Identification and Characterization of Glucosyltransferase That Forms 1-Galloyl-ß-d-Glucogallin in Canarium album L., a Functional Fruit Rich in Hydrolysable Tannins.

Hydrolysable tannins (HTs) are useful secondary metabolites that are responsible for pharmacological activities and astringent taste, flavor, and quality in fruits. They are also the main polyphenols in Canarium album L. (Chinese olive) fruit, an interesting and functional fruit that has been cultivated for over 2000 years. The HT content of C. album fruit was 2.3-13 times higher than that of berries with a higher content of HT. 1-galloyl-ß-d-glucose (ßG) is the first intermediate and the key metabolite in the HT biosynthesis pathway. It is catalyzed by UDP-glucosyltransferases (UGTs), which are responsible for the glycosylation of gallic acid (GA) to form ßG. Here, we first reported 140 UGTs in C. album. Phylogenetic analysis clustered them into 14 phylogenetic groups (A, B, D-M, P, and Q), which are different from the 14 typical major groups (A~N) of Arabidopsis thaliana. Expression pattern and correlation analysis showed that UGT84A77 (Isoform0117852) was highly expressed and had a positive correlation with GA and ßG content. Prokaryotic expression showed that UGT84A77 could catalyze GA to form ßG. These results provide a theoretical basis on UGTs in C. album, which will be helpful for further functional research and availability on HTs and polyphenols.

Characterization of a recombinant sucrose isomerase and its application to enzymatic production of isomaltulose.

OBJECTIVE: To characterize a recombinant isomerase that can catalyze the isomerization of sucrose into isomaltulose and investigate its application for the enzymatic production of isomaltulose. RESULTS: A sucrose isomerase gene from Erwinia sp. Ejp617 was synthesized and expressed in Escherichia coli BL21(DE3). The enzymatic characterization revealed that the optimal pH and temperature of the purified sucrose isomerase were 6.0 and 40 °C, respectively. The enzyme activity was slightly activated by Mn2+and Mg2+, but partially inhibited by Ca2+, Ba2+, Cu2+, Zn2+ and EDTA. The kinetic parameters of Km and Vmax for sucrose were 69.28 mM and 118.87 U/mg, respectively. The time course showed that 240.9 g/L of isomaltulose was produced from 300 g/L of sucrose, and the yield reached 80.3% after bioreaction for 180 min. CONCLUSIONS: This recombinant enzyme showed excellent capability for biotransforming sucrose to isomaltulose at the substrate concentration of 300 g/L. Further investigations should be carried out focusing on selection of suitable heterologous expression system with the aim to improve its expression level.

cDNA Cloning and Functional Analyses of Ashitaba (Angelica keiskei) Sesquiterpene Synthase Genes.

Angelica keiskei (ashitaba) is an edible plant belonging to the Apiacea family. We focused on sesquiterpenes in the leaves eaten by humans (specifically, in the Japanese population), and confirmed the presence of several sesquiterpenes by GC-MS. Thus, total RNA was extracted from the ashitaba leaves, reverse transcribed, and the resultant cDNAs were used for degenerate PCR followed by rapid amplification of cDNA ends. Consequently, we were able to isolate two full-length Tps genes (designated AkTps1 and AkTps2). Functional analysis of these two genes was carried out with Escherichia coli cells that expressed mevalonate pathway genes to increase the substrate (farnesyl diphosphate) amount of sesquiterpene synthase, revealing that AkTps1 encodes germacrene D synthase, and AkTps2 codes for an enzyme that catalyzes the generation of germacrene B and smaller amounts of germacrene D (a germacrene B and D synthase). We proposed biosynthetic routes of these two sesquiterpenes from farnesyl diphosphate (FPP) via farnesyl cation.

Coimmobilization and colocalization of a glycosyltransferase and a sucrose synthase greatly improves the recycling of UDP-glucose: Glycosylation of resveratrol 3-O-ß-D-glucoside.

