TWI327596B - - Google Patents

Description

1327596 (1) EMBODIMENT OF THE INVENTION [Technical Field] The present invention relates to a microorganism belonging to the genus Rhodococcus transformed with a nitrilase gene, and to an enzyme using a nitrilase possessed by the above microorganism A method of producing a guanamine compound from a nitrile compound by a catalytic action.

[Prior Art] In the manufacturing technique of combining a nitrile-based water of a nitrile compound to convert it into a guanamine compound corresponding to a guanamine group, a conventional copper catalyst chemical method has been used as a catalyst method using a microorganism enzyme. Replace and become the mainstream. This enzyme is commonly referred to as a nitrilase, but many enzymes have been found in various microorganisms since the initial report. For example, there are Arthrobacter genus (Agricultural and Biological Chemistry Vol. 44 p. 2 2 5 1 - 2 2 5 2, 1 890), Agrobacterium genus (Japanese Patent Laid-Open No. 05- 10368 1 ), Acinetobacter genus (JP-61-282089) 'Aeromonas genus (Special Kaiping 05-030983), Enterobacter genus (Special Kaiping OS-236975), Escherichia ( Erwinia) Genus (Special Kaiping 05-161496) 'Xanthobacter genus (Special Kaiping 05-161495), Klebsiela genus (Special Kaiping 05-030982), Corynebacterium genus (Special opening 54) -129190, later determined to be Rhodococcus), Pseudomonas (Specially Opened: Sin 58-86093), Citrobacter (Special Edition) (2) 1327596

, genus Streptomyces (Special Kaiping 05-236976), genus Bacillus (Specially Opened 51-186 1 86, and JP-A-7-255494) 'Sputum genus (Special Kaiping 01-086889) Rhodococcus genus (JP-A-63-137688, JP-A 02-227069, JP-A-2002-369697, and JP-A-2-470), Rhizobium genus (Special Kaiping 05-23 6977), Pseudonocardia (Special Kaiping 8-56684) and the like. These enzymes have been diversified in physicochemical properties based on the diversity of their amino acid sequences, and have been studied for various purposes. Among its physicochemical properties, examples of the stability of heat or guanamine compounds and nitrile compounds, such as the literature on Rhodococcus rhodochrous J1 strain (European Journal of Biochemistry Vο 1. 196 p.581- 589, 19 9 1. Applied and Microbiology Biotechnology V o 1.4 0 p.1 89- 1 95, 1 993, special open 2004-2 155 1 3 ' and special open 2004-222538 ), related to Nocardia thermophila (Pseudonocardia thermophila) JCM3095 strain (JP-A-8-87092, and Journal of Fermentation and Bioengineering Vo 1. 83 p. 474-477, 19 9 7 ), literature on Bacillus genus BR449 (WO 99) /55719, and Applied Biochemistry and Biotechnology Vol.77-79 p.67 1 -679, 1999 ), the literature on the RAPc8 strain of the genus BaciUus (Enzyme and Microbial Technology Vol.26 p.368-373, 2000 ' and Extremophiles V o 1.2 p . 3 4 7 -357,1998 ) 'Documents on Bacillus palidus Dac521 strain (

Biochimica et Biophysica Acta Vol. 1431 p.249-260, 1999 (3) 1327596 ), related to SC-J05-1 strain (Journal of Industrial

Microbiology and Biotechnology vol_20 220-226,1 998 ), literature on Comamonastestosteroni 5-MGAM-4D strain ( W02004/101768 ), literature on Rhodococcus pyridinovorans MW3 strain (Biotechnology Letters 26: 1379-13 84, 2004 ) Wait

On the other hand, although these specific nitrilases are very ideal in their own enzymes, in industrial manufacturing, many microorganisms that produce enzymes have defects, and their hosts must be genetically engineered. In the first stage, a large number of attempts were made to conduct a review of genetic selection. For example, Pseudomonas (Special Kaiping 3-25 1 1 84), Rhodococcus (Special Kaiping 2- 1 19778, Special Kaiping 4-2 1 1 379, Special Kaiping 09-00973 'Special Kaiping 07-099980, and special Open 200 1 -069978 ), Rhizobium (Special Kaiping 6-25296), Klebsiella (Special Kaiping 6-30397 1 ), Achromobacter (Special Kaiping 08-266277), Pseudonocardia (Special Kaiping 9-275978), and Bacillus (Special Kaiping 09-248 188) and the like. In the second step, the gene must be expressed on a host that is most suitable for industrial production, but many attempts are made on the established coliforms of the genetic manipulation system. At this time, it is necessary to carry out the cultivation of the microorganisms. Excessive steps such as purification of enzymes from the bacteria and immobilization. In the case of Rhodococcus and Corynebacterium, the cell wall is very hard, and even in high concentrations of indoleamine compounds and nitrile compounds, microorganisms that maintain intracellular enzyme activity can be expressed, which can solve this problem. However, in the individual <RTI ID=0.0>> (4) 1327596 SUMMARY OF THE INVENTION The problem to be solved by the invention is that the physicochemical properties of the enzyme are also highly active at a high concentration with thermal stability and a high concentration of a nitrile compound or a product of the matrix. The nitrilase is ideal. Although there has been a report based on this purpose, the absolute type of nitrile compound has changed due to the type of the enzyme used in the reaction, and it is impossible to find a yeast with all properties.

Prime. Further, when industrially producing a guanamine compound from a nitrile compound, the production cost of the enzyme in the production cost of the amide group is extremely important, and the skilled person expects to be able to obtain a cheaper price. The microorganisms established by the culture method of the simpler culture medium are produced, and it is also desired to use these microorganisms as a host to produce a gene-transformed microbial cell. In addition to the problem of the cost of manufacturing the enzyme, it is not only necessary to be able to be easily cultivated, but it is also necessary to develop a φ gene operating system having a high expression amount of the enzyme on the cell. Further, when the nitrilase enzyme present in the microorganism of the genus Ning, such as " can be stably present in a high concentration of the guanamine compound and the nitrile compound, the microorganism can be regarded as being capable of maintaining the enzyme inside. Immobilized carrier. At this time, it can react in a high concentration matrix (nitrile compound), and the cumulative concentration of the final guanamine compound also increases. In addition, it is possible to omit the steps of taking out the enzyme and binding it to the immobilized support, and using the microorganism itself as a reaction catalyst. That is, the object of the present invention is to isolate a nitrilase having high stability to heat and a high concentration of -8 - (5) 1327596 compound in nature: to provide the amino acid sequence and the base sequence of the enzyme; And by using a recombinant plastid containing the gene, a transformant is provided which enables a rare species of enzyme to reach its performance even in a host which is highly desirable in production cost. Further, another object of the present invention is to provide a method for producing the enzyme using the transformant after culturing and proliferating the above-mentioned transforming body, and to provide a guanamine compound corresponding to the nitrile compound using the transforming body. Cheap manufacturing method.

MEANS FOR SOLVING THE PROBLEMS As a result of the detailed study of the above-mentioned problems, the present inventors have isolated the microorganisms having nitrilase activity, Geobacillus thermoglucosidasius, from the soil next to the hot springs in Saitama Prefecture. Moreover, it has not been known until now that the microorganism of the genus Geobacillus has a nitrilase and shows its nitrilase activity.

Sex. Furthermore, the culture of the microorganism is generally carried out at a temperature of 65 ° C, which is higher than the general culture temperature (45 ° C to 60 ° C) of a thermophilic bacteria having a traditional nitrilase, and the enzyme has The stability of the guanamine compound and the nitrile compound. Further, the nitrilase enzyme purified by the microorganism has a high stability of heat and a high concentration of the nitrile compound and the guanamine compound. Furthermore, based on the N-terminal amino acid sequence of each subunit of the purified enzyme, the nitrilase gene is isolated from the chromosomal DNA of the microorganism, and the results of the amino acid sequence and the gene sequence are first solved, and both are found. Some nitrilases have very low identity. Furthermore, while estimating the gene sequence of the activated protein present downstream of the gene, and -9 - (6) 1327596

The present invention has been accomplished by successfully producing a genetic recombinant strain capable of expressing the enzyme in a large amount by the expression of its gene. Furthermore, in order to further improve the degree of completion of the present invention, it is directed to a microorganism of the genus Rhodococcus which has a hard cell wall and can be used as an immobilized carrier such as an enzyme. Further, Rhodococcus rhodochrous M33 strain (VKM AC-15 15D), which is cultivated on a cheap culture machine and suitable for industrial production of microorganisms, is successfully used as a host, and it can be produced in an unusually large amount. Gene-transformed strain. On the other hand, based on this result, it was confirmed that the Rhodococcus rhodochrous M33 strain has a general price as a host for expressing a nitrilase of a xenobiotic. That is, the present invention provides the inventors of the following (1) to (9). (1) A microorganism of the genus Rhodococcus characterized by being transformed with any of the following DNAs (A) or (B), (A) the DNA system containing a code corresponding to a nitrilase α The amino acid sequence described in SEQ ID NO: 1 in the sequence listing of the unit, or the amino acid sequence described in SEQ ID NO: 1 in the Sequence Listing, having one or more amines substituted, deleted or added to the amine a base sequence of a base acid sequence; and an amino acid sequence described in SEQ ID NO: 2 encoding a sequence listing corresponding to a nitrilase; 8 subunits, or an amino acid sequence described in SEQ ID NO: 2 of the Sequence Listing a base sequence having an amino acid sequence in which one or several amino acids are substituted, deleted or added; and the DNA encodes a protein having nitrilase activity; (Β) the DNA system contains The method for producing a microorganism according to any one of (1) to (5), which is characterized by the following (A) or Any DN A of (B) is transformed into a microorganism of the genus Rhodococcus (A) the DNA contains an amino acid sequence described in SEQ ID NO: 1 in the sequence listing corresponding to the cytidine hydrolase α subunit, or an amino acid sequence described in SEQ ID NO: 1 in the coding sequence table. a base sequence of an amino acid sequence in which one or several amino acids are substituted, deleted or added; and an amino acid sequence as described in SEQ ID NO: 2 encoding a sequence listing corresponding to a nitrilase/3 subunit Or the nucleotide sequence of the amino acid sequence having one or several amino acid substitutions, deletions or additions in the amino acid sequence described in SEQ ID NO: 2 of the Sequence Listing; at the same time, the DNA coding a protein having nitrilase activity;

