| Natural starch has a low content of resistant starch?RS?.Ingestion of large amounts of it can cause metabolic synthesis related diseases.The RS content of modified starch obtained by enzymatic modification has been increased,which has great benefits for human physiological health.Branching enzymes include starch branching enzyme and glycogen branching enzyme.They can catalyze the cleavage of?-1,4 bonds to form?-1,6 branching points,and have huge application potential in the preparation of modified starch.In this study,the starch branching enzyme?Ro BE?derived from Rhodothemus obamensis with high affinity for amylase and the glycogen branching enzyme?VvGBE?derived from Vibrio vulnificus with high affinity for amylopectin were expressed in E.coli BL21?DE3?,and we study their enzymatic properties and substrate specificity,explore the process conditions for preparing modified starch,and analyze the properties of modified starch.In addition,VvGBE was expressed in B.subtilis WS11,and the influence of the promoter on its expression was explored.The main findings are as follows:?1?The branching enzyme genes derived from Vibrio vulnificus and Rhodothemus obamensis were expressed in E.coli BL21?DE3?,and after shaking flask fermentation,their intracellular enzyme activities on amylopectin were 2.06 U·mL-1 and 0.76 U·m L-1,respectively,intracellular enzyme activity on amylose were 0.53 U·m L-1 and 2.76 U·m L-1,respectively.Their enzymatic properties were investigated:the optimum temperature of VvGBE was 35?,the optimum pH was 7.5,the half-life of 35?was 10 h,and the most stable at pH 9.5;the optimum temperature of RoBE was 70?,the optimum pH was 7.0,the half-life at 70?was 84 h,the most stable at pH 8.0.?2?The recombinant enzymes were applied to corn starch with concentration of 2.5%for modification,and the best conditions for starch modification applications were investigated:VvGBE was at pH 7.5,temperature 35?,the amount of enzyme was 500 U·g-1,and the reaction time was 10 h,RS content in modified starch increased from 25.87%to 51.40%;when the RoBE was at pH 7.0,the temperature was 60?,the amount of enzyme was 60 U·g-1,and the maximum RS content in the modified starch was 41.00%when the reaction time was12 h.?3?Analysis of enzyme reaction products from three aspects:chain length distribution,molecular weight distribution,and nuclear magnetic resonance.Both VvGBE and Ro BE were mainly based on the transfer of short chains of DP3-8,but the peak area occupied by the short chains of VvGBE modified starch?the highest 17.00%?significantly more than RoBE modified starch?the highest 6.00%?;small molecular weight substance in VvGBE modified starch increased by 12.91%,but RoBE modified starch increased by 2.29%;the proportion of?-1,6 bonds in VvGBE modified starch increased by 8.45%,but RoBE modified starch increased by 4.37%.?4?Explore the process of preparing modified starch by double enzymes,granulated corn starch was prepared to a concentration of 15%,and at temperature 60?and pH 7.0,60 U·g-1of RoBE was added.After 12 h,it was gelatinized for 30 min?90??,and then 500 U·g-1 of VvGBE was added,and the reaction was carried out at a temperature of 35?and pH 7.5 for10 h.After a two-step strategy,the RS content in the modified starch reached 74.33%.Identification of the products before and after the mixing of RoBE and VvGBE:The short chains of DP3-6 in the products after compounding increased significantly;the small molecular weight substance increased from 6.91%to 7.49%;the proportion of?-1,6 bonds increased from 5.52%to 13.02%.The two-step preparation of modified starch makes its industrial production possible.?5?The branching enzyme gene derived from Vibrio vulnificus was expressed in B.subtilis,and after shaking flask fermentation,it was mainly secreted to the outside,and the activity of the extracellular supernatant was 0.65 U·m L-1.In order to increase the expression level,the effects of 7 kinds of promoters PaprE,PgsiB,Phpa II,PnprE,Psrf,Pxyl,and Pxyl'on the enzyme activity were investigated,and the optimal promoter PnprE was obtained with an enzyme activity of 0.73 U·m L-1.The obtained plasmid containing the optimal promoter was expressed in B.subtilis WS11?hrcA,and its enzyme activity was 0.92 U·m L-1,which was1.42 times the activity of the initial enzyme. |
PDF Full Text Request