Identification of an α-(1,4)-Glucan-Synthesizing Amylosucrase from

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Identification of an α‑(1,4)-Glucan-Synthesizing Amylosucrase from Cellulomonas carboniz T26 Yongchun Wang,† Wei Xu,† Yuxiang Bai,† Tao Zhang,† Bo Jiang,† and Wanmeng Mu*,†,‡ State Key Laboratory of Food Science and Technology and ‡Ministry of Education, Key Laboratory of Carbohydrate Chemistry and Biotechnology, Jiangnan University, Wuxi, 214122, Jiangsu China

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ABSTRACT: Amylosucrase, catalyzing the synthesis of α-(1,4)-glucan from sucrose, has been widely studied and used in carbohydrate biotransformation because of its versatile activities. In this study, a novel amylosucrase was characterized from Cellulomonas carboniz T26. The recombinant enzyme was overexpressed in Escherchia coli and purified by nickel affinity chromatography. It was determined to be a monomeric protein with a molecular mass of 72 kDa. The optimum pH and temperature for transglucosylation were measured to be pH 7.0 and 40 °C. The transglucosylation activity was significantly higher than the hydrolytic activity. The main product generated from sucrose was structurally determined to be α-(1,4)-glucan. A small amount of glucose was produced by hydrolysis, and sucrose isomers including turanose and trehalulose were generated as minor products. The ratio of hydrolytic, polymerization, and isomerization reactions was calculated to be 5.8:84.0:10.2. The enzyme favored production of long-chain insoluble α-glucan at lower temperature. KEYWORDS: amyloscucrase, Cellulomonas carboniz, α-(1,4)-glucan, polymerization, transglucosylation

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INTRODUCTION Homopolysacchride biosynthesis from sucrose has attracted increased attention in recent years. Some microbial glycosyltransferases (EC 2.4.1) are able to polymerize the D-glucose and D-fructose moieties of sucrose to synthesize fructans and glucans, respectively. Inulosucrase (EC 2.4.1.9) and levansucrase (EC 2.4.1.10), which occur in a wide range of bacteria, catalyze the polymerization of sucrose to inulin-type and levan-type fructan, with β-(2,1) and β-(2,6) fructosyl-fructose linkages, respectively.1 Lactic acid bacteria may use sucrose to synthesize a diversity of long-chain α-glucans with different linkages by various glycoside hydrolase family 70 glucansucrases (or glucosyltransferases). 2 For instance, dextransucrase (EC 2.4.1.5) generates α-glucans mainly composed α-(1,6) linkages;3 mutansucrase (EC 2.4.1.125) catalyzes the biosynthesis of α(1,3)-glucan, termed mutan;4 alternansucrase (EC 2.4.1.140)5 and reuteransucrase (EC 2.4.1.-)6 catalyze the D-glucosyl residue polymerization from sucrose to produce α-(1,6)-α-(1,3)-glucan, termed alternan; and α-(1,6)-α-(1,4)-glucan, termed reuteran, respectively. In addition, a glycoside hydrolase family 13 enzyme, amyloscurase (AS) (sucrose:1,4-α-D-glucan 4-α-D-glucosyltransferase, EC 2.4.1.4) converts sucrose to α-glucan with only α-(1,4) linkages.7 AS catalyzes the α-D-glucosyl residue transfer from sucrose to the 4-position of the nonreducing terminal residue of an αglucan, generating an insoluble α-(1,4)-glucan accompanied by the release of D-fructose from sucrose. Like some glucansucrases, AS also has sucrose hydrolytic activity to release D-glucose and Dfructose molecules and may catalyze the transglycosylation reaction from sucrose to many acceptor molecules. In addition, in contrast to glucansucrases, AS uniquely produces a small amount of sucrose isomers, including turanose and trehalulose when the D-glucosyl moiety of sucrose is transferred onto the released D-glucose and D-fructose, respectively.8 Because of the versatile activities, AS has been widely used for production of © 2017 American Chemical Society

