Abstract: In this study, we constructed domain D, E and DE truncated mutants of a marine-derived cyclodextrin glucosyltransferase (CGTase) my20 and heterologously expressed these mutants using the pET-24a vector and E. coli BL21 (DE3) as the host, and purified the expressed enzyme by nickel affinity column chromatography. The specific cyclization activities of my20ΔD, my20ΔE, and my20ΔDE were approximately 197%, 22%, and 17% of that of my20, respectively, indicating that domain E is important for the catalytic activity of CGTase, and the presence of domain D might hinder substrate molecules from entering the catalytic domains. my20 was a heat-resistant enzyme, and the three truncated mutants showed different degrees of reduction in heat resistance, suggesting that domains D and E played an important role in the heat resistance of CGTase. The domain DE-deleted mutant showed an 8% increase in the percentage of α-cyclodextrin (CD) in the catalytic product compared with the other truncated mutants, which suggested that the loss of domain DE affected the product specificity. The results of this study provide a theoretical basis for CGTase domain modification for its application in industrial production.

Key words: cyclodextrin glucosyltransferase; domain; cyclization activity; enzymatic properties