Detection of cyclodextrin glycosyltransferase gene by synthetic oligonucleotide probes

Abstract

Cyclodextrin glycosyltransferase (CGTase) from Bacillus sp. All was purified approximately 44.8 folds with a 30% yield and specific activity of 5,000 units/mg protein by corn starch adsorption, ammonium sulfate precipitation, and DEAE-cellulose column chromatography. Detection on non-denaturing polyacrylamide gel electrophoresis of the purified enzyme revealed a major and minor bands which corresponded to those of amylolytic and CD-forming activities. The intense protein band was found when sodium dodecyl sulfate polyacrylamide gel electrophoresis was performed. When the purified enzyme was determined for its amino acid composition, 60 mol% of content was polar amino acids: aspartic acid, asparagine, glutamic acid, and glutamine was rather high (25 mol%). The amino acid sequence of 20 residues at N-terminal was determined to be ala, pro, asp, thr, ser, asn, tyr, asn, lys, glu, asn, phe, arg, tyr, asp, val, ile, gln, and ile, respectively. The two oligonucleotides containing 17 bases were designed by deducing the N-and C-terminal amino acid sequences, and named PNB 5' HO-GTA(C/T)TATGATGTCAGCG-OH 3' and PCC 5' HO-CAACAAGCA(G/C)AATTTCC-OH 3', respectively. Both synthetic oligonucleotides were labeled at 5' end with [gamma-32P] and used as probe for the first screening of CGTase by colony hybridization. Partially Sau3AI-digested chromosomal DNA from Bacillus sp. All was ligated to BamHI-digested pUC18 and transformed into E. coli strain DH5alpha. Five CGTase-producing transformants were screened and isolated by colony hybridization, starch hydrolysis and clear zone detection, dextrinizing activity, phenol red inclusion complex test, and CD-trichloroethylene assay. Transformant 909 showed the highest activity but lower activity than Bacillus sp. All. Localization of CGTase in transformant 909 was determined and 87.5%, 11.2%, and 1.3% of dextrinizing activity were found in extracellular enzyme, periplasmic space, and cell pellet, respectively. HPLC analysis of products showed that ratio of alpha-:beta-:gamma-CD was 1:4.6:1.6. Crude CGTase of transformant 909 showed the same electrophoretic patterns as that of Bacillus sp. All when analyzed by non-denaturing and SDS-polyacrylamide gel electrophoresis.