大兴安岭森林土壤中纤维素降解真菌的分离及产酶条件优化

Isolation of cellulose-degrading fungi from soils in Da Hinggan forest and optimization of enzyme production conditions

为了获得高酶活性的常温纤维素酶产生菌,试验采用纯培养的方法以羧甲基纤维素钠(CMC-Na)为碳源在28℃下对内蒙古大兴安岭森林土壤中的纤维素降解真菌进行筛选,以细菌18S rDNA通用引物PCR扩增后进行序列同源性比对确定种属,以3,5-二硝基水杨酸比色法(DNS法)测定纤维素酶活性,并对酶活较高的菌株进行产酶条件优化。结果表明:共分离到2株酶活性较高的常温纤维素降解真菌W14和DAZJ3,菌株W14为草酸青霉(Penicillium oxalicum),菌株DAZJ3为Oidiodendron tenuissimum;2株菌的最佳产酶条件:初始pH值为6、以酵母膏为碳源、培养温度分别为35℃(菌株W14)和30℃(菌株DAZJ3)、培养时间为4 d,羧甲基纤维素(CMC)酶活性可分别达到103.89 U/mL和158.36 U/mL。说明菌株W14和DAZJ3是两株具有较强应用潜力的常温纤维素降解真菌。

In order to obtain normal temperature cellulase-producing fungi with some high enzyme activity, cellulose-degrading fungi from soil in the Da Hinggan forest of Inner Mongolia were screened at 28 ℃by pure culture method using CMC-Na as carbon source. Bacterial 18 S rDNA universal primers were used for PCR amplification, and then the species were identified by sequence homology comparison. The cellulase activity was determined by DNS method, and the enzyme-producing conditions of high enzyme activity cellulase-producing strain was further optimized. The results showed that two strains of cellulose-degrading fungi W14 T DAZJ3 with high enzyme activity were isolated, They were strain W14(Penicillium oxalicum) and strain DAZJ13(Oidiodendron tenuissimum). The optimum conditions for cellulase production of the two strains were as follows: initial pH was 6, yeast extract was used as carbon source, culture temperature was 35 ℃(strain W14) or 30 ℃(strain DAZJ3), and culture time was 4 d. Under the optimum conditions, the CMC enzyme activities of W14 and DAZJ13 were 103.89 U/mL and 158.36 U/mL respectively. It indicated that the strains of W14 and DAZJ13 were two normal temperature cellulose-degrading fungi with high application potential.