Unraveling the role of flexible coil near calcium binding site of levansucrase on thermostability and product profile via proline substitution and molecular dynamics simulations.

Due to its bioactivity and versatile applications, levan has appeared as a promising biomaterial. Levansucrase is responsible for the conversion of sucrose into levan. With the goal of enhancing levan production, the strategy for enhancing the stability of levansucrase is being intensively studied. To make proteins more stable under high temperatures, proline, the most rigid residue, can be introduced into previously flexible regions. Herein, G249, D250, N251, and H252 on the flexible coil close to the calcium binding site of Bacillus licheniformis levansucrase were replaced with proline. Mutations at G249P greatly enhance both the enzyme's thermodynamic and kinetic stability, while those at H252P improve solely the enzyme's kinetic stability. GPC analysis revealed that G249P synthesize more levan, but H252P generate primarily oligosaccharides. Molecular dynamics simulations (MD) and MM/GBSA analysis revealed that G249P mutation increased not only the stability of levansucrase, but also affinity toward fructan.

Mechanistic Insights into Roseoflavin Synthesis by N,N-8-Demethyl-8-aminoriboflavin Dimethyltransferase (RosA): Molecular Dynamics Simulations and Residue Conservation Analysis.

8-Demethyl-8-dimethylaminoriboflavin (Roseoflavin or RoF) is a natural riboflavin analogue found in Streptomyces davaonensis and Streptomyces cinnabarinus. RoF displays potent antibiotic properties because it affects FMN riboswitches and flavoproteins of cellular targets. N,N-8-Demethyl-8-aminoriboflavin dimethyltransferase (RosA) is an enzyme that catalyzes the last step of RoF biosynthesis, a consecutive dimethylation of 8-demethyl-8-aminoriboflavin (AF) to generate RoF. Thus, understanding mechanistic insights into RosA structures and mechanisms could lead to the improvement of the RoF product yield. Herein, mechanistic insights into roseoflavin synthesis by RosA were evaluated using molecular dynamics simulations. The obtained results revealed that RosA possibly catalyzes the reaction by positioning the substrate binding to have proper distance and orientation to the methyl group donor, S-adenosylmethionine. No direct participation of catalytic residues in the reaction was identified. The enzyme's active site structures change drastically to accommodate the ligand binding. On the basis of the MM/GBSA calculations and conservation analysis, the amino acid residues involved in substrate binding were identified. The structural information obtained from this study could be beneficial in designing RosA to efficiently produce roseoflavin.

Energy- and evolution-based design of inulosucrase for enhanced thermostability and inulin production.

Inulosucrase from Lactobacillus reuteri 121 (LrInu) exhibits promise in the synthesis of prebiotic inulin and fructooligosaccharides. However, for its use in industry, LrInu's thermostability is a crucial consideration. In this study, the computational program FireProt was used to predict the thermostable variants of LrInu. Using rational criteria, nine variants were selected for protein expression and characterization. The G237P variant was determined to be the greatest designed candidate due to its greatly enhanced stability and activity in comparison to the wild-type enzyme. The optimum temperature of G237P increased from 50 to 60°C, with an over 5-fold increase in the half-life. Spectroscopy studies revealed that the G237P mutation could prevent the structural change in LrInu caused by heat or urea treatment. Molecular dynamics (MD) simulations showed that the enhanced thermostability of the G237P variant resulted from an increase in structural rigidity and the number of native contacts within the protein molecule. In addition, G237P variant synthesizes inulin with greater efficiency than WT. KEY POINTS: • Thermostable inulosucrase variant(s) were designed by Fireprot server. • G237P variant showed significantly improved thermostability compared to the wild type. • Inulin is synthesized more efficiently by G237P variant.

Aurisin A Complexed with 2,6-Di-O-methyl-ß-cyclodextrin Enhances Aqueous Solubility, Thermal Stability, and Antiproliferative Activity against Lung Cancer Cells.

