Structural analysis and optimised production of fructo- oligosaccharides by levansucrase from Acetobacter diazotrophicus SRT4

Major fructo-oligosaccharides (FOS) produced by levansucrase (EC 2.4.1.10) from Acetobacter diazotrophicus SRT4 were characterised as 1-kestose and nystose by acid hydrolysis and 13C-NMR spectroscopy. The highest yields of 1-kestose (481 mM; 241 g/l) and nystose (81 mM; 54 g/l) were achieved at initial sucrose concentration of 1754 mM (600 g/l), pH 5.5 and 40°C. The synthesized FOS reached 50% (w/w) of total sugars in the reaction mixture, with a conversion efficiency over 70% (w/w) based on the amount of sucrose converted to 1-kestose.     

Biochemical characterization of a β-fructofuranosidase from Rhodotorula dairenensis with transfructosylating activity

An extracellular β-fructofuranosidase from the yeast Rhodotorula dairenensis was characterized biochemically. The enzyme molecular mass was estimated to be 680 kDa by analytical gel filtration and 172 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, of which the N -linked carbohydrate accounts for 16% of the total mass. It displays optimum activity at pH 5 and 55–60 °C. The enzyme shows broad substrate specificity, hydrolyzing sucrose, 1-kestose, nystose, leucrose, raffinose and inulin. Although the main reaction catalyzed by this enzyme is sucrose hydrolysis, it also exhibits transfructosylating activity that, unlike other microbial β-fructofuranosidases, produces a varied type of prebiotic fructooligosaccharides containing β-(2→1)- and β-(2→6)-linked fructose oligomers. The maximum concentration of fructooligosaccharides was reached at 75% sucrose conversion and it was 87.9 g L⁻¹. The 17.0% (w/w) referred to the total amount of sugars in the reaction mixture. At this point, the amounts of 6-kestose, neokestose, 1-kestose and tetrasaccharides were 68.9, 10.6, 2.6 and 12.7 g L⁻¹, respectively.     

The Production of β-Fructofuranosidase from Bifidobacterium spp.

The productions of β-fructofuranosidase from Bifidohacterium longum A1, B. adolescentis G1, and four other strains of Bifidobacteria were investigated. All strains used in this study were grown in modified BL broth containing a mixture of fructooligosaccharides [1^F (1-β-D-fructofuranosyl)_n-1sucrose, GF_n (n = 2 – 5)] as the only carbon source. Hydrolyses of 1-kestose, sucrose, and inulin were detected in the extract of the cell. The highest activity on 1-kestose was detected in the extract of B. longum A1 followed by B. adolescentis G1. The other extracts weakly attacked 1-kestose. The relative activities of the extract of B. adolescentis G1 for 1-kestose, nystose, 1^F-fructosylnystose, sucrose, and inulin were 100, 82.5, 50.8, 28.3, and 15.0, respectively. The relative activities for various substrates differed from invertases (yeast β-fructofuranosidases) and exo-inulinase from Penicillium trzehinskii.     

Purification and kinetic characterization of a fructosyltransferase from Aspergillus aculeatus

A fructosyltransferase present in Pectinex Ultra SP-L, a commercial enzyme preparation from Aspergillus aculeatus, was purified to 107-fold and further characterised. The enzyme was a dimeric glycoprotein (20% (w/w) carbohydrate content) with a molecular mass of around 135 kDa for the dimer. Optimal activity/stability was found in the pH range 5.0-7.0 and at 60 degrees C. It was stable or slightly activated (upto 1.4-fold) in the presence of reducing agents, such as dithiothreitol and 2-mercaptoethanol, and detergents, such as sodium dodecylsulphate and Tween 80. The enzyme was able to transfer fructosyl groups from sucrose as donor producing the corresponding series of fructooligosaccharides: 1-kestose, nystose and fructosylnystose. Using sucrose as substrate, the k(cat) and K(m) values for transfructosylating activity were 1.62+/-0.09 x 10(4)s(-1) and 0.53+/-0.05 M, whereas for hydrolytic activity the corresponding values were 775+/-25s(-1) and 27+/-3 mM. At elevated sucrose concentrations, the fructosyltransferase from A. aculeatus showed a high transferase/hydrolase ratio that confers it a great potential for the industrial production of prebiotic fructooligosaccharides.     

