Enzymatic Strategy Research Articles

Tissue biomarkers of drug efficacy: case studies using a MALDI-MSI workflow.

MALDI MS imaging (MALDI-MSI) offers a capability to not only evaluate the distribution, localization and metabolism of drugs within tissues but also allow correlative tissue measurement of the effect of the drug on biomolecules in the targeted pathway. Particularly for MALDI-MSI, lipid molecules are readily detectable within tissues. Case study examples are provided for two different drugs targeting the sphingosine-1-phosphate/ceramide nexus in tumor xenograft tissues. A workflow combining high-resolution MALDI-MSI with on-tissue confirmation of targeted compounds using a structural library and on-tissue enzymatic digestion strategy is described. Representative images of drug metabolite distribution that correlate to an increase or decrease in sphingosine-1-phosphate or ceramide species are provided.

Leaf gas exchange and multiple enzymatic and non-enzymatic antioxidant strategies related to drought tolerance in two oil palm hybrids

The drought tolerance in young oil palm plants is related to greater efficiency in preventing oxidative damage by activating enzymatic and non-enzymatic antioxidant strategies simultaneously. Drought is a major environmental constraint limiting growth and yield of oil palm trees. In this study, two oil palm hybrids (BRS Manicore and BRS C 2501) were grown in large containers and subjected to a water deficit during 57 days. Leaf gas exchange analysis was combined with an in-depth assessment of the antioxidant system over the drought imposition. Under drought, leaf water potential at predawn (Ψ pd) decreased similarly in both hybrids. In parallel, there were decreases in the net CO2 assimilation rate (A), chlorophyll concentrations and Rubisco total activity. Overall, these decreases were more pronounced in BRS C 2501 than in BRS Manicore. BRS C 2501 plants triggered more markedly its enzymatic antioxidant system earlier (Ψ pd = −2.1 MPa) than did BRS Manicore, but these responses were accompanied by higher concentrations of H2O2 and malondialdehyde in BRS C 2510 than in BRS Manicore. With the progress of drought stress (Ψ pd = −2.9 MPa and below), BRS Manicore was better able to cope with oxidative stress through a more robust antioxidant system. In addition, significant decreases in drought-induced NAD+-malate dehydrogenase activities were only observed in stressed BRS C 2501 plants. Regardless of watering regimes, the total carotenoid, ascorbate and glutathione concentrations were higher in BRS Manicore than in BRS C 2501. In conclusion, BRS Manicore is better able to tolerate drought than BRS C 2501 by triggering multiple antioxidant strategies involved both in reactive oxygen species scavenging and dissipation of excess energy and/or reducing equivalents particularly under severe drought stress.

Closely related fungi employ diverse enzymatic strategies to degrade plant biomass

BackgroundPlant biomass is the major substrate for the production of biofuels and biochemicals, as well as food, textiles and other products. It is also the major carbon source for many fungi and enzymes of these fungi are essential for the depolymerization of plant polysaccharides in industrial processes. This is a highly complex process that involves a large number of extracellular enzymes as well as non-hydrolytic proteins, whose production in fungi is controlled by a set of transcriptional regulators. Aspergillus species form one of the best studied fungal genera in this field, and several species are used for the production of commercial enzyme cocktails.ResultsIt is often assumed that related fungi use similar enzymatic approaches to degrade plant polysaccharides. In this study we have compared the genomic content and the enzymes produced by eight Aspergilli for the degradation of plant biomass. All tested Aspergilli have a similar genomic potential to degrade plant biomass, with the exception of A. clavatus that has a strongly reduced pectinolytic ability. Despite this similar genomic potential their approaches to degrade plant biomass differ markedly in the overall activities as well as the specific enzymes they employ. While many of the genes have orthologs in (nearly) all tested species, only very few of the corresponding enzymes are produced by all species during growth on wheat bran or sugar beet pulp. In addition, significant differences were observed between the enzyme sets produced on these feedstocks, largely correlating with their polysaccharide composition.ConclusionsThese data demonstrate that Aspergillus species and possibly also other related fungi employ significantly different approaches to degrade plant biomass. This makes sense from an ecological perspective where mixed populations of fungi together degrade plant biomass. The results of this study indicate that combining the approaches from different species could result in improved enzyme mixtures for industrial applications, in particular saccharification of plant biomass for biofuel production. Such an approach may result in a much better improvement of saccharification efficiency than adding specific enzymes to the mixture of a single fungus, which is currently the most common approach used in biotechnology.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0285-0) contains supplementary material, which is available to authorized users.

