Fungi Cellulose-degrading Enzyme System Of Alpine Grassland And Its Degradation Characteristics On Rape Straw

Rape straw is usually used as fuel and becomes waste and pollution sources in northern China especially in the alpine region. Methods by using fungi system of multi-enzyme to degradation rape straw cell wall material were carried out by traditional microbiological methods. Fungal strains, produce high enzyme activity, was screened, including cellulose, xylanase and laccase. Gene sequences of rDNA-ITS, cultural characteristics, enzymatic properties of liquid crude enzyme, and the synergies between different enzyme was analyzed as well. Construction of fungi system of multi-enzymes for degradation of rape straw cell wall material and its degradation characteristics was studied. The results obtained as follows:(1) Based on preliminary work of isolating125fungal strains from the east Qilian Mountain alpine grassland soil.Fifty six fungal strains were screened by using traditional chromogenic substrate screening method, and forty nine fungal strains can grow on filter paper plate medium,thirty four fungal strains that could produce high enzyme activity of both cellulose and xylanase were screened by enzyme activity screening method in flask liquid fermentation while rape straw used as sole carbon source. Four fungal strains with high yield of cellulose and xylan enzyme activity, Embellisia sp.(strain26B), Saccharicola sp.(strains24d), Athelia sp.(strain23a), Thielavia sp.(strain H) were obtaied. One fungal strain, code310b, producing laccase was obtained, and identified as Marasmius sp., Based on rDNA-ITS gene sequence analysis.(2) To Marasmius sp., the optimum temperature of laccase activity producing is25℃with pH value4.0; Add sugar source will inhibit laccase activity of the strains; peptone induced the highest laccase activity among the nine nitrogen sources tested; O-toluidine, a-naphthol and Tween-80can promote the strains laccase activity while catechol, tannic acid, indole acetic acid inhibit the strains laccase activity, and indole acetic acid inhibit it strongest among the test acid; Guaiacol had no siginificant effect on laccase activity. Laccase activity increased with the content of Cu2+increasing, and it reached up to maximum when Cu2+was added to0.025g/L, Laccase activity arosen with the increase of rotar speed.(3) Culture characteristics of five tested strains showed that the optimum growth temperature range between25-35℃. It was conducive that the five strains can coexist under the culture conditions of25℃. The type of enzyme and enzyme activity of five fungal strains were different determined in flask liquid fermentation while rape straw used as sole carbon sources. Strain26B,23a, H,24d can produce both cellulase and xylanase, Carboxymethyl cellulose activity (CMCase) of strain26B was the highest, xylanase activity of strain24d was the highest. Three kinds of enzyme activity was measured after crude enzyme solution of single strain was mixed by equal volume in order to further understand synergies between different type of enzyme of crude enzyme of five strains. Multi-enzyme activity decreased to a certain extent. And combination strains were inoculated and mixed culture in flask liquid fermentation was measured, As opposed to single strain fermentation, enzyme activity of mixed strain fermentation was decline, the reason was probably the environmental conditions of mixture reaction system change, and was related to changes of pH value and reducing sugar content in closely.(4) To strain26B, the optimum temperature of CMCase activity producing is55℃with pH value6.0, it is unstable on the temperature.to strain23a, the optimum temperature of CMCase activity producing is30℃with pH value5.0; and to strain H, the optimum temperature of CMCase activity producing is50℃with pH value5.5; Strains23a and H are relatively stable under the condition of temperature range between0-40℃. To strain24d, the optimum temperature of xylanase activity producing is55℃with pH value5.0, it is unstable on the temperature. to strain23a, the optimum temperature of xylanase activity producing is35℃with pH value5.5, to strain H, the optimum temperature of xylanase activity producing is55℃with pH value5.0, Strain24d is instability on the temperature, the xylanase of strain23a and H is relatively stable under the condition of temperature range between0～35℃. To strain310B, the optimum temperature of laccase activity producing is30℃with pH value4.5, the laccase activity reaches its maximum, and the enzyme activity is relative stability under the condition of temperature range between0～45℃(5) In the solid fermentation system which made up of strains310b,24d and26B, the CMCase, xylanase and laccase activity could detect in the crude enzyme of fermentation. A conclusion that there was high correlation between three kinds of enzymes activity in the number of combinations of the reaction system and the relative cellulose content, the same as relative reducing sugar content did were obtained by analysis the mutual relations of the relative content of reducing sugar and enzyme activity and the relative cellulose content in the different reaction system of combinations strain. It indicated that the decomposition effect of cell wall material of rape straw was better in multi-enzymes fungi system.