Development and Application of Cyclodextrin Hydrolyzing Mutant

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Development and application of cyclodextrin hydrolyzing mutant enzyme which hydrolyzes #- and #-CD selectively Ye-Seul Koo, Dam-Seul Ko, Da-Woon Jeong, and Jae-Hoon Shim J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00269 • Publication Date (Web): 02 Mar 2017 Downloaded from http://pubs.acs.org on March 2, 2017

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Journal of Agricultural and Food Chemistry

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Development and application of cyclodextrin hydrolyzing mutant

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enzyme which hydrolyzes β- and γ-CD selectively

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Running Title: CGTase mutant hydrolyzing β - and γ-CD selectively

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Ye-Seul Koo, Dam-Seul Ko, Da-Woon Jeong, and Jae-Hoon Shim*

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Department of Food Science and Nutrition and Center for Aging and Health Care, Hallym

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University, Hallymdaehak-gil 1, Chuncheon, Gangwon-do, 24252, Korea

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*Author to whom correspondence should be addressed

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E-mail: [email protected]; Phone: 82-33-248-2137; FAX: 82-33-248-2146

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ACS Paragon Plus Environment


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ABSTRACT

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Cyclodextrins (CDs) are produced from starch by cyclodextrin glucanotransferase (CGTase),

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which has cyclization activity. Specifically, α-CD is an important biomolecule as it is a

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molecular carrier and soluble dietary fiber used in the food industry. Upon inspection of the

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conserved regions of the glycoside hydrolase (GH) 13 family amylases, the amino acids

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K232 and H233 of CGTase were identified as playing an important role in enzyme reaction

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specificity. A novel CD hydrolyzing enzyme, cyclodextrin glycosyl transferase (CGTase)-

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alpha, was developed using site-directed mutagenesis at these positions. Action pattern

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analysis using various substrates revealed that CGTase-alpha was able to hydrolyze β- and γ-

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CD, but not α-CD. This selective CD hydrolyzing property was employed to purify α-CD

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from a CD mixture solution. The α-CD that remained after treatment with CGTase-alpha and

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exo-type glucoamylase was purified using hydrophobic interaction chromatography with

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99% purity.

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Keywords: cyclodextrins, α-cyclodextrin, site-directed mutagenesis, CGTase, amylase,

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bioconversion

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INTRODUCTION

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Cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19) has multiple catalytic actions

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including disproportionation, cyclization, coupling, and hydrolysis activity.1 CGTase is an

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extracellular enzyme that has industrial significance.2 In the starch industry, CGTase plays a

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main role in the production of cyclodextrins (CDs), as it catalyzes cyclization reactions in

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starch.3, 4 However, recent developments in CGTase activity have enabled us to employ this

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enzyme for the synthesis of modified oligosaccharides using alternative acceptor substrates.1

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Also, mutant CGTases have been developed as antistaling agents and biocatalysts for the

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synthesis of size-specific maltooligosaccharides.5-7

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CDs are a family of cyclic oligosaccharides composed of six, seven, and eight glycosyl units

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with α-1,4-linkages, which are named α-, β-, and γ-CD, respectively.4 The circular

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construction of CDs offers a hydrophobic cavity that allows the formation of inclusion

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complexes with a variety of non-polar organic molecules of suitable sizes.1 Therefore, CDs

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can react with various drug molecules to yield an inclusion complex.8 Among the CDs, α-CD

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has been shown to affect the stabilization of fatty acids, decrease cholesterol levels in blood,

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and aid in the retention of flavor compounds.9 Also, it has been reported that α-CD can

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reduce carbohydrate digestion, and this in turn may reduce postprandial glycemic reactions to

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carbohydrates.10 Recently, α-CD use was permitted in Europe as a dietary fiber food additive

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and α-CGTase which produces α-CDs as main products subsequently garnered industrial

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interest.4 Therefore, α-CD has attracted a great interest in various industries including food,

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pharmaceutical, chemical, and agricultural areas. However, the market share of α-CD is still

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smaller than that of other CDs because of its low production yield and high price caused by

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its purification procedure.4 To solve these problems, genetically modified mutant CGTases,



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which increase the α-CD production ratio, have been developed; however, they result in the

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production of β- and γ-CD as well.4, 11-13

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In this study, based on amino acid sequence alignment, we generated a mutant enzyme able to

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hydrolyze β- and γ-CD selectively and investigated its use for the efficient purification of α-

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CD from a CD mixture.