Enzymatic hydrolysate-induced displacement reaction with multifunctional silica beads doped with horseradish peroxidase-thionine conjugate for ultrasensitive electrochemical immunoassay.

A novel (invertase) enzymatic hydrolysate-triggered displacement reaction strategy with multifunctional silica beads, doped with horseradish peroxidase-thionine (HRP-Thi) conjugate, was developed for competitive-type electrochemical immunoassay of small molecular aflatoxin B1 (AFB1). The competitive-type displacement reaction was carried out on the basis of the affinity difference between enzymatic hydrolysate (glucose) and its analogue (dextran) for concanavalin A (Con A) binding sites. Initially, thionine-HRP conjugates were doped into nanometer-sized silica beads using the reverse micelle method. Then monoclonal anti-AFB1 antibody and Con A were covalently conjugated to the silica beads. The immunosensor was prepared by means of immobilizing the multifunctional silica beads on a dextran-modified sensing interface via the dextran-Con A binding reaction. Gold nanoparticles functionalized with AFB1-bovine serum albumin conjugate (AFB1-BSA) and invertase were utilized as the trace tag. Upon target AFB1 introduction, a competitive-type immunoreaction was implemented between the analyte and the labeled AFB1-BSA on the nanogold particles for the immobilized anti-AFB1 antibody on the electrode. The invertase followed by gold nanoparticles hydrolyzed sucrose into glucose and fructose. The produced glucose displaced the multifunctional silica beads from the electrode based on the classical dextran-Con A-glucose system, thus decreasing the catalytic efficiency of the immobilized HRP on the electrode relative to that of the H2O2-thionine system. Under optimal conditions, the detectable electrochemical signal increased with the increasing target AFB1 in a dynamic working range from 3.0 pg mL(-1) to 20 ng mL(-1) with a detection limit of 2.7 pg mL(-1). The strong bioconjugation with two nanostructures also resulted in a good repeatability and interassay precision down to 9.3%. Finally, the methodology was further validated for analysis of naturally contaminated or spiked AFB1 peanut samples, giving results matched well with those from a commercialized AFB1 enzyme-linked immunosorbent assay kit. Importantly, the system provides a signal-on competitive-type immunosensing platform for ultrasensitive detection of small molecules.

Characterization and identification of the xylanolytic enzymes from Aspergillus fumigatus Z5.

BackgroundPlant biomass, the most abundant natural material on earth, represents a vast source of food and energy in nature. As the main component of plant biomass, xylan is a complex polysaccharide comprising a linear β(1,4)-linked backbone of xylosyl residues substituted by acetyl, arabinosyl, glucuronysyl and 4-O-methylglucuronycyl residues.ResultsAspergillus fumigatus Z5 is an efficient plant biomass depolymerization fungus. In this study, its crude xylanolytic enzymes were characterized and identified by two-dimensional gel electrophoresis (2-DE). The optimal temperature for the crude xylanases was close to 60 °C, the highest xylanase activity was achieved at pH ranged from 3 to 6, and the crude xylanases also showed a very broad region of pH (3–11) stability. The maximal xylanase activity of 21.45 U · ml−1 was observed in the fourth day of cultivation at 50 °C and 150 rpm with 2 % xylan as the sole carbon source. Zymogram analysis indicated that there were more than seven secreted proteins with xylanase activity. In the crude enzyme, two major endoxylanases, five cellulases and several associated enzymes were identified to be involved in the hydrolysis of polysaccharides. Of the total 13 xylanase genes in the Z5 genome, 11 were observed using q-PCR to be induced by xylan, one of which, An endo-1,4-β-xylanase with a low secretion level, was also expressed and characterized. The final hydrolysis products of xylan by crude enzyme mainly consisted of xylobiose.ConclusionsThis study provides a comprehensive understanding of the depolymerization of xylan by Z5 and will help to design enzymatic strategies for plant biomass utilization.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0463-z) contains supplementary material, which is available to authorized users.

