In vitro evaluation of the application of an optimized xylanase cocktail for improved monogastric feed digestibility.

Xylanases from glycoside hydrolase (GH) families 10 and 11 are common feed additives for broiler chicken diets due to their catalytic activity on the nonstarch polysaccharide xylan. This study investigated the potential of an optimized binary GH10 and GH11 xylanase cocktail to mitigate the antinutritional effects of xylan on the digestibility of locally sourced chicken feed. Immunofluorescence visualization of the activity of the xylanase cocktail on xylan in the yellow corn of the feed showed a substantial collapse in the morphology of cell walls. Secondly, the reduction in the viscosity of the digesta of the feed by the cocktail showed an effective degradation of the soluble fraction of xylan. Analysis of the xylan degradation products from broiler feeds by the xylanase cocktail showed that xylotriose and xylopentaose were the major xylooligosaccharides (XOS) produced. In vitro evaluation of the prebiotic potential of these XOS showed that they improved the growth of the beneficial bacteria Streptococcus thermophilus and Lactobacillus bulgaricus. The antibacterial activity of broths from XOS-supplemented probiotic cultures showed a suppressive effect on the growth of the extraintestinal infectious bacterium Klebsiella pneumoniae. Supplementing the xylanase cocktail in cereal animal feeds attenuated xylan's antinutritional effects by reducing digesta viscosity and releasing entrapped nutrients. Furthermore, the production of prebiotic XOS promoted the growth of beneficial bacteria while inhibiting the growth of pathogens. Based on these effects of the xylanase cocktail on the feed, improved growth performance and better feed conversion can potentially be achieved during poultry rearing.

Towards an understanding of the enzymatic degradation of complex plant mannan structures.

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 ß-mannanase, ß-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-ß-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 ß-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.

Bioconversion of spent coffee grounds to prebiotic mannooligosaccharides - an example of biocatalysis in biorefinery.

Spent coffee ground (SCG), an agro-industrial waste, have a high content of polysaccharides such as mannan, making it ideal for utilisation for the production of nutraceutical oligosaccharides. Recently, there has been growing interest in the production of mannooligosaccharides (MOS) for health promotion in humans and animals. MOS are reported to exhibit various bioactive properties, including prebiotic and antioxidant activity. In this study, SCG was Vivinal pretreated using NaOH, characterized and hydrolysed using a Bacillus sp. derived endo-ß-1,4-mannanase, Man26A, for MOS production. Structural analyses using Fourier-transform infrared spectroscopy (FT-IR) and thermogravimetric analysis (TGA) were conducted to assess the efficacy of the pretreatment. Lignin removal by the pretreatment from SCG was clearly shown by TGA. FT-IR, on the other hand, showed the presence of α-linked d-galactopyranoside (812 cm-1) and ß-linked d-mannopyranoside residues (817 cm-1) in both SCG samples, signifying the presence of mannan. Hydrolysis of pretreated SCG by Man26A produced mannobiose and mannotriose as the main MOS products. The effect of simulated gastric conditions on the MOS was investigated and showed this product to be suitable for oral administration. Finally, the prebiotic effect of the MOS on the growth of selected beneficial bacteria was investigated in vitro; showing that it enhanced Lactobacillus bulgaricus, Bacillus subtilis and Streptococcus thermophilus growth. These findings suggest that SCG is a viable source for the production of MOS which can be orally administered as prebiotics for effecting luxuriant growth of probiotic bacteria in the host's digestive tract, leading to a good health status.

Analysis of the galactomannan binding ability of ß-mannosidases, BtMan2A and CmMan5A, regarding their activity and synergism with a ß-mannanase.

