Nitrile hydratase (NHase) is an important industrial enzyme used for acrylamide production from acrylonitrile.The deactivation kinetics of NHases in free resting cells of Rhodococcus sp.was presented based on a bi-ste...Nitrile hydratase (NHase) is an important industrial enzyme used for acrylamide production from acrylonitrile.The deactivation kinetics of NHases in free resting cells of Rhodococcus sp.was presented based on a bi-steady state assumption.Effects of hydration temperature,product concentration and substrate concentration on NHase deactivation were investigated experimentally and correlated with a first order deactivation kinetics.The results showed that the hydration temperature and product concentration were major factors governing the deactivation of NHases under substrate-feeding conditions.When acrylamide concentration was higher than 250 g·L1,the deactivation of NHases became serious and the bi-steady state assumption was not applicable.When the hydration temperature was controlled at a relatively higher level such as 28°C,the total deactivation rate constant was about 2.8-fold of that at 20°C.展开更多
Inducing expression and the reaction characteristic of nitrile hydratase （NHase） from Rhodococcus sp. SHZ-1 were investigated. The results showed that the expression of NHase was greatly enhanced with the cooperatio...Inducing expression and the reaction characteristic of nitrile hydratase （NHase） from Rhodococcus sp. SHZ-1 were investigated. The results showed that the expression of NHase was greatly enhanced with the cooperation of acrylonitrile and ammonium chloride as inducer in the medium and the specific activity of NHase was increased of 44%. Then the temperature, pH, concentration of acrylonitrile and acrylamide were evaluated, which affected the activity and reaction characteristic of NHase. It was found that the temperature and concentration of acrylarnide were the most important factors for the catalyzation of NHase. The optimal catalysis temperature of NHase from Rhodococcus sp. SHZ-1 was 30℃, and the activation energy of the hydration of NHase was 90.2kJ·mol^-1 in the temperature range from 5℃ to 30℃. Kmof NHase was 0.095mol·L^-1 using acrylonitrile（AN） as substrate, and NHase activity was inhibited seriously when acrylonitrile concentration was up to 40g·L^-1, the substrate inhibition constant Ki is 0.283mol·L^-1. Moreover, the NHase from Rhodococcus sp. SHZ-1 had very strong tolerance to acrylamide, in which the final concentration of acrylamide reached to 642g·L^-1 and the residual activity of NHase still maintained 8.6% of the initial enzyme activity.展开更多
The synthesis of hydroxy fatty acids(HFAs)from renewable oil feedstock by addition of water onto C_C bonds has attracted great attention in recent years.Given that selective asymmetric hydration of non-activated C_C b...The synthesis of hydroxy fatty acids(HFAs)from renewable oil feedstock by addition of water onto C_C bonds has attracted great attention in recent years.Given that selective asymmetric hydration of non-activated C_C bonds has been proven difficult to achieve with chemical catalysts,enzymatic catalysis by fatty acid hydratases(FAHs)presents an attractive alternative approach to produce value-added HFAs with high regio-,enantioand stereospecificity,as well as excellent atom economy.Even though FAHs have just been investigated as a potential biocatalyst for a decade,remarkable information about FAHs in different aspects is available;however,a comprehensive review has not been archived.Herein,we summarize the research progresses on biochemical characterization,structural and mechanistic determination,enzyme engineering,as well as biotechnological application of FAHs.The current challenges and opportunities for an efficient utilization of FAHs in organic synthesis and industrial applications are critically discussed.展开更多
Nitrile hydratase(NHase)is a metalloenzyme that catalyzes the conversion of nitrile to amide and is widely used in the biocatalysis of bulk chemicals such as acrylamide and nicotinamide.Improving the thermostability,a...Nitrile hydratase(NHase)is a metalloenzyme that catalyzes the conversion of nitrile to amide and is widely used in the biocatalysis of bulk chemicals such as acrylamide and nicotinamide.Improving the thermostability,activity,and soluble expression of natural NHase is crucial for its industrial application.However,conventional engineering strategies are often based on the design and evaluation of single-point mutations,followed by multiple rounds of iterative combinations,which are inefficient and difficult to predict the evolutionary direction of the combinatorial mutations due to epistatic effects.In this study,we used PROSS,an automated design tool based on structural and sequence information,to design a thermophilic NHase from Pseudonocardia thermophila JCM3095(PtNHase).By sequentially applying subunit-independent mutations,subunit-synergistic mutations,and single-point revertant mutations,we obtained the superior mutant A2B1-β221.This mutant exhibited 1.4-fold and 2.3-fold higher activity towards acrylonitrile and 3-cyanopyridine,respectively,compared to the wild type.Additionally,A2B1-β221 showed a significant enhancement in thermostability.Moreover,benefiting from the enhanced soluble expression,a high-performance whole-cell catalyst for NHase was obtained.Furthermore,conventional molecular dynamics simulations and metadynamics simulations were employed to resolve the molecular mechanisms under-lying the high activity and thermostability of A2B1-β221.This study not only provided highly efficient whole-cell catalyst for NHase,but also demonstrated the efficacy of utilizing automated design tools and molecular dynamics simulations in the engineering of heterologous multimeric proteins,offering valuable insights into their applicability.展开更多
