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Journal of the Science of Food and... Mar 2021L-Glutaminase is considered to be an important industrial enzyme in both the pharmaceutical and food industries, especially for producing functional glutamyl compounds,...
BACKGROUND
L-Glutaminase is considered to be an important industrial enzyme in both the pharmaceutical and food industries, especially for producing functional glutamyl compounds, such as l-theanine. Pseudomonas nitroreducens SP.001 with intracellular l-glutaminase activity has been screened previously. In the present study, three physical permeabilization methods were used to improve l-glutaminase activity. Then, the whole-cell immobilization conditions of permeabilized cells using sodium alginate as an embedding agent were optimized to enhance the enzyme's stability and reusability. The characteristics of the immobilized cells were investigated in comparison with those of permeabilized cells.
RESULTS
The results obtained showed that cell permeabilization using osmotic shock with 155 g L sucrose markedly improved enzyme activity. Then, an effective procedure for immobilization of permeabilized P. nitroreducens cells was established. The optimum conditions for cell immobilization were: sodium alginate 40 g L , calcium chloride 30 g L , cell mass 100 g L and a curing time of 3 h. After successful immobilization, characterization studies revealed that the thermostability and pH resistance of l-glutaminase from immobilized cells were enhanced compared to those from permeabilized cells. Moreover, the immobilized biocatalyst could be reused up to 10 times and retained 80% of its activity.
CONCLUSION
The stability and reusability of the permeabilized cells were improved through the immobilization. These findings indicated that immobilized whole-cell l-glutaminase from P. nitroreducens SP.001 possesses more potential for various industrial biotechnological applications than free cells. © 2020 Society of Chemical Industry.
Topics: Alginates; Bacterial Proteins; Biocatalysis; Cells, Immobilized; Glutamates; Glutaminase; Pseudomonas
PubMed: 32790072
DOI: 10.1002/jsfa.10736 -
Biochemical and Biophysical Research... Jan 2021γ-Glutamyltranspeptidase (GGT) is a ubiquitous enzyme that catalyzes the hydrolysis of the γ-glutamyl linkage of γ-glutamyl compounds and the transfer of their...
γ-Glutamyltranspeptidase (GGT) is a ubiquitous enzyme that catalyzes the hydrolysis of the γ-glutamyl linkage of γ-glutamyl compounds and the transfer of their γ-glutamyl moiety to acceptor substrates. Pseudomonas nitroreducens GGT (PnGGT) is used for the industrial synthesis of theanine, thus it is important to determine the structural basis of hydrolysis and transfer reactions and identify the acceptor site of PnGGT to improve the efficient of theanine synthesis. Our previous structural studies of PnGGT have revealed that crucial interactions between three amino acid residues, Trp385, Phe417, and Trp525, distinguish PnGGT from other GGTs. Here we report the role of Trp525 in PnGGT based on site-directed mutagenesis and structural analyses. Seven mutant variants of Trp525 were produced (W525F, W525V, W525A, W525G, W525S, W525D, and W525K), with substitution of Trp525 by nonaromatic residues resulting in dramatically reduced hydrolysis activity. All Trp525 mutants exhibited significantly increased transfer activity toward hydroxylamine with hardly any effect on acceptor substrate preference. The crystal structure of PnGGT in complex with the glutamine antagonist, 6-diazo-5-oxo-l-norleucine, revealed that Trp525 is a key residue limiting the movement of water molecules within the PnGGT active site.
Topics: Bacterial Proteins; Catalytic Domain; Crystallography, X-Ray; Models, Molecular; Mutagenesis, Site-Directed; Mutant Proteins; Pseudomonas; Static Electricity; Substrate Specificity; Tryptophan; gamma-Glutamyltransferase
PubMed: 33288198
DOI: 10.1016/j.bbrc.2020.11.093 -
3 Biotech Apr 20205-Enolpyruvylshikimate 3-phosphate synthase (EPSPS) is the primary target for the broad-spectrum herbicide, glyphosate. Improvement of gene for high level of glyphosate...