Glycosylation is one of the most efficient biocompatible methodologies to enhance the water solubility of natural products, and therefore their bioavailability. The excellent regio- and stereoselectivity of nucleotide sugar-dependent glycosyltransferases enables single-step glycosylations at specific positions of a broad variety of acceptor molecules without the requirement of protection/deprotection steps. However, the need for stoichiometric quantities of high-cost substrates, UDP-sugars, is a limiting factor for its use at an industrial scale. To overcome this challenge, here we report tailor-made coimmobilization and colocalization procedures to assemble a bi-enzymatic cascade composed of a glycosyltransferase and a sucrose synthase for the regioselective 5-O-ß-D-glycosylation of piceid with in situ cofactor regeneration. Coimmobilization and colocalization of enzymes was achieved by performing slow immobilization of both enzymes inside the porous support. The colocalization of both enzymes within the porous structure of a solid support promoted an increase in the overall stability of the bi-enzymatic system and improved 50-fold the efficiency of piceid glycosylation compared with the non-colocalized biocatalyst. Finally, piceid conversion to resveratrol 3,5-diglucoside was over 90% after 6 cycles using the optimal biocatalyst and was reused in up to 10 batch reaction cycles accumulating a TTN of 91.7 for the UDP recycling.

Effects of enzymatically modified chestnut starch on the gut microbiome, microbial metabolome, and transcriptome of diet-induced obese mice.

Modification of chestnut starch with amylosucrase from Deinococcus geothermalis (DGAS) increases the proportion of resistant starch. DGAS-modified chestnut starch (DMCS) attenuates obesity in diet-induced obese mice via a receptor of short chain fatty acids (SCFAs), G-protein-coupled receptor 43. SCFAs are gut microbial metabolites produced by fermenting resistant starch and have key roles in the obesity-ameliorating effects of DMCS. Here, we evaluated the mechanical links among DMCS-induced changes in the gut microbiota, consequent production of microbial metabolites, and host genetic responses. Supplementation with DMCS altered the proportions of cecal microbiota, such as Ruminococcaceae and Bacteroides; microbial metabolites, such as acetic acid; and some carbohydrate metabolites. DMCS also induced changes in the expression of some genes in cecal epithelial cells, including genes involved in energy production, the cell cycle, and cellular junctions. Changes in the gut microbiota, microbial metabolites, and host gene expression were found to be significantly correlated. Our findings demonstrated the integrated and incorporated association among the gut microbiota, their beneficial metabolites, and the host transcriptome, which contributed to clarifying the anti-obesity effects of DMCS as a prebiotic. Therefore, fortifying resistant starch by modification of chestnut starch using DGAS may be a good strategy in the functional food industry.

Molecular identification of a cyclodextrin glycosyltransferase-producing microorganism and phylogenetic assessment of enzymatic activities.

Cyclodextrin glycosyltransferases (CGTases) are important enzymes in the biotechnology field because they catalyze starch conversion into cyclodextrins and linear oligosaccharides, which are used in food, pharmaceutical and cosmetic industries. The CGTases are classified according to their product specificity in α-, ß-, α/ß- and γ-CGTases. As molecular markers are the preferred tool for bacterial identification, we employed six molecular markers (16S rRNA, dnaK, gyrB, recA, rpoB and tufA) to test the identification of a CGTase-producing bacterial strain (DF 9R) in a phylogenetic context. In addition, we assessed the phylogenetic relationship of CGTases along bacterial evolution. The results obtained here allowed us to identify the strain DF 9R as Paenibacillus barengoltzii, and to unveil a complex origin for CGTase types during archaeal and bacterial evolution. We postulate that the α-CGTase activity represents the ancestral type, and that the γ-activity may have derived from ß-CGTases.

Molecular basis for branched steviol glucoside biosynthesis.

Steviol glucosides, such as stevioside and rebaudioside A, are natural products roughly 200-fold sweeter than sugar and are used as natural, noncaloric sweeteners. Biosynthesis of rebaudioside A, and other related stevia glucosides, involves formation of the steviol diterpenoid followed by a series of glycosylations catalyzed by uridine diphosphate (UDP)-dependent glucosyltransferases. UGT76G1 from Stevia rebaudiana catalyzes the formation of the branched-chain glucoside that defines the stevia molecule and is critical for its high-intensity sweetness. Here, we report the 3D structure of the UDP-glucosyltransferase UGT76G1, including a complex of the protein with UDP and rebaudioside A bound in the active site. The X-ray crystal structure and biochemical analysis of site-directed mutants identifies a catalytic histidine and how the acceptor site of UGT76G1 achieves regioselectivity for branched-glucoside synthesis. The active site accommodates a two-glucosyl side chain and provides a site for addition of a third sugar molecule to the C3' position of the first C13 sugar group of stevioside. This structure provides insight on the glycosylation of other naturally occurring sweeteners, such as the mogrosides from monk fruit, and a possible template for engineering of steviol biosynthesis.

