Screening Of Enzyme Activity Research Articles

Improving Hydrolytic Activity and Enantioselectivity of Epoxide Hydrolase from Phanerochaete chrysosporium by Directed Evolution.

Epoxide hydrolases (EHs) catalyze the conversion of epoxides into vicinal diols. The epoxide hydrolase gene from P. chrysosporium was previously cloned and subjected to site-directed mutation to study its enzyme activity, but the results were unsatisfactory. This study used error prone PCR and DNA shuffling to construct a PchEHA mutation library. We performed mutation-site combinations on PchEHA based on enzyme activity measurement results combined with directed evolution technology. More than 15,000 mutants were randomly selected for the preliminary screening of PchEHA enzyme activity alongside 38 mutant strains with increased enzyme activity or enantioselectivity. Protein expression and purification were conducted to determine the hydrolytic activity of PchEHA, and three mutants increased their activity by more than 95% compared with that of the wt. After multiple rounds of screening and site-specific mutagenesis, we found that F3 offers the best enzyme activity and enantioselectivity; furthermore, the molecular docking results confirmed this result. Overall, this study uncovered novel mutants with potential value as industrial biocatalysts.

Characterization and identification of extracellular polysaccharides-degrading enzymes (epes)-producing marine sediment bacteria

The utilization of polysaccharide-degrading enzymes (EPEs) from bacteria has been increasing, therefore many studies are exploring new producers of EPEs. This study aimed to screen and identify the EPEs-producing marine sediment bacteria collected from Panjang Island, Jepara. A total of 11 bacteria were isolated from the sediment sample. Further, seven strains were selected to conduct further analysis, such as biochemical test and screening of enzyme activity. According to the calculation of enzyme activity index (EAI), it was noted that strain with given codes PP.K.15, PP.K.21, PP.K.6, and PP.K.20 demonstrated potential for carrageenase, alginate-lyase, amylase, and agarase. In addition, molecular identification using 16S rRNA gene sequencing discovered that PP.K.6 was identified as Basillus safensis, PP.K.11 as Sinomicrobium oceani, PP.K. 20 as Salinicola zeshunii, PP.K 15 and PP.K. 21 as Micrococcus luteus, and PP.K 22 as Qipengyuania flava.

Cell-Based Assay Approaches for Glycosaminoglycan Synthase High-Throughput Screening: Development and Applications.

Glycosaminoglycan synthases have immense potential in applications involving synthesis of oligosaccharides, using enzymatic approaches and construction of cell factories that produce polysaccharides as critical metabolic components. However, the use of high-throughput activity assays to screen for the evolution of these enzymes can be challenging because there are no significant changes in fluorescence or absorbance associated with glycosidic bond formation. Here, using incorporation of azido-labeled N-acetylhexosamine analogs into bacterial capsule polysaccharides via bacterial metabolism and bioorthogonal chemistry, fluorophores were specifically introduced onto cell surfaces. Furthermore, correlations between detectable fluorescence signals and the polysaccharide-synthesizing capacity of individual bacteria were established. Among 10 candidate genes, 6 members of the chondroitin synthase family were quickly identified in a recombinant Bacillus subtilis host strain. Additionally, directed evolution of heparosan synthase was successfully performed using fluorescence-activated cell sorting of recombinant Escherichia coli O10:K5(L):H4, yielding several mutants with increased activity. Cell-based approaches that selectively detect the presence or absence of synthases within an individual colony of bacterial cells, as well as their level of activity, have broad potential in the exploration and engineering of glycosaminoglycan synthases. These approaches also support the creation of novel strategies for high-throughput screening of enzyme activity based on cell systems.

Single-Walled Carbon Nanotube Probes for the Characterization of Biofilm-Degrading Enzymes Demonstrated against Pseudomonas aeruginosa Extracellular Matrices.

