Rapid method to determine the molecular weight of dextrins and dextrans

A rapid method was developed to determine the molecular weight (M_n) of β-limit dextrin and dextrans (Leuconostoc mesenteroides) using a reducing power approach. The M_n of the β-limit dextrin was also estimated from high performance liquid chromatography (HPLC). Chromatograms were pre-calibrated with the dextrans. The three dextrins had a M_n of 2.09, 2.40 and 2.63 × 10⁵ using the reducing method and 4.80, 5.90 and 2.80 × 10⁵ by HPLC. The method could be employed to estimate M_n of dextrins where chromatographic systems were not available.     

Isolation, structure, and characterization of the putative soluble amyloses from potato, wheat, and rice starches

Amylose, a putative linear α-(1→4)-glucan and a component of most starches, was isolated from potato, rice, and wheat starches by forming the 1-butanol complex in a solution of the starches. It previously had been found that these amyloses were incompletely hydrolyzed by β-amylase, indicating that it was partially branched. Solubilization of the butanol complex in water and steam distillation of the 1-butanol, followed by cooling to 4 °C gave precipitation of the double helical, linear, retrograded amylose over a 15 h period, leaving the soluble amylose in solution. The soluble amyloses were precipitated with two volumes of ethanol, and the precipitate was solubilized and reprecipitated to remove traces of linear amylose. The precipitated, soluble amyloses, were partially branched and had properties intermediate between linear amylose and amylopectin. The water solubility of the potato amylose was 10.52 mg/mL, with a number-average degree of polymerization (DP_n) of 8440 and 2.1% branch linkages that had a DP_n of 48; the water solubility of the rice amylose was 8.83 mg/mL, with a DP_n of 2911 and 1.4% branch linkages that had a DP_n of 72; and the water solubility of wheat amylose was 6.33 mg/mL, with a DP_n of 1160 and 1.6% branch linkages that had a DP_n of 64. The three soluble amyloses have structures and properties intermediate between the nearly water insoluble (?1 mg/mL), linear amylose, and the highly water-soluble, 4-5% branched, amylopectin.     

Purification and characterization of chitinase from the stomach of silver croaker Pennahia argentatus

A chitinase was purified from the stomach of a fish, the silver croaker Pennahia argentatus, by ammonium sulfate fractionation and column chromatography using Chitopearl Basic BL-03, CM-Toyopearl 650S, and Butyl-Toyopearl 650S. The molecular mass and isoelectric point were estimated at 42 kDa and 6.7, respectively. The N-terminal amino acid sequence showed a high level of homology with family 18 chitinases. The optimum pH of silver croaker chitinase toward p-nitrophenyl N-acetylchitobioside (pNp-(GlcNAc)₂) and colloidal chitin were observed to be pH 2.5 and 4.0, respectively, while chitinase activity increased about 1.5- to 3-fold with the presence of NaCl. N-Acetylchitooligosaccharide ((GlcNAc)_n, n = 2–6) hydrolysis products and their anomer formation ratios were analyzed by HPLC using a TSK-GEL Amide-80 column. Since the silver croaker chitinase hydrolyzed (GlcNAc)_4–6 and produced (GlcNAc)_2–4, it was judged to be an endo-type chitinase. Meanwhile, an increase in β-anomers was recognized in the hydrolysis products, the same as with family 18 chitinases. This enzyme hydrolyzed (GlcNAc)₅ to produce (GlcNAc)₂ (79.2%) and (GlcNAc)₃ (20.8%). Chitinase activity towards various substrates in the order pNp-(GlcNAc)_n (n = 2–4) was pNp-(GlcNAc)₂ >> pNp-(GlcNAc)₄ > pNp-(GlcNAc)₃. From these results, silver croaker chitinase was judged to be an enzyme that preferentially hydrolyzes the 2nd glycosidic link from the non-reducing end of (GlcNAc)_n. The chitinase also showed wide substrate specificity for degrading α-chitin of shrimp and crab shell and β-chitin of squid pen. This coincides well with the feeding habit of the silver croaker, which feeds mainly on these animals.     

