Bacterial Levansucrases Research Articles

Use of Tunable Copolymers in Aqueous Biphasic Systems for Extractive Bioconversion Aimed at Continuous Fructooligosaccharide Production

Aqueous biphasic systems (ABSs) based on sodium polyacrylate (NaPA), ethylene oxide/propylene oxide (EO/PO) polymers, and (EO)x-(PO)y-(EO)x triblock copolymers were prepared and applied aiming at continuous fructooligosaccharide (FOS) production and separation. EO/PO hydrophilicity/hydrophobicity balance had a significant effect on ABS formation. To develop an integrated process including the continuous enzymatic (levansucrase) production of FOSs and their purification while improving the production yield by further glucose separation, the potential of these novel polymer-based ABSs as alternative platforms was investigated. They were used for the partitioning of different carbohydrates (FOS, sucrose, d-fructose, and d-glucose) and levansucrase. Results revealed a highly polymer-dependent partition of carbohydrates and a poorly dependent one of the enzymes. Changing EO/PO and copolymers, FOS was purified with high yields (72.94–100.0%). Using polypropylene glycol 400 + NaPA 8000-based ABS, the FOS was precipitated in the interphase and separated from the other components. Pluronic PE-6800 + NaPA 8000 was identified as the best ABS for FOS continuous production and in situ purification, while minimizing levansucrase inhibition by d-glucose. This system allowed selective partition of FOSs and d-glucose toward the top phase and that of levansucrase and its substrates toward the bottom one. COnductor-like Screening MOdel for Real Solvent (COSMO-RS) suggested that ABS formation may have been due to NaPA and polymer/copolymer competition to form hydrogen bonds with water molecules. Moreover, the partition of FOSs and sugar may have been the result of a subtle balance between hydrogen bonding of sugar and polymer/copolymer and electrostatic misfit of solute with NaPA. Finally, two integrated processes were proposed to be applied with real FOS extracts obtained by chemical or enzymatic hydrolysis of inulin or by transfructosylation of concentrated sucrose solutions using bacterial levansucrases.

Complete secretion of recombinant Bacillus subtilis levansucrase in Pichia pastoris for production of high molecular weight levan

Three signal peptides from α-mating factor (α-MF), inulinase (INU) and native levansucrase (LS) were compared for secretion efficiency of Bacillus subtilis levansucrase SacB-T305A in Pichia pastoris GS115. The first complete secretion of bacterial levansucrase in yeasts under methanol induction was achieved while using α-MF signal. The secreted recombinant Lev(α-MF) proved to be glycosylated by combination of NanoLC-MS/MS and Endo H digestion. Interestingly, glycosylation not only improved significantly the polymerase thermostability, but also reversed the products profiles to favor synthesis of high molecular weight (HMW) levan which accounted for approximately 73 % to total levan-type polysaccharides. It indicated for the first time that the glycosylation of recombinant B. subtilis levansucrase affected significantly the products molecular weight distribution. It also provided a promising enzymatic way to effectively product HMW levan from sucrose resources.

Impaired coordination of nucleophile and increased hydrophobicity in the +1 subsite shift levansucrase activity towards transfructosylation.

Bacterial levansucrases produce β(2,6)-linked levan-type polysaccharides using sucrose or sucrose analogs as donor/acceptor substrates. However, the dominant reaction of Bacillus megaterium levansucrase (Bm-LS) is hydrolysis. Single domain levansucrases from Gram-positive bacteria display a wide substrate-binding pocket with open access to water, challenging engineering for transfructosylation-efficient enzymes. We pursued a shift in reaction specificity by either modifying the water distribution in the active site or the coordination of the catalytic acid/base (E352) and the nucleophile (D95), thus affecting the fructosyl-transfer rate and allowing acceptors other than water to occupy the active site. Two serine (173/422) and two water-binding tyrosine (421/439) residues located in the first shell of the catalytic pocket were modified. Library variants of S173, Y421 and S422, which coordinate the position of D95 and E352, show increased transfructosylation (30–200%) and modified product spectra. Substitutions at position 422 have a higher impact on sucrose affinity, while changes at position 173 and 421 have a strong effect on the overall catalytic rate. As most retaining glycoside hydrolases (GHs) Bm-LS catalyzes hydrolysis and transglycosylation via a double displacement reaction involving two-transition states (TS1 and TS2). Hydrogen bonds of D95 with the side chains of S173 and S422 contribute a total of 2.4 kcal mol−1 to TS1 stabilization, while hydrogen bonds between invariant Y421, E352 and the glucosyl C-2 hydroxyl-group of sucrose contribute 2.15 kcal mol−1 stabilization. Changes at Y439 render predominantly hydrolytic variants synthesizing shorter oligosaccharides.

