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

摘要:

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Candida parapsilosis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Escherichia coli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pH 6.0、30 ℃条件下保温13 h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60 d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 (Tm) 分析证明，MBP-SCR2的Tm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。

Abstract:

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 °C. 2) The half-life of MBP-SCR2 at 50 °C 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 °C for 60 d. Structural informatics indicated that the α-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 Tm of the recombinant MBP-SCR2 was about 5 °C 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.