Aryl Alcohol Oxidase Activity Research Articles

Agar plate assay for rapid screening of aryl-alcohol oxidase mutant libraries in Pichia pastoris

Directed evolution is a powerful tool for developing biocatalysts with tailor-made properties for biocatalytic applications. High-throughput activity screening of large mutant libraries generated by e.g. means of directed evolution is usually performed in 96-well microtiter plates requiring, however, time-consuming and laborious enzyme expression, cell harvesting and activity measurements. In addition, automated liquid handling systems are needed to cope with the high number of colonies to be screened. Herein, we developed an agar plate-based assay for simple and fast screening of H2O2-producing aryl-alcohol oxidase (AAO) mutant libraries in Pichia pastoris. Buffered minimal methanol agar plates were supplemented with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), horseradish peroxidase (HRP) and the target substrate. AAO activity is visualized by formation of green zones around AAO-secreting P. pastoris colonies due to ABTS oxidation by HRP which is fueled with H2O2 by AAO in course of substrate oxidation. Colonies were screened within 24 h for AAO activity using different AAO substrates like veratryl alcohol, p-anisyl alcohol or trans,trans-2,4-hexadien-1-ol and even low AAO activity towards 5-hydroxymethylfurfural could be detected within 48 h. The developed agar plate-based assay can be extended to other substrates and might also be applied for fast and substrate-specific screening of other H2O2-producing oxidases in P. pastoris.

Process optimization and scale-up production of fungal aryl alcohol oxidase from genetically modified Aspergillus nidulans in stirred-tank bioreactor

Microbial production of aryl alcohol oxidase (AAO) has attracted increasing attention due to the central role of AAO in enzymatic lignin depolymerization. However, large-scale production of AAO has not been reached because of the low yield and inefficient fermentation process. This study aims to optimize the process parameters and scale-up production of AAO using Aspergillus nidulans in a stirred-tank bioreactor. Effects of pH and dissolved oxygen on AAO production at bioreactor scale were particularly investigated. Results revealed that pH control significantly affected protein production and increasing dissolved oxygen level stimulated AAO production. The greatest AAO activity (1906 U/L) and protein concentration (1.19 g/L) were achieved in 48 h at 60% dissolved oxygen with pH controlled at 6.0. The yield and productivity (in 48 h) were 31.2 U/g maltose and 39.7 U/L/h, respectively. In addition, crude AAO was concentrated and partially purified by ultrafiltration and verified by protein identification.

Development of a cost-effective medium for submerged production of fungal aryl alcohol oxidase using a genetically modified Aspergillus nidulans strain

Aryl alcohol oxidase (AAO), an extracellular H2O2-providing enzyme, plays a central role in lignin depolymerization. Cost-effective production of AAO has not been achieved, due to the low yield of enzyme-producing microorganisms and the high cost of fermentation media. This study aims to develop a cost-effective medium for high-yield production of AAO in submerged culture using a recombinant Aspergillus nidulans strain. Results demonstrate that corn steep liquor (CSL) was a rich but inexpensive nitrogen source for AAO production, and CSL can provide enough trace metals and vitamins (i.e. pyridoxine) for A. nidulans. A 2-level Plackett-Burman design was utilized to determine the main affecting factors in AAO production. The medium was further optimized by a 3-level Box-Behnken design to obtain the optimum medium component concentrations (61.0 g/L maltose, 26.4 g/L CSL, and 13.8 g/L NaNO3). The greatest AAO activity achieved was 1021 U/L with a protein concentration of 0.75 g/L.

Degradation of the herbicide paraquat by macromycetes isolated from southeastern Mexico.

Fifty-four macromycetes, isolated from southeastern Mexico, were used in order to evaluate their capacity for degradation and tolerance to the herbicide paraquat. Ten of these strains were capable of growing in a solid culture medium in the presence of 200ppm paraquat. Subsequently, assays to evaluate the degradation of the xenobiotic in a liquid medium were carried out. Of the ten strains evaluated, three presented the highest levels of degradation of the compound, which were Trametes pavonia (54.2%), Trametes versicolor (54.1%) and Hypholoma dispersum. They presented the highest overall degradation percentage (70.7%) after 12days culture. The presence of ligninolytic enzymes in these strains was evaluated. H. dispersum only presented aryl alcohol oxidase activity; however, with the data obtained, it was not possible to conclude whether this specific enzyme is responsible for paraquat degradation. The level of degradation obtained is above the one reported for Pseudomonas putida, one of the few reports on paraquat degradation. This is the first report on the contaminant degradation capacity of H. dispersum.

