Enzyme Screening Research Articles

A screening method for polyester films-degrading microorganisms and enzymes.

Enzymatic degradation of plastic pollution offers a promising environmentally friendly waste management strategy, however, suitable biocatalysts must be screened and developed. Traditional screening methods using soluble or solubilised polymers do not necessarily identify enzymes that are effective against solid or crystalline polymers. This study presents a simple, time-saving and cost-effective method for identifying microorganisms and enzymes capable of degrading polymeric films. The method was tested on polycaprolactone (PCL), polyethylene terephthalate (PET), polylactate (PLA) and polyhydroxybutyrate/polyhydroxyvalerate (PHB/PHV) films. It involves two steps: first, screening for PCL diol (PCLD)-degrading microorganisms on agar plates, and second, testing these microorganisms on polyester films. Using this screening method, over 100 PCLD-degrading microorganisms and 27 E. coli clones carrying genomic or metagenomic DNA fragments have been isolated. In addition, recombinant cutinases from Streptomyces scabiei and Thermobifida fusca have been tested. Approximately 66 % of the microorganisms forming halos on PCLD agar plates hydrolysed PCL and 6 % - the biaxially oriented PET film. In addition, five PLA- and four PHB/PHV-degrading esterases have been identified. The proposed method is effective for detecting both wild-type and recombinant microorganisms, as well as recombinant enzymes from in vitro transcription-translation reactions. Screening for thermostable and thermophilic enzymes, including those resistant to organic solvents or environmental inhibitors, is also easily implemented.

Isolation optimization and screening of halophilic enzymes and antimicrobial activities of halophilic archaea from the high-altitude, hypersaline Da Qaidam Salt Lake, China.

The aim of this study is to increase the diversity of culturable halophilic archaea by comparing various isolation conditions and to explore the application of halophilic archaea for enzyme-producing activities and antimicrobial properties. We systematically compared the isolation performance of various archaeal and bacterial media by isolating halophilic archaea from the Da Qaidam Salt Lake, a magnesium sulfate subtype hypersaline lake on the Qinghai-Tibet Plateau, China, using multiple enrichment culture and gradient dilution conditions. A total of 490 strains of halophilic archaea were isolated, which belonged to five families and 11 genera within the order Halobacteriales of the class Halobacteria of the phylum Euryarchaeota. The 11 genera consisted of nine known genera and two potentially new genera, the former including Halorubrum, Natranaeroarchaeum, Haloplanus, Haloarcula, Halorhabdus, Halomicrobium, Halobacterium, Natrinema, and Haloterrigene. Halorubrum was the dominant genus with a relative abundance of 78.98%. By comparing different culture conditions, we found that bacterial media 2216E and R2A showed much better isolation performance than all archaeal media, and enrichment culture after 60 d and dilution gradients of 10-1 and 10-2 were best fitted for halophilic archaea cultivation. The screening of 40 halophilic archaeal strains of different species indicated that these halophilic archaea had great extracellular enzyme activities, including amylase (62.5%), esterase (50.0%), protease (27.5%), and cellulase (15.0%), and possessed great antimicrobial activities against human pathogens. A total of 34 strains exhibited antimicrobial activity against four or more pathogens, and 19 strains exhibited antimicrobial activity against all six pathogens. The diversity of culturable halophilic archaea was significantly increased by enrichment culture and selection of bacterial media, and screening of representative strains showed that halophilic archaea have multiple extracellular enzyme activities and broad-spectrum antimicrobial activity against human pathogens.

Construction and Optimization of Engineered Saccharomyces cerevisiae for De Novo Synthesis of Phloretin and Its Derivatives.

