Cloning And Expression Of ?-D-galactosidase From Lactobacillus Kefiranofaciens ZW3 In Pichia Pastoris

Lactase(?-D-galactosidase EC3.2.1.23)hydrolyzes the lactose glycosidic linkage,and this process leads to the breaking down of lactose with generating the galactose and glucose.In addition,the lactase can catalyse the transglycosylation reaction to produce galactooligosaccharide(GOS).Currently,the lactase is mainly used for the production of the low-lactose milk.It can effectively reduce the effects of lactose intolerance.The lactase obtained from the lactic acid bacteria belongs to neutral lactase,and its optimum temperature is 40?60 ?,and it can meet the requirements of dairy production.However it does not belong to extracellular enzymes,and involving the difficulty of separation and purification techniques,it is not suitable for the industrial production in food industry.In this paper,the bioinformatics analysis of the lactase overlapping gene LacLM which from lactic acid bacteria Lactobacillus Kefiranofaciens ZW3 were conducted,and the results revealed that the gene with 2833bp encode a polypeptide which contains 948 amino acids.Besides,the ?-D-galactosidase protein belongs to hydrophobic proteins without the signal peptide,and it is a kind of the non-secretory cell proteins.This study was also focused on the amplification of the ?-galactosidase genes(LacL and LacM)which isolated from Lactobacillus Kefiranofaciens ZW3 by PCR,and were recombined into vector pPIC9K after digestion by EcoRI and SnaBI.Two recombined plasmids pPIC9K-LacL and pPIC9K-LacM were transformed into E.coli DH5?,and the nucleic acid sequence was verified by sequencing method.The two recombined plasmids pPIC9K-LacL and pPIC9K-LacM which isolated from E.coli DH5? were also transformed into Pichia pastoris through electroporation to produce pPIC9K-LacL-GS115 and pPIC9K-LacM-GS115.By the screening step,the multi-copy recombinations pPIC9K-LacL-GS115 and pPIC9K-LacM-GS115 were obtained with resistanting the 3.0mg/mL G418.However no protein band appeared from the supernatant obtained from pPIC9K-LacL-GS115 fermentation after 72h induced by methanol.But,LacL genes were detected on cDNA through reverse PCR.Moreover,the activity of ?-galactosidase from recombination pPIC9K-LacL-GS115 was only1.17U/mL.The culture conditions of the recombinant Pichia pastoris pPIC9K-LacL-GS115 were optimized in the shaking flask step.At last,it reached a maximum activity of 1.44 U/mL with 23%higher lactase enzyme production than before.