Yin Huifang, Pijning Tjaard, Meng Xiangfeng, Dijkhuizen Lubbert, van Leeuwen Sander S
Microbial Physiology and §Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen , Nijenborgh 7, 9747 AG Groningen, The Netherlands.
Biochemistry. 2017 Feb 7;56(5):704-711. doi: 10.1021/acs.biochem.7b00032. Epub 2017 Jan 25.
Microbial β-galactosidase enzymes are widely used as biocatalysts in industry to produce prebiotic galactooligosaccharides (GOS) from lactose. GOS mixtures are used as beneficial additives in infant formula to mimic the prebiotic effects of human milk oligosaccharides (hMOS). The structural variety in GOS mixtures is significantly lower than in hMOS. Since this structural complexity is considered as the basis for the multiple biological functions of hMOS, it is important to broaden the variety of GOS structures. In this study, residue R484 near +1 subsite of the C-terminally truncated β-galactosidase from Bacillus circulans (BgaD-D) was subjected to site saturation mutagenesis. Especially the R484S and R484H mutant enzymes displayed significantly altered enzyme specificity, leading to a new type of GOS mixture with altered structures and linkage types. The GOS mixtures produced by these mutant enzymes contained 14 structures that were not present in the wild-type enzyme GOS mixture; 10 of these are completely new structures. The GOS produced by these mutant enzymes contained a combination of (β1 → 3) and (β1 → 4) linkages, while the wild-type enzyme has a clear preference toward (β1 → 4) linkages. The yield of the trisaccharide β-d-Galp-(1 → 3)-β-d-Galp-(1 → 4)-d-Glcp produced by mutants R484S and R484H increased 50 times compared to that of the wild-type enzyme. These results indicate that residue R484 is crucial for the linkage specificity of BgaD-D. This is the first study showing that β-galactosidase enzyme engineering results in an altered GOS linkage specificity and product mixture. The more diverse GOS mixtures produced by these engineered enzymes may find industrial applications.
微生物β-半乳糖苷酶作为生物催化剂在工业上被广泛用于从乳糖生产益生元低聚半乳糖（GOS）。GOS混合物被用作婴儿配方奶粉中的有益添加剂，以模拟人乳寡糖（hMOS）的益生元作用。GOS混合物中的结构多样性明显低于hMOS。由于这种结构复杂性被认为是hMOS多种生物学功能的基础，因此拓宽GOS结构的多样性很重要。在本研究中，对环状芽孢杆菌C端截短的β-半乳糖苷酶（BgaD-D）+1亚位点附近的R484残基进行了位点饱和诱变。特别是R484S和R484H突变酶表现出显著改变的酶特异性，导致产生了一种结构和连接类型改变的新型GOS混合物。这些突变酶产生的GOS混合物包含14种野生型酶GOS混合物中不存在的结构；其中10种是全新的结构。这些突变酶产生的GOS包含（β1→3）和（β1→4）连接的组合，而野生型酶明显偏好（β1→4）连接。突变体R484S和R484H产生的三糖β-d-Galp-（1→3）-β-d-Galp-（1→4）-d-Glcp的产量比野生型酶增加了50倍。这些结果表明，R484残基对BgaD-D的连接特异性至关重要。这是第一项表明β-半乳糖苷酶工程导致GOS连接特异性和产物混合物改变的研究。这些工程酶产生的更多样化的GOS混合物可能会有工业应用。
