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Iranian Journal of Biotechnology Jun 2016Phytate is an anti-nutritional factor in plants, which catches the most phosphorus contents and some vital minerals. Therefore, Phytase is added mainly as an additive to...
BACKGROUND
Phytate is an anti-nutritional factor in plants, which catches the most phosphorus contents and some vital minerals. Therefore, Phytase is added mainly as an additive to the monogastric animals' foods to hydrolyze phytate and increase absorption of phosphorus.
OBJECTIVES
phytase is a new phytase with special characteristics such as high specific activity, pH stability, and thermostability. Our aim was to clone, express, and characterizea codon optimized phytase gene in .
MATERIALS AND METHODS
The phytase gene was optimized according to the codon usage in . The sequence was synthesized and sub-cloned in pET-22b (+) vector and transformed into Bl21 (DE3). The protein was expressed in the presence of IPTG at a final concentration of 1 mM at 30°C. The purification of recombinant protein was performed by Ni affinity chromatography. Phytase activity and stability were determined in various pH and temperatures.
RESULTS
The codon optimized phytase gene was sub-cloned successfully.The expression was confirmed by SDS-PAGE and Western blot analysis. The recombinant enzyme (approximately 45 kDa) was purified. Specific activity of enzyme was 3849 (U.mg) with optimal pH 5 and optimal temperature of 55°C. Thermostability (80°C for 15 min) and pH stability (3-6) of the enzyme were 56 and more than 80%, respectively.
CONCLUSIONS
The results of the expression and enzyme characterization revealed that the optimized phytase gene has a good potential to be produced commercially andto be applied in animals' foodsindustry.
PubMed: 28959328
DOI: 10.15171/ijb.1412 -
Journal of Clinical Microbiology Aug 2009The species Yersinia intermedia is a member of the genus Yersinia which belongs to the Enterobacteriaceae family. This species is divided into eight biotypes, according...
The species Yersinia intermedia is a member of the genus Yersinia which belongs to the Enterobacteriaceae family. This species is divided into eight biotypes, according to Brenner's biotyping scheme. This scheme relies on five tests (utilization of Simmons citrate and acid production from d-melibiose, d-raffinose, alpha-methyl-d-glucoside [alphaMG], and l-rhamnose). The collection of the French Yersinia Reference Laboratory (Institut Pasteur, Paris, France) contained 44 strains that were originally identified as Y. intermedia but whose characteristics did not fit into the biotyping scheme. These 44 strains were separated into two biochemical groups: variant 1 (positive for acid production from l-rhamnose and alphaMG and positive for Simmons citrate utlization) and variant 2 (positive for acid production from l-rhamnose and alphaMG). These atypical strains could correspond to new biotypes of Y. intermedia, to Y. frederiksenii strains having the atypical property of fermenting alphaMG, or to new Yersinia species. These strains did not exhibit growth or phenotypic properties different from those of Y. intermedia and Y. frederiksenii and did not harbor any of the virulence traits usually found in pathogenic species. DNA-DNA hybridizations performed between one strain each of variants 1 and 2 and the Y. intermedia and Y. frederiksenii type strains demonstrated that these variants do belong to the Y. intermedia species. We thus propose that Brenner's biotyping scheme be updated by adding two new biotypes: 9 (for variant 1) and 10 (for variant 2) to the species Y. intermedia.
Topics: Bacterial Proteins; Bacterial Typing Techniques; Carbohydrate Metabolism; Citric Acid; DNA, Bacterial; France; Nucleic Acid Hybridization; Paris; Virulence Factors; Yersinia; Yersinia Infections
PubMed: 19494062
DOI: 10.1128/JCM.02512-08 -
Genome Announcements Aug 2017like strains are usually understudied. In this work, we reported the draft genome sequences of two , two , and two strains isolated from humans, animals, food, and the...
like strains are usually understudied. In this work, we reported the draft genome sequences of two , two , and two strains isolated from humans, animals, food, and the environment in Brazil. These draft genomes will provide better molecular characterizations of these species.
PubMed: 28798182
DOI: 10.1128/genomeA.00780-17 -
BMC Microbiology Apr 2007The presence of beta-lactamases in Y. enterocolitica has been reported to vary with serovars, biovars and geographical origin of the isolates. An understanding of the...
BACKGROUND
The presence of beta-lactamases in Y. enterocolitica has been reported to vary with serovars, biovars and geographical origin of the isolates. An understanding of the beta-lactamases in other related species is important for an overall perception of antibiotic resistance in yersiniae. The objective of this work was to study the characteristics of beta-lactamases and their genes in strains of Y. intermedia and Y. frederiksenii, isolated from clinical and non-clinical sources in India.
