刺芹侧耳芳基醇氧化酶的分离纯化及其酶学性质

分离纯化刺芹侧耳Pleurotus eryngii芳基醇氧化酶,并探究其酶学性质。通过硫酸铵盐沉、DEAE-Sepharose Fast Flow弱阴离子交换层析、Sephacryl S-200 High Resolution凝胶过滤层析和Source 15Q强阴离子交换层析,得到纯化的单一酶。经肽指纹图谱鉴定,确定其为芳基醇氧化酶,酶活回收率25.5%,纯化倍数38.2。结合SDS-PAGE和IEF-PAGE分析,确定其分子量和等电点分别为70kDa和4.2。以藜芦醇为底物,该酶最适反应pH为6.0,最适反应温度为70℃,金属离子Zn²⁺、Fe²⁺和Cu²⁺对芳基醇氧化酶的活性抑制作用明显,K_m和V_max分别为0.921mmol/L和80U/mg。     

Production of hydrogen peroxide by aryl-alcohol oxidase from the ligninolytic fungusPleurotus eryngii

Summary Production of extracellular hydrogen peroxide by fungal oxidases is been investigated as a requirement for lignin degradation. Aryl-alcohol oxidase activity is described in extracellular liquid and mycelium ofPleurotus eryngii and studied under non-limiting nitrogen conditions. This aryl-alcohol oxidase catalyses conversion of primary aromatic alcohols to the corresponding aldehydes and H₂O₂, showing no activity with aliphatic and secondary aromatic alcohols. The enzyme is stable at pH 4.0–9.0, has maximal activity at 45°–50°C and pH 6.0–6.5, is inhibited by Ag⁺, Pb²⁺ and NaN₃, and has aK _m of 1.2 mM using veratryl alcohol as substrate. A single protein band with aryl-alcohol oxidase activity was found in zymograms of extracellular and intracellular crude enzyme preparations fromP. eryngii.     

Molecular characterization of a novel peroxidase isolated from the ligninolytic fungus Pleurotus eryngii

A haem peroxidase different from other microbial, plant and animal peroxidases is described. The enzyme is secreted as two isoforms by dikaryotic Pleurotus eryngii in peptone-containing liquid medium. The corresponding gene, which presents 15 introns and encodes a 361-amino-acid protein with a 30-amino-acid signal peptide, was isolated as two alleles corresponding to the two isoforms. The alleles differ in three amino acid residues and in a seven nucleotide deletion affecting a single metal response element in the promoter. When compared with Phanerochaete chrysosporium peroxidases, the new enzyme appears closer to lignin peroxidase (LiP) than to Mn-dependent peroxidase (MnP) isoenzymes (58–60% and 55% identity respectively). The molecular model built using crystal structures of three fungal peroxidases as templates, also showed high structural affinity with LiP (C_α-distance 1.2 Å). However, this peroxidase includes a Mn²⁺ binding site formed by three acidic residues (E36, E40 and D175) near the haem internal propionate, which accounts for the ability to oxidize Mn²⁺. Its capability to oxidize aromatic substrates could involve interactions with aromatic residues at the edge of the haem channel. Another possibility is long-range electron transfer, e.g. from W164, which occupies the same position of LiP W171 recently reported as involved in the catalytic cycle of LiP.     

Studies on homoveratric acid transformation by the ligninolytic fungus Pleurotus eryngii

Homoveratric acid (HVA) degradation was observed in cultures of Pleurotus eryngii lacking lignin peroxidase (LiP) activity. Extracellular enzymes seemed responsible for this transformation, and the lack of activity after ultrafiltration of the culture liquid suggests that the presence of some low-molecular-size compounds is required. This hypothesis is supported by rapid HVA transformation after addition of the synthetic laccase substrate 2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) to the ultrafiltered liquid. HVA transformation by the extracellular enzymes from P. eryngii takes place via C-C breakdown and formation of veratryl alcohol, which is further transformed into veratraldehyde. The same major compounds were found during HVA transformation by LiP from Phanerochaete chrysosporium, but this reaction was not stimulated by ABTS. Although the involvement of other enzymes cannot be ruled out, purified laccase from Pleurotus eryngii caused the same HVA transformation pattern in presence of ABTS. Moreover, veratryl alcohol oxidation by P. eryngii laccase was demonstrated in the presence of ABTS. These results suggest that enzymatic systems lacking LiP could be responsible for natural degradation of lignin.     

