Soluble and immobilized phenol oxidase of the fungusMycelia sterilia IBR 35219/2: A comparative study

Phenol oxidase (EC 1.14.18.1) from the microscopic fungusMycelia sterilia IBR 35219/2 was immobilized using glutaraldehyde on macroporous silica carriers. The enzyme immobilized on amino-Silochrome SKh-2 or aminopropyl-Silochrome 350/80 exhibited maximum activity. Soluble and immobilized phenol oxidases were compared. Compared to the soluble enzyme, the activity of which was optimum at pH 5.5, immobilized phenol oxidase exhibited optimum activity under slightly more acidic conditions (pH 5.2). Immobilization considerably increased enzyme stability. Both soluble and immobilized forms of phenol oxidase fromM. sterilia IBR 35 219/2 catalyze oxidative conversion of phenolic compounds of green tea extract.     

Consumption of the triazine herbicide atrazine by laccase and laccase-free variants of the soil fungus Mycelia sterilia INBI-2-26

Asporogenic fungus Mycelia sterilia INBI 2-26 isolated from tropical soils with high residual dioxin content (as a result of Agent Orange defoliant treatment during the Vietnamese-American war) and capable of atrazine decomposition was treated to obtain protoplasts. This technique resulted in isolation of laccase-positive and laccase-negative clones. Atrazine consumption by liquid surface cultures of Mycelia sterilia INBI 2-26 was monitored by using enzyme immune assay and reversed phase HPLC. Atrazine (20 micrograms/l) stimulated fungal growth. Laccase-positive clone consumed up to 80% of atrazine within four weeks. However, no correlation of atrazine consumption and laccase activity in the culture medium was observed. Moreover, the laccase-negative clone was also capable of consuming at least 60-70% of atrazine within three weeks. Surprisingly, in the corresponding control set (cultivation of laccase-negative clone without atrazine) an unidentified metabolite having a retention time and UV-spectrum similar to those of atrazine was also found. It was concluded that the presence of laccase was not a crucial factor in atrazine consumption by this fungus.     

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     

Decolorization of phenolic effluents by soluble and immobilized phenol oxidases

Summary Colour removal from phenplic industrial effluents by phenol oxidase enzymes and white-rot fungi was compared. Soluble laccase and horseradish peroxidase (HRP) removed colour from pulp mill (E), cotton mill hydroxide (OH) and cotton mill sulphide (S) effluents, but rapid and irreversible enzyme inactivation took place. Entrapment of laccase in alginate beads improved decolorization by factors of 3.5 (OH) and 2 (E); entrapment of HRP improved decolorization by 36 (OH), 20 (E) and 9 (S). Beads were unsuitable for continuous use because the enzymes were rapidly released into solution. Co-polymerization of laccase or HRP with L-tyrosine gave insoluble polymers with enzyme activity. Entrapment of the co-polymers in gel beads further increased the efficiency of decolorization of E by 28 (laccase) and by 132 (HRP) compared with soluble enzymes. Maximum decolorization of all three effluents by batch cultures of Coriolus versicolor (70%–80% in 8 days) was greater than the maximum enzymic decolorization (48% of OH in 3 days by entrapped laccase). Soluble laccase (222 units ml^–1) precipitated 1.2 g l^–1 phenol from artificial coal conversion effluent at pH 6.0 and the rate of precipitation and enzyme inactivation was faster at pH 6.0 than at pH 8.5.Offprint requests to: R. G. Burns     

Comparative study of immobilized and soluble NADH:FMN-oxidoreductase-luciferase coupled enzyme system

The properties of a coupled enzyme system (NAD(P)H:FMN-oxidoreductase and luciferase) from luminous bacteria were studied. The enzymes and their substrates were immobilized in polymer gels of different types: starch (polysaccharide) and gelatin (polypeptide). Maximum activity yield (100%) was achieved with the enzymes immobilized in starch gel. An increase in K _{m app} was observed in both immobilized systems as compared with the soluble coupled enzyme system. Immobilization in starch and gelatin gels increased the resistance of the NAD(P)H:FMN-oxidoreductase and luciferase coupled enzyme system to the effects of external physical and chemical factors. The optimum pH range expanded both to the acidic and alkaline regions. The resistance to concentrated salt solutions and high temperature also increased. The coupled enzyme system immobilized in starch gel (with activation energy 30 kJ/mol) was characterized by the best thermostability. The immobilized coupled enzyme system can be used to produce a stable and highly active reagent for bioluminescent analysis.     

