Transglycosylation Properties of a Novel α1 , 4-Glucanotransferase from Bacteroides thetaiotaomicron and Its Application in Developing an α-Glucosidase-Specific Inhibitor

In this study, α-glucanotransferase from Bacteroides thetaiotaomicron was expressed in Escherichia coli and characterized. Conserved amino-acid sequence alignment showed that Bacteroides thetaiotaomicron α-glucanotransferase (BtαGTase) belongs to the glycoside hydrolase family 77. .e enzyme exhibited optimal catalytic activity at 60°C and pH 3.0. BtαGTase catalyzed transglycosylation reactions that produced only glycosyl or maltosyl transfer products, which are preferable for the generation of transglycosylated products with high yield. .e 1-deoxynojirimycin (DNJ) glycosylation product G1-DNJ was generated using BtαGTase, and the inhibitory effect of G1-DNJ was analyzed. A kinetic study of inhibition revealed that G1-DNJ inhibited α-glucosidase to a greater extent than did DNJ but did not show any inhibitory effects towards α-amylase, suggesting that G1-DNJ is a potential candidate for the prevention of diabetes.


Introduction
Bacteroides thetaiotaomicron is a human colonic gramnegative obligate anaerobe found in high numbers in the human intestine that can ferment a wide diversity of polysaccharides [1].Members of this genus require carbohydrates as a source of carbon and energy.Polysaccharides are the essential source of carbohydrates for these bacteria in the human intestine [2].Carbohydrates are fermented by Bacteroides and other intestinal bacteria, resulting in the production of volatile fatty acids that are reabsorbed by the large intestine and used by the host as an energy source; these constitute a significant proportion of the host's daily energy requirements.B. thetaiotaomicron contains a system that enables the broad utilization of starch and various genes involved in starch binding and application [3].Wexler reported that its 172 glycohydrolases and 163 homologs of starch-binding proteins enable this organism to utilize a wide variety of dietary carbohydrates available in the gut [4].
e glycoside hydrolase family GH77 is a monospecific family consisting of 4-α-glucanotransferase (α-GTase, EC 2.4.1.25)and defined by an established classification system based on the sequences of all active carbohydrate enzymes from the Carbohydrate-Active Enzyme (CAZy) database [5].α-GTase catalyzes the transfer of α-1,4-glucan to an acceptor, which is usually the 4-hydroxyl group of another α-1,4-glucan or glucose [6].In this reaction, hydrolysis of the α-1,4 linkage and subsequent synthesis of a new α-1,4 linkage occur repeatedly within the same glucan molecule or between different molecules [7].Effective donors of maltooligosaccharides include amylopectin and soluble starch, which together with glucose also serve as acceptors [8].
ese enzymes are found in microorganisms and plants, in which they are involved in maltooligosaccharide metabolism or glycogen and starch metabolism, respectively [6].While the Bacteroides thetaiotaomicron GH77 family contains only one of these enzymes, other GH families contain many carbohydrate-related enzymes.
α-GTase transglycosylation activity is useful in carbohydrate chemistry.Starches that are modified by α-GTases show novel rheological and nutritional properties, such as thermoreversible gelation, fat-replacing properties, and hypocholesterolemic and hypoglycemic effects [9].α-GTases from different bacteria have successfully modified the properties of various food materials, including increased water solubility, stability, functional effects, and taste [10].In addition, intramolecular glucan transferase produces cyclic glucans (cycloamyloses) with a higher degree of polymerization compared with cyclodextrins [9].1-Deoxynojirimycin (DNJ), an aza-sugar, has structural characteristics similar to those of cyclic monosaccharides; however, the oxygen is substituted with a nitrogen atom.DNJ prevents glucose from entering the bloodstream from the intestines by inhibiting the activities of α-amylase and α-glucosidase [11,12].Intestinal α-glucosidase is one of the glucosidases of the small intestinal epithelium [13].α-Glucosidase hydrolyzes α-1-4-linked D-glucose from the nonreducing end of α-glucoside, which is the last step in the digestion of disaccharides and polysaccharides [14].
us, inhibition of intestinal α-glucosidases would prevent the rapid digestion of carbohydrate and, consequently, the sharp postprandial rise in blood glucose [13].However, antidiabetic drugs that prevent carbohydrate digestion have gastrointestinal side effects, such as abdominal distention and flatulence.ese side effects may be caused by the inhibition of α-amylase, which leads to the accumulation of undigested carbohydrates in the intestines [15,16].erefore, antidiabetic drugs that specifically inhibit α-glucosidase are required.
In this study, we cloned a novel α-GTase of the GH77 family from Bacteroides thetaiotaomicron (BtαGTase) and examined its reaction pattern using diverse substrates.Additionally, we applied the transglycosylation activity of BtαGTase to DNJ to develop a prospective candidate for the prevention of diabetes.

