The Discovery, Molecular Cloning, and Characterization of Dextransucrase LmDexA and Its Active Truncated Mutant from Leuconostoc mesenteroides NN710.

Dextransucrases play a crucial role in the production of dextran from economical sucrose; therefore, there is a pressing demand to explore novel dextransucrases with better performance. This study characterized a dextransucrase enzyme, LmDexA, which was identified from the Leuconostoc mesenteroides NN710. This bacterium was isolated from the soil of growing dragon fruit in Guangxi province, China. We successfully constructed six different N-terminal truncated variants through sequential analysis. Additionally, a truncated variant, ΔN190LmDexA, was constructed by removing the 190 amino acids fragment from the N-terminal. This truncated variant was then successfully expressed heterologously in Escherichia coli and purified. The purified ΔN190LmDexA demonstrated optimal hydrolysis activity at a pH of 5.6 and a temperature of 30 °C. Its maximum specific activity was measured to be 126.13 U/mg, with a Km of 13.7 mM. Results demonstrated a significant improvement in the heterologous expression level and total enzyme activity of ΔN190LmDexA. ΔN190LmDexA exhibited both hydrolytic and transsaccharolytic enzymatic activities. When sucrose was used as the substrate, it primarily produced high-molecular-weight dextran (>400 kDa). However, upon the addition of maltose as a receptor, it resulted in the production of a significant amount of oligosaccharides. Our results can provide valuable information for enhancing the characteristics of recombinant dextransucrase and potentially converting sucrose into high-value-added dextran and oligosaccharides.

Dextran Used in Blood Transfusion, Hematology, and Pharmaceuticals: Biosynthesis of Diverse Molecular-Specification-Dextrans in Enzyme-Catalyzed Reactions.

Dextran is an exopolysaccharide synthesized in reactions catalyzed by enzymes obtained from microbial agents of specific species and strains. Products of dextran polysaccharides with different molecular weights are suitable for diverse pharmaceutical and clinical uses. Dextran solutions have multiple characteristics, including viscosity, solubility, rheological, and thermal properties; hence, dextran has been studied for its commercial applications in several sectors. Certain bacteria can produce extracellular polysaccharide dextran of different molecular weights and configurations. Dextran products of diverse molecular weights have been used in several industries, including medicine, cosmetics, and food. This article aims to provide an overview of the reports on dextran applications in blood transfusion and clinical studies and its biosynthesis. Information has been summarized on enzyme-catalyzed reactions for dextran biosynthesis from sucrose and on the bio-transformation process of high molecular weight dextran molecules to obtain preparations of diverse molecular weights and configurations.

Brewers' spent grain as substrate for dextran biosynthesis by Leuconostoc pseudomesenteroides DSM20193 and Weissella confusa A16.

BACKGROUND: Lactic acid bacteria can synthesize dextran and oligosaccharides with different functionality, depending on the strain and fermentation conditions. As natural structure-forming agent, dextran has proven useful as food additive, improving the properties of several raw materials with poor technological quality, such as cereal by-products, fiber-and protein-rich matrices, enabling their use in food applications. In this study, we assessed dextran biosynthesis in situ during fermentation of brewers´ spent grain (BSG), the main by-product of beer brewing industry, with Leuconostoc pseudomesenteroides DSM20193 and Weissella confusa A16. The starters performance and the primary metabolites formed during 24 h of fermentation with and without 4% sucrose (w/w) were followed. RESULTS: The starters showed similar growth and acidification kinetics, but different sugar utilization, especially in presence of sucrose. Viscosity increase in fermented BSG containing sucrose occurred first after 10 h, and it kept increasing until 24 h concomitantly with dextran formation. Dextran content after 24 h was approximately 1% on the total weight of the BSG. Oligosaccharides with different degree of polymerization were formed together with dextran from 10 to 24 h. Three dextransucrase genes were identified in L. pseudomesenteroides DSM20193, one of which was significantly upregulated and remained active throughout the fermentation time. One dextransucrase gene was identified in W. confusa A16 also showing a typical induction profile, with highest upregulation at 10 h. CONCLUSIONS: Selected lactic acid bacteria starters produced significant amount of dextran in brewers' spent grain while forming oligosaccharides with different degree of polymerization. Putative dextransucrase genes identified in the starters showed a typical induction profile. Formation of dextran and oligosaccharides in BSG during lactic acid bacteria fermentation can be tailored to achieve specific technological properties of this raw material, contributing to its reintegration into the food chain.

