Maturity Status Strongly Influences the Relative Age Effect in International Elite Under-9 Soccer.

The aim of the study was to assess the role of the relative age effect (RAE) and to investigate the influence of the biological maturity status on the RAE in international under-9 soccer. The birth dates of 222 male participants of the U9 Eurochampionship Soccer Tournament in Vienna in 2016 were analyzed and divided into four relative age quarters (Q1-Q4) and the biological maturity status was assessed with the age at peak height velocity (APHV) method. Based on the mean±standard deviation of the APHV, the athletes were divided into three groups of maturity: early, normal and late maturing. Chi-Square-tests were used to assess the difference between the observed and the expected even relative age quarter distribution and to evaluate the difference between the observed distribution of early, normal and late maturing athletes and the expected normal distribution. A univariate analysis of variance was performed to assess differences in the APHV between the relative age quarters. A RAE was present (χ2 = 23.87; p < 0.001; ω = 0.33). A significant difference was found in APHV between the four relative age quarters (F = 9.906; p < 0.001); relatively older athletes were significantly less mature. A significant difference was found between the distribution of early, normal and late maturing athletes and the expected normal distribution for athletes of Q1 (high percentage of late maturing athletes: 27%; χ2 = 17.69; p < 0.001; ω = 0.46) and of Q4 (high percentage of early maturing soccer players: 31%; χ2 = 12.08; p = 0.002; ω = 0.58). These findings demonstrated that the selection process in international soccer, with athletes younger than 9 years, seems to be associated with the biological maturity status and the relative age. Relatively younger soccer players seem to have a better chance for selection for international tournaments, if they enter puberty at an earlier age, whereas relatively older athletes seem to have an increased likelihood for selection independent of their biological maturity status.

Fully Enzymatic Membraneless Glucose|Oxygen Fuel Cell That Provides 0.275 mA cm(-2) in 5 mM Glucose, Operates in Human Physiological Solutions, and Powers Transmission of Sensing Data.

Coimmobilization of pyranose dehydrogenase as an enzyme catalyst, osmium redox polymers [Os(4,4'-dimethoxy-2,2'-bipyridine)2(poly(vinylimidazole))10Cl](+) or [Os(4,4'-dimethyl-2,2'-bipyridine)2(poly(vinylimidazole))10Cl](+) as mediators, and carbon nanotube conductive scaffolds in films on graphite electrodes provides enzyme electrodes for glucose oxidation. The recombinant enzyme and a deglycosylated form, both expressed in Pichia pastoris, are investigated and compared as biocatalysts for glucose oxidation using flow injection amperometry and voltammetry. In the presence of 5 mM glucose in phosphate-buffered saline (PBS) (50 mM phosphate buffer solution, pH 7.4, with 150 mM NaCl), higher glucose oxidation current densities, 0.41 mA cm(-2), are obtained from enzyme electrodes containing the deglycosylated form of the enzyme. The optimized glucose-oxidizing anode, prepared using deglycosylated enzyme coimmobilized with [Os(4,4'-dimethyl-2,2'-bipyridine)2(poly(vinylimidazole))10Cl](+) and carbon nanotubes, was coupled with an oxygen-reducing bilirubin oxidase on gold nanoparticle dispersed on gold electrode as a biocathode to provide a membraneless fully enzymatic fuel cell. A maximum power density of 275 µW cm(-2) is obtained in 5 mM glucose in PBS, the highest to date under these conditions, providing sufficient power to enable wireless transmission of a signal to a data logger. When tested in whole human blood and unstimulated human saliva maximum power densities of 73 and 6 µW cm(-2) are obtained for the same fuel cell configuration, respectively.

Transcription analysis of pyranose dehydrogenase from the basidiomycete Agaricus bisporus and characterization of the recombinantly expressed enzyme.

Agaricus bisporus is a litter degrading basidiomycete commonly found in humic-rich environments. It is used as model organism and cultivated in large scale for food industry. Due to its ecological niche it produces a variety of enzymes for detoxification and degradation of humified plant litter. One of these, pyranose dehydrogenase, is thought to play a role in detoxification and lignocellulose degradation. It is a member of the glucose-methanol-choline family of flavin-dependent enzymes and oxidizes a wide range of sugars with concomitant reduction of electron acceptors like quinones. In this work, transcription of pdh in A. bisporus was investigated with real-time PCR revealing influence of the carbon source on pdh expression levels. The gene was isolated and heterologously expressed in Pichia pastoris. Characterization of the recombinant enzyme showed a higher affinity towards disaccharides compared to other tested pyranose dehydrogenases from related Agariceae. Homology modeling and sequence alignments indicated that two loops of high sequence variability at substrate access site could play an important role in modulating these substrate specificities.

