Efficient Biocatalytic Production of Cyclodextrins by Combined Action

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Efficient Biocatalytic Production of Cyclodextrins by Combined Action of Amylosucrase and Cyclodextrin Glucanotransferase Dong-Wan Koh, Min-Oh Park, Seong-Won Choi, Byung-Hoo Lee, and Sang-Ho Yoo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01080 • Publication Date (Web): 12 May 2016 Downloaded from http://pubs.acs.org on May 12, 2016

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Journal of Agricultural and Food Chemistry

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Efficient Biocatalytic Production of Cyclodextrins by Combined Action of Amylosucrase

2

and Cyclodextrin Glucanotransferase

3 4 5

Dong-Wan Koh,† Min-Oh Park,† Seong-Won Choi,† Byung-Hoo Lee,*,§ and Sang-Ho Yoo*,†

6 7

†

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Sejong University, Gunja-Dong, Gwangjin-Gu, Seoul 143-747, Republic of Korea

9

§

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Department of Food Science & Technology and Carbohydrate Bioproduct Research Center,

Department of Food Science & Biotechnology, College of BioNano Technology, Gachon

University, Seongnam, Gyeonggi-do 461-701, Republic of Korea

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*Corresponding Authors :

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†

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Tel: +82-2-3408-3221; E-mail: [email protected]

15

§

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Tel: +82-31-750-5405; E-mail: [email protected]

Sang-Ho Yoo

Byung-Hoo Lee

17 18

1 ACS Paragon Plus Environment


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ABSTRACT

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A novel enzymatic process for cyclodextrin (CD) production was developed by utilizing

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sucrose as raw material instead of corn starch. Cyclodextrin glucanotransferase (CGTase) from

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Bacillus macerans was applied to produce the CDs from linear α-(1,4)-glucans which were

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obtained by Neisseria polysaccharea amylosucrase (NpAS) treatment on sucrose. The greatest

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CD yield (21.1%, w/w) was achieved from a one-pot dual enzyme reaction at 40°C for 24 h. The

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maximum level of CD production (15.1 mg/mL) was achieved with 0.5 M sucrose in a

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simultaneous mode of dual enzyme reaction, whereas the reaction with 0.1 M sucrose was the

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most efficient one with regard to the conversion yield. Consequently, dual enzyme synthesis of

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CDs was successfully carried out with no need of starch material. This result can be applied as a

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novel efficient bioconversion process that does not require high temperature necessary for starch

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liquefaction by thermostable α-amylase in conventional industrial processing.

31 32

Keywords: cyclodextrins, amylosucrase, cyclodextrin glucanotransferase, one-pot synthesis

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INTRODUCTION

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Cyclodextrins (CDs) are cyclic oligosaccharides composed of a different number of D-

36

glucopyranoside units linked by α-(1,4)-glycosidic linkages. There are three main types of CDs

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distinguished by the number of glucosyl units, namely α-, β-, and γ-CDs consisting of 6, 7, and 8

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α-(1,4)-linked glucosyl units, respectively.1 These CDs have a hydrophobic central cavity, which

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is capable of solubilizing and stabilizing hydrophobic and unstable materials by forming an

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inclusion complex. This structural feature of CDs has made them frequently utilized chemical

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substances in food, pharmaceutical, cosmetic, and chemical industries.2-4

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CDs are commercially produced from starch by cyclodextrin glucanotransferase (EC

43

2.4.1.19, CGTase).5-7 CGTases generally catalyze the production of CDs via intramolecular

44

transglycosylation reactions. CGTase is a multifunctional enzyme that catalyzes three major

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different reactions: cyclization, coupling, and disproportionation as well hydrolyzation as a

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minor activity.8 Cyclization is the process through which a linear α-(1,4)-linkage is cleaved and

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each end of the cleaved fragment is joined with the formation of cyclic oligosaccharides of

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different sizes. Coupling is the reverse process of cyclization: the enzyme is able to cleave CDs,

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thereby generating a linear α-(1,4)-linkage. Disproportionation is very similar to coupling, but in

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this case, a cleaved linear α-(1,4)-linkage is joined to a second linear acceptor molecule.9,

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During industrial CD production, corn starch is liquefied by a starch-liquefying enzyme at high

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gelatinization temperature, and then saccharificated by CGTase.11 Many efforts have been made

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to improve CD production by decreasing energy consumption or increasing the production yield.

