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Papers by Mark Harrison

Research paper thumbnail of Optical Spectroscopic Investigation of the Alkaline Transition in Umecyanin from Horseradish Root

Biochemistry

Umecyanin (UMC) from horseradish root belongs to the stellacyanin subclass of the phytocyanins, a... more Umecyanin (UMC) from horseradish root belongs to the stellacyanin subclass of the phytocyanins, a family of plant cupredoxins. The protein possesses the typical type-1 His 2 Cys equatorial ligand set at its mononuclear copper site but has an axial Gln ligand in place of the usual weakly coordinated Met of the plantacyanins, uclacyanins, and most other cupredoxins. UMC exhibits, like other phytocyanins, altered visible, EPR, and paramagnetic 1 H NMR spectra at elevated pH values and also a modified reduction potential. This alkaline transition occurs with a pK a of ∼10 [Dennison, C., Lawler, A. T. (2001) Biochemistry 40, 3158-3166]. In this study, we investigate the alkaline transition by complementary optical spectroscopic techniques. The contemporary use of absorption, fluorescence, dynamic light scattering, and resonance Raman spectroscopy allows us to demonstrate that the alkaline transition induces a reorganization of the protein and that the overall size of UMC increases, but protein aggregation does not occur. The transition does not have a dramatic influence on the active-site environment of UMC, but there are subtle alterations in the Cu site geometry. Direct evidence for the strengthening of a Cu-N(His) bond is presented, which is in agreement with the hypothesis that the deprotonation of the N 2 H moiety of one of the His ligands is the cause of the alkaline transition. A weakening of the Cu-S(Cys) bond is also observed which, along with a weakened axial interaction, must be due to the enhanced Cu-N(His) interaction.

Research paper thumbnail of Exogenous Probiotics Improve Fermentation Quality, Microflora Phenotypes, and Trophic Modes of Fermented Vegetable Waste for Animal Feed

Microorganisms, 2021

The fermentation of leaf vegetable waste to produce animal feed reduces the environmental impact ... more The fermentation of leaf vegetable waste to produce animal feed reduces the environmental impact of vegetable production and transforms leaf vegetable waste into a commodity. We investigated the effect of exogenous probiotics and lignocellulose enzymes on the quality and microbial community of fermented feed (FF) produced from cabbage waste. The addition of exogenous probiotics resulted in increased crude protein (CP) content (p < 0.05), better odor (moderate organic acid and ethanol, with low ammonia-N, p < 0.05), and a lower relative abundance (RA) of pathogens (below 0.4%, p < 0.05) in FF, compared to without. With the addition of exogenous probiotics, only Pediococcus and Saccharomyces were enriched and symbiotic in FF; these were the keystone taxa to reduce the abundance of aerobic, form-biofilms, and pathogenic microorganisms, resulting in an efficient anaerobic fermentation system characterized by facultative anaerobic and Gram-positive bacterial communities, and und...

Research paper thumbnail of Valorization of sugarcane biorefinery residues using fungal biocatalysis

Biomass Conversion and Biorefinery

A sustainable future requires novel technologies that transform renewable feedstocks into liquid ... more A sustainable future requires novel technologies that transform renewable feedstocks into liquid transport fuels, materials, and fine chemicals. Existing sugarcane biorefineries (sugar mills) generate a wide range of renewable feedstocks that can be readily converted into value-added products. Emerging biorefinery technologies offer further expansion of the products that can be generated in sugarcane biorefineries but biocatalysts that can use by-products and residues from these emerging technologies are needed to fully realize the opportunity these technologies represent. Filamentous fungi are versatile biocatalysts that can transform sugarcane biorefinery by-products and residues into a wide range of valuable products. This review provides an overview of sugarcane-based biorefining, existing and emerging processing technologies with application in sugarcane biorefineries; the residues and by-products generated in such a facility; and the opportunities to use filamentous fungi as biocatalysts to produce enzymes, organic acids, single cell protein, specialized metabolites, and animal feed.

Research paper thumbnail of Effects of glycerol on enzymatic hydrolysis and ethanol production using sugarcane bagasse pretreated by acidified glycerol solution

Bioresource technology, Jan 2, 2015

In this study, for the first time the effects of glycerol on enzymatic hydrolysis and ethanol fer... more In this study, for the first time the effects of glycerol on enzymatic hydrolysis and ethanol fermentation were investigated. Enzymatic hydrolysis was inhibited slightly with 2.0wt% glycerol, leading to reduction in glucan digestibility from 84.9% without glycerol to 82.9% (72h). With 5.0wt% and 10.0wt% glycerol, glucan digestibility was reduced by 4.5% and 11.0%, respectively. However, glycerol did not irreversibly inhibit cellulase enzymes. Ethanol fermentation was not affected by glycerol up to 5.0wt%, but was inhibited slightly at 10.0wt% glycerol, resulting in reduction in ethanol yield from 86.0% in the absence of glycerol to 83.7% (20h). Based on the results of laboratory and pilot-scale experiments, it was estimated that 0.142kg ethanol can be produced from 1.0kg dry bagasse (a glucan content of 38.0%) after pretreatment with acidified glycerol solution.

