Epoxide hydrolases from yeasts and other sources: versatile tools in biocatalysis

Major characteristics, substrate specificities and enantioselectivities of epoxide hydrolases from various sources are described. Epoxide hydrolase activity in yeasts is discussed in more detail and is compared with activities in other microorganisms. Constitutively produced bacterial epoxide hydrolases are highly enantioselective in the hydrolysis of 2,2- and 2,3-disubstituted epoxides. A novel bacterial limonene-1,2-epoxide hydrolase, induced by growth on monoterpenes, showed high activities and selectivities in the hydrolysis of several substituted alicyclic epoxides. Constitutively produced epoxide hydrolases are found in eukaryotic microorganisms. Enzymes from filamentous fungi are useful biocatalysts in the resolution of aryl- and substituted alicyclic epoxides. Yeast epoxide hydrolase activity has been demonstrated for the enantioselective hydrolysis of various aryl-, alicyclic- and aliphatic epoxides by a strain of Rhodotorula glutinis. The yeast enzyme, moreover, is capable of asymmetric hydrolysis of meso epoxides and performs highly enantioselective resolution of unbranched aliphatic 1,2-epoxides. Screening for other yeast epoxide hydrolases shows that high enantioselectivity is restricted to a few basidiomycetes genera only. Resolution of very high substrate concentrations is possible by using selected basidiomycetes yeast strains.     

Biocatalytic preparation of chiral epichlorohydrins using recombinant<Emphasis Type="Italic">Pichia pastoris</Emphasis> expressing epoxide hydrolase of<Emphasis Type="Italic">Rhodotorula glutinis</Emphasis>

The use of enantioselective hydrolysis for preparing chiral epichlorohydrins was investigated using recombinantPichia pastoris with the enantioselective epoxide hydrolase ofRhodotorula glutinis. The rate of the recombinant epoxide hydrolase-catalyzed enantioselective hydrolysis of racemic epichlorohydrins was enhanced by the addition of 5% (v/v) Tween 20. Enantiopure (R)-epichlorohydrins with an enantiopurity of 100%ee and a yield of 26% were obtained within 5 min from 50 mM racemates.     

Enantioselective Degradation of the Chiral Fungicides Metalaxyl and Furalaxyl by Brevibacillus brevis

For almost four decades, the chiral fungicides metalaxyl and furalaxyl have been in use in plant protection on a global scale. Both substances are distributed as racemic mixtures, yet the desirable interference in nucleic acid synthesis of harmful fungi only occurs by the (‐)‐R‐enantiomer. As enantioselective degradation in Scheyern (Germany) and Yaoundé (Cameroon) soils has been documented, the influence of 50 isolated microorganisms on the R/S ratio was investigated. A high‐pressure liquid chromatography method with a chiral column to separate enantiomers of metalaxyl and furalaxyl, and subsequent detection by tandem mass spectrometry, was employed. Only one of these microorganisms, a strain of Brevibacillus brevis, showed an enantioselective degradation pattern in liquid culture; the respective (‐)‐R‐enantiomers were preferably degraded. Moreover, (‐)‐R‐furalaxyl was degraded faster in cultures supplemented simultaneously with both fungicides of the same concentration. Chirality 25:336–340, 2013. © 2013 Wiley‐Liss Inc. Chirality 00:000‐000:, 2013. © 2013 Wiley Periodicals, Inc.     

The role of macrofungi in environmental conservation

Abstract

The motives for the conservation of fungi are briefly outlined. The data and methods needed for successful use of fungi in environmental conservation are mentioned. A survey is given of the main habitats of threatened fungi in Western and Central Europe, including data on the significance of their mycoflora, the causes of decrease and possible measures to improve the situation. The value of macrofungi as bio-indicators for environmental quality is discussed and demonstrated with three examples: the indicator value of wood-inhabiting fungi for undisturbed forest sites; of ectomycorrhizal fungi for air pollution stress in forests and of saprotrophic fungi for the duration of undisturbed grassland use. Lists of indicator species with different indicator values for air pollution stress and grassland use are provided.     

Application of microorganisms towards synthesis of chiral terpenoid derivatives

Biotransformations are a standard tool of green chemistry and thus are following the rules of sustainable development. In this article, we describe the most common types of reactions conducted by microorganisms applied towards synthesis of chiral terpenoid derivatives. Potential applications of obtained products in various areas of industry and agriculture are shown. We also describe biological activity of presented compounds. Stereoselective hydroxylation, epoxidation, Baeyer-Villiger oxidation, stereo- and enantioselective reduction of ketones, and various kinetic resolutions carried out by bacteria and fungi have been reviewed. Mechanistic considerations regarding chemical and enzymatic reactions are presented. We also briefly describe modern approaches towards enhancing desired enzymatic activity in order to apply modified biocatalysts as an efficient tool and green alternative to chemical catalysts used in industry.     