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MATERIALS AND METHODS

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Bacterial strains

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Escherichia coli (E. coli) MC1061 [F±, araD139, recA13, ∆ (araABC-leu) 7696, galU, galK,

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∆lacX74, rpsL, thi, hsdR2, and mcrB] was utilized as the bacterial host for DNA operation,

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alteration, and expression of the CGTase enzyme.14 Escherichia coli MC1061 was grown at

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37°C in Luria-Bertani (LB) medium [1% (w/v) Bacto-tryptone, 0.5% (w/v) yeast extract, and

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0.5% (w/v) NaCl] containing 100 µg/mL of ampicillin.15, 16 All chemicals, including soluble

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starch, were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).

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Site-directed mutagenesis and sequence analysis

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Site-directed mutagenesis was performed using the QuikChange Stratagene kit (La Jolla, CA,

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USA). The CGTase I-5 gene in the pR2CGT plasmid was amplified with polymerase chain

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reaction (PCR) using the following primers: CGT-N (5’-GAC GCG GTC AAC GAA ACG

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CCA TTC GGC TGG-3’) and CGT-C (5’-GAA TGG CGT TTC GTT GAC CGC GTC CAC

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GCG-3’).14 Eighteen cycles of PCR were performed in a heat cycler under the following

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conditions: 30 s at 95°C, 30 s at 55°C, and 8 min at 68°C (SuperCycler SC200, Kyratec;

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Mansfield, Australia). The PCR products were transformed into MC1061 host cells.14 The


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transformants were cultured in LB agar medium containing 100 µg/mL of ampicillin (LBA-

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agar).17 The mutation sites were identified using dideoxy chain-termination sequencing with

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an ABI PRISM 3700 DNA analyzer (Applied Biosystems; Foster City, CA, USA).14

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Purification of the CGTase mutant

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The E. coli transformants were cultured in LB medium supplemented with ampicillin (100

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µg/mL) for 24 h at 37°C on a shaker at 200 rpm.6 The cells were harvested by centrifugation

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(6,500 × g for 20 min at 4°C), and the supernatant was supplemented in lysis buffer (50 mM

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sodium acetate buffer, pH 6.0). After cell disruption using sonication, the cell extracts were

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kept in cool in a bath (XL-2000, Qsonica, LLC; Newtown, CT; output 12, 5 min, four times)

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and centrifuged (7000 × g for 20 min at 4°C) to obtain the supernatant.14 The supernatant was

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purified using a β-CD affinity column with washing (50 mM sodium acetate buffer, pH 6.0)

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and elution (50 mM sodium acetate buffer (pH 6.0), and 1% (w/v) β-CD) buffers.6, 14 The

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purified enzymes were concentrated using a Centricon filter (Millipore, Bedford, MA, USA)

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with 50 mM sodium acetate buffer (pH 6.0).6 The purified enzyme was analyzed using

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sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with

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Coomassie Brilliant Blue R-250 (Sigma-Aldrich Co.; destaining solution was composed of

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20% methanol, 10% (v/v) acetic acid, and water).18

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Enzyme assay

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The hydrolysis enzyme reaction was performed at 45°C in sodium acetate buffer (50 mM, pH

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6.0) containing 0.5% (w/v) soluble starch and the hydrolytic activity of CGTase was

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measured with the 3,5-dinitrosalicylic acid (DNS) method using 1% (w/v) β-CD as a

