氧化葡萄糖酸杆菌中5-葡萄糖酸脱氢酶基因的克隆、表达及酶学性质分析

Cloning,Expression and Characterization of Gluconate 5-dehydrogenase from Gluconobacter oxydans

根据NCBI上的报道的基因序列设计引物,以氧化葡萄糖酸杆菌(Gluconobacter oxydans)H24的基因组为模板,获得5-葡萄糖酸脱氢酶(Ga5DH)基因,将其与表达载体pET-28a连接,构建重组质粒pET-28a-Ga5DH,并转化大肠杆菌Rosetta进行表达。SDS-PAGE检测结果显示,表达蛋白的分子大小为26.5 kD,纯化后酶活达7.83 U/mg。酶学性质分析表明,该酶的最适反应温度为40℃,最适pH为11。在pH 9-11的缓冲中保温8 h,酶活力仍有80%以上的残余。该酶对多种有机溶剂具有良好的耐受性。

Guconate 5-dehydrogenase gene from Gluconobacter oxydans H24 was amplified with primes designed based on NCBI database published sequence information. Ga5 DH was cloned and expressed in Escherichia coli Rosetta strain. Results showed that the molecular mass of the enzyme was 26.5 kD by SDS-PAGE gel analysis. After purification, the enzyme activity could reach 7.83 U/mg. It was suggested that the recombinant protein had a maximum activity at 40℃, pH 11. The characterization of purified enzyme indicated that Ga5 DH was highly resistant to organic solvents.