5-Enolpyruvylshikimate 3-phosphate synthase (EPSPS) is the primary target for the broad-spectrum herbicide, glyphosate. Improvement of gene for high level of glyphosate tolerance is important to generate glyphosate-tolerant crops. In this study, we report the isolation and characterization of genes of glyphosate-tolerant strains FY43 and FY47. Both strains FY43 and FY47, which showed glyphosate tolerance up to 8.768% (518.4 mM, 32 × higher than field application), were isolated from soil samples collected from oil palm plantation with a long history of glyphosate application. The glyphosate tolerance property of genes of strains FY43 and FY47 was functionally characterized by expressing the genes in strain BL21(DE3). Error-prone PCR was performed to mutagenize native gene of strains FY43 and FY47. Ten mutagenized EPSPS with amino acid changes (R21C, N265S, A329T, P71L, T258A, L184F, G292C, G292S, L35F and A242V) were generated through error-prone PCR. Both native and mutated genes of strains FY43 and FY47 were introduced into strain BL21(DE3) and transformants were selected on basal salt medium supplemented with 8.768% (518.4 mM) glyphosate. Mutants with mutations (R21C, N265S, A329T, P71L, T258A, L35F, A242V, L184F and G292C) showed sensitivity to 8.768% glyphosate, whereas glyphosate tolerance for mutant with G292S mutation was not affected by the mutation.
PubMed: 32257739
DOI: 10.1007/s13205-020-02176-7 -
Endocrine, Metabolic & Immune Disorders... 2023The host micronutrient milieu is a compilation of factors of both endogenous and exogenous origin. This milieu shapes the host's immune responses and can control the...
BACKGROUND AND AIMS
The host micronutrient milieu is a compilation of factors of both endogenous and exogenous origin. This milieu shapes the host's immune responses and can control the inflammatory response of the host when infected. Among vitamins, B12 plays a key role in the defense process because there is intense competition for it between pathogenic invaders and infected host cells. Alcoholic beverages and antibiotics can cause biological (in vivo) interferences that affect pathogenhost crosstalk. Ethanol is known to interfere with the absorption, distribution, and excretion of vitamin B12 in men and animals. However, the molecular mechanisms underlying this backdrop are not fully understood. Here, we explored how Gram-positive ethanol-producing and Gram-negative vitamin B12- producing microbes of the infected milieu interact to influence biomarkers of host cell defense responses in absorbing, digesting, and defensive cells.
MATERIAL AND METHODS
We investigated two different cell types of colon and liver origin, hepatic-like Huh7 cells and HT- 29/B6 colon cells. To assess the ability of secreted factors from bacteria to exert influence on co-cultured cell's secretion of host-defense markers in response to invading pathogens, cocultured human colonic HT-29/B6 and human hepatic Huh-7 (hereafter Huh7) cells were stimulated or not with Klebsiella pneumoniae 52145 for 24 h in the presence or absence of either Weissella confusa strain NRRL-B-14171 (as a Gram-positive producer of ethanol), Limosilactobacillus reuteri 20016 (as a Gram-positive producer of vitamin B12), or Pseudomonas nitroreducens 1650 (as a Gram-negative producer of vitamin B12). After stimulation, molecular functional biomarkers of host cell defense responses including total MMP-1, lysozyme activity, ALP, and IL-25 were measured.
RESULTS
While simultaneously reducing IL-25 secretion, Kp52145 alone significantly elicited MMP-1, lysozyme, and ALP secretion from co-cultured cells, as compared to no treatment. When compared with Kp 52145 stimulation alone, Pn1650 significantly potentiated MMP-1 and lysozyme secretions from Kp 52145-stimulated co-cultured cells by 29.7% and 67.4%, respectively. Simultaneously, a potentiated suppression (an overall decrease of 77.3%) in IL-25 secretion occurred 24 hours after Kn52145 plus Pn1650 administration. Compared to Kp52145-stimulation alone, treatment with W. confusa NRRL-B-14171 and Kp52145-stimulated co-cultured cells was associated with significant additive induction of MMP-1 and lysozyme secretions. However, compared to Kp52145-stimulation alone, W. confusa NRRL-B-14171 treatment significantly potentiated Kp52145-induced suppression of IL-25. Using the same condition as mentioned above and compared to Kp52145-stimulation alone, L. reuteri 20016 treatment altered the secretion pattern in response to Kp52145: L. reuteri 20016-treated cells displayed less aversive responses towards Kp52145, suggesting that L. reuteri 20016 modulation may act differently on Kp52145 - induced signaling.
