It is reported that fermentative liquids with various concentrations of La and Nd affect the fer- mentation of alginic acid from the strain 342 of Azotobacter vinelandii.The results are as follows:When the concentrati...It is reported that fermentative liquids with various concentrations of La and Nd affect the fer- mentation of alginic acid from the strain 342 of Azotobacter vinelandii.The results are as follows:When the concentration of La or Nd was up to 100 ppm,the cell growth is stimulated and the production of alginic acid is promoted.The La or Nd in concentration higher than 200 ppm or 150 ppm inhibits the fermentation, respectively.As the concentration range of La is 0～100 ppm or that of Nd is 0～150 ppm,the yield of fixed nitrogen increases,and the ratio of c_M to c_G(c_M/c_G)decreases with the raise of the concentration of La or Nd.When the concentration range of La is 100～400 ppm and that of Nd is 150～400 ppm,the conclusion is contrary to the above mentioned result.展开更多
nifB-MoFe protein （nifB-Av1）, AnifE MoFe protein （△nifE Av1） and AnifZ MoFe protein （△nifZ Av1） were obtained by chromatography on DE52, Sephacryl S-300 and Q-Sepharose columns from nifB point-mutated, nifE de...nifB-MoFe protein （nifB-Av1）, AnifE MoFe protein （△nifE Av1） and AnifZ MoFe protein （△nifZ Av1） were obtained by chromatography on DE52, Sephacryl S-300 and Q-Sepharose columns from nifB point-mutated, nifE deleted and nifZ deleted mutant stains （UW45, DJ35 and DJ194） of Azotobacter vinelandii Llpmann, respectively. When complemented with nltrogenase Fe protein （Av2）, AnifZ Av1 had partial activity and both nifB-Avl and △nifE Av1 had hardly any activity, but could be obviously activated by FeMoco extracted from wild-type MoFe protein （OP Av1） or △nifZ Av1. After being Incubated with excess O-phenanthrollne （O-phen） for 150 mln at 30 ℃ and subjected to chromatography on a Sephadex G-25 column In an Ar atmosphere, nifB- Av1C, △nifE Av1C and △nifZ Av1C were obtained, respectively. Based on a calculation of Fe atoms In the Ophen-Fe compound with ε 512nm = 11 100, lost Fe atoms of nifB-Av1, △nifE Av1 and △nifZ Av1 were estimated to be 1.35, 2.89 and 8.44 per molecule of protein, respectively. As a result of the Fe loss, △nifZ Av1 loses Its original activity. In the presence of both MgATP and Av2, these Fe-loslng proteins, but not the original proteins untreated with O-phen, could be significantly activated by reconstltuent solution （RS） composed of dlthlothreltol, ferric homocltrate, Na2S and Na2MoO4, or K2CrO4, or KMnO4. But In the absence of MgATP or Av2, the activation did not occur, with the exception that △nifZ AvlC was partially activated, and the activity was only 17%. These findings Indicate that： （I） △nifZ Avl with half P-cluster content Is somewhat different from FeMoco-deflclent nifB-Avl and ,△nifE Av1 with respect to protein conformation either before or after treatment with O-phen; （11） full activation of these proteins with RS requires pretreatment with O-phen and the simultaneous presence of MgATP and Av2.展开更多
A new protein, an approximately 59-kDa monomer containing iron atoms, was first isolated from the mutant strain DJ35 of Azotobacter vinelandii Llpmann. After analysis by matrix-assisted laser desorptlon ionization tim...A new protein, an approximately 59-kDa monomer containing iron atoms, was first isolated from the mutant strain DJ35 of Azotobacter vinelandii Llpmann. After analysis by matrix-assisted laser desorptlon ionization time-of-flight mass spectrometry, the protein was Identified as the product of a predicted gene. Thus, the protein was tentatively called HBP59. Its absorption spectra （ABS） In the reduced state exhibited three peaks at 421,517, and 556 nm and the maximal peak was shifted from 421 to 413 nm after exposure of HBP59 to air. The Soret circular dichrolsm （CD） spectrum of HBP59 In the reduced state displayed four positive peaks at 364, 382, 406, and 418 nm and two negative peaks at 398 and 433 nm; the △ε （CD extinction coefficient） values of these peaks were found to be 0.92, 0.58, 0.87, 0.72, -0.65 and -1.12 L/mol per cm, respectively. Titration with heme showed that the protein has 0.1 heme molecules/protein molecule. After HBP59 had fully Interacted with heme, Its maximal ABS value and Soret CD Intensity were increased by approximately 10-fold compared with values before Interaction. Therefore, It seems that one molecule of HBP59 can be interacted with only one heme. These results indicate that HBP59 contains heme with low spin and may be Involved In heme utilization or adhesion.展开更多
