32 results about "Nitrile hydratase activity" patented technology

The invention relates to a Rhodococcus polynucleotide cluster which contains nucleotide sequences which encode polypeptides having the activity of a nitrile hydratase, of an auxiliary protein P15K which activates this enzyme and of a cobalt transporter, to transformed microorganisms in which the nucleotide sequences encoding these proteins are present in increased quantity, and to the use of the transformed microorganisms for preparing amides from nitriles.

The present invention is directed to methods for inducing desired activity in enzymes or microorganisms capable of producing the enzymes. The invention is further directed to methods of stabilizing activity in microorganisms. In specific embodiments, the invention provides methods for inducing and stabilizing nitrile hydratase activity, amidase activity, and asparaginase I activity. The invention further provides compositions comprising enzymes or microorganisms having induced and / or stabilized activity.

The present invention is directed to methods for inducing desired activity in enzymes or microorganisms capable of producing the enzymes. The invention is further directed to methods of stabilizing activity in microorganisms. In specific embodiments, the invention provides methods for inducing and stabilizing nitrile hydratase activity, amidase activity, and asparaginase I activity. The invention further provides compositions comprising enzymes or microorganisms having induced and / or stabilized activity.

The present invention is directed to methods for inducing desired activity in enzymes or microorganisms capable of producing the enzymes. The invention is further directed to methods of stabilizing activity in microorganisms. In specific embodiments, the invention provides methods for inducing and stabilizing nitrile hydratase activity, amidase activity, and asparaginase I activity. The invention further provides compositions comprising enzymes or microorganisms having induced and / or stabilized activity.

The object of this invention is to provide a method which not only maintains the nitrile hydratase activity of a nitrile hydratase-containing cell or a treated material of the cell under conditions where the cell does not grow, but also improves the nitrile hydratase activity of a nitrile hydratase-containing cell or a treated material of the cell whose activity was once reduced. This invention relates to a method of maintaining or improving a nitrile hydratase activity which comprises bringing a nitrile hydratase-containing cell or a treated material of the cell into contact with an oxidizing agent under conditions where the cell does not grow, as well as a method of producing an amide compound from a nitrile compound, which comprises using the cell brought into contact with an oxidizing agent or a treated material of the cell.

The present invention provides: a protein having an improved nitrile hydratase activity, whereby heat resistance has been improved when compared with a wild-type nitrile hydratase activity, wherein the amino acid sequence of a nitrile hydratase is modified; a gene DNA encoding the above protein; a recombinant vector having the above gene DNA; a transformant or transductant having the above recombinant vector; a nitrile hydratase collected from a culture of the above transformant or transductant, and a production method thereof; and a method for producing an amide compound.

The object of this invention is to provide a method which not only maintains the nitrile hydratase activity of a nitrile hydratase-containing cell or a treated material of the cell under conditions where the cell does not grow, but also improves the nitrile hydratase activity of a nitrile hydratase-containing cell or a treated material of the cell whose activity was once reduced. This invention relates to a method of maintaining or improving a nitrile hydratase activity which comprises bringing a nitrile hydratase-containing cell or a treated material of the cell into contact with an oxidizing agent under conditions where the cell does not grow, as well as a method of producing an amide compound from a nitrile compound, which comprises using the cell brought into contact with an oxidizing agent or a treated material of the cell.

A process for the preparation of five-membered or six-membered ring lactams from aliphatic alpha , omega -dinitriles has been developed. In the process an aliphatic alpha , omega -dinitrile is first converted to an ammonium salt of an omega -nitrilecarboxylic acid in aqueous solution using a catalyst having an aliphatic nitrilase (EC 3.5.5.7) activity, or a combination of nitrile hydratase (EC 4.2.1.84) and amidase (EC 3.5.1.4) activities. The ammonium salt of the omega -nitrilecarboxylic acid is then converted directly to the corresponding lactam by hydrogenation in aqueous solution, without isolation of the intermediate omega -nitrilecarboxylic acid or omega -aminocarboxylic acid. When the aliphatic alpha , omega -dinitrile is also unsymmetrically substituted at the alpha -carbon atom, the nitrilase produces the omega -nitrilecarboxylic acid ammonium salt resulting from hydrolysis of the omega -nitrile group with greater than 98% regioselectivity, thereby producing only one of the two possible lactam products during the subsequent hydrogenation. A heat-treatment process to select for desirable regioselective nitrilase or nitrile hydratase activities while destroying undesirable activities is also provided.

The invention discloses modified nitrile hydratase and application thereof in the technical field of enzyme engineering and industrial microorganisms. A method for obtaining the modified nitrile hydratase disclosed by the invention is that Pro of 133 site of a alpha subunit of an amino acid sequence shown as by a sequence table SEQ ID NO:2 and Asp of 215 site of a beta subunit of an amino acid sequence shown as by a sequence table SEQ ID NO:1 are respectively replaced with Cys, so that a disulfide bond is formed between the two subunits. Stress resistance and heat resistance of the modified nitrile hydratase disclosed by the invention and product tolerance are all obviously improved, and activity of the nitrile hydratase is not lowered. The modified nitrile hydratase can be catalyzed to obtain a high-concentration acrylamide product, and can be recycled repeatedly.