Analysis of Structural and Functional Differences of Glucans Produced by the Natively Released Dextransucrase of Liquorilactobacillus hordei TMW 1.1822.

The properties of the glucopolymer dextran are versatile and linked to its molecular size, structure, branching, and secondary structure. However, suited strategies to control and exploit the variable structures of dextrans are scarce. The aim of this study was to delineate structural and functional differences of dextrans, which were produced in buffers at different conditions using the native dextransucrase released by Liquorilactobacillus (L.) hordei TMW 1.1822. Rheological measurements revealed that dextran produced at pH 4.0 (MW = 1.1 * 108 Da) exhibited the properties of a viscoelastic fluid up to concentrations of 10% (w/v). By contrast, dextran produced at pH 5.5 (MW = 1.86 * 108 Da) was gel-forming already at 7.5% (w/v). As both dextrans exhibited comparable molecular structures, the molecular weight primarily influenced their rheological properties. The addition of maltose to the production assays caused the formation of the trisaccharide panose instead of dextran. Moreover, pre-cultures of L. hordei TMW 1.1822 grown without sucrose were substantial for recovery of higher dextran yields, since the cells stored the constitutively expressed dextransucrase intracellularly, until sucrose became available. These findings can be exploited for the controlled recovery of functionally diverse dextrans and oligosaccharides by the use of one dextransucrase type.

Potential prebiotic effect of a long-chain dextran produced by Weissella cibaria: an in vitro evaluation.

Dextrans are homopolysaccharides of D-glucose units produced by lactic acid bacteria. They have several technological applications and potential utilisation in positively modulating gut microbiota is attracting increasing attention. Whereas the prebiotic activity of low polymerisation degree (DP) dextrans has been established, high DP dextrans still deserve deeper investigation. In the present study, a long linear chain dextran produced by Weissella cibaria was compared to inulin with regards to the growth of specific health-related taxa and to the production of organic acids in pH-controlled batch cultures of intestinal microbiota. qPCR quantification of Lactobacillus, Bifidobacterium, Prevotella, Bacteroides fragilis, and Faecalibacterium prausnitzii revealed differences in their relative abundance, depending on the carbon source, that reflected the pattern of fermentation products determined by HPLC. Dextran mainly enhanced the relative amount of Prevotella and Bacteroides, consistently with a favourable acetate-propionate ratio suggesting a promising utilisation as functional ingredient in the food industry.

Evaluation of efficient carbon, nitrogen sources, micro and macro nutrients for dextran production by Weissella cibaria CMGDEX3 by utilizing a modified multifactorial Placket-Burman statistical design.

In the present study previously isolated Weissella cibaria CMG DEX3 capable of producing high molecular weight, water soluble dextran (Ahmed et al., 2012) is characterized for most efficient less expensive carbon, nitrogen sources, micro and macro nutrients by utilizing a multifactorial Placket-Burman statistical design for optimization of dextran production. A twelve run Plackett-Burman experimental model with slight modification was utilized to evaluate the impact of ten diverse nutrients on the production of dextran by the bacterial isolate Weissella cibaria CMG DEX3.

Structural analysis and properties of dextran produced by Weissella confusa and the effect of different cereals on its rheological characteristics.

Weissella confusa is a commonly found species in boza, a highly viscous beverage obtained from fermented cereals. Exopolysaccharide (EPS) produced by Weissella confusa C19 was characterized and the role of different cereals on its rheological characteristics were studied. Thus, the effect of the type of cereals on textural characteristics of boza for standard boza production and the fate of W. confusa during fermentation were assessed. W. confusa C19 EPS consisted of glucose, sucrose and 1.7 mg/kg protein. Structural characterization of water-soluble dextran was determined by 1H NMR, functional groups were identified by Fourier transform infrared spectroscopy (FT-IR) and the crystal structure was determined by X-ray diffraction (XRD). After EPS characterization, different cereal-based media including maize, oat, rice and wheat were used for the growth and EPS production of the bacterium. The rheologial properties of EPS obtained from different cereal-based media showed that the steady shear behavior of EPS was pseudoplastic. W. confusa C19 is a unique strain that can adapt to the environment containing high sugar to produce high amounts of polysaccharides. Besides new information on EPS from W. confusa origin, this study showed that the amount of dextran increased using solid media fortified with different cereals.

