Effect of hydrogen peroxide pretreatment on the structural features and the enzymatic hydrolysis of rice straw

Assessment was made to evaluate the effect of hydrogen peroxide pretreatment on the change of the structural features and the enzymatic hydrolysis of rice straw. Changes in the lignin content, weight loss, accessibility for Cadoxen, water holding capacity, and crystallinity of straw were measured during pretreatment to express the modification of the lignocellulosic structure of straw. The rates and the extents of enzymatic hydrolysis, cellulase adsorption, and cellobiose accumulation in the initial stage of hydrolysis were determined to study the pretreatment effect on hydrolysis. Pretreatment at 60 degrees C for 5 h in a solution with 1% (w/w) H(2)O(2) and NaOH resulted in 60% delignification, 40% weight loss, a fivefold increase in the accessibility for Cadoxen, an one times increase in the water-holding capacity, and only a slight decrease in crystallinity as compared with that of the untreated straw. Improvement on the pretreatment effect could be made by increasing the initial alkalinity and the pretreatment temperature of hydrogen peroxide solution. A saturated improvement on the structural features was found when the weight ratio of hydrogen peroxide to straw was above 0.25 g H(2)O(2)/g straw in an alkaline H(2)O(2) solution with 1% (w/w) NaOH at 32 degrees C. The initial rates and extents of hydrolysis, cellulase adsorption, and cellobiose accumulation in hydrolysis were enhanced in accordance with the improved structural features of straw pretreated. A four times increase in the extent of the enzymatic hydrolysis of straw for 24 h was attributed to the alkaline hydrogen peroxide pretreatment.     

Effect of oxygen delignification on the rate and extent of enzymatic hydrolysis of lignocellulosic material

In this study, we examined the effect of oxygen delignification on the rate and extent of enzymatic hydrolysis (using commercial cellulase and beta-glucosidase) of a number of lignocellulosic substrates, including kraft pulp (model substrate), pulp mill primary clarifier sludge (PCS) and steam-exploded Douglas fir chips. Oxygen delignification removed up to 67% of the lignin from softwood pulp and improved the rate of, and yield from, hydrolysis by up to 111% and 174%, respectively. Glucose yield varied linearly with fractional lignin removal. Oxygen delignification of primary clarifier sludge improved hydrolysis yield by up to 90%. However steam-exploded Douglas fir was very resistant to hydrolysis at low enzyme loading, and oxygen delignification decreased hydrolysis rate and yield.     

Degradation of Lignin-Containing Materials by Xylanase in Biopreparation of Cotton

Solubilization of lignin and carbohydrates from the lignin-holocellulose structure of cotton seed-coat fragments was investigated by UV/VIS spectrometry. Xylanase (Pulpzyme HC) pre-treatment partially destroyed the lignocellulosic structure of the seed-coat fragments, producing reducing sugars and soluble lignin in the supernatant. Furthermore, the pre-treatment by enzyme enhanced the delignification in the subsequent alkaline scouring process and increased the lightness of the substrate.     

Bicarbonate is a recycling substrate for cyanase

Cyanase is an inducible enzyme in Escherichia coli that catalyzes bicarbonate-dependent decomposition of cyanate to ammonia and bicarbonate. Previous studies provided evidence that carbamate is an initial product and that the kinetic mechanism is rapid equilibrium random (bicarbonate serving as substrate as opposed to activator); the following mechanism was proposed (Anderson, P. M. (1980) Biochemistry 19, 2282-2888; Anderson, P. M., and Little, R. M. (1986) Biochemistry 25, 1621-1626). (formula; see text) Direct evidence for this mechanism was obtained in this study by 1) determining whether CO2 or HCO3- serves as substrate and is formed as product, 2) identifying the products formed from [14C]HCO3- and [14C] OCN-, 3) identifying the products formed from [13C] HCO3- and [12C]OCN- in the presence of [18O]H2O, and 4) determining whether 18O from [18O]HCO3- is incorporated into CO2 derived from OCN-. Bicarbonate (not CO2) is the substrate. Carbon dioxide (not HCO3-) is produced in stoichiometric amounts from both HCO3- and OCN-. 18O from [18O]H2O is not incorporated into CO2 formed from either HCO3- or OCN-. Oxygen-18 from [18O]HCO3- is incorporated into CO2 derived from OCN-. These results support the above mechanism, indicating that decomposition of cyanate catalyzed by cyanase is not a hydrolysis reaction and that bicarbonate functions as a recycling substrate.     