Characterization of a beta-fructofuranosidase from Schwanniomyces occidentalis with transfructosylating activity yielding the prebiotic 6-kestose

beta-Fructofuranosidases are powerful tools in industrial biotechnology. We have characterized an extracellular beta-fructofuranosidase from the yeast Schwanniomyces occidentalis. The enzyme shows broad substrate specificity, hydrolyzing sucrose, 1-kestose, nystose and raffinose, with different catalytic efficiencies (k(cat)/K(m)). Although the main reaction catalysed by this enzyme is sucrose hydrolysis, it also produces two fructooligosaccharides (FOS) by transfructosylation. A combination of (1)H, (13)C and 2D-NMR techniques shows that the major product is the prebiotic trisaccharide 6-kestose. The 6-kestose yield obtained with this beta-fructofuranosidase is, to our concern, higher than those reported with other 6-kestose-producing enzymes, both at the kinetic maximum (76gl(-1)) and at reaction equilibrium (44gl(-1)). The total FOS production in the kinetic maximum was 101gl(-1), which corresponded to 16.4% (w/w) referred to the total carbohydrates in the reaction mixture.     

Production of 1-kestose with intact mycelium of Aspergillus phoenicis containing sucrose-1F-fructosyltransferase

Summary Favourable reaction conditions for the enzymatic production of 1-kestose by sucrose-1^F-fructosyltransferase, SFT (EC 2.4.1.99) from Aspergillus phoenicis CBS 294.80 mycelium were established. The intracellular enzyme SFT works best at 60°C, exhibits a relatively high thermostability and possesses an alkaline pH optimum. An invertase also present in the mycelium of A. phoenicis possesses an acidic pH optimum. Consequently, around pH 8.0 sucrose is converted mainly to 1-kestose and nystose while fructose is only formed in relatively small amounts. Under optimal conditions (55° C, pH 8.0 and an initial sucrose concentration of 750 g 1^-1) a yield of about 300 g 1-kestose per 1.01 reaction mixture could be achieved after 8 h.Offprint requests to: J. A. M. van Balken     

Manifestation of anomeric form,ring structure,and linkage in the 13c-n.m.r. spectra of oligomers and polymers containing D-fructose: maltulose,isomaltulose, sucrose,leucrose, 1-kestose,nystose, inulin,and grass levan

¹³C-N.m.r. spectroscopy has been used to determine the equilibrium composition of solutions of maltulose and isomaltulose in deuterium oxide. Resonance assignments have been made for maltulose, isomaltulose, sucrose, leucrose, 1-kestose, nystose, inulin, and grass levan. Some earlier assignments for sucrose and grass levan are corrected. The resonances of the D-glucopyranosyl group in maltulose and isomaltulose have been observed to be sensitive to the ring and anomeric forms of the adjacent D-fructose residue. Spin-lattice relaxation-times (T1) and nuclear Overhauser enhancement factors (n.O.e.f.) for the carbon atoms of the D-fructofuranosyl residues of inulin have been measured, and used in conjunction with deuteration, to aid in resonance and linkage assignments.     

Purification and partial characterization of fructosyltransferase and invertase from Aspergillus niger AS0023

