Abstract
The initial action of cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) from an alkalophilic Bacillus sp. A2-5a on amylose was investigated. Synthetic amylose was incubated with purified CGTase then terminated in the very early stage of the enzyme reaction. When the reaction mixture was treated with glucoamylase and the resulting glucoamylase-resistant glucans were analyzed with high performance anion exchange chromatography, cyclic alpha-1,4-glucans, with degree of polymerization ranging from 9 to more than 60, in addition to well known alpha-, beta-, and gamma-cyclodextrin (CD), were detected. The time-course analysis revealed that larger cyclic alpha-1, 4-glucans were preferentially produced in the initial stage of the cyclization reaction and were subsequently converted into smaller cyclic alpha-1,4-glucans and into the final major product, beta-CD. CGTase from Bacillus macerans also produced large cyclic alpha-1, 4-glucans except that the final major product was alpha-CD. Based on these results, a new model for the action of CGTase on amylose was proposed, which may contradict the widely held view of the cyclization reaction of CGTase.
Highlights
Cyclodextrin glucanotransferase (CGTase,1 EC 2.4.1.19), found in several bacterial species, catalyzes the inter- and intramolecular transglycosylation of ␣-1,4-glucan
We reported that the glycogen-branching enzyme (EC 2.4.1.18) from Bacillus stearothermophilus catalyzes the intramolecular transglycosylation of amylose and amylopectin to produce branched cyclic glucans with DP larger than 18 [23, 24]
The enzyme reaction was terminated at the early stage of the reaction (10 min), and the reaction mixture was incubated with glucoamylase to digest the linear amylose into glucose
Summary
Chemicals and Enzymes—Synthetic amylose with an average molecular mass of 30 kDa (amylose AS-30) and soluble starch were purchased from Nakano Vinegar Co., Ltd. (Aichi, Japan) and E. Analysis of Reaction Products of CGTase on Amylose—CGTase (0.75 unit/ml) was incubated at 40 °C with amylose AS-30 (0.4% (w/v)) in 0.2 M sodium acetate buffer, pH 5.5, and reactions were terminated by boiling the solutions for 10 min. The reaction mixture containing 20 g of glucan was incubated with glucoamylase (0.2 units) in 20 mM sodium acetate buffer (pH 5.5) for 16 h at 40 °C and boiled for 5 min. “Time of Flight” Mass Spectrometry (TOF-MS)—A reaction mixture (5 ml) containing 10 mg of amylose AS-30 and CGTase (0.7 unit) was incubated in 0.2 M sodium acetate buffer, pH 5.5, at 40 °C for 1 h, and boiled for 10 min. After removing glucose with the Waters Sep-Pak C18 cartridge, the molecular masses of glucoamylase-resistant glucans were determined with a Kompact Maldi I TOF-MS system (Shimadzu, Kyoto, Japan)
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