Aurisin A (AA), an aristolane dimer sesquiterpene isolated from the luminescent mushroom Neonothopanus nambi, exhibits various biological and pharmacological effects. However, its poor solubility limits its use for further medicinal applications. This study aimed to improve the water solubility of AA via complexation with ß-cyclodextrin (ßCD) and its derivatives (2,6-di-O-methyl-ßCD (DMßCD) and 2-hydroxypropyl-ßCD (HPßCD). A phase solubility analysis demonstrated that the solubility of AA linearly enhanced with increasing concentrations of ßCDs (ranked in the order of AA/DMßCD > AA/HPßCD > AA/ßCD). Notably, ßCDs, especially DMßCD, increased the thermal stability of the inclusion complexes. The thermodynamic study indicated that the complexation between AA and ßCD(s) was a spontaneous endothermic reaction, and AA/DMßCD possesses the highest binding strength. The complex formation between AA and DMßCD was confirmed by means of FT-IR, DSC, and SEM. Molecular dynamics simulations revealed that the stability and compactness of the AA/DMßCD complex were higher than those of the DMßCD alone. The encapsulation of AA led to increased intramolecular H-bond formations on the wider rim of DMßCD, enhancing the complex stability. The antiproliferative activity of AA against A549 and H1975 lung cancer cells was significantly improved by complexation with DMßCD. Altogether, the satisfactory water solubility, high thermal stability, and enhanced antitumor potential of the AA/DMßCD inclusion complex would be useful for its application as healthcare products or herbal medicines.

Unravelling Regioselectivity of Leuconostoc citreum ABK-1 Alternansucrase by Acceptor Site Engineering.

Alternansucrase (ALT, EC 2.4.1.140) is a glucansucrase that can generate α-(1,3/1,6)-linked glucan from sucrose. Previously, the crystal structure of the first alternansucrase from Leuconostoc citreum NRRL B-1355 was successfully elucidated; it showed that alternansucrase might have two acceptor subsites (W675 and W543) responsible for the formation of alternating linked glucan. This work aimed to investigate the primary acceptor subsite (W675) by saturated mutagenesis using Leuconostoc citreum ABK-1 alternansucrase (LcALT). The substitution of other residues led to loss of overall activity, and formation of an alternan polymer with a nanoglucan was maintained when W675 was replaced with other aromatic residues. Conversely, substitution by nonaromatic residues led to the synthesis of oligosaccharides. Mutations at W675 could potentially cause LcALT to lose control of the acceptor molecule binding via maltose-acceptor reaction-as demonstrated by results from molecular dynamics simulations of the W675A variant. The formation of α-(1,2), α-(1,3), α-(1,4), and α-(1,6) linkages were detected from products of the W675A mutant. In contrast, the wild-type enzyme strictly synthesized α-(1,6) linkage on the maltose acceptor. This study examined the importance of W675 for transglycosylation, processivity, and regioselectivity of glucansucrases. Engineering glucansucrase active sites is one of the essential approaches to green tools for carbohydrate modification.

Molecular basis of the new COVID-19 target neuropilin-1 in complex with SARS-CoV-2 S1 C-end rule peptide and small-molecule antagonists.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for causing the current coronavirus 2019 (COVID-19) pandemic, uses its spike (S1) protein for host cell attachment and entry. Apart from angiotensin-converting enzyme 2, neuropilin-1 (NRP1) has been recently found to serve as another host factor for SARS-CoV-2 infection; thus, blocking S1-NRP1 interaction can be a potential treatment for COVID-19. Herein, molecular recognition between SARS-CoV-2 S1 C-end rule (CendR) heptapeptide including small-molecule antagonists (EG00229 and EG01377) and the NRP1 was investigated using molecular dynamics simulations and binding free energy calculations based on MM-PBSA method. The binding affinity and the number of hot-spot residues of EG01377/NRP1 complex were higher than those of CendR/NRP1 and EG00229/NRP1 systems, in line with the reported experimental data as well as with the lower water accessibility at the ligand-binding site. The (i) T316, P317, and D320 and (ii) S346, T349, and Y353 residues of NRP1 were confirmed to respectively form H-bonds with the positively charged guanidinium group and the negatively charged carboxyl moiety of all studied ligands. Moreover, Rosetta protein design was employed to improve the binding affinity between CendR peptide and NRP1. The newly designed peptides, especially R683G and A684M, exhibited higher binding efficiency than the native CendR heptapeptide as well as the small-molecule EG00229 by forming more H-bonds and hydrophobic interactions with NPR1, suggesting that these designed peptides could be promising NRP1 inhibitors to combat SARS-CoV-2 infection.

Improving the thermostability and modulating the inulin profile of inulosucrase through rational glycine-to-proline substitution.