Research paper thumbnail of Stability of endoglucanases from mesophilic fungus and thermophilic bacterium in acidified polyols

Enzyme and microbial technology

Recent developments in chemical pretreatments of lignocellulosic biomass using polyols as co-solv... more Recent developments in chemical pretreatments of lignocellulosic biomass using polyols as co-solvents (e.g., glycerol and ethylene glycol) at temperatures less than 100°C may allow the effective use of thermostable and non-thermostable cellulases in situ during the saccharification process. The potential of biomass saccharifying enzymes, endoglucanases (EG) from a thermophilic bacterium (Thermotoga maritima) and a mesophilic fungus (Trichoderma longibrachiatum), to retain their activity in aqueous buffer, acidified glycerol, and acidified ethylene glycol used as co-solvents at pretreatment temperatures at or below 100°C were examined. The results show that despite its origin, T. longibrachiatum EG (Tl-EG) retained 75% of its activity after exposure to 100°C for 5 min in aqueous buffer while T. maritima EG (Tm-EG) retained only 5% activity. However, at 90°C both enzymes retained over 87% of their activity. In acidified (0.1% (w/w) H2SO4) glycerol, Tl-EG retained similar activity (80%...

Research paper thumbnail of The combination of plant-expressed cellobiohydrolase and low dosages of cellulases for the hydrolysis of sugar cane bagasse

Biotechnology for biofuels, 2014

The expression of biomass-degrading enzymes (such as cellobiohydrolases) in transgenic plants has... more The expression of biomass-degrading enzymes (such as cellobiohydrolases) in transgenic plants has the potential to reduce the costs of biomass saccharification by providing a source of enzymes to supplement commercial cellulase mixtures. Cellobiohydrolases are the main enzymes in commercial cellulase mixtures. In the present study, a cellobiohydrolase was expressed in transgenic corn stover leaf and assessed as an additive for two commercial cellulase mixtures for the saccharification of pretreated sugar cane bagasse obtained by different processes. Recombinant cellobiohydrolase in the senescent leaves of transgenic corn was extracted using a simple buffer with no concentration step. The extract significantly enhanced the performance of Celluclast 1.5 L (a commercial cellulase mixture) by up to fourfold on sugar cane bagasse pretreated at the pilot scale using a dilute sulfuric acid steam explosion process compared to the commercial cellulase mixture on its own. Also, the extracts w...

Research paper thumbnail of A metallothionein containing a zinc finger within a four-metal cluster protects a bacterium from zinc toxicity

Proceedings of the National Academy of Sciences, 2001

Zinc is essential for many cellular processes, including DNA synthesis, transcription, and transl... more Zinc is essential for many cellular processes, including DNA synthesis, transcription, and translation, but excess can be toxic. A zinc-induced gene, smtA , is required for normal zinc-tolerance in the cyanobacterium Synechococcus PCC 7942. Here we report that the protein SmtA contains a cleft lined with Cys-sulfur and His-imidazole ligands that binds four zinc ions in a Zn 4 Cys 9 His 2 cluster. The thiolate sulfurs of five Cys ligands provide bridges between the two ZnCys 4 and two ZnCys 3 His sites, giving two fused six-membered rings with distorted boat conformations. The inorganic core strongly resembles the Zn 4 Cys 11 cluster of mammalian metallothionein, despite different amino acid sequences, a different linear order of the ligands, and presence of histidine ligands. Also, SmtA contains elements of secondary structure not found in metallothioneins. One of the two Cys 4 -coordinated zinc ions in SmtA readily exchanges with exogenous metal ( 111 Cd), whereas the other is iner...

Research paper thumbnail of Accumulation of recombinant cellobiohydrolase and endoglucanase in the leaves of mature transgenic sugar cane

Plant Biotechnology Journal, 2011

A major strategic goal in making ethanol from lignocellulosic biomass a cost-competitive liquid t... more A major strategic goal in making ethanol from lignocellulosic biomass a cost-competitive liquid transport fuel is to reduce the cost of production of cellulolytic enzymes that hydrolyse lignocellulosic substrates to fermentable sugars. Current production systems for these enzymes, namely microbes, are not economic. One way to substantially reduce production costs is to express cellulolytic enzymes in plants at levels that are high enough to hydrolyse lignocellulosic biomass. Sugar cane fibre (bagasse) is the most promising lignocellulosic feedstock for conversion to ethanol in the tropics and subtropics. Cellulolytic enzyme production in sugar cane will have a substantial impact on the economics of lignocellulosic ethanol production from bagasse. We therefore generated transgenic sugar cane accumulating three cellulolytic enzymes, fungal cellobiohydrolase I (CBH I), CBH II and bacterial endoglucanase (EG), in leaves using the maize PepC promoter as an alternative to maize Ubi1 for controlling transgene expression. Different subcellular targeting signals were shown to have a substantial impact on the accumulation of these enzymes; the CBHs and EG accumulated to higher levels when fused to a vacuolar-sorting determinant than to an endoplasmic reticulum-retention signal, while EG was produced in the largest amounts when fused to a chloroplast-targeting signal. These results are the first demonstration of the expression and accumulation of recombinant CBH I, CBH II and EG in sugar cane and represent a significant first step towards the optimization of cellulolytic enzyme expression in sugar cane for the economic production of lignocellulosic ethanol.

