Riaan Den Haan - Academia.edu (original) (raw)

Papers by Riaan Den Haan

Research paper thumbnail of Effect of N-linked glycosylation on the activity and stability of a β-glucosidase from Putranjiva roxburghii

International journal of biological macromolecules, 2018

In this study, the effect of N-glycosylation on the conformational and functional stability of Pu... more In this study, the effect of N-glycosylation on the conformational and functional stability of Putranjiva roxburghii family 1 β-glucosidase (PRGH1) enzyme was investigated. The deglycosylation of PRGH1 was carried out by using PNGase F enzyme and confirmed by SDS-PAGE and carbohydrate estimation. Comparative analysis with respect to enzyme activity, stability and aggregation behaviour was carried out for the glycosylated and deglycosylated PRGH1. The deglycosylation of PRGH1 affected enzyme activity to a certain extent only where Kwas not affected but a slight reduction in Vfor various substrates was observed. Circular dichroism, fluorescence studies and differential scanning calorimetry (DSC) analysis demonstrated the possible effect of glycosylation on local and/or global conformational dynamics of protein and its effect on the thermostability of PRGH1. DSC results showed deglycosylated form had lower Tas compared to the glycosylated form of PRGH1. The PRGH1 was found to be more s...

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Research paper thumbnail of Strain Breeding Enhanced Heterologous Cellobiohydrolase Secretion by Saccharomyces cerevisiae in a Protein Specific Manner

Biotechnology Journal

The yeast Saccharomyces cerevisiae has a long association with alcoholic fermentation industries ... more The yeast Saccharomyces cerevisiae has a long association with alcoholic fermentation industries and has received renewed interest as a biocatalyst for second-generation bioethanol production. Rational engineering strategies are used to create yeast strains for consolidated bioprocessing of lignocellulosic biomass. Although significant progress is made in this regard with the expression of different cellulolytic activities in yeast, cellobiohydrolase (CBH) titers remain well below ideal levels. Through classical breeding, S. cerevisiae strains with up to twofold increased CBH secretion titers is obtained in strains expressing a single gene copy. An increase of up to 3.5-fold in secreted cellobiohydrolase activity is subsequently shown for strains expressing the heterologous gene on a high copy episomal vector. To our knowledge, this is the first report of classical breeding being used to enhance heterologous protein secretion and also the most significant enhancement of CBH secretion in yeast yet reported. This enhanced secretion phenotype is specific for cellobiohydrolase I secretion, indicating that reporter protein properties might be a major determining factor for efficient protein secretion in yeast. By exploring the latent potential of different S. cerevisiae strains, the authors show that the allele pool of various strains is a valuable engineering resource to enhance secretion in yeast.

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Research paper thumbnail of Improvement of ethanol production from crystalline cellulose via optimizing cellulase ratios in cellulolytic Saccharomyces cerevisiae

Biotechnology and bioengineering, Jun 23, 2017

Crystalline cellulose is one of the major contributors to the recalcitrance of lignocellulose to ... more Crystalline cellulose is one of the major contributors to the recalcitrance of lignocellulose to degradation, necessitating high dosages of cellulase to digest, thereby impeding the economic feasibility of cellulosic biofuels. Several recombinant cellulolytic yeast strains have been developed to reduce the cost of enzyme addition, but few of these strains are able to efficiently degrade crystalline cellulose due to their low cellulolytic activities. Here, by combining the cellulase ratio optimization with a novel screening strategy, we successfully improved the cellulolytic activity of a Saccharomyces cerevisiae strain displaying four different synergistic cellulases on the cell surface. The optimized strain exhibited an ethanol yield from Avicel of 57% of the theoretical maximum, and a 60% increase of ethanol titer from rice straw. To our knowledge, this work is the first optimization of the degradation of crystalline cellulose by tuning the cellulase ratio in a cellulase cell-surf...

