ABSTRACT
A Gram-stain-negative, aerobic, non-motile, catalase- and oxidase-positive, pale orange, rod-shaped strain EF6T, was isolated from a natural wetland reserve in Hebei province, China. The strain grew at 25-37 °C (optimum, 30 °C), pH 5-9 (optimum, pH 7), and in the presence of 1.0-4.0% (w/v) NaCl (optimum, 2%). A phylogenetic analysis based on 16S rRNA gene sequence revealed that strain EF6T belongs to the genus Paracoccus, and the closest members were Paracoccus shandongensis wg2T with 98.1% similarity, Paracoccus fontiphilus MVW-1 T (97.9%), Paracoccus everestensis S8-55 T (97.7%), Paracoccus subflavus GY0581T (97.6%), Paracoccus sediminis CMB17T (97.3%), Paracoccus caeni MJ17T (97.0%), and Paracoccus angustae E6T (97.0%). The genome size of strain EF6T was 4.88 Mb, and the DNA G + C content was 65.3%. The digital DNA-DNA hybridization, average nucleotide identity, and average amino acid identity values between strain EF6T and the reference strains were all below the threshold limit for species delineation (< 32.8%, < 88.0%, and < 86.7%, respectively). The major fatty acids (≥ 5.0%) were summed feature 8 (86.3%, C18:1 ω6c and/or C18:1 ω7c) and C18:1 (5.0%) and the only isoprenoid quinone was Q-10. The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, two unidentified glycolipids, five unidentified phospholipids, and an unidentified aminolipid. Strain EF6T displays notable resistance to benzoate and selenite, with higher tolerance levels (25 g/L for benzoate and 150 mM for selenite) compared to the closely related species. Genomic analysis identified six benzoate resistance genes (acdA, pcaF, fadA, pcaC, purB, and catA) and twenty selenite resistance and reduction-related genes (iscR, ssuB, ssuD, selA, selD and so on). Additionally, EF6T possesses unique genes (catA, ssuB, and ssuC) absent in the closely related species for benzoate and selenite resistance. Its robust resistance to benzoate and selenite, coupled with its genomic makeup, make EF6T a promising candidate for the remediation of both organic and inorganic pollutants. It is worth noting that the specific resistance phenotypes described above were not reported in other novel species in Paracoccus. Based on the results of biochemical, physiological, phylogenetic, and chemotaxonomic analyses, combined with comparisons of the 16S rRNA gene sequence and the whole genome sequence, strain EF6T is considered to represent a novel species of the genus Paracoccus within the family Rhodobacteraceae, for which the name Paracoccus benzoatiresistens sp. nov. is proposed. The type strain is EF6T (= GDMCC 1.3400 T = JCM 35642 T = MCCC 1K08702T).
Subject(s)
Base Composition , DNA, Bacterial , Fatty Acids , Paracoccus , Phylogeny , RNA, Ribosomal, 16S , Wetlands , Paracoccus/genetics , Paracoccus/classification , Paracoccus/isolation & purification , Paracoccus/metabolism , Paracoccus/drug effects , RNA, Ribosomal, 16S/genetics , Fatty Acids/metabolism , Fatty Acids/chemistry , DNA, Bacterial/genetics , China , Sodium Selenite/metabolism , Bacterial Typing Techniques , Phospholipids/analysis , Sequence Analysis, DNA , Nucleic Acid Hybridization , Oxidation-Reduction , Drug Resistance, BacterialABSTRACT
This study aimed to develop an ultraminiature pressure sensor array to measure the force exerted on teeth. Orthodontic force plays an important role in effective, rapid, and safe tooth movement. However, owing to the lack of an adequate tool to measure the orthodontic force in vivo, it remains challenging to determine the best orthodontic loading in clinical and basic research. In this study, a three-dimensional (3D) orthodontic force detection system based on piezoresistive absolute pressure sensors was designed. The 3D force sensing array was constructed using five pressure sensors on a single chip. The size of the sensor array was only 4.1 × 2.6 mm, which can be placed within the bracket base area. Based on the barometric calibration, conversion formulas for the output voltage and pressure of the five channels were constructed. Subsequently, a 3D linear mechanical simulation model of the voltage and stress distribution was established using 312 tests of the applied force in 13 operating modes. Finally, the output voltage was first converted to pressure and then to the resultant force. The 3D force-detection chip was then tested to verify the accuracy of force measurement on the teeth. Based on the test results, the average output force error was only 0.0025 N (0.7169%) (p = 0.958), and the average spatial positioning error was only 0.058 mm (p = 0.872) on the X-axis and 0.050 mm (p = 0.837) on the Y-axis. The simulation results were highly consistent with the actual force applied (intraclass correlation efficient (ICC): 0.997-1.000; p < 0.001). Furthermore, through in vivo measurements and a finite element analysis, the movement trends generated when the measured orthodontic forces that acted on the teeth were simulated. The results revealed that the device can accurately measure the orthodontic force, representing the first clinical test of an orthodontic-force monitoring system. Our study provides a hardware basis for clinical research on efficient, safe, and optimal orthodontic forces, and has considerable potential for application in monitoring the biomechanics of tooth movement.
