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Appl Biochem Biotechnol DOI 10.1007/s12010-014-0889-z Biosynthesis of Glucosyl Glycerol, a Compatible Solute, Using Intermolecular Transglycosylation Activity of Amylosucrase from Methylobacillus flagellatus KT Jin-Woo Jeong & Dong-Ho Seo & Jong-Hyun Jung & Ji-Hae Park & Nam-In Baek & Myo-Jeong Kim & Cheon-Seok Park Received: 9 January 2014 / Accepted: 24 March 2014 # Springer Science+Business Media New York 2014 Abstract A putative α-amylase gene (accession number, CP000284) of Methylobacillus flagellatus KT ATCC51484 was cloned in Escherichia coli, and its gene product was expressed and characterized. The purified recombinant enzyme (MFAS) displayed a typical amylosucrase (ASase) activity by the demonstration of multiple activities of hydrolysis, isomerization, and polymerization although it was designated as an α-amylase. The optimal reaction temperature and pH for the sucrose hydrolysis activity of MFAS were determined to be 45 °C and pH 8.5, respectively. MFAS has relatively high thermostable characteristics compared with other ASases, as demonstrated by a half-life of 19.3 min at 50 °C. MFAS also showed polymerization activity using sucrose as a sole substrate. Glycerol was transglycosylated by the intermolecular transglycosylation activity of MFAS. Two major products were observed by thin-layer chromatography and isolated by paper chromatography and recycling HPLC. Using 1H and 13C NMR, their chemical structures were determined to be (2S)-1-O-α-D-glucosyl-glycerol or (2R)-1-O-α-D-glucosyl-glycerol and 2-O-α-D-glucosyl-glycerol, in which a glucose molecule is linked to glycerol via an α-glycosidic linkage. Keywords Amylosucrase . Glucosyl glycerol . Glycerol . Methylobacillus flagellatus . Transglycosylation Jin-Woo Jeong and Dong-Ho Seo contributed equally. J.<W. Jeong : D.<H. Seo : J.<H. Jung : J.<H. Park : N.<I. Baek : C.<S. Park (*) Graduate School of Biotechnology and Institute of Life Science and Resources, Kyung Hee University, Yongin 446-701, South Korea e-mail: [email protected] M.<J. Kim Food Research Institute and School of Food and Life Science and Biohealth Products Research Center, Inje University, Gimhae 621-749, South Korea Appl Biochem Biotechnol Introduction Enzymes are highly selective biocatalysts that are responsible for a variety of cellular biochemical reactions. Glycoside hydrolases (GH, EC 3.2.1) are a widespread group of enzymes that cleave the glycosidic linkages in di-, oligo-, and polysaccharides [1]. GHs are able to hydrolyze the glycosidic bonds of carbohydrate and non-carbohydrate moieties in biological molecules. The GH13 family is the major GH family and acts on substrates containing α-glycosidic linkages. Most of the known starch-modifying enzymes such as αamylases, pullulanases, α-1,6-glucosidases, maltogenic amylases, neopullulanases, and cyclodextrinases (CDases) belong to this GH13 family [2, 3]. Amylosucrase (ASase, EC 2.4.1.4) is a versatile enzyme in the GH13 family that hydrolyzes sucrose to equal amounts of glucose and fructose [4]. Simultaneously, it can transfer a released glucose to the four positions of other glucose molecules or the non-reducing end of the resulting α-1-4 glucan molecules resulting in the synthesis of an amylose-like glucan polymer [4, 5]. ASase also performs an isomerization reaction to make sucrose isomers such as turanose and trehalulose [6]. Recently, the intermolecular transglycosylation activity of ASase has gained increasing attention because it uses a relatively cheap substrate, sucrose, as well as its broad range of acceptor specificity [7, 8]. ASase can employ not only various glycones such as salicin [9] and arbutin [10] but also numerous aglycone compounds including catechin [11] and hydroquinone [12], as acceptor molecules. With the development of advanced sequencing technology, data on numerous microbial genomes are now available, and the presence of ASases and their homologues are being revealed. However, to date, only five ASases from five different bacterial species, Neisseria polysaccharea, Deinococcus radiodurans, Deinococcus geothermalis, Alteromonas macleodii, and Arthrobacter chlorophenolicus, have been studied [4, 13–16]. A previous study reported that the suh gene from Xanthomonas axonopodis pv. glycines, a causative agent of bacterial pustule disease in soybeans, showed a very strong deduced amino