Title: Functional characterization and reclassification of an enzyme previously proposed to be a limonoid UDP ‐glucosyltransferase
AbstractBACKGROUND
A major problem in the orange industry is ‘delayed’ bitterness, which is caused by limonin, a bitter compound developing from its non‐bitter precursor limonoate A‐ring lactone (LARL) during and after extraction of orange juice. The glucosidation of LARL by limonoid UDP‐glucosyltransferase (LGT) to form non‐bitter glycosyl‐limonin during orange maturation has been demonstrated as a natural way to debitter by preventing the formation of limonin.
RESULT
Here, the debittering potential of heterogeneously expressed glucosyltransferase, maltose‐binding protein (MBP) fused tocuGT fromCitrus unishiu Marc(MBP‐cuGT), which was previously regarded as LGT, was evaluated. A liquid chromatography – mass spectrometry (LC–MS) method was established to determine the concentration of limonin and its derivatives. The protocols to obtain its potential substrates, LARL and limonoate (limonin with both A and D ring open), were also developed. Surprisingly, MBP‐cuGT did not exhibit any detectable effect on limonin degradation when Navel orange juice was used as the substrate; MBP‐cuGT was unable to biotransform either LARL or limonoate as purified substrates. However, it was found that MBP‐cuGT displayed a broad activity spectrum towards flavonoids, confirming that the enzyme produced was active under the conditions evaluatedin vitro.
Javanshad, Roshan; Taylor, Christopher James; Delavari, Niusha; Barkman, Todd J.; Stull, Frederick; Venter, Andre R.(
, Rapid Communications in Mass Spectrometry)
Rationale
Purification of recombinant proteins is a necessary step for functional or structural studies and other applications. Immobilized metal affinity chromatography is a common recombinant protein purification method. Mass spectrometry (MS) allows for confirmation of identity of expressed proteins and unambiguous detection of enzymatic substrates and reaction products. We demonstrate the detection of enzymes purified on immobilized metal affinity surfaces by direct or ambient ionization MS, and follow their enzymatic reactions by direct electrospray ionization (ESI) or desorption electrospray ionization (DESI).
Methods
A protein standard, His‐Ubq, and two recombinant proteins, His‐SHAN and His‐CS, expressed inEscherichia coliwere immobilized on two immobilized metal affinity systems, Cu–nitriloacetic acid (Cu‐NTA) and Ni‐NTA. The proteins were purified on surface, and released in the ESI spray solvent for direct infusion, when using the 96‐well plate form factor, or analyzed directly from immobilized metal affinity‐coated microscope slides by DESI‐MS. Enzyme activity was followed by incubating the substrates in wells or by depositing substrate on immobilized protein on coated slides for analysis.
Results
Small proteins (His‐Ubq) and medium proteins (His‐SAHN) could readily be detected from 96‐well plates by direct infusion ESI, or from microscope slides by DESI‐MS after purification on surface from clarifiedE. colicell lysate. Protein oxidation was observed for immobilized proteins on both Cu‐NTA and Ni‐NTA; however, this did not hamper the enzymatic reactions of these proteins. Both the nucleosidase reaction products for His‐SAHN and the methylation product of His‐CS (theobromine to caffeine) were detected.
Conclusions
The immobilization, purification, release and detection of His‐tagged recombinant proteins using immobilized metal affinity surfaces for direct infusion ESI‐MS or ambient DESI‐MS analyses were successfully demonstrated. Recombinant proteins were purified to allow identification directly out of clarified cell lysate. Biological activities of the recombinant proteins were preserved allowing the investigation of enzymatic activity via MS.
González-Morales, Maria A.; Thomson, Andrea E.; Yeatts, James; Enomoto, Hiroko; Haija, Ahmed; Santangelo, Richard G.; Petritz, Olivia A.; Crespo, Rocio; Schal, Coby; Baynes, Ronald(
, Parasites & Vectors)
AbstractBackground
Bed bug infestations are re-emerging in the poultry industry throughout the USA. Although the impacts of bed bugs on birds’ health and welfare are poorly understood, adverse outcomes are expected, including stress, anemia, infections and lower production rates. Worker welfare is also an important consideration in commercial poultry farms. A limited number of insecticides are available for use in the complex spatial environment of commercial farms. Systemic drugs have the potential to overcome the limitations of existing pest management tactics. A recent study showed that fluralaner administered to chickens caused high levels of mortality in bed bugs.
Methods
To further understand the efficacy of this approach, we evaluated the pharmacokinetics of an oral solid formulation of fluralaner in 11 chickens and quantified its plasma concentration in chickens using UPLC/MS. We administered fluralaner to chickens with two doses of Bravecto®(each 0.5 mg/kg body mass) via gavage 1 week apart and evaluated its efficacy on bed bugs that fed on medicated chickens for up to 28 days post-treatment.
Results
Bed bugs that fed on fluralaner-treated chickens experienced > 50% mortality within 30 min of the administration of Bravecto and 100% mortality 2 days post-treatment. Mortality slowly declined to 66.6% by day 28. Fluralaner was quantifiable in the hens’ plasma for at least 28 days post-treatment. The treatment resulted in maximal plasma concentrations (Cmax) of 106.4 ng/ml around day 9.0 (Tmax), substantially higher than the LC90, the concentration needed to kill 90% of the bed bugs.
Conclusions
Fluralaner appears to be a promising candidate for bed bug control in poultry farms, with a treatment effect lasting at least 28 days.
