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.

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.

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.

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.

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.

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.

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.

Production of Large-Ring Cyclodextrins by Amylomaltases.

Amylomaltase is a well-known glucan transferase that can produce large ring cyclodextrins (LR-CDs) or so-called cycloamyloses via cyclization reaction. Amylomaltases have been found in several microorganisms and their optimum temperatures are generally around 60-70 °C for thermostable amylomaltases and 30-45 °C for the enzymes from mesophilic bacteria and plants. The optimum pHs for mesophilic amylomaltases are around pH 6.0-7.0, while the thermostable amylomaltases are generally active at more acidic conditions. Size of LR-CDs depends on the source of amylomaltases and the reaction conditions including pH, temperature, incubation time, and substrate. For example, in the case of amylomaltase from Corynebacterium glutamicum, LR-CD productions at alkaline pH or at a long incubation time favored products with a low degree of polymerization. In this review, we explore the synthesis of LR-CDs by amylomaltases, structural information of amylomaltases, as well as current applications of LR-CDs and amylomaltases.

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.

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.

Enhancement of large ring cyclodextrin production using pretreated starch by glycogen debranching enzyme from Corynebacterium glutamicum.

Synthesis of large-ring cyclodextrins (LR-CDs) in any significant amount has been challenging. This study enhanced the LR-CDs production by Thermus filiformis amylomaltase (TfAM) enzyme by starch pretreatment using glycogen debranching enzyme from Corynebacterium glutamicum (CgGDE). CgGDE pretreated tapioca starch gave LR-CD conversion of 31.2 ± 2.2%, compared with LR-CDs produced from non-treated tapioca starch (16.0 ± 2.4%). CgGDE pretreatment enhanced amylose content by approximately 30%. Notably, a shorter incubation time of 1 h is sufficient for CgGDE starch pretreatment to produce high LR-CD yield, compared with 6 h required for the commercial isoamylase. High-Performance Anion Exchange Chromatography coupled with Pulsed Amperometric Detection (HPAEC-PAD) and Gel Permeable Chromatography (GPC) revealed that CgGDE is more efficient than the commercial isoamylase in debranching tapioca starch and gave lower molecular weight products. In addition, lower amount of by-products (linear oligosaccharides) were detected in cyclization reaction when using CgGDE-pretreated starch. In conclusion, CgGDE is a highly effective enzyme to promote LR-CD synthesis from starch with a shorter incubation time than the commercial isoamylase.

A GH13 α-glucosidase from Weissella cibaria uncommonly acts on short-chain maltooligosaccharides.

α-Glucosidase (EC 3.2.1.20) is a carbohydrate-hydrolyzing enzyme which generally cleaves α-1,4-glycosidic bonds of oligosaccharides and starch from the nonreducing ends. In this study, the novel α-glucosidase from Weissella cibaria BBK-1 (WcAG) was biochemically and structurally characterized. WcAG belongs to glycoside hydrolase family 13 (GH13) and to the neopullanase subfamily. It exhibits distinct hydrolytic activity towards the α-1,4 linkages of short-chain oligosaccharides from the reducing end. The enzyme prefers to hydrolyse maltotriose and acarbose, while it cannot hydrolyse cyclic oligosaccharides and polysaccharides. In addition, WcAG can cleave pullulan hydrolysates and strongly exhibits transglycosylation activity in the presence of maltose. Size-exclusion chromatography and X-ray crystal structures revealed that WcAG forms a homodimer in which the N-terminal domain of one monomer is orientated in proximity to the catalytic domain of another, creating the substrate-binding groove. Crystal structures of WcAG in complexes with maltose, maltotriose and acarbose revealed a remarkable enzyme active site with accessible +2, +1 and -1 subsites, along with an Arg-Glu gate (Arg176-Glu296) in front of the active site. The -2 and -3 subsites were blocked by Met119 and Asn120 from the N-terminal domain of a different subunit, resulting in an extremely restricted substrate preference.

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.

Assessing Dynamic Changes of Taste-Related Primary Metabolism During Ripening of Durian Pulp Using Metabolomic and Transcriptomic Analyses.