Thermostable CITase from Thermoanaerobacter thermocopriae shows negative cooperativity.

OBJECTIVE: The biochemical properties of a putative thermostable cycloisomaltooligosaccharide (CI) glucanotransferase gene from Thermoanaerobacter thermocopriae were determined using a recombinant protein (TtCITase) expressed in Escherichia coli and purified to a single protein. RESULTS: The 171-kDa protein displayed maximum activity at pH 6.0, and enzyme activity was stable at pH 5.0-11.0. The optimal temperature was 60 °C in 1 h incubation, and thermal stability of the protein was 63% at 60 °C for 24 h. TtCITase produced CI-7 to CI-17, as well as CI-18, CI-19, and CI-20, which are relatively large CIs. Additionally, an unusual kinetic feature of TtCITase was its negative cooperative behavior in the dextran T2000 cleavage reaction. CONCLUSIONS: Based on our results, TtCITase can be applied to produce relatively large CIs at high temperature.

Purification and Analysis of Effector Glucosyltransferase Lgt1 from Legionella pneumophila.

Legionella pneumophila is a facultative intracellular pathogen responsible for legionellosis, a severe lung disease in humans. This bacterium uses a type 4b secretion system to deliver various effector proteins into the cytoplasm of a eukaryotic target cell. Among those is the glucosyltransferase Lgt1. This effector modifies serine-53 in eukaryotic elongation factor 1A (eEF1A) by mono-O-glucosylation. Modification of eEF1A results in inhibition of protein synthesis and death of the eukaryotic cell, processes which are thought to contribute to Legionella infection. Here we describe a protocol for isolation of the glucosyltransferase Lgt1 from L. pneumophila culture followed by assaying its enzymatic activity using 14C-UDP-glucose autoradiography.

Photoelectrochemical biosensor for hydroxymethylated DNA detection and T4-ß-glucosyltransferase activity assay based on WS2 nanosheets and carbon dots.

5-Hydroxymethylcytosine (5hmC) plays an important role in switching genes on and off in mammals, and it is implicated in both embryonic development and cancer progression. Herein, a novel photoelectrochemical (PEC) biosensor was developed for 5hmC detection based on WS2 nanosheets as the photoactive material and boronic acid functionalized carbon dots (B-CDs) for signal amplification unit. This biosensor can also be used for T4-ß-glucosyltransferase (ß-GT) activity assessment. Firstly, WS2 nanosheets and gold nanoparticles (AuNPs) were immobilized on an ITO electrode surface. Then probe DNA was immobilized on this electrode surface via Au-S bond. Afterwards, the complementary DNA containing 5hmC was then captured on the modified electrode surface by hybridization. Subsequently, ß-GT transferred glucose from uridine diphosphoglucose to the hydroxyl groups of the 5hmC residues. After glycosylation, B-CDs could further be immobilized on the modified electrode surface resulting in a strong photocurrent. The PEC biosensor afforded high selectivity, excellent sensitivity and good reproducibility, with detection limits of 0.0034 nM and 0.028 unit/mL for 5hmC and ß-GT, respectively. Results demonstrate that the photoelectrochemical strategy introduced here based on WS2 nanosheets and B-CDs offers a versatile platform for hydroxymethylated DNA detection, ß-GT activity assessment and ß-GT inhibitor screening.

Production and characterization of ß-cyclodextrin glucanotransferase from Bacillus sp. ND1.

A potent ß-CGTase producing bacterium ND1 has been isolated from sugarcane field soil in India. The biochemical, physiologicaland phylogenetic analyses based on 16S rRNA gene suggest that the isolate belongs to Bacillus cereus group. The enzyme ß-CGTase produced from isolate ND1 catalyzes production of ß-cyclodextrin utilizing starch as a substrate which has diverse applications in various fields. The enzyme production parameters pH, temperature, and substrate concentration were optimized using Central Composite Design (CCD) of Response Surface Methodology (RSM) and were found to be 8.9, 30.55 °C, and 1.88%, respectively for optimal enzyme activity. The crude enzyme was partially purified (29-fold) using ammonium sulphate precipitation followed by ion exchange chromatography. The specific activity of the purified enzyme was found to be 63.53 U mg-1 . The enzyme is monomeric in nature with a molecular weight of 97.4 kD as determined by SDS-PAGE. It is stable in a wide range of pH (6-10) and temperature (40-60 °C) values. The maximum CGTase activity was observed at pH 9 and temperature 50 °C. The Km value was found to be 2.613 ± 0.5 and Vmax was 0.309 ± 0.05 µg min-1 indicating high substrate specificity. Together; these results suggest that the enzyme may be of wide commercial value in various industrial processes.

