Reduction thermodynamics of the T1 Cu site in plant and fungal laccases

The thermodynamic parameters for reduction of the type-1 (T1) copper site in Rhus vernicifera and Trametes versicolor laccases and for the derivative of the former protein from which the type-2 copper has been selectively removed (T2D) have been determined with UV–vis spectroelectrochemistry. In all cases, the enthalpic term turns out to be the main determinant of the E ^o′ of the T1 site. Also the difference between the reduction potentials of the two laccases is enthalpy-based and reflects differences in the coordination features of the T1 sites and their protein environment. The T1 sites in native R. vernicifera laccase and its T2D derivative show the same E ^o′, as a result of compensatory differences in the reduction thermodynamics. This suggests that removal of the type-2 (T2) copper results in modification of the reduction-induced solvent reorganization effects, with no influence in the structure of the multicopper protein site. This conclusion is supported by NMR data recorded on the native, the T2D, and Hg-substituted T1 derivatives of R. vernicifera laccase, which show that the T1 and T2/T3 sites are largely noninteracting.     

多环芳烃的真菌漆酶转化及污染土壤修复技术

漆酶可以转化多种有机污染物,在环境保护领域具有广泛的应用潜力。二十年来,通过多学科协同研究,对真菌漆酶转化多环芳烃的机制、特征等各方面的认识不断深入。基于漆酶等真菌木质素分解酶的污染土壤修复技术不断发展,并逐渐走向田间应用。本文首先介绍了真菌漆酶的一般作用机制与多环芳烃转化特征,结合我们的相关研究提出了漆酶作用下多环芳烃在土壤中的迁移模式;其次介绍了利用漆酶氧化原理修复污染农田土壤的潜力,着重对利用农业废弃物进行真菌生物刺激的修复实践进行了评述;最后,就漆酶转化多环芳烃基础研究中的若干重要问题进行了思考,并展望了真菌及其漆酶系统在污染土壤修复应用中的发展方向。     

Effect of mannan oligosaccharide elicitor and ferulic acid on enhancement of laccases production in liquid cultures of basidiomycetes

The effects of mannan oligosaccharides preparation (MO), as elicitor, and ferulic acid inducer for enhancement in laccases production in liquid cultures of three strains of basidiomycetes, Pycnoporus sanguineus, Coriolopsis polyzona and Pleurotus ostreatus was studied using a full factorial 3² experimental design. MO, either individually or combined with ferulic acid, enhanced laccases levels in the three different strains of the white-rot fungi. The enhancement of laccases production was species specific with the highest increase in liquid cultures of P. sanguineus (88-fold) followed by P. ostreatus (3-fold) and C. polyzona (2-fold). Separate additions of 75 mg/l of MO to the cultures of P. sanguineus and P. ostreatus caused the increase while a combined addition of 150 mg/l of MO and 1 mM ferulic acid resulted in the optimal production of laccases in the cultures of C. polyzona.     

Improving the catalytic performance of peroxidases in organic synthesis

Peroxidases are ubiquitous enzymes that catalyze a variety of enantioselective oxygen-transfer reactions with hydrogen peroxide (H2O2). Although they have enormous potential, their industrial application is hampered by their high price and low operational stability. Recent developments, such as the controlled addition and in situ formation of the oxidant, protein engineering and the rational design of semi-synthetic peroxidases, aim to improve the operational stability of peroxidases.     

Screening mutant libraries of fungal laccases in the presence of organic solvents

Reliable screening methods are being demanded by biocatalysts' engineers, especially when some features such as activity or stability are targets to improve under non-natural conditions (i.e., in the presence of organic solvents). The current work describes a protocol for the design of a fungal laccase-expressed in Saccharomyces cerevisiae-highly active in organic cosolvents. A high-throughput screening assay based on ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)) oxidation was validated. The stability of the ABTS radical cation was not significantly altered in the presence of acetonitrile, ethanol, or DMSO. With a coefficient of variance below 10% and a sensitivity limit of 15 pg laccase/microL, the assay was reproducible and sensitive. The expression system of Myceliophthora thermophila laccase variant T2 in S. cerevisiae was highly dependent on the presence of Cu2+. Copper concentration was limited up to 10 microM CuSO4 where expression levels (approximately 14-18 mg/L) were acceptable without compromising the reliability of the assay. A mutant library was created by error-prone PCR with 1.1 to 3.5 mutations per kb. After only 1 generation of directed evolution, mutant 6C9 displayed about 3.5-fold higher activities than parent type in the presence of 20% acetonitrile or 30% ethanol. The method provided here should be generally useful to improve the activity of other redox enzymes in mixtures of water/cosolvents.     

