本实验对厚垣镰孢霉HML278采用液体发酵产纤维素酶,加入0.2%、0.4%、0.6%、0.8%、1.0%不同浓度的茶皂素,探究茶皂素对其产纤维素酶的影响。实验结果显示,加入0.6%浓度的茶皂素的纤维素酶活性最高。在酶学特性研究中,酶的最适温度为60℃...本实验对厚垣镰孢霉HML278采用液体发酵产纤维素酶,加入0.2%、0.4%、0.6%、0.8%、1.0%不同浓度的茶皂素,探究茶皂素对其产纤维素酶的影响。实验结果显示,加入0.6%浓度的茶皂素的纤维素酶活性最高。在酶学特性研究中,酶的最适温度为60℃,最适pH为6.4。在金属离子对酶活性的探究中,发现Li+、K+、Ca2+、Mn2+、Zn2+、Fe3+对酶促反应具有促进作用,其中Ca2+、Mn2+对酶促反应的促进作用最强;而Co2+、Al3+对酶促反应具有抑制作用。This experiment used liquid fermentation to produce cellulase from Fusarium graminearum HML278, and added tea saponin at different concentrations of 0.2%, 0.4%, 0.6%, 0.8%, and 1.0% to investigate the effect of tea saponin on its cellulase production. The experimental results showed that the cellulase activity was highest when 0.6% concentration of tea saponin was added. In the study of enzymatic characteristics, the optimal temperature and pH for the enzyme are 60˚C and 6.4, respectively. In the exploration of metal ions on enzyme activity, it was found that Li+, K+, Ca2+, Mn2+, Zn2+, and Fe3+ have a promoting effect on enzymatic reactions, among which Ca2+ and Mn2+ have the strongest promoting effect on enzymatic reactions;Co2+ and Al3+ have inhibitory effects on enzymatic reactions.展开更多
文摘硫酸软骨素A(chondroitin sulfate A,CSA)是关节软骨修复中不可或缺的重要成分。CSA由软骨素4-O-磺基转移酶-1(chondroitin 4-O-sulfotransferase-1,C4ST-1)催化软骨素中N-乙酰氨基半乳糖胺(N-acetylgalactosamine,GalNAc)4号位羟基磺酸化合成。然而,由于C4ST-1酶活性较低,其催化能力受限,进而阻碍了CSA的工业化生产进程。为此,该研究旨在通过结合重组菌构建和培养基优化提升C4ST-1酶活性。首先在筛选确定了以毕赤酵母GS115为底盘细胞的基础上,进一步优化了以OST1-α分泌信号肽和SUMO Pro 3促溶标签的组合方式进行C4ST-1分泌表达,经摇瓶发酵C4ST-1最高酶活性为1889.2 U/L。鉴于前期研究发现无机盐对C4ST-1酶活性的抑制以及现有培养基成本较高问题,该研究通过优化培养基组分,发现不添加昂贵成分酵母无氨基氮源(yeast nitrogen base without amino acids,YNB)时,C4ST-1酶活性较原培养基提高68.4%。此外,结合碳源、其他氮源以及生物素的筛选与优化使C4ST-1酶活性进一步提高。最终,在5L发酵罐补料分批发酵72 h时,获得最高酶活性为5040.7 U/L。该研究不仅为CSA规模化生产奠定了基础,也将为其他糖胺聚糖(如肝素、硫酸皮肤素)合成所需的磺基转移酶发酵生产提供借鉴。
文摘本实验对厚垣镰孢霉HML278采用液体发酵产纤维素酶,加入0.2%、0.4%、0.6%、0.8%、1.0%不同浓度的茶皂素,探究茶皂素对其产纤维素酶的影响。实验结果显示,加入0.6%浓度的茶皂素的纤维素酶活性最高。在酶学特性研究中,酶的最适温度为60℃,最适pH为6.4。在金属离子对酶活性的探究中,发现Li+、K+、Ca2+、Mn2+、Zn2+、Fe3+对酶促反应具有促进作用,其中Ca2+、Mn2+对酶促反应的促进作用最强;而Co2+、Al3+对酶促反应具有抑制作用。This experiment used liquid fermentation to produce cellulase from Fusarium graminearum HML278, and added tea saponin at different concentrations of 0.2%, 0.4%, 0.6%, 0.8%, and 1.0% to investigate the effect of tea saponin on its cellulase production. The experimental results showed that the cellulase activity was highest when 0.6% concentration of tea saponin was added. In the study of enzymatic characteristics, the optimal temperature and pH for the enzyme are 60˚C and 6.4, respectively. In the exploration of metal ions on enzyme activity, it was found that Li+, K+, Ca2+, Mn2+, Zn2+, and Fe3+ have a promoting effect on enzymatic reactions, among which Ca2+ and Mn2+ have the strongest promoting effect on enzymatic reactions;Co2+ and Al3+ have inhibitory effects on enzymatic reactions.
