Mannanase Activity Research Articles

Fusion expression of β-mannanase Man5A and xylanase Tlxyn11B in Pichia pastoris

Mannanase and xylanase, the main hemicellulolytic enzymes, are widely used in food, feed, textile and papermaking industries, and usually they are used in combination. Mannanase Man5A from Talaromyces leycettanus JCM12802 consist of the carbohydrate binding module (CBM), linker region and catalytic domain. The CBM coding region of Man5A was removed and fused to C-terminal of the xylanase gene Tlxyn11B. The fusion gene Tlxyn11B-linker-man5A was successfully expressed in Pichia pastoris and the fusion protein Tlxyn11B-Man5A was purified and characterized. The theoretical molecular weights of Tlxyn11B, Man5A without CBM region, and Tlxyn11B-Man5A are 21.6 kDa, 41.0 kDa, and 62.6 kDa, respectively. The fusion protein had high xylanase and mannanase activities. The optimal temperature of the fused xylanase is 70 °C, which is 5 °C higher than Tlxyn11B-w (xylanase before fusion). The fused mannanase exhibited maximal activity at 90 °C, which is similar to Man5A-w (mannanase before fusion). More than 48% of xylanase activity of Tlxyn11B-Man5A was residual after the condition of 60 °C with 1 h, which is significantly higher than Tlxyn11B-w (only 20% of activity was left at 60 °C for 20 minutes). The optimal pHs of Tlxyn11B-Man5A for xylanase and mannanase activity are 4.0 and 5.0, respectively, which are 0.5 and 1.0 units higher than those of Tlxyn11B-w and Man5A-w. The pH range of fused enzymes got wider and the pH stability is improved. The specific activities of xylanase and mannanase of Tlxyn11B-Man5A are 1 784.3 U/mg and 1 639.6 U/mg, respectively, which is lower than those of Tlxyn11B and Man5A (8 300.0 U/mg and 1 979.0 U/mg). It may be due to of the high molecular weight of fusion enzyme. The Km and Vmax of the fused xylanase and mannanase are 1.2 mg/mL and 1.7 mg/mL, 2 000.0 μmol/(min·mg) and 2 831.6 μmol/(min·mg), respectively. Tlxyn11B and Man5A were successfully fusion expressed in P. pastoris, and the good properties of fusion of xylanase and mannanase make it has great application potential in animal feed, food and other industrial production, and it provided new ideas for the improvement of enzyme performance.

A high-efficiency and eco-friendly degumming process for ramie fibers

Degumming is the most critical step in the preparation of ramie fibers for textile application. However, the traditional chemical degumming process, which uses large amounts of alkali at high temperature, has been a severe challenge to the environment. In this study, a bacterial strain Pectobacterium carotovorum HG-49, was isolated and successfully applied to the degumming of ramie fibers at an industrial scale. The gum removal ratio can reach 82.16% within 16 h of degumming, which is more efficient than most reported methods. Analyses of changes in bacterial populations and community structures over the degumming process showed that strain HG-49 is the dominant bacteria at all time points with a maximum concentration of 2.17 × 109 CFU/mL at 12 h. The maximum activities of pectinase, mannanase, and xylanase reached 110.89, 39.89, and 32.17 U/mL at 10–12 h, respectively. The residual gum content of refined ramie fibers decreased to 1.85% after degumming, and their bundle breaking tenacity and whiteness reached 5.03 cN/dtex and 52, respectively. These values exceed the first-class standards of China textile criteria. Scanning Electron Microscopy (SEM) and Attenuated Total Reflectance Fourier Transform Infrared spectroscopy (ATR FT-IR) analysis further confirmed the excellent degumming effects of the proposed method. The whole genome sequencing of strain HG-49 was completed, and 28 degumming enzyme genes were found, which explains its high-efficiency degumming abilities. Moreover, because the method abandons the use of acid and greatly reduces alkali consumption, it is eco-friendly. This study provides a promising cleaner degumming method for the ramie textile industry.

