Catalytic properties of Phe41→His mutant of horseradish peroxidase expressed inE. coli

The recombinant horseradish peroxidase and its single-point F41H mutant have been reactivated fromE. coli inclusion bodies. The influence of the mutation on the heme entrapment, stability and activity of the enzyme was demonstrated. The catalytic rate constants for H₂O₂ cleavage and ammonium 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonate (ABTS) oxidation decrease by two and one orders of magnitude, respectively. Unlike the wild-type recombinant horseradish peroxidase, the elimination of the ABTS oxidation product is not a rate-determining step for the mutant. The F41H replacement results in significant changes of kinetics of iodide ion oxidation. The reaction rate is linear to the concentrations of iodide, H₂O₂, and the enzyme. The results suggest the direct interaction of iodide with the porphyrin ring of the heme. The decrease in ABTS oxidation activity accompanied by retention of activity in iodide oxidation in the course of low-dosage radiolysis of the F41H mutant is additional evidence of the direct electron transfer from iodide to the heme, in contrast to ABTS oxidation, in which the electron transfer chain in the protein molecule is involved.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 11, pp. 2034–2038, November, 1994.     

Effect of a negative charge on the screening of the active site of horseradish peroxidase

The F143E mutant form of the recombinant horseradish peroxidase was reactivated fromE. coli inclusion bodies. The mutation inhibits heme entrapment and results in a decrease in the catalytic activity, mainly affecting the stage of the oxidation of a donor substrate (ABTS, iodide). An increase in stability of the mutant form obtained under radiation inactivation over that of the wild-type recombinant enzyme was observed. The data obtained confirms the proposed location of Phel43 at the entrance of the active center, hence its replacement by the negatively charged glutamic acid residue retards heme entrapment and substrate binding, thus protecting the active center of the enzyme against the radicals generated by radiolysis.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 2, pp. 371–374, February, 1995.     

Intensification of Biocatalytical Processes by Synergistic Substrate Conversion. Fungal Peroxidase Catalyzed <Emphasis Type="Italic">N</Emphasis>-Hydroxy Derivative Oxidation in Presence of 10-Propyl Sulfonic Acid Phenoxazine

Many industrial pollutants, xenobiotics, and industry-important compounds are known to be oxidized by peroxidases. It has been shown that highly efficient peroxidase substrates are able to enhance the oxidation of low reactive substrate by acting as mediators. To explore this effect, the oxidation of two N-hydroxy derivatives, i.e., N-hydroxy-N-phenyl-acetamide (HPA) and N-hydroxy-N-phenyl-carbamic acid methyl ester (HPCM) catalyzed by recombinant Coprinus cinereus (rCiP) peroxidase has been studied in presence of efficient substrate 3-(4a,10a-dihydro- phenoxazin-10-yl)-propane-1-sulfonic acid (PPSA) at pH 8.5. The bimolecular constant of PPSA cation radical reaction with HPA was estimated to be (2.5 ± 0.2)·10⁷ M⁻¹ s⁻¹ and for HPCM was even higher. The kinetic measurements show that rCiP-catalyzed oxidation of HPA and HPCM can increase up to 33,000 times and 5,500 times in the presence of equivalent concentration of high reactive substrate PPSA. The mathematical model of synergistic rCiP-catalyzed HPA–PPSA and HPCM–PPSA oxidation was proposed. Experimentally obtained rate constants were in good agreement with those calculated from the model confirming the synergistic scheme of the substrate oxidation. In order to explain the different reactivity of substrates, the docking of substrates in the active site of the enzyme was calculated. Molecular dynamic calculations show that the enzyme–substrate complexes are structurally stable. The high reactive PPSA exhibited higher affinity to enzyme active site than HPA and HPCM. Furthermore, the orientation of HPA and HPCM was not favorable for proton transfer to the distal histidine, and different substrate reactivity was explained by these diversities.     

