Studies On Expression Of Acid Phosphorutase PhyB Gene In Pichia Pastoris GS115

The phyB gene without signal peptides or introns has been amplified by PCR using the recombinant plasmid pBV220-PhyB as templates. The PCR products were cloned into pUCm-T vector and sequenced. The DNA sequences and their deduced amino acid sequences were compared with sequences reported in GenBank Accession No. L20567, it was found that 7 nucleotides and 3 amino acids have been changed, there is no change at active site sequences. The phyB gene contains 1383bp and encoding 460 amino acids. The recombinant plasmid pPIC9K-phyB was constructed by means of connecting phyB and plasmid together and then was transformed into Pichia pastor is GS115. Transformants with inserted target fragment were screened using RDB. MM,MD culture midium. Induced by methanol, the phyB gene was expressed and the molecular mass of the muture phyB was determined to be 75KD by SDS-PAGE analysis, while the mass is 60KD as phyB gene expressed in E.coli, which may be caused by the glycosylation of the yeast cell. The analysis of enzymatic peoperties shows that its optimal temperature is 65℃, which is 10℃ higher than phyB produced from wide type strain; and its optimal induce pH and optimal reaction pH are 5.5 and 2.5 respectively; the expression level of phyB can reach up to 10528.6U/ml, which is 2.8 times as much as wide type strain's. Under the condition of 70℃ for 5min, the phyB remains 90% of the initial activity,but only 40% remained after 60min; it retains 50% of its activity after denaturation at 75℃ for 5min, only 10% of its activity retained after 10min.