Structural Modelling and Structure-Function Analysis of <i>Zymomonas mobilis</i> Levansucrase

Levansucrases are bacterial enzymes which produce fructan polymers from sucrose via hydrolysis and transfructosylation activities. These polymers; levan and fructooligosaccharides are valuable for food and pharmaceutical industries. Levansucrases from Gram-positive bacteria such as Bacillus subtilis tend to produce levan, while those from Gram-negative bacteria preferentially produce fructooligosaccharides. Zymomonas mobilis is an efficient levansucrase producer and its extracellular levansucrase can produce both fructooligosaccharides and levan depending on the reaction parameters. In this study, the structure of Z. mobilis levansucrase was modeled in order to help to understand the structure-function relationship of the enzyme. Furthermore, amino acids previously reported to be important for levansucrase activity were mapped on the model. The structural model presents a five-bladed propeller with a deep, negatively charged central pocket, similar to other bacterial levansucrases. Mapping showed that amino acids which previously reported to affect fructan length are located on the periphery of the structure covering the active site central pocket. Thus it is showed that, for the first time, that hydrolysis and transfructosylation reactions are catalyzed on different parts of Z. mobilis levansucrase structure. The structural location of the critical amino acids will pave the way to identify other residues which control fructan length by site directed mutagenesis without altering the overall fold of the enzyme.

The crystal structure of Erwinia amylovora levansucrase provides a snapshot of the products of sucrose hydrolysis trapped into the active site.

Levansucrases are members of the glycoside hydrolase family and catalyse both the hydrolysis of the substrate sucrose and the transfer of fructosyl units to acceptor molecules. In the presence of sufficient sucrose, this may either lead to the production of fructooligosaccharides or fructose polymers. Aim of this study is to rationalise the differences in the polymerisation properties of bacterial levansucrases and in particular to identify structural features that determine different product spectrum in the levansucrase of the Gram-negative bacterium Erwinia amylovora (Ea Lsc, EC 2.4.1.10) as compared to Gram-positive bacteria such as Bacillus subtilis levansucrase. Ea is an enterobacterial pathogen responsible for the Fire Blight disease in rosaceous plants (e.g., apple and pear) with considerable interest for the agricultural industry. The crystal structure of Ea Lsc was solved at 2.77 Å resolution and compared to those of other fructosyltransferases from Gram-positive and Gram-negative bacteria. We propose the structural features, determining the different reaction products, to reside in just a few loops at the rim of the active site funnel. Moreover we propose that loop 8 may have a role in product length determination in Gluconacetobacter diazotrophicus LsdA and Microbacterium saccharophilum FFase. The Ea Lsc structure shows for the first time the products of sucrose hydrolysis still bound in the active site.

High-throughput assay of levansucrase variants in search of feasible catalysts for the synthesis of fructooligosaccharides and levan.

Bacterial levansucrases polymerize fructose residues of sucrose to β-2,6 linked fructans—fructooligosaccharides (FOS) and levan. While β-2,1-linked FOS are widely recognized as prebiotics, the health-related effects of β-2,6 linked FOS are scarcely studied as they are not commercially available. Levansucrase Lsc3 (Lsc-3) of Pseudomonas syringae pv. tomato has very high catalytic activity and stability making it a promising biotechnological catalyst for FOS and levan synthesis. In this study we evaluate feasibility of several high-throughput methods for screening and preliminary characterization of levansucrases using 36 Lsc3 mutants as a test panel. Heterologously expressed and purified His-tagged levansucrase variants were studied for: (1) sucrose-splitting activity; (2) FOS production; (3) ability and kinetics of levan synthesis; (4) thermostability in a Thermofluor assay. Importantly, we show that sucrose-splitting activity as well as the ability to produce FOS can both be evaluated using permeabilized levansucrase-expressing E. coli transformants as catalysts. For the first time we demonstrate the key importance of Trp109, His113, Glu146 and Glu236 for the catalysis of Lsc3. Cost-effective and high-throughput methods presented here are applicable not only in the levansucrase assay, but have a potential to be adapted for high-throughput (automated) study of other enzymes.

