肌酐酶(creatininase, CA)是一种应用于体外检测试剂的重要工具酶,目前主要由微生物法生产,存在产量低和活性差的问题,限制了其实际应用。该研究通过全基因合成和异源表达构建了重组大肠杆菌BL21(DE3)/pET28a-CA,获得了高性能CA。通过单因素试验和响应面设计分析确定最优培养基组成,并系统地优化了发酵条件,从而提高了CA表达量。培养基优化实验结果表明,最优培养基各组分为:葡萄糖10.33 g/L、酵母浸粉21.80 g/L、磷酸盐137.63 mmol/L和胰蛋白胨15.60 g/L。经过5 L发酵罐发酵工艺优化,极大地提高了CA的活力,在接种量4%、诱导温度25 ℃、诱导时间18 h、异丙基-β-D-硫代半乳糖苷浓度0.2 mmol/L、溶氧值30%条件下,其活力达到470.24 U/mL,比酶活力为367.38 U/mg。该研究为CA的生产和工业化应用提供了理论基础和技术支持。
Creatininase, an important tool enzyme for in vitro detection reagents, is currently produced mainly by microbiological methods, which suffer from low yield and poor activity, limiting its practical application. Herein, recombinant Escherichia coli BL21(DE3)/pET28a-CA was constructed by whole gene synthesis and heterologous expression, obtaining high-performance creatininase. The optimal medium composition was determined by single-factor test and response surface design analysis, and the fermentation conditions were systematically optimized, resulting in improved creatininase expression. The medium optimization results showed that the optimal medium consisted of glucose 10.33 g/L, yeast extract 21.80 g/L, phosphate 137.63 mmol/L, tryptone 15.60 g/L. After optimization of the fermentation process in 5 L fermenters, the enzyme activity and specific enzyme activity of creatinine was greatly improved and reached 470.24 U/mL and 367.38 U/mg, respectively, under the conditions of 4% inoculum, 25 ℃ induction temperature, 18 h induction time, 0.2 mmol/L IPTG and 30% dissolved oxygen value. This study provides a theoretical basis and technical support for the production and industrial application of creatininase.
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