Lund University, Department of Chemistry, Biotechnology, Lund, Sweden.
Appl Environ Microbiol. 2013 Jun;79(11):3400-5. doi: 10.1128/AEM.00359-13. Epub 2013 Mar 22.
Alkyl glycosides are attractive surfactants because of their high surface activity and good biodegradability and can be produced from renewable resources. Through enzymatic catalysis, one can obtain well-defined alkyl glycosides, something that is very difficult to do using conventional chemistry. However, there is a need for better enzymes to get a commercially feasible process. A thermostable β-glucosidase from the well-studied glycoside hydrolase family 1 from Thermotoga neapolitana, TnBgl1A, was mutated in an attempt to improve its value for synthesis of alkyl glycosides. This was done by rational design using prior knowledge from structural homologues together with a recently generated model of the enzyme in question. Three out of four studied mutations increased the hydrolytic reaction rate in an aqueous environment, while none displayed this property in the presence of an alcohol acceptor. This shows that even if the enzyme resides in a separate aqueous phase, the presence of an organic solvent has a great influence. We could also show that a single amino acid replacement in a less studied part of the aglycone subsite, N220F, improves the specificity for transglycosylation 7-fold and thereby increases the potential yield of alkyl glycoside from 17% to 58%.
烷基糖苷是一种很有吸引力的表面活性剂，因为它们具有高表面活性、良好的生物降解性，并且可以从可再生资源中获得。通过酶催化，可以得到具有明确结构的烷基糖苷，这在传统化学方法中是非常困难的。然而，为了获得商业上可行的工艺，需要更好的酶。来自研究充分的糖苷水解酶家族 1 的耐热β-葡萄糖苷酶 Thermotoga neapolitana 的 TnBgl1A 被突变，试图提高其用于合成烷基糖苷的价值。这是通过使用结构同源物的先验知识和最近生成的酶模型进行合理设计来实现的。在所研究的四个突变体中，有三个在水相环境中提高了水解反应速率，而在有醇接受体存在的情况下，没有一个显示出这种性质。这表明，即使酶位于单独的水相，有机溶剂的存在也有很大的影响。我们还可以证明，在糖苷配基部位研究较少的部分，即 N220F 处的单个氨基酸替换，将转糖苷反应的特异性提高了 7 倍，从而将烷基糖苷的潜在产率从 17%提高到 58%。
