Development and characterization of cyclodextrin glucanotransferase as a maltoheptaose-producing enzyme using site-directed mutagenesis

Abstract

Cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19) mainly produces cyclodextrins (CDs) using linear maltooligosaccharides. We performed site-directed saturation mutagenesis on the +1 substrate-binding residue, H233, of CGTase from alkalophilic Bacillus sp. I-5 to prepare specific-length oligosaccharides. The obtained mutant CGTase, H233Y, primarily produced maltoheptaose (G7) using β-CD via a hydrolysis reaction. A kinetic study of H233Y showed that the kcat/Km value of β-CD was 7-fold greater than that of G7, which accounts for the accumulation of G7 during the H233Y enzyme reaction. A structure comparison of CGTases with H233Y modeling suggests that the substitution of H233Y may alter the position of the +1 subsite and slow the further hydrolysis of G7 after the ring-opening reaction.

Development and Application of Cyclodextrin Hydrolyzing Mutant Enzyme Which Hydrolyzes β- and γ-CD Selectively

“…Escherichia coli (E. coli) MC1061 [F±, araD139, recA13, Δ (araABC-leu) 7696, galU, galK, ΔlacX74, rpsL, thi, hsdR2, and mcrB] was utilized as the bacterial host for DNA operation, alteration, and expression of the CGTase enzyme. 14 Escherichia coli MC1061 was grown at 37 °C in Luria−Bertani (LB) medium [1% (w/ v) Bacto-tryptone, 0.5% (w/v) yeast extract, and 0.5% (w/v) NaCl] containing 100 μg/mL of ampicillin. 15,16 All chemicals, including soluble starch, were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).…”

“…The PCR products were transformed into MC1061 host cells. 14 The transformants were cultured in LB agar medium containing 100 μg/mL of ampicillin (LBA-agar). 17 The mutation sites were identified using dideoxy chain-termination sequencing with an ABI PRISM 3700 DNA analyzer (Applied Biosystems; Foster City, CA, USA).…”

“…The CGTase enzyme reaction products were analyzed using thin-layer chromatography (TLC) and high-performance anion-exchange chromatography (HPAEC) as previously described. 14 The TLC plate (K5F, Whatman; Maidstone, UK) was preheated to 105 °C for 60 min and then cooled for 10 min. Samples were spotted onto the plate and dried.…”

Ring-Opening of Cyclodextrins: An Efficient Route to Pure Maltohexa-, Hepta-, and Octaoses

“…CD glucanotransferases (CGTase) mainly produce CDs starting from linear OM. Koo et al [19] performed site-directed saturation mutagenesis on the +1 substrate-binding residue H233 of CD glucanotransferase CGTase from alkalophilic Bacillus sp. I-5 to prepare specific-length oligosaccharides.…”

Production and Surfactant Properties of Tert-Butyl α-d-Glucopyranosides Catalyzed by Cyclodextrin Glucanotransferase

“…As a result, a low yield of the desired glycosylated product is typically obtained [23]. Several strategies have been assessed to improve the transglycosylation activity of CGTases, including protein engineering [24][25][26], chemical modification [7], immobilization [27,28], and the addition of cosolvents and additives [29][30][31]. Bacterial CGTases are produced mainly by the genus Bacillus [32], although Micrococcus and Klebsiella species had also been reported as producers.…”