Fructosyltransferase (EC.2.4.1.9) and invertase (EC.3.2.1.26) have been purified from the crude extract of Aspergillus niger AS0023 by successive chromatographies on DEAE-sephadex A-25, sepharose 6B, sephacryl S-200, and concanavalin A-Sepharose 4B columns. On acrylamide electrophoresis the two enzymes, in native and denatured forms, gave diffused glycoprotein bands with different electrophoretic mobility. On native-PAGE and SDS-PAGE, both enzymes migrated as polydisperse aggregates yielding broad and diffused bands. This result is typical of heterogeneous glycoproteins and the two enzymes have proved their glycoprotein nature by their adsorption on concanavalin A lectin. Fructosyltransferase (FTS) on native PAGE migrated as two enzymatically active bands with different electrophoretic mobility, one around 600 kDa and the other from 193 to 425 kDa. On SDS-PAGE, these two fractions yielded one band corresponding to a molecular weight range from 81 to 168 kDa. FTS seems to undergo association-dissociation of its glycoprotein subunits to form oligomers with different degrees of polymerization. Invertase (INV) showed higher mobility corresponding to a molecular range from 82 to 251 kDa, on native PAGE, and from 71 to 111 kDa on SDS-PAGE. The two enzymes exhibited distinctly different pH and temperature profiles. The optimum pH and temperature for FTS were found to be 5.8 and 50 degrees C, respectively, while INV showed optimum activity at pH 4.4 and 55 degrees C. Metal ions and other inhibitors had different effects on the two enzyme activities. FTS was completely abolished with 1 mM Hg(2+) and Ag(2+), while INV maintained 72 and 66% of its original activity, respectively. Furthermore, the two enzymes exhibited distinctly different kinetic constants confirming their different nature. The K(m) and V(m) values for each enzyme were calculated to be 44.38 mM and 1030 micromol ml(-1)min(-1) for FTS and 35.67 mM and 398 micromol ml(-1) min(-1) for INV, respectively. FTS and INV catalytic activity was dependent on sucrose concentration. FTS activity increased with increasing sucrose concentrations, while INV activity decreased markedly with increasing sucrose concentration. Furthermore, INV exhibited only hydrolytic activity producing exclusively fructose and glucose from sucrose, while FTS catalyzed exclusively fructosyltransfer reaction producing glucose, 1-kestose, nystose and fructofuranosyl nystose. In addition, at 50% sucrose concentration FTS produced fructooligosaccharides at the yield of 62% against 54% with the crude extract.     

Characterization of a novel fructosyltransferase from Lactobacillus reuteri that synthesizes high-molecular-weight inulin and inulin oligosaccharides

Fructosyltransferase (FTF) enzymes produce fructose polymers (fructans) from sucrose. Here, we report the isolation and characterization of an FTF-encoding gene from Lactobacillus reuteri strain 121. A C-terminally truncated version of the ftf gene was successfully expressed in Escherichia coli. When incubated with sucrose, the purified recombinant FTF enzyme produced large amounts of fructo-oligosaccharides (FOS) with beta-(2-->1)-linked fructosyl units, plus a high-molecular-weight fructan polymer (>10(7)) with beta-(2-->1) linkages (an inulin). FOS, but not inulin, was found in supernatants of L. reuteri strain 121 cultures grown on medium containing sucrose. Bacterial inulin production has been reported for only Streptococcus mutans strains. FOS production has been reported for a few bacterial strains. This paper reports the first-time isolation and molecular characterization of (i) a Lactobacillus ftf gene, (ii) an inulosucrase associated with a generally regarded as safe bacterium, (iii) an FTF enzyme synthesizing both a high molecular weight inulin and FOS, and (iv) an FTF protein containing a cell wall-anchoring LPXTG motif. The biological relevance and potential health benefits of an inulosucrase associated with an L. reuteri strain remain to be established.     

Microbial enzymatic production and applications of short-chain fructooligosaccharides and inulooligosaccharides: recent advances and current perspectives

The industrial production of short-chain fructooligosaccharides (FOS) and inulooligosaccharides is expanding rapidly due to the pharmaceutical importance of these compounds. These compounds, concisely termed prebiotics, have biofunctional properties and hence health benefits if consumed in recommended dosages. Prebiotics can be produced enzymatically from sucrose elongation or via enzymatic hydrolysis of inulin by exoinulinases and endoinulinases acting alone or synergistically. Exoinulinases cleave the non-reducing β-(2, 1) end of inulin-releasing fructose while endoinulinases act on the internal linkages randomly to release inulotrioses (F3), inulotetraoses (F4) and inulopentaoses (F5) as major products. Fructosyltransferases act by cleaving a sucrose molecule and then transferring the liberated fructose molecule to an acceptor molecule such as sucrose or another oligosaccharide to elongate the short-chain fructooligosaccharide. The FOS produced by the action of fructosyltransferases are 1-kestose (GF2), nystose (GF3) and fructofuranosyl nystose (GF4). The production of high yields of oligosaccharides of specific chain length from simple raw materials such as inulin and sucrose is a technical challenge. This paper critically explores recent research trends in the production and application of short-chain oligosaccharides. Inulin and enzyme sources for the production of prebiotics are discussed. The mechanism of FOS chain elongation and also the health benefits associated with prebiotics consumption are discussed in detail.     

