摘要
研究液体发酵嗜热毛壳菌(Chaetomium thermophilum)产生的一种外切葡聚糖纤维二糖水解酶的分离纯化及特性。粗酶液经硫酸铵沉淀、DEAE-Sepharose Fast Flow阴离子层析、Sephacryl S-100分子筛层析、Q Sepharose Fast Flow强阴离子层析等步骤后获得凝胶电泳均一的外切葡聚糖纤维二糖水解酶。经12.5%SDS-PAGE和凝胶过滤层析方法测得该酶的分子量大小约为66.3kDa和67.1kDa。该酶反应的最适温度和pH值分别为65℃和5.0。在60℃以下酶比较稳定,在70℃酶的半衰期为1h,在80℃下保温20min仍具有20%的活性,该酶的热稳定性较中温真菌的同类酶高,与国外报道的嗜热真菌的同类酶热稳定性接近。以pNPC为底物的Km值为0.956mmol/L。
Chaetomium thermophilum CT2 was a cellulolytic fungus. It was a widely-existing saprophyte, which growed rapidly in soil. The ceUulases synthesized by C. thermophilum CT2 was overall, consisting of three principal types of enzymes. The cellobiohydrolase was one of these three cellulases, which was associated with the endo-β-1, 4-glucanase and β-glucosidase activities. C. thermophilum CT2 produced cellobiohydrolase availably at 50℃, when grown on ferment liquid substrate, containing 1% Avicel, 0.14% （NH4）2SO4, 0.2% KH2PO4, 0.03 % CaCl2· 2H2O, 0.03 % MnSO4·7H2O, 0.1% peptone, 0.05 % yeast extract, 0.1% Tween 80 and trace element solution at 1 mL/L, containing 18mmol/L FeSO4·7H2O, 6.6mmol/L MnSO4, 4.8mmol/L ZnSO·7H2O and 15 mmol/L COCl2. A cellobiohydrolase was purified to homogeneity by an inexpensive and straightforward method for extraction of the enzyme involving fractional ammonium sulphate precipitation, ion-exchange chromatography on DEAE-Sepharose Fast Flow, gel filtration on Sephacryl S-100 and ion-exchange chromatography on Q Sepharose Fast Flow. The molecular weight of the enzyme was estimated to be 66.3kDa by 12.5 % SDS-PAGE and was to be 67. lkDa by gel filtration on Sephacryl S-100 respectively. Kinetic studies of the prified cellobiohydrolase of C. thermophilum CT2 showed that the Km for p-NPC （p-trophenylβ-dcellobioside） was 0.956mmol/L as determined from a Lineweaver-Bark plot. Optimum enzyme activity was at 65℃ and pH5.0. It was thermostable at 60℃ and remained 20% activity after 20min at 80℃. The half life time of the enzyme at 70℃ was lh. It indicated that the cellobiohydrolase possessed of excellent acid stability and thermostable property. The properties of the cellobiohydrolase make it possible to be good material in scientific researches of protein thermostable mechanism and good model for designing and constructing a new type protein in industry. The enzyme may also provide instructive insight on the diversity and mechanism of cellulose degradation by C. thermophilum CT2. As a thermophilic fungus C. thermophilum CT2 is an attractive potential source of cellulases. It indicates that C. thermophilum CT2 may be a new excellent industrialized fungus for producing cellulases through molecule biology means.
出处
《微生物学报》
CAS
CSCD
北大核心
2006年第1期143-146,共4页
Acta Microbiologica Sinica
基金
国家自然科学基金(30270013
30170013)
国家"863计划"(2003AA241162)~~
作者简介
李亚玲（1978-），女，山东招远人，博士研究生，主要从事嗜热酶酶学研究。E-mail：yalingli@163.com
通讯作者。Tel：86-538-8249071；E-mail：lidc20@sdau．edu．cn