Wild type and mutant forms of recombinant horseradish peroxidase C expressed inEscherichia coli

Two horseradish peroxidase C (HRPC) mutants with substitutions in the active center, i.e., Phe41→ His and Phel43→ Glu, were compared to the wild-type recombinant enzyme expressed in Escherichia coli in terms of the enzymatic activity and stability under irradiation. Both mutations caused a significant decrease in activity, but it was still possible to follow the effect of mutations on the key steps of the reaction mechanism. Phe41 can be considered a nonpolar barrier separating histidine residues in the active center and providing a firm noncovalent binding with the highly hydrophobic porphyrin ring. The replacement of Phe41 with the ionizable His residue destabilizes the enzyme. The Phel43→ Glu replacement creates a negative charge at the entrance of the heme-binding pocket, and protects the latter from both donor substrates and free radicals. The radiolytic inactivation of the wild-type and mutant forms of recombinant HRP suggested different binding sites for iodide, 2,2′-bis(3-ethylbenzothiasoline-6-sulfonate (ABTS), guaiacol, and o-phenylene diamine. The study of kinetics and inactivation is in agreement with the direct binding of iodide to the heme porphyrin ring. The results also suggest that the ABTS binding site is less accessible than that for o-phenylene diamine.     

Screening of laccase, manganese peroxidase, and versatile peroxidase activities of the genus Pleurotus in media with some raw plant materials as carbon sources

Species of the genus Pleurotus are among the most efficient natural species in lignin degradation belonging to the subclass of ligninolytic organisms that produce laccase (Lac), Mn-dependent peroxidase (MnP), versatile peroxidase (VP), and the H₂O₂-generating enzyme aryl-alcohol oxidase, but not lignin peroxidases. Production of Lac and oxidation of 2,6-dimethoxyphenol (DMP) in the presence and absence of Mn²⁺ were detected both in submerged fermentation (SF) of dry ground mandarine peels and in solid-state fermentation (SSF) of grapevine sawdust in all investigated Pleurotus species and strains. Evidence of cultivation methods having a distinct influence on the level of enzyme activities has been demonstrated. Most of the species and strains had higher Lac activity under SSF conditions than under SF conditions. DMP oxidation in the presence and absence of Mn²⁺ was detected in all investigated species and strains, but was lower under SF conditions than under SSF conditions for most of them. However, relative activities of DMP oxidation in the absence of Mn²⁺, as percentages of activity agasint DMP in the presence of Mn²⁺, were higher under conditions of SF than in SSF cultures in most of the investigated species and strains. The obtained results showed that strains of different origins have different efficiently ligninolytic systems and that conditions of SSF are more favorable for ligninolytic activity than those in SF owing to their similarity to natural conditions on wood substrates.     

Purification and Partial Characterization of Lignin Peroxidase from <Emphasis Type="Italic">Acinetobacter calcoaceticus</Emphasis> NCIM 2890 and Its Application in Decolorization of Textile Dyes

Lignin peroxidase was purified (72-fold) from Acinetobacter calcoaceticus NCIM 2890. The purified lignin peroxidase (55–65 kDa) showed dimeric nature. The maximum enzyme activity was observed at pH 1.0, between a broad temperature range of 50 and 70°C, at H₂O₂ concentration (40 mM) and the substrate concentration (n-propanol, 100 mM). Purified lignin peroxidase was able to oxidize a variety of substrates including Mn²⁺, tryptophan, mimosine, l-Dopa, hydroquinone, xylidine, n-propanol, veratryl alcohol, and ten textile dyes of various groups indicating as a versatile peroxidase. Most of the dyes decolorized up to 90%. Tryptophan stabilizes the lignin peroxidase activity during decolorization of dyes.     