(Β) the DNA contains a base sequence of 695th to 13thth of SEQ ID NO: 3 in the sequence of the sequence corresponding to the base sequence of the nitrilase α subunit, or hybridizes the base sequence under stringent conditions. a base sequence; and a base sequence comprising the first to the 681th nucleotide sequence number 3 of the sequence listing corresponding to the nucleotide sequence encoding the nitrilase Θ subunit, or hybridizing the base sequence under stringent conditions The base sequence; at the same time, the DNA is a protein encoding a nitrilase activity. (7) A method for producing a nitrilase or a bacterium for treating a bacterium comprising the nitrilase, which comprises culturing a microorganism according to any one of (1) to (5) in a culture medium. (8) A nitril hydrolase or a bacterial cell treated product containing the nitrilase, which is produced by the production method of (7). • 13- (10) 1327596 (9) A method for producing a guanamine compound, which comprises subjecting a nitrilase of (8) or a bacterial body treatment containing the nitrilase to a nitrile compound, and The nitrile compound is synthesized by a guanamine compound. By using the microorganism of the present invention, a nitrilase having high stability to heat and a high concentration of a nitrile compound or a guanamine compound can be produced, and by using the nitrilase, the nitrile can be efficiently and inexpensively obtained. The compound is converted into a corresponding guanamine compound. BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, embodiments of the present invention will be described in detail. First, the nitrilase which is expressed in the present invention will be described. The present invention has a nitrilase activity such as acetonitrile as acetamamine, n-propanenitrile as n-propionamine, acrylonitrile as acrylamide, and a nitrile compound plus water molecules to convert it into a guanamine compound. Active. Further, the resulting compound is separated by liquid chromatography, and then characterized by gas chromatography/mass spectrometry (GC/MS), φ infrared absorption spectroscopy (IR), and nuclear magnetic resonance spectroscopy (NMR). • When measuring the nitrilase activity in the present invention, for example, 1 μl of a nitrile compound solution (0.05 M-phosphate buffer, pH 7.7) may be added to a 10 μl nitrilase solution at a reaction temperature. After incubation at 27 ° C to 60 ° C for 10 to 60 minutes, 0.1 ml of IN HC1 was added to stop the reaction, and a portion of the reaction solution was analyzed by liquid chromatography to determine the presence of a guanamine compound. The nitrile compound as a matrix in the present invention includes, for example, a fatty acid nitrile compound such as acetonitrile, n-propionitrile-14-(11) 1327596, n-butyronitrile, isobutyronitrile, n-valeronitrile or n-hexane, and 2-chloropropionitrile. An aliphatic nitrile compound containing an unsaturated bond such as a nitrile compound of a halogen atom, acrylonitrile, crotononitrile, or crotononitrile, a hydroxynitrile compound such as a lactonitrile or a phenylglycolonitrile, or an aminonitrile such as a 2-phenylglycine nitrile An aromatic nitrile compound such as a compound, benzonitrile, nitrile or cyanopyridine, a dinitrile compound such as malononitrile, succinonitrile or adiponitrile, or a trinitrile compound. The nitrilase of the present invention, whose matrix specificity can be judged by measuring whether the substrate has nitrilase activity after various changes in the substrate under the above-described measurement of φ. If the matrix specificity can be expanded, the type of corresponding guanamine compound can be increased. It is desirable to use acrylonitrile, adiponitrile, acetonitrile, isobutyronitrile, n-valeronitrile, or even a small amount of the enzyme. Nitrile, benzonitrile, hexane nitrile as matrix. The nitrilase of the present invention is more preferably, for example, an α subunit represented by 205 amino acid sequences represented by SEQ ID NO: 1 in the Sequence Listing, and 226 amino acids represented by SEQ ID NO: 2 in the Sequence Listing. As shown in the sequence; 8 sub- φ bits constitute the one. Metal or other peptides may be contained in addition to the two subunits. The metal is particularly rich in iron and cobalt. Also, it may be a protein containing any of the subunits. Further, the amino acid sequence as each subunit may be a complex of other subunits and has a nitrile water "enzymatic activity, and the amino acid described in the above Sequence Listing No. 1 or 2; In the sequence, substitutions, deletions, or insertions of one or more amino acids may also occur, and it is foreseen that modifications are made after translation depending on the type of host. Among them, especially in the α subunit of nitrilase, after translation, most of the cysteine residues are modified into cysteine sulfinic acid or cysteine sulfenic acid-15-(13) 1327596 It will hinder the reaction and become a serious problem when a high concentration of the reaction product is obtained. However, the nitrilase has a significant decrease in the acrylonitrile concentration of the matrix even when 35% by weight of acrylamide is added to the activity measurement solution. The activity remains the same. A nitrilase having a physicochemical property as described above can be obtained, for example, by culturing a microorganism of the genus Geobacillus. And Geobacillus is a microbial aspect, such as Geobacillus caldoxylsilyticus, Geobacillus

Kaustophilus , Geobacillus lituanicus ' Geobacillus stearothermophilus, Geobacillus subterr aneu s,

Geobacillus thermocatenulatus, Geobacillus thermodenitrificans, Geobacillus thermoglucosidasius, Geobacillus thermoleovorans ' Geobacillus toebii ' and

Geobacillus uzenensis. Further, the present invention is not particularly limited to microorganisms belonging to the genus Geobacillus, and may also contain a nitrilase gene derived from other microorganisms. Such microorganisms include, for example, Agrobacterium, Achromobacter, Acinetobacter, Aeromonas, Enterobacter, Erwinia. Genus, Xanthobacter, Klebsiela, Corynebacterium, Sinorhizobium, Pseudomonas, Streptomyces, Norcardia, Bacillus (Bacillus) is a genus of 'Micrococcus' Rhodococcus, Rhodopseudomonas, Rhizobium -17- (14) (14) 1327596, Pseudonocardia (Pseudonocardia) belongs to the genus. Specifically, the present invention can be screened by the following method. First, a small amount of soil collected in various places was placed in a test tube containing water or physiological saline, and shake culture was carried out in a vibrating incubator at 65 °C for 2 to 14 days. Take out a part of the culture solution and put it into a liquid medium containing a main component of a medium generally used for microorganisms, such as glycerin, polyglycan, yeast extract, etc., at a culture temperature of 65 ° C for 1 day. Up to 7 days of culture. One of the thus obtained culture liquids was applied to a canola flat medium containing the medium for the growth of the microorganisms, and then recultured at 65 °C to form colonies, thereby separating the microorganisms. The microorganism thus obtained is a test tube or a conical flask Φ containing a liquid medium containing a nitrile compound such as n-valeronitrile or a guanamine compound such as methacrylamide, or the like, at a suitable time, for example, about 12 The cells were cultured by shaking culture at a culture temperature of 65 ° C for about 7 days, and the target microorganism was selected according to the above-described method for measuring nitrilase activity. The representative strain of this microorganism is defined by 16SrRN A and the following biochemical properties as Geobacillus thermoglucosidasius, named Geobacillus thermoglucosidasius Q-6, with the registration number of FERM P-19351 (the date of registration is May 15, 2005). It is deposited in the Industrial and Technological Research Institute of the Independent Administrative Corporation and is transferred under the jurisdiction of the Budapest Treaty by FERM BP-08658 (the date of registration is March 11th, 2006). Geobacillus thermoglucosidasius Q-6 is deposited with the registry of Taiwan at BCRC 9 10263. Although investigations have been made on various patents and literatures, the contents of the microorganisms of Geobacillus thermoglucosidasius are not described as having nitrilase activity -18-(15) 1327596. Thus, it was confirmed that the Geobacillus thermoglucosidasius Q-6 strain is a novel microorganism. The nature of the Geobacillus thermoglucosidasius Q-6 strain is as follows: (a) the nature of the form

Culture conditions: nutrient agar gel (〇\〇1 (1; 1^13 11 (1,1) 1 〇 medium 60 ° 〇 1 · cell shape and size and shape: bacilli size · · · · · · χ 2.0 ~ 3.0; zm 2 · Cell pleomorphism: a 3. Exercise: + Flagellar hairy state: Peripheral hair 4. Cyt:: Cell site: End (b) Culture properties Culture conditions: Nutrition agar gel (〇x〇id England UK) Medium 6〇.〇1 ·Color: Cream 2. Gloss: + 3. Pigment production: ~ Culture conditions: nutrient liquid (1) "^^"^ to..." medium (10) art 1 _ surface growth: ~ 2 · medium Turbidity: + Culture conditions: Gelatin puncture culture 6 〇 ^ Growth state: + -19- (16) (16) 1327596 Gelatin liquefaction: +

Culture conditions: litmus milk medium 6 (TC 1. solidification: one 2. liquefaction: one (c) physiological properties 1. Gram stain: indefinite 2. nitrate reduction: a 3. denitrification reaction: a 4. MR test: a 5. VP test: a 6. 吲哚 generation: a 7. hydrogen sulfide production: a 8. starch hydrolysis: - 9. citric acid utilization Ko s er: -

Christensen :- 9. Utilization of Inorganic Nitrogen Sources Nitrate: Monoammonium Salt: + 10. Pigment Formation: 11. 11. Urease Activity: -1 2. Oxidase: + 1 3. Hydrogen Peroxide: + 14. Growth Range -20 - (17) 1327596