Environmental impacts and limitations of third‐generation biobutanol: Life cycle assessment of n‐butanol produced by genetically engineered cyanobacteria

Photosynthetic cyanobacteria have attracted interest as production organisms for third‐generation biofuels, where sunlight and CO₂ are used by microbes directly to synthesize fuel molecules. A particularly suitable biofuel is n‐butanol, and there have been several laboratory reports of genetically engineered photosynthetic cyanobacteria capable of synthesizing and secreting n‐butanol. This work evaluates the environmental impacts and cumulative energy demand (CED) of cyanobacteria‐produced n‐butanol through a cradle‐to‐grave consequential life cycle assessment (LCA). A hypothetical production plant in northern Sweden (area 1 ha, producing 5–85 m³ n‐butanol per year) was considered, and a range of cultivation formats and cellular productivity scenarios assessed. Depending on the scenario, greenhouse gas emissions (GHGe) ranged from 16.9 to 58.6 gCO₂eq/MJ_BuOH and the CED from 3.8 to 13 MJ/MJ_BuOH. Only with the assumption of a nearby paper mill to supply waste sources for heat and CO₂ was the sustainability requirement of at least 60% GHGe savings compared to fossil fuels reached, though placement in northern Sweden reduced energy needed for reactor cooling. A high CED in all scenarios shows that significant metabolic engineering is necessary, such as a carbon partitioning of >90% to n‐butanol, as well as improved light utilization, to begin to displace fossil fuels or even first‐ and second‐generation bioethanol.     

Concise synthesis of 4-methylumbelliferyl-penta-N-acetylchitopentaoside and its inhibition effect on chitinase

The synthesis of 4-methylumbelliferyl (UMB)-penta-N-acetylchitopentaoside 4 and its inhibition effect on chitinase are described. The fluorophore-assisted carbohydrate electrophoresis (FACE) analysis showed that the partially N-acetylated chitooligosaccharide (COS) mixture mainly contained glucosamine (GlcN) and oligomers [(GlcN)_n, n = 2–7]. The peracetylated COSs [(GlcNAc)_n, n = 1–7] were synthesized by treating the partially N-acetylated COS mixture with Ac₂O–NaOAc. The peracetylated chitopentaoside 1 was obtained by isolation of peracetylated COS mixture. The peracetylated UMB chitopentaoside 3 was synthesized by treating compound 1 with 4-methylumbelliferone and a Lewis acid (SnCl₄) catalyst. NaOMe in dry methanol was used for deacetylation of the blocked derivative, to give the target compound 4 in an overall yield of 32%. In binding chitinase assay, it indicates that compound 4 is much more stable than the corresponding penta-N-acetylchitopentaose 2.     

Intermittent contact mode AFM investigation of native plasma membrane of <Emphasis Type="Italic">Xenopus laevis</Emphasis> oocyte

Intermittent contact mode atomic force microscopy (AFM) was used to visualize the native plasma membrane of Xenopus laevis oocytes. Oocyte membranes were purified via ultracentrifugation on a sucrose gradient and adsorbed on mica leaves. AFM topographs and the corresponding phase images allowed for visualization and identification of both oocyte plasma membrane patches and pure lipid bilayer regions with a height of about 5 nm within membrane patches. The quantitative analysis showed a normal distribution for the lateral dimension and height of the protein complexes centered on 16.7 ± 0.2 nm (mean ± SE, n = 263) and 5.4 ± 0.1 nm (n = 262), respectively. The phase signal, providing material-dependent information, allowed for the recognition of structural features observed in AFM topographs.     

