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Extremophilic microorganisms are valuable sources of enzymes for various industrial applications. In fact, given their optimal catalytic activity and operational stability under harsh physical and chemical conditions, they represent a suitable alternative to their mesophilic counterparts. For instance, extremophilic enzymes are important to foster the switch from fossil-based to lignocellulose-based industrial processes. Indeed, more stable enzymes are needed, because the conversion of the lignocellulosic biomass to a wide palette of value-added products requires extreme chemo-physical pre-treatments. Galactomannans are part of the hemicellulose fraction in lignocellulosic biomass. They are heteropolymers constituted by a β-1,4-linked mannan backbone substituted with side chains of α-1,6-linked galactose residues. Therefore, the joint action of different hydrolytic enzymes (i.e. β-mannanase, β-mannosidase and α-galactosidase) is needed to accomplish their complete hydrolysis. So far, numerous galactomannan-degrading enzymes have been isolated and characterized from extremophilic microorganisms. Besides applications in biorefinery, these biocatalysts are also useful to improve the quality (i.e. digestibility and prebiotic properties) of food and feed as well as in paper industries to aid the pulp bleaching process. In this review, an overview about the structure, function and applications of galactomannans is provided. Moreover, a survey of (hyper)-thermophilic galactomannans-degrading enzymes, mainly characterized in the last decade, has been carried out. These extremozymes are described in the light of their biotechnological application in industrial processes requiring harsh conditions.

The full utilization of hemicellulose sugars (pentose and exose) present in lignocellulosic material, is required for an efficient bio-based fuels and chemicals production. Two recombinant thermophilic enzymes, an endo-1,4-β-mannanase from Dictyoglomus turgidum (DturCelB) and an α-galactosidase from Thermus thermophilus (TtGalA), were assayed at 80 °C, to assess their heterosynergystic association on galactomannans degradation, particularly abundant in hemicellulose. The enzymes were tested under various combinations simultaneously and sequentially, in order to estimate the optimal conditions for the release of reducing sugars. The results showed that the most efficient degree of synergy was obtained in simultaneous assay with a protein ratio of 25% of DturCelB and 75% of TtGalA, using Locust bean gum as substrate. On the other hand, the mechanism of action was demonstrated through the sequential assays, i.e. when TtGalA acting as first to enhance the subsequent hydrolysis performed by DturCelB. The synergistic association between the thermophilic enzymes herein described has an high potential application to pre-hydrolyse the lignocellulosic biomasses right after the pretreatment, prior to the conventional saccharification step.

Exogenous fibrolytic enzymes have been shown to be a promising way to improve feed conversion efficiency (FCE). β-Mannanase is an important enzyme digesting the polysaccharide β-mannan in hemicellulose. Supplementation of diets with β-mannanase to improve FCE has been more extensively studied in nonruminants than in ruminants. The objective of this study was to investigate the effects of β-mannanase supplementation on nutrient digestibility, FCE, and nitrogen utilization in lactating Holstein dairy cows. Twelve post-peak-lactation multiparous Holstein cows producing 45.5±6.6kg/d of milk at 116±19.0d in milk were randomly allotted to 1 of 3 treatments in a 3×3 Latin square design with 3 periods of 18d (15d for adaptation plus 3d for sample collection). All cows were fed the same basal diet and the 3 treatments differed only by the β-mannanase dose: 0% dry matter (DM; control), 0.1% of DM (low supplement, LS), and 0.2% of DM (high supplement, HS) supplemented to the basal diet. Supplementation of β-mannanase enzyme at the LS dose reduced dry matter intake (DMI) but did not affect milk yield or milk composition. Cows receiving LS produced 90g more milk per kg of DMI compared with control cows. Somatic cell count (SCC) in milk was lower for cows fed the LS diet compared with cows fed control diets. Cows fed LS diet had lower DM, organic matter and crude protein digestibility compared with cows fed control diets. Starch, neutral detergent fiber, and acid detergent fiber digestibility were not affected by LS. Milk yield, DMI, SCC, and nutrient digestibility did not change for HS. Despite the reduced crude protein digestibility, reduced N intake led to similar fecal N excretions in LS cows and control cows (234 vs. 235g/cow per day). Urinary N excretions remained similar between enzyme-fed and control cows (~190g/cow per day), although the percentage of N intake partitioned to urinary N tended to be greater in LS than in control cows (31 vs. 27%). Cows fed LS significantly improved the percentage of apparently absorbed N partitioned to milk protein N (42 vs. 38%). When supplemented at 0.1% of dietary DM, β-mannanase can improve FCE and lower the SCC of dairy cows without affecting milk yield, milk composition, or total manure N excretions of dairy cows.

Abstract Background Deficiency in the enzyme β‐mannosidase was described over three decades ago. Although rare in occurrence, the presentation of childhood‐onset β‐mannosidase deficiency consists of hypotonia in the newborn period followed by global development delay, behavior problems, and intellectual disability. No effective pharmacologic treatments have been available. Methods We report 2‐year outcomes following the first umbilical cord blood transplant in a 4‐year‐old boy with early childhood‐onset disease. Results We show restoration of leukocyte β‐mannosidase activity which remained normal at 2 years posttransplant, and a simultaneous increase in plasma β‐mannosidase activity and dramatic decrease in urine‐free oligosaccharides were also observed. MRI of the brain remained stable. Neurocognitive evaluation revealed test point gains, although the magnitude of improvement was less than expected for age, causing lower IQ scores that represent a wider developmental gap between the patient and unaffected peers. Conclusion Our findings suggest that hematopoietic cell transplant can correct the biochemical defect in β‐mannosidosis, although preservation of the neurocognitive trajectory may be a challenge.

The objective of this study is to investigate the beneficial effects and underlying mechanism of dietary β-mannanase supplementation on the productive performance of laying hens fed with metabolic energy (ME)-reduced diets. A total of 448 Hy-Line gray laying hens were randomly assigned to seven groups. Each group had 8 replicates with 8 hens. The groups included a control diet (CON) with a ME of 2750 kcal/Kg, diets reduced by 100 kcal/Kg or 200 kcal/Kg ME (ME _100 or ME _200 ), and diets with 0.15 g/Kg or 0.2 g/Kg β-mannanase (ME _100 +β-M _0.15 , ME _100 +β-M _0.2 , ME _200 +β-M _0.15 , and ME _200 +β-M _0.2 ). The productive performance, egg quality, intestinal morphology, inflammatory response, mRNA expression related to the Nuclear factor kappa B (NF-κB) and AMPK pathway, and cecum microbiome were evaluated in this study. ME-reduced diets negatively impacted the productive performance of laying hens. However, supplementation with β-mannanase improved FCR, decreased ADFI, and restored average egg weight to the level of the CON group. ME-reduced diets increased the levels of interleukin-1β (IL-1β) and IL-6 while decreasing the levels of IL-4 and IL-10 in the jejunum of laying hens. However, dietary β-mannanase supplementation improved jejunum morphology, reduced pro-inflammatory cytokine concentrations, and increased levels of anti-inflammatory factors in laying hens fed with ME-reduced diets. The mRNA levels of IL-6, IFN-γ, TLR4, MyD88, and NF-κB in the jejunum of ME-reduced diets were significantly higher than that in CON, dietary β-mannanase supplementation decreased these genes expression in laying hens fed with ME-reduced diets. Moreover, dietary β-mannanase supplementation also decreased the mRNA levels of AMPKα and AMPKγ, and increased the abundance of mTOR in the jejunum of laying hens fed with ME-reduced diets. Cecum microbiota analysis revealed that dietary β-mannanase increased the abundance of various beneficial bacteria (e.g., g_Pseudoflavonifractor, g_Butyricicoccus, and f_Lactobacillaceae) in laying hens fed with ME-reduced diets. In conclusion, dietary β-mannanase supplementation could improve the productive performance of laying hens fed with a ME-reduced diet by improving intestinal morphology, alleviating intestinal inflammation, changing energy metabolism-related signaling pathways, and increasing cecum-beneficial microbiota., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