pH : 5.5 ~ 8.0

Temperature: 45 ° C ~ 72 ° C 15. Reaction to oxygen: facultative anaerobic 1 6 · Ο - F Test: - / - (d) Acid production from sugar: gas generation 1. L - arabinose - / — 2. D-Xylose + / — 3. D-Glucose + / — 4. D - Mannose + / — 5. D-Fructose + / — 6. D-galactose-/- 7. Maltose + / _ 8. Recommended sugar + / - 9. Lactose _ / a 10. Trehalose + / - 11. D-sorbitol - / - 12. D-maltitol + /-1 3. Inositol one / - 14. Glycerol - / - (e) Other properties 1. /3-galactosidase activity: a 2. Arginine dihydrolase activity: a 3. Lysine decarboxylase activity: - 4. Tryptophan deamination Enzyme activity: _ -21 - (19) (19) 1327596. In the present specification, unless otherwise specified, genetic recombination techniques, recombinant protein production techniques, and analytical methods well known in the art can be employed. The DN A encoding the nitrilase of the present invention may be based on sequence information such as the nucleotide sequence or the amino acid sequence disclosed in the specification of the present invention or an amino acid sequence determined by the above-mentioned purified enzyme as the case may be. A microorganism in which a nitrilase is invented, for example, is obtained from a strain of Geobacillus thermoglucosidasius. According to the oligonucleotide synthesized by the amino acid sequence as a probe, the chromosomal DNA of the microorganism containing the nitrilase is decomposed by a restriction enzyme, and the DN A fragment is introduced into the phage or plastid, and then the host is transformed. The DN A encoding the nitrilase of the present invention can also be obtained from a database according to dot hybridization or colony hybridization. Furthermore, the oligonucleotide is used as a probe, and the primer is produced based on the N-terminal amino acid sequence information of the two subunits determined by the purified enzyme, and a part of the nitrilase gene is used as an amplification factor of the PCR reaction. The probe can also perform the same process. The resulting DNA can be inserted into a plasmid vector such as Puc 118 for colonization, and the base is subjected to a conventional method such as the deoxygenation method (Proceedings of the National Academy of Sciences. USA, 74: 5463-5467, 1977). Base sequence definition. The gene thus produced can be identified as a DNA encoding a nitrilase by the general activity measurement method described above by performing the expression product in the transformed Escherichia coli host using the gene. Microorganisms suitable for industrial production are transformed using DNA encoding a nitrilase having such properties. Industrial microorganisms more suitable for production are ideal for microorganisms of the genus Rhodococcus. In the case of transformation, a recombinant vector characterized by a vector having the above DN A is used. -23- (20) 1327596 The recombinant vector of the present invention is linked to the 5' end of the DNA obtained by the above method, in the case of being suitable for exerting its function, downstream of a promoter region suitable for the host microorganism. If necessary, a transcription termination sequence can be inserted downstream of this, and can be prepared by recombination with an appropriate expression vector. In terms of an appropriate expression vector, it is not particularly limited as long as it can be replicated in the host microorganism. As the vector, for example, a pK4 system, a PRF30 system, a PBS305 system, a pRE-7 or the like can be used, and it is generally selected from a carrier of the circumference φ of Rhodococcus. The promoter used for the expression vector is preferably a promoter which can expect a high expression amount such as a nitrile gene or a nitrilase gene, but is not limited thereto. Furthermore, the host may be inserted into the chromosome as long as it is a gene, and there is no need to have a self-regulating reproducible region in the host. Further, the α-subunit gene and the yS subunit gene can be expressed by each promoter as an independent effector, or can be expressed by a common promoter as a polymerization. Moreover, independent sub-agents, individual subunit genes can also be used on other vectors. In this case, the host transformation was carried out using two expression vectors comprising the first φ expression vector of the α subunit gene and the second expression vector comprising the /3 subunit gene. Further, on the above recombinant vector, an amino acid sequence necessary for activating the nitrilase can also be used (license, No. 3408737, and J〇urnal 〇f Bi〇chemtry 1 25: 696-704). (*1999)) The performance of DNA coexists. Specifically, a plastid vector containing a promoter and a transcription terminator necessary for the same expression as described above, a protein-coding gene involved in the activation of a nitrilase, an α-subunit gene of a nitrilase, and/or Each of the five subunit genes is expressed as an action, and can also be expressed by a common control region as an action. Similarly, the pH of -24-(22) 1327596 is not particularly limited, but is usually pH 3 to 9 ', preferably pH 5 to 8, more preferably pH 6 to 7. In the present invention, the host microorganism is not particularly limited as long as it is a microorganism species of the genus Rhodococcus. For example, there are Rhodococcus rhodochrous 'Rhodococcus ruber, Rhodococcus erythropolis, Rhodococcus rubropertinctus and the like.

Particularly desirable is the Rhodococcus rhodochrous M33 strain (VKM Ac-1515D or VKPM S-1268). The particularly desirable reason for using Rhodococcus rhodochrous M33 strain (VKM AC-1515D or VKPM S-1268) as a host microorganism is reported (U.S. Patent No. 5,827,699), indicating that the characteristic characteristics of Rhodococcus are cell wall. In the production of the above-described guanamine compound, the cells obtained by the culture can be directly reacted with the nitrile compound to accumulate 46% (weight/liquid amount) by the action of the nitrilase possessed by the bacterium. Acrylamide. Further, Rhodococcus rhodochrous M33 strain has Rhodococcus rhodochrous M33 strain, which is a medium containing a high-priced nutrient such as yeast extract, meat extract, or vitamin, which is required for sufficient growth, such as Rhodococcus rhodochrous J1 strain. The advantages of simple and cheap synthetic media can be cultivated. Furthermore, for convenience, the present invention is deposited in the Institute of

Biochemistry and Physiology of Microorganisms of The Russian Academy of Science (IBFM) (December 6, 1993), Rhodococcus rhodochrous M33 -26- (24) (24) 1327596. When a microorganism or a transformant or the like is used, the cells themselves can be used, and the cells can be treated with a living cell body or a solvent such as acetone or toluene, or freeze-dried to increase the permeability of the compound. Depending on the situation, it can also be an enzyme-containing substance such as a bacterial cell or a bacterial extract. The method for producing a bacterial cell-containing material containing the enzyme, for example, the solid and liquid obtained by first separating the culture, the obtained wet cells can be suspended in a buffer solution such as a phosphate buffer or a trihydrochloric acid buffer, and then ultrasonic waves are used. Treatment, high-pressure crushing treatment or pulverization treatment using glass beads, or appropriate combination of cell wall lysozyme such as lysozyme or protease for cell disruption treatment, and extraction of the enzyme from the bacteria to obtain a crude liquid containing nitrilase . The crude enzyme-containing solution can be further purified by a separation or purification method of a conventional protein 'enzyme, if necessary. For example, in a crude enzyme-containing solution, an organic solvent such as acetone or ethanol is added to separate and precipitate, or thiamine is added thereto for salting out, and a portion containing a nitrilase is separated by precipitation in an aqueous solution. Further, purification can be carried out by an appropriate combination of anion exchange, cation exchange, colloidal filtration, affinity chromatography using an antibody or a chelating agent, or the like. Of course, the enzyme or the bacteria, the bacterial body treatment containing the enzyme, etc., can be filled in the column or solid on the carrier by a conventional method, especially in the case of the bacteria, can be embedded in the colloidal phase of the polypropylene guanamine. In the molecule, β is suspended in an aqueous solution such as a buffer solution such as water or a phosphate buffer solution, and a nitrile compound is added thereto to carry out a reaction. The concentration of the bacterial or bacterial cell treated material used is 0.01% by weight to 20% by weight, preferably 1:1% by weight to 10% by weight. The upper limit of the reaction temperature is preferably 90 ° C, more preferably 85 ° C, most preferably 70 ° C, and the lower limit of the reaction temperature -28 - (25) (25) 1327596 degrees is, for example, 1. (:, preferably 4 ° C, more desirably 10 ° C, the reaction pH is, for example, 5 to 〇, preferably 6 to 8, and the reaction time is, for example, 1 minute to 72 hours. Then, by a nitrile compound The guanamine compound can be accumulated to a high concentration by dripping in. The method for recovering the guanamine compound from the reaction solution, for example, after filtration or centrifugation of the bacterial cell or the bacterial cell treatment, is taken out and then recovered by crystallization or the like.

EXAMPLES The present invention is illustrated in more detail by the following examples, but these examples do not limit the scope of the invention. Example 1: Bacterial isolation A small amount of soil (about lg) sampled by a hot spring in Saitama Prefecture was placed in a test tube containing 5 ml of physiological saline, and cultured in a shock culture machine at 65 °C for 3 days. A part of the culture solution (〇. 5 ml) was taken out, and 1.0% by weight of glucose, 0.5% by weight of polyprotein gland, and 0.3% by weight of yeast extract were added to the medium (pH 7.0). Repeated shock culture for 65t. A part of the medium (0.1 mL) thus obtained was applied to a plate medium containing the aforementioned medium components, and cultured at 65 ° C for another 2 days to form colonies, and the microorganisms were isolated. The isolated microorganism was seeded in a liquid medium supplemented with 0.1% by weight of n-valeronitrile in the same composition medium as above, and cultured at 65 t for 24 hours to obtain a culture solution of a microorganism having a high nitrile group decomposing power. To 1 ml of the culture solution, 9 ml of a -29-(26) 1327596 1.1 wt% acrylonitrile solution (0.05 M-phosphate buffer, ρΗ7·7 ) was added at a reaction temperature of 27. The reaction was carried out. After 1 minute, the reaction was stopped after adding 1 ml of IN HC1. A part of the reaction solution was analyzed by HPLC, and the microorganism having nitrilase activity was selected by assaying the presence or absence of acrylamide. A microorganism having water and activity capable of converting a nitrile compound into a guanamine compound in this manner was obtained as a strain of Geobacillus thermoglucosidasius Q-6.

(Liquid chromatography analysis conditions) Main body: HITACHI D-7000 (manufactured by Hitachi, Ltd.) Pipe column; Inertsil ODS-3 (manufactured by GL Scientific Co., Ltd.) Length; 200 mm

Column temperature; 3 5 °C flow rate; 1 ml/mi η sample injection amount; 1 0 μ 1

Solution; O.lwt% phosphoric acid aqueous solution Example 2: Geobacillus thermoglucosidasius Q-6 strain Determination of nitrilase activity and its temperature dependence will contain 1% by weight of 0.2% by weight of glycerol, 〇2 % by weight of trisodium citrate dihydrate, 0.1% by weight of potassium dihydrogen phosphate, 1% by weight of dipotassium hydrogen phosphate '0.1% by weight of polyprotein gland, 1% by weight of yeast extract, 0.1% by weight % sodium chloride, 1% by weight of n-valeronitrile, 0.02% by weight of magnesium sulfate heptahydrate '〇〇〇3 wt% of ferric sulfate-30- (27) 1327596

(Π) a 500 ml triangular conical flask of a bactericidal medium (ρΗ7.0) of heptahydrate and 0.0002% cobalt chloride hexahydrate, inoculated with a culture solution of Geobacillus thermoglucosidasius Q-6 strain cultured in the same medium. 1ml. The cells were cultured at 65 ° C for one day, and shake-cultured at 200 str 〇 ke / min to obtain a bacterial culture solution. 300 ml of the culture solution of the Geobacillus thermoglucosidasius Q-6 strain was collected by centrifugation (10 μg, 15 minutes), and the cells were washed with 0.05 M phosphate buffer (pH 7.5) and suspended. 50 ml of the same buffer. With respect to the thus prepared bacterial cell suspension, the hydration activity of the nitrile compound into a guanamine compound was measured at the reaction temperature disclosed in Table 1. The unit of enzyme activity is defined as the activity of converting the acrylonitrile group of ίμιηοΐ into acrylamide in 1 minute as 1 unit (hereinafter referred to as U), and the nitrilase per unit wet cell at 27 ° C The activity (U/mg) was 9.37 U/mg. And 5 U/ml of a bacterial suspension containing 0.5% by weight of acrylonitrile was prepared at 10 ° C, and the bacterial suspension was used at 30 ° C '40 ° C, 50 ° C, 60 ° C, 70 The same nitrilase activity was determined under the conditions of °C, as shown in Table 1. As a result, the optimum temperature at the time of using the cell reaction was 6 (TC or so, and showed an extremely high activity in a high temperature region. -31 - (28) 1327596 Table 1 Reaction temperature (°c) Nitrilase activity (U/ml) 10 5.0 30 19.2 40 39.4 50 49.2 60 50.6 70 42.4

