PURIFICATION AND CHARACTERIZATION OF A CELLULOSE-BINDING BETA-GLUCOSIDASE FROM CELLULOSE-DEGRADING CULTURES OF PHANEROCHAETE-CHRYSOSPORIUM

被引:92
作者
LYMAR, ES [1 ]
LI, B [1 ]
RENGANATHAN, V [1 ]
机构
[1] OREGON GRAD INST SCI & TECHNOL,DEPT CHEM BIOCHEM & MOLEC BIOL,PORTLAND,OR 97291
关键词
D O I
10.1128/AEM.61.8.2976-2980.1995
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Extracellular beta-glucosidase from cellulose-degrading cultures of Phanerochaete chrysosporium was purified by DEAE Sephadex chromatography, by Sephacryl S-200 chromatography, and by fast protein liquid chromatography (FPLC) using a Mono Q anion-exchange column, Sodium dodecyl sulfate-polyacrylamide gel electrophoretic (SDS-PAGE) analysis of FPLC-purified beta-glucosidase indicated the presence of three enzyme forms with molecular weights of 96,000, 98,000, and 114,000. On further fractionation with a microcrystalline cellulose column, the 114,000-molecular-weight beta-glucosidase, which had 82% of the beta-glucosidase activity, was bound to cellulose, The beta-glucosidases with molecular weights of 96,000 and 98,000 did not bind to cellulose. The cellulose-bound beta-glucosidase was eluted completely from the cellulose matrix with water, Cellulose-bound beta-glucosidase catalyzed p-nitrophenylglucoside hydrolysis, suggesting that the catalytic site is not involved in cellulose binding, When the cellulose-binding form was incubated with papain for 20 h, no decrease in the enzyme activity was observed; however, approximately 74% of the papain-treated glucosidase did not bind to microcrystalline cellulose, SDS PAGE analysis of the nonbinding glucosidase produced by papain indicated the presence of three bands with molecular weights in the range of 95,000 to 97,000, On the basis of these results, we propose that the low-molecular-weight (96,000 and 98,000) non-cellulose binding beta-glucosidase forms are most probably formed from the higher-molecular-weight (114,000) cellulose-binding beta-glucosidase via extracellular proteolytic hydrolysis. Also, it appears that the extracellular beta-glucosidase from P, chrysosporium might be organized into two domains, a cellulose-binding domain and a catalytic domain, Kinetic characterization of the cellulose-binding form is also presented.
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页码:2976 / 2980
页数:5
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