Prevention of melanin formation during aryl alcohol oxidase production under growth-limited conditions using an Aspergillus nidulans cell factory

An Aspergillus nidulans cell factory was genetically engineered to produce an aryl alcohol oxidase (AAO). The cell factory initiated production of melanin when growth-limited conditions were established using stationary plates and shaken flasks. This phenomenon was more pronounced when the strain was cultured in a trickle bed reactor (TBR). This study investigated different approaches to reduce melanin formation in fungal mycelia and liquid medium in order to increase the enzyme production yield. Removal of copper from the medium recipe reduced melanin formation in agar cultures and increased enzyme activities by 48% in agitated liquid cultures. Copper has been reported as a key element for tyrosinase, an enzyme responsible for melanin production. Ascorbic acid (0.44g/L) stopped melanin accumulation, did not affect growth parameters and resulted in AAO activity that was more than two-fold greater than a control treatment with no ascorbic acid.

Peach and Cherry Agroindustrial Wastes: New and Economic Sources for the Production of Lignocellulolytic Enzymes.

Pleurotus eryngii was incubated on both peach and cherry agroindustrial wastes by solid state fermentation for 30 days without/with pretreatment conditions. The lignocellulosic substrates were pretreated with dilute acid and alkaline solutions, hot water before incubation. The maximum carboxymethyl cellulase and xylanase activities peaked on 3rd and 5th days under control conditions of both wastes, respectively. The highest laccase and manganese peroxidase activities reached to their maximum on 17th day as 2193.06 ± 50.4 UL-1 and 732.73 ± 19.8 UL-1, respectively. The highest aryl alcohol oxidase activity was obtained as 239.25 ± 7.3 UL-1 in control condition of peach cultures. The used pretreatment methods had generally negative effects on lignocellulolytic enzyme production. The highest lignocellulolytic activities were detected using peach wastes. To results, these wastes could.

Production of Enzymes and Biotransformation of Orange Waste by Oyster Mushroom, <i>Pleurotus pulmonarius</i> (Fr.) Quél.

The wood-decay fungi are able to bioconvert a wide variety of lignocellulosic residues due to the secretion of extracellular enzymes. The use of agricultural wastes as substrate for mushroom cultivation or enzymes production can help to solve environmental problems caused by inadequate discharge in the nature. The production of hydrolytic and oxidative enzymes by Pleurotus pulmonarius developed in solid state system using orange waste as substrate was evaluated in this work. Among the hydrolytic enzymes, pectinase was the main enzyme produced by the fungus, presenting the highest enzymatic activity of 9.4 U/mL after 35 days of cultivation. Considering the oxidative enzymes, laccase was the main enzyme produced with maximal activity of 12.2 U/mL obtained after 20 days of cultivation. Low enzyme levels of manganese peroxidase, β-glucosidase and β-xy-losidase were detected with activity peaks at the end of the cultivation. The enzymatic levels of amylase, carboxymethyl cellulase and xylanase were similar and less than 1.5 U/mL. No aryl-alcohol oxidase activity was detected. NDF, ADF and cellulose values increased during 45 days of cultivation. There was no lignin degradation during the study period and the fungus culture in orange solid waste caused protein enrichment in the substrate. Our results demonstrate that P. pulmonarius was an efficient producer of two important industrial enzymes, pectinase and laccase in a cheap solid state system using orange waste as substrate.

EXTRACELLULAR LIGNINOLYTIC ENZYMES PRODUCTION BY Pleurotus eryngii ON AGROINDUSTRIAL WASTES

Pleurotus eryngii (DC.) Gillet (MCC58) was investigated for its ligninolytic ability to produce laccase (Lac), manganese peroxidase (MnP), aryl alcohol oxidase (AAO), and lignin peroxidase (LiP) enzymes through solid-state fermentation using apricot and pomegranate agroindustrial wastes. The reducing sugar, protein, lignin, and cellulose levels in these were studied. Also, the production of these ligninolytic enzymes was researched over the growth of the microorganism throughout 20 days, and the reducing sugar, protein, and nitrogen levels were recorded during the stationary cultivation at 28 ± 0.5°C. The highest Lac activity was obtained as 1618.5 ± 25 U/L on day 12 of cultivation using apricot. The highest MnP activity was attained as 570.82 ± 15 U/L on day 17 in pomegranate culture and about the same as apricot culture. There were low LiP activities in both cultures. The maximum LiP value detected was 16.13 ± 0.8 U/L in apricot cultures. In addition, AAO activities in both cultures showed similar trends up to day 17 of cultivation, with the highest AAO activity determined as 105.99 ± 6.3 U/L on day 10 in apricot cultures. Decolorization of the azo dye methyl orange was also achieved with produced ligninolytic enzymes by P. eryngii using apricot and pomegranate wastes.

