Sorbate metal complexes as newer antibacterial, antibiofilm, and anticancer compounds.

BACKGROUND: The ineffectiveness of treatments for infections caused by biofilm-producing pathogens and human carcinoma presents considerable challenges for global public health organizations. To tackle this issue, our study focused on exploring the potential of synthesizing new complexes of Co(II), Cu(II), Ni(II), and Zn(II) with sorbic acid to enhance its antibacterial, antibiofilm, and anticancer properties. METHODS: Four novel complexes were synthesized as solid phases by reacting sorbic acid with Co(II), Cu(II), Ni(II), and Zn(II). These complexes were characterized by various technique, including infrared spectra, UV-Visible spectroscopy, proton nuclear magnetic resonance (1H NMR), and thermal analysis techniques, including thermogravimetry (TG). RESULTS: The data acquired from all investigated chemical characterization methods confirmed the chemical structure of the sorbate metal complexes. These complexes exhibited antibacterial and antibiofilm properties against both Gram-positive and Gram-negative bacteria. Furthermore, these complexes enhanced the antibacterial effects of commonly used antibiotics, such as gentamicin and imipenem, with fractional inhibitory concentration (FIC) indices ≤ 0.5. Notably, the Cu(II) complex displayed the most potent antibacterial and antibiofilm activities, with minimum inhibitory concentration (MIC) values of 312.5 µg/mL and 625.0 µg/mL for Bacillus cereus and Escherichia coli, respectively. Additionally, in vitro assays using the methyl thiazolyl tetrazolium (MTT) method showed inhibitory effects on the growth of the human colon carcinoma cell line (HCT-116 cells) following treatment with the investigated metal complexes. The IC50 values for Co(II), Cu(II), Zn(II), and Ni(II) were 3230 µg/mL, 2110 µg/mL, 3730 µg/mL, and 2240 µg/mL, respectively. CONCLUSION: Our findings offer potential for pharmaceutical companies to explore the development of novel combinations involving traditional antibiotics or anticancer drugs with sorbate copper complex.

Sustainable biocatalytic synthesis of substituted muconic acids.

The development of sustainable routes to organic building blocks is a critical endeavor for reducing the environmental impact of chemical synthesis. Biocatalysts are poised to play an important role in sustainable synthesis, as they perform highly selective reactions under mild conditions. The application of enzymes to organic synthesis requires an approach which is operationally simple, inexpensive to prepare, and reasonably scalable. In this work, we demonstrated the utility of a Type I ring-cleaving dioxygenase CatA (P. putida KT2440) for preparative-scale synthesis of muconic acid derivatives. Muconic acids are important precursors in the synthesis of polymers and commodity chemicals. In this work, we optimized the performance of CatA under millimolar substrate concentrations and characterized the activity of the enzyme with an array of catechol substrates. Furthermore, we developed a scalable platform using cellular lysates to produce diverse muconic acids, generating up to a gram of the desired product. A simple trituration procedure was utilized for the purification of these muconic acids that obviated the need for chromatographic purification and reduced overall solvent waste.

An integrated yeast-based process for cis,cis-muconic acid production.

Cis,cis-muconic acid (CCM) is a promising polymer building block. CCM can be made by whole-cell bioconversion of lignin hydrolysates or de novo biosynthesis from sugar feedstocks using engineered microorganisms. At present, however, there is no established process for large-scale CCM production. In this study, we developed an integrated process for manufacturing CCM from glucose by yeast fermentation. We systematically engineered the CCM-producing Saccharomyces cerevisiae strain by rewiring the shikimate pathway flux and enhancing phosphoenolpyruvate supply. The engineered strain ST10209 accumulated less biomass but produced 1.4 g/L CCM (70 mg CCM per g glucose) in microplate assay, 71% more than the previously engineered strain ST8943. The strain ST10209 produced 22.5 g/L CCM in a 2 L fermenter with a productivity of 0.19 g/L/h, compared to 0.14 g/L/h achieved by ST8943 in our previous report under the same fermentation conditions. The fermentation process was demonstrated at pilot scale in 10 and 50 L steel tanks. In 10 L fermenter, ST10209 produced 20.8 g/L CCM with a CCM yield of 0.1 g/g glucose and a productivity of 0.21 g/L/h, representing the highest to-date CCM yield and productivity. We developed a CCM recovery and purification process by treating the fermentation broth with activated carbon at low pH and low temperature, achieving an overall CCM recovery yield of 66.3% and 95.4% purity. In summary, we report an integrated CCM production process employing engineered S. cerevisiae yeast.

