Screening,Molecular Cloning And Enzymatic Properties Of Lactase With High Transgalactosylation Activity

Lactase(EC 3.2.1.23),classified as the ?-galactosidase family,is a class of enzymes that can catalyze the hydrolysis of lactose to form glucose and galactose or catalyze the transgalatoside to form oligogalactose.Currently the main method of industrial production of galacto-oligosaccharides(GOS)is enzymatic preparation usin glactose as the raw material with lactase of high transglycosidase activity.The lack of a lactase with high transglycosylation circumscribes the way for improving GOS manufacturing technology and product quality.In order to obtain a lactase with high transglycosylation activity,the enzyme-producing strains were screemed and selected,its encoding gene was cloned and expressed and its enzymatic properties was comprehensively examined,and the main achievements were obtained as follows.(1)Lactase-producing bacteria with high transglycosidic activity were obtained using higthroughput screening.Bifidobacterium longum B1172 who grew well on GOS as sole carbon source but poorly on lactose,glucose and galactose,was selected.Its GOS-biomass correlation was then established and a high-throughput screening method for transgalatoside activity was established.Five strains,Bacillus circulans B0212 and B2301,Paenibacillus pini B0809 and B2809,and Paenibacillus polymyxa B3156,producing lactase with relatively high transglycosidic activity were selected from about 3 700 isolates.In which,strain B2301 produced the highest intracellular activity transgalactosidase.(2)Gene encoding lactase was successfully cloned from B.circulans B2301.The gene,bglBc,encoding lactase was successfully cloned from the genome of B.circulans B2301 by using shotgun cloning.It coded for a complete open reading frame with 5 133 bp and comprised 1 710 amino acid residues.BglBc had no typical signal peptide with a predicted molecular weight of 189.4 ku;The amino acid sequence of Bg1Bc had the highest similarity to a ?-galactosidase derived from B.circulans ATCC 31382,with a sequence identity of 93.6%.It was also similar to those from Bacillus mesonae(87.4%),Paraliobacillus sp.(60%)but less similar to those from others.Glu420,Tyr483 and Glu503 residues in Bg1Bc were predicited to form its catalytic active center,two(Glu420 and Glu503)of which constituted its acid/base and nucleophilic catalysis.Its secondary structure contained nine domains,four of which near the N-end were consistent with that of?-galactosidase LacZ in domain structure.(3)Bg1Bc was heterologously expressed and its enzymatic properties was illustrated.The functional domain(Bg1D)composed of 782 amino acid residues from the N-terminal of Bg1Bc was expressed in Bacillus subtilis WB600.Under optimized shake flask fermentation conditions,the highest enzyme production level was 3.92 u/mL.The optimal temperature and pH of the purified Bg1D were 60? and 6.0,respectively.it was relatively stable in a temperature below 50? and pHs of 5.0-8.0.No metal ions were fiund to significantly promote the activity.The Km and Vmax against lactose were 111.24 mmol/L and 0.85 mmol/(L·min),respectively.It effectively mediated the GOS formation from lactose with the conversion rate of 52.5%(w/w).