Colorimetric Screening Method Research Articles

Enhanced biosynthesis of poly(3-hydroxybutyrate) in engineered strains of Pseudomonas putida via increased malonyl-CoA availability.

Malonyl-coenzyme A (CoA) is a key precursor for the biosynthesis of multiple value-added compounds by microbial cell factories, including polyketides, carboxylic acids, biofuels, and polyhydroxyalkanoates. Owing to its role as a metabolic hub, malonyl-CoA availability is limited by competition in several essential metabolic pathways. To address this limitation, we modified a genome-reduced Pseudomonas putida strain to increase acetyl-CoA carboxylation while limiting malonyl-CoA utilization. Genes involved in sugar catabolism and its regulation, the tricarboxylic acid (TCA) cycle, and fatty acid biosynthesis were knocked-out in specific combinations towards increasing the malonyl-CoA pool. An enzyme-coupled biosensor, based on the rppA gene, was employed to monitor malonyl-CoA levels invivo. RppA is a type III polyketide synthase that converts malonyl-CoA into flaviolin, a red-colored polyketide. We isolated strains displaying enhanced malonyl-CoA availability via a colorimetric screening method based on the RppA-dependent red pigmentation; direct flaviolin quantification identified four engineered strains had a significant increase in malonyl-CoA levels. We further modified these strains by adding a non-canonical pathway that uses malonyl-CoA as precursor for poly(3-hydroxybutyrate) biosynthesis. These manipulations led to increased polymer accumulation in the fully engineered strains, validating our general strategy to boost the output of malonyl-CoA-dependent pathways in P. putida.

Development of an oil-sealed anaerobic fermentation process for high production of γ-aminobutyric acid with Lactobacillus brevis isolated by directional colorimetric screening

γ-aminobutyric acid (GABA) is an important inhibitory neurotransmitter in the brain with many physiological functions. Therefore, this work establishes an efficient biosynthesis process to achieve the high production of food-grade GABA. Firstly, we adopted a directional colorimetric screening method and then obtained the strain Lactobacillus brevis (Lb. brevis) CGMCC 24975 with 22.61 g/L GABA in shake flasks, much higher than Lb. brevis ATCC 367 (3.07 g/L) due to its more efficient and stable expression of its glutamate decarboxylase (GAD) system. After exploring the effects of optimal fermentation conditions and oxygen on GABA production, we found that anaerobic fermentation could increase GABA production by up-regulating gad operon expression and activating GADase. Finally, we developed an oil-sealed anaerobic fermentation process with 157.90 g/L GABA in a 5 L bioreactor, which was 6.98-fold of that in shake flasks. This work includes a complete process from screening GABA-producing strains to developing efficient fermentation processes, laying the foundation for producing food-safe GABA.

Colorimetric High-Throughput Screening Assay to Engineer Fungal Peroxygenases for the Degradation of Thermoset Composite Epoxy Resins

At present, the end-of-life management of thermoset composite epoxy resins is limited to incineration and landfill storage, highlighting the demand for the development of more sustainable measures. Due to their broad spectrum of C-H oxyfunctionalization reactions, fungal unspecific peroxygenases (UPOs) are becoming important biotechnological tools in organic synthesis while their potential use in biodegradation processes should not be underestimated. Here, we present a colorimetric screening assay aimed at engineering UPOs for the degradation of epoxy resins. We based our study on Hexflow® RTM-6, a commercial epoxy resin used extensively in the aeronautics sector. UPO mutants from the short and long families were initially benchmarked by GC/MS to determine their potential N-dealkylation activity on N,N-bis(2-hydroxypropyl)-p-toluidine (NNBT), the main structural scaffold of Hexflow® RTM-6. A reliable high-throughput colorimetric screening method was developed to quantify the lactaldehyde released by UPO attack on the tertiary amine of NNBT. Based on an evolved UPO from Psathyrella aberdarensis that was expressed by yeast, a small subset of mutant libraries with different mutational loadings was constructed and screened for NNBT N-dealkylation, thereby establishing a directed evolution platform as a vehicle to engineer UPO composite degrading variants.

Involvement of NO3- in Ecophysiological Regulation of Dissimilatory Nitrate/Nitrite Reduction to Ammonium (DNRA) Is Implied by Physiological Characterization of Soil DNRA Bacteria Isolated via a Colorimetric Screening Method.

