文献类型：期刊文献

英文题名：Screening novel β-galactosidases from a sequence-based metagenome and characterization of an alkaline β-galactosidase for the enzymatic synthesis of galactooligosaccharides

作者：Liu, Pei[1];Wang, Wei[1];Zhao, Jian[1];Wei, Dongzhi[1]

机构：[1]East China Univ Sci & Technol, New World Inst Biot, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China

年份：2019

卷号：155

起止页码：104

外文期刊名：PROTEIN EXPRESSION AND PURIFICATION

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000456905400016)】；

基金：This research was supported by the National Natural Science Foundation of China [No. C010302-31500066].

语种：英文

外文关键词：beta-Galactosidase; CRISPR Cas9; Galactooligosaccharides; lacZ; Metagenome; Transglycosylation

摘要：beta galactosidases have wide industrial applications in lactose hydrolysis and transglycosylation reactions. Therefore, there is a need to mine novel and high-quality beta-galactosidases with good tolerance and novel features from harsh environments and genomic databases. In this study, an Escherichia coli -galactosidase-deficient host, Delta lacZ(DE3)pRARE, was constructed by the CRISPR-Cas9 system for screening active beta-galactosidases. Of thirty selected beta-galactosidases, twelve novel enzymes showed beta-galactosidase activity, four of which were purified for further study. BGal_375 exhibited maximal activity at pH 8 and 50 degrees C. The concentrations of two types of galactooligosaccharides, tri- and tetra-saccharides, produced by BGal_375, reached 64.53 g/l and 8.32 g/l, respectively. BGal_375 displayed a K-m value of 1.65 mM and k(cat), value of 53 s(-1) for Thnitrophenyl-beta-D-galactopyranoside (pNPG). BGal_137, BGal_144-3, and BGal_145-2 showed promising hydrolytic activity for pNPG. BGal_137 is a homodimer while BGal_144.3, BGal_145-2, and BGal_375 were all monomeric. This study provided an efficient solution for the identification of new beta-galactosidases from metagenomic data, and an alkaline beta-galactosidase efficient for the synthesis of galactooligosaccharides was obtained, which is important for potential industrial applications.