Engineering Of Nitrile Hydratase To Improve Its Stability And Study On A Novel Method Of Its Purification

Nitrile hydratase(EC 4.2.1.84, NHase), which catalyzes the hydration of nitriles to amides, is the key metalloenzyme for the production of amides in industries. Useful amides, such as acrylamide and nicotinamide, are widely applied in diverse industries, such as chemical production, fodder production, pharmacy, paper-making, textile engineering and so on. Biotechnology has advantages of lower costs, energy consumption and pollution over typical chemical production. And biotechnology has been gradually taking the place of typical chemical production in developed countries. Hence, expanding the properties of NHase to meet the requirements of different industrial application has great implication in economy and sustainable development.In this research, NHase from Pseudomonas putida NRRL-18668(Pp NHase) successfully expressed in E.coli was engineered. Such protein engineering was focused on improvement of the stability of Pp NHase and more convinency of purification of Pp NHase. Major results are above:(1) We aimed to improve the stability of NHase(Pp NHase) from Pseudomonas putida NRRL-18668 using a homologous protein fragment swapping strategy. One thermophilic NHase(Ct NHase) fragment from Comamonas testosteroni 5-MGAM-4D and two thermophilic NHase(Pt NHase) fragments from Pseudonocardia thermophila JCM3095 were selected to swap the corresponding fragments of Pp NHase. Seven chimeric NHases(1A、2B、2C、2BC、3AB、3AC、3ABC) were designed using STAR(site targeted amino recombination) software and molecular dynamics to determine the crossover sites for fragment recombination. Compared to Pp NHase, all constructed chimeric NHases showed 1.4- to 3.5- fold thermostabilities and six of them become more tolerant to high-concentration product. 3AB and 3ABC had 1.4 and 1.1- fold activities than Pp NHase(2) Notably, 3AB exhibited a 1.4- fold activity, 3.2 –fold thermostability, 1.5-fold tolerance of product and more stability in acid condition(low p H) compared to the wild-type Pp NHase. Circular dichroism spectrum analysis and homology modeling revealed that the 3AB slightly differed in secondary structure from wild-type Pp NHase. Compared to Pp NHase, 3AB has lower Km, resulting in its higher catalytic efficiency.(3) Based on "ITC(inverse transition cycling)" protein purification method, this experiment exploited a simple, fast and cheap protein purification method. After designing two kinds of ELPs, this experiment constructed two different NHase-ELP fusion proteins(NHase-[V5A2G3]-20 and NHase-[V]-20) and was able to efficiently purify the two active ELP-NHase fusion proteins by optimized ITC method. The purification fold of NHase-[V5A2G3]-20 and NHase-[V]-20 is 11.1 and 1.9, respectively. Meanwhile, both NHase-ELP fusion proteins displayed higher thermostability.