acid homology with various ASases [17]. However, it only displayed sucrose hydrolysis activity without any glucosyltransferase or isomerization activities [18]. α-D-Glucosyl glycerol (GG) was formerly known as the main compatible solute in moderately halotolerant cyanobacteria such as the Synechocystis sp. strain PCC 6803 [19]. This compound accumulates in cells to adjust the cellular osmotic potential to levels that allow water uptake. In cyanobacteria, GG is synthesized by a two-step reaction in which the enzymatic condensation of ADP-glucose and glycerol 3-phosphate by GG-phosphate synthase (GgpS) is followed by the dephosphorylation of the intermediate by GG-phosphate phosphatase (GgpP) [20]. Interestingly, GG has been found in traditional Japanese foods such as sake, miso, and mirin at concentrations of 0.1–0.5 % [21]. It was suggested that GG is formed from glycerol by the transglycosylation activity of yeast α-glucosidase (EC 3.2.1.20) [22]. GG has some industrially useful characteristics. It has approximately 0.55-fold the sweetness of sucrose, but shows high thermostability, low heat-colorability, low Maillard reactivity, low hygroscopicity, and high water-holding capacity. GG also exhibits some health benefits such as non-cariogenicity and low digestibility [23]. In this study, a putative α-amylase gene (accession number, CP000284) was cloned from an obligate methylotroph Methylobacillus flagellatus KT ATCC51484 and expressed in Escherichia coli. The catalytic properties of the recombinant enzyme (hereafter referred to as MFAS) were examined to confirm that it belongs to the ASase family. In addition, the transglycosylation activity of MFAS was employed to synthesize GG. The reaction products were purified to homogeneity, and their chemical structures were determined. Appl Biochem Biotechnol Materials and Methods Chemicals and Enzymes Sucrose and glycerol were purchased from Duchefa Biochemistry (Haarlem, Netherlands) and Sigma Chemical Co (St. Louis, MO, USA), respectively. Silica gel K5F thin-layer chromatography (TLC) plates (Whatman, Kent, UK) were used for sugar analysis. Restriction endonucleases and other modifying enzymes, such as Pfu DNA polymerase and T4 DNA ligase, were obtained from Stratagene Inc. (La Jolla, CA, USA) or Solgent (Daejeon, Korea). Preparation of genomic DNA from M. flagellatus KT ATCC51484 was carried out using the GeneAll TM total DNA purification kit (GeneAll Biotechnology, Seoul, Korea). Oligonucleotides used for polymerase chain reaction (PCR) and DNA sequencing were synthesized by Solgent Co. (Daejeon, Korea). The products of restriction enzyme digestion and PCR amplifications were purified using the QIAquick Gel extraction kit (Qiagen, Hilden, Germany). All other chemicals used in this study were of analytical reagent grade. Bacterial Strains, Media, and Plasmids Genomic DNA from M. flagellatus KT ATCC51484 was a generous gift from Dr. Mary E. Lidstrom at the University of Washington (Seattle, WA, USA). Two E. coli strains were employed for standard DNA manipulation or recombinant protein expression. E. coli DH10B [F−Φ80lacZ M15 (lacZYA-argF) U169 recA1endA1 hsdR17(rk+, mk+) phoA supE44 thi-1 gyrA96 relA1λ−] was a host for typical DNA manipulation, and BL21 [F− ompT hsdSB (rB−, mB−) gal dcm λ(DE3)plies T1R] was used as the expression vector. Selection of recombinant cells was achieved on an Lysogeny broth (LB) agar plate with 100-μg/mL ampicillin, 0.5-mM isopropyl-β-D-thiogalactopyranoside (IPTG), and 40-μg/mL 5-bromo-4-chloro-3-indolyl-β-Dgalactopyranoside (Xgal). The plasmids pGEM-T Easy vector (Promega Co., Madison, WI, USA) and pET-21a(+) vector (Novagen, Darmstadt, Germany) were used for the cloning of PCR products and construction of the expression vector, respectively. E. coli strains containing recombinant DNA were cultured in 5 mL of LB medium in the presence of ampicillin (100 μg/mL) at 37 °C overnight. Cells (1.5 mL) were collected by centrifugation at 6,000×g for 5 min in a microcentrifuge, and the plasmid DNA was obtained using the general alkaline lysis method. The final plasmid DNAs were dissolved and stored in a 50-μL TE-RNase [DNase-free RNase A 20 μg/mL in 10-mM Tris-Cl and 1-mM EDTA, pH 8.0] until further use. Construction of pETMFAS Expression Vector The gene corresponding to the ASase homologue in M. flagellatus KT ATCC51484 was amplified by PCR using the oligonucleotide primers