[Problems] The present invention provides a method for efficiently producing a corresponding amide compound from a nitrile compound by a reaction using a nitrile hydratase and a method for producing an amide-based polymer excellent in quality from the amide compound. In addition, the present invention provides a method for more efficiently producing an acrylamide with higher quality by a microbial catalyst containing a nitrile hydratase and the like and a method for producing an acrylamide-based polymer, which is excellent in hue, has a good balance between the water solubility and the high molecular weight and is also excellent in quality, by using the acrylamide.[Means for Solving the Problems] The method for producing an amide compound of the present invention is characterized in that in a method for producing an amide compound from a nitrile compound in an aqueous medium in the presence of a catalyst having a nitrile hydratase activity, the concentration of benzene in the aqueous medium is 4.0 ppm or less. In addition, the method for producing an amide-based polymer of the present invention is characterized by homopolymerizing the amide compound or by copolymerizing the amide compound and at least unsaturated monomer copolymerizable with the amide compound. Further, the method for producing acrylamide of the present invention is characterized by hydrating acrylonitrile having a concentration of acrolein of 1 ppm or less by a microbial cell containing a nitrile hydratase or a processed product of the microbial cell in an aqueous medium. Furthermore, the method for producing an acrylamide-based polymer of the present invention is characterized by homopolymerizing the acrylamide or by copolymerizing the acrylamide and at least one unsaturated monomer copolymerizable with the acrylamide.

The amino acid sequence of a mutant which is obtained by introducing a novel mutation into a Pseudonocardia thermophila JCM3095-derived nitrile hydratase consisting of two types of heterogeneous subunits, and the base sequence of the gene are provided. The nitrile hydratase is modified by specifying the region to be modified in the stereostructure / amino acid sequence of the nitrile hydratase, and applying alteration such as substitution, insertion, deletion or the like, to the amino acids in the amino acid sequence which are corresponding to the amino acid residues forming the region. Also provided is a method for modifying an enzyme having a nitrile hydratase activity.

This invention provides an aqueous solution of acrylamide that is useful as a starting material for high-quality polyacrylamide. Starting materials for polyacrylamide are an aqueous solution of acrylamide containing a saccharide and an aqueous solution of acrylamide containing a saccharide produced with the use of a biocatalyst having nitrile hydratase activity.

This invention relates to a process for the preparation of a 3-hydroxycarboxylic acid from a 3-hydroxynitrile. More specifically, 3-hydroxyvaleronitrile is converted to 3-hydroxyvaleric acid in high yield at up to 100% conversion, using as an enzyme catalyst 1) nitrile hydratase activity and amidase activity or 2) nitrilase activity of a microbial cell. 3-Hydroxyvaleric acid is used as a substitute for ε-caprolactone in the preparation of highly branched copolyester.

The present invention relates to methods for cultivating a nitrile hydratase producing microorganism, compositions for cultivating a nitrile hydratase producing microorganism, and use of compositions comprising a saccharide and an organic acid for cultivating a nitrile hydratase producing microorganism. The composition provided in and to be employed in context with the present invention is particularly suitable for inducing both, growth and nitrile hydratase production of corresponding microorganisms.

The invention provides a method for reducing a nitrile hydratase usage amount in a process of producing acrylamide by virtue of a microbiological method. In a process of producing the acrylamide by catalyzing a hydration reaction in the microbiological method, the quality of the acrylamide is optimized when a fermentation broth storage time of nitrile hydratase cells is 96-240h; and when the storage time is longer than or equal to 240h, the activity of the nitrile hydratase in a fermentation broth drops, the usage amount of the fermentation broth is increased and the quality of the obtained acrylamide is reduced. According to the method provided by the invention, by mixing the nitrile hydratase cell fermentation broth with the storage time shorter than or equal to 24h and the nitrile hydratase cell fermentation broth with the storage time longer than or equal to 240h, the activity and the quality of the nitrile hydratase are improved, so that the method for reducing the usage amount of the nitrile hydratase is achieved. With the application of the method provided by the invention, the usage amount of the nitrile hydratase in the production process is reduced, the consumption of raw materials of a fermentation medium is relieved and the stability of the quality of an acrylamide solution is improved, and the method is conducive to the subsequent production of the acrylamide. When the method provided by the invention is applied to the industrial production of the acrylamide by virtue of the microbiological method, the finished product is stable in various technical indicators.

