Intermolecular fructosyl and levanbiosyl transfers by levan fructotransferase of Arthrobacter ureafaciens

A purified levan fructotransferase preparation from the culture of the bacterium Arthrobacter ureafaciens, which produces di-D-fructose 2,6':6,2' dianhydride (difructose anhydride IV) from levan by an intramolecular levan fructosyl transfer (ILFT) reaction, was found to produce a trioligofructan and a tetraoligofructan from levan in the presence of levanbiose, indicating the intermolecular fructosyl and levanbiosyl transfer (LFT and LBT) reactions. The tri- and tetraoligofructans were identified to be levantriose and -tetraose respectively. Increase in the levanbiose concentration brought about increased production of both oligofructans with decreased formation of difructose anhydride IV, supporting the previous theory proposed by Tanaka et al. (1983) that the ILFT, LFT, and LBT reactions are catalyzed by the same enzyme. In addition, there existed a roughly stoichiometric relationship between the increase and decrease in the productions of these oligofructans, and the LBT reaction was found to occur more intensively than the LFT reaction. Acceptor specificity of the LFT and LBT reactions was studied using fifteen sugars including mono-, di-, and trisaccharides. The enzyme showed both of the reactions only with levanbiose, -triose, and kestose, indicating that the exposed non-reducing levanbiosyl residue was essential for the acceptor and suggesting the existence of a levanbiosyl acceptor site on the enzyme molecule.     

Action of levan fructotransferase of Arthrobacter ureafaciens on levanoligosaccharides

Levan fructotransferase of the bacterium Arthrobacter ureafaciens, which produces di-D-fructose 2,6':6,2' dianhydride (difructose anhydride IV) from levan by an intramolecular transfructosylation reaction, was purified to give a single protein band of pI 4.5-4.7 on isoelectric focusing. It had a molecular weight of 128,000 on gel-filtration on Sephadex G-200 and 60,000 on SDS-polyacrylamide disc gel-electrophoresis, suggesting that the enzyme is composed of two identical subunits. The shortest levanoligosaccharide chain required for the difructose anhydride IV formation was determined to be tetraose. TLC of the enzymic digest of a modified levanhexaose derived from levanhexaose by the reduction of the reducing end to an alditol residue with sodium borohydride gave the difructose anhydride IV spot, suggesting that the enzyme attacks the modified levanhexaose molecule from the direction of the non-reducing fructose end. The enzymic digests of levantetraose, -pentaose, and -hexaose as the substrate gave, in addition to the difructose anhydride IV spot, spots of oligofructans of lower mobility than the original substrate on TLC. From the digest of levantetraose, a hexaoligofructan and a smaller amount of a pentaoligofructan but no fructose were separated, indicating enzymic intermolecular levanbiosyl and fructosyl transfer reactions.     

Molecular cloning of levan fructotransferase gene from Arthrobacter ureafaciens K2032 and its expression in Escherichia coli for the production of difructose dianhydride IV

AIMS: To clone and overexpress a novel levan fructotransferase gene lftA from Arthrobacter ureafaciens K2032. METHODS AND RESULTS: The lftA gene, encoding a levan fructotransferase (LFTase) of 521 amino acids (aa) residues, was cloned from the genomic DNA of A. ureafaciens K2032, and overexpressed in Escherichia coli. The recombinant LFTase overexpressed in E. coli was then used to produce a difructose dianhydride (DFA IV) from levan. DFA IV crystals with 97% purity could be obtained from the reaction mixture in 83.7% yield by using a natural crystallization method. CONCLUSIONS: The lftA gene cloned from A. ureafaciens K2032 encode a novel levan fructotransferase which produces difructose dianhydride (DFA IV) from levan. SIGNIFICANCE AND IMPACT OF THE STUDY: Levan fructotransferase is a useful enzyme with great promise in the production of DFA IV and various fructosides.     

Molecular and enzymatic characterization of a levan fructotransferase from Microbacterium sp. AL-210

