Expression，Purification and Activity Assay of Recombinant Thermophile Lactase From Pichia pastoris

Abstract:

In order to obtain the recombinant thermostable lactase，a DNA fragment containing the mature lactase gene was amplified from Pyrococcus furious by PCR and cloned into pPICZaA，generating a fusion protein with the alpha factor from baker's yeast and integrated into the genome of Pichia pastoris strain X-33. Recombinant yeast transformants with high-level recombinant lactase production was identified by Western blotting，secreting as much as 125 U/mL induction by methanol. The recombinant thermostability lactase was purified by Ni+-NTA affinity chromatography and SDS-PAGE results shown that the purity was over 95%. The specific activity was about 1 800 U/mg and the optimal temperature was about 105 ℃. It was also showed that the purified lactase from P. pastoris has a highly thermostability and strong ability to hydrolysis lactose.