High redox potential laccases from the ligninolytic fungi Pycnoporus coccineus and Pycnoporus sanguineus suitable for white biotechnology: from gene cloning to enzyme characterization and applications.

AIMS: Exploitation of natural biodiversity in species Pycnoporus coccineus and Pycnoporus sanguineus to screen for a new generation of laccases with properties suitable for the lignin-processing sector. METHODS AND RESULTS: Thirty strains originating from subtropical and tropical environments, mainly isolated from fresh specimens collected in situ, were screened for laccase activity. On the basis of levels of enzyme activity and percentage of similarity between protein sequences, the laccases from strains BRFM 938, BRFM 66 and BRFM 902 were selected for purification and characterization. Each BRFM 938, BRFM 66 and BRFM 902 laccase gene encoded a predicted protein of 518 amino acids; the three deduced proteins showed 68.7-97.5% similarity with other Polyporale laccases. The three laccases (59.5-62.9 kDa with 7-10% carbohydrate content) had high redox potentials (0.72-0.75 V vs normal hydrogen electrode at pH 6), remained highly stable up to 75-78 degrees C and at pH 5-7 mixtures, and were resistant to methyl and ethyl alcohols, acetonitrile and dimethylsulfoxide at concentrations as high as 50% (v/v). The best laccase-1-hydroxybenzotriazole systems permitted almost 100% of various polyphenolic dye decolourization and oxidation of adlerol and veratryl alcohol. CONCLUSIONS: The three laccases showed complementary biochemical features. BRFM 938 laccase had the highest thermo- and pH stability, catalytic efficiency towards 2,2'-azino-bis-[3-ethylthiazoline-6-sulfonate] and resistance to alcoholic solvents. BRFM 66 laccase had the highest rates of dye decolourization and oxidation of nonphenolic compounds. SIGNIFICANCE AND IMPACT OF THE STUDY: This study identified P. coccineus and P. sanguineus as outstanding producers of high redox potential laccases, easy to purify and scale-up for industrial production. Three new laccases proved to be suitable models for white biotechnology processes and for further molecular breeding to create a new generation of tailor-made enzymes.

Heterologous production of the Piromyces equi cinnamoyl esterase in Trichoderma reesei for biotechnological applications.

AIMS: The objective of the study was to produce and characterize the cinnamoyl esterase EstA from the anaerobic fungus Piromyces equi for potential industrial applications. METHODS AND RESULTS: The catalytic domain EstA was produced in Trichoderma reesei. Because the two fungi displayed different genome features, including different codon usage and GC content, a synthetic gene was designed and expressed, leading to the production of the corresponding protein at around 33 mg per litre in the T. reesei culture medium. After the recombinant protein was purified, biochemical characterization showed that EstA presents peak activity at pH 6.5 and at 50-60 degrees C. Furthermore, EstA remained stable at pH 6-8 and below 50 degrees C. EstA was compared to cinnamoyl esterases FaeA and FaeB from Aspergillus niger in terms of ferulic acid (FA) release from wheat bran (WB), maize bran (MB) and sugar beet pulp (SBP). CONCLUSION: The synthetic gene was successfully cloned and overexpressed in T. reesei. EstA from P. equi was demonstrated to efficiently release FA from various natural substrates. SIGNIFICANCE AND IMPACT OF THE STUDY: Recombinant EstA produced in an industrial enzyme producer, T. reesei, was biochemically characterized, and its capacity to release an aromatic compound (FA) for biotechnological applications was demonstrated.

Technical advance in fungal biotechnology: development of a miniaturized culture method and an automated high-throughput screening.

AIMS: The goal of the study was to develop a reliable, reproducible and rapid method of culture in order to screen a large number of fungal transformants. METHODS AND RESULTS: The method is based upon miniaturized cell cultures and automated expression screening in microwell plates. For the method development, 50 recombinant Aspergillus vadensis clones producing feruloyl esterase B (FaeB) from Aspergillus niger were screened in 6 days. Then a panel of clones showing various behaviours was checked in flasks in order to demonstrate the reproducibility of the method. Using this method, a transformant of A. vadensis producing 1.2 g l(-1) of FaeB was selected (12-fold more than the A. niger overproducing strain). CONCLUSIONS: This miniaturized culture method allows to obtain reliable and reproducible results. The procedure has the advantages of being efficient, time-saving and more efficient than conventional in-flask culture screening as it can screen 800 clones per day after a culture of 3 days. SIGNIFICANCE AND IMPACT OF THE STUDY: This method could be applied to any other fungal strain culture, enzyme activity or biodiversity screening.

