Locust Bean Gum Galactomannan Research Articles

Development of Polymeric Films Based on Sunflower Seed Proteins and Locust Bean Gum.

Most polymeric food packaging materials are non-biodegradable and derived from petroleum, thus recent studies have focused on evaluating alternative biodegradable materials from renewable sources, with polysaccharides and proteins as the main types of employed biopolymers. Therefore, this study aimed to develop biopolymeric films based on sunflower proteins and galactomannans from locust bean gum. The influence of the galactomannan amount (0.10%, 0.30%, 0.50%, and 0.75% w/v) on the physicochemical, thermal, and mechanical properties of cast sunflower protein-based films was studied. Sunflower proteins gave rise to yellowish, shining, and translucid films. With the incorporation of locust bean gum-derived galactomannans, the films became more brown and opaque, although they still maintained some translucency. Galactomannans significantly changed the proteins' secondary structures, giving rise to films with increased tensile resistance and stretchability. Nevertheless, the increase in the galactomannan amount did not have a significant effect on the film's thermal stability. The protein/galactomannan-based films showed values of water vapor and oxygen permeability that were slightly higher than those of the pristine materials. Overall, blending locust bean gum galactomannans with sunflower proteins was revealed to be a promising strategy to develop naturally colored and translucid films with enhanced mechanical resistance while maintaining flexibility, fitting the desired properties for biodegradable food packaging materials.

The study of galactomannans with different molecular weights and their ability to form microparticles suitable for pulmonary delivery

The influence of locust bean gum (LBG) galactomannans (GMs) molecular weight (Mw) to assemble microparticulate systems was evaluated, and carriers for deep lung delivery were developed. A commercial batch of LBG with a mannose/galactose (M/G) ratio of 2.4 (batch 1) was used to study the influence of different microwave partial acid hydrolysis conditions on carbohydrate composition, glycosidic linkages, and aqueous solutions viscosity. The microwave treatment did not affect the composition, presenting 4-Man (36–42 %), 4,6-Man (27–35 %), and T-Gal (24–25 %) as the main glycosidic linkages. Depolymerization led to a viscosity reduction (≤0.005 Pa·s) with no major impact on polysaccharide debranching. The structural composition of the LBG galactomannans were further elucidated with sequence-specific proteins using carbohydrate microarray technologies. A second batch of LBG (M/G 3.3) was used to study the impact of GMs with different Mw on microparticle assembling, characteristics, and insulin release kinetics. The low-Mw GMs microparticles led to a faster release (20 min) than the higher-Mw (40 min) ones, impacting the release kinetics. All microparticles exhibited a safety profile to cells of the respiratory tract. However, only the higher-Mw GMs allowed the assembly of microparticles with sizes suitable for this type of administration.

Heterologous Expression, Purification and Characterization of an Alkalic Thermophilic β-Mannanase CcMan5C from Coprinopsis cinerea.

A endo-1,4-β-mannanase (CcMan5C) gene was cloned from Coprinopsis cinerea and heterologously expressed in Pichia pastoris, and the recombinant enzyme was purified by Ni-affinity chromatography and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). CcMan5C hydrolyzed only locust bean gum galactomannan (LBG) but not α-mannan from S. cerevisiae or Avicel cellulose, oat spelt xylan, or laminarin from Laminaria digitata. CcMan5C exhibited distinctive catalytic features that were different from previously reported β-mannanases. (1) CcMan5C is the first reported fungal β-mannase with an optimal alkalic pH of 8.0-9.0 for hydrolytic activity under assay conditions. (2) CcMan5C is the first reported alkalic fungal β-mannase with an optimal temperature of 70 °C for hydrolytic activity under assay conditions. (3) The organic solvents methanol, ethanol, isopropanol, and acetone at concentrations of 10% or 20% did not inhibit CcMan5C activity, while 10% or 20% isopropanol and acetone even enhanced CcMan5C activity by 9.20-34.98%. Furthermore, CcMan5C tolerated detergents such as Tween 20 and Triton X-100, and its activity was even enhanced to 26.2-45.6% by 1% or 10% Tween 20 and Triton X-100. (4) CcMan5C solution or lyophilized CcMan5C exhibited unchanged activity and even increasing activity after being stored at -20 °C or -80 °C for 12 months and retained above 50% activity after being stored at 4 °C for 12 months. These features make CcMan5C a suitable candidate for the detergent industry and paper and pulp industry.

