亲和标签调节的(S)-羰基还原酶2催化2-羟基苯乙酮的酶学性质

Characterization of the affinity-tags-regulated(S)-carbonyl reductase 2 towards 2-hydroxyacetophenone reduction

不同类型的亲和标签会影响酶的催化功能和酶学性质。近平滑假丝酵母Candidaparapsilosis来源的(S)-羰基还原酶2((S)-carbonyl reductase 2,SCR2)能催化2-羟基苯乙酮。文中在SCR2的N端添加不同类型的亲和标签,在大肠杆菌Escherichiacoli中异源表达并纯化重组蛋白his6-SCR2、strep-SCR2和MBP-SCR2,研究了重组蛋白催化2-羟基苯乙酮的酶学性质。结果表明,不同类型的亲和标签对SCR2的酶学性质有一定的影响。其中,不同类型的亲和标签对重组蛋白稳定性影响较大:1)在pH6.0、30℃条件下保温13h后,重组蛋白his6-SCR2和strep-SCR2的剩余酶活力是无融合标签SCR2的90.0%-95.2%,而MBP-SCR2的剩余酶活力是无融合标签SCR2的1.25倍。2)MBP-SCR2在50℃的半衰期比strep-SCR2、his6-SCR2和无融合标签SCR2长26.6%–48.8%。3)MBP-SCR2在-80℃存储60d后,其酶活动力学参数kcat比his6-SCR2、strep-SCR2和无融合标签SCR2高1.25–1.45倍。根据三级结构分析推出重组蛋白MBP-SCR2中MBP的C末端的α螺旋具有稳定SCR2的N端无规则卷曲的作用,从而提高酶的稳定性。圆二色谱检测结果表明MBP标签对蛋白SCR2的二级结构有一定的影响,且解折叠温度(T_(m))分析证明,MBP-SCR2的T_(m)比无融合标签SCR2提高近5℃。研究结果不仅为羰基还原酶家族增添了一种2-羟基苯乙酮的稳定高效催化剂MBP-SCR2,同时为其他短链醇脱氢酶的标签设计提供了借鉴和依据。

The influence of different affinity tags on enzyme characteristics varies.The(S)-carbonyl reductase 2(SCR2)from Candida parapsilosis can reduce 2-hydroxyacetophenone,which is a valuable prochiral ketones.Different affinity tags,i.e.his-tag,strep-tag and MBP-tag,were attached to the N terminus of SCR2.These tagged SCR2 enzymes,i.e.his6-SCR2,strep-SCR2 and MBP-SCR2,were heterologously expressed in Escherichia coli and purified to study their characteristics towards 2-hydroxyacetophenone reduction.Affinity tags did affect the characteristics of the recombinant SCR2 enzymes.Specifically,affinity tags affect the stability of recombinant SCR2 enzymes:1)At pH 6.0,the remaining enzyme activities of his6-SCR2 and strep-SCR2 were only 95.2%and 90.0%of the untagged SCR2,while that of MBP-SCR2 was 1.2 times of the untagged SCR2 after incubating for 13 h at 30℃.2)The half-life of MBP-SCR2 at 50℃ was 26.6%–48.8%longer than those of strep-SCR2,his6-SCR2 and untagged SCR2.3)The kcat of MBP-SCR2 was about 1.25–1.45 times of that of small affinity-tagged and untagged SCR2 after storing at-80℃ for 60 d.Structural informatics indicated that theα-helices at the C terminus of MBP-SCR2 contributed to the stability of the N terminus of fusion protein of SCR2.Data from circular dichroism showed that the MBP-tag has some influence on the secondary structure of SCR2,while melting temperature analysis demonstrated that the T_(m)of the recombinant MBP-SCR2 was about 5℃ higher than that of the untagged SCR2.This study obtained an efficient and stable recombinant SCR2,i.e.the MBP-SCR2.Moreover,this study could serve as a reference for other researchers to evaluate and select appropriate affinity tags for their research.