MFAS-F (5’-CAT ATG TAC GAA CAA GTC TCC CAC TCG-3’) and MFAS-R (5’-CTC GAG AAG CTG TAA CCA ATA AAA GCC-3’). The PCR condition for mfas gene amplification was previously described [4, 16]. The amplified PCR product was purified using the QIAquick Gel extraction kit (Qiagen). The PCR product was cloned into pGEM-T Easy vector (Promega), and the recombinant plasmids were digested with Nde I and Xho I after confirming the absence of PCR-introduced errors. The resulting fragments were inserted into pET-21a(+) vector treated with the same enzymes to create pETMFAS, an expression vector for MFAS in which the mfas gene was controlled by the T7 promoter. E. coli BL21(DE3) was transformed with pETMFAS, and the resulting recombinant E. coli strain was used for the efficient production of MFAS. Appl Biochem Biotechnol Expression of pETMFAS and the Purification of the Recombinant MFAS Recombinant E. coli BL21 harboring pETMFAS were grown in 1 L of LB medium supplemented with ampicillin (0.1 mg/mL) at 37 °C with agitation. The cells were harvested by centrifugation at 4 °C after a 3-h induction of the mfas gene by the addition of IPTG to a final concentration of 1 mM when the optical density at 600 nm (OD600) reached 0.5–0.6. The collected pellets were resuspended in 5 mL of lysis buffer (50-mM NaH2PO4, 300-mM NaCl, 10-mM imidazole, pH 7.0) per gram (wet weight) of cells. Lysozyme (1 mg/mL) was added to the resuspended cells, and the cell suspension was incubated for 30 min on ice before disruption by sonication (Sonifier 450, Branson, Danbury, CT, USA; output four, six times for 10 s, constant duty) in an ice bath. The cell lysate was centrifuged at 6,000×g for 10 min at 4 °C. The recombinant MFAS is then purified by affinity chromatography with nickelnitrilotriacetic acid (Ni-NTA) affinity column (Qiagen) as suggested in the manufacturer’s protocol. Protein obtained from the eluted fraction was dialyzed to remove the imidazole, and the protein concentration and enzyme activity were measured before further application. Determination of Protein Concentration and Enzyme Activity The bicinchoninic acid (BCA) assay was used to quantify protein concentration as described by Smith et al. [24]. The BCA protein assay kit (Thermo Fisher Scientific, Agawam, MA, USA) was used to prepare the working reagent solution, and bovine serum albumin (BSA) was used as a standard. Enzymatic activity of MFAS was measured by the 3,5-dinitrosalicylic acid (DNS) method for detecting reducing sugar [4] or the D-glucose/D-fructose assay kit (Roche Diagnostics, Darmstadt, Germany) [15] for measuring the fructose concentration. The reaction mixture contained 100 μL of 25 % sucrose, 100 μL of double distilled water (DDW), and 250 μL of 100-mM Tris-HCl (pH 8.5). After the preincubation of the substrate solution at 45 °C for 5 min, the reaction was initiated by the addition of 50 μL of the enzyme solution. The reaction was continued for 10 min before being terminated by the addition of 500 μL of DNS solution and inactivation by boiling. The absorbance of reducing sugar at 545 nm was measured using a spectrophotometer (UV1201, Shimadzu, Kyoto, Japan). For the D-glucose/D-fructose assay kit, the reaction mixture was boiled for 5 min to inactivate the enzyme without adding DNS solution. The enzyme reaction was used as a sample for the fructose assay. One unit of MFAS hydrolysis activity was defined as the amount of enzyme that catalyzes the production of 1 μmole of fructose per minute in the assay conditions. Determination of Optimal pH, Temperature, and Thermostability The effect of pH on enzyme activity was investigated within the range of pH 4.0 to pH 10.0 (0.1-M sodium acetate buffer for pH 4 and 5; 0.1-M sodium citrate buffer for pH 5, 6, 6.5, and 7; 0.1-M Tris-HCl buffer for pH 7, 7.5, 8, 8.5, and 9; and 0.1-M glycine-NaOH for pH 9 and 10) at 45 °C. The effect of temperature on the hydrolysis activity of MFAS was studied between 30 and 65 °C at pH 8.5. The thermostability of the purified MFAS was observed by preincubation in the absence of substrate at 45, 50, and 55 °C. At various times, the residual activity was measured under standard assay conditions. Differential scanning fluorimetry (DSF) has been used to measure the melting temperature of proteins [25]. The sample was prepared by mixing 10 μL of SYPRO orange (10×) with 10 μL of enzyme solution (0.1 mg/mL). The mixture of