1. Biochemical characterization of a novel β-galactosidase from Pedobacter sp. with strong transglycosylation activity at low lactose concentration.
- Author
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Miao M, Yao Y, Yan Q, Jiang Z, He G, and Yang S
- Subjects
- Hydrogen-Ion Concentration, Glycosylation, Escherichia coli genetics, Escherichia coli metabolism, Temperature, Recombinant Proteins genetics, Recombinant Proteins metabolism, Recombinant Proteins chemistry, Recombinant Proteins isolation & purification, Molecular Weight, Oligosaccharides metabolism, Amino Acid Sequence, Milk microbiology, Substrate Specificity, Bacterial Proteins genetics, Bacterial Proteins metabolism, Bacterial Proteins chemistry, Gene Expression, beta-Galactosidase genetics, beta-Galactosidase metabolism, beta-Galactosidase chemistry, beta-Galactosidase isolation & purification, Lactose metabolism, Pedobacter enzymology, Pedobacter genetics, Cloning, Molecular, Enzyme Stability
- Abstract
A novel β-galactosidase gene (PbBgal35A) from Pedobacter sp. CAUYN2 was cloned and expressed in Escherichia coli. The gene had an open reading frame of 1917 bp, encoding 638 amino acids with a predicted molecular mass of 62.3 kDa. The deduced amino acid sequence of the gene shared the highest identity of 41% with a glycoside hydrolase family 35 β-galactosidase from Xanthomonas campestris pv. campestris (AAP86763.1). The recombinant β-galactosidase (PbBgal35A) was purified to homogeneity with a specific activity of 65.9 U/mg. PbBgal35A was optimally active at pH 5.0 and 50 °C, respectively, and it was stable within pH 4.5‒7.0 and up to 45 °C. PbBgal35A efficiently synthesized galacto-oligosaccharides from lactose with a conversion ratio of 32% (w/w) and fructosyl-galacto-oligosaccharides from lactulose with a conversion ratio of 21.9% (w/w). Moreover, the enzyme catalyzed the synthesis of galacto-oligosaccharides from low-content lactose in fresh milk, and the GOS conversion ratios of 17.1% (w/w) and 7.8% (w/w) were obtained when the reactions were performed at 45 and 4 °C, respectively. These properties make PbBgal35A an ideal candidate for commercial use in the manufacturing of GOS-enriched dairy products., (© 2024. Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i.)
- Published
- 2024
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