ABSTRACT
<p><b>OBJECTIVE</b>To construct a recombinant adenovirus of survivin vector and provid valuable reference for gene therapy of laryngeal cancer.</p><p><b>METHODS</b>The survivin gene was cloned by PCR. After confirmation by enzyme restriction analysis and sequencing, the gene and the adenovirus vector were recombined together to construct the recombinant adenovirus vector. The recombinant adenovirus vector was confirmed via both sequencing and digestion restriction analysis, and then linearized and transfected into the HEK 293 cell line to generate recombinant adenovirus.</p><p><b>RESULTS</b>The sequence analysis demonstrated that the survivin gene sequence was the same as published in the literature, suggesting that a recombinant adenovirus vector has been successfully constructed.</p><p><b>CONCLUSIONS</b>A survivin recombinant adenovirus has been successfully constructed.</p>
Subject(s)
Humans , Adenoviridae , Genetics , Genetic Vectors , HEK293 Cells , Inhibitor of Apoptosis Proteins , Genetics , Metabolism , Plasmids , Polymerase Chain Reaction , Recombinant Fusion Proteins , Genetics , Metabolism , TransfectionABSTRACT
The bglS gene encoding endo-l,3-1,4-beta-glucanase from Bacillus subtilis was cloned and sequenced in this study. The bglS expression cassette, including PGK1 promoter, bglS gene fused to the signal sequence of the yeast mating pheromone alpha-factor (MFalpha1(S)), and ADH1 terminator with G418-resistance as the selected marker, was constructed. Then one of the PEP4 allele of Saccharomyces cerevisiae WZ65 strain was replaced by bglS expression cassette using chromosomal integration of polymerase chain reaction (PCR)-mediated homologous recombination, and the bglS gene was expressed simultaneously. The recombinant strain S. cerevisiae (SC-betaG) was preliminarily screened by the clearing hydrolysis zone formed after the barley beta-glucan was hydrolyzed in the plate and no proteinase A (PrA) activity was measured in fermenting liquor. The results of PCR analysis of genome DNA showed that one of the PEP4 allele had been replaced and bglS gene had been inserted into the locus of PEP4 gene in recombinant strains. Different endo-l,3-1,4-beta-glucanase assay methods showed that the recombinant strain SC-betaG had high endo-l,3-1,4-beta-glucanase expression level with the maximum of 69.3 U/(h.ml) after 60 h of incubation. Meanwhile, the Congo Red method was suitable for the determination of endo-l,3-1,4-beta-glucanase activity during the actual brewing process. The current research implies that the constructed yeast strain could be utilized to improve the industrial brewing property of beer.
Subject(s)
Amino Acid Sequence , Aspartic Acid Endopeptidases , Genetics , Base Sequence , Glycoside Hydrolases , Genetics , Molecular Sequence Data , Mutagenesis, Insertional , Polymerase Chain Reaction , Recombination, Genetic , Saccharomyces cerevisiae , Genetics , Saccharomyces cerevisiae Proteins , GeneticsABSTRACT
Optimization of a process for extracting astaxanthin from Phaffia rhodozyma by acidic method was investigated, regarding several extraction factors such as acids, organic solvents, temperature and time. Fractional factorial design, central composite design and response surface methodology were used to derive a statistically optimal model, which corresponded to the following optimal condition: concentration of lactic acid at 5.55 mol/L, ratio of ethanol to yeast dry weight at 20.25 ml/g, temperature for cell-disruption at 30 degrees C, and extraction time for 3 min. Under this condition, astaxanthin and the total carotenoids could be extracted in amounts of 1294.7 microg/g and 1516.0 microg/g, respectively. This acidic method has advantages such as high extraction efficiency, low chemical toxicity and no special requirement of instruments. Therefore, it might be a more feasible and practical method for industrial practice.
Subject(s)
Basidiomycota , Chemistry , Hydrochloric Acid , Lactic Acid , XanthophyllsABSTRACT
Fermentation of Phaffia rhodozyma is a major method for producing astaxanthin, an important pigment with industrial and pharmaceutical application. To improve astaxanthin productivity, single factor and mixture design experiments were used to investigate the effects of nitrogen source on Phaffia rhodozyma cultivation and astaxanthin production. Results of single factor experiments showed nitrogen source could significantly affect P. rhodozyma cultivation with respect to carbon source utilization, yeast growth and astaxanthin accumulation. Further studies of mixture design experiments using (NH(4))(2)SO(4), KNO(3) and beef extract as nitrogen sources indicated that the proportion of three nitrogen sources was very important to astaxanthin production. Validation experiments showed that the optimal nitrogen source was composed of 0.28 g/L (NH(4))(2)SO(4), 0.49 g/L KNO(3) and 1.19 g/L beef extract. The kinetic characteristics of batch cultivation were investigated in a 5-L pH-stat fermentor. The maximum amount of biomass and highest astaxanthin yield in terms of volume and in terms of biomass were 7.71 mg/L and 1.00 mg/g, respectively.