Dissimilatory nitrate/nitrite reduction to ammonium (DNRA) has recently regained attention as a nitrogen retention pathway that may potentially be harnessed to alleviate nitrogen loss resulting from denitrification. Until recently, the ecophysiology of DNRA bacteria inhabiting agricultural soils has remained largely unexplored, due to the difficulty in targeted enrichment and isolation of DNRA microorganisms. In this study, >100 DNRA bacteria were isolated from NO3--reducing anoxic enrichment cultures established with rice paddy soils using a newly developed colorimetric screening method. Six of these isolates, each assigned to a different genus, were characterized to improve the understanding of DNRA physiology. All the isolates carried nrfA and/or nirB, and the Bacillus sp. strain possessed a clade II nosZ gene conferring the capacity for N2O reduction. A common prominent physiological feature observed in the isolates was NO2- accumulation before NH4+ production, which was further examined with Citrobacter sp. strain DNRA3 (possessing nrfA and nirB) and Enterobacter sp. strain DNRA5 (possessing only nirB). Both isolates showed inhibition of NO2--to-NH4+ reduction at submillimolar NO3- concentrations and downregulation of nrfA or nirB transcription when NO3- was being reduced to NO2- In batch and chemostat experiments, both isolates produced NH4+ from NO3- reduction when incubated with excess organic electron donors, while incubation with excess NO3- resulted in NO2- buildup but no substantial NH4+ production, presumably due to inhibitory NO3- concentrations. This previously overlooked link between NO3- repression of NO2--to-NH4+ reduction and the C-to-N ratio regulation of DNRA activity may be a key mechanism underpinning denitrification-versus-DNRA competition in soil.IMPORTANCE Dissimilatory nitrate/nitrite reduction to ammonium (DNRA) is an anaerobic microbial pathway that competes with denitrification for common substrates NO3- and NO2- Unlike denitrification, which leads to nitrogen loss and N2O emission, DNRA reduces NO3- and NO2- to NH4+, a reactive nitrogen compound with a higher tendency to be retained in the soil matrix. Therefore, stimulation of DNRA has often been proposed as a strategy to improve fertilizer efficiency and reduce greenhouse gas emissions. Such attempts have been hampered by lack of insights into soil DNRA bacterial ecophysiology. Here, we have developed a new screening method for isolating DNRA-catalyzing organisms from agricultural soils without apparent DNRA activity. Physiological characteristics of six DNRA isolates were closely examined, disclosing a previously overlooked link between NO3- repression of NO2--to-NH4+ reduction and the C-to-N ratio regulation of DNRA activity, which may be a key to understanding why DNRA activity is rarely observed at substantial levels in nitrogen-rich agricultural soils.

Smartphone-based tape sensors for multiplexed rapid urinalysis

Recent advances in biofluids monitoring have positioned urine as a non-invasive candidate for clinical diagnosis. In this paper, we employ adhesive tapes with indicator-functionalized microarrays for rapid urinalysis. Such tape-based sensor combines advantages of flexibility, adhesive and waterproof functionality in a platform, which is simply prepared by physical punching, fabrics filling and indicators modification. The simulated urine flow can be captured and penetrated in the tape-based microarrays for rapid detection of multiplexed markers such as glucose, nitrite, protein and phenylpyruvate with a smartphone-assisted colorimetric screening method. As a user-friendly point-of-care testing (POCT) approach, such tape-based sensor is ideally favorable for the early disease warning toward the patients of urinary incontinence and infants.

Optimization and application of a low cost, colorimetric screening method for mercury in marine sediment.

A rapid, inexpensive, colorimetric screening method for mercury (Hg) has been optimized to provide a semi-quantitative measurement of Hg concentration in marine sediment within the range 0.038 to 1.5mgkg-1 encompassing the interim sediment quality guideline (ISQG) value of 0.13mgkg-1 (CCME 1999) and the probable effects level (PEL) of 0.7mgkg-1 for Hg in marine sediment (CCME 1999). Neither salinity (up to 41 practical salinity units (psu)) nor sediment organic matter (ΟΜ) content (up to 10%) affected the performance of the method. Accurate results were obtained for spike recovery experiments and analysis of certified reference material (CRM) BCR 580 Estuarine Sediment. The method was applied to sediment samples from Elefsina Bay, Greece. Screening results indicated Hg contamination in the bay, with concentrations exceeding the PEL value. Findings were confirmed by quantitative analysis of the samples by cold vapor atomic absorption spectrometry (CV-AAS), where results in the range 1.4-2.96mgkg-1 were determined.

Comparing different methods for fast screening of microbiological quality of beach sand aimed at rapid-response remediation

There is scientific evidence that beach sands are a significant contributor to the pathogen load to which visitors are exposed. To develop beach quality guidelines all beach zones must be included in microbiological evaluations, but monitoring methods for beach sand quality are relatively longstanding, expensive, laborious and require moderate laboratory infrastructure. This paper aimed to evaluate the microorganism activity in different beach zones applying and comparing a classical method of membrane filtration (MF) with two colorimetric screening methods based on fluorescein (FDA) and tetrazolium (TTC) salt biotransformation to evaluate a new rapid and low-cost method for beach sand microbiological contamination assessments. The colorimetric results can help beach managers to evaluate rapidly and at low cost the microbiological quality of different beach zones in order to decide whether remedial actions need to be adopted to prevent exposure of the public to microbes due to beach sand and/or water contamination.

Development of Rapid and Economical Colorimetric Screening Method for p-Phenylenediamine in Variety of Biological Matrices and its Application to Eleven Fatal Cases of p-Phenylenediamine Poisoning.

A rapid colorimetric method for detection of p-phenylenediamine (PPD) in various biological samples is developed. The o-cresol test for acetaminophen detection has been modified to detect PPD in blood, urine, gastric contents, and liver. After precipitating protein with trichloroacetic acid solution (2 mL, 10% w/v), biological specimens were required to convert PPD metabolites to PPD by acid hydrolysis. Finally, o-cresol solution (1 mL, 1% w/v), hydrogen peroxide (200 μL, 3%v/v), and concentrated ammonium hydroxide (0.5 mL) were added in the biological samples. The presence of PPD was indicated by formation of violet color which was turned to bluish green color within 10-15 min. The limit of detection was found to be 2 mg/L in blood, urine, and gastric contents and 2 mg/Kg in liver. This method is also free from any potential interference by p-aminophenol, acetaminophen, and other amine drugs under test conditions. This method was successfully employed to thirteen fatal cases of PPD poisoning.

A screening method of uranyl extractants in H2SO4 solutions

A UV–Vis spectrophotometric and colorimetric screening method was developed for the selection of powerful and selective ligands for the uranyl ion in sulfuric acid media. The coordinating properties of a series of molecules (mono and bisphosphonate) toward UO22+ were examined through ligand displacement reactions observed by UV–visible from the {ArzH4–UO2}2− reference complex where Na2ArzH6 is a chromogenic molecule displaying a strong affinity for uranium. Uranium/iron selectivity was evaluated by recovering the screening data from two independent sets of experiments using two different chromogenic chelates: arsenazo III for uranium(VI) and BrPADAP for iron(III). As a result, two bisphosphonate ligands among a set of more than 50 phosphonate molecules were found to display a good affinity for UO22+ and relevant U/Fe selectivity at pH=2.1.

A rapid colorimetric screening method for vanillic acid and vanillin-producing bacterial strains

To isolate a bacterial strain capable of biotransforming ferulic acid, a major component of lignin, into vanillin and vanillic acid by a rapid colorimetric screening method. For the production of vanillin, a natural aroma compound, we attempted to isolate a potential strain using a simple screening method based on pH change resulting from the degradation of ferulic acid. The strain Pseudomonas sp. AZ10 UPM exhibited a significant result because of colour changes observed on the assay plate on day 1 with a high intensity of yellow colour. The biotransformation of ferulic acid into vanillic acid by the AZ10 strain provided the yield (Yp/s ) and productivity (Pr ) of 1·08 mg mg(-1) and 53·1 mg L(-1) h(-1) , respectively. In fact, new investigations regarding lignin degradation revealed that the strain was not able to produce vanillin and vanillic acid directly from lignin; however, partially digested lignin by mixed enzymatic treatment allowed the strain to produce 30·7 mg l(-1) and 1·94 mg l(-1) of vanillic acid and biovanillin, respectively. (i) The rapid colorimetric screening method allowed the isolation of a biovanillin producer using ferulic acid as the sole carbon source. (ii) Enzymatic treatment partially digested lignin, which could then be utilized by the strain to produce biovanillin and vanillic acid. To the best of our knowledge, this is the first study reporting the use of a rapid colorimetric screening method for bacterial strains producing vanillin and vanillic acid from ferulic acid.