Research paper thumbnail of Multiple bacteria encode metallothioneins and SmtA-like zinc fingers

Molecular Microbiology, 2002

Zinc is essential but toxic in excess. Bacterial metallothionein, SmtA from Synechococcus PCC 794... more Zinc is essential but toxic in excess. Bacterial metallothionein, SmtA from Synechococcus PCC 7942, sequesters and detoxifies four zinc ions per molecule and contains a zinc finger structurally similar to eukaryotic GATA. The dearth of other reported bacterial metallothioneins has been surprising. Here we describe related bacterial metallothioneins (BmtA) from Anabaena PCC 7120, Pseudomonas aeruginosa and Pseudomonas putida that bind multiple zinc ions with high stability towards protons. Thiol modification demonstrates that cysteine coordinates zinc in all of these proteins. Additionally, (111)Cd-NMR, and (111)Cd-edited (1)H-NMR, identified histidine ligands in Anabaena PCC 7120 BmtA, analogous to SmtA. A related Escherichia coli protein bound only a single zinc ion, via four cysteine residues, with low stability towards protons; (111)Cd-NMR and (111)Cd-edited (1)H-NMR confirmed exclusive cysteine-coordination, and these cysteine residues reacted rapidly with 5,5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-dithiobis-(2-nitrobenzoic acid). (1)H-NMR of proteins from P. aeruginosa, Anabaena PCC 7120 and E. coli generated fingerprints diagnostic for the GATA-like zinc finger fold of SmtA. These studies reveal first the existence of multiple bacterial metallothioneins, and second proteins with SmtA-like lone zinc fingers, devoid of a cluster,and designated GatA. We have identified 12 smtA-like genes in sequence databases including four of the gatA type.

Research paper thumbnail of Expression of Potato virus Y cytoplasmic inclusion protein in tobacco results in disorganization of parenchyma cells, distortion of epidermal cells, and induces mitochondrial and chloroplast abnormalities, formation of membrane whorls and atypical lipid accumulation

Research paper thumbnail of Inert Site in a Protein Zinc Cluster: Isotope Exchange by High Resolution Mass Spectrometry

Journal of the American Chemical Society, 2003

Research paper thumbnail of Crystal Structures of Oxidized and Reduced Stellacyanin from Horseradish Roots †

Journal of the American Chemical Society, 2005

Research paper thumbnail of The Active-Site Structure of Umecyanin, the Stellacyanin from Horseradish Roots

Journal of the American Chemical Society, 2004

The type 1 copper sites of cupredoxins typically have a His(2)Cys equatorial ligand set with a we... more The type 1 copper sites of cupredoxins typically have a His(2)Cys equatorial ligand set with a weakly interacting axial Met, giving a distorted tetrahedral geometry. Natural variations to this coordination environment are known, and we have utilized paramagnetic (1)H NMR spectroscopy to study the active-site structure of umecyanin (UMC), a stellacyanin with an axial Gln ligand. The assigned spectra of the Cu(II) UMC and its Ni(II) derivative [Ni(II) UMC] demonstrate that this protein has the typical His(2)Cys equatorial coordination observed in other structurally characterized cupredoxins. The NMR spectrum of the Cu(II) protein does not exhibit any paramagnetically shifted resonances from the axial ligand, showing that this residue does not contribute to the singly occupied molecular orbital (SOMO) in Cu(II) UMC. The assigned paramagnetic (1)H NMR spectrum of Ni(II) UMC demonstrates that the axial Gln ligand coordinates in a monodentate fashion via its side-chain amide oxygen atom. The alkaline transition, a feature common to stellacyanins, influences all of the ligating residues but does not alter the coordination mode of the axial Gln ligand in UMC. The structural features which result in Cu(II) UMC possessing a classic type 1 site as compared to the perturbed type 1 center observed for other stellacyanins do not have a significant influence on the paramagnetic (1)H NMR spectra of the Cu(II) or Ni(II) proteins.

Research paper thumbnail of Structural control of copper and zinc exchange dynamics in bacterial transport and storage proteins

Journal of Inorganic Biochemistry, 2003

Research paper thumbnail of An Axial Met Ligand at a Type 1 Copper Site is Preferable for Fast Electron Transfer

ChemBioChem, 2004

The structure, spectroscopy and electron transfer (ET) reactivity of the type 1 copper sites of c... more The structure, spectroscopy and electron transfer (ET) reactivity of the type 1 copper sites of cupredoxins have been extensively studied by site-directed mutagenesis. [1-4] All of these investigations have highlighted the importance of the ligating residues, in particular the equatorial Cys ligand, [1] at the typically distorted tetrahedral metal centre (see Figure 1). The axially coordinating amino acid, which is usually a Met located~3

Research paper thumbnail of Effect of pretreatment on saccharification of sugarcane bagasse by complex and simple enzyme mixtures

Bioresource Technology, 2013

Research paper thumbnail of Investigating the Cause of the Alkaline Transition of Phytocyanins †

