Biochemical and biophysical studies on thermostable and  thermoactive enzymes and understanding the molecular  determinants of protein thermostability

Parmar, Krupali

Please use this identifier to cite or link to this item: http://10.1.7.192:80/jspui/handle/123456789/10119

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DC Field	Value	Language
dc.contributor.author	Parmar, Krupali	-
dc.date.accessioned	2021-09-23T11:36:44Z	-
dc.date.available	2021-09-23T11:36:44Z	-
dc.date.issued	2019-09	-
dc.identifier.uri	http://10.1.7.192:80/jspui/handle/123456789/10119	-
dc.description	ST000067	en_US
dc.description.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 QSepharose 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 activity in presence of non-ionic detergents (0.25% of Tween-20, Tween-80 or Triton X-100) but completely lost activity in presence of laundry detergents. No significant effect on activity was observed when the enzyme was incubated with 1 mM concentration of metal ions (Na+, K+, Ca2+, Mg2+, Zn2+, Cu2+) but significant reduction in activity was observed when the concentration was raised to 10 mM for divalent cations such as Ca2+, Mg2+, Zn2+ and Cu2+ . Michaelis-Menten constants, Km and Vmax, were determined for the hydrolysis of MUF-B and pNPA substrates by lipase. Crystals for the native lipase were obtained by sitting drop crystallization. Stability studies on the purified enzyme were carried out using chemical denaturation and thermal denaturation. Proteolysis and DSC (differential scanning calorimetry) analysis indicated presence of two structural domains in the lipase. Activity of the lipase enzyme at high temperatures, in presence of surfactants and its substantial tolerance towards various organic solvents makes it a satisfactory and promising candidate for industrial applications Six sequences coding for lipase genes were retrieved from the whole genome sequence of the microorganism and five of these sequences were selected for structural studies including Homology modelling, structure prediction and validation. Multiple sequence analysis of all lipases among Halomonas species to explore conserved regions demonstrated very low sequential identity, but high structural homology. The structural and sequential studies for all available lipases from PDB (protein data bank), propounded the imperative role of electrostatic interactions (salt bridges) in the thermostability of lipases. The collective information gathered from this study could throw some light into understanding the molecular determinants of thermostability of proteins. In the second study, two amylases screened from Bacillus atrophaeus, (of Molecular weights 64.3 kDa and 73.4 kDa) were purified partially by ion exchange chromatography. Ammonium sulphate fractionation (70%-80%) could segregate one amylase. The SDS-PAGE and zymography analysis showed that the purified amylase has molecular weight of 63.4 kDa. The optimum temperature and optimum pH for activity of the amylase were found to be 40°C and 5.0, respectively. Efforts were made to clone the genes of both amylases into E.coli DH5α strain. Amylase (64.3 kDa) was successfully cloned and expressed into E.coli BL21 strain, via ligation independent cloning. The recombinant protein showed activity at optimum temperature of 40°C and optimum pH 6.0. The recombinant protein could further be targeted to enhance the thermostability by protein engineering methods such as directed evolution. iv	en_US
dc.language.iso	en_US	en_US
dc.publisher	Institute of Science, Nirma University	en_US
dc.relation.ispartofseries	;ST000067	-
dc.subject	Science Theses	en_US
dc.subject	Theses 2019	en_US
dc.subject	13ftphds28	en_US
dc.subject	thermostable and thermoactive enzymes	en_US
dc.title	Biochemical and biophysical studies on thermostable and thermoactive enzymes and understanding the molecular determinants of protein thermostability	en_US
dc.type	Thesis	en_US
Appears in Collections:	Theses, IS

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