A Comparison of the Transglycosylation Capacity between the Guar GH27 Aga27A and Bacteroides GH36 BoGal36A α-Galactosidases

The transglycosylation behavior and capacity of two clan GH-D α-galactosidases, BoGal36A from the gut bacterium Bacteroides ovatus and Aga27A from the guar plant, was investigated and compared. The enzymes were screened for the ability to use para-nitrophenyl-α-galactoside (pNP-Gal), raffinose and locust bean gum (LBG) galactomannan as glycosyl donors with the glycosyl acceptors methanol, propanol, allyl alcohol, propargyl alcohol and glycerol using mass spectrometry. Aga27A was, in general, more stable in the presence of the acceptors. HPLC analysis was developed and used as a second screening method for reactions using raffinose or LBG as a donor substrate with methanol, propanol and glycerol as acceptors. Time-resolved reactions were set up with raffinose and methanol as the donor and acceptor, respectively, in order to develop an insight into the basic transglycosylation properties, including the ratio between the rate of transglycosylation (methyl galactoside synthesis) and rate of hydrolysis. BoGal36A had a somewhat higher ratio (0.99 compared to 0.71 for Aga27A) at early time points but was indicated to be more prone to secondary (product) hydrolysis in prolonged incubations. The methyl galactoside yield was higher when using raffinose (48% for BoGal36A and 38% for Aga27A) compared to LBG (27% for BoGal36A and 30% for Aga27A).

Antioxidant dressing therapy versus standard wound care in chronic wounds (the REOX study): study protocol for a randomized controlled trial

ABSTRACTBackgroundA wound that does not heal in the orderly stages of the healing process or does not heal within 3 months is considered a chronic wound. Wound healing is impaired when the wound remains in the inflammatory stage for too long. A range of factors can delay the healing process: imbalance between proteases and protease inhibitors in the wound bed; bacterial colonization and the presence of biofilm; and oxidative stress. Recently, wound management has improved significantly. A new antioxidant dressing has been developed, which combines an absorbent matrix obtained from locust bean gum galactomannan and a hydration solution with curcumin and N-acetylcysteine. This dressing combines the advantages of moist healing in exudate management and free radical neutralization, achieving wound reactivation. The primary aim of this study is to compare the effect of the antioxidant dressing on chronic wound healing against the use of a standard wound dressing in patients with hard-to-heal wounds.MethodsWe will conduct a multicentre, single-blind, randomized controlled trial with parallel groups. Participants will be selected from three primary public health care centres located in Andalucía (southern Spain). Patients will be randomized into an intervention group (antioxidant dressing) or a control group (standard wound dressing). Assessments will be carried out at weeks 2, 4, 6 and 8. Follow-up will be for a period of 8 weeks or until complete healing if this occurs earlier.DiscussionThe findings from this study should provide scientific evidence on the efficacy of the antioxidant dressing as an alternative for the treatment of chronic wounds. This study fills some of the gaps in the existing knowledge about patients with hard-to-heal wounds.Trial registrationClinicalTrials.gov: NCT03934671. Registered on 2 May 2019.

Secretory expression of β-mannanase in Saccharomyces cerevisiae and its high efficiency for hydrolysis of mannans to mannooligosaccharides.