Both ß-mannanases and ß-mannosidases are required for mannan-backbone degradation into mannose. In this study, two ß-mannosidases of glycoside hydrolase (GH) families 2 (BtMan2A) and 5 (CmMan5A) were evaluated for their substrate specificities and galactomannan binding ability. BtMan2A preferred short manno-oligomers, while CmMan5A preferred longer ones; DP >2, and galactomannans. BtMan2A displayed irreversible galactomannan binding, which was pH-dependent, with higher binding observed at low pH, while CmMan5A had limited binding. Docking and molecular dynamics (MD) simulations showed that BtMan2A galactomannan binding was stronger under acidic conditions (-8.4 kcal/mol) than in a neutral environment (-7.6 kcal/mol), and the galactomannan ligand was more unstable under neutral conditions than acidic conditions. Qualitative surface plasmon resonance (SPR) experimentally confirmed the reduced binding capacity of BtMan2A at pH 7. Finally, synergistic ß-mannanase to ß-mannosidase (BtMan2A or CmMan5A) ratios required for maximal galactomannan hydrolysis were determined. All CcManA to CmMan5A combinations were synergistic (≈1.2-fold), while combinations of CcManA with BtMan2A (≈1.0-fold) yielded no hydrolysis improvement. In conclusion, the low specific activity of BtMan2A towards long and galactose-containing oligomers and its non-catalytic galactomannan binding ability led to no synergy with the mannanase, making GH2 mannosidases ineffective for use in cocktails for mannan degradation.

Unraveling Synergism between Various GH Family Xylanases and Debranching Enzymes during Hetero-Xylan Degradation.

Enzymes classified with the same Enzyme Commission (EC) that are allotted in different glycoside hydrolase (GH) families can display different mechanisms of action and substrate specificities. Therefore, the combination of different enzyme classes may not yield synergism during biomass hydrolysis, as the GH family allocation of the enzymes influences their behavior. As a result, it is important to understand which GH family combinations are compatible to gain knowledge on how to efficiently depolymerize biomass into fermentable sugars. We evaluated GH10 (Xyn10D and XT6) and GH11 (XynA and Xyn2A) ß-xylanase performance alone and in combination with various GH family α-l-arabinofuranosidases (GH43 AXH-d and GH51 Abf51A) and α-d-glucuronidases (GH4 Agu4B and GH67 AguA) during xylan depolymerization. No synergistic enhancement in reducing sugar, xylose and glucuronic acid released from beechwood xylan was observed when xylanases were supplemented with either one of the glucuronidases, except between Xyn2A and AguA (1.1-fold reducing sugar increase). However, overall sugar release was significantly improved (≥1.1-fold reducing sugar increase) when xylanases were supplemented with either one of the arabinofuranosidases during wheat arabinoxylan degradation. Synergism appeared to result from the xylanases liberating xylo-oligomers, which are the preferred substrates of the terminal arabinofuranosyl-substituent debranching enzyme, Abf51A, allowing the exolytic ß-xylosidase, SXA, to have access to the generated unbranched xylo-oligomers. Here, it was shown that arabinofuranosidases are key enzymes in the efficient saccharification of hetero-xylan into xylose. This study demonstrated that consideration of GH family affiliations of the carbohydrate-active enzymes (CAZymes) used to formulate synergistic enzyme cocktails is crucial for achieving efficient biomass saccharification.

A Combination Approach in Inhibiting Type 2 Diabetes-Related Enzymes Using Ecklonia radiata Fucoidan and Acarbose.

Although there are chemotherapeutic efforts in place for Type 2 diabetes mellitus (T2DM), there is a need for novel strategies (including natural products) to manage T2DM. Fucoidan, a sulphated polysaccharide was extracted from Ecklonia radiata. The integrity of the fucoidan was confirmed by structural analysis techniques such as FT-IR, NMR and TGA. In addition, the fucoidan was chemically characterised and tested for cell toxicity. The fucoidan was investigated with regards to its potential to inhibit α-amylase and α-glucosidase. The fucoidan was not cytotoxic and inhibited α-glucosidase (IC50 19 µg/mL) more strongly than the standard commercial drug acarbose (IC50 332 µg/mL). However, the fucoidan lacked potency against α-amylase. On the other hand, acarbose was a more potent inhibitor of α-amylase (IC50 of 109 µg/mL) than α-glucosidase. Due to side effects associated with the use of acarbose, a combination approach using acarbose and fucoidan was investigated. The combination showed synergistic inhibition (>70%) of α-glucosidase compared to when the drugs were used alone. The medicinal implication of this synergism is that a regimen with a reduced acarbose dose may be used, thus minimising side effects to the patient, while achieving the desired therapeutic effect for managing T2DM.

Enzymatic Conversion of Mannan-Rich Plant Waste Biomass into Prebiotic Mannooligosaccharides.