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Research paper thumbnail of Heterologous expression of cellulase genes in natural Saccharomyces cerevisiae strains

Applied microbiology and biotechnology, Jan 28, 2016

Enzyme cost is a major impediment to second-generation (2G) cellulosic ethanol production. One st... more Enzyme cost is a major impediment to second-generation (2G) cellulosic ethanol production. One strategy to reduce enzyme cost is to engineer enzyme production capacity in a fermentative microorganism to enable consolidated bio-processing (CBP). Ideally, a strain with a high secretory phenotype, high fermentative capacity as well as an innate robustness to bioethanol-specific stressors, including tolerance to products formed during pre-treatment and fermentation of lignocellulosic substrates should be used. Saccharomyces cerevisiae is a robust fermentative yeast but has limitations as a potential CBP host, such as low heterologous protein secretion titers. In this study, we evaluated natural S. cerevisiae isolate strains for superior secretion activity and other industrially relevant characteristics needed during the process of lignocellulosic ethanol production. Individual cellulases namely Saccharomycopsis fibuligera Cel3A (β-glucosidase), Talaromyces emersonii Cel7A (cellobiohydro...

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Research paper thumbnail of Method for Fermenting Cellulosics

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Research paper thumbnail of Engineering cellulolytic ability into bioprocessing organisms

Applied Microbiology and Biotechnology, Mar 1, 2010

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Research paper thumbnail of Differential expression of the Trichoderma reesei ß-xylanase II ( xyn2 ) gene in the xylose-fermenting yeast Pichia stipitis

Appl Microbiol Biotechnol, 2001

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Research paper thumbnail of Yeast Expressing Cellulases for Simultaneous Saccharification and Fermentation Using Cellulose

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Research paper thumbnail of Yeast Expressing Saccharolytic Enzymes for Consolidated Bioprocessing Using Starch and Cellulose

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Research paper thumbnail of Enhanced xylan degradation and utilisation by Pichia stipitis overproducing fungal xylanolytic enzymes

Enzyme Microb Technol, 2003

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Research paper thumbnail of Engineering of a novel cellulose-adherent cellulolytic Saccharomyces cerevisiae for cellulosic biofuel production

Scientific reports, Jan 15, 2016

Cellulosic biofuel is the subject of increasing attention. The main obstacle toward its economic ... more Cellulosic biofuel is the subject of increasing attention. The main obstacle toward its economic feasibility is the recalcitrance of lignocellulose requiring large amount of enzyme to break. Several engineered yeast strains have been developed with cellulolytic activities to reduce the need for enzyme addition, but exhibiting limited effect. Here, we report the successful engineering of a cellulose-adherent Saccharomyces cerevisiae displaying four different synergistic cellulases on the cell surface. The cellulase-displaying yeast strain exhibited clear cell-to-cellulose adhesion and a "tearing" cellulose degradation pattern; the adhesion ability correlated with enhanced surface area and roughness of the target cellulose fibers, resulting in higher hydrolysis efficiency. The engineered yeast directly produced ethanol from rice straw despite a more than 40% decrease in the required enzyme dosage for high-density fermentation. Thus, improved cell-to-cellulose interactions pr...

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Research paper thumbnail of Differential expression of the Trichoderma reesei β-xylanase II ( xyn2 ) gene in the xylose-fermenting yeast Pichia stipitis

Appl Microbiol Biotechnol, 2001

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Research paper thumbnail of Combined cell-surface display- and secretion-based strategies for production of cellulosic ethanol with Saccharomyces cerevisiae

Biotechnology for biofuels, 2015

Engineering Saccharomyces cerevisiae to produce heterologous cellulases is considered as a promis... more Engineering Saccharomyces cerevisiae to produce heterologous cellulases is considered as a promising strategy for production of bioethanol from lignocellulose. The production of cellulase is usually pursued by one of the two strategies: displaying enzyme on the cell surface or secreting enzyme into the medium. However, to our knowledge, the combination of the two strategies in a yeast strain has not been employed. In this study, heterologous endoglucanase (EG) and cellobiohydrolase I (CBHI) were produced in a β-glucosidase displaying S. cerevisiae strain using cell-surface display, secretion, or a combined strategy. Strains EG-D-CBHI-D and EG-S-CBHI-S (with both enzymes displayed on the cell surface or with both enzymes secreted to the surrounding medium) showed higher ethanol production (2.9 and 2.6 g/L from 10 g/L phosphoric acid swollen cellulose, respectively), than strains EG-D-CBHI-S and EG-S-CBHI-D (with EG displayed on cell surface and CBHI secreted, or vice versa). After 3-...