Concentration oscillations in three-component reaction systems

Based on the sufficient conditions for the existence of oscillation modes in the mathematical models of biological systems, simple three-component schemes of chemical reactions with oscillatory behavior including only mono- and bimolecular steps are suggested.     

Improving catalytic performance of an arylacetonitrilase by semirational engineering

Arylacetonitrilases have been widely acknowledged as important alternatives to chemical catalysts for synthesizing optically pure 2-hydroxyphenylacetic acids from nitriles. In this work, two residues (Thr132 and Ser190) located at the catalytic tunnel in the active site of an arylacetonitrilase nitA from uncultured organisms were mutated separately by site-directed mutagenesis. Ser190 was demonstrated to be the critical position which has a greater influence on arylacetonitrilase nitA activity than Thr132. The replacement of serine at position 190 with glycine increases its activity toward mandelonitrile and (o, m, p)-chloromandelonitrile, whereas replacing it with leucine abolished its activity. The best mutant S190G exhibited threefold higher specific activity toward mandelonitrile compared with that of wild-type nitA, which rendered it promising for industrial application. Homology modeling and molecular docking experiments were in agreement with the kinetic assays and support the improved catalytic performance.

     

Controversial role of fungal laccases in decreasing the antibacterial effect of olive mill waste-waters

Antibacterial diffusion tests (against Bacillus megaterium) detected both bacterial growth-promoting and growth-inhibiting components in olive mill waste-water (OMW). Mixtures of OMW aromatic components showed antibacterial effects that did not show antibacterial activity when tested as individual compounds. Strains of white rot fungi (WRF) producing different patterns of lignin modifying enzymes (LMEs) have been evaluated for OMW remediation under nutritional conditions affecting the LMEs produced. The decrease of both the content in OMW phenols and in the OMW antibacterial activity was compared with fungal growth and LMEs production. OMW addition to the cultures increased fungal growth and laccase activity irrespectively of the nutritional conditions of the cultures. Laccase was the sole LME activity that increased after OMW addition to the cultures. Neither the increased growth of WRF in OMW-containing cultures, their content in laccase nor the amount of OMW phenols were direct indications of a greater decrease in OMW antibacterial effect. The higher decrease in OMW antibacterial activity was obtained in cultures of Phanerochaete flavido-alba in an N-rich media.     

Divergence of mycorrhizal fungal communities in crop production systems

Mycorrhizal fungi are present in all arable soils and colonize nearly all crops and weed pests of crops. They may be involved as mutualists or pathogens of crops in well known but poorly understood phenomena such as crop rotation and green manure effects on soil productivity. Crop change effects on mycorrhizal fungal community parameters were evaluated in three field experiments. In Experiment 1, soybean (Glycine max (L.) Merr. cv. Douglas) was grown continuously or rotated with corn (Zea mays L.), milo (Sorghum bicolor (L.) Moench), or fescue (Festuca arundinacea Schreb cv. Johnstone) for two years, then soybean was grown on all plots. Continuous soybean plots were dominated byGigaspora spp., while rotated crops were dominated byGlomus spp. Differences in communities and community indices of continuous soybean and rotated plots were reduced after growing soybeans on rotated plots. In Experiment 2, a fescue sod was plowed and pearl millet (Pennisetum americanum Leeke) or crabgrass (Digitaria sanguinalis (L.) Scop.) grown. Both hosts resulted in great changes in populations of individual species, decreases in community dominance, and increases in community diversity and equitability. Crabgrass also resulted in reduced species richness. In Experiment 3, tobacco (Nicotiana tabacum L.) or fescue was planted on adjacent tracts of land with a long-term history of either fescue (30 yr) or sorghum-sudangrass (Sorghum bicolor (L.) Moench. ×S. sudanense (Piper) Staph.) (3 yr). The long-term cropping history had major effects on the mycorrhizal fungal communities which were related to the expression of mycorrhizal stunt disease of tobacco. Changes occurred in these communities in response to either current-season crop. These experiments suggest that crop rotation causes large changes in mycorrhizal fungal communities, that these changes may be involved in the rotation effect on soil productivity, and that design of cropping systems should take mycorrhizal fungal communities into consideration.     

