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Synergistic Action Modes of Arabinan Degradation by Exo- and Endo-Arabinosyl Hydrolases
Synergistic Action Modes of Arabinan Degradation by Exo- and Endo-Arabinosyl Hydrolases
Journal of Microbiology and Biotechnology. 2015. Feb, 25(2): 227-233
Copyright © 2015, The Korean Society For Microbiology And Biotechnology
  • Received : November 20, 2014
  • Accepted : November 27, 2014
  • Published : February 28, 2015
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About the Authors
Jung-Mi Park
Department of Food Science and Biotechnology, and Brain Korea 21 Center for Bio-Resource Development, Chungbuk National University, Cheongju 362-763, Republic of Korea
Myoung-Uoon Jang
Department of Food Science and Biotechnology, and Brain Korea 21 Center for Bio-Resource Development, Chungbuk National University, Cheongju 362-763, Republic of Korea
Gyo Won Oh
Department of Food Science and Biotechnology, and Brain Korea 21 Center for Bio-Resource Development, Chungbuk National University, Cheongju 362-763, Republic of Korea
Eun-Hee Lee
Department of Food Science and Biotechnology, and Brain Korea 21 Center for Bio-Resource Development, Chungbuk National University, Cheongju 362-763, Republic of Korea
Jung-Hyun Kang
Department of Food Science and Biotechnology, and Brain Korea 21 Center for Bio-Resource Development, Chungbuk National University, Cheongju 362-763, Republic of Korea
Yeong-Bok Song
Sejeon Food Research Institute, Sejeon Co., Seongnam 462-807, Republic of Korea
Nam Soo Han
Department of Food Science and Biotechnology, and Brain Korea 21 Center for Bio-Resource Development, Chungbuk National University, Cheongju 362-763, Republic of Korea
Tae-Jip Kim
Department of Food Science and Biotechnology, and Brain Korea 21 Center for Bio-Resource Development, Chungbuk National University, Cheongju 362-763, Republic of Korea
tjkim@cbnu.ac.kr

Abstract
Two recombinant arabinosyl hydrolases, α- L -arabinofuranosidase from Geobacillus sp. KCTC 3012 ( G AFase) and endo -(1,5)-α- L -arabinanase from Bacillus licheniformis DSM13 ( Bl ABNase), were overexpressed in Escherichia coli , and their synergistic modes of action against sugar beet (branched) arabinan were investigated. Whereas G AFase hydrolyzed 35.9% of L -arabinose residues from sugar beet (branched) arabinan, endo -action of Bl ABNase released only 0.5% of L -arabinose owing to its extremely low accessibility towards branched arabinan. Interestingly, the simultaneous treatment of G AFase and Bl ABNase could liberate approximately 91.2% of L -arabinose from arabinan, which was significantly higher than any single exo -enzyme treatment (35.9%) or even stepwise exo - after endo -enzyme treatment (75.5%). Based on their unique modes of action, both exo - and endo -arabinosyl hydrolases can work in concert to catalyze the hydrolysis of arabinan to L -arabinose. At the early stage in arabinan degradation, exo -acting G AFase could remove the terminal arabinose branches to generate debranched arabinan, which could be successively hydrolyzed into arabinooligosaccharides via the endo -action of Bl ABNase. At the final stage, the simultaneous actions of exo - and endo -hydrolases could synergistically accelerate the L -arabinose production with high conversion yield.
Keywords
Introduction
L -Arabinose is one of the common components in hemicellulosic plant polysaccharides such as arabinan and arabinoxylan [7] . It has been reported that L -arabinose can inhibit intestinal sucrase activity to prevent the obesity caused by a high level of sucrose absorption. Therefore, L -arabinose is highly focused as a food additive such as a low-calorie sweetener [21] . Arabinan is a pectic polysaccharide with a backbone of α-(1,5)-linked L -arabinofuranosyl units, which are substituted for α-(1,2)- and/or α-(1,3)-linked arabinofuranosides [20] . For more efficient hydrolysis of arabinan towards L -arabinose, the side chains and the backbone of arabinan should be simultaneously degraded by a couple of hydrolases.