ABSTRACT
A Gram-stain-negative, strictly aerobic, non-motile, catalase- and oxidase-positive, pink and rod-shaped strain, designated RY-2T, was isolated from sediment of Fuyang River located in Wuqiang County, Hengshui City, Hebei Province, PR China. The strain grew at 25-45 °C (optimum, 37 °C), pH 7.0-8.0 (optimum, pH 7.0) and in the presence of 0-1.5â% (w/v) NaCl (optimum, 1â%). From the phylogenetic analysis of the 16S rRNA gene sequence, strain RY-2T was affiliated to the genus Mariniradius, and had the highest 16S rRNA gene sequence similarity to Mariniradius saccharolyticus JCM 17389T (98.3â%) and the similarity values between strain RY-2T and other type strains was all below 89.3â%. The genome size of strain RY-2T was 4.75 Mb and the DNA G+C content was 46.6â%. Values of digital DNA-DNA hybridization and average nucleotide identity between strain RY-2T and the reference strain were 63.2 and 95.5â%, respectively. The major fatty acids (≥5.0â%) were iso-C15â:â0 (37.9â%), summed feature 9 (8.4â%, iso-C17â:â1 ω9c and/or C16â:â010-methyl), anteiso-C15â:â0 (8.2â%), iso-C17â:â0 3-OH (7.6â%) and summed feature 4 (5.2â%, iso-C17â:â1 I and/or anteiso-C17â:â1 B) and its sole menaquinone was MK-7. The polar lipids consisted of phosphatidylethanolamine, an unknown phosphoglycolipid, an unidentified phospholipid, two unidentified aminolipids, three unidentified glycolipids and nine unidentified lipids. Based on the results of biochemical, physiological, phylogenomic and chemotaxonomic analyses, strain RY-2T is considered to represent a novel species of the genus Mariniradius within the family Cyclobacteriaceae, for which the name Mariniradius sediminis sp. nov. is proposed. The type strain is RY-2T (=GDMCC 1.2781T=JCM 35631T).
Subject(s)
Fatty Acids , Rivers , Fatty Acids/chemistry , Phylogeny , RNA, Ribosomal, 16S/genetics , Xenobiotics , DNA, Bacterial/genetics , Base Composition , Bacterial Typing Techniques , Sequence Analysis, DNA , Phospholipids/chemistry , Vitamin K 2/chemistryABSTRACT
A yellow, Gram-stain-positive, strictly aerobic, thermotolerant, non-motile and rod-shaped bacterial strain, designated RY-1T, was isolated from a silt sample of Fuyang River, Wuqiang County, Hengshui City, Hebei Province, PR China. Cells showed oxidase- and catalase-positive activities. Growth occurred at 20-45 °C (optimum, 37 °C) and pH 6.0-8.0 (optimum, pH 7.0), and in the presence of 0-1.5â% (w/v) NaCl (optimum, 0%). A phylogenetic tree based on 16S rRNA gene sequences revealed that strain RY-1T formed a phylogenetic lineage with Flavihumibacter members within the family Chitinophagaceae. A comparison of 16S rRNA gene sequences showed that strain RY-1T was most closely related to Flavihumibacter cheonanensis WS16T (98.6â%), Flavihumibacter sediminis CJ663T (97.7â%) and Flavihumibacter solisilvae 3-3T (97.6â%). The genome size of strain RY-1T was 4.71 Mb, and the DNA G+C content was 44.3ââ%. The average nucleotide identity, digital DNA-DNA hybridization and average amino acid identity values between strain RY-1T and reference strains were all lower than the threshold values for species delineation. Strain RY-1T contained menaquinone-7 and iso-C15â:â0, iso-C17â:â0 3-OH and iso-C15â:â1G as the sole respiratory isoprenoid quinone and major cellular fatty acids (≥5â%), respectively. The major polar lipids consisted of phosphatidylethanolamine, three unidentified aminolipids and four unidentified lipids. According to the results of phenotypic, phylogenetic and chemotaxonomic characteristics, strain RY-1T represents a novel species of the genus Flavihumibacter, for which the name Flavihumibacter fluminis sp. nov. is proposed. The type strain is RY-1T (=GDMCC 1.2775T=JCM 34870T).