Comparative studies on soluble and immobilized yeast hexokinase

Comparative studies have been carried out on soluble and immobilized yeast hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1). The enzyme was immobilized by covalent attachment to a polyacrylamide type support containing carboxylic functional groups. The effects of immobilization on the catalytic properties and stability of hexokinase were studied. As a result of immobilization, the pH optimum for catalytic activity was shifted in the alkaline direction to ～pH 9.7. The apparent optimum temperature of the immobilized enzyme was higher than that of the soluble enzyme. The apparent K_m value with D-glucose as substrate increased, while that with ATP as substrate decreased, compared with the data for the soluble enzyme. Differences were found in the thermal inactivation processes and stabilities of the soluble and immobilized enzymes. The resistance to urea of the soluble enzyme was higher at alkaline pH values, while that for the immobilized enzyme was greatest at ～pH 6.0.     

Characterization of lignosulfonate-induced phenol oxidase activity in the atypical white-rot fungus Polyporus dichrous

Polyporus dichrous, a white-rot fungus previously shown to lack phenol oxidase activity when grown on agar media in the presence of a variety of phenolic compounds, was found to exhibit phenol oxidase activity upon aging when grown on a lignosulfonate-containing agar medium. The phenol oxidase activity was compared with that of Trametes versicolor grown under the same conditions, in terms of substrate specificity, pH optimum, and temperature sensitivity. The phenol oxidase activity of P. dichrous was intracellular of tyrosinase type, with a pH optimum around 5.5, and was heat-sensitive, having a half-life of 10 min at 60°C.     

Consumption of Triazine Herbicide Atrazine by Laccase-positive and Laccase-negative Strains of Soil Fungus Mycelia sterilia INBI 2-26

Asporogenic fungus Mycelia sterilia INBI 2-26 isolated from tropical soils with high residual dioxin content (as a result of Agent Orange defoliant treatment during the Vietnamese–American war) and capable of atrazine decomposition was treated to obtain protoplasts. This technique resulted in isolation of laccase-positive and laccase-negative clones. Atrazine consumption by liquid surface cultures of Mycelia sterilia INBI 2-26 was monitored by using enzyme immune assay and reversed-phase HPLC. Atrazine (20 g/ml) stimulated fungal growth. The laccase-positive clone consumed up to 80% of atrazine within four weeks. However, no correlation of atrazine consumption and laccase activity in the culture medium was observed. Moreover, the laccase-negative clone was also capable of consuming at least 60–70% of atrazine within three weeks. Surprisingly, in the corresponding control set (cultivation of laccase-negative clone without atrazine) an unidentified metabolite having a retention time and UV-spectrum similar to those of atrazine was also found. It was concluded that the presence of laccase was not a crucial factor in atrazine consumption by this fungus.     

Production, properties and application to biocatalysis of a novel extracellular alkaline phenol oxidase from the thermophilic fungus Scytalidium thermophilum

