Phosphoglucose isomerase (PGI) is a key enzyme in early glycolysis, which catalyzes the reversible isomerization of glucose 6-phosphate (G6Ph) to fructose 6-phosphate. We have constructed an Escherichia coli K12 strai...Phosphoglucose isomerase (PGI) is a key enzyme in early glycolysis, which catalyzes the reversible isomerization of glucose 6-phosphate (G6Ph) to fructose 6-phosphate. We have constructed an Escherichia coli K12 strain with a deleted pgi gene (Δpgi) and shown that this strain in comparison with the parental strain 1) accumulates higher amount of G6Ph, 2) grows slowly, and 3) exhibits higher spontaneous mutation frequency to rifampicin resistance (Rifr), when grown on high glucose minimal medium. Intriguingly, the spontaneous mutation rate to Rifr was inversely related to the degree of E. coli chromosomal DNA modification with sugar derivatives. We measured higher concentrations of Amadori products, fluorophores (360 nm excitation/440 nm emission) and carboxymethyl residues in the chromosomal DNA of the E. coli parental strain than in DNA of the isogenic Δpgi strain. To explain this apparent paradox we hypothesized that PGI might be implicated in repair of G6Ph-derived lesions in DNA. In favor of our hypothesis, we further demonstrate that protein extract from the E. coli PGI proficient strain but not from the PGI deficient strain catalyzes the release of G6Ph from G6Ph-modified single stranded DNA oligonucleotide and from its hybrid duplex with a complementary peptide nucleic acid.展开更多
Current structural genomics projects aim to solve a large number of selected protein structures as fast as possible. High degree of automation and standardization is required at every step of the whole process to spee...Current structural genomics projects aim to solve a large number of selected protein structures as fast as possible. High degree of automation and standardization is required at every step of the whole process to speed up protein structure determination. Phase problem is a bottleneck in macromolecular structure determination and also in model building which is a time-consuming task. The simplest approach to phasing macromolecular crystal structures is the use of a SAD signal. SAD data can be collected using the in-house copper (1.54 A) wavelength source. Data collected using copper wavelength with the incorporation of anomalously scattering heavy metal atoms may serve as a powerful tool for structural biologists to solve novel protein structures as well where synchrotron beam line is not available. A short soak of protein crystals in heavy metal solution or by incorporating heavy atoms into the protein drop while crystallizing the protein (co-crystallization) leads to incorporation of these heavy metal ions into the ordered solvent shell around the protein surface. The present work aims to determine whether cerium ion can be successfully incorporated into the protein crystal through quick-soaking method while maintaining the isomorphism. The study also aims in understanding whether this metal ion can be used for phasing purpose. The intensity data are collected and analyzed for anomalous signal, substructure solution and the binding sites.展开更多
After the establishment of the transformation conditions of Streptomyces diastaticus No.7 Strain M1033, the integration plasmid pXW for homologous recombination, which contains a 600 bp fragment of incomplete Gl (G138...After the establishment of the transformation conditions of Streptomyces diastaticus No.7 Strain M1033, the integration plasmid pXW for homologous recombination, which contains a 600 bp fragment of incomplete Gl (G138P. G247D) gene, has been constructed in order to realize the stable overexpression of the Gl (G138P. G247D) which is valuable for large-scale industrial production. The G/gene’s disruption has been realized by pXW’s integration into M1033 chromosomes via homologous recombination and Gl deficient strain of Streptomyces M1033 has been obtained. The reliability of introduction of mutation has been proved by analysis of recombinant fragment and affirmance of existence of the mutation, as well as detection of the stability of the deficient strain.展开更多
Ⅰ. INTRODUCTION Glucose isomerase (EC 5.3.1.5) can catalyze the isomerization of glucose to fructose, which is the basis for the industrial production of high-fructose syrups. A number of glucose isomerases from a va...Ⅰ. INTRODUCTION Glucose isomerase (EC 5.3.1.5) can catalyze the isomerization of glucose to fructose, which is the basis for the industrial production of high-fructose syrups. A number of glucose isomerases from a variety of bacterial sources have been investigated crystallographically (see Table 1 for detail). It seems that the glucose isomerase from Streptomyces rubiginosus (abbreviated to STRPR) has not the same precise relationships between the spatial structure and biological function as the one from Streptomyces olivochromogenes (abbreviated to STRPO), although those two enzymes are very展开更多