Improvement of <Emphasis Type="Italic">Aspergillus sulphureus</Emphasis> Endo-β-1,4-Xylanase Expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis> by Codon Optimization and Analysis of the Enzymic Characterization

The gene xynB from Aspergillus sulphureus encoding the endo-β-1,4-xylanase was de novo synthesized by splicing overlap extension polymerase chain reaction according to Pichia pastoris protein’s codon bias. The synthetic DNA and wild-type DNA were placed under the control of a glyceraldehyde-3-phosphate dehydrogenase gene promoter (GAP) in the constitutive expression vector plasmid pGAPzαA and electrotransformed into the P. pastoris X-33 strain, respectively. The transformants screened by Zeocin were able to constitutively secrete the xylanase in YPD liquid medium. The maximum yield of the recombinant xylanase produced by the synthetic DNA was 105 U ml⁻¹, which was about 5-fold higher than that by wild-type DNA under the flask culture at 28 °C for 3 days. The enzyme showed optimal activity at 50 °C and pH 5.0. The residual activity remained above 90% after the recombinant xylanase was pretreated in Na₂HPO₄–citric acid buffer (pH 2.4) for 2 h. The xylanase activity was significantly improved by Zn²⁺. These biochemical characteristics suggest that the recombinant xylanase has a prospective application in feed industry as an additive.     

Prokaryotic Expression, Purification and Characterization of Aspergillus sulphureus β-Mannanase and Site-Directed Mutagenesis of the Catalytic Residues

Wild type (WT) DNA sequence, which encoded a mature β-mannanase of Aspergillus sulphureus, composed of 1,152 nucleotides (nt), was amplified from pUCm-T-mann by polymerase chain reaction (PCR). Based on this DNA fragment, mutants designated as E²⁰⁶G and E³¹⁴G were constructed by overextension PCR (OE-PCR). Glutamic acids of the 206th and 314th sites in the amino acid sequence of β-mannanase were separately replaced by glycine in these two mutants. The WT and mutant genes were ligated into prokaryotic vector pET-28a (+) and transformed into the Escherichia coli BL21 strain, respectively. The recombinant enzyme proteins were expressed by IPTG induction and detected by Western blot. The recombinant proteins purified with Ni-NTA column were dialyzed to correctly refold. The WT recombinant β-mannanase showed optimal activity at 50 °C and pH 2.4. The kinetic parameters of K _m and V _max for purified β-mannanase were 1.38 mg/ml and 72.99 U/mg, respectively. However, the mutant proteins did not show any activity. It was demonstrated that E²⁰⁶ and E³¹⁴ were the catalytic residues of β-mannanase.     

An Aromatic Dyad Motif in Dye Decolourising Peroxidases Has Implications for Free Radical Formation and Catalysis

Dye decolouring peroxidases (DyPs) are the most recent class of heme peroxidase to be discovered. On reacting with H₂O₂, DyPs form a high-valent iron(IV)-oxo species and a porphyrin radical (Compound I) followed by stepwise oxidation of an organic substrate. In the absence of substrate, the ferryl species decays to form transient protein-bound radicals on redox active amino acids. Identification of radical sites in DyPs has implications for their oxidative mechanism with substrate. Using a DyP from Streptomyces lividans, referred to as DtpA, which displays low reactivity towards synthetic dyes, activation with H₂O₂ was explored. A Compound I EPR spectrum was detected, which in the absence of substrate decays to a protein-bound radical EPR signal. Using a newly developed version of the Tyrosyl Radical Spectra Simulation Algorithm, the radical EPR signal was shown to arise from a pristine tyrosyl radical and not a mixed Trp/Tyr radical that has been widely reported in DyP members exhibiting high activity with synthetic dyes. The radical site was identified as Tyr374, with kinetic studies inferring that although Tyr374 is not on the electron-transfer pathway from the dye RB19, its replacement with a Phe does severely compromise activity with other organic substrates. These findings hint at the possibility that alternative electron-transfer pathways for substrate oxidation are operative within the DyP family. In this context, a role for a highly conserved aromatic dyad motif is discussed.     