This study was conducted to investigate the effect of graded levels of β-mannanase supplementation in broiler diets on growth performance, energy digestibility, and lesion scores in d-old birds fed mash, corn-soybean meal-based diets and raised to 42 d. Five dietary treatments were investigated: 1) positive control diet (PC) containing standard energy; 2) negative control (NC) with 100 kcal/kg diet reduction in AME compared to PC; 3) NC supplemented with 30 U/g β-mannanase (NC + 30 U); 4) NC supplemented with 60 U/g β-mannanase (NC + 60 U); and 5) NC supplemented with 90 U/g β-mannanase (NC + 90 U). Each treatment had 6 replicate pens with 52 chicks per replicate. Data was analyzed using 1-way ANOVA, and means were separated by LSMEANS. Reduction of 100 kcal/kg feed (NC) resulted in an overall body weight gain reduction of 51 g (P < 0.05) and feed conversion loss of approximately 4 points (P < 0.05) compared to PC at 42 d of age. At the same time, supplementing β-mannanase at 60 and 90 U/g improved growth performance parameters compared to NC, while 30 U/g did not result in significant improvements beyond NC; body weight gain was improved (P < 0.05) by 87, and 106 g when β-mannanase was supplemented at 60 and 90 U/g, respectively, compared to NC. This corresponded to an improvement by 6 and 7 points in feed conversion for 60 and 90 U/g supplementation, respectively, compared to NC. Furthermore, AMEn was improved (P < 0.05) by 15, 97, and 116 kcal/kg at 42 d when β-mannanase was added to NC at 30, 60, and 90 U/g, respectively. Digesta viscosity measured at 42 d was decreased (P < 0.05) by β-mannanase supplementation of 60 and 90 U/g, compared to NC, while 42 d lesion scores were improved (P < 0.05) by β-mannanase supplementation compared to NC. Data demonstrated that dietary supplementation of β-mannanase improved growth performance, energy digestibility, and reduced viscosity and lesion scores when supplemented with diets with a reduced energy content of 100 kcal/kg compared to a standard energy diet., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

For farm animals the supplementation of exogenous enzymes, like ß-mannanase, to soybean-based diets is beneficial to improve feed digestibility. In order to unravel the effect of ß-mannanase on soybean meal's cell structure, a novel imaging concept was developed which allows visualizing the spatial activity pattern of ß-mannanase with high sensitivity by fluorescence microscopy before any visible degradation of the cellular structure occurs. It is based on fluorescence labeling of newly formed reducing ends of ß-mannanase-hydrolyzed polysaccharides after the native reducing ends of all polysaccharides present were chemically reduced. It was revealed that ß-mannanase is not only active at the cell wall but also at previously unknown sites, like the middle lamella and, most prominently, at an intracellular matrix enclosing the protein storage vacuoles. Based on these findings it can be hypothesized that the evaluated ß-mannanase can degrade the enclosing matrix of encapsulated proteins and the cell wall structure and thereby improves efficiency of feed utilization., (© 2024. The Author(s).)

Linear β-manno-oligosaccharides (l-β-MOS) are widely used to investigate oligo- and poly-saccharide structures and mannanolytic enzyme activities. l-β-MOS are also being used as prebiotic agents with potential bio-active properties. In this study, we developed an efficient protocol to prepare a series of l-β-MOS by hydrolyzing cassia gum (CG) using mannanolytic enzymes (endo-1,4-β-mannanase, α-galactosidases and β-glucosidases). By using medium pressure liquid chromatography (MPLC), we purified l-β-MOS with different degrees of polymerization (DPs). HPAEC-PAD, MALDI-TOF-MS and NMR studies confirmed that these l-β-MOS species ranged from 1,4-β-d-mannobiose to 1,4-β-d-mannononaose (DP 2-9) with >95% purity. Our results provide a robust approach to preparing l-β-MOS, thus enabling l-β-MOS to be further used in the fields of chemistry, life science, and nutritional food., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

β-mannanase catalyzes the hydrolysis of mannans β-1,4-mannosidic linkages to produce industrially relevant oligosaccharides. These enzymes have numerous important applications in the detergent, food, and feed industries, particularly those that are resistant to harsh environmental conditions such as salts and heat. While, moderately salt-tolerant β-mannanases are already reported, existence of a high halotolerant β-mannanase is still elusive. This study aims to report the first purification and characterization of ManH1, an extremely halotolerant β-mannanase from the halotolerant B. velezensis strain H1. Electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS) analysis revealed a single major peak with a molecular mass of 37.8 kDa demonstrating its purity. The purified enzyme showed a good thermostability as no activity was lost after a 48 h incubation under optimal conditions of 50 °C and pH 5.5. The enzyme's salt activation nature was revealed when its maximum activity was obtained in the presence of 4 M NaCl, it doubled compared to the no-salt condition. Moreover, NaCl strengthens its resistance to thermal denaturation, as its melting temperature (T m ) increased steadily with increasing NaCl concentrations reaching 75.5 °C in the presence of 2.5 M NaCl. The K m and V max values were 5.63 mg/mL and 333.33 µmol/min/mL, respectively, using carob galactomannan (CG) as a substrate. The enzyme showed a significant ability to produce manno-oligosaccharides (MOS) from lignocellulosic biomass releasing 13 mg/mL of reducing sugars from olive mill wastes (OMW) after 24 h incubation. The results revealed that this enzyme may have significant commercial values for agro-waste treatment, and other potential applications., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Plant cell walls are composed of a heterogeneous mixture of polysaccharides that require several different enzymes to degrade. These enzymes are important for a variety of biotechnological processes, from biofuel production to food processing. Several classical mannanolytic enzyme functions of glycoside hydrolases (GH), such as β-mannanase, β-mannosidase and α-galactosidase activities, are helpful for efficient mannan hydrolysis. In this light, we bring three enzymes into the model of mannan degradation that have received little or no attention. By linking their three-dimensional structures and substrate specificities, we have predicted the interactions and cooperativity of these novel enzymes with classical mannanolytic enzymes for efficient mannan hydrolysis. The novel exo-β-1,4-mannobiohydrolases are indispensable for the production of mannobiose from the terminal ends of mannans, this product being the preferred product for short-chain mannooligosaccharides (MOS)-specific β-mannosidases. Second, the side-chain cleaving enzymes, acetyl mannan esterases (AcME), remove acetyl decorations on mannan that would have hindered backbone cleaving enzymes, while the backbone cleaving enzymes liberate MOS, which are preferred substrates of the debranching and sidechain cleaving enzymes. The nonhydrolytic expansins and swollenins disrupt the crystalline regions of the biomass, improving their accessibility for AcME and GH activities. Finally, lytic polysaccharide monooxygenases have also been implicated in promoting the degradation of lignocellulosic biomass or mannan degradation by classical mannanolytic enzymes, possibly by disrupting adsorbed mannan residues. Modelling effective enzymatic mannan degradation has implications for improving the saccharification of biomass for the synthesis of value-added and upcycling of lignocellulosic wastes., (© 2023. The Author(s).)

The effects of β-mannanase supplementation in metabolizable energy (ME)-reduced diets containing xylanase-phytase were investigated on growth performance, fecal score, ultra-sounded backfat thickness and loin depth, blood profile, apparent total tract digestibility (ATTD), digesta passage rate, and fecal microbiome in grower pigs (n = 40, 26.09 ± 0.96 kg) randomly assigned within 4 treatments: a control diet containing isolated phytase and xylanase valued at 40 kcal of ME/kg (CD0), CD0 + β-mannanase (0.3 g/kg valued at 30 kcal of ME/kg) (CD70), CD0 + β-mannanase (0.3 g/kg valued at 45 kcal of ME/kg) (CD85), and CD0 + β-mannanase (0.3 g/kg valued at 60 kcal of ME/kg) (CD100). Growth performance was not affected in pigs fed ME-reduced diets containing β-mannanase. Pigs with CD100 had lower serum IL-1β concentration, and higher IL-10 was observed in pigs on CD0 than those fed β-mannanase. Coefficients of ATTD, and ATTD of DM and CP were higher in animals fed CD85 or CD100. Pigs with CD85 had higher alpha diversity richness but lower Firmicutes:Bacteroidota ratio. Acidaminococcaceae and Ruminococcaceae were more abundant in pigs fed CD0, but lower for Christensenellaceae NSJ-63 and NSJ-63 sp014384805. Pigs in CD85 showed higher Bacteroidaceae and Prevotella abundance, and lower for Streptococcaceae and Streptococcus. In conclusion, supplementation of β-mannanase in diets containing xylanase-phytase saved 85 to 100 kcal of ME/kg by supporting growth performance and improving nutrient digestibility in grower pigs., (© 2023. The Author(s).)

