Mutation and expression of N233C-D506C of cellulase Cel6B from Thermobifida fusca in Escherichia coli

被引：9

作者：

Ai, YC ^{[1
]}

Wilson, DB

机构：

[1] Zhongshan Univ, Dept Biochem, Guangzhou 510275, Peoples R China

[2] Zhongshan Univ, Genet Engn Lab Chinese Educ Minist, Guangzhou 510275, Peoples R China

[3] Cornell Univ, Dept Mol Biol & Genet, Ithaca, NY 14853 USA

来源：

ENZYME AND MICROBIAL TECHNOLOGY | 2002年 / 30卷 / 06期

关键词：

Thermobifida fusca; cellulase; recombinant expression; mutation;

D O I：

10.1016/S0141-0229(02)00061-3

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：

A versatile system for expression and purification of cellulase Cel6B from Thermobifida fusca in E,Escherichia coli was developed. A double mutation. N233C-D506C, of Cel6B was created to try to increase its thermostability, by forming an extra-disulfide bond joining two loops of Cel6B that cover the active site cleft. The predicted mutant enzyme was slightly more thermostable with no CD spectroscopic changes, but a disulfide bond did form and there was a significant decrease in enzyme production. (C) 2002 Elsevier Science Inc. All rights reserved.

引用

页码：804 / 808

页数：5

共 18 条

[1] Identification of two functionally different classes of exocellulases [J].

Barr, BK ;

Hsieh, YL ;

Ganem, B ;

Wilson, DB .

BIOCHEMISTRY, 1996, 35 (02) :586-592

[2] Active-site binding of glycosides by Thermomonospora fusca endocellulase E2 [J].

Barr, BK ;

Wolfgang, DE ;

Piens, K ;

Claeyssens, M ;

Wilson, DB .

BIOCHEMISTRY, 1998, 37 (26) :9220-9229

[3] Comparing the thermodynamic stabilities of a related thermophilic and mesophilic enzyme [J].

Beadle, BM ;

Baase, WA ;

Wilson, DB ;

Gilkes, NR ;

Shoichet, BK .

BIOCHEMISTRY, 1999, 38 (08) :2570-2576

[4] Roles of the catalytic domain and two cellulose binding domains of Thermomonospora fusca E4 in cellulose hydrolysis [J].

Irwin, D ;

Shin, DH ;

Zhang, S ;

Barr, BK ;

Sakon, J ;

Karplus, PA ;

Wilson, DB .

JOURNAL OF BACTERIOLOGY, 1998, 180 (07) :1709-1714

[5] ACTIVITY STUDIES OF 8 PURIFIED CELLULASES - SPECIFICITY, SYNERGISM, AND BINDING DOMAIN EFFECTS [J].

IRWIN, DC ;

SPEZIO, M ;

WALKER, LP ;

WILSON, DB .

BIOTECHNOLOGY AND BIOENGINEERING, 1993, 42 (08) :1002-1013

[6] DISULFIDE ARRANGEMENT AND CHEMICAL MODIFICATION OF BETA-1,4-ENDOGLUCANASE-E2 FROM THERMOMONOSPORA-FUSCA [J].

MCGINNIS, K ;

WILSON, DB .

BIOCHEMISTRY, 1993, 32 (32) :8151-8156

[7] DISULFIDE ARRANGEMENT AND FUNCTIONAL DOMAINS OF BETA-1,4-ENDOGLUCANASE-E5 FROM THERMOMONOSPORA-FUSCA [J].

MCGINNIS, K ;

WILSON, DB .

BIOCHEMISTRY, 1993, 32 (32) :8157-8161

[8] 3-DIMENSIONAL STRUCTURE OF CELLOBIOHYDROLASE-II FROM TRICHODERMA-REESEI [J].

ROUVINEN, J ;

BERGFORS, T ;

TEERI, T ;

KNOWLES, JKC ;

JONES, TA .

SCIENCE, 1990, 249 (4967) :380-386

[9] Structure and mechanism of endo/exocellulase E4 from Thermomonospora fusca [J].

Sakon, J ;

Irwin, D ;

Wilson, DB ;

Karplus, PA .

NATURE STRUCTURAL BIOLOGY, 1997, 4 (10) :810-818

[10] CRYSTAL-STRUCTURE OF THE CATALYTIC DOMAIN OF A THERMOPHILIC ENDOCELLULASE [J].

SPEZIO, M ;

WILSON, DB ;

KARPLUS, PA .

BIOCHEMISTRY, 1993, 32 (38) :9906-9916

← 1 2 →