A Plasmodium falciparum screening assay for anti-gametocyte drugs based on parasite lactate dehydrogenase detection

Plasmodium gametocytes, responsible for malaria parasite transmission from humans to mosquitoes, represent a crucial target for new antimalarial drugs to achieve malaria elimination/eradication. We developed a novel colorimetric screening method for anti-gametocyte compounds based on the parasite lactate dehydrogenase (pLDH) assay, already standardized for asexual stages, to measure gametocyte viability and drug susceptibility. Gametocytogenesis of 3D7 and NF54 Plasmodium falciparum strains was induced in vitro and asexual parasites were depleted with N-acetylglucosamine. Gametocytes were treated with dihydroartemisinin, epoxomicin, methylene blue, primaquine, puromycin or chloroquine in 96-well plates and the pLDH activity was evaluated using a modified Makler protocol. Mosquito infectivity was measured by the standard membrane feeding assay (SMFA). A linear correlation was found between gametocytaemia determined by Giemsa staining and pLDH activity. A concentration-dependent reduction in pLDH activity was observed after 72 h of drug treatment, whereas an additional 72 h of incubation without drugs was required to obtain complete inhibition of gametocyte viability. SMFA on treated and control gametocytes confirmed that a reduction in pLDH activity translates into reduced oocyst development in the mosquito vector. The gametocyte pLDH assay is fast, easy to perform, cheap and reproducible and is suitable for screening novel transmission-blocking compounds, which does not require parasite transgenic lines.

Enzymatic production of 2-amino-2,3-dimethylbutyramide by cyanide-resistant nitrile hydratase

A novel enzymatic route for the synthesis of 2-amino-2,3-dimethylbutyramide (ADBA), important intermediate of highly potent and broad-spectrum imidazolinone herbicides, from 2-amino-2,3-dimethylbutyronitrile (ADBN) was developed. Strain Rhodococcus boritolerans CCTCC M 208108 harboring nitrile hydratase (NHase) towards ADBN was screened through a sophisticated colorimetric screening method and was found to be resistant to cyanide (5 mM). Resting cells of R. boritolerans CCTCC M 208108 also proved to be tolerant against high product concentration (40 g l(-1)) and alkaline pH (pH 9.3). A preparative scale process for continuous production of ADBA in both aqueous and biphasic systems was developed and some key parameters of the biocatalytic process were optimized. Inhibition of NHase by cyanide dissociated from ADBN was successfully overcome by temperature control (at 10°C). The product concentration, yield and catalyst productivity were further improved to 50 g l(-1), 91% and 6.3 g product/g catalyst using a 30/70 (v/v) n-hexane/water biphasic system. Furthermore, cells of R. boritolerans CCTCC M 208108 could be reused for at lease twice by stopping the continuous reaction before cyanide concentration rose to 2 mM, with the catalyst productivity increasing to 12.3 g product/g catalyst. These results demonstrated that enzymatic synthesis of ADBA using whole cells of R. boritolerans CCTCC M 208108 showed potential for industrial application.

Ferrous and ferric ions-based high-throughput screening strategy for nitrile hydratase and amidase

Rapid and direct screening of nitrile-converting enzymes is of great importance in the development of industrial biocatalytic process for pharmaceuticals and fine chemicals. In this paper, a combination of ferrous and ferric ions was used to establish a novel colorimetric screening method for nitrile hydratase and amidase with α-amino nitriles and α-amino amides as substrates, respectively. Ferrous and ferric ions reacted sequentially with the cyanide dissociated spontaneously from α-amino nitrile solution, forming a characteristic deep blue precipitate. They were also sensitive to weak basicity due to the presence of amino amide, resulting in a yellow precipitate. When amino amide was further hydrolyzed to amino acid, it gave a light yellow solution. Mechanisms of color changes were further proposed. Using this method, two isolates with nitrile hydratase activity towards 2-amino-2,3-dimethyl butyronitrile, one strain capable of hydrating 2-amino-4-(hydroxymethyl phosphiny) butyronitrile and another microbe exhibiting amidase activity against 2-amino-4-methylsulfanyl butyrlamide were obtained from soil samples and culture collections of our laboratory. Versatility of this method enabled it the first direct and inexpensive high-throughput screening system for both nitrile hydratase and amidase.

