The Breeding Of 2, 2-Dimethylcyclopropanecarboxnitrile Hydratase-Producing Strain And Optimization Of Enzyme Reaction Conditions

Cilastatin was the first used inhibitor of dehydropeptidase-â… , whichcould enhance the antibacterial activity of imipenem to cure the infectionof aerobe and anaerobe, and had wide clinic applications.2,2-dimethylcyclopropanecarboxamide is one of the importantintermediates in the synthesis of Cilastatin. This thesis is about thebreeding of 2,2-dimethylcyclopropanecarboxnitrile hydratase-producingstrain by N⁺ implantation and the optimization of fermentation andenzyme reaction conditions were studied.Five strains were isolated from soil which could grow well with2,2-dimethylcyclopropanecarboxnitrile as sole nitrogen source byenrichment technique. One strain which had higher NHase activity wasassumed to belong to Pseudomonas sp. and therefore named asPseudomonas sp. Z JUT0509 after morphology and physiological tests. Breeding of NHase-producing strain by ion implantation has rarelybeen reported. With N⁺ implantation, the strain Pseudomonas sp.ZJUT0509 was bred to improve nitrile hydratase for higher productivityof 2,2-dimethylcyclopropanecarboxamide. The results showed that theactivity of strain F60-4 that was one of the mutants from Pseudomonas sp.ZJUT0509 reached 30.1U-ml^-1, was about 9 times higher than that of itsoriginal strain. The optimal conditions for enzyme production by mutantF60-4 was as follows: 2 g·l^-1 glucose, 10 g·l^-1 yeast extract, 6.5 initialpH value of broth, 20℃fermentation temperature and 1.0 g·l^-1caprolactam as inducer. Comparing with the original strain, the mutantstrain F60-4 could use organic nitrogen source much better. The optimuminducer was caprolactam instead of2,2-dimethylcyclopropanecarboxnitrile, and the fermentation time wasshortened to 36 h, that was 6 h less than the original strain.Pseudomonas sp. ZJUT0509 could produce not only nitrile hydratasebut also negligible amount of amidase. Enzyme production conditions ofPseudomonas sp. ZJUT0509 were studied in detail employing2,2-dimethylcyclopropanecarboxnitrile as the substrate. The optimatemperature and pH were 40℃, 7.5. Nhase was stable within a broad pHrang of 7.0-9.0. In addition, its thermostability was poor with t_1/2 Of 2.52hat 20℃and thermal deactivation energy of 123.28 kJ/mol. The activationenergy for 2,2-dimethylcyclopropanecarbonitrile hydration was 45.9 kJ/mol. NHase activity was completely repressed by 1 mmol/l Cu²⁺ andFe². There were no substrate and product inhibitive effect. Reactiondynamics shows that the Michaelis-Menten constant K_m and V_max were1.406 mmol/l and 53.48 mmol/l min respectively.