Optimized sample preparation for single-molecule localization-based superresolution microscopy in yeast.

Single-molecule localization-based superresolution microscopy methods allow the resolution of cellular structures in the range of tens of nanometers. However, these techniques are of limited use in current yeast labeling protocols, owing to problems with structural preservation. Here we describe an optimized sample preparation protocol that enables single-molecule localization microscopy at high resolution combined with improved structural preservation in Saccharomyces cerevisiae. The protocol uses small binders called nanobodies and an enzymatic labeling strategy to deliver organic dyes to the target protein. These small binders readily penetrate through the yeast cell wall and thus eliminate the requirement for its prior degradation, and they allow structural preservation. In addition, the small size of the binders reduces the distance of the dye to the target protein, and thus it reduces the localization error. The preparation of S. cerevisiae cells for superresolution imaging takes 2-4 h to perform. Researchers should have skills in yeast molecular biology, immunolabeling techniques and access to a microscope equipped for single-molecule imaging.

ChemInform Abstract: Multi‐Step Synthesis Strategies Towards 1,2‐Amino Alcohols with Special Emphasis on Phenylpropanolamines

AbstractReview: 64 refs.

Intracellular Disassembly of Self-Quenched Nanoparticles Turns NIR Fluorescence on for Sensing Furin Activity in Cells and in Tumors

There has been no report on enzyme-controlled disassembly of self-quenched NIR fluorescent nanoparticles turning fluorescence on for specific detection/imaging of the enzyme's activity in vitro and in vivo. Herein, we reported the rational design of new NIR probe 1 whose fluorescence signal was self-quenched upon reduction-controlled condensation and subsequent assembly of its nanoparticles (i.e., 1-NPs). Then disassembly of 1-NPs by furin turned the fluorescence on. Employing this enzymatic strategy, we successfully applied 1-NPs for NIR detection of furin in vitro and NIR imaging furin activity in living cells. Moreover, we also applied 1-NPs for discriminative NIR imaging of MDA-MB-468 tumors in nude mice. This NIR probe 1 might be further developed for tumor-targeted imaging in routine preclinical studies or even in patients in the future.

Biocatalytic Approach for the Synthesis of Enantiopure Acebutolol as a β₁-Selective Blocker.

A new chemoenzymatic route is reported to synthesize acebutolol, a selective β1 adrenergic receptor blocking agent in enantiopure (R and S) forms. The enzymatic kinetic resolution strategy was used to synthesize enantiopure intermediates (R)- and (S)-N-(3-acetyl-4-(3-chloro-2-hydroxypropoxy)phenyl)butyramide from the corresponding racemic alcohols. The results showed that out of eleven commercially available lipase preparations, two enzyme preparations (Lipase A, Candida antarctica, CLEA [CAL CLEA] and Candida rugosa lipase, 62316 [CRL 62316]) act in enantioselective manner. Under optimized conditions the enantiomeric excess of both (R)- and (S)-N-(3-acetyl-4-(3-chloro-2-hydroxypropoxy)phenyl)butyramide were 99.9 and 96.8%, respectively. N-alkylation of both the (R) and (S) intermediates with isopropylamine gave enantiomerically pure (R and S)- acebutolol with a yield 68 and 72%, respectively. This study suggests a high yielding, easy and environmentally green approach to synthesize enantiopure acebutolol.

CAP+ selection: A combined chemical–enzymatic strategy for efficient eukaryotic messenger RNA enrichment via the 5′ cap

Eukaryotic messenger RNAs (mRNAs) are generally enriched using oligo(dT) selection. However, a significant fraction of mRNAs contain either short or no poly(A). Our technique permits the isolation of mRNAs via their unique biochemical feature, the 5′ cap. It involves RNA extraction, blocking of the 3′ ribose cis–diol by cordycepin, oxidation of the 5′ cis–diol of the CAP to a dialdehyde, coupling to a biotinylated linker, and enrichment on a streptavidin affinity matrix. We demonstrate that it efficiently pulls out a synthetic capped and non-polyadenylated transcript used to spike total cell RNA as well as endogenous histone 3c mRNA reported to be poly(A) negative.

