Cloning and expression of cyclodextrin glycosyltransferase gene from Paenibacillus sp. T16 isolated from hot spring soil in northern Thailand.
Gene encoding cyclodextrin glycosyltransferase (CGTase), from thermotolerant Paenibacillus sp. T16 isolated from hot spring area in northern Thailand, was cloned and expressed in E. coli (JM109). The nucleotide sequences of both wild type and transformed CGTases consisted of 2139 bp open reading frame, 713 deduced amino acids residues with difference of 4 amino acid residues. The recombinant cells required 24 h culture time and a neutral pH for culture medium to produce compatible amount of CGTase compared to 72 h culture time and pH 10 for wild type. The recombinant and wild-type CGTases were purified by starch adsorption and phenyl sepharose column chromatography and characterized in parallel. Both enzymes showed molecular weight of 77 kDa and similar optimum pHs and temperatures with recombinant enzyme showing broader range. There were some significant difference in pH, temperature stability and kinetic parameters. The presence of high starch concentration resulted in higher thermostability in recombinant enzyme than the wild type. The recombinant enzyme was more stable at higher temperature and lower pH, with lower K(m) for coupling reaction using cellobiose and cyclodextrins as substrates.
Charoensakdi
Ratiya
R
Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand.
Murakami
Shuichiro
S
Aoki
Kenji
K
Rimphanitchayakit
Vichien
V
Limpaseni
Tipaporn
T
D000595:Amino Acid Sequence; D001407:Bacillus / Q000145:classification / Q000201:enzymology / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005964:Glucosyltransferases / Q000235:genetics* / Q000378:metabolism; D045482:Hot Springs / Q000382:microbiology; D007700:Kinetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D017386:Sequence Homology, Amino Acid; D012988:Soil Microbiology; D013785:Thailand; D047408:gamma-Cyclodextrins / Q000378:metabolism
D001426:Bacterial Proteins; D047408:gamma-Cyclodextrins; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase