Microbial degradation of phenol and phenolic derivatives

Phenol and its derivatives are one of the largest groups of environmental pollutants due to their presence in many industrial effluents and broad application as antibacterial and antifungal agents. A number of microbial species possess enzyme systems that are applicable for the decomposition of various aliphatic and aromatic toxic compounds. Intensive efforts to screen species with high‐degradation activity are needed to study their capabilities of degrading phenol and phenolic derivatives. Most of the current research has been directed at the isolation and study of microbial species of potential ecological significance. In this review, some of the best achievements in degrading phenolic compounds by bacteria and yeasts are presented, which draws attention to the high efficiency of strains of Pseudomonas, Candida tropicalis, Trichosporon cutaneum, etc. The unique ability of fungi to maintain their degradation potential under conditions unfavorable for other microorganisms is outstanding. Mathematical models of the microbial biodegradation dynamics of single and mixed aromatic compounds, which direct to the benefit of the processes studied in optimization of modern environmental biotechnology are also presented.     

基于酶电极的乳酸检测生物传感器

乳酸(C₃H₆O₃),又名2-羟基丙酸、丙醇酸,属于羟基酸的一种。乳酸在食品工业、临床医学、生物技术等行业具有极其重要的意义,因此如何高通量检测不同样品中的乳酸成为目前业界研究的重点。传统乳酸检测方法操作繁琐、费时费力或需要昂贵的检测设备,乳酸生物传感器可以克服这些限制,不需要样品制备,能够快速、简便、可靠地定量测定食品或血浆中的乳酸,具有广阔的应用前景。乳酸酶电极生物传感器主要有两种类型——基于L-乳酸氧化酶(L-LOD)和L-乳酸脱氢酶(L-LDH)的乳酸生物传感器。本文综述了L-LOD和L-LDH结构特征、来源及催化机理,讨论了改善基于酶电极的乳酸传感器性能的3种策略(电极材料改造策略、酶固定化策略、酶分子工程改造策略),还根据用于制造乳酸生物传感器的不同载体包括膜、透明凝胶基质、水凝胶载体、纳米颗粒等对乳酸生物传感器进行了归类分析,最后本文将目前商品化应用的酶电极乳酸生物传感器特点进行了对比总结讨论,阐述了乳酸生物传感器的未来应用方向,并对未来发展前景进行了展望。     

Aptamers for pharmaceuticals and their application in environmental analytics

Aptamers are single-stranded DNA or RNA oligonucleotides, which are able to bind with high affinity and specificity to their target. This property is used for a multitude of applications, for instance as molecular recognition elements in biosensors and other assays. Biosensor application of aptamers offers the possibility for fast and easy detection of environmental relevant substances. Pharmaceutical residues, deriving from human or animal medical treatment, are found in surface, ground, and drinking water. At least the whole range of frequently administered drugs can be detected in noticeable concentrations. Biosensors and assays based on aptamers as specific recognition elements are very convenient for this application because aptamer development is possible for toxic targets. Commonly used biological receptors for biosensors like enzymes or antibodies are mostly unavailable for the detection of pharmaceuticals. This review describes the research activities of aptamer and sensor developments for pharmaceutical detection, with focus on environmental applications.     

酚酸类物质的化感作用研究进展

酚酸类物质是普遍存在于高等植物组织并与植物生长密切相关的次级代谢产物。几十年来,人们对酚酸类化合物的认识逐渐加深,但关于其在生物学、生态学以及农业上的作用机制仍不是很清楚。因此,进一步了解这些生物分子将有助于生态系统的维持与保护。重点介绍了酚酸类物质的来源及化感作用,微生物对酚酸类物质的降解机理,代谢途径及相应分子水平的研究,指出了酚酸类物质研究中存在的问题,同时展望了酚酸类物质的研究方向与前景。     

Enzyme inhibition-based biosensors for food safety and environmental monitoring

Analytical technology based on sensors is an extremely broad field which impacts on many major industrial sectors such as the pharmaceutical, healthcare, food, and agriculture industries as well as environmental monitoring. This review will highlight the research carried out during the last 5 years on biosensors that are based on enzyme inhibition for determination of pollutants and toxic compounds in a wide range of samples. Here the different enzymes implicated in the inhibition, different transducers forming the sensing devices, and the different contaminants analyzed are considered. The general application of the various biosensors developed, with emphasis on food and environmental applications, is reviewed as well as the general approaches that have been used for enzyme immobilization, the enzyme catalysis, and the inhibition mechanism.     

Bacterial tyrosinases and their applications

Tyrosinases with different physico-chemical properties have been identified from various bacterial phyla such as Actinobacteria and Proteobacteria and their production is often inducible by environmental stresses. Tyrosinases are enzymes catalysing the oxidation of mono- and di-phenolic compounds to corresponding quinones with the concomitant reduction of molecular oxygen to water. Since the quinone produced can further react non-enzymatically with other nucleophiles, e.g. amino groups, many tyrosinases have a recorded cross-linking activity on proteins. Various bacterial tyrosinases oxidise tyrosine, catechol, l/d-DOPA, caffeic acid and polyphenolic substrates such as catechins. This substrate specificity has been exploited to engineer biosensors able to detect even minimal amounts of different phenolic compounds. The physiological role of tyrosinases in the biosynthesis of melanins has been used for the production of coloured and dyeing agents. Moreover, the cross-linking activity of tyrosinases has found application in food processing and in the functionalisation of materials. Numerous tyrosinases with varying substrate specificities and stability features have been isolated from bacteria and they can constitute valuable alternatives to the well-studied tyrosinase from common mushroom.     

Biodegradation of phenol and its derivatives by engineered bacteria: current knowledge and perspectives

Biodegradation of phenolic compounds is a promising alternative to physical and chemical methods used to remove these toxic pollutants from the environment. The ability of various microorganisms to metabolize phenol and its derivatives (alkylphenols, nitrophenols and halogenated derivatives) has therefore been intensively studied. Knowledge of the enzymes catalyzing the individual reactions, the genes encoding these enzymes and the regulatory mechanisms involved in the expression of the respective genes in bacteria serves as a basis for the development of more efficient degraders of phenols via genetic engineering methods. Engineered bacteria which efficiently degrade phenolic compounds were constructed in laboratories using various approaches such as cloning the catabolic genes in multicopy plasmids, the introduction of heterologous genes or broadening the substrate range of key enzymes by mutagenesis. Efforts to apply the engineered strains in in situ bioremediation are problematic, since engineered strains often do not compete successfully with indigenous microorganisms. New efficient degraders of phenolic compounds may be obtained by complex approaches at the organism level, such as genome shuffling or adaptive evolution. The application of these engineered bacteria for bioremediation will require even more complex analysis of both the biological characteristics of the degraders and the physico-chemical conditions at the polluted sites.     

DNA生物传感器在环境污染监测中的应用

基于生物催化和免疫原理的生物传感器在环境领域中获得了广泛应用.近年来,随着分子生物学和生物技术的发展,人们开发了以核酸探针为识别元件,基于核酸相互作用原理的DNA生物传感器.该传感器可用于受感染微生物的核酸序列分析、优先控制污染物的检测以及污染物与DNA之间相互作用的研究,在环境污染监测中具有潜在的巨大应用前景.简要介绍了核酸杂交生物传感器的基本原理及其在环境微生物和优先控制污染物（priority pollutant）检测中的应用研究进展.     

Effects of phenol and natural phenolic compounds on biofilm formation by Pseudomonas aeruginosa

A biofilm is formed as a result of adhesion of microorganisms to various surfaces with the production of extracellular polymers (polysaccharides and proteins). Biofilms cause serious problems in the chemical, medical and pharmaceutical industries. Recent findings indicate that some natural phenolic compounds found in plants have an anti-biofouling effect on biofilm formation by Gram-negative bacteria. The anti-biofouling activities of 14 selected phenol and natural phenolic compounds were tested against Pseudomonas aeruginosa, using a microtiter-plate. A modified microtiter-plate assay was used because it enabled indirect measurement of bacterial cells attached to the surface of the wells. This assay involved fixing the bacterial film with methanol, staining with crystal violet dye and then releasing the bound dye with 33% glacial acetic acid. The optical density (OD) of the solution was measured at 570 nm by using an automated ICN Flow Titertek Multiscan Plus reader. Phenol and natural phenolic compounds except ethyl linoleate and tocopherol showed a significant reduction in biofilm formation by P. aeruginosa.     

Cytochrome P450 (CYP) enzymes and the development of CYP biosensors

Cytochrome P450s (CYPs) are a large family of heme-containing monooxygenase enzymes involved in the first-pass metabolism of drugs and foreign chemicals in the body. CYP reactions, therefore, are of high interest to the pharmaceutical industry, where lead compounds in drug development are screened for CYP activity. CYP reactions in vivo require the cofactor NADPH as the source of electrons and an additional enzyme, cytochrome P450 reductase (CPR), as the electron transfer partner; consequently, any laboratory or industrial use of CYPs is limited by the need to supply NADPH and CPR. However, immobilizing CYPs on an electrode can eliminate the need for NADPH and CPR provided the enzyme can accept electrons directly from the electrode. The immobilized CYP can then act as a biosensor for the detection of CYP activity with potential substrates, albeit only if the immobilized enzyme is electroactive. The quest to create electroactive CYPs has led to many different immobilization strategies encompassing different electrode materials and surface modifications. This review focuses on different immobilization strategies that have been used to create CYP biosensors, with particular emphasis on mammalian drug-metabolizing CYPs and characterization of CYP electrodes. Traditional immobilization methods such as adsorption to thin films or encapsulation in polymers and gels remain robust strategies for creating CYP biosensors; however, the incorporation of novel materials such as gold nanoparticles or quantum dots and the use of microfabrication are proving advantageous for the creation of highly sensitive and portable CYP biosensors.     

Progress in enzyme inhibition based detection of pesticides

The previous few decades have seen the development of biosensors and their use in monitoring of pesticides in food and environmental samples. Although inhibition‐based biosensors have been subject of several recent research works, their performance characteristics greatly depend on the type of immobilization and the presence of interfering compounds in the samples. Moreover, sensitivity, detection limits, and rapidity of the response are few of the other major features that need to be investigated further if they are to become operationally user‐friendly. This review will highlight research carried out in the past on biosensors that are based on enzyme inhibition for determination of organophosphorus compounds and carbamate pesticides.     

New redox mediator-modified polysulfone composite films for the development of dehydrogenase-based biosensors

This work presents polysulfone membranes as new materials for the development of compact dehydrogenase-based biosensors. Composite films were prepared by mixing polysulfone with graphite and were deposited on epoxy-graphite composite electrodes. Redox mediators were successfully immobilized in the composite film leading to highly reproducible biosensors, without leakage of the immobilized species. This results in a more reliable analytical system as, at the same time, problems of electrode fouling related to the detection of the coenzyme nicotinamide adenine dinucleotide (NADH) on which is based the amperometric detection of dehydrogenase-based biosensors are avoided. Scanning electron microscopy was used to study the morphological characteristics of the surface and the cross-section of the polysulfone-graphite composite films. Several procedures to immobilize enzymes in these membranes were demonstrated. Glutamate dehydrogenase (GlDH) was immobilized as an example of dehydrogenase enzyme, in this case for the development of an ammonium biosensor. High sensitivity, good selectivity, wide linear ranges and short response times were obtained for the optimized sensors and biosensors. Their good performance combined with the simplicity of the construction method, make the polysulfone-graphite composite films attractive matrices for the development of new enzyme-based biosensors, especially those based on dehydrogenase enzymes.     

Phenol degradation by halophilic bacteria isolated from hypersaline environments

Phenol is a toxic aromatic compound used or produced in many industries and as a result a common component of industrial wastewaters. Phenol containing waste streams are frequently hypersaline and therefore require halophilic microorganisms for efficient biotreatment without dilution. In this study three halophilic bacteria isolated from different saline environments and identified as Halomonas organivorans, Arhodomonas aquaeolei and Modicisalibacter tunisiensis were shown to be able to grow on phenol in hypersaline media containing 100 g/L of total salts at a concentration of 3 mM (280 mg/L), well above the concentration found in most waste streams. Genes encoding the aromatic dioxygenase enzymes catechol 1,2 dioxygenase and protocatechuate 3,4-dioxygenase were present in all strains as determined by PCR amplification using primers specific for highly conserved regions of the genes. The gene for protocatechuate 3,4-dioxygenase was cloned from the isolated H. organivorans and the translated protein was evaluated by comparative protein sequence analysis with protocatechuate 3,4-dioxygenase proteins from other microorganisms. Although the analysis revealed a wide range of sequence divergence among the protocatechuate 3,4-dioxygenase family, all of the conserved domain amino acid structures identified for this enzyme family are identical or conservatively substituted in the H. organivorans enzyme.     

