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It is very popular to fuse a protein drug or drug candidate to the Fc domain of immunoglobulin G (IgG) in order to prolong the in vivo half-life. In this study, we have designed, prepared, and tested an Fc-fused thermostable cocaine esterase (CocE) mutant (known as E196-301, with the T172R/G173Q/L196C/I301C substitutions on CocE) expressed in E. coli . As expected, Fc-fusion does not affect the in vitro enzyme activity and thermal stability of the enzyme and that Fc-E196-301 can favorably bind FcRn with K d = 386 ± 35 nM. However, Fc-fusion does not prolong the in vivo half-life of E196-301 at all; Fc-E196-301 and E196-301 have essentially the same PK profile ( t 1/2 = 0.4 ± 0.1 h) in rats. This is the first time demonstrating that Fc-fusion does not prolong in vivo half-life of a protein. This finding is consistent with the mechanistic understanding that E196-301 and Fc-E196-301 are all degraded primarily through rapid proteolysis in the body. The Fc fusion cannot protect E196-301 from the proteolysis in the body. Nevertheless, it has been demonstrated that PEGylation can effectively protect E196-301, as the PEGylated E196-301, i.e., PEG-E196-301, has a significantly prolonged in vivo half-life. It has also been demonstrated that both E196-301 and PEG-E196-301 have dose-dependent in vivo half-lives (e.g., 19.9 ± 6.4 h for the elimination t 1/2 of 30 mg/kg PEG-E196-301), as the endogenous proteolytic enzymes responsible for proteolysis of E196-301 (PEGylated or not) are nearly saturated by the high plasma concentration produced by a high dose of E196-301 or PEG-E196-301.

Droplet microfluidics has the ability to greatly increase the throughput of screening and sorting of enzymes by carrying reagents in picoliter droplets flowing in inert oils. It was found with the use of a specific surfactant, the interfacial tension of droplets can be very sensitive to droplet pH. This enables the sorting of droplets of different pH when confined droplets encounter a microfabricated trench. The device can be extended to sort enzymes, as a large number of enzymatic reactions lead to the production of an acidic or basic product and a concurrent change in solution pH. The progress of an enzymatic reaction is tracked from the position of a flowing train of droplets. We demonstrate the sorting of esterase isoenzymes based on their enzymatic activity. This label-free technology, that we dub droplet sorting by interfacial tension (SIFT), requires no active components and would have applications for enzyme sorting in high-throughput applications that include enzyme screening and directed evolution of enzymes., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Caffeoyl shikimate esterase (CSE) has been reported to be involved in lignin biosynthesis; however, studies of CSE in gymnosperms are lacking. In this study, CSE was successfully cloned from Larix kaempferi ( LkCSE ) based on Larix laricina transcriptome screening. LkCSE was likely to have catalytic activity based on homologous sequence alignment and phylogenetic analyses of CSEs from different species. In vitro assays with the recombinant enzyme validated the catalytic activity of LkCSE, indicating its function in converting caffeoyl shikimate into caffeate and shikimate. Additionally, the optimum reaction pH and temperature of LkCSE were determined to be 6.0 and 30 °C, respectively. The values of K m and V max of CSE for caffeoyl shikimate were 98.11 μM and 14.44 nM min -1 , respectively. Moreover, LkCSE was observed to have tissue expression specificity and was abundantly expressed in stems and leaves, especially stems, which was 50 times higher than the expression levels of roots. Lastly, translational fusion assays using LkCSE fused with green fluorescent proteins (GFP) in tobacco leaves indicated that LkCSE was localized in the plasma membrane and endoplasmic reticulum (ER). These results revealed that CSE clearly functions in gymnosperms and it is possible for LkCSE to interact with other ER-resident proteins and regulate mass flux in the monolignol biosynthesis pathway.

The effects of near freezing temperature (NFT) storage at -1.9 °C on cell wall degradation of 'Shushanggan' apricot was studied comparing to 0 °C and 5 °C storage. Our results indicated that NFT storage strongly inhibited the solubilization of Na 2 CO 3 -soluble pectin and cellulose, by the suppression of cell wall modifying enzymes (polygalacturonase, β-Galactosidase, pectin methyl esterase and cellulase) and related genes expressions. The loss of side chains was the main modification in CDTA (Cyclohexane-diamine-tetraacetic Acid)-soluble pectin during storage and made the main contribution to the softening of apricot, while the loss of side chain was suppressed by NFT storage. Microscopic observation showed that NFT storage delayed the degradation of pectin fraction and protected cell wall structure from loosing. This study proves that NFT storage is an effective technology to suppress the cell wall polysaccharides degradation and ultrastructure modification of apricot., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Main Conclusion: Ferulic acid esterases have been identified and partially purified from maize pollen. Results suggest that maize pollen FAEs may play an important role in pollen fertilization. A critical step in maize (Zea mays) seed production involves fertilization of the ovule by pollen, a process that relies on ability of the pollen tube to grow through the highly structured and feruloylated arabinoxylan/cellulose-rich tissue of the silk and stigma. It is known that different cell wall hydrolases are present on the surface of pollen. An important hydrolase reported to date is an endo-xylanase (ZmXYN1). We report presence and characterization of another hydrolase, ferulic acid esterase (FAE), in maize pollen. Using a combination of biochemical approaches, these FAEs were partially purified and characterized with respect to their biochemical properties and putative sequences. Maize pollen FAEs were shown to be expressed early during pollen development, to release significant amounts of both monomeric and dimeric ferulates esterified from maize silks and other grass cell walls, and to synergize with an externally applied fungal endo-1,4-β-xylanase on the release of cell wall ferulates and diferulates. Preliminary analysis of maize silk cell walls following pollination, showed a significant reduction of esterified ferulates up to 96 h following pollination, compared to unpollinated silks. These results suggest that maize pollen FAEs may play an important biological role in pollen fertilization and possibly in seed production.

Feruloyl esterases (FAEs) have great potential applications in paper and breeding industry. A new thermo-stable feruloyl esterase gene, TtfaeB was identified from the thermophilic fungus Thielavia terrestris h408. Deduced protein sequence shares the identity of 67% with FAEB from Neurospora crassa. The expression vector pPIC9K-TtfaeB was successfully constructed and electro-transformed into GS115 strain of Pichia pastoris. One transformant with high feruloyl esterase yield was obtained through plate screening and named TtFAEB1. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of fermentation supernatant from transformant TtFAEB1 showed a distinct protein band appearing at the position of about 35-kDa, indicating that TtfaeB gene has been successfully expressed in P. pastoris. The recombinant TtFAEB was purified by affinity chromatography and the specific activity of purified TtFAEB was 6.06 ± 0.72 U/mg. The optimal temperature and pH for purified recombinant TtFAEB was 60 °C and 7.0, respectively. TtFAEB was thermostable, retaining 96.89 and 84.16% of the maximum activity after being treated for 1 h at 50 °C and 60 °C, respectively. Additionally, the enzyme was stable in the pH range 4.5-8.0. The homology model of TtFAEB showed that it consists of a single domain adopting a typical α/β-hydrolase fold and contains a catalytic triad formed by Ser117, Asp201, and His260. TtFAEB in association with xylanase from Trichoderma reesei could release 77.1% of FA from destarched wheat bran. The present results indicated that the recombinant TtFAEB with excellent enzymatic properties is a promising candidate for potential applications in biomass deconstruction and biorefinery.