Engineering of pyranose dehydrogenase for application to enzymatic anodes in biofuel cells.

In the search for improved glucose oxidising enzymes for biofuel cells, a number of Agaricus meleagris (Am) pyranose dehydrogenase mutants (mPDHs) exhibiting different degrees of glycosylation were produced using site-directed mutagenesis and electrochemically characterised. The response of electrodes modified with different mPDHs is compared in a mediated electron transfer mode, where the electrodes are modified with each of the mutants covalently attached to redox polymers based on polyvinylimidazole-bound osmium complexes using a cross-linking agent. Coating of each of the enzymes onto the graphite electrode surface is also used to screen for their capacity for direct electron transfer. The double mutant PDH exhibits the highest response to glucose at physiological pH in both direct and mediated electron transfer modes, producing a Jmax of ≈800 µA cm(-2) at room temperature and when "wired" to the Os-polymer having the highest formal potential. From the results obtained the double mPDH is proposed as the most suitable candidate for application to bioanode fabrication.

Further insights into the catalytical properties of deglycosylated pyranose dehydrogenase from Agaricus meleagris recombinantly expressed in Pichia pastoris.

The present study focuses on fragmented deglycosylated pyranose dehydrogenase (fdgPDH) from Agaricus meleagris recombinantly expressed in Pichia pastoris . Fragmented deglycosylated PDH is formed from the deglycosylated enzyme (dgPDH) when it spontaneously loses a C-terminal fragment when stored in a buffer solution at 4 °C. The remaining larger fragment has a molecular weight of ∼46 kDa and exhibits higher volumetric activity for glucose oxidation compared with the deglycosylated and glycosylated (gPDH) forms of PDH. Flow injection amperometry and cyclic voltammetry were used to assess and compare the catalytic activity of the three investigated forms of PDH, "wired" to graphite electrodes with two different osmium redox polymers: [Os(4,4'-dimethyl-2,2'-bipyridine)2(poly(vinylimidazole))10Cl](+) [Os(dmbpy)PVI] and [Os(4,4'-dimethoxy-2,2'-bipyridine)2(poly-(vinylimidazole))10Cl](+) [Os(dmobpy)PVI]. When "wired" with Os(dmbpy)PVI, the graphite electrodes modified with fdgPDH showed a pronounced increase in the current density with Jmax 13- and 6-fold higher than that observed for gPDH- and dgPDH-modified electrodes, making the fragmented enzyme extraordinarily attractive for further biotechnological applications. An easier access of the substrate to the active site and improved communication between the enzyme and mediator matrix are suggested as the two main reasons for the excellent performance of the fdgPDH when compared with that of gPDH and dgPDH. Three of the four glycosites in PDH: N(75), N(175), and N(252) were assigned using mass spectrometry in conjunction with endoglycosidase treatment and tryptic digestion. Determination of the asparagine residues carrying carbohydrate moieties in PDH can serve as a solid background for production of recombinant enzyme lacking glycosylation.

Determining the effect of one decade on fitness of elite Austrian youth soccer players using propensity score matching.

Current trends in attacking strategies and increases in external workload have led to a need for fast and well-conditioned athletes in modern soccer. More recently, progressions in speed, coordination, power and endurance were found over a decade in elite Austrian youth players. However, possible confounders such as relative age, maturation, learning effects, and academy philosophy may have influenced these changes. The present study aimed to determine the decade effect on fitness under statistical control of players' exact age, height, body mass, test location as well as total number of pretests and time interval between test and pretest. Players annually completed a battery of anthropometric, general and soccer-specific fitness tests. MANCOVA was calculated to identify the overall impacts of the covariates on fitness. To balance the covariates of initially 2,530 "former" (2002 to 2005) and 2,611 "recent" (2012 to 2015) players, 1:1 nearest neighbor propensity score (PS) matching was used, resulting in 587 U13, 573 U14, 475 U15, 325 U16, 262 U17, and 129 U18 matched pairs. The decade effect on fitness was assessed by independent t-tests and Cohen's d separately at each age group. Superior performances of recent players were found for linear sprint across all age categories (d = 0.154-0.476) as well as for agility (d = 0.125-0.340) and change-of-direction speed (d = 0.172-0.466) in U15 to U18. Reaction speed increased in U13 (d = 0.288) and U15 (d = 0.310). Flexibility reduced over the decade in all age categories (d = -0.151 to -0.589) and upper-limb power decreased (d = -0.278 to -0.347) in U13 and U14. Balancing the covariate distribution via PS matching generally confirmed previous findings, with fitness decade effects reflecting the athletic needs for modern soccer. Since fitness performance changed over time, reference values should be periodically updated. Coaches favor both physical and cognitive fast players nowadays. Thus, training should target all aspects of speed, without disregarding flexibility, upper-limb power and other preventive strategies that keep the players on the pitch.