The effects of mannanase activity on viscosity in different gums

Beta (β)-mannanases, hydrolyzed 1,4-β-D mannosidic bonds in mannan, galactomannan, glucomannan, and galactoglucomannans, can be used to reduce the viscosity of foods for gum modification. Locust bean gum (LBG) and guar gum (GG) have a wide range of use from food to textile as a thickening agent. In this study, the effect of β-mannanase on the viscosity of the LBG and GG was investigated. The rheological analysis showed that the flow behavior of LBG and GG changed from pseudoplastic to Newtonian after hydrolysis. The highest viscosities of non-hydrolyzed LBG (%1) and GG (%1) were 1,864.30 and 1,883.80 mPa/s, and the lowest values were 1.60 mPa/s for hydrolyzed LBG (0.5 g/L) and 2.90 mPa/s for GG (0.5 g/L), respectively. A significant decrease in viscosity was observed on the gums with the use of β-mannanase. Results showed that the β-mannanase enzyme can be used for gum modification. Practical applications Beta (β)-mannanase enzymes could hydrolyze gums, which include mannan units, such as galactomannans and glucomannans. Galactomannans as locust bean gum and guar gum have large scale applications in different food, cosmetic, and pharmaceutical products. They could have been used as a thickener, stabilizer, emulsifier, and gelling agent in emulsion and suspension systems. Additionally, they are also used as food ingredients in bakery goods (icings and cake mixes) and dietary products, coffee whiteners, baby milk formulations, and prebiotic sources. Despite the valuable properties of gums, their solutions have higher viscosity. Consequently, water-soluble application in industry and cleaning of gum processing equipment usually harder. Using β-mannanase could have overcome the negative effect of viscosity. Moreover, partially hydrolyzed LBG and GG solutions using β-mannanase could be benefited as a prebiotic source in daily nutrition.

Biocatalysis of Industrial Kraft Pulps: Similarities and Differences between Hardwood and Softwood Pulps in Hydrolysis by Enzyme Complex of Penicillium verruculosum

Kraft pulp enzymatic hydrolysis is a promising method of woody biomass bioconversion. The influence of composition and structure of kraft fibers on their hydrolysis efficiency was evaluated while using four substrates, unbleached hardwood pulp (UHP), unbleached softwood pulp (USP), bleached hardwood pulp (BHP), and bleached softwood pulp (BSP). Hydrolysis was carried out with Penicillium verruculosum enzyme complex at a dosage of 10 filter paper units (FPU)/g pulp. The changes in fiber morphology and structure were visualized while using optical and electron microscopy. Fiber cutting and swelling and quick xylan destruction were the main processes at the beginning of hydrolysis. The negative effect of lignin content was more pronounced for USP. Drying decreased the sugar yield of dissolved hydrolysis products for all kraft pulps. Fiber morphology, different xylan and mannan content, and hemicelluloses localization in kraft fibers deeply affected the hydrolyzability of bleached pulps. The introduction of additional xylobiase, mannanase, and cellobiohydrolase activities to enzyme mixture will further improve the hydrolysis of bleached pulps. A high efficiency of never-dried bleached pulp bioconversion was shown. At 10% substrate concentration, hydrolysates with more than 50 g/L sugar concentration were obtained. The bioconversion of never-dried BHP and BSP could be integrated into working kraft pulp mills.

Engineering Clostridium acetobutylicum to utilize cellulose by heterologous expression of a family 5 cellulase

Innovative processes to transform plant biomass into renewable chemicals are needed to replace fossil fuels and limit climate change. Clostridium acetobutylicum is of industrial interest because it ferments sugars into acetone, butanol and ethanol (ABE). However, this organism is unable to depolymerize cellulose, limiting its use for the direct transformation of lignocellulose. In this study, the freely secreted family 5 cellulase cphy2058 from Clostridium phytofermentans was efficiently expressed in C. acetobutylicum to enhance cellulolysis. RT-qPCR confirmed the transcription of the cellulase gene, and galactomannan and carboxymethyl cellulose plate assays showed endoglucanase and mannanase activity of cphy2058. ABE titers by the recombinant, cellulase-expressing C. acetobutylicum reached 85.83 mM in cellobiose cultures, whereas ABE titers by wild-type were 44.52 mM. ABE titers by the cellulase-expressing strain were similarly elevated relative to wild-type in cellulose and galactomannan cultures. These results demonstrate a novel strategy to enhance complex substrate utilization by heterologous cellulase expression in C. acetobutylicum.