various carbohydrate-based bioactive compounds, such as modified starch,9−13 sucrose isomers,14 α-glucans,15 and some bioactive α-glucosides, including dihydrochalcone glucosides,16 α-D-glucosyl glycerol,17 (+)-catechin α-glycosides,18 salicin glycosides,19 arbutin-α-glucoside,20 and rutin derivatives.21 AS can be used to convert sucrose to cyclodextrin,22 cycloamyloses coupled with 4-α-glucanotransferase reaction,23 and trehalose coupled with maltooligosyltrehalose synthase-trehalohydrolase.24 In addition, AS can be used for the synthesis of amylose microparticles,25 amylose nanocomposite microbeads,26 and amylose magnetic microparticles27 through a self-assembly process of biosynthesized amylose. Seventy years ago, Hehre and co-workers first found that sucrose can be converted to a glycogen-like polysaccharide by certain bacteria of Neisseria genus, without dependence of Dglucose-1-phospahte as an intermediate substance, and they named this responsible enzyme AS.28 Then, AS from Neisseria perflava (NPr-AS)29 and Neisseria polysaccharea ATCC 43768 (NPo-AS)30 was identified in native and recombinant form, respectively. In the new century, six more recombinant AS enzymes have been identified from Deinococcus radiodurans ATCC 13939 (DRd-AS),31 Deinococcus geothermalis DSM 11300 (DG-AS),32,33 Alteromonas macleodii KCTC 2957 (AM-AS),34 Arthrobacter chlorophenolicus A6 (AC-AS),35 Methylobacillus flagellatus KT (MF-AS),17 and Deinococcus radiopugnans ATCC 19172 (DRp-AS),7 respectively. Unlike other members of the glycoside hydrolase family of enzymes, AS is not found in a broad range of microorganisms.7 So far, this distinct enzyme has only been characterized from less than 10 microorganisms above-mentioned. In this work, a novel Received: Revised: Accepted: Published: 2110

December 19, 2016 February 27, 2017 February 27, 2017 February 27, 2017 DOI: 10.1021/acs.jafc.6b05667 J. Agric. Food Chem. 2017, 65, 2110−2119

Article

Journal of Agricultural and Food Chemistry

total activity minus hydrolytic activity. In this work, the enzyme activity was described as total activity, unless otherwise specified. Effect of pH, Temperature, and Metal Ions on Enzymatic Activity. Effect of pH on AS activity was investigated within a range of pH 4.5−8.5 at 40 °C. Three different buffer systems were used, including acetate buffer (50 mM, pH 4.5−6.0), sodium phosphate buffer (50 mM, pH 6.0−7.5), and Tris-HCl (50 mM, pH 7.5−8.5). The effect of temperature on the enzymatic activity was studied in 50 mM sodium phosphate buffer (50 mM, pH 7.0) ranging from 30 to 50 °C. To investigate the effect of metal ions on enzymatic activity, metal ions (in the form of CuSO4, FeSO4, ZnSO4, MgSO4, MnSO4, and NiSO4) were used at a final concentration of 1 mM. Effect of Temperature on the Enzyme Stability. Thermostability was observed by preincubating the purified enzyme at different temperatures and examined by measuring the residual activity after various durations of preincubation. The melting temperature (Tm) was determined by differential scanning calorimetry (DSC) using a TA Instruments Nano DSC with a platinum capillary cell (New Castle, PA). The enzyme was redialyzed against sodium phosphate buffer overnight, and dialyzed buffer was collected to serve as a reference. The enzyme solution was degassed under vacuum (635 mmHg) for 10 min and loaded into the DSC cell. The cell was heated from 25 to 100 °C at three atmospheric pressures with a temperature ramp of 1 °C/min. Sodium phosphate buffer was used as the corresponding reference. DSC data were analyzed using TA Instruments NanoAnalyze software, and the observed thermograms were baseline-corrected. Iodine Binding Properties. Reactions were performed in duplicate in 50 mM sodium phosphate buffer (pH 7.0) containing 100 mM sucrose. After reaction at 40 °C for 24 h, one sample was checked by aqueous iodine solution treatment; the other was hydrolyzed by 20 μL amyloglucosidase-A7095 (Sigma-Aldrich, St Louis, MO) at 37 °C for 2 h, followed by iodine treatment. The iodine reactivity was performed using 40 μL of an aqueous iodine solution [2% (w/v) KI and 0.2% (w/v) I2]. Isolation of Soluble Products Produced from Sucrose by CCAS. Reaction products from sucrose by CC-AS were centrifuged at 13 000g for 15 min to remove the precipitate. The supernatant was treated with Sevag reagent (n-butanol:chloroform = 1:4, v/v) six times to remove any proteins.37 After that, three volumes of 95% ethanol were added at room temperature and the mixture was stored at 4 °C overnight. The mixture was then centrifuged at 4 °C and 13 000g for 30 min to separate the precipitate. Then the precipitate was collected, dissolved in deionized water, and freeze-dried using a 4.5 L FreeZone freeze-dry system (Labconco Corp.) Fourier-Transform Infrared Spectroscopy (FT-IR) Analysis. The basic functional group of the reaction products was determined by FT-IR analysis. The product powder was mixed with potassium bromide at a ratio of 1:100 and pressed into a tablet. An intermediate infrared region from 400 to 4000 cm−1 was used for scanning at 4 cm−1 resolution using a Thermo Nicolet NEXUS 470 FT-IR (Thermo Fisher Scientific). Nuclear Magnetic Resonance Spectroscopy (NMR) Analysis. Lyophilized sample (35 mg) was mixed with 650 μL of deuterium oxide and maintained in a water bath at 90 °C for 5 h to completely dissolve the sample. The 1H, 13C, and 1H−13C heteronuclear single quantum coherence (HSQC) NMR spectra were recorded at 60 °C using an AVANCE III 400 MHz digital NMR spectrometer (Bruker Biospin International AG). Chemical shifts (δ), expressed in ppm, were determined with respect to the signals for sodium 4,4-dimethyl-4silapentane-1-sulfonate (DSS) (δH = δC = 0.00 ppm) dissolved in the samples. High-Performance Anion Exchange Chromatography (HPAEC) Analysis. HPAEC with pulsed amperometric detection (PAD) was used for carbohydrate analysis. Samples were filtered by a 0.45-μm membrane filter and then injected into the HPAEC-PAD system (Dionex DX 600) equipped with an ED 50 electrochemical detector with a gold working electrode, GP 50 gradient pump, LC 30 chromatography oven, and AS 40 automated sampler (Dionex Corp., Sunnyvale, CA).

