苏云金芽孢杆菌重组L-异亮氨酸羟化酶的酶学性质及其在4-羟基异亮氨酸合成中的应用

Characterization of recombinant L-isoleucine-4-hydroxylase from Bacillus thuringiensis and its application in 4-hydroxyisoleucine biosynthesis

【目的】克隆并表达来源于苏云金芽孢杆菌(Bacillus thuringiensis)TCCC 11826的L-异亮氨酸羟化酶(L-isoleucine-4-hydroxylase,IDO),测定重组IDO酶学特性并构建用于4-羟基异亮氨酸(4-Hydroxyisoleucine,4-HIL)微生物转化的重组菌株,以考察该酶在4-HIL合成中的潜在应用价值。【方法】以B.thuringiensis TCCC 11826基因组为模板PCR扩增ido基因并构建该基因过表达菌株BL-IDO;采用Ni-NTA亲和层析法分离纯化重组IDO后检测其酶学特性;构建重组株菌W3110-IDO进行4-HIL的微生物转化。【结果】克隆B.thuringiensis TCCC 11826的ido基因,测序结果显示该基因含723个核苷酸,编码240个氨基酸,与已报道的B.thuringiensis 2-e-2的ido基因相似度分别为97.47%和97.91%。此IDO含有His1-X-Asp/Glu-Xn-His2基序,属于Fe2+和α-酮戊二酸依赖型羟化酶家族;酶学实验表明该酶能够特异性地催化L-异亮氨酸生成(2S,3R,4S)-4-HIL,其Km和Vm ax分别为0.18 mmol/L和2.10μmol/min/mg,最适反应温度和pH分别为35℃和7.0,该酶于35℃条件下放置5 h后仍具有85.1%的活性;在Escherichia coli W3110中过表达重组IDO,在未经优化条件下4-HIL最高转化率达89.28%。【结论】获得IDO编码基因序列(Accession No.KC884243)并首次较为系统地研究了其酶学特性,该酶反应条件温和且具有较高的活性及稳定性,在酶法或微生物转化法合成4-HIL中有较广泛的应用价值。本研究可为4-HIL及其它氨基酸衍生物的生物制造技术奠定理论基础。

[ Objective ] L-isoleucine-4-hydroxylase （IDO） encoding gene ido from Bacillus thuringiensis TCCC 11826 was cloned and expressed, followed by enzyme characterization. In addition, recombinant strain was tested for its 4- Hydroxyisoleucine （4-HIL） biotransformation. [ Methods] Ido gene was amplified from B. thuringiensis TCCC 11826 genomic DNA and expressed in BL-IDO. Recombinant IDO was extracted, purified and characterized. Recombinant strain used for biotransformation of 4-HIL was constructed. [ Results ] Composed of 723 nucleotides encoding 240 amino acids （sharing 97.47% and 97.91% identities with that of B. thuringiensis 2-e-2）, ido gene was cloned from B. thuringiensis TCCC 11826. The recombinant IDO contained a His1-X-Asp/Glu-Xn-His2 motif that is specific for Fe（II）/α-ketoglutarate- dependent hydroxylases and catalyzed L-isoleucine to 4-HIL. Normal hyperbolic kinetics was observed with L-Ile in the reaction by recombinant IDO. Lineweaver-Burk treatment of the data yielded apparent K and the Vmox was 0. 18 mmol/L and 2. 10 p.mol/min/mg, respectively. The optimum temperature and pH for the recombinant IDO was 35℃ and 7.0 respectively; moreover, the relative activity of the enzyme remain 85.10% after 5 h incubation at 35℃. In all, recombinant strain harboring ido transformed 89.28% of L-isoleucine to 4-HIL. [ Conclusion ] In this study, an ido （ Accession No. KC884243） with novel sequence was isolated and enzymatic characteristics of recombinant IDO was systematically analyzed. In addition, we successfully achieved the biotransformation of 4-HIL from L-isoleucine. This work will lay theoretical foundation and practical basis on the microbial manufacture technology of 4-HIL and other amino acid derivatives. This work will lay theoretical foundation and practical basis on the microbial manufacture technology of 4-HIL and other amino acid derivatives.