Cloning and Sequence Analysis.
e gene encoding bt_2146 was separated from the genomic DNA of B. thetaiotaomicron, and the target DNA was amplified by polymerase chain reaction (PCR) using two primers (bt-F, 5′-AAAACCATGGCCACTGTATCATTTAAC-3′ and bt-R, 5′-AAAACTCGAGTTTCTTGGGAGCTCTGCC-3′) containing the NcoI and XhoI restriction enzyme sites, respectively.e PCR conditions were as follows: denaturation for 1 min at 98 °C, followed by 30 cycles of 10 s at 98 °C, annealing for 30 s at 53 °C, and extension for 1 min 30 s at 72 °C.
e BtαGTase concentration was determined from 2 µL drops of protein solution using the NanoDrop 2000c spectrophotometer ( ermo Fisher Scientific, Waltham, MA, USA) at 280 nm with the appropriate extinction coefficient for BtαGTase (227,128 cm −1 M −1 ).

Sequence Analysis.
e characterized α-GTase sequences of GH77 family members were obtained from the CAZy database (http://www.cazy.org/).Sequence alignment was performed using the Alignment X software, a component of Vector NTI Suite 5.5 (InforMax, Bethesda, MD, USA).In addition, a phylogenetic tree was constructed using MEGA6 software based on the neighbor-joining tree method (1,000 bootstrap samples) [17].

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Journal of Chemistry 2.5.Enzymatic Assay.BtαGTase enzymatic activity was measured in an amylose and maltose mixture (0.02% (w/v) amylose solution and 0.05% (w/v) maltose solution in 50 mM citric-NaOH (pH 3.0) at 60 °C) as described previously by Lee et al. [7].After preheating at 60 °C for 5 min, the enzyme activity was measured using Lugol's solution.e standard curve consisted of ∼0.000391-0.025%(w/v) amylose solution, and one unit of BtαGTase activity was defined as the amount of enzyme that hydrolyzed 1 µg/mL reducing amylose 1 min after transfer to G2. Absorbance was measured at 620 nm using a spectrophotometer (Multiskan FC; ermo Fisher Scientific).

Effects of Temperature and pH on Enzyme Activity and Stability.
e optimal temperature for BtαGTase activity was determined using amylose and G2 as substrates in 50 mM citric-NaOH (pH 3.0) buffer, and Lugol's solution was used to measure the activity at various temperatures (40-70 °C).Similarly, the effect of pH on BtαGTase activity was measured at various pH values (2.5-6) using 50 mM citric-NaOH (pH 2.5-3.5) and 50 mM sodium acetate (pH 3.5-6.0).

Effects of Metal Ions on Enzyme Activity.
To evaluate the effects of metal ions on the activity of the purified enzyme, the reaction mixture was preincubated at 50 °C for 10 min at 5 mM final concentrations of MgCl 2 , MnCl 2 , CaCl 2 , CoCl 2 , CuCl 2 , FeCl 3 , ZnCl 2 , and EDTA.
e relative activity of BtαGTase was measured under standard conditions (50 °C, pH 3.0) using Lugol's solution.e enzyme activity in the absence of metal ions was considered to be 100%.

