Rapid improvement of lipase production in Penicillium expansum by genome shuffling / 生物工程学报

In the present study, the genome shuffling was used to improve lipase production of Penicillium expansum. A lipase producing mutant strain-Penicillium expansum FS8486 and a wild type of Aspergillus Tamarii FS-132 isolated from soil of a volcano in Xinjiang were used as the parental strains. After two rounds of genome shuffling, several elite daughter strains were screened. The lipase activity in one of the daughter strains was increased 317% over the starting strain FS8486. Comparisons of the morphology, RAPD (Random Amplification of Polymorphic DNA) polymorphism and the fatty acid compositions between the daughter and the parental strains suggested that the filial generation were generated by genome shuffling. In this study, the genome shuffling used successfully first time in eukaryotic microorganism and increases the production of the desired metabolite in short time, the study will be useful to spread the genome shuffling in eukaryotic microbial breeding.

Overexpression of Penicillium expansum lipase gene in Pichia pastoris / 生物工程学报

The alkaline lipase gene of Penicillium expansum (PEL) was coloned into the yeast integrative plasmid pPIC3.5K, which was then transformed into His4 mutant yeast GS115. Recombinant Pichia strains were obtained by minimal olive oil-methanol plates screening and confirmed by PCR. The expression producus of PEL gene was analysis by SDS-PAGE and olive oil plate, the result indicated that PEL gene was functionally overexpressed in Pichia pastoris and up to 95% of the secreted protein. Recombinant lipase had a molecular mass of 28kD, showing a range similar to that of PEL, could hydrolyze olive oil and formed clear halos in the olive oil plates. Four different strategies (different media, pH, glycerol and methanol concentration) were applied to optimize the cultivation conditions, the activity of lipase was up to 260 u/mL under the optimal cultivation conditions. It is pointed out that the absence of the expensive biotin and yeast nitrogen base in the medium increased the lipase production. The possible reason of this result is absence of yeast nitrogen base increased the medium pH during cultivation, and PEL shows a higher stability at this condition. The lipase activity of the supernatant from the culture grown at pH 7 was higher than the one from the culture in the same medium at pH 6.0 is due to the pH stability of PEL too. The results also showed that the methanol and glycerol concentration had a marked effect on the production of lipase.

PURIFICATION AND PROPERTIES OF FUMARASE FROM ASPERGILLUS WENTII / 微生物学通报

The fumarase from the culture of Aspergillus wentii F-871 was partially by means of precipitation with protamine sulphate, ammonium sulfate fractionation and column chromatography on Sephadex G-200, and then the concentrated enzyme solution was freeze-dried. The comparative enzyme activity of the fumarase was increased by 31.70 times and reached 24.6 U/mg, and the recovery was 36.64% . The optimal pH and temperature was 8.0 and 30℃, respectively. The pH stability of fumarase ranged 6.0 - 8.5, and more than 90% of the enzyme activity remained after incubated at 35℃ for 30 min.

FERMENTATION CONDITIONS OF SOYMILK-CLOTTING ENZYME PRODUCTION BY BACILLUS SP.AND STUDIES ON THE PROPERTIES OF THE ENZYM E / 微生物学通报

The optimum condition of shaking-flask p roducing enzyme were the tempe rature 26℃,initial pH 6 4,fermentation period 19 hours,medium volume 15mL m e dium/300mL Flask.soymilk-clotting enzyme was obtained from ammonium sulfate p r ecipitation.The optimum temperature and pH for the soymilk-clotting activity wa s 70℃and 5 8.The enzyme was easy to lose activity in acid or alkaline circumst a nce.About 60% of the original activity remained after 1 hour at 60℃.Ca 2+ ,Fe 2+ , Mg 2+ ,Na +increased the clotting activity,whereas Zn 2+ ,Al 3+ ca use inhibition.