Enzyme synthesis of oligosaccharides using cashew apple juice as substrate

The use of agriculture substrates in industrial biotechnological processes has been increasing because of their low cost. In this work, the use of clarified cashew apple juice was investigated as substrate for enzyme synthesis of prebiotic oligosaccharide. The results showed that cashew apple juice is a good source of reducing sugars and can be used as substrate for the production of dextransucrase by Leuconostoc citreum B-742 for the synthesis of oligosaccharides using the crude enzyme. Optimal oligosaccharide yield (approximately 80%) was obtained for sucrose concentrations lower than 60 g/L and reducing sugar concentrations higher than 100 g/L.     

Cloning of a dextransucrase gene (fmcmds) from a constitutive dextransucrase hyper-producing Leuconostoc mesenteroides B-512FMCM developed using VUV

Dextransucrase (FMCMDS) from Leuconostoc mesenteroides B-512FMCM, a dextransucrase constitutive and hyper-producing strain, catalyzes the synthesis of dextran from sucrose. The coding region for fmcmds was isolated and sequenced. It consisted of an open reading frame (ORF) of 4699 bp, coding for a 1527 amino acid protein with a molecular mass of 170 kDa. However, it showed a dextransucrase activity band at 180 kDa in SDS-PAGE. Only one nucleotide changed in the promoter site and two amino acid residues were changed in the structural gene from that of the parent L. mesenteroides NRRL B-512F dsrS; an inducible dextransucrase gene of low productivity.     

Characterization of Leuconostoc mesenteroides NRRL B-512F dextransucrase (DSRS) and identification of amino-acid residues playing a key role in enzyme activity

Dextransucrase (DSRS) from Leuconostoc mesenteroides NRRL B-512F is a glucosyltransferase that catalyzes the synthesis of soluble dextran from sucrose or oligosaccharides when acceptor molecules, like maltose, are present. The L. mesenteroides NRRL B-512F dextransucrase-encoding gene (dsrS) was amplified by the polymerase chain reaction and cloned in an overexpression plasmid. The characteristics of DSRS were found to be similar to the characteristics of the extracellular dextransucrase produced by L. mesenteroides NRRL B-512F. The enzyme also exhibited a high homology with other glucosyltransferases. In order to identify critical amino acid residues, the DSRS sequence was aligned with glucosyltransferase sequences and four amino acid residues were selected for site- directed mutagenesis experiments: aspartic acid 511, aspartic acid 513, aspartic acid 551 and histidine 661. Asp-511, Asp-513 and Asp-551 were independently replaced with asparagine and His-661 with arginine. Mutation at Asp-511 and Asp-551 completely suppressed dextran and oligosaccharide synthesis activities, showing that at least two carboxyl groups (Asp-511 and Asp-551) are essential for the catalysis process. However, glucan-binding properties were retained, showing that DSRS has a two-domain structure like other glucosyltransferases. Mutations at Asp-513 and His-661 resulted in greatly reduced dextransucrase activity. According to amino acid sequence alignments of glucosyltransferases, α-amylases or cyclodextrin glucanotransferases, His-661 may have a hydrogen-bonding function. Received: 16 April 1997 / Received revision: 17 June 1997 / Accepted: 23 June 1997     

Dextransucrase production using cashew apple juice as substrate: effect of phosphate and yeast extract addition

Cashew apples are considered agriculture excess in the Brazilian Northeast because cashew trees are cultivated primarily with the aim of cashew nut production. In this work, the use of cashew apple juice as a substrate for Leuconostoc mesenteroides cultivation was investigated. The effect of yeast extract and phosphate addition was evaluated using factorial planning tools. Both phosphate and yeast extract addition were significant factors for biomass growth, but had no significant effect on maximum enzyme activity. The enzyme activities found in cashew apple juice assays were at least 3.5 times higher than the activity found in the synthetic medium. Assays with pH control (pH = 6.5) were also carried out. The pH-controlled fermentation enhanced biomass growth, but decreased the enzyme activity. Crude enzyme free of cells produced using cashew apple juice was stable for 16 h at 30°C at a pH of 5.0.     