A Nanoprobe Based on Gated Mesoporous Silica Nanoparticles for The Selective and Sensitive Detection of Benzene Metabolite t,t-Muconic Acid in Urine.

Benzene is a highly toxic aromatic hydrocarbon. Inhaling benzene can cause dizziness, vertigo, headaches, aplasia, mutations and, in the most extreme cases, cancer. Trans,trans-muconic acid (t,t-MA) is one of the metabolization products of benzene. Although different analytical methods have been reported for the determination of t,t-MA, these are often expensive, require trained personnel, are not suitable for on-site measurements, and use hazardous organic solvents. For these reasons, the development of reliable, selective and sensitive methods for rapid and in situ detection of t,t-MA are of importance. Addressing this challenge, a nanodevice for the selective and sensitive quantification of t,t-MA in urine is reported. The nanodevice used is achieved using mesoporous silica nanoparticles loaded with a dye reporter and capped with a dicopper(II) azacryptand. Pore opening and payload release is induced rapidly (10 min) and selectively with t,t-MA in urine, using a simple fluorimeter without sample pretreatment.

Assessment of Concentrated Liquid Coffee Acceptance during Storage: Sensory and Physicochemical Perspective.

Concentrated liquid coffees (CLCs) refer to stored extracts stable at environmental temperature, used as ingredients in the retail market. Their low chemical stability affects the sensory profile. This study was performed in two CLCs, one without additives (BIB) and another with a mix of sodium benzoate and potassium sorbate additives (SD), stored at 25 °C for one year. Quantitative-Descriptive (QDA) and discriminant analyses permitted identifying the critical sensory attributes and their evolution over time. The concentrate without additives presented an acceptance limit of 196 days (evaluated at a 50% acceptance ratio), while the additives increased the shelf life up to 226 days (38.9% improvement). The rejection was related to a decreased aroma, increased acidity, and reduced bitterness. A bootstrapped feature selection version of Partial Least Square analysis further demonstrated that reactions of 5-caffeoylquinic acid (5CQA) and 3,5-dicaffeoylquinic acid (3,5diCQA) could cause changes in the aroma at the first degradation stage. In the following stages, changes in fructose and stearic acid contents, a key indicator of acceptance for both extracts possibly related to non-enzymatic reactions involving fructose and other compounds, might affect the bitterness and acidity. These results provided valuable information to understand flavor degradation in CLCs.

Properties and Application of Multifunctional Composite Polypropylene-Based Films Incorporating a Combination of BHT, BHA and Sorbic Acid in Extending Donut Shelf-Life.

To extend the shelf-life of packaged donut without the addition of preservative, polypropylene-based active composite films loaded with a combination of sorbic acid, BHA and BHT were prepared by the extrusion moulding method: T1 (Control-pure PP-film), T2 (PP-BHT1%-SA2%), T3 (PP-BHA3%-SA2%) and T4 (PP-BHT1%-BHA1%-SA2%). The incorporation of active additives enhanced water vapour permeability (WVP) and increased oxygen permeability of films. Active films had higher antioxidant activity than pure PP in the order T4 > T2 > T3 (89.11, 83.40 and 79.16%). In vitro examinations demonstrated a significant antibacterial effect on Escherichia coli and S. aureus growth. Overall migration was not significantly different for watery food simulants, while in acidic and fatty foods increased it significantly. The effect of the active films on the fried and packaged donut samples showed significantly higher moisture contents and peroxide values, while acidity was lower. T2 film is proposed due to the preservation of the intrinsic properties of the film, increasing the storage period up to 25 to 50 days.

Effect of Potassium Sorbate and Ultrasonic Treatment on the Properties of Fish Scale Collagen/Polyvinyl Alcohol Composite Film.

