腈水合酶(nitrile hydratase,NHase,EC 4.2.1.84)是一种金属酶,能将腈类物质催化水合生成酰胺类物质,在工业上主要应用于烟酰胺和丙烯酰胺的生产。腈水合酶在应用过程中,普遍存在热稳定性较差的缺陷,而腈类的水合催化属于放热反应,在工业催化过程中,过高的反应温度使得腈水合酶的催化效率、重复利用率均较低,故寻找一种耐热型腈水合酶对工业应用以及理论研究具有重要意义。该研究以提高腈水合酶热稳定性为出发点,从美国国立生物技术信息中心通过BLAST筛选到一种新型耐热腈水合酶,该腈水合酶来源为温泉热碱芽孢杆菌(Caldalkalibacillus thermarum TA2.A1),该菌株最适生长温度为65~70 ℃。将来源于Cal. thermarum TA2.A1的腈水合酶基因序列进行密码子优化,基因合成后在大肠杆菌BL21(DE3)宿主中异源表达,通过在该腈水合酶β亚基的C端添加strep标签成功纯化该酶,测定其在65 ℃的半衰期为3 h,在30 ℃下催化烟腈的比酶活为395 U/mg,其全细胞催化反应速率比已报道数据将近快1倍,烟酰胺的最终产量提高了25%,更有利于工业应用。
Nitrile hydratase (NHase, EC 4.2.1.84) is a metalloenzyme that catalyzes the hydration of nitriles to amides and is mainly used in the industry to produce nicotinamide and acrylamide. In the application process, the common defect of nitrile hydratase is its poor thermal stability. As the process of catalyzing nitrile is an exothermic reaction, during industrial applications, the catalytic efficiency, recovery rate and recycling rate of nitrile hydratase are hindered in large extent. Therefore, discovering new NHase with high thermostability is of great significance for industrial applications and scientific research. In this study, a novel nitrile hydratase was found by BLAST in the Genebank of NCBI from the starting point of heat resistance. The strain was Caldalkalibacillus thermarum TA2.A1,of which the optimum growth temperature was 65-70 ℃. After codon optimization, the NHase gene was heterologously expressed in E. coli BL21 (DE3). The protein was purified by adding strep tag at the C-terminus of the β subunit. The half-life of this novel NHase at 65 ℃ was 3 h and the specific activity towards 3-cyanopyridine was 395 U/mg at 30 ℃. The reaction rate of the whole cell catalysis process at 30 ℃ was nearly twice as fast as the reported data, and the final yield of nicotinamide was increased by 25%, which was more favorable for industrial applications.
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