The flexibility of protein structure plays a crucial role in enzyme stability and catalysis. Among the amino acids, glycine is particularly important in conferring flexibility to proteins. In this study, the effects of flexible glycine residues in Lactobacillus reuteri 121 inulosucrase (LrInu) on stability and inulin profile were investigated through glycine-to-proline substitutions. Molecular dynamics (MD) simulations were employed to discover the flexible glycine residues, and eight glycine residues, including Gly217, Gly298, Gly330, Gly416, Gly450, Gly624, Gly627, Gly629, were selected for site-directed mutagenesis. The results demonstrated significant changes in both thermostability and inulin profiles of the variants. Particularly, the G624P and G627P variants showed reduced production of long-chain oligosaccharides compared to the WT. This can be ascribed to the increased rigidity of the active site, which is crucial for the induction-fit mechanism. Overall, this study provides valuable insights into the role of flexible glycine residues in the activity, stability, and inulin synthesis of LrInu.

Endo- and exo-levanases from Bacillus subtilis HM7: Catalytic components, synergistic cooperation, and application in fructooligosaccharide synthesis.

Levan-type fructooligosaccharides (LFOS) exhibit significant biological activities and selectively promote the growth of certain beneficial bacteria. Levanase is an important enzyme for LFOS production. In this study, two isoforms of levanases, exo- and endo-type depolymerizing enzymes, from Bacillus subtilis HM7 isolated from Dynastes hercules larvae excrement were cloned, expressed, and characterized. The synergistic effect on the levan hydrolysis and kinetic properties of both isoforms were evaluated, indicating their cooperation in levan metabolism, where the endo-levanase catalyzes a rate-limiting step. In addition, homology models and molecular dynamics simulations revealed the key amino residues of the enzymes for levan binding and catalysis. It was found that both isoforms possessed distinct binding residues in the active sites, suggesting the importance of the specificity of the enzymes. Finally, we demonstrated the potential of endo-type levanase in LFOS synthesis using a one-pot reaction with levansucrase. Overall, this study fills the knowledge gap in understanding levanase's mechanism, making an important contribution to the fields of food science and biotechnology.

A theoretical study on binding and stabilization of galactose and novel galactose analogues to the human α-galactosidase A variant causing Fabry disease.

α-galactosidase A (α-Gal A) catalyzes the hydrolysis of terminal α-galactosyl moieties from globotriaosylceramide, and mutations in this enzyme lead to the lipid metabolism disorder "Fabry disease". Mutation in α-Gal A possibly causes the protein misfolding, which reduces catalytic activity and stability of the enzyme. A recent study demonstrated that the binding of galactose on the α-Gal A catalytic site significantly increases its stability. Herein, the effect of mutation on secondary structure, structural energy, and galactose affinity of α-Gal A (wild type and A143T variant) was investigated using molecular dynamics simulations and free energy calculations based on MM/GBSA method. The results showed that A143T mutation caused the formation of unusual H-bonds that induced the change in secondary structure and binding affinities toward galactose. The amino acid residues involved in galactose binding were identified. The molecular binding mechanism obtained from this study could be helpful for optimizations and designs of new galactose analogs as pharmacological chaperones against Fabry disease.

Cassava pullulanase and its synergistic debranching action with isoamylase 3 in starch catabolism.

Pullulanase (EC 3.2.1.41, PUL), a debranching enzyme belonging to glycoside hydrolase family 13 subfamily 13, catalyses the cleavage of α-1,6 linkages of pullulan and ß-limit dextrin. The present work studied PUL from cassava Manihot esculenta Crantz (MePUL) tubers, an important economic crop. The Mepul gene was successfully cloned and expressed in E. coli and rMePUL was biochemically characterised. MePUL was present as monomer and homodimer, as judged by apparent mass of ~ 84 - 197 kDa by gel permeation chromatography analysis. Optimal pH and temperature were at pH 6.0 and 50 °C, and enzyme activity was enhanced by the addition of Ca2+ ions. Pullulan is the most favourable substrate for rMePUL, followed by ß-limit dextrin. Additionally, maltooligosaccharides were potential allosteric modulators of rMePUL. Interestingly, short-chain maltooligosaccharides (DP 2 - 4) were significantly revealed at a higher level when rMePUL was mixed with cassava isoamylase 3 (rMeISA3), compared to that of each single enzyme reaction. This suggests that MePUL and MeISA3 debranch ß-limit dextrin in a synergistic manner, which represents a major starch catabolising process in dicots. Additionally, subcellular localisation suggested the involvement of MePUL in starch catabolism, which normally takes place in plastids.

Synthesis of Cationic Quaternized Nanolevan Derivative for Small Molecule and Nucleic Acid Delivery.