proteins and SYPRO orange was incubated in a real-time polymerase chain reaction (RT-PCR) system using a temperature Appl Biochem Biotechnol gradient from 30 to 99 °C with 1 °C increments. All fluorescence measurements were carried out using an Applied Biosystems 7500 Fast RT-PCR system (Applied Biosystems, Grand Island, NY, USA). Sigma Plot 12.0 software (Systat Software Inc., Chicago, IL, USA) and Microsoft Excel program (Redmond, WA, USA) were used for all graphical and statistical analyses. High-Performance Anion-Exchange Chromatography (HPAEC) Analysis HPAEC analysis was carried out with analytical columns (CarboPac PA100 or CarboPac MA1, Dionex Co., Sunnyvale, CA, USA) to detect carbohydrate in the reaction. Filtered samples were applied and eluted with a linear gradient from 100 % buffer A (100 mM of NaOH in water) to 60 % buffer B (500 mM of sodium acetate in buffer A) over 70 min. The flow rate of the mobile phase was maintained at 1.0 mL/min. Detection was carried out using a Dionex ED50 module. The areas of the peaks were calculated for quantitative analysis. Gel Permeation Chromatography (GPC) Analysis GPC (GE Healthcare, Little Chalfont, UK) analysis with a Superdex 200HR (GE Healthcare) was used to confirm the molecular size of MFAS. To calculate the molecular weight of the enzyme, standard proteins were loaded as controls, including carbonic anhydrase (29 KDa), BSA (66 KDa), β-amylase (200 KDa), apoferritin (443 KDa), and thyroglobulin (669 KDa). The concentration of injected protein was 1.0 mg/ml, and the flow rate was 0.4 mL/min. Bioconversion of Glycerol to Glucosyl Glycerols (GGs) GG biosynthesis reactions were carried out in 50-mM Tris-HCl buffer (pH 8.5) at 35 °C instead of 45 °C because the transglycosylation activity of MFAS was similar to that of other AS enzymes, which are known to have a higher transglycosylation activity at lower temperatures, in contrast to the hydrolysis activity [10]. The effect of different molar ratios of sucrose and glycerol on the production of GG was examined. MFAS (1 unit) was added to 5 mL of substrate solution containing 100 mM of sucrose as a glucosyl donor and 10, 20, 50, 100, 200, 500, or 1,000 mM of glycerol as an acceptor (molar ratios of 1:0.1, 1:0.2, 1:0.5, 1:1, 1:2, 1:5, and 1:10, respectively) in 50-mM Tris-HCl (pH 8.5). The reaction mixture was incubated at 35 °C for 12 h. The reaction was stopped by heating in boiling water for 10 min, and the reaction products were analyzed by TLC and HPAEC. In addition, a time course of the transglycosylation reaction was generated over 24 h with sample collection every 3 h. The reaction was stopped by boiling for 10 min, and the mixture was centrifuged at 15,300×g for 10 min to remove residual protein before analysis. Thin-Layer Chromatography (TLC) Analysis TLC was used to detect and identify the transglycosylation products synthesized by MFAS. One microliter aliquots of the reaction mixture were spotted onto Whatman K5F silica gel plates that were activated at 110 °C for 30 min and developed with a solvent system of isopropyl alcohol/ethyl acetate/water (3:1:1, v/v/v) in a TLC developing tank. Ascending development was performed once at room temperature. The plate was allowed to air-dry in a hood and was developed by soaking rapidly in 0.5 % (w/v) N-(1-naphthyl)-ethylenediamine Appl Biochem Biotechnol and 5 % (v/v) H2SO4 in methanol. The plate was dried and placed in a 110 °C oven for 10 min to visualize the reaction spots. Purification of Transglycosylated GGs Paper chromatography was performed to purify transglycosylated GG products by multiple descending techniques. Approximately 250 μL of the enzyme reaction product was loaded onto Whatman 3-MM paper (23×55 cm). The paper was irrigated with a 3:1:1 (v/v/v) mixture of isopropyl alcohol/ethyl acetate/water for 24 h in a glass paper chromatography developing tank. After paper ascending development, the paper was allowed to air-dry in a hood. Spots on the paper were detected using an AgNO3 reagent to verify the separation of purified carbohydrates. The paper was sectioned and eluted with deionized water. The separated compounds in the elutant were freeze-dried (Il-shin Lab, Suwon, Korea) and collected for further analysis. A recycling preparative HPLC system (LC-9104, JAI, Tokyo, Japan) was used for the isolation of GGs. Samples (3 μL) were applied to a JAIGEL-W252 (2×50 cm, JAI) column connected