A novel dimeric exoglucanase (GH5_38)
- Mafa, Mpho S, Dirr, Heinrich W, Malgas, Samkelo, Krause, Rui W M, Pletschke, Brett I
- Authors: Mafa, Mpho S , Dirr, Heinrich W , Malgas, Samkelo , Krause, Rui W M , Pletschke, Brett I
- Date: 2020
- Subjects: To be catalogued
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/193976 , vital:45412 , xlink:href="https://doi.org/10.3390/molecules25030746"
- Description: 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.
- Full Text:
- Date Issued: 2020
- Authors: Mafa, Mpho S , Dirr, Heinrich W , Malgas, Samkelo , Krause, Rui W M , Pletschke, Brett I
- Date: 2020
- Subjects: To be catalogued
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/193976 , vital:45412 , xlink:href="https://doi.org/10.3390/molecules25030746"
- Description: 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.
- Full Text:
- Date Issued: 2020
Combination of CTec2 and GH5 or GH26 Endo-Mannanases for Effective Lignocellulosic Biomass Degradation
- Malgas, Samkelo, Pletschke, Brett I
- Authors: Malgas, Samkelo , Pletschke, Brett I
- Date: 2020
- Subjects: To be catalogued
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/429397 , vital:72607 , xlink:href="https://doi.org/10.3390/catal10101193"
- Description: Among endo-mannanases, glycoside hydrolase (GH) family 26 enzymes have been shown to be more catalytically active than GH5 enzymes on mannans. However, only GH5 endo-mannanases have been used for the formulation of enzyme cocktails. In this study, Bacillus sp.-derived GH5 and GH26 endo-mannanases were comparatively analysed biochemically for their synergistic action with a commercial cellulase blend, CTec2, during pre-treated lignocellulose degradation. Substrate specificity and thermo-stability studies on mannan substrates showed that GH26 endo-mannanase was more catalytically active and stable than GH5. GH26 also exhibited higher binding affinity for mannan than GH5, while GH5 showed more affinity for lignocellulosic substrates than GH26. Applying the endo-mannanases in combination with CTec2 for lignocellulose degradation led to synergism with a 1.3-fold increase in reducing sugar release compared to when CTec2 was used alone. This study showed that using the activity of endo-mannanases displayed with model substrates is a poor predictor of their activity and synergism on complex lignocelluloses.
- Full Text:
- Date Issued: 2020
- Authors: Malgas, Samkelo , Pletschke, Brett I
- Date: 2020
- Subjects: To be catalogued
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/429397 , vital:72607 , xlink:href="https://doi.org/10.3390/catal10101193"
- Description: Among endo-mannanases, glycoside hydrolase (GH) family 26 enzymes have been shown to be more catalytically active than GH5 enzymes on mannans. However, only GH5 endo-mannanases have been used for the formulation of enzyme cocktails. In this study, Bacillus sp.-derived GH5 and GH26 endo-mannanases were comparatively analysed biochemically for their synergistic action with a commercial cellulase blend, CTec2, during pre-treated lignocellulose degradation. Substrate specificity and thermo-stability studies on mannan substrates showed that GH26 endo-mannanase was more catalytically active and stable than GH5. GH26 also exhibited higher binding affinity for mannan than GH5, while GH5 showed more affinity for lignocellulosic substrates than GH26. Applying the endo-mannanases in combination with CTec2 for lignocellulose degradation led to synergism with a 1.3-fold increase in reducing sugar release compared to when CTec2 was used alone. This study showed that using the activity of endo-mannanases displayed with model substrates is a poor predictor of their activity and synergism on complex lignocelluloses.
- Full Text:
- Date Issued: 2020
Delineating functional properties of a cello-oligosaccharide and Bglucan specific cellobiohydrolase (GH5_38): Its synergism with Cel6A and Cel7A for B-(1,3)-(1,4)-glucan degradation
- Mafa, Mpho S, Malgas, Samkelo, Rashamuse, Konanani, Pletschke, Brett I
- Authors: Mafa, Mpho S , Malgas, Samkelo , Rashamuse, Konanani , Pletschke, Brett I
- Date: 2020
- Subjects: To be catalogued
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/429425 , vital:72609 , xlink:href="https://doi.org/10.1016/j.carres.2020.108081"
- Description: 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 (β-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 β-glucan (38.94 U/mg protein), soluble wheat flour β-glucan (12.71 U/mg protein) and oat β-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 β-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-β-(1,3)-(1,4)-glucan.
- Full Text:
- Date Issued: 2020
- Authors: Mafa, Mpho S , Malgas, Samkelo , Rashamuse, Konanani , Pletschke, Brett I
- Date: 2020
- Subjects: To be catalogued
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/429425 , vital:72609 , xlink:href="https://doi.org/10.1016/j.carres.2020.108081"
- Description: 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 (β-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 β-glucan (38.94 U/mg protein), soluble wheat flour β-glucan (12.71 U/mg protein) and oat β-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 β-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-β-(1,3)-(1,4)-glucan.
