With response surface analysis,the predicted maximum enzyme activity was 549.65 U/ml at the condition of 19.8 g/L maltose concentration,18.2 h culture time and 0.2 ml inoculate. The demonstration test showed that the enzyme activity were 529 and 544 U/ml,which means this model could predict precisely.
Estimated optimal conditions of the three factors were as the follows: soybean hydrolysate 24.0 g·L-1,glycerol 24.4 g·L-1 and ammonium sulfate 5.40 g·L-1.The enzyme activity was increased by 16.4%,from 6.27 μ·mL-1 to 7.30 μ·mL-1 using the optimal flask-shaking batch fermentation medium.
D-hydantoinase was separated and purified from Burkholderic cepecia 1003 following the steps of the crude separation(the cellular fragmentation 、the precipitation of (NH_4)_2SO_4), the purification of chromatography(Phenyl CL-4B HIC、DEAE Sepharose Fast Flow negative IEX) in this experiment. The purification multiple was 25.11 and the recycle yield of enzymatic activity was 11.87%.
本实验将Burkholderic cepecia 1003 菌株发酵产菌经过粗分离(细胞破碎、(NH_4)_2SO_4沉淀)、层析纯化(Phenyl CL-4B疏水层析、DEAE Sepharose Fast Flow阴离子交换层析)等步骤后得到纯化好的D-海因酶,其纯化倍数为25.11,酶活回收率为11.87%。
hydrophobic chromatography and Sephadex G-25 Fine. The final specific activity of LDH could beup to 1025.1 U/mg and purity was 88%. 28.7-fold purification was obtained with 67.9%enzymatic activity recovery.
For culturing of phytase,bran were used glucose(0.02g),(NH 4) 2SO 4(0.01g),MgCl 2(0.002g)and distilled water(40ml) were added. Dymamic analysis during fermentation indicated that the enzymatic activity could reach 5616u/g. ssc after the bacterial strain had been incubated at 29℃ for 8～10 days under the circustances.
The optimum activity of ADH is observed at the temperature25℃ and at pH7.5. The range of temperature stability is below 35℃, the range of pH stability is from 6.5 to 7.5 .The activity of enzyme is activated by Zn~(2+) dramatically and inhibited by Mg~(2+) slightly.
Compared with the former one, the new medium not only decreased effectively the remaining bean powder and the foaming formation during fermentation, but also increased the activity of enzyme by 10% higher than before.