Arabinan-degrading enzymes can be classified into the α- L -arabinofuranosidases (AFases; E.C. 3.2.1.55) and the endo -1,5-α- L -arabinanases (ABNases; E.C. 3.2.1.99) on the basis of their modes of action. AFases are typical exo -acting enzymes, which hydrolyze the terminal nonreducing L -arabinose residues from arabinose-containing polysaccharides. This enzyme works in concert with other hemicellulases to completely degrade the hemicellulose backbone, which makes it an essential enzyme for the industrial production of L -arabinose [18] . AFases belong to glycoside hydrolase (GH) families 3, 43, 51, 54, and 62. A variety of AFases have been purified from fungi, bacteria, and plants [2 - 4 , 6 , 15 , 17 , 19] . The three-dimensional structure of AFase GH 51 from Geobacillus stearothermophilus revealed that it is a hexamer with a (β/α) 8 -barrel structure [5] . Meanwhile, ABNases are endo -acting hydrolases that can cleave the internal α-(1,5)- L -arabinofuranosidic linkages of arabinan [1] . Microbial arabinanases have been studied from several Bacillus , Cellvibrio , and Aspergillus spp. [1 , 9 , 11 , 16] . Most ABNases GH 43 are known to possess the monomeric structure with the 5-bladed β-propeller topology [14 , 23] .
In general, AFases are know n to act as the accessory enzymes necessary for the complete hydrolysis of hemicellulosic biomass including arabinoxylan. These enzymes can synergistically hydrolyze the glycosidic bonds of polymers in combination with other hemicellulases [10 , 22] . Some AFases with both exo - and endo -activity on arabinan have been reported [11 , 13] . The same effect has been shown when these enzymes act synergistically on arabinoxylan. AFases also act synergistically with endo -arabinanase and cinnamoyl esterase from Aspergillus niger [8] . Compared with xylan degradation, however, the synergistic effects of exo - and endo -arabinosyl hydrolases on arabinan degradation have not been studied well. The synergistic L -arabinose production from arabinan was recently reported by using the combined treatment of thermostable enzymes from Caldicellulosiruptor saccharolyticus [10] . However, their detailed modes of action in synergism have not been studied yet.
In the present study, the synergistic effect of both exo - and endo -acting arabinosyl hydrolases on the degradation of arabinan was investigated on the basis of their modes of action. Moreover, we proposed the cost-effective enzymatic production of L -arabinose as a functional five-carbon sugar.
Materials and Methods
- Substrates and Chemicals
Sugar beet (branched) arabinan, debranched (linear) arabinan, and arabinooligosaccharides were purchased from Megazyme (Wicklow, Ireland). Oat spelt xylan was obtained from Sigma-Aldrich (MO, USA). Other chemicals were supplied by Duchefa Biochemie (Haarlen, The Netherlands), Sigma-Aldrich, or Merck (Germany).
- Gene Expression and Enzyme Purification
In order to produce Geobacillus sp. AFase ( G AFase), Escherichia coli MC1061 harboring plasmid pHCXGAF [15] was grown in LBA broth (0.5% bacto-tryptone, 1% yeast extract, 1% NaCl, 100 µg/ml of ampicillin) at 37℃ for 14 h. E. coli BL21 (DE3) harboring pETBLABN, encoding the Bacillus licheniformis ABNase gene [16] , was cultivated in LBA broth at 37℃ for 4 h, and IPTG at the final concentration of 0.1 mM was added to induce the gene expression. After additional cultivation for 12 h, the grown cells were harvested and disrupted by ultrasonication (VCX750; Sonics & Materials, Newtown, CT, USA). The recombinant enzymes with the C-terminal six-histidines were purified by using an AKTA Prime system with a HisTrap-FF column (GE Healthcare, Uppsala, Sweden). The protein concentration was measured using the BCA protein assay kit (Pierce Biotechnology, Rockford, IL, USA) with bovine serum albumin as a standard.