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substrate.6 Absorbance at 570 nm was measured using a spectrophotometer (Multiskan FC,

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Thermoscientific, Waltham, MI, USA). One unit (U) of hydrolysis activity was defined as the



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amount of CGTase enzyme that was able to split a 1-µmol equivalent of glycosidic linkages

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in β-CD in one minute. The enzyme quantity was measured using the Bradford method.19

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Thin-layer chromatography and high-performance anion-exchange chromatography

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analysis

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The CGTase enzyme reaction products were analyzed using thin-layer chromatography

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(TLC) and high-performance anion-exchange chromatography (HPAEC) as previously

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described.14 The TLC plate (K5F, Whatman; Maidstone, UK) was preheated to 105°C for 60

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min and then cooled for 10 min. Samples were spotted onto the plate and dried. TLC analysis

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was performed using the following solvent mixture: n-butanol: ethanol: water = 5:5:3, v/v/v.

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For the detection of the enzyme reaction products, the TLC plate was dried and visualized

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using a dipping solution containing 0.3% (w/v) N-(1-naphthyl)-ethylenediamine and 5% (v/v)

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H2SO4 in methanol, followed by heating for 10 min at 105°C. HPAEC analysis was

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performed as previously described.14, 20 A CarboPac PA1 column (0.4 × 25 cm; Dionex,

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Sunnyvale, CA, USA) and a DX-500 system equipped with a pulsed amperometric detector

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(ED40, Dionex) were used for the analysis. Samples were eluted with a gradient of 600 mM

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sodium acetate in 150 mM NaOH at a flow rate of 1.0 mL/min. Gradients of 600 mM sodium

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acetate solution were carried out as follows: linear gradients of 10–30% for 0–10 min, 30–

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40% for 10–16 min, 40–50% for 16–27 min, 50–60% for 27–44 min, and 60–64% for 44–60

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min.14

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Kinetic parameter analysis

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The kinetic parameters of the CGTase mutant for the β- and γ-CD substrates were calculated

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as previously described.14 The reaction mixture was composed of 500 µL of enzyme solution

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and 500 µL of substrate in 50 mM sodium acetate buffer (pH 6.0). The enzyme reaction was


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performed at pH 6.0 at 45°C. The substrate concentrations used in the kinetic study were 0.5–

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10-fold the Michaelis constant (Km) values of the hydrolysis activity. Samples (100 µL) were

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taken every 3 min and the enzyme reaction was terminated by boiling for 5 min. The amount

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of hydrolyzed substrate was measured using HPAEC. The Km and kcat values were calculated

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by fitting a Michaelis-Menten equation in the GraFit software (ver. 7.0; Erithacus Software

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Ltd., Staines, UK).

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Purification of α-CD from a CD mixture

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A mixture solution containing α-, β-, and γ-CD (10 mM each) with 50 mM sodium acetate

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buffer was reacted with the CGTase mutant enzyme (1.4 U) at 45°C for 2 h. The enzyme

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reaction was terminated by boiling for 5 min. Then, 0.3 U of exo-type glucoamylase (AMG

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300 L; Novozyme, Bagsvaerd, Denmark) was added to the reaction mixture. The AMG

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reaction was carried out at 60°C for 30 min and 60 min and the remaining α-CD was purified

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using HyperSep C18 cartridges (Thermo Fisher Scientific, Fair Lawn, NJ, USA). The

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samples were injected into the C18 cartridges and washed using distilled water. After

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washing, α-CD was eluted using methanol (100%). HPAEC was used to determine the yield

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and purity of purified α-CD.

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Sequence alignment of CGTases

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Amino acid sequences of alpha-amylase families of the GH13 family were obtained from the

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Carbohydrate-Active enZYmes (CAZy) database (http://www.cazy.org/GH13.html) and

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sequence alignment of the enzymes was performed using Align X software (Invitrogen,

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Carlsbad, CA, USA).

