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Title: Molecular Evolution and characterization of and#946; galactosidase enzyme from thermophilic bacteria
Researcher: Rani. Ms Varsha
Guide(s): Dev, Dr Kamal
Keywords: Arthrobacter
Thermus thermophilus
University: Shoolini University of Biotechnology and Management Sciences
Completed Date: 08-01-2016
Abstract: vii newlineAbstract newlineand#946; galactosidase is ubiquitously present in all life forms including extremophiles So it is very interesting to study the evolution and adaptation of and#946; galactosidase under different environmental conditions. Tattapni hotspring in Mandi district of Himachal Pradesh, India was unexplored for the and#61538; galactosidase production. We have isolated thermophilic bacteria form Tattapani hotspring and screened for the production of and#61538; galactosidase. In order to study the evolution and adaptation at molecular basis, and#61538; galactosidase was studied by in silico approach. Fifty and#946; galactosidase proteins from thermophiles, psychrophiles, plants and animals were analyzed. Conserved domain analysis revealed that and#946;-galactosidase belongs to glycosyl hydrolase family. Thermophiles showed a different pattern of domains organization as compared to the psychrophiles and mesophiles among bacteria while plants and animals had a common domain named glyco_hydro35. Thermophilic and#61538; galactosidases have higher percentage of -helix region, which may be responsible for temperature tolerance, while psychrophilic and mesophilic and#61538; galactosidases showed higher percentage of beta sheets. Docking analysis predicted that lactose showed maximum interaction with and#61538; galactosidase of Escherichia coli, followed by Arthrobacter sp. C2-2 and Homo sapiens. and#61538; galactosidase of Thermus thermophilus showed maximum interaction with ONPG. Brassica oleracea dosen t show interaction with lactose and ONPG. Competitive inhibition was observed among ONP, ONPG, PNPG and lactose in and#61538; galactosidase of Homo sapiens. Lactose, galactose and PNPG showed competitive inhibition in and#61538; galactosidase of Thermus Thermophilus. Glucose and ONP showed competitive inhibition. and#61538; galactosidase of Arthrobacter sp. C2-2 and Brassica oleracea. Glucose, galactose and ONP were uncompetitive inhibitors for and#61538; galactosidase of Escherichia coli. Phylogenetic analysis showed that mesophilic and#946; galactosidase does not show any divergence and are closely related to their group. newlineNineteen thermophilic bacterial isolates were isolated and screened for and#946;-galactosidase activity. Out of 19 thermophilic isolates, only PW10 and PS7 produced extracellular and auto inducible and#946;-galactosidase. Both PW10 and PS7 were Gram s positive, rod shaped bacteria and exhibit growth between 50-80 °C and pH 5-9. On the other hand, optimum and#946;- galactosidase activity of 32083.33 U/mg/min was observed at 60 °C at pH 7 for PS7 and 2666.66 U/mg/min at 60 °C at pH 9 for PW10. 16S rDNA sequencing of PW10 isolate newlineviii newlineshowed 99% similarity with Anoxybacillus flavithermus and PS7 showed 99% similarity with Bacillus licheniformis and submitted in GenBank database under accession no. KF039883 and KF039882 respectively. Carbon sources like galactose, starch, sucrose, inositol and lactose enhanced and#946;-galactosidase production by 5.2, 5.2, 1.1, 1.2, 3.6 and 7.6 folds respectively in Bacillus licheniformis PS7. Galactose, sucrose, xylose, trehalose and lactose enhanced and#946;-galactosidase production by 1.4, 2.2, 1.2 and 2.5 folds respectively for Anoxybacillus flavithermus PW10. Yeast extract enhanced and#946;-galactosidase production by 3.5 fold in Bacillus licheniformis PS7 and by 1.4 fold in Anoxybacillus flavithermus PW10. Solvents like, ethanol (0.5 and 1%), and hydrogen peroxide (0.05 and 0.1%) does not affect the growth in Bacillus licheniformis PS7, while butanol, cyclohexane, phenol and toluene (0.05 and 0.1% concentration) decreased the growth by 8.8, 19.5, 4.7 and 1.2 fold respectively at 0.1% concentration. Ethanol, butanol, cyclohexane, phenol and toluene decreased the growth of Anoxybacillus flavithermus PW10 by 7.3 folds at 1% concentration, 2.5, 1.4, 14.7 and 1.2 fold respectively at 0.1% concentration. Hydrogen peroxide inhibited the growth in Anoxybacillus flavithermus PW10. and#946; galactosidase activity of Bacillus licheniformis PS7 was found metal independent, while Anoxybacillus flavithermus PW10 was newlinefound to be metal (Zn2+, Ca2+, Cu2+, Fe2+, Na+ and Mg2+) activated. Interestingly, lactose and newlineglucose activated the and#946;-galactosidase by 1 and 2 fold respectively for PS7 isolate, whereas newlineand 2 fold increase for PW10 isolate. Glucose did not affect the and#61538; galactosidase activity of both the isolates. Maximum and#946;-galactosidase production was observed at ~ 72 h of incubation. Kinetic determinations at 60° C established a Km of 8.0 mM for ONPG in Bacillus licheniformis PS7 and 1.3 mM in Anoxybacillus flavithermus PW10. and#946;-galactosidase was stable at 4 and 25 °C for 5 6 days in Bacillus licheniformis PS7 and Anoxybacillus flavithermus PW10. newline
Pagination: viii-126
Appears in Departments:Faculty Of Biotechnology

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