The hydrogen peroxide reactivity of peptidylglycine monooxygenase supports a Cu(II)-superoxo catalytic intermediate

We have investigated the reaction of peptidylglycine monooxygenase with hydrogen peroxide to determine whether Cu(II)-peroxo is a likely intermediate. When the oxidized enzyme was reacted with the dansyl-YVG substrate and H(2)O(2), the alpha-hydroxyglycine product was formed. The reaction was catalytic and did not require the presence of additional reductant. When (18)O-labeled H(2)O(2) was reacted with peptidylglycine monooxygenase and substrate anaerobically, oxygen in the product was labeled with (18)O and must therefore be derived from H(2)O(2). However, when the reaction was carried out with H (16)(2)O(2) in the presence of (18)O(2), 60% of the product contained the (18)O label. Therefore, the reaction must proceed via an intermediate that can react directly with dioxygen and thus scramble the label. Under strictly anaerobic conditions (in the presence of glucose and glucose oxidase, where no oxygen was released into the medium from nonenzymatic peroxide decomposition), product formation and peroxide consumption were tightly coupled, and the rate of product formation was identical to that measured under aerobic conditions. Peroxide reactivity was eliminated by a mutation at the Cu(H) center, which should not be involved in the peroxide shunt. Our data lend support to recent proposals that Cu(II)-superoxide is the active species.     

Polymerization of lignin fragments contained in a model effluent by polyphenoloxidases and horseradish peroxidase/hydrogen peroxide system*

An effluent containing soluble lignin fragments was treated with potato-polyphenoloxidases (PPO) or horseradish peroxidase/hydrogen peroxide system (HRP/H(2)O(2)). In both cases the reaction was evidenced by the formation of a brown precipitate that was a consequence of the polymerization of lignin fragments. The effect of reaction time, pH, and amount of soluble lignin per unit of enzyme activity on the insolubilization yield was evaluated for PPO-initiated reactions. For HRP-initiated reactions, the amount of H(2)O(2) per unit of enzyme activity was also evaluated. Mathematical models were calculated to predict the insolubilization yield as a function of the significant variables. Based on these models, the insolubilization reaction was optimized and reached maximal values of ca. 50% in both reaction systems. Higher insolubilization yields were not achieved. Chemical characterization of the soluble lignin fragments indicated that the insolubilization yield could not be improved, because the lignin fragments had limited amounts of free phenolic substructures available for the initial steps of the polymerization reaction.     

Alkaline peroxide delignification of agricultural residues to enhance enzymatic saccharification

Approximately one-half of the lignin and most of the hemicellulose present in agricultural residues such as wheat straw and corn stover are solubilized when the residue is treated at 25 degrees C in an alkaline solution of hydrogen peroxide. The delignification reaction is most efficient when the ratio of hydrogen peroxide to substrate is at least 0.25 (w/w) and the pH is 11.5. The supernatant fraction from a given pretreatment, after addition of makeup peroxide and readjustment of the pH, can be recycled to treat at least six additional batches of substrate, resulting in a substantial concentration of hemicellulose and soluble lignin degradation products. Hydrolysis of the insoluble fraction with Trichoderma reesei cellulase after alkaline peroxide treatment yields glucose with almost 100% efficiency, based upon the cellulose content of the residue before treatment. These data indicate that alkaline peroxide pretreatment is a simple and efficient method for enhancing the enzymatic digestibility of lignocellulosic crop residues to levels approaching the theoretical maximum.     

A comparison of chemical pretreatment methods for improving saccharification of cotton stalks