We previously described the fungus Penicillium chrysogenum 31B, which has high performance to produce the ferulic acid esterase (FAE) for de-esterifying ferulic acids (FAs) from sugar beet pulp. However, the characteristics of this fungus have not yet been determined. Therefore, in this study, we evaluated the molecular characteristics and natural substrate specificity of the Pcfae1 gene from Penicillium chrysogenum and examined its synergistic effects on sugar beet pectin. The Pcfae1 gene was cloned and overexpressed in Pichia pastoris KM71H, and the recombinant enzyme, named PcFAE1, was characterized. The 505 amino acids of PcFAE1 possessed a GCSTG motif (Gly164 to Gly168), characteristic of the serine esterase family. By comparing the amino acid sequence of PcFAE1 with that of the FAE (AoFaeB) of Aspergillus oryzae, Ser166, Asp379, and His419 were identified as the catalytic triad. PcFAE1 was purified through two steps using anion-exchange column chromatography. Its molecular mass without the signal peptide was 75 kDa. Maximum PcFAE1 activity was achieved at pH 6.0-7.0 and 50 °C. The enzyme was stable up to 37 °C and at a pH range of 3-8. PcFAE1 activity was only inhibited by Hg 2+ , and the enzyme had activity toward methyl FA, methyl caffeic acid, and methyl p-coumaric acid, with specific activities of 6.97, 4.65, and 9.32 U/mg, respectively, but not on methyl sinapinic acid. These results indicated that PcFAE1 acted similar to FaeB type according the Crepin classification. PcFAE1 de-esterified O-[6-O-feruloyl-β-d-galactopyranosyl-(1→4)]-d-galactopyranose, O-[2-O-feruloyl-α-l-arabinofuranosyl-(1→5)]-l-arabinofuranose, and O-[5-O-feruloyl-α-l-arabinofuranosyl-(1→3)]-O-β-d-xylopyranosyl-(1→4)-d-xylopyranose, indicating that the enzyme could de-esterify FAs decorated with both β-d-galactopyranosidic and α-l-arabinofuranosidic residues in pectin and xylan. PcFAE1 acted in synergy with endo-α-1,5-arabinanase and α-l-arabinofuranosidase, which releases FA linked to arabinan, to digest the sugar beet pectin. Moreover, when PcFAE1 was allowed to act on sugar beet pectin together with Driselase, approximately 90% of total FA in the substrate was released. Therefore, PcFAE1 may be an interesting candidate for hydrolysis of lignocellulosic materials and could have applications as a tool for production of FA from natural substrates., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In plant innate immunity, enhanced disease susceptibility 1 (EDS1) integrates all pathogen-induced signals transmitted by TIR-type NLR receptors. Driven by an N-terminal α/β-hydrolase-fold domain with a protruding interaction helix, EDS1 assembles with two homologs, phytoalexin-deficient 4 (PAD4) and senescence-associated gene 101 (SAG101). The resulting heterodimers are critical for EDS1 function and structurally well characterized. Here, we resolve solution and crystal structures of unbound Arabidopsis thaliana EDS1 (AtEDS1) using nanobodies for crystallization. These structures, together with gel filtration and immunoprecipitation data, show that PAD4/SAG101-unbound AtEDS1 is stable as a monomer and does not form the homodimers recorded in public databases. Its PAD4/SAG101 anchoring helix is disordered unless engaged in protein/protein interactions. As in the complex with SAG101, monomeric AtEDS1 has a substrate-inaccessible esterase triad with a blocked oxyanion hole and without space for a covalent acyl intermediate. These new structures suggest that the AtEDS1 monomer represents an inactive or pre-activated ground state., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Molecular information about family VIII esterases, which have similarities with class C β-lactamases and penicillin-binding proteins, remains largely unknown. In this study, a novel family VIII esterase with β-lactamase activity ( Ps EstA) from Paenibacillus sp. was characterized using several biochemical and biophysical methods. Ps EstA was effective on a broad range of substrates including tertiary butyl acetate, glyceryl tributyrate, glucose pentaacetate, olive oil, and p -nitrophenyl esters. Additionally, Ps EstA hydrolyzed nitrocefin, cefotaxime, and 7-aminocephalosporanic acid. Interestingly, two forms of immobilized Ps EstA (CLEAs- Ps EstA and mCLEAs- Ps EstA) showed high recycling property and enhanced stability, but hybrid nanoflowers (hNFs) of Ps EstA require improvement. This study provides a molecular understanding of substrate specificities, catalytic regulation, and immobilization of Ps EstA, which can be efficiently used in biotechnological applications.

Therapeutic treatment of cocaine toxicity or addiction is a grand medical challenge. As a promising therapeutic strategy for treatment of cocaine toxicity and addiction to develop a highly efficient cocaine hydrolase (CocH) capable of accelerating cocaine metabolism to produce physiologically/biologically inactive metabolites, our previously designed A199S/S287G/A328W/Y332G mutant of human butyrylcholinesterase (BChE), known as cocaine hydrolase-1 (CocH1), possesses the desirably high catalytic activity against cocaine. The C-terminus of CocH1, truncated after amino acid #529, was fused to human serum albumin (HSA) to extend the biological half-life. The C-terminal HSA-fused CocH1 (CocH1-HSA), known as Albu-CocH1, Albu-CocH, AlbuBChE, Albu-BChE, or TV-1380 in literature, has shown favorable preclinical and clinical profiles. However, the actual therapeutic value of TV-1380 for cocaine addiction treatment is still limited by the short half-life. In this study, we designed and tested a new type of HSA-fused CocH1 proteins, i.e., N-terminal HSA-fused CocH1, with or without a linker between the HSA and CocH1 domains. It has been demonstrated that the catalytic activity of these new fusion proteins against cocaine is similar to that of TV-1380. However, HSA-CocH1 (without a linker) has a significantly longer biological half-life (t 1/2  = 14 ± 2 h) compared to the corresponding C-terminal HSA-fused CocH1, i.e., CocH1-HSA (TV-1380 with t 1/2  = 5-8 h), in rats. Further, the N-terminal HSA-fused CocH1 proteins with a linker have further prolonged biological half-lives: t 1/2  = 17 ± 2 h for both HSA-EAAAK-CocH1 and HSA-PAPAP-CocH1, and t 1/2  = 18 ± 3 h for HSA-(PAPAP) 2 -CocH1. These N-terminal HSA-fused CocH1 proteins may serve as more promising protein drug candidates for cocaine addiction treatment.

Feruloyl esterases (EC 3.1.1.73), belonging to carbohydrate esterase family 1 (CE1), hydrolyze ester bonds between ferulic acid (FA) and arabinose moieties in arabinoxylans. Recently, some CE1 enzymes identified in metagenomics studies have been predicted to contain a family 48 carbohydrate-binding module (CBM48), a CBM family associated with starch binding. Two of these CE1s, wastewater treatment sludge (wts) Fae1A and wtsFae1B isolated from wastewater treatment surplus sludge, have a cognate CBM48 domain and are feruloyl esterases, and wtsFae1A binds arabinoxylan. Here, we show that wtsFae1B also binds to arabinoxylan and that neither binds starch. Surface plasmon resonance analysis revealed that wtsFae1B's K d for xylohexaose is 14.8 μm and that it does not bind to starch mimics, β-cyclodextrin, or maltohexaose. Interestingly, in the absence of CBM48 domains, the CE1 regions from wtsFae1A and wtsFae1B did not bind arabinoxylan and were also unable to catalyze FA release from arabinoxylan. Pretreatment with a β-d-1,4-xylanase did enable CE1 domain-mediated FA release from arabinoxylan in the absence of CBM48, indicating that CBM48 is essential for the CE1 activity on the polysaccharide. Crystal structures of wtsFae1A (at 1.63 Å resolution) and wtsFae1B (1.98 Å) revealed that both are folded proteins comprising structurally-conserved hydrogen bonds that lock the CBM48 position relative to that of the CE1 domain. wtsFae1A docking indicated that both enzymes accommodate the arabinoxylan backbone in a cleft at the CE1-CBM48 domain interface. Binding at this cleft appears to enable CE1 activities on polymeric arabinoxylan, illustrating an unexpected and crucial role of CBM48 domains for accommodating arabinoxylan., (© 2019 Holck et al.)