Production and properties of a dextransucrase from Leuconostoc mesenteroides IBT-PQ isolated from ‘pulque’, a traditional Aztec alcoholic beverage

Dextransucrase was produced from a Leuconostoc mesenteroides isolated from pulque, a traditional Aztec alcoholic beverage produced from agave juice containing sucrose as the main carbon source. Almost all the dextransucrase activity (87%) was associated with the cells, and was unusually high (1.04 U mg⁻¹ of cells). The culture medium composition was optimized through a Box-Behnken method resulting in a process yielding 2.2 U ml⁻¹ of insoluble glucosyltransferase activity. The enzyme had a molecular weight of 166 kDa. Optimal temperature was 35°C with a half-life of 137 min at the same temperature. As with dextransucrase from the industrial strain L. mesenteroides NRRL B-512F, the enzyme showed Michaelis–Menten kinetic behavior with excess substrate inhibition (K _m and K _i values of 0.026 M and 1.23 M respectively); produced soluble linear dextran with glucose molecules linked mainly in α(1–6) with branching in α(1–3) in a proportion of 4:1 as shown by NMR studies; and produced a high yield of isomalto-oligosaccharides in the presence of maltose. Received 4 February 1998/ Accepted in revised form 25 July 1998     

Structural and functional features of fructansucrases present in <Emphasis Type="Italic">Leuconostoc mesenteroides</Emphasis> ATCC 8293

Glycosyltransferases produced by Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 (equivalent to NRRL B-1118) were identified. Two glucansucrases and one fructansucrases were observed in batch culture while levC and levL genes, corresponding to two fructansucrases, were isolated from information obtained from the released draft sequence of this Leuconostoc strain genome and cloned in Escherichia coli. The recombinant enzymes were shown to be fructansucrases producing a polymer identified by NMR as levan, confirming our recent report stating that these are also mosaic levansucrases bearing structural features of glucansucrases in the amino and carboxy terminal regions, as is also the case of inulosucrase (IslA) from Leuconostoc citreum CW28 and levansucrase (LevS) from L. mesenteroides NRRL B-512F. The recombinant levansucrase LevC was purified and characterized in terms of pH, temperature, and kinetic properties. The enzyme exhibits Michaelis–Menten kinetic properties with a K _m = 27.3 mM and a k _cat = 282.9 s⁻¹. This levansucrase behaves mainly as a transferase as only 30% of the substrate is hydrolyzed in a wide range of sucrose concentrations, with higher hydrolytic activities at low substrate concentrations. With this report we experimentally confirm the unusual structural pattern displayed by fructansucrases present in Leuconostoc species that group as a novel sub family of fructansucrases.     

An overview of purification methods of glycoside hydrolase family 70 dextransucrase

The enzyme dextransucrase (sucrose:1, 6-α-D-glucan 6-α-glucosyltransferase, EC 2.4.1.5) catalyses the synthesis of exopolysaccharide, dextran from sucrose. This class of polysaccharide has been extensively exploited in pharmaceutical industry as blood volume expander, as stabiliser in food industry and as a chromatographic medium in fine chemical industry because of their nonionic nature and stability. Majority of the dextrans are synthesized from sucrose by dextransucrase secreted mainly by bacteria belonging to genera Leuconostoc, Streptococcus and Lactobacillus. Bulk of the information on purification of extracellular dextransucrase has been generated from Leuconostoc species. Various methods such as precipitation by ammonium sulphate, ethanol or polyethylene glycol, phase partitioning, ultrafiltration and chromatography have been used to purify the enzyme. Purification of dextransucrase is rendered difficult by the presence of viscous dextran in the medium. However, processes like ultra-filtration, salt and PEG precipitation, chromatography and phase partitioning have been standardized and successfully used for higher scale purification of the enzyme. A recombinant dextransucrase from Leuconostoc mesenteroides B-512F with a histidine tag has been expressed in E. coli cells and purifi ed by immobilized metal ion chromatography. This review reports the available information on purifi cation methods of dextransucrase from Leuconostoc mesenteroides strains.     

Isomaltose formation by free and immobilized dextransucrase

The acceptor reaction of dextransucrase from Leuconostoc mesenteroides NRRL-B512F with glucose as acceptor is of technical interest for isomaltooligosaccharide (IMOs) synthesis. Different experimental conditions were investigated for free and immobilized enzyme. The data for oligosaccharide formation up to a degree of polymerization 4 were correlated with a model developed earlier, and optimal reaction conditions for immobilized dextransucrase design and application were identified for later continuous application. Furthermore, stability was investigated for free and immobilized enzyme including stabilization by sugars.     