Composite films containing different amounts of potassium sorbate (KS) were prepared by using fish scale collagen (Col) and polyvinyl alcohol (PVA). Fourier transform infrared spectroscopy (FTIR), light transmittance, mechanical, water vapor transmission rate (WVTR), and the antibacterial properties of the composite films were analyzed. The results showed that the addition of Col significantly reduced the light transmittance of the composite film, but KS had no significant effect on the light transmission. The tensile strength decreased first and then increased with the addition of KS, while the WVTR increased first and then decreased. The composite film exhibited a certain degree of antibacterial properties against E. coli and S. aureus. In addition, we found that ultrasonic treatment reduced the WVTR, and also improved tensile strength and elongation at break of the composite films, but had no significant effect on other properties. The KS/Col/PVA films have the potential to be used as antimicrobial food packaging.

Characterization of a non-phosphotransferase system for cis,cis-muconic acid production in Corynebacterium glutamicum.

Cis,cis-muconic acid (CCM) is a biochemical material that can be used for the production of various plastics and polymers and is particularly gaining attention as an adipic acid precursor for the synthesis of nylon-6,6. In the current study, the production of CCM was first attempted by introducing a newly developed protocatechuate (PCA) decarboxylase from Corynebacterium glutamicum 13032 to inha103, which completed the biosynthetic pathway therein. To improve CCM productivity, a phosphoenol pyruvate (PEP)-dependent phosphotransferase system (PTS) that consumed the existing glucose was developed, in the form of a strain with a non-PTS that did not consume PEP. To improve glucose uptake, we developed P25 strain, in which iolR (a transcriptional regulator gene) was additionally deleted. Strain P28, a P25 derivative expressing PCA decarboxylase, produced 4.01 g/L of CCM, which was 14% more than that produced by the parental strain. Moreover, strains P29 and P30, with an active pentose phosphate pathway and overexpressing important genes (qsuB) in the metabolic pathway, produced 4.36 and 4.5 g/L of CCM, respectively. Particularly, the yield per glucose in strain P30 was similar to that of the fed-batch culture of Escherichia coli, which has the highest reported yield of 22% (mol/mol). These results are underpinned by the characteristics of the non-PTS with increased PEP availability and a strain with deletion of the iolR gene, which greatly increased glucose uptake.

An expanded enzyme toolbox for production of cis, cis-muconic acid and other shikimate pathway derivatives in Saccharomyces cerevisiae.

A wide range of commercially relevant aromatic chemicals can be synthesized via the shikimic acid pathway. Thus, this pathway has been the target of diverse metabolic engineering strategies. In the present work, an optimized yeast strain for production of the shikimic acid pathway intermediate 3-dehydroshikimate (3-DHS) was generated, which is a precursor for the production of the valuable compounds cis, cis-muconic acid (CCM) and gallic acid (GA). Production of CCM requires the overexpression of the heterologous enzymes 3-DHS dehydratase AroZ, protocatechuic acid (PCA) decarboxylase AroY and catechol dioxygenase CatA. The activity of AroY limits the yield of the pathway. This repertoire of enzymes was expanded by a novel fungal decarboxylase. Introducing this enzyme into the pathway in the optimized strain, a titer of 1244 mg L-1 CCM could be achieved, yielding 31 mg g-1 glucose. This represents the highest yield of this compound reported in Saccharomyces cerevisiae to date. To demonstrate the applicability of the optimized strain for production of other compounds from 3-DHS, we overexpressed AroZ together with a mutant of a para-hydroxybenzoic acid hydroxylase with improved substrate specificity for PCA, PobAY385F. Thereby, we could demonstrate the production of GA for the first time in S. cerevisiae.

QM/MM study of the reaction mechanism of Cl-cis,cis-muconate with muconate lactonizing enzyme.

The lactonization process of Cl-cis,cis-muconate catalyzed by anti-muconate lactonizing enzyme (anti-MLE) was studied theoretically with the aid of a combined quantum mechanics/molecular mechanics (QM/MM) approach. Two elementary processes steps involved in the lactanization process were investigated. The calculated energy barriers agree well with the experimental values. The present work provided the explicit structures of the enolate anion intermediates. The electrostatic influence analysis highlighted residues Arg51, Gln294 and TIP383 for the MLE-Cl-2 system and the residue Asn193 for the MLE-Cl-4 system as the possible mutation targets for rational design of anti-MLE in future enzyme modification.

