Study On The Production Of L-serine By Biotransformation

L-serine production by biotransformation method, includes isolation and identification of a bacterium, breeding the bacterium, optimization of medium composition, fermentation and transformation conditions, was studied in this paper.The serine hydroxymethyltransferase(SHMT) was produced by a newly isolated methylotrophic bacterium, strain ML206, which was isolated from methanol polluted soil, the SHMT activity attained to 0.0302 U/mL. Based on the analysis of morphology, biochemical and physiological characteristics, strain ML206 was identified as Methylobacterium aminovorans combined with the blasted in the GenBank of its 16S rDNA gene sequence.A L-serine producing methylotroph mutant ML216 (Methanolr, Glyr, SGr) was derived from the wild bacterium by combination treatment with diethylsulfate and N-methyl-N'-nitro -N-nitrosoguanidine. The screenings were methanol, glycine and sulfaguanidine(SG) which could increase the level of L-serine production. The SHMT activity which attained to 0.0467 U/mL from 0.0302 U/mL was increased by 54.6 %.P1ackett-Burman design was undertaken to evaluate the effects of the sixteen factors. The response surface methodology was used to optimize the above critical internal factors, to find out the optimization concentration levels and the relationships between these factors. By solving the quadratic regression model equation using appropriate statistic methods,the optimal concentration of the variables were determined as: (NH4)2HPO4 8.6g/L, FeSO4·7H2O 38 mg/L, methanol 2.2 %, rotate speed 205 r/min. The activity of SHMT was increased from 0.0467 U/mL to 0.0562 U/mL, was increased by 20.3 %. Through changed the mode of adding methanol to fed-batch fermentation, the fermentation time was reduced from 72 h to 50 h. Finally, the SHMT activity was increased by 31.0 %. The fermentation time was reduced by 30.6 %, the biomass was increased by 133.3 %.The method of detecting L-serine concentration with paper chromatography was established. The new developing agent was n-butanol: acetone: water: ammonium hydroxide =100:40:38:2. Compared to the Amino Acid Analyzer, the standard deviation was between 0.015 and 0.028, the biggest coefficient of variation was 1.65%, the rate of correlation was between 99.6% and 100.4%.The biotransformation conditions of SHMT were optimized. The optimal pH and temperature of SHMT were 8.5 and 30℃. The optimal concentration of methanol and glycine were 2.5 % and 8 % separately. When 25μmol/L pyridoxal was added, the production attained to 7.46 g/L, increased by 12.3 %. The time of transformation was reduced by 58.3 %. When four times transformed continuously with certain biomass, the production was less changed.