Subject(s)
Bacteroidetes , Phylogeny , Rivers , Bacterial Typing Techniques , Base Composition , DNA, Bacterial/genetics , Fatty Acids/chemistry , Rivers/microbiology , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA , Vitamin K 2/chemistry , Bacteroidetes/classification , Bacteroidetes/isolation & purification , ChinaABSTRACT
Environmental pollution by heavy metals is becoming an increasing problem and has become a matter of great concern due to the adverse effects worldwide. In this study, we report a novel strain of multi-metal resistant bacteria. A Gram-stain-negative, strictly aerobic, non-motile, yellow, rod-shaped strain 17AT, was isolated from the shallow silt of Fuyang River located in Longdian town, Hengshui city, Hebei province, China. Strain 17AT grew at 20-35 °C (optimum, 30 °C), pH 5-10 (optimum, pH 7) and 0-2% (w/v) NaCl (optimum, 1%). Phylogenetic analyses of 16S rRNA gene sequences showed that strain 17AT was closely related to members of the genus Flavobacterium, and had the highest 16S rRNA gene sequence similarity with 'Flavobacterium panacis' DCY106T (97.5%), followed by Flavobacterium johnsoniae subsp. johnsoniae UW101T (97.3%), Flavobacterium cutihirudinis E89T (97.2%), Flavobacterium limi THG-AG6.4T (97.2%), Flavobacterium hibisci THG-HG1.4T (97.2%) and Flavobacterium johnsoniae subsp. aurantiacum DSM 6792T (97.1%). The genome size of strain 17AT was 5.4 Mb and the DNA G + C content was 34.0%. The average nucleotide identity, digital DNA-DNA hybridization and average amino acid identity values among strain 17AT and reference strains were in the ranges of 79.8-86.1%, 24.1-31.4% and 80.5-88.6%, respectively, lower than the threshold values for species delineation. Strain 17AT contained iso-C15:0 and C16:0 3-OH as the predominant fatty acids (≥ 10%). The main isoprenoid quinone of strain 17AT was identified as MK-6. The polar lipids consisted of phosphatidylethanolamine, three unidentified aminolipids, two unidentified aminophospholipids and six unidentified lipids. Comparative genomics analysis between strain 17AT and its reference type strains revealed that there are a number of metal-resistant genes in strain 17AT, which are located in 15 gene clusters responsible for the copper homeostasis, cobalt-zinc-cadmium resistance, copper resistance, and arsenic/antimony resistance, with the copper resistance protein NlpE being unique to 17AT. Combined data from phenotypic, phylogenetic and chemotaxonomic studies demonstrated that strain 17AT is a representative of a novel species within the genus Flavobacterium, for which the name Flavobacterium potami sp. nov. is proposed. The type strain is 17AT (= GDMCC 1.2723T = JCM 34833T).