A growing demand in novel food products for well-being and preventative medicine has attracted global attention on nutraceutical prebiotics. Various plant agro-processes produce large amounts of residual biomass considered "wastes", which can potentially be used to produce nutraceutical prebiotics, such as manno-oligosaccharides (MOS). MOS can be produced from the degradation of mannan. Mannan has a main backbone consisting of ß-1,4-linked mannose residues (which may be interspersed by glucose residues) with galactose substituents. Endo-ß-1,4-mannanases cleave the mannan backbone at cleavage sites determined by the substitution pattern and thus give rise to different MOS products. These MOS products serve as prebiotics to stimulate various types of intestinal bacteria and cause them to produce fermentation products in different parts of the gastrointestinal tract which benefit the host. This article reviews recent advances in understanding the exploitation of plant residual biomass via the enzymatic production and characterization of MOS, and the influence of MOS on beneficial gut microbiota and their biological effects (i.e., immune modulation and lipidemic effects) as observed on human and animal health.

Production and in vitro evaluation of prebiotic manno-oligosaccharides prepared with a recombinant Aspergillus niger endo-mannanase, Man26A.

In this study, a GH26 endo-mannanase (Man26A) from an Aspergillus niger ATCC 10864 strain, with a molecular mass of 47.8 kDa, was cloned in a yBBH1 vector and expressed in Saccharomyces cerevisiae Y294 strain cells. Upon fractionation by ultra-filtration, the substrate specificity and substrate degradation pattern of the endo-mannanase (Man26A) were investigated using ivory nut linear mannan and two galactomannan substrates with varying amounts of galactosyl substitutions, guar gum and locust bean gum. Man26A exhibited substrate specificity in the order: locust bean gum ≥ ivory nut mannan > guar gum; however, the enzyme generated more manno-oligosaccharides (MOS) from the galactomannans than from linear mannan during extended periods of mannan hydrolysis. MOS with a DP of 2-4 were the major products from mannan substrate hydrolysis, while guar gum also generated higher DP length MOS. All the Man26A generated MOS significantly improved the growth (approximately 3-fold) of the probiotic bacterial strains Streptococcus thermophilus and Bacillus subtilis in M9 minimal medium. Ivory nut mannan and locust bean gum derived MOS did not influence the auto-aggregation ability of the bacteria, while the guar gum derived MOS led to a 50 % reduction in bacterial auto-aggregation. On the other hand, all the MOS significantly improved bacterial biofilm formation (approximately 3-fold). This study suggests that the prebiotic characteristics exhibited by MOS may be dependent on their primary structure, i.e. galactose substitution and DP. Furthermore, the data suggests that the enzyme-generated MOS may be useful as potent additives to dietary foods.

Feruloyl esterase (FAE-1) sourced from a termite hindgut and GH10 xylanases synergy improves degradation of arabinoxylan.

Cereal feedstocks have high arabinoxylan content as their main hemicellulose, which is linked to lignin by hydroxycinnamic acids such as ferulic acid. The ferulic acid is linked to arabinoxylan by ester bonds, and generally, the high substitution of ferulic acid leads to a loss of activity of xylanases targeting the arabinoxylan. In the current study, a feruloyl esterase (FAE-1) from a termite hindgut bacteria was functionally characterised and used in synergy with xylanases during xylan hydrolysis. The FAE-1 displayed temperature and pH optima of 60 â„ƒ and 7.0, respectively. FAE-1 did not release reducing sugars from beechwood xylan (BWX), wheat arabinoxylan (WAX) and oat spelt xylan (OX), however, displayed high activity of  164.74 U/mg protein on p-nitrophenyl-acetate (pNPA). In contrast, the GH10 xylanases; Xyn10 and XT6, and a GH11 xylanase, Xyn2A, showed more than two-fold increased activity on xylan substrates with low sidechain substitutions; BWX and OX, compared to the highly branched substrate, WAX. Interestingly, the FAE-1 and GH10 xylanases (Xyn10D and XT6) displayed a degree of synergy (DS) that was higher than 1 in all enzyme loading combinations during WAX hydrolysis. The 75%XT6:25%FAE-1 synergistic enzyme combination increased the release of reducing sugars by 1.34-fold from WAX compared to the control, while 25%Xyn10D:75%FAE-1 synergistic combination released about 2.1-fold of reducing sugars from WAX compared to controls. These findings suggest that FAE-1 can be used in concert with xylanases, particularly those from GH10, to efficiently degrade arabinoxylans contained in cereal feedstocks for various industrial settings such as in animal feeds and baking.

