耐低温纤维素降解真菌的筛选、鉴定及酶活性研究

Screening and Identification of Cold-adapted Fungi with Cellulose Degradation and Its Enzymatic Properties

[目的]为筛选低温下高效降解纤维素的菌种资源,促进秸秆在低温条件的快速腐解。[方法]本研究利用低温稀释平板涂布法分离、刚果红定性、内切β-葡聚糖酶活力定量等分析方法进行低温纤维素降解真菌的筛选,依据菌株形态特征及rDNA-ITS序列分析鉴定菌种,探究其在不同温度下的产酶能力,并对粗酶提取物进行酶学性质的初步研究。[结果](1)筛选到一株低温下高效产纤维素酶的真菌ZY-50,鉴定为球芽异普可尼亚菌(Metapochoniabulbillosa),目前尚无该菌产纤维素酶的报道;(2)球芽异普可尼亚菌ZY-50可在低温条件下生长并具有较强产纤维素酶能力,其内切β-葡聚糖酶活力在28℃、16℃、10℃温度下分别为76.03 U mL^(-1)、128.34 U mL^(-1)和115.67 U mL^(-1),该菌在16℃下产外切β-葡聚糖酶和β-葡萄糖苷酶的能力较稳定,最高分别为2.31 U mL^(-1)、2.43 U mL^(-1),16℃下滤纸酶活力最高为7.89 U mL^(-1);(3)粗酶提取物的内切β-葡聚糖酶最适作用温度为60℃,在10~40℃温度下,在1.5 h内相对酶活为90%,最适pH为5.0,10 mmol L^(-1)Co^(2+)对其活性有显著的促进作用。[结论]球芽异普可尼亚菌ZY-50可在低温条件下生长且产纤维素酶活力较强,在促进秸秆低温腐熟方面具有较好的的应用潜力。

[Objective]The aim was to screen strain resources for efficient cellulose degradation and to promote rapid decomposition of straw at low temperature.[Method]Low-temperature cellulose degrading fungi were screened by using low-temperature dilution plate coating method,Congo red characterization and quantification of endo-β-glucanase activities.The strains were identified based on morphological characteristics and rDNA-ITS sequence analysis,their enzyme production capacities at different temperatures were investigated,and preliminary studies on the enzymatic properties of crude enzyme extracts were conducted.[Result]①A cold-adapted fungus,namely ZY-50,was obtained and identified as Metapochonia-bulbillosa.There are no reports of cellulase production by this fungus.②Metapochonia-bulbillosa ZY-50 could grow at low temperatures and has strong cellulase production ability.Its Endoβglucanase activities were 76.03 U mL^(-1),128.34 U mL^(-1),and 115.67 U mL^(-1) at 28℃,16℃and 10℃respectively,and the abilities of the fungus to produce Exoβ-glucanase andβ-Glucosidase were more stable at 16℃.The highest activities of them were 2.31 U mL^(-1) and 2.43 U mL^(-1).The synergistic effect of the three cellulases was stronger at 16℃,with the highest filter paper activity of 7.89 U mL^(-1).③The endo-β-glucanase of the crude enzyme extract had an optimum action temperature of 60℃.The relative enzyme activity was 90%within 1.5 h at temperatures between 10 and 40℃.The optimum pH was 5.0,and its activity was promoted by 10 mmol L^(-1) Co^(2+).[Conclusion]Metapochonia-bulbillosa ZY-50 can grow at low temperature and has a strong cellulase production ability,which has a high potential to promote straw decomposition at low temperature.