Site-directed mutagenesis and CBM engineering of Cel5A (Thermotoga maritima)

被引：50

作者：

Mahadevan, Shobana Arumugam ^{[1
]}

Wi, Seung Gon ^{[1
]}

Lee, Dae-Seok ^{[1
]}

Bae, Hyeun-Jong ^{[1
]}

机构：

[1] Chonnam Natl Univ, Dept Forest Prod & Technol, Program BK21, Kwangju 500757, South Korea

来源：

FEMS MICROBIOLOGY LETTERS | 2008年 / 287卷 / 02期

关键词：

cellulose-binding module; hyperthermostable; endoglucanase; site-directed mutagenesis; domain engineering;

D O I：

10.1111/j.1574-6968.2008.01324.x

中图分类号：

Q93 [微生物学];

学科分类号：

071005 ; 100705 ;

摘要：

In order to make cost-effective bioethanol from dynamic lignocellulosic material, we require potentially acting and stable cellulolytic enzymes. In our investigation, the hyperthermostable endoglucanase Cel5A from Thermotoga maritima was subjected to site-directed mutagenesis and carbohydrate-binding module (CBM) engineering. For this purpose, amino acids around the active-site region were targeted. Results indicated that five single mutants showed a shift in optimal pH from 5 to 5.4. The N147E mutant displayed 10% higher activity than native Cel5A. Domain engineering was performed with fungal and bacterial CBM. In addition, CBM1 from (CBHII) Trichoderma reesei and CBM6 from Clostridium stercorarium xylanase A were fused with Cel5A. Both the CBM-engineered Cel5A showed 14-18-fold higher hydrolytic activity towards Avicel. Immuno-gold labeling assay of engineered enzymes further indicated the relativity that exists between binding ability and activity.

引用

页码：205 / 211

页数：7

共 25 条

[1]

Bae HJ, 2004, FEMS MICROBIOL LETT, V233, P325, DOI 10.1111/j.1574-6968.2004.tb09499.x

[2] A comparative study between an endoglucanase IV and its fused protein complex Cel5-CBM6 [J].

Bae, HJ ;

Turcotte, G ;

Chamberland, H ;

Karita, S ;

Vézina, LP .

FEMS MICROBIOLOGY LETTERS, 2003, 227 (02) :175-181

[3] THE BIOLOGICAL DEGRADATION OF CELLULOSE [J].

BEGUIN, P ;

AUBERT, JP .

FEMS MICROBIOLOGY REVIEWS, 1994, 13 (01) :25-58

[4] PURIFICATION OF THERMOTOGA-MARITIMA ENZYMES FOR THE DEGRADATION OF CELLULOSIC MATERIALS [J].

BRONNENMEIER, K ;

KERN, A ;

LIEBL, W ;

STAUDENBAUER, WL .

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 1995, 61 (04) :1399-1407

[5]

CHAUVAUX S, 1992, J BIOL CHEM, V267, P4472

[6] Biochemical characterization of Thermotoga maritima endoglucanase Ce174 with and without a carbohydrate binding module (CBM) [J].

Chhabra, SR ;

Kelly, RM .

FEBS LETTERS, 2002, 531 (02) :375-380

[7] Regulation of endo-acting glycosyl hydrolases in the hyperthermophilic bacterium Thermotoga maritima grown on glucan- and mannan-based polysaccharides [J].

Chhabra, SR ;

Shockley, KR ;

Ward, DE ;

Kelly, RM .

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2002, 68 (02) :545-554

[8] Carbohydrate-induced differential gene expression patterns in the hyperthermophilic bacterium Thermotoga maritima [J].

Chhabra, SR ;

Shockley, KR ;

Conners, SB ;

Scott, KL ;

Wolfinger, RD ;

Kelly, RM .

JOURNAL OF BIOLOGICAL CHEMISTRY, 2003, 278 (09) :7540-7552

[9] Microbial biochemistry, physiology, and biotechnology of hyperthermophilic Thermotoga species [J].

Conners, Shannon B. ;

Mongodin, Emmanuel F. ;

Johnson, Matthew R. ;

Montero, Clemente I. ;

Nelson, Karen E. ;

Kelly, Robert M. .

FEMS MICROBIOLOGY REVIEWS, 2006, 30 (06) :872-905

[10] NON-HYDROLYTIC DISRUPTION OF CELLULOSE FIBERS BY THE BINDING DOMAIN OF A BACTERIAL CELLULASE [J].

DIN, N ;

GILKES, NR ;

TEKANT, B ;

MILLER, RC ;

WARREN, AJ ;

KILBURN, DG .

BIO-TECHNOLOGY, 1991, 9 (11) :1096-1099

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