Fucoidan Structure and Its Impact on Glucose Metabolism: Implications for Diabetes and Cancer Therapy.

Fucoidans are complex polysaccharides derived from brown seaweeds which consist of considerable proportions of L-fucose and other monosaccharides, and sulphated ester residues. The search for novel and natural bioproduct drugs (due to toxicity issues associated with chemotherapeutics) has led to the extensive study of fucoidan due to reports of it having several bioactive characteristics. Among other fucoidan bioactivities, antidiabetic and anticancer properties have received the most research attention in the past decade. However, the elucidation of the fucoidan structure and its biological activity is still vague. In addition, research has suggested that there is a link between diabetes and cancer; however, limited data exist where dual chemotherapeutic efforts are elucidated. This review provides an overview of glucose metabolism, which is the central process involved in the progression of both diseases. We also highlight potential therapeutic targets and show the relevance of fucoidan and its derivatives as a candidate for both cancer and diabetes therapy.

Delineating functional properties of a cello-oligosaccharide and ß-glucan specific cellobiohydrolase (GH5_38): Its synergism with Cel6A and Cel7A for ß-(1,3)-(1,4)-glucan degradation.

Cellulase cocktails formulated to degrade crystalline cellulose generally contain cellobiohydrolases (CBHs), referred to as CBHI (Cel7A) and CBHII (Cel6A), as the major constituents. The combined hydrolytic activities of CBHI and CBHII improve the release of fermentable sugars (ß-1,4-cellobiose as the main product) from crystalline cellulose. In this study, a novel cellobiohydrolase (Exg-D) sourced from a metagenome of hindgut bacterial symbionts of a termite was heterologouly expressed, purified, and functionally characterised. Exg-D specific activity was higher on insoluble barley ß-glucan (38.94 U/mg protein), soluble wheat flour ß-glucan (12.71 U/mg protein) and oat ß-glucan (8.89 U/mg protein) compared to cellulosic substrates; Avicel and CMC. We further explored Exg-D activity on the unpretreated or NaOH-pretreated (mercerised) Avicel and compared its activity to commercially available CBHI and CBHII on these celluloses. CBHI displayed the highest activity of 4.74 U/mg protein on mercerised cellulose followed by CBHII (2.14 U/mg protein), while Exg-D activity on untreated and mercerised cellulose was 1.66 and 1.67 U/mg protein, respectively. The high activity of CBHI was supported by binding assays, which revealed that CBHI has a higher binding capacity towards crystalline cellulose compared to Exg-D and CBHII. Only CBHI and CBHII showed synergism during the hydrolysis of mercerised Avicel, showing a degree of synergy (DS) of about 1.299 and yielded about 1.43 µmol/ml of reducing sugars higher than control. In contrast, Exg-D and CBHII displayed synergism during ß-glucan degradation, displaying a DS of about 1.22. Thus, we propose that Exg-D should only be used synergistically with other CBHs to degrade mixed linked-ß-(1,3)-(1,4)-glucan.

Tryptic Mapping Based Structural Insights of Endo-1, 4-ß-Xylanase from Thermomyces lanuginosus VAPS-24.

An endo-1,4-ß-xylanase, XynA, from Thermomyces lanuginosus VAPS-24, was purified to homogeneity and exhibited a molecular mass of approximately 20 kDa. The protein sequence of XynA was found to be similar to those of other Thermomyces lanuginosus derived xylanases and, as a result, could be used as a model enzyme for understanding the protein structure-activity relationship and facilitating protein engineering to design enzyme variants with desirable properties. Therefore, this xylanase will be an attractive candidate for applications in the biofuel and fine chemical industries for the degradation of xylans in steam pre-treated biomass.

Lignocellulosic pretreatment-mediated phenolic by-products generation and their effect on the inhibition of an endo-1,4-ß-xylanase from Thermomyces lanuginosus VAPS-24.

