Defining the frontiers of synergism between cellulolytic enzymes for improved hydrolysis of lignocellulosic feedstocks

Lignocellulose has economic potential as a bio-resource for the production of value-added products (VAPs) and biofuels. The commercialization of biofuels and VAPs requires efficient enzyme cocktail activities that can lower their costs. However, the basis of the synergism between enzymes that compose cellulolytic enzyme cocktails for depolymerizing lignocellulose is not understood. This review aims to address the degree of synergism (DS) thresholds between the cellulolytic enzymes and how this can be used in the formulation of effective cellulolytic enzyme cocktails. DS is a powerful tool that distinguishes between enzymes’ synergism and anti-synergism during the hydrolysis of biomass. It has been established that cellulases, or cellulases and lytic polysaccharide monooxygenases (LPMOs), always synergize during cellulose hydrolysis. However, recent evidence suggests that this is not always the case, as synergism depends on the specific mechanism of action of each enzyme in the combination. Additionally, expansins, nonenzymatic proteins responsible for loosening cell wall fibers, seem to also synergize with cellulases during biomass depolymerization. This review highlighted the following four key factors linked to DS: (1) a DS threshold at which the enzymes synergize and produce a higher product yield than their theoretical sum, (2) a DS threshold at which the enzymes display synergism, but not a higher product yield, (3) a DS threshold at which enzymes do not synergize, and (4) a DS threshold that displays anti-synergy. This review deconvolutes the DS concept for cellulolytic enzymes, to postulate an experimental design approach for achieving higher synergism and cellulose conversion yields.The Central Research Fund initiative (CRF) of the Natural and Agricultural Science Deans Office at University of the Free State, the University of Pretoria and Rhodes University.https://www.mdpi.com/journal/catalystsam2022BiochemistryGeneticsMicrobiology and Plant Patholog

Evaluating Feruloyl Esterase—Xylanase Synergism for Hydroxycinnamic Acid and Xylo-Oligosaccharide Production from Untreated, Hydrothermally Pre-Treated and Dilute-Acid Pre-Treated Corn Cobs:

Agricultural residues are considered the most promising option as a renewable feedstock for biofuel and high valued-added chemical production due to their availability and low cost. The efficient enzymatic hydrolysis of agricultural residues into value-added products such as sugars and hydroxycinnamic acids is a challenge because of the recalcitrant properties of the native biomass. Development of synergistic enzyme cocktails is required to overcome biomass residue recalcitrance, and achieve high yields of potential value-added products. In this study, the synergistic action of two termite metagenome-derived feruloyl esterases (FAE5 and FAE6), and an endo-xylanase (Xyn11) from Thermomyces lanuginosus, was optimized using 0.5% (w/v) insoluble wheat arabinoxylan (a model substrate) and then applied to 1% (w/v) corn cobs for the efficient production of xylo-oligosaccharides (XOS) and hydroxycinnamic acids

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) β-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 β-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.The National Research Foundation of South Africa (NRF) and the Department of Science and Technology (DST)/Council for Scientific and Industrial Research (CSIR) Masters and PhD bursarie. Article Processing charges (APC) were funded by The University of the Free State.https://www.mdpi.com/journal/moleculesam2022BiochemistryGeneticsMicrobiology and Plant Patholog

Covalent immobilisation of an Aspergillus niger derived endo-1,4-beta-mannanase, man26A, on glutaraldehyde-activated chitosan nanoparticles for the effective production of prebiotic MOS from soybean meal