in tandem with a JAIGEL-W251 (2×50 cm, JAI) and guard column. The sample was eluted with deionized water at a flow rate of 3 mL/min. The fractions corresponding to GGs were collected and freeze-dried. The purity of each sample was determined using TLC analysis. NMR Analysis 1 H and 13C NMR spectra of the purified GGs were obtained with a Varian Inova AS 400 MHz NMR spectrometer (Varian, Palo Alto, CA). The sample was dissolved in CD3OD at 24 °C with tetramethylsilane (TMS) as the chemical shift reference. Results and Discussion Sequence Analysis, Cloning, and Expression of Putative mfas Gene The putative mfas gene was designated an α-amylase gene (accession number, CP000284) in NCBI Genbank. The gene consists of 1,953 nucleotides, which encode a predicted 650-amino acid protein with a calculated molecular mass of 74,556 Da. The deduced amino acid analysis revealed that the putative MFAS protein shared not only catalytic residues, but also five consensus regions (CR I-V), with other ASases (Fig. 1 and Table 1), although the overall protein showed a low level of amino acid sequence homology when compared with known ASases (ACAS, AMAS, DGAS, DRAS, and NPAS). The topology of A, B, and B’ domains creates an active pocket in ASases [5, 26, 27]. Interestingly, the amino acid homology of B and B’ domains in MFAS with that of other ASases was higher than the overall sequence identity (Table 1). Multiple amino acid sequence alignments revealed that the MFAS protein has retained the key conserved residues required for ASase activity (Fig. 1). These include two catalytic residues (Asp284 and Glu326) belonging to CR III and CR IV, respectively, and Asp141 and Arg520 (located in CR I and CR V, respectively), which are able to form a salt bridge that assists in the formation of an active pocket in ASases [26, 27]. Furthermore, the residues of CR IV (395-HDDIG-399) that were proposed to play an important role in the polymerization activity of ASase are present in the putative MFAS protein [28]. Therefore, we suspected that the putative mfas gene might encode an ASase rather than an α-amylase. Appl Biochem Biotechnol Fig. 1 Amino acid sequence alignment of MFAS with previously identified ASases from various microorganisms. Fully conserved amino acid residues are blocked in black, and five conserved regions (CR), CR I, CR II, CR III, CR IV, and CR V, are indicated by arrows. Triangles indicate amino acid residues required for the catalytic active site. The solid line box indicates the B-domain of ASase, and the dotted line box indicates the B’-domain. Arrows show residues that form a salt bridge, and the asterisk indicates oligosaccharide binding sites The putative mfas gene was successfully amplified by PCR from M. flagellatus KT genomic DNA and expressed in E. coli. An inducible expression vector pET21a was used to construct pETMFAS for efficient expression in E. coli. When DGAS, DRAS, and NPAS were previously expressed in a pGEX expression system, the expression levels of the recombinant proteins were lower than in other systems such as pET and pHCE protein expression systems [4, 12–14]. Recently, ACAS has been successfully expressed in the constitutive and inducible expression systems pET21a and pHCXHD, respectively [16]. Although it was easier to achieve protein expression with the constitutive system (pHCAC AS) than with the inducible system (pETACAS), the production level of pHCACAS was lower than that of pETACAS [16]. As expected, MFAS was expressed in E. coli BL21(DE3) as a dominant protein with a molecular mass of 74 kDa. The expressed recombinant MFAS was efficiently purified by Ni-NTA affinity chromatography, and SDS-PAGE analysis showed a single band at approximately 74 kDa, which matched the theoretical molecular mass of MFAS. Biochemical Properties of MFAS The optimum pH of MFAS was investigated using various buffers, including sodium acetate (pH 4.0 and 5.0), sodium citrate (pH 5.0, 6.0, 6.5, and 7.0), Tris-HCl (pH7.0, 7.5, 8.0, 8.5, and 9.0), and glycine-NaOH (pH9.0 and 10.0) (Fig. 2a). The pH optimum of MFAS was found to Table 1 Alignment results for amylosucrase showing the percentage identity with Methylobacillus flagellatus ASase (MFAS) for the whole protein, the B-domain, and the B’-domain Enzyme Overall amino acids (%) B-domain amino acids B’-domain amino acids ACAS 37.48 53.52 51.52 AMAS 39.29 55.56 52.78 DGAS DRAS 37.42 39.38 56.94 