Rational Design Of Aminotransferase BtrR And Cloning And Characterization Of Glucosylase AcCel16

Diabetes has great harm to human health.And so the effective anti-diabetic drugs study has been found implications for life-sustaining activities.As a novel anti-diabetic drug,?-glucosidase inhibitors are widely used clinically.They have a nitrogen-containing pseudo-saccharide structure,which can inhibit the formation of glycosidic bonds and weaken the?-glucosidase activity.Valienamine is an?-glucosidase inhibitor with a similar configuration to?-D-glucose.Valienamine requires a series of complicated process synthesis in vivo,which takes a long time and high cost.Recently,it was found that aminotransferase BtrR can be used to achieve one-step synthesis of valienamine by transamination.However,the wild-type BtrR has the disadvantages of low catalytic efficiency and poor stability.Bioinformatics calculations showed that the amino acid at position 92 of the wild-type BtrR plays a crucial role in the enzyme activity.In order to obtain high transaminase activity,site-directed mutagenesis was used to mutate the Trp92 of wild-type.We expressed and purified BtrR and its mutants,respectively.GDH coupling method was used to determine the enzyme activity of BtrR and its mutants.The results showed that the activity of mutant R was 1.12-fold higher than that of wild-type,while the activity of other mutants K,L,M,H,G,A and F was lower compared to the wild-type and the activity of other mutants was similar to that of wild-type.This result indicates that the Trp92 sequence has a great influence on the enzyme activity.Our results provide an important clue for the construction of an efficient synthesis pathway for valienamine.With the loss of renewable resources on the earth,looking for renewable energy has become a concern worldwide.The use of cellulase to catalyze the degradation of lignocellulose is one of the effective ways to obtain renewable resources.In order to obtain high activity and stability glycosylase,the glucanase ABK51904.1 gene was cloned from Acidothermus cellulolyticus 11B,belonging to 16 families of glycoside hydrolases?GH16?.The pET-28a expression vector was used to construct the Escherichia coli engineering bacteria,and the recombinant protein AcCel16 was obtained by soluble expression.After heat treatment and the Ni-NTA column affinity chromatography,the target protein of electrophoresis purity was obtained.Enzymatic properties analysis showed the enzyme that had the highest hydrolysis activity for Laminarin,the optimum pH is 5.0 and the optimal temperature is 55?.The thermal stability showed that it still retained about 60%of enzyme activity when incubated at70?for 1 hour.The experiment found that the enzyme is a non-metal ion-dependent enzyme with a Km value and Vmax of 1.92 mg·mL^-11 and 44.6 U·mg^-1,respectively.The experimental results proved that the enzyme has good properties in acid tolerance and thermal stability.In addition,the substrate specificity is widely and has application potential in biomass utilization.