An Aspergillus niger endo-1,4- -mannanase, Man26A, was confirmed by FTIR and XRD to be immobilised on glutaraldehyde-activated chitosan nanoparticles via covalent bonding. The immobilisation (%) and activity yields (%) were 82.25% and 20.75%, respectively. The biochemical properties (pH, temperature optima, and stability) were then comparatively evaluated for both the free and immobilised Man26A. The optimal activity of Man26A shifted to a lower pH after immobilisation (pH 2.0–3.0, from pH 5 for the free enzyme), with the optimum temperature remaining unchanged (60 C). The two enzymes exhibited identical thermal stability, maintaining 100% activity for the first 6 h at 55 C. Substrate-specific kinetic analysis showed that the two enzymes had similar affinities towards locust bean gum (LBG) with varied Vmax values. In contrast, they showed various affinities towards soybean meal (SBM) and similar Vmax values. The immobilised enzyme was then employed in the enhancement of the functional feed/prebiotic properties of SBM from poultry feed, increasing mannooligosaccharides (MOS) quantities. The SBM main hydrolysis products were mannobiose (M2) and mannose (M1). The SBM-produced sugars could be utilised as a carbon source by probiotic bacteria; Streptococcus thermophilus, Bacillus subtilis, and Lactobacillus bulgaricus. The results indicate that the immobilised enzyme has the potential for use in the sustainable and cost-effective production of prebiotic MOS from agricultural biomass.Rhodes University’s Research Council (RC) grant and a Rated Researcher Grant (RRG).https://www.mdpi.com/journal/agronomyam2023BiochemistryGeneticsMicrobiology and Plant Patholog

Accelerated sludge solubilisation under sulphate reducing conditions: the effect of hydrolytic enzymes on sludge floc size distribution and EPS composition

Extracellular polymeric substances (EPS) are the construction materials for microbial aggregates such as biofilms, flocs and sludge, and greatly contribute to the structural integrity of sludge flocs in wastewater treatment processes. The loss of integrity of the sewage sludge floc is believed to be due to enhanced hydrolysis of important structural components such as lignin, protein and cellulose in the sludge floc matrix. The mechanism of enhanced sludge floc fracture, due to the action of enzymes hydrolysing these structural components, remains a key element in our understanding of how the floc integrity in systems utilising a sulphate reducing system is compromised. A range of relatively non-specific exogenous enzymes (ß-glucosidase, cellulase, proteases: trypsin, pronase E and chymotrypsin) were added to a sulphidogenic bioreactor- (containing both sulphate reducing bacteria (SRB) and a methanogenic bacterial system) and a (control) methanogenic bioreactor sample, and the effect of these enzymes on sludge floc size (diameter) distribution and EPS composition was investigated. Sludge samples from the bioreactors were examined under bright field and differential interference contrast light microscopy. Proteolytic and glucohydrolytic activity of the enzymes were monitored using standard enzyme assaying techniques, and Bradford, Somogyi-Nelson, and total carbohydrate assays were performed to establish the composition of the EPS (after extraction with 3% (v/v) glutaraldehyde and Sephacryl S-400 size exclusion chromatography). Sludge flocs present in the sulphidogenic environment of the sulphidogenic bioreactor were found to have smaller diameters than their counterparts present in the methanogenic bioreactor. The addition of hydrolytic (i.e. proteolytic and glycohydrolytic) enzymes resulted in an increased rate of matrix hydrolysis, leading to increased rates of floc fracture and deflocculation. The presence of ß-glucosidase, cellulase, and proteases naturally residing within the sludge floc was confirmed. We propose that the addition of commercially available enzymes may be prohibitively costly on a large scale, and that the activity of the enzymes naturally residing within the floc matrix be optimised or enhanced. As the bulk of the EPS was shown to be composed of mainly polysaccharides, we propose that by increasing the activity of the naturally occurring ß-glucosidases residing within the floc matrix, the process of deflocculation may be enhanced. As sulphide has been shown to increase the activity of this very important key enzyme, we propose that this is one of the contributing factors why sludge solubilisation is accelerated under sulphate reducing conditions

Enzymatic Hydrolysis of Softwood Derived Paper Sludge by an In Vitro Recombinant Cellulase Cocktail for the Production of Fermentable Sugars