- Enzyme Activity Assay
Purified enzyme was reacted with 0.5% of each substrate in 50 mM sodium acetate buffer (pH 6.0) at 55℃ for an appropriate time. The 3,5-dinitrosalicylic acid (DNS) reducing sugar method [12] was employed for the determination of the hydrolyzing activity against sugar beet arabinan, debranched arabinan, or oat spelt xylan (2.5%). One unit of enzyme activity on each substrate was defined as the amount of enzyme producing 1 µmol equivalent of L -arabinose per minute.
- Analysis of Enzyme Reaction Products
Thin-layer chromatography (TLC) and high-performance anion-exchange chromatography (HPAEC) analyses were performed to determine the hydrolysis products generated from various substrates. The appropriate amount of Bl ABNase was reacted with 0.5% of each substrate at its optimal reaction conditions. For TLC analysis, the resulting products were separated on the 60F 254 silica gel plate (Merck, Darmstadt, Germany) with the solvents of ethyl acetate/acetic acid/water (2:1:1 (v/v/v)). The plate was visualized by dipping it into a solution containing 0.3% (w/v) N -(1-naphthyl)-ethylenediamine and 5% (v/v) H 2 SO 4 in methanol, and then heating it for 10 min at 110℃. A CarboPac PA1 column (0.4 × 25 cm; Thermo Fisher Scientific, Sunnyvale, CA, USA) was used for HPAEC analysis (Bio-LC ICS-3000; Thermo Fisher Scientific, Rockford, IL, USA) with an electrochemical detector (ED40; Thermo). Samples were eluted with a linear gradient from 100% buffer A (150 mM NaOH in water; Thermo) to 50% buffer B (600 mM of sodium acetate in buffer A; Sigma-Aldrich) over 50 min. The flow rate of the mobile phase was maintained at 1.0 ml/min.
- Enzymatic Degradation of Arabinan
The reaction mixture (1 ml) containing 0.5% of sugar beet arabinan was reacted with a single enzyme (0.26 U) or in combination of dual enzymes (0.13 U each). The hydrolysis of arabinans in use of G AFase and Bl ABNase were incubated in 50 mM sodium acetate buffer (pH 6.0) at 55℃ for 6 h. The released reducing sugars and L -arabinose were measured as L -arabinose equivalents by DNS assay and HPAEC analysis, respectively. For the stepwise enzyme treatments, either endo - or exo -acting enzyme was incubated with a substrate for 3 h at the first stage. The first enzyme (0.26 U) was inactivated by boiling for 3 min, and the other enzyme (0.26 U) was newly added and reacted for an additional 3 h at the second stage. In order to determine the hydrolysis yield and L -arabinose yield, the resulting products at each time interval were quantitatively analyzed by the DNS reducing sugar assay and HPAEC analysis, respectively.
Results and Discussion
- Gene Expression and Purification of Arabinosyl Hydrolases
Both exo - and endo -arabinosyl hydrolase genes had been cloned previously [15 , 16] . In this study, the recombinant G AFase and Bl ABNase with the C-terminal six-histidines were produced in E. coli via the constitutive and the inducible expression systems, respectively. By using Ni-NTA column chromatography, each enzyme was purified to apparent homogeneity. Gel permeation chromatography analyses revealed that G AFase (58.5 kDa of monomer) and Bl ABNase (36.4 kDa) exist as a hexamer and a monomer, respectively (data not shown). G AFase and Bl ABNase showed the highest activity at 60℃ and 55℃ in 50 mM sodium acetate buffer (pH 5.0 and 6.0), respectively ( Table 1 ). These arabinosyl hydrolases shared similar optimal reaction conditions, which could make them good candidates to be treated simultaneously for efficient L -arabinose production.
Summarized characteristics ofexo- andendo-arabinosyl hydrolases used in this study.