CONCLUSION
Gram-negative and Gram-positive vitamin B12- producing bacteria differently affect the secretion of key immune biomarkers in co-cultured HT-29/B6 and Huh7 cells following exposure to Kp52145. Ethanol-producing bacteria additively potentiate pathogenicity and inflammatory responses upon infection. To confirm the biological consequences of these effects on human gut microbiota and health, further studies are warranted, incorporating ex vivo studies of human colon samples and host biomarkers such as cytohistological, molecular, or biochemical measurements.
Topics: Male; Animals; Humans; Ethanol; Matrix Metalloproteinase 1; Muramidase; Colon; Vitamin B 12
PubMed: 36998141
DOI: 10.2174/1871530323666230330111355 -
3 Biotech May 2022A novel glufosinate-tolerant sp. LA21, was isolated from soil samples of an oil palm plantation with a long history of glufosinate application. The genome of sp. LA21...
UNLABELLED
A novel glufosinate-tolerant sp. LA21, was isolated from soil samples of an oil palm plantation with a long history of glufosinate application. The genome of sp. LA21 was sequenced with 150 bp paired-end conducted using Illumina sequencing technology. De novo genome assembly was performed using SPAdes, ABySS, and Velvet assemblers. Phylogenetic analysis using 16S rRNA gene sequence showed that sp. LA21 was closely related to ATCC 33634. Multilocus sequence analysis (MLSA) based on four bacterial housekeeping genes (16S rRNA, , , and ) was conducted together with 138 reference genomes of species. The phylogenetic tree derived from MLSA analysis using concatenated 16S rRNA- sequences grouped sp. LA21 under group and subgroup. Detailed phylogenomic analysis using average nucleotide identity (ANI) and genome-to-genome distance calculator (GGDC) approaches showed that sp. LA21 could be classified as a novel species.
SUPPLEMENTARY INFORMATION
The online version contains supplementary material available at 10.1007/s13205-022-03185-4.
PubMed: 35547011
DOI: 10.1007/s13205-022-03185-4 -
Bioscience, Biotechnology, and... Nov 2020Maltol derivatives are used in a variety of fields due to their metal-chelating abilities. In the previous study, it was found that cytochrome P450 monooxygenase,...
Maltol derivatives are used in a variety of fields due to their metal-chelating abilities. In the previous study, it was found that cytochrome P450 monooxygenase, P450nov, which has the ability to effectively convert the 2-methyl group in a maltol derivative, transformed 3-benzyloxy-2-methyl-4-pyrone (BMAL) to 2-(hydroxymethyl)-3-(phenylmethoxy)-4-pyran-4-one (BMAL-OH) and slightly to 3-benzyloxy-4-oxo-4 -pyran-2-carboxaldehyde (BMAL-CHO). We isolated SB32154 with the ability to convert BMAL-CHO to BMAL-COOH from soil. The enzyme responsible for aldehyde oxidation, a BMAL-CHO dehydrogenase, was purified from SB32154 and characterized. The purified BMAL-CHO dehydrogenase was found to be a xanthine oxidase family enzyme with unique structure of heterodimer composed of 75 and 15 kDa subunits containing a molybdenum cofactor and [Fe-S] clusters, respectively. The enzyme showed broad substrate specificity toward benzaldehyde derivatives. Furthermore, one-pot conversion of BMAL to BMAL-COOH via BMAL-CHO by the combination of the BMAL-CHO dehydrogenase with P450nov was achieved.
Topics: Aldehyde Dehydrogenase; Biocatalysis; Molybdenum; Oxidation-Reduction; Pseudomonas; Pyrones
PubMed: 32729393
DOI: 10.1080/09168451.2020.1799749 -
RdmA Is a Key Regulator in Autoinduction of DSF Quorum Quenching in Pseudomonas nitroreducens HS-18.MBio Feb 2023Diffusible signal factor (DSF) represents a family of widely conserved quorum-sensing (QS) signals which regulate virulence factor production and pathogenicity in...
