Department of Biochemical and Microbial Technology, Ghent University, Ghent, Belgium.
Carbohydr Res. 2011 Sep 27;346(13):1860-7. doi: 10.1016/j.carres.2011.06.024. Epub 2011 Jun 24.
In this study, the transglucosylation potential of six sucrose phosphorylase (SP) enzymes has been compared using eighty putative acceptors from different structural classes. To increase the solubility of hydrophobic acceptors, the addition of various co-solvents was first evaluated. All enzymes were found to retain at least 50% of their activity in 25% dimethylsulfoxide, with the enzymes from Bifidobacterium adolescentis and Streptococcus mutans being the most stable. Screening of the enzymes' specificity then revealed that the vast majority of acceptors are transglucosylated very slowly by SP, at a rate that is comparable to the contaminating hydrolytic reaction. The enzyme from S. mutans displayed the narrowest acceptor specificity and the one from Leuconostoc mesenteroides NRRL B1355 the broadest. However, high activity could only be detected on l-sorbose and l-arabinose, besides the native acceptors d-fructose and phosphate. Improving the affinity for alternative acceptors by means of enzyme engineering will, therefore, be a major challenge for the commercial exploitation of the transglucosylation potential of sucrose phosphorylase.
在这项研究中，使用了来自不同结构类别的 80 种假定受体，比较了六种蔗糖磷酸化酶 (SP) 酶的转葡糖基潜力。为了提高疏水性受体的溶解度，首先评估了各种共溶剂的添加。结果发现，所有酶在 25%的二甲基亚砜中保留至少 50%的活性，其中双歧杆菌和变异链球菌的酶最稳定。然后对酶的特异性进行筛选，结果表明，大多数受体被 SP 非常缓慢地转葡糖基化，其速率与污染的水解反应相当。来自变异链球菌的酶表现出最窄的受体特异性，而来自肠膜明串珠菌 NRRL B1355 的酶则具有最宽的受体特异性。然而，除了天然受体 d-果糖和磷酸盐外，只有在 l-山梨糖和 l-阿拉伯糖上才能检测到高活性。因此，通过酶工程提高对替代受体的亲和力将是商业利用蔗糖磷酸化酶转葡糖基潜力的主要挑战。