Durian is an economically important fruit of Southeast Asia. There is, however, a lack of in-depth information on the alteration of its metabolic networks during ripening. Here, we annotated 94 ripening-associated metabolites from the pulp of durian cv. Monthong fruit at unripe and ripe stages, using capillary electrophoresis- and gas chromatography- time-of-flight mass spectrometry, specifically focusing on taste-related metabolites. During ripening, sucrose content increased. Change in raffinose-family oligosaccharides are reported herein for the first time. The malate and succinate contents increased, while those of citrate, an abundant organic acid, were unchanged. Notably, most amino acids increased, including isoleucine, leucine, and valine, whereas aspartate decreased, and glutamate was unchanged. Furthermore, transcriptomic analysis was performed to analyze the dynamic changes in sugar metabolism, glycolysis, TCA cycle, and amino acid pathways to identify key candidate genes. Taken together, our results elucidate the fundamental taste-related metabolism of durian, which can be exploited to develop durian metabolic and genetic markers in the future.

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.

Conserved Calcium-Binding Residues at the Ca-I Site Involved in Fructooligosaccharide Synthesis by Lactobacillus reuteri 121 Inulosucrase.

Inulosucrase is an enzyme that synthesizes inulin-type ß-2,1-linked fructooligosaccharides (IFOS) from sucrose. Previous studies have shown that calcium is important for the activity and stability of Lactobacillus reuteri 121 inulosucrase (LrInu). Here, mutational analyses of four conserved calcium-binding site I (Ca-I) residues of LrInu, Asp418, Gln449, Asn488, and Asp520 were performed. Alanine substitution for these residues not only reduced the stability and activity of LrInu, but also modulated the pattern of the IFOS produced. Circular dichroism spectroscopy and molecular dynamics simulation indicated that these mutations had limited impact on the overall conformation of the enzyme. One of Ca-I residues most critical for controlling LrInu-mediated polymerization of IFOS, Asp418, was also subjected to mutagenesis, generating D418E, D418H, D418L, D418N, D418S, and D418W. The activity of these mutants demonstrated that the IFOS chain length could be controlled by a single mutation at the Ca-I site.

Levansucrase from Bacillus amyloliquefaciens KK9 and Its Y237S Variant Producing the High Bioactive Levan-Type Fructooligosaccharides.

Levan-typed fructooligosaccharide (LFOS), a ß-2,6 linked oligofructose, displays the potential application as a prebiotic and therapeutic dietary supplement. In the present study, LFOS was synthesized using levansucrase from Bacillus amyloliquefaciens KK9 (LsKK9). The wild-type LsKK9 was cloned and expressed in E. coli, and purified by cation exchanger chromatography. Additionally, Y237S variant of LsKK9 was constructed based on sequence alignment and structural analysis to enhance the LFOS production. High-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) analysis indicated that Y237S variant efficiently produced a higher amount of short-chain LFOS than wild type. Also, the concentration of enzyme and sucrose in the reactions was optimized. Finally, prebiotic activity assay demonstrated that LFOS produced by Y237S variant had higher prebiotic activity than that of the wild-type enzyme, making the variant enzyme attractive for food biotechnology.

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.

Galactomannan Pentasaccharide Produced from Copra Meal Enhances Tight Junction Integration of Epithelial Tissue through Activation of AMPK.

Mannan oligosaccharide (MOS) is well-known as an effective fed supplement for livestock to increase their nutrients absorption and health status. Pentasaccharide of mannan (MOS5) was reported as a molecule that possesses the ability to increase tight junction of epithelial tissue, but the structure and mechanism of action remains undetermined. In this study, the mechanism of action and structure of MOS5 were investigated. T84 cells were cultured and treated with MOS5 compared with vehicle and compound C, a 5'-adenosine monophosphate-activated protein kinase (AMPK) inhibitor. The results demonstrated that the ability of MOS5 to increase tight junction integration was inhibited in the presence of dorsomorphine (compound C). Phosphorylation level of AMPK was elevated in MOS5 treated group as determined by Western blot analysis. Determination of MOS5 structure was performed using enzymatic mapping together with 1H, 13C NMR, and 2D-NMR analysis. The results demonstrated that the structure of MOS5 is a ß-(1,4)-mannotetraose with α-(1,6)-galactose attached at the second mannose unit from non-reducing end.