The amino acid composition of α and β structural class of proteins from five species, Escherichia coli, Thermotoga maritima, Thermus thermophilus, yeast, and humans were investigated. Amino acid residues of proteins...The amino acid composition of α and β structural class of proteins from five species, Escherichia coli, Thermotoga maritima, Thermus thermophilus, yeast, and humans were investigated. Amino acid residues of proteins were classified into interior or surface residues based on the relative accessible surface area. The hydrophobic Leu, Ala, Val, and Ile residues were rich in interior residues, and hydrophilic Glu, Lys, Asp, and Arg were rich in surface residues both in α and β proteins. The amino acid composition of α proteins was different from that of β proteins in five species, and the difference was derived from the different contents of their interior residues between α and β proteins. α-helix content of α proteins was rich in interior residues than surface ones. Similarly, β-sheet content of β proteins was rich in interior residues than surface ones. The content of Leu residues was very high, approximately 20%, in interior residues of α proteins. This result suggested that the Leu residue plays an important role in the folding of α proteins.展开更多
We analyzed the amino acid residues present in the water-soluble and transmembrane proteins of 6 thermophilic and 6 mesophilic species of the domains Archaea and Eubacteria, and characterized them as favorable or unfa...We analyzed the amino acid residues present in the water-soluble and transmembrane proteins of 6 thermophilic and 6 mesophilic species of the domains Archaea and Eubacteria, and characterized them as favorable or unfavorable. The characterization was performed by comparing the observed number of each amino acid residue to the expected number calculated from the percentage of nucleotides present in each gene. Amino acids that were more or less abundant than expected were considered as favorable or unfavorable, respectively. Comparisons of amino acid compositions indicated that the water-soluble proteins were rich in charged residues such as Glu, Asp, Lys, and His, whereas hydrophobic residues such as Trp, Phe, and Leu were abundant in transmembrane proteins. Interestingly, our results found that although the Trp residue was abundant in transmembrane proteins, it was not defined as favorable by our calculations, indicating that increased numbers of a particular amino acid does not necessary indicate it is a favorable residue. Amino acids with high G + C content such as Ala, Gly, and Pro were frequently observed as favorable in species with low G + C content. Comparatively, amino acids with low G + C content such as Phe, Tyr, Lys, Ile, and Met were frequently observed as favorable in species with high G + C content. These are the examples to increase the supply of amino acids than expected. Amino acids with neutral G + C content, i.e., Glu and Asp were favorable in water-soluble proteins from all species analyzed, and Cys was unfavorable both in water-soluble and transmembrane proteins. These results indicate that amino acid compositions are essentially determined by the nucleotide sequence of the genes, and the amino acid content is altered by a deviation from expectation.展开更多
A response surface method was employed to study the effect of α-amylase concentration, hydrolysis temperature and time on the production of high protein glutinous rice flour(HPGRF). The suspension of glutinous rice f...A response surface method was employed to study the effect of α-amylase concentration, hydrolysis temperature and time on the production of high protein glutinous rice flour(HPGRF). The suspension of glutinous rice flour(15%) that contained 6.52% protein was gelatinized and subsequently hydrolyzed by thermostable α-amylase. The hydrolysis yielded 0.144–0.222 g/g HPGRF with 29.4%–45.4% protein content. Hydrolysis time exerted a significant effect, while enzyme concentration and hydrolysis temperature showed insignificant effect on the protein content and production yield of HPGRF. The result of response surface method showed that the optimum condition for the production of HPGRF that contained at least 36% protein was treating gelatinized 15% glutinous rice flour suspension with 0.90 Kilo Novo α-amylase Unit(KNU)/g α-amylase at 80 oC for 99 min. By carrying out the predicted hydrolysis condition, HPGRF with 35.9% protein and 61.8% carbohydrates was resulted. The process yielded 0.172 g/g HPGRF. HPGRF contained higher amount of essential amino acids compared to glutinous rice flour. HPGRF had higher solubility and lower swelling power, and also showed no pasting peak compared with glutinous rice flour.展开更多
