黑曲霉葡萄糖淀粉酶的定向进化以提高热稳定性。

Directed evolution of Aspergillus niger glucoamylase to increase thermostability.

机构信息

Department of Food Science and Human Nutrition, Iowa State University, Ames, IA 50011, USA.

出版信息

Microb Biotechnol. 2008 Nov;1(6):523-31. doi: 10.1111/j.1751-7915.2008.00055.x.

DOI:10.1111/j.1751-7915.2008.00055.x

PMID:21261873

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC3815294/

Abstract

Using directed evolution and site-directed mutagenesis, we have isolated a highly thermostable variant of Aspergillus niger glucoamylase (GA), designated CR2-1. CR2-1 includes the previously described mutations Asn20Cys and Ala27Cys (forming a new disulfide bond), Ser30Pro, Thr62Ala, Ser119Pro, Gly137Ala, Thr290Ala, His391Tyr and Ser436Pro. In addition, CR2-1 includes several new putative thermostable mutations, Val59Ala, Val88Ile, Ser211Pro, Asp293Ala, Thr390Ser, Tyr402Phe and Glu408Lys, identified by directed evolution. CR2-1 GA has a catalytic efficiency (k(cat)/K(m)) at 35°C and a specific activity at 50°C similar to that of wild-type GA. Irreversible inactivation tests indicated that CR2-1 increases the free energy of thermoinactivation at 80°C by 10 kJ mol(-1) compared with that of wild-type GA. Thus, CR2-1 is more thermostable (by 5 kJ mol(-1) at 80°C) than the most thermostable A. niger GA variant previously described, THS8. In addition, Val59Ala and Glu408Lys were shown to individually increase the thermostability in GA variants by 1 and 2 kJ mol(-1), respectively, at 80°C.

摘要

通过定向进化和定点突变，我们分离到了一种高度耐热的黑曲霉葡萄糖淀粉酶（GA）变体，命名为 CR2-1。CR2-1 包含先前描述的突变：Asn20Cys 和 Ala27Cys（形成新的二硫键）、Ser30Pro、Thr62Ala、Ser119Pro、Gly137Ala、Thr290Ala、His391Tyr 和 Ser436Pro。此外，CR2-1 还包含几个新的潜在耐热突变，Val59Ala、Val88Ile、Ser211Pro、Asp293Ala、Thr390Ser、Tyr402Phe 和 Glu408Lys，这些突变是通过定向进化发现的。CR2-1 GA 在 35°C 时的催化效率（kcat/Km）和在 50°C 时的比活度与野生型 GA 相似。不可逆失活动力学测试表明，与野生型 GA 相比，CR2-1 在 80°C 时增加了热失活动力学的自由能 10 kJ mol(-1)。因此，CR2-1 在 80°C 时比以前描述的最耐热的黑曲霉 GA 变体 THS8 更耐热（提高 5 kJ mol(-1)）。此外，Val59Ala 和 Glu408Lys 分别使 GA 变体在 80°C 时的耐热性提高了 1 和 2 kJ mol(-1)。