The postharvest fermentation process of coffee has rapidly advanced in the last few years due to the search for quality and diversity of sensorial profiles. A new type of fermentation, named self-induced-anaerobic fermentation (SIAF), is a promising process that has been increasingly used. This study aims to evaluate the sensorial improvement of coffee beverages during SIAF and the influence of microorganism's community and enzymatic activity. The SIAF process was conducted in Brazilian farms for up to 8 days. The sensorial quality of coffee was evaluated by Q-graders; the microbial community was identified by the high-throughput sequencing of 16S rRNA and ITS regions; and the enzymatic activity (invertase, polygalacturonase, and endo-β-mannanase) was also investigated. SIAF increased up to 3.8 points in the total score of sensorial evaluation (compared to the non-fermented sample), in addition to presenting more flavor diversity (especially within the fruity and sweetness descriptors). The high-throughput sequencing identified 655 bacterial and 296 fungal species during the three processes. The bacteria Enterobacter sp., Lactobacillus sp., Pantoea sp., and the fungi Cladosporium sp. and Candida sp. were the predominant genera. Fungi that are potential producers of mycotoxin were identified throughout the process, which indicates a risk of contamination since some of them are not degraded in the roasting process. Thirty-one species of microorganisms were described for the first time in coffee fermentation. The microbial community was influenced by the place where the process was carried out, mainly in relation to the diversity of fungi. Washing the coffee fruits before fermenting led to a fast reduction of pH; a fast development of Lactobacillus sp. and a fast dominance of Candida sp.; a reduction of the fermentation time necessary to achieve the best sensorial score; an increase in the invertase activity in the seed; a more expressive invertase activity in the husk; and a decreasing trend in polygalacturonase activity in the coffee husk. The increase in endo-β-mannanase activity suggests that coffee starts germinating during the process. SIAF has a huge potential to increase the quality and add value to coffee, but further studies must be conducted to access its safety. The study allowed a better knowledge of the spontaneous microbial community and the enzymes that were present in the fermentation process., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

β-Mannanase capable of hydrolyzing β-1,4-linkages in guar gum was immobilized as cross-linked enzyme aggregates (M-CLEAs). The aggregation and cross-linking process was optimized by response surface methodology (RSM) for maximum activity. The resulting M-CLEAs were characterized by FTIR, DSC, SEM, and SDS-PAGE. The M-CLEAs showed higher pH stability, improved thermal and storage stability, and reusability than free β-mannanase. For example, M-CLEAs were stable over broader pH range (5.5-8.5) with highest relative of activity of 98.17% at pH 6.5 and retained almost double activity than free mannanase at 50 °C after 4 h. Moreover, K m and V max of M-CLEAs were altered significantly, with a 1.5-fold increase and 0.98-fold decrease, respectively, than free β-mannanase. The prepared M-CLEAs could hydrolyze native guar gum (MW = 588,147 Da) to yield partially hydrolyzed guar gum (PHGG) (MW = 8023 Da)., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Spent coffee grounds (SCG) are inexpensive materials with a complex composition that makes them promising feedstocks for a biorefinery.Here, conversion of SCG into a wide range of high value-added products (coffee oil, bio-ethanol, D-mannose, manno-oligosaccharide (MOS), cafestol and kahweol) using a novel integrated system was evaluated. The process involves oil extraction, MOS production by mannanase obtained from Penicillium purpurogenum, NaOH (Na) and hydrogen peroxide (HP) pretreatment for the degradation of lignin and phenolic compounds, diterpenes extraction, enzymatic hydrolysis, and fermentation, which can be performed using environmentally friendly technologies. Approximately 97 mL of coffee oil, 164 g of D-mannose, 102 g of MOS, 99 g of bioethanol and a dash of cafestol/kahweol were produced from 1 kg of dry SCG. Producing high-value co-products from SCG using an integrated approach as demonstrated here may be an efficient strategy to reduce waste generation, while improving the economics of the biorefinery production process., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

β-mannan is a nonstarch polysaccharide found in hulled and dehulled soybeans that can survive drying-toasting phase of processing soybeans and have antinutritive effects in poultry. β-mannanase is an active enzyme (endohydrolase) that can hydrolyze β-mannan to reduce its antinutritional effects. Two experiments were conducted to evaluate the effects of β-mannanase supplementation in low energy/protein diets on egg production, egg quality, and apparent ileal digestibility of the dry matter (DM), crude protein (CP), and amino acids in 21-week-old Single Comb White Leghorn hens (Hy-Line W-36). A total of 192 hens (8 replicates of 6 hens per treatment) for a production study (Exp. 1) and a total of 64 hens (8 replicates of 2 hens per treatment) for a digestibility study (Exp. 2) were randomly allocated to 4 experimental treatments in a 2 × 2 factorial arrangement. Four dietary treatments were control (CS) based on corn and 44% CP soybean meal (ME: 2,850 kcal/kg CP: 18.5%) and CS-low energy/protein (CSL) (ME: 2,750 kcal/kg CP: 17.5%), with or without 0.05% β-mannanase enzyme. Hens were fed the experimental diets for 14 d for the digestibility study and 8 wk for the production study. Hen-day egg production (HDEP), weekly feed intake, FCR, and biweekly egg quality parameters were measured. Significant interaction on feed intake (P < 0.01) was observed between energy/protein and enzyme. At 3, 6 and 8 wk, the feed intake and FCR of CSL with enzyme were significantly lower than those of CSL without enzymes. The main effects indicated that birds fed diets without inclusion of β-mannanase had higher feed intake than those fed diets with enzymes at 4, 7, and 8 wk. The inclusion of β-mannanase significantly increased (P < 0.05) HDEP at 2, 3, 5, and 7 wk. However, there was no significant effect of nutrient density or enzyme supplementation on egg quality parameters. The digestibility study showed that the inclusion of β-mannanase significantly improved (P < 0.01) apparent ileal digestibility of lysine, histidine and tryptophan in the diet. The results of these experiments indicate that supplementation of β-mannanase could reduce the feed intake and FCR and improve HDEP and apparent ileal digestibility of key amino acids in corn/soy diets fed to laying hens., Competing Interests: DISCLOSURES The authors declare no conflicts of interest., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