Influence of chelators and iron ions on the production and degradation of H2O2 by β-amyloid–copper complexes

β-Amyloid peptide (Aβ) 1–42, involved in the pathogenesis of Alzheimer’s disease, binds copper ions to form Aβ · Cu_n complexes that are able to generate H₂O₂ in the presence of a reductant and O₂. The production of H₂O₂ can be stopped with chelators. More reactive than H₂O₂ itself, hydroxyl radicals HO (generated when a reduced redox active metal complex interacts with H₂O₂) are also probably involved in the oxidative stress that creates brain damage during the disease. We report in the present work a method to monitor the effect of chelating agents on the production of hydrogen peroxide by metallo-amyloid peptides. The addition of H₂O₂ associated to a pre-incubation step between ascorbate and Aβ · Cu_n allows to study the formation of H₂O₂ but also, at the same time, its transformation by the copper complexes. Aβ · Cu_n peptides produce but do not efficiently degrade H₂O₂. The reported analytic method, associated to precipitation experiments of copper-containing amyloid peptides, allows to study the inhibition of H₂O₂ production by chelators. The action of a ligand such as EDTA is probably due to the removal of the copper ions from Aβ · Cu_n, whereas bidentate ligands such as 8-hydroxyquinolines probably act via the formation of ternary complexes with Aβ · Cu_n. The redox activity of these bidentate ligands can be modulated by the incorporation or the modification of substituents on the quinoline heterocycle.     

Methylated N-aryl chitosan derivative/DNA complex nanoparticles for gene delivery: Synthesis and structure–activity relationships

In this study, three kinds of methylated chitosan containing different aromatic moieties were synthesized by two steps, reductive amination and methylation, respectively. The chemical structures of all methylated derivatives, methylated N-(4-N,N-dimethylaminocinnamyl) chitosan chloride (MDMCMChC), methylated N-(4-N,N-dimethylaminobenzyl) chitosan chloride (MDMBzChC), and methylated N-(4-pyridinylmethyl) chitosan chloride (MPyMeChC) were characterized by ATR–FTIR and ¹H NMR spectroscopy. The complexes between the chitosan derivatives and plasmid DNA at different N/P ratios were characterized by gel electrophoresis, dynamic light scattering, and atomic force microscopic techniques. The smallest particle sizes of these complexes were obtained at N/P ratio of 5 and ranged from 95 to 124 nm while the zeta-potentials were in the range of 18–27 mV. Transfection efficiencies of these complexes were investigated by expression of the plasmid DNA encoding green fluorescence protein (pEGFP-C2) on human hepatoma cells (Huh 7 cells) compared to N,N,N-trimethyl chitosan chloride (TMChC). The rank of transfection efficiency was MPyMeChC > MDMBzChC > TMChC > MDMCMChC, respectively. The cytotoxicity of these complexes was also studied by MTT assay where the MPyMeChC complex exhibited less toxicity than other derivatives even at high N/P ratios. Therefore, MPyMeChC demonstrated potential as its safe and efficient gene carrier.     

Biohydrogen production from cellulosic hydrolysate produced via temperature-shift-enhanced bacterial cellulose hydrolysis

A “temperature-shift” strategy was developed to improve reducing sugar production from bacterial hydrolysis of cellulosic materials. In this strategy, production of cellulolytic enzymes with Cellulomonas uda E3-01 was promoted at a preferable temperature (35 °C), while more efficient enzymatic cellulose hydrolysis was achieved under an elevated culture temperature (45 °C), at which cell growth was inhibited to avoid consumption of reducing sugar. This temperature-shift strategy was shown to markedly increase the reducing sugar (especially, monosaccharide and disaccharide) concentration in the hydrolysate while hydrolyzing pure (carboxymethyl-cellulose, xylan, avicel and cellobiose) and natural (rice husk, rice straw, bagasse and Napier-grass) cellulosic materials. The cellulosic hydrolysates from CMC and xylan were successfully converted to H₂ via dark fermentation with Clostridium butyricum CGS5, attaining a maximum hydrogen yield of 4.79 mmol H₂/g reducing sugar.     