Improving the performance of short‐duration basmati rice in water‐saving production systems by boron nutrition

Over exploitation of groundwater and decreasing canal water resources are threating the productivity of conventional rice production systems in Asia which is the main rice bowl. Therefore, strategies are needed to produce more rice with less water in the shortest possible duration without compromising the yield to feed the increasing world population. Panicle sterility is one of the major obstacles in wide‐scale adoption of water‐saving rice production systems. Boron (B) deficiency, in water‐saving rice production systems, has been identified as a possible reason for panicle sterility. This 2‐year field study was aimed to investigate the potential of pre‐optimised boron application through various methods in improving the productivity of short‐duration basmati rice (Shaheen Basmati) in water‐saving production systems, as delivered through seed priming (0.1 mM boron), foliar spray (200 mM boron) or soil application (1 kg boron ha^?1), while hydropriming and no boron application were taken as control. Boron nutrition, by either way, improved the growth, water relations, morphology, yield‐related traits, panicle fertility, grain yield, grain quality and grain boron contents of short‐duration basmati rice; nonetheless, boron application as seed priming was superior and cost effective with maximum marginal rate of return. In conclusion, boron nutrition through seed priming is cost effective and may help improving the productivity, quality, and boron grain contents in short‐duration basmati rice under water‐saving production systems.     

漆酶空间结构、反应机理及应用

漆酶 (EC 1.10.3.2) 属于多铜氧化酶家族,可以催化氧化酚类和芳香类化合物,利用自由基反应机理完成4个电子的转移,并将分子氧还原成水。漆酶具有非常保守的拓扑学结构,结合作者自身工作实践,对漆酶结构与功能的最新研究进展进行综述,其中对漆酶的三维结构、活性中心、催化机理研究和最新的应用进展作重点阐述。     

Water-in-oil microemulsions as the reaction medium for the solvent-sensitive yellow laccases

To determine the applicability of water-in-oil microemulsions for enzymatic conversions catalysed by yellow laccase from Pleurotus ostreatus (YLPO) D1 the following were studied: (i) the catalytic activity of the YLPO D1 in the oxidation of typical phenolic substrates: catechol (CAT), 2,6-dimethoxyphenol (DMOP) and 2,2^'-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS), at the appropriate pH optimum values in aqueous buffer solutions and in 62 mM bis-2-(ethylhexyl) sulfosuccinate sodium salt (AOT) in isooctane water-in-oil microemulsions; (ii) the effect of acetonitrile (ACN) on the kinetic parameters of DMOP oxidation catalysed by this laccase; (iii) the optimum conditions for the laccase catalytic activity in AOT in isooctane w/o microemulsions (w, laccase, AOT concentration); (iiv) the possibility of using the optimum water-in-oil microemulsions for the oxidation of aromatic alcohols (veratryl alcohol (VA) and benzyl alcohol (BA)) and the oxidative degradation of selected pollutants (3-chlorophenol, anthracene (ANT) and fluorene (FLU)).     

Evaluation of some fungal diseases and yield of groundnut in groundnut-based cropping systems

A study on the evaluation of some fungal diseases and yield of groundnut in groundnut-based cropping systems was conducted in 2002 and 2003 planting seasons. Analysis of variance indicated that intercropping was highly significant on leaf spot disease severity 0.76; 0.75, rust 2.75; 2.69, as well as percentage defoliation 78.42%; 78.10% in 2002 and 2003, respectively. Plant population was significant on leaf spot severity 4.52, 4.60 rust 2.76; 366 and defoliation 226.5; 441.1 fungal as well as yield, while interactions were not significant on the fungal diseases and yield. Sole groundnut recorded significantly high severity of the fungal diseases investigated but low yield, when groundnut was intercropped with maize and melon and recorded the lowest yield in 2002 and 2003 respectively. 250,000 plants/ha recorded the lowest severity of the diseases investigated, while 444,444 plants/ha recorded the highest. Sole groundnut also recorded the highest percentage defoliation 79.37%, 79.25% when groundnut was intercropped with maize recorded the lowest 77.06%; 77.60%. 250,000 plants/ha had the lowest defoliation when 444,444 plants/ha had the highest 80.75% 82.13% in 2002 and 2003, respectively. Sole groundnut recorded the lowest in the majority of yields when intercropped with maize and with melon recorded a high yield and yield components in 2002 and 2003, respectively. The microorganisms identified were Cercospora spp., Aspergillus linked and Blastomyces.     

Affinity chromatography as a rapid and convenient method for purification of fungal laccases

A rapid and convenient method for graduation, isolation, and purification of laccase from Trametes versicolor and Fomes fomentarius culture fluids was developed. For purification affinity chromatography on syringyl- and vanillyl-controlled porosity glass (CPG) columns was applied. The purified laccase of F. fomentarius was immobilized on porous glass. Some properties of the immobilized enzyme in comparison to the free one are discussed.     