β-mannanase with high specific activity is a prerequisite for the industrial preparation of prebiotic mannooligosaccharides. Three mutants, namely MEI, MER, and MEIR, were constructed by cooperative substitution based on three predominant single-point site mutations (K291E, L211I, and Q112R, respectively). Heterologous expression was facilitated in Pichia pastoris and the recombinase was characterized completely. The specific activities of MER (7481.9 U mg -1 ) and MEIR (9003.1 U mg -1 ) increased by 1.07- and 1.29-fold from the initial activity of ME (6970.2U mg -1 ), respectively. MEIR was used for high-cell-density fermentation to further improve enzyme activity, and the expression levels achieved in the 10-L fermenter were significantly high (105,836 U mL -1 ). The prebiotic mannooligosaccharides (<2000 Da) were prepared by hydrolyzing konjac gum and locust bean gum with MEIR, with 100% and 76.40% hydrolysis rates, respectively. These characteristics make MEIR highly attractive for prebiotic development in food and related industries., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Mannooligosaccharides (MOS) were derived using Aspergillus oryzae β-mannanase (ManAo) from different mannan-rich agro-wastes, palm kernel cake (PKC), guar gum and copra meal (CM). Guar gum (GG) released higher amount of MOS (56.31% w/w) from which purification of mannobiose (0.68 mg) and mannotriose (1.26 mg) was demonstrated using size-exclusion chromatography. FTIR analysis of mannan hydrolysates showed characteristic peaks in 1200-900 cm - 1 region indicating the presence of MOS. 1 H & 13 C NMR spectra showed presence of anomeric sugar forms of MOS in different mannan hydrolysates. MOS from locust bean gum and guar gum had both α- and β-anomers while PKC and CM had only α-anomer. Growth promotional activities of different MOS were demonstrated using two probiotic Lactobacilli. Besides, enzymatically derived MOS also showed metal (Fe 2+ ) chelating and anti-oxidant activities, wherein best anti-glycating agent was evaluated as MOS from PKC. PKC derived MOS showed highest cytotoxicity (74.19%) against human colon adenocarcinoma cell line (Caco-2). This study demonstrated the prebiotic potential of agro-waste derived MOS and possibility of their utilization as a functional food ingredient., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Deficiency in the enzyme β‐mannosidase was described over three decades ago. Although rare in occurrence, the presentation of childhood‐onset β‐mannosidase deficiency consists of hypotonia in the newborn period followed by global development delay, behavior problems, and intellectual disability. No effective pharmacologic treatments have been available. Methods: We report 2‐year outcomes following the first umbilical cord blood transplant in a 4‐year‐old boy with early childhood‐onset disease. Results: We show restoration of leukocyte β‐mannosidase activity which remained normal at 2 years posttransplant, and a simultaneous increase in plasma β‐mannosidase activity and dramatic decrease in urine‐free oligosaccharides were also observed. MRI of the brain remained stable. Neurocognitive evaluation revealed test point gains, although the magnitude of improvement was less than expected for age, causing lower IQ scores that represent a wider developmental gap between the patient and unaffected peers. Conclusion: Our findings suggest that hematopoietic cell transplant can correct the biochemical defect in β‐mannosidosis, although preservation of the neurocognitive trajectory may be a challenge. [ABSTRACT FROM AUTHOR]

An engineered thermophilic and acidophilic β-mannanase (ManAK) from Aspergillus kawachii IFO 4308 was highly expressed in Pichia pastoris. Through high cell density fermentation, the maximum yield reached 11,600 U/mL and 15.5 g/L, which is higher than most extreme β-mannanases. The recombinant ManAK was thermostable with a temperature optimum of 80 °C, and acid tolerant with a pH optimum of 2.0. ManAK could efficiently degrade locust bean gum, konjac gum, and guar gum into small molecular mannooligosaccharide (<2000 Da), even at high initial substrate concentration (10%), and displayed different Mw distributions in their end products. Docking analysis demonstrated that the catalytic pocket of ManAK could only accommodate a galactopyranosyl residue in subsite -1, which might be responsible for the distinct hydrolysis product compositions from locust bean gum and guar gum. These superior properties of ManAK strongly facilitate mannooligosaccharide preparation and application in food and feed area., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

A multi-tolerant β-mannanase (ManAo) was produced by Aspergillus oryzae on copra meal, a low-cost agro waste. Under statistically optimized conditions, 4.3-fold increase in β-mannanase production (434 U/gds) was obtained. Purified ManAo had MW ∼34 kDa and specific activity of 335.85 U/mg with optimum activity at 60 °C and at pH 5.0. Activity of ManAo was enhanced by most metal ions and modulators while maximum enhancement was noticed with Ag + and Triton X-100. K m and V max were 2.7 mg/mL and 1388.8 µmol/min/mg for locust bean gum while the enzyme showed lower affinity towards konjac gum (8.8 mg/mL, 555.5 µmol/min/mg). Evaluation of various thermodynamic parameters indicated high-efficiency of the ManAo with activation energy 12.42 KJ/mol and 23.31 KJ/mol towards LBG and konjac gum, respectively. End product analysis of β-mannanase action by fluorescence assisted carbohydrate electrophoresis (FACE) revealed the generation of sugars from DP 1-4 with some higher DP MOS from different mannans., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The polysaccharides of different germplasm resources of Astragalus membranaceus var. mongholicus〓(cultured Astragalus Radix (RA) and natural RA) and A. membranaceus (MJ) (cultured RA and natural RA) were studied by using the optimal enzymatic conditions of endo-1,4-β-mannanase. Saccharide fingerprints were obtained for the identification and evaluation of the germplasm resources of RA by Fluorophore-assisted Carbohydrate Electrophoresis (FACE). The data were analyzed by principal component analysis to obtain the difference between RA of different germplasm resources. The results showed that trisaccharide, tetrasaccharide and pentasaccharide of endo-1,4-β-mannanase hydrolyzate could be used as the differential fragments to distinguish MG (cultured RA and natural RA); the pentasaccharide and hexasaccharide can be used as differentially expressed carbohydrate fragments that distinguish MJ (cultured RA and natural RA); the trisaccharide and tetrasaccharide can be used as the differential fragments to distinguish the cultured MG and cultured MJ. Studies have shown that polysaccharide products degraded by endo-1,4-β-mannanase can well distinguish RA species (MG and MJ), growth mode (cultured RA and natural RA). This study laid the foundation for the quality evaluation of Astragalus medicinal herbs and screening of active oligosaccharides., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Chinese Pharmaceutical Association.)

An engineered β-mannanase (mRmMan5A) from Rhizomucor miehei was successfully expressed in Pichia pastoris. Through high cell density fermentation, the expression level of mRmMan5A reached 79,680 U mL -1 . The mRmMan5A showed maximum activity at pH 4.5 and 65 °C, and exhibited high specific activities towards mannans. To produce manno-oligosaccharides, palm kernel cake (PKC) was pretreated by steam explosion at 200 °C for 7.5 min, and then hydrolyzed by mRmMan5A. As a result, the total manno-oligosaccharide yield reached 34.8 g/100 g dry PKC, indicating that 80.6% of total mannan in PKC was hydrolyzed. Moreover, the kilo-scale production of manno-oligosaccharides was carried out to verify the feasibility of mass production. A total of 261.3 g manno-oligosaccharides were produced from 1.0 kg of dry PKC. An effective β-mannanase for the bioconversion of mannan-rich biomasses and an efficient method for the production of manno-oligosaccharides from PKC are provided in this paper., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Abstract: β-Mannosidosis (OMIM 248510) is an inborn lysosomal storage disorder caused by deficiency of β-mannosidase activity. This enzyme is encoded by a single gene (MANBA), located on chromosome 4q22–25. This autosomal recessive disorder is characterized by a wide range of symptoms including mental retardation, behavioural problems, hearing loss, recurrent respiratory infections, angiokeratoma, facial dysmorphism, skeletal deformation, seizures, hypotonia, demyelinating polyneuropathy, and hepatosplenomegaly. The age of symptom onset is variable. We describe a 14-year clinical follow-up of a patient with β-mannosidase deficiency with symptoms of mental retardation, progressive spasticity and cerebellar ataxia, a clinical spectrum that so far has never been reported in β-mannosidosis. A novel mutation in the MANBA gene was found in our patient. Evoked potentials were in favour of a demyelinating pathology of the central nervous system. Serial MRI showed generalized cortical and subcortical atrophy in the absence of white matter changes suggesting an additional axonal pathophysiological component. [Copyright &y& Elsevier]

The effects of an enzymatic treatment with cellulase and mannanase on the properties of marine microalgae Nannochloropsis sp. were investigated. The combined use of these enzymes synergistically promoted the recovery of lipids from the microalgae, increasing the extraction yield from 40.8 to over 73%. Untreated and enzymatically treated microalgae were characterized by chemical analysis and by TGA/DTG, FTIR, XRD and SEM. Significant changes were observed in the chemical composition and thermal behavior of the microalgae. The enzymatic treatment also resulted in an increase of the crystalline-to-amorphous cellulose ratio. SEM images revealed dramatic changes in cell morphology, extensive cell damage and release of intracellular material. Overall, the results obtained indicate that the enzymes used are capable of disrupting the microalgal cell wall and that a combination of common analytical techniques can be used to assess the enzyme-induced damage., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Currently, the traditional fermentation using konjac powder for β-mannanase production presents operational difficulties and high energy consumption, because of the individual hot air sterilization for konjac powder. A simply enzymatic hydrolysis pretreatment for konjac powder was developed to solve the problems of the traditional process in a 7-L fermenter. In the new process, when hydrolysis yield of konjac powder was above 50%, the media became liquid state from gelatinous state and could be sterilized immediately, avoiding the hot air sterilization and solving the operational difficulties. Interestingly, the new process didn't have negative influence on β-mannanase production. Additionally, it could save close to 23% of power consumption during the whole fermentation. For another example, it did work well using locust bean gum for β-mannanase production in a 7-L fermenter. Therefore, the new process might be scaled up for industrial production using mannan-based bioresource as substrate., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