Management of infected wounds is one of the most costly procedures in the health care sector. Burn wounds are of significant importance due to the high infection risk that can possibly lead to severe consequences such as sepsis. Because antibiotic wound treatments have caused increasing antibiotic resistance in bacteria, there is currently a strong need for alternative strategies. Therefore, we developed new antimicrobial wound dressings consisting of pH-responsive human serum albumin/silk fibroin nanocapsules immobilized onto cotton/polyethylene terephthalate (PET) blends loaded with eugenol, which is an antimicrobial phenylpropanoid. Ultrasound-assisted production of eugenol-loaded nanocapsules resulted in particle sizes (hydrodynamic radii) between 319.73 ± 17.50 and 574.00 ± 92.76 nm and zeta potentials ranging from -10.39 ± 1.99 mV to -12.11 ± 0.59 mV. Because recent discoveries have indicated that the sweat glands contribute to wound reepithelialisation, release studies of eugenol were conducted in different artificial sweat formulas that varied in pH. Formulations containing 10% silk fibroin with lower degradation degree exhibited the highest release of 41% at pH 6.0. After immobilization, the functionalized cotton/PET blends were able to inhibit 81% of Staphylococcus aureus and 33% of Escherichia coli growth. Particle uniformity, silk fibroin concentration, and high surface-area-to-volume ratio of the produced nanocapsules were identified as the contributing factors leading to high antimicrobial activities against both strains. Therefore, the production of antimicrobial textiles using nanocapsules loaded with an active natural compound that will not contribute to antibiotic resistance is seen as a potential future alternative to commercially available antiseptic wound dressings.

1. Schisandra chinensis, also called wuweizi in Chinese, is the fruit of Schisandra chinensis (Turcz.) Baill., and has been officially utilized as an astringent tonic for more than two thousand years in China. This study aims to evaluate the inhibition of carboxylesterases (CESs) by the major ingredients isolated from Schisandra chinensis, including Anwuligan, Schisandrol B, Schisanhenol, deoxyschizandrin, and Schisandrin B. 2. In vitro human liver microsomes (HLMs)-catalyzed hydrolysis of 2-(2-Benzoyl-3-methoxyphenyl) benzothiazole (BMBT) and fluorescein diacetate (FD) was employed as the probe reaction for CES1 and CES2, respectively. Initial screening, inhibition kinetics determination (inhibition type and parameters (K i )), and in silico docking method were carried out. 3. Schisandrin B showed strong inhibition on the activity of CES1, and the activity of CES2 was strongly inhibited by Anwuligan and Schisandrin B. Schisandrin B exhibited noncompetitive inhibition towards CES1 and CES2. Anwuligan showed competitive inhibition towards CES2. The inhibition kinetic parameters (K i ) were calculated to be 29.8, 0.6, and 8.1 uM for the inhibition of Schisandrin B on CES1, Anwuligan on CES2, and Schisandrin B on CES2. In silico docking showed that hydrogen bonds and hydrophobic interactions contributed to the inhibition of Schisandrin B on CES1, Anwuligan on CES2, and Schisandrin B on CES2. All these information will be helpful for understanding the adverse effects of Schisandra chinensis due to the inhibition of CESs-catalyzed metabolism.

Rohapect 10L is an enzyme cocktail commercialized for juice clarification. Here, we characterized the activity and stability of five enzymatic activities present in this cocktail: total pectinase (PE), polygalacturonase (PG), pectin lyase (PL), pectin methyl esterase (PME), and total cellulase (CE) activities. All these enzyme activities have the maximum activity and stability at pH 4, conditions near those found in most fruit juices. However, if the enzymes need to be handled under different conditions (e.g., to immobilize them), their stability becomes extremely low in some cases, just at pH values slightly higher than the optimal one. For example, at pH 10 only CE was reasonably stable at 25°C, while many other enzyme activities were rapidly almost inactivated, even at 4°C. For these cases, different additives were evaluated, and we found that polyethylene glycol was positive or very positive for all enzyme stabilities, allowing keeping reasonable activities after several hours at pH 10 and 25°C. Another additive, that is, dextran, has a small positive effect for PE, PG, and CE, and a very positive effect for PL, albeit significantly destabilizing PME. Thus, the handling and use of this extract requires some care when is performed out of optimal conditions., (© 2019 American Institute of Chemical Engineers.)

Plant nucleotide binding/leucine-rich repeat (NLR) immune receptors are activated by pathogen effectors to trigger host defenses and cell death. Toll-interleukin 1 receptor domain NLRs (TNLs) converge on the ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) family of lipase-like proteins for all resistance outputs. In Arabidopsis ( Arabidopsis thaliana ) TNL-mediated immunity, At EDS1 heterodimers with PHYTOALEXIN DEFICIENT4 ( At PAD4) transcriptionally induced basal defenses. At EDS1 uses the same surface to interact with PAD4-related SENESCENCE-ASSOCIATED GENE101 ( At SAG101), but the role of At EDS1- At SAG101 heterodimers remains unclear. We show that At EDS1- At SAG101 functions together with N REQUIRED GENE1 ( At NRG1) coiled-coil domain helper NLRs as a coevolved TNL cell death-signaling module. At EDS1- At SAG101- At NRG1 cell death activity is transferable to the Solanaceous species Nicotiana benthamiana and cannot be substituted by At EDS1- At PAD4 with At NRG1 or At EDS1- At SAG101 with endogenous Nb NRG1. Analysis of EDS1-family evolutionary rate variation and heterodimer structure-guided phenotyping of At EDS1 variants and At PAD4- At SAG101 chimeras identify closely aligned ɑ-helical coil surfaces in the At EDS1- At SAG101 partner C-terminal domains that are necessary for reconstituted TNL cell death signaling. Our data suggest that TNL-triggered cell death and pathogen growth restriction are determined by distinctive features of EDS1-SAG101 and EDS1-PAD4 complexes and that these signaling machineries coevolved with other components within plant species or clades to regulate downstream pathways in TNL immunity., (© 2019 American Society of Plant Biologists. All rights reserved.)

Herein, we report four series of mutual prodrugs of epalrestat by combining it with different antioxidants using glycine, beta-alanine, and phenylalanine as a linker and by directly linking them through an ester linkage. The designed prodrugs were subjected to pharmacophore and docking-based virtual screening to determine the susceptibility of these prodrugs to be hydrolyzed by human carboxylesterases (hCES1 and hCES2). Five best prodrugs from each series were further submitted to detailed mechanistic study of hCES1 and hCES2 based hydrolytic activation using quantum mechanics and molecular dynamics approach. Additionally, Verloop'ssterimol parameters (B1-B5, L) were calculated in order to investigate the steric constraint of prodrugs to the catalytic sites of hCES1 and hCES2 enzymes. Our results indicated that, among these prodrugs, EP-D4, EP-E13, EP-F5, and EP-F14 are best substrates for hCES1 while EP-G3 and EP-G15 are potential substrates for hCES2. On the other hand, EP-D19 serves as good substrates for both enzymes., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objectives: To discover novel feruloyl esterases (FAEs) by the function-driven screening procedure from soil metagenome., Results: A novel FAE gene bds4 was isolated from a soil metagenomic library and over-expressed in Escherichia coli. The recombinant enzyme BDS4 was purified to homogeneity with a predicted molecular weight of 38.8 kDa. BDS4 exhibited strong activity (57.05 U/mg) toward methyl ferulate under the optimum pH and temperature of 8.0 and 37°C. Based on its amino acid sequence and model substrates specificity, BDS4 was classified as a type-C FAE. The quantity of the releasing ferulic acid can be enhanced significantly in the presence of xylanase compared with BDS4 alone from de-starched wheat bran. In addition, BDS4 can also hydrolyze several phthalates such as diethyl phthalate, dimethyl phthalate and dibutyl phthalate., Conclusion: The current investigation discovered a novel FAE with phthalate-degrading activity and highlighted the usefulness of metagenomic approaches as a powerful tool for discovery of novel FAEs.