Levan-type FOS production using a Bacillus licheniformis endolevanase

In the present work we describe an enzymatic production method to obtain β2-6 fructose oligosaccharides (levan-type FOS) through a sequential reaction in which a bacterial endolevanase is applied to levan produced from sucrose by bacterial levansucrases. A putative gene encoding an endolevanase, designated as LevBl, was identified through a bioinformatics search, isolated from a strain of Bacillus licheniformis IBt1 from our own collection and expressed in Escherichia coli. LevB1 showed a specific activity of 1.8U/mg protein at 35°C in 50mM phosphate buffer pH 6.0. A first order kinetic behavior was found when up to 150g/L of low molecular weight levan (8.3kDa) was used as the substrate. The product profile was determined by HPAEC-PAD and consisted of levan-type FOS with a polymerization degree between 2 and 8, with levanbiose as the major product after long reaction times. Yields of 97% of levan-type FOS were obtained when 1.0U/mL of LevB1 reacted with 100g/L of levan produced by the levansucrase from Bacillus subtilis. Finally, it was observed that levan-type FOS are efficiently fermented by probiotic lactic acid bacteria.

Polysaccharide Synthesis of the Levansucrase SacB from Bacillus megaterium Is Controlled by Distinct Surface Motifs

Despite the widespread biological function of carbohydrates, the polysaccharide synthesis mechanisms of glycosyltransferases remain largely unexplored. Bacterial levansucrases (glycoside hydrolase family 68) synthesize high molecular weight, β-(2,6)-linked levan from sucrose by transfer of fructosyl units. The kinetic and biochemical characterization of Bacillus megaterium levansucrase SacB variants Y247A, Y247W, N252A, D257A, and K373A reveal novel surface motifs remote from the sucrose binding site with distinct influence on the polysaccharide product spectrum. The wild type activity (k(cat)) and substrate affinity (K(m)) are maintained. The structures of the SacB variants reveal clearly distinguishable subsites for polysaccharide synthesis as well as an intact active site architecture. These results lead to a new understanding of polysaccharide synthesis mechanisms. The identified surface motifs are discussed in the context of related glycosyltransferases.

High levan accumulation in transgenic tobacco plants expressing the Gluconacetobacter diazotrophicus levansucrase gene

Bacterial levansucrase (EC 2.4.1.10) converts sucrose into non-linear levan consisting of long β(2,6)-linked fructosyl chains with β(2,1) branches. Bacterial levan has wide food and non-food applications, but its production in industrial reactors is costly and low yielding. Here, we report the constitutive expression of Gluconacetobacter diazotrophicus levansucrase (LsdA) fused to the vacuolar targeting pre-pro-peptide of onion sucrose:sucrose 1-fructosyltransferase (1-SST) in tobacco, a crop that does not naturally produce fructans. In the transgenic plants, levan with degree of polymerization above 10 4 fructosyl units was detected in leaves, stem, root, and flowers, but not in seeds. High levan accumulation in leaves led to gradual phenotypic alterations that increased with plant age through the flowering stage. In the transgenic lines, the fructan content in mature leaves varied from 10 to 70% of total dry weight. No oligofructans were stored in the plant organs, although the in vitro reaction of transgenic LsdA with sucrose yielded β(2,1)-linked FOS and levan. Transgenic lines with levan representing up to 30 mg g −1 of fresh leaf weight produced viable seeds and the polymer accumulation remained stable in the tested T1 and T2 progenies. The lsdA-expressing tobacco represents an alternative source of highly polymerized levan.

Effect of ethylene glycol and glycerol fructosides on the activity and product specificity of bacterial and plant fructosyltransferases

Fructosyltransferases (FTs) are key enzymes in plants and bacteria to synthesize fructans. To gain insight on the specificity of the hexose subsites in the active site of FTs, ethylene glycol fructoside (EGF) and glycerol fructoside (GF), containing fructose in the furanose configuration, were synthesized in vitro and used as substrates to study the effect on the activity of bacterial levansucrase (BsLS), chicory root sucrose:sucrose 1-fructosyltransferase (1-SST) and fructan:fructan 1-fructosyltransferase (1-FFT). The results demonstrated that EGF and GF, at physiologically relevant concentrations, were efficient acceptor substrates for BsLS and 1-FFT, but not for 1-SST. EGF and GF cannot be used as donor substrates for BsLS, 1-SST and 1-FFT. A model is proposed to explain the subsite specificity differences between the three FTs involved in this study.