Enzymatic degradation of ochratoxin A in the gastrointestinal tract of piglets.

Animal feeds are often contaminated with ochratoxin A (OTA), a potent natural mycotoxin hazardous to animal and human health that accumulates in blood and tissues. To the best of our knowledge, this study is the first to investigate the in vivo application of an enzyme (OTA amidohydrolase; OAH) that degrades OTA into the nontoxic molecules phenylalanine and ochratoxin α (OTα) in the gastrointestinal tract (GIT) of pigs. Piglets were fed six experimental diets over 14 days, varying in OTA contamination level (50 or 500 µg/kg; OTA50 and OTA500) and presence of OAH; a negative control diet (no OTA added) and a diet containing OTα at 318 µg/kg (OTα318). The absorption of OTA and OTα into the systemic circulation (plasma and dried blood spots, DBS), their accumulation in kidney, liver, and muscle tissues, and excretion through feces and urine were assessed. The efficiency of OTA degradation in the digesta content of the GIT was also estimated. At the end of the trial, accumulation of OTA in blood was significantly higher in OTA groups (OTA50 and OTA500) in comparison to enzyme groups (OAH50 and OAH500, respectively). The supplementation of OAH explicitly reduced the absorption of OTA (P < 0.005) into plasma by 54% and 59% (from 40.53 ± 3.53 to 18.66 ± 2.28 ng/mL in piglets fed the 50 µg OTA/kg diets and from 413.50 ± 71.88 to 168.35 ± 41.02 ng/mL in piglets fed the 500 µg OTA/kg diets, respectively) and in DBS by 50% and 53% (from 22.79 ± 2.63 to 10.67 ± 1.93 ng/mL in piglets fed the 50 µg OTA/kg diets and from 232.85 ± 35.16 to 105.71 ± 24.18 ng/mL in piglets fed the 500 µg OTA/kg diets, respectively). The OTA concentrations in plasma were positively associated with the OTA levels detected in all tissues analyzed; adding OAH reduced OTA levels in the kidney, liver, and muscle (P < 0.005) by 52%, 67%, and 59%, respectively. The analysis of GIT digesta content showed that OAH supplementation led to OTA degradation in the proximal GIT where natural hydrolysis is inefficient. Overall, the data of present in vivo study demonstrated that supplementation of swine feeds with OAH successfully reduced OTA levels in blood (plasma and DBS) as well as in kidney, liver, and muscle tissues. Therefore, an approach to use enzymes as feed additives might be most promising to mitigate the harmful effects of OTA on the productivity and welfare of pigs and at the same time improving the safety of pig-derived food products.

Ochratoxin A (OTA) is a potent toxin frequently present in animal feeds, which accumulates in the animal tissues for human consumption. This results in critical animal welfare issues, as well as food safety issues. To the best of our knowledge, the present study is the first to show that OTA can be degraded by an enzyme supplemented with pigs' feed. In the pig gut, this enzyme successfully breaks down the toxic OTA molecule into a nontoxic form that is excreted through urine and feces, which is applicable in supporting the pig's health, welfare, and productivity as well as assuring the safety of the pig meat for human consumption.

Ochratoxin A degrading enzymes of Stenotrophomonassp. 043-1a.

Ochratoxin A is a secondary metabolite that acts as a mycotoxin and is produced by Aspergillus, Penicillium, and other fungal species. It is a threat to animal and human health due to nephrotoxic, carcinogenic, and genotoxic properties and its widespread incidence in agricultural products. To reduce this threat, biological remediation processes are of growing interest. The aerobic gram-negative bacterium Stenotrophomonassp. 043-1a, isolated from soil, was previously shown to degrade ochratoxin A into the non-toxic ochratoxin α and l-phenylalanine (Schatzmayr et al. 2002). However, the enzyme or enzymes catalyzing this reaction in this strain remained elusive. Here, we report the targeted purification of Stenotrophomonassp. 043-1a lysate via ammonium sulfate precipitation, size-exclusion chromatography, and hydrophobic interaction chromatography to identify the ochratoxin A degrading enzymes by subsequent peptide fragment fingerprinting. The metallo-dependent hydrolase Chr1_3858681_3267 and a member of the peptidase S9 family, Chr1_3858681_771, were shown to degrade ochratoxin A. This was, to our knowledge, the first report of an ochratoxin A degrading enzyme from the peptidase S9 family.