Induction and Purification by Three-Phase Partitioning of Aryl Alcohol Oxidase (AAO) from Pleurotus ostreatus

Aryl alcohol oxidase (AAO) is an extracellular flavoenzyme involved in lignin degradation by white rot fungi. Screening of lignolytic and AAO activity from twenty different fungal species were carried out. Among them, seven species showed lignolytic activity and three of them (Pleurotus ostreatus, Pleurotus eous, and Pleurotus platypus) were found to be AAO positive. Maximal AAO activity was observed in batch cultures of P. ostreatus and was found to be induced by aromatic amino acids and aryl alcohols up to a level of 289U/l. Purification of AAO was carried out by three-phase partitioning (TPP). The 67kDa enzyme was purified up to 10.19-fold by TPP with an overall recovery of 10.95%. Optimum pH and temperature for P. ostreatus AAO activity was found to be around 6 and 40°C, respectively. From the LB plot, K (m) value of AAO for oxidizing veratryl alcohol was determined to be 0.6mM. Results of the study indicate that P. ostreatus is the best producers of AAO, and they could be employed as promising fungal species for biotechnological applications.

Efficient Dye Decolorization and Production of Dye Decolorizing Enzymes by the Basidiomycete Thanatephorus cucumeris Dec 1 in a Liquid and Solid Hybrid Culture

Thanatephorus cucumeris Dec 1, a basidiomycete, is a promising decomposer of several xenobiotics. Air-membrane surface bioreactor culture (AMS culture), a hybrid between solid-state and submerged culture, was used for Dec 1 growth. The optimum temperature for AMS culture (25 degrees C) was lower than that for submerged culture (30 degrees C). Fungal growth was almost the same in AMS culture as in submerged culture at optimum temperatures. However, protein secretion, dye decolorizing peroxidase (DyP) activity, and aryl alcohol oxidase activity, were 18-, 233-, and 36.5-fold higher in AMS culture than in submerged culture, respectively, indicating that AMS culture of Dec 1 was superior to submerged culture. In in vivo dye decolorization tests, 13 of 16 dyes were decolorized by more than 90% within 10 days. Interestingly, under AMS culture, a biofilm was formed; biofilm formation was not, however, essential for DyP and manganese dependent peroxidase (MnP) activities. Although the correlation between DyP activity and water activity was unclear, MnP activity seemed to increase in activity with decreasing water activity, even when no biofilm formation was observed.

Solid-state cultures of Fusarium oxysporum transform aromatic components of olive-mill dry residue and reduce its phytotoxicity

The present study mainly investigated the ability of solid-state cultures of the non-pathogenic Fusarium oxysporum strain BAFC 738 to transform aromatic components to reduce the phytotoxicity in olive-mill dry residue (DOR), the waste from the two-phase manufacturing process. Lignin, hemicellulose, fats and water-soluble extractives contents of DOR colonized by the fungus for 20 weeks were reduced by 16%, 25%, 71% and 13%, respectively, while the cellulose content increased by 25%. In addition, the ethyl acetate-extractable phenolic fraction of the waste was reduced by 65%. However, mass-balance ultra-filtration and size-exclusion chromatography experiments suggested that the apparent removal of that fraction, mainly including 2-(3,4-dihydroxyphenyl)ethyl alcohol and 2-(4-hydroxyphenyl)ethyl alcohol, was due to polymerization. Mn-peroxidase and Mn-independent peroxidase activities were found in F. oxysporum solid-state cultures, while laccase and aryl alcohol oxidase activities were not detected. Tests performed with seedlings of tomato ( Lycopersicum esculentum L.), soybean ( Glycine maximum Merr.), and alfalfa ( Medicago sativa L.) grown on soils containing 6% (w/w) of bioconverted DOR (kg soil) −1 showed that the waste’s phytotoxicity was removed by 20 weeks-old fungal cultures. By contrast, the same material exhibited a high residual toxicity towards lettuce ( Lactuca sativa L.).

