嗜热子囊菌光孢变种Thermoascus aurantiacus var. levisporus内切β-葡聚糖酶的分离纯化及特性研究

采用培养分离、室内测定等方法，对嗜热子囊菌光孢变种Thermoascus aurantiacus var. levisporus产生的内切β-葡聚糖酶进行了分离纯化及特性研究.粗酶液经硫酸铵沉淀、DEAE-Sepharose Fast Flow阴离子层析、Phenyl-Sepharose疏水层析等步骤获得了凝胶电泳均一的内切β-葡聚糖酶.结果表明，经12% SDS-PAGE测得酶的单亚基分子量约为31.5 kD，凝胶过滤层析测得酶的分子量约为34.5 kD.该酶反应的最适温度为55 ℃,最适pＨ为2.5～3.0该酶在pH3.0条件下60 ℃较为稳定；80 ℃保温30 min有20%原酶活性.金属离子对内切β-葡聚糖酶活性影响较大, 其中K⁺、 Ca²⁺、Mn²⁺对酶有激活作用；Al⁺、Cu²⁺ 、Al³⁺对酶有显著抑制作用.该酶对羧甲基纤维素具有很强的底物特异性.

Purification and Properties of Endocllulase from Thermoascus aurantiacus var.levisporus

Endoglucanase was purified from culture supernatant of Thermoascus aurantiacus var.levisporus to homogeneity, using ammonium sulfate fractionation, DEAE-Sepharose, Pheny1-Sepharos, and Sephacry1 S-100 fast-flow chromatography. The obtained endoglucanase ia a single subunit molecule about 31.5 kD or 34.5 kD as determined by 12% SDS-PAGE and gel filtration, respectively. The maximum endoglucanase activity was observed at pH 2.5～3.0 and 55 ℃. The　endoglucanase appeared to be thermostable at 60 ℃, and exposure in 80 ℃ for 30 min resulted in 80% of activity loss. Metal ions　like K⁺, Ca²⁺　and Mn²⁺ enhanced the endoglucanase activity, whereas Ag_⁺,Cu²⁺ and Al³⁺ inhibited the activity significantly. The　endoglucanase possessed a strong specificity to carboxymethyl cellulose.