Molecular mass and chain conformations of Rhizoma Panacis Japonici polysaccharides

The molecular mass and size of five water-soluble polysaccharides isolated from Rhizoma Panacis Japonici (RPJ) were determined with laser light scattering (LLS), size-exclusion chromatography (SEC) combined with LLS (SEC–LLS), dynamic light scattering (DLS), as well as transmission electron microscope (TEM). Their weight-average molecular masses (M_w) were 3.5 × 10⁴, 1.47 × 10⁵, 1.24 × 10⁶, 9.26 × 10⁵ and 1.36 × 10⁶, radii of gyration (<s²>_z^1/2) were 14.7, 31.7, 50.8, 41.8 and 40.4 nm, and hydrodynamic radii (R_h) were 19.9, 37.5, 66.2, 52.1, and 55.2 nm, respectively. The results showed that molecular masses and sizes of the polysaccharides were influenced by the pH and temperature of the extraction mediums. The conformation parameters were calculated from the above data according to polymer solution theory. The values of ρ (= <s²>_z^1/2/R_h) were from 0.7 to 0.8, exponents (ν) of <s²>_z^1/2 = k M_w^ν were from 0.31 to 0.43, and fractal dimension (d_f) were from 2.3 to 3.2, respectively. The results revealed that all of the polysaccharides existed as spheres in 0.15 M NaCl aqueous solution. TEM and atomic force microscope (AFM) further confirmed the spherical morphologies of these molecules. The spherical conformations of the polysaccharides were a result of their highly branched structures.     

Physical and stability characteristics of Burkholderia cepacia lipase encapsulated in κ-carrageenan

In this work, a novel approach for lipase immobilization was exploited. Lipase from Burkholderia cepacia was encapsulated into κ-carrageenan by co-extrusion method to form a liquid core capsule. The diameter of the encapsulated lipase was found to be in the range of 1.3–1.8 mm with an average membrane thickness of 200 μm and 5% coefficient of variance. The encapsulation efficiency was 42.6% and 97% moisture content respectively. The encapsulated lipase was stable between pH 6 and 9 and temperature until 50 °C. The encapsulated lipase was stable until disintegration of the carrier when stored at 27 °C and retained 72.3% of its original activity after 6 cycles of hydrolysis of p-NPP. The encapsulated lipase was stable in various organic solvents including methanol, ethanol, iso-propanol, n-hexane and n-heptane. Kinetic parameters K_m and V_max were found to be 0.22 mM and 0.06 μmol/min for free lipase and 0.25 mM and 0.05 μmol/min for encapsulated lipase respectively.     

Sinus node dysfunction in ATX-II-induced in-vitro murine model of long QT3 syndrome and rescue effect of ranolazine

The aim of this study was to characterize the role of the late Na⁺ current (I_Na,L) as a mechanism for induction of both tachy and bradyarrhythmias in murine heart and sino-atrial node tissue. The sea anemone toxin ATX-II and ranolazine were used to increase and inhibit, respectively, I_Na,L. In sixteen hearts studied, exposure to 1–10 nM ATX-II caused a slowing of intrinsic heart rate and prolongations of the P–R and QT intervals, the duration of the monophasic action potential, and the sinus node recovery time, accompanied by frequent occurrences of early afterdepolarisations, delayed afterdepolarisations and rapid, repetitive ventricular tachy and sino-atrial bradyarrhythmias. ATX-II also slowed sinus node pacemaking, and induced bradycardic arrhythmias in isolated sino-atrial preparations (n = 5). The ATX-II-induced alteration of electrophysiological properties and occurrence of arrhythmic events were significantly attenuated by 10 μM ranolazine in intact hearts (n = 11) and isolated sino-atrial preparations (n = 5). In conclusion, the I_Na,L enhancer ATX-II causes both tachy and bradyarrhythmias in the murine heart, and these arrhythmias are markedly attenuated by the I_Na,L blocker, ranolazine (10 μM). The results suggest that I_Na,L blockade may be the mechanism underlying the reductions of both brady and tachyarrhythmias by ranolazine that were observed during the MERLIN-TIMI clinical outcomes trial.     

