Center for Bioprocess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Søltofts Plads, Building 229, 2800, Kongens Lyngby, Denmark.
Faculty of Chemical and Natural Resources Engineering, University Malaysia Pahang, Lebuhraya Tun Razak, 26300, Gambang, Kuantan, Pahang, Malaysia.
Chembiochem. 2018 Sep 4;19(17):1858-1865. doi: 10.1002/cbic.201800181. Epub 2018 Jul 18.
Certain enzymes of the glycoside hydrolase family 20 (GH20) exert transglycosylation activity and catalyze the transfer of β-N-acetylglucosamine (GlcNAc) from a chitobiose donor to lactose to produce lacto-N-triose II (LNT2), a key human milk oligosaccharide backbone moiety. The present work is aimed at increasing the transglycosylation activity of two selected hexosaminidases, HEX1 and HEX2, to synthesize LNT2 from lactose and chitobiose. Peptide pattern recognition analysis was used to categorize all GH20 proteins in subgroups. On this basis, we identified a series of proteins related to HEX1 and HEX2. By sequence alignment, four additional loop sequences were identified that were not present in HEX1 and HEX2. Insertion of these loop sequences into the wild-type sequences induced increased transglycosylation activity for three out of eight mutants. The best mutant, HEX1 , had a transglycosylation yield of LNT2 on the donor that was nine times higher than that of the wild-type enzyme. Homology modeling of the enzymes revealed that the loop insertion produced a more shielded substrate-binding pocket. This shielding is suggested to explain the reduced hydrolytic activity, which in turn resulted in the increased transglycosylation activity of HEX1 .
某些糖苷水解酶家族 20（GH20）的酶具有转糖基化活性，并催化β-N-乙酰葡萄糖胺（GlcNAc）从壳二糖供体转移到乳糖上，生成乳糖-N-三糖 II（LNT2），这是一种关键的人乳寡糖主链部分。本工作旨在提高两种选定的己糖胺酶（HEX1 和 HEX2）的转糖基化活性，以从乳糖和壳二糖合成 LNT2。肽模式识别分析用于将所有 GH20 蛋白质分为亚组。在此基础上，我们确定了一系列与 HEX1 和 HEX2 相关的蛋白质。通过序列比对，发现了另外四个不在 HEX1 和 HEX2 中存在的环序列。将这些环序列插入野生型序列中，诱导了 8 个突变体中的 3 个转糖基化活性增加。最好的突变体 HEX1 在供体上的 LNT2 转糖基化产率比野生型酶高 9 倍。酶的同源建模表明，环插入产生了一个更屏蔽的底物结合口袋。这种屏蔽被认为可以解释水解活性的降低，从而导致 HEX1 的转糖基化活性增加。
