Isolation And Identification Of Nitrile-hydrolyzing Microorganisms And Biotransformation Of β-aminopropionitrile To β-alanine Production By Stenotrophomonas Maltophilia A39

β-alanine is an important intermediate for pantothenic acid (vitamin B5) and coenzyme A (Co A) production. A new method has been developed forβ-alanine production by microbial hydrolysis ofβ-aminopropionitrile. Isolation, identification, improvement and cultivation process of the microorganism were investigated in this paper. The optimization ofβ-aminopropionitrile biotransformation with a39, a nitrile hydrolyzing bacterium and the quantification assay ofβ-alanine by HPLC were also investigated.Four microorganism strains capable ofβ-alanine production byβ-aminopropionitrile hydrolysis were isolated from various samples. And one strain numbered a39 was identified as Stenotrophomonas maltophilia based on morphology, physiological and biochemical characterization, combined with ATB system (ID32 GN). Strain was performed by two consecutive implantations of low energy ion beam for improving biotransformation ability and one positive mutant was selected for further study.A rapid method (TLC-scanning method) was established to determineβ-alanine in reaction mixture. For more precise assay, a high performance liquid chromatography (HPLC) method based on 2,4-dinitrofluorobenzene (DNFB) derivatization ofβ-alanine was studied. When determined at 360nm, the average recovery rate of the method was 99.3%. The linear range of the assay was from 2μg/mL to 1000μg/mL. The minimal detection limit was 0. 02 ng. The cultivation conditions of strain were optimized using response surface methodology (RSM). The results showed that the optimal carbon source and nitrogen source were sucrose and yeast extract, respectively, and the optimal incubation media were as follows: sucrose 1.29%, yeast extract 0.71%,β-aminopropionitrile 0.52%. Specific activity of 22.51 U/g was obtained in this study.The conditions of biotransformation process were also optimized by RSM. The results showed that the optimal biotransformation conditions were: time 8h,β-aminopropionitrile concentration 2.5%, and cell weight of 0.86 g. Under the above conditions, theβ-alanine average yield of 102.81 mg was acquired in 10 mL reaction mixture. The enzymatic characteristics were investigated with resting cells as crude nitrilase. The preliminary data indicated that the optimal temperature and pH of the crude nitrilase were 35℃and 8.4, respectively.Finally,β-alanine production byβ-aminopropionitrile biotransformation with immobilized cells by Ca-alginate gel was studied. The results showed that the optimal temperature and concentration ofβ-aminopropionitrile forβ-alanine production by the immobilized cell were 40℃and 1.5%, respectively. The operation stabilities of immobilized cells were investigated, and the immobilized cell could keep stable catalyzing ability after more than ten batches.