AS was identified from a Gram-positive aerobic strain, Cellulomonas carboniz T26, with a high α-(1,4)-glucan-producing activity. The recombinant C. carboniz AS (CC-AS), heterologously expressed in Escherichia coli, was purified and characterized, and its enzymatic properties were investigated and compared with the reported ones from other bacteria. To our best knowledge, it is the first report on the identification of AS from a Cellulomonas species strain.

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MATERIALS AND METHODS

Cloning, Expression, and Enzyme Purification. The full-length nucleotide sequence of the AS-encoding gene (locus_tag: N868_11335) from C. carboniz T2638 was commercially synthesized by Shanghai Generay Biotech Co., Ltd. (Shanghai, China). The gene fused with a 6× histidine-tag sequence at the 3′-terminus was inserted into a pET-22b(+) vector with NdeI and XhoI restriction sites. The generated reconstructed plasmid, termed pET-CC-AS, was transformed into host E. coli BL21(DE3). A selected colony of recombinant E. coli BL21(DE3) harboring pETCC-AS was inoculated in 200 mL of Luria−Betani (LB) medium (5 g/L yeast extract, 10 g/L tryptone, and 5 g/L NaCl) supplemented with 100 μg/mL ampicillin for growth at 37 °C and 200 rpm. When the optical density at 600 nm reached 0.6, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM to induce the expression at 28 °C for another 6 h. The pelleted cells were collected by centrifugation at 4900g for 20 min, resuspended in lysis buffer (50 mM Tris-HCl buffer, 100 mM NaCl, pH 7.5), kept in an ice bath, and then disrupted by sonication for 15 min (pulse on for 1 s and pulse off for 3 s) using a Vibra-Cell 72405 sonicator (Bioblock, Illkirch, France). After being centrifugated at 19 000g for 30 min, the supernatant-dissolved AS was filtered through a 0.45-μm filter, and then was loaded onto a Ni2+-chelating Sepharose Fast Flow column (Uppsala, Sweden) for nickel affinity chromatography. The column was equilibrated with a binding buffer (50 mM sodium phosphate buffer, 500 mM NaCl, pH 7.0), followed by using a washing buffer (50 mM sodium phosphate buffer, 500 mM NaCl, 50 mM imidazole, pH 7.0) and an elution buffer (50 mM sodium phosphate buffer, 500 mM NaCl, 500 mM imidazole, pH 7.0) to remove miscellaneous or unbound proteins and obtain recombinant AS. All the purification steps were carried out at 4 °C. The purified enzyme was then dialyzed against 50 mM sodium phosphate buffer (pH 7.0). Protein Concentration and Molecular Mass. The protein concentration was calculated according to the method of Bradford.36 The molecular mass of subunit and native enzyme was examined by sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS− PAGE) and gel filtration, respectively. For SDS−PAGE, a 5% stacking gel and a 12% separating gel were used, and the separate protein bands were fixed with trichloroacetic acid (12%, w/v), stained with Coomassie Brilliant Blue R250, and finally destained until the background was colorless. For gel filtration, the total molecular mass of the protein was estimated using a gel filtration chromatography [column, TSK G2000SWxl (Tosoh Bioscience LLC, Minato-ku, Tokyo, Japan); mobile phase, 100 mM phosphate buffer (pH 6.7) containing 100 mM Na2SO4 and 0.05% (w/v) NaN3; flow rate, 1 mL/min; detection, UV at 280 nm; standard samples, thyroglobulin (porcine thyroid gland, MW 669 kDa), β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), albumin (66 kDa), and carbonic anhydrase (29 kDa)]. Enzyme Assay. Enzymatic activity was assayed at 40 °C with 100 mM sucrose as the sole substrate in 50 mM phosphate buffer (pH 7.0) for 20 min. Total activity was measured on the basis of the release of fructose from sucrose, since fructose generation reflects total consumption of sucrose. Glucose is produced from sucrose due to sucrose hydrolysis with water as an acceptor, and thus, hydrolytic activity was determined by calculating the release of glucose. Transglycosylation activity was measured as total activity minus hydrolytic activity and was calculated by subtracting the amount of glucose from that of fructose.34 One unit of total activity and hydrolytic activity was defined as the amount of enzyme catalyzing the release of 1 μmol of fructose and glucose per min, respectively. Transglycosylation activity was defined as 2111