Phloretin and its derivatives are dihydrochalcone compounds with diverse pharmacological properties and biological activities, offering significant potential for applications in the food and pharmaceutical industries. Due to their structural similarity to flavonoids, their extraction and isolation were highly challenging. Although the biosynthesis of phloretin via three distinct pathways has been reported, a systematic comparison within the same host has yet to be conducted. In this study, we employed rational design and synthetic biology approaches to engineer Saccharomyces cerevisiae for de novo synthesis of phloretin and its derivatives. We constructed and evaluated three biosynthetic pathways for phloretin in S. cerevisiae, demonstrating that effective phloretin synthesis is achievable only via the p-coumaryl-CoA pathway. Additionally, by optimizing enzyme screening, strain engineering, and coordinating heterologous pathways with endogenous metabolism, we achieved the highest reported de novo titer of 287.2 mg/L for phloretin, 184.6 mg/L for phlorizin, 103.1 mg/L for trilobatin, and 164.5 mg/L for nothofagin and the first-time synthesis of 4-methylphloretin and hesperetin dihydrochalcone. This study was committed to addressing the growing demand for dihydrochalcones while laying the foundation for the biosynthesis of more complex derivatives.

AcidBasePred: a protein acid-base tolerance prediction platform based on deep learning

The structures and activities of enzymes are influenced by pH of the environment. Understanding and distinguishing the adaptation mechanisms of enzymes to extreme pH values is of great significance for elucidating the molecular mechanisms and promoting the industrial applications of enzymes. In this study, the ESM-2 protein language model was used to encode the secreted microbial proteins with the optimal performance above pH 9 and below pH 5, which yielded 47 725 high-pH protein sequences and 66 079 low-pH protein sequences, respectively. A deep learning model was constructed to identify protein acid-base tolerance based on amino acid sequences. The model showcased significantly higher accuracy than other methods, with the overall accuracy of 94.8%, precision of 91.8%, and a recall rate of 93.4% on the test set. Furthermore, we built a website (https://enzymepred.biodesign.ac.cn), which enabled users to predict the acid-base tolerance by submitting the protein sequences of enzymes. This study has accelerated the application of enzymes in various fields, including biotechnology, pharmaceuticals, and chemicals. It provides a powerful tool for the rapid screening and optimization of industrial enzymes.

Functional and Genomic Insights into the Biotechnological Potential of Vibrio spp. Isolated from Deeply Polluted and Pristine Environments.

Vibrio spp. are remarkably diverse bacteria, being worthy of investigation not only for their antibiotic resistance and virulence, but also for their biotechnological potential. Indeed, there is increasing evidence that these bacteria display industrially relevant traits, particularly as producers of antimicrobial substances, tensioactive/emulsifying compounds, and enzymes. Here, our aim was to investigate the potential of Vibrio strains isolated from two different marine sources to produce such biotechnologically applicable substances. From the eighteen analyzed strains, five were isolated from plastic particles from a heavily polluted urban estuary and 13 from calcareous sponges inhabiting submarine caves in an isolated volcanic archipelago in the Atlantic Ocean. Enzymatic screening revealed that most strains were agarolytic and cellulolytic. Overall, six strains showed antimicrobial activity against Staphylococcus aureus ATCC 29,213, with four of them active towards Escherichia coli ATCC 25,922 as well. Additionally, eight strains were positive for the production of bioemulsifiers. Genomic analyses of four strains further revealed insights regarding the enzymatic arsenal, as shown by the detection of several key gene clusters pertaining to the chitin degradation pathway, and also encoding diverse classes of antimicrobial-active metabolites. Our findings highlight the biotechnological potential of Vibrio spp., evidencing their functional diversity and the need for continued and sustained prospecting of this bacterial genus to uncover its potential high-value-added bioproducts.

Evaluation and Comparison of Candida albicans vs Mammalian 6-O-Phospho-Kinases: Substrate Specificity and Applications.

Sensing of peptidoglycan fragments is essential for inducing downstream signaling in both mammalian and fungal systems. The hexokinases NagK and Hxk1 are crucial enzymes for the phosphorylation of peptidoglycan molecules in order to activate specific cellular responses; however, biochemical characterization of their enzymatic specificity and efficiency has yet to be investigated in depth. Here a mass spectrometry enzymatic screen was implemented to assess substrate specificity, and an ATP coupled assay provided the quantitative kinetic profiles of these two homologous, eukaryotic enzymes. The data show, that while homologous, NagK and Hxk1 have vastly different substrate profiles. NagK accepts a variety of different peptidoglycan-based substrates albeit with reduced efficiency but are still valuable as a tool in large scale chemoenzymatic settings. Conversely, Hxk1 has a smaller substrate scope but can turnover these alternative substrates at similar levels to its natural substrate. These results allow for deeper understanding into the biosynthetic machinery responsible for essential cellular processes including UDP-GlcNAc regulation and immune recognition events in the cell.