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RESULTS

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Mutation and preparation of CGTase

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A multiple sequence alignment of the alpha-amylase super family allowed us to identify

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features as targets for CGTase mutation (Figure 1). Two amino acid residues in the second

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conserved region were strictly conserved among CD-hydrolyzing enzymes such as

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cyclodextrinase (CDase), maltogenic amylase, and neopullulanase, which possessed

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conserved Asn (N) and Glu (E) residues. The corresponding sites of enzymes without CD-

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hydrolyzing activity were conserved as Lys (K) and His (H), respectively. Based on these

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results, the K and H residues of CGTase I-5 were replaced with N and E using the

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QuikChange site-directed mutagenesis kit. The mutation was confirmed by DNA sequencing

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and the corresponding gene was used for enzyme expression. The CGTase mutant was

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purified using β-CD affinity column chromatography. The molecular mass of the CGTase

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mutant was determined using SDS-PAGE analysis (Figure 2) and the purification data are

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shown in Table 1. The mutant CGTase was named CGTase-alpha.

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Characterization of recombinant CGTase-alpha

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We used the DNS method with β-CD as a substrate to determine the optimum temperature

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and pH for CGTase-alpha activity. The optimum temperature and pH conditions at which the

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highest CGTase-alpha activity was found were 45°C and pH 7.5, respectively (data not

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shown).

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The hydrolysis patterns of CGTase-alpha were measured using α-, β-, and γ-CD. The time

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course of CD degradation during a CGTase-alpha treated reaction was analyzed using TLC.


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β- and γ-CD were hydrolyzed gradually to small oligosaccharides by CGTase-alpha. However,

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CGTase-alpha was unable to hydrolyze α-CD (Figure 3).

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CGTase-alpha was reacted in a CD mixture solution with the same molar ratio of each CD for

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24 h. Consequently, β- and γ-CD were linearized and hydrolyzed to maltose (G2), maltotriose

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(G3), and maltotetraose (G4), but α-CD was not (Figure 4). To further examine these effects,

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we analyzed the resistance of α-CD to enzymatic hydrolysis using HPAEC. After a 2-h

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reaction, approximately 100% of the original α-CD remained; however, all of the β- and γ-

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CD were hydrolyzed to maltooligosaccharides (Figure 5).

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Kinetic study of the CGTase-alpha mutant

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To observe the catalytic properties of CGTase-alpha, we determined its kinetic parameters

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using different types of CDs (Table 2). CGTase-alpha did not show any catalytic activity

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when α-CD was used as a substrate. However, the kcat/Km value of γ-CD was roughly 2-fold

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higher than that of β-CD, which correlated with the time course analysis results (Figures 3

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and 5).

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Application of the CGTase-alpha mutant for α-CD purification

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Although CGTase-alpha can hydrolyze β- and γ-CD in a CD mixture selectively, the small

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oligosaccharides produced by the enzymatic reaction must be removed for the purification of

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α-CD. To maximize the difference in chemical properties of α-CD and other reaction products,

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it is necessary to hydrolyze linearized maltooligosaccharides to glucose (G1), which is small

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and hydrophilic. Therefore, treatment with AMG was performed after the CGTase-alpha

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reaction.

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maltooligosaccharides into G1 from the non-reduced end.21 As shown in Figure 6, all linear

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maltooligosaccharides in the mixture were hydrolyzed within 30 min by AMG (Figure 6).

AMG

is

an

exo-type

glucoamylase

enzyme


that

degrades

linear-


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Subsequently, α-CD and G1 were successfully separated using one-step hydrophobic

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interaction chromatography (Figure 7). The eluted α-CD from the C18 cartridge column was

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gathered and analyzed using HPAEC. The purity and yield of the obtained α-CD were 99 and

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90%, respectively.

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DISCUSSION

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In general, the products of CGTase reactions contain α-, β-, and γ-CD.1, 4 The purification of

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a single type of cyclodextrin from a CD mixture requires several steps, which can be time

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consuming and disadvantageous for human health and the environment.1 Therefore, to enable

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an efficient purification process, CGTases with high product specificity have been developed.