Anisaldehyde production and aryl-alcohol oxidase and dehydrogenase activities in ligninolytic fungi of the genus Pleurotus.

A variety of simple aromatic compounds were identified in liquid cultures of the basidiomycetes Pleurotus cornucopiae, P. eryngii, P. floridanus, P. pulmonarius, P. ostreatus, and P. sajor-caju by using gas chromatography-mass spectrometry. Such compounds were detected in fungal cultures on lignin- and straw-containing media, but it was found that they were also produced in the absence of aromatic precursors. Anisylic and hydroxybenzylic compounds (such as alcohols, aldehydes, and acids) were identified, p-anisaldehyde being the most characteristic extracellular metabolite synthesized by these ligninolytic fungi. Small amounts of 3-chloro-p-anisaldehyde were also detected in several species. It is postulated that the balance between the more-or-less-oxidized aromatic compounds can be explained in terms of the activity of fungal enzymes, including aryl-alcohol oxidase and dehydrogenase. The former enzyme shows high affinity for p-anisyl alcohol, which is oxidized to p-anisaldehyde with production of H2O2. The aryl-alcohol dehydrogenase was detected only in the mycelium, where it reduces aromatic aldehydes in the presence of NADPH. Both enzymes could be involved in the redox cycling of these aromatic compounds, providing H2O2 to ligninolytic peroxidases.     

Stability and activity of a phenol oxidase from the ligninolytic fungus Pleurotus ostreatus

Three different phenol oxidases produced by the basidiomycete fungus Pleurotus ostreatus have been isolated and their main structural, enzymatic and physico-chemical properties characterized. Studies have forcaused on the most abundantly secreated of these proteins, a copper-e nzyme specific towards ortho-diphenol substrates. This protein was purified to homogeneity and part of its primary structure determined by direct protein sequencing. The ingluence of pH, temperature and presence of water-soluible or water-insoluble organic solvents on the activity and stability of the enzyme were also investigated. These data can be used for applying bioarectors to problems of environmental concern such as waste-water treatment Correspondens to: G. Sannia     

Expression of Pleurotus eryngii aryl-alcohol oxidase in Aspergillus nidulans: purification and characterization of the recombinant enzyme

Aryl-alcohol oxidase (AAO) is an extracellular flavoenzyme involved in lignin biodegradation by some white-rot fungi. The enzyme catalyzes the extracellular oxidation of aromatic alcohols to the corresponding aldehydes. The electron acceptor is molecular oxygen yielding H(2)O(2) as the product. Herein we describe, for the first time, the expression of AAO from Pleurotus eryngii in the ascomycete Aspergillus nidulans. The activity of the recombinant enzyme in A. nidulans cultures is much higher than found in the extracellular fluid of P. eryngii. The recombinant enzyme showed the same molecular mass, pI and catalytic properties as that of the mature protein secreted by P. eryngii. The enzymic properties are also similar to those reported from other Pleurotus and Bjerkandera species.     

Hydrogen-peroxide-producing system ofPleurotus eryngii involving the extracellular enzyme aryl-alcohol oxidase

The effect of benzyl alcohol, benzaldehyde and benzoic acid on the production of extracellular hydrogen peroxide (H₂O₂) by the ligninolytic fungusPleurotus eryngii was investigated. It was found that an equilibrium between oxidative and reductive reactions of these compounds is established, leading to the continuous production of H₂O₂. A multienzymatic cyclic system is proposed in which H₂O₂ is produced extracellularly by the action of aryl-alcohol oxidase on benzyl alcohol, the most abundant compound after redox reactions, and to a lower extent on benzaldehyde. The oxidation products of these reactions, benzaldehyde and benzoic acid, are reduced by intracellular dehydrogenases.     