Synthesis, biological, and theoretical evaluations of new 1,2,3-triazoles against the hemolytic profile of the Lachesis muta snake venom

The current treatment used against envenomation by Lachesis muta venom still presents several side effects. This paper describes the synthesis, pharmacological and theoretical evaluations of new 1-arylsulfonylamino-5-methyl-1H-[1,2,3]-triazole-4-carboxylic acid ethyl esters (8a–f) tested against the hemolytic profile of the L. muta snake venom. Their structures were elucidated by one- and two-dimensional NMR techniques (¹H, APT, HETCOR ¹J_CH and ⁿJ_CH, n = 2, 3) and high-resolution electrospray ionization mass spectrometry. The series of triazole derivatives significantly neutralized the hemolysis induced by L. muta crude venom presenting a dose-dependent inhibitory profile (IC₅₀ = 30−83 μM) with 1-(4′-chlorophenylsulfonylamino)-5-methyl-1H-[1,2,3]-triazole-4-carboxylic acid ethyl ester (8e) being the most potent compound. The theoretical evaluation revealed the correlation of the antiophidian profile with the coefficient distribution and density map of the Highest Occupied Molecular Orbitals (HOMO) of these molecules. The elucidation of this new series may help on designing new and more efficient antiophidian molecules.     

Engineering alternative butanol production platforms in heterologous bacteria

Alternative microbial hosts have been engineered as biocatalysts for butanol biosynthesis. The butanol synthetic pathway of Clostridium acetobutylicum was first re-constructed in Escherichia coli to establish a baseline for comparison to other hosts. Whereas polycistronic expression of the pathway genes resulted in the production of 34 mg/L butanol, individual expression of pathway genes elevated titers to 200 mg/L. Improved titers were achieved by co-expression of Saccharomyces cerevisiae formate dehydrogenase while overexpression of E. coli glyceraldehyde 3-phosphate dehydrogenase to elevate glycolytic flux improved titers to 580 mg/L. Pseudomonas putida and Bacillus subtilis were also explored as alternative production hosts. Polycistronic expression of butanol biosynthetic genes yielded butanol titers of 120 and 24 mg/L from P. putida and B. subtilis, respectively. Production in the obligate aerobe P. putida was dependent upon expression of bcd-etfAB. These results demonstrate the potential of engineering butanol biosynthesis in a variety of heterologous microorganisms, including those cultivated aerobically.     

Nitrogen utilization by the raphidophyte Heterosigma akashiwo: Growth and uptake kinetics in laboratory cultures

The nitrogen uptake and growth capabilities of the potentially harmful, raphidophycean flagellate Heterosigma akashiwo (Hada) Sournia were examined in unialgal batch cultures (strain CCMP 1912). Growth rates as a function of three nitrogen substrates (ammonium, nitrate and urea) were determined at saturating and sub-saturating photosynthetic photon flux densities (PPFDs). At saturating PPFD (110 μE m⁻² s⁻¹), the growth rate of H. akashiwo was slightly greater for cells grown on NH₄⁺ (0.89 d⁻¹) compared to cells grown on NO₃⁻ or urea, which had identical growth rates (0.82 d⁻¹). At sub-saturating PPFD (40 μE m⁻² s⁻¹), both urea- and NH₄⁺-grown cells grew faster than NO₃⁻-grown cells (0.61, 0.57 and 0.46 d⁻¹, respectively). The N uptake kinetic parameters were investigated using exponentially growing batch cultures of H. akashiwo and the ¹⁵N-tracer technique. Maximum specific uptake rates (V_max) for unialgal cultures grown at 15 °C and saturating PPFD (110 μE m⁻² s⁻¹) were 28.0, 18.0 and 2.89 × 10⁻³ h⁻¹ for NH₄⁺, NO₃⁻ and urea, respectively. The traditional measure of nutrient affinity—the half saturation constants (K_s) were similar for NH₄⁺ and NO₃⁻ (1.44 and 1.47 μg-at N L⁻¹), but substantially lower for urea (0.42 μg-at N L⁻¹). Whereas the α parameter (α = V_max/K_s), which is considered a more robust indicator for substrate affinity when substrate concentrations are low (<K_s), were 19.4, 12.2 and 6.88 × 10⁻³ h⁻¹/(μg-at N L⁻¹) for NH₄⁺, NO₃⁻ and urea, respectively. These laboratory results demonstrate that at both saturating and sub-saturating N concentrations, N uptake preference follows the order: NH₄⁺ > NO₃⁻ > urea, and suggests that natural blooms of H. akashiwo may be initiated or maintained by any of the three nitrogen substrates examined.     