Example 3: Thermal stability of nitrilase in cells of Geobacillus thermoglucosidasius Q-6 strain The thermal stability of nitrilase activity in the cells of Geobacillus thermoglucosidasius Q-6 strain was investigated, and the culture method of Example 2 was examined. The obtained cells were suspended in distilled water to a temperature of 1 〇U/ml, and subjected to a predetermined temperature incubation for 30 minutes to measure the residual viability. Into 5 ml of a 1 wt% acrylonitrile solution (0.05 M potassium phosphate buffer, pH 7.5), 0.5 ml of the sterilized bacterial body solution was added, and the reaction was started at 27 ° C with stirring. After 5 minutes, 100 μL of 1N hydrochloric acid was added to stop the reaction. The activity after the preservation treatment with respect to the activity before the preservation treatment was calculated, and the conversion obtained based on the activity before storage treatment (1〇〇) was as shown in Table 2. From this result, it was found that the nitrilase activity in the cells of the Geobacillus thermoglucosidasius Q-6 strain can be maintained at a high temperature, and can maintain an activity of 80% or more at a high temperature of 70 t at 80 ° C. It can maintain more than 30% activity at high temperature. -32- (29)1327596 Table 2 Treatment temperature (t) Activity (U/ml) Residual activity (%) 30 5 100 40 4.8 96 50 4.5 90 60 4.4 88 70 4.2 84 80 1.6 32

Example 4: Reaction to various nitrile compounds The nitrilase activities of the respective guanamine compounds converted to the respective nitrile compounds described in Table 3 below were investigated. 1 ml of a specific suspension was added to 9 ml of a 1.1% nitrile solution (0.05 M potassium phosphate buffer, ρΗ7·5), and the reaction was started at a reaction temperature of 30 °C. After 10 minutes, 1 ml of IN HC1 was added to stop the reaction. The results showed that all had nitrile hydrolase activity. Table 3 provides the tested nitrile compound adiponitrile n-butyronitrile acetonitrile acetonitrile isopropyl nitrile benzonitrile n-valeronitrile-33- (30) 1327596 Example 5 The nitrilase from Geobacillus thermoglucosidasius Q-6 strain described below The amine-based acid sequence and the base sequence of the α subunit (ORF2), the cold subunit (ORF1), and the nitrilase activating factor (ORF3) are described below in the flow of the present invention. The φ cells obtained from the cultivation of Geobacillus thermoglucosidasius Q-6 strain were pulverized, precipitated with ammonium sulfate, and subjected to anion exchange column chromatography, DEAE column, hydroxyapatite column for dialysis by colloidal filtration chromatography. The nitrilase is purified. Defining an amino acid sequence of the α subunit of the purified nitrilase and about 30 residues at the N-terminus of the yS subunit, and using the amino acid codon based on the genus of the genus to produce a gene amplification oligo The nucleotide degrading primer is subjected to degenerate PCR using the chromosomal DNA extracted from the cell as a mold to obtain an amplified DNA fragment. After the amplified DNA fragment is selected, the base sequence of the % insert is defined. The amino acid sequence* predicted by the base sequence is compared with the N-terminal amino acid sequence of the nitrilase α subunit and the subunit purified by the Geobacillus thermoglucosidasius Q-6 strain. The sequence that was selected was the one encoding the nitrilase. As a result, it was confirmed that in the Geobacillus thermoglucosidasius Q-6 strain, the nitrilase gene upstream of the 5'-end side was adjacent to each other in the order of 9 subunits and α subunits. An oligonucleotide-degrading primer for gene amplification is produced by a well-identified sequence of a downstream gene of various nitrilase α subunits, which is extracted by the strain -34-(31) (31)1327596 The chromosomal DNA is subjected to PCR degradation as a mold to obtain an amplified DNA fragment. The resulting increase in the alpha subunit portion of the cell is determined by colonization to define its base sequence. The nitrilase α subunit and the quinone subunit of the Geobacillus thermoglucosidasius Q-6 strain obtained above were introduced into an appropriate expression vector. Examples of the use of the constructed plastids to transform the appropriate host strain into a host include, for example, the genus Rhodococcus, the genus Corynebacterium, and Escherichia coli. Among them, a host having no guanaminease is preferred. Further, the cells obtained by culturing the obtained transformant were contacted with acrylonitrile in an aqueous medium to form acrylamide, and the production efficiency and nitrilase activity were compared. Next, +, as the probe, the DNA fragment obtained as described above was subjected to colony hybridization to select a peripheral gene containing the nitrilase α subunit of Geobacillus thermoglucosidasius Q-6 strain and the downstream gene of the subunit. The downstream gene was expressed simultaneously with the nitrilase α subunit and the subunit, and compared with the nitrilase activity. As a result, a downstream gene line and a gene related to a significant increase in nitrilase activity were found. Example 5: Physical and chemical properties of purified enzyme [Step 1] Purification of nitrilase enzyme The Geobacillus thermoglucosidasius Q-6 strain was cultured and supplied to various columns to purify the nitrilase active fraction. The method for measuring the active portion of the nitrilase in the chromatography is carried out as follows. 1 part by weight of acrylonitrile was added to each fraction of the solution-35-(32) 1327596 diluted with HEPES buffer (100 mM, pH 7.2), and reacted at 27 ° C for 1 minute. The reaction was stopped by adding IN HC1 to a 1% liquid amount % reaction solution, and the resulting acrylamide concentration was measured by the above HPLC analysis method.

To purify the nitrilase enzyme from Geobacillus thermoglucosidasius Q-6 strain, first in V/F medium containing 0.1% by weight of n-valeronitrile (0.2% by weight of glycerol, 0.2% by weight of trisodium citrate dihydrate, 0.1) % by weight of potassium dihydrogen phosphate, 0.1% by weight of dipotassium hydrogen phosphate, 0.1% by weight of polyprotein gland, 0.1% by weight of yeast extract, 0.1% by weight of sodium chloride, 0.1% by weight of n-valeronitrile, 0.02% by weight of magnesium sulfate heptahydrate, 0.003% by weight of iron (II) sulfate heptahydrate, 0.0002% by weight of cobalt chloride hexahydrate, and Geobacillus thermoglucosidasius Q-6 strain, cultured at 65 ° C 24 hour. The culture system used a 96-well 2 ml deep-bottomed Petri dish (COSTAR). After the completion of the culture, the cells were collected by centrifugation at 8000 g for 10 minutes, and 3 g of the obtained wet cells were resuspended in 20 ml of HEPES buffer (100 mM 'pH 7.2). After the cells were cooled and crushed using an ultrasonic breaker, ammonium sulfate (30% saturated concentration) was added to the cell disrupted solution, and the mixture was gently stirred at 4 ° C for 30 minutes, and centrifuged at 20,000 g for 10 minutes to obtain a clear liquid. Add the ammonium sulfate (70% saturated concentration) to the centrifugation solution and gently stir at 4 ° C for 30 minutes, then carry out 2000 〇g, and centrifuge for 10 minutes to obtain a precipitate in 9 ml of HEPES buffer (100 mM, ρΗ7· 2) Re-dissolution was carried out, and 1 L of the same solution was subjected to dialysis at 4 ° C for 24 hours, and provided by anion exchange chromatography (Amersham Biochemical Science Co.; HiTrap DEAE FF (column volume 5 mL x 5 roots)). Extension -36- (33) 1327596 The developing solution was flooded with HEPES buffer (100 mM, pH 7.2) and the concentration of potassium chloride was linearly increased from 0.0 M to 0.5 M to obtain a nitrilase-containing portion. This fraction was supplied to an apatite column chromatography (manufactured by BIO-RAD Co., Ltd.; CHT2-I (column volume: 2 mL)). 〇.〇1Μ potassium phosphate aqueous solution (

pH 7.2) As an extension liquid, the potassium phosphate concentration was linearly increased from 0.01 M to 0.3 M to be dissolved, and a fraction containing a nitril hydrolase was obtained. This fraction was supplied to a colloidal filtration chromatography (Amersham Biochemical Science Co.; Superdex 200 HR 10/30) using a 0.05 M aqueous sodium phosphate solution (pH 7_2) containing 0.15 M NaCl as an extension liquid to obtain a nitrilase active fraction. The following examples were carried out using the nitrilase active portion of the thus obtained colloidal filtration chromatography. [Step 2] Reaction temperature dependence of purified nitrilase for nitrilase active partial solution (3.2 mg/mL, 0.05 M phosphate buffer (ρΗ7·5)) from Geobacillus thermoglucosidasius Q-6 strain, as shown in the table The nitrilase activity of converting the nitrile compound into a guanamine compound was measured at the reaction temperature shown in 4. A solution of the nitrile hydrolase active portion was added to 1 ml of a 0.5 wt% acrylonitrile solution (0.05 M potassium phosphate buffer, pH 7.5), and the reaction was started with stirring at each temperature. After 2 minutes, the reaction was stopped after the addition of ΙΟΟμί of 1N hydrochloric acid. The activity unit of the enzyme is defined as 1 unit (hereinafter referred to as U) for converting acrylonitrile to acrylamide in 1 minute, and the hydration activity (U/mg) per unit weight of the enzyme is as shown in Table 4. Show. From the results, it was found that the activity of the nitrilase in the nitrilase-37-(34) 1327596 fraction purified by Geobacillus thermoglucosidasius Q-6 strain before the high temperature of 60 degrees, as the reaction temperature increased rise. The optimum temperature is about 60 °C in the same manner as when the cells are reacted, and a relatively high nitrile hydrolase activity can be exhibited at a high temperature of 7 degrees. Table 4 Reaction temperature (°c) Activity (U / m g ) 20 2 10.4 27 550.7 40 1135.3 50 2228.8 60 2823.3 70 278 1.1

[Step 3] Thermal stability of purified nitrilase When the thermal stability of the nitrilase purified by Geobacillus thermoglucosidasius Q-6 strain was investigated, the nitrilase active partial solution (3_2 mg/ml, 0.05 M phosphate buffer) was used. The liquid (ρΗ7·5)) was subjected to heat treatment at a temperature of 30 ° C and its residual activity was measured. To the 0.5% by weight acrylonitrile solution (〇.〇5M potassium phosphate buffer * pH 7.5) of ImL, 5 μl of the heat-treated nitrilase solution was added, and the reaction was started while stirring at 27 °C. After 2 minutes, the reaction was stopped by the addition of IN HC1 of ι〇〇μΐ. The activity (residuality) after storage for the activity before the storage treatment was calculated, and the activity before the storage treatment was used as a reference (100), and the conversion was as shown in Table 5-38-(35) 1327596. From the results, it was found that the nitrilase activity of the active portion of the nitrilase active fraction purified by G eobaci 11 usthermog 1 ucosidasius Q-6 strain can be maintained at a high temperature, and is also at a high temperature of 6 (TC). It can maintain more than 60% activity, and can maintain more than 35% activity at 70 ° C. Table 5 Treatment temperature (°c) Residual activity (%) 20 89.2 27 85.6 40 82.3 50 78.9 60 66.9 70 38.8

