Response surface methodology was applied to optimize medium components for production of recombinant calf chymosin by Kluyveromyces lactis GG799.The previous data indicated that the most suitable carbon source,nitroge...Response surface methodology was applied to optimize medium components for production of recombinant calf chymosin by Kluyveromyces lactis GG799.The previous data indicated that the most suitable carbon source,nitrogen source,salt and vitamin were glucose,yeast extract,KH2PO4 and Ca D-Pantothenate,respectively.The concentration of four media components were optimized by using central composite design of response surface methodology.The optimum medium composition for recombinant calf chymosin production was found to contain glucose 29.84 g· L-1,yeast extract 19.85 g·L-1,KH2PO4 0.1 g·L-1 and Ca D-Pantothenate 4.49 mg·L-1.The enzyme activity of recombinant calf chymosin was 722 U· mL-1,which was in an excellent agreement with the predicted value（723 U·mL-1）.The production of recombinant calf chymosin from Kluyveromyces lactis GG799 was effectively increased by response surface methodology.展开更多
We cloned and expressed bile salt hydrolase gene ofLactobacillus plantarum M1-UVS29 in Lactococcus lactis NZ9000 successfully. Gene-specific primers for amplification of L. plantarum bsh were designed by using sequenc...We cloned and expressed bile salt hydrolase gene ofLactobacillus plantarum M1-UVS29 in Lactococcus lactis NZ9000 successfully. Gene-specific primers for amplification of L. plantarum bsh were designed by using sequence which availabled from GenBank. The production of PCR amplicon was confirmed by sequencing and cloned into pMD18-T vector, and then recombined into expression vector pNZ8148 and yielding vector pNZ8148-BSH, pNZ8148-BSH was transferred into Lactococcus lactis NZ9000. Sequencing indicated that the cloned bsh fragment contained 995 nucleotides, and shared 99.3% sequence homology with bsh gene from L. plantarum MBUL10. Cloned bsh fragment was successfully transduced into NICE expression system and confirmed by PCR and restriction digest. Recombinant BSH protein was analyzed by SDS-PAGE. The molecular weight of BSH protein was approximately 37 ku. Activity of the expressed protein was 0.77 μmol· min^-1. The successfully expressed proteins by genetic engineering technology made the function of lactic acid bacteria be abundant and laid the foundation for further researches into cholesterol-lowering lactic acid bacterium food and probiotics.展开更多
基金Supported by Science and Technology Fund of Heilongjiang Province Education Department (11541018)
文摘Response surface methodology was applied to optimize medium components for production of recombinant calf chymosin by Kluyveromyces lactis GG799.The previous data indicated that the most suitable carbon source,nitrogen source,salt and vitamin were glucose,yeast extract,KH2PO4 and Ca D-Pantothenate,respectively.The concentration of four media components were optimized by using central composite design of response surface methodology.The optimum medium composition for recombinant calf chymosin production was found to contain glucose 29.84 g· L-1,yeast extract 19.85 g·L-1,KH2PO4 0.1 g·L-1 and Ca D-Pantothenate 4.49 mg·L-1.The enzyme activity of recombinant calf chymosin was 722 U· mL-1,which was in an excellent agreement with the predicted value（723 U·mL-1）.The production of recombinant calf chymosin from Kluyveromyces lactis GG799 was effectively increased by response surface methodology.
基金Supported by the National Natural Science Fund Project(31171657)Heilongjiang Province Natural Fund Project(ZD201207)Heilongjiang Province Postdoctoral Special Funds(LBH-Q13133)
文摘We cloned and expressed bile salt hydrolase gene ofLactobacillus plantarum M1-UVS29 in Lactococcus lactis NZ9000 successfully. Gene-specific primers for amplification of L. plantarum bsh were designed by using sequence which availabled from GenBank. The production of PCR amplicon was confirmed by sequencing and cloned into pMD18-T vector, and then recombined into expression vector pNZ8148 and yielding vector pNZ8148-BSH, pNZ8148-BSH was transferred into Lactococcus lactis NZ9000. Sequencing indicated that the cloned bsh fragment contained 995 nucleotides, and shared 99.3% sequence homology with bsh gene from L. plantarum MBUL10. Cloned bsh fragment was successfully transduced into NICE expression system and confirmed by PCR and restriction digest. Recombinant BSH protein was analyzed by SDS-PAGE. The molecular weight of BSH protein was approximately 37 ku. Activity of the expressed protein was 0.77 μmol· min^-1. The successfully expressed proteins by genetic engineering technology made the function of lactic acid bacteria be abundant and laid the foundation for further researches into cholesterol-lowering lactic acid bacterium food and probiotics.