DOI: 10.1021/acs.jafc.6b05667 J. Agric. Food Chem. 2017, 65, 2110−2119

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Journal of Agricultural and Food Chemistry Table 1. Comparison of the Amino Acid Sequences of Identified AS Enzymes from Various Microorganismsa percentage of identity no.

1

2

3

4

5

6

7

8

1 2 3 4 5 6 7 8

100.00 67.03 58.85 47.61 45.48 44.16 43.70 40.22

67.03 100.00 56.22 46.43 44.01 44.44 44.69 39.46

58.85 56.22 100.00 49.28 43.56 42.21 42.63 37.84

47.61 46.43 49.28 100.00 42.02 40.38 41.55 40.35

45.48 44.01 43.56 42.02 100.00 75.74 74.96 38.64

44.16 44.44 42.21 40.38 75.74 100.00 75.70 40.47

43.70 44.69 42.63 41.55 74.96 75.70 100.00 37.93

40.22 39.46 37.84 40.35 38.64 40.47 37.93 100.00

a

The Arabic numerals from 1 to 8 represent CC-AS (GenBank accession no. KGM11272.1), AC-AS (ACL41561.1), NPo-AS (CAA09772.1), AMAS (BAG82876.1), DRd-AS (NP_294657), DG-AS (ABF44874.1), DRp-AS,7 and MF-AS (ABE50875.1), respectively.

Figure 1. Multiple sequence alignment of AS enzymes and their homologues. The strictly conserved residues are shown on a red background, and the highly conserved residues were shown in red type and boxed in blue. The secondary structure elements were symbolized on the basis of the previously determined structure of NPo-AS (PDB ID 1G5A). Two catalytic residues, Glu and Asp, were emphasized using the star symbol above the residues. The alignment was performed using ESPript software. Regions 1−7 involved in DG-AS dimerization from structural analysis41 are shown under the sequence alignment. acetate, ultrapure water, and 250 mM NaOH as eluent A, B, and C, respectively. Relatively long-chain oligosaccharides were eluted with a linear gradient (flow rate, 0.5 mL/min; column temperature, 30 °C;

Dionex CarboPac PA1 and PA200 columns (Dionex Corp.) were used for analysis of short-chain and relatively long-chain oligosaccharides, respectively. Three eluents were used, including 1 M sodium 2112

DOI: 10.1021/acs.jafc.6b05667 J. Agric. Food Chem. 2017, 65, 2110−2119

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Journal of Agricultural and Food Chemistry