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Several studies have indicated that the reaction of CGTase with starch generates around 80% of

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waste material that cannot be further used as a substrate for the CGTase reaction.12, 13

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Amylosucrase is a glucosyltransferase (EC 2.4.1.4), first discovered in cultures of 3 ACS Paragon Plus Environment


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Neisseria perflava, which catalyzes the synthesis of linear α-(1,4)-glucan polymers from sucrose

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molecules.14 Amylosucrase from Neisseria polysaccharea (NpAS) was cloned and expressed in

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Escherichia coli.15 When sucrose is used as sole substrate, the transferring activity of NpAS

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elongates the linear α-(1,4)-glucan chains. In this NpAS-catalyzed reaction, the concentration of

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sucrose influences the chain length of the glucan products.

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Industrial CD production processes require energy-intensive, time-consuming pre-

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gelatinization, and liquefaction steps. In this study, a novel method of CD production was

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designed and optimized using sucrose as a highly water-soluble starting raw material. We

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utilized CGTase to produce CDs from linear α-(1,4)-glucan molecules that was supplied by

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amylosucrase treatment on sucrose. The strategic objective of this study was to investigate the

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efficiency of combining the action of elongation and cyclization glycosyltransferases to improve

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CD production in the absence of heat-transfer processing. The novel approach can be applied to

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establish mass-production of CDs that will be utilized in wide range of industries including food

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and pharmaceutical area.

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MATERIALS AND METHODS

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Materials. Soluble starch was purchased from Yakuri Pure Chemicals Co. (Kyoto, Japan).

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Sucrose was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Cyclodextrins

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(α-, β-, and γ-CDs) were purchased from Wacker Chemie AG (Munich, Germany). Thermostable

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α-amylase (Liquozyme supra®, Novozymes A/S, Bagsvaerd, Denmark), fungal α-amylase

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(Fungamyl 800L, Novozymes A/S), and CGTase from Bacillus macerans (Amano

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Pharmaceutical Co., Nagoya, Japan) were used for CD production from sucrose. Glucoamylase

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from Rhizopus sp. was obtained from Toyobo Co. (Osaka, Japan). 4 ACS Paragon Plus Environment

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CGTase assay. One unit (U) of CGTase was defined as the amount of enzyme that produced a

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10% reduction in the intensity of blue color of amylose complex for 1 min. The enzyme solution

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was reacted with 1.2% soluble potato starch (Sigma-Aldrich Chemical Co, St. Louis, MO) in 1

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mL of a 50 mM sodium acetate buffer (pH 5.5 at 40°C) for 10 min. To stop the enzyme reaction,

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0.5 mL of 0.5 N acetic acid and 0.5 N HCl (5:1, v/v) was added to an aliquot (0.25 mL) of the

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reaction mixture. Then, 5 mL of 0.005% I2 in a 0.05% KI solution was added to 0.1 mL of this

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mixture, and the absorbance of the final solution was measured at 660 nm with a

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spectrophotometer (DU 730, Beckman Coulter, Fullerton, CA).16

89 90

Neisseria polysaccharea amylosucrase assay. The recombinant NpAS was cloned and

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expressed as previously described.17 One unit of NpAS was defined as the amount of enzyme

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that releases 1 µmol of fructose per min from 0.1 M sucrose and 0.1% (w/v) waxy corn starch in

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a 50 mM Tris-HCl buffer, pH 7.0 at 35°C. The reaction was stopped by boiling for 10 min and

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the amount of fructose was measured by the DNS method with fructose used as the standard.18

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Synthesis of linear α-glucans of different sizes in reaction mixtures containing NpAS and

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sucrose at various concentrations. During the transglucosylating reaction, NpAS (8.4 U/mL)

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was incubated with solutions containing different concentrations of sucrose (0.1 to 1.0 M) in 1

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mL of a 50 mM tris-HCl buffer, pH 7.0 at 40°C for 24 h. The reaction was stopped by boiling for

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10 min and the reaction mixture was centrifuged at 9,000 × g for 10 min at 25°C to separate

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insoluble α-(1,4)-glucan products. The synthesized α-(1,4)-glucans (100 µL, 1% w/v) were

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dispersed in 0.9 mL of dimethyl sulfoxide. The resulting clear solution was mixed with 6 5 ACS Paragon Plus Environment


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volumes of 99% ethanol and centrifuged at 4,500 × g for 10 min. The precipitates were dissolved

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in 1 mL of the pre-heated 50 mM Tris-HCl buffer (pH 7.0) and then boiled for 30 min.