A prescription for engineering PFAS biodegradation.

Per- and polyfluorinated chemicals (PFAS) are of rising concern due to environmental persistence and emerging evidence of health risks to humans. Environmental persistence is largely attributed to a failure of microbes to degrade PFAS. PFAS recalcitrance has been proposed to result from chemistry, specifically C-F bond strength, or biology, largely negative selection from fluoride toxicity. Given natural evolution has many hurdles, this review advocates for a strategy of laboratory engineering and evolution. Enzymes identified to participate in defluorination reactions have been discovered in all Enzyme Commission classes, providing a palette for metabolic engineering. In vivo PFAS biodegradation will require multiple types of reactions and powerful fluoride mitigation mechanisms to act in concert. The necessary steps are to: (1) engineer bacteria that survive very high, unnatural levels of fluoride, (2) design, evolve, and screen for enzymes that cleave C-F bonds in a broader array of substrates, and (3) create overall physiological conditions that make for positive selective pressure with PFAS substrates.

KAHA ligation as a platform for the rapid discovery of Protein Tyrosine phosphatase 1B (PTP1B) inhibitors.

We have successfully designed and assembled a 66-member library of protein tyrosine phosphatases (PTP) inhibitor candidates using α-ketoacid-hydroxylamine (KAHA) ligation. Subsequent in situ enzymatic screening revealed a potent hit (IC50=1.67μM) against PTP1B, which displayed 6.8- to 50-fold selectivity over other phosphatases.

Design of a Bioluminescent Assay Platform for Quantitative Measurement of Histone Acetyltransferase Enzymatic Activity.

Protein acetylation and acylation are widespread post-translational modifications (PTMs) in eukaryotic and prokaryotic organisms. Histone acetyltransferase (HATs) enzymes catalyze the addition of short-chain acyl moieties to lysine residues on cellular proteins. Many HAT members are found to be dysregulated in human diseases, especially oncological processes. Screening potent and selective HAT inhibitors has promising application for therapeutic innovation. A biochemical assay for quantification of HAT activity utilizing luminescent output is highly desirable to improve upon limitations associated with the classic radiometric assay formats. Here we report the design of a bioluminescent technological platform for robust and sensitive quantification of HAT activity. This platform utilizes the metabolic enzyme acetyl-CoA synthetase 1 (ACS1) for a coupled reaction with firefly luciferase to generate luminescent signal relative to the HAT-catalyzed acetylation reaction. The biochemical assay was implemented in microtiter plate format and our results showed this assay sensitively detected catalytic activity of HAT enzyme p300, accurately measured its steady-state kinetic parameters of histone acetylation and measured the inhibitory potency of HAT inhibitor. This platform demonstrated excellent robustness, reproducibility, and signal-to-background ratios, with a screening window Z'=0.79. Our new bioluminescent design provides an alternative means for HAT enzymatic activity quantitation and HAT inhibitor screening.

Electrochemical sensor for the detection of serine β-lactamase catalytic activity

A new approach is proposed based on the use of electrodes modified with carbon nanomaterials to determine enzymatic activity and screening for inhibitors of serine β-lactamases such as extended spectrum β-lactamases (ESBLs). These enzymes are responsible for the development of antibiotic resistance of pathogenic bacteria to β-lactam antibiotics. Electrochemical oxidation of cephalosporin antibiotic cefotaxime was effectively registered at a potential E from +596 to +625 mV (relative to Ag/AgCl). This property makes it possible to determine the change in cefotaxime concentration in solution upon interaction with serine β-lactamases. By analyzing the electrochemical characteristics of the cefotaxime oxidation reaction, the kinetic parameters of its hydrolysis catalyzed by the serine β-lactamase CTX-M-116 were determined. The Michaelis constant was KM = 50 µM and the maximum rate of the catalytic reaction was 1.67∙10–6 M/min. A comparative analysis of the electrochemical parameters of the enzyme/substrate cefotaxime and enzyme/substrate cefotaxime/inhibitor sulbactam (SBT) systems was carried out. Inhibition of β-lactamase by sulbactam was characterized by an IC50 value of 2.5 μM. The proposed approach can be used for screening new substrates and inhibitors of β-lactamases.