Degradation of mannans is a key process in the production of foods and prebiotics. β-Mannanase is the key enzyme that hydrolyzes 1,4-β-D-mannosidic linkages in mannans. Heterogeneous expression of β-mannanase in Pichia pastoris systems is widely used; however, Saccharomyces cerevisiae expression systems are more reliable and safer. We optimized β-mannanase gene from Aspergillus sulphureus and expressed it in five S. cerevisiae strains. Haploid and diploid strains, and strains with constitutive promoter TEF1 or inducible promoter GAL1, were tested for enzyme expression in synthetic auxotrophic or complex medium. Highest efficiency expression was observed for haploid strain BY4741 integrated with β-mannanase gene under constitutive promoter TEF1, cultured in complex medium. In fed-batch culture in a fermentor, enzyme activity reached ~ 24U/mL after 36h, and production efficiency reached 16U/mL/day. Optimal enzyme pH was 2.0-7.0, and optimal temperature was 60°C. In studies of β-mannanase kinetic parameters for two substrates, locust bean gum galactomannan (LBG) gave Km = 24.13mg/mL and Vmax = 715U/mg, while konjac glucomannan (KGM) gave Km = 33mg/mL and Vmax = 625U/mg. One-hour hydrolysis efficiency values were 57% for 1% LBG, 74% for 1% KGM, 39% for 10% LBG, and 53% for 10% KGM. HPLC analysis revealed that the major hydrolysis products were the oligosaccharides mannose, mannobiose, mannotriose, mannotetraose, mannopentaose, and mannohexaose. Our findings show that this β-mannanase has high efficiency for hydrolysis of mannans to mannooligosaccharides, a type of prebiotic, suggesting strong potential application in food industries.

Synthetic Glycopeptides Allow for the Quantitation of Scarce Nonfucosylated IgG Fc N-Glycans of Therapeutic Antibody.

Glycans attached to the IgG Fc domain affect strongly biological activities such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of therapeutic antibodies. However, molecular mechanism in the glycoform-dependent functional modulation of the IgGs remains elusive. The present study communicates that selected reaction monitoring (SRM)-based assay of tryptic IgG Fc glycopeptides is a promising approach for the characterization of antibodies when combined with structure-defined synthetic Fc peptides having a focused N-glycoform as a calibration standard. We describe a novel synthetic approach to the human IgG1 Fc peptide having a bisected decasaccharide and its nonbisected counterpart compound, the signatures of antibodies involving Fc domain with rare N-glycans expected to show much higher ADCC/CDC than abundant IgG N-glycans, and their application to the SRM-based quantitative glycoproteomics. Use of a key intermediate, phenyl (2-O-benzyl-4,6-O-benzylidine-β-d-mannopyranosyl)-(1 → 4)-3,6-di-O-benzyl-2-azido-2-deoxy-1-thio-β-d-glucopyranoside, derived from locust bean gum galactomannan, facilitated greatly the synthesis of a bisected nonasaccharide as a stable precursor of oxazoline derivative needed for the enzymatic trans-glycosylation with Fc nonapeptide carrying a GlcNAc at Asn297 residue, while the coupling reaction catalyzed by mutant endo-M-N175Q proceeded very slowly. Strikingly, SRM assay using the synthetic Fc glycopeptides as calibration standards uncovered the occurrence of the targeted IgG1 Fc fragment carrying a nonfucosylated and bisected (315 fmol, 0.20%) and its nonbisected counterpart (1154 fmol, 0.73%) in the tryptic digests from 158 pmol of anticancer antibody Herceptin (trastuzumab). The results suggest that aberrantly glycosylated IgG Fc variants may contribute to the total biological activities of the therapeutic antibodies.

Characterization of an inhibitor-resistant endo-1,4-β-mannanase from the gut microflora metagenome of Hermetia illucens.