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Distributions of molecular weights of insoluble linear α-(1,4)-glucans were determined

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by high-performance size-exclusion chromatography (HPSEC) with a refractive index detector

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on Shodex OHpak SB-804HQ and OHpak SB-802.5 HQ columns (8 × 300 mm each, Showa

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Denko, Tokyo, Japan). A total of 10 mg of freeze-dried α-glucans was completely wetted with

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0.1 mL of distilled water for 10 min and then dispersed in 0.9 mL of dimethyl sulfoxide. The

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precipitates were dissolved in 1 mL of heated distilled water and then boiled for 30 min. The

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resulting solutions were filtered through a 0.45-µm nylon membrane filter. The aliquot (100 µL)

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was injected and eluted with distilled water at a flow rate of 0.8 mL for 1 min at 50°C.19

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One-pot synthesis of cyclodextrins from sucrose by the dual enzyme treatment. To

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synthesize CDs, NpAS (8.4 U/mL) and CGTase (0.75 U/mL) were co-incubated with sucrose at

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different concentrations (0.1 to 1.0 M) in 1 mL of a 50 mM Tris-HCl buffer, pH 7.0 at 40°C for

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24 h. The reaction was stopped by boiling for 10 min. After cooling, the reaction mixture was

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centrifuged at 9,000 × g at 25°C for 10 min. Then, 0.1 mL of the supernatant was mixed with 0.4

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mL of a 50 mM sodium acetate buffer (pH 5.5) and treated with glucoamylase (8 U/mL) to

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hydrolyze the residual linear α-(1,4)-glucans into glucose.

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Cyclodextrin analysis by MALDI-TOF-MS and HPAEC. The cyclodextrin products in

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distilled water (0.5 mg/10 µL, w/v) and 2,5-dihydroxybenzoic acid (10 mg/mL, w/v) as a matrix

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were mixed in equal volumes (1.0 µL each). The mixture was dropped on a sample plate, and the

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molecular-mass spectrum of CDs produced by CGTase reaction was obtained by matrix-assisted 6 ACS Paragon Plus Environment

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laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS, AXIMA-LNR

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system; Shimadzu, Kyoto, Japan).

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CD content (%) was measured by high-performance anion-exchange chromatography

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with pulsed amperometric detection (HPAEC-PAD) using a CarboPac™ PA-100 analytical

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column (4 × 250 mm, Dionex, Sunnyvale, CA, USA). The reaction sample (25 µL) was injected

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and eluted with a gradient of sodium nitrate eluent (0–2 min, 8 mM; 2–22 min, 16 mM; 22–30

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min, 17 mM; 30–40 min, 200 mM) in 150 mM NaOH with a flow rate of 1 mL/min at 30°C. The

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amount of CDs was quantified by comparing respective peak areas to those of CD standard

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materials.20

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Statistical analysis. All measurements were made in duplicate. The analysis of variance

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(ANOVA) was initially used for the statistical treatment of the experimental results followed by

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Tukey’s HSD tests, if ANOVA reported a significant F value. All statistical analyses were carried

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out using IBM® SPSS® Statistics for Windows (Version 21.0, IBM Corporation, Armonk, NY).

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Statistical significance was indicated at a confidence level of 95%.

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RESULTS AND DISCUSSION

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Characterization of cyclodextrins produced by the CGTase reaction. To determine the ratio

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of individual CDs generated by CGTase from Bacillus macerans in a control CD production

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process, we used the liquefied normal corn starch slurry (15%, w/v) treated with 0.75 U/mL

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CGTase at 40°C for 12 h. The production yield of individual CDs was determined by HPAEC.

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We found that in these reaction conditions, the synthesized α-, β-, and γ-CDs comprised 54.3%,

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35.8%, and 9.9%, respectively. The type of CDs produced by the CGTase reaction were 7 ACS Paragon Plus Environment


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confirmed by the MALDI-TOF-MS based on their molecular masses (Fig. 1). Three major peaks

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were detected at the same 162-dalton molecular mass interval (equal to that of a single glycosyl

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residue), and their molecular masses corresponded to those of α-, β-, and γ-CDs.