Subject(s)
Bioreactors , Microbiology , Cell Culture Techniques , Methods , Cell Proliferation , Computer Simulation , Mitosporic Fungi , Physiology , Models, Biological , Nitrogen , Metabolism , XanthophyllsABSTRACT
The work is intended to achieve optimum culture conditions of alpha-galactosidase production by a mutant strain Aspergillus foetidus ZU-G1 in solid-state fermentation (SSF). Certain fermentation parameters involving moisture content, incubation temperature, cultivation period of seed, inoculum volume, initial pH value, layers of pledget, load size of medium and period of cultivation were investigated separately. The optimal cultivating conditions of alpha-galactosidase production in SSF were 60% initial moisture of medium, 28 degrees C incubation temperature, 18 h cultivation period of seed, 10% inoculum volume, 5.0 approximately 6.0 initial pH of medium, 6 layers of pledget and 10 g dry matter loadage. Under the optimized cultivation conditions, the maximum alpha-galactosidase production was 2 037.51 U/g dry matter near the 144th hour of fermentation.
Subject(s)
Aspergillus , Classification , Cell Culture Techniques , Methods , Enzyme Activation , Enzyme Stability , Fermentation , Hydrogen-Ion Concentration , Species Specificity , Temperature , alpha-Galactosidase , ChemistryABSTRACT
Sequential methodology based on the application of three types of experimental designs was used to optimize the fermentation conditions for elastase production from mutant strain ZJUEL31410 of Bacillus licheniformis in shaking flask cultures. The optimal cultivation conditions stimulating the maximal elastase production consist of 220 r/min shaking speed, 25 h fermentation time, 5% (v/v) inoculums volume, 25 ml medium volume in 250 ml Erlenmeyer flask and 18 h seed age. Under the optimized conditions, the predicted maximal elastase activity was 495 U/ml. The application of response surface methodology resulted in a significant enhancement in elastase production. The effects of other factors such as elastin and the growth factor (corn steep flour) on elastase production and cell growth were also investigated in the current study. The elastin had no significant effect on enzyme-improved production. It is still not clear whether the elastin plays a role as a nitrogen source or not. Corn steep flour was verified to be the best and required factor for elastase production and cell growth by Bacillus licheniformis ZJUEL31410.
Subject(s)
Bacillus , Fermentation , Pancreatic Elastase , Research DesignABSTRACT
Expression strain of des-pGlu1-brazzein was constructed and the conditions using lactose as inducer was also optimized. The Influences of three factors which were lactose concentration, induction time and inducing temperature on the growth of strain and on the yield of des-pGlul-Brazzein was analyzed in detail. The result indicated that high lactose concentration inhibit the growth of strains (P < 0.01) but made no difference on expression of target protein between 0.5%-5% (P > 0.05), Biomass would be improved as time passed (P < 0.01), but the yield of target protein didn't increase obviously at 30 degrees C compared with at 37 degrees C. Further result showed that the greater expressed level of des-pGlul-Brazzein, as high as about 20% of total cell protein, could be achieved after the strain had been induced with 0.5% lactose under 28 degrees C - 30 degrees C for 4 h.