RESULTS
The enzymes, Bla-A (a constitutive class A penicillinase) and Bla-B (an inducible class C cephalosporinase) were found to be present in all the clinical and non-clinical strains of Y. intermedia and Y. frederiksenii by double disc diffusion method. The results showed differential expression of Bla-A as indicated by presence/absence of synergy whereas expression of Bla-B was quite consistent. The presence of these enzymes was also reflected in the high minimum inhibitory concentrations, MIC50 (126-1024 mg/L) and MIC90 (256-1024 mg/L) of beta-lactam antibiotics against these species. Restriction fragment length polymorphism (RFLP) revealed heterogeneity in both blaA and blaB genes of Y. intermedia and Y. frederiksenii. The blaA gene of Y. intermedia shared significant sequence identity (87-96%) with blaA of Y. enterocolitica biovars 1A, 1B and 4. The sequence identity of blaA of Y. frederiksenii with these biovars was 77-79%. The sequence identity of blaB gene of Y. intermedia and Y. frederiksenii was more (85%) with that of Y. enterocolitica biovars 1A, 1B and 2 compared to other species viz., Y. bercovieri, Y. aldovae and Y. ruckeri. Isoelectric focusing data further revealed that both Y. intermedia and Y. frederiksenii produced Bla-A (pI 8.7) and "Bla-B like" (pI 5.5-7.1) enzymes.
CONCLUSION
Both Y. intermedia and Y. frederiksenii showed presence of blaA and blaB genes and unequivocal expression of the two beta-lactamases. Limited heterogeneity was detected in blaA and blaB genes as judged by PCR-RFLP. Phylogenetic relationships showed that the two species shared a high degree of identity in their bla genes. This is the first study reporting characteristics of beta-lactamases and their genes in strains of Y. intermedia and Y. frederiksenii isolated from Asian region.
Topics: Amino Acid Sequence; Cephalosporinase; DNA, Bacterial; Drug Resistance, Bacterial; Genes, Bacterial; Isoelectric Focusing; Microbial Sensitivity Tests; Molecular Sequence Data; Molecular Weight; Penicillinase; Phylogeny; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Species Specificity; Yersinia; beta-Lactamases
PubMed: 17407578
DOI: 10.1186/1471-2180-7-25 -
Applied and Environmental Microbiology Jun 2006In this study, 231 strains of Yersinia enterocolitica, 25 strains of Y. intermedia, and 10 strains of Y. bercovieri from human and porcine sources (including reference...
Genotyping of human and porcine Yersinia enterocolitica, Yersinia intermedia, and Yersinia bercovieri strains from Switzerland by amplified fragment length polymorphism analysis.
In this study, 231 strains of Yersinia enterocolitica, 25 strains of Y. intermedia, and 10 strains of Y. bercovieri from human and porcine sources (including reference strains) were analyzed using amplified fragment length polymorphism (AFLP), a whole-genome fingerprinting method for subtyping bacterial isolates. AFLP typing distinguished the different Yersinia species examined. Representatives of Y. enterocolitica biotypes 1A, 1B, 2, 3, and 4 belonged to biotype-related AFLP clusters and were clearly distinguished from each other. Y. enterocolitica biotypes 2, 3, and 4 appeared to be more closely related to each other (83% similarity) than to biotypes 1A (11%) and 1B (47%). Biotype 1A strains exhibited the greatest genetic heterogeneity of the biotypes studied. The biotype 1A genotypes were distributed among four major clusters, each containing strains from both human and porcine sources, confirming the zoonotic potential of this organism. The AFLP technique is a valuable genotypic method for identification and typing of Y. enterocolitica and other Yersinia spp.
Topics: Animals; Chromosome Banding; Chromosomes, Bacterial; Genotype; Humans; Polymorphism, Genetic; Swine; Switzerland; Yersinia; Yersinia enterocolitica
PubMed: 16751516
DOI: 10.1128/AEM.01996-05 -
Applied and Environmental Microbiology Jun 2022The good thermostability of enzymes is an important basis for their wide application in industry. In this study, the phytase APPA from Yersinia intermedia was designed...