Specific screening method for dextran and hydroxyethyl starch in human urine by size exclusion chromatography–in-source collision-induced dissociation–time-of-flight mass spectrometry

The use of plasma volume expanders (PVE), such as dextran (DEX) and hydroxyethyl starch (HES), is prohibited in sports. DEX is a naturally occurring glucose polymer, whereas HES is synthetically produced from amylopectin starch by substitution with hydroxyethyl groups. In doping control, the commonly applied enzymatic and colorimetric screening methods are lacking adequate specificity for DEX and HES. Also, gas chromatographic-mass spectrometic (GC-MS) screening methods have specificity issues with DEX. In addition, due to the nature of the target compounds, time-consuming derivatisation steps are required in GC-MS. Based on the high molecular weight of carbohydrate polymers excreted in urine after administration of DEX and HES, a screening method was developed involving size exclusion chromatography (SEC) combined with time-of-flight mass spectrometry (TOFMS). By using solely a SEC guard column as an analytical column allowed sufficient chromatographic resolution in a minimal amount of time and with reasonable repeatability (average RSD of 10%). Detector response was linear throughout the measurement range with R(2) > 0.99 for both analytes. Limits of detection were 100 and 250 μg mL(-1) for DEX and HES, respectively. Ion suppression was found to be 52% at maximum. In-source collision-induced dissociation (ISCID) was used to produce characteristic fragments at a mass accuracy better than 2 mDa. The specificity of the SEC-ISCID-TOFMS method was demonstrated with 120 PVE negative doping control samples analyzed in parallel with a routine GC-MS screening method. In addition, seven urine samples from diabetic athletes, causing interpretation problems in routine GC-MS, showed here a definitely negative profile.

Screening and confirmatory analysis of glyoxylate: A biomarker of plants resistance against herbicides

The evidence that glyoxylate is a biomarker of tolerance or susceptibility to the action of herbicides belonging to the glycine family makes necessary to develop simple methods for the determination of this metabolite. Glyoxylate level allows both to know the presence/absence of members of the glycine family in plants and plant response to these herbicides. With this aim, a colorimetric-screening method has been developed for determination of glyoxylate based on formation of a phenylhydrazone, then oxidised to red coloured 1,5-diphenylformazan. Simultaneous optimization of ultrasound-assisted extraction of glyoxylate from plants and derivatization by a multivariate design has allowed the determination of the target analyte in fresh plants without interferences from pheophytines and compounds with carbonyl groups. Limits of detection and quantification are 0.05 μg ml −1 and 0.17 μg ml −1, respectively, with precision, expressed as relative standard deviation, of 3.3% for repeatability and 5.6% for the within-day laboratory reproducibility. Only 50 mg of plant is necessary for determination of glyoxylate within 32 min. Confirmatory analysis by capillary electrophoresis-diode array detection in samples of Lolium spp. subjected to treatment with glyphosate shows that the relative error of the proposed method is always lower than 7%.

A pH‐Based High‐Throughput Screening of Sucrose‐Utilizing Transglucosidases for the Development of Enzymatic Glucosylation Tools

AbstractSucrose‐utilizing transglucosidases are valuable enzymatic tools for the diversification of carbohydrate‐based molecules. Among them, recombinant amylosucrase from Neisseria polysaccharea is a glucansucrase that naturally catalyzes the synthesis of an amylose‐like polymer as well as the transglucosylation of exogenous hydroxylated acceptors. A semirational engineering approach was recently undertaken to redesign the enzyme active site and adapt it to the glucosylation of a nonnatural acceptor, allyl 2‐N‐acetyl‐2‐deoxy‐α‐D‐glucopyranoside (α‐D‐GlcpNAcOAll), to produce a key building block in the chemoenzymatic synthesis of Shigella flexneri 1b serotype O‐antigen repeating unit. This prior work shows the beneficial effect of single amino acid mutations at two positions (228 and 290) on the recognition of the acceptor by amylosucrase. On the basis of these first results, a library of about 8000 amylosucrase variants combining mutations at these two positions is constructed by saturation mutagenesis. The library is prescreened using a novel pH‐sensitive colorimetric screening method for the detection of sucrose‐utilizing amylosucrase variants, thereby reducing by about 95 % the size of the library to be subsequently screened for acceptor glucosylation. Active clones (5 % of the initial library) are then screened for acceptor recognition, leading to the isolation of 20 variants of potential interest for the production of the target disaccharide α‐D‐Glcp‐(1→4)‐α‐D‐GlcpNAc.