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Effects of the length of linear α-(1,4)-glucans on CD formation catalyzed by CGTase. Linear

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α-(1,4)-glucans, which were synthesized by NpAS-catalyzed reaction from sucrose used as a sole

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substrate, were analyzed by HPSEC (Fig. 2) and then utilized as the substrate for the CD

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production. The molecular size of each linear α-(1,4)-glucan product was represented by the

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degree of polymerization (DP) value at the apex of the peaks in the HPSEC chromatogram. The

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DP values obtained in experiments with different starting concentrations of sucrose (0.1, 0.3, 0.5,

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0.7, and 1.0 M) were 166, 116, 44, 38, and 29, respectively. Corresponding production yields of

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linear α-glucans were 19.6%, 24.8%, 29.8%, 26.1%, and 20.3%, respectively. Thus, the sucrose

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concentration affected the DP value of linear α-(1,4)-glucans.19 Moreover, yields positively

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correlated with the sucrose concentration when the latter was in the range of 0.1–0.5 M. The

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decrease of linear α-(1, 4)-glucan production at sucrose concentrations higher than 0.5 M was

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likely caused by the utilization of sucrose in alternative reactions, which generated sucrose

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isomers, such as turanose and trehalulose, instead of linear α-(1,4)-glucans. 21

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In the present study, linear α-glucans of different sizes synthesized by NpAS were used in

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CD production by CGTase in a sequential manner to determine the effect of the linear α-glucan

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length on the properties of final products. The CD production yield was evaluated by HPAEC

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analysis after glucoamylase treatment to remove linear α-glucan residues. The maximum

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conversion yield of 85.1% was observed in the case of linear α-(1,4)-glucans with major peaks of

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DP 65 and 166 that were produced from 0.1 M sucrose (Table 1). The production yield of CDs 8 ACS Paragon Plus Environment

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decreased along with the linear α-(1,4)-glucan DP values. This result suggested that the

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maximum yield of CDs synthesized by CGTase could be achieved using 0.1 M sucrose as the

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optimal initial substrate concentration.

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One-pot synthesis of CDs from sucrose by a simultaneous treatment with NpAS and

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CGTase. CDs were synthesized from 0.1 M sucrose as a sole substrate instead of starch

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materials by a simultaneous treatment with NpAS (8.4 U/mL) and CGTase (0.75 U/mL). The

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time course experiment revealed that the greatest yield of CD production (17.98%) was observed

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after 24 h (Fig. 3). However, the amount of produced CDs became lower after 48 h due to

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CGTase-catalyzed hydrolysis of α-(1,4)-linkages that represents a minor effect of this enzyme.22

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Depending on the initial concentration of sucrose, NpAS catalyzes the formation of linear α-

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(1,4)-glucans of different sizes,19, 23 which can be subsequently utilized as the starting material

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for CGTase-catalyzed CD production. In this study, the production yield of CDs by CGTase

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negatively correlated with the starting sucrose concentration (Table 2). To produce CDs by

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CGTase reaction, this enzyme requires the minimal size of the linear α-(1,4)-linked glucans as a

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subtrate material which can be easily accessed through the binding pocket of the active site.4, 24 A

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previous study showed that longer α-glucan chains were synthesized from low starting sucrose

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concentrations by the amylosucrase reaction.23 This result demonstrates that CD production from

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reaction mixtures that contain low concentrations of sucrose and a combination of NpAS and

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CGTase shows a high conversion yield because the reaction condition during one-pot synthesis

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can produce the substrate of the sufficient size for CGTase. The total amount of CDs obtained

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from reaction mixtures with 0.3 and 0.5 M sucrose was significantly higher than that generated

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in other reaction conditions (p < 0.05). At the same time, it appears that the reaction with 0.1 M 9 ACS Paragon Plus Environment


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sucrose is the most efficient one with regard to the conversion yields after the combined action of

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NpAS and CGTase.

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In summary, in the present study, we developed and evaluated a novel process to produce

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CDs from sucrose by a combined action of NpAS and CGTase. When 0.75 U/ml CGTase and 8.4

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U/ml NpAS reacted with 0.1 M sucrose, the maximum CD production yield of 21.1% was

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achieved. We found that longer linear α-(1,4)-glucans were preferred for CD production.

