α-环糊精糖基转移酶活性区域突变提高选择形成γ-环糊精能力  

Increasing of product specificity of γ-cyclodextrin by mutating the active domain of α-cyclodextrin glucanotransferase from Paenibacillus macerans sp. 602-1

作　　者：谢婷[1,2] 岳洋[1,3] 宋炳红[1] 钞亚鹏[1] 钱世钧[1] 

机构地区：[1]中国科学院微生物研究所工业酶国家工程实验室,北京100101 [2]中国科学院大学,北京100049 [3]北京航空航天大学生物与医学工程学院,北京100191

出　　处：《生物工程学报》2013年第9期1234-1244,共11页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(No.31171643)资助~~

摘　　要：探讨α-环糊精糖基转移酶（CGT酶）活性区域-3亚位点（47位赖氨酸残基），-7亚位点（146～152位氨基酸残基）以及环化中心位点（195位酪氨酸残基）对其催化底物形成Y一环糊精（CD）能力的影响。将α-CGT酶相应位点分别进行如下突变：K47T，Y195I，以及146～152位氨基酸残基替换为异亮氨酸（命名为△6），并在大肠杆菌BL21中实现异源活性表达。以可溶性淀粉作为底物进行转化，利用HPLC分析各种突变酶的催化产物中3种环糊精产量和比例。结果表明，和野生酶相比，所有突变酶的淀粉水解活性和环糊精总生成量都有不同程度的下降。在产物的组成方面，突变酶Y1951的催化产物中，α-CD的含量由68％降为30％，β—CD由22．2％提高为33．3％；而γ-CD由8．9％提高为36．7％，含量提高了4倍，取代α-CD成为产物中的主要成分；γ-CD的实际产量为1．1g／L，是野生酶（0．4g／L）的3倍。突变酶K47T和△6的转化产物中α-CD比例有不同程度下降，但仍然是产物中的主要组分，β-和γ-CD的比例都有所增加。由此可见，活性区域中195位氨基酸对于α-CGT酶的活力和催化选择性具有重要的影响，Y195I突变体酶最有利于选择性形成γ—CD。纯化后突变酶Y1951的酶学性质试验表明，其最适反应温度和野生酶相同，但最适反应pH有所提高，且比野生酶具有更好的pH稳定性。因此，突变酶Y195I具有生产制备γ-CD的潜力。We studied the mutation effect of subsites -3（Lys47）, -7（146-152）, and cyclization center （Tyr195） in active domain on product specificity of α-cyclodextrin glucanotransferase （α-CGTase） from Paenibacillus macerans sp. 602-1. The Lys47 was replaced by Thr47 and Tyr195 by Ile195, and the amino acids from 146 to 152 were replaced by lie （named as A6）. All these mutant α-CGTases were actively expressed in E. coli BL21. Compared with the wild-type α-CGTase, the starch-degrading activities of all the mutant enzymes were declined. For mutant Y195I, the percentage of α-CD was decreased from 68% to 30%, and β-CD was raised from 22.2% to 33.3%. Interestingly, y-CD was increased from 8.9% to 36.7% and became the main product, while the actual yield was increased from 0.4 g/L to 1.1 g/L. Mutant K47T and A6 still produced α-CD as main product though the percentage of β- and y-CD increased. Purified Y195I CGTase showed similar optimum temperature with the wild-type α-CGTase, but its optimum pH shifted from 5.0 to 6.0 with better pH stability. In summary, mutant Y195I CGTase has the potential to produce y-CD as the main product.

关 键 词：α-环糊精糖基转移酶 α-CD Β-CD γ-CD 突变 产物选择性 

分 类 号：TQ925[轻工技术与工程—发酵工程]