Esterase Gene Research Articles

Characterization of a novel family VIII esterase EstM2 from soil metagenome capable of hydrolyzing estrogenic phthalates

BackgroundMicrobes are rich sources of enzymes and esterases are one of the most important classes of enzymes because of their potential for application in the field of food, agriculture, pharmaceuticals and bioremediation. Due to limitations in their cultivation, only a small fraction of the complex microbial communities can be cultured from natural habitats. Thus to explore the catalytic potential of uncultured organisms, the metagenomic approach has turned out to be an effective alternative method for direct mining of enzymes of interest. Based on activity-based screening method, an esterase-positive clone was obtained from metagenomic libraries.ResultsFunctional screening of a soil metagenomic fosmid library, followed by transposon mutagenesis led to the identification of a 1179 bp esterase gene, estM2, that encodes a 392 amino acids long protein (EstM2) with a translated molecular weight of 43.12 kDa. Overproduction, purification and biochemical characterization of the recombinant protein demonstrated carboxylesterase activity towards short-chain fatty acyl esters with optimal activity for p-nitrophenyl butyrate at pH 8.0 and 37 °C. Amino acid sequence analysis and subsequent phylogenetic analysis suggested that EstM2 belongs to the family VIII esterases that bear modest similarities to class C β-lactamases. EstM2 possessed the conserved S-x-x-K motif of class C β-lactamases but did not exhibit β-lactamase activity. Guided by molecular docking analysis, EstM2 was shown to hydrolyze a wide range of di- and monoesters of alkyl-, aryl- and benzyl-substituted phthalates. Thus, EstM2 displays an atypical hydrolytic potential of biotechnological significance within family VIII esterases.ConclusionsThis study has led to the discovery of a new member of family VIII esterases. To the best of our knowledge, this is the first phthalate hydrolase (EstM2), isolated from a soil metagenomic library that belongs to a family possessing β-lactamase like catalytic triad. Based on its catalytic potential towards hydrolysis of both phthalate diesters and phthalate monoesters, this enzyme may find use to counter the growing pollution caused by phthalate-based plasticizers in diverse geological environment and in other aspects of biotechnological applications.

Facile One-Pot Immobilization of a Novel Esterase and Its Application in Cinnamyl Acetate Synthesis

Cinnamyl acetate has a wide application in food industries. This work focuses on cinnamyl acetate synthesis with a novel esterase. estGUZ753, an esterase gene from Geobacillus uzenensis DSMZ 13551, was first cloned and expressed in Pichia pastoris KM71. The optimal activity of EstGUZ753 towards p-NP caprylate was at pH 8.0 and 70 °C, and the half-life at 70 °C was 28 h. Furthermore, EstGUZ753 showed marked tolerance in methanol. The activity of EstGUZ753 increased up to 1.16-fold in 90% methanol after 72 h, and the half-life in it was 336 h. The crude fermentation broth of EstGUZ753 was immobilized directly onto the epoxy resin (Lx-105s), and the immobilized EstGUZ753 exhibited a 99% conversion for cinnamyl acetate synthesis at a high cinnamyl alcohol concentration in 6 h (cinnamyl alcohol: 1.0 M, enzyme dosage: 3%). These characteristics of EstGUZ753 indicated its great potential for organic synthesis. A novel esterase gene from Geobacillus uzenensis DSMZ 13551 was first cloned and expressed in Pichia pastoris KM71. Esterase EstGUZ753 showed marked thermostability at high temperature and tolerance in methanol. Furthermore, the crude fermentation broth of EstGUZ753 was immobilized directly onto the epoxy resin, and immobilized EstGUZ753 exhibited a 99% conversion for cinnamyl acetate synthesis.

Resistance risk assessment of the novel complex II inhibitor pyflubumide in the polyphagous pest Tetranychus urticae

