Cloning And Expression Of Lactase From Aspergillus Niger In Pichia Pastoris

The milk and other dairy products are the ideal foods for improving man's constitution. There is about 5% lactose in the milk. Many people (about 75%～90%) performed "lactose intolerance" in China, which affected the application of dairy products. The beta-D-galactosidase (EC 3.2.1.23, also referred to as lactase) hydrolysis the disaccharide lactose to glucose and galactose. Lactase can be used for treatment of "lactose intolerance" and production of low-lactose milk.DNA and cDNA sequences of lactase from Aspergillus niger DL116 were isolated by PCR and RT-PCR methods in the thesis. Sequences analysis revealed that the DNA sequences were 3368 bp containing eight introns. The cDNA was 2967 bp, encoding a protein of 988 amino acids. 12 potential N-glycosylation sites were assumed. The gene had been registered in Genbank with accession No.EF103141.The lactase gene was cloned into pPIC9K and constituted the recombinant vector of pPIC9K-lac. 156 transformants were selected in MD, MM plates and 65 positive transformants screened by PCR analysis were used for determining the lactase activity. A high-expression strain LAC-47 was obtained and the recombinant lactase was expressed highly. The expressed product had the normal bioactivity. The enzyme activity in the medium was reached 271.15 U/mL after inducing by methanol for 12 d.Lactase was purified by using ultrafiltration, dialysis, DEAE-cellulose ion-exchange chromatography and Sephacryl^TM S-200 gel filtration. The result of SDS-PAGE showed a single protein band with 130～140 kD. The specific activity of lactase had reached 532.9 U/mg.The recombinant expressed lactase shows optimum pH value of 3.0 to 4.2 and good pH stability from 2.5 to 9.0, optimum temperature was 60℃and good temperature stability at 60℃. Most metal ions and chemicals have no effect on the lactase. K⁺, Pb²⁺, Mg²⁺, Fe³⁺ can slightly activate the enzyme, and SDS would slightly inhibit the activity. According to the effect of EDTA, we concluded that the lactase doesn't strongly depend on metal ions. At 60℃and pH3.8, the Km was 2.43 mmol/L and the Vmax was 1.52μmol/min.