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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 -
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 -
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 -
PLoS Neglected Tropical Diseases Jan 2017Enteropathogenic Yersinia circulate in the pig reservoir and are the third bacterial cause of human gastrointestinal infections in Europe. In West Africa, reports of...
BACKGROUND
Enteropathogenic Yersinia circulate in the pig reservoir and are the third bacterial cause of human gastrointestinal infections in Europe. In West Africa, reports of human yersiniosis are rare. This study was conducted to determine whether pathogenic Yersinia are circulating in pig farms and are responsible for human infections in the Abidjan District.
METHODOLOGY/PRINCIPAL FINDINGS
From June 2012 to December 2013, pig feces were collected monthly in 41 swine farms of the Abidjan district. Of the 781 samples collected, 19 Yersinia strains were isolated in 3 farms: 7 non-pathogenic Yersinia intermedia and 12 pathogenic Yersinia enterocolitica bioserotype 4/O:3. Farm animals other than pigs and wild animals were not found infected. Furthermore, 2 Y. enterocolitica 4/O:3 strains were isolated from 426 fecal samples of patients with digestive disorders. All 14 Y. enterocolitica strains shared the same PFGE and MLVA profile, indicating their close genetic relationship. However, while 6 of them displayed the usual phage type VIII, the other 8 had the highly infrequent phage type XI. Whole genome sequencing and SNP analysis of individual colonies revealed that phage type XI strains had unusually high rates of mutations. These strains displayed a hypermutator phenotype that was attributable to a large deletion in the mutS gene involved in DNA mismatch repair.
CONCLUSIONS/SIGNIFICANCE
This study demonstrates that pathogenic Y. enterocolitica circulate in the pig reservoir in Côte d'Ivoire and cause human infections with a prevalence comparable to that of many developed countries. The paucity of reports of yersiniosis in West Africa is most likely attributable to a lack of active detection rather than to an absence of the microorganism. The identification of hypermutator strains in pigs and humans is of concern as these strains can rapidly acquire selective advantages that may increase their fitness, pathogenicity or resistance to commonly used treatments.
Topics: Animals; Animals, Wild; Cote d'Ivoire; Feces; Humans; Phylogeny; Swine; Swine Diseases; Yersinia Infections; Yersinia enterocolitica
PubMed: 28081123
DOI: 10.1371/journal.pntd.0005216 -
PloS One 2017The limits of life of aerobic microorganisms are well understood, but the responses of anaerobic microorganisms to individual and combined extreme stressors are less...
The limits of life of aerobic microorganisms are well understood, but the responses of anaerobic microorganisms to individual and combined extreme stressors are less well known. Motivated by an interest in understanding the survivability of anaerobic microorganisms under Martian conditions, we investigated the responses of a new isolate, Yersinia intermedia MASE-LG-1 to individual and combined stresses associated with the Martian surface. This organism belongs to an adaptable and persistent genus of anaerobic microorganisms found in many environments worldwide. The effects of desiccation, low pressure, ionizing radiation, varying temperature, osmotic pressure, and oxidizing chemical compounds were investigated. The strain showed a high tolerance to desiccation, with a decline of survivability by four orders of magnitude during a storage time of 85 days. Exposure to X-rays resulted in dose-dependent inactivation for exposure up to 600 Gy while applied doses above 750 Gy led to complete inactivation. The effects of the combination of desiccation and irradiation were additive and the survivability was influenced by the order in which they were imposed. Ionizing irradiation and subsequent desiccation was more deleterious than vice versa. By contrast, the presence of perchlorates was not found to significantly affect the survival of the Yersinia strain after ionizing radiation. These data show that the organism has the capacity to survive and grow in physical and chemical stresses, imposed individually or in combination that are associated with Martian environment. Eventually it lost its viability showing that many of the most adaptable anaerobic organisms on Earth would be killed on Mars today.
Topics: Cold Temperature; Desiccation; Dose-Response Relationship, Radiation; Mars; Oxidation-Reduction; RNA, Ribosomal, 16S; Salts; Stress, Physiological; X-Rays; Yersinia
PubMed: 29069099
DOI: 10.1371/journal.pone.0185178 -
Journal of Zhejiang University....The storage and transportation of raw milk at low temperatures promote the growth of psychrotrophic bacteria and the production of thermo-stable enzymes, which pose...
The storage and transportation of raw milk at low temperatures promote the growth of psychrotrophic bacteria and the production of thermo-stable enzymes, which pose great threats to the quality and shelf-life of dairy products. Though many studies have been carried out on the spoilage potential of psychrotrophic bacteria and the thermo-stabilities of the enzymes they produce, further detailed studies are needed to devise an effective strategy to avoid dairy spoilage. The purpose of this study was to explore the spoilage potential of psychrotrophic bacteria from Chinese raw milk samples at both room temperature (28 °C) and refrigerated temperature (7 °C). Species of Yersinia, Pseudomonas, Serratia, and Chryseobacterium showed high proteolytic activity. The highest proteolytic activity was shown by Yersinia intermedia followed by Pseudomonas fluorescens (d). Lipolytic activity was high in isolates of Acinetobacter, and the highest in Acinetobacter guillouiae. Certain isolates showed positive β-galactosidase and phospholipase activity. Strains belonging to the same species sometimes showed markedly different phenotypic characteristics. Proteases and lipases produced by psychrotrophic bacteria retained activity after heat treatment at 70, 80, or 90 °C, and proteases appeared to be more heat-stable than lipases. For these reasons, thermo-stable spoilage enzymes produced by a high number of psychrotrophic bacterial isolates from raw milk are of major concern to the dairy industry. The results of this study provide valuable data about the spoilage potential of bacterial strains in raw milk and the thermal resistance of the enzymes they produce.
