Cyclomaltodextrin glucanotransferase from Bacillus circulans E 192. I. Purification and characterization of the enzyme

Abstract

The cyclomaltrodextrin glucanotransferase (CGTase) [1,4-alpha-D-glucan:4-alpha-D-(1,4-alpha-D-glucano)-transferase (cyclizing), EC 2.4.1.19] from Bacillus circulans E 192 has been purified to homogeneity by Cetavlon treatment, ammonium sulfate precipitation, DEAE Trisacryl M chromatography, Q Fast Flow chromatography, and affinity on beta-cyclodextrin-Sepharose 4B. Two isoenzymes were separated by FPLC on a Mono Q column. Their isoelectric points were estimated as 6.7 and 6.9 and they represented 13 and 87%, respectively, of the initial activity. Their molecular weight, pH, and temperature optima were estimated as 78,000, 5.5, and 60 degrees C, respectively. Kinetic parameters indicated that both enzymes had the same properties; they preferentially modified high-molecular-weight substrates to produce cyclodextrins. The apparent Vmax and Km values for soluble starch were 43 mumol of beta-cyclodextrin/min/mg of protein and 0.57% (w/v), respectively. Although this CGTase is not markedly thermostable, it is protected against heat denaturation by substrate, product, and/or calcium ions. The ratios of alpha-, beta-, and gamma-cyclodextrins produced have been determined as 1/7/2 in the initial phase of the reaction and 3/3/1 at equilibrium.

Cyclodextrin glycosyltransferase production by new Bacillus sp. strains isolated from brazilian soil

“…Cyclodextrins are macrocyclic carbohydrates, and all three types are non-hygroscopic crystalline substances. In aqueous solution, their macro-rings are cylindrical, with a hydrophilic external surface and a hydrophobic internal cavity, allowing for the accommodation of organic and inorganic molecules in their interior (4,26). CDs were initially studied by Villiers in 1891 (25) and several published works exist, including reviews regarding elements derived from CDs, complexation, catalytic reactivity and photochemical studies (8,26,29,30).…”

“…Such molecules have also been used in the food, agriculture, pharmaceutical, cosmetic and other industries (5,22,23,24). The CGTase enzyme is produced by different species of microorganisms and the most commonly cited in the literature is the Bacillus genera (1,4,15). According to Van der Veen and Uitdehaag (29), CGTases catalyze the following reactions: a) hydrolysis reaction of the a link (1-4), described as: G(n) or cyclic G(x) → maltooligosaccharides b) cyclization reaction to form CD rings (a CGTase specific reaction), described as: G(n) ↔ cyclic G(x) + G(n-x) c) disproportionation reaction, which is a glucotransferase reaction, where a maltooligosaccharide is hydrolyzed and transferred to another receiving linear oligosaccharide, described as: G(n) +G(m) → G(n-x) + G(m+x) d) coupling reaction, which represents reverse cyclization, in which the CD ring is hydrolyzed and transferred to a receiving linear oligosaccharide, described as:…”

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“…17-1, 36 while 70-75% sequence identity was found with CGTases of B. circulans 8, E192, and B. macerans. 14,34,37 When compared with the N-terminus of CGTases from B. ohbensis, 38 and B. stearothermophilus, 39 only 50% sequence identity was observed. No homology was found with Nterminal sequence of K. oxytoca M5a1 CGTase.…”

“…12 Techniques of higher separating performance ie, isoelectric focusing or FPLC on a Mono Q column which could separate different subforms of CGTases with very close pI values were later reported. [13][14] Purification of the enzyme from Bacillus circulans A11 required at least two consecutive columns after starch adsorption or ammonium sulphate precipitation. [15][16][17] The attempt to use specific antibody-linked chromatographic resin as an immunoaffinity column to improve CGTase purification was raised.…”

Cyclodextrin glycosyltransferase from Bacillus licheniformis: optimization of production and its properties

“…Bacillus is the main genera, with various producing species (1,2,3,8,17,19,20,21,27). They are also known to catalyze four different transferase reactions: cyclization, coupling, disproportionation, and hydrolysis (4). All known CGTases produce α-, β-and γ-CDs from starch in different reactions.…”