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11, 13, 22-27

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expression levels and their product ratio is usually lower than 90%, which limits the potential

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application of α-CD in many fields.4, 12, 24, 28 To overcome this problem, another approach,

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‘the solvent process’, has been developed. This method involves the addition of organic

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solvent as a complexing agent to extract target CDs selectively.29-31 For example, in the

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presence of 1-butanol, the α:β CD formation ratio of the CGTase increased dramatically to

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97:3 with a 42% (w/w) conversion rate. However, this solvent process is relatively expensive,

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flammable, and toxic. Also, the high boiling point of the solvent is disadvantageous for

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recovery of the formed molecules.4 Therefore, more environmentally friendly and economic

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methods are warranted.

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In this study, instead of developing an α-CGTase that increases the α-CD production ratio,

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we created a CGTase mutant with selective CD hydrolysis activity and suggested an

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alternative way to increase the α-CD ratio by degrading other types of CDs. We investigated

However, compared to other kind of CGTases, α-CGTases show relatively low


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the applicability of CGTase-alpha as an enhancing agent of α-CD production in CD mixtures.

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Even in a CD mixture containing the same amount of all CD types, CGTase-alpha

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successfully hydrolyzed β- and γ-CD, while α-CD remained intact (Figure 5). Additional

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treatment with AMG degraded the maltooligosaccharides linearized by CGTase-alpha to

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glucose, which would be useful for any subsequent purification steps, such as hydrophobicity

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interaction chromatography, size exclusion chromatography, or immobilized yeast treatment.

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Our results suggest that CGTase-alpha can be adapted to the α-CD purification process as an

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agent that enhances the α-CD production ratio. In addition, this treatment is environmental

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friendly.

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In the GH13 family, the catalytic subsites –1 to +3 are known to be responsible for enzyme

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reaction specificity, and these motifs are useful in guiding ‘inter-conversion’ within the GH13

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enzymes.32 This study focused on the H233 and K232 residues, which are located at the

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subsites +1 and +2, respectively.1, 33, 34 To introduce CD hydrolysis specificity, we replaced

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the K232 and H233 residues of CGTase with N and E residues, respectively, which are

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strictly conserved amino acids in the CD hydrolysis enzyme sequence (Figure 1).

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It has been reported that α-CD is structurally more rigid and more resistant to hydrolysis by

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amylases than other CDs.4,

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hydrolysis activity, it did not have α-CD hydrolysis capability due to an incomplete catalytic

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structure. Therefore, this mutant CGTase was able to hydrolyze β- and γ-CD only.

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In conclusion, our CGTase mutant, which is able to hydrolyze β- and γ-CD, was prepared by

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making mutations at the +1 and +2 subsite binding residues, and showed applicability for α-

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CD purification. Our results suggest that CGTase-alpha may contribute to enhanced

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production of α-CD.

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Although our mutant CGTase-alpha gained β- and γ-CD



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ACKNOWLEDGMENTS

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This study was supported by the Basic Science Research Program (NRF-2014-

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R1A1A2057436) of the National Research Foundation, funded by the Korean Government.

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Goo, B. G.; Hwang, Y. J.; Park, J. K., Bacillus thuringiensis: a specific gamma-cyclodextri Chen, F.; Xie, T.; Yue, Y.; Qian, S.; Chao, Y.; Pei, J., Molecular dynamic analysis of mut

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Jeang, C. L.; Lin, D. G.; Hsieh, S. H., Characterization of cyclodextrin glycosyltransferase

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352 353



354

Figure Legends

355

Figure 1. Multiple sequence alignment of the α-amylase super family at its conserved

356

regions. CD I-5, cyclodextrinase from Bacillus sp. I-5; TVA II, neopullulanase-type α-

357

amylase from Thermoactinomyces vulgaris; BSMA, maltogenic amylase from Geobacillus

