重组表达D-泛解酸内酯水解酶乳酸克鲁维酵母的发酵工艺优化
Optimization of fermentation strategy for recombinant D- lactonohydrolasetype in Kluyveromyces lactis
本文以研究室前期构建的重组表达D-泛解酸内酯水解酶的乳酸克鲁维酵母（Kluyveromyces lactis yeast）GG799为对象，在5 L发酵罐中，基于考察pH、搅拌转速、接种量三种单因素条件对产酶的影响结果，进一步优化了补料策略，初步确定了促进重组D-泛解酸内酯水解酶高效表达的补料发酵方式。单因素条件研究结果表明，当pH 6.0、搅拌转速300 r/min、接种量2 %时，可以获得最高表达水平，酶活达5.12 U/mL，较摇瓶最佳结果提高了2倍。补料发酵研究表明，当通过补料维持发酵液恒糖浓度为5 g/L时，酶活可达到8.20 U/mL，较分批发酵酶活提高了60.1 %；当分批发酵结束后，以恒速5 g/h进行补料，酶活最高可达10.70 U/mL，较恒糖补料方式酶活提高了30.5 %，较分批发酵酶活提高了98.6 %。通过分批发酵和补料分批发酵的研究，显著提高了D-泛解酸内酯水解酶的酶活，为进一步的放大生产奠定了一定的基础。
生物工程学生物化学
D-泛解酸内酯水解酶pH搅拌转速接种量分批发酵补料发酵
谢颂洋,张梁.重组表达D-泛解酸内酯水解酶乳酸克鲁维酵母的发酵工艺优化[EB/OL].(2021-03-15)[2025-09-26].http://www.paper.edu.cn/releasepaper/content/202103-151.点此复制
The object of the current research is Kluyveromyces lactis GG799, which has been restructured for the expression of D- lactonohydrolasetype in previous study. In this work, three conditional parameters (pH, agitation speed and inoculum volume) were controlled to investigate the key factors for the production of enzyme and thereafter to optimize the feeding strategy for promoting the effective expression of D- lactonohydrolasetype in a 5-L fermenter. According to the results from the single-factor experiment, the maximum expression level with the enzyme activity of 5.12 U/mL could be achieved when the pH = 6.0, agitation speed = 300 r/min and inoculum volume = 2%. It was 2 times higher than that of shaking flask .For the optimization of feeding strategy, when the concentration of glucose was maintained as 5 g/L via continuously feeding, the enzyme activity could reach 8.20 U/mL, which was 60.1% higher than that acquired from the batch fermentation process. When controlling the supply at a rate of 5 g/h after the end of batch fermentation, the enzyme activity could be as high as 10.70 U/mL, which was increased by 30.5% and 98.6%, corresponding to constant concentration and feeding rate, respectively. Based on the above research, the enzyme activity of D- lactonohydrolasetype was significantly improved and hence to provide meaningful information for further scale-up production.
D-lactonohydrolasepHAgitation speedInoculum volumeBatch fermentationFed-batch fermentation
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