University of Nottingham, School of Chemistry, University Park, Nottingham, NG7 2RD, United Kingdom.
Dipartimento di Bioscienze, Università di Milano, Via Celoria 26, Milan, 20133, Italy.
J Biotechnol. 2021 Mar 10;329:21-28. doi: 10.1016/j.jbiotec.2021.01.017. Epub 2021 Jan 27.
Current methods for the production of natural vanilla extract are long and tedious, and the efficiency of the vanillin extraction is usually conditioned by different factors during the traditional curing process (temperatures and weather conditions). As an important fraction of vanillin is present in the form of glucovanillin in green beans, endogenous β-glucosidases contribute to its hydrolysis; however, these enzymes lose efficiency during the curing process. The use of extremophilic organisms as a source of an appropriate exogenous enzyme can offer a valid alternative when producing natural vanillin. Here, a β-glucosidase from the thermo-acidophilic organism Alicyclobacillus acidiphilus (AacGH1) was cloned, expressed in E. coli BL21, and fully characterized in respect to both function and crystal structure. Notably, AacGH1 was stable at a temperature up to 50 °C and exhibited good tolerance to glucose, fructose and organic solvents, in particular it maintained full activity in the presence of up to 20 % (v/v) ethanol. The enzyme was then successfully applied to an ethanol-water (20 % (v/v)) extract of green vanilla beans and the complete hydrolysis of glucovanillin (1.7 mM) to vanillin, and other flavour compounds commonly found in vanilla, was achieved using 0.5 mg/mL of enzyme in just 15 min at 30 °C.
目前生产天然香草精的方法冗长而繁琐，香草醛的提取效率通常受到传统陈化过程中（温度和天气条件）的不同因素的制约。由于绿咖啡豆中存在相当一部分香草醛以葡糖基香草醛的形式存在，内源性β-葡萄糖苷酶有助于其水解；然而，这些酶在陈化过程中效率降低。当生产天然香草醛时，使用极端微生物作为合适的外源酶源可以提供有效的替代方法。在这里，我们从嗜热嗜酸菌（Alicyclobacillus acidiphilus）中克隆了一种β-葡萄糖苷酶（AacGH1），在大肠杆菌 BL21 中表达，并对其功能和晶体结构进行了全面表征。值得注意的是，AacGH1 在高达 50°C 的温度下稳定，并且对葡萄糖、果糖和有机溶剂具有良好的耐受性，特别是在高达 20%（v/v）乙醇存在下仍保持全部活性。然后，该酶成功地应用于绿香草豆的乙醇-水（20%（v/v））提取物中，在 30°C 下仅用 0.5mg/mL 的酶在 15 分钟内即可将葡糖基香草醛（1.7mM）完全水解为香草醛和其他常见的香草风味化合物。