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Research paper thumbnail of Enhanced xylan degradation and utilisation by Pichia stipitis overproducing fungal xylanolytic enzymes. Enzyme Microb Technol

Enzyme and Microbial Technology

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Research paper thumbnail of Lignocellulosic Hydrolysate Inhibitors Selectively Inhibit/Deactivate Cellulase Performance

Enzyme and Microbial Technology, 2015

In this study, we monitored the inhibition and deactivation effects of various compounds associat... more In this study, we monitored the inhibition and deactivation effects of various compounds associated with lignocellulosic hydrolysates on individual and combinations of cellulases. Tannic acid representing polymeric lignin residues strongly inhibited cellobiohydrolase 1 (CBH1) and β-glucosidase 1 (BGL1), but had a moderate inhibitory effect on endoglucanase 2 (EG2). Individual monomeric lignin residues had little or no inhibitory effect on hydrolytic enzymes. However, coniferyl aldehyde and syringaldehyde substantially decreased the activity of CBH1 and deactivated BGL1. Acetic and formic acids also showed strong inhibition of BGL1 but not CBH1 and EG2, whereas tannic, acetic and formic acid strongly inhibited a combination of CBH1 and EG2 during Avicel hydrolysis. Diminishing enzymatic hydrolysis is largely a function of inhibitor concentration and the enzyme-inhibitor relationship, rather than contact time during the hydrolysis process (i.e. deactivation). This suggests that decreased rates of hydrolysis during the enzymatic depolymerisation of lignocellulosic hydrolysates may be imparted by other factors related to substrate crystallinity and accessibility.

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Research paper thumbnail of Next-generation cellulosic ethanol technologies and their contribution to a sustainable Africa

Interface focus, Jan 6, 2011

The world is currently heavily dependent on oil, especially in the transport sector. However, ris... more The world is currently heavily dependent on oil, especially in the transport sector. However, rising oil prices, concern about environmental impact and supply instability are among the factors that have led to greater interest in renewable fuel and green chemistry alternatives. Lignocellulose is the only foreseeable renewable feedstock for sustainable production of transport fuels. The main technological impediment to more widespread utilization of lignocellulose for production of fuels and chemicals in the past has been the lack of low-cost technologies to overcome the recalcitrance of its structure. Both biological and thermochemical second-generation conversion technologies are currently coming online for the commercial production of cellulosic ethanol concomitantly with heat and electricity production. The latest advances in biological conversion of lignocellulosics to ethanol with a focus on consolidated bioprocessing are highlighted. Furthermore, integration of cellulosic etha...

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Research paper thumbnail of Heterologous expression of cellobiohydrolases in yeast

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Research paper thumbnail of Expression of human papillomavirus type 16 (HPV16) L1 protein in Pichia pastoris

AFRICAN JOURNAL OF BIOTECHNOLOGY

To investigate the production of human papillomavirus type 16 (HPV16) L1 structural protein in Pi... more To investigate the production of human papillomavirus type 16 (HPV16) L1 structural protein in Pichia pastoris, the effect of different gene constructs and varying cultivation conditions on production levels were assessed. The highest volumetric production levels in P. pastoris were obtained with a human codon-optimised gene construct. This construct displayed both the lowest codon adaptation index (CAI) and the highest guanine-cytosine (GC) content of the three constructs investigated. Dissolved oxygen (DO) controlled cultivations produced superior results compared to a constant methanol feeding (CMF) strategy, delivering the highest volumetric HPV L1 protein concentration (104.8 µg.l -1) at a 20% DO set-point. This was concomitant with an increased biomass concentration in the DO controlled cultivations.