A general model of reaction kinetics in biological systems

Dynamic mathematical models in biotechnology require, besides the information about the stoichiometry of the biological reaction system, knowledge about the reaction kinetics. Modulation phenomena like limitation, inhibition and activation occur in different forms of competition with the key enzymes responsible for the respective metabolic reaction steps. The identification of a priori unknown reaction kinetics is often a critical task due to the non-linearity and (over-) parameterization of the model equations introduced to account for all the possible modulation phenomena. The contribution of this paper is to propose a general formulation of reaction kinetics, as an extension of the Michaelis-Menten kinetics, which allows limitation/activation and inhibition effects to be described with a reduced number of parameters. The versatility of the new model structure is demonstrated with application examples.     

Male powerlifting performance described from the viewpoint of complex systems

This paper reflects on the factors that condition performance in powerlifting and proposes that the result-generating process is inadequately described by the allometric equations commonly used. We analysed the scores of 1812 lifters belonging to all body mass categories, and analysed the changes in the results achieved in each weight category and by each competitor. Current performance-predicting methods take into account biological variables, paying no heed to other competition features. Performance in male powerlifting (as in other strength sports) behaves as a self-organised system with non-linear interactions between its components. Thus, multiple internal and external elements must condition changes in a competitor's score, the most important being body mass, body size, the number of practitioners, and the concurrency of favourable factors in one individual. It was observed that each behaved in a specific form in the high level, according to the individuals’ circumstances, which make up the main elements of the competitive system in every category. In powerlifting, official weight categories are generally organised in three different groups: light (<52.0 to <60 kg), medium (<67.5 to <90.0 kg) and heavy (<100 to >125 kg) lifter categories, each one of them with specific allometric exponents. The exponent should be revised periodically, especially with regard to the internal dynamics of the category, and adjusted according to possible changes affecting competition.     

Crop rotation and tillage systems as a proactive strategy in the control of peanut fungal soilborne diseases

Soil management practices can affect the population dynamics of soil microbial communities. Cultural practices can be adequately combined to benefit natural populations of microorganisms that may have a role in biological control (actinomycetes, Trichoderma spp., and Gliocladium spp.), thus contributing to the management of peanut fungal soilborne diseases in a sustainable manner within ecological boundaries. During six agricultural cycles, rhizosphere soil samples were taken from a field subjected to crop rotation (soybean, peanut, and maize), peanut being under two tillage systems (no till, reduced tillage) with the aim of quantifying populations of soil microorganisms. The incidence of diseases caused by soilborne fungi in peanut was determined at harvest. The highest amount of actinomycetes, Trichoderma spp., and Gliocladium spp. were recorded when maize was the preceding crop. Regarding tillage systems, the populations of the three groups of microorganisms were higher in peanut under no tillage than under reduced tillage. Under these conditions, the lowest incidence of peanut blight (Sclerotinia minor) and root rot (strains of Fusarium solani) was observed, suggesting a possible natural control of peanut soilborne pathogens. The quantification of actinomycetes, Trichoderma spp., and Gliocladium spp. was used as a tool to explore the impacts of different management systems on microbial groups that may be involved in the biological control of soilborne diseases, with the aim of combining those practices that improve native populations of possible beneficial microorganisms. This manipulation can provide sustainable management strategies in the control of soilborne diseases, avoiding the use of artificial inoculations of microorganisms, and reducing agrochemical application.     

Molecular characterization and enzymatic activity of laccases in two Pleurotus spp. with different pathogenic behaviour

Pleurotus eryngii and P. ferulae, two species belonging to the P. eryngii complex, synthesize laccases, ligninolytic enzymes that play a role in the host-pathogen interaction in the first step of infection. Ecological studies have shown that although both fungi have been recognized as saprophytes, P. eryngii weakly pathogenic when colonizing the roots and stems of Eryngium campestre, whereas P. ferulae is mostly pathogenic to Ferula communis. The paper describes the genomic organization of four putative laccase genes (lac1, lac2, lac3, and lac5-like gene; gene names were assigned on the basis of sequence homologies) of P. eryngii and P. ferulae. The mRNA expression and enzymatic activity of the laccases were analysed under culture conditions where a source of lignin (wheat bran) or lyophilized roots of E. campestre or F. communis were present. These experiments indicated that the four lac-like genes were differentially regulated in the two mushrooms. Specifically, the addition of the lyophilized roots of the respective host plant to the culture media induced an advance in the mRNA expression of the four lac-like genes and a seven-fold higher total laccase activity in P. ferulae than in P. eryngii. The results obtained are discussed in relation to the possible role of laccases in the interaction of P. eryngii and P. ferulae with their respective host.