Hermetia illucens is a voracious insect scavenger that efficiently decomposes food waste. To exploit novel hydrolytic enzymes from this insect, we constructed a fosmid metagenome library using unculturable H. illucens intestinal microorganisms. Functional screening of the library on carboxymethyl cellulose plates identified a fosmid clone with a product displaying hydrolytic activity. Fosmid sequence analysis revealed a novel mannan-degrading gene (ManEM17) composed of 1371 base pairs, encoding 456 amino acids with a deduced 54 amino acid N-terminal signal peptide sequence. Conceptual translation and domain analysis revealed that sequence homology was highest (46%) with endo-1,4-β-mannosidase of Anaerophaga thermohalophila. Phylogenetic and domain analysis indicated that ManEM17 belongs to a novel β-mannanase containing a glycoside hydrolase family 26 domain. The recombinant protein (rManEM17) was expressed in Escherichia coli, exhibiting the highest activity at 55°C and pH 6.5. The protein hydrolyzed substrates with β-1,4-glycosidic mannoses; maximum specific activity (5467Umg-1) occurred toward locust bean gum galactomannan. However, rManEM17 did not hydrolyze p-Nitrophenyl-β-pyranosides, demonstrating endo-form mannanase activity. Furthermore, rManEM17 was highly stable under stringent conditions, including polar organic solvents as well as chemical reducing and denaturing reagents. ManEM17 is an attractive candidate for mannan degradation under the high-organic-solvent and protein-denaturing processes in food and feed industries.

Locust bean gum galactomannan hydrolyzed by thermostable β-d-mannanase may reduce the secretion of pro-inflammatory factors and the release of granule constituents

Locust bean gum (LBG) galactomannan has been claimed to have applications in the biopharmaceutical field. However, the effects of LBG galactomannan on immunomodulatory aspects are not yet clear. The purpose of this study was to over-express thermostable β-d-mannanase from the thermophilic actinomycete Thermobifida fusca BCRC 19214 using a Pichia pastoris expression system. The maximum intracellular β-d-mannanase activity obtained from the cell-free extract was approximately 40.0U/mL after 72h of cultivating a P. pastoris transformant (pPICZ-man) induced with methanol. Hydrolysis of native LBG galactomannan with 8U/mL β-d-mannanase for 24h significantly decreased the weight-average molecular weight of LBG galactomannan from 5,580,010 to 3188. Native and hydrolyzed LBG galactomannan in a range of 0–0.2% did not trigger significant cytotoxicity after 24h of treatment compared with the control. The native LBG galactomannan stimulated RAW 264.7 cells to produce cytokine TNF-α dose-dependently, but there was no significant IL-1β or nitric oxide production. The native LBG galactomannan also stimulated β-hexosaminidase secretion in RBL-2H3 cells. After the native LBG galactomannan was hydrolyzed with β-d-mannanase, all of the immunological properties disappeared. These results suggest the possible immunomodulatory effects of native LBG galactomannan.

A multi-tolerant low molecular weight mannanase from Bacillus sp. CSB39 and its compatibility as an industrial biocatalyst.

Bacillus sp. CSB39, isolated from popular traditional Korean food (Kimchi), produced a low molecular weight, thermostable mannanase (MnCSB39); 571.14U/mL using locust bean gum galactomannan as a major substrate. It was purified to homogeneity using a simple and effective two-step purification strategy, Sepharose CL-6B and DEAE Sepharose Fast Flow, which resulted in 25.47% yield and 19.32-fold purity. The surfactant-, NaCl-, urea-, and protease-tolerant monomeric protein had a mass of ∼30kDa as analyzed by SDS-PAGE and galactomannan zymography. MnCSB39 was found to have optimal activity at pH 7.5 and temperature of 70°C. The enzyme showed ˃55% activity at 5.0-15% (w/v) NaCl, and ˃93% of the initial activity after incubation at 37°C for 60min. Trypsin and proteinase K had no effect on MnCBS39. The enzyme showed ˃80% activity in up to 3M urea. The N-terminal amino acid sequence, ALKGDGX, did not show identity with reported mannanases, which suggests the novelty of our enzyme. Activation energy for galactomannan hydrolysis was 26.85kJmol(-1) with a Kcat of 142.58×10(4)s(-1). MnCSB39 had Km and Vmax values of 0.082mg/mL and 1099±1.0Umg(-1), respectively. Thermodynamic parameters such as ΔH, ΔG, ΔS, Q10, ΔGE-S, and ΔGE-T supported the spontaneous formation of products and the high hydrolytic efficiency and feasibility of the enzymatic reaction, which strengthen its novelty. MnCSB39 activity was affected by metal ions, modulators, chelators, and detergents. Mannobiose was the principal end-product of hydrolysis. Bacillus subtilis CSB39 produced a maximum of 1524.44U mannanase from solid state fermentation of 1g wheat bran. MnCSB39 was simple to purify, was active at a wide pH and temperature range, multi-stress tolerant and catalyzes a thermodynamically possible reaction, characteristics that suggests its suitability for application as an industrial biocatalyst.

