Enzyme Screening Research Articles

Advances in abscisic acid biosynthesis

Abscisic acid, a plant hormone that inhibits growth, is a key factor in balancing plant endogenous hormones and regulating growth and metabolism. Abscisic acid can improve the drought resistance and salt tolerance of crops, reduce fruit browning, reduce the incidence rate of malaria and stimulate insulin secretion, so it has a broad application potential in agriculture and medicine. Compared with traditional plant extraction and chemical synthesis, abscisic acid synthesis by microorganisms is an economic and sustainable route. At present, a lot of progress has been made in the synthesis of abscisic acid by natural microorganisms such as Botrytis cinerea and Cercospora rosea, while the research on the synthesis of abscisic acid by engineered microorganisms is rarely reported. Saccharomyces cerevisiae, Yarrowia lipolytica and Escherichia coli are common hosts for heterologous synthesis of natural products due to their advantages of clear genetic background, easy operation and friendliness for industrial production. Therefore, the heterologous synthesis of abscisic acid by microorganisms is a more promising production method. The author reviews the research on the heterologous synthesis of abscisic acid by microorganisms from five aspects: selection of chassis cells, screening and expression enhancement of key enzymes, regulation of cofactors, enhancement of precursor supply and promotion of abscisic acid efflux. Finally, the future development direction of this field is prospected.

Quantitative Proteomics-Based Substrate Screening Revealed Cyclophilin Stabilization Regulated by Deubiquitinase Ubp7.

Quantitative proteomics has emerged as a crucial approach to identifying ubiquitinated substrates to investigate the functions of ubiquitination in cells. In this regard, although the substrate screening of certain enzymes in the ubiquitin system has been based on proteome or ubiquitinome level measurements, the direct comparison of these two approaches has not been determined to date. To quantitatively compare the efficiency and effectiveness of substrate screening from the entire proteomics to the ubiquitinomics filter, we used yeast deubiquitinating enzyme, Ubp7, as an example to evaluate it in this study. A total of 112 potential ubiquitinated substrates were identified from the ubiquitinomics level, whereas only 27 regulated substrates were identified from the entire proteomic screening, demonstrating the increased efficiency of ubiquitinomics quantitative analysis. Subsequently, we selected cyclophilin A (Cpr1) protein as an example, which was filtered out at the proteomics level but was a promising candidate according to the ubiquitinomics filter. Additional investigations revealed that Cpr1 possessed a K48-linked ubiquitin chain regulated by Ubp7, which may affect its homeostasis and, consequently, sensitivity to the therapeutic drug cyclosporine (CsA).

Screening and characterization of Lignolytic Enzymes Produced by Escherichia coli exposed to polyethylene

Screening and characterization of Lignolytic Enzymes Produced by Escherichia coli exposed to polyethylene

Screening and characterization of Lignolytic Enzymes Produced by Escherichia coli exposed to polyethylene

Screening and characterization of Lignolytic Enzymes Produced by Escherichia coli exposed to polyethylene

Machine Learning: A Suitable Method for Biocatalysis

Biocatalysis is currently a workhorse used to produce a wide array of compounds, from bulk to fine chemicals, in a green and sustainable manner. The success of biocatalysis is largely thanks to an enlargement of the feasible chemical reaction toolbox. This materialized due to major advances in enzyme screening tools and methods, together with high-throughput laboratory techniques for biocatalyst optimization through enzyme engineering. Therefore, enzyme-related knowledge has significantly increased. To handle the large number of data now available, computational approaches have been gaining relevance in biocatalysis, among them machine learning methods (MLMs). MLMs use data and algorithms to learn and improve from experience automatically. This review intends to briefly highlight the contribution of biocatalysis within biochemical engineering and bioprocesses and to present the key aspects of MLMs currently used within the scope of biocatalysis and related fields, mostly with readers non-skilled in MLMs in mind. Accordingly, a brief overview and the basic concepts underlying MLMs are presented. This is complemented with the basic steps to build a machine learning model and followed by insights into the types of algorithms used to intelligently analyse data, identify patterns and develop realistic applications in biochemical engineering and bioprocesses. Notwithstanding, and given the scope of this review, some recent illustrative examples of MLMs in protein engineering, enzyme production, biocatalyst formulation and enzyme screening are provided, and future developments are suggested. Overall, it is envisaged that the present review will provide insights into MLMs and how these are major assets for more efficient biocatalysis.