Pyflubumide is a novel selective carboxanilide acaricide that inhibits mitochondrial complex II of spider mite species such as Tetranychus urticae. We explored the baseline toxicity and potential cross-resistance risk of pyflubumide in a reference panel of T. urticae strains resistant to various acaricides with different modes of action. A cyenopyrafen-resistant strain (JPR) was identified as the only strain with low-to-moderate level of cross-resistance to pyflubumide (LC50 = 49.07 mg/L). In a resistance risk assessment approach, JPR was subsequently selected which led to two highly resistant strains JPR-R1 (RR = 466.7) and JPR-R2 (RR = 614.8). Interestingly, compared to adult females, resistance was much less pronounced in adult males and eggs of the two JPR-R strains. In order to elucidate resistance mechanisms, we first sequenced the complex II subunits in susceptible and resistant strains, but target-site insensitivity could not be detected. In contrast, synergism/antagonism experiments strongly suggested that cytochrome P450 monooxygenases are involved in pyflubumide resistance. We therefore conducted genome-wide gene expression experiments to investigate constitutive and induced expression patterns and documented the overexpression of five cytochrome P450 and four carboxyl/choline esterase genes in the JPR-R strains after pyflubumide exposure. Together, we provide a first resistance risk assessment of a novel complex II inhibitor and provide first evidence for metabolic resistance mediated by cytochrome P450s in T. urticae.

Characterization of Novel Salt-Tolerant Esterase Isolated from the Marine Bacterium Alteromonas sp. 39-G1.

An esterase gene, estA1, was cloned from Alteromonas sp. 39-G1 isolated from the Beaufort Sea. The gene is composed of 1,140 nucleotides and codes for a 41,190 Da protein containing 379 amino acids. As a result of a BLAST search, the protein sequence of esterase EstA1 was found to be identical to Alteromonas sp. esterase (GenBank: PHS53692). As far as we know, no research on this enzyme has yet been conducted. Phylogenetic analysis showed that esterase EstA1 was a member of the bacterial lipolytic enzyme family IV (hormone sensitive lipases). Two deletion mutants (Δ20 and Δ54) of the esterase EstA1 were produced in Escherichia coli BL21 (DE3) cells with part of the N-terminal of the protein removed and His-tag attached to the C-terminal. These enzymes exhibited the highest activity toward p-nitrophenyl (pNP) acetate (C2) and had little or no activity towards pNP-esters with acyl chains longer than C6. Their optimum temperature and pH of the catalytic activity were 45°C and pH 8.0, respectively. As the NaCl concentration increased, their enzyme activities continued to increase and the highest enzyme activities were measured in 5 M NaCl. These enzymes were found to be stable for up to 8 h in the concentration of 3-5 M NaCl. Moreover, they have been found to be stable for various metal ions, detergents and organic solvents. These salt-tolerant and chemical-resistant properties suggest that the enzyme esterase EstA1 is both academically and industrially useful.

Characterization and purification via nucleic acid aptamers of a novel esterase from the metagenome of paper mill wastewater sediments

A new esterase gene est906 was identified from paper mill wastewater sediments via a function-based metagenomic approach. The gene encoded a protein of 331 amino acids, that shared 86% homology with known esterases. Based on the results of multiple sequence alignment and phylogenetic analysis, it was confirmed that Est906 contained a characteristic hexapeptide motif (G-F-S-M-G-G), which classified it as a lipolytic enzyme family V protein. Est906 displayed the highest hydrolysis activity to ρ-nitrophenyl caproate (C6), and its optimal temperature and pH were 54 °C and 9.5, respectively. Additionally, this enzyme had good stability under strong alkaline conditions (pH 10.0–11.0) in addition to moderate heat resistance and good tolerance against several metal ions and organic solvents. Furthermore, a specific nucleic acid aptamer (Apt1) bound to Est906 was obtained after five rounds of magnetic bead SELEX screening. Apt1 displayed high specific recognition and capture ability to Est906. In conclusion, this study not only identified a new esterase of family V with potential industrial application by metagenomic technology but also provided a new method to purify recombinant esterases via nucleic acid aptamers, which will facilitate the isolation and purification of target proteins in the future.

A Moraxella Virulence Factor Catalyzes an Essential Esterase Reaction of Biotin Biosynthesis.