Using microbial fermentation to increase the content of free amino acids and short peptides in the organic fertilizer for castor bean meal can effectively promote plant growth and improve fruit quality.Using free amin...Using microbial fermentation to increase the content of free amino acids and short peptides in the organic fertilizer for castor bean meal can effectively promote plant growth and improve fruit quality.Using free amino acid and short peptide content as an indicator,through single factor and response surface optimization experiments,the process parameters(moisture content,fermentation time and inoculum quantity)of castor meal solid-state fermentation were optimized.The best process parameters for the solid-state fermentation were:the moisture content 62%,the fermentation time 20 d,and the inoculum quantity 0.23%.The moisture content had the greatest impact on the conversion rate of free amino acids and short peptides,and the protein conversion rate reached 65.6%.The scale-up experiment under the optimal conditions showed that the solid-state fermentation using the inoculum had a significant beneficial effect compared with other fermentation methods.The fermentation of castor cake fertilizer provides a theoretical and practical basis for production feasibility,and has important guiding significance for the effective utilization of castor bean meal.展开更多
While phthalate acid esters(PAEs)cannot fluoresce alone,they can be detected by fluorescence spectroscopy after chelation with bovine serum albumin(BSA).In this study,the types of amino acid residues at the active sit...While phthalate acid esters(PAEs)cannot fluoresce alone,they can be detected by fluorescence spectroscopy after chelation with bovine serum albumin(BSA).In this study,the types of amino acid residues at the active site of PAEs chelated with BSA were determined using molecular docking technology.A modification scheme of BSA with higher detection sensitivity fluorescence spectroscopy for PAEs was proposed based on the docking results and constructed for a novel BSA structure with a higher detection sensitivity of fluorescence spectroscopy using a homologous modeling method.Density functional theory(DFT)was employed to explore the influence before and after BSA modification on PAEs’detection through fluorescence spectroscopy.The results showed that the docking scores between BSAs and dimethyl phthalate(DMP),dibutyl phthalate(DBP)and di-n-octyl phthalate(DNOP)were increased up to 26.45%,16.82%and 16.30%,respectively,indicating that the active site modification of BSA could enhance the binding affinity between BSA and PAEs.The fluorescence intensity of PAEs chelated with modified BSAs were calculated.The fluorescence intensity of fluorescence spectroscopy for DMP,DBP and DNOP chelated with BSAs after modification was increased up to 2.8-,104.51-and 62.43-fold,respectively,which achieved the purpose of theoretically modifying BSA to improve the detection sensitivity of fluorescence spectroscopy for PAEs.展开更多
<strong>Object</strong>: To analyze porcine reproductive and respiratory syndrome virus (PRRSV) strains from 13 infection cases via the N protein gene and its encoded amino acid sequence and to provide a t...<strong>Object</strong>: To analyze porcine reproductive and respiratory syndrome virus (PRRSV) strains from 13 infection cases via the N protein gene and its encoded amino acid sequence and to provide a theoretical basis for the epidemiological study, prevention and control of porcine reproductive and respiratory syndrome (PRRS). <strong>Methods</strong>: In clinically suspected PRRSV infections, viruses were isolated by extracting viral nucleic acid and amplifying the N protein gene by RT-PCR. Then, the product was purified and sequenced to acquire the whole gene sequence of the N protein and its encoded amino acid sequence. DNASTAR software was used to analyze the homology, the genetic evolution and the derivation of the variability of amino acids of the N protein gene from 13 PRRSV strains and classical domestic and foreign strains. <strong>Results</strong>: Among the thirteen strains of PRRSV isolated from this study, ten strains had the greatest homology with the JXA1 strain (98.9% - 100%), and they belonged to the sublineage 8.7. The remaining three strains had the greatest homology with the NADC30 strain (95.4% - 97.1%), and they belonged to lineage one. The analysis of the variability of N protein amino acids showed that there were high frequency mutations in the five loci of 13 isolated strains of PRRSV as follows: 15th amino acid (10/13), 46<sup>th</sup> amino acid (11/13), 91st amino acid (10/13), 109th amino acid (10/13), and 117th amino acid (10/13). <strong>Conclusion</strong>: In recent years, sublineage 8.7 was the dominant pedigree in field PRRSV epidemic strains in China with lineage one occupying a certain proportion of the field. Four high frequency mutations existed in N protein antigen epitopes of isolated strains from the region. The nuclear localization signal (NLS) structure, specifically the 46<sup>th</sup> amino acid residue of the N protein, was mutated and genetically stable.展开更多