- Full Text:
- Date Issued: 2020
Evaluating Feruloyl Esterase—Xylanase Synergism for Hydroxycinnamic Acid and Xylo-Oligosaccharide Production from Untreated, Hydrothermally Pre-Treated and Dilute-Acid Pre-Treated Corn Cobs:
- Mkabayi, Lithalethu, Malgas, Samkelo, Wilhelmi, Brendan S, Pletschke, Brett I
- Authors: Mkabayi, Lithalethu , Malgas, Samkelo , Wilhelmi, Brendan S , Pletschke, Brett I
- Date: 2020
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/149240 , vital:38818 , https://doi.org/10.3390/agronomy10050688
- Description: 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.
- Full Text:
- Date Issued: 2020
- Authors: Mkabayi, Lithalethu , Malgas, Samkelo , Wilhelmi, Brendan S , Pletschke, Brett I
- Date: 2020
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/149240 , vital:38818 , https://doi.org/10.3390/agronomy10050688
- Description: 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.
- Full Text:
- Date Issued: 2020
Fucoidan from Ecklonia maxima is a powerful inhibitor of the diabetes-related enzyme, Éø-glucosidase
- Daub, Chantal D, Mabate, Blessing, Malgas, Samkelo, Pletschke, Brett I
- Authors: Daub, Chantal D , Mabate, Blessing , Malgas, Samkelo , Pletschke, Brett I
- Date: 2020
- Subjects: To be catalogued
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/425982 , vital:72304 , xlink:href="https://doi.org/10.1016/j.ijbiomac.2020.02.161"
- Description: 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.
- Full Text:
- Date Issued: 2020
Fucoidan from Ecklonia maxima is a powerful inhibitor of the diabetes-related enzyme, Éø-glucosidase
- Authors: Daub, Chantal D , Mabate, Blessing , Malgas, Samkelo , Pletschke, Brett I
- Date: 2020
- Subjects: To be catalogued
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/425982 , vital:72304 , xlink:href="https://doi.org/10.1016/j.ijbiomac.2020.02.161"
- Description: 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.
- Full Text:
- Date Issued: 2020
The Effects of Alkaline Pretreatment on Agricultural Biomasses (Corn Cob and Sweet Sorghum Bagasse) and Their Hydrolysis by a Termite-Derived Enzyme Cocktail:
- Mafa, Mpho S, Malgas, Samkelo, Bhattacharya, Abhishek, Rashamuse, Konanani, Pletschke, Brett I
- Authors: Mafa, Mpho S , Malgas, Samkelo , Bhattacharya, Abhishek , Rashamuse, Konanani , Pletschke, Brett I
- Date: 2020
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/160273 , vital:40430 , https://doi.org/10.3390/agronomy10081211
- Description: Sweet sorghum bagasse (SSB) and corncob (CC) have been identified as promising feedstocks for the production of second-generation biofuels and other value-added chemicals. In this study, lime (Ca(OH)2) and NaOH pretreatment efficacy for decreasing recalcitrance from SSB and CC was investigated, and subsequently, the pretreated biomass was subjected to the hydrolytic action of an in-house formulated holocellulolytic enzyme cocktail (HEC-H). Compositional analysis revealed that SSB contained 29.34% lignin, 17.75% cellulose and 16.28% hemicellulose, while CC consisted of 22.51% lignin, 23.58% cellulose and 33.34% hemicellulose. Alkaline pretreatment was more effective in pretreating CC biomass compared to the SSB biomass.
- Full Text:
- Date Issued: 2020
- Authors: Mafa, Mpho S , Malgas, Samkelo , Bhattacharya, Abhishek , Rashamuse, Konanani , Pletschke, Brett I
- Date: 2020
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/160273 , vital:40430 , https://doi.org/10.3390/agronomy10081211
- Description: Sweet sorghum bagasse (SSB) and corncob (CC) have been identified as promising feedstocks for the production of second-generation biofuels and other value-added chemicals. In this study, lime (Ca(OH)2) and NaOH pretreatment efficacy for decreasing recalcitrance from SSB and CC was investigated, and subsequently, the pretreated biomass was subjected to the hydrolytic action of an in-house formulated holocellulolytic enzyme cocktail (HEC-H). Compositional analysis revealed that SSB contained 29.34% lignin, 17.75% cellulose and 16.28% hemicellulose, while CC consisted of 22.51% lignin, 23.58% cellulose and 33.34% hemicellulose. Alkaline pretreatment was more effective in pretreating CC biomass compared to the SSB biomass.
- Full Text:
- Date Issued: 2020
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