24 research outputs found
Formulation of an enzyme cocktail, HoloMix, using cellulolytic and xylanolytic enzyme core sets for effective degradation of various pre-treated hardwoods
Expected release date-April 202
Towards an understanding of the enzymatic degradation of complex plant mannan structures
DATA AVAILABILITY: Data sharing is not applicable to this article as no new data were created or analysed in this study.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.Open access funding provided by University of Pretoria. The Central Research Fund (CRF) from the Dean of Natural and Agricultural Sciences (Entity: 1114A5534) and the National Research Foundation of South Africa – Thuthuka grant, the Research Development Programme (RDP) from the University of Pretoria.http://www.springer.comchemistry/biotech/journal/11274BiochemistryGeneticsMicrobiology and Plant Patholog
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
Revisiting the phenomenon of cellulase action : not all endo-and exo-cellulase interactions are synergistic
The conventional endo–exo synergism model has extensively been supported in literature,
which is based on the perception that endoglucanases (EGs) expose or create accessible sites on the
cellulose chain to facilitate the action of processive cellobiohydrolases (CBHs). However, there is
a lack of information on why some bacterial and fungal CBHs and EGs do not exhibit synergism.
Therefore, the present study evaluated and compared the synergistic relationships between cellulases
from different microbial sources and provided insights into how different GH families govern
synergism. The results showed that CmixA2 (a mixture of TlCel7A and CtCel5A) displayed the
highest effect with BaCel5A (degree of synergy for reducing sugars and glucose of 1.47 and 1.41,
respectively) in a protein mass ratio of 75–25%. No synergism was detected between CmixB1/B2
(as well as CmixC1/C2) and any of the EGs, and the combinations did not improve the overall
cellulose hydrolysis. These findings further support the hypothesis that “not all endo-to exo-cellulase
interactions are synergistic”, and that the extent of synergism is dependent on the composition of
cellulase systems from various sources and their compatibility in the cellulase cocktail. This method
of screening for maximal compatibility between exo- and endo-cellulases constitutes a critical step
towards the design of improved synergistic cellulose-degrading cocktails for industrial-scale biomass
degradation.National Research Foundation (NRF); Department of Science & Technology of South Africa and Rhodes University.http://www.mdpi.com/journal/catalystspm2021BiochemistryGeneticsMicrobiology and Plant Patholog
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
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
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 -amylase and -glucosidase. The fucoidan was not cytotoxic and inhibited
-glucosidase (IC50 19 g/mL) more strongly than the standard commercial drug acarbose (IC50
332 g/mL). However, the fucoidan lacked potency against -amylase. On the other hand, acarbose
was a more potent inhibitor of -amylase (IC50 of 109 g/mL) than -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 -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.The German Academic Exchange Service (DAAD) In-Region Scholarship; the South African National Research Foundation (NRF); Henderson Scholarship; Pearson- Young Memorial scholarship; the University of Pretoria; Rhodes University and KelpX.https://www.mdpi.com/journal/pharmaceuticsam2022BiochemistryGeneticsMicrobiology and Plant Patholog