Alkyl glycosides are well-characterized nonionic surfactants, and can be prepared by transglycosylation reactions with retaining GH1 glycosidases being normally used for this purpose. The produced alkyl glycosides can also be hydrolyzed by the glycosidase, and hence, the yields of alkyl glycosides can be too low for industrial use. To improve the transglycosylation-to-hydrolysis ratio for a β-glucosidase from Thermotoga maritima (TmBglA) for the synthesis of alkyl glycoside, six mutants (N222F, N223C, N223Q, G224A, Y295F, and F414S) were produced. N222F, N223C, N223Q, G224A improved catalytic activity, F295Y and F414S are hydrolytically crippled with p-nitrophenol-β-d-glucopyranoside (pNPG) as substrate with an 85 and 70-fold decrease in apparent kcat, respectively; N222F shows the highest kcat/km value for pNPG. The substrate selectivity altered from pNPG to pNP-β-d-fucoside for N222F, F295Y, and F414S and from cellubiose to gentiobiose for N222F and F414S. Using pNPG (34 mM) and hexanol 80% (vol/vol), N222F, Y295F, and F414S synthesized hexyl-β-glycoside (HG) yields of 84.7%, 50.9%, and 54.1%, respectively, HG increased from 14.49 (TmBglA) to 22.8 mM (N222F) at 2 hr by 57.42%. However, this higher transglycosylation effect depended on that three mutants creates an environment more suited for hexanol in the active site pocket, and consequently suppressed its HG hydrolysis.

中文翻译：

---

工程化嗜热袍菌β-葡萄糖苷酶通过定点诱变改进烷基糖苷的合成

烷基糖苷是很好表征的非离子表面活性剂，可以通过转糖基化反应制备，保留通常用于此目的的 GH1 糖苷酶。产生的烷基糖苷也可以被糖苷酶水解，因此烷基糖苷的产率对于工业用途来说可能太低。为了提高来自海栖热袍菌 (TmBglA) 的 β-葡萄糖苷酶 (TmBglA) 用于合成烷基糖苷的转糖基化与水解比率，制备了六种突变体（N222F、N223C、N223Q、G224A、Y295F 和 F414S）。N222F、N223C、N223Q、G224A 提高了催化活性，F295Y 和 F414S 被对硝基苯酚-β-d-吡喃葡萄糖苷 (pNPG) 作为底物水解，表观 kcat 分别降低了 85 倍和 70 倍；N222F 显示 pNPG 的最高 kcat/km 值。N222F、F295Y 和 F414S 的底物选择性从 pNPG 变为 pNP-β-d-岩藻糖苷，N222F 和 F414S 的底物选择性从纤维素二糖变为龙胆二糖。使用 pNPG (34 mM) 和己醇 80% (vol/vol)、N222F、Y295F 和 F414S 合成的己基-β-糖苷 (HG) 产率分别为 84.7%、50.9% 和 54.1%，HG 从 14.49 ( TmBglA) 至 22.8 mM (N222F) 在 2 小时 57.42%。然而，这种更高的转糖基化作用取决于三个突变体在活性位点口袋中创造了一个更适合己醇的环境，从而抑制了其 HG 水解。49 (TmBglA) 至 22.8 mM (N222F) 在 2 小时时下降 57.42%。然而，这种更高的转糖基化作用取决于三个突变体在活性位点口袋中创造了一个更适合己醇的环境，从而抑制了其 HG 水解。49 (TmBglA) 至 22.8 mM (N222F) 在 2 小时时下降 57.42%。然而，这种更高的转糖基化作用取决于三个突变体在活性位点口袋中创造了一个更适合己醇的环境，从而抑制了其 HG 水解。