Topics: Animals; Bacteria; Bacterial Proteins; Biofilms; Cold Temperature; Dairy Products; Endopeptidases; Enzyme Stability; Food Microbiology; Hot Temperature; Lipase; Milk; Peptide Hydrolases; Phospholipases; RNA, Ribosomal, 16S; Raw Foods; beta-Galactosidase
PubMed: 30070086
DOI: 10.1631/jzus.B1700352 -
Frontiers in Microbiology 2018Growth in sodium chloride (NaCl) is known to induce stress in non-halophilic microorganisms leading to effects on the microbial metabolism and cell structure....
Growth in sodium chloride (NaCl) is known to induce stress in non-halophilic microorganisms leading to effects on the microbial metabolism and cell structure. Microorganisms have evolved a number of adaptations, both structural and metabolic, to counteract osmotic stress. These strategies are well-understood for organisms in NaCl-rich brines such as the accumulation of certain organic solutes (known as either compatible solutes or osmolytes). Less well studied are responses to ionic environments such as sulfate-rich brines which are prevalent on Earth but can also be found on Mars. In this paper, we investigated the global metabolic response of the anaerobic bacterium MASE-LG-1 to osmotic salt stress induced by either magnesium sulfate (MgSO) or NaCl at the same water activity (0.975). Using a non-targeted mass spectrometry approach, the intensity of hundreds of metabolites was measured. The compatible solutes L-asparagine and sucrose were found to be increased in both MgSO and NaCl compared to the control sample, suggesting a similar osmotic response to different ionic environments. We were able to demonstrate that MASE-LG-1 accumulated a range of other compatible solutes. However, we also found the global metabolic responses, especially with regard to amino acid metabolism and carbohydrate metabolism, to be salt-specific, thus, suggesting ion-specific regulation of specific metabolic pathways.
PubMed: 29535699
DOI: 10.3389/fmicb.2018.00335 -
Current Issues in Molecular Biology 2020Five bacterial (facultatively) anaerobic strains, namely Buttiauxella sp. MASE-IM-9, Clostridium sp. MASE-IM-4, Halanaerobium sp. MASE-BB-1, Trichococcus sp. MASE-IM-5,...
Five bacterial (facultatively) anaerobic strains, namely Buttiauxella sp. MASE-IM-9, Clostridium sp. MASE-IM-4, Halanaerobium sp. MASE-BB-1, Trichococcus sp. MASE-IM-5, and Yersinia intermedia MASE-LG-1 isolated from different extreme natural environments were subjected to Mars relevant environmental stress factors in the laboratory under controlled conditions. These stress factors encompassed low water activity, oxidizing compounds, and ionizing radiation. Stress tests were performed under permanently anoxic conditions. The survival rate after addition of sodium perchlorate (Na-perchlorate) was found to be species-specific. The inter-comparison of the five microorganisms revealed that Clostridium sp. MASE-IM-4 was the most sensitive strain (D10-value (15 min, NaClO4) = 0.6 M). The most tolerant microorganism was Trichococcus sp. MASE-IM-5 with a calculated D10-value (15 min, NaClO4) of 1.9 M. Cultivation in the presence of Na-perchlorate in Martian relevant concentrations up to 1 wt% led to the observation of chains of cells in all strains. Exposure to Na-perchlorate led to a lowering of the survival rate after desiccation. Consecutive exposure to desiccating conditions and ionizing radiation led to additive effects. Moreover, in a desiccated state, an enhanced radiation tolerance could be observed for the strains Clostridium sp. MASE-IM-4 and Trichococcus sp. MASE-IM-5. These data show that anaerobic microorganisms from Mars analogue environments can resist a variety of Martian-simulated stresses either individually or in combination. However, responses were species-specific and some Mars-simulated extremes killed certain organisms. Thus, although Martian stresses would be expected to act differentially on microorganisms, none of the expected extremes tested here and found on Mars prevent the growth of anaerobic microorganisms.
Topics: Bacteria, Anaerobic; Carnobacteriaceae; Cell Survival; Clostridium; Desiccation; Enterobacteriaceae; Extraterrestrial Environment; Extreme Environments; Firmicutes; Mars; Oxidative Stress; Perchlorates; Radiation Tolerance; Sodium Compounds; Stress, Physiological; Time Factors; Yersinia
PubMed: 31967578
DOI: 10.21775/cimb.038.103