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Isolation and Identification of Nitrile-Hydrolyzing Microorganisms and Biotransformation of β-Aminopropionitrile to β-Alanine Production by Stenotrophomonas Maltophilia a39

Author: XuJianMiao
Tutor: ZhengYuGuo
School: Zhejiang University of Technology
Course: Biochemical Engineering
Keywords: β-amino propionic acid Filter Culture conditions Biotransformation Response Surface
CLC: TQ922
Type: Master's thesis
Year: 2006
Downloads: 15
Quote: 0
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Abstract


beta-alanine can be used to prepare the product of pantothenic acid, calcium pantothenate and coenzyme A, is an important pharmaceutical raw materials and intermediates. The purpose of this study was to develop the a microbial catalyzed hydrolysis of the β-amino-propionitrile, producing β-production aminopropionates biological conversion process, the establishment of a quantitative measurement method of the β-amino propionic acid tlc scanning and reverse-phase liquid chromatography, Breeding capable of producing nitrilase and can be transformed into the microbial strains of the production of β-producing amino acid, and was identified. Transforming the seed culture of the production of β-producing amino acid β-amino-propionitrile biological conversion process condition optimization study. Filter to 4 having the ability of the conversion of β-aminopropionitrile microbial strain from soil samples. On which a strain a39 physiological and biochemical detection and ATB system (ID 32GN) identified, results showed that the bacteria: Stenotrophomonas oligotrophic Aeromonas (Stenotrophomonas maltophilia). Strain a39 mutagenesis by low energy ion implantation, the catalytic hydrolysis of β-aminopropionitrile specific activity increased by 51.6%. Established a method of rapid detection of β-amino propionic acid content in the fermentation liquor: TLC scanning and reverse-phase high performance liquid chromatography method. RP-HPLC method as derivatization reagent 2,4 - dinitrofluorobenzene (DNFB), detection wavelength of 360nm, the sample average recovery was 99.3%, the linear range: 2μg/mL ~ 1000μg/mL The minimum detection limit for 0.02ng. The response surface method for the optimization of the fermentation medium components, test results show that the best source of carbon and optimal nitrogen source were sucrose and yeast extract. When sucrose, yeast extract, β-aminopropionitrile concentrations were 1.29%, 0.71% and 0.52%, compared with the enzyme activity of reach 22.51U / g. The response surface method maltophilia widowed raising Aeromonas (Stenotrophomonas maltophilia) free cell transformation conditions were optimized, when the conversion time, substrate concentration and cell volume were 8h, 2.5% and 0.86g, 10mL conversion solution β-amino propionic acid production was 102.81mg. Preliminary study results showed that the enzyme characteristics as a biological catalyst resting cells: the enzyme reaction temperature is 35 ° C, the optimum pH is 8.4. Immobilized cells into β-amino-propionitrile, test results show that: the optimum temperature of the enzyme reaction of the cells immobilized calcium alginate and substrate concentrations were 40 ° C and 1.5% of immobilized cells of at least continuous catalytic 10 Batch or more.

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