Sucrose-based biosynthetic process for chain-length-defined α-glucan and functional sweetener by Bifidobacterium amylosucrase.

A unique thermostable amylosucrase from Bifidobacterium thermophilum was produced as a recombinant protein with the half-life of 577 h at 50 °C. By adding 1.0 M fructose, turanose yield was improved from 22.7% to 43.3% with 1.0 M sucrose, and from 23.7% to 39.4% with 1.5 M sucrose. Sucrose consumption rate was greatest at 55 °C, but the lowest amount of turanose was produced. Thus, turanose yield from sucrose biomass was inversely proportional to reaction temperature and was highly dependent on [fructose]. Meanwhile, insoluble α-glucan yield was clearly reduced as [fructose] increased. With 1.0 M fructose + 1.0 M sucrose, glucan byproduct yield significantly decreased from 29.4% to 1.1%. Molecular weights of linear glucans were almost identical among various [sucrose]s and were homogenous with very low polydispersity. This unique dual reaction patterns of amylosucrase enzyme would be very useful for massive productions of two different biomaterials simply by changing sucrose biomass concentration.

Biochemical Characterization of Alkaliphilic Cyclodextran Glucanotransferase from an Alkaliphilic Bacterium, Paenibacillus daejeonensis.

Cycloisomaltooligosaccharide glucanotransferase (CITase) was isolated from alkaliphilic Paenibacillus daejeonensis via an amino acid homology search for the reported CITase. The recombinant alkaliphilic CITase (PDCITase) from P. daejeonensis was expressed in an Escherichia coli expression system and purified as a single protein band of 111 kDa. PDCITase showed optimum activity at pH 8.0 and retained 100% of activity within a broad pH range (7.0-11.5) after 18 h, indicating alkaliphilic or alkalistable CITase properties. In addition, PDCITase produced CI-7 to CI-17, CI-18, and CI-19, which are relatively large cycloisomaltooligosaccharides yet to be reported. Therefore, these large cycloisomaltooligosaccharides can be applied to the improvement of water solubility of pharmaceutical biomaterials.

Enzymatic Synthesis of Unnatural Ginsenosides Using a Promiscuous UDP-Glucosyltransferase from Bacillus subtilis.

Glycosylation, which is catalyzed by UDP-glycosyltransferases (UGTs), is an important biological modification for the structural and functional diversity of ginsenosides. In this study, the promiscuous UGT109A1 from Bacillus subtilis was used to synthesize unnatural ginsenosides from natural ginsenosides. UGT109A1 was heterologously expressed in Escherichia coli and then purified by Ni-NTA affinity chromatography. Ginsenosides Re, Rf, Rh1, and R1 were selected as the substrates to produce the corresponding derivatives by the recombinant UGT109A1. The results showed that UGT109A1 could transfer a glucosyl moiety to C3-OH of ginsenosides Re and R1, and C3-OH and C12-OH of ginsenosides Rf and Rh1, respectively, to produce unnatural ginsenosides 3,20-di-O-ß-d-glucopyranosyl-6-O-[α-l-rhamnopyrano-(1→2)-ß-d-glucopyranosyl]-dammar-24-ene-3ß,6α,12ß,20S-tetraol (1), 3,20-di-O-ß-d-glucopyranosyl-6-O-[ß-d-xylopyranosyl-(1→2)-ß-d-glucopyranosyl]-dammar-24-ene-3ß,6α,12ß,20S-tetraol (6), 3-O-ß-d-glucopyranosyl-6-O-[ß-d-glucopyranosyl-(1→2)-ß-d-glucopyranosyl]-dammar-24-ene-3ß,6α,12ß,20S-tetraol (3), 3,12-di-O-ß-d-glucopyranosyl-6-O-[ß-d-glucopyranosyl-(1→2)-ß-d-glucopyranosyl]-dammar-24-ene-3ß,6α,12ß,20S-tetraol (2), 3,6-di-O-ß-d-glucopyranosyl-dammar-24-ene-3ß,6α,12ß,20S-tetraol (5), and 3,6,12-tri-O-ß-d-glucopyranosyl-dammar-24-ene-3ß,6α,12ß,20S-tetraol (4). Among the above products, 1, 2, 3, and 6 are new compounds. The maximal activity of UGT109A1 was achieved at the temperature of 40 °C, in the pH range of 8.0⁻10.0. The activity of UGT109A1 was considerably enhanced by Mg2+, Mn2+, and Ca2+, but was obviously reduced by Cu2+, Co2+, and Zn2+. The study demonstrated that UGT109A1 was effective in producing a series of unnatural ginsenosides through enzymatic reactions, which could pave a way to generate promising leads for new drug discovery.