Plant development inhibitory genes in binary vector backbone improve quality event efficiency in soybean transformation

Conventional Agrobacterium-mediated plant transformation often produces a significant frequency of transgenic events containing vector backbone sequence, which is generally undesirable for biotechnology applications. We tested methods to reduce the frequency of transgenic plants containing vector backbone by incorporating genes into the backbone that inhibit the development of transgenic plants. Four backbone frequency reduction genes, bacterial levansucrase (sacB), maize cytokinin oxidase (CKX), Phaseolus GA 2-oxidase (GA 2-ox), and bacterial phytoene synthase (crtB), each expressed by the enhanced CaMV 35S promoter, were placed individually in a binary vector backbone near the left border (LB) of binary vectors. In transformed soybean plants, the lowest frequency of backbone presence was observed when the constitutively expressed CKX gene was used, followed by crtB. Higher backbone frequencies were found among the plants transformed with the GA 2-oxidase and sacB vectors. In some events, transfer of short backbone fragments appeared to be caused by LB readthrough and termination within the backbone reduction gene. To determine the effect of the backbone genes on transformation frequency, the crtB and CKX vectors were then compared to a control vector in soybean transformation experiments. The results revealed that there was no significant transformation frequency difference between the crtB and control vectors, but the CKX vector showed a significant transformation frequency decrease. Molecular analysis revealed that the frequency of transgenic plants containing one or two copies of the transgene and free of backbone was significantly increased by both the CKX and crtB backbone reduction vectors, indicating that there may be a correlation between transgene copy number and backbone frequency.

Insights into the fine architecture of the active site of chicory fructan 1-exohydrolase: 1-kestose as substrate vs sucrose as inhibitor.

* Invertases and fructan exohydrolases (FEHs) fulfil important physiological functions in plants. Sucrose is the typical substrate for invertases and bacterial levansucrases but not for plant FEHs, which are usually inhibited by sucrose. * Here we report on complexes between chicory (Cichorium intybus) 1-FEH IIa with the substrate 1-kestose and the inhibitors sucrose, fructose and 2,5 dideoxy-2,5-imino-D-mannitol. Comparisons with other family GH32 and 68 enzyme-substrate complexes revealed that sucrose can bind as a substrate (invertase/levansucrase) or as an inhibitor (1-FEH IIa). * Sucrose acts as inhibitor because the O2 of the glucose moiety forms an H-linkage with the acid-base catalyst E201, inhibiting catalysis. By contrast, the homologous O3 of the internal fructose in the substrate 1-kestose forms an intramolecular H-linkage and does not interfere with the catalytic process. Mutagenesis showed that W82 and S101 are important for binding sucrose as inhibitor. * The physiological implications of the essential differences in the active sites of FEHs and invertases/levansucrases are discussed. Sucrose-inhibited FEHs show a K(i) (inhibition constant) well below physiological sucrose concentrations and could be rapidly activated under carbon deprivation.

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

Levan or high molecular β-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) α 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 PPARα mRNA expression was dose dependently up-regulated by dietary levan, whereas PPARγ 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 PPARα gene expression.

Properties of fructan:fructan 1-fructosyltransferases from chicory and globe thistle, two Asteracean plants storing greatly different types of inulin.

