Screening,enzyme-producing Conditions Optimized And Enzymatic Characteristics Of Cellulose-producing Strain With High Cellulase Activity

Isolation and identification of strains hydrolyzing crystalline cellulose and enzyme Collceted Isolation and identification of the soil samples from turpan of Xinjiang province,and isolated 11 strains from the samples.According to multiparous enzyme function, The morphological observation,chemotaxonomic assays and 16 Sr RNA sequence analysis showed that strain C-8 was identified as Bacillus sp. The celluloses produced by C-8 could hydrolyze cellulose, the optimum p H was 6.0 and temperature was 40℃; And it have good thermal stability and p H stability.In the different metal ions after the treatment, Fe3+ activate.Fermentation optimization of cellulase production from the C-8 strain In order to improve the ability of Cellulose Producing Strain C-8, the fermentation conditions were optimized using response surface method. Screening out the effects of the main effect of enzyme production condition factors using Plackett-Burman design, the steepest ascent experiment approximation maximum response area,optimization of fermentation conditions by response surface Box-Behnken. The results show that : the initial p H, CMC-Na% and culture temperature was the main influencing factor, the response of three factors and three levels of analysis to obtain the optimal conditions for the initiation of p H 8.0, CMC-Na was 2.5%, the culture temperature was 28 ℃, under the condition of cellulose activity can reach to193.89U/ml, compared with before optimization, the enzyme activity increased 2.35 times.Screen high cellulase-producing strains by ultraviolet ray(UV) and nitrosoguanidine(NTG)Screen 20 cellulase-producing strains by ultraviolet ray(UV)and nitrosoguanidine(NTG).The optimal mutation conditions were sequential treatment with 0.6mg/m L NTG for 10 minutes and then illumination with 25 W UV lamp for 20 seconds at a distance of 20 cm. The cellulase activity of this high-yield strain reached 266.261U/ ml, which was 2.32 times of that of initial strain.The activity of the enzyme remained basically unchanged, the stability is better. Enzymatic properties analysis shows:the strains produced cellulase optimum temperature of 50℃, the optimal reaction p H was 6.0, the comparative stability of p H value, but the temperature stability is poor.Optimization of fermentation conditions of strain Three factors quadratic orthogonal rotation design was used to investigated the factors influencing the yields of cellulase production from UV-NTG-10 strain. The results show that:maltose was 30g/L,Yeast extract was 15g/L,Twain-80 was5g/L.Under this optimum conditions obtained by fermentation, cellulase enzyme activityreached 453.20U/m L.Purification and characterization of cellulase Using ammonium sulfate precipitation,dialysis and Sephadex G-50 gel column chromatography of concentrated fermentation crude enzyme solution of endoglucanase ofseparation and purification,the purified cellulase activity increased by14.65 times,the recovery of exo-1,4-β-D-glucanase or cellobiohydrolase was 23%, SDS-PAGE was a clear bandwas about 29 KD.The optimal reaction temperature of 50 degrees Celsius,the most suitable for p H7.0.Kinetic parameters of Km 2.606 umol/L and Vmax 0.606mmol/L·min. Histidine residue and tryptophan residue are the necessary groups of the enzyme. Arginine residues, amino and sulfydryl may be associated with enzymatic activity, but not the required groups.