Zhao, Hua; Campbell, Chante' D.(
, Journal of Chemical Technology & Biotechnology)
AbstractBACKGROUND
Knoevenagel condensation is an important tool for building carbon–carbon (CC) bonds, especially when catalyzed by enzymes to enable a potentially high chemo‐, regio‐ and/or stereoselectivity. Although many Knoevenagel condensation reactions are carried out in aqueous solutions, insoluble hydrophobic substrates often lead to poor catalytic efficiencies. The use of water‐miscible organic solvents improves the substrate solubilization, but usually induces activity suppression or inactivation of enzymes. There is a great need to develop alternative solvents for both substrate dissolution and enzyme compatibility in CC bond formation reactions.
RESULTS
Our group previously developed dual‐functionalized water‐mimicking ionic liquids (ILs) for the activation and stabilization of hydrolases (e.g. lipase and protease). In the present study, we evaluated the Knoevenagel condensation of 4‐chlorobenzaldehyde with acetylacetone, and found that porcine pancreas lipase in water‐mimicking ILs carrying ammonium, imidazolium and benzimidazolium cations enabled higher reaction rates (up to 3.22 μmol min−1 g−1lipase) and better yields thantert‐butanol, glymes and [BMIM][Tf2N]. Interestingly, tertiary amide solvents such asN‐methyl‐2‐pyrrolidone (NMP),N,N‐dimethylformamide (DMF) andN,N‐dimethylacetamide (DMAc) led to 8.2‐ to 11.1‐fold increases in the initial rate (up to 35.66 μmol min−1 g−1lipase) when compared with dual‐functionalized ILs, which is likely due to some synergistic effect of these tertiary amides with the lipase.
The genomes of broad host range insect pathogenic fungi, includingCordyceps fumosorosea, encode for a suite of secreted proteases implicated in targeting, penetration, and degradation of the host exoskeleton or cuticle. These cuticle‐degrading proteases act as critical virulence factors, but their functions within the biological context, particularly in relation to host specificity, remain poorly characterized.
RESULT
AC. fumosoroseaprotease gene,Cfcdp1, was identified and a targeted gene‐knockout strain constructed. Minor growth defects were observed for theΔCfcdp1strain when compared to the wild‐type parent and complemented (ΔCfcdp1::Cfcdp1) strains, with delayed and decreased sporulation noted for the mutant. Decreased subtilisin‐like protease activity was seen for theΔCfcdp1strain, although total secreted protease activity was similar between the mutant and wild‐type strains. Insect bioassays using whitefly,Bemisia tabaci, and cabbageworm,Pieris rapae, showed decreased infectivity, i.e. 2.4–3.4‐fold increase in lethal dose (LC50) and an increased time to death (LT50), for theΔCfcdp1strain. In contrast, insect bioassays using the diamondback moth,Plutella xylostella, or the brown planthopper,Nilaparvata lugens, showed increased infectivity, i.e. a 3–5‐fold decrease in LC50, and a decreased LT50. Differential effects were also seen on the fecundity ofB. tabaciinfected by the different fungal strains.
Stable isotope probing (SIP) approaches are a critical tool in microbiome research to determine associations between species and substrates, as well as the activity of species. The application of these approaches ranges from studying microbial communities important for global biogeochemical cycling to host-microbiota interactions in the intestinal tract. Current SIP approaches, such as DNA-SIP or nanoSIMS allow to analyze incorporation of stable isotopes with high coverage of taxa in a community and at the single cell level, respectively, however they are limited in terms of sensitivity, resolution or throughput.
Results
Here, we present an ultra-sensitive, high-throughput protein-based stable isotope probing approach (Protein-SIP), which cuts cost for labeled substrates by 50–99% as compared to other SIP and Protein-SIP approaches and thus enables isotope labeling experiments on much larger scales and with higher replication. The approach allows for the determination of isotope incorporation into microbiome members with species level resolution using standard metaproteomics liquid chromatography-tandem mass spectrometry (LC–MS/MS) measurements. At the core of the approach are new algorithms to analyze the data, which have been implemented in an open-source software (https://sourceforge.net/projects/calis-p/). We demonstrate sensitivity, precision and accuracy using bacterial cultures and mock communities with different labeling schemes. Furthermore, we benchmark our approach against two existing Protein-SIP approaches and show that in the low labeling range used our approach is the most sensitive and accurate. Finally, we measure translational activity using18O heavy water labeling in a 63-species community derived from human fecal samples grown on media simulating two different diets. Activity could be quantified on average for 27 species per sample, with 9 species showing significantly higher activity on a high protein diet, as compared to a high fiber diet. Surprisingly, among the species with increased activity on high protein were severalBacteroidesspecies known as fiber consumers. Apparently, protein supply is a critical consideration when assessing growth of intestinal microbes on fiber, including fiber-based prebiotics.
Conclusions
We demonstrate that our Protein-SIP approach allows for the ultra-sensitive (0.01 to 10% label) detection of stable isotopes of elements found in proteins, using standard metaproteomics data.
Cui, Youtian, Allmon, Steven D., and Siegel, Justin B. Functional characterization and reclassification of an enzyme previously proposed to be a limonoid UDP ‐glucosyltransferase. Journal of the Science of Food and Agriculture 100.13 Web. doi:10.1002/jsfa.10547.
Cui, Youtian, Allmon, Steven D., & Siegel, Justin B. Functional characterization and reclassification of an enzyme previously proposed to be a limonoid UDP ‐glucosyltransferase. Journal of the Science of Food and Agriculture, 100 (13). https://doi.org/10.1002/jsfa.10547
Cui, Youtian, Allmon, Steven D., and Siegel, Justin B.
"Functional characterization and reclassification of an enzyme previously proposed to be a limonoid UDP ‐glucosyltransferase". Journal of the Science of Food and Agriculture 100 (13). Country unknown/Code not available: Wiley Blackwell (John Wiley & Sons). https://doi.org/10.1002/jsfa.10547.https://par.nsf.gov/biblio/10456876.
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