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aGlycoside hydrolase (GH) family from Carbohydrate-Active enZYmes (http://www.cazy.org). bBoth enzymes showed the highest activity in 50 mM sodium acetate buffer.
- Enzymatic Characteristics ofGAFase andBlABNase
G AFase showed similar activities on both sugar beet and debranched arabinans, which proposed that this hydrolase is able to attack the α-(1,5)-linked arabinofuranosyl backbone as well as α-(1,2)- or α-(1,3)-linked arabinosyl branches ( Table 2 ). G AFase can weakly hydrolyze arabinoxylan from oat spelt as well. Meanwhile, Bl ABNase could exclusively hydrolyze debranched arabinan, but not sugar beet arabinan. These results imply that Bl ABNase has an extremely lower accessibility towards the branched structure of sugar beet arabinan than the α-(1,5)-linked arabinofuranosyl backbone of debranched arabinan.
Specific activities ofGAFase andBlABNase on different substrates.
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aEnzyme activity on 0.5% of arabinan or 2.5% of xylan substrate was determined by the DNS reducing sugar assay. One unit of activity was defined as the amount of enzyme to release 1 µmol of L-arabinose equivalent per minute under the optimal reaction conditions. bEnzyme activity was not detected.
In order to confirm the modes of action, each enzyme was reacted with 0.5% (w/v) of arabinan substrate under the optimal reaction conditions for 6 h. At any given time interval, the resulting reaction products were taken and analyzed by TLC. Exo -acting G AFase could exclusively release L -arabinose from sugar beet and debranched arabinans through the reaction ( Fig. 1 A). On the contrary, Bl ABNase could hydrolyze only debranched arabinan to produce a series of arabinooligosaccharides (AOs) as intermediates longer than arabinotetraose, as the same as typical endo -acting enzymes. Even though arabinotetraose and arabinotriose are the major products from debranched arabinan hydrolysis, the excess amount (5.0 U/ml) of Bl ABNase could catalyze the further degradation of AOs into arabinobiose and arabinotriose ( Fig. 1 B). Time-course hydrolysis of debranched arabinan by Bl ABNase was quantitatively monitored by HPAEC analysis ( Fig. 2 ). At the early stage, Bl ABNase mainly produced a series of AOs longer than arabinohexaose from debranched arabinan. As the reaction proceeded, however, the long-chain AOs were gradually degraded into arabinotriose and arabinotetraose as major products.
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TLC analyses of the enzymatic hydrolysates from sugar beet (branched) arabinan by GAFase (A) and from debranched arabinan by BlABNase (B). Each substrate (0.5% (w/v)) was reacted with 0.1 unit of the corresponding enzyme. AO, arabinooligosaccharide standards from arabinose (A1) to arabinohexaose (A6); H, the complete hydrolysate reacted with 5.0 unit of BlABNase for 6 h.
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Time-course analysis of the hydrolysis of 0.5% (w/v) debranched arabinan by 1.0 unit of BlABNase. The concentration of each arabinooliosaccharides (AOs) was quantitatively determined by HPAEC analysis. AOs are shown as abbreviations from arabinose (A1) to arabinohexaose (A6).
- Arabinan Degradation by Single Enzyme Treatments
In order to examine the detailed hydrolysis pattern and efficacy in arabinan degradation, 0.26 U of G AFase or Bl ABNase was reacted with 0.5% (w/v) of sugar beet arabinan under their co-optimal conditions at 55℃ in 1 ml of 50 mM sodium acetate buffer (pH 6.0) for 6 h. The sugar beet arabinan (Megazyme) used in this study has an approximately 95% purity, and consists of L -arabinose (88%), galactose (3%), rhamnose (2%), and galacturonate (7%). The DNS reducing sugar assay was chosen for the simultaneous detection of both actions of exo - and endo -hydrolases, compared with L -arabinose as a standard. The resultant reaction products were taken at appropriate time intervals, and the changes of L -arabinose equivalent value were monitored by measuring the reducing sugar content.
