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

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

Mutations in the Different Residues between Dextransucrase Gtf-DSM and Reuteransucrase GtfO for the Investigation of Linkage Specificity Determinants

The dextransucrase Gtf-DSM has 99.3% sequence identity with the reuteransucrase GtfO, and only 11 out of 1045 residues are different between their N-terminally truncated recombinant forms. Gtf-DSM is capable of synthesizing a dextran with 1% (α1 → 2), 6% (α1 → 4), 24% (α1 → 3), and 69% (α1 → 6) linkages, while GtfO produces a reuteran with 21% (α1 → 6) and 79% (α1 → 4) linkages. In this work, using recombinant Gtf-DSM and GtfO as templates, parallel substitutions targeting these 11 distinguishing residues were performed to investigate their linkage specificity determinants. The combinatorial mutation (I937L/D977A/D1083V/Q1086K/K1087G) at the acceptor binding subsites +1 and +2 nearly converted the linkage specificity of Gtf-DSM to that of GtfO. Surprisingly, all of the individual or combinatorial mutations in four residues from domains IV and V of Gtf-DSM significantly altered the linkage specificity of Gtf-DSM. Additionally, all mutations in the 11 distinguishing residues of Gtf-DSM resulted in a dramatically reduced transferase/hydrolysis activity ratio, which was closer to that of GtfO. These mutation results suggested that the linkage specificity differences between Gtf-DSM and GtfO are determined by the distinct micro-physicochemical environments, formed by the concerted action of a series of residues not only from the acceptor binding subsites +1 and +2 but also from domains IV and V

High-Level Production of l‑Fucose by Plasmid-Free or Antibiotic-Independent Metabolically Engineered Escherichia coli Strains

l-Fucose is an important monosaccharide unit that exists in various biomasses, especially in microalgae. Microbial l-fucose using metabolically engineered strains has attracted attention due to its high yield and industrial feasibility. Previously, we engineered Escherichia coli MG1655 to efficiently produce 2′-fucosyllactose by genomic integration. Herein, this plasmid-free strain was further engineered to produce l-fucose by integrating a specific α-l-fucosidase gene and deleting the l-fucose degradation pathway. Its effectiveness of l-fucose biosynthesis by plasmid-free and inducer-free fermentation was demonstrated by both shake-flask and fed-batch cultivation with titers of 2.74 and 21.15 g/L, respectively. The precursor GDP-l-fucose supply was strengthened to obviously enhance l-fucose biosynthesis by introducing a single plasmid expressing four pathway genes. The hok/sok system was introduced to promote the plasmid stabilization without antibiotic. The final engineered strain efficiently could produce l-fucose without antibiotics, with titers of 6.83 and 35.68 g/L by shake-flask and fed-cultivation cultivation, respectively

Thermal (A) and urea-induced (B) unfolding of <i>C.</i><i>scindens</i> DPEase.

<p>The symbols ▪ and • represented the unfolding curves of <i>C. scindens</i> DPEase in the absence and presence of Mn²⁺, which were monitored by CD. The CD measurements and unfolding analysis procedures were shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062987#s2" target="_blank">Materials and Methods</a>.</p

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

Enhancement of the d‑Allulose 3‑Epimerase Expression in Bacillus subtilis through Both Transcriptional and Translational Regulations

d-Allulose, a functional bulk sweetener, has recently attracted increasing attention because of its low-caloric-ness properties and diverse health effects. d-Allulose is industrially produced by the enzymatic epimerization of d-fructose, which is catalyzed by ketose 3-epimerase (KEase). In this study, the food-grade expression of KEase was studied using Bacillus subtills as the host. Clostridium sp. d-allulose 3-epimerase (Clsp-DAEase) was screened from nine d-allulose-producing KEases, showing better potential for expression in B. subtills WB600. Promoter-based transcriptional regulation and N-terminal coding sequence (NCS)-based translational regulation were studied to enhance the DAEase expression level. In addition, the synergistic effect of promoter and NCS on the Clsp-DAEase expression was studied. Finally, the strain with the combination of a PHapII promoter and gln A-Up NCS was selected as the best Clsp-DAEase-producing strain. It efficiently produced Clsp-DAEase with a total activity of 333.2 and 1860.6 U/mL by shake-flask and fed-batch cultivations, respectively

Efficient Synthesis of Glucosyl-β-Cyclodextrin from Maltodextrins by Combined Action of Cyclodextrin Glucosyltransferase and Amyloglucosidase

Instead of β-cyclodextrin (β-CD), branched β-CDs have been increasingly used in many aspects as they possess better solubility and higher bioadaptability. But most commercialized branched β-CDs were chemically synthesized. Thus, the glucosyl-β-cyclodextrin (G₁-β-CD) prepared via enzymatic approach could be a nice substitute. However, the yield of G₁-β-CD was low. Here, we reported a controlled two-step reaction to efficiently prepare G₁-β-CD from maltodextrins by β-cyclodextrin glucosyltransferase (β-CGTase) and amyloglucosidase (AG). Compared to the single β-CGTase reaction, controlled two-step reaction caused a yield increase of G₁-β-CD by 130%. Additionally, the percentage of G₁-β-CD was enhanced from 2.4% to 24.0% and the side products α-CD and γ-CD were hydrolyzed because of the coupling activity of β-CGTase. Thus, this controlled two-step reaction might be an efficient approach for industrial production of pure G₁-β-CD

Multiple sequence alignment of DTEase family enzymes and their homologs.

<p>Amino acid sequence for DPEase from <i>C. scindens</i> 35704 (Csc-DPEase; GeneBank accession No: CLOSCI_02528) was aligned with <i>C. cellulolyticum</i> H10 (Cce-DPEase; ACL75304), <i>Ruminococcus</i> sp. 5_1_39BFAA (Rsp-DPEase; ZP_04858451), <i>P. cichorii</i> DTEase (Pci-DTEase; BAA24429), <i>A. tumefaciens</i> DPEase (Atu-DPEase; AAL45544), and <i>R. sphaeroides</i> DTEase (Rsp-DTEase; ACO59490). The alignment was performed using ClustalW2 program (<a href="http://www.ebi.ac.uk/Tools/clustalw2/index.html" target="_blank">http://www.ebi.ac.uk/Tools/clustalw2/index.html</a>). Amino acid residues that are identical in all the displayed sequences are marked by asterisks (*), strongly conserved or weakly conserved residues are indicated by colons (:) or dots (.), respectively. The symbol ▪, •, and ▴ represented the residues involved in the metal coordinating site, those responsible for the interaction between the enzyme and O1, O2, and O3 of D-fructose, and those providing a hydrophobic environment around the substrate around the O4, O5, and O6 of D-fructose, respectively (according to the crystal structures of <i>C. cellulolyticum</i> DPEase, <i>A. tumefaciens</i> DPEase, and <i>P. cichorii</i> DTEase).</p

SDS-PAGE analysis of proteins stained with Coomassie blue.

<p>A represented the SDS-PAGE of whole-cell protein. Lane 1, protein marker; lane 2, the cell without IPTG induction; and lane 3–8, the cell induced by IPTG for 1–6 h. B showed the SDS-PAGE analysis of the purified recombinant <i>C. scindens</i> DPEase (lane 2).</p