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Importantly, the enzymatic production of CDs using a combined treatment with NpAS and

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CGTase did not need prior liquefaction of starch at high temperatures, which is required in the

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majority of industrial CD production routines. Consequently, our innovative process is an

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effective and promising tool to utilize sucrose using green technology. As CDs have been widely

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used as novel commercial ingredients in food and pharmaceutical products, our suggested

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method is an important step toward ecologically safe production of this class of chemical

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substances, the demand for which is likely to be growing in the near future.

208 209

ACKNOWLEDGMENTS

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This research was supported by High Value-added Food Technology Development Program

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(Project number: 115035-03-1-HD020), Ministry for Food, Agriculture, Forestry and Fisheries,

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Republic of Korea. This research was also supported by the Gachon University research fund of

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2015 (GCU-2015-0058).

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FIGURE LEGENDS

Figure 1. MALDI-TOF-MS analysis of α-, β-, and γ-cyclodextrins produced by CGTase from Bacillus macerans. Molecular mass of each peak is equivalent to 162n + 23 Da as determined by the number of glucosyl residues (n) and the mass of the sodium ion (Na+, 23 Da).

Figure 2. Distributions of molecular weights of linear α-(1,4)-glucans synthesized following the NpAS treatment of reaction mixtures containing sucrose at different concentrations measured by HPSEC. DP, Degree of Polymerization.

Figure 3. Time course study of the total CD production and the ratio of α-, β-, and γ-CDs in the presence of 8.4 U/mL NpAS, 0.75 U/mL CGTase, and 0.1 M sucrose during 48-h reaction.


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Table 1. Production Yield of CDs Synthesized by CGTase from Linear α-(1,4)-Glucans of Different Sizes Generated by a Reaction of Sucrose with 8.4 U/mL NpAS Peak DP values of glucan sample

CD type fractions (%) α-CD

β-CD

γ-CD

Total amount of CDs (mg/mL)1

29

83.8±1.0e

15.8±0.2a

0.4±0.1a

0.3±0.0a

28.9±0.3a

38

70.3±0.4b

25.7±0.3d

4. 0±0.1b

0.6±0.0b

57.2±0.3b

44

71.8±0.5c

24.2±0.3c

4.0±0.2b

0.6±0.0c

63.6±0.1c

53, 116

75.4±0.1d

21.0±0.1b

3.5±0.0b

0.6±0.0b

57.2±1.0b

65, 166

74.7±0.2d

20.7±0.1b

4.6±0.1c

0.9±0.0e

85.1±1.1e

Conversion yield (%)2

1

Mean value ± standard deviation of measurement of experiments performed in duplicate. Values denoted by different letters within the same column are significantly different from each other at p < 0.05 in ANOVA and Tukey’s HSD multiple comparison tests.

2

Conversion yield (%) of the CGTase reaction = (total amount of CD products) / (initial amount of the linear α-(1,4)-glucan substrate) × 100



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Table 2. Quantitative Analysis of CD Products Synthesized by One-pot Dual Enzyme Reaction Using NpAS (8.4 U/mL) and CGTase (0.75 U/mL) Sucrose concentration (M)

CD type fractions (%) α-CD β-CD γ-CD

Total amont of CDs (mg/mL)1

Conversion yield (%)2

0.1

45.89±3.93b

42.02±3.39a

12.09±0.54a

7.23±1.26a

21.13±3.69c

0.3

43.73±2.23ab

41.10±1.70a

15.18±0.53b

10.79±0.95b

10.50±0.92b

0.5

41.20±2.34ab

42.73±2.16a

16.07±0.18b

10.56±1.19b

6.17±0.70ab

0.7

40.03±1.05ab

44.14±1.36a

15.83±0.28b

9.59±0.61ab

4.00±0.25a

1.0

38.34±2.29a

46.34±1.35a

15.32±0.94b

7.71±0.44a

2.25±0.13a

1

Mean value ± standard deviation of measurement of experiments performed in duplicate. Values denoted by different letters within the same column are significantly different from each other at p < 0.05 in ANOVA and Tukey’s HSD multiple comparison tests.

2

Conversion yield (%) of the CGTase reaction = (total amount of CD products) / (initial amount of the linear α-(1,4)-glucan substrate) × 100


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Figure 1.



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Figure 2.


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Figure 3.



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ACS Paragon Plus Environment

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Efficient Biocatalytic Production of Cyclodextrins by Combined Action

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