Fungal tyrosinases: new prospects in molecular characteristics, bioengineering and biotechnological applications.

Tyrosinases are type-3 copper proteins involved in the initial step of melanin synthesis. These enzymes catalyse both the o-hydroxylation of monophenols and the subsequent oxidation of the resulting o-diphenols into reactive o-quinones, which evolve spontaneously to produce intermediates, which associate in dark brown pigments. In fungi, tyrosinases are generally associated with the formation and stability of spores, in defence and virulence mechanisms, and in browning and pigmentation. First characterized from the edible mushroom Agaricus bisporus because of undesirable enzymatic browning problems during postharvest storage, tyrosinases were found, more recently, in several other fungi with relevant insights into molecular and genetic characteristics and into reaction mechanisms, highlighting their very promising properties for biotechnological applications. The limit of these applications remains in the fact that native fungal tyrosinases are generally intracellular and produced in low quantity. This review compiles the recent data on biochemical and molecular properties of fungal tyrosinases, underlining their importance in the biotechnological use of these enzymes. Next, their most promising applications in food, pharmaceutical and environmental fields are presented and the bioengineering approaches used for the development of tyrosinase-overproducing fungal strains are discussed.

Characterization of a new tyrosinase from Pycnoporus species with high potential for food technological applications.

AIMS: Tyrosinase production by Pycnoporus cinnabarinus and Pycnoporus sanguineus was screened among 20 strains originating from various geographical areas, particularly from tropical environments. The tyrosinase from the most efficient strain was purified and characterized and tested for food additive applications. METHODS AND RESULTS: Monophenolase and diphenolase activities of tyrosinase were measured from cell lysate from the 20 Pycnoporus strains, for 8-10 days of cultivation. The strain P. sanguineus CBS 614.73 showed the highest productivity (45.4 and 163.6 U g(-1) protein per day for monophenolase and diphenolase respectively). P. sanguineus CBS 614.73 tyrosinase was purified from concentrated cell lysate, anion-exchange, size-exclusion and hydroxyapatite chromatography, with a final yield of 2% and a purification factor of 35-38. The pure enzyme was a monomere with a molecular mass of 45 kDa and it showed four isoforms or isoenzymes with pI between 4.5-5. No N-glycosylation was found. The N-terminal amino acid sequence was IVTGPVGGQTEGAPAPNR. The enzyme was shown to be almost fully active in a pH range of 6-7, in a large temperature range (30-70 degrees C), and was stable below 60 degrees C. The main kinetic constants were determined. The tyrosinase was able to convert p-tyrosol and p-coumaric acid into hydroxytyrosol and caffeic acid, respectively, and it could also catalyse the cross-linking formation of a model protein. CONCLUSIONS: Among the genus Pycnoporus, known for the production of laccase, the strain P. sanguineus CBS 614.73 was shown to produce one other phenoloxidase, a new monomeric tyrosinase with a specific activity of 30 and 84 U mg(-1) protein for monophenolase and diphenolase respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: This study identified P. sanguineus CBS 614.73 as a potential producer of a tyrosinase which demonstrated effectiveness in the synthesis of antioxidant molecules and in protein cross-linking.

Natural and recombinant fungal laccases for paper pulp bleaching.

Three laccases, a natural form and two recombinant forms obtained from two different expression hosts, were characterized and compared for paper pulp bleaching. Laccase from Pycnoporus cinnabarinus, a well known lignolytic fungus, was selected as a reference for this study. The corresponding recombinant laccases were produced in Aspergillus oryzae and A. niger hosts using the lacI gene from P. cinnabarinus to develop a production process without using the expensive laccase inducers required by the native source. In flasks, production of recombinant enzymes by Aspergilli strains gave yields close to 80 mg l(-1). Each protein was purified to homogeneity and characterized, demonstrating that the three hosts produced proteins with similar physico-chemical properties, including electron paramagnetic resonance spectra and N-terminal sequences. However, the recombinant laccases have higher Michaelian (Km) constants, suggesting a decrease in substrate/enzyme affinity in comparison with the natural enzyme. Moreover, the natural laccase exhibited a higher redox potential (around 810 mV), compared with A. niger (760 mV) and A. oryzae (735 mV). Treatment of wheat straw Kraft pulp using laccases expressed in P. cinnabarinus or A. niger with 1-hydroxybenzotriazole as redox mediator achieved a delignification close to 75%, whereas the recombinant laccase from A. oryzae was not able to delignify pulp. These results were confirmed by thioacidolysis. Kinetic and redox potential data and pulp bleaching results were consistent, suggesting that the three enzymes are different and each fungal strain introduces differences during protein processing (folding and/or glycosylation).