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Research paper thumbnail of Expression of fungal hydrolases in �Saccharomyces cerevisiae�

Conversion of plant biomass to ethanol or other commodities through direct microbial conversion c... more Conversion of plant biomass to ethanol or other commodities through direct microbial conversion could be economically feasible if microbes that have optimal processing and product formation properties could be identified or engineered. The yeast Saccharomyces cerevisiae holds several advantages over other candidate organisms for this development such as its process robustness, long industrial history and genetic malleability. As this yeast cannot utilize polymeric sugar substrates, the heterologous production of the hydrolytic activities required for the degradation of the cellulose and hemicellulose components of plant biomass is imperative. Several researchers have attempted the expression of genes encoding lignocellulolytic hydrolases in S. cerevisiae over the past three decades. This chapter will assess the progress made in this field and will highlight some of the successes and future challenges.

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Research paper thumbnail of Engineering Recombinant Organisms for Next-generation Ethanol Production, In: Biofuels: From Microbes to Molecules

ABSTRACT Development of microbes for the production of fuel ethanol continues at a rapid pace, fo... more ABSTRACT Development of microbes for the production of fuel ethanol continues at a rapid pace, for established first generation processes relying on sugar- and starch-based foodcrops, and second generation processes for conversion of lignocellulose to ethanol by pretreatment-hydrolysis-fermentation. The production of bio-ethanol is dominated by Saccharomyces cerevisiae as fermentative organism, although Escherichia coli, Zymomonas mobilis and Kluyveromyces marxianus, among others, were considered for second generation processes. For S. cerevisiae, key developments for fermentation of raw and cooked starch, as well as lignocellulose, include consolidated bioprocessing (CBP) where enzyme production for substrate hydrolysis and fermentation of released sugars are performed by a single organism. Further yeast developments required for second generation ethanol include engineering of xylose utilization capability, to increase yields from lignocellulose, and increasing the resistance of yeasts to inhibitory compounds formed during lignocellulose pretreatment prior to hydrolysis and fermentation. The present chapter will review the full scope of organism development for ethanol production, followed by examination of S. cerevisiae strain development for xylose utilization, CBP and hardening, as well as metabolic engineering, application of –omics technologies, and synthetic biology as examples of global efforts in organism development for ethanol production.

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Research paper thumbnail of Effect of N-linked glycosylation on the activity and stability of a β-glucosidase from Putranjiva roxburghii

International journal of biological macromolecules, 2018

In this study, the effect of N-glycosylation on the conformational and functional stability of Pu... more In this study, the effect of N-glycosylation on the conformational and functional stability of Putranjiva roxburghii family 1 β-glucosidase (PRGH1) enzyme was investigated. The deglycosylation of PRGH1 was carried out by using PNGase F enzyme and confirmed by SDS-PAGE and carbohydrate estimation. Comparative analysis with respect to enzyme activity, stability and aggregation behaviour was carried out for the glycosylated and deglycosylated PRGH1. The deglycosylation of PRGH1 affected enzyme activity to a certain extent only where Kwas not affected but a slight reduction in Vfor various substrates was observed. Circular dichroism, fluorescence studies and differential scanning calorimetry (DSC) analysis demonstrated the possible effect of glycosylation on local and/or global conformational dynamics of protein and its effect on the thermostability of PRGH1. DSC results showed deglycosylated form had lower Tas compared to the glycosylated form of PRGH1. The PRGH1 was found to be more s...

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Research paper thumbnail of Strain Breeding Enhanced Heterologous Cellobiohydrolase Secretion by Saccharomyces cerevisiae in a Protein Specific Manner