Two isolates of Fusarium proliferatum from different habitats and global locations have similar abilities to degrade lignin

Two isolates of Fusarium proliferatum from different global locations and habitats mineralized several natural and synthetic lignins. MUCL 31970 was isolated from a forest soil whereas the second strain, NRRL 31071, was a wheat endophyte causing disease in stressed seedlings. Onset and the fastest rate of lignin mineralization occurred during logarithmic and early stationary-phase of culture. Reduction of glucose in the medium shortened log-growth phase and advanced the onset of mineralization for both isolates. Mineralization correlated with the detection of extracellular laccase and aryl alcohol oxidase activities. The carbon–nitrogen ratio in the medium influenced laccase isozyme production and secretion by both strains. These studies suggest that both F. proliferatum strains degrade lignin via comparable routes, despite their different habitats and saprophytic or endophytic strategies.

Characterization of Aryl Alcohol Oxidase Produced by Dye-Decolorizing Fungus, Geotrichum candidum Decl

Aryl alcohol oxidase (AAO) produced by Geotrichum candidum Dec1 (Dec1), a newly isolated decolorizing fungus, was purified by ultrafiltration and by using diethylaminoethyl (DEAE) Sephacel, Butyl-Toyopearl and Mono-Q columns. H 2O 2 produced by concomitant AAO oxidation of veratryl alcohol (VA) to veratraldehyde was consumed by a peroxidase (DyP) purified from Dec1 culture, leading to the decolorization of a dye in vitro. In the liquid culture of Dec1, the existence of H 2O 2 and veratraldehyde was confirmed during cultivation, when dye-decolorization and AAO activities were maintained. This indicates that VA produced by Dec1 was oxidized by AAO to veratraldehyde, generating H 2O 2, which supported dye-decolorizing activity of Decl in vivo. The prevention of polymerization of DyP oxidation products of a dye in the presence of AAO was shown.

Production of ligninolytic enzymes by Fusarium solani strains isolated from different substrata

A comparative study on the extracellular ligninolytic enzymatic activity of five strains of Fusarium solani in a carbon-limited medium under shaking, revealed a differential production of these enzymes. Aryl alcohol oxidase (AAO) activity was observed only in the supernatant of strain CLPS no. 568 with levels higher than 57 mU ml−1. Free extracellular laccase activity was detected in strains CLPS nos. 493, 568 and 570, strain no. 568 being the one which showed the highest activity (over 8.6 mU ml−1). Free extracellular lignin peroxidase (LiP) activity was not detected in any isolate tested, whereas low levels of manganese-dependent peroxidase (MnP) and manganese-independent peroxidase (MIP) activities were detected in certain isolates used. The AAO activity of F. solani on primary α-alcohols such as veratryl alcohol, is reported for the first time; this enzyme activity is hydrogen-peroxide independent. This is also the first report for extracellular MnP and MIP activities of F. solani.

Regulation of the synthesis of aryl metabolites by phospholipid sources in the white-rot fungus Bjerkandera adusta

The white-rot basidiomycete Bjerkandera adusta was cultivated in a liquid medium enriched with l-phenylalanine and various phospholipid sources (lecithin, egg yolk and asolectin). Three aromatic metabolites (benzaldehyde, benzyl alcohol and benzoic acid) were produced under these culture conditions. High concentrations of benzaldehyde (404 mg l–1) were obtained when the cultures were supplemented with 10 g lecithin l–1. Benzyl alcohol production was promoted when the strain was grown with 5 or 10 g lecithin l–1. In the absence of or with a low concentration of lecithin (2.5 g l–1), benzoic acid was the major aryl metabolite synthesized. The results presented here indicate that aryl alcohol oxidase, an extracellular enzyme catalyzing the oxidation of benzyl alcohol into benzaldehyde, was maximally detected when significant amounts of benzaldehyde were produced. Aryl alcohol oxidase activity was significantly enhanced in the presence of elevated concentrations of phospholipid sources. Together with lignin peroxidase, methoxylated and hydroxylated aryl metabolites were also synthesized under these culture conditions. The possible involvement of phospholipids in the synthesis of aryl metabolites is discussed.

Production of aryl metabolites in solid-state fermentations of the white-rot fungus Bjerkandera adusta

Bjerkandera adusta produced aromatic compounds such as benzaldehyde (bitter almond aroma), benzyl alcohol and benzoic acid from L-phenylalanine (3 g kg−1). Two supports for the fungus, wheat bran (organic support) and Perlite (mineral support), gave optimal production with water contents of 66% and 60%, respectively. Benzyl alcohol (4.53 g kg−1) and benzaldehyde (1.56 g kg−1) were produced after 4 days on wheat bran respectively with 20 and 30 g L-phenylalanine kg−1. Aryl alcohol oxidase activity, which oxidises benzyl alcohol to benzaldehyde, was only detected when the fungus was grown on wheat bran, the support which promotes the most benzaldehyde production. Results are compared with those obtained in submerged liquid cultures.