Effects of feeding broiler breeder hens a coextruded full-fat flaxseed and pulses mixture without or with multienzyme supplement

The effects of coextruded full-fat flaxseed and pulses (FFF; 1:1 wt/wt) mixture on n-3 polyunsaturated fatty acids (PUFA) enrichment in egg yolk, hepatic attributes, apparent retention (AR) of components, and ceca metabolites were evaluated in broiler breeder hens. The diets were as follows: 1) corn–soybean control, 2) control diet plus 18% FFF (FFF−), and 3) FFF plus enzyme supplement (FFF+) containing galactanase, protease, mannanase, glucanase, xylanase, amylase, and cellulase activities. Twenty-six-week-old Cobb 500 broiler breeder hens were allocated to 30 identical cages (2 hens/cage) and given 1-week adaptation period. The 3 diets were assigned to 10 replicate cages based on postadaptation BW and fed based on breeder curve for 30 D. Excreta samples were collected from day 24 to 27 for determination of AR of components, and eggs were collected from day 28 to 30 for yolk polyunsaturated fatty acids analyses. On day 30, birds were weighed, killed via cervical dislocation, liver weighed, and stored for fat analyses. Ceca digesta samples were taken for concentration of short-chain fatty acids. Liver and yolk weights as well as total yolk FA were not influenced by diets (P > 0.05). Control birds had lower yolk concentration of α-linolenic acid than birds fed either FFF− or FFF+ (P < 0.01) corresponding to 7.5, 36.8, and 37.3 mg/g for the control, FFF−, and FFF+, respectively. Control birds also exhibited lower yolk concentration of docosahexaenoic acid (P < 0.01). Control birds had higher hepatic concentration of crude fat and apparent retention of dry matter and crude protein compared with either the FFF− or FFF+ birds (P < 0.05). Birds fed FFF- diet had lower ceca digesta concentration of lactic acid than control and FFF+ (P < 0.05) birds. Results showed broiler breeder hens enriched egg yolk with n-3 polyunsaturated fatty acids without effects on the liver while the supplemental enzyme did not improve the utilization of FFF.

Delipidation and Deproteination of Coconut Dregs Fermented with Aspergillus niger to Produce Prebiotic compounds

Coconut dregs are the waste product of oil extraction, either to produce conventional cooking oil or virgin coconut oil (VCO). These coconut dregs contain mannan compound in the form of galactomannan. Fermentation of this compound could generate mannan-oligosaccharides, having prebiotic properties, due to the presence of mannanase enzyme during fermentation. A study on fermentation of coconut dregs was carried out with a delipidation process using n-hexane and deproteination using NaOH 1M. The fermentation process was done using Aspergi l lus niger with different duration of fermentation (48, 72, 96, 120 and 144 hours). The fermentation product was placed in the free-air container to kill the fungi and keep the biodegradation process through enzymatic process for 0, 24, 48 and 72 hours. The products were tested for prebiotic properties through spread plate method using Lactobacillus casei and Escherichia coli . The results of the delipidation process could decreased lipid content to 0.77% and the deproteination process could bring down crude protein of coconut dregs to 1.45%. The highest mannanase activity was found when coconut dregs were fermented for 120 hours with a value of 1.0331U/ml. Hydrolyzate produced during incubation had a prebiotic activity by increasing the population Lactobacillus casei and decreasing population of Escherich i a coli . Lactobacillus casei bacteria increased from 2x10 7 CFU/ml to between 30.6x10 7 CFU/ml and 35.0x10 7 CFU/ ml, whereas Escherich i a coli bacteria decreased from 2x10 7 CFU / ml to between 5.3x10 6 CFU/ml and 8.3x10 6 CFU/ml. Keyword s : Coconut dregs, Aspergil l us niger , Prebiotics DOI: 10.7176/JBAH/10-6-03 Publication date: March 31 st 2020

Characteristics of mannanase enzyme in actinomycetes isolated from Taman Nasional Bukit Duabelas Jambi