Biological control of brown rot (Moniliana laxa Ehr.) on apricot (Prunus armeniaca L. cv. Hacıhaliloğlu) by Bacillus, Burkholdria, and Pseudomonas application under in vitro and in vivo conditions

In 2002 and 2003, a study was conducted to determine the effect of bacterial strains, Burkholdria OSU 7, Bacillus OSU 142, and Pseudomonas BA 8, on biological control of brown rot disease (Monilinia laxa Ehr.) on apricot cv. Hacıhaliloğlu in Malatya province of Turkey. Apricot orchard at full blooming stage was inoculated with conidial suspension (1 × 10⁶ spores/ml) of M. laxa Ehr. After inoculation, two apricot trees for each application were treated with each of the three biological control agents (Burkholdria gladii OSU 7, Bacillus subtilis OSU 142, and Pseudomonas putida BA 8) by spraying (1 × 10⁹ cfu/ml) on inoculated branches. Disease incidence was evaluated for untreated (control 1) and four different treatment groups including commercial disease management (control 2, positive control: 3% Bourdox in fall, 50% Cupper at pink flower, 30 g/100 l Corus at first blooming, and 300 g/100 l Captan at last blooming stage) and treatments including each of the three bacterial strains (OSU 7, OSU 142, and BA 8). The results showed that disease incidence for negative control (control 1) was 9.94, which was significantly higher than disease incidence for commercial application (2.57%) or bacterial treatments (2.82–5.00%) in the first year. In 2003, the lowest disease incidence observed in OSU 7 treatment (6.80%), while disease incidence rate for positive control and negative control were 9.45% and 28.46%, respectively. This result may suggest that OSU 7 has potential to be used as biopesticide for effective management of brown rot disease on apricot.     

Inhibition of Fungal Plant Pathogens by Synergistic Action of Chito-Oligosaccharides and Commercially Available Fungicides

Chitosan is a linear heteropolymer consisting of β 1,4-linked N-acetyl-D-glucosamine (GlcNAc) and D-glucosamine (GlcN). We have compared the antifungal activity of chitosan with DP_n (average degree of polymerization) 206 and F _A (fraction of acetylation) 0.15 and of enzymatically produced chito-oligosaccharides (CHOS) of different DP_n alone and in combination with commercially available synthetic fungicides, against Botrytis cinerea, the causative agent of gray mold in numerous fruit and vegetable crops. CHOS with DP_n in the range of 15–40 had the greatest anti-fungal activity. The combination of CHOS and low dosages of synthetic fungicides showed synergistic effects on antifungal activity in both in vitro and in vivo assays. Our study shows that CHOS enhance the activity of commercially available fungicides. Thus, addition of CHOS, available as a nontoxic byproduct of the shellfish industry, may reduce the amounts of fungicides that are needed to control plant diseases.     