Levan is a biopolymer composed of fructose chains covalently linked by ß-2,6 glycosidic linkages. This polymer self-assembles into a nanoparticle of uniform size, making it useful for a wide range of applications. Also, levan exhibits various biological activities such as antioxidants, anti-inflammatory, and anti-tumor, that make this polymer very attractive for biomedical application. In this study, levan synthesized from Erwinia tasmaniensis was chemically modified by glycidyl trimethylammonium chloride (GTMAC) to produce cationized nanolevan (QA-levan). The structure of the obtained GTMAC-modified levan was determined by FT-IR, 1H-NMR and elemental (CHN) analyzer. The size of the nanoparticle was calculated using the dynamic light scattering method (DLS). The formation of DNA/QA-levan polyplex was then investigated by gel electrophoresis. The modified levan was able to increase the solubility of quercetin and curcumin by 11-folds and 205-folds, respectively, compared to free compounds. Cytotoxicity of levan and QA-levan was also investigated in HEK293 cells. This finding suggests that GTMAC-modified levan should have a potential application for drug and nucleic acid delivery.

ECDD-S16 targets vacuolar ATPase: A potential inhibitor compound for pyroptosis-induced inflammation.

BACKGROUND: Cleistanthin A (CA), extracted from Phyllanthus taxodiifolius Beille, was previously reported as a potential V-ATPase inhibitor relevant to cancer cell survival. In the present study, ECDD-S16, a derivative of cleistanthin A, was investigated and found to interfere with pyroptosis induction via V-ATPase inhibition. OBJECTIVE: This study examined the ability of ECDD-S16 to inhibit endolysosome acidification leading to the attenuation of pyroptosis in Raw264.7 macrophages activated by both surface and endosomal TLR ligands. METHODS: To elucidate the activity of ECDD-S16 on pyroptosis-induced inflammation, Raw264.7 cells were pretreated with the compound before stimulation with surface and endosomal TLR ligands. The release of lactate dehydrogenase (LDH) was determined by LDH assay. Additionally, the production of cytokines and the expression of pyroptosis markers were examined by ELISA and immunoblotting. Moreover, molecular docking was performed to demonstrate the binding of ECDD-S16 to the vacuolar (V-)ATPase. RESULTS: This study showed that ECDD-S16 could inhibit pyroptosis in Raw264.7 cells activated with surface and endosomal TLR ligands. The attenuation of pyroptosis by ECDD-S16 was due to the impairment of endosome acidification, which also led to decreased Reactive Oxygen Species (ROS) production. Furthermore, molecular docking also showed the possibility of inhibiting endosome acidification by the binding of ECDD-S16 to the vacuolar (V-)ATPase in the region of V0. CONCLUSION: Our findings indicate the potential of ECDD-S16 for inhibiting pyroptosis and prove that vacuolar H+ ATPase is essential for pyroptosis induced by TLR ligands.

Fisetin glycosides synthesized by cyclodextrin glycosyltransferase from Paenibacillus sp. RB01: characterization, molecular docking, and antioxidant activity.

Fisetin is a flavonoid that exhibits high antioxidant activity and is widely employed in the pharmacological industries. However, the application of fisetin is limited due to its low water solubility. In this study, glycoside derivatives of fisetin were synthesized by an enzymatic reaction using cyclodextrin glycosyltransferase (CGTase) from Paenibacillus sp. RB01 in order to improve the water solubility of fisetin. Under optimal conditions, CGTase was able to convert more than 400 mg/L of fisetin to its glycoside derivatives, which is significantly higher than the previous biosynthesis using engineered E. coli. Product characterization by HPLC and LC-MS/MS revealed that the transglycosylated products consisted of at least five fisetin glycoside derivatives, including fisetin mono-, di- and triglucosides, as well as their isomers. Enzymatic analysis by glucoamylase and α-glucosidase showed that these fisetin glycosides were formed by α-1,4-glycosidic linkages. Molecular docking demonstrated that there are two possible binding modes of fisetin in the enzyme active site containing CGTase-glysosyl intermediate, in which O7 and O4' atoms of fisetin positioned close to the C1 of glycoside donor, corresponding to the isomers of the obtained fisetin monoglucosides. In addition, the water solubility and the antioxidant activity of the fisetin monoglucosides were tested. It was found that their water solubility was increased at least 800 times when compared to that of their parent molecule while still maintaining the antioxidant activity. This study revealed the potential application of CGTase to improve the solubility of flavonoids.

Production and bioactivities of nanoparticulated and ultrasonic-degraded levan generated by Erwinia tasmaniensis levansucrase in human osteosarcoma cells.