Study of the substrate specificities of the extracellular lignin peroxidases of the wood-destroying fungusPleurotus ostreatus

The substrate specificities of two forms of purified extracellular lignin peroxidase isolated from a total enzyme preparation of the fungus Pleurotus ostreatus — LGP-1 and LGP-II — have been determined. The substrate specificities of the isoenzymes differ considerably: LGP-I preferentially destroys model compounds of lignin — the -guaiacyl ether of I-veratrylpropanol, coniferyl alcohol, and pyrocatechol, while LGP-II is most specific in relation to veratryl alcohol, veratrylpropane-1,3-diol, and vanillyl alcohol. Both forms partially oxidize syringaldazine and ABTS. The isoenzymes possess peroxidase and oxidase properties simultaneously, since veratryl, vanillyl, and coniferyl alcohols were oxidized by both forms of the enzyme only in the presence of H₂O₂, which confirms their peroxidase natures. At the same time, ABTS, syringaldazine, pyrocatechol, and o -phenylenediamine were also oxidized by the lignin peroxidase in the absence of H₂O₂, which confirms their oxidase function. The isoenzymes also possess Mn-peroxidase activity in relation to NADH. Since almost all substrates were oxidized by the enzymes only in the presence of hydrogen peroxide, they cannot be assigned to the class of oxidases. On the other hand, the LGP ofP. ostreatus is not Mn-dependent, since the presence of manganese ions had no effect whatever on the oxidation of aromatic substrates by the enzyme. Moreover, both forms of the enzyme oxidized veratryl alcohol — a specific substrate for ligninases, which permits the extracellular isoenzymes of P. ostreatus, LGP-I and LGP-II, to be assigned to the class of ligninases.Institute of Microbiology, Academy of Sciences of the Republic of Uzbekistan, Tashkent, fax (3712) 41 71 29. Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 596–601, July–August, 1996. Original article submitted November 11, 1994.     

Cloning,Expression, and Characterization of a Wide-pH-Range Stable Phosphite Dehydrogenase from <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. K in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A phosphite dehydrogenase gene (ptdhK) consisting of 1,011-bp nucleotides which encoding a peptide of 336 amino acid residues was cloned from Pseudomonas sp. K. gene ptdhK was expressed in Escherichia coli BL21 (DE3) and the corresponding recombinant enzyme was purified by metal affinity chromatography. The recombinant protein is a homodimer with a monomeric molecular mass of 37.2 kDa. The specific activity of PTDH-K was 3.49 U mg⁻¹ at 25 °C. The recombinant PTDH-K exhibited maximum activity at pH 3.0 and at 40 °C and displayed high stability within a wide range of pHs (5.0 to 10.5). PTDH-K had a high affinity to its natural substrates, with K _m values for sodium phosphite and NAD of 0.475 ± 0.073 and 0.022 ± 0.007 mM, respectively. The activity of PTDH-K was enhanced by Na⁺, NH₄⁺, Mg²⁺, Fe²⁺, Fe³⁺, Co²⁺, and EDTA, and PTDH-K exhibited different tolerance to various organic solvents.     

Oxidation of crocein orange G by lignin peroxidase isoenzymes

The ligninolytic enzyme system ofPhanerochaete chrysosporiun is able to decolorize several recalcitrant dyes. Three lignin peroxidase isoenzymes, LiP 3.85, LiP 4.15, and LiP 4.65, were purified by preparative isoelectric focusing from the carbon-limited culture medium ofP. chrysosporium. Based on amino terminal sequences, the purified isoenzymes correspond to the isoenzymes H8, H6, and H2, respectively, from theN-limited culture. The purified isoenzymes were used for decolorization of an azo dye, Crocein Orange G (COG). According to the kinetic data obtained, the oxidation of COG by lignin peroxidase appeared to follow Michaelis-Menten kinetics. Kinetic parameters for each isoenzyme were determined. The inactivating effect of ascending H₂O₂ concentrations on COG oxidation is shown to be exponential within the used concentration range. The best degree of decolorization of 100 μM COG was obtained when the H₂O₂ concentration was 150 μM. This was also the lowest H₂O₂ concentration for maximal decolorization of 100 μM COG, regardless of the amount of lignin peroxidase used in the reaction.     

