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GH2 family beta-galactosidases evolution using degenerate oligonucleotide gene shuffling

文献类型： 外文期刊

作者： Sun, Jingjing 1 ; Wang, Wei 1 ; Hao, Jianhua 1 ;

作者机构： 1.Chinese Acad Fishery Sci, Yellow Sea Fisheries Res Inst, Key Lab Sustainable Dev Polar Fishery, Minist Agr & Rural Affairs, Qingdao 266071, Peoples R China

2.Qingdao Natl Lab Marine Sci & Technol, Lab Marine Drugs & Bioprod, Lab Marine Fisheries Sci & Food Prod Proc, Qingdao 266071, Peoples R China

3.Jiangsu Collaborat Innovat Ctr Exploitat & Utiliza, Lianyungang 222005, Peoples R China

关键词： beta-Galactosidase; Enzyme evolution; Family shuffling; GH2 family; Transglycosylation

期刊名称：BIOTECHNOLOGY LETTERS （ 2022影响因子：2.7； 五年影响因子：2.5 ）

ISSN： 0141-5492

年卷期： 2023 年 45 卷 5-6 期

页码：

收录情况： SCI

摘要： Objectives To improve the biochemical characteristics of the GH2 family Chi-galactosidases using a family shuffling method based on degenerate oligonucleotide gene shuffling. Results Four beta-galactosidase genes from the genus Alteromonas were divided into 14 gene segments, and each included the homologous sequence in the adjacent segments. The gene segments were regenerated into complete beta-galactosidase genes and amplified by PCR. The obtained chimeric genes were cloned into a plasmid and screened for beta-galactosidase activity. Approximately 320 positive clones were observed on the screening plate, of which nine sequenced genes were chimera. Additionally, the M22 and M250 mutants were expressed, purified, and characterized. The optimal temperature and substrate specificity of the recombinant M22 and M250 were consistent with those of the wild-type enzymes. The catalytic efficiency of recombinant M22 enzyme was higher than that of the wild-type enzymes, and the recombinant M250 displayed weak transglycosylation activity. Conclusions The chimeric genes of GH2 beta-galactosidase were obtained using a controlled family shuffling that will provide an enzyme evolutionary method to obtain the beta-galactosidases with excellent characteristics for laboratory and industrial purposes.

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