Hydrolase co-therapies that degrade biofilm extracellular polymeric substances (EPS) allow for a better diffusion of antibiotics and more effective treatment; current methods for quantitatively measuring the enzymatic degradation of EPS are not amendable to high-throughput screening. Herein, we present biofilm EPS-functionalized single-walled carbon nanotube (SWCNT) probes for rapid screening of hydrolytic enzyme selectivity and activity on EPS. The extent of biofilm EPS degradation is quantified by monitoring the quenching of the SWCNT fluorescence. We used this platform to screen 16 hydrolases with varying bond breaking selectivity against a panel of wild-type Pseudomonas aeruginosa and mutants deficient or altered in one or more EPS. Next, we performed concentration-dependent studies of six enzymes on two common strains found in cystic fibrosis (CF) environments and, for each enzyme, extracted three first-order rate constants and their relative contributions by fitting a parallel, multi-site degradation model, with a good model fit (R2 from 0.65 to 0.97). Reaction rates (turnover rates) are dependent on the enzyme concentration and range from 6.67 × 10-11 to 2.80 × 10-3 *s-1 per mg/mL of enzymes. Lastly, we confirmed findings from this new assay using an established crystal-violet staining assay for a subset of hydrolase panels. In summary, our work shows that this modular sensor is amendable to the high-throughput screening of EPS degradation, thereby improving the rate of discovery and development of novel hydrolases.

Use of Waste Substrates for the Lipid Production by Yeasts of the Genus Metschnikowia-Screening Study.

Oleogenic yeasts are characterized by the ability to accumulate increased amounts of lipids under certain conditions. These microbial lipids differ in their fatty acid composition, which allows them to be widely used in the biotechnology industry. The interest of biotechnologists is closely linked to the rising prices of fossil fuels in recent years. Their negative environmental impact is caused by significantly increased demand for biodiesel. The composition of microbial lipids is very similar to vegetable oils, which provides great potential for use in the production of biodiesel. In addition, some oleogenic microorganisms are capable of producing lipids with a high proportion of unsaturated fatty acids. The presented paper’s main aim was to study the production of lipids and lipid substances by yeasts of the genus Metschnikowia, to cultivate crude waste animal fat to study its utilization by yeasts, and to apply the idea of circular economy in the biotechnology of Metschnikowia yeasts. The work focuses on the influence of various stress factors in the cultivation process, such as reduced temperature or nutritional stress through the use of various waste substrates, together with manipulating the ratio of carbon and nitrogen sources in the medium. Yeast production properties were monitored by several instrumental techniques, including gas chromatography and Raman spectroscopy. The amount of lipids and in particular the fatty acid composition varied depending on the strains studied and the culture conditions used. The ability of yeast to produce significant amounts of unsaturated fatty acids was also demonstrated in the work. The most suitable substrate for lipid production was a medium containing glycerol, where the amount of accumulated lipids in the yeast M. pulcherrima 1232 was up to 36%. In our work, the crude animal fat was used for the production of high-value lipids, which to the best of our knowledge is a new result. Moreover, quantitative screening of lipase enzyme activity cultivated on animal fat substrate on selected yeasts of the genus Metschnikowia was performed. We found that for the yeast utilizing glycerol, animal fat seems to be an excellent source of carbon. Therefore, the yeast conversion of crude processed animal fat to value-added products is a valuable process for the biotechnology and food industry.

Biofoundry-assisted expression and characterization of plant proteins.

Many goals in synthetic biology, including the elucidation and refactoring of biosynthetic pathways and the engineering of regulatory circuits and networks, require knowledge of protein function. In plants, the prevalence of large gene families means it can be particularly challenging to link specific functions to individual proteins. However, protein characterization has remained a technical bottleneck, often requiring significant effort to optimize expression and purification protocols. To leverage the ability of biofoundries to accelerate design–built–test–learn cycles, we present a workflow for automated DNA assembly and cell-free expression of plant proteins that accelerates optimization and enables rapid screening of enzyme activity. First, we developed a phytobrick-compatible Golden Gate DNA assembly toolbox containing plasmid acceptors for cell-free expression using Escherichiacoli or wheat germ lysates as well as a set of N- and C-terminal tag parts for detection, purification and improved expression/folding. We next optimized automated assembly of miniaturized cell-free reactions using an acoustic liquid handling platform and then compared tag configurations to identify those that increase expression. We additionally developed a luciferase-based system for rapid quantification that requires a minimal 11–amino acid tag and demonstrate facile removal of tags following synthesis. Finally, we show that several functional assays can be performed with cell-free protein synthesis reactions without the need for protein purification. Together, the combination of automated assembly of DNA parts and cell-free expression reactions should significantly increase the throughput of experiments to test and understand plant protein function and enable the direct reuse of DNA parts in downstream plant engineering workflows.

