Sulfur speciation transformation during bioleaching of pyrite-containing sphalerite concentrate by thermophile Sulfolobus metallicus (S.metallicus) at 65 °C was investigated by X-ray diffraction (XRD),diffuse ref...Sulfur speciation transformation during bioleaching of pyrite-containing sphalerite concentrate by thermophile Sulfolobus metallicus (S.metallicus) at 65 °C was investigated by X-ray diffraction (XRD),diffuse reflectance Fourier transform infrared spectroscopy (FT-IR) and sulfur K-edge X-ray absorption near edge structure spectroscopy (XANES).The results show that the presence of S.metallicus effectively enhances the dissolution of the mineral.The yield of zinc increases from 0.5 g/L in sterile control to 2.7 g/L in bioleaching.The pyrite in the concentrate facilitates zinc dissolution in the early stage,but has hindrance role in the late stage for the formation of jarosite.Sulfur speciation analyses show that jarosite and elemental sulfur are main products in bioleaching process,and the accumulation of jarosite is mainly responsible for the decline of leaching efficiency.展开更多
In order to improve the extracellular endo-1,4-β-mannosidase(MAN) activity of recombinant Pichia pastoris, optimization of signal peptides was investigated. At first, five potential signal peptides(W1, MF4 I, INU1 A,...In order to improve the extracellular endo-1,4-β-mannosidase(MAN) activity of recombinant Pichia pastoris, optimization of signal peptides was investigated. At first, five potential signal peptides(W1, MF4 I, INU1 A, αpre, HFBI) were chosen to be analyzed by Signal P 4.0, among which W1 was designed. Then, the widely used signal peptide α-factor in expression vector p GAPZαA was replaced by those five signal peptides to reconstruct five new expression vectors. MAN activity was assayed after expression vectors were transformed into Pichia pastoris. The data show that the relative efficiencies of W1, MF4 I, INU1 A, αpre, and HFBI signal peptides are 23.5%, 203.5%, 0, 79.7%, and 120.3% compared with α-factor, respectively. The further gene copy number determination by the quantitative real-time PCR reveals that the MAN activities mediated by α-factor from 1 to 6 gene copy number levels are 12.95, 43.33, 126.63, 173.53, 103.23 and 88.63 U/m L, while those mediated by MF4 I are 79.22, 133.89, 260.14, 347.5, 206.15 and 181.89 U/m L, respectively. The maximum MAN activity reached 347.5 U/m L with 4 gene copies mediated by MF4 I. These results indicate that replacing the signal peptide α-factor with MF4 I and increasing MAN gene copies to a proper number can greatly improve the secretory expression of MAN.展开更多
A simple optimized microplate-based method to assay endo-1,4-β-mannosidase activity was described as an improved high-throughput screening method.A series of experimental conditions were optimized.It is revealed that...A simple optimized microplate-based method to assay endo-1,4-β-mannosidase activity was described as an improved high-throughput screening method.A series of experimental conditions were optimized.It is revealed that the optimum measurement procedure is as follows:adding 50 μL of diluted enzyme sample and 50 μL substrate,incubating at 45 °C for exactly 5 min in micro-plate,mixing with 100 μL 3,5-dinitrosalicylic acid (DNS) reagent,maintaining at boiling point for 15 min,cooling down to room temperature before determining the ABS value at 540 nm using an ELISA micro-plate reader.The reaction volume of the optimized microplate-assay is reduced to 200 μL from 2 500 μL used in the standard β-mannanase macro-assay.The optimized micro-assay is significantly more sensitive in all of the 643 candidates during endo-1,4-β-mannosidase screening.Statistical analyses show that the sensitivity of the optimized micro-method is significantly greater than that of the macro-assay.The optimized method is convenient,fast,and cheap for high throughput enzyme screening.展开更多
PCR-based DNA fingerprinting, REP-PCR(repetitive element PCR), RAPD(randomly amplified polymorphic DNA) and16 S r DNA sequence analyses were used to characterize 23 Acidithiobacillus ferrooxidans strains isolated from...PCR-based DNA fingerprinting, REP-PCR(repetitive element PCR), RAPD(randomly amplified polymorphic DNA) and16 S r DNA sequence analyses were used to characterize 23 Acidithiobacillus ferrooxidans strains isolated from different environments.(GTG)5 and BOXA1 R primer were selected for REP-PCR. Twenty arbitrary primers were used for RAPD to acquire DNA profiles from A. ferrooxidans. Both RAPD and REP-PCR produce complex banding patterns and show good discriminatory ability in differentiating closely related strains of A. ferrooxidans. The strains are clustered into 4 or 5 major groups and reveal genomic diversity using(GTG)5-PCR, BOX-PCR and RAPD analysis. Phylogenetic tree based on 16 S r DNA sequences of 23 strains and related strains shows that they are clustered into two distinct groups. Twelve strains are highly related to a new Acidithiobacillus named Acidithiobacillus ferrivorans. The results indicate that PCR-based methods are effective in revealing genetic diversity among A. ferrooxidans.展开更多
