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

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γ-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Y195I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1.1 g/L，是野生酶 (0.4 g/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Y195I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。

Abstract:

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 Ile (named as Δ6). 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, γ-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 Δ6 still produced α-CD as main product though the percentage of β- and γ-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 γ-CD as the main product.