Kinetic analysis of cellulose synthase of Gluconacetobacter hansenii in whole cells and in purified form.

The Gram-negative bacterium, Gluconacetobacter hansenii, has been long studied and is a model for cellulose synthesis. It produces cellulose, using the enzyme AcsA-AcsB, of exceptionally high crystallinity in comparison to the cellulose of higher plants. We determined the rate of cellulose synthesis in whole cells measured as moles of glucose incorporated into cellulose per second per mole of enzyme. This was determined by quantifying the rate of cellulose synthesis (over a short time span, such that the enzyme concentration is not changing due to cell growth) and the amount of enzyme in the whole cell by quantitative western blotting. We found that the whole cell rate of 24 s-1 is much faster than the kcat, measured from steady-state kinetic analysis, of 1.7 s-1. Our whole cell rates are consistent with previous studies using microscopy. We postulate that the rationale for this difference is the presence of an alternative in vivo priming mechanism. This in turn can increase the rate of initiation, which we previously postulated to be the rate-limiting step in catalysis.

Combination of sequence-based and in silico screening to identify novel trehalose synthases.

A combined approach of sequence-based screening from metagenomic soil DNA and subsequent in silico screening was established to identify novel trehalose synthases (TS, EC 5.4.99.16). Metagenomic DNA was isolated from diverse soil samples and used as template for PCR-based screening targeted against conserved regions of trehalose synthases. This resulted in four metagenomic TS-like fragments with broad sequence diversity (41-67% identity to each other). The encoded open reading frames were used as templates for further in silico screening. Two trehalose synthases were discovered using this novel approach and their enzymatic properties were further investigated. The trehalose synthase from Micrococcus terreus MtTS exhibited a broad pH optimum between 6.5 and 7.5 with highest reaction velocity at 35 °C and a protruding stability at this temperature (t1/2 = 50 h). Characteristic of enzymes from thermophilic organisms, the trehalose synthase from Thermobaculum terrenum had a distinct temperature optimum at 50 °C, exhibiting also a prominent half time with t1/2 = 45 h at pH 6.5. Both bioconversions resulted in final trehalose levels of 60%, whereas TtTS produced reduced amounts of the byproduct glucose (10%) compared with MtTS (15%), which is favorable for trehalose production. This combined screening approach intended to circumvent the bottleneck of metagenomic enzyme mining, regarding time and cost of intensive screening procedures for industrial relevant biocatalysts such as trehalose synthases.

Cellulose Synthase Stoichiometry in Aspen Differs from Arabidopsis and Norway Spruce.

Cellulose is synthesized at the plasma membrane by cellulose synthase complexes (CSCs) containing cellulose synthases (CESAs). Genetic analysis and CESA isoform quantification indicate that cellulose in the secondary cell walls of Arabidopsis (Arabidopsis thaliana) is synthesized by isoforms CESA4, CESA7, and CESA8 in equimolar amounts. Here, we used quantitative proteomics to investigate whether the CSC model based on Arabidopsis secondary cell wall CESA stoichiometry can be applied to the angiosperm tree aspen (Populus tremula) and the gymnosperm tree Norway spruce (Picea abies). In the developing xylem of aspen, the secondary cell wall CESA stoichiometry was 3:2:1 for PtCESA8a/b:PtCESA4:PtCESA7a/b, while in Norway spruce, the stoichiometry was 1:1:1, as observed previously in Arabidopsis. Furthermore, in aspen tension wood, the secondary cell wall CESA stoichiometry changed to 8:3:1 for PtCESA8a/b:PtCESA4:PtCESA7a/b. PtCESA8b represented 73% of the total secondary cell wall CESA pool, and quantitative polymerase chain reaction analysis of CESA transcripts in cryosectioned tension wood revealed increased PtCESA8b expression during the formation of the cellulose-enriched gelatinous layer, while the transcripts of PtCESA4, PtCESA7a/b, and PtCESA8a decreased. A wide-angle x-ray scattering analysis showed that the shift in CESA stoichiometry in tension wood coincided with an increase in crystalline cellulose microfibril diameter, suggesting that the CSC CESA composition influences microfibril properties. The aspen CESA stoichiometry results raise the possibility of alternative CSC models and suggest that homomeric PtCESA8b complexes are responsible for cellulose biosynthesis in the gelatinous layer in tension wood.

