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.

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 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.

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.

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.

Synergism of fungal and bacterial cellulases and hemicellulases: a novel perspective for enhanced bio-ethanol production.

The complex structure of lignocellulose requires the involvement of a suite of lignocellulolytic enzymes for bringing about an effective de-polymerization. Cellulases and hemicellulases from both fungi and bacteria have been studied extensively. This review illustrates the mechanism of action of different cellulolytic and hemi-cellulolytic enzymes and their distinctive roles during hydrolysis. It also examines how different approaches can be used to improve the synergistic interaction between fungal and bacterial glycosyl hydrolases with a focus on fungal cellulases and bacterial hemicellulases. The approach entails the role of cellulosomes and their improvement through incorporation of novel enzymes and evaluates the recent break-through in the construction of designer cellulosomes and their extension towards improving fungal and bacterial synergy. The proposed approach also advocates the incorporation and cell surface display of designer cellulosomes on non-cellulolytic solventogenic strains along with the innovative application of combined cross-linked enzyme aggregates (combi-CLEAs) as an economically feasible and versatile tool for improving the synergistic interaction through one-pot cascade reactions.

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.

The effects of Cannabis sativa and cannabinoids on the inhibition of pancreatic lipase - An enzyme involved in obesity.

INTRODUCTION: Obesity is a chronic noncommunicable disease characterized by excessive body fat that can have negative health consequences. Obesity is a complex disease caused by a combination of genetic, environmental, and lifestyle factors. It is characterized by a discrepancy between caloric intake and expenditure. Obesity increases the risk of acquiring major chronic diseases, including heart disease, stroke, cancer, and Type 2 diabetes mellitus (T2DM). Currently, the inhibition of pancreatic lipases (PL) is a promising pharmacological therapy for obesity and weight management. In this study, the inhibition of pancreatic lipase by Cannabis sativa (C. sativa) plant extract and cannabinoids was investigated. METHODS: The inhibitory effect was assessed using p-nitrophenyl butyrate (pNPB), and the results were obtained by calculating the percentage relative activity and assessed using one-way analysis of variance (ANOVA). Kinetic studies and spectroscopy techniques were used to evaluate the mode of inhibition. Diet-induced; and diabetic rat models were studied to evaluate the direct effects of C. sativa extract on PL activity. RESULTS: Kinetic analyses showed that the plant extracts inhibited pancreatic lipase, with tetrahydrocannabinol (THC) and cannabinol (CBN) being the potential cause of the inhibition noted for the C. sativa plant extract. CBN and THC inhibited the pancreatic lipase activity in a competitive manner, with the lowest residual enzyme activity of 52 % observed at a 10 µg/mL concentration of CBN and 39 % inhibition at a 25 µg/mL concentration of THC. Circular dichroism (CD) spectroscopy revealed that the inhibitors caused a change in the enzyme's secondary structure. At low concentrations, THC showed potential for synergistic inhibition with orlistat. C.sativa treatment in an in vivo rat model confirmed its inhibitory effects on pancreatic lipase activity. CONCLUSION: The findings in this study provided insight into the use of cannabinoids as pancreatic lipase inhibitors and the possibility of using these compounds to develop new pharmacological treatments for obesity.

Inhibitory effects of selected cannabinoids against dipeptidyl peptidase IV, an enzyme linked to type 2 diabetes.

Ethnopharmacological relevance: In recent times the decriminalisation of cannabis globally has increased its use as an alternative medication. Where it has been used in modern medicinal practises since the 1800s, there is limited scientific investigation to understand the biological activities of this plant. Aim of the study: Dipeptidyl peptidase IV (DPP-IV) plays a key role in regulating glucose homeostasis, and inhibition of this enzyme has been used as a therapeutic approach to treat type 2 diabetes. However, some of the synthetic inhibitors for this enzyme available on the market may cause undesirable side effects. Therefore, it is important to identify new inhibitors of DPP-IV and to understand their interaction with this enzyme. Methods: In this study, four cannabinoids (cannabidiol, cannabigerol, cannabinol and Δ9-tetrahydrocannabinol) were evaluated for their inhibitory effects against recombinant human DPP-IV and their potential inhibition mechanism was explored using both in vitro and in silico approaches. Results: All four cannabinoids resulted in a dose-dependent response with IC50 values of between 4.0 and 6.9 µg/mL. Kinetic analysis revealed a mixed mode of inhibition. CD spectra indicated that binding of cannabinoids results in structural and conformational changes in the secondary structure of the enzyme. These findings were supported by molecular docking studies which revealed best docking scores at both active and allosteric sites for all tested inhibitors. Furthermore, molecular dynamics simulations showed that cannabinoids formed a stable complex with DPP-IV protein via hydrogen bonds at an allosteric site, suggesting that cannabinoids act by either inducing conformational changes or blocking the active site of the enzyme. Conclusion: These results demonstrated that cannabinoids may modulate DPP-IV activity and thereby potentially assist in improving glycaemic regulation in type 2 diabetes.

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.

Marine Cellulases and their Biotechnological Significance from Industrial Perspectives.