Scytalidium thermophilum produces an extracellular phenol oxidase on glucose-containing medium. Certain phenolic acids, specifically gallic acid and tannic acid, induce the expression of the enzyme. Production at 45°C in batch cultures is growth-associated and is enhanced in the presence of 160 μM CuSO₄.5 H₂O and 3 mM gallic acid. The highest enzyme activity is observed at pH 7.5 and 65°C, on catechol. When incubated for 1 h at pH 7 and pH 8, 95% and 86% of the activity is retained. Thermostability decreases gradually from 40°C to 80°C. Estimated molecular mass is c. 83 kDa, and pI is acidic at c. 5.4. Substrate specificity and inhibition analysis in culture supernatants suggest that the enzyme has unique properties showing activity towards catechol; 3,4-dihydroxy-l-phenylalanine (l-DOPA); 4-amino-N, N-diethylaniline (ADA); p-hydroquinone; gallic acid; tannic acid and caffeic acid, and no activity towards l-tyrosine, guaiacol, 2,2′-azino-bis(3-ethyl-benzthiazoline-6-sulphonic acid) (ABTS) and syringaldazine. Inhibition is observed in the presence of salicyl hydroxamic acid (SHAM) and p-coumaric acid. Enzyme activity is enhanced by cetyltrimethylammonium bromide (CTAB) and polyvinylpyrrolidone (PVP), and the organic solvents dimethyl sulfoxide (DMSO) and ethanol. No inhibition is observed in the presence of carbon monoxide. Benzoin, benzoyl benzoin and hydrobenzoin are converted into benzil, and stereoselective oxidation is observed on hydrobenzoin. The reported enzyme is novel due to its catalytic properties resembling mainly catechol oxidases, but displaying some features of laccases at the same time.     

The importance of phenol oxidase activity in lignin degradation by the white-rot fungus Sporotrichum pulverulentum

The lignin degradation abilities of wildtype, a phenol oxidase-less mutant and a phenol oxidase-positive revertant of Sporotrichum pulverulentum were compared to determine if phenol oxidase activity is necessary for lignin degradation by white-rot fungi. The phenol oxidase-less mutant was unable to degrade kraft lignin or wood. The phenol oxidase-positive revertant, however, regained the ability of the wildtype to degrade kraft lignin and all of the major components of wood. It was found that kraft lignin and lignin-related phenols decreased cellulase and xylanase production by the phenol oxidase-less mutant. Addition of highly purified laccase increased the production of endo-1,4--glucanase in the phenol oxidase-less mutant in the presence of vanillic acid and kraft lignin. After addition of laccase to kraft lignin agar plates, the phenol oxidase-less mutant could degrade kraft lignin.It is proposed that phenol oxidase function in regulating the production of both lignin-and polysaccharide-degrading enzymes by oxidation of lignin and lignin-related phenols when S. pulverulentum is growing on wood.Abbreviation WT wildtype Sporotrichum pulverulentum Research supported by a grant from Stiftelsen Nils and Dorthi Troëdssons forskningsfond     

Characterisation of phenol oxidase and peroxidase from maize silk

Silk of some maize genotypes contains a high level of phenolics that undergo enzymatic oxidation to form quinones, which condense among themselves or with proteins to form brown pigments. Two phenolic oxidizing enzymes, peroxidase (POD; EC 1.11.1.7) and polyphenol oxidase (PPO; EC 1.10.3.1), from maize (Zea mays L.) silk were characterised with respect to their preferred substrate, different isoforms and specific effectors. One browning silk sample with high, and two non‐browning samples with low phenolic content were investigated. Although POD oxidizes a wide range of phenolic substrates in vitro, its activity rate was independent of silk phenolic content. PPO activity, detected with o‐diphenolic substrates, was abundant only in browning silk, and low or absent in non‐browning silk. Pollination increased POD but not PPO activity. Isoelectric‐focusing (IEF) and specific staining for POD and PPO showed a high degree of polymorphism that varied with silk origin. The IEF pattern of POD revealed a number of anionic and several cationic isoenzymes, with the most pronounced having neutral pI 7 and a basic isoform with pI 10. Detected isoforms of PPO were anionic, except for one neutral form found only in browning silk, and occupied positions different from those of POD. Different inhibitory effects of NaN₃, EDTA, KCN, and L‐cysteine, as well as different impacts of a variety of cations on the oxidation of chlorogenic acid, mediated by PPO or POD, were detected. The findings are discussed in terms of a possible roles of these enzymes in defence and pollination.     