Bl ABNase rapidly hydrolyzed only debranched arabinan, but showed an extremely low activity on sugar beet arabinan with branches. As expected, endo -acting Bl ABNase could convert only 3.9% of arabinan into L -arabinose for 6 h, due to its poor activity towards the highly branched α-(1,5)-arabinofuranosyl backbone of arabinan. Meanwhile, exo -acting G AFase could consistently increase the release of reducing sugars, and its hydrolysis yield reached up to 54.0% ( Fig. 3 ). As shown in Table 2 , G AFase has similar preferences to branched and debranched arabinans. It means that this exo -hydrolase can simultaneously attack both arabinofuranosyl linkages in the backbone and the branches of sugar beet arabinan. The cooperative actions between exo - and endo -hydrolases should be considered for the improvement of the hydrolysis yield from arabinan to L -arabinose.
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Time-course hydrolysis of 0.5% (w/v) sugar beet arabinan via single, stepwise, or simultaneous enzymatic treatment. G, treated with 0.26 U/ml of GAFase; B, with 0.26 U/ml of BlABNase; G+B, simultaneous treatment with 0.13 U/ml of GAFase and BlABNase, respectively; G→B or B→G, stepwise treatment of GAFase and BlABNase for 3 h each, or vice versa.
- Synergistic Arabinan Degradation by Dual Enzymes
The cooperative reactions in use of exo -acting G AFase and endo -acting Bl ABNase were comparatively studied here for the hydrolysis of sugar beet arabinan to arabinose units. The types of treatment with dual enzymes were designed as the stepwise (or sequential) or the simultaneous treatment. The stepwise treatment with dual enzymes, endo - Bl ABNase after exo - G AFase, could liberate 61.3% of L -arabinose from sugar beet arabinan. At the first stage for 3 h, 12.2 µmol of L -arabinose equivalent was produced by 0.26 U of G AFase treatment. However, very limited further hydrolysis, up to 18.0 µmol of L -arabinose equivalent, was observed during the second stage treated with 0.26 U of Bl ABNase for 3 h. It suggests that the structure of G AFase-treated arabinan is not suitable for the efficient endo -action of Bl ABNase during the second stage. On the other hand, the treatment of exo - G AFase after endo - Bl ABNase resulted in an enhanced L -arabinose equivalent (21.4 µmol) and hydrolysis yield (73.1%), respectively. This result reveals that the pretreatment of endo -hydrolase on arabinan can significantly facilitate the saccharifying action of exo -hydrolase. Although Bl ABNase treatment could not release any detectable short-chain AOs or free L -arabinose, the resultant hydrolysates at the first stage would be more favorable for the successive G AFase treatment ( Fig. 3 ).
Interestingly, the simultaneous treatment of exo - and endo -hydrolases could remarkably increase both the reaction rate at the early stage and the hydrolysis yield (97.7%) at the end point. This result proposed that the simultaneous treatment of exo - and endo -arabinosyl hydrolases can highly accelerate the complete hydrolysis of branched arabinan via their synergistic actions, compared with the single enzyme treatments or the stepwise dual enzyme treatments.
In order to verify the synergistic effects of the cooperative enzyme actions on arabinan degradation, the exact amount of L -arabinose was quantitatively analyzed by the HPAEC method ( Table 3 ). The single treatment with G AFase and Bl ABNase was able to liberate 35.9% and 0.5% of L -arabinose from sugar beet arabinan, respectively. The stepwise treatments of endo - after exo -hydrolase and exo - after endo -hydrolase showed 43.4% and 75.5% of hydrolysis yield, respectively. The simultaneous treatment gave the highest hydrolysis yield (91.2%) by the synergistic actions of exo -and endo -hydrolases. Even though HPAEC analysis showed a relatively low L -arabinose content, the tendency of the synergism among the enzyme treatments was almost the same as the L -arabinose equivalent values obtained from the DNS assay ( Fig. 4 ).