Diffusible signal factor (DSF) represents a family of widely conserved quorum-sensing (QS) signals which regulate virulence factor production and pathogenicity in numerous Gram-negative bacterial pathogens. We recently reported the identification of a highly potent DSF-quenching bacterial isolate, Pseudomonas nitroreducens HS-18, which contains an operon with four DSF-inducible genes, , or , that are responsible for degradation of DSF signals. However, the regulatory mechanisms that govern the response to DSF induction have not yet been characterized. In this study, we identified a novel transcriptional regulator we designated RdmA (regulator of DSF metabolism) which negatively regulates the expression of and represses DSF degradation. In addition, we found that a gene cluster located adjacent to was also negatively regulated by RdmA and played a key role in DSF degradation; this cluster was hence named (DSF metabolism genes). An electrophoretic mobility shift assay and genetic analysis showed that RdmA represses the transcriptional expression of the genes in a direct manner. Further studies demonstrated that DSF acts as an antagonist and binds to RdmA, which abrogates RdmA binding to the target promoter and its suppression on transcriptional expression of the genes. Taken together, the results from this study have unveiled a central regulator and a gene cluster associated with the autoinduction of DSF degradation in HS-18, and this will aid in the understanding of the genetic basis and regulatory mechanisms that govern the quorum-quenching activity of this potent biocontrol agent. DSF family quorum-sensing (QS) signals play important roles in regulation of bacterial physiology and virulence in a wide range of plant and human bacterial pathogens. Quorum quenching (QQ), which acts by either degrading QS signals or blocking QS communication, has proven to be a potent disease control strategy, but QQ mechanisms that target DSF family signals and associated regulatory mechanisms remain largely unknown. Recently, we identified four autoinduced DSF degradation genes () in HS-18. By using a combination of transcriptome and genetic analysis, we identified a central regulator that plays a key role in autoinduction of expression, as well as a new gene cluster () involved in DSF degradation. The significance of our study is in unveiling the autoinduction mechanism that governs DSF signal quorum quenching for the first time, to our knowledge, and in identification of new genes and enzymes responsible for DSF degradation. The findings from this study shed new light on our understanding of the DSF metabolism pathway and the regulatory mechanisms that modulate DSF quorum quenching and will provide useful clues for design and development of a new generation of highly potent QQ biocontrol agents against DSF-mediated bacterial infections.
Topics: Humans; Quorum Sensing; Pseudomonas; Virulence; Virulence Factors; Bacterial Proteins
PubMed: 36537811
DOI: 10.1128/mbio.03010-22 -
Frontiers in Microbiology 2021Quorum quenching (QQ) is a novel, promising strategy that opens up a new perspective for controlling quorum-sensing (QS)-mediated bacterial pathogens. QQ is performed by...
Quorum quenching (QQ) is a novel, promising strategy that opens up a new perspective for controlling quorum-sensing (QS)-mediated bacterial pathogens. QQ is performed by interfering with population-sensing systems, such as by the inhibition of signal synthesis, catalysis of degrading enzymes, and modification of signals. In many Gram-negative pathogenic bacteria, a class of chemically conserved signaling molecules named -acyl homoserine lactones (AHLs) have been widely studied. AHLs are involved in the modulation of virulence factors in various bacterial pathogens including . is the causal agent of plant-rot disease of bananas, rice, maize, potatoes, etc., causing enormous economic losses of crops. In this study, a highly efficient AHL-degrading bacterial strain W-7 was isolated from activated-sludge samples and identified as . Strain W-7 revealed a superior ability to degrade -(3-oxododecanoyl)-l-homoserine lactone (OdDHL) and completely degraded 0.2 mmol/L of OdDHL within 48 h. Gas chromatography-mass spectrometry (GC-MS) identified -cyclohexyl-propanamide as the main intermediate metabolite during AHL biodegradation. A metabolic pathway for AHL in strain W-7 was proposed based on the chemical structure of AHL and intermediate products. In addition to the degradation of OdDHL, this strain was also found to be capable of degrading a wide range of AHLs including -(3-oxohexanoyl)-l-homoserine lactone (OHHL), -(3-oxooctanoyl)-l-homoserine lactone (OOHL), and -hexanoyl-l-homoserine lactone (HHL). Moreover, the application of strain W-7 as a biocontrol agent could substantially attenuate the soft rot caused by EC1 to suppress tissue maceration in various host plants. Similarly, the application of crude enzymes of strain W-7 significantly reduced the disease incidence and severity in host plants. These original findings unveil the biochemical aspects of a highly efficient AHL-degrading bacterial isolate and provide useful agents that exhibit great potential for the control of infectious diseases caused by AHL-dependent bacterial pathogens.