Biochemistry, 2005

The phytocyanins are a family of plant cupredoxins that have been subdivided into the stellacyani... more The phytocyanins are a family of plant cupredoxins that have been subdivided into the stellacyanins, plantacyanins, and uclacyanins. All of these proteins possess the typical type 1 His(2)Cys equatorial ligand set at their mononuclear copper sites, but the stellacyanins have an axial Gln ligand in place of the weakly coordinated Met of the plantacyanins, uclacyanins, and most other cupredoxins. The stellacyanins exhibit altered visible, EPR, and paramagnetic (1)H NMR spectra at elevated pH values and also modified reduction potentials. This alkaline transition occurs with a pK(a) of approximately 10 [Dennison, C., Lawler, A. T. (2001) Biochemistry 40, 3158-3166]. In this study we demonstrate that the alkaline transition has a similar influence on the visible, EPR, and paramagnetic NMR spectra of cucumber basic protein (CBP), which is a plantacyanin. The mutation of the axial Gln95 ligand into a Met in umecyanin (UMC), the stellacyanin from horseradish roots, and the axial Met89 into a Gln in CBP have very limited, yet similar, influence on the pK(a) for the alkaline transition as judged from alterations in visible spectra. The complete removal of the axial ligand in the Met89Val variant of CBP results in a slightly larger decrease in the pK(a) for this effect, but similar spectral alterations are still observed at elevated pH. Thus, the axial Gln ligand is not the cause of the alkaline transition in Cu(II) stellacyanins, and alterations in the active site structures of the phytocyanins have a limited effect on this feature. The conserved Lys residue found adjacent to the axial ligand in the sequences of all phytocyanins, and implicated as the trigger for the alkaline transition, has been mutated to an Arg in UMC. The influence of increasing pH on the spectroscopic properties of Lys96Arg UMC is almost identical to those of the wild type protein, and thus, this residue is not responsible for the alkaline transition. However, a positively charged residue in this position seems to be important for the correct folding of UMC. Other possible triggers for the effects seen in the phytocyanins at elevated pH are discussed along with the relevance of the alkaline transition.

Research paper thumbnail of Alkaline transition of phytocyanins: a comparison of stellacyanin and umecyanin

Biochemical Journal, 2003

The effect of pH on Cu(I) and Cu(II) forms of the isolated soluble domain of the stellacyanin fro... more The effect of pH on Cu(I) and Cu(II) forms of the isolated soluble domain of the stellacyanin from Rhus vernicifera (SCu), the Japanese lacquer tree, has been studied by electronic and NMR spectroscopy and using direct electrochemical measurements. A pKa value of 10.1–10.4 is observed for the alkaline transition in this oxidized phytocyanin and results in a slightly altered active-site structure, as indicated by changes in the visible and paramagnetic 1H NMR spectra. Electrochemical studies show that the pKa value for this transition in SCu(I) (reduced SCu) is 11.0. These results are compared with those recently obtained for other phytocyanins, and in particular umecyanin. In all cases, the alkaline transition is caused by the deprotonation of the surface lysine residue adjacent to the axial ligand. This lysine residue is completely conserved in known phytocyanin sequences. Also highlighted in these studies are the remarkable active-site similarities between stellacyanin and umecyanin.

Research paper thumbnail of The Enterococcus hirae copper chaperone CopZ delivers copper(I) to the CopY repressor

FEBS Letters, 1999

Expression of the cop operon which effects copper homeostasis in Enterococcus hirae is controlled... more Expression of the cop operon which effects copper homeostasis in Enterococcus hirae is controlled by the copper responsive repressor CopY. Purified Zn(II)CopY binds to a synthetic cop promoter fragment in vitro. Here we show that the 8 kDa protein CopZ acts as a copper chaperone by specifically delivering copper(I) to Zn(II)CopY and releasing CopY from the DNA. As shown by gel filtration and luminescence spectroscopy, two copper(I) are thereby quantitatively transferred from Cu(I)CopZ to Zn(II)CopY, with displacement of the zinc(II) and transfer of copper from a non-luminescent, exposed, binding site in CopZ to a luminescent, solvent shielded, binding site in CopY.

Research paper thumbnail of Optical Spectroscopic Investigation of the Alkaline Transition in Umecyanin from Horseradish Root †

Biochemistry, 2005

Umecyanin (UMC) from horseradish root belongs to the stellacyanin subclass of the phytocyanins, a... more Umecyanin (UMC) from horseradish root belongs to the stellacyanin subclass of the phytocyanins, a family of plant cupredoxins. The protein possesses the typical type-1 His 2 Cys equatorial ligand set at its mononuclear copper site but has an axial Gln ligand in place of the usual weakly coordinated Met of the plantacyanins, uclacyanins, and most other cupredoxins. UMC exhibits, like other phytocyanins, altered visible, EPR, and paramagnetic 1 H NMR spectra at elevated pH values and also a modified reduction potential. This alkaline transition occurs with a pK a of ∼10 [Dennison, C., Lawler, A. T. Biochemistry 40, [3158][3159][3160][3161][3162][3163][3164][3165][3166]. In this study, we investigate the alkaline transition by complementary optical spectroscopic techniques. The contemporary use of absorption, fluorescence, dynamic light scattering, and resonance Raman spectroscopy allows us to demonstrate that the alkaline transition induces a reorganization of the protein and that the overall size of UMC increases, but protein aggregation does not occur. The transition does not have a dramatic influence on the active-site environment of UMC, but there are subtle alterations in the Cu site geometry. Direct evidence for the strengthening of a Cu-N(His) bond is presented, which is in agreement with the hypothesis that the deprotonation of the N 2 H moiety of one of the His ligands is the cause of the alkaline transition. A weakening of the Cu-S(Cys) bond is also observed which, along with a weakened axial interaction, must be due to the enhanced Cu-N(His) interaction.