Analysis of the Reaction Products Using in-Layer Chromatography (TLC).
e TLC was performed using a K5F silica-gel plate (Whatman, Maidstone, UK).After the samples were spotted, the silica-gel plate was dried and placed in developing solvent (n-butanol:ethanol:water, 5 : 5 : 3, v/v/ v).To analyze the reaction products, the TLC plate was dried, saturated in dipping solution (0.33% w/v N-[1-naphthyl]ethylenediamine and 5% (v/v) H 2 SO 4 in methanol), and heated at 110 °C for 10 min.

Preparation of the Transglycosylation Product.
For the transglycosylation reaction, a substrate solution containing 1.5% (w/v) DNJ and 1% (w/v) soluble starch was prepared in 50 mM sodium acetate buffer (pH 6.0).BtαGTase was added to this substrate solution and was incubated for 7 h before terminating the enzyme reaction by boiling the solution for 10 min.

Purification of the Transglycosylation Product Using Preparative-High Performance Liquid Chromatography (Prep-HPLC).
e DNJ transglycosylation product was purified using LC-Forte/R preparative-high-performance liquid chromatography (prep-HPLC; YMC Korea, Seongnam, Korea) equipped with a Triart-C18 column (250 × 20 mm; YMC Korea) and an ultraviolet detector (200 and 210 nm).For the isocratic solvent system, 0.1% ammonium in deionized water was used at a flow rate of 12.0 mL/min at room temperature, and 1 mL of the sample was injected.
e mixture (1 µL) was applied to a MALDI-TOF mass spectrometry probe and dried slowly at room temperature.An accelerating voltage of 20,000 V was used.

Inhibition Kinetics of α-Glucosidase and α-Amylase.
To determine the inhibition mechanism, a modified Dixon plot was produced [18].e inhibitory mode of DNJ and the DNJ transfer product (G1-DNJ) against α-glucosidase and α-amylase were analyzed using the substrates pNPG and pNPG2, respectively.e increase in absorption due to the hydrolysis of pNPG substrates was observed at 405 nm using an enzyme-linked immunosorbent assay (ELISA) plate reader (Multiskan FC; ermo Fisher Scientific).e reaction mixture containing 100 µL of the substrate, 50 mM sodium acetate buffer (pH 7.0), 50 mM potassium phosphate buffer (pH 6.9, 40 µL), and various concentrations of inhibitor (0.1-1 µmol) dissolved in 20 µL distilled water was preheated for 5 min before adding the enzyme (40 µL).

Cloning and Expression of Alpha-Glucanotransferase str. IM2 in E. coli.
e α-glucanotransferase-encoding gene bt_2146 was amplified successfully from B. thetaiotaomicron using PCR (Figure S1 in Supplementary Data).e amplified gene (∼2.7 kb) was ligated into the pTKNd119 vector.e resulting recombinant plasmid, pTKNdBtαgtase, was transformed into E. coli MC1061, and the expressed enzyme was subsequently purified by Ni-NTA affinity chromatography.e expression of BtαGTase was analyzed by SDS-PAGE (Figure S2 in Supplementary Data).e predicted size of the expressed enzyme was 105 kDa.Table 1 shows the purification results.

Preparation of the DNJ Transfer Product. As shown in
Figure 4, DNJ and soluble starch were reacted with BtαGTase, and the enzyme reaction was analyzed by HPAEC.e enzyme reaction product was puri ed using prep-HPLC and analyzed by HPAEC and MALDI/TOF-MS (Figures 5 and 6).With a molecular mass of 349.33 Da, the transfer product was identi ed as a glucosyl DNJ (G1-DNJ).

