Azoreductases are involved in the bioremediation by bacteria of azo dyes found in waste water.In the gut flora,they activate azo pro-drugs,which are used for treatment of inflammatory bowel disease,releasing the activ...Azoreductases are involved in the bioremediation by bacteria of azo dyes found in waste water.In the gut flora,they activate azo pro-drugs,which are used for treatment of inflammatory bowel disease,releasing the active component 5-aminosalycilic acid.The bacterium P.aeruginosa has three azoreductase genes,paAzoR1,paAzoR2 and paAzoR3,which as recombinant enzymes have been shown to have different substrate specificities.The mechanism of azoreduction relies upon tautomerisation of the substrate to the hydrazone form.We report here the characterization of the P.aeruginosa azoreductase enzymes,including determining their thermostability,cofactor preference and kinetic constants against a range of their favoured substrates.The expression levels of these enzymes during growth of P.aeruginosa are altered by the presence of azo substrates.It is shown that enzymes that were originally described as azoreductases,are likely to act as NADH quinone oxidoreductases.The low sequence identities observed among NAD(P)H quinone oxidoreductase and azoreductase enzymes suggests convergent evolution.展开更多
Acidithiobacillus caldus,a typical sulfur oxidizer,derives the majority of its energy from sulfur oxidation.And the essential enzyme for sulfide oxidation catalysis is sulfide quinone oxidoreductase(SQR),an ancient fl...Acidithiobacillus caldus,a typical sulfur oxidizer,derives the majority of its energy from sulfur oxidation.And the essential enzyme for sulfide oxidation catalysis is sulfide quinone oxidoreductase(SQR),an ancient flavoprotein.Here,the catalytic mechanism of SQR generated from A.caldus was investigated(SQR^(Ac)).According to phylogenetic study,SQR^(Ac)(ACAty RS11315)is closely related to SQR(BAD99305)of Acidithiobacillus ferrooxidans NASF-1 and is classified as a type I Sqr enzyme.SQR^(Ac)heterologously produced in Escherichia coli exhibits the distinctive absorption peaks(375,450 nm)of the flavoproteins family of proteins in its absorption spectrum.Utilizing site-directed mutagenesis,the function of conserved cysteines in the catalytic pathway was determined.Based on the sulfide quinone redox reactions in vitro of SQR^(Ac)and variations,Cys160 and Cys356 have been identified as enzyme-active residues.Mutation of another cysteine present in all type I SQRs(Cys128)decreased enzyme activity by 56%,indicating that this residue plays an important but non-essential role in enzyme function.In addition,the binding affinities of SQR^(Ac),the visualization of its 3D structure,and the interaction between receptors and ligands were investigated.Finally,a suitable sulfide quinone redox catalytic mechanism for A.caldus was proposed.展开更多
污水生物脱氮过程中N_2O的产生与活性污泥中细菌的羟氨氧化还原酶(Hydroxylamine oxidoreductase,HAO)活性有着密切关系.但目前活性污泥中细菌的HAO提取和活性测定方法尚未建立.本文首先探索了在25℃、酶活性反应液电子受体供体配比1∶...污水生物脱氮过程中N_2O的产生与活性污泥中细菌的羟氨氧化还原酶(Hydroxylamine oxidoreductase,HAO)活性有着密切关系.但目前活性污泥中细菌的HAO提取和活性测定方法尚未建立.本文首先探索了在25℃、酶活性反应液电子受体供体配比1∶1和终止剂选用2 mol·L^(-1)HCl条件下超声或高压破碎细胞法对HAO粗酶活性的影响,结果表明高压破碎比超声破碎获取的粗酶活性高(p<0.01).在此基础上,我们进一步优化了高压破碎下压力大小、破碎次数和裂解液用量的参数.粗酶提取液中DNA含量、酶活力及酶比活力结果进行多因素方差分析表明压力大小(50、110或160 MPa)、破碎次数(1、2或3次)和裂解液用量(2、5或10 m L)均对脱氮活性污泥破碎效果、酶活性和比活力有显著影响(p<0.01);综合DNA含量、酶活力及酶比活力结果看,110 MPa压力条件下,加5 m L裂解液破碎2次更适合污水生物处理中HAO的提取和活性测定,既节省时间,又能较好的保持酶活性.展开更多
文摘Azoreductases are involved in the bioremediation by bacteria of azo dyes found in waste water.In the gut flora,they activate azo pro-drugs,which are used for treatment of inflammatory bowel disease,releasing the active component 5-aminosalycilic acid.The bacterium P.aeruginosa has three azoreductase genes,paAzoR1,paAzoR2 and paAzoR3,which as recombinant enzymes have been shown to have different substrate specificities.The mechanism of azoreduction relies upon tautomerisation of the substrate to the hydrazone form.We report here the characterization of the P.aeruginosa azoreductase enzymes,including determining their thermostability,cofactor preference and kinetic constants against a range of their favoured substrates.The expression levels of these enzymes during growth of P.aeruginosa are altered by the presence of azo substrates.It is shown that enzymes that were originally described as azoreductases,are likely to act as NADH quinone oxidoreductases.The low sequence identities observed among NAD(P)H quinone oxidoreductase and azoreductase enzymes suggests convergent evolution.
