产纤维素酶细菌的分离、鉴定与酶学性质研究

Isolation and identification of cellulase-producing strains and characterization of their cellulases

目的筛选并鉴定纤维素酶高产菌株,初步研究其所产纤维素酶的酶学性质,为纤维素资源的综合利用提供菌种资源。方法通过刚果红染色和酶活测定,从广州凉茶中药渣堆肥和黑龙江玉米地土壤中筛选出纤维素酶高产菌株,进行基于16S rDNA分析的分子鉴定,同时采用单因素试验方法确定菌株最佳培养时间,判断温度、pH值、金属离子、有机溶剂和盐浓度对所产纤维素酶活性及稳定性的影响。结果筛选获得3株纤维素酶高产菌株,其中Z-1鉴定为甲基营养型芽孢杆菌(Bacillus methylotrophicus),Z-2和H-3鉴定为枯草芽孢杆菌(B.subtilis),其所产CMCase酶学活性分别为977.92 U/mL、1 089.79 U/mL和1 538.44 U/mL。所产纤维素酶最适反应温度为50℃,最适pH值范围为pH4～5,该纤维素酶可被Ag^+、Co^(2+)和Mg^(2+)离子激活,也可被一定浓度的Tris-100和甲醇激活。经浓度为1.5 mol/L的NaCl处理4 h后,其酶活力仍剩余56.34%以上。结论 B.methylotrophicus Z-1,B.subtilis Z-2以及B.subtilis H-3为纤维素酶高产菌株,其所产纤维素酶耐热、耐酸、耐盐,并可被部分金属离子和有机物质所激活,具有潜在的开发价值。

Objective To provide strain resources for the comprehensive utilization of cellulose resources by screening of cellulase-producing strains and studying of the enzymatic properties of cellulase. Methods CMC-Congo red staining and enzyme activity assay were used to screen the cellulase-producing strains from compost of traditional Chinese herbal tea residue in Guangzhou and soil of cornfields in Heilongjiang province. 16S rDNA sequence analysis was used for gene identification of the strains. Single factor tests were used to determine influence of temperature,pH,metal ions,organic solvents and salt concentration on the cellulase activity and stability. Results 3 strains of Bacillus Z-1,Z-2 and H-3 were screened out,and their cellulase activities were 977.92 U/mL,1 089.79 U/mL,and 1 538.44 U/mL,respectively. The optimal reaction temperature was 50 ℃,and the optimal pH was pH 4.0~5.0. The cellulases were activated by Ag ^+,Co^ 2+,Mg^ 2+,Tris-100,and methanol in a certain concentration range. The relative cellulase activity still reached to 56.34% under 1.5 mol/L sodium chloride solution treatment for 4 h. Conclusion The cellulases produced by B. methylotrophicus Z-1, B. subtilis Z-2,and B. subtilis H-3 are resistant to heat,acid and salt,and can be activated by some metal ions and organic substances,which exhibit the potential development value.