This work aims to produce glutathione directly from mannan-based bioresources using engineered Saccharomyces cerevisiae. Mannan proved to be a valuable carbon source for glutathione production by this organism. Mannan-hydrolyzing S. cerevisiae was developed by heterologous expression of mannanase/mannosidase on its cell surface. This strain efficiently produced glutathione from mannose polysaccharide, β-1,4-mannan. Furthermore, it produced glutathione from locust bean gum (LBG), a highly dense and inexpensive mannan-based bioresource, as sole carbon source. Glutathione productivity from LBG was enhanced by engineering the glutathione metabolism of mannan-hydrolyzing S. cerevisiae. Expression of extracellular mannanase/mannosidase protein combined with intracellular metabolic engineering is potentially applicable to the efficient, environmentally friendly bioproduction of targeted products from mannan-based bioresources., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Apparent ileal digestibility (AID) of energy, dry matter (DM), nitrogen (N) and amino acids and standardized ileal digestibility (SID) of amino acids in low quality soybean meals with different CP concentration (SBM 44% CP and SBM 48% CP) with or without 400 U β-mannanase/kg supplementation were evaluated in 20 cannulated barrows ((Landrace×Yorkshire)×Duroc) with an average BW of 25.08±3.42 kg. A N-free diet was used to determine basal endogenous losses of amino acids. The supplementation of β-mannanase improved (P0.05) AID of N and energy. The type of SBM (SBM 44% CP v. SBM 48% CP) had no effect on AID of DM, N and energy. β-mannanase improved (P<0.05) AID of sum of essential amino acids, arginine, histidine, lysine, valine and glycine. The SID of lysine was higher (P<0.05) in enzyme supplemented than in non-supplemented diets. Larger AID and SID of threonine and proline (P<0.05) were observed in SBM 48% CP than in SBM 44% CP. In conclusion, the supplementation of enzyme improved AID of arginine, histidine, lysine, valine and glycine, but it did not cause marked difference in SID of these amino acids except for lysine. The low nutrient digestibility of the SBM sources used in the present experiment might have favoured the positive effect of β-mannanase supplementation.

Plant mannanases are enzymes that carry out fundamentally important functions in cell wall metabolism during plant growth and development by digesting manno-polysaccharides. In this work, the Arabidopsis mannanase 5-2 (AtMan5-2) from a previously uncharacterized subclade of glycoside hydrolase family 5 subfamily 7 (GH5&#95;7) has been heterologously produced in Pichia pastoris. Purified recombinant AtMan5-2 is a glycosylated protein with an apparent molecular mass of 50kDa, a pH optimum of 5.5-6.0 and a temperature optimum of 25°C. The enzyme exhibits high substrate affinity and catalytic efficiency on mannan substrates with main chains containing both glucose and mannose units such as konjac glucomannan and spruce galactoglucomannan. Product analysis of manno-oligosaccharide hydrolysis shows that AtMan5-2 requires at least six substrate-binding subsites. No transglycosylation activity for the recombinant enzyme was detected in the present study. Our results demonstrate diversification of catalytic function among members in the Arabidopsis GH5&#95;7 subfamily., (Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.)

Spent coffee ground (SCG) is the main residue generated during the production of instant coffee by thermal water extraction from roasted coffee beans. This waste is composed mainly of polysaccharides such as cellulose and galactomannans that are not solubilised during the extraction process, thus remaining as unextractable, insoluble solids. In this context, the application of an enzyme cocktail (mannanase, endoglucanase, exoglucanase, xylanase and pectinase) with more than one component that acts synergistically with each other is regarded as a promising strategy to solubilise/hydrolyse remaining solids, either to increase the soluble solids yield of instant coffee or for use as raw material in the production of bioethanol and food additives (mannitol). Wild fungi were isolated from both SCG and coffee beans and screened for enzyme production. The enzymes produced from the selected wild fungi and recombinant fungi were then evaluated for enzymatic hydrolysis of SCG, in comparison to commercial enzyme preparations. Out of the enzymes evaluated on SCG, the application of mannanase enzymes gave better yields than when only cellulase or xylanase was utilised for hydrolysis. The recombinant mannanase (Man1) provided the highest increments in soluble solids yield (17 %), even when compared with commercial preparations at the same protein concentration (0.5 mg/g SCG). The combination of Man1 with other enzyme activities revealed an additive effect on the hydrolysis yield, but not synergistic interaction, suggesting that the highest soluble solid yields was mainly due to the hydrolysis action of mannanase.

This paper reports the production of very high levels of cellulase free xylanase and associated hemicellulases by an indigenous thermophilic isolate of Thermomyces lanuginosus (D(2)W(3)) using solid-state fermentation. Sorghum straw, an inexpensive and abundant source of carbon supported maximal xylanase activity (11,855 units/g dry substrate). Culturing T. lanuginosus D(2)W(3) on sorghum straw and optimizing other culture conditions (media types, particle size of carbon source, inoculum level, inoculum age and additives), yielded increased levels of xylanase (39,726 units/g dry substrate). Further optimization of enzyme production was carried out using Box-Behnken design of experiments with three independent variables (inoculum level, glycerol and ammonium sulphate concentrations) which resulted in very high levels of xylanase, 48,000+/-1774 units/g dry substrate, and 2.6+/-0.2, 13.4+/-0.56, 68+/-1.7, 1.4+/-0.08, 1.2+/-0.05 (units/g dry substrate) of beta-xylosidase, alpha-galactosidase, pectinase, beta-mannosidase and alpha-L-arabinofuranosidase, respectively.

Natural intoxication of livestock by the ingestion of Ipomoea carnea (Convolvulaceae) sometimes occurs in tropical regions of the world. Polyhydroxylated alkaloids were isolated from the leaves, flowers, and seeds of the poisonous plant and characterized. Chromatographic separation of the leaf extract resulted in the isolation of swainsonine (1), 2-epi-lentiginosine (2), calystegines B(1) (3), B(2) (4), B(3) (5), and C(1) (6), and N-methyl-trans-4-hydroxy-l-proline (7). The contents of 1 in the fresh leaves and flowers were 0.0029 and 0.0028%, respectively, whereas the contents of 1, 3, and 4 in the seeds were approximately 10 times higher than those in the leaves and flowers. Alkaloids 3, 4, and 6 showed a potent inhibitory activity toward rat lysosomal beta-glucosidase, with IC(50) values of 2.1, 0.75, and 0.84 microM, respectively, and alkaloid 5 was a moderate inhibitor of alpha- and beta-mannosidases. Although alkaloid 1 is known as a powerful inhibitor of lysosomal alpha-mannosidase (IC(50) = 0.02 microM), alkaloid 2, which has been thought to be an intermediate in the biosynthesis of 1, was also a potent inhibitor of alpha-mannosidase with an IC(50) value of 4.6 microM.

