Strain-Screening For Producing D-Pseudoephedrine And Its Transformation Research

With the booming progress of modern biotechnology, the method of microbialtransformation emerges and develops dramatically as a new way of preparing for opticallypure compounds, which has such advantages as high selectivity, easy accessibility and shortperiod of production. This new technique plays an increasingly significant role in developingnew drugs, synthesizing pharmaceutical intermediates and many other aspects, one of whichis the field of producing ephedrine and pseudoephedrine. According to previous achievements,this study screened a series of strains with the ability of producing optically pured-pseudoephedrine by means of predictive analysis of bioinformatics, verified the functions oftheir respective enzymes by molecular biology, and subsequently investigated the courses ofstrain growth and enzyme production over the one with optimal transformation capacity.In early experiences of our laboratory, Morganella morganii J-8which could transform1-phenyl-2-methylamine-acetone（MAK） to optically pure d-pseudoephedrine had beenobtained, and its functional carbonyl reductase（MLDH） was isolated and purified by proteinengineering followed by researches from perspective of molecular biology. This paperfocused on screening new strains that had the same function as M.morganii J-8. Beginningwith the gene and amino acid sequences, five strains were selected from the CICIM-CU bycomparisons of sequence and functional domain homology and further HPLC detection.There were Bacillus thuringiensis B0081, Bacillus megaterium B0108, Oceanobacillusiheyensis B1109, Bacillus clausii B0658and Xanthoonas campestris B1070. Their functionalenzymes were all extracellular and one of them, B.clausii B0658, got the highesttransformation capacity. With the genome DNAs of the five strains as templates andpET28a（+） as the vector, each of the genes was cloned and expressed in E.coli BL21（DE3）,and recombinants were validated to have the function of transforming MAK.Finally, to obtain optimal yield of d-pseudoephedrine, processes of B.clausii B0658growth and its leucine dehydrogenase production and biotransformation were investigated.The components of optimal medium was as follows（g/L）: yeast extract40, glycerol15, NaCl20, KH₂PO₄2.5,（NH₄）₂HPO₄2.5, while the optimal culture condition was:37℃, pH7.5, LBvolume12%, inoculum size2.5%, shaking revolution150r/min. Taking advantage of2mLcrude enzyme extract after17h cultivation for biotransformation, with0.20mg/L MAK,1.5mmol/L NADH and35mmol/L glucose under37℃and pH7.5for8h, the yield ofd-pseudoephedrine and the mole ratio of MAK could reach128.3mg/L and63.38%,respectively.The study started from the sequence-known enzyme, screened five strains which couldtransform MAK to d-pseudoephedrine optically, testified the same function of differentenzymes and further verified the possibility of obtaining strains by bioinformatics method.Meanwhile, a relatively optimal strain was chosen and its growth and enzyme productionwere optimized. Comparing with M.morganii J-8, B.clausii B0658grows faster and its leucinedehydrogenase is extracellular which can make fermentation operations more convenient. Tosome extent, the new strain is much safer and more suitable for industrial application.