Identification of a single and non-essential cysteine residue in dextransucrase of Leuconostoc mesenteroides NRRL B-512F

Amino acid analysis of purified dextransucrase (sucrose: 1,6-α-D-glucan 6-α-D-glucosyltransferase EC 2.4.1.5) from Leuconostoc mesenteroides NRRL B-512F was carried out. The enzyme is virtually devoid of cysteine residue there being only one cysteine residue in the whole enzyme molecule comprising over 1500 amino acid residues. The enzyme is rich in acidic amino acid residues. The number of amino acid residues was calculated based on the molecular weight of 188,000 (Goyal and Katiyar 1994). Amino sugars were not found, implying that the enzyme is not a glycoprotein. It has been shown earlier that the cysteine residue in dextransucrase is not essential for enzyme activity (Goyal and Katiyar 1998). The presence of only one cysteine residue per enzyme molecule illustrates that its tertiary structure is solely dependent on other types of non-covalent interactions such as hydrogen bonding, ionic and nonpolar hydrophobic interactions.     

Enzymatic synthesis of l-DOPA α-glycosides by reaction with sucrose catalyzed by four different glucansucrases from four strains of Leuconostoc mesenteroides

l-DOPA α-glycosides were synthesized by reaction of l-DOPA with sucrose, catalyzed by four different glucansucrases from Leuconostoc mesenteroides B-512FMC, B-742CB, B-1299A, and B-1355C. The glucansucrases catalyzed the transfer of d-glucose from sucrose to the phenolic hydroxyl position-3 and -4 of l-DOPA. The glycosides were fractionated and purified by Bio-Gel P-2 column chromatography, and the structures were determined by ¹H NMR spectroscopy. The major glycoside was 4-O-α-d-glucopyranosyl l-DOPA, and the minor glycoside was 3-O-α-d-glucopyranosyl l-DOPA. The two glycosides were formed by all four of the glucansucrases. The ratio of the 4-O-α-glycoside to the 3-O-α-glycoside produced by the B-512FMC dextransucrase was higher than that for the other three glucansucrases. The glycosylation of l-DOPA significantly reduced the oxidation of the phenolic hydroxyl groups, which prevents their methylation, potentially increasing the use of l-DOPA in the treatment of Parkinson’s disease. The use of one enzyme, glucansucrase, and sucrose as the d-glucosyl donor makes the synthesis considerably simpler and cheaper than the formerly published procedure using cyclomaltodextrin and cyclomaltodextrin glucanyltransferase, followed by glucoamylase, and β-amylase hydrolysis.     

Construction of Chimeric Glucansucrases for Analyzing Substrate-binding Regions That Affect the Structure of Glucan Products

A gene that encodes dextransucrase S (dsrS) from Leuconostoc mesenteroides NRRL B-512F encodes a glucansucrase dextransucrase S (DSRS) which mainly produces water-soluble glucan (dextran), while the dsrT5 gene derived from dsrT of the B-512F strain encodes an enzyme dextransucrase T5 (DSRT5), which mainly produces water-insoluble glucan. Tyr340-Asn510 of DSRS and Tyr307-Asn477 of DSRT5 (Site 1), Lys696-Gly768 of DSRS and Lys668-Gly740 of DSRT5 (Site 2), and Asn917-Lys1131 of DSRS and Asn904-Lys1118 of DSRT5 (Site 3) were exchanged and six different chimeric enzymes were constructed. Water-soluble glucan produced by recombinant DSRS was composed of 64% 6-linked glucopyranoside (Glcp), 9% 3,6-linked Glcp, and 13% 4-linked Glcp. Water-insoluble glucan produced by recombinant DSRT5 was composed of 47% 6-linked Glcp and 43% 3-linked Glcp. All of the chimeric enzymes produced glucans different from the ones produced by their parental enzymes. Some of the glucans produced by chimeric enzymes were extremely changed. The Site 1 chimeric enzyme of DSRS (STS1) produced water-soluble glucan composed mostly of 6-linked Glcp. That of DSRT5 (TST1) produced water-insoluble glucan composed mostly of 4-linked Glcp. The Site 3 chimeric enzyme of DSRS (STS3) produced mainly water-insoluble glucan, DSRT5 (TST3) produced mainly water-soluble glucans, and all of the glucan fractions consisted of 3-Glcp, 4-Glcp, and 6-Glcp. The amounts of the three linkages in the water-soluble glucan produced by TST3 were about 1:1:1. Site 1 was assumed to be important for making or avoiding making α-1,4 linkages, while Site 3 was assumed to be important for determining the kinds of glucosyl linkages made.     

