Breeding Of Aminoacylase Producer And Its Conditions Optimization In Fermentation

Aspergillus oryzae L-09, an aminoacylase-highproducting strain was used in this study. This strain had subjected mutagensis of UV and NTG, and was isolated using the paper chromatography methodology. The enzyme activity was enhanced 60% higher than the original strain by the mutagenesis of UV, and was further enhanced 30.5% by NTG. As a result, high enzyme activity of 539.7 U/g was obtained after 2 days culture at 30℃.To determine the effect of the fermentation conditions and medium ingredinents on enzyme production, flask solid-state fermentation experiments were carried out. It was found that both of them affected the enzymes formation in various degrees. The optimal medium compositionand fermentation conditions on aminoacylase production were determined as: compounding ratio of wheat bran bean flour 8.0: 2.0, maltose 5g/L, tween-80 5g/L, phytic acid 0.5g/L, pH 7.0, compounding ratio of solid state and water 1:1. As a result, high enzyme activity of 1254.4 U/g was obtained after 2 days culture at 30℃.The extracting conditions of the fermented medium were studied. The results showed that after extracting in10 times of pH 7.0, 0.1mol/L phosphate buffer at 40℃for 2 h, the activity of enzyme was highest.In the process of aminoacylase purification, saltout with 30% and 70% of Ammonium sulfat were used repectively. Throught the salting out by dialysis and concentrate, the enzymes were obtained.It was found that temperature, pH, and metal ions affected the transamination reaction. Conditions of the reaction were chosen as followed: temperature 55℃, pH 7.0, substrate concentration 200mmol/L. The metal ions such as Co²⁺could accelerate the reaction; Ac- and high substrate concentration had some degree of inhibitive effects. For the dissociated aminoacyase the Michaelis-Menten constant at 37℃and 55℃were K_m=30.49 mmol/L, Vmax=3.94μmol/min; K_m=33.3mmol/L, V_max=7.93μmol/min, respectively.