Subject(s)
Flavobacterium , Phospholipids , Phospholipids/chemistry , Rivers/microbiology , Phylogeny , RNA, Ribosomal, 16S/genetics , Copper , Fatty Acids/chemistry , DNA , Sequence Analysis, DNA , DNA, Bacterial/genetics , Bacterial Typing Techniques , Vitamin K 2/chemistryABSTRACT
A Gram-stain-negative, yellow-pigmented, motile, flagellated and rod-shaped bacterium, designated as 13AT, was isolated from a river sediment sample of Fuyang River in Hengshui City, Hebei Province, PR China. Strain 13AT grew at 10-37 °C (optimum, 30 °C), at pH 5.0-11.0 (optimum, pH 7.0) and at 0-7â% (w/v) NaCl concentration (optimum, 0â%). Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain 13AT belongs to the genus Lysobacter, and was most closely related to Lysobacter spongiicola DSM 21749T (97.8â%), Lysobacter concretionis DSM 16239T (97.5â%), Lysobacter daejeonensis GIM 1.690T (97.3â%) and Lysobacter arseniciresistens CGMCC 1.10752T (96.9â%). Meanwhile, the type species Lysobacter enzymogenes ATCC 29487T was selected as a reference strain (95.2â%). The genomic size of strain 13AT was 3.0 Mb and the DNA G+C content was 69.0â%. The average nucleotide identity values between strain 13AT and each of the reference type strains L. spongiicola DSM 21749T, L. concretionis DSM 16239T, L. daejeonensis GIM 1.690T, L. arseniciresistens CGMCC 1.10752T and L. enzymogenes ATCC 29487T were 75.9, 76.1, 77.7, 78.0 and 73.2â%, respectively. The digital DNA-DNA hybridization values between strain 13AT and each of the reference type strains were 21.7, 22.2, 21.9, 22.7 and 23.2â%, respectively. The average amino acid identity values between strain 13AT and each of the reference type strains were 72.5, 72.9, 72.3, 75.0 and 69.2â%, respectively. The major fatty acids were iso-C15â:â0, iso-C16â:â0 and summed feature 9 (iso-C17â:â1 ω9c and/or C16â:â0 10-methyl). The sole respiratory quinone was identified as ubiquinone-8. The polar lipid profile contained phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, an unidentified aminolipid, an unidentified lipid, four unidentified phospholipids and two unidentified glycolipids. Based on the phenotypic, physiological, phylogenetic and chemotaxonomic data, strain 13AT represents a novel species of the genus Lysobacter, for which the name Lysobacter selenitireducens sp. nov. is proposed. The type strain is 13AT (=JCM 34786T=GDMCC 1.2722T).
Subject(s)
DNA, Bacterial , Lysobacter , Lysobacter/genetics , RNA, Ribosomal, 16S/genetics , Ubiquinone/chemistry , Phylogeny , Phosphatidylethanolamines/metabolism , Base Composition , Rivers , Sodium Chloride , Cardiolipins , Soil Microbiology , DNA, Bacterial/genetics , Fatty Acids/chemistry , Bacterial Typing Techniques , Sequence Analysis, DNA , Phospholipids/chemistry , Glycolipids/analysis , Amino Acids/metabolism , NucleotidesABSTRACT
A Gram-stain-negative, non-motile, aerobic, yellow, convex, rod-shaped mesophilic bacterial strain, designated strain D33T, was isolated from rhizosphere soil of ancient mulberry in Dezhou city, Shandong province, PR China. The strain grew at 8-37 °C (optimum, 30 °C), pH 4-9 (optimum, pH 7) and growth occurred at 0.5-5.5â% (w/v) NaCl (optimally at 1â%). The results of the phylogenetic analyses of 16S rRNA gene and whole genome sequences indicated that D33T was closely related to members of the genus Flavobacterium and had the highest 16S rRNA gene sequence similarity with 'Flavobacterium agri' KACC 19300 (95.4â%), Flavobacterium ichthyis NST-5T (94.6â%), Flavobacterium ahnfeltiae KCTC 32467T (93.6â%) and Flavobacterium longum JCM 19141T (93.6â%). The genome size of D33T was 3.8 Mb and the DNA G+C content was 48.0 mol%. The average nucleotide identity (ANI), digital DNA-DNA hybridization (dDDH) and average amino acid identity (AAI) values among D33T and reference strains were lower than the threshold values for species delineation. The only respiratory quinone of D33T was menaquinone 6 (MK-6). The predominant fatty acids (>5â%) were C15:0, C16â:â0, C18â:â0, iso-C15:0, iso-C17â:â0 3-OH, anteiso-C15â:â0 and summed feature 9 . The polar lipid profile contained phosphatidylethanolamine, two unidentified aminophospholipids, three unidentified aminolipids and two unidentified lipids. Combined data from phenotypic, phylogenetic and chemotaxonomic studies indicated that D33T is a representative of a novel species of the genus Flavobacterium, for which the name Flavobacterium selenitireducens sp. nov. is proposed. The type strain is D33T (=GDMCC 1.1946T=KACC 22131T).