Isolation and characterization of a multifunctional flavonoid glycosyltransferase from Ornithogalum caudatum with glycosidase activity.

Glycosyltransferases (GTs) are bidirectional biocatalysts catalyzing the glycosylation of diverse molecules. However, the extensive applications of GTs in glycosides formation are limited due to their requirements of expensive nucleotide diphosphate (NDP)-sugars or NDP as the substrates. Here, in an effort to characterize flexible GTs for glycodiversification of natural products, we isolated a cDNA, designated as OcUGT1 from Ornithogalum caudatum, which encoded a flavonoid GT that was able to catalyze the trans-glycosylation reactions, allowing the formation of glycosides without the additions of NDP-sugars or NDP. In addition, OcUGT1 was observed to exhibit additional five types of functions, including classical sugar transfer reaction and three reversible reactions namely NDP-sugar synthesis, sugars exchange and aglycons exchange reactions, as well as enzymatic hydrolysis reaction, suggesting OcUGT1 displays both glycosyltransferase and glycosidase activities. Expression profiles revealed that the expression of OcUGT1 was development-dependent and affected by environmental factors. The unusual multifunctionality of OcUGT1 broadens the applicability of OcUGT1, thereby generating diverse carbohydrate-containing structures.

Expression, purification, and characterization of a novel amylosucrase from Neisseria subflava.

Amylosucrase (ASase) is a glucosyltransferase, which catalyzes the de novo synthesis of amylose-like polymers from sucrose. In the present study, ASase from Neisseria subflava (NsAS) was cloned, sequenced, and expressed in Escherichia coli. The production of NsAS was achieved by inducting gene expression with 0.2 mM isopropyl-ß-d-thiogalactopyranoside. The molecular mass of the Ni-NTA column purified NsAS analyzed by SDS-PAGE was determined to be 72 kDa. NsAS exhibited maximal activity at 45 °C and pH 8.0, and showed strong thermal stability at 40 °C with a half-life of 385 h. The reaction pattern of NsAS at [sucrose] range of 0.1-1.0 M showed that at 0.7 M of [sucrose], the production yield of insoluble linear α-(1,4)-glucans reached 24% maximum, and any further increase in [sucrose] resulted in a slight decrease in yield. Meanwhile, the production yield of turanose significantly increased from 16 to 29% by increasing [sucrose] from 0.1 to 1.0 M. The synthesized glucan had degrees of polymerization (DP); for 0.1, 0.4, 0.7, and 1.0 M sucrose, the DP values were 77, 49, 39, and 31 respectively. These results suggested that NsAS would be a promising candidate for food industrial production of linear α-(1,4)-glucans and turanose as a next generation sweetener.

New dextransucrase purification process of the enzyme produced by Leuconostoc mesenteroides IBUN 91.2.98 based on binding product and dextranase hydrolysis.

This paper examines a new dextransucrase (DS) purification process of the extracellular enzyme (EC 2.4.1.5) produced by Leuconostoc mesenteroides IBUN 91.2.98. The enzyme was purified using a methodology which combines the immobilization of the enzyme in the produced biopolymer dextran, followed by a concentration step by ultrafiltration, using a membrane with a pore size of 300kDa and subsequent hydrolysis of dextran by action of a dextranase and finally enzyme purification by anion exchange chromatography. The obtained enzyme has a purification factor of 118 and a yield of 26% from the initial extract. The purified dextransucrase has a specific activity of 335.1U/mg, electrophoretic analysis shows absence of subunits, and a molecular weight of 170.1kDa, a Vmax of 28.1U/ml and a Km of 48mM. Optimal conditions of pH, temperature and substrate concentration were 5, 30°C and 584mM sucrose respectively in a ratio of 0.4U/mole of substrate. The produced dextran has a molecular size of 800-1000kDa. Both the hydrolytic and transference activity are inhibited by Fe+3 (96.5%) and Al+3 (99.1%), whereas Mg+2 and K produce activation of 36.7% and 27.2%, respectively.