以黄素腺嘌呤二核苷酸(flavin adenine dinucleotide,FAD)为辅基的葡萄糖脱氢酶(glucose dehydrogenase with FAD,FAD-GDH,EC 1.1.99.10),与辅基结合紧密,催化效率高,是临床检测血糖指标的新型诊断用酶。将Burkholderia cepacia的FAD-GDH基因(gdh)构建含单启动PHpaⅡ的穿梭质粒pMA5-1,在蛋白酶缺陷型菌株Bacillus subtilis WB600中表达。为了获得该酶的高效表达,采用启动子串联及改造策略考察产酶情况。将4种启动子(PamyQ’,P43,PgsiB,Popuaa)分别与质粒上自带的启动子PHpaⅡ串联,结果表明PHpaⅡ-PamyQ’串联组合获得的FAD-GDH胞内酶活最高,为2 497 U/L,是串联前单启动子的2.7倍。为了减少发酵过程中,葡萄糖和甘油对产酶的抑制作用,在串联组合的基础上删去PamyQ’启动子中与碳代谢调控蛋白结合的cre位点,使胞内产酶水平提高至3 626 U/L,说明cre位点的去除能够减少碳代谢产物对启动子转录的抑制。本研究为新型诊断用酶FAD-GDH的菌种改造和工业化生产应用提供参考与借鉴。
Glucose dehydrogenase (FAD-GDH, EC1.1.99.10),conjugated tightly with flavin adenine dinucleotide, is a novel diagnostic enzyme for the clinical detection of blood glucose. A protease-defective strain Bacillus subtilis WB600 was used as a host to construct a shuttle plasmid pMA5-1 containing a single promoter PHpaII for expression of FAD-GDH gene (gdh) from Burkholderia cepacia. The use of promoters in series and transformation strategies to investigate enzyme production. Four promoters (PamyQ', P43, PgsiB, Popuaa) were ligated with the promoter PHpaII on the plasmid,respectively. The results showed that the intracellular enzymatic activity of FAD-GDH was highest with PHpaII-PamyQ' tandem, which was 2 497 U/L. In order to reduce the inhibitory effect of glucose and glycerol on enzyme production during fermentation, on the basis of tandem combination, the cre sites binding to carbon metabolism regulatory proteins in PamyQ 'promoter were deleted, and the intracellular enzyme production level was increased to 3 626 U/L, indicating that the removal of cre sites can reduce the inhibition of carbon metabolism products on promoter transcription. This study provides a reference for genetic modification and industrial production of a new diagnostic enzyme (FAD-GDH)
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