An extremely alkaline mannanase from Streptomyces sp. CS428 hydrolyzes galactomannan producing series of mannooligosaccharides.

An alkaline-thermostable mannanase from Streptomyces sp. CS428 was produced, purified, and biochemically characterized. The extracellular mannanase (Mn428) was purified to homogeneity with 12.4 fold, specific activity of 2406.7 U/mg, and final recovery of 37.6 %. The purified β-mannanase was found to be a monomeric protein with a molecular mass of approximately 35 kDa as analyzed by SDS-PAGE and zymography. The first N-terminal amino acid sequences of mannanase enzyme were HIRNGNHQLPTG. The optimal temperature and pH for enzyme were 60 °C and 12.5, respectively. The mannanase activities were significantly affected by the presence of metal ions, modulators, and detergents. Km and Vmax values of Mn428 were 1.01 ± 3.4 mg/mL and 5029 ± 85 µmol/min mg, respectively when different concentrations (0.6-10 mg/mL) of locust bean gum galactomannan were used as substrate. The substrate specificity of enzyme showed its highest specificity towards galactomannan which was further hydrolyzed to produce mannose, mannobiose, mannotriose, and a series of mannooligosaccharides. Mannooligosaccharides can be further converted to ethanol production, thus the purified β-mannanase isolated from Streptomyces sp. CS428 was found to be attractive for biotechnological applications.

Understanding β-mannanase from Streptomyces sp. CS147 and its potential application in lignocellulose based biorefining.

Hydrolytic enzymes such as cellulase and hemicellulase have been attracted in lignocellulose based biorefinery. Especially, mannanase has been a growing interest in industrial applications due to its importance in the bioconversion. In this study, an extracellular endo-β-1,4-D-mannanase was produced by Streptomyces sp. CS147 (Mn147) and purified 8.5-fold with a 43.4% yield using Sephadex G-50 column. The characterization of Mn147 was performed, and the results were as follows: molecular weight of ∼25 kDa with an optimum temperature of 50°C and pH of 11.0. The effect of metal ions and various reagents on Mn147 was strongly activated by Ca(+2) but inhibited by Mg(+2) , Fe(+2) , hydrogen peroxide, EDTA and EGTA. Km and Vmax values of Mn147 were 0.13 mg/mL and 294 μmol/min mg, respectively, when different concentrations (3.1 to 50 mg/mL) of locust bean gum galactomannan were used as substrate. In enzymatic hydrolysis of heterogeneous substrate (spent coffee grounds), Mn147 shows a similar conversion compared to commercial enzymes. In addition, lignocellulosic biomass can be hydrolyzed to oligosaccharides (reducing sugars), which can be further utilized for the production of biomaterials. These results showed that Mn147 is attractive in quest of potential bioindustrial applications.

A New Spectrophotometric Method for Determining the Enzymatic Activity of endo-β-mannanase in Seeds

The objective of this study was to develop a new, highly sensitive, efficient and easy touse spectrophotometric method to determine the enzymatic activity of endo-β-mannanase in seeds. The method was optimized and applied to determine the site of enzyme activity in different parts of seeds of Butia capitata. It consists in using the enzyme extracted from seeds to hydrolyze Locust bean gum galactomannan. The reducing sugars react with p-hydroxybenzoic acid hydrazide and are quantified by spectrophotometry in the visible region. The optimized and validated method showed low limits of quantification for the reducing sugars (0.6 µg mannose) and endo-β-mannanase activity (1 mU). Only 5 mg of germinating seeds are needed to perform the method.

