Identification of a Recombinant Inulin Fructotransferase (Difructose Dianhydride III Forming) from Arthrobacter sp. 161MFSha2.1 with High Specific Activity and Remarkable Thermostability

Difructose dianhydride III (DFA III) is a functional carbohydrate produced from inulin by inulin fructotransferase (IFTase, EC 4.2.2.18). In this work, an IFTase gene from Arthrobacter sp. 161MFSha2.1 was cloned and expressed in Escherachia coli. The recombinant enzyme was purified by metal affinity chromatography. It showed significant inulin hydrolysis activity, and the produced main product from inulin was determined as DFA III by nuclear magnetic resonance analysis. The molecular mass of the purified protein was calculated to be 43 and 125 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and gel filtration, respectively, suggesting the native enzyme might be a homotrimer. The recombinant enzyme showed maximal activity as 2391 units/mg at pH 6.5 and 55 °C. It displayed the highest thermostability among previously reported IFTases (DFA III forming) and was stable up to 80 °C for 4 h of incubation. The smallest substrate was determined as nystose. The conversion ratio of inulin to DFA III reached 81% when 100 g/L inulin was catalyzed by 80 nM recombinant enzyme for 20 min at pH 6.5 and 55 °C. All of these data indicated that the IFTase (DFA III forming) from Arthrobacter sp. 161MFSha2.1 had great potential for industrial DFA III production

Probing the Role of Two Critical Residues in Inulin Fructotransferase (DFA III-Producing) Thermostability from Arthrobacter sp. 161MFSha2.1

Inulin fructotransferase (IFTase) is an important enzyme that produces di-d-fructofuranose 1,2′:2,3′ dianhydride (DAF III), which is beneficial for human health. Present investigations mainly focus on screening and characterizing IFTase, including catalytic efficiency and thermostability, which are two important factors for enzymatic industrial applications. However, few reports aimed to improve these two characteristics based on the structure of IFTase. In this work, a structural model of IFTase (DFA III-producing) from Arthrobacter sp. 161MFSha2.1 was constructed through homology modeling. Analysis of this model reveals that two residues, Ser-309 and Ser-333, may play key roles in the structural stability. Therefore, the functions of the two residues were probed by site-directed mutagenesis combined with the Nano-DSC method and assays for residual activity. In contrast to other mutations, single mutation of serine 309 (or serine 333) to threonine did not decrease the enzymatic stability, whereas double mutation (serine 309 and serine 333 to threonine) can enhance thermostability (by approximately 5 °C). The probable mechanisms for this enhancement were investigated

Improving the Catalytic Behavior of DFA I‑Forming Inulin Fructotransferase from Streptomyces davawensis with Site-Directed Mutagenesis

Previously, a α-d-fructofuranose-β-d-fructofuranose 1,2′:2,1′-dianhydride (DFA I)-forming inulin fructotransferase (IFTase), namely, <i>Sd</i>IFTase, was identified. The enzyme does not show high performances. In this work, to improve catalytic behavior including activity and thermostability, the enzyme was modified using site-directed mutagenesis on the basis of structure. The mutated residues were divided into three groups. Those in group I are located at central tunnel including G236, A257, G281, T313, and A314S. The group II contains residues at the inner edge of substrate binding pocket including I80, while group III at the outer edge includes G121 and T122. The thermostability was reflected by the melting temperature (<i>T</i>_m) determined by Nano DSC. Finally, the <i>T</i>_m values of G236S/G281S/A257S/T313S/A314S in group I and G121A/T122L in group III were enhanced by 3.2 and 4.5 °C, and the relative activities were enhanced to 140.5% and 148.7%, respectively. The method in this work may be applicable to other DFA I-forming IFTases

Enzymatic Production of Melibiose from Raffinose by the Levansucrase from Leuconostoc mesenteroides B‑512 FMC

Melibiose, which is an important reducing disaccharide formed by α-1,6 linkage between galactose and glucose, has been proven to have beneficial applications in both medicine and agriculture. In this study, a characterized levansucrase from Leuconostoc mesenteroides B-512 FMC was further used to study the bioproduction of melibiose from raffinose. The reaction conditions were optimized for melibiose synthesis. The optimal pH, temperature, substrate concentration, ratio of substrates, and units of enzymes were determined as pH 6.0, 45 °C, 210 g/L, 1:1 (210 g/L:210 g/L), and 5 U/mL, respectively. The transfructosylation product of raffinose was determined to be melibiose by FTIR and NMR. A high raffinose concentration was found to strongly favor the production of melibiose. When 210 g/L raffinose and 210 g/L lactose were catalyzed using 5 U/mL purified levansucrase at pH 6.0 and 45 °C, the maximal yield of melibiose was 88 g/L

Molecular docking of DFA III to the putative active pocket of <i>Aa</i>DFA IIIase mutants.

