Identification Of B17 Strain And Preliminary Study On Its Cellulase

The extent utilization of cellulose is not very large.How to improve the utilization of cellulose has become an important direction of our research,and finding microorganism who can effective degrade cellulose is an effective way to obtain efficient cellulolytic enzymes.The aim of this study was to identify the species of white and filamentous fungi,to study their growth characteristics and the cellulase.The main results were as follows:1.The morphological and ITS sequence of the fungus B17 strain are identificated and homologous in Genebank.The results of the morphological showed that was vigorous on the solid medium,the mycelium of the B17 strain was thick and dense as well as has strong ability of climbing wall.The microscopic observation of Hyphae and spores showed the branches with more diaphragm,the end of the spatula with sporulation and slightly curved;the tip of the spatula to produce conidia colorless,spherical to the egg shape.The result of ITS sequencing showed that B17 strain was identical to Trichoderma koningiopsis,the similarity rate was 100%.Morphological identification combined with molecular biology identification,the B17 strain is Trichoderma koningiopsis in Trichoderma.2.The optimal growth conditions of B17 strain were determined by L9(34)orthogonal test,the results as follows: the culture temperature was 25 ?,the carbon source was starch,the nitrogen source was peptone,and the initial p H value was 5.0,when B17 strain of speed of growth was the fastest.In optimized liqid medium,the B17 strain incubated at 25 ? for 3 d,the activity of cellulase was 62.95 ± 1.13 U/m L,the activity of amylase was 95.37 ± 1.52 U/m L,and the activity of xylanase was 14.53 ± 0.14 U/m L,the activity of pectinase was 6.36 ± 0.26U/m L,the activity of chitinase was 39.15 ± 1.23 U/m L,the activity of protease was 383.81 ±6.19 U/m L,the activity of laccase was 2.60 ± 0.03 U/m L,and the enzyme activity was 0.13 ±0.002 U/m L.The mycelial protein content was about 46.30%.3.The optimum fermentation conditions by response surface methodology were determined the result as follows: fermentation temperature was 25 ?,inoculation amount was 2.0%,rotation speed was 200 r/min,fermentation initial p H valve was 4.8 culture,Fermentation time was 3 d.The enzyme activity of cellulase was 290.32 U/m L,what was 1.75 times higher than before optimization.4.Cellulase in the fermentation broth of strain B17 is purified by ammonium sulfate precipitation method,DEAE-Sepharose Fast Flow anion exchange chromatography and Sephadex G-75 Gel Column Chromatography technology.And the enzymatic properties of the purify cellulase is studied.The result showed that the cellulase was purified by 9.57 times,the total enzyme mass was 12.21 ± 0.45 mg/m L,the enzyme activity was 49.08 ± 0.90 U/m L,the specific activity was 4.02 U/mg,the recovery was 16.91%,and the relative molecular weight was about 66.2 k Da.The optimal temperature for enzymatic hydrolysis of the cellulase was60 ?,the optimum p H valve was 5.0,which has good thermal stability and p H stability.The Km value of cellulase was 3.80 mg/m L and Vmax was 3.09 mg/(min·m L),which indicated that the affinity of cellulase and sodium carboxymethyl cellulose was high,and that cellulase degraded cellulose ability was strong and high.