Enzymatic Properties Of Cold-Active ?-Amylase From Marine Bacillus Sp.

Starch is a natural biomass resource in nature,which is second only to cellulose in quantity.Amylase can decompose starch into glucose and its derivatives,which have high added value and are widely used in food,paper making,textile,chemical industry,pharmaceutical and other fields.The ocean contains a large amount of cold-active microorganisms that can produce valuable cold-active enzymes.Due to its high catalytic efficiency,high structural flexibility and thermal instability,the cold-active enzymes have advantages over medium and high-termperature enzymes in low-temperature applications,and moreover mild heat treatment can inactivate cold-active enzymes,which can quickly and economically terminate the reaction.Therefore,the study of marine cold-active a-amylase and its properties is of great significance for the further development and utilization of new amylase resources and energy consumption.The contents of this dissertation were as follows:Firstly,we identified a cold-active a-amylase strain with industrial application potential,designated as dsh19-1.The bacterial strain was isolated from sediment samples in the Yellow Sea area of Dalian and cultured using soluble starch as the only carbon source.Based on the evidences obtained from morphological characteristics,phylogenetic tree construction and fatty acid composition and the analysis of the 16S rRNA sequence,this strain was determined to belong to the genus Bacillus and was named Bacillus sp.dsh19-1.Secondly,the fermentation medium and conditions of the cold-active a-amylase strain Bacillus sp.dsh19-1 were optimized based on the experimental designs using single-factor experiments and response surface methods.The results demonstrated that the optimal medium contained the following components:4.25%of soluble starch,1.52%of beef extract.1.59%of NaCl,0.03%of MgSO4 and 0.05%of K2HPO4.The optimum fermentation conditions were as follows:fermentation time 42.2 h,fermentation temperature 20.6 ?,inoculum volume 4.1%,the initial pH 6.4,and the medium volume 125 mL/500 mL.The enzyme activity of the cold-active alpha-amylase was 3.06 U/mL with the optimized procedures,which was 17.69%higher than that without optimization.The natural cold-active a-amylase protein AmyD was purified by ammonium sulfate precipitation,dialysis,ultrafiltration,DEAE-anion exchange column chromatography and Sephadex G-100 column chromatography.The purification yielded 22.27 mg of AmyD with a purification factor of 10.8 and a yield of 6.47%.The specific activity of AmyD was 8.86 U/mg.The enzyme had a molecular weight of 50.1 kDa,an optimum temperature of 20 ?,and an optimum pH of 6.0.Both Na+ and Ca2+ could increase its activity.The TLC analysis of AmyD hydrolyzed product demonstrated the AmyD has the hydrolyzing properties of typical a-amylase.Thirdly,the a-amylase gene of Bacillus sp.dsh19-1 was successfully cloned by PCR.The prokaryotic expression vector pColdI-Amy was constructed and Amy soluble expression was achieved in E.coli.The recombinant protein AmyD-1 was purified by TALON superfluous metal affinity chromatography to obtain 19.5 mg of purified protein from a 2 L fermentation broth,which is 8,2-fold purification with a yield of 71.3%.The specific activity of AmyD-1 was 16.4 U/mg.The optimum temperature of AmyD-1 found to be 20 ?,indicating the enzyme has the feature of cold activity.The optimum pH was 6.0,and the Km,Vmax,and kcat values of soluble starch were 2.8 mg/kg mL,21.8 mg/mL/min,933.5 1/s at 20 ?,respectively.AmyD-1 showed strong salt tolerance and could remain 50%of the highest enzyme activity at 5 M NaCl.The analysis of AmyD-1 degraded substrate with mass spectrometry demonstrated that AmyD-1 is an alpha-amylase.Finally,starch branching enzyme gene(Sbe)was successfully cloned from the Bacillus sp.dsh19-1 by PCR.The prokaryotic expression vector pColdl-Sbe was constructed and the non-soluble Sbe gene was expressed in E.coli.The study of ?-amylase and starch branching enzymes of marine bacteria Bacillus sp.dsh19-1 will provide theoretical support for the low-temperature hydrolysis and low-temperature modification of starch and will lay a foundation for the application of cold-adapted enzymes in low-temperature washing and food production.