Comparative Analysis of Universal Protein Extraction Methodologies for Screening of Lipase Activity from Agricultural Products

Protein extraction techniques are absolutely required for the research of biological catalysts. The present study compared four universal protein extraction methodologies (ammonium sulfate precipitation, TCA/acetone precipitation, and two commercial kits) to provide practical information on protein extraction in order to discover a novel lipase in agricultural products. Yields of protein extraction from 24 domestic agricultural products and their specific activities were evaluated and compared with each other. TCA/acetone precipitation showed a relatively higher extraction yield (on average, 3.41 ± 1.08 mg protein/0.1 g sample) in crude protein extraction, whereas the Pierce™ Plant Total Protein Extraction Kit showed the highest specific lipase activity on average in both spectrophotometric (266.61 ± 235.78 μU/mg protein) and fluorometric (41.52 ± 32.63 μU/mg protein) assays. Our results suggest that commercial kits for the rapid extraction of soluble functional proteins would be a better choice than conventional precipitation techniques to perform the high-throughput screening of enzyme activity from plant sources. Finally, several agricultural products such as cordyceps, pepper, bracken, and hemp, all of which exhibited an excellent specific lipase activity, were proposed as promising candidates for a source of novel lipases.

Surface display as a functional screening platform for detecting enzymes active on PET

Poly(ethylene terephthalate) (PET) is the world’s most abundant polyester plastic, and its ongoing accumulation in nature is causing a global environmental problem. Currently, the main recycling processes utilize thermomechanical or chemical means, resulting in the deterioration of the mechanical properties of PET. Consequently, polluting de novo synthesis remains preferred, creating the need for more efficient and bio-sustainable ways to hydrolyze the polymer. Recently, a PETase enzyme from the bacterium Ideonella sakaiensis was shown to facilitate PET biodegradation, albeit at slow rate. Engineering of more efficient PETases is required for industrial relevance, but progress is currently hampered by the dependency on intracellular expression in Escherichia coli. To create a more efficient screening platform in E. coli, we explore different surface display anchors for fast and easy assaying of PETase activity. We show that PETases can be functionally displayed on the bacterial cell surface, enabling screening of enzyme activity on PET microparticles – both while anchored to the cell and following solubilization of the enzymes.

Fast and accurate enzyme activity measurements using a chip-based microfluidic calorimeter

Recent developments in microfluidic and nanofluidic technologies have resulted in development of new chip-based microfluidic calorimeters with potential use in different fields. One application would be the accurate high-throughput measurement of enzyme activity. Calorimetry is a generic way to measure activity of enzymes, but unlike conventional calorimeters, chip-based calorimeters can be easily automated and implemented in high-throughput screening platforms. However, application of chip-based microfluidic calorimeters to measure enzyme activity has been limited due to problems associated with miniaturization such as incomplete mixing and a decrease in volumetric heat generated. To address these problems we introduced a calibration method and devised a convenient protocol for using a chip-based microfluidic calorimeter. Using the new calibration method, the progress curve of alkaline phosphatase, which has product inhibition for phosphate, measured by the calorimeter was the same as that recorded by UV-visible spectroscopy. Our results may enable use of current chip-based microfluidic calorimeters in a simple manner as a tool for high-throughput screening of enzyme activity with potential applications in drug discovery and enzyme engineering.

Direct screening of enzyme activity using infrared matrix-assisted laser desorption electrospray ionization.