Biochemical characterization, cDNA cloning and protein crystallization of aryl-alcohol oxidase from Pleurotus pulmonarius

Aryl-alcohol oxidase (AAO) involved in lignin degradation by Pleurotus pulmonarius has been purified and characterized. The enzyme was produced in glucose-peptone medium and isolated in a sole chromatographic step using Sephacryl S-200. The purified enzyme is an extracellular glycoprotein with 14% N-carbohydrate content and an estimated molecular mass of 70.5 kDa and pI of 3.95. The kinetic studies showed the highest enzyme affinity against p-anisyl alcohol, with constants similar to those of Pleurotus eryngii and Bjerkandera adusta AAO but different from the intracellular AAO described in Phanerochaete chrysosporium, which present the highest activity on m-anisyl alcohol. Simultaneously, the cDNA of P. pulmonarius AAO has been cloned and sequenced. The translation of this sequence consisted of 593 amino acids including a signal peptide of 27 amino acids. The comparison with other alcohol oxidases, 35% amino acid identity with glucose oxidase, showed highly conserved amino acid sequences in N-terminal and C-terminal regions, in spite of differences in substrate specificity. Crystallization of AAO, carried out for the first time using the P. pulmonarius enzyme, will permit to obtain a molecular model for this oxidase and establish some characteristic of its catalytic site and general structure.     

Substrate specificity of ascorbate oxidase.

Ascorbate oxidase oxidizes leuco 2, 6-dichloroindophenol to the blue quinoid dye and produces spectral changes in the UV spectra of certain substituted polyhydric and amino phenols at pH 5.7. The new peaks produced by the addition of enzyme to the dichlorohydroquinones (2,5 and 2,6) and hydroxyhydroquinone correspond to the respective p-quinones of these substrates. At pH 5.7, the enzyme does not oxidize hydroquinone, barely oxidizes chlorohydroquinone, but oxidizes 2,6- and 2,5-dichlorohydroquinone and hydroxyhydroquinone at a rate about $\text{1}\text{12}$ that of ascorbic acid, with the uptake of one gram atom of oxygen per mole of substrate. A correlation has been found between the concentration of anion present in solution at pH 5.7 and the rate of oxidation of compounds of the hydroquinone series by the enzyme. The results indicate that an anionic form of the substrate is an important requirement of the enzyme specificity.     

Purification and properties of an aryl-alcohol dehydrogenase from the white-rot fungus Phanerochaete chrysosporium

An intracellular aryl-alcohol dehydrogenase (previously referred to as aryl-aldehyde reductase) was purified from the white-rot fungus Phanerochaete chrysosporium. The enzyme reduced veratraldehyde to veratryl alcohol using NADPH as a cofactor. Other aromatic benzaldehydes were also reduced, but not aromatic ketones. Methoxy-substituted rings were better substrates than hydroxylated ones. The enzyme was also able to reduce a dimeric aldehyde (4-benzyloxy-3-methoxybenzaldehyde). The highest reduction rate was measured when 3,5-dimethoxybenzaldehyde was used as a substrate. On SDS/PAGE the purified enzyme showed one major band with a molecular mass of 47 kDa, whereas gel filtration suggested a molecular mass of 280 kDa. Polyclonal antibodies raised against the gel purified 47-kDa protein were able to immunoprecipitate the aryl-alcohol dehydrogenase indicating that its activity possibly resides entirely in this protein fragment. The pI of the enzyme was 5.2 and it was most active at pH 6.1. The aryl-alcohol dehydrogenase was partially inhibited by typical oxidoreductase inhibitors.     