The yeast Pichia pastoris(P. pastoris) has been used for the expression of heterologous proteins with the significant success. However, it is time-consuming to screen the high expression level of the recombinant P. pa...The yeast Pichia pastoris(P. pastoris) has been used for the expression of heterologous proteins with the significant success. However, it is time-consuming to screen the high expression level of the recombinant P. pastoris directly. Thus, for β-mannanase production, developing the accurate, rapid and inexpensive screening method to substitute random screening is certainly required. A simple method based on the size of hydrolysis hole was described here, but this method was not very accurate that could only be used in preliminary screening. To further improve the accuracy, a micro-plate screening method is established, which appears to be more accurate and effective. The efficiency of this screening method is about 10 times higher than that of the general screening strategy of cultivation in shaking flasks. Two methods presented here can also be used for screening of recombinant Pichia strains with high-level expression of other heterologous protein after modification.展开更多
Our previously described environmental DNA extraction method has been widely used in environmental microbial community analysis.However,residual humic substances may remain with obtained environmental DNA,which interf...Our previously described environmental DNA extraction method has been widely used in environmental microbial community analysis.However,residual humic substances may remain with obtained environmental DNA,which interferes downstream molecular analyses.To remedy this situation,two DNA extraction buffers (PIPES and Tris-HCl) and four purification strategies including our new modified low melting point gel purification method and three commercial kits from QIAEX,Omega and Promega were evaluated with diverse soil samples.The PIPES buffer (pH 6.5) is found to be more effective for removing the humic substances,but it leads to lower DNA yield and causes more severe DNA shearing than using the Tris-HCl buffer (pH 8.0).Gel purification and the Promega purification kit achieve much higher DNA recoveries than QIAEX or Omega kit,and higher purity of DNA is obtained by gel purification than by the Promega kit with both DNA extraction buffers mentioned above.Considering all results together,two alternative methods for DNA extraction and purification are proposed:one uses Tris-HCl buffer extraction and gel purification as the primary approach when the amount of soil or biomass is not a major concern,and the other uses PIPES buffer extraction and the Promega kit purification when severe DNA shearing and/or limited biomass occurs.Purified DNA samples by both methods are amenable for use as templates for whole community genome amplifications and PCR amplifications of bacterial 16S rRNA genes.It is demonstrated that these two alternative methods could be applied to a wide variety of environmental samples.展开更多
基金Project(50974140) supported by the National Natural Science Foundation of ChinaProject(VR-09157) supported by Beijing Synchrotron Radiation Facility (BSRF) Public User Program,China
文摘Sulfur speciation transformation during bioleaching of pyrite-containing sphalerite concentrate by thermophile Sulfolobus metallicus (S.metallicus) at 65 °C was investigated by X-ray diffraction (XRD),diffuse reflectance Fourier transform infrared spectroscopy (FT-IR) and sulfur K-edge X-ray absorption near edge structure spectroscopy (XANES).The results show that the presence of S.metallicus effectively enhances the dissolution of the mineral.The yield of zinc increases from 0.5 g/L in sterile control to 2.7 g/L in bioleaching.The pyrite in the concentrate facilitates zinc dissolution in the early stage,but has hindrance role in the late stage for the formation of jarosite.Sulfur speciation analyses show that jarosite and elemental sulfur are main products in bioleaching process,and the accumulation of jarosite is mainly responsible for the decline of leaching efficiency.
基金Project(13JJ9002)supported by Hunan Provincial Natural Science Foundation of ChinaProject(2012XK4081)supported by the Key Science Technology Plan Project of Hunan Provincial Science&Technology Department,ChinaProject(CX2012B124)supported by the Graduate Degree Thesis Innovation Program of Hunan Province,China
文摘In order to improve the extracellular endo-1,4-β-mannosidase(MAN) activity of recombinant Pichia pastoris, optimization of signal peptides was investigated. At first, five potential signal peptides(W1, MF4 I, INU1 A, αpre, HFBI) were chosen to be analyzed by Signal P 4.0, among which W1 was designed. Then, the widely used signal peptide α-factor in expression vector p GAPZαA was replaced by those five signal peptides to reconstruct five new expression vectors. MAN activity was assayed after expression vectors were transformed into Pichia pastoris. The data show that the relative efficiencies of W1, MF4 I, INU1 A, αpre, and HFBI signal peptides are 23.5%, 203.5%, 0, 79.7%, and 120.3% compared with α-factor, respectively. The further gene copy number determination by the quantitative real-time PCR reveals that the MAN activities mediated by α-factor from 1 to 6 gene copy number levels are 12.95, 43.33, 126.63, 173.53, 103.23 and 88.63 U/m L, while those mediated by MF4 I are 79.22, 133.89, 260.14, 347.5, 206.15 and 181.89 U/m L, respectively. The maximum MAN activity reached 347.5 U/m L with 4 gene copies mediated by MF4 I. These results indicate that replacing the signal peptide α-factor with MF4 I and increasing MAN gene copies to a proper number can greatly improve the secretory expression of MAN.