Research paper thumbnail of Optical Spectroscopic Investigation of the Alkaline Transition in Umecyanin from Horseradish Root

Biochemistry

Umecyanin (UMC) from horseradish root belongs to the stellacyanin subclass of the phytocyanins, a... more Umecyanin (UMC) from horseradish root belongs to the stellacyanin subclass of the phytocyanins, a family of plant cupredoxins. The protein possesses the typical type-1 His 2 Cys equatorial ligand set at its mononuclear copper site but has an axial Gln ligand in place of the usual weakly coordinated Met of the plantacyanins, uclacyanins, and most other cupredoxins. UMC exhibits, like other phytocyanins, altered visible, EPR, and paramagnetic 1 H NMR spectra at elevated pH values and also a modified reduction potential. This alkaline transition occurs with a pK a of ∼10 [Dennison, C., Lawler, A. T. (2001) Biochemistry 40, 3158-3166]. In this study, we investigate the alkaline transition by complementary optical spectroscopic techniques. The contemporary use of absorption, fluorescence, dynamic light scattering, and resonance Raman spectroscopy allows us to demonstrate that the alkaline transition induces a reorganization of the protein and that the overall size of UMC increases, but protein aggregation does not occur. The transition does not have a dramatic influence on the active-site environment of UMC, but there are subtle alterations in the Cu site geometry. Direct evidence for the strengthening of a Cu-N(His) bond is presented, which is in agreement with the hypothesis that the deprotonation of the N 2 H moiety of one of the His ligands is the cause of the alkaline transition. A weakening of the Cu-S(Cys) bond is also observed which, along with a weakened axial interaction, must be due to the enhanced Cu-N(His) interaction.

Research paper thumbnail of Exogenous Probiotics Improve Fermentation Quality, Microflora Phenotypes, and Trophic Modes of Fermented Vegetable Waste for Animal Feed

Microorganisms, 2021

The fermentation of leaf vegetable waste to produce animal feed reduces the environmental impact ... more The fermentation of leaf vegetable waste to produce animal feed reduces the environmental impact of vegetable production and transforms leaf vegetable waste into a commodity. We investigated the effect of exogenous probiotics and lignocellulose enzymes on the quality and microbial community of fermented feed (FF) produced from cabbage waste. The addition of exogenous probiotics resulted in increased crude protein (CP) content (p < 0.05), better odor (moderate organic acid and ethanol, with low ammonia-N, p < 0.05), and a lower relative abundance (RA) of pathogens (below 0.4%, p < 0.05) in FF, compared to without. With the addition of exogenous probiotics, only Pediococcus and Saccharomyces were enriched and symbiotic in FF; these were the keystone taxa to reduce the abundance of aerobic, form-biofilms, and pathogenic microorganisms, resulting in an efficient anaerobic fermentation system characterized by facultative anaerobic and Gram-positive bacterial communities, and und...

Research paper thumbnail of Valorization of sugarcane biorefinery residues using fungal biocatalysis

Biomass Conversion and Biorefinery

A sustainable future requires novel technologies that transform renewable feedstocks into liquid ... more A sustainable future requires novel technologies that transform renewable feedstocks into liquid transport fuels, materials, and fine chemicals. Existing sugarcane biorefineries (sugar mills) generate a wide range of renewable feedstocks that can be readily converted into value-added products. Emerging biorefinery technologies offer further expansion of the products that can be generated in sugarcane biorefineries but biocatalysts that can use by-products and residues from these emerging technologies are needed to fully realize the opportunity these technologies represent. Filamentous fungi are versatile biocatalysts that can transform sugarcane biorefinery by-products and residues into a wide range of valuable products. This review provides an overview of sugarcane-based biorefining, existing and emerging processing technologies with application in sugarcane biorefineries; the residues and by-products generated in such a facility; and the opportunities to use filamentous fungi as biocatalysts to produce enzymes, organic acids, single cell protein, specialized metabolites, and animal feed.

Research paper thumbnail of Effects of glycerol on enzymatic hydrolysis and ethanol production using sugarcane bagasse pretreated by acidified glycerol solution

Bioresource technology, Jan 2, 2015

In this study, for the first time the effects of glycerol on enzymatic hydrolysis and ethanol fer... more In this study, for the first time the effects of glycerol on enzymatic hydrolysis and ethanol fermentation were investigated. Enzymatic hydrolysis was inhibited slightly with 2.0wt% glycerol, leading to reduction in glucan digestibility from 84.9% without glycerol to 82.9% (72h). With 5.0wt% and 10.0wt% glycerol, glucan digestibility was reduced by 4.5% and 11.0%, respectively. However, glycerol did not irreversibly inhibit cellulase enzymes. Ethanol fermentation was not affected by glycerol up to 5.0wt%, but was inhibited slightly at 10.0wt% glycerol, resulting in reduction in ethanol yield from 86.0% in the absence of glycerol to 83.7% (20h). Based on the results of laboratory and pilot-scale experiments, it was estimated that 0.142kg ethanol can be produced from 1.0kg dry bagasse (a glucan content of 38.0%) after pretreatment with acidified glycerol solution.