AIM: To establish a convenient immunoassay method based on recombinant antigen preS1(21-119aa) to detect anti-preS1 antibodies and evaluate the clinical significance of antibodies in hepatitis B. METHODS: The expressi...AIM: To establish a convenient immunoassay method based on recombinant antigen preS1(21-119aa) to detect anti-preS1 antibodies and evaluate the clinical significance of antibodies in hepatitis B. METHODS: The expression plasmid pET-28a-preS1 was constructed, and a large quantity of preS1(21-119aa) fragment of the large HBsAg protein was obtained. The preS1 fragment purified by Ni(2+)-IDA affinity chromatography was used as coated antigen to establish the indirect ELISA based on streptavidin-biotin system for detection of the anti-preS1 antibodies in sera from HBV-infected patients. For follow-up study, serial sera were collected during the clinical course of 21 HBV-infected patients and anti-preS1 antibodies, preS1 antigen, HBV-DNA and other serological HBV markers were analyzed. RESULTS: preS1(21-119aa) fragment was highly expressed from the plasmid pET-28a-preS1 in a soluble form in E.Coli (30mg.L(-1)), and easily purified to high purity over 90% by one step of Ni(2+)-IDA-sepharose 6B affinity chromatography. The purity and antigenicity of the purified preS1(21-119aa) protein was determined by 150g.L(-1) SDS-PAGE, Western blot and a direct ELISA. Recombinant preS1(21-119aa) protein was successfully applied in the immunoassay which could sensitively detect the anti-preS1 antibodies in serum specimens of acute or chronic hepatitis B patients. Results showed that more than half of 19 acute hepatitis B patients produced anti-preS1 antibodies during recovery of the disease, however, the response was only found in a few of chronic patients. In the clinical follow-up study of 11 patients with anti-preS1 positive serological profile, HBsAg and HBV-DNA clearance occurred in 6 of 10 acute hepatitis B patients in 5-6 months, and seroconversion of HBeAg and disappearance of HBV-DNA occurred in 1 chronic patients treated with lavumidine, a antiviral agent. CONCLUSION: The high-purity preS1(21-119aa) coated antigen was successfully prepared by gene expression and affinity chromatography. Using this antigen, a conveniently detective system of anti-preS1 antibodies in sera was established. Preliminarily clinical trial the occurrence of anti-preS1 antibodies in acute hepatitis B patients suggests the clearance of HBV from serum in a short-term time, and anti-preS1 positive in chronic patients means health improvement or recovery from the disease.展开更多
Cytochrome P450s(P450s)are the most versatile catalysts utilized by plants to produce structurally and functionally diverse metabolites.Given the high degree of gene redundancy and challenge to functionally characteri...Cytochrome P450s(P450s)are the most versatile catalysts utilized by plants to produce structurally and functionally diverse metabolites.Given the high degree of gene redundancy and challenge to functionally characterize plant P450s,protein engineering is used as a complementarystrategy to study the mechanisms of P450-mediated reactions,or to alter their functions.We previously proposed an approach of engineering plant P450s based on combining high accuracy homology models generated by Rosetta combined with data-driven design using evoluti onary information of these enzymes.With this strategy,we repurposed a multi-functional P450(CYP87D20)into a monooxygenase after red esigning its active site.Since most plant P450s are membrane-anchored proteins that are adapted to the micro-environments of plant cells,expressing them in heterologous hosts usually results in problems of expression or activity.Here,we applied compu-tational design to tackle these issues by simultaneous optimization of the protein surface and active site.After screening 17 variants,effective su bstitutions of surface residues were observed to improve both expression and activity of CYP87D20.In addition,the identified substitutions were additive and by com-bining them a highly eficient C11 hydroxylase of cucurbitadienol was created to participate in the mogrol biosynthesis.This study shows the importance of considering the interplay between surface and active site residues for P450 engineering.Our integrated strategy also opens an avenue to create more tai loring enzymes with desired functions for the metabolic engineering of high-valued compounds like mogrol,the precursor of natural sweetener mogrosides.展开更多