基金Project(31000350)supported by the National Natural Science Foundation of China
文摘A simple optimized microplate-based method to assay endo-1,4-β-mannosidase activity was described as an improved high-throughput screening method.A series of experimental conditions were optimized.It is revealed that the optimum measurement procedure is as follows:adding 50 μL of diluted enzyme sample and 50 μL substrate,incubating at 45 °C for exactly 5 min in micro-plate,mixing with 100 μL 3,5-dinitrosalicylic acid (DNS) reagent,maintaining at boiling point for 15 min,cooling down to room temperature before determining the ABS value at 540 nm using an ELISA micro-plate reader.The reaction volume of the optimized microplate-assay is reduced to 200 μL from 2 500 μL used in the standard β-mannanase macro-assay.The optimized micro-assay is significantly more sensitive in all of the 643 candidates during endo-1,4-β-mannosidase screening.Statistical analyses show that the sensitivity of the optimized micro-method is significantly greater than that of the macro-assay.The optimized method is convenient,fast,and cheap for high throughput enzyme screening.
基金Project(2010CB630901)supported by the National Basic Research Program of China
文摘PCR-based DNA fingerprinting, REP-PCR(repetitive element PCR), RAPD(randomly amplified polymorphic DNA) and16 S r DNA sequence analyses were used to characterize 23 Acidithiobacillus ferrooxidans strains isolated from different environments.(GTG)5 and BOXA1 R primer were selected for REP-PCR. Twenty arbitrary primers were used for RAPD to acquire DNA profiles from A. ferrooxidans. Both RAPD and REP-PCR produce complex banding patterns and show good discriminatory ability in differentiating closely related strains of A. ferrooxidans. The strains are clustered into 4 or 5 major groups and reveal genomic diversity using(GTG)5-PCR, BOX-PCR and RAPD analysis. Phylogenetic tree based on 16 S r DNA sequences of 23 strains and related strains shows that they are clustered into two distinct groups. Twelve strains are highly related to a new Acidithiobacillus named Acidithiobacillus ferrivorans. The results indicate that PCR-based methods are effective in revealing genetic diversity among A. ferrooxidans.
基金Project(CX2012B124)supported by the Graduate Degree Thesis Innovation Program of Hunan ProvinceChina+3 种基金Project(13JJ9002)supported by the Natural Science Foundation of Hunan ProvinceChinaProject(2012XK4081)supported by the Key Science and Technology Plan of Hunan Provincial Science&Technology DepartmentChina
文摘The yeast Pichia pastoris(P. pastoris) has been used for the expression of heterologous proteins with the significant success. However, it is time-consuming to screen the high expression level of the recombinant P. pastoris directly. Thus, for β-mannanase production, developing the accurate, rapid and inexpensive screening method to substitute random screening is certainly required. A simple method based on the size of hydrolysis hole was described here, but this method was not very accurate that could only be used in preliminary screening. To further improve the accuracy, a micro-plate screening method is established, which appears to be more accurate and effective. The efficiency of this screening method is about 10 times higher than that of the general screening strategy of cultivation in shaking flasks. Two methods presented here can also be used for screening of recombinant Pichia strains with high-level expression of other heterologous protein after modification.
基金Projects(51934009, 52074353) supported by the National Natural Science Foundation of ChinaProject(20225199K001) supported by the Zijin Mining State Key Laboratory of Biometallurgy Open Fund,China。
基金Projects(51934009,52074353)supported by the National Natural Science Foundation of ChinaProject(2019YFC1803600)supported by the National Key Research and Development Program of ChinaProject(2021JJ30855)supported by the Natural Science Foundation of Hunan Province,China。
基金Project(51104189)supported by the National Natural Science Foundation of ChinaProject(2010CB630901)supported by the National Basic Research Program of China+1 种基金Project(1343-77341)supported by the Graduate Education Innovative Program of Central South University,ChinaProject(DOE-ER64125)supported by Department of Energy,Office of Science under the Environmental Remediation Science Program of the United States
文摘Our previously described environmental DNA extraction method has been widely used in environmental microbial community analysis.However,residual humic substances may remain with obtained environmental DNA,which interferes downstream molecular analyses.To remedy this situation,two DNA extraction buffers (PIPES and Tris-HCl) and four purification strategies including our new modified low melting point gel purification method and three commercial kits from QIAEX,Omega and Promega were evaluated with diverse soil samples.The PIPES buffer (pH 6.5) is found to be more effective for removing the humic substances,but it leads to lower DNA yield and causes more severe DNA shearing than using the Tris-HCl buffer (pH 8.0).Gel purification and the Promega purification kit achieve much higher DNA recoveries than QIAEX or Omega kit,and higher purity of DNA is obtained by gel purification than by the Promega kit with both DNA extraction buffers mentioned above.Considering all results together,two alternative methods for DNA extraction and purification are proposed:one uses Tris-HCl buffer extraction and gel purification as the primary approach when the amount of soil or biomass is not a major concern,and the other uses PIPES buffer extraction and the Promega kit purification when severe DNA shearing and/or limited biomass occurs.Purified DNA samples by both methods are amenable for use as templates for whole community genome amplifications and PCR amplifications of bacterial 16S rRNA genes.It is demonstrated that these two alternative methods could be applied to a wide variety of environmental samples.