Enzymes in stereoselective pharmacokinetics of endogenous substances.

The use of enzymes to assay individual components of the L-carnitine family in pharmaceuticals, foodstuffs, and biological fluids with various forms of detection is reviewed. The most useful enzyme in the assay of compounds of the L-carnitine family is carnitine acetyl transferase (CAT), which catalyses the reversible interconversion of L-carnitine and its short-chain acyl esters. CAT can be used in one or more coupled reactions combined with U.V., or radiolabelled detection, or combined with HPLC, allowing, enantioselective, structurally specific, and, in the case of radiolabelled tracing, highly sensitive assays to be carried out. When compared with chromatographic separation of enantiomers or diastereoisomers, enantioselective enzyme mediated assays may be cheaper, more sensitive, and simpler, but they do not allow the nonpreferred isomer to be assayed. Consequently, they are appropriate for the specific assay of endogenous enantiomeric substrates of the enzyme concerned, in biological samples. The analysis of the other enantiomer in raw materials or in pharmaceuticals must be more properly approached by enantioselective chromatographic methods.     

Using invasion theory to predict the fate of arbuscular mycorrhizal fungal inoculants

The agricultural use of commercial “biofertilizers” containing arbuscular mycorrhizal (AM) fungi, obligate symbionts that help plants take up nutrients, has resulted in the global introduction of AM fungi to non-native environments. Despite their growing use and evidence for endemism among AM fungi, the risk of AM fungi becoming invasive through “biofertilizer” application has not been studied in the context of general invasion theory. Understanding their dispersal by multiple vectors and in different environments is key to understanding how quickly they could spread. In this review, we consider the risk of invasion by commercial AM fungi in terms of a theoretical framework. We propose traits that make an isolate good for use as a commercial product, such competitiveness and high sporulation rate, may also increase invasiveness.     

The screening and selection of inoculant arbuscular-mycorrhizal and ectomycorrhizal fungi

The initiation of a programme of screening and selection of arbuscular-mycorrhizal fungi (AM fungi) and ectomycorrhizal fungi (ECM fungi) for use as inoculant fungi in agriculture, horticulture of forestry will depend on whether inoculation is more appropriate as a management option than manipulation of the indigenous mycorrhizal populations. The greatest immediate potential for the successful use of mycorrhizal inoculants will be in soils and growth media where phosphorus (P) limits plant growth and where the indigenous mycorrhizal fungi are either ineffective at increasing P uptake by the plant or their numbers have been drastically reduced by human influence or natural disturbance. In recent investigations, however, additional benefits to the plant following colonization of roots by effective AM or ECM fungi have been demonstrated. These additional benefits of an effective mycorrhizal association have necessitated a re-evaluation of the optimum screening procedures for isolates of AM and ECM fungi. Both current methodologies and suggestions for alternative approaches to the screening of AM and ECM fungi are discussed in this paper. The problems inherent in choosing suitable experimental conditions to compare different isolates at the screening stage are also addressed. The review also stresses the importance of continued monitoring of introduced mycorrhizal fungi to learn more about their longer-term ecological role, particularly within reforestation or revegetation studies.     

Lipases: Useful biocatalysts for the preparation of pharmaceuticals

Biocatalysis offers a clean and ecological way to perform chemical processes, in mild reaction conditions and with high degree of selectivity. The use of enzymes, specially lipases, in organic solvents proves an excellent methodology for the preparation of single-isomer chiral drugs. This review covers some general aspects and representative examples of the use of lipases for the enantioselective or regioselective preparation of alcohol and amine intermediates in the synthesis of pharmaceuticals.     

Pro-antibiotic Substrates for the Identification of Enantioselective Hydrolases

A new growth-based screening method for the identification of enantioselective hydrolases, such as lipases and esterases, using pro-antibiotic substrates was devised. An enantioselective hydrolase could be identified by measuring growth rates of cells in liquid media containing (R)- or (S)-2–phenylbutyric chloramphenicol esters. This method can be applied to the screening of novel enantioselective microbes and to the high-throughput screening for the directed evolution of enantioselective hydrolytic enzymes.     

Estimating chromatographic enantioselectivity (α) from gradient enantioselective chromatography data

Calculation of chromatographic enantioselectivity (α) is critically important in enantioselective chromatographic method development studies. The fact that α can only be calculated from isocratic elution conditions, whereas gradient elution conditions are predominantly used in method development screening, presents some problems for the use of α as a scoring indicator for automated, intelligent enantioselective chromatography method development systems. In this study, an empirical algorithm was developed to estimate α at isocratic conditions based upon information collected from a gradient elution. The algorithm was validated for SFC applications and has been shown to accurately predict enantioselectivity for a wide variety of racemic test analytes eluted on different chiral column and mobile phase conditions. Chirality, 2011. © 2010 Wiley‐Liss, Inc.     