Research paper thumbnail of Stability of endoglucanases from mesophilic fungus and thermophilic bacterium in acidified polyols

Enzyme and microbial technology

Recent developments in chemical pretreatments of lignocellulosic biomass using polyols as co-solv... more Recent developments in chemical pretreatments of lignocellulosic biomass using polyols as co-solvents (e.g., glycerol and ethylene glycol) at temperatures less than 100°C may allow the effective use of thermostable and non-thermostable cellulases in situ during the saccharification process. The potential of biomass saccharifying enzymes, endoglucanases (EG) from a thermophilic bacterium (Thermotoga maritima) and a mesophilic fungus (Trichoderma longibrachiatum), to retain their activity in aqueous buffer, acidified glycerol, and acidified ethylene glycol used as co-solvents at pretreatment temperatures at or below 100°C were examined. The results show that despite its origin, T. longibrachiatum EG (Tl-EG) retained 75% of its activity after exposure to 100°C for 5 min in aqueous buffer while T. maritima EG (Tm-EG) retained only 5% activity. However, at 90°C both enzymes retained over 87% of their activity. In acidified (0.1% (w/w) H2SO4) glycerol, Tl-EG retained similar activity (80%...

Research paper thumbnail of The combination of plant-expressed cellobiohydrolase and low dosages of cellulases for the hydrolysis of sugar cane bagasse

Biotechnology for biofuels, 2014

The expression of biomass-degrading enzymes (such as cellobiohydrolases) in transgenic plants has... more The expression of biomass-degrading enzymes (such as cellobiohydrolases) in transgenic plants has the potential to reduce the costs of biomass saccharification by providing a source of enzymes to supplement commercial cellulase mixtures. Cellobiohydrolases are the main enzymes in commercial cellulase mixtures. In the present study, a cellobiohydrolase was expressed in transgenic corn stover leaf and assessed as an additive for two commercial cellulase mixtures for the saccharification of pretreated sugar cane bagasse obtained by different processes. Recombinant cellobiohydrolase in the senescent leaves of transgenic corn was extracted using a simple buffer with no concentration step. The extract significantly enhanced the performance of Celluclast 1.5 L (a commercial cellulase mixture) by up to fourfold on sugar cane bagasse pretreated at the pilot scale using a dilute sulfuric acid steam explosion process compared to the commercial cellulase mixture on its own. Also, the extracts w...

Research paper thumbnail of A metallothionein containing a zinc finger within a four-metal cluster protects a bacterium from zinc toxicity

Proceedings of the National Academy of Sciences, 2001

Zinc is essential for many cellular processes, including DNA synthesis, transcription, and transl... more Zinc is essential for many cellular processes, including DNA synthesis, transcription, and translation, but excess can be toxic. A zinc-induced gene, smtA , is required for normal zinc-tolerance in the cyanobacterium Synechococcus PCC 7942. Here we report that the protein SmtA contains a cleft lined with Cys-sulfur and His-imidazole ligands that binds four zinc ions in a Zn 4 Cys 9 His 2 cluster. The thiolate sulfurs of five Cys ligands provide bridges between the two ZnCys 4 and two ZnCys 3 His sites, giving two fused six-membered rings with distorted boat conformations. The inorganic core strongly resembles the Zn 4 Cys 11 cluster of mammalian metallothionein, despite different amino acid sequences, a different linear order of the ligands, and presence of histidine ligands. Also, SmtA contains elements of secondary structure not found in metallothioneins. One of the two Cys 4 -coordinated zinc ions in SmtA readily exchanges with exogenous metal ( 111 Cd), whereas the other is iner...

Research paper thumbnail of Accumulation of recombinant cellobiohydrolase and endoglucanase in the leaves of mature transgenic sugar cane

Plant Biotechnology Journal, 2011

A major strategic goal in making ethanol from lignocellulosic biomass a cost-competitive liquid t... more A major strategic goal in making ethanol from lignocellulosic biomass a cost-competitive liquid transport fuel is to reduce the cost of production of cellulolytic enzymes that hydrolyse lignocellulosic substrates to fermentable sugars. Current production systems for these enzymes, namely microbes, are not economic. One way to substantially reduce production costs is to express cellulolytic enzymes in plants at levels that are high enough to hydrolyse lignocellulosic biomass. Sugar cane fibre (bagasse) is the most promising lignocellulosic feedstock for conversion to ethanol in the tropics and subtropics. Cellulolytic enzyme production in sugar cane will have a substantial impact on the economics of lignocellulosic ethanol production from bagasse. We therefore generated transgenic sugar cane accumulating three cellulolytic enzymes, fungal cellobiohydrolase I (CBH I), CBH II and bacterial endoglucanase (EG), in leaves using the maize PepC promoter as an alternative to maize Ubi1 for controlling transgene expression. Different subcellular targeting signals were shown to have a substantial impact on the accumulation of these enzymes; the CBHs and EG accumulated to higher levels when fused to a vacuolar-sorting determinant than to an endoplasmic reticulum-retention signal, while EG was produced in the largest amounts when fused to a chloroplast-targeting signal. These results are the first demonstration of the expression and accumulation of recombinant CBH I, CBH II and EG in sugar cane and represent a significant first step towards the optimization of cellulolytic enzyme expression in sugar cane for the economic production of lignocellulosic ethanol.