Remarkably, within the Asteraceae, a species-specific fructan pattern can be observed. Some species such as artichoke (Cynara scolymus) and globe thistle (Echinops ritro) store fructans with a considerably higher degree of polymerization than the one observed in chicory (Cichorium intybus) and Jerusalem artichoke (Helianthus tuberosus). Fructan:fructan 1-fructosyltransferase (1-FFT) is the enzyme responsible for chain elongation of inulin-type fructans. 1-FFTs were purified from chicory and globe thistle. A comparison revealed that chicory 1-FFT has a high affinity for sucrose (Suc), fructose (Fru), and 1-kestose as acceptor substrate. This makes redistribution of Fru moieties from large to small fructans very likely during the period of active fructan synthesis in the root when import and concentration of Suc can be expected to be high. In globe thistle, this problem is avoided by the very low affinity of 1-FFT for Suc, Fru, and 1-kestose and the higher affinity for inulin as acceptor substrate. Therefore, the 1-kestose formed by Suc:Suc 1-fructosyltransferase is preferentially used for elongation of inulin molecules, explaining why inulins with a much higher degree of polymerization accumulate in roots of globe thistle. Inulin patterns obtained in vitro from 1-kestose and the purified 1-FFTs from both species closely resemble the in vivo inulin patterns. Therefore, we conclude that the species-specific fructan pattern within the Asteraceae can be explained by the different characteristics of their respective 1-FFTs. Although 1-FFT and bacterial levansucrases clearly differ in their ability to use Suc as a donor substrate, a kinetic analysis suggests that 1-FFT also works via a ping-pong mechanism.

In situ production of exopolysaccharides during Sourdough fermentation by cereal and intestinal isolates of lactic acid bacteria.

EPS formed by lactobacilli in situ during sourdough fermentation may replace hydrocolloids currently used as texturizing, antistaling, or prebiotic additives in bread production. In this study, a screening of >100 strains of cereal-associated and intestinal lactic acid bacteria was performed for the production of exopolysaccharides (EPS) from sucrose. Fifteen strains produced fructan, and four strains produced glucan. It was remarkable that formation of glucan and fructan was most frequently found in intestinal isolates and strains of the species Lactobacillus reuteri, Lactobacillus pontis, and Lactobacillus frumenti from type II sourdoughs. By the use of PCR primers derived from conserved amino acid sequences of bacterial levansucrase genes, it was shown that 6 of the 15 fructan-producing lactobacilli and none of 20 glucan producers or EPS-negative strains carried a levansucrase gene. In sourdough fermentations, it was determined whether those strains producing EPS in MRS medium modified as described by Stolz et al. (37) and containing 100 g of sucrose liter(-1) as the sole source of carbon also produce the same EPS from sucrose during sourdough fermentation in the presence of 12% sucrose. For all six EPS-producing strains evaluated in sourdough fermentations, in situ production of EPS at levels ranging from 0.5 to 2 g/kg of flour was demonstrated. Production of EPS from sucrose is a metabolic activity that is widespread among sourdough lactic acid bacteria. Thus, the use of these organisms in bread production may allow the replacement of additives.

Identification of functionally important amino acid residues in Zymomonas mobilis levansucrase.

The catalytic residues of levansucrase (sucrose:2,6-beta-D-fructan 6-beta-D-fructosyltransferase, EC 2.4.1.10) from Zymomonas mobilis were analyzed by random mutation and site-directed mutagenesis. We found that substitution of Glu278 with Asp and His reduced the k(cat) for sucrose hydrolysis 30- and 210-fold, respectively, strongly suggesting Glu278 plays a key role in catalyzing this reaction. Given the likelihood that another acidic amino residue was also involved, we constructed variants in which acidic amino acids located within homologous regions among bacterial levansucrases and fructosyltransferases were substituted, and found that substitution of Asp194, located in homologous region III, abolished sucrose hydrolysis. In addition, Glu278 was determined to be situated within the DXXER motif in homologous region IV conserved among bacterial levansucrases and fructosyltransferases, while Asp194 was within the triplet RDP motif conserved among bacterial levansucrases, fructosyltransferases and fructofuranosidases. Finally, comparison of our findings with published data on other site-directed mutated enzymes indicated His296, also located in homologous region IV, is crucial for catalysis of the transfructosylation reaction.

Prediction of a common beta-propeller catalytic domain for fructosyltransferases of different origin and substrate specificity.

The three-dimensional (3D) structure of fructan biosynthetic enzymes is still unknown. Here, we have explored folding similarities between reported microbial and plant enzymes that catalyze transfructosylation reactions. A sequence-structure compatibility search using TOPITS, SDP, 3D-PSSM, and SAM-T98 programs identified a beta-propeller fold with scores above the confidence threshold that indicate a structurally conserved catalytic domain in fructosyltransferases (FTFs) of diverse origin and substrate specificity. The predicted fold appeared related to that of neuraminidase and sialidase, of glycoside hydrolase families 33 and 34, respectively. The most reliable structural model was obtained using the crystal structure of neuraminidase (Protein Data Bank file: 5nn9) as template, and it is consistent with the location of previously identified functional residues of bacterial levansucrases (Batista et al., 1999; Song & Jacques, 1999). The sequence-sequence analysis presented here reinforces the recent inclusion of fungal and plant FTFs into glycoside hydrolase family 32, and suggests a modified sequence pattern H-x (2)-[PTV]-x (4)-[LIVMA]-[NSCAYG]-[DE]-P-[NDSC][GA]3 for this family.