The inhibitory effect of eight model lignin derivatives (ferulic acid, guaiacol, kraft lignin (alkali, low sulfonate content), p-coumaric acid, gallic acid, syringic acid, vanillin and vanillic acid) on XynA activity was evaluated. The model lignin derivatives viz. gallic acid, vanillic acid and vanillin were inhibitory to XynA activity, with an over 50% reduction in activity at concentrations as low as 0.5 mg/ml. However, enzyme deactivation studies in the absence of substrate showed that these pretreatment by-products do not interact with the enzyme except when in the presence of its substrate. The effect of the main structural properties of the pretreatment-derived phenolics, for example their hydroxyl and carbonyl group types, on XynA enzyme inhibition was investigated. The presence of carbonyl groups in phenolics appeared to confer stronger inhibitory effects than hydroxyl groups on XynA activity. The hydrolytic potential of XynA was not inhibited by a mixture of phenolics derived after steam pretreatment of woody biomass (Douglas fir and Black wattle). It appears as if the liquors from steam-pretreated woody biomass did not possess high enough phenolic content to confer XynA inhibition. The xylanase (XynA from Thermomyces lanuginosus) is, therefore, a striking choice for application in biofuel and fine chemical industries for the xylan degradation in steam-pretreated biomass.

A Novel Dimeric Exoglucanase (GH5_38): Biochemical and Structural Characterisation towards its Application in Alkyl Cellobioside Synthesis.

An exoglucanase (Exg-D) from the glycoside hydrolase family 5 subfamily 38 (GH5_38) was heterologously expressed and structurally and biochemically characterised at a molecular level for its application in alkyl glycoside synthesis. The purified Exg-D existed in both dimeric and monomeric forms in solution, which showed highest activity on mixed-linked ß-glucan (88.0 and 86.7 U/mg protein, respectively) and lichenin (24.5 and 23.7 U/mg protein, respectively). They displayed a broad optimum pH range from 5.5 to 7 and a temperature optimum from 40 to 60 °C. Kinetic studies demonstrated that Exg-D had a higher affinity towards ß-glucan, with a Km of 7.9 mg/mL and a kcat of 117.2 s-1, compared to lichenin which had a Km of 21.5 mg/mL and a kcat of 70.0 s-1. The circular dichroism profile of Exg-D showed that its secondary structure consisted of 11% α-helices, 36% ß-strands and 53% coils. Exg-D performed transglycosylation using p-nitrophenyl cellobioside as a glycosyl donor and several primary alcohols as acceptors to produce methyl-, ethyl- and propyl-cellobiosides. These products were identified and quantified via thin-layer chromatography (TLC) and liquid chromatography-mass spectrometry (LC-MS). We concluded that Exg-D is a novel and promising oligomeric glycoside hydrolase for the one-step synthesis of alkyl glycosides with more than one monosaccharide unit.

Fucoidan from Ecklonia maxima is a powerful inhibitor of the diabetes-related enzyme, α-glucosidase.

Ecklonia maxima, an endemic South African seaweed, is a potential source of beneficial bioactive compounds. Among these compounds, fucoidan, a sulphated polysaccharide has a wide range of bioactivities including anti-diabetic activity. In this study, fucoidan was extracted from E. maxima by the hot water extraction method and then characterised by colorimetric assays for sugar composition. The extraction from E. maxima yielded 6.89% fucoidan which was found to contain 4.45 ± 0.25% L-fucose and 6.01 ± 0.53% sulphate. The water extracted E. maxima fucoidan had a low molecular weight of approximately 10 kDa. Structural studies (FT-IR, NMR and XRD) confirmed the structure and integrity of the fucoidan to be similar to previously studied fucoidans in literature. Finally, the activities of starch digestive enzymes; α-amylase and α-glucosidase, were investigated in the presence of the E. maxima fucoidan extract. Fucoidan from E. maxima was observed to be a potent mixed-type inhibitor of α-glucosidase with an IC50 range of 0.27-0.31 mg.ml-1, which was significantly lower than the commercial anti-diabetic standard, acarbose. Our present study demonstrated that fucoidan from E. maxima is a more powerful inhibitor compared to some standard anti-diabetic compounds and thus shows great potential for managing type 2 diabetes.

A mini review of xylanolytic enzymes with regards to their synergistic interactions during hetero-xylan degradation.

