources, NaCl and metallic ions were determined as 36 hrs, 37°C, pH 11.0, 1% glucose, 1% yeast extract, 1M NaCl, and 1mM Zn2+, respectively for protease production further the P.aeruginosa protease was purified to homogeneity using ammonium sulphate precipitation, dialysis and ion exchange chromatography with a fold purification of 2.28 and a recovery of 35.91%. The enzyme has a relative molecular weight of 30 kDa, pH and temperature optima for this protease were 9.0 and 40ᵒC. The activity was stable between a pH ranged of 8.0-10.0. Metal ions such as Ca2+ and Mg2+ stimulated the protease activity up to 115 and 109% respectively. The activity was completely inhibited by EDTA indicating the presence of metalloprotease. The protease was found to be stable in some commonly used commercial detergents tested (Wheel, Rin, Ariel and Tide). It retained 70% residual activity after 3hrs of incubation with Wheel, 71% with Rin, 73% with Ariel and 52% with Tide. The maximum stability was observed with Ariel. The 9 supplementation of the enzyme preparation in detergents could significantly improve the cleansing performance towards the blood and grass stains and suggest its possible use as a laundry additive. The protease was degrading 60% of poultry feathers in 48hrs showing its keratin degrading potential and it can be used as an additive in poultry industry as waste treatment. The A. Variabilis protease was purified to homogeneity using ammonium sulphate precipitation, dialysis and ion exchange chromatography with a fold purification of 2.01 and a recovery of 41.27%. The enzyme has a relative molecular weight of 28.5 kDa, pH and temperature optima for this protease were 11.0 and 40ᵒC. The activity was stable between a pH ranged of 9.0-12.0. Metal ions such as Ca2+ and Mg2+ stimulated the protease activity up to 110 and 104% respectively. The activity was completely inhibited by EDTA indicating the presence of metalloprotease. The protease was found to be stable in some commonly used commercial detergents tested (Wheel, Rin, Ariel and Tide). It retained 66% residual activity after 3hrs of incubation with Wheel, 72% with Rin, 76% with Ariel and 66% with Tide. The maximum stability was observed with Ariel. The supplementation of the enzyme preparation in detergents could significantly improve the cleansing performance towards the blood and grass stains and suggest its possible use as a laundry additive. The protease was degrading 60% of poultry feathers in 48hrs showing its keratin degrading potential and it can be used as an additive in poultry industry as waste treatment. The results of the application studies viz, compatibility with detergents, washing performance and feather degradation revealed that the bacterial strains P.aeruginosa and A. variabilis are potent source of alkaline protease. In consequence, these two strains have immense scope in various industries.
Description: Ph. D. Thesis
Subject: Biochemistry
Theme: Partial purification and industrial applications of alkaline protease from selected bacterial strains
Research Problem: Partial purification and industrial applications of alkaline protease from selected bacterial strains
These Type: Ph.D
Issue Date: 2019
Appears in Collections:Thesis

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