Ion-pair-based hollow-fiber liquid-phase microextraction combined with high-performance liquid chromatography for the simultaneous determination of urinary benzene, toluene, and styrene metabolites.

In the current study, a novel technique for extraction and determination of trans,trans-muconic acid, hippuric acid, and mandelic acid was developed by means of ion-pair-based hollow fiber liquid-phase microextraction in the three-phase mode. Important factors affecting the extraction efficiency of the method were investigated and optimized. These metabolites were extracted from 10 mL of the source phase into a supported liquid membrane containing 1-octanol and 10% w/v of Aliquat 336 as the ionic carrier followed by high-performance liquid chromatography analysis. The organic phase immobilized in the pores of a hollow fiber was back-extracted into 24 µL of a solution containing 3.0 mol/L sodium chloride placed inside the lumen of the fiber. A very high preconcentration of 212- to 440-fold, limit of detection of 0.1-7 µg/L, and relative recovery of 87-95% were obtained under the optimized conditions of this method. The relative standard deviation values for within-day and between-day precisions were calculated at 2.9-8.5 and 4.3-11.2%, respectively. The method was successfully applied to urine samples from volunteers at different work environments. The results demonstrated that the method can be used as a sensitive and effective technique for the determination of the metabolites in urine.

Structure-activity relationship investigation of tertiary amine derivatives of cinnamic acid as acetylcholinesterase and butyrylcholinesterase inhibitors: compared with that of phenylpropionic acid, sorbic acid and hexanoic acid.

In the present investigation, 48 new tertiary amine derivatives of cinnamic acid, phenylpropionic acid, sorbic acid and hexanoic acid (4d-6g, 10d-12g, 16d-18g and 22d-24g) were designed, synthesized and evaluated for the effect on AChE and BChE in vitro. The results revealed that the alteration of aminoalkyl types and substituted positions markedly influences the effects in inhibiting AChE. Almost of all cinnamic acid derivatives had the most potent inhibitory activity than that of other acid derivatives with the same aminoalkyl side chain. Unsaturated bond and benzene ring in cinnamic acid scaffold seems important for the inhibitory activity against AChE. Among them, compound 6g revealed the most potent AChE inhibitory activity (IC50 value: 3.64 µmol/L) and highest selectivity over BChE (ratio: 28.6). Enzyme kinetic study showed that it present a mixed-type inhibition against AChE. The molecular docking study suggested that it can bind with the catalytic site and peripheral site of AChE.

Kinetic insights into ϵ-caprolactone synthesis: Improvement of an enzymatic cascade reaction.

A computational approach for the simulation and prediction of a linear three-step enzymatic cascade for the synthesis of ϵ-caprolactone (ECL) coupling an alcohol dehydrogenase (ADH), a cyclohexanone monooxygenase (CHMO), and a lipase for the subsequent hydrolysis of ECL to 6-hydroxyhexanoic acid (6-HHA). A kinetic model was developed with an accuracy of prediction for a fed-batch mode of 37% for substrate cyclohexanol (CHL), 90% for ECL, and >99% for the final product 6-HHA. Due to a severe inhibition of the CHMO by CHL, a batch synthesis was shown to be less efficient than a fed-batch approach. In the fed-batch synthesis, full conversion of 100 mM CHL was 28% faster with an analytical yield of 98% compared to 49% in case of the batch synthesis. The lipase-catalyzed hydrolysis of ECL to 6-HHA circumvents the inhibition of the CHMO by ECL enabling a 24% higher product concentration of 6-HHA compared to ECL in case of the fed-batch synthesis without lipase. Biotechnol. Bioeng. 2017;114: 1215-1221. © 2017 Wiley Periodicals, Inc.

Preservation effect of meat product by natural antioxidant tea polyphenol.

