Screening Of The Strain Producing β-Galactosidase With Transglycosylation Activity, Gene Cloning, Expression And Elementatl Enzyme Properties

β-galactosidase (EC 3.2.1.23), commonly named lactase which not only can hydrolyze theβ-D-1,4-galactosidic linkage but also has transglycosylation activity, is a very importmant glycosidase and plays an important role in food, medicine, analytical chemistry and other fields. In recent years, with the development of glycobiology,β-galactosidase has become an important tool for saccharide synthesis, various glycoside compounds such as galactosyl-oligosaccharides and lactulose are synthesized by its function. In this rearch, our purpose was to obtain a strain that has higher hydrolysis activity, higher transglycosylation activity and better growth conditions and identified it. Then, the gene from the strain edcoding the P-galactosidase was cloned and expressed in the system of Escherichia coli. The enzyme properties, the induced expression conditions and its ability to synthesize oligosaccharides were also discussed in details.With four-stage screening, we obtained a strain that has higher hydrolysis activity, higher transglycosylation activity and better growth conditions. Based on its morphological, biochemical properties as well as 16S rDNA sequence analysis, it was identified as Klebsiella pneumoniae 285.Medium and fermentation conditions were optimized by single factor and orthogonal experiments. The optimum medium composition was lactose 50g/L, peptone 20g/L, yeast extract 10g/L and NaCl 3g/L. The optimum conditions were inoculation time 6h, initial pH 7.0, inoculation volume 3%, liquid volume 40mL/250mL, incubation time 8h under 30℃. Under the optimum conditions, the enzyme activity reached 7.21 U/mL.Using lactose as the substrates to synthesize oligosaccharides by Klebsiella pneumoniae 285β-galactosidase, the products included fructose, galactose, glucose, lactose, TD and TOS. In the production, the percents of glucose, lactose and oligosaccharides were 9.7%,26.9% and 53.7%, respectively. While using lactose and fructose as the substracts, the percents of fructose, glucose, lactose and oligosaccharides were 21.4%,5.1%,23.4% and 45.0%, respectively.The bgaB gene and LacZ gene of Klebsiella pneumoniae 285 were amplified and then cloned into pMD18-T plasmid seperately, then sequenced and homology analyzed. The result showed that both of the two sequences had a high homology with those of different subspecies.The bgaB gene and LacZ gene were cloned into pET-28a(+) plasmid separately and transformed E. coli BL21(DE3) to express the foreign protein successfully. The induced expression conditions were optimized by single factor experiments. The optimum conditions for BL21/pET-28a(+)-bgaB recombinant strain were induction time 135min, IPTG concentration 0.2mmol/L, induction duration 8h. The optimum conditions for BL21/pET-28a(+)-LacZ recombinant strain were induction time 120min, IPTG concentration 0.6mmol/L, induction duration 9h.The two recombinant enzymes were separated by Ni-Sepharose high performance. The optimum imidazole elution concentration for both was 350mmol/L, and a single band was obtained in SDS-PAGE.The specific activity of the purified bgaB recombinantβ-galactosidase was 185.45U/mg, it was 91.46 times higher than the enzyme before purification; The specific enzyme activity of the purified LacZ recombinantβ-galactosidase was 62.05U/mg, it was 8.09 times higher than the enzyme before purification.Properties of the two recombinant enzymes were studied. The result showed that, for bgaB recombinantβ-galactosidase, the optimum temperature was 50℃, it showed good stability at 40℃and 50℃; The optimum pH was 8.0, it showed good stability between from pH7.5 to pH9.0; Cu2+ and chelating agents such as EDTA displayed evident inhibition against the enzyme, other metal ions had little effects on the enzyme activity even for some promotion effects. For LacZ recombinantβ-galactosidase, the optimum temperature was 40℃, it showed good stability at 40℃and 50℃; The optimum pH was 7.5,it showed good stability between from pH7.5 to pH9.0; Pb2+, Cu2+ and chelating agents such as EDTA displayed evident inhibition against the enzyme, other metal ions had little effects on the enzyme activity even for certain promotion effects.Using ONPG as the substrate, the apparent Vmax and Km of bgaB recombinantβ-galactosidase were 227.27μmol/(mg protein-min) and 0.82mmol/L; the apparent Vmax and Km of LacZ recombinantβ-galactosidase were 58.82μmol/(mg protein-min) and 0.72mmol/L.Using lactose as the substrate to synthesize oligosaccharides by the two recombinantβ-galactosidase, for bgaB recombinant P-galactosidase, the percents of glucose, lactose and oligosaccharides were6.3%,45.2% and 42.3%, respectively. For LacZ recombinantβ-galactosidase, the percents of glucose, lactose and oligosaccharides were12.8%,14.1% and 60.4%, respectively.Using lactose and fructose as the substrate to synthesize oligosaccharides by the two recombinantβ-galactosidase, for bgaB recombinantβ-galactosidase, the percents of fructose, glucose, lactose and oligosaccharides were 21.2%,2.8%,28.3% and 44.9%, respectively. For LacZ recombinantβ-galactosidase, the percents of fructose, glucose, lactose and oligosaccharides were 21.5%,11.7%,8.9% and 46.4%, respectively.