Microbacterium sp. AL-210 producing a novel levan fructotransferase (LFTase) was screened from soil samples. The LFTase was purified to homogeneity by (NH4)2SO4 fractionation, column chromatography on Resource Q, and Superdex 200HR. The molecular weight of the purified enzyme was estimated to be approximately 46 kDa by both SDS-PAGE and gel filtration, and the enzyme's isoelectric point was pH 4.8. The major product produced from the levan hydrolysis by the enzyme reaction was identified by atmospheric pressure ionization mass spectrometry and NMR analysis as di-D-fructose-2,6':6,2'-dianhydride (DFA IV). The optimum pH and temperature for DFA IV production were 7.0 and 40 degrees C, respectively. The enzyme was stable at a pH range 7.0-8.0 and up to 40 degrees C. The enzyme activity was inhibited by FeCl2 and AgNO3. The enzyme converted the levan to DFA IV, with a conversion yield of approximately 44%. A gene encoding the LFTase (lftM) from Microbacterium sp. AL-210 was cloned and sequenced. The nucleotide sequence included an ORF of 1593 nucleotides, which is translated into a protein of 530 amino acid residues. The predicted amino acid sequence of the enzyme shared 79% of the identity and 86% of the homology with that of Arthrobacter nicotinovorans GS-9.     

Enzymic hydrolysis of di-D-fructofuranose 1, 2'; 2, 3' dianhydride with Arthrobacter ureafaciens.

Enzymic hydrolysis of di-D-fructofuranose 1, 2'; 2, 3' dianhydride with the bacteria Arthrobacter ureafaciens was studied to elucidate its mechanism. Hydrolysis of the difructose dianhydride to D-fructose, which did not occur with yeast invertase [EC 3.2.1.26], was found to occur on incubation with an enzyme preparation from an autolysate of the above bacteria. However, incubation with enzyme which had been treated at 60 degrees for 30 min yielded an intermediate hydrolysis product. The product isolated was found to be inulobiose and to be hydrolyzed to D-fructose by the original enzyme, as well as by yeast invertase. It was thus shown that the hydrolysis of the difructose dianhydride to D-fructose with the crude enzyme took place not in a single step but in two separate steps at 2, 3' and 1, 2' linkages. It was not determined whether the entire process is mediated by one and the same beta-fructofuranosidase or by different enzymes.     

Probing the critical residues for intramolecular fructosyl transfer reaction of a levan fructotransferase

Levan fructotransferase (LFTase) preferentially catalyzes the transfructosylation reaction in addition to levan hydrolysis, whereas other levan-degrading enzymes hydrolyze levan into a levan-oligosaccharide and fructose. Based on sequence comparisons and enzymatic properties, the fructosyl transfer activity of LFTase is proposed to have evolved from levanase. In order to probe the residues that are critical to the intramolecular fructosyl transfer reaction of the Microbacterium sp. AL-210 LFTase, an error-prone PCR mutagenesis process was carried out, and the mutants that led to a shift in activity from transfructosylation towards hydrolysis of levan were screened by the DNS method. After two rounds of mutagenesis, TLC and HPLC analyses of the reaction products by the selected mutants revealed two major products; one is a di-D-fructose- 2,6':6,2'-dianhydride (DFAIV) and the other is a levanbiose. The newly detected levanbiose corresponds to the reaction product from LFTase lacking transferring activity. Two mutants (2-F8 and 2-G9) showed a high yield of levanbiose (38-40%) compared with the wild-type enzyme, and thus behaved as levanases. Sequence analysis of the individual mutants responsible for the enhanced hydrolytic activity indicated that Asn-85 was highly involved in the transfructosylation activity of LFTase.     

Structural and functional basis for substrate specificity and catalysis of levan fructotransferase

Levan is β-2,6-linked polymeric fructose and serves as reserve carbohydrate in some plants and microorganisms. Mobilization of fructose is usually mediated by enzymes such as glycoside hydrolase (GH), typically releasing a monosaccharide as a product. The enzyme levan fructotransferase (LFTase) of the GH32 family catalyzes an intramolecular fructosyl transfer reaction and results in production of cyclic difructose dianhydride, thus exhibiting a novel substrate specificity. The mechanism by which LFTase carries out these functions via the structural fold conserved in the GH32 family is unknown. Here, we report the crystal structure of LFTase from Arthrobacter ureafaciens in apo form, as well as in complexes with sucrose and levanbiose, a difructosacchride with a β-2,6-glycosidic linkage. Despite the similarity of its two-domain structure to members of the GH32 family, LFTase contains an active site that accommodates a difructosaccharide using the -1 and -2 subsites. This feature is unique among GH32 proteins and is facilitated by small side chain residues in the loop region of a catalytic β-propeller N-domain, which is conserved in the LFTase family. An additional oligosaccharide-binding site was also characterized in the β-sandwich C-domain, supporting its role in carbohydrate recognition. Together with functional analysis, our data provide a molecular basis for the catalytic mechanism of LFTase and suggest functional variations from other GH32 family proteins, notwithstanding the conserved structural elements.     

Action of Arthrobacter ureafaciens inulinase II on several oligofructans and bacterial levans.