Through the anaerobic chromatography on the columns of DEAE 52, Q-Sepharose and Sephacryl S-200, a nitrogenase MoFe protein (?nifZ Av1) was obtained from a nifZ deleted mutant of Azotobacter vinelandii (stain DJ194). ...Through the anaerobic chromatography on the columns of DEAE 52, Q-Sepharose and Sephacryl S-200, a nitrogenase MoFe protein (?nifZ Av1) was obtained from a nifZ deleted mutant of Azotobacter vinelandii (stain DJ194). The results of Western blotting after anoxic native electro-phoresis and SDS-PAGE showed that ?nifZ Av1 was similar to wild type MoFe protein (OP Av1) at the electrophoretic mobility, molecular weight and subunit composition. Fur-thermore, ?nifZ Av1 was also similar to OP Av1 at the mo-lybdenum content, EPR signal (g≈4.3, 3.65 and 2.01), and the molar extinction coefficient (?ε) of circular dichroism (CD) at 660 nm region. All of these indicated that, besides having the same α2β2 composition as OP Av1, the ?nifZ Av1 also contained equal amount of reductive FeMoco in the spin state of S=3/2 to OP Av1. However, the iron content and sub-strate (C2H2, H+ and N2)-reduction activity of ?nifZ Av1 were 74% and 46%―50% of those of OP Av1, respectively. Fur-thermore, the ?ε at around 450 nm, which reflects P-cluster in Av1, was obviously lower than that of OP Av1. It suggested that the difference between ?nifZ Av1 and OP Av1 resulted from P-cluster rather than FeMoco, and from the half num-ber of P-cluster in ?nifZ Av1, but the composition or redox state of P-cluster in ?nifZ Av1 were not changed. Thus it could propose that ?nifZ Av1 is composed of two different αβ subunit pairs. One is a FeMoco- and P-cluster-containing pair, and the other is a P-cluster-deficient but FeMoco-con- taining pair. Since the deletion of nifZ gene leads to the defi-ciency of only one of two P-clusters in a α2β2 tetramer, the assembly of P-cluster may not simply depend on one gene product, and so a possible mechanism of NifZ is supposed here.展开更多
A MoFe protein (ΔnifE Av1) with a purity of ～80% was purified from a nifE-deleted mutant of Azotobacter vinelandii DJ35. Compared with MoFe protein purified from wild-type strain OP (OP Av1), ΔnifE Av1 had the same...A MoFe protein (ΔnifE Av1) with a purity of ～80% was purified from a nifE-deleted mutant of Azotobacter vinelandii DJ35. Compared with MoFe protein purified from wild-type strain OP (OP Av1), ΔnifE Av1 had the same subunits composition, and had immune reaction with antibody to OP Av1, but its relative mobility in anaerobic native polyacrylamide gel electrophoresis (PAGE) was a little larger than that of OP Av1. Metal analysis showed that Mo and Fe contents of ΔnifE Av1 both apparently decreased. When complemented with OP Fe protein, ΔnifE Av1 had no C2H2-reduction activity, but it could be in vitro activated by FeMoco extracted from OP Av1. The circular dichroism (CD) spectrum of ΔnifE Av1 at ~450 nm was similar to that of OP Av1, while the EPR signal at g≈3.7 was absolutely silent, and the signal intensities at g≈4.3 and 2.0 decreased by 75% and 50%, respectively. The results indicated that ΔnifE Av1 purified from DJ35 was a FeMoco-deficient but P-cluster-con- taining MoFe protein.展开更多
文摘It is reported that fermentative liquids with various concentrations of La and Nd affect the fer- mentation of alginic acid from the strain 342 of Azotobacter vinelandii.The results are as follows:When the concentration of La or Nd was up to 100 ppm,the cell growth is stimulated and the production of alginic acid is promoted.The La or Nd in concentration higher than 200 ppm or 150 ppm inhibits the fermentation, respectively.As the concentration range of La is 0～100 ppm or that of Nd is 0～150 ppm,the yield of fixed nitrogen increases,and the ratio of c_M to c_G(c_M/c_G)decreases with the raise of the concentration of La or Nd.When the concentration range of La is 100～400 ppm and that of Nd is 150～400 ppm,the conclusion is contrary to the above mentioned result.