High-Efficiency Enzyme Assay and Screening of Enzyme-Inhibiting Nanomaterials Using Capillary Electrophoresis with Hierarchically Porous Metal-Organic Framework-Based Immobilized Enzyme Microreactor.

Enzyme-inhibiting nanomaterials have significant potential for regulating enzyme activity. However, a universal and efficient method for systematically screening and evaluating the inhibitory effects of various nanomaterials on drug target enzymes has not been established. While the integrated technique of immobilized enzyme microreactor (IMER) with capillary electrophoresis (CE) serves as an effective tool for enzyme analysis, it still faces challenges such as low enzyme loadability, unsatisfactory stability, and limited applicability. Herein, hierarchical porous metal-organic frameworks (HP-MOFs) were explored as high-performance enzyme immobilization carriers and stationary phases to develop a novel HP-MOFs-based IMER-CE microanalysis system for efficient online enzyme assay and systematic screening of enzyme-inhibiting nanomaterials. As a proof-of-concept demonstration, the model enzyme xanthine oxidase (XOD) was immobilized on a HP-UiO-66-NH2 coated capillary, serving as an efficient and durable IMER for screening potential XOD-inhibiting nanomaterials. The hierarchically micro- and mesoporous structure and superior enzyme loadability of as-prepared HP-UiO-66-NH2-IMER was intensively characterized, followed by systematic evaluation of the separation performance of HP-UiO-66-NH2 coated column and the enzyme kinetics of the immobilized XOD. Compared to the microporous UiO-66-NH2-IMER, the HP-UiO-66-NH2-IMER-CE system showed significant improvements in enzyme loading, maximum reaction rate, repeatability, and long-term stability. Furthermore, the established method was effectively employed to screen the XOD inhibitory activity of various nanomaterials, revealing that graphene oxide, single wall carbon nanotube and three other nanomaterials exhibited inhibitory potentials. The HP-MOFs-based microanalysis system can be easily expanded by modifying the types of immobilized enzymes and holds the potential to accelerate the identification and rational design of effective enzyme-inhibiting nanomaterials.

Challenges and perspectives in using unspecific peroxygenases for organic synthesis

In the past 20 years, unspecific peroxygenases (UPOs) have emerged as promising biocatalysts for various organic transformations. Particularly, we have witnessed great attention being paid to the screening of new enzymes and expansion of the substrates/products. However, challenges such as enzyme stability, low turnover numbers, and substrate specificity hinder their widespread utilization in practical organic synthesis. This review article provides a concrete and mini-overview of the challenges associated with using UPOs in organic synthesis and discusses strategies for enzyme engineering to overcome these limitations. The article highlights recent advancements in UPO research and presents potential solutions to enhance their catalytic efficiency, stability, substrate specificity, and regioselectivity. Additionally, the review outlines the current methodologies employed for directed evolution and protein engineering of UPOs, along with computational modeling approaches for rational enzyme design. By addressing the challenges and exploring avenues for enzyme engineering, this review aims to shed light on the prospects of UPOs in organic synthesis.

Ene‐Reductase‐Catalysed Oxidation Reactions

Ene‐reductases from the Old Yellow Enzyme (OYE) family have been traditionally employed in the reduction of conjugated C=C double bonds. This study explores the underutilised oxidative potential of OYEs, demonstrating their capability to catalyse the enantioselective desaturation of carbonyl compounds. Utilising a deprotonated tyrosine residue as a catalytic base, we developed a method to enable OYE‐catalysed desaturation at ambient temperature and alkaline pH, without the need for high‐temperature conditions. Through screening of various OYE enzymes, we identified several candidates from different genera with enhanced desaturase activity across different substrates. This work broadens the scope of biocatalytic applications for OYEs, introducing a novel approach to the synthesis of chiral α,β‐unsaturated carbonyl compounds.