[Step 4] Acrylonitrile concentration dependence and concentration tolerance of purified nitrilase For investigation of dependence on acrylonitrile concentration and tolerance of nitrilase purified by Geobacillus thermoglucosidasius Q-6 strain, 4μ1 nitrilase active partial solution (3.2mg/ml, 0.05M phosphate buffer (pH7.5)) was added to various weight% 5ml acrylonitrile solution (0.05M potassium phosphate buffer, ρΗ7·5 ) at 27° The reaction was started by stirring at C. After 5 minutes, 10 minutes, 20 minutes, and 40 minutes, 1 ml was taken out, and the reaction was stopped by adding HCμΙ of IN HC1. The concentration of acrylamide was -33- (36) 1327596, which was quantified by HPLC, as shown in Table 6. Show. From the results, it was found that the nitrilase activity of the aqueous solution of the active hydrolyzase of the purified protein of Geobacillus thermoglucosidasius Q-6 was stable at a high acrylonitrile concentration. Even in the 6% high concentration acrylonitrile solution, even if it was reacted for 40 minutes, no decrease in activity was observed as compared with the lower acrylonitrile concentration, and conversely, as the matrix concentration was increased. Table 6 Acrylonitrile concentration at the start of the reaction (% by weight) The concentration of acrylamide after 5 minutes (% by weight) The concentration of acrylamide after 10 minutes ί 量 %) The concentration of acrylamide after 20 minutes f ) Acrylamide concentration after 40 minutes (% by weight) 0.5% 0.018 0.032 0.052 0.087 2.0% 0.023 0.042 0.070 0.107 4.0% 0.026 0.046 0.077 0.137 6.0% 0.030 0.051 0.082 0.169

[Step 5] The concentration of acrylamide concentration of the nitrilase purified by Geobacillus thermoglucosidasius Q-6 strain is resistant to the production of acrylamide by the nitrilase purified by Geobacillus thermoglucosidasius Q-6 strain. For investigation, a 1 μl nitrilase active partial solution (3.2 mg/ml, 0.05 guanidine phosphate buffer (pH 7.5)) was added to 1 ml of a solution containing 0.5% by weight of acrylonitrile and 35% by weight of acrylamide ( 0.05M potassium phosphate buffer, ρΗ7·5), stirred at 27 ° C for 10 minutes, the concentration of acrylamide in the liquid after the reaction was quantified by HPLC, all acrylonitrile was turned -40- (37) 1327596 Change to acrylamide. From the results, it was found that the nitrilase activity in the aqueous solution purified by the Geobacillus thermoglucosidasius Q-6 strain remained active at a concentration of 35% of a high concentration of acrylamide. Example 6 - Gene selection of nitrilase cold subunit, alpha subunit and ORF3 portion from Geobacillus thermoglucosidasius Q-6 strain

Colonization [Step 1] Confirmation of nitrilase from Geobacillus thermoglucosidasius Q-6 strain and N-terminal amino acid sequence definition The nitrilase active partial solution of colloidal filtration chromatography obtained in Example 5 was supplied under reducing conditions. The protein staining of Coomassie brilliant blue (CBB) was carried out after electrophoresis in reduced SDS-polyacrylamide electrophoresis, and it was confirmed to be two main fragments having a molecular weight of about 25 K Dalton and about 28 K Dalton after decolorization. The two main purified proteins were transferred to a PVDF membrane (MILLIPORE) using a Blotting apparatus (BIO-RAD), and after CBB staining, the two fragments of the adsorption target were cut by a PVDF membrane. Out. Next, two types of protein N-terminal amino acid sequences were interpreted using the fully automated protein primary structure analyzer PPSQ-23A (Shimadzu Corporation). As a result, the N-terminal amino acid sequence of the protein having a molecular weight of 25 K Dalton is the sequence described in SEQ ID NO: 23 of the Sequence Listing, and the N-terminal amino acid sequence of the protein having a molecular weight of 28 K Darton is the sequence number 24 of the Sequence Listing. The preface of the record! J. -41 - (38) 1327596 Compared with the amino acid sequence of the known nitrilase, the 25K Dalton polypeptide chain is the nitrilase α subunit, and the 28K Dalton polypeptide chain is the nitrilase. The /3 subunit, although low, still shows identity and is the one that encodes the protein. [Step 2] Synthesis of an oligonucleotide primer corresponding to the amino terminal amino acid sequence

The following 12 types of degenerate PCR oligonucleotide primers were synthesized using the codons of the genus of the genus of the two types of proteins as described above. The primer 1 (aF1) described in the sequence number 5 of the sequence table, the primer 2 ( a F 2 ) described in the sequence number 6 of the sequence table, and the primer 3 ( a F 3 ) ' sequence described in the sequence number 7 of the sequence table. The primer 4 ( a R1 ) described in the sequence number 8 of the list, the primer 5 ( a R2 ) described in the sequence number 9 of the sequence table, and the primer 6 ( α R3 ) described in the sequence number 1 of the sequence table, the sequence table The primer 7 (cold F 1 ) described in the sequence number 1 1 , the primer 8 ( /3 F 2 ) described in the sequence number 1 2 of the sequence table, and the primer 9 (y?) described in the sequence number 13 of the sequence listing. F3), the primer number 1 〇 ( yg R 1 ) described in the sequence number of the sequence table, and the sequence number of the sequence list of the primer 1 1 ( Θ R 2 )' listed in the sequence number of the sequence table 1 6 The introduced primer 12 (泠R3). And y represents c or t, r represents a or g 'm represents a or c, k represents g or t, s represents c or g, w represents a or t, d represents a, g or t, and n represents a , c, g or t. At the same time, taking into account the coding 〇: subunit and A subunit genes at the chromosomal location to make a primer 0 - 42 · (39) (39)

1327596

[Step 3] Chromosome DNA extraction and degradation PCR of Geobacillus thermoglucosidasius Q The Geobacillus thermoglucosidasius Q-6 strain was cultured and recovered in the same manner as in Example 2 using the QIAGEN Gong Genomic-tip System (500/G) kit. The DNA is extracted from the body. PCR was carried out using O. lpg genomic DNA of Geobacillus thermoglucosidasius Q-6 strain dissolved in TE solution as a prayer type. The degenerate PCR was carried out by combining primers 1 to 6' of sequence numbers 5 to 10 of the sequence listing table and 36 types of primers 7 to 12 of sequence numbers 1 1 to 16 of the sequence listing. Two kinds of primers of lOOpmol The growth of the degraded PCR reaction fragment was carried out by using 5 U of Takara DNA polymerase of Takara Co., Ltd. and a total amount of 1 〇〇μ1 of the reaction solution. The reaction conditions are as follows. 96 ° C, 3 minutes of heat, 9 6 ° C, 30 seconds of thermal denaturation, 4 2 ° C, 30 seconds of calcination at 72 ° C for 1 minute and 30 seconds of elongation reaction, repeated 35 After that, the elongation reaction was carried out at 72 ° C for 5 minutes, and then kept cold at 4 ° C. Each PCR product was provided by 1% by weight of 5 agarose electrophoresis to confirm the DNA amplification, only the serial number of the combined sequence table. The primer 5 ( a R2 ) of 9 and the arch (F 1 ) described in the sequence number 1 1 of the sequence listing, and the primer a R2 described in the sequence number 9 of the combination sequence table are listed in the sequence number 12 of the sequence listing. Primer 8 (When the PCR reaction was carried out under cold, it was confirmed that the color spectrum of 6 strains of the DNA fragment of about 700 bp was degraded, and the buffer DNA was denatured and recycled. In the case, the increase of the reporter 7 5 ( F2 ) -43- (40) 1327596 [Step 4] Decoding of Degenerate PCR Products and Interpretation of Base Sequences of Amplified DNA Fragments

The amplified DNA fragment was excised from the colloid and extracted using QIAquick Gel . Extraction Kit (QIAGEN), PGEM-T

Vector (proniega) uses T4 DNA ligase (Takara) to connect. The result of the PCR reaction obtained by Ex Taq was 3, and the A at the φ end was a property of using 1 base. After the ligation reaction, the Escherichia coli JM109 strain was transformed into LB agar medium (50 pg/ml ampicillin '〇·5 wt% Bato-yeast extract, 1 wt% Bacto-trypsin' 0.5% by weight NaCl, 2.0% by weight of Bacto Agar (ρΗ7·5 )) was cultured overnight at 37 ° C, and the transformant was selected with ansicillin. The plastid DNA was extracted from the transformant cultured in LB medium containing ancillin according to the usual method, and the insert sequence of about 700 bp was used as a primer to interpret the base sequence using the sequence of the SP6 and T7 promoters on the vector. As a result, it was confirmed that the amplified DNA fragment had an open reading frame of 681 bp (hereinafter referred to as ORF1). The translation stop codon of ORF1 is 13 bp between the translation start codon ATG of the following open reading frame (hereinafter referred to as ORF2). The N-terminal 25 amino acid sequence deduced from the ORF1 thiol sequence is identical to the 25 amino acid sequence on the N-terminal side of the purified 28 K Dalton poly-peptide chain, corresponding to the sequence listing. Sequence No. 1 to No. 25 of the amino acid sequence described in SEQ ID NO: 2. The amino acid sequence of 0RF1 and the amino-44-(41) 1327596 acid sequence of the subunit of the known nitrilase show low identity but still have the same identity and indicate that the protein is encoded.

Geobacillus thermoglucosidasius Q-6 strain of hydrolytic enzyme cold

The subunit is an amino acid sequence encoding a 226 amino acid and having a homologous protein in the existing data, and the order of identity is 'the nitrilase/5 of the Klebsiela genus MC12609 strain. The subunit is 43%. The degree of similarity, the nitrilase point subunit of the genus Agrobacterium is 42% identical, and the nitrilase of the Rhodopseudomonas genus CGA3095 has no subunit of 40%. Very low degree of identity. Further, the degree of identity with the amino acid from the Bacillus protein belonging to the genus Geobacillus is nitrilase with the thermophilic strain Bacillus BR449; 3 subunits are 35.0%, and the thermophilic bacteria Bacillus smithii SC-] The nitrilase/3 subunit of the 05-1 strain was extremely low at 34.5%. On the other hand, the nitrilase yS subunit of the thermophilic Bacillus BR449 strain was as high as 85.6 % identical to the nitrilase subunit of the thermophilic Bacillu smithii SC-J05-1 strain. [Step 5] The gene of Geobacillus thermoglucosidasius Q-6 strain nitrilase α subunit is selected as the peripheral gene and α subunit of the gene of the cytohydrolase subunit of Geobacillus thermoglucosidasius Q-6 strain obtained above. Part of the gene is subjected to degenerate PCR. The following two types of degenerate PCR oligonucleotide primers were made with reference to a gene located downstream of the known nitrilase α subunit. The sequence number of the sequence listing is 1 7 and the primer 1 3 ( pR 1 ) -45- (42) 1327596 and the sequence number of the sequence listing 1 8 are described as the primer 1 4 (PR2). Further, in the nitrilase subunit of the Geobacillus thermoglucosidasius Q-6 strain which interprets the base sequence, the following two types of oligonucleotide primers for PCR amplification are prepared. The sequence number of the sequence table is 19, and the primer 16 (Q6abF1) is described in the primer number 15 (Q6AposF) and the sequence number 20 of the sequence table. 0. lpg

