土壤中纤维素降解真菌的筛选及其纤维素酶活性的研究

Isolation of cellulose-decomposing fungi from the soil and analyzing its cellulase activity

通过对小麦/玉米秸秆还田土壤样品进行富集培养,利用刚果红纤维素培养基筛选得到产纤维素酶的真菌XJ-25,并对该菌株进行形态学观察和18S-ITS-5.8S区序列系统发育分析,初步鉴定为格孢腔菌属（Phaeosphaeria）,定名为Phaeosphaeria sp.XJ-25。在研究液体发酵培养基中碳源、氮源、培养时间、起始pH、培养温度、接种量以及通气量对菌株XJ-25的羧甲基纤维素酶（CMC）的酶活及滤纸酶（FPA）的酶活影响的基础上,利用响应面法对发酵条件进行优化。结果显示,CMC酶活达到最大的条件为玉米秸秆粉1.34 g·L^-1（麸皮0.16 g·L^-1）、尿素10.06 g·L^-1、培养基初始pH 3.25、接种量4.13%,CMC酶活达到23.871 U·mL^-1。FPA酶活最大的发酵最佳条件为玉米秸秆粉1.34 g·L^-1（麸皮0.16 g·L^-1）、尿素10.12 g·L^-1、培养基初始pH 3.41、接种量4.22%,FPA酶活为8.653 U·mL^-1。粗酶液的最适反应温度为50℃,酶液的热稳定较差,当温度超过40℃,该酶活力显著下降。最适反应pH为5,在pH 4-6的范围内酶活性较稳定。

A highly active cellulose-decomposing strain XJ-25 was isolated from the soil in the wheat and maize straw returned fields using enrichment culture and Congo red staining test. The isolated strain was identified as Phaeosphaeria sp. XJ-25 by its morphological characteristics as well as by phylogenetic relationships based on 18S-ITS-5.8S ribosomal gene sequences. Conditions for liquid fermentation, a process that produces cellulase were optimized by the response surface methodology（RSM）. The results showed that the conditions for CMCase activity reaching the maximum were： the corn stalk powder, 1.34 g·L^-1（bran was 0.16 g·L^-1）; urea, 10.06 g·L^-1; initial pH of culture medium, 3.25; inoculation quantity, 4.13%; and CMCase activity reached 23.871 U·mL^-1. The conditions of FPase activity reaching the maximum were： the corn stalk powder, 1.34 g·L^-1（bran was of 0.16 g·L^-1）; urea, 10.12 g·L^-1; initial pH of culture medium, 3.41; inoculation quantity, was 4.22%; and FPase activity reached 8.653 U·mL^-1. The optimal reaction temperature of the crude enzyme solution was 50℃. When the temperature was over 40℃, the thermal stability of enzyme solution was poor, and the enzyme activity significantly decreased. The optimum reaction pH was 5. In the range of pH 4-6, the enzyme activity was more stable.