The bglS gene encoding endo-1,3-1,4-β-glucanase from Bacillus subtil＆ was cloned and sequenced in this study. The bglS expression cassette, including PGK1 promoter, bglS gene fused to the signal sequence of the yeas...The bglS gene encoding endo-1,3-1,4-β-glucanase from Bacillus subtil＆ was cloned and sequenced in this study. The bglS expression cassette, including PGK1 promoter, bglS gene fused to the signal sequence of the yeast mating pheromone a-factor （MFals）, and ADH1 terminator with G418-resistance as the selected marker, was constructed. Then one of the PEP4 allele of Saccharomyces cerevisiae WZ65 strain was replaced by bglS expression cassette using chromosomal integration of polymerase chain reaction （PCR）-mediated homologous recombination, and the bglS gene was expressed simultaneously. The recombinant strain S. cerevisiae （SC-βG） was preliminarily screened by the clearing hydrolysis zone formed after the barley β-glucan was hydrolyzed in the plate and no proteinase A （PrA） activity was measured in fermenting liquor. The results of PCR analysis of genome DNA showed that one of the PEP4 allele had been replaced and bglS gene had been inserted into the locus of PEP4 gene in recombinant strains. Different endo-1,3-1,4-β-glucanase assay methods showed that the recombinant strain SC-βG had high endo-1,3-1,4-β-glucanase expression level with the maximum of 69.3 U/（h·ml） after 60 h of incubation. Meanwhile, the Congo Red method was suitable for the determination of endo-1,3-1,4-β-glucanase activity during the actual brewing process. The current research implies that the constructed yeast strain could be utilized to improve the industrial brewing property of beer.展开更多
In order to refine the products of wheat germ agglutinin(WGA),several ion exchangers,such as D261,732,DEAE-cellulose-32 and CM-cellulose-32, have been used to removed proteins and pigments with different colors,such a...In order to refine the products of wheat germ agglutinin(WGA),several ion exchangers,such as D261,732,DEAE-cellulose-32 and CM-cellulose-32, have been used to removed proteins and pigments with different colors,such as brown,red,yellow,green and black,in the extracts of wheat germ.The WGA obtained from this procedure has higher hemagglutination activity than that available from sigma Co. The minimum hemagglutination dose of the purified WGA for an equal volume of a 2% type A red blood cells is 4μg/ml.WGA is a mixture of isolectins with different isoelectric points,5.9,6.2 and 6.8.Their molecular weight identified by SDS-polyacrylamide gel electrophoresis are 15,000 dalton,18,000 dalton and 35,000 dalton,respectively.展开更多
基金the National Hi-Tech Research and Develop-ment Program (863) of China (No. 2007AA10Z315)the Natural Science Foundation of Zhejiang Province, China (No. Z304076)
文摘The bglS gene encoding endo-1,3-1,4-β-glucanase from Bacillus subtil＆ was cloned and sequenced in this study. The bglS expression cassette, including PGK1 promoter, bglS gene fused to the signal sequence of the yeast mating pheromone a-factor （MFals）, and ADH1 terminator with G418-resistance as the selected marker, was constructed. Then one of the PEP4 allele of Saccharomyces cerevisiae WZ65 strain was replaced by bglS expression cassette using chromosomal integration of polymerase chain reaction （PCR）-mediated homologous recombination, and the bglS gene was expressed simultaneously. The recombinant strain S. cerevisiae （SC-βG） was preliminarily screened by the clearing hydrolysis zone formed after the barley β-glucan was hydrolyzed in the plate and no proteinase A （PrA） activity was measured in fermenting liquor. The results of PCR analysis of genome DNA showed that one of the PEP4 allele had been replaced and bglS gene had been inserted into the locus of PEP4 gene in recombinant strains. Different endo-1,3-1,4-β-glucanase assay methods showed that the recombinant strain SC-βG had high endo-1,3-1,4-β-glucanase expression level with the maximum of 69.3 U/（h·ml） after 60 h of incubation. Meanwhile, the Congo Red method was suitable for the determination of endo-1,3-1,4-β-glucanase activity during the actual brewing process. The current research implies that the constructed yeast strain could be utilized to improve the industrial brewing property of beer.
文摘In order to refine the products of wheat germ agglutinin(WGA),several ion exchangers,such as D261,732,DEAE-cellulose-32 and CM-cellulose-32, have been used to removed proteins and pigments with different colors,such as brown,red,yellow,green and black,in the extracts of wheat germ.The WGA obtained from this procedure has higher hemagglutination activity than that available from sigma Co. The minimum hemagglutination dose of the purified WGA for an equal volume of a 2% type A red blood cells is 4μg/ml.WGA is a mixture of isolectins with different isoelectric points,5.9,6.2 and 6.8.Their molecular weight identified by SDS-polyacrylamide gel electrophoresis are 15,000 dalton,18,000 dalton and 35,000 dalton,respectively.
