钝齿棒杆菌N-乙酰鸟氨酸转氨酶的克隆表达分析及其重组菌的精氨酸发酵

Cloning,expression and characterization of N-Acetylornithine aminotransferase from Corynebacterium crenatum and its effects on L-arginine fermentation

N-乙酰鸟氨酸转氨酶(EC 2.6.1.11,ACOAT)是钝齿棒杆菌Corynebacterium crenatum精氨酸合成途径中的第4个酶,催化底物N-乙酰谷氨酸半醛生成产物N-乙酰鸟氨酸。为研究N-乙酰鸟氨酸转氨酶在钝齿棒杆菌中精氨酸合成中的作用,考察其酶学性质,对培养基成分和发酵过程工艺条件的优化提高精氨酸产量提供依据。从精氨酸高产菌株钝齿棒杆菌SYPA 5-5染色体扩增获得ACOAT编码基因argD,全长1 176 bp,编码390个氨基酸,在Escherichia coli BL21(DE3)及C.crenatum SYPA中成功表达。采用Ni柱亲和层析纯化后获得的重组蛋白比酶活达108.2 U/g,对其部分酶学性质进行初步研究。构建重组钝齿棒杆菌C.crenatum SYPA(pJCtac-CcargD),加强精氨酸合成途径ACOAT蛋白表达量,对重组菌产精氨酸进行初步发酵分析。多次发酵结果表明重组钝齿棒杆菌与出发菌株相比胞内ACOAT酶活得以增强;重组菌CCD1精氨酸平均产量为39.7 g/L,产酸提高14.7%。结果还表明在重组菌发酵精氨酸的同时不仅ACOAT得到了加强表达,同时还提高了重组菌在发酵过程中菌体自身的氧利用率。

N-Acetylornithine aminotransferase（EC 2.6.1.11,ACOAT） catalyzes the conversion of N-acetylglutamic semialdehyde to N-acetylornithine,the forth step involved in the L-arginine biosynthetic pathways.We studied the enzyme properties to set up reliable theoretical basis for the arginine fermentation optimization.ACOAT encoding gene argD was cloned from an industrial L-arginine producer Corynebacterium crenatum SYPA 5-5.Analysis of argD sequences revealed that only one ORF existed,which coded a peptide of 390 amino acids with a calculated molecular weight of 41.0 kDa.The argD gene from C.crenatum SYPA 5-5 was expressed both in Escherichia coli BL21 and C.crenatum SYPA.Then ACOAT was purified by Ni-NTA affinity chromatography and its specific enzyme activity was 108.2 U/g.Subsequently,the expression plasmid pJCtac-CcargD was transformed into C.crenatum SYPA and the specific activity of ACOAT was improved evidently in the recombinant C.crenatum CCD.Further fermentative character of CCD1 was also analyzed.The results showed that the L-arginine producing ability of the recombinant strain was 39.7 g/L improved by 14.7%.