Indonesia has about 14.03 million hectars of palm oil plantation. Palm kernel cake (PKC) is a byproduct of palm oil processing majority consist of β-mannan. Mannan is a component of hemicellulose which have an economic value. Mannan can be converted to the manooligosaccharides (MOS) using bacterial enzyme. This study aimed to produce MOS from PKC using actinomycetes from Taman Nasional Bukit Duabelas Jambi. The selection was begun with the production of the clear zone on medium containing PKC then enzyme activity was measured. Isolate HJ4-5B had the highest mannanolytic index up to 0.4. The enzyme activity of isolate HJ4-5B increased significantly up to 0.895 U/mL at 7th day of culture incubation. The highest mannanase activity of isolate HJ4-5B occurs at pH 6 and 70 °C with an incubation time of 7th day which reached 1.186 U/mL.

Rheological and physicochemical properties of polysaccharides extracted from stems of Dendrobium officinale

The physicochemical and rheological properties of polysaccharides extracted from stems of Dendrobium officinale (DOP) were investigated. Two samples were prepared: the first sample (HEPDOP) was prepared by pretreating D. officinale stems by hot water scalding, followed by water extraction and ethanol precipitation; and the second sample (EPDOP) was obtained by water extraction and ethanol precipitation without pretreatment. The contents of total carbohydrate and protein, mannanase activity, infrared spectra, molecular weight distribution, thermal properties, and hydrophobicity of the two samples were analyzed. The results showed that mannanase activity of HEPDOP (0.0247 U) was much lower than that of EPDOP (0.1158 U). HEPDOP had higher molecular weight and carbohydrate content, and lower polydispersity index (PDI) value and protein content compared to EPDOP. The results further showed that the decomposition temperature of HEPDOP was 311 °C, which is higher than that of EPDOP (306 °C); and its hydrophobicity was significantly lower than that of EPDOP. Interestingly, at the same concentration (2% (w/w)), HEPDOP had higher viscosity than EPDOP, and acidity, alkalinity, the addition of salt ions, and temperature was found to reduce their viscosity. Finally, we observed that increasing temperature caused the decrease of viscosity and modulus of both DOPs.

A novel multifunctional GH9 enzyme from Paenibacillus curdlanolyticus B-6 exhibiting endo/exo functions of cellulase, mannanase and xylanase activities.

PcMulGH9, a novel glycoside hydrolase family 9 (GH9) from Paenibacillus curdlanolyticus B-6, was successfully expressed in Escherichia coli. It is composed of a catalytic domain of GH9, two domains of carbohydrate-binding module family 3 (CBM3) and two domains of fibronectin type 3 (Fn3). The PcMulGH9 enzyme showed broad activity towards the β-1,4 glycosidic linkages of cellulose, mannan and xylan, including cellulose and xylan contained in lignocellulosic biomass, which is rarely found in GH9. The enzyme hydrolysed substrates with bifunctional endo-/exotypes cellulase, mannanase and xylanase activities, but predominantly exhibited exo-activities. This enzyme released cellobiose as a major product from cellohexaose, while mannotriose and xylotriose were major hydrolysis products from mannohexaose and xylohexaose, respectively. Moreover, PcMulGH9 could hydrolyse untreated corn hull and rice straw into xylo- and cello-oligosaccharides. Enzyme kinetics, site-directed mutagenesis and molecular docking revealed that Met394, located at the binding subsite + 2, was involved in broad substrate specificity of PcMulGH9 enzyme. This study offers new knowledge of the multifunctional cellulase/mannanase/xylanase in GH9. The PcMulGH9 enzyme showed a novel function of GH9, which increases its potential for saccharification of lignocellulosic biomass into value-added products, especially oligosaccharides.