Biotechnology Journal

The yeast Saccharomyces cerevisiae has a long association with alcoholic fermentation industries ... more The yeast Saccharomyces cerevisiae has a long association with alcoholic fermentation industries and has received renewed interest as a biocatalyst for second-generation bioethanol production. Rational engineering strategies are used to create yeast strains for consolidated bioprocessing of lignocellulosic biomass. Although significant progress is made in this regard with the expression of different cellulolytic activities in yeast, cellobiohydrolase (CBH) titers remain well below ideal levels. Through classical breeding, S. cerevisiae strains with up to twofold increased CBH secretion titers is obtained in strains expressing a single gene copy. An increase of up to 3.5-fold in secreted cellobiohydrolase activity is subsequently shown for strains expressing the heterologous gene on a high copy episomal vector. To our knowledge, this is the first report of classical breeding being used to enhance heterologous protein secretion and also the most significant enhancement of CBH secretion in yeast yet reported. This enhanced secretion phenotype is specific for cellobiohydrolase I secretion, indicating that reporter protein properties might be a major determining factor for efficient protein secretion in yeast. By exploring the latent potential of different S. cerevisiae strains, the authors show that the allele pool of various strains is a valuable engineering resource to enhance secretion in yeast.

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Research paper thumbnail of Improvement of ethanol production from crystalline cellulose via optimizing cellulase ratios in cellulolytic Saccharomyces cerevisiae

Biotechnology and bioengineering, Jun 23, 2017

Crystalline cellulose is one of the major contributors to the recalcitrance of lignocellulose to ... more Crystalline cellulose is one of the major contributors to the recalcitrance of lignocellulose to degradation, necessitating high dosages of cellulase to digest, thereby impeding the economic feasibility of cellulosic biofuels. Several recombinant cellulolytic yeast strains have been developed to reduce the cost of enzyme addition, but few of these strains are able to efficiently degrade crystalline cellulose due to their low cellulolytic activities. Here, by combining the cellulase ratio optimization with a novel screening strategy, we successfully improved the cellulolytic activity of a Saccharomyces cerevisiae strain displaying four different synergistic cellulases on the cell surface. The optimized strain exhibited an ethanol yield from Avicel of 57% of the theoretical maximum, and a 60% increase of ethanol titer from rice straw. To our knowledge, this work is the first optimization of the degradation of crystalline cellulose by tuning the cellulase ratio in a cellulase cell-surf...

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Research paper thumbnail of Heterologous expression of cellulase genes in natural Saccharomyces cerevisiae strains

Applied microbiology and biotechnology, Jan 28, 2016

Enzyme cost is a major impediment to second-generation (2G) cellulosic ethanol production. One st... more Enzyme cost is a major impediment to second-generation (2G) cellulosic ethanol production. One strategy to reduce enzyme cost is to engineer enzyme production capacity in a fermentative microorganism to enable consolidated bio-processing (CBP). Ideally, a strain with a high secretory phenotype, high fermentative capacity as well as an innate robustness to bioethanol-specific stressors, including tolerance to products formed during pre-treatment and fermentation of lignocellulosic substrates should be used. Saccharomyces cerevisiae is a robust fermentative yeast but has limitations as a potential CBP host, such as low heterologous protein secretion titers. In this study, we evaluated natural S. cerevisiae isolate strains for superior secretion activity and other industrially relevant characteristics needed during the process of lignocellulosic ethanol production. Individual cellulases namely Saccharomycopsis fibuligera Cel3A (β-glucosidase), Talaromyces emersonii Cel7A (cellobiohydro...

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Research paper thumbnail of Method for Fermenting Cellulosics

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Engineering cellulolytic ability into bioprocessing organisms

Applied Microbiology and Biotechnology, Mar 1, 2010

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Research paper thumbnail of Differential expression of the Trichoderma reesei ß-xylanase II ( xyn2 ) gene in the xylose-fermenting yeast Pichia stipitis

Appl Microbiol Biotechnol, 2001

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Yeast Expressing Cellulases for Simultaneous Saccharification and Fermentation Using Cellulose

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Yeast Expressing Saccharolytic Enzymes for Consolidated Bioprocessing Using Starch and Cellulose

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Enhanced xylan degradation and utilisation by Pichia stipitis overproducing fungal xylanolytic enzymes

Enzyme Microb Technol, 2003

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Engineering of a novel cellulose-adherent cellulolytic Saccharomyces cerevisiae for cellulosic biofuel production