Particulate and soluble forms of o-diphenol oxidase from potato tubers

A soluble and two different particulate forms of o-diphenol oxidase have been obtained from aged or fresh potato slices by differential and density gradient centrifugation. The particulate enzymes were shown to sediment with microsomes and peroxisomes, respectively. Over half the enzyme activity of aged slices was found to be particle bound, with approximately twice as much enzyme in the microsomes as in the peroxisomal fraction. Very similar distribution patterns have been obtained with fresh potatoes, which have an o-diphenol oxidase activity approximately one-third that of aged slices.     

Thermostability of soluble and immobilized alpha-amylase from Bacillus licheniformis

In view of a possible application of the alpha-amylase from Bacillus licheniformis as a time-temperature integrator for evaluation of heat processes,(11) thermal inactivation kinetics of the dissolved and covalently immobilized enzyme were studied in the temperature range 90-108 degrees C. The D-values (95 degrees C) for inactivation of alpha-amylase, dissolved in tris-HCl buffer, ranged from 6 to 157 min, depending on pH, ionic strength, and Ca(2+) and enzyme concentration. The z-value fluctuated between 6.2 and 7.6 degrees C. On immobilization of the alpha-amylase by covalent coupling with glutaraldehyde to porous glass beads, the thermoinactivation kinetics became biphasic under certain circumstances. For immobilized enzyme, the D-values (95 degrees C) ranged between 17 and 620 min, depending largely on certain environmental conditions. The z-value fluctuated between 8.1 and 12.9 degrees C. In each case of biphasic inactivation, the z-value of the stable fraction (with the higher D-values) was lower than the z-value of the labile fraction. (c) 1992 John Wiley & Sons, Inc.     

Removal of phenols from wastewater by soluble and immobilized tyrosinase

An enzymatic method for removal of phenols from industrial wastewater was investigated. Phenols in an aqueous solution were removed after treatment with mushroom tyrosinase. The reduction order of substituted phenols is catechol > p-cresol > p-chlorophenol > phenol > p-methoxyphenol. In the treatment of tyrosinase alone, no precipitate was formed but a color change from colorless to dark-brown was observed. The colored products were removed by chitin and chitosan which are available abundantly as shellfish waste. In addition, the reduction rate of phenols was observed to be accelerated in the presence of chitosan. Tyrosinase, immobilized by using amino groups in the enzyme on cation exchange resins, can be used repeatedly. By treatment with immobilized tyrosinase, 100% of phenol was removed after 2 h, and the activity was reduced very little even after 10 repeat treatments. (c) 1993 John Wiley & Sons, Inc.     

Purification and characterization of formate oxidase from a formaldehyde-resistant fungus

A formate oxidase activity was found in the crude extract of a formaldehyde-resistant fungus isolated from soil. The fungus was classified and designated as Aspergillus nomius IRI013, which could grow on a medium containing up to 0.45% formaldehyde and consumed formaldehyde completely. The specific activity of formate oxidase in the extract of the fungus grown on formaldehyde was found to be considerably higher than that in the extracts of the fungus grown on formate and methanol. Formate oxidase from the fungus grown on formaldehyde was purified to homogeneity. The enzyme had a relative molecular mass of 100000 and was composed of two apparently identical subunits that had a relative molecular mass of 59000. The enzyme showed the highest activity using formate as substrate. Hydrogen peroxide was formed during the oxidation of formate. The Michaelis constant for formate was 15.9 mM; highest enzyme activity was found at pH 4.5-5.0. The enzyme activity was strongly inhibited by NaN(3), p-chloromercuribenzoate and HgCl(2).     

Biodegradation of phenol by enzymes from Pseudomonas sp. immobilized onto ultrafiltration membranes

A method of immobilizing enzymes from Pseudomonas sp. that decompose phenol on polymeric ultrafiltration membranes is described. Transport-separation properties of neutral and enzymic membranes have been compared and the optimal ultrafiltration process parameters of a model phenol solution have been determined. The immobilized enzyme system was applied to the biodegradation of phenol in coke wastewaters.