文摘The amino acid composition of α and β structural class of proteins from five species, Escherichia coli, Thermotoga maritima, Thermus thermophilus, yeast, and humans were investigated. Amino acid residues of proteins were classified into interior or surface residues based on the relative accessible surface area. The hydrophobic Leu, Ala, Val, and Ile residues were rich in interior residues, and hydrophilic Glu, Lys, Asp, and Arg were rich in surface residues both in α and β proteins. The amino acid composition of α proteins was different from that of β proteins in five species, and the difference was derived from the different contents of their interior residues between α and β proteins. α-helix content of α proteins was rich in interior residues than surface ones. Similarly, β-sheet content of β proteins was rich in interior residues than surface ones. The content of Leu residues was very high, approximately 20%, in interior residues of α proteins. This result suggested that the Leu residue plays an important role in the folding of α proteins.
文摘We analyzed the amino acid residues present in the water-soluble and transmembrane proteins of 6 thermophilic and 6 mesophilic species of the domains Archaea and Eubacteria, and characterized them as favorable or unfavorable. The characterization was performed by comparing the observed number of each amino acid residue to the expected number calculated from the percentage of nucleotides present in each gene. Amino acids that were more or less abundant than expected were considered as favorable or unfavorable, respectively. Comparisons of amino acid compositions indicated that the water-soluble proteins were rich in charged residues such as Glu, Asp, Lys, and His, whereas hydrophobic residues such as Trp, Phe, and Leu were abundant in transmembrane proteins. Interestingly, our results found that although the Trp residue was abundant in transmembrane proteins, it was not defined as favorable by our calculations, indicating that increased numbers of a particular amino acid does not necessary indicate it is a favorable residue. Amino acids with high G + C content such as Ala, Gly, and Pro were frequently observed as favorable in species with low G + C content. Comparatively, amino acids with low G + C content such as Phe, Tyr, Lys, Ile, and Met were frequently observed as favorable in species with high G + C content. These are the examples to increase the supply of amino acids than expected. Amino acids with neutral G + C content, i.e., Glu and Asp were favorable in water-soluble proteins from all species analyzed, and Cys was unfavorable both in water-soluble and transmembrane proteins. These results indicate that amino acid compositions are essentially determined by the nucleotide sequence of the genes, and the amino acid content is altered by a deviation from expectation.
文摘A response surface method was employed to study the effect of α-amylase concentration, hydrolysis temperature and time on the production of high protein glutinous rice flour(HPGRF). The suspension of glutinous rice flour(15%) that contained 6.52% protein was gelatinized and subsequently hydrolyzed by thermostable α-amylase. The hydrolysis yielded 0.144–0.222 g/g HPGRF with 29.4%–45.4% protein content. Hydrolysis time exerted a significant effect, while enzyme concentration and hydrolysis temperature showed insignificant effect on the protein content and production yield of HPGRF. The result of response surface method showed that the optimum condition for the production of HPGRF that contained at least 36% protein was treating gelatinized 15% glutinous rice flour suspension with 0.90 Kilo Novo α-amylase Unit(KNU)/g α-amylase at 80 oC for 99 min. By carrying out the predicted hydrolysis condition, HPGRF with 35.9% protein and 61.8% carbohydrates was resulted. The process yielded 0.172 g/g HPGRF. HPGRF contained higher amount of essential amino acids compared to glutinous rice flour. HPGRF had higher solubility and lower swelling power, and also showed no pasting peak compared with glutinous rice flour.
