GH2 family β-galactosidases evolution using degenerate oligonucleotide gene shuffling.

Abstract

To improve the biochemical characteristics of the GH2 family β-galactosidases using a family shuffling method based on degenerate oligonucleotide gene shuffling. Four β-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 β-galactosidase genes and amplified by PCR. The obtained chimeric genes were cloned into a plasmid and screened for β-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. The chimeric genes of GH2 β-galactosidase were obtained using a controlled family shuffling that will provide an enzyme evolutionary method to obtain the β-galactosidases with excellent characteristics for laboratory and industrial purposes.