Background: There are seven well-known lysosomal storage diseases that produce angiokeratoma corporis diffusum clinically. beta-Mannosidosis (MANB1; OMIM248510), first reported in humans in 1986, is a rare hereditary lysosomal storage disease caused by a deficiency of the enzyme beta-mannosidase. Since then, 13 cases of beta-mannosidase deficiency in ten families have been described. A human beta-mannosidase mutation has been reported only by Alkhayat et al. in 1998., Objectives: To clarify its pathogenesis we did electron microscopic, biochemical and molecular biological investigations of a Japanese patient with beta-mannosidosis., Methods: Ultrastructural analyses, enzyme assays, cell culture and mRNA and genomic DNA were sequenced to find mutations in the beta-mannosidase gene., Results: Electron microscopy of skin biopsy specimens from the patient showed cytoplasmic vacuolation of lysosomes in blood and lymph vessels, endothelial cells, fibroblasts, secretory portions of eccrine sweat glands, neural cells and basal keratinocytes in the epidermis. This vacuolation was also observed in cultured keratinocytes and fibroblasts. Assays of seven enzyme activities in plasma and cultured skin fibroblasts showed a marked decrease of beta-mannosidase activity. Sequencing the beta-mannosidase cDNA revealed a four-base (ATAA) insertion between exons 7 and 8, resulting in a frameshift at codon 321 and termination at codon 325. Analysis of the patient's genomic DNA revealed a novel homozygous A(+1)-->G splice site mutation in intron 7., Conclusions: To our knowledge, this is the first case of beta-mannosidosis reported in Japan and the second report in which a gene mutation is identified. The biological importance of beta-mannose moieties in glycoproteins in basal keratinocytes is suggested.

The chemical mechanism of a retaining beta-mannosidase from Cellulomonas fimi has been characterized through steady-state kinetic analyses with a range of substrates, coupled with chemical rescue studies on both the wild-type enzyme and mutants in which active site carboxyl groups have been replaced. Studies with a series of aryl beta-mannosides of vastly different reactivities (pK(a)(lg) = 4-10) allowed kinetic isolation of the glycosylation and deglycosylation steps. Substrate inhibition was observed for all but the least reactive of these substrates. Brønsted analysis of k(cat) revealed a downward breaking plot (beta(lg) = -0.54 +/- 0.05) that is consistent with a change in rate-determining step (glycosylation to deglycosylation), and this was confirmed by partitioning studies with ethylene glycol. The pH dependence of k(cat)/K(m) follows an apparent single ionization of a group of pK(a) = 7.65 that must be protonated for catalysis. The tentative assignment of E429 as the acid-base catalyst of Man2A on the basis of sequence alignments with other family 2 glycosidases was confirmed by the increased turnover rate observed for the mutant E429A in the presence of azide and fluoride, leading to the production of beta-mannosyl azide and beta-mannosyl fluoride, respectively. A pH-dependent chemical rescue of E429A activity is also observed with citrate. Substantial oxocarbenium ion character at the transition state was demonstrated by the alpha-deuterium kinetic isotope effect for Man2A E429A of alpha-D(V) = 1.12 +/- 0.01. Surprisingly, this isotope effect was substantially greater in the presence of azide (alpha-D(V) = 1.166 +/- 0.009). Likely involvement of acid/base catalysis was revealed by the pH dependence of k(cat) for Man2A E429A, which follows a bell-shaped profile described by pK(a) values of 6.1 and 8.4, substantially different from that of the wild-type enzyme. The glycosidic bond cleaving activity of Man2A E519A and E519S nucleophile mutants is restored with azide and fluoride and appears to correlate with the corresponding "glycosynthase" activities. The contribution of the substrate 2-hydroxyl to stabilization of the Man2A glycosylation transition state (DeltaDeltaG() = 5.1 kcal mol(-1)) was probed using a 2-deoxymannose substrate. This value, surprisingly, is comparable to that found from equivalent studies with beta-glucosidases despite the geometric differences at C-2 and the importance of hydrogen bonding at that position. Modes of stabilizing the mannosidase transition state are discussed.

Mycobacteria are the causative agents of tuberculosis and several other significant diseases in humans. All species of mycobacteria synthesize abundant cell-wall mannolipids (phosphatidylinositol mannosides, lipoarabinomannan), a cytoplasmic methylmannose polysaccharide and O-mannosylated glycoproteins. To investigate whether these molecules are essential for mycobacterial growth, we have generated a Mycobacterium smegmatis mannose auxotroph by targeted deletion of the gene encoding phosphomannose isomerase (PMI). The PMI deletion mutant displayed a mild hyperseptation phenotype, but grew normally in media containing an exogenous source of mannose. When this mutant was suspended in media without mannose, ongoing synthesis of both the mannolipids and methylmannose polysaccharides was halted and the hyperseptation phenotype became more pronounced. These changes preceded a dramatic loss of viability after 10 h in mannose-free media. Mannose starvation did not lead to detectable changes in cell-wall ultrastructure or permeability to hydrophobic drugs, or to changes in the rate of biosynthesis of other plasma-membrane or wall-associated phospholipids. These results show that mannose metabolism is required for growth of M. smegmatis and that one or more mannose-containing molecules may play a role in regulating septation and cell division in these bacteria.

beta-1,4-Mannanases (mannanases), which hydrolyse mannans and glucomannans, are located in glycoside hydrolase families (GHs) 5 and 26. To investigate whether there are fundamental differences in the molecular architecture and biochemical properties of GH5 and GH26 mannanases, four genes encoding these enzymes were isolated from Cellvibrio japonicus and the encoded glycoside hydrolases were characterized. The four genes, man5A, man5B, man5C and man26B, encode the mannanases Man5A, Man5B, Man5C and Man26B, respectively. Man26B consists of an N-terminal signal peptide linked via an extended serine-rich region to a GH26 catalytic domain. Man5A, Man5B and Man5C contain GH5 catalytic domains and non-catalytic carbohydrate-binding modules (CBMs) belonging to families 2a, 5 and 10; Man5C in addition contains a module defined as X4 of unknown function. The family 10 and 2a CBMs bound to crystalline cellulose and ivory nut crystalline mannan, displaying very similar properties to the corresponding family 10 and 2a CBMs from Cellvibrio cellulases and xylanases. CBM5 bound weakly to these crystalline polysaccharides. The catalytic domains of Man5A, Man5B and Man26B hydrolysed galactomannan and glucomannan, but displayed no activity against crystalline mannan or cellulosic substrates. Although Man5C was less active against glucomannan and galactomannan than the other mannanases, it did attack crystalline ivory nut mannan. All the enzymes exhibited classic endo-activity producing a mixture of oligosaccharides during the initial phase of the reaction, although their mode of action against manno-oligosaccharides and glucomannan indicated differences in the topology of the respective substrate-binding sites. This report points to a different role for GH5 and GH26 mannanases from C. japonicus. We propose that as the GH5 enzymes contain CBMs that bind crystalline polysaccharides, these enzymes are likely to target mannans that are integral to the plant cell wall, while GH26 mannanases, which lack CBMs and rapidly release mannose from polysaccharides and oligosaccharides, target the storage polysaccharide galactomannan and manno-oligosaccharides.

Thermobifida fusca TM51, a thermophilic actinomycete isolated from composted horse manure, was found to produce a number of lignocellulose-degrading hydrolases, including endoglucanases, exoglucanases, endoxylanases, beta-xylosidases, endomannanases, and beta-mannosidases, when grown on cellulose or hemicellulose as carbon sources. beta-Mannosidases (EC 3.2.1.25), although contributing to the hydrolysis of hemicellulose fractions, such as galacto-mannans, constitute a lesser-known group of the lytic enzyme systems due to their low representation in the proteins secreted by hemicellulolytic microorganisms. An expression library of T. fusca, prepared in Streptomyces lividans TK24, was screened for beta-mannosidase activity to clone genes coding for mannosidases. One positive clone was identified, and a beta-mannosidase-encoding gene (manB) was isolated. Sequence analysis of the deduced amino acid sequence of the putative ManB protein revealed substantial similarity to known mannosidases in family 2 of the glycosyl hydrolase enzymes. The calculated molecular mass of the predicted protein was 94 kDa, with an estimated pI of 4.87. S. lividans was used as heterologous expression host for the putative beta-mannosidase gene of T. fusca. The purified gene product obtained from the culture filtrate of S. lividans was then subjected to more-detailed biochemical analysis. Temperature and pH optima of the recombinant enzyme were 53 degrees C and 7.17, respectively. Substrate specificity tests revealed that the enzyme exerts only beta-D-mannosidase activity. Its kinetic parameters, determined on para-nitrophenyl beta-D-mannopyranoside (pNP-betaM) substrate were as follows: K(m) = 180 micro M and V(max) = 5.96 micro mol min(-1) mg(-1); the inhibition constant for mannose was K(i) = 5.5 mM. Glucono-lacton had no effect on the enzyme activity. A moderate trans-glycosidase activity was also observed when the enzyme was incubated in the presence of pNP-alphaM and pNP-betaM; under these conditions mannosyl groups were transferred by the enzyme from pNP-betaM to pNP-alphaM resulting in the synthesis of small amounts (1 to 2%) of disaccharides.