Mutants of the strain producing natamycin, Streptomyces gilvosporeus, were obtained after space-flight mutation. With respect to the sand spores and slant spores, the mutation ratios were up to 67.6% and 78.3% and the...Mutants of the strain producing natamycin, Streptomyces gilvosporeus, were obtained after space-flight mutation. With respect to the sand spores and slant spores, the mutation ratios were up to 67.6% and 78.3% and the survival ratio was 43.1% and 3.0%, respectively. An improved mutant producing natamycin, S. gilvosporeus LK-45, was screened, which showed natamycin productivity of 1420mg·L^-1. A mutant resistant to 2-deoxy glucose, S.gilvosporeus LK-119, was further obtained using a＇rational screening procedure. The natamycin productivity of 1940mg·L^-1 was achieved when glucose was used as the carbon source.展开更多
The crystal structures of Streptomyces diastaticus No. 7 strain M1033 xylose isomerase (SDXyI) have been analysed and refined at 0.19nm. The crystal space group is I222, with unit cell dimensions of a=9.884 ran, b=9.3...The crystal structures of Streptomyces diastaticus No. 7 strain M1033 xylose isomerase (SDXyI) have been analysed and refined at 0.19nm. The crystal space group is I222, with unit cell dimensions of a=9.884 ran, b=9.393nm and c=8.798nm. Based on the coordinates of the Streptomyces rubiginosus xylose isomerase (SRXyI), the initial model of SDXyl was built up by the dose packing analysing and R-factor searching and refined by PROLSQ to a final R-factor of 0.177 with the rms deviations of bond lengths and bond angles of 0.001 9nm and 2.1°, respectively. No significant global conformation change existed between SRXyI and SDXyI except the local conformation in the active site.展开更多
Glucose isomerase(GI)is an enzyme with high potential applications.Characterization of GI producing bacteria with interesting properties from an industrial point of view is essential.Bacillus sp.,Paenarthrobacter sp.,...Glucose isomerase(GI)is an enzyme with high potential applications.Characterization of GI producing bacteria with interesting properties from an industrial point of view is essential.Bacillus sp.,Paenarthrobacter sp.,Chryseobacterium sp.,Hymenobacter sp.,Mycobacterium sp.,and Stenotrophomonas sp.were isolated from soil samples.Optimization of enzyme production yield was investigated in various fermentation conditions using response surface methodology.All isolates exhibited maximum GI activity at 40℃,pH 6–8 after 4 days of incubation.A mixture of peptone/yeast extract or tryptone/peptone enhanced higher enzyme production.The same trend was observed in fermentation medium containing 1%xylose or 2%–2.5%wheat straw.This study advanced the knowledge of these bacterial isolates in promoting wheat straw as feedstock for the bio-based industry.展开更多
Objective: To investigate whether glucose-6-phosphate isomerase (G6PI) antigen and anti-G6PI antibodies could be applied for the clinical diagnostic markers of rheumatoid arthritis (RA) and its associations with RA ac...Objective: To investigate whether glucose-6-phosphate isomerase (G6PI) antigen and anti-G6PI antibodies could be applied for the clinical diagnostic markers of rheumatoid arthritis (RA) and its associations with RA activity states. Methods: The levels of G6PI antigens and anti-G6PI Abs in sera from 176 RA patients in different states, 35 non-RA patients and 100 healthy donors and in synovia fluids from 33 patients and 11 non-RA patients were measured by ELISA. Results: The sensitivity and specificity of G6PI antigens in the RA patients were 75.0% and 93.3%, respectively. The levels of serum G6PI antigens in 176 RA patients were significantly higher than non-RA patients and the health controls. Especially, there was a significant difference between the active phase and the inactive phase in G6PI antigens levels. The levels of G6PI antigens in synovia fluid were also significantly higher in RA groups than in non-RA patients. With the values of the anti-G6PI Abs in sera, there were no marked differences among RA, non-RA patients and health controls. Also, there was no significant difference between the active phase and the inactive phase in RA patients. However, there were no significant differences of G6PI and anti-G6PI between RA patients and health controls in synovial fluid. Conclusions: G6PI is highly correlated with the activity states of RA, and could be applied for a clinical biomarker with high sensitivity and specificity for the diagnosis of RA.展开更多
The glucose isomerase(GI) was a metal activating enzyme It was most activated by Co 2+ and Mg 2+ ,and Mg 2+ was the best activator,whether the glucose or the xylose was the substrate When the glucose was substrate,the...The glucose isomerase(GI) was a metal activating enzyme It was most activated by Co 2+ and Mg 2+ ,and Mg 2+ was the best activator,whether the glucose or the xylose was the substrate When the glucose was substrate,the dissociation constant of Mg 2+ GI,Co 2+ GI and Mn 2+ -GI was 115 μmol/L,40 μmol/L, and 15 μmol/L respectively. The maximum activity of Mg 2+ GI,Co 2- GI and Mn 2+ GI was 100%,85%,and 20% respectively. When the xylose was substrate,the order of dissociation constant and maximum activity of the metal enzymes was the same Ca 2+ was a competitive inhibitor versus Mg 2+ ( K i 7 4 μmol/L)or Co 2+ ( K i 99 μmol/L). Compared with Mg 2+ GI,the K m of Co 2+ GI was more,and the V M of Co 2+ GI less The process of activity recovery from apo GI to metal GI showed that it was slow and of two展开更多