基金Special Fund for International Cooperative Research of Qilu University of Technology(QLUTGJHZ2018010)。
文摘Using microbial fermentation to increase the content of free amino acids and short peptides in the organic fertilizer for castor bean meal can effectively promote plant growth and improve fruit quality.Using free amino acid and short peptide content as an indicator,through single factor and response surface optimization experiments,the process parameters(moisture content,fermentation time and inoculum quantity)of castor meal solid-state fermentation were optimized.The best process parameters for the solid-state fermentation were:the moisture content 62%,the fermentation time 20 d,and the inoculum quantity 0.23%.The moisture content had the greatest impact on the conversion rate of free amino acids and short peptides,and the protein conversion rate reached 65.6%.The scale-up experiment under the optimal conditions showed that the solid-state fermentation using the inoculum had a significant beneficial effect compared with other fermentation methods.The fermentation of castor cake fertilizer provides a theoretical and practical basis for production feasibility,and has important guiding significance for the effective utilization of castor bean meal.
文摘While phthalate acid esters(PAEs)cannot fluoresce alone,they can be detected by fluorescence spectroscopy after chelation with bovine serum albumin(BSA).In this study,the types of amino acid residues at the active site of PAEs chelated with BSA were determined using molecular docking technology.A modification scheme of BSA with higher detection sensitivity fluorescence spectroscopy for PAEs was proposed based on the docking results and constructed for a novel BSA structure with a higher detection sensitivity of fluorescence spectroscopy using a homologous modeling method.Density functional theory(DFT)was employed to explore the influence before and after BSA modification on PAEs’detection through fluorescence spectroscopy.The results showed that the docking scores between BSAs and dimethyl phthalate(DMP),dibutyl phthalate(DBP)and di-n-octyl phthalate(DNOP)were increased up to 26.45%,16.82%and 16.30%,respectively,indicating that the active site modification of BSA could enhance the binding affinity between BSA and PAEs.The fluorescence intensity of PAEs chelated with modified BSAs were calculated.The fluorescence intensity of fluorescence spectroscopy for DMP,DBP and DNOP chelated with BSAs after modification was increased up to 2.8-,104.51-and 62.43-fold,respectively,which achieved the purpose of theoretically modifying BSA to improve the detection sensitivity of fluorescence spectroscopy for PAEs.
文摘<strong>Object</strong>: To analyze porcine reproductive and respiratory syndrome virus (PRRSV) strains from 13 infection cases via the N protein gene and its encoded amino acid sequence and to provide a theoretical basis for the epidemiological study, prevention and control of porcine reproductive and respiratory syndrome (PRRS). <strong>Methods</strong>: In clinically suspected PRRSV infections, viruses were isolated by extracting viral nucleic acid and amplifying the N protein gene by RT-PCR. Then, the product was purified and sequenced to acquire the whole gene sequence of the N protein and its encoded amino acid sequence. DNASTAR software was used to analyze the homology, the genetic evolution and the derivation of the variability of amino acids of the N protein gene from 13 PRRSV strains and classical domestic and foreign strains. <strong>Results</strong>: Among the thirteen strains of PRRSV isolated from this study, ten strains had the greatest homology with the JXA1 strain (98.9% - 100%), and they belonged to the sublineage 8.7. The remaining three strains had the greatest homology with the NADC30 strain (95.4% - 97.1%), and they belonged to lineage one. The analysis of the variability of N protein amino acids showed that there were high frequency mutations in the five loci of 13 isolated strains of PRRSV as follows: 15th amino acid (10/13), 46<sup>th</sup> amino acid (11/13), 91st amino acid (10/13), 109th amino acid (10/13), and 117th amino acid (10/13). <strong>Conclusion</strong>: In recent years, sublineage 8.7 was the dominant pedigree in field PRRSV epidemic strains in China with lineage one occupying a certain proportion of the field. Four high frequency mutations existed in N protein antigen epitopes of isolated strains from the region. The nuclear localization signal (NLS) structure, specifically the 46<sup>th</sup> amino acid residue of the N protein, was mutated and genetically stable.