A systematic approach to study the pH-dependent release, productivity and product specificity of dextransucrases.

BACKGROUND: Dextransucrases are extracellular enzymes, which catalyze the formation of α-1→6-linked glucose polymers from sucrose. These enzymes are exclusively expressed by lactic acid bacteria, which commonly acidify the extracellular environment due to their physiology. Dextransucrases are thus confronted with steadily changing reaction conditions in regards to the environmental pH, which can further affect the amount of released dextransucrases. In this work, we studied the effect of the environmental pH on the release, the productivity and the product specificity of the dextransucrase expressed by Lactobacillus (L.) hordei TMW 1.1822. Dextransucrases were recovered as crude extracts at pH 3.5-pH 6.5 and then again used to produce dextrans at these pH values. The respectively produced dextran amounts and sizes were determined and the obtained results finally systematically correlated. RESULTS: Maximum dextran amounts were produced at pH 4.0 and pH 4.5, while the productivity of the dextransucrases significantly decreased at pH 3.5 and pH 6.5. The distribution of dextran amounts produced at different pH most likely reflects the pH dependent activity of the dextransucrases released by L. hordei, since different transglycosylation rates were determined at different pH using the same dextransucrase amounts. Moreover, similar hydrolysis activities were detected at all tested conditions despite significant losses of transglycosylation activities indicating initial hydrolysis prior to transglycosylation reactions. The molar masses and rms radii of dextrans increased up to pH 5.5 independently of the stability of the enzyme. The gelling properties of dextrans produced at pH 4.0 and pH 5.5 were different. CONCLUSIONS: The presented methodological approach allows the controlled production of dextrans with varying properties and could be transferred and adapted to other microbes for systematic studies on the release and functionality of native sucrases or other extracellular enzymes.

Isolation and characterization of dextran produced by Lactobacillus sakei L3 from Hubei sausage.

An exopolysaccharide (EPS)-producing bacterial strain L3 was isolated from Hubei sausage and identified as Lactobacillus sakei via morphological, physiological, biochemical and 16S rDNA analysis. FT-IR spectroscopy and NMR revealed that L3 EPS was a dextran containing d-glucose residues with α-1,6 glycosidic linkage. Rheological studies showed that it had high viscosity at high concentration, low temperature, and acidic pH (pH 3.0). Scanning electron microscopy of the L3 dextran demonstrated a porous and branched morphology, and atomic force microscopy showed lumps of varying height on the rough surface of the L3 EPS polymer. The EPS was thermally stable up to 272°C and could coagulate sucrose-supplemented milk. Together, these results suggested that L3 EPS might have potential applications in food processing and other areas.

High Levels of CO2 Induce Spoilage by Leuconostoc mesenteroides by Upregulating Dextran Synthesis Genes.

During nonventilated storage of carrots, CO2 gradually accumulates to high levels and causes modifications in the carrot's microbiome toward dominance of Lactobacillales and Enterobacteriales The lactic acid bacterium Leuconostoc mesenteroides secretes a slimy exudate over the surface of the carrots. The objective of this study was to characterize the slime components and the potential cause for its secretion under high CO2 levels. A proteomic analysis of the exudate revealed bacterial glucosyltransferases as the main proteins, specifically, dextransucrase. A chemical analysis of the exudate revealed high levels of dextran and several simple sugars. The exudate volume and dextran amount were significantly higher when L. mesenteroides was incubated under high CO2 levels than when incubated in an aerated environment. The treatment of carrot medium plates with commercial dextransucrase or exudate protein extract resulted in similar sugar profiles and dextran production. Transcriptome analysis demonstrated that dextran production is related to the upregulation of the L. mesenteroides dextransucrase-encoding genes dsrD and dsrT during the first 4 to 8 h of exposure to high CO2 levels compared to aerated conditions. A phylogenetic analysis of L. mesenteroides YL48 dsrD revealed a high similarity to other dsr genes harbored by different Leuconostoc species. The ecological benefit of dextran production under elevated CO2 requires further investigation. However, this study implies an overlooked role of CO2 in the physiology and fitness of L. mesenteroides in stored carrots, and perhaps in other food items, during storage under nonventilated conditions.IMPORTANCE The bacterium Leuconostoc mesenteroides is known to cause spoilage of different types of foods by secreting a slimy fluid that damages the quality and appearance of the produce. Here, we identified a potential mechanism by which high levels of CO2 affect the spoilage caused by this bacterium by upregulating dextran synthesis genes. These results have broader implications for the study of the physiology, degradation ability, and potential biotechnological applications of Leuconostoc.