Enzymatic Screening of β-Amyloid Precursor Protein-Based Substrates

We performed an enzymatic screening of synthetic peptides based on β-amyloid precursor protein substrates. The template peptide sequence was a decapeptide derived from our previous screening study, which determined several effective unnatural amino acids. In this study, new libraries containing some unnatural amino acid compounds were prepared in the solid phase and digested with the β-site amyloid precursor protein-cleaving enzyme. The reaction mixture was analyzed using high-performance liquid chromatography combined with mass spectrometry. The peptides that showed a higher cleavage than the template sequence were determined and reported.

Screening and isolation of milk-clotting enzymes from seaweed

Cheese-making involves milk coagulation as a crucial step where calf rennet has been used traditionally as the milk-clotting enzyme in the cheese industry. This study investigated milk-clotting enzymes from seven species of seaweed and evaluated the parameters for their isolation and partial purification including post-harvest processing, extraction and purification methods. The saturation degree of ammonium sulphate and the precipitation stages were evaluated to obtain optimal purification conditions, and three desalting methods, namely dialysis, desalting column and the combination of dialysis and desalting column, were investigated to determine the most suitable method for ammonium sulphate removal. Protein extracts of all seven species of seaweeds exhibited some caseinolytic activity, but the extract of unprocessed (whole) dried samples had higher protein yields and stronger caseinolytic activities. The extract from one species, Gracilaria edulis, demonstrated the ability to clot milk. The evaluation of the purification method for G. edulis extract revealed an optimum single step of 50% saturation and ammonium sulphate precipitation with dialysis as the desalting method.

Discovery of novel phenyl triazole analogs as TRK/ALK dual inhibitors with prospective antitumor effects

The exploration of novel anaplastic lymphoma kinase (ALK) and tropomyosin receptor kinase (TRK) dual inhibitors tended to serve as targeted treatment of cancer. Herein, a series of phenyl triazole derivatives were designed and synthesized as ALK/TRK dual regulators based on structure-based drug design (SBDD) strategy and were evaluated for antiproliferative activity by MTT assay. Accordingly, all compounds showed surprising cytotoxicity with IC50 values below 10 μM on KM12, H2228 and KARPAS299 cell lines. Among them, compound 13a bearing (2-(4-methylpiperazin-1-yl)phenyl)morpholinomethanone moiety was identified as the optimal hit in enzymatic screening with IC50 values of 1.9 nM (TRKA), 7.2 nM (ALK) and 65.2 nM (ALKL1196M), respectively. Furthermore, 13a could inhibit KM12 cell migration and colony formation in a dose dependent manner. Meanwhile, AO/EB staining indicated that the pro-apoptotic effect of 13a was comparable to that of Entrectinib at the dose of 200 nM. Ultimately, the binding model of 13a with TRKA and ALK well established its mode of action which accounted for the superior activities as a promising antitumor candidate.

Characterization of Thermostable Lignolytic Enzymes from Penicillium italicum during Biomineralization of Polyethylene

Penicillium italicum isolated from a local dumpsite in Ilara- mokin, Ondo state, Nigeria was used to bio-transform low-density polyethylene (LDPE). Biodegradation was carried out using surface-sterilized and autoclaved LDPE over 3 months experimental period. Bio-remediation was evaluated by determining the weight loss, and physical structure changes which were revealed by scanning electron microscopy. The result showed P. italicum was able to degrade autoclaved LD-PE (25%) more efficiently than surface-sterilized LD-PE (12%). Screening and characterization of lignolytic enzymes (manganese peroxidase (MnP) and laccase (Lac)) produced by P. italicum were also investigated using autoclaved LDPE as a carbon source over 3 months degradation period. The result showed that P. italicum produced optimum MnP and Lac activities in the 6th week of cultivation (90 U/mL and 100 U/mL respectively). Study of their physicochemical properties showed that MnP and Lac had optimum activities (100%) at pH 7.0 and pH 5.0 and were stable at the same pH with 74% residual activities each respectively. The enzymes also had optimum activity (100%) at 70oC and were stable over a wide temperature range (40oC–80oC) with 78% residual activities each after 120 min of incubation. These results show the ability of P. italicum to produce thermostable lignolytic enzymes with the potentials to bio-mineralize recalcitrant plastic polymers such as LDPE.