Substrate specificity of polyphenol oxidase

Abstract

The ubiquitous type-3 copper enzyme polyphenol oxidase (PPO) has found itself the subject of profound inhibitor research due to its role in fruit and vegetable browning and mammalian pigmentation. The enzyme itself has also been applied in the fields of bioremediation, biocatalysis and biosensing. However, the nature of PPO substrate specificity has remained elusive despite years of study. Numerous theories have been proposed to account for the difference in tyrosinase and catechol oxidase activity. The “blocker residue” theory suggests that bulky residues near the active site cover CuA, preventing monophenol coordination. The “second shell” theory suggests that residues distant (～8?Å) from the active site, guide and position substrates within the active site based on their properties e.g., hydrophobic, electrostatic. It is also hypothesized that binding specificity is related to oxidation mechanisms of the catalytic cycle, conferred by coordination of a conserved water molecule by other conserved residues. In this review, we highlight recent developments in the structural and mechanistic studies of PPOs and consolidate key concepts in our understanding toward the substrate specificity of PPOs.     

Identification of naphthalene metabolism by white rot fungus Pleurotus eryngii

The use of biomaterials or microorganisms in PAHs degradation had presented an eye-catching performance. Pleurotus eryngii is a white rot fungus, which is easily isolated from the decayed woods in the tropical rain forest, used to determine the capability to utilize naphthalene, a two-ring polycyclic aromatic hydrocarbon as source of carbon and energy. In the meantime, biotransformation of naphthalene to intermediates and other by-products during degradation was investigated in this study. Pleurotus eryngii had been incubated in liquid medium formulated with naphthalene for 14 days. The presence of metabolites of naphthalene suggests that Pleurotus eryngii begin the ring cleavage by dioxygenation on C1 and C4 position to give 1,4-naphthaquinone. 1,4-Naphthaquinone was further degraded to benzoic acid, where the proposed terepthalic acid is absent in the cultured extract. Further degradation of benzoic acid by Pleurotus eryngii shows the existence of catechol as a result of the combination of decarboxylation and hydroxylation process. Unfortunately, phthalic acid was not detected in this study. Several enzymes, including manganese peroxidase, lignin peroxidase, laccase, 1,2-dioxygenase and 2,3-dioxygenase are enzymes responsible for naphthalene degradation. Reduction of naphthalene and the presence of metabolites in liquid medium showed the ability of Pleurotus eryngii to utilize naphthalene as carbon source instead of a limited glucose amount.     

Substrate specificity of cholesterol oxidase from Streptomyces cinnamomeus--a monolayer study.

The substrate specificity of cholesterol oxidase from Streptomyces cinnamomeus was examined in oriented sterol monolayers at the air/water interface. Of the cholesterol analogues with structural alterations in the A- or B-ring that were examined, it was observed that 5 alpha-cholestan-3 beta-ol was oxidized almost as fast as cholesterol itself. When the delta-5 double bond in cholesterol was instead at the delta-4 position, the oxidation rate became 3.2-fold slower. A similar reduction in the average oxidation rate was observed when the delta-5 double bond in cholesterol was instead at the delta-7 position (5 alpha-cholest-7-en-3 beta- ol). 5,7-Cholestadien-3 beta-ol was oxidized 5.1-fold slower compared to cholesterol, whereas 3 beta-hydroxy-5-cholesten-7-one and 5 beta-cholestan-3 beta-ol were not substrates of the enzyme (also verified from the lack of H2O2-production). With C(17) side chain analogues of cholesterol, it was observed that the complete lack of the C(17) side chain (5-androsten-3 beta-ol), or the insertion of an unsaturation at delta-24 (desmosterol), or even an ethyl group at C(24)(24b-ethyl-5,22- cholestadien-3 beta-ol) had no appreciable effects on sterol oxidation rate, implying that the enzyme did not recognize the side chain in oriented sterol monolayers. This study has shown that the sterol monolayer system is a good technique to examine sterol/cholesterol oxidase interactions, since both the orientation of the substrate molecules, and the quality of the interface can be mastered.