Endo-beta-1,4-D-mannanases (beta-mannanase; EC 3.2.1.78) are endohydrolases that participate in the degradation of hemicellulose, which is closely associated with cellulose in plant cell walls. The beta-mannanase from Trichoderma reesei (Man5A) is composed of an N-terminal catalytic module and a C-terminal carbohydrate-binding module (CBM). In order to study the properties of the CBM, a construct encoding a mutant of Man5A lacking the part encoding the CBM (Man5ADeltaCBM), was expressed in T. reesei under the regulation of the Aspergillus nidulans gpdA promoter. The wild-type enzyme was expressed in the same way and both proteins were purified to electrophoretic homogeneity using ion-exchange chromatography. Both enzymes hydrolysed mannopentaose, soluble locust bean gum galactomannan and insoluble ivory nut mannan with similar rates. With a mannan/cellulose complex, however, the deletion mutant lacking the CBM showed a significant decrease in hydrolysis. Binding experiments using activity detection of Man5A and Man5ADeltaCBM suggests that the CBM binds to cellulose but not to mannan. Moreover, the binding of Man5A to cellulose was compared with that of an endoglucanase (Cel7B) from T. reesei.

Endophytic fungi were isolated from the stems, petioles, midribs, and leaves of seedlings of six native tree species collected from Doi Suthep-Pui National Park, Thailand. Endophytes were isolated from all tissue samples investigated, and taxa included five ascomycetes, eight anamorphic taxa, and numerous sterile mycelia. Twenty-six strains were tested for their ability to produce cellulase, mannanase, proteinase, and xylanase. The ability to produce these enzymes was distributed amongst the strains tested. Rainforest seedlings supported a diverse array of endophytes that have a wide range of enzymatic activities. The implication of enzyme production in relation to lifestyle abilities of the endophytes is discussed.

Beta-mannosidosis is an autosomal recessive lysosomal storage disease resulting from a deficiency of the lysosomal enzyme beta-mannosidase. The clinical manifestations of this disease in reported human cases are very heterogeneous ranging from relatively mild to moderately severe. This is in contrast with the severe prenatal onset seen in ruminant beta-mannosidosis. In humans, mental retardation, hearing loss, frequent infections, and behavioral problems are relatively common. Dysmorphology and skeletal involvement such as those seen in ruminants are unusual. The purpose of this study is to determine the range of clinical expression in human beta-mannosidosis resulting from null mutations. We determined that the beta-mannosidase gene consists of 17 exons. Intron-based PCR primers were designed and used to amplify each of the exons in genomic DNA isolated from patient fibroblasts. We identified two patients with null mutations. Results of the analysis showed that one patient was heterozygous for nonsense mutations G334T (E83X) in exon 2 and C1363T (Q426X) in exon 10, resulting in truncation of the deduced peptide sequence from 879 to 82 and 425 amino acids, respectively. The second patient was homozygous for a deletion mutation in exon 11 (1541delAT). This deletion causes a reading frame shift and 26 out of frame amino acids before a stop codon occurs in exon 12, resulting in truncation of the deduced peptide sequence from 879 to 510 amino acids. Because disease presentation in these patients with null mutations is very variable, ranging from mild to severe, we conclude that beta-mannosidosis in humans may indeed be milder than typical of other lysosomal storage disorders.

A molecular envelope of the beta-mannosidase from Trichoderma reesei has been obtained by combined use of solution small-angle X-ray scattering (SAXS) and protein crystallography. Crystallographic data at 4 A resolution have been used to enhance informational content of the SAXS data and to obtain an independent, more detailed protein shape. The phased molecular replacement technique using a low resolution SAXS model, building, and refinement of a free atom model has been employed successfully. The SAXS and crystallographic free atom models exhibit a similar globular form and were used to assess available crystallographic models of glycosyl hydrolases. The structure of the beta-galactosidase, a member of a family 2, clan GHA glycosyl hydrolases, shows an excellent fit to the experimental molecular envelope and distance distribution function of the beta-mannosidase, indicating gross similarities in their three-dimensional structures. The secondary structure of beta-mannosidase quantified by circular dichroism measurements is in a good agreement with that of beta-galactosidase. We show that a comparison of distance distribution functions in combination with 1D and 2D sequence alignment techniques was able to restrict the number of possible structurally homologous proteins. The method could be applied as a general method in structural genomics and related fields once protein solution scattering data are available.

The current experimental model for galactomannan biosynthesis in membrane-bound enzyme systems from developing legume-seed endosperms involves functional interaction between a GDP-mannose (Man) mannan synthase and a UDP-galactose (Gal) galactosyltransferase. The transfer specificity of the galactosyltransferase to the elongating mannan chain is critical in regulating the distribution and the degree of Gal substitution of the mannan backbone of the primary biosynthetic product. Detergent solubilization of the galactosyltransferase of fenugreek (Trigonella foenum-graecum) with retention of activity permitted the partial purification of the enzyme and the cloning and sequencing of the corresponding cDNA with proof of functional identity. We now document the positional specificity of transfer of ((14)C)Gal from UDP-((14)C)Gal to manno-oligosaccharide acceptors, chain lengths 5 to 8, catalyzed by the detergent-solubilized galactosyltransferase. Enzymatic fragmentation analyses of the labeled products showed that a single Gal residue was transferred per acceptor molecule, that the linkage was (1-->6)-alpha, and that there was transfer to alternative Man residues within the acceptor molecules. Analysis of the relative frequencies of transfer to alternative Man residues within acceptor oligosaccharides of different chain length allowed the deduction of the substrate subsite recognition requirement of the galactosyltransferase. The enzyme has a principal recognition sequence of six Man residues, with transfer of Gal to the third Man residue from the nonreducing end of the sequence. These observations are incorporated into a refined model for enzyme interaction in galactomannan biosynthesis.

Beta-mannosidase, a high-salt-soluble enzyme, increases in activity in seeds of tomato prior to the completion of germination. This increase occurs in both the lateral and micropylar endosperm and becomes more evident during post-germinative seedling growth. The beta-mannosidase activity profile is similar to that of endo beta-mannanase although it is the first to increase in the lateral endosperm. Tomato seed beta-mannosidase was purified to homogeneity and its cDNA (LeMside1) obtained by 3'-RACE PCR using oligonucleotide sequences based on four peptide sequences obtained from the purified enzyme. The derived amino acid sequence of the tomato beta-mannosidase shows the enzyme is a member of the Glycosyl Hydrolases Family 1 (GHF1) but has a very low sequence identity with that of beta-mannosidases from non-plant sources; no other plant sequence for the enzyme is known. There appears to be only one gene encoding beta-mannosidase in tomato, the sequence of which has been determined (LeMSide2). Its expression occurs first in the micropylar endosperm, and then declines after germination. This is followed by an increase in its expression in the lateral endosperm, which precedes that of the gene for endo beta-mannanase. Expression of the beta-mannosidase gene increases appreciably in the growing seedling embryo. With this report, the cloning of all three of the enzymes involved in galactomannan mobilization (endo beta-mannanase, alpha-galactosidase and beta-mannosidase) in tomato seeds has now been achieved.

