Development and Application of Cyclodextrin Hydrolyzing Mutant

Mar 2, 2017 - Cyclodextrins (CDs) are produced from starch by cyclodextrin glucanotransferase (CGTase), which has cyclization activity. Specifically, ...
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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 is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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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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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

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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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n producer strain. Carbohydr Res 2014, 386, 12-17.

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ant Y195I alpha-cyclodextrin glycosyltransferase with switched product specificity from alpha-cy

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clodextrin to gamma-cyclodextrin. J. Mol. Model. 2015, 21, 208.

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Bradford, M. M., A rapid and sensitive method for the quantitation of microgram qua

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Song, K. M.; Shim, J. H.; Park, J. T.; Kim, S. H.; Kim, Y. W.; Boos, W.; Park, K. H., Trans

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

Song, B.; Yue, Y.; Xie, T.; Qian, S.; Chao, Y., Mutation of tyrosine167histidine at remote

substrate binding subsite -6 in alpha-cyclodextrin glycosyltransferase enhancing alpha-cyclode

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xtrin specificity by directed evolution. Mol. Biotechnol. 2014, 56, 232-239.

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menez Bonino, M.; Ferrarotti, S. A., The residue 179 is involved in product specificity of the B

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acillus circulans DF 9R cyclodextrin glycosyltransferase. Appl. Microbiol. Biotechnol. 2012, 94, 1

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sine 195 leading to diverse product specificities of an alpha-cyclodextrin glycosyltransferase fr

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om Paenibacillus sp. 602-1. J. Biotechnol. 2014, 170, 10-16.

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ucanotransferase from an alkaliphilic Bacillus sp. FEBS Open Bio. 2015, 5, 528-534.

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Xie, T.; Song, B.; Yue, Y.; Chao, Y.; Qian, S., Site-saturation mutagenesis of central tyro

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

of the same gene expressed from Bacillus macerans, Bacillus subtilis, and Escherichia coli. J.

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ing-cyclodextrin glycosyltransferase from Klebsiella pneumoniae AS- 22. Enzyme Microb. Techn

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ol. 2001, 28, 735-743.

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n by enzymatic degradation of starch. Ann. N. Y. Acad. Sci. 1984, 434, 70-77.

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Flaschel, E.; Landert, J. P.; Spiesser, D.; Renken, A., The production of alpha-cyclodextri Kumar, V., Identification of the sequence motif of glycoside hydrolase 13 family mem Penninga, D.; Strokopytov, B.; Rozeboom, H. J.; Lawson, C. L.; Dijkstra, B. W.; Bergsma,

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m Bacillus circulans strain 251 affect activity and product specificity. Biochemistry 1995, 34, 33

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r the catalytic mechanism of glycosidases. Biochemistry 1995, 34, 2234-2240.

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rin by Aspergillus oryzae α-amylase. Starch 1984, 36, 140-143.

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352 353

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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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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).

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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

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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

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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)

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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)

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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)

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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)

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