PubMed: 34413838
DOI: 10.3389/fmicb.2021.694161 -
Frontiers in Microbiology 2023The root-knot nematodes (RKN), especially spp., are globally emerging harmful animals for many agricultural crops.
INTRODUCTION
The root-knot nematodes (RKN), especially spp., are globally emerging harmful animals for many agricultural crops.
METHODS
To explore microbial agents for biological control of these nematodes, the microbial communities of the rhizosphere soils and roots of sponge gourd () infected and non-infected by nematodes, were investigated using culture-dependent and -independent methods.
RESULTS
Thirty-two culturable bacterial and eight fungal species, along with 10,561 bacterial and 2,427 fungal operational taxonomic units (OTUs), were identified. Nine culturable bacterial species, 955 bacterial and 701 fungal OTUs were shared in both four groups. More culturable bacterial and fungal isolates were detected from the uninfected soils and roots than from the infected soils and roots (except no fungi detected from the uninfected roots), and among all samples, nine bacterial species ( sp., sp., , Enterobacteriaceae sp., , sp., Micrococcaceae sp., Rhizobiaceae sp., and sp.) were shared, with sp. and sp. being dominant. was exclusively present in the infested soils, while , , and sp., together with , sp., , and sp. were found only in the uninfected soils. , sp., , and sp. were only in the uninfected roots while sp. only in infected roots. After infestation, 319 bacterial OTUs (such as ) and 171 fungal OTUs (such as ) were increased in rhizosphere soils, while 181 bacterial OTUs (such as ) and 166 fungal OTUs (such as ) rose their abundance in plant roots. Meanwhile, much more decreased bacterial or fungal OTUs were identified from rhizosphere soils rather than from plant roots, exhibiting the protective effects of host plant on endophytes. Among the detected bacterial isolates, sp. TR27 was discovered to exhibit nematocidal activity, and , sp. P35, and to show repellent potentials for the second stage juveniles, which can be used to develop RKN bio-control agents.
DISCUSSION
These findings provided insights into the interactions among root-knot nematodes, host plants, and microorganisms, which will inspire explorations of novel nematicides.
PubMed: 37303801
DOI: 10.3389/fmicb.2023.1168179 -
Genes Aug 2020The sewage sludge isolate HBP-1 was the first bacterium known to completely degrade the fungicide 2-hydroxybiphenyl. PacBio and Illumina whole-genome sequencing...
The sewage sludge isolate HBP-1 was the first bacterium known to completely degrade the fungicide 2-hydroxybiphenyl. PacBio and Illumina whole-genome sequencing revealed three circular DNA replicons: a chromosome and two plasmids. Plasmids were shown to code for putative adaptive functions such as heavy metal resistance, but with unclarified ability for self-transfer. About one-tenth of strain HBP-1's chromosomal genes are likely of recent horizontal influx, being part of genomic islands, prophages and integrative and conjugative elements (ICEs). carries two large ICEs with different functional specialization, but with homologous core structures to the well-known ICE of B13. The variable regions of ICE1 (96 kb) code for, among others, heavy metal resistances and formaldehyde detoxification, whereas those of ICE2 (171 kb) encodes complete -cleavage pathways for catabolism of 2-hydroxybiphenyl and salicylate, a protocatechuate pathway and peripheral enzymes for 4-hydroxybenzoate, ferulate, vanillin and vanillate transformation. Both ICEs transferred at frequencies of 10-10 per HBP-1 donor into , where they integrated site specifically into -gene targets, as expected. Our study highlights the underlying determinants and mechanisms driving dissemination of adaptive properties allowing bacterial strains to cope with polluted environments.
Topics: Computational Biology; Conjugation, Genetic; DNA Transposable Elements; DNA, Bacterial; Disinfectants; Fatty Acids; Gene Order; Gene Transfer, Horizontal; Genome, Bacterial; Genomic Islands; Genomics; High-Throughput Nucleotide Sequencing; Molecular Sequence Annotation; Plasmids; Prophages; Pseudomonas
PubMed: 32806781
DOI: 10.3390/genes11080930