Levan is a bioactive polysaccharide that can be synthesized by various microorganisms. In this study, the physicochemical properties and bioactivity of levan synthesized by recombinant levansucrase from Erwinia tasmaniensis were investigated. The synthesis conditions, including the enzyme concentration, substrate concentration, and temperature, were optimized. The obtained levan generally appeared as a cloudy suspension. However, it could transform into a hydrogel at concentrations exceeding 10 % (w/v). Then, ultrasonication was utilized to reduce the molecular weight and increase the bioavailability of levan. Dynamic light scattering (DLS) and gel permeation chromatography (GPC) indicated that the size of levan was significantly decreased by ultrasonication, whereas Fourier transform infrared spectroscopy, 1H-nuclear magnetic resonance, and X-ray powder diffraction revealed that the chemical structure of levan was not changed. Finally, the bioactivities of both levan forms were examined using human osteosarcoma (Saos-2) cells. The result clearly illustrated that sonicated levan had higher antiproliferative activity in Saos-2 cells than original levan. Sonicated levan also activated Toll-like receptor expression at the mRNA level. These findings suggested the important beneficial applications of sonicated levan for the development of cancer therapies.

Synergistic enzyme cocktail between levansucrase and inulosucrase for superb levan-type fructooligosaccharide synthesis.

Inulosucrase (ISC) and levansucrase (LSC) utilise sucrose and produce inulin- and levan-type fructans, respectively. This study aims to propose a new strategy to improve levan-type fructooligosaccharide (L-FOS) production. The effect of ISC/ LSC -mixed reaction was elucidated on L-FOS production. The presence of ISC in the LSC reaction significantly leads to the higher production of L-FOSs as the main products. Furthermore, the different ratios between ISC and LSC affected the distribution of L-FOSs. A greater amount of ISC compared to LSC promoted the synthesis of short-chain L-FOSs. Conversely, when LSC was increased, the synthesis of longer-chain L-FOSs was enhanced. The addition of trisaccharide mixtures obtained from either a single ISC or LSC reaction could enhance L-FOSs synthesis in the LSC reaction. Analysis of these trisaccharides revealed that most species of the oligosaccharides were similar, with 1-kestose being the major one. The supplement of only 1-kestose in the LSC reaction showed similar results to those of the reaction in the presence of trisaccharide mixtures. Moreover, the results were supported by molecular dynamics simulations. This work not only provides an improvement in L-FOS production but also revealed and supported some insights into the mechanism of fructansucrases.

High surfactant-tolerant ß-mannanase isolated from Dynastes hercules larvae excrement, and identification of its hotspot using site-directed mutagenesis and molecular dynamics simulations.

The ß-mannanase from Bacillus subtilis HM7 (Man26HM7) isolated from Dynastes hercules larvae excrement was cloned and expressed in Escherichia coli. Biochemical characterization shows that optimal pH and temperature for catalysis are 6.0 and 50 °C, respectively. Man26HM7 displayed excellent surfactant stability by retaining 70% of initial activity in 1%(w/v) SDS, and more than 90% of initial activity in 1%(w/v) Triton X-100 and Tween 80. Results from amino acid sequence alignment and molecular modeling suggest residue 238 of ß-mannanase as a hotspot of SDS-tolerance. Mutagenesis at the equivalent residue of another homolog, ß-mannanase from Bacillus subtilis CAe24 (Man26CAe24), significantly enhanced the SDS stability of this enzyme. Comparative computational analysis, including molecular docking and molecular dynamics simulation, were then performed to compute the binding free energy of SDS to Man26HM7, Man26CAe24, and variant enzymes. The results suggest that residue 238 of Man26HM7 is involved in SDS binding to the hydrophobic surface of ß-mannanase. This study provides not only the promising application of Man26HM7 in detergent and cleaning products but also valuable information for enhancing the surfactant stability of ß-mannanase by enzyme engineering.

Fisetin Inhibits Osteogenic Differentiation of Mesenchymal Stem Cells via the Inhibition of YAP.