The good thermostability of enzymes is an important basis for their wide application in industry. In this study, the phytase APPA from Yersinia intermedia was designed by evolution-guided design. Through the collection of homologous sequences in the NCBI database, we obtained a sequence set composed of 5,569 sequences, counted the number and locations of motif N-X-T/S, and selected the sites with high frequency in evolution as candidate sites for experiments. Based on the principle that -glycosylation modification sites are located on the protein surface, 13 mutants were designed to optimize the number and location of -glycosylation sites. Through experimental verification, 7 single mutants with improved thermostability were obtained. The best mutant, M14, with equal catalytic efficiency as the wild-type was obtained through combined mutation. The half-life () value of mutant M14 was improved from 3.32 min at 65°C to 25 min of at 100°C, allowing it to withstand boiling water treatment, retaining approximately 75% initial activity after a 10-min incubation at 100°C. Differential scanning calorimetry analysis revealed that while the mutants' thermodynamic stability was nearly unchanged, their kinetic stability was greatly improved, and the combined mutant exhibited strong refolding ability. The results of a digestibility test indicated that the application effect of mutant M14 was about 4.5 times that of wild-type APPA, laying a foundation for its industrial application. Due to the harsh reaction conditions of industrial production, the relative instability of enzymes limits their application in industrial production, such as for food, pharmaceuticals, and feed. For example, the pelleting process of feed includes a brief high temperature (80 to 85°C), which requires the enzyme to have excellent thermostability. Therefore, a simple and effective method to improve the thermostability of enzymes has important practical value. In this study, we make full use of the existing homologous sequences (5,569) in the database to statistically analyze the existence frequency of N-X-T/S motifs in this large sequence space to design the phytase APPA with improved thermostability and a high hit rate (~50%). We obtained the best combination mutant, M14, that can tolerate boiling water treatment and greatly improved its kinetic stability without damaging its specific activity. Simultaneously, we proved that its performance improvement is due to its enhanced refolding ability, which comes from -glycan modification rather than amino acid replacement. Our results provide a feasible and effective method for the modification of enzyme thermostability.
Topics: 6-Phytase; Catalysis; Enzyme Stability; Hot Temperature; Kinetics; Temperature
PubMed: 35546578
DOI: 10.1128/aem.00506-22 -
PloS One 2016API 20E strip test, the standard for Enterobacteriaceae identification, is not sufficient to discriminate some Yersinia species for some unstable biochemical reactions...
API 20E strip test, the standard for Enterobacteriaceae identification, is not sufficient to discriminate some Yersinia species for some unstable biochemical reactions and the same biochemical profile presented in some species, e.g. Yersinia ferderiksenii and Yersinia intermedia, which need a variety of molecular biology methods as auxiliaries for identification. The 16S rRNA gene is considered a valuable tool for assigning bacterial strains to species. However, the resolution of the 16S rRNA gene may be insufficient for discrimination because of the high similarity of sequences between some species and heterogeneity within copies at the intra-genomic level. In this study, for each strain we randomly selected five 16S rRNA gene clones from 768 Yersinia strains, and collected 3,840 sequences of the 16S rRNA gene from 10 species, which were divided into 439 patterns. The similarity among the five clones of 16S rRNA gene is over 99% for most strains. Identical sequences were found in strains of different species. A phylogenetic tree was constructed using the five 16S rRNA gene sequences for each strain where the phylogenetic classifications are consistent with biochemical tests; and species that are difficult to identify by biochemical phenotype can be differentiated. Most Yersinia strains form distinct groups within each species. However Yersinia kristensenii, a heterogeneous species, clusters with some Yersinia enterocolitica and Yersinia ferderiksenii/intermedia strains, while not affecting the overall efficiency of this species classification. In conclusion, through analysis derived from integrated information from multiple 16S rRNA gene sequences, the discrimination ability of Yersinia species is improved using our method.
Topics: Chromosomes, Bacterial; Cloning, Molecular; DNA Copy Number Variations; Genes, Bacterial; Phylogeny; RNA, Ribosomal, 16S; Yersinia
PubMed: 26808495
DOI: 10.1371/journal.pone.0147639 -
Iranian Journal of Biotechnology Mar 2016Bacterial resistance to the commonly used antibacterial agents is an increasing challenge in the medicine, and a major problem for the health care systems; the control...
BACKGROUND
Bacterial resistance to the commonly used antibacterial agents is an increasing challenge in the medicine, and a major problem for the health care systems; the control of their spread is a constant challenge for the hospitals.
OBJECTIVES
In this study, we have investigated the antimicrobial activity of the Zinc Oxide nanoparticles against clinical sample; bacteria.