Electrochemical biosensing of pesticide residues based on affinity biocomposite platforms

A novel and very sensitive electrochemical immunosensing strategy for the detection of atrazine based on affinity biocomposite transducers is presented. Firstly, the graphite-epoxy composite transducer was bulk-modified with different universal affinity biomolecules, such as avidin and Protein A. Two strategies for the immobilization of the anti-atrazine antibodies on both biocomposite transducers were evaluated: 'wet-affinity' and 'dry-assisted affinity' immobilization. Finally, the performance of a novel anti-atrazine immunocomposite bulk-modified with anti-atrazine antibodies was also evaluated. The better immobilization performance of the anti-atrazine antibodies was achieved by 'dry-assisted affinity' immobilization on Protein A (2%) graphite-epoxy biocomposite (ProtA(2%)-GEB) as a transducer. The immunological reaction for the detection of atrazine performed on the ProtA(2%)-GEB biosensors is based on a direct competitive assay using atrazine-HRP tracer as the enzymatic label. The electrochemical detection is thus achieved through a suitable substrate and a mediator for the enzyme HRP. This novel strategy was successfully evaluated using spiked orange juice samples. The detection limit for atrazine in orange juices using the competitive electrochemical immunosensing assay was found to be 6 x 10(-3) microgL-1 (0.03 nmolL-1) thus this biosensing method accomplishes by far the LODs required for the European Community directives for potable water and food samples (0.1 microgL-1). This strategy offers great promise for rapid, simple, cost effective, and on-site biosensing of biological, food, and environmental samples.     

Bioengineering of Nitrilases Towards Its Use as Green Catalyst: Applications and Perspectives

Nitrilases are commercial biocatalysts used for the synthesis of plastics, paints, fibers in the chemical industries, pharmaceutical drugs and herbicides for agricultural uses. Nitrilase hydrolyses the nitriles and dinitriles to their corresponding carboxylic acids and ammonia. They have a broad range of substrate specificities as well as enantio-, regio- and chemo-selective properties which make them useful for biotransformation of nitriles to important compounds because of which they are considered as ‘Green Catalysts’. Nitriles are widespread in nature and synthesized as a consequence of anthropogenic and biological activities. These are also present in certain plant species and are known to cause environmental pollution. Biotransformation using native organisms as catalysts tends to be insufficient since the enzyme of interest has very low amount in the total cellular protein, rate of reaction is slow along with the instability of enzymes. Therefore, to overcome these limitations, bioengineering offers an alternative approach to alter the properties of enzymes to enhance the applicability and stability. The present review highlights the aspects of producing the recombinant microorganisms and overexpressing the enzyme of interest for the enhanced stability at high temperatures, immobilization techniques, extremes of pH, organic solvents and hydrolysing dintriles to chiral compounds which may enhance the possibilities for creating specific enzymes for biotransformation.     

Low potential detection of glutamate based on the electrocatalytic oxidation of NADH at thionine/single-walled carbon nanotubes composite modified electrode

A glutamate biosensor based on the electrocatalytic oxidation of reduced nicotinamide adenine dinucleotide (NADH), which was generated by the enzymatic reaction, was developed via employing a single-walled carbon nanotubes/thionine (Th-SWNTs) nanocomposite as a mediator and an enzyme immobilization matrix. The biosensor, which was fabricated by immobilizing glutamate dehydrogenase (GlDH) on the surface of Th-SWNTs, exhibited a rapid response (ca. 5s), a low detection limit (0.1 microM), a wide and useful linear range (0.5-400 microM), high sensitivity (137.3+/-15.7) microA mM(-1)cm(-2), higher biological affinity, as well as good stability and repeatability. In addition, the common interfering species, such as ascorbic acid, uric acid, and 4-acetamidophenol, did not cause any interference due to the use of a low operating potential (190 mV vs. NHE). The biosensor can be used to quantify the concentration of glutamate in the physiological level. The Th-SWNTs system represents a simple and effective approach to the integration of dehydrogenase and electrodes, which can provide analytical access to a large group of enzymes for wide range of bioelectrochemical applications including biosensors and biofuel cells.     