Background: Cocaine is a commonly abused drug and there is no approved medication specifically to treat its addiction or overdose. Bacterial cocaine esterase (CocE)-derived RBP-8000 is currently under clinical development for cocaine overdose treatment. It is proven to be effective for human use to accelerate cocaine metabolism into physiologically inactive products. Besides cocaine, RBP-8000 may hydrolyze the neurotransmitter acetylcholine (ACh), however, no study has reported its cholinesterase activity. The present study aims to examine RBP-8000's cholinesterase activity and substrate selectivity to address the potential concern that this enzyme therapy might produce cholinergic side-effects., Methods: Both computational modeling and experimental kinetic analysis were carried out to characterize the potential cholinesterase activity of RBP-8000. Substrates interacting with RBP-8000 were modeled for their enzyme-substrate binding complexes. In vitro enzymatic kinetic parameters were measured using Ellman's colorimetric assay and analyzed by Michaelis-Menten kinetics., Results: It is the first demonstration that RBP-8000 catalyzes the hydrolysis of acetylthiocholine (ATC). However, its catalytic efficiency (k cat /K M ) against ATC is 1000-fold and 5000-fold lower than it against cocaine at 25 °C and 37 °C, respectively, suggesting RBP-8000 has the desired substrate selectivity for cocaine over ACh., Conclusion: Given the fact that clinically relevant dose of RBP-8000 displays insignificant cholinesterase activity relative to endogenous cholinesterases in human, administration of RBP-8000 is unlikely to produce any significant cholinergic side-effects. This study provides supplemental evidences in support of further development of RBP-8000 towards a clinically used pharmacotherapy for cocaine overdose., (Copyright © 2019. Published by Elsevier B.V.)

Dialdehyde starch (DAS) is a kind of modified starch which contains many active aldehyde groups and has good biocompatibility. In this study, magnetic dialdehyde starch nanoparticles were successfully used to immobilize lipase. The lipase was immobilized onto magnetic nanoparticles by using DAS instead of glutaraldehyde as a crosslinker. The parameters like DAS dosage, enzyme concentration and immobilization time were optimized. Enzymatic properties studies exhibited that after DAS cross-linking, the storage stability of the immobilized enzyme reached 82.5%, and the recycling rate reached 53.6%, whereas in case of glutaraldehyde cross linker, it was 79.4% and 46.8%, the former also exhibited better stability and durability. Compared with the free enzyme, the immobilized enzyme indicated higher acid-base tolerance and thermal stability, and had good enzymatic properties. Magnetic dialdehyde starch nanoparticles may have application prospects as an excellent enzyme carrier, which provides a reference for the preparation of other immobilized enzymes with excellent performance., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In this study, maltoheptaose (G7)-based sugar esters were synthesized from maltoheptaose and fatty acids (C10-C16) using a commercial lipase. With the exception of dimethyl sulfoxide (DMSO; 76.4%, w/v), G7 showed only limited solubility in organic solvents. Among the fatty acids, palmitic acid (PA) was the best substrate for G7-based ester formation. G7-PA ester was successfully synthesized as the monoester structure exclusively in 10% DMSO of t-butanol with a 22% conversion yield. NMR and enzymatic analyses of the purified monoester product revealed that the ester bond in the G7 was located at C-6 of the glucose at the reducing end. The G7-PA monoester showed the melting temperature at 56.3 °C that was 6.5 °C lower than that of the free PA and exhibited a different endothermic pattern from the free G7. The G7-PA monoester exhibited excellent emulsifier potential with more even droplet size distribution compared with the commercial sucrose esters for an oil-in-water emulsion system., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Paraoxonase 1 (PON1) is an A-esterase calcium-dependent enzyme that is associated with high-density lipoprotein (HDL) and capable of hydrolyzing a wide variety of substrates, including organophosphate (OP) pesticides. The PON1 phenotype can be modulated by multiple internal and external factors, thereby affecting the catalytic capacity of the enzyme. The aim of this study was to evaluate factors that could modulate PON1 activity in a sample occupationally exposed to pesticides. A cross-sectional, descriptive, and analytical study was carried out with 240 workers. The participants were stratified according to their level of pesticide exposure as reference, moderate-exposure, and high-exposure groups. PON1 activities (arylesterase/AREase, CMPAase, and ssPONase (salt-stimulated)) were determined by spectrophotometry, and the Q192R and L55MPON1 genotypes by real-time PCR. The most frequent genotypes were heterozygous (QR) and homozygous (LL) for PON1Q192R and PON1L55M polymorphisms, respectively. The internal factors associated with the activity of PON1 were the PON1 genotypes (55 and 192) and biochemical parameters related to the lipid profile, in contrast, various external factors related to diet and harmful habits as well as with exposure to pesticides were associated with the activity of PON1. However, using a multivariate mixed ordinal regression model, we found a significant reduction of ssPONase activity in the high-exposure group compared with the reference group only in haplotypes QQLL and RRLL.

A cutinase from Saccharomonospora viridis AHK190, Cut190, can hydrolyze polyethylene terephthalate and has a unique feature that the activity and stability are regulated by Ca2+ binding. Our recent structural and functional analyses showed three Ca2+ binding sites and their respective roles. Here, we analysed the binding thermodynamics of Mn2+, Zn2+ and Mg2+ to Cut190 and their effects on the catalytic activity and thermal stability. The binding affinities of Mn2+ and Zn2+ were higher than that of Mg2+ and are all entropy driven with a binding stoichiometry of three, one and one for Zn2+, Mn2+ and Mg2+, respectively. The catalytic activity was measured in the presence of the respective metals, where the activity of 0.25 mM Mn2+ was comparable to that of 2.5 mM Ca2+. Our 3D Reference Interaction Site Model calculations suggested that all the ions exhibited a high occupancy rate for Site 2. Thus, Mn2+ and Mg2+ would most likely bind to Site 2 (contributes to stability) with high affinity, while to Sites 1 and 3 (contributes to activity) with low affinity. We elucidate the metal-dependent structural and functional properties of Cut190 and show the subtle balance on structure stability and flexibility is controlled by specific metal ions., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

The ferulic acid esterase (FAE) gene from Geobacillus thermoglucosidasius DSM 2542 T was cloned into pET28a(+) expression vector and characterized and is being reported in this study for the first time in Geobacillus. The enzyme, designated as GthFAE, was purified by heat shock and ion-exchange column chromatography. In addition, a second clone containing a Histidine tag was expressed and purified by affinity column chromatography demonstrating future potential for scale-up. FAE gene contains an open reading frame (ORF) of 759-bp encoding a hypothetical 252 amino acid protein, a molecular mass of 28.11 kDa and an isoelectric point of 5.53. From this study it was found that GthFAE had optimal activity at 50 °C and pH of 8.5. Furthermore, the enzyme has been found to retain 64% of its activity after two days incubation at 50 °C and exhibited a high level of functionality with p-nitrophenyl caprylate (C8). K m , V max , k cat and k cat /K m values for p-nitrophenyl caprylate were determined as 0.035 mM, 11,735 µmol/min/mg protein, 5491 (1/s) and 156,885 s -1  mM -1 respectively. The combination of higher activity and stability compared to previously reported FAEs makes GthFAE a potential candidate for use in the paper manufacturing industry.

The human carboxylesterase 1 (CES1), responsible for the biotransformation of many diverse therapeutic agents, may contribute to the occurrence of adverse drug reactions and therapeutic failure through drug interactions. The present study is designed to address the issue of potential drug interactions resulting from the inhibition of CES1. Based on an ensemble of 10 crystal structures complexed with different ligands and a set of 294 known CES1 ligands, we used docking (Autodock Vina) and machine learning methodologies (LDA, QDA and multilayer perceptron), considering the different energy terms from the scoring function to assess the best combination to enable the identification of CES1 inhibitors. The protocol was then applied on a library of 1114 FDA-approved drugs and eight drugs were selected for in vitro CES1 inhibition. An inhibition effect was observed for diltiazem (IC50 = 13.9 µM). Three others drugs (benztropine, iloprost and treprostinil), exhibited a weak CES1 inhibitory effects with IC50 values of 298.2 µM, 366.8 µM and 391.6 µM respectively. In conclusion, the binding site of CES1 is relatively flexible and can adapt its conformation to different types of ligands. Combining ensemble docking and machine learning approaches improves the prediction of CES1 inhibitors compared to a docking study using only one crystal structure.

