Designing of a novel [beta]-galactosidase for production of functional oligosaccharides

• Published in: European food research & technology Vol. 243; no. 6; p. 979
• Main Authors: Liao, Xueyi, Huang, Jiajun, Zhou, Qianling, Guo, Liqiong, Lin, Junfang, You, Linfeng, Liu, Sha, Yang, Jingxian
• Format: Journal Article
• Language: English
• Published: Heidelberg Springer Nature B.V 01.06.2017
• Subjects: Amino acids; Cloning; DNA polymerase; E coli; Enzymes; Food; Genomes; Lactose; Metal ions; Mutagenesis; Plasmids; R&D; Research & development
• DOI: 10.1007/s00217-016-2813-y
• ISSN: 1438-2377

[beta]-galactosidase from L. plantarum FMNP01 was subjected to mutagenesis by domain recombination. A novel [beta]-galactosidase L.pFMNP01Gal-2 was constructed and expressed using the expression vector pET-32a(+) in Escherichia coli BL21 (DE3). The L.pFMNP01Gal-2 was composed of 2406 bp encoding a polypeptide containing 802 amino acids with a predicted molecular mass of 88.22 kDa. When ONPG was used as substrate, the specific activity of L.pFMNP01Gal-2 was 2620 U/g protein, with a K m of 2.87 mmol/L and a K cat of 62.68/s. The optimal temperature and pH for L.pFMNP01Gal-2 were 40 °C and 7.0, respectively. After pre-incubation for 4 h at 40 and 50 °C, respectively, the residual activity of L.pFMNP01Gal-2 retained over 88%. The metal ions Cu2+ and Na+ had a great inhibitory effect on the activity of L.pFMNP01Gal-2, while other tested metal ions had slight influence. In the presence of lactose and fructose, when L.pFMNP01Gal-2 was used as catalyst, two transfer products were formed during transgalactosylation reaction. It is notable that when L.pFMNP01Gal-2 was used as catalyst with lactose and sucrose as substrates, four transfer products were observed.