Scientific reports, Jan 15, 2016

Cellulosic biofuel is the subject of increasing attention. The main obstacle toward its economic ... more Cellulosic biofuel is the subject of increasing attention. The main obstacle toward its economic feasibility is the recalcitrance of lignocellulose requiring large amount of enzyme to break. Several engineered yeast strains have been developed with cellulolytic activities to reduce the need for enzyme addition, but exhibiting limited effect. Here, we report the successful engineering of a cellulose-adherent Saccharomyces cerevisiae displaying four different synergistic cellulases on the cell surface. The cellulase-displaying yeast strain exhibited clear cell-to-cellulose adhesion and a "tearing" cellulose degradation pattern; the adhesion ability correlated with enhanced surface area and roughness of the target cellulose fibers, resulting in higher hydrolysis efficiency. The engineered yeast directly produced ethanol from rice straw despite a more than 40% decrease in the required enzyme dosage for high-density fermentation. Thus, improved cell-to-cellulose interactions pr...

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Research paper thumbnail of Differential expression of the Trichoderma reesei β-xylanase II ( xyn2 ) gene in the xylose-fermenting yeast Pichia stipitis

Appl Microbiol Biotechnol, 2001

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Research paper thumbnail of Combined cell-surface display- and secretion-based strategies for production of cellulosic ethanol with Saccharomyces cerevisiae

Biotechnology for biofuels, 2015

Engineering Saccharomyces cerevisiae to produce heterologous cellulases is considered as a promis... more Engineering Saccharomyces cerevisiae to produce heterologous cellulases is considered as a promising strategy for production of bioethanol from lignocellulose. The production of cellulase is usually pursued by one of the two strategies: displaying enzyme on the cell surface or secreting enzyme into the medium. However, to our knowledge, the combination of the two strategies in a yeast strain has not been employed. In this study, heterologous endoglucanase (EG) and cellobiohydrolase I (CBHI) were produced in a β-glucosidase displaying S. cerevisiae strain using cell-surface display, secretion, or a combined strategy. Strains EG-D-CBHI-D and EG-S-CBHI-S (with both enzymes displayed on the cell surface or with both enzymes secreted to the surrounding medium) showed higher ethanol production (2.9 and 2.6 g/L from 10 g/L phosphoric acid swollen cellulose, respectively), than strains EG-D-CBHI-S and EG-S-CBHI-D (with EG displayed on cell surface and CBHI secreted, or vice versa). After 3-...

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Research paper thumbnail of Enhanced xylan degradation and utilisation by Pichia stipitis overproducing fungal xylanolytic enzymes. Enzyme Microb Technol

Enzyme and Microbial Technology

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Research paper thumbnail of Lignocellulosic Hydrolysate Inhibitors Selectively Inhibit/Deactivate Cellulase Performance

Enzyme and Microbial Technology, 2015

In this study, we monitored the inhibition and deactivation effects of various compounds associat... more In this study, we monitored the inhibition and deactivation effects of various compounds associated with lignocellulosic hydrolysates on individual and combinations of cellulases. Tannic acid representing polymeric lignin residues strongly inhibited cellobiohydrolase 1 (CBH1) and β-glucosidase 1 (BGL1), but had a moderate inhibitory effect on endoglucanase 2 (EG2). Individual monomeric lignin residues had little or no inhibitory effect on hydrolytic enzymes. However, coniferyl aldehyde and syringaldehyde substantially decreased the activity of CBH1 and deactivated BGL1. Acetic and formic acids also showed strong inhibition of BGL1 but not CBH1 and EG2, whereas tannic, acetic and formic acid strongly inhibited a combination of CBH1 and EG2 during Avicel hydrolysis. Diminishing enzymatic hydrolysis is largely a function of inhibitor concentration and the enzyme-inhibitor relationship, rather than contact time during the hydrolysis process (i.e. deactivation). This suggests that decreased rates of hydrolysis during the enzymatic depolymerisation of lignocellulosic hydrolysates may be imparted by other factors related to substrate crystallinity and accessibility.

