Microcalorimetric bioassay for acute cellular toxicity is based on metabolicheat production from cultured cells. The biological response to toxicants is the inhibition of theheat production rate in cells, and toxicity...Microcalorimetric bioassay for acute cellular toxicity is based on metabolicheat production from cultured cells. The biological response to toxicants is the inhibition of theheat production rate in cells, and toxicity is expressed as the concentration of toxicant that is50% effective to this inhibition (IC_(50)). In this paper, the effect of Na_2SeO_3 on Bacillussubtilis growth was investigated at 37℃ by microcalorimetry. The relationship between growth rateconstants (k) and concentration of Na_2SeO_3(c) shows a logarithmic normal distribution, and IC_(50)is 20.3 μg/mL. All these thermokinetic information is readily obtained by an LKB 2277-204 heatconduction microcalorimeter. Microcalorimetry is a quantitative, inexpensive, and versatile methodfor toxicology research.展开更多
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
文摘Microcalorimetric bioassay for acute cellular toxicity is based on metabolicheat production from cultured cells. The biological response to toxicants is the inhibition of theheat production rate in cells, and toxicity is expressed as the concentration of toxicant that is50% effective to this inhibition (IC_(50)). In this paper, the effect of Na_2SeO_3 on Bacillussubtilis growth was investigated at 37℃ by microcalorimetry. The relationship between growth rateconstants (k) and concentration of Na_2SeO_3(c) shows a logarithmic normal distribution, and IC_(50)is 20.3 μg/mL. All these thermokinetic information is readily obtained by an LKB 2277-204 heatconduction microcalorimeter. Microcalorimetry is a quantitative, inexpensive, and versatile methodfor toxicology research.
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.