Isolation and Characterization of L-Asparaginase Producing Endophytic Fungi from Medicinal Plants of Rutaceae Family

Endophytic fungi synthesize a diverse set of enzymes and biomolecules with wide array of bioactivities. Thirty-five distinct fungal endophytes have been isolated from medicinal plants of Rutaceae and screened for their ability to produce various industrially important enzymes. Primary enzyme screening revealed that 65% of isolates produced L-Asparaginase, 62% of isolates produced cellulase, 60% of isolates produced amylase, 48% of isolates produced both lipase and pectinase, 45% of isolates produced laccase and 42% of isolates produced protease. Among the different enzymes screened, L-Asparaginase producing isolates were dominant and subjected to quantification using the nesslerization method. The endophytic isolates LCJ315, LCJ324, LCJ326 and LCJ335 were capable of producing high L-Asparaginase activity and their enzyme activity ranged between 7.58 U/ml to 8.84 U/ml. The four isolates were further subjected to molecular identification using 18S rRNA sequencing. The results were then subjected to BLAST analysis. The phylogenetic tree was created by MEGA software. The isolated endophytic fungal strains were identified as LCJ315 (Aspergillus sp.), LCJ326 (Colletotrichum sp.), LCJ324 and LCJ335 (Fusarium sp.). The fungal source of L-Asparaginase is preferred over other microbial sources of L-Asparaginase due to reduced side effects. Given the fact that L-Asparaginase is a crucial anticancer enzyme that is used as the initial therapy for acute lymphoblastic leukemia. Hence, the present study reveals that these endophytic fungal strains isolated from Rutaceae members can be used as an alternate source for L-Asparaginase production.

β-Lactamase Sensitive Probe for Rapid Detection of Antibiotic-Resistant Bacteria with Gas Chromatography-Tandem Mass Spectrometry.

β-Lactamase (Bla) produced by bacteria to resist β-lactam antibiotics is a serious public health threat. Developing efficient diagnostic protocols for drug-resistant bacteria is of great significance. In this work, based on gas molecules in bacteria, a novel research strategy was proposed to develop a gas molecule-based probe by grafting 2-methyl-3-mercaptofuran (MF) onto cephalosporin intermediates via a nucleophilic substitution reaction. The probe can release the corresponding MF by reacting with Bla. The released MF, as a marker of drug-resistant bacteria, was analyzed by headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry. The Bla concentration as low as 0.2 nM can be easily observed, providing an efficient method for detecting enzyme activity and screening drug-resistant strains in vivo. Importantly, the method is universal, and probes with different properties can be prepared by changing different substrates to further identify different types of bacteria, thereby broadening the research methods and ideas for monitoring physiological processes.

Structural characterization of corn fiber hemicelluloses extracted by organic solvent and screening of degradation enzymes

An integrated treatment coupling peracetic acid delignification, dimethyl sulfoxide extraction, and ethanol precipitation were performed to isolate hemicellulose from de-starched corn fiber. Based on chemical composition, molecular weight distribution, methylation, and nuclear magnetic resonance spectroscopy, it is proposed that hemicelluloses in corn fiber were composed of two polysaccharides, glucuronoarabinoxylan (about 80 %) and xyloglucan (about 20 %). Xylose (about 46 %) and arabinose (about 32 %) were the main components in glucuronoarabinoxylan. More than half of the xylose units in the glucuronoarabinoxylan backbone chain were substituted at O-2 and/or O-3 by various monomers or oligomeric side chains. Based on structure analysis, five hemicellulases were selected and added to Penicillium oxalicum MCAX enzymes for enzymatic hydrolysis of corn fiber. The results showed that the addition of hemicellulases increased the sugar yield of corn fiber. These results demonstrate the effectiveness of enzyme consortium constructed by elucidating the chemical structure of hemicellulose in corn fiber for the degradation of corn fiber and also provide a general solution for the rational construction of targeted and efficient enzyme systems for the degradation of lignocellulosic biomass.

Isolation and characterization of detergent-compatible amylase-, protease-, lipase-, and cellulase-producing bacteria.