Subject(s)
Dose-Response Relationship, Drug , Escherichia coli , Genetics , Isopropyl Thiogalactoside , Pharmacology , Lactose , Pharmacology , Plant Proteins , Genetics , Plasmids , Genetics , Temperature , Time Factors , Up-RegulationABSTRACT
<p><b>OBJECTIVE</b>To optimize extracting parameters of flavonoids from Humulus lupulus.</p><p><b>METHOD</b>Based on the single factors test on ethanol concentration, material and solvent ratio, extracting temperature and extracting time, orthogonal test was performed and the best combination was confirmed.</p><p><b>RESULT</b>With the optimized technology, the maximal extracting amount of flavonoids from H. lupulus was 78 mg x g(-1).</p><p><b>CONCLUSION</b>The optimal techniques obtained are 45% ethanol extracting at 60 degrees C with material and solvent ratio 1:25 for 90 min.</p>
Subject(s)
Ethanol , Flavonoids , Flowers , Chemistry , Humulus , Chemistry , Plants, Medicinal , Chemistry , Technology, Pharmaceutical , Methods , Temperature , TimeABSTRACT
The solubilization of elastin by Bacillus licheniformis elastase cannot be analyzed by conventional kinetic methods because the biologically relevant substrate is insoluble and the concentration of enzyme-substrate complex has no physical meaning. In this paper we report the optimization of elastolysis conditions and analysis of elastolytic kinetics. Our results indicated that the hydrolyzing temperature and time are very important factors affecting elastolysis rate. The optimized conditions using central composite design were as follows: elastolysis temperature 50 degrees C, elastase concentration 1 x 10(4) U/ml, elastin 80 mg, elastolytic time 4 h. Investigation of the effects of substrate content, elastase concentration and pH was also revealed that low or high elastin content inhibits the elastolysis process. Increasing elastase improves elastin degradation, but high elastase may change the kinetics characterization. Alkaline environment can decrease elastin degradation rate and pH may affect elastolysis by changing elastase reaction pH. To further elucidate the elastolysis process, the logistic model was used to elastolysis kinetics study showing clearly that the logistic model can reasonably explain the elastolysis process, especially under lower elastase concentration. However, there is still need for more investigations with the aid of other methods, such as biochemical and molecular methods.
Subject(s)
Bacillus , Colorimetry , Elastin , Metabolism , Hydrogen-Ion Concentration , Kinetics , Pancreatic Elastase , Metabolism , Regression Analysis , TemperatureABSTRACT
Octenyl succinic anhydride (OSA) modified early Indica rice starch was prepared in aqueous slurry systems using response surface methodology. The paste properties of the OSA starch were also investigated. Results indicated that the suitable parameters for the preparation of OSA starch from early Indica rice starch were as follows: reaction period 4 h, reaction temperature 33.4 degrees C, pH of reaction system 8.4, concentration of starch slurry 36.8% (in proportion to water, w/w), amount of OSA 3% (in proportion to starch, w/w). The degree of substitution was 0.0188 and the reaction efficiency was 81.0%. The results of paste properties showed that with increased OSA modification, the starch derivatives had higher paste clarity, decreased retrogradation and better freeze-thaw stability.
Subject(s)
Biochemistry , Methods , Chemical Phenomena , Chemistry, Physical , Freezing , Hydrogen-Ion Concentration , Light , Models, Statistical , Oryza , Metabolism , Starch , Chemistry , Succinic Anhydrides , Chemistry , TemperatureABSTRACT
Angiotensin I-converting enzyme (ACE) inhibitory peptides have been shown to have antihypertensive effects and have been utilized for physiologically functional foods and pharmaceuticals. The ACE inhibitory ability of a hydrolysate is determined by its peptide composition. However, the peptide composition of a hydrolysate depends on proteolytic enzyme and the hydrolysis conditions. In this study, the effect of process conditions on the ACE inhibitory activity of rice dregs hydrolyzed with a trypsin was investigated systematically using response surface methodology. It was shown that the ACE inhibitory activity of rice dregs hydrolysates could be controlled by regulation of five process conditions. Hydrolysis conditions for optimal ACE inhibition were defined using the response surface model of fractional factorial design (FFD), steepest ascent design, and central composite design (CCD).
Subject(s)
Angiotensin-Converting Enzyme Inhibitors , Chemistry , Combinatorial Chemistry Techniques , Methods , Drug Evaluation, Preclinical , Enzyme Activation , Hydrolysis , Oryza , Chemistry , Peptidyl-Dipeptidase A , Chemistry , Plant Extracts , Chemistry , Plant Proteins , Chemistry , Protein Hydrolysates , ChemistryABSTRACT
A derivative ratio spectrophotometric method was used for the simultaneous determination of beta-carotene and astaxanthin produced from Phaffia rhodozyma. Absorbencies of a series of the standard carotenoids in the range of 441 nm to 490 nm demonstrated that their absorptive spectra accorded with Beer's law and that the additivity when the concentrations of beta-carotene and astaxanthin and their mixture were within the range of 0 to 5 microg/ml, 0 to 6 microg/ml, and 0 to 6 microg/ml, respectively. When the wavelength interval (lambda) at 2 nm was selected to calculate the first derivative ratio spectra values, the first derivative amplitudes at 461 nm and 466 nm were suitable for quantitatively determining beta-carotene and astaxanthin, respectively. Effect of divisor on derivative ratio spectra could be neglected; any concentration used as divisor in range of 1.0 to 4.0 microg/ml is ideal for calculating the derivative ratio spectra values of the two carotenoids. Calibration graphs were established for beta-carotene within 0-6.0 microg/ml and for astaxanthin within 0-5.0 microg/ml with their corresponding regressive equations in: y=-0.0082x-0.0002 and y=0.0146x-0.0006, respectively. R-square values in excess of 0.999 indicated the good linearity of the calibration graphs. Sample recovery rates were found satisfactory (>99%) with relative standard deviations (RSD) of less than 5%. This method was successfully applied to simultaneous determination of beta-carotene and astaxanthin in the laboratory-prepared mixtures and the extract from the Phaffia rhodozyma culture.