CITATION:Malgas S, Rose SH, van Zyl WH, Pletschke BI. Enzymatic Hydrolysis of Softwood Derived Paper Sludge by an In Vitro Recombinant Cellulase Cocktail for the Production of Fermentable Sugars. Catalysts. 2020; 10(7):775. https://doi.org/10.3390/catal10070775Abstract: Paper sludge is an attractive biomass feedstock for bioconversion to ethanol due to its low cost and the lack of pretreatment required for its bioprocessing. This study assessed the use of a recombinant cellulase cocktail (mono-components: S. cerevisiae-derived PcBGL1B (BGL), TeCel7A (CBHI), ClCel6A (CBHII) and TrCel5A (EGII) mono-component cellulase enzymes) for the efficient saccharification of softwood-derived paper sludge to produce fermentable sugars. The paper sludge mainly contained 74.3% moisture and 89.7% (per dry mass (DM)) glucan with a crystallinity index of 91.5%. The optimal protein ratio for paper sludge hydrolysis was observed at 9.4: 30.2: 30.2: 30.2% for BGL: CBHI: CBHII: EGII. At a protein loading of 7.5 mg/g DW paper sludge, the yield from hydrolysis was approximately 80%, based on glucan, with scanning electron microscopy micrographs indicating a significant alteration in the microfibril size (length reduced from ≥ 2 mm to 93 µm) of the paper sludge. The paper sludge hydrolysis potential of the Opt CelMix (formulated cellulase cocktail) was similar to the commercial Cellic CTec2® and Celluclast® 1.5 L cellulase preparations and better than Viscozyme® L. Low enzyme loadings (15 mg/g paper sludge) of the Opt CelMix and solid loadings ranging between 1 to 10% (w/v) rendered over 80% glucan conversion. The high glucose yields attained on the paper sludge by the low enzyme loading of the Opt CelMix demonstrated the value of enzyme cocktail optimisation on specific substrates for efficient cellulose conversion to fermentable sugars

Increasing the scale of peroxidase production by Streptomyces sp. strain BSII#1

AIMS : To optimise peroxidase production by Streptomyces sp. strain BSII#1, up to 3 l culture volumes. METHODS AND RESULTS : Peroxidase production by Streptomyces sp. strain BSII#1 was optimised in terms of production temperature and pH and the use of lignin-based model chemical inducers. The highest peroxidase activity (1.30±0.04 U ml-1) in 10 ml culture volume was achieved in a complex production medium (pH 8) at 37°C in the presence of 0.1 mmol l-1 veratryl alcohol, which was greater than those reported previously. Scale-up to 100 ml and 400 ml culture volumes resulted in decreased peroxidase production (0.53±0.10 U ml- 1 and 0.26±0.08 U ml-1 respectively). However, increased aeration improved peroxidase production with the highest production achieved using an airlift bioreactor (4.76±0.46 U ml-1 in 3 l culture volume). CONCLUSIONS : Veratryl alcohol (0.1 mmol l-1) is an effective inducer of peroxidase production by Streptomyces sp. strain BSII#1. However, improved aeration increased peroxidase production in larger volumes without the use of an inducer, surpassing induced yields in an optimised small-scale process. SIGNIFICANCE AND IMPACT OF THE STUDY : Only a limited number of reports in literature have focused on the up-scaling of bacterial peroxidase production. There remains opportunity for feasible large-scale production of bacterial peroxidases with potentially novel biocatalytic properties.National Research Foundation (NRF) of South Africa,Water Research Commission.http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1365-2672hb201

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

Both β-mannanases and β-mannosidases are required for mannan-backbone degradation into mannose. In this study, two β-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 β-mannanase to β-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.The National Research Foundation (NRF) of South Africa, Rhodes University and the University of Pretoria.http://www.elsevier.com/locate/csbjhj2023BiochemistryGeneticsMicrobiology and Plant Patholog

A novel dimeric exoglucanase (GH5_38)

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 β-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 β-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% β-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

Vaccine and antibody production in plants : developments and computational tools

K.K.D. and P.S. sincerely acknowledge the support and facility provided by the M.D. University, Rohtak, Haryana. P.S. acknowledges the infrastructural support from Department of Science and Technology, Govt. of India through FIST grant (Grant No. 1196 SR/FST/LS-I/2017/4).Plants as bioreactors have been widely used to express efficient vaccine antigens against viral, bacterial and protozoan infections. To date, many different plant-based expression systems have been analyzed, with a growing preference for transient expression systems. Antibody expression in diverse plant species for therapeutic applications is well known, and this review provides an overview of various aspects of plant-based biopharmaceutical production. Here, we highlight conventional and gene expression technologies in plants along with some illustrative examples. In addition, the portfolio of products that are being produced and how they relate to the success of this field are discussed. Stable and transient gene expression in plants, agrofiltration and virus infection vectors are also reviewed. Further, the present report draws attention to antibody epitope prediction using computational tools, one of the crucial steps of vaccine design. Finally, regulatory issues, biosafety and public perception of this technology are also discussed.PostprintPeer reviewe