[Importance and application of injury prevention in Austrian football - a survey among 687 coaches]. / Stellenwert und Anwendung der Verletzungsprävention im österreichischen Fußball ­ eine Befragung unter 687 Trainer/innen.

BACKGROUND: Soccer is one of the most popular sports in the world but associated with a high risk of injury. For this reason, the study of the etiology of injuries is of great interest and numerous prevention programs have been developed in recent years. Since these prevention programs must be embedded into training routine, trainers are primarily responsible for their implementation. The present study aimed to survey Austrian soccer coaches engaged in professional, amateur or youth teams with respect to their opinions on injuries as well as the application of respective prevention programs. METHODS: An online questionnaire was developed and sent to all coaches registered in the Austrian Football Association.In addition to personal data, the trainers' attitudes to the subject of injury prevention were also asked. It was also asked which preventive measures the trainers consider important and which they also use in their training and to what extent. RESULTS: A total of 687 trainers took part in the survey. 2,3%, 37,5% and 43,6% of trainers were engaged in professional, amateur and youth clubs, respectively. The rest gave no information. The majority of respondents (56%) considered injuries a major concern in soccer. Inadequate fitness (75,7%) as well as insufficient preparation (60,7%) and poor regeneration (59,2%) were seen as the greatest risk factors for injuries. Appropriate warm-up (66,8%), regeneration (59,4%) and core stabilization training (58,2%) were considered the most effective preventative measures. More than 50% of the participants were not familiar with any of the most widely applied injury prevention programs, and only 15.4% actually implemented those programs in their training.The substantial interest in injury prevention notwithstanding, the respective standard of knowledge amongst Austrian coaches is poor. In light of the high prevalence of injuries, it is most important to inform trainers about injury prevention programs and possibilities to implement them in training practice.

Using physiological data to predict future career progression in 14- to 17-year-old Austrian soccer academy players.

The crux of any soccer-specific talent identification programme is to determine early predictors of future playing success (Williams & Reilly, 2000 ). We compare physiological characteristics among 14- to 17-year-old soccer academy players in terms of subsequent career progression ('drafted' vs. 'non-drafted'). In a longitudinal design (2001-2010), players passed through 10 fitness tests at four age levels: 14 (n = 410); 15 (n = 504); 16 (n = 456); and 17 years (n = 272). MANOVAs showed statistically significant (P < 0.05) superior performances for drafted players in all components ('speed', 'power and flexibility', 'coordination and endurance') and age categories. ANOVAs revealed significantly (P < 0.013) better performances for drafted players in almost all tests, with the largest effect sizes for shuttle sprint (η²= 0.07-0.09), 20 m sprint (η²= 0.04-0.05) and medicine ball throw (η²=0.05-0.11). Follow up discriminant analyses confirmed that a combination of three variables correctly classified 62.7 to 66.2% of the players. Soccer-specific speed and power of upper limbs best discriminated future playing status, irrespective of age category. It is concluded that physiological measurements at adolescence can provide useful information in terms of predicting future career progression.

Changes in Anthropometric and Fitness Characteristics Over a Decade of Young Elite Alpine Ski Racers.

PURPOSE: The aim of this study was to evaluate whether anthropometric and fitness characteristics have changed between former and current elite male and female Austrian young ski racers (U11-U15). METHODS: A battery of anthropometric, general, and skiing-specific fitness tests was conducted annually. In total, 1517 participants (846 males, 671 females) who were tested in 2005-2009 ("former athletes" n = 805) and 2015-2019 ("current athletes" n = 712) were included. Independent t tests and Cohen d were calculated to compare the two 5-y periods, separated by sex and age group. The level of significance was set at P < .05. RESULTS: No significant change in anthropometric characteristics was found over the decade. Current young ski racers performed significantly better in the maximal core flexion strength test in all age categories (ES = 0.88-1.50; P < .02). Core extension strength values were higher in current male U12 and female U12 and U13 athletes (ES = 0.54-0.71; P < .01) and better postural stability values in the lateral direction were found in the age categories U12 and U14 (ES = 0.36-0.68; P < .05), as well as in the forward/backward direction in the age categories U12-U14 (ES = 0.38-1.12; P < .03). Lower-leg extension strength values were apparent in the current U13-U15 age categories (ES = 0.36-1.03; P ≤ .001) and lower drop-jump reactive strength indices in the U13-U15 male athletes (ES = 0.49-0.80; P < .01). CONCLUSIONS: Current and former young ski racers differ significantly in some fitness parameters, which might lead to the assumption that some aspects (such as core strength) have gained more focus in athletic training during the last years compared with 15 y ago.