基金the National Key Research and Development Program of China(2022YFC3401300)the National Natural Science Foundation of China(No.21878128,21,776,113,31,701,582+2 种基金21,606,110)the Fundamental Research Funds for the Central Universities(No.2050205)Program of Introducing Talents of Discipline to Universities(No.111-2-06).
文摘Acidithiobacillus caldus,a typical sulfur oxidizer,derives the majority of its energy from sulfur oxidation.And the essential enzyme for sulfide oxidation catalysis is sulfide quinone oxidoreductase(SQR),an ancient flavoprotein.Here,the catalytic mechanism of SQR generated from A.caldus was investigated(SQR^(Ac)).According to phylogenetic study,SQR^(Ac)(ACAty RS11315)is closely related to SQR(BAD99305)of Acidithiobacillus ferrooxidans NASF-1 and is classified as a type I Sqr enzyme.SQR^(Ac)heterologously produced in Escherichia coli exhibits the distinctive absorption peaks(375,450 nm)of the flavoproteins family of proteins in its absorption spectrum.Utilizing site-directed mutagenesis,the function of conserved cysteines in the catalytic pathway was determined.Based on the sulfide quinone redox reactions in vitro of SQR^(Ac)and variations,Cys160 and Cys356 have been identified as enzyme-active residues.Mutation of another cysteine present in all type I SQRs(Cys128)decreased enzyme activity by 56%,indicating that this residue plays an important but non-essential role in enzyme function.In addition,the binding affinities of SQR^(Ac),the visualization of its 3D structure,and the interaction between receptors and ligands were investigated.Finally,a suitable sulfide quinone redox catalytic mechanism for A.caldus was proposed.
文摘污水生物脱氮过程中N_2O的产生与活性污泥中细菌的羟氨氧化还原酶(Hydroxylamine oxidoreductase,HAO)活性有着密切关系.但目前活性污泥中细菌的HAO提取和活性测定方法尚未建立.本文首先探索了在25℃、酶活性反应液电子受体供体配比1∶1和终止剂选用2 mol·L^(-1)HCl条件下超声或高压破碎细胞法对HAO粗酶活性的影响,结果表明高压破碎比超声破碎获取的粗酶活性高(p<0.01).在此基础上,我们进一步优化了高压破碎下压力大小、破碎次数和裂解液用量的参数.粗酶提取液中DNA含量、酶活力及酶比活力结果进行多因素方差分析表明压力大小(50、110或160 MPa)、破碎次数(1、2或3次)和裂解液用量(2、5或10 m L)均对脱氮活性污泥破碎效果、酶活性和比活力有显著影响(p<0.01);综合DNA含量、酶活力及酶比活力结果看,110 MPa压力条件下,加5 m L裂解液破碎2次更适合污水生物处理中HAO的提取和活性测定,既节省时间,又能较好的保持酶活性.