The need to develop competitive and eco-friendly processes in the cosmetic industry leads to the search for new enzymes with improved properties for industrial bioconversions in this sector. In the present study, a complete methodology to generate, express and screen diversity for the type C feruloyl esterase from Fusarium oxysporium FoFaeC was set up in a high-throughput fashion. A library of around 30,000 random mutants of FoFaeC was generated by error prone PCR of fofaec cDNA and expressed in Yarrowia lipolytica. Screening for enzymatic activity towards the substrates 5-bromo-4-chloroindol-3-yl and 4-nitrocatechol-1-yl ferulates allowed the selection of 96 enzyme variants endowed with improved enzymatic activity that were then characterized for thermo- and solvent- tolerance. The five best mutants in terms of higher activity, thermo- and solvent- tolerance were selected for analysis of substrate specificity. Variant L432I was shown to be able to hydrolyze all the tested substrates, except methyl sinapate, with higher activity than wild type FoFaeC towards methyl p-coumarate, methyl ferulate and methyl caffeate. Moreover, the E455D variant was found to maintain completely its hydrolytic activity after two hour incubation at 55 °C, whereas the L284Q/V405I variant showed both higher thermo- and solvent- tolerance than wild type FoFaeC. Small molecule docking simulations were applied to the five novel selected variants in order to examine the binding pattern of substrates used for enzyme characterization of wild type FoFaeC and the evolved variants., (Copyright © 2019. Published by Elsevier B.V.)

Quorum quenching lactonases are enzymes capable of hydrolyzing lactones, including N-acyl homoserine lactones (AHLs). AHLs are molecules known as signals in bacterial communication dubbed quorum sensing. Bacterial signal disruption by lactonases was previously reported to inhibit behavior regulated by quorum sensing, such as the expression of virulence factors and the formation of biofilms. Herein, we report the enzymatic and structural characterization of a novel lactonase representative from the metallo-β-lactamase superfamily, dubbed GcL. GcL is a broad spectrum and highly proficient lactonase, with k cat /K M values in the range of 10 4 to 10 6  m -1  s -1 . Analysis of free GcL structures and in complex with AHL substrates of different acyl chain length, namely, C4-AHL and 3-oxo-C12-AHL, allowed their respective binding modes to be elucidated. Structures reveal three subsites in the binding crevice: 1) the small subsite where chemistry is performed on the lactone ring; 2) a hydrophobic ring that accommodates the amide group of AHLs and small acyl chains; and 3) the outer, hydrophilic subsite that extends to the protein surface. Unexpectedly, the absence of structural accommodation for long substrate acyl chains seems to relate to the broad substrate specificity of the enzyme., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The limited thermostability of Yarrowia lipolytica lipase 2 (Lip2) hampers its industrial application. To improve its thermostability, we combined single disulfide bonds which our group identified previously. In this study, combining different regional disulfide bonds had greater effect than combining same regional disulfide bonds. Furthermore, mutants with 4, 5, and 6 disulfide bonds exhibited dramatically enhanced thermostability. Compared with the wild-type, sextuple mutant 6s displayed a 22.53 and 31.23 ℃ increase in the melting temperature (T m ) and the half loss temperature at 15 min (T15 50), respectively, with greater pH stability and a wider reaction pH range. Molecular dynamics simulation revealed that multiple disulfide bonds resulted in more rigid structures of mutants 4s, 5s and 6s, and prolonged enzyme unfolding times. Moreover, secretions of mutants 5s and 6s were significantly increased by 60% and 80% by co-expressing with the chaperone protein disulfide isomerase (PDI), which mitigated the reduced production issue caused by multiple disulfide bonds. Results of this study indicated that enhanced heat endurance giving more potential for industrial application., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Potato proteins are well known for their nutritional, emulsifying, foaming, gel forming or antioxidant properties that all make from them valuable protein source for food industry. Antifungal, antimicrobial and also antiviral properties, described for potato proteins in the review, enrich the possibilities of potato protein usage. Potato proteins were divided into patatin, protease inhibitors and fraction of other proteins that also included, besides others, proteins involved in potato defence physiology. All these proteins groups provide proteins and peptides with antifungal and/or antimicrobial actions. Patatins, obtained from cultivars with resistance to Phytophthora infestans, were able to inhibit spore germination of this pathogen. Protease inhibitors represent the structurally heterogeneous group with broad range of antifungal and antimicrobial activities. Potato protease inhibitors I and II reduced the growth of Phytophthora infestans, Rhizoctonia solani and Botrytis cinerea or of the fungi of Fusarium genus. Members of Kunitz family (proteins Potide-G, AFP-J, Potamin-1 or PG-2) were able to inhibit serious pathogens such as Staphylococcus aureus, Listeria monocytogenes, Escherichia coli or Candida albicans. Potato snakins, defensins and pseudothionins are discussed for their ability to inhibit serious potato fungi as well as bacterial pathogens. Potato proteins with the ability to inhibit growth of pathogens were used for developing of pathogen-resistant transgenic plants for crop improvement. Incorporation of potato antifungal and antimicrobial proteins in feed and food products or food packages for elimination of hygienically risk pathogens brings new possibility of potato protein usage.

N -Acylhomoserine lactonase degrades the lactone ring of N -acylhomoserine lactones (AHLs) and has been widely suggested as a promising candidate for use in bacterial disease control. While a number of AHL lactonases have been characterized, none of them has been developed as a commercially available enzymatic product for in vitro AHL quenching due to their low stability. In this study, a highly stable AHL lactonase (AhlX) was identified and isolated from the marine bacterium Salinicola salaria MCCC1A01339. AhlX is encoded by a 768-bp gene and has a predicted molecular mass of 29 kDa. The enzyme retained approximately 97% activity after incubating at 25 °C for 12 days and ~100% activity after incubating at 60 °C for 2 h. Furthermore, AhlX exhibited a high salt tolerance, retaining approximately 60% of its activity observed in the presence of 25% NaCl. In addition, an AhlX powder made by an industrial spray-drying process attenuated Erwinia carotovora infection. These results suggest that AhlX has great potential for use as an in vitro preventive and therapeutic agent for bacterial diseases., Competing Interests: The authors declare no conflicts of interest.

Cutinases are enzymes known to degrade polyester-type plastics. Est119, a plastic-degrading type of cutinase from Thermobifida alba AHK119 (herein called Ta_cut), shows a broad substrate specificity toward polyesters, and can degrade substrates including polylactic acid (PLA). However, the PLA-degrading mechanism of cutinases is still poorly understood. Here, we report the structure complexes of cutinase with ethyl lactate (EL), the constitutional unit. From this complex structure, the electron density maps clearly showed one lactate (LAC) and one EL occupying different positions in the active site cleft. The binding mode of EL is assumed to show a figure prior to reaction and LAC is an after-reaction product. These complex structures demonstrate the role of active site residues in the esterase reaction and substrate recognition. The complex structures were compared with other documented complex structures of cutinases and with the structure of PETase from Ideonella sakaiensis. The amino acid residues involved in substrate interaction are highly conserved among these enzymes. Thus, mapping the precise interactions in the Ta_cut and EL complex will pave the way for understanding the plastic-degrading mechanism of cutinases and suggest ways of creating more potent enzymes by structural protein engineering., (© 2019 Federation of European Biochemical Societies.)

