Iterative Saturation Mutagenesis of −6 Subsite Residues in Cyclodextrin Glycosyltransferase from Paenibacillus macerans To Improve Maltodextrin Specificity for 2-O-d-Glucopyranosyl-l-Ascorbic Acid Synthesis
Iterative Saturation Mutagenesis of −6 Subsite Residues in Cyclodextrin Glycosyltransferase from Paenibacillus macerans To Improve Maltodextrin Specificity for 2-O-d-Glucopyranosyl-l-Ascorbic Acid Synthesis
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DOI：
10.1128/aem.02918-13
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发表时间：
2013-09
影响因子：
4.4
通讯作者：
Ruizhi Han;Long Liu;Hyun‐dong Shin;Rachel Chen;Jianghua Li;G. Du;Jian Chen
中科院分区：
文献类型：
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作者：
Ruizhi Han;Long Liu;Hyun‐dong Shin;Rachel Chen;Jianghua Li;G. Du;Jian Chen
摘要 2-O-d-吡喃葡萄糖基-L-抗坏血酸 (AA-2G) 是一种稳定的 L-抗坏血酸衍生物，通常由环糊精糖基转移酶 (CGTase) 合成，该酶包含 9 个底物结合亚位点（从 +2 到 -7）。在本研究中，对浸软类芽孢杆菌 CGTase 中的 -6 个亚位点残基（Y167、G179、G180 和 N193）进行迭代饱和诱变 (ISM)，以提高其对麦芽糖糊精的特异性，麦芽糖糊精是用于 AA-2G 合成的廉价且易溶的糖基供体。首先对四个位点（Y167、G179、G180 和 N193）进行位点饱和诱变，结果表明四个突变体（Y167S、G179R、N193R 和 G180R）产生的 AA-2G 产量高于其他突变体和野生型 CGT 酶。然后以最佳阳性突变体为模板进行 ISM。在最佳条件下，突变体Y167S/G179K/N193R/G180R产生最高的AA-2G滴度，为2.12克/升，比野生型CGTase产生的AA-2G滴度（1.15克/升）高84%。使用突变型 CGTase 合成 AA-2G 的动力学分析证实了麦芽糖糊精特异性的增强，并表明与野生型 CGTase 相比，突变体没有环化活性，但具有较高的水解和歧化活性。通过突变型和野生型 CGTase 的结构建模，还分析了增强底物特异性的可能机制。这些结果表明-6亚位点在CGTase的底物结合和催化反应中起着至关重要的作用，并且所获得的CGTase突变体，特别是Y167S/G179K/N193R/G180R，是通过蛋白质工程进一步开发的有希望的起点。
ABSTRACT 2-O-d-Glucopyranosyl-l-ascorbic acid (AA-2G), a stable l-ascorbic acid derivative, is usually synthesized by cyclodextrin glycosyltransferase (CGTase), which contains nine substrate-binding subsites (from +2 to −7). In this study, iterative saturation mutagenesis (ISM) was performed on the −6 subsite residues (Y167, G179, G180, and N193) in the CGTase from Paenibacillus macerans to improve its specificity for maltodextrin, which is a cheap and easily soluble glycosyl donor for AA-2G synthesis. Site saturation mutagenesis of four sites—Y167, G179, G180, and N193—was first performed and revealed that four mutants—Y167S, G179R, N193R, and G180R—produced AA-2G yields higher than those of other mutant and wild-type CGTases. ISM was then conducted with the best positive mutant as a template. Under optimal conditions, mutant Y167S/G179K/N193R/G180R produced the highest AA-2G titer of 2.12 g/liter, which was 84% higher than that (1.15 g/liter) produced by the wild-type CGTase. Kinetics analysis of AA-2G synthesis using mutant CGTases confirmed the enhanced maltodextrin specificity and showed that compared to the wild-type CGTase, the mutants had no cyclization activity but high hydrolysis and disproportionation activities. A possible mechanism for the enhanced substrate specificity was also analyzed through structure modeling of the mutant and wild-type CGTases. These results indicated that the −6 subsite played crucial roles in the substrate binding and catalytic reactions of CGTase and that the obtained CGTase mutants, especially Y167S/G179K/N193R/G180R, are promising starting points for further development through protein engineering.