Overproduction of the Aspergillus niger feruloyl esterase for pulp bleaching application.

A well-known industrial fungus for enzyme production, Aspergillus niger, was selected to produce the feruloyl esterase FAEA by homologous overexpression for pulp bleaching application. The gpd gene promoter was used to drive FAEA expression. Changing the nature and concentration of the carbon source nature (maltose to glucose; from 2.5 to 60 g l(-1)), improved FAEA activity 24.5-fold and a yield of 1 g l(-1) of the corresponding protein in the culture medium was achieved. The secreted FAEA was purified 3.5-fold to homogeneity in a two-step purification procedure with a recovery of 69%. The overproduced protein was characterised and presented properties in good agreement with those of native FAEA. The recombinant FAEA was tested for wheat straw pulp bleaching, with or without a laccase mediator system and xylanase. Best results were obtained using a bi-sequential process with a sequence including xylanase, FAEA and laccase, and yielded very efficient delignification--close to 75%--and a kappa number of 3.9. This is the first report on the potential application of recombinant FAEA in the pulp and paper sector.

Overproduction of laccase by a monokaryotic strain of Pycnoporus cinnabarinus using ethanol as inducer.

AIMS: Laccase production by the monokaryotic strain Pycnoporus cinnabarinus ss3 was studied using ethanol as inducer in the culture medium. METHODS AND RESULTS: The effect of ethanol was tested at 10, 20, 30, 35 and 45 g l-1 and compared with that of ferulic acid, known until now as the most efficient inducer for laccase expression by P. cinnabarinus ss3. In the presence of 35 g l-1 ethanol, laccase activity (266 600 U l-1) and productivity (19 000 U l-1 day-1) were nine and fivefold higher compared with ferulic acid-induced cultures, and 155- and 65-fold higher compared with non-induced cultures, respectively. In vivo, ethanol added to the culture medium of P. cinnabarinus ss3 favoured a continuous and high expression of laccase gene. Under these conditions, P. cinnabarinus ss3 produced preferentially the isoenzyme LAC I. Ethanol added in vitro to the purified P. cinnabarinus ss3 laccase typically inhibited the enzymatic activity. CONCLUSIONS: In spite of an initial inhibitory effect on mycelial growth, ethanol was shown to be a very strong inducer for laccase expression by P. cinnabarinus ss3 allowing an average yield of 1-1.5 g l-1 laccase. SIGNIFICANCE AND IMPACT OF THE STUDY: This study identified P. cinnabarinus ss3 as an outstanding producer of laccase in the presence of ethanol as inducer. Ethanol is an inexpensive agricultural by-product and the process is simple to scale-up for industrial production.

Pycnoporus cinnabarinus laccases: an interesting tool for food or non-food applications.

The effects of the addition of ferulic acid and ethanol in P. cinnabarinus ss3 culture medium in fermentor were compared in 15-L fermentor. In the presence of 30 g l(-1) ethanol, laccase activity (270,000 U/L1) was 3-fold higher as compared with ferulic acid-induced cultures, and 150-fold higher as compared with non-induced cultures, respectively. High-quality flax pulp was bleached in a totally-chlorine free (TCF) sequence using a laccase-mediator system constituted by laccase from Pycnoporus cinnabarinus and 1-hydroxybenzotriazole (HBT) as mediator. Up to 90% delignification and strong brightness increase were attained after the laccase-mediator treatment followed by H2O2 bleaching. This TCF sequence was further improved by applying H2O2 under pressurized O2. In this way, up to 82% ISO brightness was obtained (compared with 37% in the initial pulp and 60% in the peroxide-bleached control) as well as very low kappa number. A positive evaluation of the laccase has been also performed in a food application. The colour of a tea-based beverage was significantly improved by incubating an infusion of green tea with the Pycnoporus laccase.

Increased phospholipid transfer protein activity in Aspergillus oryzae grown on various industrial phospholipid sources.

