D-氨基酸脱氢酶在不同宿主中发酵产酶条件优化

Optimization of fermentation conditions of D-amino acid dehydrogenase in different hosts

大肠杆菌产D-氨基酸脱氢酶存在破胞问题,酶活越高,破胞问题越严重,为解决这一问题,本实验室构建出一株高表达D-氨基酸脱氢酶的大肠杆菌菌株,并把连接目的基因的重组质粒转化到毕赤酵母菌中,优化了D-氨基酸脱氢酶在两种宿主中的发酵产酶条件。对大肠杆菌产D-氨基酸脱氢酶诱导温度、诱导pH、起始诱导OD600值和发酵时间进行优化,在25℃、pH 6.5、OD600为35的条件下诱导,酶活为86.0 U/mL,比优化前提高了34.4%。选择38 h左右放罐,此时D-氨基酸脱氢酶发酵液酶活为79.0 U/mL,上清液酶活占20%。通过对毕赤酵母产D-氨基酸脱氢酶诱导温度、诱导pH和溶氧值进行优化,确定诱导条件为28℃、pH 6.0和溶氧值为20%,此时D-氨基酸脱氢酶酶活为140.5 U/mL,比优化前提高了58.9%,且无破胞现象。

The production of D-amino acid dehydrogenase by Escherichia coli has a cell-breaking problem. The higher the enzyme activity, the more serious the problem. In order to solve this issue, an E.coli strain with high expression of D-amino acid dehydrogenase was constructed in our laboratory, and the recombinant plasmid containing the target gene was transformed into Pichia pastorisas well,to optimize the fermentation conditions of D-amino acid dehydrogenase in both hosts. After optimization of the induction temperature, pH, OD600 value and fermentation timein E. coli, the enzyme activity of D-amino acid dehydrogenase reached 86.0 U/ml at 25 ℃, pH 6.5 and inductionat OD600 35, which was 34.4% higher than before. After fermentation for 38 h, the enzyme activity of fermentation broth reached 79.0 U/mL and that in supernatant accounted for 20% of total activity. The optimal fermentation conditions for D-amino acid dehydrogenase in Pichia pastoriswere determined to be 28 ℃, pH 6.0 and dissolved oxygen value of 20%, under which, the enzyme activity was 140.5 U/mL, which was 58.9% higher than that without optimization, and no cell breakagewas observed.