Pimeloyl-acyl carrier protein (ACP) methyl ester esterase catalyzes the last biosynthetic step of the pimelate moiety of biotin, a key intermediate in biotin biosynthesis. The paradigm pimeloyl-ACP methyl ester esterase is the BioH protein of Escherichia coli that hydrolyses the ester bond of pimeloyl-ACP methyl ester. Biotin synthesis in E. coli also requires the function of the malonyl-ACP methyltransferase gene (bioC) to employ a methylation strategy to allow elongation of a temporarily disguised malonate moiety to a pimelate moiety by the fatty acid synthetic enzymes. However, bioinformatics analyses of the extant bacterial genomes showed that bioH is absent in many bioC-containing bacteria. The genome of the Gram-negative bacterium, Moraxella catarrhalis lacks a gene encoding a homolog of any of the six known pimeloyl-ACP methyl ester esterase isozymes suggesting that this organism encodes a novel pimeloyl-ACP methyl ester esterase isoform. We report that this is the case. The gene encoding the new isoform, called btsA, was isolated by complementation of an E. coli bioH deletion strain. The requirement of BtsA for the biotin biosynthesis in M. catarrhalis was confirmed by a biotin auxotrophic phenotype caused by deletion of btsA in vivo and a reconstituted in vitro desthiobiotin synthesis system. Purified BtsA was shown to cleave the physiological substrate pimeloyl-ACP methyl ester to pimeloyl-ACP by use of a Ser117-His254-Asp287 catalytic triad. The lack of sequence alignment with other isozymes together with phylogenetic analyses revealed BtsA as a new class of pimeloyl-ACP methyl ester esterase. The involvement of BtsA in M. catarrhalis virulence was confirmed by the defect of bacterial invasion to lung epithelial cells and survival within macrophages in the ΔbtsA strains. Identification of the new esterase gene btsA exclusive in Moraxella species that links biotin biosynthesis to bacterial virulence, can reveal a new valuable target for development of drugs against M. catarrhalis.

Spatial induction of genes encoding secreted proteins in micro-colonies of Aspergillus niger

Aspergillus niger is used by the industry to produce enzymes and metabolites such as citric acid. In liquid cultures, it can grow as a dispersed mycelium or as micro-colonies with a width in the micrometer to millimeter range. Here, it was assessed whether expression of genes encoding secreted enzymes depends on mycelium morphology. To this end, expression of the reporter gene gfp from the promoters of the glucoamylase gene glaA, the feruloyl esterase gene faeA and the α-glucuronidase gene aguA was causally related to micro-colony size within a liquid shaken culture. Data could be fitted by hyperbolic functions, implying that the genes encoding these secreted proteins are expressed in a shell at the periphery of the micro-colony. The presence of such a shell was confirmed by confocal microscopy. Modelling predicted that the width of these zones was 13 to 156 µm depending on growth medium and micro-colony diameter. Together, data indicate that the highest productive micro-colonies are those colonies that have a radius ≤ the width of the peripheral expression zone.

Sex-specific molecular specialization and activity rhythm-dependent gene expression in honey bee antennae.

We performed an RNA-seq-based comparison of gene expression levels in the antennae of honey bee drones and time-trained foragers (workers) collected at different times of the day and different activity states. Interestingly, olfaction-related genes [i.e. odorant receptor (Or) genes, odorant binding protein (Obp) genes, carboxyl esterase (CEst) genes, etc.] showed stable gene expression differences between drone and worker antennae. Drone antennae showed higher expression of 24 Or genes, of which 21 belong to the clade X which comprises the receptor for the major queen pheromone compound 9-ODA. This high number of drone-biased Or genes suggests that more than previously thought play a role in sex-pheromone communication. In addition, we found higher expression levels for many non-olfaction-related genes including nitric oxide synthase (NOS), and the potassium channel Shaw In contrast, workers showed higher expression of 67 Or genes, which belong to different Or clades that are involved in pheromone communication as well as the perception of cuticular hydrocarbons and floral scents. Further, drone antennae showed higher expression of genes involved in energy metabolism, whereas worker antennae showed higher expression of genes involved in neuronal communication, consistent with earlier reports on peripheral olfactory plasticity. Finally, drones that perform mating flight in the afternoon (innate) and foragers that are trained to forage in the afternoon (adapted) showed similar daily changes in the expression of two major clock genes, period and cryptochrome2 Most of the other genes showing changes with time or onset of daily flight activity were specific to drones and foragers.

Characterization of a new esterase for enantioselective resolution of (R, S)-methyl mandelate

A new esterase gene (EST35) was cloned from Dactylosporangium sp. BB08 and expressed in E. coli BL21 (DE3). Optimum catalytic activity of EST35 was at 30 C and it could be activated at 0 °C. EST35 remained high activity in 20% (V/V) cyclohexane, hexane, heptane, methanol and DMSO. Interestingly the enzyme exhibits good enantioselectivity towards (R, S)-methyl mandelate leaving with an optical purity of 97% (R)-methyl mandelate and make EST35 a promising enzyme for biotechnology application.