Objective: To elucidate the role of S-100B and neuron specific enolase (NSE) in predicting the outcomes of patients with severe head injury. Methods: Forty patients with severe head injury were included in this study....Objective: To elucidate the role of S-100B and neuron specific enolase (NSE) in predicting the outcomes of patients with severe head injury. Methods: Forty patients with severe head injury were included in this study. The serum concentrations of S-100B and NSE were measured within 12 hours after head injury to investigate the correlation between serum levels of S-100B and NSE and outcome. Validity of both S-100B and NSE in outcome prediction was assessed with Receiver Operator Characteristic (ROC) curve. Results: The serum concentrations of S-100B and NSE of both groups, with favorable or unfavorable outcomes, were significantly higher than those of the normal group. The serum concentrations within 12 hours after head injury were closely correlated with the prognosis. Furthermore, according to the ROC curves of S-100B and NSE, S-100B was found better in predicting outcomes than NSE. Conclusions: S-100B and NSE may play important roles in outcome prediction after severe head injury. Moreover, S-100B is clearly superior to NSE in terms of predictive value and appears to be a more promising serum marker in outcome prediction after severe head injury.展开更多
Catalyzed by Rhodococcus erythropolis AJ270, a nitrile hydratase and amidase containing microbial whole-cell catalyst, at 10 ℃ and with the use of methanol as a co-solvent, nitrile and amide biotransformations produc...Catalyzed by Rhodococcus erythropolis AJ270, a nitrile hydratase and amidase containing microbial whole-cell catalyst, at 10 ℃ and with the use of methanol as a co-solvent, nitrile and amide biotransformations produce 2S-1,4-benzodioxane-2-carboxamide and 2R-1,4-benzodioxane-2-carboxylic acid in high yields with excellent enantioselectivity.展开更多
A new approach to optically active β-alkyl-α-methylene-γ-butyrolactone derivatives was reported from the Rhodococcus sp. AJ270-catalyzed hydrolysis of appropriate nitriles. The inversion of enantioselectivity of th...A new approach to optically active β-alkyl-α-methylene-γ-butyrolactone derivatives was reported from the Rhodococcus sp. AJ270-catalyzed hydrolysis of appropriate nitriles. The inversion of enantioselectivity of the amidase has been observed when a methyl protection was introduced into the hydroxy group of the parent substrate.展开更多
基金Supported by the Foundation for the Authors of National Excellent Doctoral Dissertation of China (200345)the National High Technology Research and Development Program of China (2007AA02Z201)the National Basic Research Program of China (2007CB714304)
文摘Nitrile hydratase (NHase) is an important industrial enzyme used for acrylamide production from acrylonitrile.The deactivation kinetics of NHases in free resting cells of Rhodococcus sp.was presented based on a bi-steady state assumption.Effects of hydration temperature,product concentration and substrate concentration on NHase deactivation were investigated experimentally and correlated with a first order deactivation kinetics.The results showed that the hydration temperature and product concentration were major factors governing the deactivation of NHases under substrate-feeding conditions.When acrylamide concentration was higher than 250 g·L1,the deactivation of NHases became serious and the bi-steady state assumption was not applicable.When the hydration temperature was controlled at a relatively higher level such as 28°C,the total deactivation rate constant was about 2.8-fold of that at 20°C.
基金Supported by the National Natural Science Foundation of China （No.20466002）, the Program for New Century Excellent Talents in University （NCET-04-089） and the Key Research Projects in the Uygur Autonomous Region of Xinjiang （No.200332109）.
文摘Inducing expression and the reaction characteristic of nitrile hydratase （NHase） from Rhodococcus sp. SHZ-1 were investigated. The results showed that the expression of NHase was greatly enhanced with the cooperation of acrylonitrile and ammonium chloride as inducer in the medium and the specific activity of NHase was increased of 44%. Then the temperature, pH, concentration of acrylonitrile and acrylamide were evaluated, which affected the activity and reaction characteristic of NHase. It was found that the temperature and concentration of acrylarnide were the most important factors for the catalyzation of NHase. The optimal catalysis temperature of NHase from Rhodococcus sp. SHZ-1 was 30℃, and the activation energy of the hydration of NHase was 90.2kJ·mol^-1 in the temperature range from 5℃ to 30℃. Kmof NHase was 0.095mol·L^-1 using acrylonitrile（AN） as substrate, and NHase activity was inhibited seriously when acrylonitrile concentration was up to 40g·L^-1, the substrate inhibition constant Ki is 0.283mol·L^-1. Moreover, the NHase from Rhodococcus sp. SHZ-1 had very strong tolerance to acrylamide, in which the final concentration of acrylamide reached to 642g·L^-1 and the residual activity of NHase still maintained 8.6% of the initial enzyme activity.