1% of tea polyphenol, chitosan solution and potassium sorbate were used as film-forming materials to coat chilled mutton. Total coliforms, TVB-N value and pH value were determined and used as the mutton fresh-keeping indexes. The results showed that after 12th day at the end of the storage, mutton coated with tea polyphenol had best effects comparing chitosan solution and potassium sorbate. pH value of mutton coated by tea polyphenol was 6.0, TVB-N and the total coliforms were both significantly lower than the meat coated by chitosan solution and potassium sorbate. Also, mutton coated by tea polyphenol accorded with the requirements of national standards about fresh meat quality. In summary, the tea polyphenol film was the most suitable film on chilled mutton coating preservation among the three chemicals used in this research.

Structural basis for the substrate specificity and the absence of dehalogenation activity in 2-chloromuconate cycloisomerase from Rhodococcus opacus 1CP.

2-Chloromuconate cycloisomerase from the Gram-positive bacterium Rhodococcus opacus 1CP (Rho-2-CMCI) is an enzyme of a modified ortho-pathway, in which 2-chlorophenol is degraded using 3-chlorocatechol as the central intermediate. In general, the chloromuconate cycloisomerases catalyze not only the cycloisomerization, but also the process of dehalogenation of the chloromuconate to dienelactone. However Rho-2-CMCI, unlike the homologous enzymes from the Gram-negative bacteria, is very specific for only one position of the chloride on the substrate chloromuconate. Furthermore, Rho-2-CMCI is not able to dehalogenate the 5-chloromuconolactone and therefore it cannot generate the dienelactone. The crystallographic structure of the homooctameric Rho-2-CMCI was solved by molecular replacement using the coordinates of the structure of chloromuconate cycloisomerase from Pseudomonas putida PRS2000. The structure was analyzed and compared to the other already known structures of (chloro)muconate cycloisomerases. In addition to this, molecular docking calculations were carried out, which allowed us to determine the residues responsible for the high substrate specificity and the lack of dehalogenation activity of Rho-2-CMCI. Our studies highlight that a histidine, located in a loop that closes the active site cavity upon the binding of the substrate, could be related to the dehalogenation inability of Rho-2-CMCI and in general of the muconate cycloisomerases.

Engineering E. coli-E. coli cocultures for production of muconic acid from glycerol.

BACKGROUND: cis, cis-Muconic acid is an important chemical that can be biosynthesized from simple substrates in engineered microorganisms. Recently, it has been shown that engineering microbial cocultures is an emerging and promising approach for biochemical production. In this study, we aim to explore the potential of the E. coli-E. coli coculture system to use a single renewable carbon source, glycerol, for the production of value-added product cis, cis-muconic acid. RESULTS: Two coculture engineering strategies were investigated. In the first strategy, an E. coli strain containing the complete biosynthesis pathway was co-cultivated with another E. coli strain containing only a heterologous intermediate-to-product biosynthetic pathway. In the second strategy, the upstream and downstream pathways were accommodated in two separate E. coli strains, each of which was dedicated to one portion of the biosynthesis process. Compared with the monoculture approach, both coculture engineering strategies improved the production significantly. Using a batch bioreactor, the engineered coculture achieved a 2 g/L muconic acid production with a yield of 0.1 g/g. CONCLUSIONS: Our results demonstrate that coculture engineering is a viable option for producing muconic acid from glycerol. Moreover, microbial coculture systems are shown to have the potential for converting single carbon source to value-added products.

Probing Photosensitization by Functionalized Carbon Nanotubes.

Carbon nanotubes (CNTs) photosensitize the production of reactive oxygen species that may damage organisms by biomembrane oxidation or mediate environmental transformations of CNTs. Photosensitization by derivatized carbon nanotubes from various synthetic methods, and thus with different intrinsic characteristics (e.g., diameter and electronic properties), has been investigated under environmentally relevant aquatic conditions. We used the CNT-sensitized photoisomerization of sorbic acid ((2E,4E)-hexa-2,4-dienoic acid) and singlet oxygen formation to quantify the triplet states ((3)CNT*) formed upon irradiation of selected single-walled carbon nanotubes (SWCNTs) and multiwalled carbon nanotubes (MWCNTs). The CNTs used in our studies were derivatized by carboxyl groups to facilitate their dispersion in water. Results indicate that high-defect-density (thus well-stabilized), small-diameter, and semiconducting-rich CNTs have higher-measured excited triplet state formation and therefore singlet oxygen ((1)O2) yield. Derivatized SWCNTs were significantly more photoreactive than derivatized MWCNTs. Moreover, addition of sodium chloride resulted in increased aggregation and small increases in (1)O2 production of CNTs. The most photoreactive CNTs exhibited comparable photoreactivity (in terms of (3)CNT* formation and (1)O2 yield) to reference natural organic matter (NOM) under sunlight irradiation with the same mass-based concentration. Selected reference NOM could therefore be useful in evaluating environmental photoreactivity or intended antibacterial applications of CNTs.