358

stearothermophilus; ThMA, maltogenic amylase from Thermus sp. IM6501; GsNPL,

359

neopullulase from G. stearothermophilus; BciCGT, CGTase from Bacillus circulans; PaCGT,

360

CGTase from Paenibacillus sp. JB-13; BceCGT, CGTase from Bacillus cereus CGTase; CGT

361

I-5, CGTase from Bacillus sp. I-5; BlA, α-amylase from Bacillus licheniformis; HsA, α-

362

amylase from Homo sapiens; GsA, α-amylase from G. stearothermophilus; AoA, α-amylase

363

from Aspergillus oryzae; UBCD, CDase from uncultured bacterium; BSCD, CDase from

364

Bacillus sp. A2-5A; AMM, amylase from Alteromonas macleodii B7; ABDT 3-1, amylase

365

from Anoxybacillus sp. DT3-1; and BLMA, amylase from Bacillus lehensis G1.

366

Figure 2. Purification of CGTase-alpha. Lane S, protein size standards; lane 1, cellular

367

proteins from the crude extract; lane 2, soluble fraction; lane 3, insoluble fraction; and lane 4,

368

purified CGTase-alpha.

369

Figure 3. Hydrolysis pattern of the CGTase-alpha mutant with various substrates. α-, β-,

370

and γ-CD were reacted with CGTase-alpha. The reaction products were analyzed using TLC.

371

During the enzymatic reactions, β- and γ-CD were hydrolyzed to small maltooligosaccharides

372

but α-CD was not degraded by CGTase-alpha.

373

Figure 4. Hydrolysis pattern of an α, β, and γ-CD mixture by the CGTase-alpha mutant.

374

In a CD mixture solution, the CGTase-alpha mutant hydrolyzed β- and γ-CD selectively. Std,

375

maltooligosaccharide standards (G1–G7); lane 1, α-CD; lane 2, β-CD; lane 3, γ-CD; and


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376

lanes 4–8, hydrolysis products of the α, β, and γ-CD mixture solution at different reaction

377

times (0, 60, 90, 120, and 240 min, respectively).

378

Figure 5. Time course analysis of the residual amount of CDs during the CGTase-alpha

379

reaction. The remaining amount of CD was analyzed using HPAEC. In the CD mixture

380

solution, only β- and γ-CD were degraded gradually.

381

Figure 6. Degradation of linear maltooligosaccharides using AMG. After CGTase-alpha

382

treatment, linear maltooligosaccharides, which were generated from β- and γ-CD, were

383

hydrolyzed to glucose by AMG. α-CD, the circular maltooligosaccharide, was not affected by

384

AMG. Std, maltooligosaccharide standards (G1–G7); lane 1, CGTase-alpha treated solution

385

(before AMG treatment); lane 2, AMG treatment for 0.5 h; and lane 3, AMG treatment for 1 h.

386

Figure 7. Purification of α-CD using hydrophobic interaction chromatography. α-CD in

387

the AMG-treated mixture solution was purified using a C18 cartridge. Glucose, which is

388

hydrophilic, was washed using distilled water and α-CD was subsequently eluted using

389

methanol. Lane S, G1–G7 standard; and lane M, G1 and α-CD mixture (after AMG

390

treatment).

391



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392

393

Table 1. Purification of the CGTase-alpha mutant

Step

Volume (mL)

Enzyme activity (U)

Protein concentration (mg/mL)

Protein amount (mg)

Specific activity (U/mg)

Yield (%)

Purification fold

Cell extract

103

2158.5

16.9

1740.7

1.24

100

1

β-CD column

0.775

5.34

0.405

0.3138

17

0.25

13.7

394


Page 19 of 27


395

396

Table 2. Kinetic parameters of CGTase-alpha using CDs as substrates Substrate α-CD

397

Kinetic parameters –1

kcat (min )

Km (mM)

kcat /Km (min–1 mM–1)

n.d.

n.d.

n.d.