Enhancing the thermostability of a novel beta-agarase AgaB through directed evolution

To increase the thermostability of β-agarase AgaB by directed evolution, the mutant gene libraries were generated by error-prone polymerase chain reaction (PCR) and deoxyribonucleic acid (DNA) shuffling. Mutants with high thermostability were screened by a simple method based on agarase-degrading agar to generate a clear zone on the agar plate. A mutant S2 was obtained through two rounds of error-prone PCR and a single round of DNA shuffling and selection. It has higher thermostability and slightly increased catalytic activity than wild-type AgaB. Melting temperature (T _m) of S2, as determined by circular dichroism, is 4.6 °C higher than that of wild-type AgaB, and the half-life of S2 is 350 min at 40 °C, which is 18.4-fold longer than that of the wild-type enzyme. Saturation mutagenesis and hydrophobic cluster analysis indicated that hydrophobic interaction might be the key factor that enhances the enzyme stability.     

Comparative study of horseradish mutant forms by radioenzymology

Radioenzymology was used to study the recombinant and mutant forms of horseradish peroxidase, namely, F221W, Q176E, Q176A, S35K, E64P, E64S, S35A, and S35KQ176E. Both removal of the Trp residue and introduction of an additional one result in a simpler dose response; the insertion of polar residues stabilizes the enzyme molecule through realization of a more closed conformation. The greatest oscillation changes were found for the replacement by Ala. It was assumed that the binding site of guaiacol as a substrate is located near the residue 64, which is structurally related to the residue 176 and the heme. A scheme of formation of the intermediate through rotation of the Trp aromatic ring was proposed.     

Preparation and properties of glucose isomerase immobilized on Indion 48-R

Partially purified glucose isomerase fromStreptomyces thermonitrificans when coupled to glutaraldehyde-activated Indion 48-R, retained 30–40% activity of the soluble enzyme. However, an approximately twofold increase in the activity could be achieved by binding the enzyme in the presence of glucose. Binding the enzyme to matrices presaturated with either glucose or fructose and influence of lysine modification on the activity of the soluble enzyme revealed that the comparatively low activity observed in case of the enzyme bound in the absence of substrate is the result of the nonspecific binding of either substrate or product to the matrix. Immobilization did not affect the pH and temperature optima of the enzyme, but it lowered the temperature stability. Immobilization resulted in a marginal increase in theK _m and a threefold decrease in theV _max . Substrate concentrations as high as 36% glucose could be converted to 18.5% fructose in 5 h, at pH 7.0 and 70‡C. The bound enzyme, however, showed inferior stability to repeated use and lost approx 40% of its initial activity after five cycles of use. Indion 48-R bound glucose isomerase could be stored, in wet state, for 30 d without any apparent loss in its initial activity.     

Production and partial characterization of extracellular peroxidases produced bystreptomyces avermitilis UAH30