55.56 52.78 50.00 NPAS 37.26 52.11 43.28 ACAS=Arthrobacter chlophenoilcus, AMAS=Altermonas macleodii, DGAS=Deinococcus geothermalis, DRAS=Deinococcus radiodurans, NPAS=Neisseria polysaccharea Appl Biochem Biotechnol Fig. 2 Biochemical properties of MFAS. a Optimum pH was determined in different buffers at 45 °C: filled circle represents sodium acetate at pH 4.0–5.0, empty circle sodium citrate at pH 5.0–7.0, filled inverted triangle Tris-HCl at pH 7.0–9.0, and empty triangle glycine-NaOH at pH 9.0–10.0. b Optimal temperature was determined between 30 and 65 °C at pH 8. c Half-life of MFAS measured at various temperatures in 50-mM Tris-HCl, pH 8.5: filled circle represents 45 °C, empty circle 50 °C, and filled inverted triangle 55 °C. d Melting temperature was confirmed by differential scanning fluorimetry (DSF). A few proteins displayed a shoulder in the peak of the DSF graph similar to MFAS due to the buffer conditions such as pH, salt concentration, and presence or absence of ligand [39–41] be 8.0–8.5. The pH profile of MFAS was similar to that of other ASases such as ACAS, AMAS, and DGAS [4, 15, 16]. The optimum temperature of MFAS at pH 8.5 was determined within the range of 30 to 60 °C (Fig. 2b). Highest enzymatic activity of MFAS was observed at 45 °C. The optimum temperature of MFAS was similar to that of DGAS, which has the highest optimum temperature and thermostability among known ASases. Correlation between thermal stability and enzyme activity for MFAS is shown in Fig. 2c. The half-life of MFAS was calculated as 19.5 and 4.3 min at 50 and 55 °C, respectively, and the melting temperature of MFAS was determined as 50.6 °C (Fig. 2d). These results indicate that the order of thermostability among known ASases is DGAS>MFAS≫AMAS >NPAS>ACAS >DRAS [4, 13–16]. Recently, the three-dimensional structure of DGAS was solved, revealing that it exists as a dimeric form in solution [27]. The authors assumed that dimerization might contribute to the enzyme stabilization of DGAS. To investigate the oligomeric state of MFAS in solution, MFAS was subjected to gel permeation chromatography (GPC). GPC analysis showed that MFAS displayed a peak at a molecular mass of approximately 137.9 kDa, implying that it also exists in a dimeric conformation in solution. The reaction products of MFAS were characterized using sucrose as a sole substrate and compared with those of DGAS (Table 2 and Fig. 3). Although MFAS and DGAS are the two most thermostable enzymes among known ASases, MFAS could not produce a significant Appl Biochem Biotechnol Table 2 Yields of three ASase reactions (hydrolysis, polymerization, and isomerization) for different enzymes with sucrose as the sole substrate Enzymes Substrate concentration (mM) The ratio of reaction products (%) Hydrolysis (glucose and fructose) References Isomerization Polymerization (maltooligosaccharides (trehalulose and insoluble glucan) and turanose) MFAS 100 9.5 75.5 15.0 This study ACAS 100 1.9 78.4 19.7 [16] DGAS 100 7.0 71.0 22.0 [4] DRAS NPAS 100 100 9.6 5.4 56.9 80.1 33.5 14.5 [14] [14] amount of insoluble glucans from sucrose at a relatively high temperature (45 °C) [4]. Previous studies showed that the polymerization activity of ASase decreased as temperature increased [4, 10]. However, when sucrose was reacted with MFAS and DGAS at 40 °C for 48 h, MFAS produced substantial amounts of turbid insoluble glucans. Moreover, the reaction mixture of MFAS displayed more turbidity than that of DGAS, indicating that MFAS synthesized more insoluble α-1,4-glucan from sucrose than DGAS (Fig. 3b). The comparison of the side-chain distributions (DP>4) in the relative peak area of HPAEC chromatogram confirmed that MFAS synthesized relatively longer glucans (DP>33) than DGAS. These results indicated that the polymerization activity of MFAS was higher than that of DGAS at a high temperature. In addition, the intermolecular transglycosylation activity of MFAS was examined with glycerol as an acceptor molecule. Bioconversion of Glycerol to GG by MFAS GGs were enzymatically synthesized from glycerol through the intermolecular transglycosylation activity of MFAS. In TLC analysis, few spots appeared in the reaction with Fig. 3 High-performance anion-exchange chromatography (HPAEC) analysis of α-1,4 glucan synthesized by MFAS. a HPAEC analysis of sucrose isomers and short maltooligosaccharides (shorter than maltohexaose), b HPACE