The effect of industrial carbon sources on phospholipid transfer protein production was investigated. Phospholipid fractions of different composition were prepared from various plant oils (i.e., soybean, rapeseed, and sunflower) according to the Lucas Meyer extraction and purification process. The effect of these fractions on phospholipid transfer protein activity of cell extracts from Aspergillus oryzae grown on medium containing these phospholipids as sole carbon source was studied. It was shown that phospholipid transfer activity was markedly increased by extracts containing a particular phospholipid composition. However, this stimulation depends mainly upon the phospholipid composition of the fraction used as fermentation substrate. Fractions enriched mainly in phosphatidylinositol (Epikuron 110), at the expense of phosphatidylcholine, were the most efficient sources for phospholipid transfer protein production by A. oryzae. Maximal phospholipid transfer activity, as well as biomass production, were increased 4.1- and 9.7-fold, respectively, when cultures were supplemented with Epikuron 110 prepared from sunflower lecithin, as compared to glucose-control cultures.

Shifting the biotransformation pathways of L-phenylalanine into benzaldehyde by Trametes suaveolens CBS 334.85 using HP20 resin.

AIMS: The biotransformation of L-phenylalanine into benzaldehyde (bitter almond aroma) was studied in the strain Trametes suaveolens CBS 334.85. METHODS AND RESULTS: Cultures of this fungus were carried out in the absence or in the presence of HP20 resin, a highly selective adsorbent for aromatic compounds. For the identification of the main catabolic pathways of L-phenylalanine, a control medium (without L-phenylalanine) was supplemented with each of the aromatic compounds, previously detected in the culture broth, as precursors. Trametes suaveolens CBS 334.85 was shown to biosynthesize benzyl and p-hydroxybenzyl derivatives, particularly benzaldehyde, and large amounts of 3-phenyl-1-propanol, benzyl and p-hydroxybenzyl alcohols as the products of both cinnamate and phenylpyruvate pathways. CONCLUSION: The addition of HP20 resin, made it possible to direct the catabolism of L- phenylalanine to benzaldehyde, the desired target compound, and to trap it before its transformation into benzyl alcohol. In these conditions, benzaldehyde production was increased 21-fold, from 33 to 710 mg l-1 corresponding to a molar yield of 31%. SIGNIFICANCE AND IMPACT OF THE STUDY: These results showed the good potential of Trametes suaveolens as a biotechnological agent to synthesize natural benzaldehyde which is one of the most important aromatic aldehydes used in the flavour industry.

Cloning and expression in phospholipid containing cultures of the gene encoding the specific phosphatidylglycerol/phosphatidylinositol transfer protein from Aspergillus oryzae: evidence that the pg/pi-tp is tandemly arranged with the putative 3-ketoacyl-CoA thiolase gene.

The phosphatidylglycerol/phosphatidylinositol transfer protein (PG/PI-TP) is a new and original phospholipid transfer protein (PLTP) isolated from the Deuteromycete, Aspergillus oryzae. We have isolated a genomic clone of the A. oryzae pg/pi-tp using a probe derived from the corresponding cDNA and sequenced the complete gene. The DNA sequence analysis revealed that pg/pi-tp gene is composed of three exons encoding a 18,823 Da protein of 175 amino acids as previously described and of two introns as deduced by cDNA and genomic sequence alignment. The isolated pg/pi-tp gene do not show similarity with other PLTP genes or the deduced PG/PI-TP protein with proteins already known. Comparison of the encoded PG/PI-TP with other deduced proteins from recent genomic or cDNA sequence from databases revealed that the PG/PI-TP was close to two encoded proteins deduced from the cDNA database of Aspergillus nidulans (54% identity and 68% similarity) and the second from Neurospora crassa (53% identity and 76% similarity). Therefore, we suggested that both proteins might belong to the PLTP family. Southern blot analysis of A. oryzae genomic DNA show that the PG/PI-TP was encoded by a single gene. Expression of pg/pi-tp was performed in phospholipid containing cultures with increasing carbon source concentrations in order to study the regulation of the PLTPs in the filamentous fungus cell. This was done to know if a high density culture could yield a high amount of biomass with high phospholipid transfer activity. Results showed that phospholipids as compared to glucose in standard cultures stimulated mycelial growth and global phospholipid transfer activity, but not the pg/pi-tp transcript accumulation. However, high concentration of both carbon sources yielded an inhibition of the expression of the pg/pi-tp gene and of the global phospholipid transfer activity. In conclusion, both carbon sources are not suitable to increase the PLTP production in high density cultures for biotechnological applications. Finally, using the gene walking sequencing method it is demonstrated that the pg/pi-tp is tandemly arranged on opposite DNA strands in a tail-to-tail orientation with a putative gene encoding the 3-ketoacyl-CoA thiolase (EC 2.3.1.16). Unlike the pg/pi-tp gene, this thiolase gene show a putative 'beta-oxidation box' and encodes a putative 44,150 Da protein of 321 amino acids composed of a putative N-terminal PTS2 (Peroxisomal Targeting Signal) consensus sequence for the peroxisome targeting. Comparison of the amino acid sequence of the A. oryzae thiolase to that of the Yarrowia lipolytica showed a 50% identity and a 69% similarity.