The effect of a number of environmental parameters (pH, temperature, carbon and nitrogen ratio of nutrient) on the production of extracellular peroxidase enzymes byStreptomyces avermitilis UAH30 was examined. Maximum specific peroxidase activity (0.12 U/mg of protein) was obtained after 72 hours of 1 incubation at 45‡C in a minimal salt medium (pH 7.5) containing 0.6% (w/v) yeast extract and 0.6% (w/v) xylan corresponding to a C:N ratio of 4 to 1. A study of the effect of incubation on peroxidase activity showed that the enzyme was stable and active for at least one hour after incubation at 50‡C while at higher temperatures the stability and activity of the peroxidase was reduced such that at 60‡C the peroxidase activity has a half life of 20 min while at 80‡C the half life was reduced to 5 min. The activation energy for deactivation as a result of thermal denaturation of the enzyme was calculated to be 80 ±7 kJ/mol. The optimum pH for the activity occurred between a pH range of 6.5–8.5 with pKa₁ and pKa₂ of 5.1 ±0.1 and 9.7 ±0.1, respectively. The K_m and V_max for the peroxidase activity were determined to be 1.45 mM and 0.31 unit per mg protein respectively using 2,4dicholorophenol (2,4-DCP) as a substrate. Characterization of the peroxidase activity revealed activity against L,3–4 dihydroxyphenylalanine and guaiacol, while no inhibition of peroxidase activity could be detected with the haem inhibitors such as potassium cyanide and sodium azide, suggesting the lack of haem component in the tertiary structure. Aspects of using the crude peroxidase preparation in the pulp and paper industry are discussed.     

Tetra(p‐tolyl)borate‐Functionalized Solvent Polymeric Membrane: A Facile and Sensitive Sensing Platform for Peroxidase and Peroxidase Mimetics

The determination of peroxidase activities is the basis for enzyme‐labeled bioaffinity assays, peroxidase‐mimicking DNAzymes‐ and nanoparticles‐based assays, and characterization of the catalytic functions of peroxidase mimetics. Here, a facile, sensitive, and cost‐effective solvent polymeric membrane‐based peroxidase detection platform is described that utilizes reaction intermediates with different pK_a values from those of substrates and final products. Several key but long‐debated intermediates in the peroxidative oxidation of o‐phenylenediamine (o‐PD) have been identified and their charge states have been estimated. By using a solvent polymeric membrane functionalized by an appropriate substituted tetraphenylborate as a receptor, those cationic intermediates could be transferred into the membrane from the aqueous phase to induce a large cationic potential response. Thus, the potentiometric indication of the o‐PD oxidation catalyzed by peroxidase or its mimetics can be fulfilled. Horseradish peroxidase has been detected with a detection limit at least two orders of magnitude lower than those obtained by spectrophotometric techniques and traditional membrane‐based methods. As an example of peroxidase mimetics, G‐quadruplex DNAzymes were probed by the intermediate‐sensitive membrane and a label‐free thrombin detection protocol was developed based on the catalytic activity of the thrombin‐binding G‐quadruplex aptamer.     

Gene Cloning,Expression, and Characterization of a Family 51 α-<Emphasis Type="SmallCaps">l</Emphasis>-Arabinofuranosidase from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. S9

An α-l-arabinofuranosidase gene, abf51S9, was cloned from Streptomyces sp. S9 and successfully expressed in Escherichia coli BL21 (DE3). The full-length gene consisted of 1,506 bp and encoded 501 amino acids with a calculated mass of 55.2 kDa. The deduced amino acid sequence was highly homologous with the α-l-arabinofuranosidases belonging to family 51 of the glycoside hydrolases. The recombinant protein was purified to electrophoretic homogeneity by Ni-NTA affinity chromatography and subsequently characterized. The optimal pH and temperature for the recombinant enzyme were 6.0 and 60∼65 °C, respectively. The enzyme showed a broad pH range of stability, retaining over 75% of the maximum activity at pH 5.0 to 11.0. The specific activity, K _m, and V _max with p-nitrophenyl-α-l-arabinofuranoside as substrate were 60.0 U mg⁻¹, 1.45 mM, and 221 μmol min⁻¹ mg⁻¹, respectively. Abf51S9 showed a mild but significant synergistic effect in combination with xylanase on the degradation of oat-spelt xylan and soluble wheat arabinoxylan substrates with a 1.19- and 1.21-fold increase in the amount of reducing sugar released, respectively. These favorable properties make Abf51S9 a good candidate in various industrial applications.     

