Improved production of (R)-1-phenyl-1,2-ethanediol using Candida parapsilosis (R)-carbonyl reductase expressed in Pichia pastoris
Improved production of (R)-1-phenyl-1,2-ethanediol using Candida parapsilosis (R)-carbonyl reductase expressed in Pichia pastoris
复制标题
使用巴斯德毕赤酵母中表达的近平滑念珠菌 (R)-羰基还原酶提高 (R)-1-苯基-1,2-乙二醇的产量
DOI：
10.1016/j.procbio.2010.11.016
复制
发表时间：
2011-03-01
影响因子：
4.4
通讯作者：
Zhang, Botao
中科院分区：
文献类型：
--
作者：
Zhang, Rongzhen;Xu, Yan;Zhang, Botao
(R)-特异性羰基还原酶（RCR）是锌依赖性中链脱氢酶/还原酶家族的一员，催化2-羟基苯乙酮还原为(R)-1-苯基-1,2-乙二醇（PED）。在这项工作中，我们从假丝酵母中克隆了一个6倍组氨酸标记的RCR，并在甲醇诱导的启动子AOXI下在毕赤酵母GS115中表达。RCR酶被有效地分泌到培养基中。Western blotting显示重组RCR与小鼠抗hisx标签单克隆抗体特异性结合。在优化的培养参数（pH 7.0，初始A（600）为2.0，每天添加浓度为1%的甲醇，诱导时间为3.5 ~ 4.0 d）下，RCR的产率为185.0 mg/L。通过一步Ni2+亲和层析纯化，酶的比活性为1.35 U/mg，是大肠杆菌RCR的近3倍。Zn2+的加入大大提高了相变效率。(R)-PED的光学纯度为95.4%，收率为87.2%。与大肠杆菌RCR相比，光学纯度和收率分别提高了17.8%和43.4%。这一有效的表达体系将为RCR的进一步生化研究及其在手性醇制备中的应用提供参考。(C) 2010 Elsevier Ltd.版权所有。
(R)-Specific carbonyl reductase (RCR), a member of the Zn-dependent medium-chain dehydrogenase/reductase family, catalyzes the reduction of 2-hydroxyacetophenone to (R)-1-phenyl-1,2-ethanediol (PED). In this work, we cloned a 6x histidine-tagged RCR from Candida parapsilosis and expressed it in Pichia pastoris GS115 under the methanol-inducible promoter, AOXI. The RCR enzyme was efficiently secreted into the culture media. Western blotting showed that the recombinant RCR was bound specifically by a mouse anti-Hisx tag monoclonal antibody. Under optimized culture parameters (pH 7.0, initial A(600) of 2.0, daily addition of methanol at a concentration of 1% and an induction duration of 3.5-4.0 days), RCR was produced at 185.0 mg/L The enzyme was purified through one-step Ni2+ affinity chromatography and had a specific activity of 1.35 U/mg, which was nearly 3-fold that of Escherichia coli RCR. The addition of Zn2+ improved the transformation efficiency considerably. The (R)-PED product had the desired optical purity of 95.4% and a yield of 87.2%. Compared to E. coli RCR, the optical purity and yield were increased by 17.8% and 43.4%, respectively. This effective expression system will facilitate further biochemical studies of RCR and its use in chiral alcohol preparation in industry. (C) 2010 Elsevier Ltd. All rights reserved.