基金Supported by the State Key Basic Research and Development Plan of China （001CB1089-06） and the National Natural Science Foundation of China （30270296）.
文摘nifB-MoFe protein （nifB-Av1）, AnifE MoFe protein （△nifE Av1） and AnifZ MoFe protein （△nifZ Av1） were obtained by chromatography on DE52, Sephacryl S-300 and Q-Sepharose columns from nifB point-mutated, nifE deleted and nifZ deleted mutant stains （UW45, DJ35 and DJ194） of Azotobacter vinelandii Llpmann, respectively. When complemented with nltrogenase Fe protein （Av2）, AnifZ Av1 had partial activity and both nifB-Avl and △nifE Av1 had hardly any activity, but could be obviously activated by FeMoco extracted from wild-type MoFe protein （OP Av1） or △nifZ Av1. After being Incubated with excess O-phenanthrollne （O-phen） for 150 mln at 30 ℃ and subjected to chromatography on a Sephadex G-25 column In an Ar atmosphere, nifB- Av1C, △nifE Av1C and △nifZ Av1C were obtained, respectively. Based on a calculation of Fe atoms In the Ophen-Fe compound with ε 512nm = 11 100, lost Fe atoms of nifB-Av1, △nifE Av1 and △nifZ Av1 were estimated to be 1.35, 2.89 and 8.44 per molecule of protein, respectively. As a result of the Fe loss, △nifZ Av1 loses Its original activity. In the presence of both MgATP and Av2, these Fe-loslng proteins, but not the original proteins untreated with O-phen, could be significantly activated by reconstltuent solution （RS） composed of dlthlothreltol, ferric homocltrate, Na2S and Na2MoO4, or K2CrO4, or KMnO4. But In the absence of MgATP or Av2, the activation did not occur, with the exception that △nifZ AvlC was partially activated, and the activity was only 17%. These findings Indicate that： （I） △nifZ Avl with half P-cluster content Is somewhat different from FeMoco-deflclent nifB-Avl and ,△nifE Av1 with respect to protein conformation either before or after treatment with O-phen; （11） full activation of these proteins with RS requires pretreatment with O-phen and the simultaneous presence of MgATP and Av2.
基金Supported by the State Key Basic Research and Development Plan of China （001CB1089-06） and the National Natural Science Foundation of China （30270296）.Acknowledgements The authors thank Professors YX Jing and JG Li for their valuable advice.
文摘A new protein, an approximately 59-kDa monomer containing iron atoms, was first isolated from the mutant strain DJ35 of Azotobacter vinelandii Llpmann. After analysis by matrix-assisted laser desorptlon ionization time-of-flight mass spectrometry, the protein was Identified as the product of a predicted gene. Thus, the protein was tentatively called HBP59. Its absorption spectra （ABS） In the reduced state exhibited three peaks at 421,517, and 556 nm and the maximal peak was shifted from 421 to 413 nm after exposure of HBP59 to air. The Soret circular dichrolsm （CD） spectrum of HBP59 In the reduced state displayed four positive peaks at 364, 382, 406, and 418 nm and two negative peaks at 398 and 433 nm; the △ε （CD extinction coefficient） values of these peaks were found to be 0.92, 0.58, 0.87, 0.72, -0.65 and -1.12 L/mol per cm, respectively. Titration with heme showed that the protein has 0.1 heme molecules/protein molecule. After HBP59 had fully Interacted with heme, Its maximal ABS value and Soret CD Intensity were increased by approximately 10-fold compared with values before Interaction. Therefore, It seems that one molecule of HBP59 can be interacted with only one heme. These results indicate that HBP59 contains heme with low spin and may be Involved In heme utilization or adhesion.