β-CD

5.2 ± 0.03

0.6 ± 0.01

8.7

γ-CD

31.27 ± 3.35

1.6 ± 0.48

19.5

n.d., not detected.

398



Figure 1. Multiple sequence alignment of the α-amylase super family at its conserved regions. CD I-5, cyclodextrinase from Bacillus sp. I-5; TVA II, neopullulanase-type α-amylase from Thermoactinomyces vulgaris; BSMA, maltogenic amylase from Geobacillus stearothermophilus; ThMA, maltogenic amylase from Thermus sp. IM6501; GsNPL, neopullulase from G. stearothermophilus; BciCGT, CGTase from Bacillus circulans; PaCGT, CGTase from Paenibacillus sp. JB-13; BceCGT, CGTase from Bacillus cereus CGTase; CGT I-5, CGTase from Bacillus sp. I-5; BlA, α-amylase from Bacillus licheniformis; HsA, α-amylase from Homo sapiens; GsA, α-amylase from G. stearothermophilus; AoA, α-amylase from Aspergillus oryzae; UBCD, CDase from uncultured bacterium; BSCD, CDase from Bacillus sp. A2-5A; AMM, amylase from Alteromonas macleodii B7; ABDT 3-1, amylase from Anoxybacillus sp. DT3-1; and BLMA, amylase from Bacillus lehensis G1. 185x120mm (150 x 150 DPI)


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Figure 2. Purification of CGTase-alpha. Lane S, protein size standards; lane 1, cellular proteins from the crude extract; lane 2, soluble fraction; lane 3, insoluble fraction; and lane 4, purified CGTase-alpha. 132x87mm (150 x 150 DPI)



Figure 3. Hydrolysis pattern of the CGTase-alpha mutant with various substrates. α-, β-, and γ-CD were reacted with CGTase-alpha. The reaction products were analyzed using TLC. During the enzymatic reactions, β- and γ-CD were hydrolyzed to small maltooligosaccharides but α-CD was not degraded by CGTase-alpha. 235x118mm (150 x 150 DPI)


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Figure 4. Hydrolysis pattern of an α, β, γ-CD mixture by the CGTase-alpha mutant. In a CD mixture solution, the CGTase-alpha mutant hydrolyzed β- and γ-CD selectively. Std, maltooligosaccharide standards (G1–G7); lane 1, α-CD; lane 2, β-CD; lane 3, γ-CD; and lanes 4–8, hydrolysis products of the α, β, and γ-CD mixture solution at different reaction times (0, 60, 90, 120, and 240 min, respectively). 111x96mm (150 x 150 DPI)



Figure 5. Time course analysis of the residual amount of CDs during the CGTase-alpha reaction. The remaining amount of CD was analyzed using HPAEC. In the CD mixture solution, only β- and γ-CD were degraded gradually. 142x103mm (150 x 150 DPI)


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Figure 6. Degradation of linear maltooligosaccharides using AMG. After CGTase-alpha treatment, linear maltooligosaccharides, which were generated from β- and γ-CD, were hydrolyzed to glucose by AMG. α-CD, the circular maltooligosaccharide, was not affected by AMG. Std, maltooligosaccharide standards (G1–G7); lane 1, CGTase-alpha treated solution (before AMG treatment); lane 2, AMG treatment for 0.5 h; and lane 3, AMG treatment for 1 h. 66x108mm (150 x 150 DPI)



Figure 7. Purification of α-CD using hydrophobic interaction chromatography. α-CD in the AMG-treated mixture solution was purified using a C18 cartridge. Glucose, which is hydrophilic, was washed using distilled water and α-CD was subsequently eluted using methanol. Lane S, G1–G7 standard; and lane M, G1 and αCD mixture (after AMG treatment). 139x95mm (150 x 150 DPI)


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TOC Graphic. 383x190mm (150 x 150 DPI)


Development and Application of Cyclodextrin Hydrolyzing Mutant

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