Novel chemo-enzymatic route to a key intermediate for the taxol side-chain through enantioselective O-acylation. Unexpected acyl migration

A new enzymatic strategy has been devised for the preparation of a key intermediate for the taxol side-chain from N-hydroxymethylated cis-3-acetoxy-4-phenylazetidin-2-one. Burkholderia cepacia (PS-IM)-catalyzed acylation of the primary OH group with an excess of vinyl butyrate (10equiv.) furnished the corresponding diester and unreacted monoester with excellent enantiomeric excess values (ee≥98%). Traces of undesirable enantiomeric diacetylated product and diol due to intramolecular acyl migration (∼6% mole fractions) were also detected. Finally, (2R,3S)-3-phenylisoserine hydrochloride (ee=99%), a key intermediate for the taxol side-chain, was prepared from the corresponding diester enantiomer through acidic hydrolysis.

Antimicrobial lubricant formulations containing poly(hydroxybenzene)-trimethoprim conjugates synthesized by tyrosinase.

Poly(hydroxybenzene)-trimethoprim conjugates were prepared using methylparaben as substrate of the oxidative enzyme tyrosinase. MALDI-TOF MS analysis showed that the enzymatic oxidation of methylparaben alone leads to the poly(hydroxybenzene) formation. In the presence of trimethoprim, the methylparaben tyrosinase oxidation leads poly(hydroxybenzene)-trimethoprim conjugates. All of these compounds were incorporated into lubricant hydroxyethyl cellulose/glycerol mixtures. Poly(hydroxybenzene)-trimethoprim conjugates were the most effective phenolic structures against the bacterial growth reducing by 96 and 97% of Escherichia coli and Staphylococcus epidermidis suspensions, respectively (after 24 h). A novel enzymatic strategy to produce antimicrobial poly(hydroxybenzene)-antibiotic conjugates is proposed here for a wide range of applications on the biomedical field.

Introducing biobased ionic liquids as the nonaqueous media for enzymatic synthesis of phosphatidylserine.

Biobased ionic liquids with cholinium as the cation and amino acids as the anions, which could be prepared from renewable biomaterials by simple neutralization reactions, have recently been described as promising and green solvents. Herein, they were successfully used as the reaction media for enzyme-mediated transphosphatidylation of phosphatidylcholine with l-serine for phosphatidylserine synthesis for the first time. Our results indicated that the highest phosphatidylserine yield of 86.5% was achieved. Moreover, 75% original activity of the enzyme was maintained after being used for 10 batches. The present work could be considered an alternative enzymatic strategy for preparing phosphatidylserine. Additionally, the excellent results make the biobased ionic liquids more promising candidates for use as environmentally friendly solvents in biocatalysis fields.

Multi-step synthesis strategies towards 1,2-amino alcohols with special emphasis on phenylpropanolamines

Chiral vicinal amino alcohols are molecules with broad applications in the pharmaceutical as well as in the chemical industry. Due to their high potential, various multi-step chemical, chemo-enzymatic and enzymatic reaction synthesis strategies have been developed within the last decades. This review summarises the asymmetric synthetic routes towards vicinal amino alcohols in general and provides exemplary in-depth looks into multi-step phenylpropanolamine synthesis with special focus on recently published cascade approaches.

Enzymatic strategies and biocatalysts for amide bond formation: tricks of the trade outside of the ribosome.

Amide bond-containing (ABC) biomolecules are some of the most intriguing and functionally significant natural products with unmatched utility in medicine, agriculture and biotechnology. The enzymatic formation of an amide bond is therefore a particularly interesting platform for engineering the synthesis of structurally diverse natural and unnatural ABC molecules for applications in drug discovery and molecular design. As such, efforts to unravel the mechanisms involved in carboxylate activation and substrate selection has led to the characterization of a number of structurally and functionally distinct protein families involved in amide bond synthesis. Unlike ribosomal synthesis and thio-templated synthesis using nonribosomal peptide synthetases, which couple the hydrolysis of phosphoanhydride bond(s) of ATP and proceed via an acyl-adenylate intermediate, here we discuss two mechanistically alternative strategies: ATP-dependent enzymes that generate acylphosphate intermediates and ATP-independent transacylation strategies. Several examples highlighting the function and synthetic utility of these amide bond-forming strategies are provided.

