Biochemical properties and cyclodextrin production profiles of isoforms of cyclodextrin glycosyltransferase

Abstract

Cyclodextrin glycosyltransferase (CGTase) catalyzes the conversion of starch to cyclodextrin (CD), an important host molecule for the study of host–guest interactions. CGTase from Paenibacillus sp. RB01 and its recombinant form showed the same isoform pattern. The three isoforms, two major (isoforms I and II) and one minor (isoform III), all had a different net charge but the same molecular mass. The aim of this work was to characterize the three isoforms, and especially to compare their CD production profiles. Isoforms I and II were separated on a FPLC Mono Q column and showed the same optimum pH (pH 5 for dextrinizing and pH 6–7 for cyclization activity) and optimum temperature (65–70 °C for both activities). However, the two isoforms differed in their catalytic efficiency of the coupling reaction with variable concentrations of the β-CD donor in the presence of a fixed amount of cellobiose acceptor, with kcat/Km values of 3.46 × 10−3 and 2.20 × 10−3 mM−1 min−1, for isoforms I and II, respectively. Both isoforms I and II were found to have β-CGTase activity and gave a similar CD6:CD7:CD8 product ratio of 0.2:1.0:0.6, with an increase in the ratio of the small-ring to the large-ring CDs from 1.0:0.5 to 1.0:0.3 from a 6 to 24 h reaction time. However, in terms of maximal CD yields, the two isoforms differed in their optimal reaction temperature and time required, the optimal conditions being at 40 °C for 6 h for isoform I and at 60 °C for 24 h for isoform II.