Overexpression of an alternative allele of carboxyl/choline esterase 4 (CCE04) of Tetranychus urticae is associated with high levels of resistance to the keto-enol acaricide spirodiclofen.

Spirodiclofen is an acaricide that targets lipid biosynthesis by inhibiting acetyl-coenzyme A carboxylase. Spirodiclofen resistance in spider mites has been previously documented and was associated with overexpression of CYP392E10, a cytochrome P450 mono-oxygenase that metabolizes spirodiclofen. However, additional mechanisms have been suggested in several studies and a carboxyl/choline esterase gene, CCE04, was shown to be overexpressed in two genetically different strains, SR-VP and SR-TK, both exhibiting high spirodiclofen resistance levels. We identified two different CCE04 alleles in both resistant strains, CCE04SR-VP and CCE04London , with CCE04SR-VP being highly overexpressed. Isoelectric focusing analysis confirmed the overexpression of a single esterase isozyme, while copy number and random fragment length polymorphism analysis revealed that CCE04SR-VP overexpression was more likely due to selection for the CCE04SR-VP allele rather than gene amplification. Both CCE04 alleles were functionally expressed using the Pichia expression system. Functional enzyme assays revealed only limited kinetic differences between CCE04 isoforms for model substrates. In addition, inhibition/competition experiments with spirodiclofen suggested a similar interaction with both enzymes, whereas its active metabolite, spirodiclofen enol, did not inhibit enzyme activity. Our study suggests that selection with spirodiclofen results in enrichment of a specific allele of CCE04 (CCE04SR-VP ) in two genetically independent strains, which is highly overexpressed. Based on kinetic enzyme data, however, quantitative rather than qualitative differences between CCE04SR-VP and CCE04London seem more likely to be involved in resistance. Our findings are discussed in the light of a possible spirodiclofen resistance mechanism, with sequestration of spirodiclofen by CCE04SR-VP being a likely hypothesis. © 2019 Society of Chemical Industry.

Multiple detoxification genes confer imidacloprid resistance to Sitobion avenae Fabricius

The English grain aphid, Sitobion avenae Fabricius, is one of the major wheat pests distributed worldwide. Insecticide application is typically effective for controlling S. avenae, However, S. avenae has developed resistance to multiple insecticides. Thus, it is crucial to elucidate resistance mechanisms in S. avenae. In this study, the mechanisms of resistance to imidacloprid in S. avenae and the risk of cross resistance to other insecticides were investigated. An imidacloprid resistant strain (SA-R) of S. avenae was established via continuous imidacloprid selection with 42.7-fold resistance compared with the susceptible population (SA-S). There was cross resistance in SA-R between imidacloprid and other insecticides tested (acetamiprid, abamectin, chlorpyrifos, and omethoate), with ratios ranging from 10.7- to 24.7-fold higher than SA-S. S, S, S-tributyl phosphorotrithioate (TPP), piperonyl butoxide (PBO), and diethyl maleate (DEM) had synergism with imidacloprid in the SA-R strain with 2.85-, 3.59-, and 2.14-fold reductions, respectively, in LC50 values. Additionally, the activities of cytochrome P450 (P450), glutathione-S-transferases (GST), and carboxylesterase (COE) were significantly higher by 2.69-, 1.42-, and 1.39-fold in the SA-R strain than in the SA-S strain. Based on the S. avenae transcriptome database, 11 P450, two COE, four GST, and two esterase (EST) genes that were significantly induced by imidacloprid were selected to compare gene expression levels between SA-R and SA-S. Fourteen detoxification genes of the 19 tested from the SA-R strain were overexpressed compared to those of the SA-S strain. Silencing of CYP6A14-1, CYP307A1, GST1-1-1, and COE2 by dsRNA feeding resulted in increased susceptibility of SA-R aphids to imidacloprid. These results provide evidence that detoxification genes including CYP6A14-1, CYP307A1, GST1-1-1, and COE2 contribute to resistance to imidacloprid in S. avenae, which was confirmed by RNA interference (RNAi) feeding.

New Insights Into Cinnamoyl Esterase Activity of Oenococcus oeni.