Development of an enzymatic pretargeting strategy for dual-modality imaging.

A pretargeted imaging strategy based on the HaloTag dehalogenase enzyme is described. Here, a HaloTag-Trastuzumab conjugate has been used as the primary agent targeting HER2 expression, and three new radiolabelled HaloTag ligands have been used as secondary agents, two of which offer dual-modality (SPECT/optical) imaging capability.

Label-free, isothermal and ultrasensitive electrochemical detection of DNA and DNA 3'-phosphatase using a cascade enzymatic cleavage strategy.

Label-free and ultrasensitive electrochemical assays of target DNA and T4 polynucleotide kinase phosphatase (PNKP) were developed, which took full advantage of three enzymes to realize the signal readout and amplification. It can ultimately achieve the low detection limits of 10 fM and 1 mU mL(-1) for target DNA and PNKP, respectively.

PNGase F-mediated incorporation of18O into glycans for relative glycan quantitation

PNGase F-catalyzed glycosylation site (18)O-labeling is a widely used method for glycoprotein quantitation owing to its efficiency and simplicity. However, PNGase F-catalyzed glycan (18)O-labeling, which offers advantages for glycomics, has not yet been developed. In this study, PNGase F-mediated incorporation of (18)O into glycans during the N-glycan release from glycoproteins by PNGase F was finally realized, named as PCGOL (PNGase F-catalyzed glycan (18)O-labeling), which offers a potential strategy for relative glycan quantitation. This new method showed good linearity and high reproducibility within at least 2 orders of magnitude in the dynamic range. Furthermore, PCGOL combined with our previously developed TOSIL method (tandem (18)O stable isotope labeling for N-glycoproteome quantitation) can be used for comprehensive N-glycosylation quantification, achieving simultaneous quantification of glycans, glycopeptides and glycoproteins in a single workflow, which was also used to analyze glycosylation changes in immunoglobulin G (IgG) associated with hepatocellular carcinoma in the present work.

Effect of commercial cellulases and refining on kraft pulp properties: Correlations between treatment impacts and enzymatic activity components

The importance of enzymes as biotechnological catalysts for paper industry is now recognized. In this study, five cellulase formulations were used for fibre modification. The number of PFI revolutions decreased by about 50% while achieving the same freeness value (decrease in CSF by 200mL) with the enzymatic pretreatment. The physical properties of handsheets were modified after enzymatic pretreatment followed by PFI refining. A slight decrease in tear strength was observed with enzymes C1 and C4 at pH 7 while the most decrease in tear was observed after C2, C3, C5 treatments. C1 and C4 which had xylanase activity improved paper properties, while other enzymes had a negative impact. Therefore, the intricate balance between cellulolytic and hemicellulolytic activity is the key to optimizing biorefining and paper properties. It was also observed that C1 impact was pH dependent, which supports the importance of pH in developing an enzymatic strategy for refining energy reduction.

A tandem enzymatic approach for detecting and imaging tumor-associated Thomsen-Friedenreich antigen disaccharide.

The disaccharide galactose-β1,3-N-acetylgalactosamine (Galβ1,3-GalNAc) attached to serine and/or threonine residues of proteins, also known as the Thomsen-Friedenreich (TF) antigen, is highly expressed in various types of human carcinomas. It has been shown to contribute to tumor development, progression, and metastasis. However, current methods have limited power in detecting and imaging TF antigens among a variety of complex cell-surface glycans. Here we describe a tandem enzymatic strategy to detect and label TF antigen disaccharide with high sensitivity and selectivity. We demonstrate that this strategy enables detection of TF antigens on proteins, profiling and identification of unknown TF antigen-modified glycoproteins, and simultaneous labeling of multiple forms of complex glycan motifs on the same cell. This approach expands the capability of glycan labeling to probe the functional role of TF antigens in cancer biology.