Enzymic demethylation of lignin model polymers

We have studied the demethylation of [O¹⁴CH₃]-polyguaiacol byPhanerochaete chrysosporium as a model for the fungal demethylation of lignin. Demethylating activity of whole-cell ligninolytic cultures was compared to demethylating activities of various oxygen-activating systems. Some of these systems demethylated polyguaiacol (e.g., Fenton’s reagent, rose bengal sensitized photolysis, and horseradish peroxidase + H₂O₂). Other systems did not (e.g., xanthine/xanthine oxidase). Even where oxygen-activating systems did demethylate polyguaiacol, we found no convincing evidence that these systems are used byPhanerochaete. We have detected in concentrated extracellular culture filtrates of ligninolyticPhanerochaete cultures an enzymatic activity that demethylates [O¹⁴CH₃]-polyguaiacol. The activity was stabilized greatly by concentrating culture filtrates by pressure dialysis (20,000 MW cutoff membrane). Concentrated enzyme preparations could be filter sterilized and stored at 4‡C for several days without extensive loss of activity. The methoxyl label released by our enzyme preparation was nongaseous (e.g., not¹⁴CO₂,¹⁴CO, or¹⁴CH₄), but volatile (e.g., CH₃OH or CH₂O). The amount of labeled methoxyl released by the enzyme preparation was about the same as that released by intact cultures. The enzyme preparation contained ∼50 Μg/mL of protein and had laccase activity against catechol or hydroquinone. Unsupplemented preparations lacked activity againsto-dianisidine, a dye used to assay peroxidase. However, when H₂O₂ was provided (0.8 mM),o-dianisidine was oxidized rapidly. This indicates that the preparation contained peroxidase, but lacked substrate levels of H₂O₂. Demethylation of polyguaiacol by the enzyme preparation was not stimulated by NADH, NADPH, FAD, or FMN. Demethylation was stimulated by >50% upon addition of H₂O₂ (0.5 mM). Concentrated culture filtrates ofPhanerochaete produced ethylene from methional, a reaction that has been used as an indicator of hydroxyl radical generating systems. However, the ethylene-generating activity and the demethylase activity in such preparations showed different purification and stability characteristics. Pure horseradish peroxidase and H₂O₂ demethylated polyguaiacol and produced ethylene from methional.Phanerochaete does produce H₂O₂, so our demethylase activity appears to be similar to a peroxidase, although we have not yet determined the identity of the methyl product of either enzyme preparation. We suspect that the demethylase operates by a freeradical mechanism, and that the methyl product released is likely to be methanol. Confirmation of these hypotheses provides the basis for our future work with this novel fungal enzyme system.     

Effects of Low-Shear Modeled Microgravity on the Characterization of Recombinant β-D-Glucuronidase Expressed in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

In this study, we used a high-aspect-ratio vessel (HARV), which could model environment of microgravity on ground to investigate for the first time the effects of low-shear modeled microgravity (LSMMG) on the characterization of recombinant β-D-glucuronidase expressed in Pichia pastoris. The β-D-glucuronidase gene (GenBank accession no. EU095019) derived from Penicillium purpurogenum Li-3 encoding β-D-glucuronidase (PGUS) was expressed in P. pastoris GS115 in two different environments of LSMMG and normal gravity (NG). Results manifested that both LSMMG and NG conditions had insignificant effects on temperature and pH activity (optimal temperature and pH were 55 and 5.0 °C, respectively) and characteristic stability of recombinant PGUS. However, the catalytic activity of recombinant PGUS expressed under LSMMG was less affected by metal ions and EDTA as compared with that of NG. Furthermore, K _m value of the recombinant PGUS expressed under LSMMG was nearly one fifth of that under NG (1.72 vs. 7.72), whereas catalytic efficiency (k _cat/K _m) of PGUS expressed under LSMMG (13.55) was 3.7 times higher than that of NG (3.61). The results initially reveal the significant alterations in catalytic properties of recombinant enzyme in response to LSMMG environment and have potential application in bioprocessing and biocatalysis.