Mesenchymal stem cells (MSCs) are self-renewal and capable of differentiating to various functional cell types, including osteocytes, adipocytes, myoblasts, and chondrocytes. They are, therefore, regarded as a potential source for stem cell therapy. Fisetin is a bioactive flavonoid known as an active antioxidant molecule that has been reported to inhibit cell growth in various cell types. Fisetin was shown to play a role in regulating osteogenic differentiation in animal-derived MSCs; however, its molecular mechanism is not well understood. We, therefore, studied the effect of fisetin on the biological properties of human MSCs derived from chorion tissue and its role in human osteogenesis using MSCs and osteoblast-like cells (SaOs-2) as a model. We found that fisetin inhibited proliferation, migration, and osteogenic differentiation of MSCs as well as human SaOs-2 cells. Fisetin could reduce Yes-associated protein (YAP) activity, which results in downregulation of osteogenic genes and upregulation of fibroblast genes. Further analysis using molecular docking and molecular dynamics simulations suggests that fisetin occupied the hydrophobic TEAD pocket preventing YAP from associating with TEA domain (TEAD). This finding supports the potential application of flavonoids like fisetin as a protein-protein interaction disruptor and also suggesting an implication of fisetin in regulating human osteogenesis.

Characterization of a nanoparticulate exopolysaccharide from Leuconostoc holzapfelii KM01 and its potential application in drug encapsulation.

Fermentation of Lactic Acid Bacteria (LAB) is considered to be a sustainable approach for polysaccharide production. Herein, exopolysaccharide (EPS)-producing LAB strain KM01 was isolated from Thai fermented dessert, Khao Mak, which was then identified as Leuconostoc holzapfelii. High-performance anion-exchange chromatography, nuclear magnetic resonance spectroscopy and Fourier-transform infrared spectroscopy suggested that the KM01 EPS comprises α-1,6-linked glucosides. The molecular weight of KM01 EPS was around 500 kDa, but it can form large aggregates formation (MW > 2000 kDa) in an aqueous solution, judged by transmission electron microscopy and dynamic light scattering to be around 150 nm in size. Furthermore, this KM01 EPS form highly viscous hydrogels at concentrations above 5% (w/v). The formation of hydrogels and nanoparticle of KM01 EPS was found to be reversible. Finally, the suitability of KM01 EPS for biomedical applications was demonstrated by its lack of cytotoxicity and its ability to form complexes with quercetin. Unlike the common α-1,6-linked dextran, KM01 EPS can enhance the solubility of quercetin significantly.

Computational design of oligosaccharide producing levansucrase from Bacillus licheniformis RN-01 to improve its thermostability for production of levan-type fructooligosaccharides from sucrose.

Levansucrase catalyzes production of levan and levan-type fructooligosaccharides (LFOs) with potential applications in food and pharmaceutical industries such as prebiotics and anti-tumor agents. Previous study found that Y246S mutant of Bacillus licheniformis RN-01 levansucrase (oligosaccharide producing levansucrase, OPL) could effectively produce LFOs but its thermostability is limited at high temperature. In this study, molecular dynamics (MD) and computational protein design were used to create mutants with higher thermostability than OPL by rigidifying highly flexible residues on enzyme surface. MD results show that highly flexible residues suitable for design are K82, N83, D179, and Q308. Two approaches were employed to improve their interactions by allowing them to be amino acids that could potentially form favorable interactions with their neighboring residues or natural amino acids except G, P and C. Flexibilities of designed residues of K82H, N83R, Q308S and K82H/N83R mutants are lower than those of OPL. Experimental results show that characteristics and product patterns of designed mutants are relatively similar to those of OPL. K82H/N83R mutant has higher thermostability than OPL with 1.7-fold increase in t1/2. Circular dichroism result suggests that designed mutations do not drastically affect secondary structures. This study shows how computational technique can engineer enzyme for thermostability improvement.

Characterisation of insoluble α-1,3-/α-1,6 mixed linkage glucan produced in addition to soluble α-1,6-linked dextran by glucansucrase (DEX-N) from Leuconostoc citreum ABK-1.

Glucansucrases catalyse the formation of glucans from sucrose. The glucansucrase-encoding gene from Leuconostoc citreum ABK-1, dex-N, was successfully cloned and expressed in E. coli BL21 Star (DE3). DEX-N produces 2 types of glucans: soluble (S-dextran) and insoluble (I-glucan) glucans. The S-dextran was determined to be ca. 10 kDa in size and contained >90% α-1,6 linkages; along with its water solubility, this is similar to commercial dextran. On the other hand, I-glucan was water-insoluble, harbouring a block-wise pattern of α-1,3 and α-1,6 linkages in its structure. Notably, the FTIR and powder X-ray diffraction pattern of I-glucan exhibited a combination of features found in α-1,6-linked dextran and α-1,3-linked mutan. Although both I-glucan and mutan are insoluble glucans, their physical characteristics are notably dissimilar.