The invention discloses a method for improving the nitrile hydratase activity of bacillus subtilis. The method comprises the following steps: (1) carrying out slant culture; (2) carrying out seed culture; and (3) carrying out fermentation culture. The method has the advantages that on one hand, the nutrition components in the fermentation medium are changed, namely, the fermentation medium contains Tween-80 0.1-1 g / ml%, NH4Cl 0.1-1 g / ml%, and urea 0.01-1 g / ml%, a glucose-Co<2+> coupling fed batch process is adopted, and a supplementary material formed by mixing glucose and inducer IND is added; on the other hand, the growth environment of bacteria can be controlled, namely, in the fermentation process, by adding ammonium hydroxide, the pH is controlled to be 6.8-7.5, the temperature is controlled to be 25-35 DEG C, the oxygen quantity is maintained to be 1-3 vvm, and thus the purpose of greatly improving the enzymatic activity during the nitrile hydratase fermentation production is achieved.

This invention relates to a process for producing an amide compound from a nitrile compound using a microbial catalyst, wherein a microbial cell having nitrile hydratase activity of 50 U or higher per mg of dry cell at a reaction temperature of 10° C. is brought into contact with a nitrile compound in an aqueous medium without being immobilized. This method utilizes a microbial cell that exhibits high nitrile hydratase activity in the reaction without being entrap-immobilized. Thus, an amide compound can be effectively produced from a nitrile compound without problems of decreased reaction speed or lowered amount produced per unit cell amount, which are caused by entrap-immobilization. Accordingly, an amide compound can be produced within a very short period of time in the case of a batch reaction and with a very small-scale facility in the case of a continuous reaction.

The present invention provides a cheap and simple microbe for replacing that of the prior art, and a preserving method thereof. The invention relates to the preserving method of microbe and is characterized in that: the microbes with nitrile hydratase activity are dispersed in a dispersion medium with a concentration that the mass of dry microbe is 4-20wt%.

The invention discloses a high-throughput screening method of a signal peptide library based on a fluorescent probe Rho-IDA-CoII. Compared with a traditional screening method, the method has the advantages that the histidine tag fusion protein in the range of 50-500 ng can be accurately measured by using a fluorescent probe labeling quantitative method, high background fluorescence caused by complex samples and environmental pollution is avoided, and the application range is wide. The method comprises the following steps: screening bacillus subtilis recombinants capable of secreting and expressing active nitrile hydratase from a bacillus subtilis expression nitrile hydratase signal peptide library by virtue of a fluorescent high-throughput screening method, and detecting that the nitrile hydratase activity is 12.97 + / -0.77 U / mL in culture supernate of the bacillus subtilis recombinants. The recombinant capable of secreting and expressing the nitrile hydratase can be applied to industrial production of the nitrile hydratase and downstream application of the nitrile hydratase.

The invention relates to a method for preserving and / or storing microorganisms which exhibit at least one nitrile hydratase or nitrilase enzyme activity, with the preservation and / or storage being effected in an aqueous medium which comprises at least one aldehyde, with the total aldehyde concentration being in a range from 0.1 to 100 mM / l.

A method for storing microbial cells having nitrile hydratase activity in a hermetically sealable container, and a method for transporting microbial cells having nitrile hydratase using the container are provided. The methods are characterized in that the vapor phase portion within the container is set to 2-20% (inclusive) of the total interior volume of the container, said vapor phase portion being filled with a suspension that contains the microbial cells.

The invention discloses a Escherichia coli recombinant strain heterologously expressing heat-resistant nitrile hydratase and application of the Escherichia coli recombinant strain, and belongs to thefield of bioengineering. According to the invention, the Escherichia coli recombinant strain having a high nitrile hydratase activity is obtained by constructing of recombinant Escherichia coli, and the recombinant nitrile hydratase obtained by fermentation has better thermal stability. In addition, the Escherichia coli recombinant strain can be used as a catalyst, and nicotinonitrile or acrylonitrile can be used as a substrate to carry out a whole-cell catalytic reaction to prepare nicotinamide or acrylamide. Compared with a chemical production method, the production process is safe and clean, and has no environmental pollution. Compared with an enzymatic method, the production process employs a cheap substrate, simplifies separation and purification steps of products, has high catalyticefficiency, and has a yield of the final product nicotinamide or acrylamide of 95% or above.

The invention discloses a method for synthesizing a low-conductivity amide compound aqueous solution. The method for synthesizing the low-conductivity amide compound aqueous solution comprises the following steps that in a solution system with a biocatalyst with nitrile hydratase activity, a corresponding amide compound is synthesized from a nitrile compound, and the temperature in the synthesis process ranges from 9 DEG C to 14 DEG C. According to the method, acrylamide is synthesized through a microbiological method at a low temperature; for a 42% acrylamide product, the by-product crylic acid content is low (0.0846%), and the conductivity of the product is 400 micro Scm<-1>.

The problem is to provide a method for inexpensively and simply preserving an enzyme of a microbial biomass obtained by culturing and improve the enzyme activity during preservation. Provided is a method for preserving nitrile hydratase, characterized in that microbes having nitrile hydratase activity are cultured while being protected from light, and the resulting biomass is crushed using a high-pressure homogenizer and preserved while being protected from light.