Structural features of β-(1→4)-d-galactomannans of plant origin as a probe for β-(1→4)-mannanase polymeric substrate specificity

Statistical modeling was applied for describing structural features of β-(1→4)-d-galactomannans. According to the model suggested theoretical ratios of limiting degrees of locust bean, tara gum and guar gum galactomannan conversions by two β-(1→4)-mannanases of different origin (Myceliophthora thermophila and Trichoderma reesei) were calculated. Then the enzymes were tested for enzymatic hydrolysis of three considered galactomannans. Experimentally observed results were compared with theoretically calculated ones. It was shown that T. reesei β-mannanase attacks sequences of four and more unsubstituted mannopyranosyl residues in a row, while M. thermophila β-mannanase is a more specific enzyme and attacks sequences of five and more mannopyranosyl residues in a row. Considered statistical model and approach allows to characterize both galactomannan structures and enzyme requirements for regions of unsubstituted mannose residues for substrate hydrolysis.

Recombinant production and characterisation of two related GH5 endo-β-1,4-mannanases from Aspergillus nidulans FGSC A4 showing distinctly different transglycosylation capacity

The glycoside hydrolase family 5 (GH5) endo-β-1,4-mannanases ManA and ManC from Aspergillus nidulans FGSC A4 were produced in Pichia pastoris X33 and purified in high yields of 120 and 145mg/L, respectively, from the culture supernatants. Both enzymes showed increasing catalytic efficiency (kcat/KM) towards β-1,4 manno-oligosaccharides with the degree of polymerisation (DP) from 4 to 6 and also hydrolysed konjac glucomannan, guar gum and locust bean gum galactomannans. ManC had up to two-fold higher catalytic efficiency for DP 5 and 6 manno-oligosaccharides and also higher activity than ManA towards mannans. Remarkably, ManC compared to ManA transglycosylated mannotetraose with formation of longer β-1,4 manno-oligosaccharides 8-fold more efficiently and was able to use mannotriose, melezitose and isomaltotriose out of 36 tested acceptors resulting in novel penta- and hexasaccharides, whereas ManA used only mannotriose as acceptor. ManA and ManC share 39% sequence identity and homology modelling suggesting that they have very similar substrate interactions at subsites +1 and +2 except that ManC Trp283 at subsite +1 corresponded to Ser289 in ManA. Site-directed mutagenesis to ManA S289W lowered KM for manno-oligosaccharides by 30–45% and increased transglycosylation yield by 50% compared to wild-type. Conversely, KM for ManC W283S was increased, the transglycosylation yield was reduced by 30–45% and furthermore activity towards mannans decreased below that of ManA. This first mutational analysis in subsite +1 of GH5 endo-β-1,4-mannanases indicated that Trp283 in ManC participates in discriminating between mannan substrates with different extent of branching and has a role in transglycosylation and substrate affinity.

Demonstration of the presence of acetylation and arabinose branching as structural features of locust bean gum galactomannans

Galactomannans from locust bean gum (LBG) are polysaccharides containing single Galp residues as side chains in a β-(1→4)-Manp backbone. Nevertheless, sugars other than mannose and galactose are always detected, namely, arabinose and glucose, although in small amounts. In order to demonstrate that LBG galactomannans contain other structural features than those usually reported in literature, the galactomannans from LBG obtained from two different commercial sources were submitted to a selective degradation by Aspergillus niger endo-β-d-mannanase and the oligosaccharides (OS) obtained were fractioned by size exclusion chromatography using a Biogel-P2 (100–1800Da). A. niger endo-β-d-mannanase is able to hydrolyse the β-(1→4)-linked mannan backbone when the mannose residues are not hindered by the presence of substituents. This enables collection of fragments of the substituted mannan polysaccharide in the different chromatographic fractions that can be analysed by Electrospray Tandem Mass Spectrometry (ESI-MSn). Both samples showed the presence of hexose OS (Hex3–6), acetylated hexoses (AcHex3–6), hexoses substituted with a pentose unit (PentHex3–6), and OS composed by acetylated hexoses substituted with a pentose unit (PentAcHex3–6). These structures were confirmed by GC–qMS of reduced and permethylated oligosaccharides. Sugar and glycosidic-linkage analysis allowed to assign the pentose unit to a terminally-linked arabinofuranose residue at the O-6 of a (1→4)-Manp residue. The presence of arabinose substituted OS and acetylated mannoses have never been reported to occur in LBG.

