Expression Of Superoxide Dismutase Gene From Bacillus Cereus M22 In Pichia Pastoris

Superoxide dismutase (EC 1.15.1.1;SOD) is a metal-containing enzyme that widely exist in organisms. It catalyzes the dismutation of superoxide free radical and produces hydrogen peroxide and oxygen.It plays an important role in protecting the plasmalemma from the reactive oxygen species.A recombinant plasmid pPICZA-sodM18 was constructed and after its linearization was transferred into chromosome of Pichia pastoris GS115 by electroporation. The positive transformants with the integrates Mn-sod gene was identified by Zeocin-resistance, PCR screening and expression in P. pastoris. The recombinant Mn-SOD protein was successfully expressed in Pichia pastoris based on the evidences that a relative molecular weight about 23kD appeard in SDS-PAGE, the obvious activity of SOD existed in Native-PAGE and enzymatic activity test ,and Mn-SOD activity was specific base on the inhibition with the mixture of Chloroform-enthanol (3:5/V:V) and potassium cyanide.Two secreted plasmids pPICZaA-sodM18 and pPICZaA-sodC were constructed and after there linearization were transferred into chromosome of Pichia pastoris GS115 by electroporation. The positive transformants with the integrates Mn-sod gene and CuZn-sod gene were identified by Zeocin-resistance, PCR screening and expression in P. pastoris. The recombinant Mn-SOD protein and CuZn-SOD protein was successfully expressed in Pichia pastoris based on the evidences that the obvious activity of SOD existed in Native-PAGE and enzymatic activity test.This reseacher will laid down a foundation for industrial scale production of SOD.