ISOLATION OF 4 MAJOR SUBUNITS FROM CLOSTRIDIUM-THERMOCELLUM CELLULOSOME AND THEIR SYNERGISM IN THE HYDROLYSIS OF CRYSTALLINE CELLULOSE

被引:26
作者
BHAT, S
GOODENOUGH, PW
BHAT, MK
OWEN, E
机构
[1] INST FOOD RES, DEPT PROT ENGN, READING LAB, READING RG6 2EF, BERKS, ENGLAND
[2] UNIV READING, DEPT AGR, READING RG6 2AT, BERKS, ENGLAND
关键词
DISSOCIATION; SEPARATION; SYNERGISM;
D O I
10.1016/0141-8130(94)90066-3
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The cellulosome of Clostridium thermocellum, purified by affinity chromatography, was dissociated under mild conditions and separated by SDS-PAGE. Two major p-nitrophenylcellobiosidases (PNPCases I and II) corresponding to the S-5 (103 kDa) and S-8 (78 kDa) subunits and one major carboxymethylcellulase (CMCase) coinciding with the S-11 (60.5 kDa) subunit were isolated and characterized using carboxymethylcellulose (CMC), H3PO4-swollen cellulose and cello-oligosaccharides. Both PNPCases showed little effect on the viscosity of CMC and released twice as much total sugar as reducing sugar from H3PO4-swollen cellulose. The CMCase released ten times more total sugar than reducing sugar from H(3)P(O)4-swollen cellulose and reduced the viscosity of CMC rapidly. None of these enzymes was active on cellotriose. Both PNPCases released cellobiose from cellotetraose, and cellobiose and cellotriose from cellopentaose. In contrast, CMCase was active only on cellopentaose and released mainly glucose. Use of MeUmb(Glc)(n) revealed that both PNPCases cleaved preferentially either the second or fourth linkage from the non-reducing end while the CMCase was specific for the internal glycosidic bonds. Thus, the PNPCases and CMCase behaved as typical exo- and endoglucanases, respectively. When tested individually, all three enzymes degraded Avicel only to a small extent. A 1.5-2.0-fold increase in sugar release was observed when CMCase was combined with either PNPCase I, II or both. Combining S-1 with either PNPCase II or CMCase resulted in fourfold synergism in the hydrolysis of Avicel. Synergism was sevenfold when all three enzymes were combined with S-1.
引用
收藏
页码:335 / 342
页数:8
相关论文
共 53 条
[1]  
BEGUIN P, 1990, 6TH P INT S GEN IND, V3, P947
[2]   THE ENDO-(1-]4)-BETA-D-GLUCANASE SYSTEM OF PENICILLIUM-PINOPHILUM CELLULASE - ISOLATION, PURIFICATION, AND CHARACTERIZATION OF 5 MAJOR ENDOGLUCANASE COMPONENTS [J].
BHAT, KM ;
MCCRAE, SI ;
WOOD, TM .
CARBOHYDRATE RESEARCH, 1989, 190 (02) :279-297
[3]  
BHAT KM, 1992, CARBOHYD RES, V227, P293, DOI 10.1016/0008-6215(92)85079-F
[4]   STUDY OF THE MODE OF ACTION AND SITE-SPECIFICITY OF THE ENDO-(1-]4)-BETA-D-GLUCANASES OF THE FUNGUS PENICILLIUM-PINOPHILUM WITH NORMAL, 1-H-3-LABELED, REDUCED AND CHROMOGENIC CELLO-OLIGOSACCHARIDES [J].
BHAT, KM ;
HAY, AJ ;
CLAEYSSENS, M ;
WOOD, TM .
BIOCHEMICAL JOURNAL, 1990, 266 (02) :371-378
[5]  
BHAT S, 1993, FEMS MICROBIOL LETT, V111, P73, DOI 10.1111/j.1574-6968.1993.tb06364.x
[6]  
BRADFORD MM, 1976, ANAL BIOCHEM, V72, P248, DOI 10.1016/0003-2697(76)90527-3
[7]   STUDIES OF THE CELLULOLYTIC SYSTEM OF THE FILAMENTOUS FUNGUS TRICHODERMA-REESEI QM-9414 - SUBSTRATE-SPECIFICITY AND TRANSFER ACTIVITY OF ENDOGLUCANASE-I [J].
CLAEYSSENS, M ;
VANTILBEURGH, H ;
KAMERLING, JP ;
BERG, J ;
VRSANSKA, M ;
BIELY, P .
BIOCHEMICAL JOURNAL, 1990, 270 (01) :251-256
[8]   FUNGAL CELLULASE SYSTEMS - COMPARISON OF THE SPECIFICITIES OF THE CELLOBIOHYDROLASES ISOLATED FROM PENICILLIUM-PINOPHILUM AND TRICHODERMA-REESEI [J].
CLAEYSSENS, M ;
VANTILBEURGH, H ;
TOMME, P ;
WOOD, TM ;
MCRAE, SI .
BIOCHEMICAL JOURNAL, 1989, 261 (03) :819-825
[9]  
Coughlan M. P., 1988, Biochemistry and genetics of cellulose degradation, P11
[10]   THE 3-DIMENSIONAL CRYSTAL-STRUCTURE OF THE CATALYTIC CORE OF CELLOBIOHYDROLASE-I FROM TRICHODERMA-REESEI [J].
DIVNE, C ;
STAHLBERG, J ;
REINIKAINEN, T ;
RUOHONEN, L ;
PETTERSSON, G ;
KNOWLES, JKC ;
TEERI, TT ;
JONES, TA .
SCIENCE, 1994, 265 (5171) :524-528