Marine microorganisms represent virtually unlimited sources of novel biological compounds and can survive extreme conditions. Cellulases, a group of enzymes that are able to degrade cellulosic materials, are in high demand in various industrial and biotechnological applications, such as in the medical and pharmaceutical industries, food, fuel, agriculture, and single-cell protein, and as probiotics in aquaculture. The cellulosic biopolymer is a renewable resource and is a linearly arranged polysaccharide of glucose, with repeating units of disaccharide connected via ß-1,4-glycosidic bonds, which are broken down by cellulase. A great deal of biodiversity resides in the ocean, and marine systems produce a wide range of distinct, new bioactive compounds that remain available but dormant for many years. The marine environment is filled with biomass from known and unknown vertebrates and invertebrate microorganisms, with much potential for use in medicine and biotechnology. Hence, complex polysaccharides derived from marine sources are a rich resource of microorganisms equipped with enzymes for polysaccharides degradation. Marine cellulases' extracts from the isolates are tested for their functional role in degrading seaweed and modifying wastes to low molecular fragments. They purify and renew environments by eliminating possible feedstocks of pollution. This review aims to examine the various types of marine cellulase producers and assess the ability of these microorganisms to produce these enzymes and their subsequent biotechnological applications.

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.

Interaction of silver nanoparticles with catechol O-methyltransferase: Spectroscopic and simulation analyses.

Catechol O-methyltransferase, an enzyme involved in the metabolism of catechol containing compounds, catalyzes the transfer of a methyl group between S-adenosylmethionine and the hydroxyl groups of the catechol. Furthermore it is considered a potential drug target for Parkinson's disease as it metabolizes the drug levodopa. Consequently inhibitors of the enzyme would increase levels of levodopa. In this study, absorption, fluorescence and infrared spectroscopy as well as computational simulation studies investigated human soluble catechol O-methyltransferase interaction with silver nanoparticles. The nanoparticles form a corona with the enzyme and quenches the fluorescence of Trp143. This amino acid maintains the correct structural orientation for the catechol ring during catalysis through a static mechanism supported by a non-fluorescent fluorophore-nanoparticle complex. The enzyme has one binding site for AgNPs in a thermodynamically spontaneous binding driven by electrostatic interactions as confirmed by negative ΔG and ΔH and positive ΔS values. Fourier transform infrared spectroscopy within the amide I region of the enzyme indicated that the interaction causes relaxation of its ß-structures, while simulation studies indicated the involvement of six polar amino acids. These findings suggest AgNPs influence the catalytic activity of catechol O-methyltransferase, and therefore have potential in controlling the activity of the enzyme.

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.

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.

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.

Production and characterisation of a novel actinobacterial DyP-type peroxidase and its application in coupling of phenolic monomers.

The extracellular peroxidase from Streptomyces albidoflavus BSII#1 was purified to near homogeneity using sequential steps of acid and acetone precipitation, followed by ultrafiltration. The purified peroxidase was characterised and tested for the ability to catalyse coupling reactions between selected phenolic monomer pairs. A 46-fold purification of the peroxidase was achieved, and it was shown to be a 46 kDa haem peroxidase. Unlike other actinobacteria-derived peroxidases, it was only inhibited (27 % inhibition) by relatively high concentrations of sodium azide (5 mM) and was capable of oxidising eleven (2,4-dichlorophenol, 2,6-dimethoxyphenol, 4-tert-butylcatechol, ABTS, caffeic acid, catechol, guaiacol, l-DOPA, o-aminophenol, phenol, pyrogallol) of the seventeen substrates tested. The peroxidase remained stable at temperatures of up to 80 °C for 60 min and retained >50 % activity after 24 h between pH 5.0-9.0, but was most sensitive to incubation with hydrogen peroxide (H2O2; 0.01 mM), l-cysteine (0.02 mM) and ascorbate (0.05 mM) for one hour. It was significantly inhibited by all organic solvents tested (p ≤ 0.05). The Km and Vmax values of the partially purified peroxidase with the substrate 2,4-DCP were 0.95 mM and 0.12 mmol min-1, respectively. The dyes reactive blue 4, reactive black 5, and Azure B, were all decolourised to a certain extent: approximately 30 % decolourisation was observed after 24 h (1 µM dye). The peroxidase successfully catalysed coupling reactions between several phenolic monomer pairs including catechin-caffeic acid, catechin-catechol, catechin-guaiacol and guaiacol-syringaldazine under the non-optimised conditions used in this study. Genome sequencing confirmed the identity of strain BSII#1 as a S. albidoflavus strain. In addition, the genome sequence revealed the presence of one peroxidase gene that includes the twin arginine translocation signal sequence of extracellular proteins. Functional studies confirmed that the peroxidase produced by S. albidoflavus BSII#1 is part of the dye-decolourising peroxidase (DyP-type) family.

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.

The degradation of lignocellulose in a chemically and biologically generated sulphidic environment.

Acid mine drainage waters are characterised by a low pH, high concentrations of heavy metals, high levels of sulphate salts and low concentrations of organic material. The biological treatment of these waters has been a subject of increasing focus as an alternative to physico-chemical treatment. The utilisation of lignocellulose as a carbon source has been restricted by the amount of reducing equivalents available within the lignocellulose matrix. This present study demonstrated that lignocellulose could be utilised as a carbon source for sulphate reduction. It was shown that the initial reduction of sulphate observed using lignocellulose as a carbon source was due to the easily extractable components. This degradation resulted in the production of sulphide ( approximately 500 mg/l), which further aided in the degradation of lignin (observed as a release of aromatic compounds), allowing greater access to cellulose (and release of reducing sugars).