Interactions between fava bean protein and dextrans produced by Leuconostoc pseudomesenteroides DSM 20193 and Weissella cibaria Sj 1b.

The aim of this study was to study the interactions between dextran and fava bean protein. Two dextrans produced by Leuconostoc pseudomesenteroides DSM 20193 and Weissella cibaria Sj 1b were purified and mixed with fava bean protein isolate (FPI) in water or in different buffers. The two isolated dextrans presented a typical dextran structure, mainly α-(1 → 6) linkages (above 95%) and few α-(1 → 3) branches, but they differed in molar mass and conformation. Dry-heating incubation of FPI and dextran mixture facilitated the conjugation of dextran to FPI through the Maillard reaction. Both mixed and conjugated systems were further heat-treated, and different influences of the formed covalent bonds on rheological properties were observed. The W. cibaria Sj 1b dextran had a much higher gel-strengthening ability than the Ln. pseudomesenteroides DSM 20193 dextran. The intermolecular FPI-dextran interactions played an important role in stabilizing the mixed systems at different pH.

Characterization of the inserted mutagenesis dextransucrases from Leuconostoc mesenteroides 0326 to produce hyperbranched dextran.

Dextran produced by dextransucrase hold strong potential for industrial applications. The exact determinants of the linkage specificity of glucansucrase enzymes have remained largely unknown. Previous studies have investigated the relationships between structure and linkage specificity of the dextransucrase DSR from Leuconostoc mesenteroides by the site-directed mutagenesis of the catalytic pocket. The glycosidic linkage of dextran produced by mutant enzymes changed slightly by 3% to 20%. The mutagenesis dextransucrases were constructed by inserting an amino acid into a catalytic pocket to investigate the product specificities of dextransucrase thoroughly. The sequence and structural analysis of glycoside-hydrolase family 70 enzymes led to two sequences (Motif II and Motif IV) being targeted, which were inserted by saturation mutagenesis and simultaneously recombined between A552 and V553, D662, and S663. Variants with catalytic activity were screened of the library, which synthesizes high molecular weight α-glucans with different proportions of α(1-4) linkages, which ranges from 0% to 52%. Mutant sequence analysis, biochemical characterization, and molecular modeling studies revealed the mechanism of product specificities. The mutant dextransucrase, which synthesizes hyperbranched dextran, were obtained by the novel mutagenesis method. The different properties of dextran provide the foundation for subsequent studies and application.

Characterization of highly branched dextran produced by Leuconostoc citreum B-2 from pineapple fermented product.

A strain Leuconostoc citreum B-2 was isolated from homemade fermentation product of pineapple and its polysaccharide yield was 28.3g/L after cultivating the strain in Man-Rogosa-Sharpe (MRS) medium with 75g/L sucrose. The exopolysaccharide (EPS) was characterized by gas chromatography (GC), Fourier-transform infrared (FT-IR) spectra, high-performance size-exclusion chromatography (HPSEC), nuclear magnetic resonance (NMR) spectroscopy and scanning electron microscope (SEM) analysis in present study. The monosaccharide composition of EPS was glucose and molecular weight was 3.77×106Da. FT-IR and NMR spectra revealed that the B-2 EPS was composed of 75% α-(1→6) linked d-glucopyranose units existing in the main chain with 19% α-(1→3) branching and only a few α-(1→2) branching. The SEM of the dried EPS appeared irregular sheets with glittering surface and compact structure. Water solubility index and water holding capacity of B-2 EPS were 80% and 450%, respectively. All the mentioned characteristics suggested that the EPS has a potential application in the food, cosmetic and pharmaceuticals industry.

Dextran Utilization During Its Synthesis by Weissella cibaria RBA12 Can Be Overcome by Fed-Batch Fermentation in a Bioreactor.