재조합 대장균으로부터 생산된 Bacillus licheniformis WL-12의 Mannanase 특성

가정에서 제조된 된장으로부터 분리된 Bacillus licheniformis WL-12의 mannanase 유전자를 크로닝하여 그 염기서열을 결정한 결과 mannanase 유전자는 360 아미노산으로 구성된 단백질을 코드하며 1,080 뉴클레오티드로 이루어졌다. 아미노산 잔기배열을 분석한 결과 WL-12의 mannanase는 GH family 26에 속하는 B. licheniformis DSM13의 mannanase와 동일하였다. B. lichenifromis WL-12의 mannanase 유전자를 함유한 재조합대장균의 균체파쇄상등액으로부터 부분정제된 효소를 사용하여 반응특성을 조사하였다. pH 6.0과 <TEX>$65^{\circ}C$</TEX>에서 최대 반응활성을 보였으며, locust bean gum (LBG)과 konjac의 분해능은 높으나 guar gum의 분해능은 낮았다. Mannanase로 LBG와 mannooligosaccharides를 분해하였을 때 mannose, mannobiose와 mannotriose가 주된 최종 반응산물로 관찰되었으며 mannobiose는 분해하지 못하였으나 이보다 중합도가 큰 mannooligosaccharides은 분해하였다. A gene encoding the mannanase of Bacillus licheniformis WL-12, which had been isolated from Korean soybean paste, was cloned into Escherichia coli and nucleotide sequence of the mannanase gene was subsequently determined. The mannanase gene consisted of 1,080 nucleotides encoding a polypeptide of 360 amino acid residues. The deduced amino acid sequence was identical to that of putative mannanase from B. liceniformis DSM13 belonging to GH family 26. The mannanase was partially purified from cell-free extract of the recombinant Escherichia coli carrying a WL-12 mannanase gene by ammonium sulfate fractionation and DEAE-Sepharose column chromatography. Optimal conditions for the partially purified enzyme occurred at pH 6.0 and <TEX>$65^{\circ}C$</TEX>. The enzyme showed higher activity on locust bean gum (LBG) galactomannan and konjac glucomannan than on guar gum galactomannan. The predominant products resulting from the mannanase hydrolysis were mannose, mannobiose and mannotriose for LBG or mannooligosaccharides. The enzyme could hydrolyze mannooligosaccharides larger than mannobiose.

Cloning, expression in Pichia pastoris, and characterization of a thermostable GH5 mannan endo-1,4-beta-mannosidase from Aspergillus niger BK01.

