摘要
研究应用于甾体9α-羟基化的高效催化剂和催化体系是实现甾体药物9α位羟基高效合成的关键。本文中,笔者对来源于分支杆菌(Mycobacterium tuberculosis) H37Rv和红平红球菌(Rhodococcus erythropolis) SQ1的3-甾酮-9α-羟基化酶基因(reksh A,mtksh A)进行优化,并对2个基因的催化活性进行检测。通过构建工程表达菌株,以雄甾-4-烯-3,17-二酮(AD)为底物,对制备9α-羟基雄甾-4-烯-3,17-二酮(9α-OH-AD)的催化反应进行了探索。结果表明:基因序列优化显著提升了Re Ksh A和MtKshA蛋白的可溶性表达,其中Re Ksh A具有较好的雄甾-4-烯-3,17-二酮9α位羟基化活性。用TB培养基培养Re Ksh A的工程表达菌株BL21(DE3)-p ET28a-reksh A-yh,在30℃、500μmol/L底物浓度、0. 8%(体积分数)乙醇为助溶剂、0. 1 mmol/L IPTG诱导浓度、底物与诱导剂同时加入到工程菌液中的条件下,9α-OH-AD得率达到97. 09%。
In order to develop a biocatalyst with high activity for 3.ketosteroid 9α.hydroxylation,recombinant strains BL21 （ DE3 ）-pET28a-kshA-reyh and BL21 （ DE3 ）-pET28a-kshA-mtyh were constructed by codon optimization according to the cDNA sequence of 3.ketosteroid 9α.hydroxylase ofRhodococcus erythropolis SQ1 （rekshA） and Mycobacterium tuberculosis H37Rv （mtkshA） in the GenBankdatabase. The soluble expression of kshA was significantly improved compared to unoptimized codingsequences. The recombinant strain was used to convert AD to 9α-OH-AD. The results showed that thekshA from Rhodococcus erythropolis SQ1 has significantly higher 9αhydroxylation activity towardsanostenedione compared to that from Mycobacterium tuberculosis H37Rv. The yield reached 97. 09% whenBL21 （ DE3）-pET28a-kshA-reyh was used as catalyst under the conditions of 500 μmol / L AD, 0. 1mmol / L IPTG,0. 8% ethanol as cosolvent. The reaction was performed at 30 ℃,the substrate and IPTGwere added at the same time.
作者
王康
吴志革
赵伟睿
胡升
麻菊美
王进波
姚善泾
梅乐和
WANG Kang;WU Zhige;ZHAO Weirui;HU Sheng;MA Jumei;WANG Jinbo;YAO Shanjing;MEI Lehe(School of Biotechnology and Chemical Engineering,Ningbo Institute of Technology,Zhejiang University,Ningbo 315100,China;Department of Chemical and Biological Engineering,Zhejiang University,Hangzhou 310027,China;College of Biosystems Engineering and Food Science,Zhejiang University,Hangzhou 310058,China)
出处
《生物加工过程》
CAS
2018年第6期13-18,共6页
Chinese Journal of Bioprocess Engineering
基金
国家自然科学基金(81502421
31670804)
中国博士后科学基金(2016M592003)
国家星火计划(2015GA701022)
浙江省自然科学基金重点项目(LZ13B060002)
浙江省自然科学基金(Q14C070004
LY16B060008)
宁波市科技惠民计划(2015C50042)
宁波市自然科学基金(2014A610214)
