Development of an Escherichia coli expression system and thermostability screening assay for libraries of mutant xylanase

被引:8
作者
Ebanks, R [1 ]
Dupont, M [1 ]
Shareck, F [1 ]
Morosoli, R [1 ]
Kluepfel, D [1 ]
Dupont, C [1 ]
机构
[1] Inst Armand Frappier, INRS, Ctr Microbiol & Biotechnol, Laval, PQ HV7 1B7, Canada
关键词
xylanase; thermostability; screening assay; mutagenesis; Streptomyces lividans;
D O I
10.1038/sj.jim.7000102
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 thermostability screening assay was developed using an Escherichia coli expression system to express Streptomyces lividans xylanase A (XInA). The screening system was tested using mutants randomized at position 49 of the S. lividans XInA gene, a position previously shown to confer thermostability with a 149P point mutation. The library was cloned into an E. coli expression vector and transformed into XL1- Blue bacteria. The resulting crones were screened for increased thermostability with respect to wild-type XInA. Using this assay, we isolated the 149P mutant previously shown to be thermostable, as well as novel 149A and 149C mutants. The 149A and 149C mutants were shown to have 2.8- to 8-fold increase in thermostability over that of wild-type XInA. The results show that the screening assay can selectively enrich for clones with increased thermostability and is suitable for screening small- to medium-sized libraries of 5000-20,000 clones.
引用
收藏
页码:310 / 314
页数:5
相关论文
共 16 条
[1]   Structures of the psychrophilic Alteromonas haloplanctis α-amylase give insights into cold adaptation at a molecular level [J].
Aghajari, N ;
Feller, G ;
Gerday, C ;
Haser, R .
STRUCTURE, 1998, 6 (12) :1503-1516
[2]   SENSITIVE DETECTION OF ENDO-1,4-BETA-GLUCANASES AND ENDO-1,4-BETA-XYLANASES IN GELS [J].
BIELY, P ;
MARKOVIC, O ;
MISLOVICOVA, D .
ANALYTICAL BIOCHEMISTRY, 1985, 144 (01) :147-151
[3]  
COUTINHO PM, 1999, CARBOHYDRATE ACTIVE
[4]   CRYSTAL-STRUCTURE OF RECOMBINANT TRIOSEPHOSPHATE ISOMERASE FROM BACILLUS-STEAROTHERMOPHILUS - AN ANALYSIS OF POTENTIAL THERMOSTABILITY FACTORS IN 6 ISOMERASES WITH KNOWN 3-DIMENSIONAL STRUCTURES POINTS TO THE IMPORTANCE OF HYDROPHOBIC INTERACTIONS [J].
DELBONI, LF ;
MANDE, SC ;
RENTIERDELRUE, F ;
MAINFROID, V ;
TURLEY, S ;
VELLIEUX, FMD ;
MARTIAL, JA ;
HOL, WGJ .
PROTEIN SCIENCE, 1995, 4 (12) :2594-2604
[5]  
DEREWENDA U, 1994, J BIOL CHEM, V269, P20811
[6]   Substrate-binding domains of glycanases from Streptomyces lividans:: characterization of a new family of xylan-binding domains [J].
Dupont, C ;
Roberge, M ;
Shareck, F ;
Morosoli, R ;
Kluepfel, D .
BIOCHEMICAL JOURNAL, 1998, 330 :41-45
[7]   Psychrophilic enzymes: molecular basis of cold adaptation [J].
Feller, G ;
Gerday, C .
CELLULAR AND MOLECULAR LIFE SCIENCES, 1997, 53 (10) :830-841
[8]   PRODUCTION AND SECRETION OF PROTEINS BY STREPTOMYCETES [J].
GIBERT, M ;
MOROSOLI, R ;
SHARECK, F ;
KLUEPFEL, D .
CRITICAL REVIEWS IN BIOTECHNOLOGY, 1995, 15 (01) :13-39
[9]   A CELLULASE XYLANASE-NEGATIVE MUTANT OF STREPTOMYCES-LIVIDANS-1326 DEFECTIVE IN CELLOBIOSE AND XYLOBIOSE UPTAKE IS MUTATED IN A GENE ENCODING A PROTEIN HOMOLOGOUS TO ATP-BINDING PROTEINS [J].
HURTUBISE, Y ;
SHARECK, F ;
KLUEPFEL, D ;
MOROSOLI, R .
MOLECULAR MICROBIOLOGY, 1995, 17 (02) :367-377
[10]  
KLUEPFEL D, 1988, METHOD ENZYMOL, V160, P180