Subject(s)
Flavobacterium , Morus , Bacterial Typing Techniques , Base Composition , DNA, Bacterial/genetics , Fatty Acids/chemistry , Morus/genetics , Phylogeny , RNA, Ribosomal, 16S/genetics , Rhizosphere , Sequence Analysis, DNA , SoilABSTRACT
In this paper, the authors review origins, motivations, and generalizations of a series of inequalities involving finitely many exponential functions and sums. They establish three new inequalities involving finitely many exponential functions and sums by finding convexity of a function related to the generating function of the Bernoulli numbers. They also survey the history, backgrounds, generalizations, logarithmically complete monotonicity, and applications of a series of ratios of finitely many gamma functions, present complete monotonicity of a linear combination of finitely many trigamma functions, construct a new ratio of finitely many gamma functions, derive monotonicity, logarithmic convexity, concavity, complete monotonicity, and the Bernstein function property of the newly constructed ratio of finitely many gamma functions. Finally, they suggest two linear combinations of finitely many trigamma functions and two ratios of finitely many gamma functions to be investigated.
ABSTRACT
The traditional discipline-centered curriculum design can neither keep up with developments of modern medical science nor reach requirements of the education reform in the new century.Since 2011,College of Stomatology in School of Medicine in Shanghai Jiao Tong University had developed ‘ disease oriented integrated curriculum system reform’ for students of long-term stomatology education.In view of the problems existing in the original curriculum system,the integrated curriculum system was set up by coalescing clinical medicine curriculum according to the related systems and oral medicine curriculum according to the developmental rules of diseases.Lectures were combined with discussion classes in the reform and performance appraisal system was changed from simplex judgments into comprehensive evaluations.At last,further considerations of promoting the reform based on the practice were proposed.
ABSTRACT
OBJECTIVE: To investigate the absorbability of 8 kinds of disposable filter membranes for infusion apparatus to 10 injectable drugs, thus to provide information for clinical medication. METHODS: Each drug was separately dissolved in 250 mL 5% dextrose solution. Samples of drug solutions were collected before and after being filtered by the membranes. These samples were analyzed by HPLC-MS/MS methods. RESULTS: The 8 kinds of membranes had different adsorbabilities to the 10 injectable drugs. CN-CA membrane (CN-CA) and CN-CA Enhance membrane (CN-CA-E) adsorbed more drugs than other membranes (P<0.05). The average adsorption of PES membrane, NL-B membrane and ION membrane to the 10 drugs were lower than 1%. PP membrane and NL membrane to the 10 drugs were lower than 1.5%. CONCLUSION: PES membrane, ION membrane and NL-B membrane have lower adsorbabilityies than other membranes, thus may have little influence on medical treatment. They may be suitable for clinical application. Copyright 2012 by the Chinese Pharmaceutical Association.
