Semi-rational Design Of An Amine Dehydrogenase In The Asymmetric Reductive Synthesis Of Chiral Amino Alcohols

Optically pure amine alcohols are important building blocks in many bioactive molecules.Among them,(R)-3-aminobutanol is a crucial chiral intermediate in the synthesis of anti-HIV drug Dolutegravi.At present,(R)-3-aminobutanol is mainly synthesized by chemical methods.However,the chemical catalysis has many disadvantages of not being very friendly to the environment,long reaction route and poor product selectivity.Therefore,a sustainable yet efficient and productive biosynthesis method is urgently needed.In previous work,several amine dehydrogenases(AmDH)have been screened by our team.They can catalyze the asymmetric reductive amination of 4-hydroxy-2-butanone(la)to synthesize(R)-3aminobutanol((R)-1b),but the catalytic activity is low.Among them,the amine dehydrogenase GsAmDH(mh13)derived from Geobacillus stearothermophilus showed the highest catalytic efficiency.For this reason,mh13 was used as the template in this study.We used a semi-rational design strategy of iterative saturation mutagenesis to construct mutant libraries.Then,the enzymatic characterization and substrate scope of the dominant mutants were also investigated.The main research contents are as follows:(1)Semi-rational design and basic enzymatic characterization of GsAmDHAfter optimization,the optimal reaction conditions were:1 M ammonia ion concentration,pH 9.0 and temperature 40℃,respectively.On this basis,the conversion of mh13 reached 45%at 30 mM la for 24 h.But after optimizing the conditions,the catalytic efficiency of GsAmDH for asymmetric reduction amination of la was still low.Therefore,we used the combined active-site saturation test(CAST)strategy to couple the glucose dehydrogenase-NAD+coenzyme regeneration system to directionally evolve and screen GsAmDH.After three rounds of combinatorial iterations,the conversion of the obtained optimal mutant(mh174,E114V/T134C/P146V/H187D/V294C)was≥99%,and the ee of the product≥99%(R).For the catalytic performance of enzymes,compared with the starting template mh13,the Km of the optimal mutant mh174 was decreased by 3.2-fold,the kcat was increased by 1.6-fold,and the kcat/Km was increased by about 5.0-fold.For the thermostability of the enzyme,the Tm value of the dominant mutant in every round did not fluctuate greatly,and increased enzymatic activity did not result in decreased thermostability.(2)Preparative reaction,substrate scope and molecular docking analysis of GsAmDHThe optimal mutant mh 174 was tested in a 10 mL scale-up system and 1a loading of 100 mM to explore whether the mutant has industrial application potential.It was found that the conversion can reach 99%in 36 h,and the yield of(R)-1b purified by ion exchange chromatography is as high as 90%.To further study on the substrate specificity of mh174,ten hydroxyketone substrates were also tested.The results showed that all substrates could be transformed into corresponding products with high ee(≥99%).In addition,homology modeling and molecular docking analysis were used to further clarify the mechanism of the activity improvement of mh174.As the results,some additional interactions were formed in mh174,which affected the asymmetric reductive amination activity of GsAmDH.In this study,the AmDH for the catalytic synthesis of(R)-1b screened in the laboratory is evolved,which enriches the toolbox of biocatalyst for the synthesis of industrial chiral amine alcohol compounds,and provides guidance for the biosynthesis of similar high value-added pharmaceutical intermediates.