Enzyme modification of starch granules: formation and retention of cyclomaltodextrins inside starch granules by reaction of cyclomaltodextrin glucanosyltransferase with solid granules

doi:10.1016/S0008-6215(00)00136-1

Carbohydrate Research

Volume 328, Issue 4, 6 October 2000, Pages 509-515

https://doi.org/10.1016/S0008-6215(00)00136-1 Get rights and content

Abstract

Cyclomaltodextrin glucanosyltransferase (CGTase) was adsorbed into starch granules and allowed to react at 37 °C. The reaction was conducted with the granules removed from an aqueous environment, but containing 50% w/w water inside the granule. Reaction for 20 h gave a maximum of 1.4%, w/w of cyclodextrins (CDs) inside the granule. Waxy maize and maize starches gave the highest amounts of CDs (1.3 and 1.4%, respectively), with tapioca and amylomaize-7 starches giving about 50% less (0.9 and 0.6%, respectively). Reaction of a combination of CGTase and isoamylase with solid starch granules gave a 2.6-fold increase in the formation of CDs, with a maximum yield of 3.4 and 100% retention inside waxy maize starch granules.

Introduction

Cyclomaltodextrin glucanosyltransferase (CGTase, EC 2.4.1.19) elaborated by Bacillus macerans produces cyclic maltodextrins (CDs) from starch that have six, seven and eight α-(1→4)-linked d-glucose residues. These CDs are commonly called α-, β- and γ-cyclodextrins. The CDs have interior cavities whose size and shape are determined by the number of d-glucose units [1]. The interior cavity has hydrophobic and electrophilic properties and will complex with many organic substances [1], [2], [3]. CDs, thus, can have a wide range of applications in complexing materials in foods, pharmaceuticals, plastics and agricultural products in which they can act as emulsifiers, anti-oxidants and stabilizing agents [3], [4].

Glucoamylase [5], [6], isoamylase [7] and CGTase [8] are enzymes that have a starch-binding domain, separate from their active sites. The starch-binding domain will bind with starch chains within the granules and produce products. The reaction takes place inside the granule and a certain percentage (8–32%) of the products remain inside the granule [9], [10]. We have reported that glucoamylase acting inside solid waxy maize starch granules can produce 5–50% (w/w) of d-glucose that is 100% retained inside the granule [11]. This reaction can be controlled to give different amounts of d-glucose inside the granule by inactivating the enzyme at different times of reaction [11]. The present study reports the formation of cyclomaltodextrins by the reaction of CGTase with raw starch granules in suspension and with solid starch granules and by the reaction of a combination of CGTase and isoamylase with solid starch granules in which the reaction with solid starch granules gave 100% retention of the CDs inside the granules.

Section snippets

Enzymes

CGTase [EC 2.4.1.19] from B. macerans was obtained as an aq solution from Amano International Enzyme Co. (Troy, VA, USA), and it was dialyzed against 10 mM imidazolium–HCl (pH 6.0) containing 1 mM CaCl₂ to remove peptides, monosaccharides and other unwanted material. The dialysis membrane was obtained from Fisher Scientific and was Spectra/Por 3.1 regenerated cellulose, with a molecular weight cut-off of 3500 Da.

Enzyme activity of CGTase was determined by a modification of the method of Thoma

Results

The α-, β-, and γ-CDs were readily separated by the TLC system and gave a linear densitometric response between 50 and 800 ng. Each type of CD gave a separate linear curve. Standard curves (Fig. 1) were prepared for each CD to quantitatively determine the amounts of CD produced in the enzyme reaction digests.

The amount of CGTase that was taken up by the starch granules was dependent on the pH of the enzyme solution and the temperature of incubation. When waxy maize starch was at pH 8.0 and 4 °C,

Discussion

Enzymes can enter starch granules through pores in the granules and catalyze reactions inside the granules [9], [10], [11], [15]. The pores are approximately 1000 Å in diameter on average and can easily allow large macromolecules, such as enzymes, to enter the granule and perform catalysis. Starch granules contain 10–15% w/w water of hydration. When starch granules are suspended in an aqueous solution of enzyme, water and enzyme molecules diffuse into the granule. When the granules are removed

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¹: Present address: National Food Research Institute, Kannondai, Tsukuba, Ibaraki 305, Japan.

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Enzyme modification of starch granules: formation and retention of cyclomaltodextrins inside starch granules by reaction of cyclomaltodextrin glucanosyltransferase with solid granules

Abstract

Introduction

Section snippets

Enzymes

Results

Discussion

Enzyme Microb. Technol.

J. Biol. Chem.

Carbohydr. Res.

Carbohydr. Res.

Carbohydr. Res.

Anal. Biochem.

Anal. Biochem.

Carbohydr. Res.