Optimization of BS4 Enzyme production with Different Substrate Thickness and Type of Trays

BS4 enzyme that is produced from solid substrate fermentation (SSF) on coconut cake with Eupenicillium javanicum BS4 in tray bioreactor has been applied as a feed additive. It increases the nutritional value of animal feedstuff. The BS4 production on SSF may be influenced by the better aeration through the perforated trays or by the thinner substrate. The aim of this research is to optimize the production of BS4 with different substrate thicknesses and types of trays. The trial was carried out using a factorial randomized design (2x2x3) with 6 replicates. The first factor was the type of trays: i.e., non-perforated and perforated tray. The second factor was the thickness of the substrate: i.e., 1.5 and 3.0 cm, while the third factor was the duration of fermentation: i.e. 5, and 7 days. The variables observed were moisture content, dry matter loss (DML), mannanase and saccharification activities, soluble protein content, their specific activities, and yield. Statistical analysis showed no interactions between the three factors, but there were interactions between types of trays and substrate thicknesses, as well as type of trays and incubation times on the mannanase activity and yield of mannanase. The results showed that DML was observed on day 7 were around 31.43- 36.89. The highest mannanase activity was observed on the non-perforated tray with 3 cm thickness on day 7. The saccharification activity towards palm kernel meal was better in the non-perforated tray on day 7 but not influenced by The yield value of mannanase and saccharification activities on a non-perforated tray with 3.0 cm thickness on day 7 was also the highest. Based on energy efficiency and the cost of production, it can be concluded that the optimum condition to produce the BS4 enzyme was observed in the non-perforated tray with 3 cm thickness and fermented for 7 days.

A comparative study of phenols composition, antioxidant, and antifungal potency of leaves extract from five Moroccan Vitis vinifera L. varieties

AbstractVitis vinifera (L) crops, covers an area of about 54,000 ha corresponding to a production of 230,000 t of grapes, representing 5.5% of the national perennial corps. However, this plant is sensitive to various ravages and diseases, among others, the cryptogrammic by fungi. The degrading enzymes of the cell wall are among the factors of pathogenicity the more important than fungi use to colonize their host plants. In this study, the first step was devoted to the antioxidant activity and phenols composition (phenylalanine‐ammonia‐lyase [PAL], tyrosine‐ammonia‐lyase [TAL], flavonoids, and tannins) as well as high‐performance liquid chromatography analysis in five vineyard leaves extract, which are “Superior, Early sweet and Flame” (Allochthonous), “Abbou and Adari” (endemic). In the second step, the lytic activities “pectinase, polygalacturonase, xylanase, mannanase, glucanase, and cellulase” were investigated among six epiphytic fungus to compare their hydrolytic aggressiveness. Subsequently, as a third part, phenols antifungal potency and Trichoderma biocontrol were displayed in the study. The phenolic compounds observed in the attributes leaves were different among varieties (p &lt; .05), and show that the endemic variety “Abbou” leads the phytochemical amount, which has the highest quantities of PAL, and TAL, as well as the DPPH and FRAP antioxidant activity, while the introduced variety show a higher amount in total phenol and flavonoids. The polygalacturonase, mannanase, and pectinase activities are probably the main factors responsible for the aggressiveness of the studied fungus. Phenols potency showed a good mycelial inhibition with 82.09% as compared to the biocontrol (64.45%). Given the efficacy of the phytochemical control in this study, the phenols could be a part of integrated management strategies for sustainable control of vineyard disease.

Isolasi Bakteri Mananolitik dan Karakterisasi Mananasenya

Isolate RA05 has the highest mannanolytic index and mannanase activity which isolated from copra soil waste from Pasaman, West Sumatra. The best growth condition that produces best mannanase activity of isolate RA05 was achieved from 500 ml flask containing 100 ml medium with 100 rpm agitation. Isolate RA05 showed its mannanase activity in medium containing Locust Bean Gum and coconut meal but not in medium containing kolang kaling. This mannanase had the highest activity on medium containing 2% of coconut meal with optimum condition temperatur 800C and pH 2.5. Adding of 5 mM MnCl2 on the crude enzym increased the activity near 300%. Other kation (Ca2+, Zn2+, Cu2+, Mg2+, Fe2+ dan Co2+) did not display great effect on the activity.

Characterization and homology modelling of a novel multi-modular and multi-functional Paenibacillus mucilaginosus glycoside hydrolase.