Human butyrylcholinesterase (BChE) is known as a safe and effective protein for detoxification of organophosphorus (OP) nerve agents. Its rationally designed mutants with considerably improved catalytic activity against cocaine, known as cocaine hydrolases (CocHs), are recognized as the most promising drug candidates for the treatment of cocaine abuse. However, it is a grand challenge to efficiently produce active recombinant BChE and CocHs with a sufficiently long biological half-life. In the present study, starting from a promising CocH, known as CocH3 (i.e. A199S/F227A/S287G/A328W/Y332G mutant of human BChE), which has a ~2000-fold improved catalytic activity against cocaine compared to wild-type BChE, we designed an N-terminal fusion protein, Fc(M3)-(PAPAP) 2 -CocH3, which was constructed by fusing Fc of human IgG1 to the N-terminal of CocH3 and further optimized by inserting a linker between the two protein domains. Without lowering the enzyme activity, Fc(M3)-(PAPAP) 2 -CocH3 expressed in Chinese hamster ovary (CHO) cells has not only a long biological half-life of 105 ± 7 h in rats, but also a high yield of protein expression. Particularly, Fc(M3)-(PAPAP) 2 -CocH3 has a ~21-fold increased protein expression yield in CHO cells compared to CocH3 under the same experimental conditions. Given the observations that Fc(M3)-(PAPAP) 2 -CocH3 has not only a high catalytic activity against cocaine and a long biological half-life, but also a high yield of protein expression, this new protein entity reported in this study would be a more promising candidate for therapeutic treatment of cocaine overdose and addiction., (Copyright © 2019 Elsevier B.V. All rights reserved.)

One of the largest commercial applications of enzymes and surfactants is as main components in modern detergents. The high concentration of surfactant compounds usually present in detergents can, however, negatively affect the enzymatic activity. To remedy this drawback, it is of great importance to characterize the interaction between the enzyme and the surfactant molecules at an atomistic resolution. The protein enzyme cutinase from the thermophilic and saprophytic fungus called Humicola insolens (HiC) is a promising candidate for use in detergents thanks to its hydrolase activity targeting mostly biopolyesters (e.g., cutin). HiC is, however, inhibited by low concentrations of sodium dodecyl sulfate (SDS), an ubiquitous surfactant. In this work, we investigate the interaction between HiC and SDS using molecular dynamics simulations. Simulations of HiC dissolved in different aqueous concentrations of SDS show the interaction between HiC and SDS monomers, as well as the formation and dynamics of SDS micelles on the surface of the enzyme. These results suggest a mechanism of cutinase inhibition by SDS, which involves the nucleation of aggregates of SDS molecules on hydrophobic patches on the cutinase surface. Notably, a primary binding site for monomeric SDS is identified near the active site of HiC constituting a possible nucleation point for micelles and leading to the blockage of the entrance to the enzymatic site. Detailed analysis of the simulations allow us to suggest a set of residues from the SDS binding site on HiC to probe as engineered mutations aimed at reducing SDS binding to HiC, thereby decreasing SDS inhibition of HiC.

MomL is a marine-derived quorum-quenching (QQ) lactonase which can degrade various N -acyl homoserine lactones (AHLs). Intentional modification of MomL may lead to a highly efficient QQ enzyme with broad application potential. In this study, we used a rapid and efficient method combining error-prone polymerase chain reaction (epPCR), high-throughput screening and site-directed mutagenesis to identify highly active MomL mutants. In this way, we obtained two candidate mutants, MomL I144V and MomL V149A . These two mutants exhibited enhanced activities and blocked the production of pathogenic factors of Pectobacterium carotovorum subsp. carotovorum (Pcc) . Besides, seven amino acids which are vital for MomL enzyme activity were identified. Substitutions of these amino acids (E238G/K205E/L254R) in MomL led to almost complete loss of its QQ activity. We then tested the effect of MomL and its mutants on Pcc -infected Chinese cabbage. The results indicated that MomL and its mutants (MomL L254R , MomL I144V , MomL V149A ) significantly decreased the pathogenicity of Pcc . This study provides an efficient method for QQ enzyme modification and gives us new clues for further investigation on the catalytic mechanism of QQ lactonase.

Microbial colonization can be detrimental to the integrity of metal surfaces and lead to microbiologically influenced corrosion (MIC). Biocorrosion is a serious problem for aquatic and marine industries in the world. In Minnesota (USA), where this study was conducted, biocorrosion severely affects the maritime transportation industry. The anticorrosion activity of a variety of compounds, including chemical (magnesium peroxide) and biological (surfactin, capsaicin, and gramicidin) molecules were investigated as coating additives. We also evaluated a previously engineered, extremely stable, non-biocidal enzyme known to interfere in bacterial signaling, SsoPox (a quorum quenching lactonase). Experimental steel coupons were submerged in water from the Duluth Superior Harbor (DSH) for 8 weeks in the laboratory. Biocorrosion was evaluated by counting the number and the coverage of corrosion tubercles on coupons and also by ESEM imaging of the coupon surface. Three experimental coating additives significantly reduced the formation of corrosion tubercles: surfactin, magnesium peroxide and the quorum quenching lactonase by 31%, 36% and 50%, respectively. DNA sequence analysis of the V4 region of the bacterial 16S rRNA gene revealed that these decreases in corrosion were associated with significant changes in the composition of bacterial communities on the steel surfaces. These results demonstrate the potential of highly stable quorum quenching lactonases to provide a reliable, cost-effective method to treat steel structures and prevent biocorrosion., Competing Interests: The authors have declared that no competing interests exist.

In the present study, it was presented a strategy to maximize the cutinase production by solid-state fermentation from different microorganisms and substrates. The best results were observed using Fusarium verticillioides, rice bran being the main substrate. Maximum yield of cutinase obtained by the strain was 16.22 U/g. For concentration, ethanol precipitation was used, and the purification factor was 2.4. The optimum temperature and pH for enzyme activity were 35 °C and 6.5, respectively. The enzyme was stable at a wide range of temperature and at all pH values tested. The concentrated cutinase was used as an adjuvant in a formulation containing cutinase + bioherbicide. The use of enzyme increased the efficiency of bioherbicide, since cutinase was responsible to remove/degrade the cutin that recovery the weed leaves and difficult the bioherbicide absorption. Cutinase showed to be a promising product to be used in formulation of bioherbicides.

Quorum sensing (QS) in Gram-negative bacteria is frequently regulated by the diffusible signal N-acylhomoserine lactone (AHL) along with the production of virulence factors in pathogens. To inhibit QS, we fabricated heat-resistant, long-term-stable AHL-lactonase AiiM by electrospinning (ES) aqueous polyvinyl alcohol (PVA) solution containing genetically engineered AiiM with a maltose-binding protein (MBP) tag. MBP-AiiM was immobilized via its inclusion within a dense PVA shell formed during the drying process of ES, followed by cross-linking between hydroxyl groups on PVA. Secondary structure analysis via circular dichroism suggested no conformational change in the MBP-AiiM during ES. Even after pre-heating of MBP-AiiM/PVA fiber mats at 70 °C for 24 h, QS-dependent prodigiosin production in the model pathogen Serratia marcescens AS-1 was effectively inhibited to 0.13% that of the control. Additionally, relative prodigiosin production was reduced to ~20% that of the control after 5-month storage in buffer solution. These results suggest that a shear-thinning process using an entangled PVA aggregate during elongational changes to fibrous domains and a drying process during ES contributes not to enzymatic inactivation caused by conformational changes, but rather to the fabrication of a dense PVA shell around the MBP-AiiM molecules to protect them from disruptors including heating. The developed quorum-quenching enzyme has high potential to inhibit AHL-mediated QS frequently appearing in various Gram-negative bacteria., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The evolution of release factors catalyzing the hydrolysis of the final peptidyl-tRNA bond and the release of the polypeptide from the ribosome has been a longstanding paradox. While the components of the translation apparatus are generally well-conserved across extant life, structurally unrelated release factor peptidyl hydrolases (RF-PHs) emerged in the stems of the bacterial and archaeo-eukaryotic lineages. We analyze the diversification of RF-PH domains within the broader evolutionary framework of the translation apparatus. Thus, we reconstruct the possible state of translation termination in the Last Universal Common Ancestor with possible tRNA-like terminators. Further, evolutionary trajectories of the several auxiliary release factors in ribosome quality control (RQC) and rescue pathways point to multiple independent solutions to this problem and frequent transfers between superkingdoms including the recently characterized ArfT, which is more widely distributed across life than previously appreciated. The eukaryotic RQC system was pieced together from components with disparate provenance, which include the long-sought-after Vms1/ANKZF1 RF-PH of bacterial origin. We also uncover an under-appreciated evolutionary driver of innovation in rescue pathways: effectors deployed in biological conflicts that target the ribosome. At least three rescue pathways (centered on the prfH/RFH, baeRF-1, and C12orf65 RF-PH domains), were likely innovated in response to such conflicts.