This review examines the recent models describing the mode of action of various xylanolytic enzymes and how these enzymes can be applied (sequentially or simultaneously) with their distinctive roles in mind to achieve efficient xylan degradation. With respect to homeosynergy, synergism appears to be as a result of ß-xylanase and/or oligosaccharide reducing-end ß-xylanase liberating xylo-oligomers (XOS) that are preferred substrates of the processive ß-xylosidase. With regards to hetero-synergism, two cross relationships appear to exist and seem to be the reason for synergism between the enzymes during xylan degradation. These cross relations are the debranching enzymes such as α-glucuronidase or side-chain cleaving enzymes such as carbohydrate esterases (CE) removing decorations that would have hindered back-bone-cleaving enzymes, while backbone-cleaving-enzymes liberate XOS that are preferred substrates of the debranching and side-chain-cleaving enzymes. This interaction is demonstrated by high yields in co-production of xylan substituents such as arabinose, glucuronic acid and ferulic acid, and XOS. Finally, lytic polysaccharide monooxygenases (LPMO) have also been implicated in boosting whole lignocellulosic biomass or insoluble xylan degradation by glycoside hydrolases (GH) by possibly disrupting entangled xylan residues. Since it has been observed that the same enzyme (same Enzyme Commission, EC, classification) from different GH or CE and/or AA families can display different synergistic interactions with other enzymes due to different substrate specificities and properties, in this review, we propose an approach of enzyme selection (and mode of application thereof) during xylan degradation, as this can improve the economic viability of the degradation of xylan for producing precursors of value added products.

The effect of an oligosaccharide reducing-end xylanase, BhRex8A, on the synergistic degradation of xylan backbones by an optimised xylanolytic enzyme cocktail.

Xylan, the most abundant hemicellulose in lignocellulosic biomass, requires a consortium of xylanolytic enzymes to achieve its complete de-polymerisation. As global interest in using xylan-containing lignocellulosic feedstocks for biofuel production increases, an accompanying knowledge on how to efficiently depolymerise these feedstocks into fermentable sugars is required. Since it has been observed that the same enzyme [i.e. an enzyme with the same EC (Enzyme Commission) classification] from different GH families can display different substrate specificities and properties, we evaluated GH10 (XT6) and 11 (Xyn2A) xylanase performance alone, and in combination, during xylan depolymerisation. Synergistic enhancement with respect to reducing sugar release was observed when Xyn2A at 75% loading was supplemented with 25% loading of XT6 for both beechwood glucuronoxylan (1.14-fold improvement) and wheat arabinoxylan (1.1-fold improvement) degradation. Following this, the optimised xylanase mixture was dosed with an oligosaccharide reducing-end xylanase (Rex8A) from either Bifidobacterium adolescentis or Bacillus halodurans for further synergistic enhancement. Dosing 75% of the xylanase mixture (Xyn2A:XT6 at 75:25%) with 25% loading of Rex8A led to an enhancement of reducing sugar (up to an 1.1-fold improvement) and xylose release (up to an 1.5-fold improvement); however, this effect was both xylan and Rex8A specific. Using thin layer chromatography, synergism appeared to be a result of the GH10 and 11 xylanases liberating xylo-oligomers that are preferred substrates of the processive Rex8As. Rex8As then hydrolysed xylo-oligomers to xylose - and xylobiose which was the preferred substrate for xylosidase, SXA. This likely explains why there was a significant improvement in xylose release in the presence of Rex8As. Here, it was shown that Rex8As are key enzymes in the efficient saccharification of hetero-xylan into xylose, a major component of lignocellulosic substrates.

Time dependence of enzyme synergism during the degradation of model and natural lignocellulosic substrates.