Alternansucrase acceptor products

The regioselectivity of alternansucrase (EC 2.4.1.140) differs from dextransucrase (EC 2.4.1.5) in ways that can be useful for the synthesis of novel oligosaccharide structures. For example, it has been recently shown that the major oligosaccharides produced when maltose is the acceptor include one trisaccharide structure, two tetrasaccharides, one pentasaccharide, two hexasaccharides, one heptasaccharide, and at least two octasaccharides, containing no adjacent α-(1→3) linkages and no more than two consecutive α-(1→6) linkages. This may shed some light on how the enzyme works to produce the alternating structure. Another characteristic of alternansucrase that distinguishes it from dextransucrase is its greater ability to use leucrose as an acceptor. Leucrose, produced by glucosyl transfer to fructose released from the initial sucrose substrate, represents a very poor substrate for Leuconostoc mesenteroides NRRL B-512F dextransucrase. Alternansucrase, however, continues to transfer glucosyl units to leucrose, resulting in some unusual glucosyl-fructose oligosaccharides.     

Aggregated Form of Dextransucrases from Leuconostoc mesenteroides NRRL B-512F and Its Constitutive Mutant

Purified dextransucrases [EC 2.4.1.5], DSW-D and DSW-G, from Leuconostoc mesenteroides B-512F were obtained from affinity chromatography with DEAE-Sephadex A-50 by elution with clinical dextran and guanidine-HCl, respectively, DSM-G was purified from the B-512F mutant strain SH 3002, which produces dextransucrase constitutively. Although the sugar contents of the purified enzymes were different, their molecular masses by SDS–PAGE were all 170kDa. DSW-D and DSW-G were highly aggregated and the all the activities were eluted at the void volume (V₀) on Sepharose 6B, while the DSM-G was eluted at 1.2 × V₀ volume. On rechromatography, DSM-G was separated into three peaks corresponding to the aggregated form, monomeric form, and partially digested form, respectively. The aggregation of Leuconostoc dextransucrase was looser than that of streptococcal glucosyltransferases, but the structures of these enzymes had high homology with each other.     

Immobilization and properties of Leuconostoc mesenteroids dextransucrase

Dextransucrase from Leuconostoc mesenteroides (NRRL B-512F) was purified by ultrafiltration and gel filtration chromatography in 54% yield. The specific activity of a heart cut was 58.6 U/mg; cumulative purification of that preparation was 247?fold. Of 13 carriers surveyed, only alkylamine porous silica gave immobilization efficiencies consistently above 15 %. Immobilization to silica changed the properties of dextransucrase relatively little, the optimum pH for activity remaining at 5.2, while that for stability decreased from pH 5.5?6 to pH 5.2. In short assays, highest activities of both soluble and immobilized dextransucrase occurred at 30°C. Activation energies below that temperature were 8.6 kcal/mol for the former form and 1.7 kcal/mol for the latter. Maximum stabilization of soluble dextransucrase was attained by 5mM Ca²⁺.     

Effect of phosphate concentration on the production of dextransucrase by<Emphasis Type="Italic"> Leuconostoc mesenteroides</Emphasis> NRRL B512F

Leuconostoc mesenteroides NRRL B512F is the main strain used in industrial fermentations to produce dextransucrase and dextran. This process has been studied since the Second World War, when it was used as blood plasma expander. A study about the effect of phosphate concentration on cell propagation in a semicontinuous shake-flask culture is described in this work. Dextransucrase is obtained by fermentation of the Leuconostoc mesenteroides NRRL B512F in the presence of sucrose as substrate, a nitrogen source (corn liquor or yeast extract) and minerals. Phosphate is currently used in order to buffer the culture medium. Cell propagation can be done through a repeated batch culture, where dilution in a fresh medium is made with relatively short periods. The standard medium for dextransucrase production is prepared using 0.1 M of K₂HPO₄. In this work the level of phosphate was increased to 0.3 M, and an increase on biomass and on the enzyme activity was found when phosphate enriched medium was used. Higher phosphate buffer concentration was also able to keep the pH values above 5.0 during the entire process, avoiding enzyme denaturation.     

A novel dextransucrase is produced by <Emphasis Type="Italic">Leuconostoc</Emphasis><Emphasis Type="Italic">citreum</Emphasis> strain B/110-1-2: an isolate used for the industrial production of dextran and dextran derivatives