High-throughput screening (HTS) is a critical step in the drug discovery process. However, most mass spectrometry (MS)-based HTS methods require sample cleanup steps prior to analysis. In this work we present the utility of infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) for monitoring an enzymatic reaction directly from a biological buffer system with no sample cleanup and at high throughput. IR-MALDESI was used to directly analyze reaction mixtures from a well plate at different time points after reaction initiation. The percent conversion of precursors to products was used to screen the enzyme activity. The reaction was performed with two different concentrations of precursors and enzyme in order to assess the dynamic range of the assay. Eventually, a pseudo-HTS study was designed to investigate the utility of IR-MALDESI screening enzyme activity in a high-throughput manner. IR-MALDESI was able to readily monitor the activity of IDH1 over time at two different concentrations of precursors and enzyme. The calculated Z-factors of 0.65 and 0.41 confirmed the suitability of the developed method for screening enzyme activity in HTS manner. Finally, in a single-blind pseudo-HTS analysis IR-MALDESI was able to correctly predict the identity of all samples, where 8/10 samples were identified with high confidence and the other two samples with lower confidence. The enzymatic activity of IDH1 was screened by directly analyzing the reaction content from the buffer in well plates with no sample cleanup steps. This proof-of-concept study demonstrates the robustness of IR-MALDESI for direct analysis of enzymatic reactions from biological buffers with no sample cleanup and its immense potential for HTS applications.

Self-immolative versatile fluorogenic probes for screening of hydrolytic enzyme activity.

Enzyme triggered probes with a self-immolative linker for rapid and sensitive hydrolase detection through a cascade reaction have been reported. Their utility was proved by the preparation of three model compounds and their evaluation as enzyme substrates and demonstration of their applicability as fluorogenic probes for screening lipase, esterase and protease activities. These probes represent a new class of fluorogenic compounds, are stable under aqueous conditions and not susceptible to nonspecific degradation. The utilization of the carbamate cleavable linkage in a probe structure allows moving away of the bulky fluorophore from the enzyme recognition unit and targets different classes of enzymes with the same substrate.

Accurate label-free reaction kinetics determination using initial rate heat measurements.

Accurate label-free methods or assays to obtain the initial reaction rates have significant importance in fundamental studies of enzymes and in application-oriented high throughput screening of enzyme activity. Here we introduce a label-free approach for obtaining initial rates of enzyme activity from heat measurements, which we name initial rate calorimetry (IrCal). This approach is based on our new finding that the data recorded by isothermal titration calorimetry for the early stages of a reaction, which have been widely ignored, are correlated to the initial rates. Application of the IrCal approach to various enzymes led to accurate enzyme kinetics parameters as compared to spectroscopic methods and enabled enzyme kinetic studies with natural substrate, e.g. proteases with protein substrates. Because heat is a label-free property of almost all reactions, the IrCal approach holds promise in fundamental studies of various enzymes and in use of calorimetry for high throughput screening of enzyme activity.

Use of a genetically encoded hydrogen peroxide sensor for whole cell screening of enzyme activity

We report the use of HyPer, a genetically encoded, fluorescent sensor that reacts with hydrogen peroxide (H2O2), in a novel screen to engineer enzymes for enhanced production of H2O2. We co-expressed HyPer with cytochrome P450 BM3 variants and, using HyPer's ratiometric signal, found variants that produce greater amounts of H2O2 than the wild-type enzyme through the leakage reaction. The screen avoids lysis procedures and the addition of reagents to assay intracellular contents. Less laborious screening procedures will be useful in engineering more powerful H2O2 generators as tools in quantitative redox biology, and increasing the utility of enzymes that produce H2O2 as a by-product alongside a valuable compound.

AND logic gate application based on colorimetric screening of enzyme activity

A simple and novel DNA and gold nanoparticle (AuNPs) based colorimetric method has been developed for efficient detection of Deoxyribonuclease I (DNase-I) activity. For the detection process, the sample that contains DNase-I, which will cleave double stranded DNA into small pieces. Furthermore, the small pieces DNA sequences and water-soluble conjugated polymer cause the AuNPs aggregation (the solution change the color from red to blue). Thus, DNase-I could be detected by naked eyes or UV/Vis. Molecular substrates can be viewed as computational devices that process physical or chemical ‘inputs’ to generate ‘outputs’ based on a set of logical operators. Based on the assay, we have presented a DNA/AuNP based, multi-readout AND logic operations.