1. Arthrobacter ureafaciens inulinase II which converts inulin to di-D-fructofuranose 1,2' : 2,3' dianhydride (difructose anhydride III) leaving a small amount of oligosaccharides, was investigated in order to characterize its mode of action. 2. After the enzymatic reaction on the glucose-terminated inulin molecules had been completed, the oligosaccharides left in the enzyme digest were isolated, and identified to be the fructose-glucose oligosaccharides; O-beta-D-fructofuranosyl-(2 leads to 1)-O-beta-D-fructofuranosyl alpha-D-glucopyranoside (1-kestose), O-beta-D-fructofuranosyl-[(2 leads to 1)-O-beta-D-fructofuranosyl]2 alpha-D-glucopyranoside and O-beta-D-fructofuranosyl-[(2 leads to 1)-O-beta-D-fructofuranosyl]3 alpha-D-glucopyranoside. The difructose anhydride formation from the three fructose-glucose oligosaccharides in the separate reaction system with an increased substrate concentration was observed only with the latter two substrates, but not with the first one. 3. The difructose anhydride formation with several (2 leads to 1)-beta-linked fructose oligosaccharides and bacterial (2 leads to 6)-beta-fructans was examined. The (2 leads to 1)-beta-linked fructose oligosaccharides were effective as substrates for the enzyme with the exception of inulobiose, but the (2 leads to 6)-beta-fructans remained unaffected. 4. It was concluded that the enzyme attacks (2 leads to 1)-beta-linked fructan molecules from the nonreducing fructose ends and requires the presence of at least two adjacent (2 leads to 1)-beta-fructofuranosyl linkages.     

Optimal conditions for levan formation by an overexpressed recombinant levansucrase

Summary A genetically modified levansucrase, which contained His-affinity tag in its C-terminal, was constructed by PCR reaction using two synthetic primers. This modified protein was produced up to 30 % in total cell protein of E. coli, and was purified by a one-step affinity chromatography. The optimum pH for levan production was pH 5 and the optimum temperature was 0 °C. The higher velocity of levan formation within shorter enzyme reaction times was achieved by increasing the levels of enzyme concentration. The optimal sucrose concentration for levan production was around 20 %. Under these conditions, more than 50 g levan/l was produced.     

Purification and characterization of 2,6-beta-D-fructan 6-levanbiohydrolase from Streptomyces exfoliatus F3-2

Streptomyces exfoliatus F3-2 produced an extracellular enzyme that converted levan, a beta-2,6-linked fructan, into levanbiose. The enzyme was purified 50-fold from culture supernatant to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of this enzyme were 54,000 by SDS-PAGE and 60,000 by gel filtration, suggesting the monomeric structure of the enzyme. The isoelectric point of the enzyme was determined to be 4.7. The optimal pH and temperature of the enzyme for levan degradation were pH 5.5 and 60 degrees C, respectively. The enzyme was stable in the pH range 3.5 to 8.0 and also up to 50 degrees C. The enzyme gave levanbiose as a major degradation product from levan in an exo-acting manner. It was also found that this enzyme catalyzed hydrolysis of such fructooligosaccharides as 1-kestose, nystose, and 1-fructosylnystose by liberating fructose. Thus, this enzyme appeared to hydrolyze not only beta-2,6-linkage of levan, but also beta-2,1-linkage of fructooligosaccharides. From these data, the enzyme from S. exfoliatus F3-2 was identified as a novel 2,6-beta-D-fructan 6-levanbiohydrolase (EC 3.2.1.64).     

Production of levan using recombinant levansucrase immobilized on hydroxyapatite

Levansucrase of Zymomonas mobilis was immobilized onto the surface of hydroxyapatite by ionic binding. Optimum conditions for the immobilization were: pH 6.0, 4 h of immobilization reaction time, and 20 U of enzyme/g of matrix. The enzymatic and biochemical properties of the immobilized enzyme were similar to those of the native enzyme, especially towards the effect of salts and detergents. The immobilized enzyme showed sucrose hydrolysis activity higher as that of the native enzyme, but levan formation activity was 70% of the native enzyme. HPLC analysis of levan produced by immobilized enzyme showed the presence of two different types of levan: high-molecular-weight levan and low-molecular-weight levan. The proportion of low-molecular-weight levan to total levan produced by the immobilized enzyme was much higher than that with the native enzyme, indicating that immobilized levansucrase could be applied to produce low-molecular-weight levan. Immobilized levansucrase retained 65% of the original activity after 6 times of repeated uses and 67% of the initial activity after 40 d when stored at 4 °C.     