Preparation of dicationic palladium catalysts for asymmetric catalytic reactions

The synthesis of Pd(OTf)(2)·2H(2)O is described. This was used to generate two different types of chiral dicationic palladium complexes for highly enantioselective addition of aromatic amines to α, β-unsaturated conjugate alkenes ([(R-BINAP)Pd(OH(2))(2)][OTf](2) and [(R-BINAP)Pd(μ-OH)](2)[OTf](2)). The resulting optically active N-arylated β-amino acid derivatives are valuable synthetic intermediates for the synthesis of biologically active molecules and peptidomimetics. The reaction of (2E)-but-2-enoylcarbamate and aniline is shown as an example of the use of these catalysts for enantioselective aza-Michael addition. For the preparation of palladium(II) triflate, the time scale is 20 h 50 min, plus 5 h 15 min for the monomeric complex and plus 6 h 45 min for the dimeric complex.     

Enantioselective ene‐reduction of E‐2‐cyano‐3‐(furan‐2‐yl) acrylamide by marine and terrestrial fungi and absolute configuration of (R)‐2‐cyano‐3‐(furan‐2‐yl) propanamide determined by calculations of electronic circular dichroism (ECD) spectra

This work reports the green organic chemistry synthesis of E‐2‐cyano‐3(furan‐2‐yl) acrylamide under microwave radiation (55 W), as well as the use of filamentous marine and terrestrial‐derived fungi, in the first ene‐reduction of 2‐cyano‐3‐(furan‐2‐yl) acrylamide to (R)‐2‐cyano‐3‐(furan‐2‐yl)propanamide. The fungal strains screened included Penicillium citrinum CBMAI 1186, Trichoderma sp. CBMAI 932 and Aspergillus sydowii CBMAI 935, and the filamentous terrestrial fungi Aspergillus sp. FPZSP 146 and Aspergillus sp. FPZSP 152. A compound with an uncommon CN‐bearing stereogenic center at the α‐C position was obtained by enantioselective reactions mediated in the presence of the microorganisms yielding the (R)‐2‐cyano‐3‐(furan‐2‐yl) propanamide 3a . Its isolated yield and e.e. ranged from 86% to 98% and 39% to 99%, respectively. The absolute configuration of the biotransformation products was determined by time‐dependent density functional theory (TD‐DFT) calculations of electronic circular dichroism (ECD) spectra. Finally, the tautomerization of 2‐cyano‐3‐(furan‐2‐yl) propanamide 3a to form an achiral ketenimine was observed and investigated in presence of protic solvents.     

Are white-rot fungi a real biotechnological option for the improvement of environmental health?

The use of white-rot fungi as a biotechnological tool for cleaning the environment of recalcitrant pollutants has been under evaluation for several years. However, it is still not possible to find sufficiently detailed investigations of this subject to conclude that these fungi can decontaminate the environment. In the present review, we have summarized and discussed evidence about the potential of white-rot fungi to degrade such pollutants as polycyclic aromatic hydrocarbons, dyes or antibiotics as an example of the complex structures that these microorganisms can attack. This review also discusses field experiment results and limitations of white-rot fungi trials from contaminated sites. Moreover, the use of catabolic potential of white-rot fungi in biopurification systems (biobeds) is also discussed. The current status and future perspectives of white-rot fungi, as a viable biotechnological alternative for improvement of environmental health are noted.     

Biotransformation of (1-phenyl)ethyl hydroperoxide with Aspergillus niger: a model study on enzyme selectivity and on the induction of peroxidase activity

The biocatalytic enantioselective reduction of (1-phenyl)ethyl hydroperoxide (1) by the fungus Aspergillus niger to the corresponding alcohol 2 involves a multi-enzyme biotransformation of the hydroperoxide 1, as revealed by the change in the enantioselectivity as a function of incubation times. This unusual behavior is not exhibited by other fungi and seems to be restricted to A. niger. Furthermore, the peroxidase and other oxidoreductase activities of A. niger depend on the availability of metal ions such as Fe2+, Mn2+ and Zn2+ in the growth medium, since the addition of Fe2+ ions substantially (threefold) increases the enantioselectivity, whereas addition of Mn2+ and Zn2+ ions decreases it. Finally, the cold shock (4 degrees C) significantly enhances the reduction of the hydroperoxide by the microorganism A. niger.     