ABSTRACT
OBJECTIVE: To investigate the relationship between -308 genotype polymorphism in the promoter region of the tumor necrosis factor alpha (TNFalpha) gene and asthenospermia in infertile men. METHODS: Allele-specific polymerase chain reaction (ASPCR) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) were used to analyze the genotype at position -308 in the promoter region of the TNFalpha gene in 187 infertile male patients, who were divided into Groups A (asthenospermia, n = 60), B (oligoasthenozoospermia, n = 65) and C (infertile patients with normal sperm, n = 62). The levels of TNFalpha in the seminal plasma from these patients were measured by radioimmunoassay, and all the data were statistically analyzed by SPSS16.0. RESULTS: Groups A and B exhibited significant differences from C in the frequency of GA/AA at position 308 in the promoter region of the TNFalpha gene (21.67% and 26.15% versus 8.06%, P < 0.05). Spearman analysis showed a negative correlation between the GA + AA type of the TNFalpha-308 allele and the percentage of grade a + b sperm (r = -0.690, P < 0.05). The level of TNFalpha in the seminal plasma was significantly elevated in Groups A ([4.23 +/- 0.45] ng/ml) and B ([4.29 +/- 0.47] ng/ml) as compared with C ([4.03 +/- 0.66] ng/ml, P < 0.05), but with no significant differences between Groups A and B (P > 0.05). It was also significantly higher in the GA+AA ([4.61 +/- 0.29] ng/ml) than in the GGtype ([4.06 +/- 0.45] ng/ml, P < 0.05). CONCLUSION: Regardless of sperm density, the frequently of TNFalpha-308 GA/AA is negatively correlated with the percentage of grade a + b sperm, which may be associated with the level of TNFalpha in the seminal plasma. Accordingly, anti-TNFalpha therapy might be effective for asthenospermia, and the measurement of the TNFalpha level in the seminal plasma can be an auxiliary diagnostic marker for male infertility.
Subject(s)
Asthenozoospermia/genetics , Polymorphism, Genetic , Tumor Necrosis Factor-alpha/genetics , Adult , Alleles , Case-Control Studies , Gene Frequency , Genotype , Humans , Infertility, Male/genetics , Male , Promoter Regions, GeneticABSTRACT
<p><b>OBJECTIVE</b>To summarize and compare the short-term and long-term clinical efficacy of percutaneous transhepatic biliary drainage (PTBD) and percutaneous transhepatic biliary stent (PTBS) in the treatment of malignant obstructive jaundice.</p><p><b>METHODS</b>210 cases of malignant obstructive jaundice underwent interventional therapy, of which 161 cases of drainage catheters placement and 49 cases of metallic stent implantation. Follow-up information was obtained through telephone review or check-up records.</p><p><b>RESULTS</b>The technical success rate of technique was 100%. At 3 - 5 days after treatment, the serum total bilirubin in 15 metallic stent-treated patients was decreased by (178.04 +/- 42.32) micromol/L, and direct bilirubin by (83.97 +/- 23.63) micromol/L. Compared with those of 28 cases treated with drainage catheters: (95.67 +/- 34.28) micromol/L and (49.84 +/- 28.21) micromol/L, there were statistically significant differences between the two groups (P = 0.017 and P = 0.035). At 6 - 9 days after treatment, the serum total bilirubin in 28 cases of metallic stent group was decreased by (188.22 +/- 79.90) micromol/L, and that in 126 cases of drainage catheter group decreased by (141.39 +/- 65.32) micromol/L. The difference was statistically significant (P = 0.014). But the decline value of direct bilirubin had no significant difference. The median patency period and the median survival time of the drainage catheter group were 60 and 148 days, respectively, those of metallic stent group were 197 days and 245 days. There were statistically significant differences between the two groups (P < 0.05).</p><p><b>CONCLUSION</b>The results of this study indicate that the short-term and long-term efficacies of metallic stent implantation are better than those of catheter drainage technique.</p>
Subject(s)
Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Bile Duct Neoplasms , Bilirubin , Blood , Drainage , Methods , Gallbladder Neoplasms , Jaundice, Obstructive , Blood , Therapeutics , Liver Neoplasms , Pancreatic Neoplasms , Stents , Survival RateABSTRACT
An incubation test was conducted with mollisol applied with recommended amount of acetochlor under the conditions of sterilization, microbial inhibitor addition, and non-sterilization. During incubation, the residual amount of acetochlor and the dynamics of soil phospholipid fatty acids (PLFAs) were determined to study the relative contribution of soil microbes on the degradation of applied acetochlor, and the effects of acetochlor on the soil microbial community structure. The results showed that acetochlor was easy to be degraded by soil microbes, and bacteria contributed more than fungi. After applying acetochlor, the contents of various PLFAs changed evidently, and the soil microbial biomass indicated by C14:0, C16:0 and C18:0 was decreased. The bacterial PLFAs decreased significantly at the beginning of the incubation, but had less difference with CK (no acetochlor application) later, suggesting that bacterial activity was restored along with the degradation of acetochlor. The content of fungal PLFAs in the soil samples applied with acetochlor was lower than that of CK, implying that the inhibition of the herbicide to fungi was chronic and irreversible.
