Screening, cloning and expression of β-galactosidase from lactic acid bacteria and semi-rational design for the improvement of transgalactosylation activity

Process Biochemistry(2024)

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摘要
In this study, a heterodimeric β-galactosidase (PpenLac2) with high transgalactosylation activity from Pediococcus pentosaceus was screened from 64 lactic acid bacteria, and heterologously expressed in Escherichia coli. The optimal pH and temperature of PpenLac2 were 8.0 and 45 °C, respectively. The galactooligosaccharides (GOS) produced by PpenLac2 showed a preference for β-(1,6) linkages, and the primary products were allolactose and 6′-galactosyl lactose. Based on homology modeling and molecular docking, mutation sites were designed to modify PpenLac2 to enhance GOS yield. The key residue impacting the transgalactosylation activity of the enzyme was Phe600. Saturated mutation at the site of Phe600 revealed that the majority of mutants had higher yields of 6′-galactosyl lactose. F600H gained the highest GOS yield increasing from 37.6% to 46.7%, owing to the mutation driving the rotation of Trp 922 and generating Pi-Pi stacking between the pyran ring of substrate and the aromatic ring of Trp922, which promoted the transgalactosylation reaction without affecting hydrolysis reaction. Following the optimization of the reaction conditions, F600H achieved an even higher GOS yield of 52.4%. This study provides a promising enzyme for the synthesis of GOS and serves as a guide for the rational design for the improvement of transgalactosylation efficiency of GH2 family members.
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关键词
β-galactosidase,Lactic acid bacteria,Transgalactosylation activity,Galactooligosaccharides,Semi-rational design
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