生物转化产d-伪麻黄碱菌株的筛选及其关键酶基因的验证

Strain-screening in biotransformation of d-pseudoephedrine and expression of its bcdh gene

利用GenBank和UniProt数据库比对Morganella morganii J-8羰基还原酶基因和氨基酸序列,以同源性为依据,结合高效液相色谱(HPLC)检测验证,筛选出5株同样具有转化1-苯基-2-甲氨基丙酮(MAK)产d-伪麻黄碱功能的菌株。选取其中1株Bacillus clausii B0658,对其d-伪麻黄碱的生物转化过程进行考察,发现在最优条件下d-伪麻黄碱产量达到128.3 mg/L。进一步对B.clausii B0658的亮氨酸脱氢酶基因bcdh进行扩增,以pET28a(+)为载体构建重组质粒并在Escherichia coli BL21(DE3)中实现表达,通过重组菌的生物转化实验验证该酶的催化功能。

Based on the results of blasting gene and amino acid sequences of carbonyl reductase from Morganella morganii J-8 in GenBank and Uniprot database, 5 strains for transforming 1-phenyl-2- methylamine-acetone（MAK） to d-pseudoephedrine were screened out from the CICIM-CU by HPLC detection. The transformation process for d-pseudoephedrine production from Bacillus clausii B0658 was investigated. Under the optimal transformation condition, the yield of d-pseudoephedrine could reach 128.3 mg/L. Moreover, leucine dehydrogenase gene （bcdh） from B. clausii B0658 was amplified. A recombinant plasmid pET28a-bcdh was constructed and transformed into E. coli BL21 （DE3）. The catalytic function of leucine dehydrogenase from B. clausii B0658 was subsequently verified as bcdh was expressed, d-pseudoephedrine could be detected in the reaction system catalyzed by the recombinant E. coll.