文摘Phosphoglucose isomerase (PGI) is a key enzyme in early glycolysis, which catalyzes the reversible isomerization of glucose 6-phosphate (G6Ph) to fructose 6-phosphate. We have constructed an Escherichia coli K12 strain with a deleted pgi gene (Δpgi) and shown that this strain in comparison with the parental strain 1) accumulates higher amount of G6Ph, 2) grows slowly, and 3) exhibits higher spontaneous mutation frequency to rifampicin resistance (Rifr), when grown on high glucose minimal medium. Intriguingly, the spontaneous mutation rate to Rifr was inversely related to the degree of E. coli chromosomal DNA modification with sugar derivatives. We measured higher concentrations of Amadori products, fluorophores (360 nm excitation/440 nm emission) and carboxymethyl residues in the chromosomal DNA of the E. coli parental strain than in DNA of the isogenic Δpgi strain. To explain this apparent paradox we hypothesized that PGI might be implicated in repair of G6Ph-derived lesions in DNA. In favor of our hypothesis, we further demonstrate that protein extract from the E. coli PGI proficient strain but not from the PGI deficient strain catalyzes the release of G6Ph from G6Ph-modified single stranded DNA oligonucleotide and from its hybrid duplex with a complementary peptide nucleic acid.
文摘Current structural genomics projects aim to solve a large number of selected protein structures as fast as possible. High degree of automation and standardization is required at every step of the whole process to speed up protein structure determination. Phase problem is a bottleneck in macromolecular structure determination and also in model building which is a time-consuming task. The simplest approach to phasing macromolecular crystal structures is the use of a SAD signal. SAD data can be collected using the in-house copper (1.54 A) wavelength source. Data collected using copper wavelength with the incorporation of anomalously scattering heavy metal atoms may serve as a powerful tool for structural biologists to solve novel protein structures as well where synchrotron beam line is not available. A short soak of protein crystals in heavy metal solution or by incorporating heavy atoms into the protein drop while crystallizing the protein (co-crystallization) leads to incorporation of these heavy metal ions into the ordered solvent shell around the protein surface. The present work aims to determine whether cerium ion can be successfully incorporated into the protein crystal through quick-soaking method while maintaining the isomorphism. The study also aims in understanding whether this metal ion can be used for phasing purpose. The intensity data are collected and analyzed for anomalous signal, substructure solution and the binding sites.
文摘After the establishment of the transformation conditions of Streptomyces diastaticus No.7 Strain M1033, the integration plasmid pXW for homologous recombination, which contains a 600 bp fragment of incomplete Gl (G138P. G247D) gene, has been constructed in order to realize the stable overexpression of the Gl (G138P. G247D) which is valuable for large-scale industrial production. The G/gene’s disruption has been realized by pXW’s integration into M1033 chromosomes via homologous recombination and Gl deficient strain of Streptomyces M1033 has been obtained. The reliability of introduction of mutation has been proved by analysis of recombinant fragment and affirmance of existence of the mutation, as well as detection of the stability of the deficient strain.
文摘Ⅰ. INTRODUCTION Glucose isomerase (EC 5.3.1.5) can catalyze the isomerization of glucose to fructose, which is the basis for the industrial production of high-fructose syrups. A number of glucose isomerases from a variety of bacterial sources have been investigated crystallographically (see Table 1 for detail). It seems that the glucose isomerase from Streptomyces rubiginosus (abbreviated to STRPR) has not the same precise relationships between the spatial structure and biological function as the one from Streptomyces olivochromogenes (abbreviated to STRPO), although those two enzymes are very
文摘Mutants of the strain producing natamycin, Streptomyces gilvosporeus, were obtained after space-flight mutation. With respect to the sand spores and slant spores, the mutation ratios were up to 67.6% and 78.3% and the survival ratio was 43.1% and 3.0%, respectively. An improved mutant producing natamycin, S. gilvosporeus LK-45, was screened, which showed natamycin productivity of 1420mg·L^-1. A mutant resistant to 2-deoxy glucose, S.gilvosporeus LK-119, was further obtained using a＇rational screening procedure. The natamycin productivity of 1940mg·L^-1 was achieved when glucose was used as the carbon source.