The effectiveness of sulfuric acid (H(2)SO(4)), sodium hydroxide (NaOH), hydrogen peroxide (H(2)O(2)), and ozone pretreatments for conversion of cotton stalks to ethanol was investigated. Ground cotton stalks at a solid loading of 10% (w/v) were pretreated with H(2)SO(4), NaOH, and H(2)O(2) at concentrations of 0.5%, 1%, and 2% (w/v). Treatment temperatures of 90 degrees C and 121 degrees C at 15 psi were investigated for residence times of 30, 60, and 90 min. Ozone pretreatment was performed at 4 degrees C with constant sparging of stalks in water. Solids from H(2)SO(4), NaOH, and H(2)O(2) pretreatments (at 2%, 60 min, 121 degrees C/15 psi) showed significant lignin degradation and/or high sugar availability and hence were hydrolyzed by Celluclast 1.5L and Novozym 188 at 50 degrees C. Sulfuric acid pretreatment resulted in the highest xylan reduction (95.23% for 2% acid, 90 min, 121 degrees C/15 psi) but the lowest cellulose to glucose conversion during hydrolysis (23.85%). Sodium hydroxide pretreatment resulted in the highest level of delignification (65.63% for 2% NaOH, 90 min, 121 degrees C/15 psi) and cellulose conversion (60.8%). Hydrogen peroxide pretreatment resulted in significantly lower (p相似文献   

Mass spectrometric determination of hydroxylamine photooxidation by illuminated chloroplasts.

A mass spectrometer with a special inlet was used to directly monitor the products evolved when hydroxylamine-treated chloroplasts were exposed to short saturating light flashes. We found that: 1. Molecular dinitrogen was the sole product of hydroxylamine photooxidation, and was formed in an amount equal to twice the O2 evolved during H2O photooxidation. 2. This reaction was driven by Photosystem II, and did not involve Photo-system I-generated superoxide or peroxide. 3. In the presence of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, N2 was evolved only on the first flash. These results suggested that N2 was formed by the combination of two single-electron oxidation products of hydroxylamine.     

Screening of ecologically diverse fungi for their potential to pretreat lignocellulosic bioenergy feedstock

A widespread and hitherto by far underexploited potential among ecologically diverse fungi to pretreat wheat straw and digestate from maize silage in the future perspective of using such lignocellulosic feedstock for fermentative bioenergy production was inferred from a screening of nine freshwater ascomycetes, 76 isolates from constructed wetlands, nine peatland isolates and ten basidiomycetes. Wheat straw pretreatment was most efficient with three ascomycetes belonging to the genera Acephala (peatland isolate) and Stachybotrys (constructed wetland isolates) and two white-rot fungi (Hypholoma fasciculare and Stropharia rugosoannulata) as it increased the amounts of water-extractable total sugars by more than 50 % and sometimes up to 150 % above the untreated control. The ascomycetes delignified wheat straw at rates (lignin losses between about 31 and 40 % of the initial content) coming close to those observed with white-rot fungi (about 40 to 57 % lignin removal). Overall, fungal delignification was indicated as a major process facilitating the digestibility of wheat straw. Digestate was generally more resistant to fungal decomposition than wheat straw. Nevertheless, certain ascomycetes delignified this substrate to extents sometimes even exceeding delignification by basidiomycetes. Total sugar amounts of about 20 to 60 % above the control value were obtained with the most efficient fungi (one ascomycete of the genus Phoma, the unspecific wood-rot basidiomycete Agrocybe aegerita and one unidentified constructed wetland isolate). This was accompanied by lignin losses of about 47 to 56 % of the initial content. Overall, digestate delignification was implied to be less decisive for high yields of fermentable sugars than wheat straw delignification.     

NH(4)OH-KOH pulping mechanisms and kinetics of rice straw

The mechanisms and kinetics of NH(4)OH-KOH mixture pulping rice straw were studied. When aqueous ammonia was mixed with a small amount of caustic potash (ratio of 1:5), three distinct delignification phases were observed in the pulping process: a bulk delignification phase from the beginning of the cooking period to 100 degrees C, a supplementary delignification phase from 100 degrees C to 155 degrees C lasting a further 45 min, and a residual delignification phase until the end of the cooking period. There were two silica removal phases; the first phase was from the beginning of the cooking period to 100 degrees C and the second phase was from 100 degrees C to the end of the cooking period. The rate of delignification reaction was first order with respect to residual lignin and 0.3 order with respect to [OH(-)]. The silica removal was pseudo-first-order with respect to residual silica and 0.6 order with respect to [OH(-)]. The activation energies of the delignification and removal of silica reactions were 35.6 and 30.9 kJ/mol, respectively.     

