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Title: Biochemical and biophysical studies on thermostable and thermoactive enzymes and understanding the molecular determinants of protein thermostability
Researcher: Parmar, Krupali
Guide(s): Das, Mili
Keywords: Biochemistry and Molecular Biology
Biology and Biochemistry
Life Sciences
University: Nirma University
Completed Date: 2020
Abstract: This research work compiles biophysical and biochemical studies on two extracellular enzymes, a lipase from Halomonas shengliensis and an amylase from Bacillus atrophaeus. Both these enzymes belong to the group of candidate enzymes which possess promising industrial applications. The initial study is focused on purification, characterization, cloning and stability studies of lipase from a moderate haloalkalophile. The production of thermo-active lipase was carried out in optimized production medium containing; olive oil 2.0% (w/v), yeast extract 0.5% (w/v) and NaCl 6% (w/v), pH 8.5 at 35°C with agitation for 4 days. The protein exhibited high affinity towards anion exchanger resins. For purification, it was bound to Q-Sepharose Fast Flow column and eluted in 0.05 M-1M NaCl gradient. The crude lipase was purified with around 70% final yield and 10 fold purification. The molecular mass of lipase determined from SDS-PAGE was 41.35 kDa, while mass spectroscopy analysis estimated it to be 35.19 kDa, an anomaly which leads us to presume that the anomalous migration of lipase on SDS-PAGE could be due to the presence of glycan moiety on it. Primary glycan screens such as phenol-H2SO4 and stains-all staining of native protein on SDS-PAGE, supported our assumption. The purified lipase was found to be relatively thermostable, retaining its activity even at temperatures of up to 80°C, with optimal activity at 70°C. It was observed to be active at pH 6.0-8.5, with optimum activity at pH 7.5. This lipase could actively hydrolyse most of the p-nitrophenylester substrates, with slightly greater preference for short to medium chain fatty acid substrates (C2, C4, C6). Strong inhibition of lipase activity by phenylmethylsulfonidefluoride (PMSF), indicated the active role of serine residue at its catalytic site. The enzyme retained around 70% of its initial activity after exposure to 15% organic solvents (methanol, ethanol, acetone, benzene, acetonitrile, dimethylformmamide and n-hexane). Lipase showed slightly enhanced
Appears in Departments:Institute of Science

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01_title.pdfAttached File24.08 kBAdobe PDFView/Open
02_certificate.pdf239.17 kBAdobe PDFView/Open
03_abstract.pdf130.67 kBAdobe PDFView/Open
04_declaration.pdf216.03 kBAdobe PDFView/Open
05_acknowlegdement.pdf131.4 kBAdobe PDFView/Open
06_contents.pdf182.92 kBAdobe PDFView/Open
07_list_of_tables.pdf12.88 kBAdobe PDFView/Open
08_list_of_figures.pdf183.36 kBAdobe PDFView/Open
09_abbreviations.pdf5.96 kBAdobe PDFView/Open
10_chapter 1.pdf335.08 kBAdobe PDFView/Open
10chapter 7.pdf412.7 kBAdobe PDFView/Open
11_chapter 2.pdf377.22 kBAdobe PDFView/Open
12_chapter 3.pdf1.49 MBAdobe PDFView/Open
13_chapter 4.pdf1.62 MBAdobe PDFView/Open
14_chapter5.pdf1.02 MBAdobe PDFView/Open
15_summary & conclusions.pdf198.01 kBAdobe PDFView/Open
16_bibliography.pdf3.79 kBAdobe PDFView/Open
80_recommendation.pdf219.91 kBAdobe PDFView/Open
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