Subject(s)
Fatty Acids/chemistry , Phospholipids/chemistry , Soil Microbiology , Soil Pollutants/metabolism , Toluidines/metabolism , Biodegradation, Environmental , Herbicides/chemistry , Herbicides/metabolism , Soil Pollutants/chemistry , Toluidines/chemistryABSTRACT
In order to improve the fermentation potency of phytase in recombinant host and decrease the production cost, the pichia expression vector pGAPZalpha-A was modified by introduction of an AOX1 promoter from vector pPIC9 and the resulted vector pAOXZalpha is an methanol induced vector. After that, a phytase gene appA-m was cloned into pAOXZalpha to construct the recombinant vector pAOXZalpha-appA-m. The recombinant Pichia pastoris 74#, which already contains one copy of appA-m and its fermentation potency exceeded 7.5 x 10(6) IU/mL, was used as the host strain for the transformation of pAOXZalpha-appA-m. The Pichia pastoris transformants were gained by electroporation. PCR results indicated that the appA-m expression box has integrated into the genome of Pichia pastoris and the original construction of phytase gene has not changed. SDS-PAGE analysis revealed that phytase was overexpressed and secreted into the medium supernatant. Recombinants with high expression level were screened and used for fermentation. In 5L fermentor, the expression level of phytase protein achieved 4 mg/mL and the phytase activity (fermentation potency) exceeded 1.2 x 10(7) IU/mL, which was about 1.6-fold compared with that of the host strain 74#. Moreover, the improved recombinant Pichia pastoris is excellent at expression stability and heredity stability.
Subject(s)
6-Phytase , Genetics , Fermentation , Gene Dosage , Pichia , Genetics , Plasmids , Polymerase Chain Reaction , Recombination, GeneticABSTRACT
The hybrid xylanase TB was constructed by the substitution of the N-terminus segment of the Streptomyces olivaceoviridis xylanase XYNB with corresponding region of Thermomonosporafusca xylanase TfxA. The hybrid gene tb, encoding the TB, was correctly expressed in Escherichia coli BL21 and Pichia pastoris GS115. TB was purified and its enzymatic properties were determined. The results revealed that the optimal temperature and optimal pH of TB were at 70 degrees C and 6.0, which have been obviously improved compared with those of XYNB. The thermostability of TB were all about six-fold of XYNB's after incubating the properly diluted enzyme solutions at 80 degrees C and 90 degrees C for 3min, respectively. The pH stability of TB was 5 to approximately 9, which was narrower than that of XYNB. Still, TB remains a high specific activity as XYNB does. Analysis of a homology modeling and sequence similarity were used to reveal the factors influencing the enzymatic properties of TB and the discussion for the relationship between structure and function of xylanase was given.
Subject(s)
Amino Acid Sequence , Base Sequence , Desulfurococcaceae , Genetics , Endo-1,4-beta Xylanases , Genetics , Metabolism , Enzyme Stability , Escherichia coli , Genetics , Hot Temperature , Molecular Sequence Data , Pichia , Genetics , Protein Engineering , Methods , Recombinant Fusion Proteins , Genetics , Metabolism , Streptomyces , Genetics , Structure-Activity RelationshipABSTRACT
A homology modeling of xylanase XYNB from Streptomyces olivaceoviridis A1 was made by Swiss-Model. The hydrophobic Interaction between beta-sheet B1 and B2 in the tertiary structure model of XYNB was compared with other thermophilic xylanase. A T11Y mutation was introduced in XYNB by site-dirrected mutagenesis to improve the thermostability of the enzyme. The XYNB and mutant xylanase (XYNB') expressed in Pichia pastoris were purified and their enzymatic properties were determined. The result revealed that the thermostability of XYNB' was obviously higher than that of XYNB. The optimal temperature of XYNB' for its activity was 60 degrees C, similar to XYNB. But, compare to XYNB, the optimal pH value, the Km value and the specific activity of XYNB' had also been changed. The research results suggested that the aromatic interaction between beta-sheet B1 and B2 in xylanase should increase enzyme thermostability. The mutant xylanase XYNB' is a good material for further research in the relationship between structure and function of xylanase.