Detergent-compatible enzymes are the new trend followed by most in the detergent industry. Cellulases, lipases, proteases, and amylases are among the enzymes frequently used in detergents. Detergent-compatible enzymes can be obtained from many organisms, but the stability, cheapness, and availability of microbial enzymes make them preferable in industrial areas. In the present study, soil samples contaminated with household waste were collected from different regions of Trabzon (Turkey) for amylase-, cellulase-, protease-, and lipase-producing bacteria. A total of 55 bacterial isolates differing in colony morphology were purified from the samples and 25 of the isolates gave positive results in enzyme screening. The enzyme screening experiments revealed that 10 isolates produced amylase, 9 produced lipase, 7 produced cellulase, and 6 produced protease. While 2 isolates showed both protease and lipase activity, for 2 different isolates cellulose and amylase activity were detected together. It was also observed that one isolate, C37PLCA, produced all four enzymes. The morphological, physiological, and biochemical analyses of the bacteria from which we obtained the enzymes were performed and species close to them were determined using 16S rRNA sequences. Based on the results obtained, our enzymes show tremendous promise for the detergent industry.

Enterobacteriaceae isolates from clinical and household tap water samples: antibiotic resistance, screening for extended-spectrum, metallo- and ampC-beta-lactamases, and detection of blaTEM,blaSHV and blaCTX-M in Uyo, Nigeria.

Infections caused by multidrug-resistant (MDR) bacteria, extended spectrum β-lactamase (ESBL), metallo-β-lactamase (MBL) and AmpC-β-lactamase (AmpC-βL)-producers are increasing globally. This study identified bacteria in clinical and tap water samples and determined the prevalence of MDR, and β-lactamase enzymes and genes. Isolates were identified by the Vitek 2 (bioMérieux, France) automated system. Antibiotic resistance and screening for β-lactamase enzymes and genes was done using disc diffusion method and Vitek 2 automated system, CHROMagar-ESBL, combined double disc, inhibition-based method and multiplex polymerase chain reaction, respectively. The Enterobacteriaceae isolates obtained were Escherichia coli, Klebsiella pneumoniae, Citrobacter freundii, Salmonella spp., Proteus mirabilis, Enterobacter aerogenes, Shigella sonnei, Proteus vulgaris, Enterobacter sakazakii, Klebsiella oxytoca, Citrobacter diversus, and Serratia liquefaciens. Of the 674 isolates from clinical samples, 36.5%, 28.5%, and 19.9% were ESBL, MBL, and AmpC-βL producers, respectively. A low prevalence of AmpC-βL and MBL producers were obtained, with no significant difference (p<0.05) between the prevalence of ESBL and non-ESBL producers. Isolates exhibited varied levels of resistance to gentamicin, amoxicillin-clavulanic acid, ciprofloxacin, and tetracycline. The results showed that 54.6% of ESBL producers, 57.9% of MBL producers, and 62.8% of AmpC-βL producers were MDR strains. Of the 141 representative isolates tested, 36.9%, 15.6%, and 20.6% had only blaTEM, blaSHV, and blaCTX-M, respectively; 5.7% possessed both blaTEM and blaSHV; 7.1% possessed both blaTEM and blaCTX-M and 4.3% had both blaSHV and blaCTX-M. This study found a high prevalence of β-lactamase producers, indicating the need for further research on the molecular epidemiology of β-lactamase producers and their impacts in the region.

Production and screening of fungal L-asparaginase enzymes as anticancer agents from high-contrast soil environments in Egypt

Production and screening of fungal L-asparaginase enzymes as anticancer agents from high-contrast soil environments in Egypt

Discovery and Enzymatic Screening of Genome-Mined Microbial Levanases to Produce Second-Generation β-(2,6)-Fructooligosaccharides: Catalytic Properties.

Evidence suggests that β-(2,6)-levan-type fructooligosaccharides (FOSs) possess higher prebiotic potential and selectivity than their β-(2,1)-inulin-type counterparts. The focus of the present work was to develop an enzymatic approach for the synthesis of levan-type FOSs, employing levanases (EC 3.2.1.65), specifically those performing endo-hydrolysis on levans. To identify new levanases, a selection of candidates was obtained via in silico exploration of the levanase family biodiversity through a sequence-driven approach. A collection of 113 candidates was screened according to their specific activities on low- and high-molecular-weight (MW) levan as well as thermal stability. The most active levanases were able to hydrolyze both types of levan with similar efficiency. This ultimately revealed 10 active, highly evolutionary distant and diverse candidate levanases, which demonstrated preferential hydrolysis of levan over inulin. The end-product profile differed significantly depending on levanase with levanbiose, levantriose, and levantetraose being the major FOSs. Among them, the catalytic properties of 5 selected potential new levanases (LEV9 from Belliella Baltica, LEV36 from Dyadobacter fermentans, LEV37 from Capnocytophaga ochracea, LEV79 from Vibrio natriegens, LEV91 from Paenarthrobacter aurescens) were characterized, especially in terms of pH and temperature profiles, thermal stability, and kinetic parameters. The identification of these novel levanases is expected to contribute to the production of levan-type FOSs with properties surpassing those of commercial preparations.