基金Project supported by the National 863 Protein Engineering Program,the fund of President of the Chinese Academy Sciences and the grant of State Key Laboratory of Biomacromolecules.
文摘The crystal structures of Streptomyces diastaticus No. 7 strain M1033 xylose isomerase (SDXyI) have been analysed and refined at 0.19nm. The crystal space group is I222, with unit cell dimensions of a=9.884 ran, b=9.393nm and c=8.798nm. Based on the coordinates of the Streptomyces rubiginosus xylose isomerase (SRXyI), the initial model of SDXyl was built up by the dose packing analysing and R-factor searching and refined by PROLSQ to a final R-factor of 0.177 with the rms deviations of bond lengths and bond angles of 0.001 9nm and 2.1°, respectively. No significant global conformation change existed between SRXyI and SDXyI except the local conformation in the active site.
基金supported by the Natural Science and Engineering Research Council of Canada(NSERC)Discovery Grant(RGPIN-2017-05366)to WQ.
文摘Glucose isomerase(GI)is an enzyme with high potential applications.Characterization of GI producing bacteria with interesting properties from an industrial point of view is essential.Bacillus sp.,Paenarthrobacter sp.,Chryseobacterium sp.,Hymenobacter sp.,Mycobacterium sp.,and Stenotrophomonas sp.were isolated from soil samples.Optimization of enzyme production yield was investigated in various fermentation conditions using response surface methodology.All isolates exhibited maximum GI activity at 40℃,pH 6–8 after 4 days of incubation.A mixture of peptone/yeast extract or tryptone/peptone enhanced higher enzyme production.The same trend was observed in fermentation medium containing 1%xylose or 2%–2.5%wheat straw.This study advanced the knowledge of these bacterial isolates in promoting wheat straw as feedstock for the bio-based industry.
文摘Objective: To investigate whether glucose-6-phosphate isomerase (G6PI) antigen and anti-G6PI antibodies could be applied for the clinical diagnostic markers of rheumatoid arthritis (RA) and its associations with RA activity states. Methods: The levels of G6PI antigens and anti-G6PI Abs in sera from 176 RA patients in different states, 35 non-RA patients and 100 healthy donors and in synovia fluids from 33 patients and 11 non-RA patients were measured by ELISA. Results: The sensitivity and specificity of G6PI antigens in the RA patients were 75.0% and 93.3%, respectively. The levels of serum G6PI antigens in 176 RA patients were significantly higher than non-RA patients and the health controls. Especially, there was a significant difference between the active phase and the inactive phase in G6PI antigens levels. The levels of G6PI antigens in synovia fluid were also significantly higher in RA groups than in non-RA patients. With the values of the anti-G6PI Abs in sera, there were no marked differences among RA, non-RA patients and health controls. Also, there was no significant difference between the active phase and the inactive phase in RA patients. However, there were no significant differences of G6PI and anti-G6PI between RA patients and health controls in synovial fluid. Conclusions: G6PI is highly correlated with the activity states of RA, and could be applied for a clinical biomarker with high sensitivity and specificity for the diagnosis of RA.
文摘The glucose isomerase(GI) was a metal activating enzyme It was most activated by Co 2+ and Mg 2+ ,and Mg 2+ was the best activator,whether the glucose or the xylose was the substrate When the glucose was substrate,the dissociation constant of Mg 2+ GI,Co 2+ GI and Mn 2+ -GI was 115 μmol/L,40 μmol/L, and 15 μmol/L respectively. The maximum activity of Mg 2+ GI,Co 2- GI and Mn 2+ GI was 100%,85%,and 20% respectively. When the xylose was substrate,the order of dissociation constant and maximum activity of the metal enzymes was the same Ca 2+ was a competitive inhibitor versus Mg 2+ ( K i 7 4 μmol/L)or Co 2+ ( K i 99 μmol/L). Compared with Mg 2+ GI,the K m of Co 2+ GI was more,and the V M of Co 2+ GI less The process of activity recovery from apo GI to metal GI showed that it was slow and of two