Bioimprinting strategies: From soft lithography to biomimetic sensors and beyond

Imprinting is a straightforward, yet a reliable technique to develop dynamic artificial recognition materials—so called as synthetic antibodies. Surface imprinting strategies such as soft lithography allow biological stereotyping of polymers and sol–gel phases to prepare extremely selective receptor layers, which can be combined with suitable transducer systems to develop high performance biomimetic sensors. This article presents an overview of the remarkable technical advancements in the field of surface bioimprinting with particular emphasis on surface imprinted bioanalyte detection systems and their applications in rapid bioanalysis and biotechnology. Herein, we discuss a variety of surface imprinting strategies including soft lithography, template immobilization, grafting, emulsion polymerization, and others along with their biomimetic sensor applications, merits and demerits. The pioneering research works on surface patterned biosensors are described with selected examples of detecting biological agents ranging from small biomolecules and proteins to living cells and microorganisms.     

The Sonogel-Carbon materials as basis for development of enzyme biosensors for phenols and polyphenols monitoring: a detailed comparative study of three immobilization matrixes

Three amperometric biosensors based on immobilization of tyrosinase on a new Sonogel-Carbon electrode for detection of phenols and polyphenols are described. The electrode was prepared using high energy ultrasounds (HEU) directly applied to the precursors. The first biosensor was obtained by simple adsorption of the enzyme on the Sonogel-Carbon electrode. The second and the third ones, presenting sandwich configurations, were initially prepared by adsorption of the enzyme and then modification by mean of polymeric membrane such as polyethylene glycol for the second one, and the ion-exchanger Nafion in the case of the third biosensor. The optimal enzyme loading and polymer concentration, in the second layer, were found to be 285 U and 0.5%, respectively. All biosensors showed optimal activity at the following conditions: pH 7, -200 mV, and 0.02 mol l(-1) phosphate buffer. The response of the biosensors toward five simple phenols derivatives and two polyphenols were investigated. It was found that the three developed tyrosinase Sonogel-Carbon based biosensors are in satisfactory competitiveness for phenolic compounds determination with other tyrosinase based biosensors reported in the literature. The detection limit, sensitivity, and the apparent Michaelis-Menten constant K(m)(app) for the Nafion modified biosensor were, respectively, 0.064, 0.096, and 0.03 micromol, 82.5, 63.4, and 194 nA micromol(-1)l(-1), and 67.1, 54.6, and 12.1 micromol l(-1) for catechol, phenol, and 4-chloro-3-methylphenol. Hill coefficient values (around 1 for all cases), demonstrated that the immobilization method does not affect the nature of the enzyme and confirms the biocompatibility of the Sonogel-Carbon with the bioprobe. An exploratory application to real samples such as beers, river waters and tannery wastewaters showed the ability of the developed Nafion/tyrosinase/Sonogel-Carbon biosensor to retain its stable and reproducible response.     

Gluconobacter in biosensors: applications of whole cells and enzymes isolated from gluconobacter and acetobacter to biosensor construction

Bacteria belonging to the genus Acetobacter and Gluconobacter, and enzymes isolated from them, have been extensively used for biosensor construction in the last decade. Bacteria used as a biocatalyst are easy to prepare and use in amperometric biosensors. They contain multiple enzyme activities otherwise not available commercially. The range of compounds analyzable by Gluconobacter biosensors includes: mono- and poly-alcohols, multiple aldoses and ketoses, several disaccharides, triacylglycerols, and complex parameters like utilizable saccharides or biological O₂ demand. Here, the recent trends in Gluconobacter biosensors and current practical applications are summarized. An erratum to this article can be found at