Cloning, expression, and characterization of Bacillus sp. snu-7 inulin fructotransferase

A gene encoding inulin fructotransferase (di-D-fructofuranose 1,2': 2,3' dianhydride [DFA III]-producing IFTase, EC 4.2.2.18) from Bacillus sp. snu-7 was cloned. This gene was composed of a single, 1,353-bp open reading frame encoding a protein composed of a 40-amino acid signal peptide and a 410-amino acid mature protein. The deduced amino acid sequence was 98% identical to Arthrobacter globiformis C11-1 IFTase (DFA III-producing). The enzyme was successfully expressed in E. coli as a functionally active, His-tagged protein, and it was purified in a single step using immobilized metal affinity chromatography. The purified enzyme showed much higher specific activity (1,276units/mg protein) than other DFA III-producing IFTases. The recombinant and native enzymes were optimally active in very similar pH and temperature conditions. With a 103-min half-life at 60 degrees C, the recombinant enzyme was as stable as the native enzyme. Acidic residues and cysteines potentially involved in the catalytic mechanism are proposed based on an alignment with other IFTases and a DFA IIIase.     

Characterization of a thermostable levansucrase from Bacillus sp. TH4-2 capable of producing high molecular weight levan at high temperature

A thermoactive and thermostable levansucrase was purified from a newly isolated thermophilic Bacillus sp. from Thailand soil. The purification was achieved by alcohol precipitation, DEAE-Cellulose and gel filtration chromatographies. The enzyme was purified to homogeneity as determined by SDS-PAGE, and had a molecular mass of 56 kDa. This levansucrase has some interesting characteristics regarding its optimum temperature and heat stability. The optimum temperature and pH were 60 degrees C and 6.0, respectively. The enzyme was completely stable after treatment at 50 degrees C for more than 1 h, and its activity increased four folds in the presence of 5 mM Fe(2+). The optimum temperature for levan production was 50 degrees C. Contrary to other levansucrases, the one presented in this study is able to produce high molecular weight levan at 50 degrees C.     

Altered mRNA expression of hepatic lipogenic enzyme and PPARalpha in rats fed dietary levan from Zymomonas mobilis

Levan or high molecular beta-2,6-linked fructose polymer is produced extracellularly from sucrose-based substrates by bacterial levansucrase. In the present study, to investigate the effect of levan feeding on serum leptin, hepatic lipogenic enzyme and peroxisome proliferation-activated receptor (PPAR) alpha expression in high-fat diet-induced obese rats, 4-week-old Sprague-Dawley male rats were fed high-fat diet (beef tallow, 40% of calories as fat), and, 6 weeks later, the rats were fed 0%, 1%, 5% or 10% levan-supplemented diets for 4 weeks. Serum leptin and insulin level were dose dependently reduced in levan-supplemented diet-fed rats. The mRNA expressions of hepatic fatty acid synthase and acetyl CoA carboxylase, which are the key enzymes in fatty acid synthesis, were down-regulated by dietary levan. However, dietary levan did not affect the gene expression of hepatic malic enzyme, phosphatidate phosphohydrolase and HMG CoA reductase. Also, the lipogenic enzyme gene expression in the white adipose tissue (WAT) was not affected by the diet treatments. However, hepatic PPARalpha mRNA expression was dose dependently up-regulated by dietary levan, whereas PPARgamma in the WAT was not changed. The results suggest that the in vivo hypolipidemic effect of dietary levan, including anti-obesity and lipid-lowering, may result from the inhibition of lipogenesis and stimulation of lipolysis, accompanied with regulation of hepatic lipogenic enzyme and PPARalpha gene expression.     

Rheological characterization of levan polysaccharides from Microbacterium laevaniformans

Levan polysaccharides were produced from Microbacterium laevaniformans and its rheological behaviors were characterized as a function of concentration and temperature. The intrinsic viscosity of the purified levan was determined to be 0.38dL/g at 25 degrees C which was relatively higher than that of levans from other microbial sources. The flow behaviors of the levan solutions were characterized by the increase in the shear stress, giving more increments in the shear rate. Thus, the levan solutions exhibited the pseudoplastic behavior, which was characterized by the power law model. In addition, the flow behaviors of the levans were satisfactorily fitted to the Arrhenius equation where the activation energy of flow (Ea) decreased from 24.07 to 13.53kJ/mol (R2=0.98-0.99) with increasing concentrations. Moreover, the exponential equation was favorably applied to describe the effect of concentration on the apparent viscosity of the levan polysaccharides.     