Complexation induced fluorescence and acid-base properties of dapoxyl dye with γ-cyclodextrin: a drug-binding application using displacement assays.

Host-guest complexation of dapoxyl sodium sulphonate (DSS), an intramolecular charge transfer dye with water-soluble and non-toxic macrocycle γ-cyclodextrin (γ-CD), has been investigated in a wide pH range. Steady-state absorption, fluorescence and time-resolved fluorescence measurements confirm the positioning of DSS into the hydrophobic cavity of γ-CD. A large fluorescence enhancement ca. 30 times, due to 1 : 2 complex formation and host-assisted guest-protonation have been utilised for developing a method for the utilisation of CD based drug-delivery applications. A simple fluorescence-displacement based approach is implemented at physiological pH for the assessment of binding strength of pharmaceutically useful small drug molecules (ibuprofen, paracetamol, methyl salicylate, salicylic acid, aspirin, and piroxicam) and six important antibiotic drugs (resazurin, thiamphenicol, chloramphenicol, ampicillin, kanamycin, and sorbic acid) with γ-CD.

Metabolic engineering of Klebsiella pneumoniae for the production of cis,cis-muconic acid.

cis,cis-Muconic acid (ccMA), a metabolic intermediate of Klebsiella pneumoniae, can be converted to adipic acid and terephthalic acid, which are important monomers of synthetic polymers. However, wild-type K. pneumoniae does not produce ccMA because intracellular carbon flow does not favor ccMA biosynthesis. In this study, several metabolic engineering strategies were used in an attempt to modify the wild-type strain to induce it to produce ccMA. First, by blocking the synthesis of aromatic amino acids, 343 mg/L of catechol, a precursor of ccMA, was produced. Then, the native catechol 1,2-dioxygenasegene (catA) was overexpressed, which caused the strain to convert the catechol to ccMA. The production of ccMA was further improved by deletion of the muconate cycloisomerase gene (catB) and by deleting a feedback inhibitor of the aromatic amino acid pathway. Further improvement was achieved by adjusting the pH of the culture broth. The developed strain produced 2.1 g/L of ccMA in flask cultivation. The results showed the potential of K. pneumoniae as a ccMA producer.

Small-angle X-ray scattering and in silico modeling approaches for the accurate functional annotation of an LysR-type transcriptional regulator.

Xylella fastidiosa is a xylem-limited, Gram-negative phytopathogen responsible for economically relevant crop diseases. Its genome was thus sequenced in an effort to characterize and understand its metabolism and pathogenic mechanisms. However, the assignment of the proper functions to the identified open reading frames (ORFs) of this pathogen was impaired due to a lack of sequence similarity in the databases. In the present work, we used small-angle X-ray scattering and in silico modeling approaches to characterize and assign a function to a predicted LysR-type transcriptional regulator in the X. fastidiosa (XfLysRL) genome. XfLysRL was predicted to be a homologue of BenM, which is a transcriptional regulator involved in the degradation pathway of aromatic compounds. Further functional assays confirmed the structural prediction because we observed that XfLysRL interacts with benzoate and cis,cis-muconic acid (also known as 2E,4E-hexa-2,4-dienedioic acid; hereafter named muconate), both of which are co-factors of BenM. In addition, we showed that the XfLysRL protein is differentially expressed during the different stages of X. fastidiosa biofilm formation and planktonic cell growth, which indicates that its expression responds to a cellular signal that is likely related to the aromatic compound degradation pathway. The assignment of the proper function to a protein is a key step toward understanding the cellular metabolic pathways and pathogenic mechanisms. In the context of X. fastidiosa, the characterization of the predicted ORFs may lead to a better understanding of the cellular pathways that are linked to its bacterial pathogenicity.