Research paper thumbnail of Multiple bacteria encode metallothioneins and SmtA-like zinc fingers

Molecular Microbiology, 2002

Zinc is essential but toxic in excess. Bacterial metallothionein, SmtA from Synechococcus PCC 794... more Zinc is essential but toxic in excess. Bacterial metallothionein, SmtA from Synechococcus PCC 7942, sequesters and detoxifies four zinc ions per molecule and contains a zinc finger structurally similar to eukaryotic GATA. The dearth of other reported bacterial metallothioneins has been surprising. Here we describe related bacterial metallothioneins (BmtA) from Anabaena PCC 7120, Pseudomonas aeruginosa and Pseudomonas putida that bind multiple zinc ions with high stability towards protons. Thiol modification demonstrates that cysteine coordinates zinc in all of these proteins. Additionally, (111)Cd-NMR, and (111)Cd-edited (1)H-NMR, identified histidine ligands in Anabaena PCC 7120 BmtA, analogous to SmtA. A related Escherichia coli protein bound only a single zinc ion, via four cysteine residues, with low stability towards protons; (111)Cd-NMR and (111)Cd-edited (1)H-NMR confirmed exclusive cysteine-coordination, and these cysteine residues reacted rapidly with 5,5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-dithiobis-(2-nitrobenzoic acid). (1)H-NMR of proteins from P. aeruginosa, Anabaena PCC 7120 and E. coli generated fingerprints diagnostic for the GATA-like zinc finger fold of SmtA. These studies reveal first the existence of multiple bacterial metallothioneins, and second proteins with SmtA-like lone zinc fingers, devoid of a cluster,and designated GatA. We have identified 12 smtA-like genes in sequence databases including four of the gatA type.

Research paper thumbnail of Expression of Potato virus Y cytoplasmic inclusion protein in tobacco results in disorganization of parenchyma cells, distortion of epidermal cells, and induces mitochondrial and chloroplast abnormalities, formation of membrane whorls and atypical lipid accumulation

Research paper thumbnail of Inert Site in a Protein Zinc Cluster: Isotope Exchange by High Resolution Mass Spectrometry

Journal of the American Chemical Society, 2003

Research paper thumbnail of Crystal Structures of Oxidized and Reduced Stellacyanin from Horseradish Roots †

Journal of the American Chemical Society, 2005

Research paper thumbnail of The Active-Site Structure of Umecyanin, the Stellacyanin from Horseradish Roots

Journal of the American Chemical Society, 2004

The type 1 copper sites of cupredoxins typically have a His(2)Cys equatorial ligand set with a we... more The type 1 copper sites of cupredoxins typically have a His(2)Cys equatorial ligand set with a weakly interacting axial Met, giving a distorted tetrahedral geometry. Natural variations to this coordination environment are known, and we have utilized paramagnetic (1)H NMR spectroscopy to study the active-site structure of umecyanin (UMC), a stellacyanin with an axial Gln ligand. The assigned spectra of the Cu(II) UMC and its Ni(II) derivative [Ni(II) UMC] demonstrate that this protein has the typical His(2)Cys equatorial coordination observed in other structurally characterized cupredoxins. The NMR spectrum of the Cu(II) protein does not exhibit any paramagnetically shifted resonances from the axial ligand, showing that this residue does not contribute to the singly occupied molecular orbital (SOMO) in Cu(II) UMC. The assigned paramagnetic (1)H NMR spectrum of Ni(II) UMC demonstrates that the axial Gln ligand coordinates in a monodentate fashion via its side-chain amide oxygen atom. The alkaline transition, a feature common to stellacyanins, influences all of the ligating residues but does not alter the coordination mode of the axial Gln ligand in UMC. The structural features which result in Cu(II) UMC possessing a classic type 1 site as compared to the perturbed type 1 center observed for other stellacyanins do not have a significant influence on the paramagnetic (1)H NMR spectra of the Cu(II) or Ni(II) proteins.

Research paper thumbnail of Structural control of copper and zinc exchange dynamics in bacterial transport and storage proteins

Journal of Inorganic Biochemistry, 2003

Research paper thumbnail of An Axial Met Ligand at a Type 1 Copper Site is Preferable for Fast Electron Transfer

ChemBioChem, 2004

The structure, spectroscopy and electron transfer (ET) reactivity of the type 1 copper sites of c... more The structure, spectroscopy and electron transfer (ET) reactivity of the type 1 copper sites of cupredoxins have been extensively studied by site-directed mutagenesis. [1-4] All of these investigations have highlighted the importance of the ligating residues, in particular the equatorial Cys ligand, [1] at the typically distorted tetrahedral metal centre (see Figure 1). The axially coordinating amino acid, which is usually a Met located~3

Research paper thumbnail of Effect of pretreatment on saccharification of sugarcane bagasse by complex and simple enzyme mixtures

Bioresource Technology, 2013

Research paper thumbnail of Investigating the Cause of the Alkaline Transition of Phytocyanins †