The chromosomal DNA of the thermoglucosidasius Q-6 strain was subjected to denaturing PCR as a mold. The degenerate PCR reaction is carried out at a calcination temperature of 5 (TC, in the combination of the primers 13 and 14 described in SEQ ID NO: 17' 18 in the sequence listing table, and four types of primers 15' 16 described in SEQ ID NO: 18' 19 in the Sequence Listing. As a result, when the primer 13 (pR 1 ) described in the SEQ ID NO: 17 of the sequence listing table and the primer 15 (Q6AposF) described in SEQ ID NO: 18 of the Sequence Listing were subjected to a PCR reaction, it was confirmed that an amplified DNA of about 0.8 kb was present. Further, when the primer 13 (pR 1 ) described in the SEQ ID NO: 16 of the sequence listing is subjected to a PCR reaction with the primer 16 (Q6abF1) described in SEQ ID NO: 19 of the Sequence Listing, it is confirmed that there is about i.5k of amplified DNA. In addition, when the PCR reaction is carried out in other combinations, the presence of the amplified DNA product cannot be confirmed. The primer 1 3 ( pR 1 ) described in SEQ ID NO: 16 of the sequence listing and the primer shown in SEQ ID NO: 19 of the Sequence Listing are included. 15 (Q6AposF) The 〇.8kb DNA fragment amplified by the degenerate PCR reaction was excised from agarose gel, and the DNA was extracted according to the usual method and inserted into pGEM-TVector (

Promega, Inc., transformed E. coli JM109 strain, and selected recombinants by 50 pg/ml of ancillin. In LB-46- (43) 1327596 containing ancillin

The transformant was cultured in the medium, and the plastid DNA was extracted according to the usual method, and the base sequence of the insert portion of about 0.8 kb was read. As a result, an open reading frame of 618 bp (hereinafter referred to as ORF2) was confirmed. The N-terminal side 29 amino acid sequence deduced from the base sequence of ORF2 is identical to the 29 amino acid sequence on the N-terminal side of the purified 25 K Dalton poly-peptide chain, equivalent to the sequence. The sequence of No. 1 to No. 29 of the amino acid sequence described in SEQ ID NO: 1 in the list. The amino acid sequence of ORF2 and the amino acid sequence of the known nitrile hydrolase alpha subunit, although low, still exhibit identity, are those encoding the protein.

The nitrilase α subunit of Geobacillus thermoglucosidasius Q-6 strain is an amino acid sequence encoding 205 amino acid, which has high homology with the existing data, and the degree of homology is the same as that of thermophilic bacteria.

The nitrilase/3 subunit of Bacillus BR4 49 strain was 66.3% lower, and the nitrilase/5 subunit of the thermophilic Bacillus smithii SC-J05-1 strain was 63.9% lower. On the other hand, the nitrilase/3 subunit of the thermophilic Bacillus BR449 strain and the nitrilase/3 subunit of the thermophilic Bacillus smithii SC-J05-1 strain have a high homology of 88.88%. According to the above, it was confirmed that the Geobacillus thermoglucosidasius Q-6 strain was flown from the 5' end side, and the gene encoding the 28K Dalton nitrilase yS subunit and the gene encoding the 25K Dalton nitrilase α subunit were used. The order exists adjacently. [Step 6] The nitrilase α subunit of the Geobacillus thermoglucosidasius Q-6 strain and the -48-(44) 1327596 of the PET28a (+) vector of the 泠 subunit portion are inserted in the above-described interpreted base sequence, and the following The oligonucleotides of the two nitrilase α subunits and the yS subunit fraction were amplified by PCR. The primer 18 (Q6ab-FbT) described in SEQ ID NO: 21 of the Sequence Listing and the primer 18 (Q6ABall-Rl-BglII-Τ) described in SEQ ID NO: 22 of the Sequence Listing. The primer 17 described in SEQ ID NO: 21 of the Sequence Listing is designed to be a restriction enzyme site N d e I, G e 〇 b a c i 11 u s

The translational start codon of the nitrilase/3 subunit of the thermoglucosidasius Q-6 strain. In the primer 18 described in SEQ ID NO: 2 2 of the Sequence Listing, the translational stop codon of the nitrilase α subunit of the GeobaciUus thermoglucosidasius Q-6 strain is directly introduced into the restriction enzyme Bg III site. The chromosomal DNA of the Geobacillus thermoglucosidasius Q-6 strain was used as a mold, and the PCR reaction was carried out using the primers 17 and 18 described in the sequence numbers 21 and 22 of each sequence of 100 pmol. Using Ex Taq DNA polymerase, the whole amount was ΙΟΟμΙ at 96 ° C, heat denaturation for 3 minutes, then heat denaturation at 96 ° C for 30 seconds, calcination at 60 ° C for 30 seconds, 72 ° C for 1 minute and 30 seconds. After 30 cycles of PCR under the conditions of the elongation reaction, an elongation reaction at 72 ° C for 5 minutes was carried out, followed by cooling. The solution after the PCR reaction was subjected to 1.5% by weight of agarose gel electrophoresis, and an increase of about 1.3 kb DNA fragment was recognized. The amplified DNA product was extracted by agarose gel and ligated to Promega's pGEM-Teasy Vector to transform Escherichia coli JM109 strain. The plastid DNA was extracted from the transformant, and the base sequence of the inserted portion was read, and it was confirmed that no increase was caused by the PCR amplification. -48- (45) (45) 1327596 Next, the plastid was digested with Ndel and EcoRl restriction enzymes and provided in 1.5% by weight agarose gel electrophoresis. The inserted DNA of about i.3Kb was excised from the agarose gel. Extracted according to the usual method. The performance vector uses Novagene's pET-28a ( + ) Vector. The DNA was digested with Ndel and EcoRl, and was supplied in 1% by weight agarose gel electrophoresis. A DNA fragment of about 5.3 Kb was extracted according to the usual method. These inserts were ligated with the vector according to the usual method, and 109 strains of Escherichia coli were transformed, and the plastid DN A was extracted with a transformant selected from kanamycin resistance, and the plastid into which the insert was introduced was selected. From the above, the expression plastid introduced by using the nitrilase subunit and the subunit portion of the Geobacillus thermoglucosidasius Q-6 strain as an insert was obtained. The completed plastid is called pET-28a ( + )-β a . [Step 7] A nitrilase peripheral gene derived from Geobacillus thermoglucosidasius Q-6 strain was obtained by colony hybridization. (1) Preparation of the fluorescent marker DIG probe The nitrilase G of the Geobacillus thermoglucosidasius Q-6 strain described in SEQ ID NO: 25 of the Sequence Listing: the DNA of the subunit portion was used as a mold, DIG-DNA produced by Roche The identification kit is made into a fluorescent marker probe. The manufacturing method is based on Roche's DIG manual. (2) Chromosome Southern Hybridization The chromosomal DNA of the Geobacillus thermoglucosidasius Q-6 strain prepared in the third step of Example 6 was digested with various restriction enzymes and provided in 1% by weight agarose gel electrophoresis. Will agarose gel -49- (46) 1327596

After the inside DNA was transcribed into the nylon membrane Hybond-N+ (AMERSHAM), the chromosome Southern hybridization method was carried out using the previously prepared fluorescent marker DIG probe. Each piece of DN A-transcribed 'fixed film was immersed in 1 〇ml of hybridization buffer (containing 1% by weight of skim milk powder, 0.1% by weight of N-lauroyl sarcosine, 0.02% by weight of SDS, 50% by weight 5xSSC of methotrexate was pre-hybridized at 42 °C for 2 hours. 100 ng of the fluorescent labeling probe prepared in the same manner as described above was subjected to boiling and quenching treatment at 95 ° C for 1 minute, and then thermally denatured, and then added to the pre-hybridization buffer at 42 ° C. One night of hybridization. The hybridized membrane was washed twice with 150 ml of 2xSSC containing 0.1% by weight of SDS at room temperature. This was followed by heating at 65 ° C and washing twice with 150 ml of lxSSC containing 0.1% by weight of SDS at room temperature for 5 minutes. Continue to wash with 100 ml of maleic acid buffer (0.1 M maleic acid, 0.15 M NaCl, NaOH adjusted pH 7.5) for 5 minutes, then in a 50 ml blocking solution (locking solution containing 0.3% by weight of Tween20, 0.15) M NaCl and 1% by weight of skim milk powder in 0.1 M maleic acid buffer; ρΗ7·5) were blocked at room temperature for 30 minutes. Dilute with 20ml of blocking solution to hydroxydigoxigenin-AP at 75mU/ml, and perform antibody reaction at room temperature for 30 minutes, then in 100ml of washing buffer (containing 0.3% by weight of Tween20, 0·15M NaCl) The membrane was washed 5 times in 0.1 M maleic acid buffer; pH 7.5), and the unbound antibody was washed away. After 5 minutes of equilibration in 20 ml of assay buffer (0.1 M Tris-HCl, 0.1 M NaCl, pH 9.5), 34 μl of 100 mg/ml of NBT (nitroso blue) in 10 ml of assay buffer Bisazo-5-(47) 1327596) solution, prepared as a chromogenic substrate solution NBT/ diluted with 35 μl of 50 mg/ml BCIP (5-bromo-4-chloro-3-indolyl phosphate) solution BCIP, the membrane is completely immersed therein, and cultured at a constant temperature in the dark for about 1 minute to 16 hours. In constant temperature culture, do not move or shake the table to confirm its color. As a result, it was found that a downstream gene containing a nitrilase α subunit portion was found in a gene fragment of about 2.3 kb which was decomposed by the restriction enzyme Hind III.

(3) Target selection by colony hybridization (i) Production of plastid gene pool for colony hybridization Next, colony hybridization was carried out using the same fluorescent marker DIG probe. The chromosomal DNA of 10 g/g g of Geobacillus thermog 1 ucosidasius Q-6 strain was subjected to 1% by weight agarose gel electrophoresis, and a DNA fragment containing about 2.0 kb to 2.6 kb was excised from the agarose gel. The DNA fragment was extracted and purified in the same manner as described above. The obtained DNA fragment was introduced into the Hind III restriction enzyme site in the multicloning site of the pUC 118 plastid vector (manufactured by Takara Shuzo Co., Ltd.) using a DNA ligation kit (manufactured by Takara Shuzo Co., Ltd.). The pUC1 18 plastid vector DNA used for the ligation was purified by restriction enzyme Hind III, purified by phenol/chloroform treatment and ethanol precipitation, and further subjected to dephosphorylation at the 5' end using alkaline phosphatase (manufactured by Takara Shuzo Co., Ltd.). Thereafter, the phenol/chloroform treatment and ethanol precipitation were carried out again, and the mixture was subjected to agarose gel electrophoresis, and then extracted and re-purified by agarose gel. A large intestine rod-51 - (48) 1327596 strain JM109 was used by using a solution in which the chromosomal DNA of the fragmented Geobacillus thermoglucosidasius Q-6 strain of about 2.Okb to 2.6 kb was ligated to the Hind III restriction enzyme site of the pUC118 plastid vector. The transformation of the strain was carried out in an ancifluorin containing 5 〇pg/ml, 1 butyl 0 and 2% by weight of 乂-〇 & 1 (5-bromo-4-chloro-3-indole) LB-Agar medium (0.5% by weight of Bacto-yeast extract, 1% by weight of Bacto-trypsin, 0.5% by weight of NaCl, 2.0% by weight of Agar) ; pH 7.5), via 37. (: (: after the next night of cultivation. The result) was obtained in most petri dishes, which showed 50 to 500 white colonies per dish.