Biochemistry of the oxidation of lignin by Phanerochaete chrysosporium

The objective of this research was to identify the biochemical agents responsible for the oxidative degradation of lignin by the white-rot fungus Phanerochaete chrysosporium. We examined the hypothesis that activated oxygen species are involved, and we also sought the agent in ligninolytic cultures responsible for a specific oxidative degradative reaction in substructure model compounds. Results of studies of the production of activated oxygen species by cultures, of the effect of their removal on ligninolytic activity, and of their action on substructure model compounds support a role for hydrogen peroxide (H(2)O(2)) and possibly superoxide (O(2)(*)(-)) in lignin degradation. Involvement of hydroxyl radical (*OH) or singlet oxygen (1O(2)) is not supported by our data. The actual biochemical agent responsible for one important oxidative C-C bond cleavage reaction in non-phenolic lignin substructure model compounds, and in lignin itself, was found to be an enzyme. The enzyme is extracellular, has a molecular weight of 42,000 daltons, is azide-sensitive, and requires H(2)O(2) for activity.     

Restriction of the enzymatic hydrolysis of steam-pretreated spruce by lignin and hemicellulose

The presence of lignin is known to reduce the efficiency of the enzymatic hydrolysis of lignocellulosic raw materials. On the other hand, solubilization of hemicellulose, especially of xylan, is known to enhance the hydrolysis of cellulose. The enzymatic hydrolysis of spruce, recognized among the most challenging lignocellulosic substrates, was studied by commercial and purified enzymes from Trichoderma reesei. Previously, the enzymatic hydrolysis of steam pretreated spruce has been studied mainly by using commercial enzymes and no efforts have been taken to clarify the bottlenecks by using purified enzyme components.Steam-pretreated spruce was hydrolyzed with a mixture of Celluclast and Novozym 188 to obtain a hydrolysis residue, expectedly containing the most resistant components. The pretreated raw material and the hydrolysis residue were analyzed for the enrichment of structural bottlenecks during the hydrolysis. Lignin was removed from these two materials with chlorite delignification method in order to eliminate the limitations caused by lignin. Avicel was used for comparison as a known model substrate. Mixtures of purified enzymes were used to investigate the hydrolysis of the individual carbohydrates: cellulose, glucomannan and xylan in the substrates. The results reveal that factors limiting the hydrolysis are mainly due to the lignin, and to a minor extent by the lack of accessory enzymes. Removal of lignin doubled the hydrolysis degree of the raw material and the residue, and reached close to 100% of the theoretical within 2 days. The presence of xylan seems to limit the hydrolysability, especially of the delignified substrates. The hydrolysis results also revealed significant hemicellulose impurities in the commonly used cellulose model substrate, making it questionable to use Avicel as a model cellulose substrate for hydrolysis experiments.     

Advances in microbial delignification

Microbial delignification is a new field of applied research. The progress will therefore run parallel to the development of new basic knowledge on the physiological demands of white-rot fungi to degrade lignin and on new knowledge on enzyme mechanisms involved in lignin degradation.In the last few years both basic and applied research on microbial conversion of lignocellulosic materials have vastly expanded. In certain areas, such as microbial delignification, considerable progress has recently been made. Basidiospores from Sporotrichum pulverulentum and some CEL(-) mutants have been obtained. Crossing of mycelium from single basidiospore cultures of wild-type and CEL(-) mutants will eventually give rise to much better CEL(-) mutants than those which have been used in the past. An understanding of which enzymes are the most important for lignin degradation to take place is also beginning to develop. This review discusses present knowledge and future possibilities in this field.     

Modification of wheat straw in a high-shear mixer

Wheat straw (Ws)was treated in a pilot-scale continuous mixer to disrupt the lignin-hemicellulose-cellulose (LHC) complex. An efficient and practical method was desired to remove lignin and hemicellulose (pentosans)rapidly and efficiently from the lignocellulose complex and to make the cellulose accessible to enzymatic hydrolysis. Milled WS in the presence of various chemicals in aqueous solutions was extruded from the mixer under several processing conditions. Chemicals used were sodium hydroxide (NaOH) sodium sulfide (Na(2)S), anthraquinone (AQ), anthrahydroquinone (AHQ), hexamethylenediamine (HMDA), hexamethylenetetramine (HMTA) hydrogen peroxide (H(2)O(2)), and ferrous ammonium sulfate (FAS), which were used alone and in selected combinations. Concomitantly, WS was treated in laboratory batches using similar reaction conditions, except for mixing and shearing. In extrusion treatments of WS at 20% concentration at 97 degrees C for 5.5 min with NaOH (15.7%, dry WS basis), NaOH (15.7%) + AHQ (0.3%), and NaOH (12.7%) + Na(2)S (5.0%), 64-72% of the WS lignin and 36-43% of the pentosans were removed from aqueously washed extrudates (residues). This compares with 46-56% and 23-27%, respectively, for batch treatments. AHQ and Na(2)S enhanced delignification. Cellulase treatment of the residues, which contained about 99%of the WS cellulose, converted 90-92%of the cellulose to glucose compared with 61-69%for the batch pretreatments. Treatments of WS with amines and H(2)O(2) (alone or combined with NaOH)were less effective for LHC disruption. In all instances the relatively high-shear extrusion treatments were superior to the laboratory-batch treatments.     