Continuous Flow Immobilized Enzyme Reactor–Tandem Mass Spectrometry for Screening of AChE Inhibitors in Complex Mixtures

A method is described for identifying bioactive compounds in complex mixtures based on the use of capillary-scale monolithic enzyme-reactor columns for rapid screening of enzyme activity. A two-channel nanoLC system was used to continuously infuse substrate coupled with automated injections of substrate/small molecule mixtures, optionally containing the chromogenic Ellman reagent, through sol-gel derived acetylcholinesterase (AChE) doped monolithic columns. This is the first report of AChE encapsulated in monolithic silica for use as an immobilized enzyme reactor (IMER), and the first use of such IMERs for mixture screening. AChE IMER columns were optimized to allow rapid functional screening of compound mixtures based on changes in the product absorbance or the ratio of mass spectrometric peaks for product and substrate ions in the eluent. The assay had robust performance and produced a Z' factor of 0.77 in the presence of 2% (v/v) DMSO. A series of 52 mixtures consisting of 1040 compounds from the Canadian Compound Collection of bioactives was screened and two known inhibitors, physostigmine and 9-aminoacridine, were identified from active mixtures by manual deconvolution. The activity of the compounds was confirmed using the enzyme reactor format, which allowed determination of both IC(50) and K(I) values. Screening results were found to correlate well with a recently published fluorescence-based microarray screening assay for AChE inhibitors.

Multifaceted microbes

Multifaceted microbes

Conjugated polyelectrolyte as signal amplifier for fluorogenic probe based enzyme activity study

A cationic conjugated polyelectrolyte (CPE) is used as a signal amplifier to enhance the sensory response of a fluorogenic substrate (fluorescein diacetate) for esterase activity study. As fluorescein diacetate is nonfluorescent, it can be cleaved by esterase to yield a negatively charged fluorescent dye. Electrostatic interaction between the cationic CPE and negatively charged fluorescent dye leads to complexes which allow fluorescence resonance energy transfer to amplify the dye signal by a factor of up to ∼19-fold. The presence of CPE does not obviously influence the enzyme efficiency and the assay allows real-time monitoring of enzyme activity. As compared with common fluorogenic assays, the CPE amplified system has the advantage of shorter readout time with higher sensitivity, which could have wide applications in high throughput screening of enzyme activity.

Screening of enzyme activity for assessing the condition of larvae in the seven-band grouper Epinephelus septemfasciatus and devil stinger Inimicus japonicus

We conducted screening tests to determine whether enzyme activity is a suitable biomarker for assessing the physiological condition of marine fish larvae. The rearing experiments consisted of three trials, of which two were conducted using the seven-band grouper Epinephelus septemfasciatus for a period of 5 days after hatching (DAH), and one was conducted using the devil stinger Inimicus japonicus for 10 DAH. The trials were conducted under three different rearing-tank environments (shallow tank, intermediate tank, deep tank) in a water volume of 100 l and an aeration rate of 50 ml/min. We determined survival, surface death, growth, and enzyme activities (trypsin, esterase, and alkaline phosphatase). The highest survival rates and lowest surface deaths in both species occurred among the larvae grown in the deep tank. There was a significant and negative correlation between survival at 5 DAH and alkaline phosphatase activity at 0 DAH in the seven-band grouper. The same correlation was found between survival at 10 DAH and trypsin and alkaline phosphatase activity at 1 DAH in the devil stinger. Based on these results, we conclude that the activity of a specific enzyme is a candidate for assessing the physiological condition of marine fish larvae.

Colorimetric screening of bacterial enzyme activity and inhibition based on the aggregation of gold nanoparticles

A simple and novel gold nanoparticle (Au-NPs) based colorimetric method has been developed for efficient screening of class A beta-lactamase (Bla) activity and inhibitors in vitro and in bacterial strains.

Sequestration of the Active Site by Interdomain Shifting: CRYSTALLOGRAPHIC AND SPECTROSCOPIC EVIDENCE FOR DISTINCT CONFORMATIONS OF l-3-HYDROXYACYL-CoA DEHYDROGENASE

Sequestration of the Active Site by Interdomain Shifting: CRYSTALLOGRAPHIC AND SPECTROSCOPIC EVIDENCE FOR DISTINCT CONFORMATIONS OF l-3-HYDROXYACYL-CoA DEHYDROGENASE