Tyrosinase Inhibitory Peptide from the Hydrolysates of Eel Bone: Optimization of Preparation, Inhibition Kinetics and Molecular Docking

ABSTRACT The main purpose of this study was to discover new tyrosinase inhibitory peptides from eel bone hydrolysates. After enzyme screening, alkaline protease was utilized, and optimal conditions were achieved through response surface optimization: solid-liquid ratio (1:25), time (5 h), and enzymolysis base ratio (3500 U/g). Inhibitory peptides of 1–3 kDa were isolated (IC50 = 2.162 mg/mL), and the inhibition was determined to be non-competitive and reversible. Subsequent LC-MS/MS and molecular docking revealed that the peptide with the lowest binding energy exhibited hydrogen bonding, hydrophobic interactions, and provided a potential explanation for the mechanism of inhibition by the peptides.

Nitrile hydratase as a promising biocatalyst: recent advances and future prospects.

Amides are an important type of synthetic intermediate used in the chemical, agrochemical, pharmaceutical, and nutraceutical industries. The traditional chemical process of converting nitriles into the corresponding amides is feasible but is restricted because of the harsh conditions required. In recent decades, nitrile hydratase (NHase, EC 4.2.1.84) has attracted considerable attention because of its application in nitrile transformation as a prominent biocatalyst. In this review, we provide a comprehensive survey of recent advances in NHase research in terms of natural distribution, enzyme screening, and molecular modification on the basis of its characteristics and catalytic mechanism. Additionally, industrial applications and recent significant biotechnology advances in NHase bioengineering and immobilization techniques are systematically summarized. Moreover, the current challenges and future perspectives for its further development in industrial applications for green chemistry were also discussed. This study contributes to the current state-of-the-art, providing important technical information for new NHase applications in manufacturing industries.

Discovery of alkaline laccases from basidiomycete fungi through machine learning-based approach

BackgroundLaccases can oxidize a broad spectrum of substrates, offering promising applications in various sectors, such as bioremediation, biomass fractionation in future biorefineries, and synthesis of biochemicals and biopolymers. However, laccase discovery and optimization with a desirable pH optimum remains a challenge due to the labor-intensive and time-consuming nature of the traditional laboratory methods.ResultsThis study presents a machine learning (ML)-integrated approach for predicting pH optima of basidiomycete fungal laccases, utilizing a small, curated dataset against a vast metagenomic data. Comparative computational analyses unveiled the structural and pH-dependent solubility differences between acidic and neutral-alkaline laccases, helping us understand the molecular bases of enzyme pH optimum. The pH profiling of the two ML-predicted alkaline laccase candidates from the basidiomycete fungus Lepista nuda further validated our computational approach, showing the accuracy of this comprehensive method.ConclusionsThis study uncovers the efficacy of ML in the prediction of enzyme pH optimum from minimal datasets, marking a significant step towards harnessing computational tools for systematic screening of enzymes for biotechnology applications.Graphical

Development and Optimization of a Bromothymol Blue-Based PLA2 Assay Involving POPC-Based Self-Assemblies.

Phospholipase A2 (PLA2) is a superfamily of phospholipase enzymes that dock at the water/oil interface of phospholipid assemblies, hydrolyzing the ester bond at the sn-2 position. The enzymatic activity of these enzymes differs based on the nature of the substrate, its supramolecular assemblies (micelle, liposomes), and their composition, reflecting the interfacial nature of the PLA2s and requiring assays able to directly quantify this interaction of the enzyme(s) with these supramolecular assemblies. We developed and optimized a simple, universal assay method employing the pH-sensitive indicator dye bromothymol blue (BTB), in which different POPC (3-palmitoyl-2-oleoyl-sn-glycero-1-phosphocholine) self-assemblies (liposomes or mixed micelles with Triton X-100 at different molar ratios) were used to assess the enzymatic activity. We used this assay to perform a comparative analysis of PLA2 kinetics on these supramolecular assemblies and to determine the kinetic parameters of PLA2 isozymes IB and IIA for each supramolecular POPC assembly. This assay is suitable for assessing the inhibition of PLA2s with great accuracy using UV-VIS spectrophotometry, being thus amenable for screening of PLA2 enzymes and their substrates and inhibitors in conditions very similar to physiologic ones.