Preliminary screening method for the determination of inorganic arsenic in urine

A simple and rapid method was developed for the routine determination and classification of inorganic arsenic based on its clinical and forensic properties. Inorganic arsenic was isolated from urine by using copper granules, which was then made to react with ammonium molybdate in order to detect its presence with the naked eye. Based on studies of extraction and reaction conditions, e.g., reaction temperature and time, a colorimetric screening method was established. The reaction mixture was measured by a spectrophotometer, and there was linearity from 0.05 to 2.0 μg/ml and the correlation coefficients of the calibration curves were greater than 0.99. The coefficients of intra-day variation at 0.2 and 2.0 μg/ml of inorganic arsenic in urine were 9.6 and 4.2%, respectively (n = 5). The minimum detectable level in urine is 0.03 μg/ml, and it is possible to detect the lowest level of poisoning according to the published reports. The proposed method was applied to a poisoning case wherein the patient ingested NEOARSEN BLACK® with alcohol, which contained 45% of arsenic trioxide. This method produced positive results in all the urine samples tested, and this method is useful for the screening of inorganic arsenic based on its clinical properties because it enables the detection of inorganic arsenic in urine without expensive equipment.

R-enantioselective hydrolysis of 2,2-dimethylcyclopropanecarboxamide by amidase from a newly isolated strainBrevibacterium epidermidisZJB-07021

To isolate new micro-organisms with R-stereospecific amidase activity and to examine their potential as biocatalysts in enantioselective hydrolysis of 2,2-dimethylcyclopropanecarboxamide (1). A novel R-stereospecific amidase-producing strain ZJB-07021 was isolated through a sophisticated colorimetric screening method. Based on morphology, physiological tests, Biolog system (GP2) and 16S rRNA sequence, the new isolate was identified as Brevibacterium epidermidis. After 70 min of bioconversion at 35 degrees C, kinetic resolution of (R,S)-1 by the amidase afforded (S)-1 in 41.1% yield (>99% ee) and (R)-2 in 49.9% yield (69.7% ee) with an average E-value of 23. The enantioselectivity was found to be temperature dependent and enhanced from 12.6 at 45 degrees C to 65.9 at 14 degrees C. A novel bacterial strain of B. epidermidis ZJB-07021 producing R-stereospecific amidase was isolated and characterized. The isolate exhibited high E values for kinetic resolution of racemic-1 to (S)-1. To our knowledge, this was the first report on the species B. epidermidis that harboured R-stereospecific amidase. Strain ZJB-07021 could be further improved as a suitable biocatalyst for the stereoselective bioconversion of racemic-1 after optimization of culture and biotransformation process.

Expression in yeast of secreted lignin peroxidase with improved 2,4-dichlorophenol degradability by DNA shuffling

Lignin peroxidase (LiP) from Phanerochaete chrysosporium was shown to mineralize a variety of recalcitrant aromatic compounds and oxidize a number of polycyclic aromatic and phenolic compounds. The major problem of the wild type LiP is that it can be inactivated by excess H 2O 2 and high concentrations of aromatic compounds. We applied a directed evolution technique coupled with a rapid colorimetric screening method to obtain mutant genes with improved H 2O 2 stability and polychlorinated phenol degradability, and they were successfully expressed as the secretive LiPs in recombinant Saccharomyces cerevisiae. The resulting variants showed approximately 1.6-fold improved 2,4-dichlorophenol (2,4-DCP) degradation activity and stability against H 2O 2 compared with the parent strain. The kinetic properties of the variants toward 2,4-DCP and H 2O 2 were also increased compared with the wild type for all three mutants studied. Amino acid sequence analysis indicated that the greatest number of amino acid substitutions was located near the surface or Ca 2+ binding sites of the enzyme.

Functionality improvement of fungal lignin peroxidase by DNA shuffling for 2,4-dichlorophenol degradability and H 2O 2 stability

One of the major problems of wild-type lignin peroxidase (LiP) is its inactivity at the presence of excess H 2O 2 and high concentration of aromatic compounds. Little is known about the substrate-binding site of LiP, and functionality improvement of LiP was not actively tried by genetic engineering and directed evolution. In order to improve LiPs functionality, we performed directed evolution with a colorimetric screening method. Finally, three types of LiP mutants were screened. The catalytic efficiency of the variants toward 2,4-dichlorophenol (DCP) degradation activity and the stability against H 2O 2 was increased over the wild type. The K m value of the variants toward H 2O 2 was increased, but K m value toward 2,4-DCP degradation was reduced. Overall, The K cat/ K m values of the mutants toward 2,4-DCP was increased ca. 4-fold, and that toward H 2O 2 was increased ca. 89-fold. Amino acid sequence analysis indicated that the most of the mutations were located on the enzyme surface. We expect that these results coupled with recombining mutation can be successfully applied to the molecular evolution cycles for screening of LiPs and other oxidative enzymes with improved functionality and stability.