Mutational analysis of the active center of plant fructosyltransferases: Festuca 1-SST and barley 6-SFT

The active center of the glycoside hydrolase family 32 contains the three characteristic motifs (N/S)DPNG, RDP, and EC. We replaced the N-terminal region including the (N/S)DPNG motif of barley 6-SFT (sucrose:fructan 6-fructosyltransferase) by the corresponding region of Festuca 1-SST (sucrose:sucrose 1-fructosyltransferase). The chimeric enzyme, expressed in Pichia, retained the specificity of 6-SFT. Attempts to replace a larger piece at the N-terminus including also the RDP motif failed. A point mutation introduced in the RDP motif of 1-SST abolished enzymatic activity. Interestingly, point mutations of the EC-motif resulted in an enzyme which had lost the capability to form 1-kestose and glucose from sucrose but still accepted 1-kestose, producing fructose and sucrose as well as nystose.     

Effect of pH on transfructosylation and hydrolysis by β-fructofuranosidase from Aspergillus oryzae

 β-Fructofuranosidase was purified from commercial alkaline protease (Aspergillus oryzae origin). The optimal pH of its transfructosylating activity was more alkaline (pH 8) than that of its hydrolyzing activity (pH 5). In the case of a 24-h reaction with sucrose, the hydrolysis and transfructosylation reaction were optimal at pH 4–5 and pH 8, respectively. In the reaction at pH 8 1-kestose and nystose were the main fructooligosaccharides produced. The transfer ratio was hardly different between pH 5 and pH 8 early in the reaction, but the transfer products (1-kestose and nystose) were decreased at pH 5 as the reaction proceeded because of their hydrolysis. Received: 18 January 1995/Received last revision: 23 August 1995/Accepted: 13 September 1995     

Method for Enrichment of Rhodopseudomonas sphaeroides from Fresh Water

An inulinase was highly purified from the culture broth of Penicillium purpurogenum by chromatographies on DEAE-Sepharose CL-6B, Toyopearl HW-65, and Bio-Gel P-100. The enzyme was homogeneous by disc electrophoretic analysis. The molecular weight was 6.4 × 10⁴ by SDS-disc electrophoresis and gel filtration on Bio-Gel P-150. The isoelectric point was pH 3.6 by isoelectric focusing. The enzyme hydrolyzed inulin rapidly, but did not affect sucrose. By paper chromatography analysis, the major products from inulin were tri-, tetra-, penta-, and hexa-saccharides. The substrate specificity of the enzyme on hydrolyses of fructo-oligosaccharides[1^F(1-β-d-fructofuranosyl)n sucrose (n = 1 to 6 and n (average of polymerization degree) = 8)] were examined. The Km values and relative maximum velocities for the hydrolyses of inulin and fructo-oligosaccharides (GF_n, n = 2 to 7 and n = 9) were as follows: inulin, (DP = 35) 0.21 mM and 100; GF₉, 0.24 mM and 86.5; GF₇, 0.33 mM and 132; GF₆, 0.85 mM and 71.2; GF₅, 3.8 mM and 25.4; GF₄, 2.8 mM and 28.8; GF₃, (nystose) 16 mM and 0.8; GF₂ (1-kestose), 8.4 mM and 0.2. The molecular activities for the hydrolyses of fructo-oligosaccharides (GF_n, n = 2 to 6) were increased depending on the degree of polymerization of fructosyl residues, and were nearly constant if the polymerization degree was over seven. These results strongly suggested that the endo-type inulinase from Penicillium purpurogenum had a subsite structure consisting of at least seven subsites.     