基金supported by the State Key Basic Research and Developmental Plan of China(Grant No.001CB1089-06)the National Natural Science Foundation of China(Grant No.30270296)the National Science Foundation for distinguished Young Scholars of China(Grant No.20403024).
文摘Through the anaerobic chromatography on the columns of DEAE 52, Q-Sepharose and Sephacryl S-200, a nitrogenase MoFe protein (?nifZ Av1) was obtained from a nifZ deleted mutant of Azotobacter vinelandii (stain DJ194). The results of Western blotting after anoxic native electro-phoresis and SDS-PAGE showed that ?nifZ Av1 was similar to wild type MoFe protein (OP Av1) at the electrophoretic mobility, molecular weight and subunit composition. Fur-thermore, ?nifZ Av1 was also similar to OP Av1 at the mo-lybdenum content, EPR signal (g≈4.3, 3.65 and 2.01), and the molar extinction coefficient (?ε) of circular dichroism (CD) at 660 nm region. All of these indicated that, besides having the same α2β2 composition as OP Av1, the ?nifZ Av1 also contained equal amount of reductive FeMoco in the spin state of S=3/2 to OP Av1. However, the iron content and sub-strate (C2H2, H+ and N2)-reduction activity of ?nifZ Av1 were 74% and 46%―50% of those of OP Av1, respectively. Fur-thermore, the ?ε at around 450 nm, which reflects P-cluster in Av1, was obviously lower than that of OP Av1. It suggested that the difference between ?nifZ Av1 and OP Av1 resulted from P-cluster rather than FeMoco, and from the half num-ber of P-cluster in ?nifZ Av1, but the composition or redox state of P-cluster in ?nifZ Av1 were not changed. Thus it could propose that ?nifZ Av1 is composed of two different αβ subunit pairs. One is a FeMoco- and P-cluster-containing pair, and the other is a P-cluster-deficient but FeMoco-con- taining pair. Since the deletion of nifZ gene leads to the defi-ciency of only one of two P-clusters in a α2β2 tetramer, the assembly of P-cluster may not simply depend on one gene product, and so a possible mechanism of NifZ is supposed here.
基金supported by the State Key Basic Research and Developmental Plan of China(Grant No.001CB1089-06)the National Natural Science Foundation of China(Grant No.30270296)the National Science Foundation for Distin-guished Young Scholars of China(Grant No.20403024).
文摘A MoFe protein (ΔnifE Av1) with a purity of ～80% was purified from a nifE-deleted mutant of Azotobacter vinelandii DJ35. Compared with MoFe protein purified from wild-type strain OP (OP Av1), ΔnifE Av1 had the same subunits composition, and had immune reaction with antibody to OP Av1, but its relative mobility in anaerobic native polyacrylamide gel electrophoresis (PAGE) was a little larger than that of OP Av1. Metal analysis showed that Mo and Fe contents of ΔnifE Av1 both apparently decreased. When complemented with OP Fe protein, ΔnifE Av1 had no C2H2-reduction activity, but it could be in vitro activated by FeMoco extracted from OP Av1. The circular dichroism (CD) spectrum of ΔnifE Av1 at ~450 nm was similar to that of OP Av1, while the EPR signal at g≈3.7 was absolutely silent, and the signal intensities at g≈4.3 and 2.0 decreased by 75% and 50%, respectively. The results indicated that ΔnifE Av1 purified from DJ35 was a FeMoco-deficient but P-cluster-con- taining MoFe protein.
基金supported by the State Key Basic Research and Development Plan of China(Grant No.001CB1089-06)the National Science Foundation for Distinguished Young Scholars of China(Grant No.20403024)the National Natural Science Foundation of China(Grant No.30270296).