Some strains of Oenococcus oeni possess cinnamoyl esterase activity that can be relevant in the malolactic stage of wine production liberating hydroxycinnamic acids that are precursors of volatile phenols responsible for sensory faults. The objective of this study was to better understand the basis of the differential activity between strains. After initial screening, five commercial strains of O. oeni were selected, three were found to exhibit cinnamoyl esterase activity (CE+) and two not (CE−). Although the use of functional annotation of genes revealed genotypic variations between the strains, no specific genes common only to the three CE+ strains could explain the different activities. Pasteurized wine was used as a natural source of tartrate esters in growth and metabolism experiments conducted in MRS medium, whilst commercial trans-caftaric acid was used as substrate for enzyme assays. Detoxification did not seem to be the main biological mechanism involved in the activity since unlike its phenolic cleavage products and their immediate metabolites (trans-caffeic acid and 4-ethylcatechol), trans-caftaric acid was not toxic toward O. oeni. In the case of the two CE+ strains OenosTM and CiNeTM, wine-exposed samples showed a more rapid degradation of trans-caftaric acid than the unexposed ones. The CE activity was present in all cell-free extracts of both wine-exposed and unexposed strains, except in the cell-free extracts of the CE− strain CH11TM. This activity may be constitutive rather than induced by exposure to tartrate esters. Trans-caftaric acid was totally cleaved to trans-caffeic acid by cell-free extracts of the three CE+ strains, whilst cell-free extracts of the CE− strain CH16TM showed significantly lower activity, although higher for the strains in experiments with no prior wine exposure. The EstB28 esterase gene, found in the genomes of the 5 strains, did not reveal any difference on the upstream regulation and transport functionality between the strains. This study highlights the complexity of the basis of this activity in wine related O. oeni population. Variable cinnamoyl esterases or/and membrane transport activities in the O. oeni strains analyzed and a possible implication of wine molecules could explain this phenomenon.

Potential of esterase DmtH in transforming plastic additive dimethyl terephthalate to less toxic mono-methyl terephthalate

Dimethyl terephthalate (DMT) is a primary ingredient widely used in the manufacture of polyesters and industrial plastics; its environmental fate is of concern due to its global use. Microorganisms play key roles in the dissipation of DMT from the environment; however, the enzymes responsible for the initial transformation of DMT and the possible altered toxicity due to this biotransformation have not been extensively studied. To reduce DMT toxicity, we identified the esterase gene dmtH involved in the initial transformation of DMT from the AOPP herbicide-transforming strain Sphingobium sp. C3. DmtH shows 24–41% identity with α/β-hydrolases and belongs to subfamily V of bacterial esterases. The purified recombinant DmtH was capable of transforming DMT to mono-methyl terephthalate (MMT) and potentially transforming other p-phthalic acid esters, including diallyl terephthalate (DAT) and diethyl terephthalate (DET). Using C. elegans as an assay model, we observed the severe toxicity of DMT in inducing reactive oxygen species (ROS) production, decreasing locomotion behavior, reducing lifespan, altering molecular basis for oxidative stress, and inducing mitochondrial stress. In contrast, exposure to MMT did not cause obvious toxicity, induce oxidative stress, and activate mitochondrial stress in nematodes. Our study highlights the usefulness of Sphingobium sp. C3 and its esterase DmtH in transforming p-phthalic acid esters and reducing the toxicity of DMT to organisms.

Silencing of the phytoene desaturase (PDS) gene affects the expression of fruit-ripening genes in tomatoes

BackgroundPast research has shown that virus-induced phytoene desaturase (PDS) gene silencing via agroinjection in the attached and detached fruit of tomato plants results in a pale-yellow fruit phenotype. Although the PDS gene is often used as a marker for gene silencing in tomatoes, little is known about the role of PDS in fruit ripening. In this study, we investigated whether the pepper PDS gene silenced endogenous PDS genes in the fruit of two tomato cultivars, Dotaerang Plus and Legend Summer.ResultsWe found that the pepper PDS gene successfully silenced endogenous PDS in tomato fruit at a silencing frequency of 100% for both cultivars. A pale-yellow silenced area was observed over virtually the entire surface of individual fruit due to the transcriptional reduction in phytoene desaturase (PDS), zeta-carotene (ZDS), prolycopene isomerase (CrtlSO), and beta-carotene hydroxylase (CrtR-b2), which are the carotenoid biosynthesis genes responsible for the red coloration in tomatoes. PDS silencing also affected the expression levels of the fruit-ripening genes Tomato AGAMOUS-LIKE1 (TAGL1), RIPENING INHIBITOR (RIN), pectin esterase gene (PE), lipoxygenase (LOX), FRUITFULL1/FRUITFUL2 (FUL1/FUL2), and the ethylene biosynthesis and response genes 1-aminocyclopropane-1-carboxylate oxidase 1 and 3 (ACO1 and ACO3) and ethylene-responsive genes (E4 and E8).ConclusionThese results suggest that PDS is a positive regulator of ripening in tomato fruit, which must be considered when using it as a marker for virus-induced gene silencing (VIGS) experiments in order to avoid fruit-ripening side effects.