Magnetic nanoparticles supported ionic liquids for lipase immobilization: Enzyme activity in catalyzing esterification

Candida rugosa lipase was immobilized on magnetic nanoparticles supported ionic liquids having different cation chain length (C₁, C₄ and C₈) and anions (Cl⁻, BF₄⁻ and PF₆⁻). Magnetic nanoparticles supported ionic liquids were obtained by covalent bonding of ionic liquids–silane on magnetic silica nanoparticles. The particles are superparamagnetic with diameter of about 55 nm. Large amount of lipase (63.89 mg/(100 mg carrier)) was loaded on the support through ionic adsorption. Activity of the immobilized lipase was examined by the catalysis of esterification between oleic acid and butanol. The activity of bound lipase was 118.3% compared to that of the native lipase. Immobilized lipase maintained 60% of its initial activity even when the temperature was up to 80 °C. In addition, immobilized lipase retained 60% of its initial activity after 8 repeated batches reaction, while no activity was detected after 6 cycles for the free enzyme.     

Skewed birth sex ratio and premature mortality in elephants

Sex allocation theories predict equal offspring number of both sexes unless differential investment is required or some competition exists. Left undisturbed, elephants reproduce well and in approximately even numbers in the wild. We report an excess of males are born and substantial juvenile mortality occurs, perinatally, in captivity. Studbook data on captive births (CB, n = 487) and premature deaths (PD, <5 years of age; n = 164) in Asian and African elephants in Europe and North America were compared with data on Myanmar timber (Asian) elephants (CB, n = 3070; PD, n = 738). Growth in CB was found in three of the captive populations. A significant excess of male births occurred in European Asian elephants (ratio: 0.61, P = 0.044) and in births following artificial insemination (0.83, P = 0.003), and a numerical inclination in North American African elephants (0.6). While juvenile mortality in European African and Myanmar populations was 21–23%, it was almost double (40–45%) in all other captive populations. In zoo populations, 68–91% of PD were within 1 month of birth with stillbirth and infanticide being major causes. In Myanmar, 62% of juvenile deaths were at >6 months with maternal insufficient milk production, natural hazards and accidents being the main causes. European Asian and Myanmar elephants PD was biased towards males (0.71, P = 0.024 and 0.56, P < 0.001, respectively). The skewed birth sex ratio and high juvenile mortality hinder efforts to help captive populations become self-sustaining. Efforts should be invested to identify the mechanism behind these trends and seek solutions for them.     

Detoxification of model phenolic compounds in lignocellulosic hydrolysates with peroxidase for butanol production from <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis>

In the present study, we investigated the peroxidase-catalyzed detoxification of model phenolic compounds and evaluated the inhibitory effects of the detoxified solution on butanol production by Clostridium beijerinckii National Collection of Industrial and Marine Bacteria Ltd. 8052. The six phenolic compounds, p-coumaric acid, ferulic acid, 4-hydroxybenzoic acid, vanillic acid, syringaldehyde, and vanillin, were selected as model fermentation inhibitors generated during pretreatment and hydrolysis of lignocellulose. The enzyme reaction was optimized as a function of the reaction conditions of pH, peroxidase concentration, and hydrogen peroxide to substrate ratio. Most of the tested phenolics have a broad optimum pH range of 6.0 to 9. Removal efficiency increased with the molar ratio of H₂O₂ to each compound up to 0.5–1.25. In the case of p-coumaric acid, ferulic acid, vanillic acid, and vanillin, the removal efficiency was almost 100% with only 0.01 μM of enzyme. The tested phenolic compounds (1 g/L) inhibited cell growth by 64–74%, while completely inhibiting the production of butanol. Although syringaldehyde and vanillin were less toxic on cell growth, the level of inhibition on the butanol production was quite different. The detoxified solution remarkably improved cell growth and surprisingly increased butanol production to the level of the control. Hence, our present study, using peroxidase for the removal of model phenolic compounds, could be applied towards the detoxification of lignocellulosic hydrolysates for butanol fermentation.