Effect of initial moisture content and chip size on the bioconversion efficiency of softwood lignocellulosics

Previous optimization strategies for the bioconversion of lignocellulosics by steam explosion technologies have focused on the effects of temperature, pH, and treatment time, but have not accounted for changes in severity brought about by properties inherent in the starting feedstock. Consequently, this study evaluated the effects of chip properties, feedstock size (40-mesh, 1.5 x 1.5 cm, 5 x 5 cm), and moisture content (12% and 30%) on the overall bioconversion process, and more specifically on the efficacy of removal of recalcitrant lignin from the lignocellulosic substrates following steam explosion. Increasing chip size resulted in an improvement in the solids recovery, with concurrent increases in the water soluble, hemicellulose-derived sugar recovery (7.5%). This increased recovery is a result of a decrease in the "relative severity" of the pretreatment as chip size increases. Additionally, the decreased relative severity minimized the condensation of the recalcitrant residual lignin and therefore increased the efficacy of peroxide fractionation, where a 60% improvement in lignin removal was possible with chips of larger initial size. Similarly, increased initial moisture content reduced the relative severity of the pretreatment, generating improved solids and hemicellulose-derived carbohydrate recovery. Both increased chip size and higher initial moisture content results in a substrate that performs better during peroxide delignification, and consequently enzymatic hydrolysis. Furthermore, a post steam-explosion refining step increased hemicellulose-derived sugar recovery and was most effectively delignified (to as low as 6.5%). The refined substrate could be enzymatically hydrolyzed to very high levels (98%) and relatively fast rates (1.23 g/L/h).     

Benzene-induced uncoupling of naphthalene dioxygenase activity and enzyme inactivation by production of hydrogen peroxide

Naphthalene dioxygenase (NDO) is a multicomponent enzyme system that oxidizes naphthalene to (+)-cis-(1R,2S)-1,2-dihydroxy-1, 2-dihydronaphthalene with consumption of O2 and two electrons from NAD(P)H. In the presence of benzene, NADH oxidation and O2 utilization were partially uncoupled from substrate oxidation. Approximately 40 to 50% of the consumed O2 was detected as hydrogen peroxide. The rate of benzene-dependent O2 consumption decreased with time, but it was partially increased by the addition of catalase in the course of the O2 consumption by NDO. Detailed experiments showed that the total amount of O2 consumed and the rate of benzene-induced O2 consumption increased in the presence of hydrogen peroxide-scavenging agents, and further addition of the terminal oxygenase component (ISPNAP) of NDO. Kinetic studies showed that ISPNAP was irreversibly inactivated in the reaction that contained benzene, but the inactivation was relieved to a high degree in the presence of catalase and partially relieved in the presence of 0.1 mM ferrous ion. Benzene- and naphthalene-reacted ISPNAP gave almost identical visible absorption spectra. In addition, hydrogen peroxide added at a range of 0.1 to 0.6 mM to the reaction mixtures inactivated the reduced ISPNAP containing mononuclear iron. These results show that hydrogen peroxide released during the uncoupling reaction acts both as an inhibitor of benzene-dependent O2 consumption and as an inactivator of ISPNAP. It is proposed that the irreversible inactivation of ISPNAP occurs by a Fenton-type reaction which forms a strong oxidizing agent, hydroxyl radicals (. OH), from the reaction of hydrogen peroxide with ferrous mononuclear iron at the active site. Furthermore, when [14C]benzene was used as the substrate, cis-benzene 1,2-dihydrodiol formed by NDO was detected. This result shows that NDO also couples a trace amount of benzene to both O2 consumption and NADH oxidation.     

