Purification and biochemical characterization of cyclodextrin glycosyltransferase from thermotolerant Paenibacillus sp. strain RB01

Abstract

Cyclodextrin glycosyltransferase (CGTase) catalyzes the conversion of starch to cyclodextrin (CD). The enzyme from a thermotolerant Paenibacillus sp. strain RB01 isolated from hot spring area in Ratchaburi province, Thailand, was purified and characterized. Purification steps involved starch adsorption, DEAE-cellulose and Bio-Gel P-100 column chromatography. Final purification fold was 47.5 and the yield obtained was 35%. The purified CGTase showed a single band on SDS-PAGE with the molecular weight of 65 kDa. On native PAGE, one major with two minor protein bands which corresponded to bands obtained from dextrinizing activity were observed. The result suggests the existence of 3 isoforms of the same size of this CGTase. However, they showed small difference in net charge as evidenced by their closed relative mobilities on native PAGE. In addition, by isoelectric focusing, two major bands were found at pI of 5.2 and 5.3 with one minor band at 5.1. The enzyme was found to be a glycoprotein due to positive staining with PAS. By HPLC analysis of reaction products, it was found that this CGTase hydrolyzed soluble starch to form α:β:γ-CD in the ratio of 1.0:1.8:0.4. Optimum pH and temperature for dextrinizing activity were 5.0 and 65 ℃, for cyclizing activity were 7.0 and 70 ℃, and for CD-TCE activity were 7.0-9.0 and 55 ℃. The enzyme was stable at pH 7-9 and temperature of 45-55 ℃ within 60 min incubation period. At 70 ℃, in the presence of soluble starch substrate, no activity was lost after 30 min incubation while only 20% was lost after 60 min. The enzyme was chemically modified with a series of group-specific reagents to identify essential amino acid residues. Incubation of the enzyme for 5 min with 5, 1.5 and 0.005 mM of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), diethylpyrocarbonate (DEP) and N-bromosuccinimide (NBS) which were specific for carboxyl, histidine, and tryptophan residues, respectively, led to complete loss of enzyme activity. In the presence of protective substance, α-, β-, or γ-CD, the loss of activities were reduced. These results suggested that carboxyl, histidine and tryptophan were located at the active site. In addition, it was proved that cystein, tyrosine, lysine, and serine were not important residues for enzyme activity. The enzyme was found to be specific for substrates with α-1, 4 glucosidic linkage, and G3 was the smallest saccharide acting as substrate. For kinetic parameters on CD coupling activity, enzyme efficiency (kcat/Km) was higher for native than for modified CDs. Turnover number (kcat) showed similar trend as that of β- ≅ γ- > α > modified CDs. The result from urea-induced denaturation of CGTase suggested that multiple forms of the enzyme did not arise from different tertiary structure of protein.