Biochemical and Molecular Analysis of Acetylcholine Esterase to Rule out Organophosphorus Detoxification Level in Field Populations of Culex Quiquefasciatus Say

Background & Objectives: Acetylcholinesterase, responsible for neurotransmitter degradation at the cholinergic nerve synapse, is the target of both organophosphate and carbamate insecticides. The current study envisage to assess acetylcholine esterase levels and mutation in the acetylcholine esterase gene1 of Cx. quinquefasciatus of Kozhikode, Cochin, Malappuram, Thrissur, and Palakkad town areas of Kerala, India. Methods: The samples were collected from regularly insecticide spraying area and scarcely spraying area of the five locations in the year 2014, 15 and 16. The assays were done according to the protocol provided in techniques to detect insecticide resistance mechanism; Field and laboratory Manual. Site specific mutation in acetylcholine esterase gene 1 was identified by RFLP using Alu1 enzyme. Results: The % remaining activity in propoxur inhibited fraction in the regularly and scarcely insecticide treating field populations of Cx.quinquefasciatus were higher than 30% and lesser than 30% respectively. The value greater than 30% indicates the chance to develop resistance in field populations. The RFLP using Alu1 on the ace1 also showed the presence of heterozygous genotype in all the five field populations except in Ernakulam where the Cx.quinquefasciatus population possessed homozygous resistant genotype, which is an indication to the excessive and extensive usage of Temephos as a larvicide. Interpretation & Conclusion: The evidence of development of resistance to synthetic insecticides in mosquitoes observed in the present study points to the need of employing new or alternate insecticides which would be easily degradable and have less harmful effect on other organisms.

A54 Genomic analysis of camel-HKU23 in Nigeria dromedary camels reveals strain-specific cross-species recombination

Coronaviruses (CoVs) are enveloped, single stranded, positive-sense RNA viruses with a large genomic size of 26–32 kilobases. The first human CoV identified in the 1960s was isolated from patients presenting with common cold symptoms. Subsequent epidemic outbreaks of novel zoonotic CoV transmission were reported, examples including HCoV-229E (229E), HCoV-OC43 (OC43), severe acute respiratory syndrome, and Middle East respiratory syndrome (MERS). The ongoing outbreak of MERS in the Middle East is originating from a zoonotic source of dromedary camels. Surveillance later revealed that three CoV species—HCoV-229E (229E), camel-HKU23, and MERS-CoV—were co-circulating in Saudi Arabia dromedary camels. Camel-HKU23 belongs to Group 2a CoV, which also includes human coronavirus OC43, bovine coronavirus, and porcine hemagglutinating encephalomyelitis virus. Recombination, resulting in the generation of different novel genotypes, has been reported previously among these CoVs. Our surveillance of dromedary camels slaughtered in a major abattoir in Nigeria identified camel-HKU23 from nasal swab samples with a prevalence of 2.2 per cent. Phylogenetic analysis showed Nigeria camel-HKU23 is distinct from those previously identified in Saudi Arabia, while still genetically similar, as they share a monophyletic origin. Recombination analysis of Nigeria camel-HKU23 revealed two recombination breakpoints at positions of 22774–24100 base pairs (bp) and 28224–29362 bp. Recombination breakpoint at position 22774, encoding the Group 2a CoV-specific hemagglutinin esterase gene, exhibited high bootstrap support for clustering with RbCoV HKU14, which was previously detected in domestic rabbits in China. The recombination signal is only observed in Nigeria camel-HKU23, suggesting a regional varied evolutionary history of camel-HKU23. Our findings extended the knowledge of the evolutionary relationship among Group 2a CoVs. Further surveillance in other African camels will be important to elucidate the evolution of camel-HKU23.