Jelly fig ( Ficus awkeotsang Makino) is used to prepare drinks and desserts in Asia, owing to the gelling capability of its pectin via endogenous pectin methylesterase (PE) catalyzation. Meanwhile, substances with PE inhibitory activity (S PEI ) in jelly fig achenes (JFA) residue were noticed to be able to impede the gelation. In this study, we characterized and isolated S PEI from JFA by a series of PE inhibition-guided isolations. Crude aqueous extract of JFA residue was mixed with acetone, and 90% acetone-soluble matter was further fractionated by Diaion HP-20 chromatography. The retained fraction with dominant PE inhibitory activity was collected from 100% methanol eluate. Results from high-performance liquid chromatography mass spectrometry (HPLC/MS) and hydrolysis-induced chromogenic transition revealed the S PEI as complex tannins. Total tannins content was determined in each isolated fraction, and was closely related to PE inhibitory activity. In addition, S PEI in this study could inhibit activities of digestive enzymes in vitro and may, therefore, be assumed to act as non-specific protein binding agent.

Background: Bark beetles rely on detoxifying enzymes to resist the defensive terpenoids of their host trees. Research on carboxylesterases (CarEs) has focused on their multiple functions in the metabolic detoxification of pesticides and plant allelochemicals, drug resistance, and juvenile hormone and pheromone degradation., Result: We identified eight new CarE genes in the Chinese white pine beetle (Dendroctonus armandi) and carried out bioinformatics analysis on the deduced full-length amino acid sequences. Differential transcript levels of CarE genes were observed between sexes; within these levels, significant differences were found among the different development stages, and between insects fed on the phloem of Pinus armandi and exposed to five stimuli [(-)-α-pinene, (-)-β-pinene, (+)-3-carene, limonene and turpentine] at 8 and 24 h., Conclusion: Transcription levels of CarE genes suggest some relationship with the detoxification of terpenoids released by host trees. The functions of bark beetle esterase are mainly in hydrolyzing the host chemical defense and degrading odorant molecules during host selection and colonization. © 2018 Society of Chemical Industry., (© 2018 Society of Chemical Industry.)

Green tea processed from autumn leaves is more bitter and astringent than that from spring leaves, mainly due to gallated catechins. The present study aimed to improve the taste of autumn green tea and green tea infusion by using tannase to treat tea leaves and tea infusion. The results showed that, after hydrolysis, the sweet aftertaste and overall acceptability improved, and the ratio of gallated catechins decreased, as did the bitterness and astringency of the autumn green tea. The pH value was significantly correlated with the concentrations of gallated catechins (r = 0.930, p < 0.01), non-gallated catechins (r = -0.893, p < 0.01), and gallic acid (r = 0.915, p < 0.01), as well as with the intensities of bitterness, astringency, and sweet aftertaste during hydrolysis. Gallic acid contributed to the sweet aftertaste of green tea infusion. These results will help to improve autumn green tea products with tannase., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Biofilm formation is a critical determinant in the pathopoiesis of Pseudomonas aeruginosa It could significantly increase bacterial resistance to drugs and host defense. Thus, inhibition of biofilm matrix production could be regarded as a promising attempt to prevent colonization of P. aeruginosa and the subsequent infection. PpgL, a periplasmic gluconolactonase, has been reported to be involved in P. aeruginosa quorum-sensing (QS) system regulation. However, the detailed function and catalysis mechanism remain elusive. Here, the crystal structure of PpgL is described in the current study, along with biochemical analysis, revealing that PpgL is a typical β-propeller enzyme with unique metal-independent lactone hydrolysis activity. Consequently, comparative analysis of seven-bladed propeller lactone-catalyzing enzymes and mutagenesis studies identify the critical sites which contribute to the diverse catalytic and substrate recognition functions. In addition, the reduced biofilm formation and attenuated invasion phenotype resulting from deletion of ppgL confirm the importance of PpgL in P. aeruginosa pathogenesis. These results suggest that PpgL is a potential target for developing new agents against the diseases caused by P. aeruginosa ., (Copyright © 2019 American Society for Microbiology.)

Peptidyl-tRNA hydrolase (Pth) cleaves the ester bond between the peptide and the tRNA of peptidyl-tRNA molecules, which are the products of defective translation, to recycle the tRNA for further rounds of protein synthesis. Pth is ubiquitous in nature, and its activity is essential for bacterial viability. Here, we have determined the crystal structure of Pth from Thermus thermophilus (TtPth) at 1.00 Å resolution. This is the first structure of a Pth from a thermophilic bacterium and the highest resolution Pth structure reported so far. The present atomic resolution data enabled the calculation of anisotropic displacement parameters for all atoms, which revealed the directionality of the fluctuations of key regions for the substrate recognition. Comparisons between TtPth and mesophilic bacterial Pths revealed that their structures are similar overall. However, the structures of the N- and C-terminal, loop-helix α4, and helix α6 regions are different. In addition, the helix α1 to strand β4 region of TtPth is remarkably different from those of the mesophilic bacterial Pths, because this region is 9 or 10 amino acid residues shorter than those of the mesophilic bacterial Pths. This shortening seems to contribute to the thermostability of TtPth. To further understand the determinants for the thermostability of TtPth, we compared various structural factors of TtPth with those of mesophilic bacterial Pths. The data suggest that the decreases in accessible surface area and thermolabile amino acid residues, and the increases in ion pairs, hydrogen bonds, and proline residues cooperatively contribute to the thermostability of TtPth., (© 2018 Wiley Periodicals, Inc.)

The effects of high pressure treatments (200, 400, 600 MPa for 5 min) and a thermal treatment (85 °C for 5 min) were evaluated on cubes of two pumpkin species (Cucurbita maxima L. var. Delica and Cucurbita moschata Duchesne var. Butternut) up to 2 months of refrigerated storage. Increasing the pressure, small parenchyma cells from the pumpkin tissue exhibited collapses and separations, especially for Butternut. This species showed a lower hardness than Delica at time 0. For both species, 400 MPa and thermal treatment were the most effective in the inactivation of pectinmethylesterase, which reactivated after 2 months, especially for Butternut. Colorimetric parameters decreased after all treatments. Antioxidant activity resulted affected by pressure, showing a significant increase during storage especially for the samples treated at 200 MPa after 2 months, comparable to the thermal treated ones. Among the tested treatments, 400 MPa may be considered as the best option for the quality retention during storage., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The possibility of improving brain function coupled with its preferential uptake in the brain has garnered attention for docosahexaenoic acid-bound lysophosphatidylcholine (DHA-LPC). However, studies focusing on the health benefits of dietary DHA-LPC are lacking. We prepared a dietary oil rich in DHA-LPC (DHA-LPC rich oil) via enzymatic modification of phospholipids (PL) extracted from squid (Todarodes pacificus) meal and purification of active carbon, ion exchange resin, and silica gel. We then examined the effects of dietary DHA-LPC rich oil on male Wistar rats by evaluating serum and liver lipid profiles, fatty acid (FA) metabolizing enzyme activity, and the FA composition of serum and brain. The rats were fed a basal diet containing either soybean oil alone (7%) or soybean oil (4.5%) with DHA-LPC rich oil (2.5%) for 28 days, and then evaluated. The rats fed the diet containing DHA-LPC rich oil showed reduced triacylglycerol concentration due, in part, to the enhancement of carnitine palmitoyltransferase 2 and acyl-CoA oxidase activities and suppression of acetyl-CoA carboxylase and glucose-6-phosphate dehydrogenase activities in the liver. Moreover, the dietary DHA-LPC rich oil moderately increased DHA in the FA composition of the rat hippocampus, which may be due to elevated DHA composition in serum LPC. These results suggest that DHA-LPC rich oil has hypolipidemic effect and moderate increase in hippocampal DHA amount in normal rats.