Figure 2. SDS−PAGE and gel filtration analysis of the recombinant CC-AS. (A) SDS−PAGE analysis of CC-AS expression and purification. Lanes 1−4 reprensent whole recombinant E. coli cells harboring CC-AS encoding gene expressed by IPTG induction at 28 °C for 0, 2, 4, and 6 h, respectively. Lane 5 represents the purified recombinant CC-AS purified by nickel affinity chromatography. Lane M represents the standard molecular weight protein marker. (B) Molecular mass estimation of the native purified CC-AS based on the gel filtration analysis using a TSK G2000SWxl column. The retention times at 5.77, 8.15, 8.74, 10.95, and 12.97 min represent the standard molecular mass proteins, including thyroglobulin (porcine thyroid gland, 669 kDa), β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), albumin (66 kDa), and carbonic anhydrase (29 kDa), respectively. The native purified recombinant CC-AS was eluted at 10.68 min with a calculated molecular mass of 72 kDa. elution procedure, 38% elution C and 58% elution B at 0.0 min, 24% elution C and 36% elution B at 40.0 min, and 38% elution C and 58% elution B at 40.1 min). Short-chain oligosaccharides were eluted by elution C at 1.0 mL/min and 30 °C.

Expression and Purification of the Recombinant CCAS. The CC-AS encoding gene, which was annotated using locus_tag: N868_11335 in the C. carboniz T26 genome, was identified as an open reading frame of 1935 base pairs of nucleotides encoding a protein of 644 amino acids with a calculated molecular mass of 71 934 Da and a theoretical isoelectric point of 5.39 calculated by the ExPASy Computer pI/ Mw tool. The full-length gene was synthesized commercially and cloned into a pET-22b(+) expression vector containing an inframe 6× histidine-tag sequence at the 3′-terminus. The recombinant plasmid was transformed into host cell E. coli BL21(DE3) and the foreign gene was expressed by IPTG induction. As shown in Figure 2A, IPTG significantly induced the recombinant CC-AS expression, forming a strong protein band at approximately 70 kDa on the SDS−PAGE gel, and the expression amount reached the maximum at 6 h. Because of fusion with a 6× histidine-tag, the recombinant CC-AS was easily purified to homogeneity by one-step nickel affinity chromatography. As shown in Figure 2A, the purified enzyme showed a molecular mass of approximately 70 kDa, which was close to the predicted molecular mass based on the known amino sequence. In previous works, some recombinant AS enzymes, including NPo-AS,43 DRd-AS,31 DG-AS,32,33 and AM-AS,34 were expressed in fusion with a 26-kDa glutathione Stransferase (GST) tag, but GST fusion probably affected the catalytic activity of AS and the purified recombinant GST-fused AS generally required a GST removal step for further characterization. GST-free DG-AS showed 2.4 times higher specific activity than the GST-fused one.32 The cleavage of the GST-tag also improved the activity of GST-fused DRd-AS.31 In addition, GST fusion probably affected the recombinant AS expression level. It was reported that GST-fused DG-AS was mainly produced as insoluble and inactive protein forming inclusion bodies.32 Jeong et al. mentioned that AS expression level expressed in a pGEX system with GST fusion recombination was lower than that in pET and pHCE expression system.17 Recently, AC-AS,35 DRp-AS,7 and MF-AS17 were expressed in fusion with a 0.7-kDa 6× histidine-tag using pET vectors, with relatively high expression level and no need of treatment for the fusion tag. In this work, a remarkable expression level of the recombinant CC-AS was achieved using the pET-22b(+) vector under the T7 promoter, and the enzyme

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RESULTS AND DISCUSSION Sequence Analysis. C. carboniz T26 was a Gram-positive, aerobic, motile, and rod-shaped bacterium isolated from the subsurface soil of a Chinese coal mine.38 Its whole genomic sequence has just recently been determined and deposited at DDBJ/EMBL/GenBank under accession number AXCY00000000. 39 The genome annotation showed the presence of a putative gene (locus_tag: N868_11335) encoding the hypothetical protein (protein ID: KGM11272.1). In this work, this hypothetical protein was identified as an AS with high α-(1,4)-glucan-producing activity, termed CC-AS. Among all the eight reported AS enzymes, only NPo-AS was from the wild microorganism with unknown amino acid sequence information. Overall, various reported AS enzymes showed 35−75% identity with one another (Table 1). Herein, CC-AS was found to be relatively homologous in amino acid sequence with AC-AS (GenBank accession no. ACL41561.1) and NPo-AS (CAA09772.1) with 67.03% and 58.85%, respectively, and shared less than 50% identity with other ones, including NPo-AS (CAA09772.1), AM-AS (BAG82876.1), DRd-AS (NP_294657), DG-AS (ABF44874.1), DRp-AS,7 and MF-AS (ABE50875.1) (Table 1). The crystal structures of NPo-AS,40 DG-AS,41 and DRd-AS42 have recently been determined and deposited in the Protein Databank (PDB) with IDs 1G5A, 3UCQ, and 4AYS, respectively. All the structure information supported the enzyme as a glycoside hydrolase family 13 member containing a catalytic (β/α)8-barrel domain. The active sites were highly conversed in all reported AS emzymes (Figure 1). Two strictly reserved residues, Glu334 and Asp292, in the (β/α)8-barrel domain of CC-AS were hypothesized to be the general acid/base and the nucleophilic catalytic residues, respectively, and played an important role in the formation of the β-glucosyl enzyme intermediate through an α-retaining mechanism. These two residues were found to be completely conserved as the corresponding residues Glu328 and Asp286 in NPo-AS,40 Glu326 and Asp284 in DG-AS,41 and Glu318 and Asp276 in DRd-AS,42 respectively. 2113