基金Financial Supports from Novo Nordisk Foundation(NNF16OC0021740)Aarhus Universitets Forskningsfond,AUFFNOVA(AUFF-E-2015-FLS-9-12)。
文摘The synthesis of hydroxy fatty acids(HFAs)from renewable oil feedstock by addition of water onto C_C bonds has attracted great attention in recent years.Given that selective asymmetric hydration of non-activated C_C bonds has been proven difficult to achieve with chemical catalysts,enzymatic catalysis by fatty acid hydratases(FAHs)presents an attractive alternative approach to produce value-added HFAs with high regio-,enantioand stereospecificity,as well as excellent atom economy.Even though FAHs have just been investigated as a potential biocatalyst for a decade,remarkable information about FAHs in different aspects is available;however,a comprehensive review has not been archived.Herein,we summarize the research progresses on biochemical characterization,structural and mechanistic determination,enzyme engineering,as well as biotechnological application of FAHs.The current challenges and opportunities for an efficient utilization of FAHs in organic synthesis and industrial applications are critically discussed.
基金supported by the National Natural Science Foundation of China(32201034,32271301)China Innovation Challenge(NingBo)Major Project(2023T020),the Natural Science Foundation of Jiangsu(BK20210470)。
文摘Nitrile hydratase(NHase)is a metalloenzyme that catalyzes the conversion of nitrile to amide and is widely used in the biocatalysis of bulk chemicals such as acrylamide and nicotinamide.Improving the thermostability,activity,and soluble expression of natural NHase is crucial for its industrial application.However,conventional engineering strategies are often based on the design and evaluation of single-point mutations,followed by multiple rounds of iterative combinations,which are inefficient and difficult to predict the evolutionary direction of the combinatorial mutations due to epistatic effects.In this study,we used PROSS,an automated design tool based on structural and sequence information,to design a thermophilic NHase from Pseudonocardia thermophila JCM3095(PtNHase).By sequentially applying subunit-independent mutations,subunit-synergistic mutations,and single-point revertant mutations,we obtained the superior mutant A2B1-β221.This mutant exhibited 1.4-fold and 2.3-fold higher activity towards acrylonitrile and 3-cyanopyridine,respectively,compared to the wild type.Additionally,A2B1-β221 showed a significant enhancement in thermostability.Moreover,benefiting from the enhanced soluble expression,a high-performance whole-cell catalyst for NHase was obtained.Furthermore,conventional molecular dynamics simulations and metadynamics simulations were employed to resolve the molecular mechanisms under-lying the high activity and thermostability of A2B1-β221.This study not only provided highly efficient whole-cell catalyst for NHase,but also demonstrated the efficacy of utilizing automated design tools and molecular dynamics simulations in the engineering of heterologous multimeric proteins,offering valuable insights into their applicability.
文摘Researches in recent years have shown that cellular immune factor plays an important role in the generation and development of cerebral hemorrhage1-3.
文摘Objective: To elucidate the role of S-100B and neuron specific enolase (NSE) in predicting the outcomes of patients with severe head injury. Methods: Forty patients with severe head injury were included in this study. The serum concentrations of S-100B and NSE were measured within 12 hours after head injury to investigate the correlation between serum levels of S-100B and NSE and outcome. Validity of both S-100B and NSE in outcome prediction was assessed with Receiver Operator Characteristic (ROC) curve. Results: The serum concentrations of S-100B and NSE of both groups, with favorable or unfavorable outcomes, were significantly higher than those of the normal group. The serum concentrations within 12 hours after head injury were closely correlated with the prognosis. Furthermore, according to the ROC curves of S-100B and NSE, S-100B was found better in predicting outcomes than NSE. Conclusions: S-100B and NSE may play important roles in outcome prediction after severe head injury. Moreover, S-100B is clearly superior to NSE in terms of predictive value and appears to be a more promising serum marker in outcome prediction after severe head injury.
基金Project supported by the Major State Basic Research Development Program （No. 2003CB716005）, the Ministry of Science and Technology, the National Science Foundation of China, and the Chinese Academy of Sciences.
文摘Catalyzed by Rhodococcus erythropolis AJ270, a nitrile hydratase and amidase containing microbial whole-cell catalyst, at 10 ℃ and with the use of methanol as a co-solvent, nitrile and amide biotransformations produce 2S-1,4-benzodioxane-2-carboxamide and 2R-1,4-benzodioxane-2-carboxylic acid in high yields with excellent enantioselectivity.
文摘A new approach to optically active β-alkyl-α-methylene-γ-butyrolactone derivatives was reported from the Rhodococcus sp. AJ270-catalyzed hydrolysis of appropriate nitriles. The inversion of enantioselectivity of the amidase has been observed when a methyl protection was introduced into the hydroxy group of the parent substrate.