Biochemistry, 2005

The phytocyanins are a family of plant cupredoxins that have been subdivided into the stellacyani... more The phytocyanins are a family of plant cupredoxins that have been subdivided into the stellacyanins, plantacyanins, and uclacyanins. All of these proteins possess the typical type 1 His(2)Cys equatorial ligand set at their mononuclear copper sites, but the stellacyanins have an axial Gln ligand in place of the weakly coordinated Met of the plantacyanins, uclacyanins, and most other cupredoxins. The stellacyanins exhibit altered visible, EPR, and paramagnetic (1)H NMR spectra at elevated pH values and also modified reduction potentials. This alkaline transition occurs with a pK(a) of approximately 10 [Dennison, C., Lawler, A. T. (2001) Biochemistry 40, 3158-3166]. In this study we demonstrate that the alkaline transition has a similar influence on the visible, EPR, and paramagnetic NMR spectra of cucumber basic protein (CBP), which is a plantacyanin. The mutation of the axial Gln95 ligand into a Met in umecyanin (UMC), the stellacyanin from horseradish roots, and the axial Met89 into a Gln in CBP have very limited, yet similar, influence on the pK(a) for the alkaline transition as judged from alterations in visible spectra. The complete removal of the axial ligand in the Met89Val variant of CBP results in a slightly larger decrease in the pK(a) for this effect, but similar spectral alterations are still observed at elevated pH. Thus, the axial Gln ligand is not the cause of the alkaline transition in Cu(II) stellacyanins, and alterations in the active site structures of the phytocyanins have a limited effect on this feature. The conserved Lys residue found adjacent to the axial ligand in the sequences of all phytocyanins, and implicated as the trigger for the alkaline transition, has been mutated to an Arg in UMC. The influence of increasing pH on the spectroscopic properties of Lys96Arg UMC is almost identical to those of the wild type protein, and thus, this residue is not responsible for the alkaline transition. However, a positively charged residue in this position seems to be important for the correct folding of UMC. Other possible triggers for the effects seen in the phytocyanins at elevated pH are discussed along with the relevance of the alkaline transition.

Research paper thumbnail of Alkaline transition of phytocyanins: a comparison of stellacyanin and umecyanin

Biochemical Journal, 2003

The effect of pH on Cu(I) and Cu(II) forms of the isolated soluble domain of the stellacyanin fro... more The effect of pH on Cu(I) and Cu(II) forms of the isolated soluble domain of the stellacyanin from Rhus vernicifera (SCu), the Japanese lacquer tree, has been studied by electronic and NMR spectroscopy and using direct electrochemical measurements. A pKa value of 10.1–10.4 is observed for the alkaline transition in this oxidized phytocyanin and results in a slightly altered active-site structure, as indicated by changes in the visible and paramagnetic 1H NMR spectra. Electrochemical studies show that the pKa value for this transition in SCu(I) (reduced SCu) is 11.0. These results are compared with those recently obtained for other phytocyanins, and in particular umecyanin. In all cases, the alkaline transition is caused by the deprotonation of the surface lysine residue adjacent to the axial ligand. This lysine residue is completely conserved in known phytocyanin sequences. Also highlighted in these studies are the remarkable active-site similarities between stellacyanin and umecyanin.

Research paper thumbnail of The Enterococcus hirae copper chaperone CopZ delivers copper(I) to the CopY repressor

FEBS Letters, 1999

Expression of the cop operon which effects copper homeostasis in Enterococcus hirae is controlled... more Expression of the cop operon which effects copper homeostasis in Enterococcus hirae is controlled by the copper responsive repressor CopY. Purified Zn(II)CopY binds to a synthetic cop promoter fragment in vitro. Here we show that the 8 kDa protein CopZ acts as a copper chaperone by specifically delivering copper(I) to Zn(II)CopY and releasing CopY from the DNA. As shown by gel filtration and luminescence spectroscopy, two copper(I) are thereby quantitatively transferred from Cu(I)CopZ to Zn(II)CopY, with displacement of the zinc(II) and transfer of copper from a non-luminescent, exposed, binding site in CopZ to a luminescent, solvent shielded, binding site in CopY.

Research paper thumbnail of Optical Spectroscopic Investigation of the Alkaline Transition in Umecyanin from Horseradish Root †

Biochemistry, 2005

Umecyanin (UMC) from horseradish root belongs to the stellacyanin subclass of the phytocyanins, a... more Umecyanin (UMC) from horseradish root belongs to the stellacyanin subclass of the phytocyanins, a family of plant cupredoxins. The protein possesses the typical type-1 His 2 Cys equatorial ligand set at its mononuclear copper site but has an axial Gln ligand in place of the usual weakly coordinated Met of the plantacyanins, uclacyanins, and most other cupredoxins. UMC exhibits, like other phytocyanins, altered visible, EPR, and paramagnetic 1 H NMR spectra at elevated pH values and also a modified reduction potential. This alkaline transition occurs with a pK a of ∼10 [Dennison, C., Lawler, A. T. Biochemistry 40, [3158][3159][3160][3161][3162][3163][3164][3165][3166]. In this study, we investigate the alkaline transition by complementary optical spectroscopic techniques. The contemporary use of absorption, fluorescence, dynamic light scattering, and resonance Raman spectroscopy allows us to demonstrate that the alkaline transition induces a reorganization of the protein and that the overall size of UMC increases, but protein aggregation does not occur. The transition does not have a dramatic influence on the active-site environment of UMC, but there are subtle alterations in the Cu site geometry. Direct evidence for the strengthening of a Cu-N(His) bond is presented, which is in agreement with the hypothesis that the deprotonation of the N 2 H moiety of one of the His ligands is the cause of the alkaline transition. A weakening of the Cu-S(Cys) bond is also observed which, along with a weakened axial interaction, must be due to the enhanced Cu-N(His) interaction.