Glycoside hydrolases, particularly cellulases, xylanases and mannanases, are essential for the depolymerisation of lignocellulosic substrates in various industrial bio-processes. In the present study, a novel glycoside hydrolase from Paenibacillus mucilaginosus (PmGH) was expressed in E. coli, purified and characterised. Functional analysis indicated that PmGH is a 130kDa thermophilic multi-modular and multi-functional enzyme, comprising a GH5, a GH6 and two CBM3 domains and exhibiting cellulase, mannanase and xylanase activities. The enzyme displayed optimum hydrolytic activities at pH 6 and 60°C and moderate thermostability. Homology modelling of the full-length protein highlighted the structural and functional novelty of native PmGH, with no close structural homologs identified. However, homology modelling of the individual GH5, GH6 and the two CBM3 domains yielded excellent models based on related structures from the Protein Data Bank. The catalytic GH5 and GH6 domains displayed a (β/α)8 and a distorted seven stranded (β/α) fold, respectively. The distinct homology at the domain level but low homology of the full-length protein suggests that this protein evolved by exogenous gene acquisition and recombination.

Redox metabolism and cell wall modifications as global and local targets respectively, of cyanide induced dormancy release of walnut kernels

The HCN-induced seed dormancy release necessitates alterations in reactive oxygen species (ROS) metabolism and radicle cell wall loosening. Little is known about the interaction of ROS metabolism with cell wall hydrolytic enzymes during HCN-induced seed dormancy release. Thus dormant walnut (Juglans regia L.) kernels were exposed to HCN (4 h) and studied for redox metabolism and cell wall-modifying enzymes during 10 days of incubation (DI) i.e. before radicle emergence. HCN increased ROS especially in the embryonic axes (EAs) but decreased ROS-generating NADPH oxidase and ROS scavenging superoxide dismutase (SOD) and peroxidase (POX) with no effects on catalase (CAT), ascorbate peroxidase (APX) and cell wall-modifying enzymes activities in short term up to 2 DI. In long term roughly from 4 DI onwards, HCN-exposed EA displayed greater superoxide anions and enhanced activities of POX, APX, NADPH oxidase, cell wall peroxidase (CW-POX), β- 1, 4-D glucanase, mannanase, polygacturonase and xylanase. Meanwhile HCN increased greater expression of POX and mannanase isoforms as revealed by in-gel activity assay. Except for higher activities of CAT, POX and APX, cotyledonary activities of CW-POX, mannanase and polygacturonase and to some extent β- 1, 4-D glucanase remained unaffected by HCN. Thus short term ROS accumulation in HCN-treated EA is due to declined SOD and POX activities. In long term the enhanced activities of both NADPH oxidase: CW-POX couple and cell wall-modifying enzymes in EA bring about wall loosening in preparation for radicle emergence. Evidences for the simultaneous operation of both mechanisms are provided in walnut EAs during dormancy release.

Biochemical characterization and thermodynamic study of β-mannanase from Enterobacter asburiae

This study aimed for the partial purification, biochemical characterization, determination of bioprocess and thermodynamic parameters of mannanase from newly isolated strain Enterobacter asburiae SD26 under submerged culture. From different variables, locust bean gum and combination of peptone + yeast extract was found as best carbon and nitrogen sources respectively. The profiles of mannanases production and growth kinetics of E. asburiae SD26 were similar. The maximum specific growth rate (μ max ) and productivity ( γ Pmax ) of enzyme was 0.02 h -1 and 0.006 g l - 1 hr -1 . The highest mannanases activity was achieved at pH 6.0 and temperature 50°C. The kinetic parameters of mannanases was K m = 25 mg.ml -1 , V max = 2500 μmol.min -1 .ml -1 and thermodynamic parameters of enzyme, Δ H d ° , Δ G d ° , Δ S d ° were 67.74 KJmol -1 , 105.19 KJmol -1 , − 115.89 J.mol -1 .K -1 respectively, recorded for mathematical description of the mannanase action at different process conditions. • A bacterial strain Enterobacter asburiae SD26 as mannanase producer was identified. • Bioprocess parameters for growth of bacterial strain and mannanase production were estimated. • Optimum pH and temperature conditions for high mannanase activity were determined. • Kinetics and thermodynamics study of mannanase action were estimated.