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Research paper thumbnail of Next-generation cellulosic ethanol technologies and their contribution to a sustainable Africa

Interface focus, Jan 6, 2011

The world is currently heavily dependent on oil, especially in the transport sector. However, ris... more The world is currently heavily dependent on oil, especially in the transport sector. However, rising oil prices, concern about environmental impact and supply instability are among the factors that have led to greater interest in renewable fuel and green chemistry alternatives. Lignocellulose is the only foreseeable renewable feedstock for sustainable production of transport fuels. The main technological impediment to more widespread utilization of lignocellulose for production of fuels and chemicals in the past has been the lack of low-cost technologies to overcome the recalcitrance of its structure. Both biological and thermochemical second-generation conversion technologies are currently coming online for the commercial production of cellulosic ethanol concomitantly with heat and electricity production. The latest advances in biological conversion of lignocellulosics to ethanol with a focus on consolidated bioprocessing are highlighted. Furthermore, integration of cellulosic etha...

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Research paper thumbnail of Heterologous expression of cellobiohydrolases in yeast

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Expression of human papillomavirus type 16 (HPV16) L1 protein in Pichia pastoris

AFRICAN JOURNAL OF BIOTECHNOLOGY

To investigate the production of human papillomavirus type 16 (HPV16) L1 structural protein in Pi... more To investigate the production of human papillomavirus type 16 (HPV16) L1 structural protein in Pichia pastoris, the effect of different gene constructs and varying cultivation conditions on production levels were assessed. The highest volumetric production levels in P. pastoris were obtained with a human codon-optimised gene construct. This construct displayed both the lowest codon adaptation index (CAI) and the highest guanine-cytosine (GC) content of the three constructs investigated. Dissolved oxygen (DO) controlled cultivations produced superior results compared to a constant methanol feeding (CMF) strategy, delivering the highest volumetric HPV L1 protein concentration (104.8 µg.l -1) at a 20% DO set-point. This was concomitant with an increased biomass concentration in the DO controlled cultivations.

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Expression of fungal hydrolases in �Saccharomyces cerevisiae�

Conversion of plant biomass to ethanol or other commodities through direct microbial conversion c... more Conversion of plant biomass to ethanol or other commodities through direct microbial conversion could be economically feasible if microbes that have optimal processing and product formation properties could be identified or engineered. The yeast Saccharomyces cerevisiae holds several advantages over other candidate organisms for this development such as its process robustness, long industrial history and genetic malleability. As this yeast cannot utilize polymeric sugar substrates, the heterologous production of the hydrolytic activities required for the degradation of the cellulose and hemicellulose components of plant biomass is imperative. Several researchers have attempted the expression of genes encoding lignocellulolytic hydrolases in S. cerevisiae over the past three decades. This chapter will assess the progress made in this field and will highlight some of the successes and future challenges.

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Engineering Recombinant Organisms for Next-generation Ethanol Production, In: Biofuels: From Microbes to Molecules

ABSTRACT Development of microbes for the production of fuel ethanol continues at a rapid pace, fo... more ABSTRACT Development of microbes for the production of fuel ethanol continues at a rapid pace, for established first generation processes relying on sugar- and starch-based foodcrops, and second generation processes for conversion of lignocellulose to ethanol by pretreatment-hydrolysis-fermentation. The production of bio-ethanol is dominated by Saccharomyces cerevisiae as fermentative organism, although Escherichia coli, Zymomonas mobilis and Kluyveromyces marxianus, among others, were considered for second generation processes. For S. cerevisiae, key developments for fermentation of raw and cooked starch, as well as lignocellulose, include consolidated bioprocessing (CBP) where enzyme production for substrate hydrolysis and fermentation of released sugars are performed by a single organism. Further yeast developments required for second generation ethanol include engineering of xylose utilization capability, to increase yields from lignocellulose, and increasing the resistance of yeasts to inhibitory compounds formed during lignocellulose pretreatment prior to hydrolysis and fermentation. The present chapter will review the full scope of organism development for ethanol production, followed by examination of S. cerevisiae strain development for xylose utilization, CBP and hardening, as well as metabolic engineering, application of –omics technologies, and synthetic biology as examples of global efforts in organism development for ethanol production.

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