基金the grants No.KY951-Al-301 and No.KY95T-06-03 from the 9th Five Years Plan Key Research Programs of the Chinese Academy of Sciences.
文摘AIM: To establish a convenient immunoassay method based on recombinant antigen preS1(21-119aa) to detect anti-preS1 antibodies and evaluate the clinical significance of antibodies in hepatitis B. METHODS: The expression plasmid pET-28a-preS1 was constructed, and a large quantity of preS1(21-119aa) fragment of the large HBsAg protein was obtained. The preS1 fragment purified by Ni(2+)-IDA affinity chromatography was used as coated antigen to establish the indirect ELISA based on streptavidin-biotin system for detection of the anti-preS1 antibodies in sera from HBV-infected patients. For follow-up study, serial sera were collected during the clinical course of 21 HBV-infected patients and anti-preS1 antibodies, preS1 antigen, HBV-DNA and other serological HBV markers were analyzed. RESULTS: preS1(21-119aa) fragment was highly expressed from the plasmid pET-28a-preS1 in a soluble form in E.Coli (30mg.L(-1)), and easily purified to high purity over 90% by one step of Ni(2+)-IDA-sepharose 6B affinity chromatography. The purity and antigenicity of the purified preS1(21-119aa) protein was determined by 150g.L(-1) SDS-PAGE, Western blot and a direct ELISA. Recombinant preS1(21-119aa) protein was successfully applied in the immunoassay which could sensitively detect the anti-preS1 antibodies in serum specimens of acute or chronic hepatitis B patients. Results showed that more than half of 19 acute hepatitis B patients produced anti-preS1 antibodies during recovery of the disease, however, the response was only found in a few of chronic patients. In the clinical follow-up study of 11 patients with anti-preS1 positive serological profile, HBsAg and HBV-DNA clearance occurred in 6 of 10 acute hepatitis B patients in 5-6 months, and seroconversion of HBeAg and disappearance of HBV-DNA occurred in 1 chronic patients treated with lavumidine, a antiviral agent. CONCLUSION: The high-purity preS1(21-119aa) coated antigen was successfully prepared by gene expression and affinity chromatography. Using this antigen, a conveniently detective system of anti-preS1 antibodies in sera was established. Preliminarily clinical trial the occurrence of anti-preS1 antibodies in acute hepatitis B patients suggests the clearance of HBV from serum in a short-term time, and anti-preS1 positive in chronic patients means health improvement or recovery from the disease.
基金the National Key Research and Development Program of China(2018YFA0901800)Yunnan Science Fund(202005AE160015 and 2019FJ004)This work was also supported from Shenzhen Municipal Governments.
文摘Cytochrome P450s(P450s)are the most versatile catalysts utilized by plants to produce structurally and functionally diverse metabolites.Given the high degree of gene redundancy and challenge to functionally characterize plant P450s,protein engineering is used as a complementarystrategy to study the mechanisms of P450-mediated reactions,or to alter their functions.We previously proposed an approach of engineering plant P450s based on combining high accuracy homology models generated by Rosetta combined with data-driven design using evoluti onary information of these enzymes.With this strategy,we repurposed a multi-functional P450(CYP87D20)into a monooxygenase after red esigning its active site.Since most plant P450s are membrane-anchored proteins that are adapted to the micro-environments of plant cells,expressing them in heterologous hosts usually results in problems of expression or activity.Here,we applied compu-tational design to tackle these issues by simultaneous optimization of the protein surface and active site.After screening 17 variants,effective su bstitutions of surface residues were observed to improve both expression and activity of CYP87D20.In addition,the identified substitutions were additive and by com-bining them a highly eficient C11 hydroxylase of cucurbitadienol was created to participate in the mogrol biosynthesis.This study shows the importance of considering the interplay between surface and active site residues for P450 engineering.Our integrated strategy also opens an avenue to create more tai loring enzymes with desired functions for the metabolic engineering of high-valued compounds like mogrol,the precursor of natural sweetener mogrosides.
