Centre for Synthesis Biology (CSB) Department of Biotechnology, Ghent University, Coupure Links 653, 9000, Gent, Belgium.
Chembiochem. 2021 Sep 14;22(18):2777-2782. doi: 10.1002/cbic.202100192. Epub 2021 Jun 2.
2-O-Glucosylglycerol is accumulated by various bacteria and plants in response to environmental stress. It is widely applied as a bioactive moisturising ingredient in skin care products, for which it is manufactured via enzymatic glucosylation of glycerol by the sucrose phosphorylase from Leuconostoc mesenteroides. This industrial process is operated at room temperature due to the mediocre stability of the biocatalyst, often leading to microbial contamination. The highly thermostable sucrose phosphorylase from Bifidobacterium adolescentis could be a better alternative in that regard, but this enzyme is not fit for production of 2-O-glucosylglycerol due to its low regioselectivity and poor affinity for glycerol. In this work, the thermostable phosphorylase was engineered to alleviate these problems. Several engineering approaches were explored, ranging from site-directed mutagenesis to conventional, binary, iterative or combinatorial randomisation of the active site, resulting in the screening of ∼3,900 variants. Variant P134Q displayed a 21-fold increase in catalytic efficiency for glycerol, as well as a threefold improvement in regioselectivity towards the 2-position of the substrate, while retaining its activity for several days at elevated temperatures.
2-O-葡萄糖基甘油在各种细菌和植物中积累，以应对环境压力。它被广泛应用于护肤品中的生物活性保湿成分，通过肠膜明串珠菌的蔗糖磷酸化酶对甘油进行酶促葡萄糖基化来制造。由于生物催化剂的稳定性较差，该工业过程在室温下进行，这通常会导致微生物污染。双歧杆菌的高度耐热蔗糖磷酸化酶在这方面可能是更好的选择，但由于其对甘油的低区域选择性和差亲和力，该酶不适合生产 2-O-葡萄糖基甘油。在这项工作中，对耐热磷酸化酶进行了工程改造，以缓解这些问题。探索了几种工程方法，从定点突变到活性位点的常规、二元、迭代或组合随机化，筛选了约 3900 个变体。变体 P134Q 对甘油的催化效率提高了 21 倍，对底物 2 位的区域选择性提高了三倍，同时在高温下保持数天的活性。