Cellulosic ethanol production relies on the biochemical (enzymatic) conversion of lignocellulose to fermentable sugars and ultimately to bioethanol. However, the cost of lignocellulolytic enzymes is a limiting factor in the commercialisation of this technology. This therefore necessitates the optimisation of lignocellulolytic enzyme cocktails through the elucidation of synergistic interactions between enzymes so as to improve lignocellulose hydrolysis and also lower protein loadings in these reactions. However, many factors affect the synergism that occurs between these lignocellulolytic enzymes, such as enzyme ratios, substrate characteristics, substrate loadings, enzyme loadings and time. This review examines the effect of time on the synergistic dynamics between lignocellulolytic enzymes during the hydrolysis of both complex (true) lignocellulosic substrates and model substrates. The effect of sequential and simultaneous application of the lignocellulolytic enzymes on the synergistic dynamics during the hydrolysis of these substrates is also explored in this review. Finally, approaches are further proposed for efficient and synergistic hydrolysis of both complex lignocellulosic substrates and model substrates. With respect to the synergistic enzymatic hydrolysis of lignocellulosic biomass, this review exposed knowledge gaps that should be covered in future work in order to fully understand how enzyme synergism works: e.g. elucidating protein to protein interactions that exist between these enzymes in establishing synergy; and the effect of lignocellulose degradation products of one enzyme on the behaviour of the other enzyme and ultimately their synergistic relationship.

A review of the enzymatic hydrolysis of mannans and synergistic interactions between ß-mannanase, ß-mannosidase and α-galactosidase.

Mannan is an important polysaccharide found in softwoods and many other plant sources. Mannans from various sources display large differences in composition, structure and complexity. To hydrolyse mannan into its monomer sugars requires a number of enzymes working in synergy. This review examines mannan structure and the enzymes required for its hydrolysis. Several studies have investigated the effect of supplementing ß-mannanases with ß-mannosidases and α-galactosidases in binary and ternary combinations. Synergistic enhancement of hydrolysis has been found in some, but not all cases. In the case of mannosidases, they sometimes display an anti-synergistic effect with mannanases, most likely due to competition for binding sites. Most importantly, in the case of α-galactosidases, the same enzyme from different families display differences in synergistic interactions due to different specificities. An improved understanding of enzyme interactions will aid in achieving enhanced hydrolysis of mannans and higher sugar yields. This review highlights areas which require further research in order to gain a better understanding of mannan hydrolysis and utilisation. Such knowledge is very important as this can be used in the optimisation of commercial or purified enzyme mixtures to improve the economic viability of the conversion of high mannan-containing biomass such as softwoods into fermentable sugars for bioethanol production.

ß-mannanase (Man26A) and α-galactosidase (Aga27A) synergism - a key factor for the hydrolysis of galactomannan substrates.

This study investigated the behavior of mannan-degrading enzymes, specifically focusing on differences with respect to their substrate specificities and their synergistic associations with enzymes from different glycoside hydrolase (GH) families. Galactosidases from Cyamopsis tetragonolobus seeds (Aga27A, GH27) and Aspergillus niger (AglC, GH36) were evaluated for their abilities to synergistically interact with mannanases from Clostridium cellulovorans (ManA, GH5) and A. niger (Man26A, GH26) in hydrolysis of guar gum and locust bean gum. Among the mannanases, Man26A was more efficient at hydrolyzing both galactomannan substrates, while among the galactosidases; Aga27A was the most effective at removing galactose substituents on both galactomannan substrates and galactose-containing oligosaccharides. An optimal protein mass ratio of glycoside hydrolases required to maximize the release of both reducing sugar and galactose residues was determined. Clear synergistic enhancement of locust bean gum hydrolysis with respect to reducing sugar release was observed when both mannanases at 75% enzyme dosage were supplemented with 25% enzyme protein dosage of Aga27A. At a protein ratio of 75% Man26A to 25% Aga27A, the presence of Man26A significantly enhanced galactose release by 25% Aga27A (2.36 fold) with locust bean gum, compared to when Aga27A was used alone at 100% enzyme protein dosage. A dosage of Aga27A at 75% and ManA at 25% protein content liberated the highest reducing sugar release on guar gum hydrolysis. A dosage of Man26A and Aga27A at 75-25% protein content, respectively, liberated reducing sugar release equivalent to that when Man26A was used alone at 100% protein content. From the findings obtained in this study, it was observed that the GH family classification of an enzyme affects its substrate specificity and synergistic interactions with other glycoside hydrolases from different families (more so than its EC classification). The GH26 Man26A and GH27 Aga27A enzymes appeared to be more promising for applications that involve the hydrolysis of galactomannan containing biomass. This method of screening for maximal compatibility between various GH families can ultimately lead to a more rational development of tailored enzyme cocktails for lignocellulose hydrolysis.