For the plastid gene pool of these chromosomal DNAs, a previously modulated fluorescent marker DIG probe was used to perform colony hybridization, and a clone containing a downstream gene of the target nitrilase α subunit was selected. (ii) Obtaining the clone of interest by colony hybridization First, about 1000 clones of white colonies will appear, and the sterilized inoculation needles are used to draw the line on the LB agar medium, at this time, the LB agar medium which hybridizes the membrane, and The cells were cultured in the same manner as the LB vegetable medium, and cultured at 30 ° C overnight.

Next, the nylon membrane Hyb〇nd-N+ manufactured by AMERSHAM was placed on a petri dish where colonies were formed, and was slowly taken out using tweezers after 1 minute. The peeled film was immersed in a denaturation solution (0.5 M NaOH aqueous solution containing 1.5 M NaCl) with the bacterial adhering side facing up for 7 minutes, and immersed in a neutralization solution (containing 1.5 M NaCl and 1 mM EDTA·2Na). 0.5 M Tris-hydrochloric acid solution; ρ Η 7 · 2) for 3 minutes, and then immersed in the new neutralization solution for 3 minutes. Next, the solution was again washed once in a 2xSSC solution (1.87 g of NaCl and 4.41 g of sodium citrate in 1 L of lxSSC), and then the film was air-dried on a dried filter paper. Further, DNA fixation on the membrane was carried out by UV irradiation of 120 mJ/cm 2 -52-(49) (49) 1327596. (iii) by the detection of the DIG antibody and the separation of the target clone, the membrane which was fixed after the above treatment and immobilized with DNA was immersed in 10 ml of the hybridization buffer per tablet (containing 1% by weight of skim milk powder, 〇.1% by weight) N-lauric acid creatinine, 0.02% by weight of SDS, 50% by weight of 5xSSC of formamide, was pre-hybridized at 42 ° C for 2 hours. 100 ng of the fluorescent marker probe prepared in the same manner as above was boiled and quenched at 95 ° C for 10 minutes to be thermally denatured, and then added to the pre-hybridization buffer to carry out 42 ° C - late Hybridization. The hybridized membrane was washed twice with 150 ml of 2xSSC containing 0.1% by weight of SDS at room temperature. Next, it was heated at 65 ° C, and washed twice with 150 ml of lxSSC containing 0.1% by weight of SDS at room temperature for 5 minutes. Continue to wash with 100 ml of maleic acid buffer (0.1 M maleic acid, 0.15 M NaCl, and NaOH to ρΗ7·5) for 5 minutes, then in a 50 ml blocking solution (containing 0.3% by weight of Tween20). 0.15 M NaCl and 1% by weight of skim milk powder in 0.1 M maleic acid buffer; pH 7.5), and blocked for 30 minutes at room temperature. Dilute with 20ml of blocking solution to hydroxydigoxigenin-AP at 75rnU/inl, perform antibody reaction at room temperature for 30 minutes, and then wash in 100ml of washing buffer (containing 0.3% by weight of Tween20, 0·15M NaCl). The membrane was washed 5 times in 0.1 M maleic acid buffer; pH 7.5), and the unbound antibody was washed away. After 5 minutes of equilibration in 20 ml of assay buffer (0.1 M Tris-HCl, 0.1 M NaCl, pH 9.5), 34 μl of 100 mg/ml of NBT (nitrogen) in 10 ml of assay buffer Base blue bisazo chloride-53-(50) 1327596 compound) The chromogenic substrate solution NBT/BCIP diluted with 35 μl of 50 mg/ml BCIP solution, the membrane was completely immersed therein, and cultured in the dark for about 1 minute. Up to 16 hours. In constant temperature culture, do not move or shake the table to confirm its color. As a result, in the 1000 clones on the membrane, four positive signals were found, and the positive clones overlapping with the position were determined from the original culture dish. (4) Containing Geobacillus thermoglucosidasius Q-6 strain

Positive cloning analysis of the downstream gene of the nitrilase α subunit portion The positive clone was confirmed to be inoculated on a LB liquid medium containing ancillin by a culture dish, and subjected to shaking culture at 37 ° C for one night at 250 rpm. The cells were recovered by centrifugation, and the plastid DNA was extracted according to the usual method to restriction enzyme Hind III digestion, and then provided in 1.5% by weight of agarose gel electrophoresis. When the size of the inserted fragment was confirmed, it was found to be about The size of 2.3kb. Furthermore, the inserts were determined to contain the nitrilase alpha subunit portion of the Geobacillus thermoglucosidasius Q-6 strain by several forms of PCR and restriction enzyme digestion. The plasmid obtained from the above was named pUC118-Q6Hin2.3, and the entire base sequence of the insert was defined. The nitrilase of the Geobacillus thermoglucosidasius Q-6 strain and the restriction enzyme map and gene organization of the downstream gene group are shown in Fig. 1. As a result, the base sequence of 3 3 9 bp in the insert was confirmed to open reading frame (hereinafter referred to as 0RF3), and it was present in the same direction downstream of the 5' end side of the nitrilase α subunit (0RF2). The translation stop codon of ORF2 is 12 bp between the translation start codon of ORF3 and the translation start codon of ORF3 is -54-(51) (51)1327596. The translation start codon and the translation of the ORF at the downstream position are 145 bp between the start codons. . 0RF3 encodes a 1 12 amino acid which is extremely similar to the following proteins in the existing database. In the existing database, the ratio of the amino acid to the sequence with the same high identity was 31% for the P12K of the Bacillus strain BR449, and 31% for the Rhhococcus rhodochrous J1 strain with the Rhodococcus rhodochrous J1. The strain has a NhlE of 21% with Pseudomonas thermophilus (

Pseudonocardia thermophila) JCM3095 strain has a P16 of 23.2% [Step 8] Geobacillus thermoglucosidasius Q-6 strain nitrilase α subunit and yS subunit and downstream gene ORF3 expression plastid remodeling pET-28a (+ - The cold alpha plastid is decomposed by the restriction enzyme Hindlll and subjected to dephosphorization oxidation reaction, and dephosphorylated by extraction with phenol chloroform. This digested product was subjected to electrophoresis on a 1% by weight agarose gel, and a DNA fragment of about 6.1 kb was extracted according to a usual method. The DNA fragment contains the pET vector and the Hindlll restriction enzyme site of the 60th amino acid of the nitrilase/3 subunit and subunit of Geobacillus thermoglucosidasius Q-6 strain. Next, the pUC118-Q6Hin2.3 plastid was decomposed with the restriction enzyme Hindlll, and then subjected to electrophoresis on a 1 weight agarose gel to extract an insert DNA of about 2.3 kb. After the insert was first ligated to the extracted fragment, the E. coli JM109' was selected from the transformant selected by kanamycin to select a plastid containing the insert-55-(52) 1327596 into the DNA. The direction of insertion was confirmed by PCR, and the nitrilase α subunit and the cold subunit of the Geobacillus thermoglucosidasius Q-6 strain and the plastid of the downstream gene 0RF3 and the downstream region were obtained on the PET-28a (+) vector. Hereinafter, the plastid thus completed is referred to as pET-28a ( + ) — β a \ 2 . Example 7: Nitrile of Q6 strain using nitrilase gene promoter

The performance of the hydrolase gene on the rhodococcus rhodochrous M33 strain [Step 1] The nitrilase gene region of the M33 strain was selected to make the nitrilase of the Q6 strain appear in the M3 3 strain, and the M3 was selected. The nitrilase gene region of the 3 strain.

(1) Extraction of chromosomal DNA from M33 strain Rhodococcus rhodochrous M33 strain was cultured in YMPD medium (glucose 1.0% by weight, polyprotein gland 0.5% by weight, yeast extract 0.3% by weight, malt extract 0.3% by weight) In pH 7.2), QIAGEN's Genomic-tip System (500/G) kit was used to extract chromosomal DNA from the recovered cells. (2) Lamda phage database of M33 strain The chromosomal DNA of Rhodococcus rhodochrous M33 strain was decomposed with restriction enzyme Sau3 A 1 and then provided in 0.7% by weight agarose gel electrophoresis, from agarose gel. The fraction containing about 20 kb of the DN A fragment was excised and extracted and purified. The DNA of the chromosomal DNA was used in a DNA-binding kit Ver.l (manufactured by Takara Shuzo Co., Ltd.), and -56 - (53) 1327596

The carrier DN A in Lamda DASH II/BamHI Vector Kits (manufactured by S. tratagene) was combined, and a plastid database was prepared according to the manual of the same set. The Lamda phage library for these chromosomal DNAs was subjected to dot blot hybridization using the following fluorescent marker DIG probe (3) fluorescent marker DIG probe preparation

Using the primer NH5 described in SEQ ID NO: 32 of the Sequence Listing and the primer NH3 described in SEQ ID NO: 33 of the Sequence Listing, the nitrilase gene is about 530 bp by the chromosomal DNA of the Rhodococcus rhodochrous Μ 3 3 strain of Rhodococcus rhodochrousus. After the DNA was amplified by the PCR reaction, a DIG-DNA marker set made by Roche was used to prepare a fluorescent labeling probe. The manufacturing method is based on Roche's DIG manual. (4) Target clones were obtained by spotted southern hybridization. Next, dot blot hybridization was performed according to the DIG manual. First, about 1000 spots appearing were copied onto the membrane screen, and three phage spots showing positive signals were obtained using the above-mentioned adjusted fluorescent marker DIG probe. (5) Analysis of positive clones containing up-stream of nitrilase gene After separating and purifying each spot, phage DNA was adjusted using Wizard Lamda Prep (Promeg a). After the three phage DN A produced were cleaved with various restriction enzymes, the longest λ 311 containing the upper part of the nitrilase gene was selected. [Step 2] Construction of nitrilase expression vector (1) Production of M33KpnISacI/pGEM3zf -57- (54) 1327596

