Study On Biocatalytic Preparation Of Aldehydes Flavor Compounds

This dissertation mainly studied biocatalytic preparation of aldehydes flavor compounds.In the second part of this dissertation, a simple, rapid and safe method for determining of the content of organic acids based on the ferric hydroxamate spectrophotometry was established. The presented method could be preliminary used for the rapid determination of fatty acids in the third part of this dissertation and lay the foundation for accumulating for aldehydes in high yield. A hydroxamic acid from carboxylic acid can be formed in the presence of hydroxylamine hydrochloride and DCC. And the formation of purple ferric hydroxamate could be spectrophotometrically assayed from hydroxamic acid in the presence of ferric trichloride. Strategies for the determination of the content of organic acids were established based on this spectrophotometry mechanism of ferric hydroxamate. In order to obtain the optimum conditions for this color-generating reaction, the concentrations of hydroxylamine hydrochloride, DCC, ferric trichloride, incubation time, water bath temperature, water bath time and the interference factors were investigated. The optimum hydroxylamine hydrochloride concentration, DCC concentration, ferric trichloride concentration, incubation time, water bath temperature and water bath time were 4 m M, 0.2 M, 0.5 M, 10 min, 60 ? and 20 min, respectively. The accuracy of the method established was validated by the bioconversion of 1-cyano-cyclohexyl acetonitrile hydrolysis with the resting cells of Escherichia coli. The RSD and the recovery rate for the ferric hydroxamate spectrophotometry determination were 0.6892 ? ~ 1.8250 ? and 99 ? ~ 105 ?,respectively. Km and Vmax were explored by using ferric hydroxamate spectrophotometry and HPLC determination which were 9.7968 m M and 9.8219 m M,0.5772 m M/min and 0.5784 m M/min, respectively. Results indicate that the accuracy of this method for assaying the content of organic acids is as high as that of the HPLC method. The research showed that owing to its simple, fast and accurate properties.In the third part of this dissertation, the long chain fatty alcohol oxidase gene FAO1 from Candida sp. was inserted into the p ET-21 a plasmid and the recombinant strain Rosetta BL21(DE3) was achieved. By extraction of plasmid digestion, sequence analysis and agarose gel electrophoresis, as well as SDS-polyacrylamidegelelectrophoresis, the FAO1 gene of the recombinant strain was proved to be correctly expressed. The optimum conditions for the cell disruption were 20 g/L dry cell weight and 10 min for ultrasound, respectively. In addition, the optimal conditions for the determination of alcohol oxidase activity were 0.2 mg protein and300 ?g HRP, respectively. The biotransformation processes using growth cells was carried out, the optimum fermentation and biotransformation conditions were 3 g/L lactose, induction time 2 h, 5 ? inoculum, p H 7.0, fermentation temperature 30 ?, 4? DMSO content, reaction time 8 h and 30 g/L dry cell, respectively, The appropriate ratio for the biphasic system reaction system was 1:4(cyclohexane:water) and the appropriate reducing agent was Vitamin C. The highest yield of 99 % for n-octanoic acid and n-octaldehyde were obtained respectively under optimal conditions.Furthermore, Hexanal, capraldehyde and dodecanal were accumulated by studying substrate spectrum.In the fourth part of this dissertation, high-producing strains that can produce vanillin from ferulic acid were screened by enrichment and habituated culture. Two bacterial strains, E4-1 and E6-1, were selected out by this way. E4-1 and E6-1 were respectively identified as Bacillus altitudinis and Bloodthirsty bacillus by common methods such as morphological characteristics, physiological and biochemical identification, 16 S r DNA agarose gel electrophoresis, etc. Strain E4-1 was chosen as the best one and the fermentation technology of biocatalytic preparation of vanillin from ferulic acid was studied. The optimal nutrient media components and concentration, as well as the other best fermentation conditions were investigated. The results showed that the optimal cultural medium consisted of 10 g/L sucrose, 15 g/L yeast extract, 50 m L loading volume, initial p H value 7.0 and incubation temperature30 ?. After a series of exploration, for the first time a bacterial strain that can accumulate relatively high yield of vanillin was achieved by biocatalytic method.