Sortase A介导的羰基还原酶寡聚体热稳定性加强及高效转化手性醇

发布时间：2018-05-18 00:00

  本文选题：Sortase + A介导　； 参考：《江南大学》2017年硕士论文

【摘要】：来自于近平滑假丝酵母(Candida parapsilosis CCTCC M203011)中的短链脱氢酶(S)-羰基还原酶II(SCRII)在辅酶NADPH的参与下,能够特异性催化2-羟基苯乙酮合成(S)-苯基乙二醇,但催化效率较低、热稳定性不高。本研究利用sortase A(SrtA)的转肽作用,通过在SCRII的C末端引入SrtA识别序列,在SrtA的介导下构建功能和稳定性均加强的SCRII寡聚体。以2-羟基苯乙酮作为底物,NADPH作为辅酶,SCRII寡聚体为催化剂高效制备(S)-苯基乙二醇。随后,将SrtA介导的寡聚化扩展至其它8种羰基还原酶,建立了增强羰基还原酶稳定性,提高酶催化效率的平台技术。主要研究内容包括:(1)从金黄色葡萄球菌Staphylococcus aureus ST451的基因组中钓取SrtA基因核心序列并构建pET21a-srt A质粒,实现了SrtA在大肠杆菌Escherichia coli BL21(DE3)中的高效表达;通过设计引物在SCRII的C末端引入GGGGSLPETGG序列,构建pET28a-scr II-mtf质粒,诱导表达结果表明加入GGGGSLPETGG标签对酶的表达没有影响;优化了SrtA介导SCRII-mtf连接的条件,当SrtA的酶浓度为1 mg·m L-1的情况下,Ca2+的最佳浓度为10 mmol·L-1,连接反应的最佳温度为25℃,连接时间为36 h时,几乎所有的SCRII-mtf都被消耗,此时连接产物产量最高,且连接产物成分主要为二聚体和三聚体。(2)测定了SCRII寡聚体的酶学性质,在35℃,pH 6.0的条件下,SCRII-mtf较SCRII酶活有略微提高,而SrtA介导产生的SCRII寡聚体对2-羟基苯乙酮的比活达到了38.5 U·mg-1,与SCRII相比提高了6倍;SCRII寡聚体在50℃时相对酶活最高,SCRII寡聚体和SCRII在50℃放置1 h,活性分别维持在90%以上和50%左右,表明SCRII寡聚化后,温度稳定性显著提高;SCRII、SCRII-mtf和SCRII寡聚体对于pH的依赖性非常相似,但SCRII寡聚体的pH稳定性明显高于其它两种酶;与SCRII相比,SCRII寡聚体的Km值降低了3.3倍,说明经过寡聚化,SCRII寡聚体与底物2-羟基苯乙酮的亲和力有所增加。(3)确定了SCRII寡聚体生物转化(S)-苯基乙二醇的最适反应条件,从整体上来看,SCRII寡聚体在35℃,pH 6.0,5g·L-1 2-羟基苯乙酮的条件下,2 h内SCRII寡聚体转化生成(S)-苯基乙二醇的产率高达90%以上;在3 h内生成(S)-苯基乙二醇的产率和光学纯度均达到了100%。(4)选取了另外8种氧化还原酶,利用SrtA构建氧化还原酶寡聚体,8种寡聚体在SrtA的介导下均出现不同程度的寡聚化,圆二色谱显示寡聚体的Tm值较原始酶升高了5℃-12℃,寡聚体酶活力较单体提高了5-11倍,生物转化反应6 h后,对应手性产物的产率提高了25%-285%。
[Abstract]:The short chain dehydrogenase (SCRII) from Candida parapsilosis CCTCC M203011), with the participation of coenzyme NADPH, can specifically catalyze the synthesis of thio-phenylglycol from 2-hydroxyacetophenone, but the catalytic efficiency is low and the thermal stability is not high. The aim of this study was to construct a SCRII oligomer with enhanced function and stability by introducing SrtA recognition sequence into the C terminal of SCRII by using the transpeptide of sortase An SrtA. Using 2-hydroxyacetophenone as substrate, NADPH was used as coenzyme SCRII oligomer as catalyst. Subsequently, the SrtA mediated oligomerization was extended to the other eight carbonyl reductase, and the platform technology was established to enhance the stability of the carbonyl reductase and improve the catalytic efficiency of the enzyme. The main research contents include: 1) the core sequence of SrtA gene was isolated from the genome of Staphylococcus aureus Staphylococcus aureus ST451 and the plasmid pET21a-srt A was constructed. The high expression of SrtA in E. coli Escherichia coli BL21DDE3) was achieved. The pET28a-scr II-mtf plasmid was constructed by introducing GGGGSLPETGG sequence into the C terminal of SCRII by designing primers. The induced expression results showed that the addition of GGGGSLPETGG tag had no effect on the expression of the enzyme, and the conditions of SrtA mediated SCRII-mtf ligation were optimized. When the enzyme concentration of SrtA was 1 mg mL ~ (-1), the optimal concentration of Ca ~ (2 2) was 10 mmol L ~ (-1), the optimal reaction temperature was 25 鈩，

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