天津科技大学学报2017，Vol.32Issue(5)：34-38,78,6.DOI:10.13364/j.issn.1672-6510.20160130
赭曲霉甾体11α-羟化酶基因AOH在毕赤酵母中的表达及功能分析
Expression and Function of Steroid 11α-Hydroxylase Gene AOH from Aspergillus ochraceus in Pichia pastoris
摘要
Abstract
11α-hydroxy-13-ethyl-gon-4-en-3,17-dione(11α-OH-GD)is a key intermediate in the synthesis of desogestrel,a major ingredient of the third generation of oral contraceptive.Filamentous fungusAspergillus ochraceusTCCC41060 can convert 13-ethyl-gon-4-en-3,17-dione(GD)into 11α-OH-GD.However,low reaction specificity and high-level side prod-ucts limit its application in large-scale preparation of 11α-hydroxy-GD.In order to investigate the mechanism of low reaction specificity ofA.ochraceus TCCC41060 on substrate GD,cDNA of 11α-hydroxylase geneAOH was cloned and ligated to plasmid pPIC3.5,K in order to generate expression vector pPIC3.5,K-AOH;and then recombinantP.pastoris strains express-ing AOH were constructed.SDS-PAGE and Western blot analyses indicated that AOH was 6.12×104,which was consistent with the predicted size.TLC and HPLC analyses showed that the AOH recombinantP.pichia strain was capable of trans-forming GD into the target product 11α-OH-GD and three types of side products.The results indicated that the 11α-hydroxylase withAOH gene has poor specificity of conversion reaction on GD.关键词
赭曲霉/11α-左旋乙基甾烯双酮/毕赤酵母Key words
Aspergillus ochraceus/11α-hydroxy-13-ethyl-gon-4-en-3,17-dione/Pichia pastoris分类
生物科学引用本文复制引用
郭凯,曹通,黄琳琳,刘晓光,王正祥,路福平..赭曲霉甾体11α-羟化酶基因AOH在毕赤酵母中的表达及功能分析[J].天津科技大学学报,2017,32(5):34-38,78,6.基金项目
国家高技术研究发展计划(863计划)资助项目(2011AA02A211) （863计划）