Laccase-HBT bleaching of eucalyptus kraft pulp: influence of the operating conditions

Different operating conditions (viz. pulp consistency, oxygen pressure and treatment time) in the biobleaching of eucalyptus kraft pulp with the laccase-HBT system was tested in order to describe their effect and normalize a biobleaching protocol. A high O(2) pressure (0.6MPa) was found to result in improved laccase-assisted delignification of the pulp. Also, a high pulp consistency (10%) and a short treatment time (2h) proved the best choices with a view to obtaining good pulp properties (kappa number and ISO brightness) under essentially mild conditions. The laccase-HBT treatment was found to result in slight delignification (in the form of a 20-27% decrease in kappa number); however, an alkaline extraction stage raised delignification to 41-45%, a much higher level than those obtained in the control tests (16-23%). Also, the use of hydrogen peroxide in the extraction stage resulted in improved brightness (14-19%), but in scarcely improved delignification (4-7%). Treating the pulp with the laccase-HBT system reduced the amount of hydrogen peroxide required for subsequent alkaline bleaching by a factor of 3-4 relative to control tests.     

Biological Pretreatment of Lignocellulosic Substrates for Enhanced Delignification and Enzymatic Digestibility

Sheer enormity of lignocellulosics makes them potential feedstock for biofuel production but, their conversion into fermentable sugars is a major hurdle. They have to be pretreated physically, chemically, or biologically to be used by fermenting organisms for production of ethanol. Each lignocellulosic substrate is a complex mix of cellulose, hemicellulose and lignin, bound in a matrix. While cellulose and hemicellulose yield fermentable sugars, lignin is the most recalcitrant polymer, consisting of phenyl-propanoid units. Many microorganisms in nature are able to attack and degrade lignin, thus making access to cellulose easy. Such organisms are abundantly found in forest leaf litter/composts and especially include the wood rotting fungi, actinomycetes and bacteria. These microorganisms possess enzyme systems to attack, depolymerize and degrade the polymers in lignocellulosic substrates. Current pretreatment research is targeted towards developing processes which are mild, economical and environment friendly facilitating subsequent saccharification of cellulose and its fermentation to ethanol. Besides being the critical step, pretreatment is also cost intensive. Biological treatments with white rot fungi and Streptomyces have been studied for delignification of pulp, increasing digestibility of lignocellulosics for animal feed and for bioremediation of paper mill effluents. Such lignocellulolytic organisms can prove extremely useful in production of bioethanol when used for removal of lignin from lignocellulosic substrate and also for cellulase production. Our studies on treatment of hardwood and softwood residues with Streptomyces griseus isolated from leaf litter showed that it enhanced the mild alkaline solubilisation of lignins and also produced high levels of the cellulase complex when growing on wood substrates. Lignin loss (Klason lignin) observed was 10.5 and 23.5% in case of soft wood and hard wood, respectively. Thus, biological pretreatment process for lignocellulosic substrate using lignolytic organisms such as actinomycetes and white rot fungi can be developed for facilitating efficient enzymatic digestibility of cellulose.     

Suicide-peroxide inactivation of microperoxidase-11: a kinetic study

The kinetics of microperoxidase-11 (MP-11) in the oxidation reaction of guaiacol (AH) by hydrogen peroxide was studied, taking into account the inactivation of enzyme during reaction by its suicide substrate, H2O2. Concentrations of substrates were so selected that: 1) the reaction was first-order in relation to benign substrate, AH and 2) high ratio of suicide substrate to the benign substrate, [H2O2] > [AH]. Validation and reliability of the obtained kinetic equations were evaluated in various nonlinear and linear forms. Fitting of experimental data into the obtained integrated equation showed a close match between the kinetic model and the experimental results. Indeed, a similar mechanism to horseradish peroxidase was found for the suicide-peroxide inactivation of MP-11. Kinetic parameters of inactivation including the intact activity of MP-11, alphai, and the apparent inactivation rate constant, ki, were obtained as 0.282 +/- 0.006 min(-1) and 0.497 +/- 0.013(-1) min at [H2O2] = 1.0 mM, 27 degrees C, phosphate buffer 5.0 mM, pH = 7.0. Results showed that inactivation of microperoxidase as a peroxidase model enzyme can occur even at low concentrations of hydrogen peroxide (0.4 mM).