Industrial dye decolorization by laccases from ligninolytic fungi.

White-rot fungi were studied for the decolorization of 23 industrial dyes. Laccase, manganese peroxidase, lignin peroxidase, and aryl alcohol oxidase activities were determined in crude extracts from solid-state cultures of 16 different fungal strains grown on whole oats. All Pleurotus ostreatus strains exhibited high laccase and manganese peroxidase activity, but highest laccase volumetric activity was found in Trametes hispida. Solid-state culture on whole oats showed higher laccase and manganese peroxidase activities compared with growth in a complex liquid medium. Only laccase activity correlated with the decolorization activity of the crude extracts. Two laccase isoenzymes from Trametes hispida were purified, and their decolorization activity was characterized.

Influence of cell immobilization on the production of benzaldehyde and benzyl alcohol by the white-rot fungi Bjerkandera adusta , Ischnoderma benzoinum and Dichomitus squalens

Three white-rot basidiomycetes, Bjerkandera adusta, Ischnoderma benzoinum and Dichomitus squalens, were cultivated on a liquid medium supplemented with l-phenylalanine, a precursor for benzaldehyde (bitter almond aroma) and benzyl alcohol. Remarkable amounts of benzaldehyde (587 mg l−1) were found in cultures of B. adusta. Immobilization of this fungus on polyurethane foam cubes allowed an 8.3-fold increase of the production of benzaldehyde and a 15-fold increase of the productivity as compared with non-immobilized cells. Aryl-alcohol oxidase activity was only detected in B. adusta. This activity was also significantly enhanced in immobilized cells, suggesting that it plays an important role in benzaldehyde biosynthesis. Conversely, consistent amounts of benzyl alcohol (340 mg l−1 for B. adusta and I. benzoinum and 100 mg l−1 for D. squalens) were produced by the three fungi when immobilized. Laccase activity was found only in the strains I. benzoinum and D. squalens. This activity was markedly enhanced in free cells cultures. Immobilization of the fungi did not promote benzyl alcohol production by comparison with free cell cultures (500 mg l−1).

Screening of 68 species of basidiomycetes for enzymes involved in lignin degradation

A survey for enzymes involved in lignin degradation was carried out from 90 strains of 68 species of different groups of basidiomycetes. Laccase activity was found in 50% of the fungi tested, whereas aryl-alcohol oxidase (AAO) and Mn-dependent peroxidase (MnP) were detected in 40% and 29% of species, respectively. Laccase activity of more than 200 U l−1 was obtained in cultures of Trametes versicolor, Phellinus torulosus, Cerrena unicolor and Pleurotus eryngii, whereas the AAO and MnP levels were comparatively lower, and lignin peroxidase was not detected in the different fungi tested. Previously known AAO-producing fungi, P. eryngii and Bjerkandera adusta, showed the highest AAO activities, and the enzyme was detected for the first time in fungi from very different taxonomic groups, including the gasteromycete Cyathus olla. In contrast to laccase and AAO, MnP seemed to be restricted to species of Aphyllophorales, with the exception of the tremellaceous fungus Exidia glandulosa. It is interesting to note that MnP was found in most Phellinus species, P. ribis, P. trivialis and P. torulosus producing the highest activities among the fungi studied. Simultaneous production of MnP and H2O2-producing AAO was found in several polyporaceous fungi, but the latter enzyme was absent from the above Phellinus species.

Manganese regulation of manganese peroxidase expression and lignin degradation by the white rot fungus Dichomitus squalens

Extracellular manganese peroxidase and laccase activities were detected in cultures of Dichomitus squalens (Polyporus anceps) under conditions favoring lignin degradation. In contrast, neither extracellular lignin peroxidase nor aryl alcohol oxidase activity was detected in cultures grown under a wide variety of conditions. The mineralization of 14C-ring-, -side chain-, and -methoxy-labeled synthetic guaiacyl lignins by D. squalens and the expression of extracellular manganese peroxidase were dependent on the presence of Mn(II), suggesting that manganese peroxidase is an important component of this organism's lignin degradation system. The expression of laccase activity was independent of manganese. In contrast to previous findings with Phanerochaete chrysosporium, lignin degradation by D. squalens proceeded in the cultures containing excess carbon and nitrogen.