BackgroundMannans are key components of lignocellulose present in the hemicellulosic fraction of plant primary cell walls. Mannan endo-1,4-β-mannosidases (1,4-β-D-mannanases) catalyze the random hydrolysis of β-1,4-mannosidic linkages in the main chain of β-mannans. Biodegradation of β-mannans by the action of thermostable mannan endo-1,4-β-mannosidase offers significant technical advantages in biotechnological industrial applications, i.e. delignification of kraft pulps or the pretreatment of lignocellulosic biomass rich in mannan for the production of second generation biofuels, as well as for applications in oil and gas well stimulation, extraction of vegetable oils and coffee beans, and the production of value-added products such as prebiotic manno-oligosaccharides (MOS).ResultsA gene encoding mannan endo-1,4-β-mannosidase or 1,4-β-D-mannan mannanohydrolase (E.C. 3.2.1.78), commonly termed β-mannanase, from Aspergillus niger BK01, which belongs to glycosyl hydrolase family 5 (GH5), was cloned and successfully expressed heterologously (up to 243 μg of active recombinant protein per mL) in Pichia pastoris. The enzyme was secreted by P. pastoris and could be collected from the culture supernatant. The purified enzyme appeared glycosylated as a single band on SDS-PAGE with a molecular mass of approximately 53 kDa. The recombinant β-mannanase is highly thermostable with a half-life time of approximately 56 h at 70°C and pH 4.0. The optimal temperature (10-min assay) and pH value for activity are 80°C and pH 4.5, respectively. The enzyme is not only active towards structurally different mannans but also exhibits low activity towards birchwood xylan. Apparent Km values of the enzyme for konjac glucomannan (low viscosity), locust bean gum galactomannan, carob galactomannan (low viscosity), and 1,4-β-D-mannan (from carob) are 0.6 mg mL-1, 2.0 mg mL-1, 2.2 mg mL-1 and 1.5 mg mL-1, respectively, while the kcat values for these substrates are 215 s-1, 330 s-1, 292 s-1 and 148 s-1, respectively. Judged from the specificity constants kcat/Km, glucomannan is the preferred substrate of the A. niger β -mannanase. Analysis by thin layer chromatography showed that the main product from enzymatic hydrolysis of locust bean gum is mannobiose, with only low amounts of mannotriose and higher manno-oligosaccharides formed.ConclusionThis study is the first report on the cloning and expression of a thermostable mannan endo-1,4-β-mannosidase from A. niger in Pichia pastoris. The efficient expression and ease of purification will significantly decrease the production costs of this enzyme. Taking advantage of its acidic pH optimum and high thermostability, this recombinant β-mannanase will be valuable in various biotechnological applications.

Spruce galactoglucomannan films show promising barrier properties

Films were prepared from sorbitol-plasticized spruce galactoglucomannans (GGM), which are a potential new product from forest biorefineries. GGM were also mixed with konjac glucomannan (KGM), poly(vinyl alcohol) (PVOH), and cellulose nanowhiskers (CNW). Permeability properties of GGM-based films were compared to those of films from commercial mannans, namely KGM, guar gum galactomannan (GG), and locust bean gum galactomannan (LBG). GGM-based films had lower water vapor permeability than the films formed from any of the other mannans. The oxygen permeability of GGM films was of the same magnitude as that of commercial polyethylene/ethylene vinyl alcohol/polyethylene laminate film. The aroma permeability of GGM films was low. All films were transparent in the visible region, but GGM films blocked the light transmission in the ultraviolet region of the spectra.

Immunostimulatory properties of coffee mannans

Coffee infusion mannans are acetylated polysaccharides containing single Galp and Araf residues as side chains of a beta-(1 --> 4)-Manp backbone. These mannans are structurally similar to the bioactive acetylated mannans from Aloe vera (AV). In this study, acetylated mannans were obtained from two coffee infusions prepared from light and dark roasted beans. These samples were tested for their immunostimulatory activity and compared with an extract of AV mannan and with locust bean gum (LBG) galactomannans. The coffee samples, as well as the AV extract, stimulated murine B- and T-lymphocytes, as evaluated by the in vitro expression of the surface lymphocyte activation marker CD69, more marked on B- than on T-lymphocytes. In coffee samples, contrarily to the AV, no proliferative effect was noticed. LBG sample did not show any immunostimulatory activity. Because the material that remains in the residue of the hot water extraction was still very rich in mannans, a sequential extraction was performed and a main fraction was recovered with a 4 M NaOH solution. Because this material was insoluble in water, a partial acetylation was performed. These polysaccharides also showed immunostimulatory activity, opening the possibility of exploitation of coffee infusion and coffee residue as sources of bioactive polysaccharides.