Characterization of an exo-beta-D-fructosidase from Streptococcus mutans Ingbritt.

An extracellular enzyme beta-D-fructosidase was purified from the culture supernatant of Streptococcus mutans Ingbritt and characterized. The molecular weight of the enzyme was 127,000 as determined by SDS-polyacrylamide gel electrophoresis. The enzyme was specific for levan which mainly consists of beta-(2,6)-linked D-fructose and was also able to hydrolyze inulin, sucrose and raffinose at the activities of 13, 9 and 5% of that hydrolyzing levan, respectively. The pH optima for levan, inulin and sucrose were approximately 5.5, 6.0 and 5.0, respectively. The enzyme was optimally reactive at 55 C for levan. The enzyme was inhibited by Fe3+, Hg2+ and Zn2+ and not by either anionic or non-ionic detergents. Paper chromatographic analysis revealed that the enzyme attacked levan by an exo-type mechanism.     

Biosynthesis of levan and a new method for the assay of levansucrase activity

The polysaccharide levan was synthesized in a solidified agar medium containing sucrose as a source of fructose. The biosynthesis was achieved by the enzyme levansucrase (2,6-fructan–d-glucose 6-fructosyltransferase, EC 2.4.1.10), a small quantity of which was placed in circular wells cut in the agar gel. The enzyme slowly diffused through the agar–sucrose medium and the synthesis of levan was observed as circular white areas, the size of which was dependent on the time of incubation and the concentration of enzyme used.     

Levansucrase from <Emphasis Type="Italic">Leuconostoc mesenteroides</Emphasis> NTM048 produces a levan exopolysaccharide with immunomodulating activity

Objectives

A levansucrase from Leuconostoc mesenteroides NTM048 was cloned and expressed and its enzymatic product was characterized.

Results

The fructansucrase gene from Leuconostoc mesenteroides was cloned and expressed in Escherichia coli. The recombinant enzyme was purified as a single protein and its properties investigated. The polymer produced by the recombinant enzyme was identified as levan by various means including TLC and NMRs, and the enzyme was identified as a GH68 levansucrase. The enzyme was optimal at pH 5.5–6 and 30 °C, and its activity was stimulated by Ca²⁺. The levan produced by this strain induced IgA production in mice.

Conclusion

Leuconostoc mesenteroides, a probiotic strain, possessed levansucrase which catalyzed the produced levan that had immunomodulating activity.

     

Levan果聚糖的应用与生产研究进展

Levan是一类果聚糖,由大量的果糖单元以β-（2,6）果糖苷键连接构成聚糖主链并含有少量β-（2,1）果糖苷键连接的支链组成。部分微生物来源的Levan具有抗肿瘤、抗病毒、降血糖、降血脂、免疫增强等重要的生物活性,在医药和功能性食品方面具有巨大的应用潜能。微生物发酵液提取和酶法合成是目前大量获得Levan果聚糖的两种方法,其中微生物发酵液提取的Levan果聚糖产量和蔗糖转化率一般较低,且发酵液中同时存在的其他高聚物不利于Levan的规模化纯化;而利用Levan蔗糖酶以蔗糖为底物转果糖基合成的Levan果聚糖产量已经高达200g/L、蔗糖转化率高达50%,并且Levan蔗糖酶合成Levan过程中酶的活性受到pH值、温度、螯合剂、金属离子等多种因素的影响,可以通过控制反应条件促进多糖合成反应的进行。因此,酶法合成将是工业化获得Levan果聚糖的主要方式。     

Purification of an isoflavonoid 7-O-beta-apiosyl-glucoside beta-glycosidase and its substrates from Dalbergia nigrescens Kurz

A beta-glycosidase was purified from the seeds of Dalbergia nigescens Kurz based on its ability to hydrolyse p-nitrophenyl beta-glucoside and beta-fucoside. This enzyme did not hydrolyze various glycosidic substrates efficiently, so it was used to identify its own natural substrates. Two substrates were identified, isolated and their structures determined as: compound 1, dalpatein 7-O-beta-D-apiofuranosyl-(1-->6)-beta-D-glucopyranoside and compound 2, 6,2',4',5'-tetramethoxy-7-hydroxy-7-O-beta-D-apiofuranosyl-(1-->6)-beta-D-glucopyranoside (dalnigrein7-O-beta-D-apiofuranosyl-(1-->6)-beta-D-glucopyranoside). The beta-glycosidase removes the sugar from these glycosides as a disaccharide, despite its initial identification as a beta-glucosidase and beta-fucosidase.