Objective In Corynebacterium crenatum,the adjacent D311 and D312 of N-acetyl-L-glutamate kinase(NAGK),as a key rate-limiting enzyme of L-arginine biosynthesis under substrate regulatory control by arginine,were initia...Objective In Corynebacterium crenatum,the adjacent D311 and D312 of N-acetyl-L-glutamate kinase(NAGK),as a key rate-limiting enzyme of L-arginine biosynthesis under substrate regulatory control by arginine,were initially replaced with two arginine residues to investigate the L-arginine feedback inhibition for NAGK.Methods NAGK enzyme expression was evaluated using a plasmid-based method.Homologous recombination was employed to eliminate the pro B.Results The IC50 and enzyme activity of NAGK M4,in which the D311 R and D312 R amino acid substitutions were combined with the previously reported E19 R and H26 E substitutions,were 3.7-fold and 14.6% higher,respectively,than those of the wild-type NAGK.NAGK M4 was successfully introduced into the C.crenatum MT genome without any genetic markers;the L-arginine yield of C.crenatum MT-M4 was 26.2% higher than that of C.crenatum MT.To further improve upon the L-arginine yield,we constructed the mutant C.crenatum MT-M4 ?pro B.The optimum concentration of L-proline was also investigated in order to determine its contribution to L-arginine yield.After L-proline was added to the medium at 10 mmol/L,the L-arginine yield reached 16.5 g/L after 108 h of shake-flask fermentation,approximately 70.1% higher than the yield attained using C.crenatum MT.Conclusion Feedback inhibition of L-arginine on NAGK in C.crenatum is clearly alleviated by the M4 mutation of NAGK,and deletion of the pro B in C.crenatum from MT to M4 results in a significant increase in arginine production.展开更多
A novel complex [Zn（Im）2（A-glu）]-0.5H2O（Im = imidazole, A-glu = N-acetyi- L-glutamic acid） has been synthesized from the reaction of A-glu with Zn（CH3COO）2·2H20 in the presence of Im at 65 ℃, and structu...A novel complex [Zn（Im）2（A-glu）]-0.5H2O（Im = imidazole, A-glu = N-acetyi- L-glutamic acid） has been synthesized from the reaction of A-glu with Zn（CH3COO）2·2H20 in the presence of Im at 65 ℃, and structurally characterized by single-crystal X-ray diffraction. The complex crystallizes in tetragonai, space group P43212 with a = b = 8.9078（6）, c = 43.458（6） A, C26H36N10O11Zn2, Mr = 795.39, V= 3448.3（6） A^3, De = 1.532 g/cm^3, Z = 4,μ（MoKα） = 1.461 mm^-1, F（000） = 1640, the final R = 0.0453 and wR = 0.0992. X-ray analysis reveals that the crystal structure is constructed by mixed iigands. A-glu adopts the bis-monodentate coordination mode linking two adjacent metal ions to form a one-dimensional chain. Zinc（Ⅱ） ions are four-coordinated with a distorted tetrahedral geometry. Luminescent properties of the complex have been inves- tigated.展开更多
基金supported by Natural Science Foundation of China,No.31360219 and No.30960012the Open Project Program of Key Laboratory of Functional Small Organic Molecule,Ministry of Education,Jiangxi Normal University(No.KLFS-KF-201414)
文摘Objective In Corynebacterium crenatum,the adjacent D311 and D312 of N-acetyl-L-glutamate kinase(NAGK),as a key rate-limiting enzyme of L-arginine biosynthesis under substrate regulatory control by arginine,were initially replaced with two arginine residues to investigate the L-arginine feedback inhibition for NAGK.Methods NAGK enzyme expression was evaluated using a plasmid-based method.Homologous recombination was employed to eliminate the pro B.Results The IC50 and enzyme activity of NAGK M4,in which the D311 R and D312 R amino acid substitutions were combined with the previously reported E19 R and H26 E substitutions,were 3.7-fold and 14.6% higher,respectively,than those of the wild-type NAGK.NAGK M4 was successfully introduced into the C.crenatum MT genome without any genetic markers;the L-arginine yield of C.crenatum MT-M4 was 26.2% higher than that of C.crenatum MT.To further improve upon the L-arginine yield,we constructed the mutant C.crenatum MT-M4 ?pro B.The optimum concentration of L-proline was also investigated in order to determine its contribution to L-arginine yield.After L-proline was added to the medium at 10 mmol/L,the L-arginine yield reached 16.5 g/L after 108 h of shake-flask fermentation,approximately 70.1% higher than the yield attained using C.crenatum MT.Conclusion Feedback inhibition of L-arginine on NAGK in C.crenatum is clearly alleviated by the M4 mutation of NAGK,and deletion of the pro B in C.crenatum from MT to M4 results in a significant increase in arginine production.
基金This work was supported by the National Natural Science Foundation of China (No. 20471026), the Natural Science Foundation of Henan Province (No. 0311021200) and the Foundation of Education Committee of Henan Province (2006150017)
文摘A novel complex [Zn（Im）2（A-glu）]-0.5H2O（Im = imidazole, A-glu = N-acetyi- L-glutamic acid） has been synthesized from the reaction of A-glu with Zn（CH3COO）2·2H20 in the presence of Im at 65 ℃, and structurally characterized by single-crystal X-ray diffraction. The complex crystallizes in tetragonai, space group P43212 with a = b = 8.9078（6）, c = 43.458（6） A, C26H36N10O11Zn2, Mr = 795.39, V= 3448.3（6） A^3, De = 1.532 g/cm^3, Z = 4,μ（MoKα） = 1.461 mm^-1, F（000） = 1640, the final R = 0.0453 and wR = 0.0992. X-ray analysis reveals that the crystal structure is constructed by mixed iigands. A-glu adopts the bis-monodentate coordination mode linking two adjacent metal ions to form a one-dimensional chain. Zinc（Ⅱ） ions are four-coordinated with a distorted tetrahedral geometry. Luminescent properties of the complex have been inves- tigated.