An In Silico Target Fishing Approach to Identify Novel Ochratoxin A Hydrolyzing Enzyme.

Ochratoxin A (OTA), a mycotoxin that is of utmost concern in food and feed safety, is produced by fungal species that mainly belong to the Aspergillus and Penicillium genera. The development of mitigation strategies to reduce OTA content along the supply chains is key to ensuring safer production of food and feed. Enzyme-based strategies are among the most promising methods due to their specificity, efficacy, and multi-situ applicability. In particular, some enzymes are already known for hydrolyzing OTA into ochratoxin alpha (OTα) and phenylalanine (Phe), eventually resulting in detoxification action. Therefore, the discovery of novel OTA hydrolyzing enzymes, along with the advancement of an innovative approach for their identification, could provide a broader basis to develop more effective mitigating strategies in the future. In the present study, a hybrid in silico/in vitro workflow coupling virtual screening with enzymatic assays was applied in order to identify novel OTA hydrolyzing enzymes. Among the various hits, porcine carboxypeptidase B was identified for the first time as an effective OTA hydrolyzing enzyme. The successful experimental endorsement of findings of the workflow confirms that the presented strategy is suitable for identifying novel OTA hydrolyzing enzymes, and it might be relevant for the discovery of other mycotoxin- mitigating enzymes.

Changes Over a Decade in Anthropometry and Fitness of Elite Austrian Youth Soccer Players.

Increases in physical (e.g., high-intensity running and sprinting), technical (e.g., passing rate), and tactical (e.g., player density) aspects made elite level soccer more challenging within the past years. The aim of the study was to evaluate whether these evolutions are also been reflected in changes in anthropometric and fitness characteristics between former (2002 to 2005) and current (2012 to 2015) elite Austrian youth development center (U13 to U14) and soccer academy (U15 to U18) players. A battery of anthropometric, general and soccer-specific fitness tests was conducted annually at the end of each year. Independent t-test and Cohen's d (ES) were calculated to compare the two four-year periods (2530 vs. 2611 players) at each age group separately. Current players were significantly faster in 20 m sprint (ES = 0.26-0.50) and reaction test (ES = 0.15-0.39, except for U18), but less flexible at sit-and-reach (ES = -0.19 to -0.55), in all age categories. Whereas height (ES = 0.26-0.32), body mass (ES = 0.11-0.18) and countermovement jump (ES = 0.24-0.26) increased significantly at youth development center level, current academy players performed superior at shuttle sprint (ES = 0.21-0.59), hurdles agility run (ES = 0.24-0.49), and endurance run (ES = 0.11-0.20). These changes over time in speed, change-of-direction ability, lower-body power, coordination, and endurance were attributed to modern training approaches (e.g., modified games and change-of-direction drills) and modifications in selection politics (e.g., coaches favor speed and decision-making skills).

Maturity status influences the relative age effect in national top level youth alpine ski racing and soccer.

Since the relative age effect (RAE) characterizes a problem in all age categories of alpine ski racing and soccer and the fact that, yet, to date the underlying factors have not been well investigated, the aim of the present study was to assess the influence of the biological maturity status on the RAE among youth alpine ski racers (YSR) and soccer players (SP). In total, 183 male and female YSR selected for national final races and 423 male SP selected for Elite Youth Development Centres were investigated. Additionally, a comparison group of 413 non-athletes was evaluated. The birth months were split into four relative age quarters. The biological maturity status was assessed by the age at peak height velocity (APHV) method; according to the M±SD of the comparison group, the athletes were divided into normal, early and late maturing. Chi2-tests indicated a significant RAE among YSR (χ2(3,N = 183) = 18.0; p<0.001; ω = 0.31) and SP (χ2(3,N = 423) = 33.1; p<0.001; ω = 0.28). In total, only a small number of late maturing athletes were present (0.5-2.3%). Among relatively younger athletes, high percentages of early maturing athletes were found (43.1-43.3%). The findings indicate that relatively younger and less mature athletes are marginalized or totally excluded in alpine ski racing and soccer. Thus, selection criteria in both sports are effectively based on early biological development and relatively older age, both of which should be considered in future in the talent selection process. In this context, the easy feasible method of assessing the APHV can be used.