Weissella cibaria RBA12 produced a maximum of 9 mg/ml dextran (with 90% efficiency) using shake flask culture under the optimized concentration of medium components viz. 2% (w/v) of each sucrose, yeast extract, and K2HPO4 after incubation at optimized conditions of 20 °C and 180 rpm for 24 h. The optimized medium and conditions were used for scale-up of dextran production from Weissella cibaria RBA12 in 2.5-l working volume under batch fermentation in a bioreactor that yielded a maximum of 9.3 mg/ml dextran (with 93% efficiency) at 14 h. After 14 h, dextran produced was utilized by the bacterium till 18 h in its stationary phase under sucrose depleted conditions. Dextran utilization was further studied by fed-batch fermentation using sucrose feed. Dextran on production under fed-batch fermentation in bioreactor gave 35.8 mg/ml after 32 h. In fed-batch mode, there was no decrease in dextran concentration as observed in the batch mode. This showed that the utilization of dextran by Weissella cibaria RBA12 is initiated when there is sucrose depletion and therefore the presence of sucrose can possibly overcome the dextran hydrolysis. This is the first report of utilization of dextran, post-sucrose depletion by Weissella sp. studied in bioreactor.

Characterization of a dextran produced by Leuconostoc pseudomesenteroides XG5 from homemade wine.

A water-souble exopolysaccharide (EPS) produced by Leuconostoc pseudomesenteroides XG5 from homemade wine was investigated. The EPS yield of 35.5g/L was achieved at 30°C for 48h in De Man-Rogosa-Sharpe (MRS) medium containing 12.5% sucrose. The EPS was a dextran composed exclusively of glucose and the molecular weight was 2.6×106Da. Fourier transform infrared spectra and nuclear magnetic resonance spectra revealed that the EPS was a dextran containing D-glucose residues in a linear chain with consecutive α-(1→6) linkages. Scanning electron microscopy of the EPS appeared a highly branched and porous structure. Rheological studies showed that the EPS had higher viscosity in 0.1M KCl solution, at lower temperature, or at acidic pH. Thermal gravimetric analysis and differential scanning calorimetric indicated that the EPS had excellent thermal stability with a degradation temperature of 313.80°C and melting point at 274.14°C. Water solubility index and water holding capacity of XG5 dextran were 90.2% and 412% respectively. The results suggest that L. pseudomesenteroides XG5 might be widely used in the production of linear dextran which has potential to serve as natural agent applied in food and other fields.

Biosynthesis of dextran by Weissella confusa and its In vitro functional characteristics.

The aim of this study was to monitor the influence of the fermentation conditions on the exopolysaccharides (EPS) biosynthesis. For this, different culture media compositions were tested on an isolated lactic acid bacteria (LAB) strain, identified by 16S rDNA sequence as being Weissella confusa. It was proved that this bacterial strain culture in MRS medium supplemented with 80g/L sucrose and dissolved in UHT milk produced up to 25.2g/L of freeze-dried EPS, in static conditions, after 48h of fermentative process. Using FTIR and NMR analysis, it was demonstrated that the obtained EPS is a dextran. The thermal analysis revealed a dextran structure with high purity while GPC analysis depicted more fractions, which is normal for a biological obtained polymer. A concentration up to 3mg/mL of dextran proved to have no cytotoxic effect on normal human dermal fibroblasts (NHDF). Moreover, at this concentration, dextran breaks up to 70% of the biofilms formed by the Candida albicans SC5314 strain, and has no antimicrobial activity against standard bacterial strains. Due to their characteristics, these EPS are suitable as hydrophilic matrix for controlled release of drugs in pharmaceutical industry.

A dextran with unique rheological properties produced by the dextransucrase from Oenococcus kitaharae DSM 17330.

A gene encoding a novel dextransucrase was identified in the genome of Oenococcus kitaharae DSM17330 and cloned into E. coli. With a kcat of 691s-1 and a half-life time of 111h at 30°C, the resulting recombinant enzyme -named DSR-OK- stands as one of the most efficient and stable dextransucrase characterized to date. From sucrose, this enzyme catalyzes the synthesis of a quasi linear dextran with a molar mass higher than 1×109g·mol-1 that presents uncommon rheological properties such as a higher viscosity than that of the most industrially used dextran from L. mesenteroides NRRL-B-512F, a yield stress that was never described before for any type of dextran, as well as a gel-like structure. All these properties open the way to a vast array of new applications in health, food/feed, bulk or fine chemicals fields.