蔗糖:蔗糖-1-果糖基转移酶的表面展示及酶学性质分析

【目的】蔗糖:蔗糖-1-果糖基转移酶催化1分子蔗糖上的果糖基转移到另一个蔗糖分子上,形成1-蔗果三糖和葡萄糖。在低聚果糖中,1-蔗果三糖益生素活性最高。本研究将该酶展示在酵母菌细胞表面上,并用于1-蔗果三糖的制备。【方法】将来自莴苣的蔗糖:蔗糖-1-果糖基转移酶基因克隆到用于酵母细胞表面展示的表达载体上,并在解脂亚罗酵母菌中进行异源表达,表达的酶展示在该细胞表面上,然后以蔗糖为底物,研究表面展示的蔗糖:蔗糖-1-果糖基转移酶的性质。【结果】免疫荧光实验结果表明蔗糖:蔗糖-1-果糖基转移酶已展示在酵母菌的细胞表面上,高效液相色谱结果表明酵母表面展示的该酶具有转移酶的催化活性。该酶的最适作用温度、最适作用p H分别为45°C和7.5;该酶的催化活性受Zn2+和Cu2+的抑制,受Ca2+激活;该酶重复使用7次后,酶活下降50%。表面展示的蔗糖:蔗糖-1-果糖基转移酶和3%蔗糖混合后在40°C条件下孵育30 min后,所产1-蔗果三糖含量最高为20.8 mmol/L。【结论】蔗糖:蔗糖-1-果糖基转移酶在解脂亚罗酵母菌中得到成功表达,并展示在其细胞表面上,生化研究表明该重组蛋白具有果糖基转移酶活性,且催化蔗果三糖的生成。表面展示的蔗糖:蔗糖-1-果糖基转移酶作为一种全细胞催化剂能够用于1-蔗果三糖的制备。     

Production, purification and identification of fructooligosaccharides produced by β-fructofuranosidase from Aspergillus niger IMI 303386

Aspergillus niger IMI 303386 produced higher levels of intra- and extracellular -fructofuranosidase and inulinase on inulin than on sucrose. Intracellular -fructofuranosidase from sucrose medium catalysed the best transfructosylation reaction. The concentration of fructooligosaccharides (FOS) reached a maximum in 72 h with 25% (w/v) sucrose. The FOS were purified and the main products were kestose and nystose. Inulinase hydrolysed inulin in an exofashion and released mainly fructose.     

Production of high-content fructo-oligosaccharides by an enzymatic system from Penicillium rugulosum

Summary The production of high-content fructo-oligosaccharides from sucrose by a crude FTF from a new strain of Penicillium isolated in our Laboratory was investigated. The optimum conditions for the production of the enzyme and for the enzymic reaction have been determined. It has been demonstrated that the crude enzyme acts as a mixed enzyme system of fructosyl transferase (FTF; Class 2 of Enzyme Nomenclature) and glycosidases (Class 3 of Enyme Nomenclature). Under optimum conditions: pH 5.5, temperature 55°C, sucrose concentration 750 g/l, enzyme concentration 5 FTF units/g sucrose, conversion yield up to 80% were obtained and high concentration of nystose (412 g/l) and fructofuranosyl-nystose (176 g/l) were accumulated.     

Secretion of fructosyltransferase by Streptococcus salivarius involves the sucrose-dependent release of the cell-bound form

Three strains of Streptococcus salivarius including a recent clinical isolate were found to possess Ca2(+)-dependent fructosyltransferase (FTF) activity. The extracellular FTF activity of cells grown on sucrose increased as much as 9-fold compared with cells grown on either glucose, fructose or galactose. This increase in activity was due not to induction of FTF by sucrose, but to the release of the cell-bound form of the enzyme. Studies with washed cells of S. salivarius ATCC 25975 showed that the extent of release of the cell-bound FTF activity was dependent upon the sucrose concentration up to 4 mM, at which concentration maximum release (95%) of cell-bound FTF occurred. Several lines of evidence suggested that either substrate binding or de novo synthesis of fructan is required for the release of the cell-bound FTF activity.