De Groeve Manu R M, De Baere Miet, Hoflack Lieve, Desmet Tom, Vandamme Erick J, Soetaert Wim
Department of Biochemical and Microbial Technology, Ghent University, Ghent, Belgium.
Protein Eng Des Sel. 2009 Jul;22(7):393-9. doi: 10.1093/protein/gzp017. Epub 2009 May 31.
Disaccharide phosphorylases are interesting enzymes for the production of sugar phosphates from cheap starting materials and for the synthesis of novel glycosides. Cellobiose phosphorylase (CP) from Cellulomonas uda was subjected to directed evolution in order to create enzyme variants with significantly increased lactose phosphorylase (LP) activity, useful for the production of alpha-D-galactose 1-phosphate. In a first round, random mutagenesis was performed on part of the CP gene and the resultant library was selected on minimal lactose medium. One clone containing six amino acid mutations was found with increased LP activity compared with the wild-type CP enzyme. The negative and neutral mutations were eliminated by site-directed mutagenesis and the resultant enzyme variant containing two amino acid substitutions (T508A/N667T) showed more LP activity than the parent mutant. Saturation mutagenesis of the beneficial sites and screening for improved mutants allowed us to identify the T508I/N667A mutant which has 7.5 times higher specific activity on lactose than the wild-type. The kinetic parameters of the mutants were determined and showed that the increased LP activity was caused by a higher k(cat) value. This is the first report of an engineered CP with modified substrate specificity.
双糖磷酸化酶是一类有趣的酶，可用于从廉价起始原料生产糖磷酸酯以及合成新型糖苷。对来自 uda 纤维单胞菌的纤维二糖磷酸化酶（CP）进行定向进化，以创建乳糖磷酸化酶（LP）活性显著提高的酶变体，这对于生产 α-D-半乳糖 1-磷酸酯很有用。在第一轮中，对 CP 基因的一部分进行随机诱变，并在基本乳糖培养基上选择所得文库。发现一个含有六个氨基酸突变的克隆，与野生型 CP 酶相比，其 LP 活性有所提高。通过定点诱变消除了负向和中性突变，所得含有两个氨基酸取代（T508A/N667T）的酶变体显示出比亲本突变体更高的 LP 活性。对有益位点进行饱和诱变并筛选改良突变体，使我们鉴定出 T508I/N667A 突变体，其对乳糖的比活性比野生型高 7.5 倍。测定了突变体的动力学参数，结果表明 LP 活性的提高是由更高的 k(cat) 值引起的。这是关于具有修饰底物特异性的工程化 CP 的首次报道。
