Enzymatic Characteristics Of Cold-adapted α-amylase From Antarctic Sea Ice Bacterium Pseudoalteromonas Sp. M175

Cold-adapted α-amylase is an important hydrolase,which can perform hydrolysis function at low temperature and has great application potential in food processing,textile desizing,bio-fuel manufacturing and other aspects.However,there are few commercial cold-adapted α-amylases now,so the tapping and research of novel cold-adapted α-amylase is very important to meet the great demand of industry.In this thesis,M175,a high-yield strain of cold-adapted α-amylase from Antarctic sea ice,was selected as the research object.A novel cold-adaptedα-amylase was mined by genetic engineering,site-directed mutagenesis,and molecular dynamics simulation and other methods.Its enzymatic properties were explored,which laid a foundation for industrial production of cold-adaptedα-amylase.M175,a high-yield cold-adapted α-amylase strain,identified by physiological,biochemical and phylogenetic tree belonged to Pseudoalteromonas and was named as Pseudoalteromonas sp.M175.The wild-type α-amylase(wtAmy175)from M175 strain was purified by ammonium sulfate precipitation,ion exchange chromatography and gel exclusion chromatography.The specific activity of purified enzyme was 289.79 U/mg.Enzymatic properties of wtAmy175 were studied.Its optimal temperature was 30 °C and it had poor stability above 40 ℃,so it was a typical cold-adapted enzyme.In addition,its optimal pH was 7.5,and it was well tolerated to SDS.The hydrolytic ability analysis showed that wtAmy175 had dual activities of α-amylase and α-glucosidase.Cold-adapted α-amylase gene(amy175)of M175 strain was cloned and sequenced.This protein had low homology with other reported α-amylases,so it was a novel α-amylase.The protein had a low arginine content(2.7%)and no disulfide bonds.In addition,β-6 and β-7 of TIM in the tertiary structure were replaced by loops,which showed high flexibility in its structure.The amy175 gene was expressed in prokaryotic system and expression conditions were optimized.After optimization,the maximum amylase activity of the supernatant could reach 141.1U/m L.The recombinant α-amylase(Amy175)was purified by Ni affinity chromatography.The result of mass spectrometry proved that it was the recombinant protein corresponding to wtAmy175.Enzymatic properties of Amy175 were studied.Its optimal temperature(25 ℃)was lower than that of wild-type enzyme,optimal pH was 8.0,and optimal salinity was 1 mol/L NaCl.Therefore,Amy175 was a cold-adapted and salt-tolerant α-amylase.Similar to wtAmy175,Amy175 could specifically hydrolyze α-1,4-glycosidic bond,and had both α-amylase andɑ-glucosidase activity.Amy175 belonged to GH13＿36 subfamily.Molecular modification of Amy175 was performed by site-directed mutagenesis to improve its thermal stability.Glycines at 181 and 451 sites of Amy175 were mutated to prolines.The results showed that the optimal temperature and optimal pH of G181 P,G451P and G181P/G451 P didn’t change,but the thermal stability was significantly improved.After incubated at 40 °C for 60 min,the residual activities of the mutants were 1.7,2.0 and 2.7 times of that of Amy175,respectively.The molecular dynamics simulations were performed at 313 K.Compared with Amy175,the RMSD value,the PMSF value,the Rg value and the SASA value of the mutants decreased in varying degrees,the number of hydrogen bonds increased,the distances between the active sites were shortened,and the double mutant was the most obvious.It was further proved that the thermal stability of the protein could be improved when glycines at 181 and 451 sites were mutated to prolines.The combination of Amy175 with maltose and maltotriose was studied by molecular docking.The result showed that the electrostatic force and van der Waals force were the main binding forces for Amy175 and substrates.V142,R242 and N370 were the most important amino acids for Amy175 to bind maltose,while R242,W288 and H371 played a key role in binding maltotriose.Catalytic triads D244,E286 and D372 were the major amino acids involved in the formation of hydrogen bonds between Amy175 and substrates.Above results helped us to better understand the action mechanism of amylases.Enzymatic properties of wild-type α-amylase(wtAmy175)and recombinantα-amylase(Amy175)from Antarctic sea ice bacterium Pseudoalteromonas sp.M175 were studied in detail.Additionally,Amy175 was modified to improve its thermal stability,which laid a foundation for the accumulation,in-depth study and application of cold-adapted α-amylase.