Han S J, Yoo Y J, Kang H S
Department of Microbiology, College of Natural Sciences, Seoul National University, Korea.
J Biol Chem. 1995 Oct 27;270(43):26012-9. doi: 10.1074/jbc.270.43.26012.
Bacillus sp. D04 secreted a bifunctional cellulase that had a molecular weight of 35,000. This cellulase degraded Cm-cellulose, cellotetraose, cellopentaose, p-nitrophenyl-beta-D-cellobioside, and avicel PH101. Based on the high performance liquid chromatography analysis of the degradation products, this cellulase randomly cleaved internal beta-1, 4-glycosidic bonds in cellotetraose and cellopentaose as an endoglucanase. It also hydrolyzed the aglycosidic bond in p-nitrophenyl-beta-D-cellobioside and cleaved avicel to cellobiose as an exoglucanase. Cellobiose competitively inhibited the p-nitrophenyl-beta-D-cellobioside degrading activity but not Cm-cellulose degrading activity. Ten mM p-chloromercuribenzoate inhibited p-nitrophenyl-beta-D-cellobioside degrading activity completely, but Cm-cellulose degrading activity incompletely. Cm-cellulose increased p-nitrophenyl-beta-D-cellobioside degrading activity, and vice versa, whereas methylumbelliferyl-beta-D-cellobiose strongly inhibited p-nitrophenyl-beta-D-cellobioside degrading activity. The cellulase gene (cel gene), 1461 base pairs, of Bacillus sp. D04 was cloned. The nucleotide sequence of the cel gene was highly homologous to those of Bacillus subtilis DLG and B. subtilis BSE616. The cel gene was overexpressed in Escherichia coli, and its product was purified. The substrate specificity and substrate competition pattern of the purified recombinant cellulase were the same as those of the purified cellulase from Bacillus sp. D04. These results suggest that a single polypeptide cellulase had both endo- and exoglucanase activities and each activity exists in a separate site.
芽孢杆菌属D04分泌一种分子量为35000的双功能纤维素酶。这种纤维素酶能降解羧甲基纤维素、纤维四糖、纤维五糖、对硝基苯基-β-D-纤维二糖和微晶纤维素PH101。基于对降解产物的高效液相色谱分析，这种纤维素酶作为内切葡聚糖酶可随机切割纤维四糖和纤维五糖内部的β-1,4-糖苷键。它还能水解对硝基苯基-β-D-纤维二糖中的糖苷键，并作为外切葡聚糖酶将微晶纤维素切割成纤维二糖。纤维二糖竞争性抑制对硝基苯基-β-D-纤维二糖的降解活性，但不抑制羧甲基纤维素的降解活性。10 mM对氯汞苯甲酸完全抑制对硝基苯基-β-D-纤维二糖的降解活性，但不完全抑制羧甲基纤维素的降解活性。羧甲基纤维素增加对硝基苯基-β-D-纤维二糖的降解活性，反之亦然，而甲基伞形酮基-β-D-纤维二糖强烈抑制对硝基苯基-β-D-纤维二糖的降解活性。克隆了芽孢杆菌属D04的纤维素酶基因（cel基因），其长度为1461个碱基对。cel基因的核苷酸序列与枯草芽孢杆菌DLG和枯草芽孢杆菌BSE616的高度同源。cel基因在大肠杆菌中过表达，并对其产物进行了纯化。纯化的重组纤维素酶的底物特异性和底物竞争模式与从芽孢杆菌属D04纯化的纤维素酶相同。这些结果表明，单一多肽纤维素酶具有内切和外切葡聚糖酶活性，且每种活性存在于一个独立的位点。