Analysis of Agaricus meleagris pyranose dehydrogenase N-glycosylation sites and performance of partially non-glycosylated enzymes.

Pyranose Dehydrogenase 1 from the basidiomycete Agaricus meleagris (AmPDH1) is an oxidoreductase capable of oxidizing a broad variety of sugars. Due to this and its ability of dioxidation of substrates and no side production of hydrogen peroxide, it is studied for use in enzymatic bio-fuel cells. In-vitro deglycosylated AmPDH1 as well as knock-out mutants of the N-glycosylation sites N75 and N175, near the active site entrance, were previously shown to improve achievable current densities of graphite electrodes modified with AmPDH1 and an osmium redox polymer acting as a redox mediator, up to 10-fold. For a better understanding of the role of N-glycosylation of AmPDH1, a systematic set of N-glycosylation site mutants was investigated in this work, regarding expression efficiency, enzyme activity and stability. Furthermore, the site specific extend of N-glycosylation was compared between native and recombinant wild type AmPDH1. Knocking out the site N252 prevented the attachment of significantly extended N-glycan structures as detected on polyacrylamide gel electrophoresis, but did not significantly alter enzyme performance on modified electrodes. This suggests that not the molecule size but other factors like accessibility of the active site improved performance of deglycosylated AmPDH1/osmium redox polymer modified electrodes. A fourth N-glycosylation site of AmPDH1 could be confirmed by mass spectrometry at N319, which appeared to be conserved in related fungal pyranose dehydrogenases but not in other members of the glucose-methanol-choline oxidoreductase structural family. This site was shown to be the only one that is essential for functional recombinant expression of the enzyme.

Constitutive expression of Botrytis aclada laccase in Pichia pastoris.

The heterologous expression of laccases is important for their large-scale production and genetic engineering--a prerequisite for industrial application. Pichia pastoris is the preferred expression host for fungal laccases. The recently cloned laccase from the ascomycete Botrytis aclada (BaLac) has been efficiently expressed in P. pastoris under the control of the inducible alcohol oxidase (AOX1) promoter. In this study, we compare these results to the constitutive expression in the same organism using the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. The results show that the amounts of BaLac produced with the GAP system (517 mgL(-1)) and the AOX1 system (495 mgL(-1)) are comparable. The constitutive expression is, however, faster, and the specific activity of BaLac in the culture supernatant is higher (41.3 Umg(-1) GAP, 14.2 Umg(-1) AOX1). In microtiter plates, the constitutive expression provides a clear advantage due to easy manipulation (simple medium, no methanol feeding) and fast enzyme production (high-throughput screening assays can already be performed after 48 h).

A chloride tolerant laccase from the plant pathogen ascomycete Botrytis aclada expressed at high levels in Pichia pastoris.

Fungal laccases from basidiomycetous fungi are thoroughly investigated in respect of catalytic mechanism and industrial applications, but the number of reported and well characterized ascomycetous laccases is much smaller although they exhibit interesting catalytic properties. We report on a highly chloride tolerant laccase produced by the plant pathogen ascomycete Botrytis aclada, which was recombinantly expressed in Pichia pastoris with an extremely high yield and purified to homogeneity. In a fed-batch fermentation, 495 mg L(-1) of laccase was measured in the medium, which is the highest concentration obtained for a laccase by a yeast expression system. The recombinant B. aclada laccase has a typical molecular mass of 61,565 Da for the amino acid chain. The pI is approximately 2.4, a very low value for a laccase. Glycosyl residues attached to the recombinant protein make up for approximately 27% of the total protein mass. B. aclada laccase exhibits very low K(M) values and high substrate turnover numbers for phenolic and non-phenolic substrates at acidic and near neutral pH. The enzyme's stability increases in the presence of chloride ions and, even more important, its substrate turnover is only weakly inhibited by chloride ions (I(50)=1.4M), which is in sharp contrast to most other described laccases. This high chloride tolerance is mandatory for some applications such as implantable biofuel cells and laccase catalyzed reactions, which suffer from the presence of chloride ions. The high expression yield permits fast and easy production for further basic and applied research.