analysis of long maltooligosaccharides. G2 maltose, G3 maltotriose, G4 maltotetraose, G5 maltopentaose, G6 maltohexaose, G7 maltoheptaose, DP degree of polymerization. The image on the upper right corner of b is the reaction mixtures of MFAS and DGAS. The reaction was performed for 48 h at 40 °C with sucrose as a sole substrate. The sample was diluted 10-fold and filtered before injection Appl Biochem Biotechnol MFAS compared with the reaction mixture without glycerol (Fig. 4). Although the main spots in TLC analysis with MFAS were maltooligosaccharides, as expected from a typical ASase reaction with sucrose as the sole substrate, two spots that were not identical to any standard molecule or maltooligosaccharide were clearly evident (GT1 and GT2 in Fig. 4a). In addition, two new peaks other than glycerol, glucose, and fructose were observed in the HPAEC analysis (Fig. 4a). These analytical experiments indicated that two unidentified glycerol transglycosylation products (GT1 and GT2) were synthesized by MFAS. The amount of GT2 produced was higher than that of GT1. Various enzymatic methods were previously employed to produce GGs [23, 29–31]. Two β-GGs were synthesized by β-glucosidase from Pyrococcus furiosus using 2-nitophenyl-β-D-glucopyranoside or cellobiose as a donor and glycerol as an acceptor [31]. When the transglycosylation reaction of cyclodextrin glucanotransferase (CGTase) was used to synthesize GGs using soluble starch as a donor and glycerol as an acceptor, (2R/S)-1-O-α-D-glucosyl-glycerol and 2-O-α-D-glucosyl-glycerol were isolated as the major and minor smallest transfer products, respectively [29]. 2-O-α-DGlucosyl-glycerol, (2R)-1-O-α-D-glucosyl-glycerol, and (2S)-1-O-α-D-glucosyl-glycerol were synthesized by transglucosidase L “Amano” (Amano Enzyme Inc, Nagoya, Japan) with maltose as a donor and glycerol as an acceptor. Among these three GGs, (2R)-1-O-α-Dglucosyl-glycerol was the main constituent [23]. 2-O-α-D-glucosyl-glycerol as a major product was mainly synthesized through the transglycosylation reaction of sucrose phosphorylase with sucrose or glucose-1-phosphate as a donor and glycerol as an acceptor [30]. Similar to these reactions, two transglycosylation products were detected in the MFAS reaction and one major product was predominantly synthesized, indicating that MFAS might have regioselectivity for the transglycosylation reaction using glycerol as an acceptor. Purification and Structural Determination of GGs The GG products synthesized by MFAS were purified by paper chromatography and a preparative recycling HPLC system. Two isolated GG products were each detected as a single spot on TLC analysis, indicating that the purification was successful (Fig. 4b). Interestingly, the isolated GT1 was detected as a double peak on HPAEC analysis whereas the isolated GT2 Fig. 4 HPAEC and TLC analyses of transglycosylation reaction by MFAS with glycerol as an acceptor and sucrose as a donor (a) and purification of GGs synthesized by MFAS (b). a TLC analysis, Lane M standard markers from G1 (glucose) to G7 (maltoheptaose); Lane 1 only sucrose reaction with MFAS; Lane 2 glycerol transglycosylation reaction without MFAS; Lane 3 glycerol transglycosylation reaction with MFAS; GT1 and GT2 glucosyl-glycerols; Gly glycerol. b TLC analysis, Lane M standard marker from G1 (glucose) to G7 (maltoheptaose); Lane 1 glycerol transglycosylation reaction with MFAS; Lane 2 purified GT1; Lane 3 purified GT2. HPAEC analysis, solid lines represents purified GT1and dotted lines purified GT2 Appl Biochem Biotechnol appeared as a single peak. Previous HPAEC analysis showed that stereoisomers appeared as a double peak because they are not completely separated by HPAEC [32, 33]. This result led us to assume that GT1 might be a mixture of GG stereoisomers. 