Optimization of dextran production by Weissella cibaria NITCSK4 using Response Surface Methodology-Genetic Algorithm based technology.

The most influencing factor on dextran production by Weissella cibaria NITCSK4 were screened using Plackett Burman design at 95% confidence limit with higher value of co-efficient of determination (R2) 99.58%. The combined effects of significant factors, namely, sucrose, temperature, dipotassium hydrogen phosphate (K2HPO4) and yeast extract were studied and optimized using Response Surface Methodology (RSM). The input parameters of non-linear models predicted by RSM were subsequently optimized using the genetic algorithm (GA) for obtaining a maximum dextran yield. The maximum yield was obtained with sucrose concentration of 15.78%, yeast extract 1.27%, K2HPO4 1.25%, and at 26°C. The predicted conditions were experimentally validated and 43.79mg/ml of dextran was produced. The dextran yield was 51% higher as compared to unoptimized medium. The molecular weight of resulting dextran produced at 26°C is >2000kDa. The NMR spectroscopic analysis demonstrated that the NITCSK4 produced linear dextran with predominant α (1-6) linkage.

Rheology and bioactivity of high molecular weight dextrans synthesised by lactic acid bacteria.

Dextrans synthesised by three Leuconostoc mesenteroides strains, isolated from mammalian milks, were studied and compared with dextrans produced by Lc. mesenteroides and Lactobacillus sakei strains isolated from meat products. Size exclusion chromatography coupled with multiangle laser light scattering detection analysis demonstrated that the dextrans have molecular masses between 1.74×108Da and 4.41×108Da. Rheological analysis of aqueous solutions of the polymer revealed that all had a pseudoplastic behaviour under shear conditions and a random, and flexible, coil structure. The dextrans showed at shear zero a difference in viscosity, which increased as the concentration increased. Also, the purified dextrans were able to immunomodulate in vitro human macrophages, partially counteracting the inflammatory effect of Escherichia coli O111:B4 lipopolysaccharide. During prolonged incubation on a solid medium containing sucrose, dextran-producing bacteria showed two distinct phenotypes not related to the genus or species to which they belonged. Colonies of Lc. mesenteroides CM9 from milk and Lb. sakei MN1 from meat formed stable and compact mucoid colonies, whereas the colonies of the other three Leuconostoc strains became diffuse after 72h. This differential behaviour was also observed in the ability of the corresponding strains to bind to Caco-2 cells. Strains forming compact mucoid colonies showed a high level of adhesion when grown in the presence of glucose, which decreased in the presence of sucrose (the condition required for dextran synthesis). However no influence of the carbon source was detected for the adhesion ability of the other Lc. mesenteroides strains, which showed variable levels of binding to the enterocytes.

Functional analysis of truncated and site-directed mutagenesis dextransucrases to produce different type dextrans.

Dextrans with distinct molecular size and structure are increasingly being used in the food and pharmaceutical industries. Dextran is produced by dextransucrase (DSR, EC2.4.5.1), which is produced by Leuconostoc mesenteroides. DSR belongs to glycosyl hydrolase family (GH70) and synthesizes branched α-glucan (dextran) with both 5% α(1-3) and 95% α(1-6) glycosidic linkages. The DSR gene dex-YG (Genebank, Accession No. DQ345760) was cloned from the wild strain Leuconostoc mesenteroides 0326. This study generated a series of C-terminally truncated variants of dextransucrase and substituting the amino-acid residues in the active site of DSR. With shorter length of DSR, its polysaccharide-synthesizing capability was impaired heavily, whereas oligosaccharide (acting as prebiotics)-synthesizing capability increased significantly, efficiently producing special sizes of dextran. All truncated mutant enzymes were active. Results demonstrated that the catalytic domain dextransucrase was likely in 800 aa or less. Based on the three-dimensional structure model of dextransucrase built through homology modeling methods, the DSR and its mutants with the acceptor substrate of maltose and donor substrate of sucrose were studied by molecular-docking method. Substituting these amino-acid residues significantly affected enzyme activities. Compared with the wild-type dextran, mutant enzymes catalyzed the synthesis of a-glucan with 1-9% α(1-3) and 90-98% α(1-6) branching linkages. Some mutants introduced a small amount of α(1-4) linkages and α(1-2) linkages. This strategy can be effectively used for the rational protein design of dextransucrase.