Aspergillus niger I-1472 and Pycnoporus cinnabarinus MUCL39533, selected for the biotransformation of ferulic acid to vanillin, are also able to produce cell wall polysaccharide-degrading enzymes and feruloyl esterases.

The filamentous fungal strains Aspergillus niger I-1472 and Pycnoporus cinnabarinus MUCL39533, previously selected for the bioconversion of ferulic acid to vanillic acid and vanillin respectively, were grown on sugar beet pulp. A large spectrum of polysaccharide-degrading enzymes was produced by A. niger and very few levels of feruloyl esterases were found. In contrast, P. cinnabarinus culture filtrate contained low amount of polysaccharide-degrading enzymes and no feruloyl esterases. In order to enhance feruloyl esterases in A. niger cultures, feruloylated oligosaccharide-rich fractions were prepared from sugar beet pulp or cereal bran and used as carbon sources. Number of polysaccharide-degrading enzymes were induced. Feruloyl esterases were much higher in maize bran-based medium than in sugar beet pulp-based medium, demonstrating the ability of carbon sources originating from maize to induce the synthesis of feruloyl esterases. Thus, A. niger I-1472 could be interesting to release ferulic acid from sugar beet pulp or maize bran.

Evidence of a new biotransformation pathway of p-coumaric acid into p-hydroxybenzaldehyde in Pycnoporus cinnabarinus.

Pycnoporus cinnabarinus MUCL39533 was shown to be able to convert p-coumaric acid into p-hydroxybenzaldehyde, a component of high organoleptic note present in natural vanilla aroma. Use of phospholipid-enriched medium led to high-density cultures of P. cinnabarinus, since dry mycelial biomass was increased three-fold as compared to glucose medium. In the presence of phospholipids, 155 mg l(-1) p-hydroxybenzaldehyde was produced as the major compound on culture day 13 with a molar yield of 26%. The degradation pathways of p-coumaric acid were investigated. Based on the different metabolites identified, an oxidative side-chain degradation pathway of p-coumaric acid conversion to p-hydroxybenzoic acid was suggested. This acid was further reduced to p-hydroxybenzaldehyde and p-hydroxybenzyl alcohol, or hydroxylated and reduced to protocatechyl derivatives. Additionally, a reductive pathway of p-coumaric acid with 3-(4-hydroxyphenyl)-propanol as the terminal product occurred.

Isolation of a new laccase isoform from the white-rot fungi Pycnoporus cinnabarinus strain ss3.

Two extracellular laccase isoforms (Lac I and Lac II) produced by the white-rot fungus Pycnoporus cinnabarinus from the monokaryotic strain ss3 were purified from ferulic-acid-induced liquid culture medium using ammonium sulphate precipitation, followed by anion-exchange chromatography on a Mono Q column. Strain ss3 is the first generation of the parental strain P. cinnabarinus I-937. The new isolated isoform, Lac II, consists of an 86,000 molecular weight protein as determined by SDS polyacrylamide gel electrophoresis. The N-terminal amino acid sequences of both isoforms were determined, and compared to known laccase protein sequences of other organisms.

Molecular cloning of the cDNA encoding laccase from Pycnoporus cinnabarinus I-937 and expression in Pichia pastoris.