Metabolic Engineering of Candida tropicalis for the De Novo Synthesis of β-Ionone.

β-ionone, a norisoprenoid, is a natural aromatic compound derived from plants, which displays various biological activities including anticancer, antioxidant and deworming properties. Due to its large biomass and strong environmental tolerance, the nonconventional oleaginous yeast Candida tropicalis was selected to efficiently synthesize β-ionone. We initially investigated the capacity of the cytoplasm and subcellular compartments to synthesize β-ionone independently. Subsequently, through adaptive screening of enzymes, functional identification of subcellular localization signal peptides and subcellular compartment combination strategies, a titer of 152.4 mg/L of β-ionone was achieved. Finally, directed evolution of rate-limiting enzyme and overexpression of key enzymes were performed to enhance β-ionone production. The resulting titer was 400.5 mg/L in shake flasks and 730 mg/L in a bioreactor. This study demonstrates the first de novo synthesis of β-ionone in C. tropicalis, providing a novel cellular chassis for terpenoid fragrances with considerable industrial potential.

Comparative evaluation of oxidative and biochemical parameters of red cell concentrates (RCCs) prepared from G6PD deficient donors and healthy donors during RCC storage

IntroductionGlucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common inherited enzyme disorder in red blood cell (RBC). Due to the importance of G6PD enzyme as an antioxidant in RBC, we tried to investigate the oxidative damage in red cell concentrates (RCCs) prepared from donors with G6PD enzyme deficiency in comparison with healthy donors. Material methodThis cross-sectional study was conducted on 20 male donors. Ten of the donors had G6PD deficiency (as a case) and the others had normal enzyme activity (as a control). Biochemical and oxidative damage parameters were examined in RCCs prepared from two groups on days 0, 7, 14, 21, 28 and 35 of RCCs storage; data comparison was analyzed by SPSS statistical software. ResultsAccording to the result, lactate concentration increased significantly from the 7th day to the 35th day of RCC storage in G6PD-deficient donors compared to the control (P < 0.05). In addition, malondialdehyde (MDA) concentration in G6PD-deficient RCC showed a significant increase compared to the control in all days of storage (P < 0.05). Among the hematological parameters, mean corpuscular volume (MCV) and mean cell hemoglobin (MCH) increased significantly in all days of RCC storage in G6PD-deficient donors compared to the control (P < 0.05). ConclusionOur study showed that oxidative changes in G6PD-deficient donors were significantly increased compared to the healthy donors, which probably leads to RCC storage lesion and an increase in blood transfusion complications. Due to the high prevalence of G6PD enzyme deficiency in pandemic areas, it seems that enzyme screening should be included in donor screening programs.

Functionalized Polysaccharides Improve Sensitivity of Tyramide/Peroxidase Proximity Labeling Assays through Electrostatic Interactions.

High-throughput assays that efficiently link genotype and phenotype with high fidelity are key to successful enzyme engineering campaigns. Among these assays, the tyramide/peroxidase proximity labeling method converts the product of an enzymatic reaction of a surface expressed enzyme to a highly reactive fluorescent radical, which labels the cell surface. In this context, maintaining the proximity of the readout reagents to the cell surface is crucial to prevent crosstalk and ensure that short-lived radical species react before diffusing away. Here, we investigated improvements in tyramide/peroxidase proximity labeling for enzyme screening. We modified chitosan (Cs) chains with horseradish peroxidase (HRP) and evaluated the effects of these conjugates on the efficiency of proximity labeling reactions on yeast cells displaying d-amino acid oxidase. By tethering HRP to chitosan through different chemical approaches, we localized the auxiliary enzyme close to the cell surface and enhanced the sensitivity of tyramide-peroxidase labeling reactions. We found that immobilizing HRP onto chitosan through a 5 kDa PEG linker improved labeling sensitivity by over 3.5-fold for substrates processed with a low turnover rate (e.g., d-lysine), while the sensitivity of the labeling for high activity substrates (e.g., d-alanine) was enhanced by over 0.6-fold. Such improvements in labeling efficiency broaden the range of enzymes and conditions that can be studied and screened by tyramide/peroxidase proximity labeling.