Hydrolysis of sugar beet arabinan toL-arabinoseviasingle or dual enzymatic treatments.
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aEach hydrolase (0.26 U/ml) was reacted with 0.5% sugar beet arabinan at 50℃ for 6 h. bEach hydrolase (0.26 U/ml) was reacted with sugar beet arabinan for 3 h each. cBoth hydrolases (0.13 U/ml each) were simultaneously treated on sugar beet arabinan for 6 h. dThe amount of L-arabinose liberated from sugar beet arabinan was quantitatively determined by HPAEC analysis. eL-arabinose (29.3 µmol) in 0.5% sugar beet arabinan was considered as 100%.
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Synergistic effects of enzymatic treatments on sugar beet arabinan degradation. The relative yield (%) for arabinan hydrolysis was comparatively determined by the DNS reducing sugar assay and HPAEC analysis. The simultaneous treatment with the highest hydrolysis yield was considered as 100%. G, GAFase; B, BlABNase; G+B, simultaneous treatment with GAFase and BlABNase; G→B or B→G, stepwise treatment of GAFase and BlABNase.
- Synergistic Modes of Action in Arabinan Degradation
When polymeric substrates are degraded by enzymes, treatment of both exo - and endo -hydrolases commonly results in their efficient saccharification. Specifically, the industrial enzymatic sccharification of starch materials to glucose can be a well-known example for the synergism of exo - and endo -hydrolases. Liquefying α-amylase randomly cleaves the internal α-(1,4)-glycosidic linkages in starch, which is followed by successive saccharification of the resulting dextrins via treatment of exo -acting glucoamylase or α-glucosidase.
However, the very short and abundant L -arabinosyl side chains can make sugar beet arabinan distinguished from starch with longer side chains. Although a single-enzyme process with G AFase or Bl ABNase showed the detectable hydrolytic activity on arabinan polymer, the simultaneous treatment of both the hydrolases released 2.5 times more L -arabinose (26.7 µmol) than the total sum of L -arabinose (10.6 µmol) produced from single-enzyme treatments ( Table 3 ). It revealed that the simultaneous use of both endo -acting Bl ABNase and exo -acting G AFase could work synergistically with a much higher conversion of L -arabinose from sugar beet arabinan than that obtained from any single-enzyme process. It was recently reported that the combined use of thermostable arabinanases could synergistically produce L -arabinose with 80% of hydrolysis yield from sugar beet and debranched arabinan [10] . However, their synergistic modes of action have not been investigated on the basis of enzymatic characteristics in detail.
In the present study, therefore, both the stepwise and the simultaneous treatments on arabinan degradation were intensively compared with each other, and their synergistic modes of action were schematically proposed ( Fig. 5 ). At the first stage of simultaneous treatment on arabinan, endo - Bl ABNase can cleave any specific region of branched arabinan to generate very limited amounts of branched long-chain AOs. Meanwhile, G AFase can remove L -arabinofuranosyl residues from the nonreducing ends of sugar beet arabinan and long-chain AOs. As G AFase continues to produce more debranched AOs and arabinan, the resulting debranched substances at the second stage are able to become more suitable substrates for the endo -action of Bl ABNase to produce various short-chain AOs. At the final stage, the debranched short-chain AOs can be rapidly degraded into L -arabinose via the exo -action of G AFase.
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Schematic model of the synergistic degradation of sugar beet arabinan via the simultaneous treatment with exo- and endo-arabinosyl hydrolases, GAFase (solid arrows) and BlABNase (dashed arrows).
In conclusion, the simultaneous treatment of both endo-Bl ABNase and exo - G AFase could maximize their cooperative and complementary modes of action, which could shorten the operation time and enhance the conversion yield from sugar beet arabinan to L -arabinose.
Acknowledgements
This research was supported by the grants from the National Research Foundation (NRF) of Korea through the Basic Science Research Program and funded by the Ministry of Education (Grant No. 2010-0023566 and NRF-2013R1A1A2063848).
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