MATERIALS AND METHODS
Nanoparticle susceptibility constants and death kinetic were used to evaluate the antimicrobial characteristics of the Zinc Oxide (ZnO) against the bacteria. Antimicrobial tests were performed with 10 cfu.mL at baseline. At first, Minimum Inhibitory Concentration (MIC) of ZnO was determined and then nanoparticle suspension at one and two times of the MIC was used for death kinetic and susceptibility constant assay at 0 to 360 min treatment time.
RESULTS
ZnO nanoparticles with size ranging from 10 to 30 nm showed the highest susceptibility reaction against (Z=39.06 mL.μg). The process of death in ZnO suspension was assumed to follow the first-order kinetics and the survival ratio of bacteria decreased with the increasing treatment time. An increased concentration of the nanoparticle was seen to enhance the bactericidal action of the nanoparticle. Then we performed the best ratio of the nanoparticles on semi-sensitive and resistance antibiotic for the bacteria. However, based on experimental results, synergy of ZnO nanoparticles and Oxacilin was determined and showed a higher sensitivity compared to the ZnO nanoparticles alone.
CONCLUSIONS
The results of the present study illustrates that ZnO has a strong antimicrobial effect and could potentially be employed to aid the bacterial control. It could also improve- antibacterial effects in combination with the antibiotics.
PubMed: 28959316
DOI: 10.15171/ijb.1184 -
Journal of Clinical Microbiology Oct 1979Yersinia enterocolitica and related Yersinia species share many temperature-dependent biological attributes. The present report documents temperature-dependent...
Yersinia enterocolitica and related Yersinia species share many temperature-dependent biological attributes. The present report documents temperature-dependent production of a bacteriocin-like substance only among Yersinia strains which ferment L-rhamnose, raffinose, and melibiose and which have been tentatively designed Y. intermedia. When tested by the "lawn-spotting" technique at 25 and 37 degrees C, 7 of 15 Y. intermedia strains produced a bacteriocin only at 25 degrees C with a uniform spectrum of activity against 2 strains of Y. enterocolitica, 1 rhamnose-positive, raffinose-negative, melibiose-negative strain tentatively designated Y. frederiksenii, 8 Y. intermedia, and 3 sucrose- and acetylmethylcarbinol-negative yersinial isolates tested. Bacteriocin-like activity was not detected among the Y. enterocolitica, Y. frederiksenii, or sucrose-negative yersinial strains tested. The exclusive activity of the Y. intermedia antibacterial substance only against yersiniae and not against other representative Enterobacteriaceae tested supports the placement of these microorganisms within the genus Yersinia and further establishes the singularity of Y. intermedia.
Topics: Bacteriocins; Drug Resistance, Microbial; Species Specificity; Temperature; Yersinia
PubMed: 528679
DOI: 10.1128/jcm.10.4.433-436.1979 -
Journal of Clinical Microbiology May 1987Since May 1983, our laboratory has, upon request, cultured stools for Yersinia spp. by using direct plating on cefsulodin-irgasan-novobiocin agar and a 3-week cold...
Since May 1983, our laboratory has, upon request, cultured stools for Yersinia spp. by using direct plating on cefsulodin-irgasan-novobiocin agar and a 3-week cold enrichment procedure. We isolated bacteria identified as Y. intermedia from six adult patients. All isolates were recovered only by the cold enrichment procedure and misidentified as Y. enterocolitica by the API 20E system (Analytab Products, Plainview, N.Y.). Final identification was made on the basis of results obtained with conventional tube biochemical tests. The isolates were tested for the following characteristics associated with virulence in Y. enterocolitica: lack of pyrazinamidase activity, autoagglutinability, presence of a 40- to 50-megadalton plasmid, production of heat-stable enterotoxin, and mouse lethality. All isolates tested had pyrazinamidase activity, and none were autoagglutinable. However, one isolate possessed a 40-megadalton plasmid. None produced enterotoxin or were lethal for mice. Review of the medical histories of the patients revealed that four of the six had diarrhea; however, none had disease typical of that caused by Y. enterocolitica. Our data confirmed the limited pathogenic potential of Y. intermedia and suggested that its isolation was without clinical significance in our patients. Conventional biochemical tests were required for reliable identification of Y. intermedia.
Topics: Adult; Aged; Aged, 80 and over; Diarrhea; Feces; Female; Humans; Male; Yersinia; Yersinia Infections
PubMed: 3584421
DOI: 10.1128/jcm.25.5.859-862.1987