The Overlapping Gene Cloning, Expression And Characterization Of A β-galactosidase From Lactobacillus Crispatus

P-galactosidase commonly referres to lactase. The enzyme usually origins from plant, fungus and the gut bacteria of animal.It can hydrolyzes the disaccharide lactose to glucose and galactose, also has the function of transglycosidation. It has been obtained from various plants and animals’ intestine. The application of the enzyme is mainly on the removal of lactose from milk products for lactose intolerant people and the production of oligosaccharides products. In recent years, there is a rising concern on lactase’s application in galacto-oligosaccharide production field and the demand of market is increasing year by year. This study, therefore, presents important theoretical and practical values.In this study, a lactase gene containg overlapping sequences from Lactobacillus crispatus was expressed in pichia pastoria GS115, which was feasiblefor the further industrial production. The lactase gene bg2-470in Lactobacillus crispatus B470was cloned via PCR approach,.thermal asymmetric interlaced (TAIL) PCR. Sequence analysis indicated that the lactase gene with a43%GC content encoded a polypeptide of946amino acid residues, contained2815bp and a stop codon, without signal peptide. Sequence comparison showed that bg2-470belonged to the glycosyl hydrolase second family. Futher, using BLAST tool, the deduced amino acid sequence of this lactase showed99%identities to the previously reported genes from Lactobacillus crispatus ST1.The lactase gene bg2-470was expressed in Escherichia coli BL21(DE3) and Pichia pastoris GS115. A strain of high level expression was selected and fermentation experimrnts were carried out in a3L container. The fermentation product was the mix of target proteins. A large mass of target protein with high purity was obtained through the microfiltration, ultrafiltration, molecular sieve and ion exchange purification. The properties of eukaryotic expressed lactase was characterized. The result showed that its optimum pH and temperature were at pH7.0and50℃respectively, the Km,Vmax and specific activity of this enzyme was0.964mmol/L,466nmol/min and221U/mg, respectively.