1 H and 13C NMR analyses were used to determine the structure of isolated GT1 and GT2 (Table 3). In 1H and 13C NMR analyses, the signals of C1’, C1, C3, C4, and H1 in the GT1 compound were observed as double peaks, and the chemical shift of C1 in the glycerol molecule changed greatly from 64 to 72 (8 ppm). The chemical shift of C1’ and C3 was slightly changed. This results were comparable to 1H and 13C NMR spectra of a mixture of (2R)-1-O-β-D-glucosyl-glycerol and (2S)-1-O-β-D-glucosyl-glycerol, suggesting GT1 was a mixture of these two stereoisomers [34]. The bond between glucose and glycerol in GT1 was determined to be an α-glycosidic linkage according to the coupling constant (J=3.6 Hz) in 1H NMR analysis. These results indicated that GT1 (1-GGs) was a mixture of (2R)-1-O-α-Dglucosyl-glycerol (R-1-GG) and (2S)-1-O-α-D-glucosyl-glycerol (S-1-GG) (Fig. 5a). GT2 was determined to be 2-O-α-D-glucosyl-glycerol (2-GG) by 1H and 13C NMR analyses (Fig. 5a). The chemical shift of C2 in the glycerol molecule changed greatly from 72 to 81.5 (9.5 ppm) in GT2, confirming that the transferred glucose molecule was connected to C2 in the glycerol molecule. The result of 13C NMR analysis was identical to 13C NMR results of 2-O-β-D-glucosyl-glycerol synthesized by β-glucosidase [35]. In addition, 1H NMR analysis revealed that the glucose molecule was transferred to C2 in the glycerol molecule with an α-anomeric configuration based on the coupling constant (J=4.0 Hz) of the glucose anomeric proton signal observed at 4.9 ppm. Effect of Acceptor Concentration on Bioconversion of Glycerol to GGs The production yield of GGs by MFAS was analyzed at various glycerol concentrations with a fixed concentration of sucrose (100 mM) by HPAEC (Fig. 5b). As the glycerol concentration increased, the production level of GGs increased. Interestingly, the production level of 2-GG was higher than that of 1-GGs under all reaction conditions. The ratio of 2-GG over 1-GGs was calculated to be 4.5–5.3 when no less than 100 mM of glycerol was used. The production yield of MFAS was 48 % (based on acceptor), which Table 3 13 C (100 MHz) and 1H NMR (400 MHz) data for GGs synthesized by MFAS (units—ppm, in CD3OD) 1-GGs 13 C NMR Glycerol 1 H NMR 13 C NMR 2-GG 1 H NMR 13 C NMR 1 63.8 72.3, 72.1 2 73.2 71.8 81.5 3 63.8 64.2, 64.1 62.3 α-D-Glucopyranose 1’ 101.2 100.6, 100.3 2’ 73.7 73.7 3’ 75.3 75.2 4’ 72.0 70.9, 70.1 5’ 73.9 73.8 6’ 62.7 62.6 Reference [37, 38] 1 H NMR 62.6 4.80, 4,79 (d, J=3.6 Hz) 3.39 100.1 4.98 (d, J=4.0 Hz) 73.8 3.37 (dd, J=10.4, 4.0 Hz) 75.1 3.28 (dd, J=9.2, 9.2 Hz) 71.8 3.26 (dd, J=9.2, 9.2 Hz) 73.9 62.9 This study This study Appl Biochem Biotechnol Fig. 5 Molecular structures of GGs synthesized by MFAS (a) and production of GGs in the transglycosylation reaction with various concentrations of glycerol as the acceptor and 100 mM of sucrose as a donor (b). a Structure of (2R)-1-O-α-D-glucosyl-glycerol and (2S)-1-O-α-D-glucosyl-glycerol and 2-O-α-D-glucosyl-glycerol. b Open bar represents the concentration of 2-O-α-D-glucosyl-glycerol whereas closed bar stands for the concentration of (2R/S)-1-O-α-D-glucosyl-glycerol was higher than those of other enzymes. The production yield of 19.9 % was observed with CGTase reaction whereas 5.1 % with α-glucosidase [23, 29]. The most comparable production yield (35 %) was obtained with sucrose phosphorylase [30]. When αglucosidase produced three GGs (R-1-GG, S-1-GG, and 2-GG) using maltose and glycerol, the ratio of R-1-GG, S-1-GG, and 2-GG was 49:41:10 [23]. Likewise, CGTase was shown to predominantly transfer glucose to the one (or three) position of glycerol [29]. On the other hand, sucrose phosphorylase from Leuconostoc mesenteroides synthesized mainly 2-GG using 800 mM of sucrose as a donor and 2,000 mM of glycerol as an acceptor [30]. Similar to sucrose phosphorylase, MFAS predominantly synthesized 2-GG over 1-GGs. This result suggests that the active site of MFAS provided a regioselectivity of glucosyl transfer using glycerol as an acceptor. It is quite interesting that previous studies showed that both ASase and sucrose phosphorylase produced the same transglycosylation products using hydroquinone and catechin as an acceptor [11, 12, 36]. This indicates that these two enzymes share similar regioselectivity when they perform the intermolecular transglycosylation reaction. Our results indicate that MFAS can be employed to make various biological materials with high efficiency using inexpensive donor molecules. Appl Biochem Biotechnol Conclusions The enzymatic properties of a putative amylosucrase (ASase) gene of M. flagellatus KT ATCC51484 (mfas) were confirmed in this study. MFAS was relatively thermostable and produced significant amounts of insoluble glucans at a high temperature (40 °C) compared with other previously characterized ASases. The intermolecular transglycosylation activity was employed in the production of GG, a compatible solute and enzyme stabilizer. 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