Characterization of an acetyl xylan esterase from the marine bacterium Ochrovirga pacifica and its synergism with xylanase on beechwood xylan

BackgroundAcetyl xylan esterase plays an important role in the complete enzymatic hydrolysis of lignocellulosic materials. It hydrolyzes the ester linkages of acetic acid in xylan and supports and enhances the activity of xylanase. This study was conducted to identify and overexpress the acetyl xylan esterase (AXE) gene revealed by the genomic sequencing of the marine bacterium Ochrovirga pacifica.ResultsThe AXE gene has an 864-bp open reading frame that encodes 287 aa and consists of an AXE domain from aa 60 to 274. Gene was cloned to pET-16b vector and expressed the recombinant AXE (rAXE) in Escherichia coli BL21 (DE3). The predicted molecular mass was 31.75 kDa. The maximum specific activity (40.08 U/mg) was recorded at the optimal temperature and pH which were 50 °C and pH 8.0, respectively. The thermal stability assay showed that AXE maintains its residual activity almost constantly throughout and after incubation at 45 °C for 120 min. The synergism of AXE with xylanase on beechwood xylan, increased the relative activity 1.41-fold.ConclusionResulted higher relative activity of rAXE with commercially available xylanase on beechwood xylan showed its potential for the use of rAXE in industrial purposes as a de-esterification enzyme to hydrolyze xylan and hemicellulose-like complex substrates.

Cloning, Characterization, and Functional Expression of a Thermostable Type B Feruloyl Esterase from Thermophilic Thielavia Terrestris.

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.

Effects of dietary supplementation with lysozyme on the structure and function of the cecal microbiota in broiler chickens.

Lysozyme is known to eliminate intestinal pathogens in poultry and improve their growth performance. However, whether it can replace antibiotic growth promoters without the associated risk of the emergence of antibiotic-resistant bacterial strains is not known, and the effects of lysozyme supplementation on the composition, biodiversity, and function of the chicken gut microbiota remain unclear. Here, we used the 16S rRNA gene and ITS fragment Illumina sequencing combined with transcriptomic analysis to address this issue. A total of 400 1-d-old Di Gao chicks were allocated randomly to five groups, each consisting of four replicates (20 birds/group). The chicks were fed a starter (1–21 d) and a grower (22–42 d) diet supplemented with 0 (control), 40 (LYS40), 100 (LYS100), or 200 ppm (LYS200) lysozyme, or 400 ppm flavomycin as an antibiotic control for 6 weeks. Lysozyme administration did not contribute significantly (P > 0.05) to the growth of the broiler chickens. No significant (P > 0.05) differences in the diversity and composition of the bacterial and fungal communities in the cecal microbiota of chickens in the different diet groups were found. However, lysozyme supplementation led to a significant (P < 0.05) enrichment of genes involved in the synthesis/degradation of bacterial outer membranes and cell walls, cross-cell substrate transport, and carbohydrate metabolic processes, thus possibly promoting the cecal microbiota carbon and energy metabolism. Bacteroides contributed 31.9% of glycoside hydrolase genes (17,681–24,590), 26.1% of polysaccharide lyase genes (479–675), 20.7% of carbohydrate esterase genes (3,509–4,101), 8.8% of auxiliary activity genes (705–1,000), 16.2% of glycosyltransferase genes (5,301–6,844), and 13.9% of carbohydrate-binding module genes (8838–15,172) identified in the cecal samples. Thus, they were the main players in the breakdown of non-starch polysaccharides in the cecum, although Parabacteroides, Alistipes, Prevotella, Clostridium, Blastocystis, Barnesiella, Blautia, Faecalibacterium, Subdoligranulum, Megamonas, Eubacterium, Ruminococcus, Paenibacillus, Bifidobacterium, Akkermansia, and other bacteria also participated.

Biochemical characterization of a novel thermostable feruloyl esterase from Geobacillus thermoglucosidasius DSM 2542T.

The ferulic acid esterase (FAE) gene from Geobacillus thermoglucosidasius DSM 2542T 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 28.11kDa 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). Km, Vmax, kcat and kcat/Km values for p-nitrophenyl caprylate were determined as 0.035mM, 11,735µmol/min/mg protein, 5491 (1/s) and 156,885s-1mM-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.