Engineering Modular and Highly Sensitive Cell-Based Biosensors for Aromatic Contaminant Monitoring and High-Throughput Enzyme Screening.

Although a variety of whole-cell-based biosensors have been developed for different applications in recent years, most cannot meet practical requirements due to insufficient sensing performance. Here, we constructed two sets of modular genetic circuits by serial and parallel modes capable of significantly amplifying the input/output signal in Escherichia coli. The biosensors are engineered using σ54-dependent phenol-responsive regulator DmpR as a sensor and enhanced green fluorescent protein as a reporter. Cells harboring serial and parallel genetic circuits displayed nearly 9- and 16-fold higher sensitivity than the general circuit. The genetic circuits enabled rapid detection of six phenolic contaminants in 12 h and showed the low limit of detection of 2.5 and 2.2 ppb for benzopyrene (BaP) and tetracycline (Tet), with a broad detection range of 0.01-1 and 0.005-5 μM, respectively. Furthermore, the positive rate was as high as 73% when the biosensor was applied to screen intracellular enzymes with ester-hydrolysis activity from soil metagenomic libraries using phenyl acetate as a phenolic substrate. Several novel enzymes were isolated, identified, and biochemically characterized, including serine peptidases and alkaline phosphatase family protein/metalloenzyme. Consequently, this study provides a new signal amplification method for cell-based biosensors that can be widely applied to environmental contaminant assessment and screening of intracellular enzymes.

A Genetically Encoded Biosensor for the Detection of Levulinic Acid.

Levulinic acid (LA) is a valuable chemical used in fuel additives, fragrances, and polymers. In this study, we proposed possible biosynthetic pathways for LA production from lignin and poly(ethylene terephthalate). We also created a genetically encoded biosensor responsive to LA, which can be used for screening and evolving the LA biosynthesis pathway genes, by employing an LvaR transcriptional regulator of Pseudomonas putida KT2440 to express a fluorescent reporter gene. The LvaR regulator senses LA as a cognate ligand. The LA biosensor was first examined in an Escherichia coli strain and was found to be non-functional. When the host of the LA biosensor was switched from E. coli to P. putida KT2440, the LA biosensor showed a linear correlation between fluorescence intensity and LA concentration in the range of 0.156-10 mM LA. In addition, we determined that 0.156 mM LA was the limit of LA detection in P. putida KT2440 harboring an LA-responsive biosensor. The maximal fluorescence increase was 12.3-fold in the presence of 10 mM LA compared to that in the absence of LA. The individual cell responses to LA concentrations reflected the population-averaged responses, which enabled high-throughput screening of enzymes and metabolic pathways involved in LA biosynthesis and sustainable production of LA in engineered microbes.

Screening and Production of Fungal L-Asparaginase Enzymes as Anticancer Agents from High-Contrast Soil Environments in Egypt

Screening and Production of Fungal L-Asparaginase Enzymes as Anticancer Agents from High-Contrast Soil Environments in Egypt

Analysis of glycans in a Burnt-on/Baked-on (BoBo) model food soil using Microarray Polymer Profiling (MAPP) and immunofluorescence microscopy

Burning of food materials during cooking can increase the difficulty in removal from solid surfaces, forming residual food soils. Using molecular probe-based technologies, the aim of this work was to elucidate the composition and relative abundance of glycans within a Burnt-On/Baked-On (BoBo) model food soil and investigate enzyme systems that may facilitate soil breakdown. Microarray Polymer Profiling identified xylan, arabinoxylan, mixed-linkage glucan and mannan as target substrates for the enzymatic cleaning of BoBo residues from surfaces. Indirect immunofluorescence microscopy revealed that burning resulted in extensive structural modifications and degradation of the three-dimensional architecture of constituent polysaccharide matrices. Results from high-throughput enzyme screening indicate that inclusion of xylan depolymerising enzymes in automatic dishwashing detergents may improve cleaning of recalcitrant, plant glycan-rich BoBo soils. Collectively, this study provides new insight into the composition and removal chemistry of complex, multi-component food soils.