Manno‐oligosaccharide preparation by the hydrolysis of konjac flour with a thermostable endo‐mannanase from Talaromyces cellulolyticus

A thermostable endo-mannanase from the fungus Talaromyces cellulolyticus was identified to facilitate manno-oligosaccharide preparation from Konjac (Amorphophallus konjac) flour. A putative endo-1,4-β-mannanase from the T. cellulolyticus was obtained and efficiently expressed by improving its gene dosage in the genome of the host. After cultivation in a bench-top bioreactor for about 120h, the protein content and enzyme activity of mannanase increased to 3·4gl-1 and 17500Uml-1 respectively. Enzymatic characterization showed that this enzyme has an optimal temperature of 80°C, optimal pH of 5·0. Under the optimized hydrolysis conditions of pH 5·0, 70°C, and an enzyme concentration of 200Ul-1 solution, this enzyme could efficiently hydrolyse 0·5% konjac flour into manno-oligosaccharides (MOSs) with the degree of polymerization range from 3 to 7. The possible mechanism by which the enzyme produced MOSs was also discussed. Talaromyces cellulolyticus endo-mannanase is thermostable and has a broad pH range adaptability. Method of improving the dosage of mannanase gene in the genome could realized its high-level impression. This enzyme could efficiently hydrolyse konjac flour into manno-oligosaccharide products. This study has enriched endo-mannanase resources, facilitated its bulk production and provided a strong reference for its application in manno-oligosaccharide preparation from the natural glucomannan of konjac flour.

Mannanase activity produced through fermentation of coconut flour at various pH by Aspergilus niger

Coconut flour is one of agriculture waste product, containing high concentration of mannan polysaccharide. This waste product can be used as a substrate to produce mannanase enzyme through fermentation by using inoculum of Aspergilus niger. The aim of the study was to produce mannanase enzyme with high activity from the non-lipid and non-protein coconut flour. The coconut flour was extracted by using 1M NaOH obtain free-protein coconut flour. The non protein coconut flour was then extracted by using hexane to release lipid. The non-protein and non-lipid substrates were then fermented with Aspergilus niger. A completely randomized design was used in this study with various pH (pH 6 7 8 and 9). The result of the study indicated that pH 9 of the substrate produce the highest mannanase activity, being 0.0313 U/ml. In conclusion, optimal activity of mannanase was reached when the pH of substrate was 9.

Constitutive expression and cell-surface display of a bacterial \u03b2-mannanase in Lactobacillus plantarum

BackgroundLactic acid bacteria (LAB) are important microorganisms in the food and beverage industry. Due to their food-grade status and probiotic characteristics, several LAB are considered as safe and effective cell-factories for food-application purposes. In this present study, we aimed at constitutive expression of a mannanase from Bacillus licheniformis DSM13, which was subsequently displayed on the cell surface of Lactobacillus plantarum WCFS1, for use as whole-cell biocatalyst in oligosaccharide production.ResultsTwo strong constitutive promoters, Pgm and SlpA, from L. acidophilus NCFM and L. acidophilus ATCC4356, respectively, were used to replace the inducible promoter in the lactobacillal pSIP expression system for the construction of constitutive pSIP vectors. The mannanase-encoding gene (manB) was fused to the N-terminal lipoprotein anchor (Lp_1261) from L. plantarum and the resulting fusion protein was cloned into constitutive pSIP vectors and expressed in L. plantarum WCFS1. The localization of the protein on the bacterial cell surface was confirmed by flow cytometry and immunofluorescence microscopy. The mannanase activity and the reusability of the constructed L. plantarum displaying cells were evaluated. The highest mannanase activities on the surface of L. plantarum cells obtained under the control of the Pgm and SlpA promoters were 1200 and 3500 U/g dry cell weight, respectively, which were 2.6- and 7.8-fold higher compared to the activity obtained from inducible pSIP anchoring vectors. Surface-displayed mannanase was shown to be able to degrade galactomannan into manno-oligosaccharides (MOS).ConclusionThis work demonstrated successful displaying of ManB on the cell surface of L. plantarum WCFS1 using constitutive promoter-based anchoring vectors for use in the production of manno-oligosaccharides, which are potentially prebiotic compounds with health-promoting effects. Our approach, where the enzyme of interest is displayed on the cell surface of a food-grade organism with the use of strong constitutive promoters, which continuously drive synthesis of the recombinant protein without the need to add an inducer or change the growth conditions of the host strain, should result in the availability of safe, stable food-grade biocatalysts.

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.