DOI: 10.1021/acs.jafc.6b05667 J. Agric. Food Chem. 2017, 65, 2110−2119

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Journal of Agricultural and Food Chemistry

Figure 3. Enzymatic properties of recombinant CC-AS. Effect of pH on (A) the catalytic activity and (B) the T/H ratio of CC-AS. Three buffer systems were used, including acetate buffer (50 mM, pH 4.5−6.0), sodium phosphate buffer (50 mM, pH 6.0−7.5), and Tris-HCl (50 mM, pH 7.5−8.5). Effect of temperature on (C) the catalytic activity and (D) T/H ratio of CC-AS. (E) Effect of various metal ions on the total activity. (F) Effect of temperature on the stability of CC-AS. Values are means of three replications ± standard deviation. (G) Nano-DSC measurement of CC-AS. The fitting curve (red line) was determined by the two state scaled method from the raw data (blue line).

in Figure 1.41 The NPo-AS displays relatively shorter loops in regions 4, 6, and 7 compared to DG-AS40 and thus probably prevents the formation of dimer.41 By comparison, CC-AS showed regions 4, 6, and 7 similar to those of NPo-AS but different from those of DG-AS (Figure 1), and this was the possible reason that CC-AS was a monomeric protein like NPoAS. Effect of pH and Temperature on the Catalytic Activity of the Recombinant CC-AS. The total, hydrolytic, and transglucosylation activities of the recombinant CC-AS were measured at 40 °C with pH from 4.5 to 8.5. As shown in Figure 3A, the highest total and transglucosylation activities were shown at pH 7.0; however, the highest hydrolytic activity was shown at pH 5.0. Overall, the enzyme showed remarkably higher transglucosylation than hydrolytic activity above pH 5.5, with

was easily purified by affinity chromatography for further investigation. The total molecular mass of the native recombinant CC-AS was measured to be approximately 72 kDa on the basis of the gel filtration results (Figure 2B). Thus, it was indicated that CC-AS should be a monomer. In previous studies, NPo-AS crystallized as a monomer;40 however, both DG-AS41 and DRd-AS42 crystal structures showed the homodimeric proteins, and the biochemical analysis indicated that the monomeric NPo-AS and dimeric DG-AS were stable over a few weeks at 4 °C.41 AC-AS was a monomeric protein like NPo-AS and CC-AS,35 and MFAS17 and DRp-AS7 were determined to be homodimeric proteins. On the basis of the reported crystal structure information, DGAS dimerizes via an interface composed of seven regions shown 2114

DOI: 10.1021/acs.jafc.6b05667 J. Agric. Food Chem. 2017, 65, 2110−2119

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Journal of Agricultural and Food Chemistry

Figure 4. Structural determination of the products produced from sucrose by CC-AS. (A) The amylose−iodine color reaction. (B) The FT-IR spectrum. The (C) 13C NMR, (D) 1H NMR, and (E) HSQC spectrum, respectively.

ratio than AM-AS, indicating that CC-AS has a great potential for application. In addition, DG-AS,33 AC-AS,35 MF-AS,17 and DRp-AS7 showed optimum pH and temperature at 8.0 and 45 °C, 8.0 and 45 °C, 8.5 and 45 °C, and 8.0 and 40 °C, respectively. But the T/ H ratio of these enzymes was not studied in detail. Effect of Metal Ions on the Catalytic Activity of the Recombinant CC-AS. The total activity of recombinant CC-AS was measured in the presence of various metal ions at the final concentration of 1 mM (Figure 3E). All the tested metal ions showed negative effects on the activity. The Fe2+, Mg2+, and Mn2+ caused marginal decreases in the enzyme activity (