Subject(s)
Algorithms , Basidiomycota , Metabolism , Spectrophotometry, Ultraviolet , Methods , Xanthophylls , beta Carotene , ChemistryABSTRACT
The partition behaviors of beta-1,3-1,4-glucanase, alpha-amylase and neutral proteases from clarified and whole fermentation broths of Bacillus subtilis ZJF-1A5 were investigated. An aqueous two-phase system (polyethylene glycol (PEG)/MgSO(4)) was examined with regard to the effects of PEG molecular weight (MW) and concentration, MgSO(4) concentration, pH and NaCl concentration on enzyme partition and extraction. The MW and concentration of PEG were found to have significant effects on enzyme partition and extraction with low MW PEG showing the greatest benefit in the partition and extraction of beta-glucanase with the PEG/MgSO(4) system. MgSO(4) concentration influenced the partition and extraction of beta-glucanase significantly. pH had little effect on beta-glucanase or proteases partition but affected alpha-amylase partition when pH was over 7.0. The addition of NaCl had little effect on the partition behavior of beta-glucanase but had very significant effects on the partitioning of alpha-amylase and on the neutral proteases. The partition behaviors of beta-glucanase, alpha-amylase and proteases in whole broth were also investigated and results were similar to those obtained with clarified fermentation broth. A two-step process for purifying beta-glucanase was developed, which achieved beta-glucanase recovery of 65.3% and specific activity of 14027 U/mg, 6.6 times improvement over the whole broth.
Subject(s)
Bacillus subtilis , Chemical Fractionation , Methods , Endo-1,3(4)-beta-Glucanase , Chemistry , Extracellular Fluid , Chemistry , Phase Transition , Water , ChemistryABSTRACT
Waste hops are good sources of flavonoids. Extraction of flavonoids from waste hops (SC-CO(2) extracted hops) using supercritical fluids technology was investigated. Various temperatures, pressures and concentrations of ethanol (modifier) and the ratio (w/w) of solvent to material were tested in this study. The results of single factor and orthogonal experiments showed that at 50 degrees C, 25 MPa, the ratio of solvent to material (50%), ethanol concentration (80%) resulted in maximum extraction yield flavonoids (7.8 mg/g). HPLC-MS analysis of the extracts indicated that flavonoids obtained were xanthohumol, the principal prenylflavonoid in hops.
Subject(s)
Chromatography, High Pressure Liquid , Chromatography, Supercritical Fluid , Methods , Ethanol , Chemistry , Flavonoids , Humulus , Chemistry , Mass SpectrometryABSTRACT
The production of butyric acid by Clostridium butyricum ZJUCB at various pH values was investigated. In order to study the effect of pH on cell growth, butyric acid biosynthesis and reducing sugar consumption, different cultivation pH values ranging from 6.0 to 7.5 were evaluated in 5-L bioreactor. In controlled pH batch fermentation, the optimum pH for cell growth and butyric acid production was 6.5 with a cell yield of 3.65 g/L and butyric acid yield of 12.25 g/L. Based on these results, this study then compared batch and fed-batch fermentation of butyric acid production at pH 6.5. Maximum value (16.74 g/L) of butyric acid concentration was obtained in fed-batch fermentation compared to 12.25 g/L in batch fermentation. It was concluded that cultivation under fed-batch fermentation mode could enhance butyric acid production significantly (P<0.01) by C. butyricum ZJUCB.
