为解决细菌葡萄糖氧化酶(glucose oxidase, GOD)活力较低的问题,提取和纯化了实验室保藏海洋细菌Citrobacters sp. 8-III的基因组DNA,并与现存GOD基因比对获得目的序列,以此序列为模板获得GOD基因。将人工合成并密码子优化的GOD基因亚克隆至载体pET28a(+),构建重组表达载体pET28a(+)-GOD并转化到E. coli BL21(DE3)中实现表达。经镍柱亲和层析得到较纯的重组GOD,并对其进行酶学性质研究。实验成功构建了产GOD的E. coli BL21(DE3)/pET-28a(+)-GOD,目的蛋白约46 kDa。重组GOD酶活为2.04 U/mL,该酶最适作用温度为25 ℃;最适作用pH为6.0;K+、Ni2+对GOD的活性有明显促进作用;重组GOD添加至饲料中可加快雏鸡生长,且具备一定防腐功效。该研究首次将海洋细菌GOD基因导入大肠杆菌中,开拓GOD外源表达新的宿主。同时重组GOD具备低温酶特性,为其在饲料添加剂和低温领域应用奠定了基础。
The purpose of this study was to solve the problem that glucose oxidase (GOD) from marine bacteria has low activity. The genomic DNA of Citrobacters sp. 8-III was extracted and purified, and the target sequence was obtained by comparing against existing GOD genes. The synthetic and codon-optimized GOD gene was subcloned into the vector pET28a(+), and the recombinant expression vector pET28a(+)-GOD was constructed and transformed into Escherichia coli BL21(DE3) for expression. The recombinant GOD was purified by nickel affinity chromatography, and its enzymatic properties were studied. It was found that E. coli BL21(DE3)/pET-28a(+)-GOD producing GOD was successfully constructed. The purified GOD was 46 kDa. The activity of recombinant GOD was 2.04 U/mL, and its optimal reaction temperature and pH were 25 ℃ and 6.0, respectively. Additionally, K+ and Ni2+ significantly promoted its activity. Moreover, recombinant GOD could significantly accelerate the growth of chicken and it had good antiseptic effects in feeds of puppies. In conclusion, this study firstly introduced the marine bacterial GOD gene into E. coli to reveal a new host of GOD. Besides, recombinant GOD has cold-active enzyme properties, which lays a foundation for its applications in feed additives and cryogenics.
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