<p>A, B, C, and D represented the molecular docking of D207A, D207N, E218A, and E218N mutants, respectively. The dotted and solid pink lines represented strong and relatively weak hydrogen bonds, respectively. The distances of H bonds were labeled with pink Arabic numbers.</p

Effect of pH (A) and temperature (B) on the activity of <i>Aa</i>DFA IIIase.

<p>(A) The relative activity was investigated at 55°C and different pH values. (B) The relative activity was investigated at temperatures varying from 30–60°C at pH 5.5. Data were plotted as ln (relative activity, %) versus T^-1. Relative activity was expressed as a percentage of the maximal enzyme activity. Values were the means of three replications ± standard deviation.</p

Identification of a Novel Di-D-Fructofuranose 1,2’:2,3’ Dianhydride (DFA III) Hydrolysis Enzyme from <i>Arthrobacter aurescens</i> SK8.001

<div><p>Previously, a di-D-fructofuranose 1,2’:2,3’ dianhydride (DFA III)-producing strain, <i>Arthrobacter aurescens</i> SK8.001, was isolated from soil, and the gene cloning and characterization of the DFA III-forming enzyme was studied. In this study, a DFA III hydrolysis enzyme (DFA IIIase)-encoding gene was obtained from the same strain, and the DFA IIIase gene was cloned and expressed in <i>Escherichia coli</i>. The SDS-PAGE and gel filtration results indicated that the purified enzyme was a homotrimer holoenzyme of 145 kDa composed of subunits of 49 kDa. The enzyme displayed the highest catalytic activity for DFA III at pH 5.5 and 55°C, with specific activity of 232 U mg^-1. <i>K</i>_m and <i>V</i>_max for DFA III were 30.7 ± 4.3 mM and 1.2 ± 0.1 mM min^-1, respectively. Interestingly, DFA III-forming enzymes and DFA IIIases are highly homologous in amino acid sequence. The molecular modeling and docking of DFA IIIase were first studied, using DFA III-forming enzyme from <i>Bacillus</i> sp. snu-7 as a template. It was suggested that <i>A</i>. <i>aurescens</i> DFA IIIase shared a similar three-dimensional structure with the reported DFA III-forming enzyme from <i>Bacillus</i> sp. snu-7. Furthermore, their catalytic sites may occupy the same position on the proteins. Based on molecular docking analysis and site-directed mutagenesis, it was shown that D207 and E218 were two potential critical residues for the catalysis of <i>A</i>. <i>aurescens</i> DFA IIIase.</p></div

Efficient Biosynthesis of Lactosucrose from Sucrose and Lactose by the Purified Recombinant Levansucrase from <i>Leuconostoc mesenteroides</i> B‑512 FMC

Lactosucrose, a rare trisaccharide formed from sucrose and lactose by enzymatic transglycosylation, is a type of indigestible carbohydrate with a good prebiotic effect. In this study, lactosucrose biosynthesis was efficiently carried out by a purified levansucrase from <i>Leuconostoc mesenteroides</i> B-512. The target gene was cloned and expressed in <i>Escherichia coli</i>, and the recombinant enzyme was purified to homogeneity by nickel affinity and gel filtration chromatography. The effects of pH, temperature, substrate concentration, substrate ratio, and enzyme amount on lactosucrose biosynthesis were studied in detail, and the optimized conditions were determined to be pH 6.5, 50 °C, 27% (W/V) sucrose, 27% (W/V) lactose, and 5 U mL^–1 of the purified recombinant enzyme. Under the optimized reaction conditions, the maximal lactosucrose yield reached 224 g L^–1 after reaction for 1 h. Therefore, <i>L. mesenteroides</i> levansucrase could be considered a potential candidate for future industrial production of lactosucrose

Estimation of the molecular mass of <i>Aa</i>DFA IIIase by SDS-PAGE and gel filtration.

<p>(A) SDS-PAGE analysis of <i>Aa</i>DFA IIIase. The protein and markers were stained with Coomassie Blue. (B) Gel filtration analysis of <i>Aa</i>DFA IIIase. The marker proteins include thyroglobulin (bovine, Mw: 670 kDa), γ-globulin (bovine, Mw: 158 kDa), ovalbumin (chicken, Mw: 44 kDa), myoglobin (horse, Mw: 17 kDa), and vitamin B12 (Mw: 1.35 kDa), respectively. The retention times of above corresponding markers are 7.0, 8.3, 9.7, 11.1 and 13.9 min, respectively, and 8.5 min for <i>Aa</i>DFA IIIase.</p

Primers for site-directed mutagenesis.

<p>The underlined sequences represented mutated codons.</p