Four experiments were conducted to determine the effects of adding a beta-mannanase preparation (Hemicell, ChemGen, Gaithersburg, MD) to corn-soybean meal-based diets on growth performance and nutrient digestibility of weanling and growing-finishing pigs. In Exp. 1, 156 weanling pigs (20 d, 6.27 kg BW) were allotted to four dietary treatments in a randomized complete block design. Treatments were a factorial arrangement of diet complexity (complex vs simple) and addition of 3-mannanase preparation (0 vs 0.05%). Pigs were fed in three dietary phases (Phase 1, d 0 to 14; Phase 2, d 14 to 28; and Phase 3, d 28 to 42). Pigs fed complex diets gained faster and were more efficient (P < 0.05) during Phase 1 compared with pigs fed simple diets. Overall, gain:feed ratio (G:F) tended to be improved (P < 0.10) for pigs fed complex diets and it was improved (P < 0.01) for those fed diets with beta-mannanase. In Exp. 2, 117 pigs (44 d, 13.62 kg BW) were allotted randomly to three dietary treatments. Dietary treatments were 1) a corn-soybean meal-based control, 2) the control diet with soybean oil added to increase metabolizable energy (ME) by 100 kcal/kg, and 3) the control diet with 0.05% beta-mannanase preparation. Beta-mannanase or soybean oil improved (P < 0.05) G:F compared with pigs fed the control diet. In Exp. 3, 60 pigs (22.5 kg BW) were allotted randomly to the three dietary treatments used in Exp. 2. Dietary treatments were fed in three phases (23 to 53 kg, 53 to 82 kg, and 82 to 109 kg with 0.95, 0.80, and 0.65% lysine, respectively). Overall, the addition of soybean oil tended to improve G:F (P < 0.10) compared with that of pigs fed the control diet, and G:F was similar (P > 0.54) for pigs fed diets with soybean oil or beta-mannanase. Also, addition of beta-mannanase increased ADG (P < 0.05) compared with that of pigs fed the control or soybean oil diets. There were no differences (P > or = 0.10) in longissimus muscle area or backfat; however, on a fat-free basis, pigs fed the diet with beta-mannanase had greater (P < 0.05) lean gain than pigs fed the control or soybean oil diets. In Exp. 4, 12 barrows (93 kg BW) were allotted randomly to one of the three dietary treatments used in Exp. 3. Addition of 3-mannanase had no effect (P > 0.10) on energy, nitrogen, phosphorus, or dry matter digestibility. These results suggest that beta-mannanase may improve growth performance in weanling and growing-finishing pigs but has minimal effects on nutrient digestibility.

A beta-mannosidase gene (PH0501) was identified in the Pyrococcus horikoshii genome and cloned and expressed in E. coli. The purified enzyme (BglB) was most specific for the hydrolysis of p-nitrophenyl-beta-D-mannopyranoside (pNP-Man) (Km: 0.44 mM) with a low turnover rate (kcat: 4.3 s(-1)). The beta-mannosidase has been classified as a member of family 1 of glycoside hydrolases. Sequence alignments and homology modeling showed an apparent conservation of its active site region with, remarkably, two unique active site residues, Gln77 and Asp206. These residues are an arginine and asparagine residue in all other known family 1 enzymes, which interact with the catalytic nucleophile and equatorial C2-hydroxyl group of substrates, respectively. The unique residues of P. horikoshii BglB were introduced in the highly active beta-glucosidase CelB of Pyrococcus furiosus and vice versa, yielding two single and one double mutant for each enzyme. In CelB, both substitutions R77Q and N206D increased the specificity for mannosides and reduced hydrolysis rates 10-fold. In contrast, BglB D206N showed 10-fold increased hydrolysis rates and 35-fold increased affinity for the hydrolysis of glucosides. In combination with inhibitor studies, it was concluded that the substituted residues participate in the ground-state binding of substrates with an equatorial C2-hydroxyl group, but contribute most to transition-state stabilization. The unique activity profile of BglB seems to be caused by an altered interaction between the enzyme and C2-hydroxyl of the substrate and a specifically increased affinity for mannose that results from Asp206.

Using PCR, cloning and sequencing techniques, a 1.1-kb complementary DNA fragment encoding for a beta-mannanase (mannan endo-1,4-beta-mannosidase, EC 3.2.1.78) has been identified in the digestive gland of blue mussel, Mytilus edulis. The cDNA sequence shows significant sequence identity to several beta-mannanases in glycoside hydrolase family 5. The beta-mannanase gene has been isolated and sequenced from gill tissue of blue mussel and contains five introns. The beta-mannanase has been expressed extracellularly in Pichia pastoris using the Saccharomyces cerevisiae alpha-factor signal sequence. The beta-mannanase was produced in a 14-L fermenter with an expression level of 900 mg.L-1. The expression level is strongly affected by the induction temperature. A two-step purification procedure, composed of a combination of immobilized metal ion affinity chromatography and ion exchange chromatography, is required to give a pure beta-mannanase. However, due to post-translational modifications, structural varieties regarding molecular mass and isoelectric point were obtained. The specific activity of the purified recombinant M. edulis beta-mannanase was close to that of the wild-type enzyme. Also pH and temperature optima were the same as for the native protein. In conclusion, P. pastoris is regarded as a suitable host strain for the production of blue mussel beta-mannanase. This is the first time a mollusc beta-mannanase has been characterized at the DNA level.

The glycohydrolase family 5 beta-mannanase Man5A from Mytilus edulis has been expressed in Pichia pastoris and purified in a form suitable for X-ray crystallographic analysis. Crystals were grown by the hanging-drop technique at 293 K using polyethylene glycol 5000 monomethylether as precipitant and dioxane as additive. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 61.8, b = 64.8, c = 90.7 A. Diffraction to 1.4 A resolution has been obtained at 100 K. Expression was also performed in the presence of selenomethionine. The incorporation of SeMet was estimated at 40% by amino-acid analysis and its presence in crystals was confirmed from the X-ray absorption scanning spectrum.

A method to characterize plant cell wall polysaccharides is presented. The complexity of the polymer structures and the large number of different charged and uncharged monosaccharides that make up plant polysaccharides have previously made analysis technically demanding and laborious. Polysaccharide analysis using carbohydrate gel electrophoresis (PACE) relies on derivatization of reducing ends of sugars and oligosaccharides with a fluorophore, followed by electrophoresis under optimized conditions in polyacrylamide gels. We show that PACE is a sensitive and simple tool for studying the monosaccharide composition of polysaccharides and of cell wall preparations. In combination with specific hydrolases, it can be used to analyze the structure of polysaccharides. Moreover, the specificity and kinetics of the plant polysaccharide hydrolases themselves can be quickly and effectively studied. PACE can detect as little as 500 fmol of monosaccharides and 100 fmol of oligosaccharides, and it is fast and quantitative., ((c)2001 Elsevier Science.)

The enzymatic hydrolysis of mannan-based hemicelluloses is technologically important for applications ranging from pulp and paper processing to food processing to gas and oil well stimulation. In many cases, thermostability and activity at elevated temperatures can be advantageous. To this end, the genes encoding beta-mannosidase (man2) and beta-mannanase (man5) from the hyperthermophilic bacteria Thermotoga neapolitana 5068 and Thermotoga maritima were isolated, cloned, and expressed in Escherichia coli. The amino acid sequences for the mannosidases from these organisms were 77% identical and corresponded to proteins with an M(r) of approximately 92 kDa. The translated nucleotide sequences for the beta-mannanase genes (man5) encoded polypeptides with an M(r) of 76 kDa that exhibited 84% amino acid sequence identity. The recombinant versions of Man2 and Man5 had similar respective biochemical and biophysical properties, which were also comparable to those determined for the native versions of these enzymes in T. neapolitana. The optimal temperature and pH for the recombinant Man2 and Man5 from both organisms were approximately 90 degrees C and 7.0, respectively. The presence of Man2 and Man5 in these two Thermotoga species indicates that galactomannan is a potential growth substrate. This was supported by the fact that beta-mannanase and beta-mannosidase activities were significantly stimulated when T. neapolitana was grown on guar or carob galactomannan. Maximum cell densities increased by at least tenfold when either guar or carob galactomannan was added to the growth medium. For T. neapolitana grown on guar at 83 degrees C, Man5 was secreted into the culture media, whereas Man2 was intracellular. These localizations were consistent with the presence and lack of signal peptides for Man5 and Man2, respectively. The identification of the galactomannan-degrading enzymes in these Thermotoga species adds to the list of biotechnologically important hemicellulases produced by members of this hyperthermophilic genera., (Copyright 2001 John Wiley & Sons, Inc.)