Tannin acyl hydrolases, also known as tannases, are a group of enzymes critical for the transformation of tannins. The study of these enzymes, which initially evolved in different organisms to detoxify and/or use these plant metabolites, has nowadays become relevant in microbial enzymology research due to their relevant role in food tannin transformation. Microorganisms, particularly bacteria, are major sources of tannase. Cloning and heterologous expression of bacterial tannase genes and structural studies have been performed in the last few years. However, a systematic compilation of the information related to all recombinant tannases, their classification, and characteristics is missing. In this review, we explore the diversity of heterologously produced bacterial tannases, describing their substrate specificity and biochemical characterization. Moreover, a new classification based on sequence similarity analysis is proposed. Finally, putative tannases have been identified in silico for each group of tannases taking advantage of the use of the "tannase" distinctive features previously proposed.

A cosmid metagenomic library containing 1.3 × 10 5 clones was created from a soil sample. A novel gene (fae-xuan) encoding a feruloyl esterase was identified through functional screening. Primary sequence analysis showed that the gene consisted of 759 base pairs and encoded a protein of 252 amino acids. The gene was expressed in Escherichia coli BL21 (DE3) and the corresponding purified recombinant enzyme exhibited a molecular weight of 29 kDa. The FAE-Xuan showed high activity (40.0 U/mg) toward methyl ferulate with an optimal temperature and pH of 30 °C and 5.0, respectively. Besides methyl ferulate, FAE-Xuan can also hydrolyze methyl sinapate and methyl p-coumarate. The substrate utilization preferences and phylogenetic analysis indicated that FAE-Xuan belongs to type A FAE. FAE-Xuan was quite stable over a broad pH range from 3.0 to 10.0. The activity reduced remarkably in presence of Cu 2+ . FAE-Xuan can enhance the quantity of ferulic acid from de-starched wheat bran in presence of xylanase. The work presented here highlighted the effectiveness of metagenomic strategy in identifying novel FAEs with diverse properties for potential use in industrial production.

Background: Carboxylesterases (CarEs) are important in pesticide resistance. Four overexpressed CarE genes with inducible character were screened out in fenpropathrin-resistant Tetranychus cinnabarinus, but their functional roles remained to be further analyzed by RNAi and protein expression., Results: Feeding a single double-stranded (ds)RNA of each of four genes led to gene-specific downregulation of mRNA, decreased esterase activity and diminished resistance in T. cinnabarinus. More interestingly, feeding four dsRNAs simultaneously led to a more significant decrease in enzymatic activity and fold resistance than feeding a single dsRNA individually, suggesting that these CarE genes were involved in fenpropathrin-resistance and had cooperative roles. The gene CarE6 was regarded as the primary and representative candidate to be functionally expressed, because silencing of CarE6 led to the most significant decrease in resistance level. The activity of CarE6 protein was competitively inhibited by fenpropathrin. It could effectively decompose 41.7 ± 0.09% of fenpropathrin within 3 h, proving that CarE6 protein was capable of metabolizing fenpropathrin effectively in T. cinnabarinus., Conclusion: The results confirm that four CarE genes are cooperatively involved in fenpropathrin resistance and the metabolic enzymes encoded by these overexpressed genes do indeed metabolize acaricide in resistant T. cinnabarinus in the evolution of acaricide resistance. © 2018 Society of Chemical Industry., (© 2018 Society of Chemical Industry.)

The characterization of soluble cholinesterases (ChEs) together with carboxylesterases (CEs) in Ficopomatus enigmaticus as suitable biomarkers of neurotoxicity was the main aim of this study. ChEs of F. enigmaticus were characterized considering enzymatic activity, substrate affinity (acetyl-, butyryl-, propionylthiocholine), kinetic parameters ( K m and V max ) and in vitro response to model inhibitors (eserine hemisulfate, iso-OMPA, BW284C51), and carbamates (carbofuran, methomyl, aldicarb, and carbaryl). CEs were characterized based on enzymatic activity, kinetic parameters and in vitro response to carbamates (carbofuran, methomyl, aldicarb, and carbaryl). Results showed that cholinesterases from F. enigmaticus showed a substrate preference for acetylthiocholine followed by propionylthiocholine; butyrylthioline was not hydrolyzed differently from other Annelida species. CE activity was in the same range of cholinesterase activity with acetylthiocholine as substrate; the enzyme activity showed high affinity for the substrate p-nytrophenyl butyrate. Carbamates inhibited ChE activity with propionylthiocholine as substrate to a higher extent than with acetylthiocoline. Also CE activity was inhibited by all tested carbamates except carbaryl. In vitro data highlighted the presence of active forms of ChEs and CEs in F. enigmaticus that could potentially be inhibited by pesticides at environmentally relevant concentration.

Human carboxylesterase 1 (hCE1) is a key enzyme responsible for the hydrolysis of a wide range of endogenous and xenobiotic esters, but the highly selective inhibitors against hCE1 are rarely reported. This study aimed to assess the inhibitory effects of natural flavonoids against hCE1 and to find potential specific hCE1 inhibitors. To this end, fifty-eight natural flavonoids were collected and their inhibitory effects against both hCE1 and hCE2 were assayed. Among all tested compounds, nevadensin, an abundant natural constitute from Lysionotus pauciflorus Maxim., displayed the best combination of inhibition potency and selectivity towards hCE1. The inhibition mechanism of nevadensin on hCE1 was further investigated using two site-specific hCE1 substrates including D-luciferin methyl ester (DME) and 2‑(2‑benzoyloxy‑3‑methoxyphenyl)benzothiazole (BMBT). Furthermore, docking simulations demonstrated that the binding area of nevadensin on hCE1 was highly overlapped with that of DME but was far away from that of BMBT, which was highly consistent with the inhibition modes of nevadensin. These findings found a natural occurring specific inhibitor of hCE1, which could be served as a lead compound for the development of novel hCE1 inhibitor with improved properties, and also hold great promise for investigating hCE1-ligand interactions., (Copyright © 2018. Published by Elsevier B.V.)

A pectin methylesterase gene, pme-zj5a, from Aspergillus niger ZJ5 was cloned and high-level expressed in Pichia pastoris. The highest PME activity was 71.11 U/ml after induction with methanol for 20 h at 30 °C. The molecular mass of purified PME-ZJ5A was estimated to be 37 kDa by SDS-PAGE, and its K m , V max and k cat values of PME-ZJ5A were determined to be 3.27 mg/ml, 5.36 μmol/min/mg, and 22.33 s -1 with pectin. Purified recombinant PME-ZJ5A exhibited optimal activity at pH 3.8 and 45 °C. It retained more than 60% of its maximum activity at 10 °C. Moreover, recombinant PME-ZJ5A can increase the transmittance of pineapple juice by 60.8%, and increase the firmness of pineapple cubes nearly double when combined with CaCl 2 , which showed good potential in fruit processing., (Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)