Screening And Fermentation Optimization Of Branched-Chain Amino Acid Aminotransferase-Producing Strain

Branched-chain amino acid could be specially utilized by branched-chain amino acid aminotransferase. The enzyme was widely used in the field of biosynthesis ofα-keto acid, separate of chiral amino acid enantiomer and biosynthesis of amino acid. This paper mainly studied on the screening of the enzyme-producing strain, optimization of fernentation and transamination conditions, kinetics parameters of the strain, which laid a foundation for the industry application of the branched-chain amino acid aminotransferase.A strain named as WJ44 with capability of producing branched-chain amino acid aminotransferase was isolated from the soil. The enzyme activity of strain WJ44 was especially high when L-leucine was used as a substrate. According to the morphology observation, 16S rRNA sequence and phylogenetic tree, physiological and biochemical characteristics, the strain was identified as Bacillus cereus, named as Bacillus cereus WJ44.With the method of single factor test and response surface methodology, the optimized constitutes of the culture were: 20.99 g/L glucose, 16.85 g/L tryptone, 5 g/L beef extract, 18.24 g/L corn steep liquor, 3 g/L KH2PO4, and 0.5 g/L MgSO4·7H2O. The optimum culture conditions were: original pH value 7.0, cultural temperature 37℃, liquid volume 20 mL/250 mL, shaker's rotating speed 200 r/min, inoculation volume 3%. The highest enzyme activity of 45.79 U/mL was achieved after 10 h under the optimal condition, and the dry weight of cell was 8.64 g/L, increased by 40% and 10% respectively. This result showed that WJ44 was a branched-chain amino acid aminotransferase-producing strain with fine capability.The optimum conditions of transamination were obtained by single factor test and orthogonal experiment: L-leucine15 g/L, pyruvic acid 12 g/L, the proportions of zymotic fluid and substrate fluid 1:1(V:V), surface active agent hexadecyl trimethyl ammonium bromide 3 g/L, coenzyme 5'-pyridoxal phosphate 0.04 mmol/L, the initial pH of the substrate and reaction temperature were 9.0 and 40℃respectively. The metal ions could suppress the transamination, especially Cu2+. With the optimal transamination condition, the concentrations ofα-ketoisocaproate could achieved at 5.63 g/L after 8 h.The kinetics characteristics of Bacillus cereus WJ44 in the continuous culture were studied. The specific growth rate and the concentrations of the restricted substrate were consonant with the Monod equation when the glucose was used as a restricted substrate. The maxmium specific growth rateμmax was 0.438 h-1, and the saturation constant Ks was 0.510 g·L-1. The maxmium productivity of the biomass (DX)m was 0.909 g·(L·h)-1. The best theory dilution rate Dm was 0.388 h-1. The yield of the substrate Y x/s and the maintenance coefficient m were 0.159 g·g-1 and 0.0249 g·(g·h)-1 respectively.