In vitro digestibility and fermentability of levan and its hypocholesterolemic effects in rats

This study describes the in vitro digestibility and fermentability of high molecular weight (ca. 2,000,000) levan and its effect on the metabolism of lipids in growing rats fed cholesterol-free diets. Levan was synthesized from sucrose using bacterial levansucrase immobilized on a honeycomb-shaped ceramic support. Although body weight gain, weight of visceral organs, morphologic changes in the digestive tract, and the serum triacylglycerol and glucose concentrations were not affected by feeding levan diets for 4 weeks, a significant hypocholesterolemic effect was observed. Serum cholesterol level was decreased to 83% or 59% by feeding a 1% or 5% levan diet, respectively. The hypocholesterolemic effect was accompanied by a significant increase in fecal excretion of sterols and lipids. High molecular weight levan, though not hydrolyzed by the salivary amylases, was hydrolyzed by artificial gastric juice and was changed to a low molecular weight (ca. 4,000) levan with a small amount of fructose, but did not produce any fructooligosaccharides. Low molecular weight (ca. 6,000) levan was not hydrolyzed by either pancreatic juice or small intestinal enzymes. This suggests that, in vivo, low molecular weight levan derived from the high molecular weight material is not further digested and reaches the colon intact. The fermentation of low molecular weight levan (ca. 6,000) by several strains of bifidobacteria was not observed. These results showed that the hypocholesterolemic effect of levan may result from the prevention of intestinal sterol absorption, and not from the action of the fermentation products of levan.

Polymerase and hydrolase activities of Bacillus subtilis levansucrase can be separately modulated by site-directed mutagenesis

The levansucrase (sucrose:2,6-beta-D-fructan 6-beta-D-fructosyltransferase, EC 2.4.1.10) structural gene from a Bacillus subtilis mutant strain displaying a low polymerase activity was sequenced. Only one missense mutation changing Arg331 to His was responsible for this modified catalytic property. From this allele we created new mutations by directed mutagenesis, which modified the charge and polarity of site 331. Examination of the kinetics of the purified levansucrase variants revealed that transfructosylation activities are affected differently by the substitution chosen. His331----Arg completely restored the properties of the wild-type enzyme. The most striking feature of the other variants, namely Lys331, Ser331 and Leu331, was that they lost the ability of the wild-type enzyme to synthesize levan from sucrose alone. They were only capable of catalysing the first step of levan chain elongation, which is the formation of the trisaccharide ketose. The variant His331----Lys presented a higher kcat. for sucrose hydrolysis than the wild-type, and only this hydrolase activity was preserved in a solvent/water mixture in which the wild-type acted as a true polymerase. The two other substitutions reduced the efficiency of transfructosylation activities of the enzyme via the decrease of the rate of fructosyl-enzyme intermediate formation. For all variants, the sucrose affinity was slightly affected. This strong modulation of the enzyme specificities from a single amino acid substitution led us to postulate the hypothesis that bacterial levansucrases and plant fructosyltransferases involved in fructan synthesis may possess a common ancestral form.

Extraction and characterization of the enzymes of fructan biosynthesis in timothy (Phleum pratense)

A preparation of phlein sucrase from seedling shoots of timothy (Phleum pratense L.) is described which catalyzed the synthesis of fructan with a mean molecular size of 34 000 using sucrose as the substrate. Activity was fully sedimentable at 25 000 × g, had a pH optimum of 7.0, and a Km for sucrose of 0.15 M. Activity was inhibited by β-mercaptoethanol and sodium diethyl dithiocarbamate. Raffinose and stachyose, but not members of the kestose series of oligofructans, could act as fructosyl donors in addition to sucrose. Formation of oligosaccharides during high molecular weight fructan synthesis was minimal, with synthesis occurring by a mechanism apparently analogous to bacterial levansucrase. These observations are discussed in relation to the in vivo patterns of fructan biosynthesis observed in different species of higher plants.