食药用菌酸乳制作中深层发酵滤液添加量的比较

利用深层发酵培养基对两种食药用菌（香菇、猴头菌）进行深层发酵培养,然后分别将香菇（猴头菌）发酵滤液、奶粉、无菌水按不同比例配制成相应的复原奶,并接种乳酸菌制成酸乳。同时检测不同比例发酵滤液添加量所制得的酸乳的营养和理化特性,选择出了最佳比例的滤液添加量,即香菇滤液添加量以3份较好,而猴头菌则以4份滤液添加量最佳。     

Using Species Distribution Models For Fungi

Species distribution models (SDMs) are an emerging tool in the study of fungi, and their use is expanding across species and research topics. To summarise progress to date and to highlight important considerations for future users, we review 283 studies that apply SDMs to fungi. We found that macrofungi, lichens, and pathogenic microfungi are most often studied. While many studies only aim to model species response to environmental covariates, the use of SDMs for explicitly predicting fungal occurrence in space and time is growing. Many studies collect fungal occurrence data, but the use of pre-collected records from reference collections and citizen science programs is increasing. Challenges of applying SDMs to fungi include detection and sampling biases, and uncertainties in identification and taxonomy. Further, finding environmental covariates at appropriate spatial and temporal scales is important, as fungi can respond to fine-scale environmental patterns. Fine-scale covariate data can be difficult to gather across space, but we show remote-sensing measurements are viable for fungi SDMs. For those fungi interacting with host species, host information is also important, and can be used as covariates in SDMs. We also highlight that competition among fungi, and dispersal, can affect observed distributions, with the latter particularly prominent for invasive fungi. We show how one can account for these processes in models, when suitable data are available. Finally, we note that environmental DNA records create new opportunities and challenges for future modelling efforts, and discuss the difficulties in predicting invasions and climate change impacts. The application of SDMs to fungi has already provided interesting lessons on how to adapt modelling tools for specific questions, and fungi will continue to be relevant test subjects for further technical development of SDMs.     

Investigation of ionic liquids as reaction media for enzymatic enantioselective acylation of amines

The use of ionic liquids as reaction media for lipase-catalyzed enantioselective acylation of 1-phenylethylamine (1) and 2-phenyl-1-propylamine (2) with 4-pentenoic acid was investigated. The best performing ionic liquid for each of these amines as well as its solvent properties were very different. Preparative scale kinetic resolution of 1 was performed efficiently in 1-butyl-2,3-dimethylimidazolium trifluoromethanesulphonate.     

同位素示踪技术在丛枝菌根真菌生态学研究中的应用

丛枝菌根(arbuscular mycorrhizal,AM)真菌是生态系统中重要的土壤微生物之一。AM真菌菌丝体网络是由AM真菌菌丝体在土壤生态系统中连接两株或两株以上植物根系所形成的菌丝体网络。随着菌根学研究的深入,如何直观的揭示AM真菌的生态学功能已经成为相关领域关注的热点问题。研究发现,利用同位素示踪技术可以开展AM真菌与宿主植物对土壤矿质营养的吸收、转运等方面的研究,以及菌丝体网络对不同宿主植物之间营养物质的分配研究和AM真菌在生态系统生态学中的功能研究。基于此,为了阐明同位素示踪技术在AM真菌研究中的价值,围绕菌根学最新研究进展,系统回顾了利用同位素示踪技术探究AM共生体对不同元素吸收和转运的机制、同位素示踪技术在AM真菌菌丝体网络研究中的价值和利用同位素示踪技术研究AM真菌在生态系统中的功能,为AM真菌生态学功能的研究提供理论基础,并对本领域未来的研究方向和应用前景进行展望。     

Potential analysis of grass endophytes Neotyphodium as biocontrol agents

Many grasses in the subfamily Pooideae develop symbioses with Neotyphodium fungal endophytes, which exist widely in nature. The stably symbiotic relationship not only ensures accessible nutrients required by Neotyphodium fungal endophytes, but also significantly increases the resistance of host grasses to biological stresses through the production of secondary metabolites. Previous studies show that infected grasses with endophytic fungi have prominently enhanced resistance to pests, plant diseases, companion plants and other biological stresses. Grass endophytic fungi show remarkable resistant to at least 79 species of pests from three classes; arachnida, nematode and insecta, and to at least 22 species of pathogenic fungi. Although the biotechnological application of endophytic fungi in grass breeding for variety selection and quality improvement has progressed well, opportunities remain for further exploring the use of fungal endophytes among different host grasses coupled with the examination of genetic stability of Neotyphodium in novel host grasses. In the future application of endophytic fungi as a bio-control method, researchers should not only consider specificities of host grasses, but also need to have comprehensive analysis and knowledge about the mutual relationships among grasses, endophytic fungi and ecological environments, which will help use endophytic fungi to better serve humanity.