Kinetic Study of the Inhibitory Activity of DNJ and G1-DNJ.
e inhibitory e ects of DNJ and G1-DNJ against α-glucosidase and α-amylase were analyzed in a kinetic study.As shown in Figure 7(a), both the DNJ and G1-DNJ plots showed a series of lines converging on the same point above the x-axis, indicating that DNJ and G1-DNJ have competitive inhibitory e ects on α-glucosidase.e K i value of G1-DNJ was lower than that of DNJ, suggesting that G1-DNJ is a better inhibitor of α-glucosidase (Table 2).In the α-amylase inhibition assay, the DNJ plot showed a competitive inhibitory pattern, while the G1-DNJ plot did   in-layer chromatography (TLC) analysis of the transferred products of the BtαGTase reaction.TLC analysis of the hydrolytic products generated by BtαGTase.S, standard (G1-G7); lane 1, glucose (G1); lane 2, maltose (G2); lane 3, maltotriose (G3); lane 4, maltotetraose (G4); lane 5, maltopentaose (G5); lane 6, maltohexaose (G6); and lane 7, maltoheptaose (G7).a, before the reaction; b, after the reaction.Hydrolysis was performed at 60 °C at pH 3.0 for 12 h.

Discussion
αGTase belongs to the α-amylase superfamily [8,19].is enzyme catalyzes disproportionation reactions, which hydrolyze α-glycosidic linkage and subsequently synthesize new α-glycosidic linkage within the same or di erent glucan molecules [7,8].Recently, the use of α-GTase has received considerable attention, speci cally for the development of many starch products such as cycloamylose, cyclic cluster dextrin, slowly digestible starch, and thermoreversible starch [20][21][22][23].In our study, a novel α-GTase from B. thetaiotaomicron was cloned and characterized.An amino acid sequence alignment indicated that BtαGTase contained conserved regions and three catalytic sites that belong to the GH77 family (Supplementary Data 4).Interestingly, BtαGTase demonstrated low disproportionation activity, resulting in only one or two types of transfer products using various maltooligosaccharides as acceptor molecules    Journal of Chemistry (Figure 2).Although BtαGTase is not able to elongate several glucosyl units, this low disproportionation activity could be advantageous for the preparation of a single glucosyltransfer product and to enhance the availability of the acceptor molecule.In addition, the production of various products from an enzyme reaction is not bene cial for the puri cation of a speci c product.erefore, BtαGTase may be a valuable enzyme for transglycosylation reactions.Generally, antidiabetic drugs used to treat type II diabetes mellitus, such as acarbose and DNJ, inhibit the activities of α-amylase and α-glucosidase [11].Inhibition of α-amylase leads to the accumulation of undigested carbohydrates in the intestines, which may cause abdominal distention and atulence [15,16].In this study, the transfer product G1-DNJ inhibited only α-glucosidase (Table 2, Figure 7), which may reduce the side e ects of other antidiabetic drugs.
In conclusion, we assessed the properties and industrial applicability of BtαGTase.is enzyme is a novel α-glucanotransferase of the glycoside hydrolase family GH77, and its transglycosylation properties render it e cient in preparing molecules with the transfer of single-glucosyl residues.G1-DNJ, prepared using BtαGTase, showed stronger inhibitory e ects than those of DNJ, but it did not a ect α-amylase activity, suggesting that this molecule may be a potential drug candidate for the treatment of type II diabetes mellitus.
Data Availability e data used to support the ndings of this study are available from the corresponding author upon request.

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Journal of Chemistry not demonstrate any convergence of lines, indicating that G1-DNJ does not have an inhibitory e ect on α-amylase (Figure 7(b)).

Figure S1 :Figure 7 :
Figure S1: generation of the DNA construct for BtαGTase expression.(A) e bt_2146 gene was ampli ed by polymerase chain reaction (PCR).Lane S, DNA marker; lane 1, ampli ed BtαGTase gene.(B) e pKTNd_bt2146 construct was generated by ligating bt_2146 into the pTKNd vector.Figure S2: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of BtαGTase at each puri cation step.Lane S, protein size standards; lane 1, cellular protein from the crude extract; lane 2, soluble fraction;

Table 1 :
Puri cation of the BtαGTase enzyme.