The industrial Leuconostoc strain B/110-1-2 producing dextran and dextran derivatives was taxonomically identified by 16S rRNA as L. citreum. Its dextransucrase enzymes were characterized according to their cellular location and reaction specificity. In the presence of sucrose, the strain B/110-1-2 produced two cell-associated dextransucrases (31.54% of the total glucosyltransferase activity) with molecular weights of 160 and 240 kDa and a soluble dextransucrase (68.46%) at 160–180 kDa. Two open reading frames (ORF) coding for L. citreum strain B/110-1-2 dextransucrases were identified. One of them shared a 52% identity with the alternansucrase ASR of L. citreum NRRL B-1355 and with a putative annotated alternansucrase sequence found in the genome of L. citreum KM20. The structural analysis (HPAEC-PAD, HPSEC, and ¹³C-NMR) of the polymer and oligodextrans produced by the B/110-1-2 dextransucrases suggest this novel glucansucrase has specificity similar to a dextransucrase but not to an alternansucrase, producing a soluble linear dextran with glucose molecules linked mainly in α-1,6 and α-1,3 with α-1,4 branches. These results enhance the understanding of this industrially significant strain and will aid in distinguishing between physiologically similar Leuconostoc spp. strains.     

Proteolytic modification of <Emphasis Type="Italic">Leuconostoc mesenteroides</Emphasis> B-512F dextransucrase

Multiple active lower molecular weight forms from Leuconostoc mesenteroides B512F dextransucrase have been reported. It has been suggested that they arise from proteolytic processing of a 170 kDa precursor. In this work, the simultaneous production of proteases and dextransucrase was studied in order to elucidate the dextransucrase proteolytic processing. The effect of the nitrogen source on protease and dextransucrase production was studied. Protease activity reaches a maximum early in the logarithmic phase of dextransucrase synthesis using the basal culture medium but the nitrogen source plays an important effect on growth: the highest protease concentration was obtained when ammonium sulfate, casaminoacids or tryptone were used. Two active forms of 155 and 129 kDa were systematically obtained from dextransucrase precursor by proteolysis. The amino termini of these forms were sequenced and the cleavage site deduced. Both forms of the enzyme obtained had the same cleavage site in the amino terminal region (F209–Y210). From dextransucrase analysis, various putative cleavage sites with the same sequence were found in the variable region and in the glucan binding domain. Although no structural differences were found in dextrans synthesized with both the precursor and the proteolyzed 155 kDa form under the same reaction conditions, their rheological behaviour was modified, with dextran of a lower viscosity yielded by the smaller form.Martha Argüello-Morales and Mónica Sánchez-González equally contributed to this work.     

Proteolytic processing of dextransucrase of Leuconostoc mesenteroides

Various dextransucrase molecular mass forms found in enzyme preparations may sometimes be products of proteolytic activity. Extracellular protease in Leuconostoc mesenteroides strains NRRL B-512F and B-512FMC dextransucrase preparations was identified. Protease had a molecular mass of 30 kDa and was the predominant form derived from a high molecular mass precursor. The production and activity of protease in culture medium was strongly dependent on pH. When L. mesenteroides dextransucrase (173 kDa) was hydrolyzed by protease, at pH 7 and 37 degrees C, various dextransucrase forms with molecular masses as low as 120 kDa conserving dextransucrase activity were obtained.     

A facile purification of Leuconostoc mesenteroides B-512FM dextransucrase

Leuconostoc mesenteroides NRRL B-512F has been mutated by treatment with N-nitrosoguanidine. The resulting mutant (designated as B-512FM) produces 300 times as much enzyme as the parent strain. B-512FM dextransucrase was treated extensively with Sigma crude dextranase, followed by column chromatography on Bio-Gel A-5m. The purified dextransucrase had a specific activity of 84 IU/mg, a 100-fold purification with 42% yield, and was shown by SDS-PAGE to have a single protein of molecular weight of 158,000 with dextransucrase activity. The procedure has been used to produce purified enzyme for sequencing. The molecular weight of 158,000 agrees with that calculated from its amino acid sequence.     

One-step synthesis of isomalto-oligosaccharide syrups and dextrans of controlled size using engineered dextransucrase

Isomalto-oligosaccharides and dextrans of controlled molecular weight of about 10 and 40 kDa were produced using a simple one-step process using engineered L. mesenteroides NRRL B-512F dextransucrase variants. Isomalto-oligosaccharides were produced in a 58% yield by the acceptor reaction with glucose, and reached a degree of polymerization of at least 27 glucosyl units. Reaction conditions for optimal synthesis of dextrans of controlled molecular weight were defined, in respect of initial sucrose concentration and reaction temperature. Thus, we achieved synthesis with impressive yields of 69 and 75% for the 40 and 10 kDa dextran species, respectively. These two dextran sizes are particularly suitable for clinical applications, and are of great industrial demand. Compared with the traditional processes based on chemical hydrolysis and fractionation, which achieve only low yields, the new enzymatic methods offer improvement in quantity, quality and efficiency.