Laccases are multicopper-containing enzymes which catalyse the oxidation of phenolic and nonphenolic compounds with the concomitant reduction of molecular oxygen. In this study, a full-length cDNA coding for laccase (lac1) from Pycnoporus cinnabarinus I-937 was isolated and characterized. The corresponding open reading frame is 1557 nucleotides long and encodes a protein of 518 amino acids. The cDNA encodes a precursor protein containing a 21 amino-acid signal sequence corresponding to a putative signal peptide. The deduced amino-acid sequence of the encoded protein was similar to that of other laccase proteins, with the residues involved in copper coordination sharing the greatest extent of similarity. The cDNA encoding for laccase was placed under the control of the alcohol oxidase (Aox 1) promoter and expressed in the methylotropic yeast Pichia pastoris. The laccase leader peptide, as well as the Saccharomyces cerevisiae alpha-factor signal peptide, efficiently directed the secretion into the culture medium of laccase in an active form. Moreover, the laccase activity was directly detected in plates. The identity of the recombinant product was further confirmed by protein immunoblotting. The expected molecular mass of the mature protein is 81 kDa. However, the apparent molecular mass of the recombinant protein is 110 k Da, thus suggesting that the protein expressed in P. pastoris may be hyperglycosylated.

Selection of Pycnoporus cinnabarinus strains for laccase production.

A comparison of Pycnoporus cinnabarinus strains for laccase production was carried out. A dikaryotic strain, I-937 strain, producing a high level of laccase (9500 U l(-1)) was selected. The study of the life cycle in vitro of this dikaryotic strain led to isolation of monokaryons. Forty-eight monokaryotic strains were isolated and screened for laccase production. One of these strains, ss3, produced a higher level of laccase than the parental strain I-937. The maximum production reached 29000 U l(-1) in medium supplemented with ferulic acid.

Design of a fungal bioprocess for vanillin production from vanillic acid at scalable level by Pycnoporus cinnabarinus.

The biotechnological process of vanillin production from vanillic acid by Pycnoporus cinnabarinus was scaled-up at the laboratory level. Vanillin production was studied in two types of bioreactors, a mechanically agitated and an air-lift bioreactor. In the mechanically agitated bioreactor where vanillin was produced in greater quantities, oxygen availability was studied during the growth and production phases. A maximal aeration rate (90l/h equivalent to 0.83 volume of air/volume of medium/min or vvm) during the growth phase and a minimal aeration rate (30 l/h equivalent to 0.28 vvm) during the production phase were necessary to increase vanillin production to 1260 mg/l. Vanillic acid bioconversion to vanillin occurred under the conditions of reduced dissolved oxygen concentration, gentle agitation, high carbon dioxide production and low specific growth rate. However, under these conditions, vanillin production was accompanied by a significant amount of methoxyhydroquinone. Vanillin over a concentration of 1000 mg/l was shown to be highly toxic to the growth of P. cinnabarinus on agar medium. The application of selective XAD-2 resin led to a reduction of vanillin concentration in the medium, thus limiting its toxicity towards the fungal biomass as well as the formation of unwanted by-products such as methoxyhydroquinone and allowed the concentration of vanillin produced to reach 1575 mg/l.

Enhanced benzaldehyde formation by a monokaryotic strain of Pycnoporus cinnabarinus using a selective solid adsorbent in the culture medium.

A monokaryotic strain of the white-rot fungus Pycnoporus cinnabarinus was shown to produce, in a 2-L bioreactor culture, 100 mg.L-1 benzaldehyde (bitter almond aroma) from L-phenylalanine with a productivity of 33 mg.L-1.day-1. The addition of HP20 resin, a styrene divinylbenzene copolymer highly selective for benzaldehyde, enabled an eightfold increase in the production of benzaldehyde and a twofold increase in productivity. In the presence of HP20 resin, the production of 790 mg.L-1 benzaldehyde was concomitant with the synthesis of cinnamic acid derivatives of high organoleptic notes such as cinnamaldehyde, cinnamyl alcohol, and methyl cinnamate.

Design and scale up of a process for manganese peroxidase production using the hypersecretory strain Phanerochaete chrysosporium I-1512.

Manganese peroxidase (MnP) production was performed in a airlift bioreactor in which Phanerochaete chrysosporium I-1512, an MnP hypersecretory strain, was immobilized on a stainless steel mesh. Production was scaled up from a 2.5-L bench scale to a 100-L bioreactor. The yield of MnP was increased 2-fold and reached 6600 U L(-1). These results indicate the feasibility of MnP production on a medium scale, which promises sufficient MnP availability for its use in pulp bleaching at industrial scale.