Abstract
The cloning of a polymerase chain reaction (PCR) gene fragment from Bacillus sp. NR5 UPM isolated from the soil in Malaysia into an Escherichia coli expression vector was successfully carried out. Analysis of the nucleotide sequences revealed the presence of an open reading frame of 2112 bp which encoded a protein containing 704 amino acids with a putative molecular weight of 78.6 kDa. The deduced amino acids sequence showed about 98% homology with the CGTase from Bacillus sp. KC201. Compared to the wild type, the CGTase that was produced in E. coli cells only required one-fourth of culture time and neutral pH to produce CGTase. After 12 h of cultivation, the CGTase activity in the culture medium reached 29.6 U/ml, which was approximately 2.5-fold higher than the CGTase from the parental strain. The CGTase was produced extracellularly by E. coli (94%) indicating the signal peptide was functional in E. coli. Key words: Molecular cloning, nucleotide sequence, cyclodextrin glycosyltransferase,Bacillus sp. NR5 UPM.
Highlights
Cyclodextrin glycosyltransferase (CGTases, 1,4-α-Dglucopyranosyltransferase, EC 2.4.1.19) is an important enzyme that catalyzes the formation of α-CD, β-CD and γ-CD, containing 6, 7 and 8 glucose residues linked with α-1,4-glucosidic bonds, respectively
NR5 UPM isolated from the soil in Malaysia into an Escherichia coli expression vector was successfully carried out
The CGTase was produced extracellularly by E. coli (94%) indicating the signal peptide was functional in E. coli
Summary
Cyclodextrin glycosyltransferase (CGTases, 1,4-α-Dglucopyranosyltransferase (cyclizing), EC 2.4.1.19) is an important enzyme that catalyzes the formation of α-CD, β-CD and γ-CD, containing 6, 7 and 8 glucose residues linked with α-1,4-glucosidic bonds, respectively. Due to their unique abilities to form inclusion complexes with a variety of hydrophobic materials and to entrap volatile compounds, these CDs have found extensive applications in food, pharmaceuticals, agricultural chemicals, cosmetics, industrial chemicals and others (Hashimoto, 2002). The isolated CGTase gene was cloned into an E. coli expression vector and over expressed to study the improved properties of the enzyme
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