Subject(s)
Bioreactors , Microbiology , Butyric Acid , Metabolism , Cell Culture Techniques , Methods , Cell Proliferation , Clostridium butyricum , Metabolism , Glucose , Metabolism , Hydrogen-Ion ConcentrationABSTRACT
This paper presents the evaluation of an aqueous two-phase system (ATPS) for extracting elastase produced by Bacillus sp. EL31410. The elastase and cell partition behavior in polyethylene glycol (PEG)/salt systems was investigated. The suitable system for elastase extraction was PEG/KH(2)PO(4)-K(2)HPO(4), in which elastase is mainly partitioned into the PEG-rich phase, while the cells remained in the other phase. The influence of defined system parameters (e.g. PEG molecular mass, pH, NaCl addition) on the partitioning behavior of elastase is described. The concentration of phase forming components, PEG and KH(2)PO(4)-K(2)HPO(4), was optimized for elastase recovery by means of response surface methodology, and it was found that they greatly influenced extraction recovery. The optimal ATPS was 23.1% (w/w) PEG 2 000 and 11.7% (w/w) KH(2)PO(4)-K(2)HPO(4). The predicted recovery was about 89.5%, so this process is suggested to be a rapid and convenient method for elastase extraction.
Subject(s)
Bacillus , Bioreactors , Microbiology , Cell Culture Techniques , Methods , Chemical Fractionation , Methods , Combinatorial Chemistry Techniques , Culture Media , Chemistry , Metabolism , Fermentation , Physiology , Hydrogen-Ion Concentration , Pancreatic Elastase , Chemistry , Phase Transition , Polyethylene Glycols , Chemistry , Water , ChemistryABSTRACT
2,4-Dichlorophenol is toxic and biorefratory organic pollutant. A 2,4-dichlorophenol degrading bacterial strain GT241-1, identified as Pseudomonas sp., was isolated from soil samples which was collected from drainage area of several 2,4-dichlorophenol producing factories. Strain GT241-1 had strong 2,4-dichlorophenol degrading ability, it could decompose 91% 2, 4-dichlorophenol of 90 mg/L within 48 hours at 25 - 30 degrees C, and could utilize 2,4-dichlorophenol, 2,4-dichlorophenoxyacetic acid (2,4-D), benzoate and catechol as sole carbon and energy source. Southern blot showed that 2,4-dichlorophenol hydroxylase gene (dcpA) of strain GT241-1 locates on the about 10kb EcoR I/Xba I fragment. This fragment was recovered, linked to the vecter pUC19 and transformed into the E. coli DH5alpha. A aim transformant, Z539, was obtained by dot blotting from about 1200 transformants. PCR and the sequencing results shew that the whole dcpA gene is contained within the 10kb EcoR I /Xba I fragment of pZ539. This fragment was shortened to about 2.4kb by HindmIII. The shorted fragment was subcloned to vecter pRSET-B to get a transformant BS1-12. The subcloned fragment was sequenced. Sequencing results showed that the whole length of the subcloned fragment containing dcpA is 2389bp and the nucleotide span of coding region is from number 276 to number 2072 (1797 bp), with ATG and TAA as start and stop codon respectively. The sequence analysis of dcpA and the deduced amino acid encoded by dcpA showed that they are different from the relative sequences registered in the GenBank. The subcloned fragment carry the promoter of dcpA, this can deduce from the fact that the upflow length of dcpA coding region is 275bp, and further confirmed by the 2,4-dichlorophenol hydroxylase activity measurement results. The 2,4-dichlorophenol hydroxylase activity of transformant Z539 and BS1-12 were detected, the results showed these transformants have 2,4-dichlorophenol hydroxylase activity. By comparison, the activity of these transformants were lower than that of the strain GT241-1.
Subject(s)
Amino Acid Sequence , Bacterial Proteins , Genetics , Metabolism , Biodegradation, Environmental , Chlorophenols , Metabolism , Cloning, Molecular , Environmental Pollutants , Metabolism , Mixed Function Oxygenases , Genetics , Metabolism , Molecular Sequence Data , Pseudomonas , Genetics , Soil MicrobiologyABSTRACT
A 2,4 -dichlorophenol degrading Pseudomonas strain GI241-1 was isolated from a soil sample. The dienelactone hydrolase gene, designated as dcpD which encodes dienelactone hydrolase involved in transforming cis-2-chloro-dienelactone into 2-chloromaleylacetic acid, was cloned from this bacterium strain. The gene cloning strategy was to construct genomic library after location of its neighbouring gene by Southem blot and to screen the aim transformant by dot blotting. Sequencing results showed that length of dcpD is 702bp. The sequence of dcpD and the deduced amino acid are different from the relative sequences registered in the GenBank.