In order to re-select the nitrilase gene of M3 3 strain and its peripheral region to the plastid vector, the 3-1Kbp fragment was recovered by agarose electrophoresis by cleavage of 3ρηΙ and SacI; I 3 1 1 , pGEM3zf (+) (manufactured by Promega) was cleaved with ΚρηΙ and Sacl, and then mixed, and then ligated with T4 DNA ligase. This plastid was named M33KpnISacI/pGEM3zf, and the entire base sequence of the insert was defined, and the result is shown in SEQ ID NO: 4 in the Sequence Listing. Further, the restriction enzyme map and the gene composition are as shown in Fig. 2 . (2) Preparation of KSNH-pGEM3z The promoter and terminator of the nitrilase gene of M33 strain remained on M33KpnISacI/pGEM3zf, and the KSNH-pGEM3z vector was prepared according to the following procedure to insert Ndel and Bglll 'Clal positions between the vectors. And the expression gene can be inserted. The IN V 1 0 1 strain was transformed with M33KpnISacI/pGEM3zf to obtain a plastid that was not methylated by dam, and then cleaved with Clal and then partially decomposed with PstI, and the Pstl-Clal fragment containing the nitrilase construct gene was subtracted. The 6545 bp fragment was recovered by electrophoresis on agarose. On the other hand, using M33KpnISacI/pGEM3zf as a mold, PCR amplification was carried out using the primer M33-F05 described in SEQ ID NO: 26 of the Sequence Listing and the primer M33-R18 described in SEQ ID NO: 27, and the fragment of 707 bp was subjected to agarose electrophoresis. Recycling. M33-R18 is designed to limit the positions of enzymes Ndel, Bglll and Clal in the order of translation of the nitrilase A subunit protein of M33 strain. The PCR product was cut with PstI and Clal, and then subjected to agarose electrophoresis to recover a 400 bp fragment, which was mixed with the above-mentioned 6545 bp fragment, and ligated with -58-(55) 1327596 T4 DNA ligase to prepare KSNH- pGEM3z. (3) Preparation of M33KSNH-pHSG298 KSNH-pHSG298 was prepared according to the following method in order to replace the plastid with pHS298 (manufactured by Takara Shuzo Co., Ltd.) by KSNH-pGEM3z. The KSNH-pGEM3z was cut with ΚρηΙ and SacI, and the 3774 bp fragment was recovered by agarose electrophoresis. After the PHSG298 was cleaved with ΚρηΙ and SacI, the two were ligated with T4 DNA ligase to prepare a plastid of about 6.44 kb.

M33KSNH-pHSG298. (4) Introduction of the nitrilase/3, α subunit and downstream gene ORF3 of Geobacillus thermoglucosidasius Q-6 strain to M33KSNH_PHSG298 The alpha-maeF1 and sequence number 29 of the sequence listing are shown in the sequence number reef 28 of the sequence listing. The described orfl-atoRl was used as the primer, and pET-28a ( + ) -; Sal2 produced in Example 6 was used as the cast DNA, and the eyesight hydrolase cold 'α subunit and its downstream gene of Geobacillus thermoglucosidasius Q-6 strain were amplified. Approximately 1.5 kb fragment of 0RF3. After confirming that there is no error due to the increase, the Ndel restriction enzyme site designed on the translation start codon of the yS subunit and the translation stop codon of the ORF3 gene are directly decomposed by the restriction enzyme Bg III, and about 1.5 kb is recovered. DNA fragment. On the other hand, the plastid M33KSNH-PHSG298 of about 6.44 kb was decomposed by the restriction enzyme Ndel and then decomposed by Bglll to provide agarose electrophoresis, and the DNA fragment was recovered. The carrier region of the plastid M33KSNH-PHSG298 of about 6.44 kb, and the nitrilase derived from the Q-6 strain of about 1.5 kb; 8, the alpha subunit and its downstream gene 〇rF3 -59-(56) 1327596 The insertion region was connected to make a M7KSNH-pHSG298-Q6; Sa orf3 plastid of about 7.94 kb.

The M33KSNH-pHSG298-Q6 cold a orf3 plastid is about 2.64 kb from the vector portion, and the nitrilase promoter region of M33 in the inserted portion is about 3.8 kb, and the nitrilase α and the stone subunit of the Q-6 strain and The downstream gene 0RF3 is about 1.5 kb, and is composed of a total of about 5.3 kb. As a result, the translation start code ATG portion of the nitrilase/3 subunit of the M33 strain was substituted with the translation start position of the nitrilase/3 subunit of the Q-6 strain, and the nitrilase of the M33 strain was used to initiate the translation. The subunit is constructed into a gene unit represented by the nitrilase of the Q-6 strain and the downstream gene ORF3. (5) The nitrilase expression unit on the shuttle vector pRE_7 between Escherichia coli and Rhodococcus is constructed. The above-mentioned performance unit is designed to lead to approximately 5.9 kb of coliform and red with kanamycin resistance. The shuttle vector pRE-7 is used between the genus Cocci. Further, 'pRE-7 was prepared by the method of Zheng et al. (Plasmid 38 180-187, 1997) to prepare pBluescript (manufactured by Stratagne Co., Ltd.), pACYC177 (manufactured by New England Biolabs Co., Ltd.), and p〇TS. This is the same as pl03 described in TAKAI et al. (Infection and Imunity, Dec. 2000, p6848-6847), and is almost identical to p33701 in the same literature, especially for the production of pRE_7. The whole phase is asked, and p33701 can be isolated from the Rhodococcus equi ATCC33701 strain. First, the Ad5F--60- (57) 1327596 will be on the serial number 30 of the sequence listing.

SacKpnNotl (5,-pCCGGTACCGC-3 ') and Ad5R-SacKpnNotI (5'-pGGCCGCGGTACCGGAGCT-3,) on the sequence number 31 of the sequence listing (p indicates phosphorylation at the 5' end) The glycoside was calcined for the purpose of changing the restriction site SacI to the Notl site to prepare the adaptor Ad5-SacKpnNotI.

The approximately 7.94 kb of M33KSNH-PHSG2.98-Q6 constructed as described above was decomposed into a orf3 plastids by SacI and ΚρηΙ, and an insert portion of about 5.3 kb was excised, and the DNA fragment was extracted. The adaptor Ad5-SacKpnNotI was ligated to this SacI restriction enzyme site. Using a multi-selection site of the shuttle vector pRE-7, it was decomposed with Kpnl and Notl and provided in agarose gel electrophoresis to obtain a DNA fragment of a vector portion of about 5.9 kb. The vector was ligated to the insert portion by restriction enzyme sites Notl, Kpnl, coliform was transformed, and the transformant was selected in LB medium supplemented with kanamycin. As a result, a nitrilase promoter region (3.8 kb) into which the M33 strain was introduced into the shuttle vector pRE-7 (5.9 kb) and a nitrilase/3, α subunit and its downstream gene orf3 of the Q-6 strain were obtained. (1.5 kb) of the 11.2 kb pRE-7-M33pro-Q6 β a orf3 plastid. [Step 3] Transformation of M33 strain using gRE-7-M33pro-Q6 cold a orf3 plastid / LB medium containing l〇〇ml (0.5% by weight Bac to yeast extract ' 1% by weight Bacto pancreas, 0.5% by weight of NaCl (pH 7.5)) in a 500 ml volumetric flask, inoculated with a Rhododendron Rhodocarpus Rhodochrous M33 strain, and cultured at 30 ° C for 17 small -61 - (58) 1327596, The cells were collected by centrifugation at 1 °C for 15 minutes at 4 °C. The cells were washed twice with ice-cold distilled water (centrifuged at 10,000 x g for 15 minutes at 4 ° C), and washed with 10% by weight of glycerol sterilized by a filter to prepare a turbidity of 40 at a wavelength of 600 nm. suspension. The glycerin suspension 50/M was mixed with 2 yg of pRE-7-M3 3pro-Q6 lux a orf3 suspended in 1//1 water, and rapidly poured into an electrode tube for a 1 mm wide cell shock. The gene was introduced using ECM630 manufactured by Azbil Corporation under the conditions of 1.5 KV, 25 μF, and 800 Ω. Thereafter, it was kept in an ice bath for 10 minutes, and the whole amount was added to 1 ml of LB medium and cultured at 30 ° C for 4 hours on LB acacia medium containing 100 // g/ml of kanamycin. After the line was drawn, the culture was carried out at 30 ° C for 3 days to 5 days. 'The colony with the presence of kanamycin resistance was selected to obtain the Rhodococcus rhodochrous M33 strain. [Step 4] Culture of M33 strain transform

Contains 100ml of production medium (KH2PO40.5g/L, K2HPO4 0.4g/L, CoC126H20 0.02g/L, FES047H20 0·003

g/L, EDTA-2Na 0.006g/L, NaCl 0 · 0 1 g/L, C aC 12 2 Η 2 O 0.01g/L, glucose 20g/L, MgS047H20 1.2g/L, urea 7.5g/L, In a 500 ml volumetric flask of kanamycin lOmg/L), a transformant of Rhodococcus rhodochrous M33 strain of Rhododendron rhodochrous M33 was inoculated and cultured at 30 ° C for 2 days. 90 ml of the culture solution thus obtained was transplanted into a 5 L volume fermentation tank containing 3 l of a production medium, and cultured at 30 ° C for 48 hours at pH 7.2 and a stirring speed of 500 rpm. -62- (59) (59) 1327596 The culture solution was centrifuged at 25 ° C, 1 Torr, 〇〇〇 xg for 15 minutes, and collected. The same cells were centrifuged at 1,500 x g for 15 minutes in 25 ml of water at 25 ° C and washed twice to obtain a cell for production reaction. [Step 5] Using a propylene amide production reaction of the M33 strain transforming body, 800 ml of distilled water containing 0.9 g of the rhodococcus Rhodococcus rhodochrous M33 strain obtained by the above step 4 culture (the equivalent amount of the dry weight of the cells) is poured. In a 1.5 L reactor, continue to adjust the temperature inside the vessel at 20 ° C to 25 ° C and stir. Then, the liquid acrylonitrile was dropped thereinto and the concentration thereof was not more than 2%, the sample was appropriately taken out, the supernatant after centrifugation was diluted, and the concentration of acrylamide was measured by a high-speed liquid chromatography method. As a result, a Rhodococcus rhodochrous M33 strain having a nitrile hydrolase gene of the Q-6 strain accumulated 52% (weight/solution) of acrylamide. Comparative Example 1 In the comparative example, the parent strain Rhodococcus rhodochrous M33 was cultured in the same manner as in the above step 4 except that kanamycin was not added to the medium, and the strain obtained from the culture solution was used. When the acrylamide production reaction was carried out in the same manner as in the step 5, it was confirmed that the cumulative amount of the nitrilase gene 'acrylamide amide accumulated from the Q6 strain was significantly accumulated in the above M33 transformant. The results of comparison between this example and the comparative example are shown in Fig. 3, and the results of Fig. 3 are also shown in Table 3. -63- (60) 1327596 (60)

Table 7 Reaction time (Hrs) Rhodococcus rhodochrous M33 strain Rhodococcus rhodochrous M33 strain 0 0.0 0.0 1 14.2 19.4 2 25.8 32.6 3 34.0 39.2 4 38.5 43.0 5 40.8 45.7 6 41.9 47.8 7 42.9 49.6 8 43.2 50.6 9 43.2 51.3 10 43.2 51.8

Comparative Example 2: Confirmation of the amount of nitrilase in each of the bacteria by negative-type polyacrylamide gel electrophoresis. The culture solution of the parent strain M3 3 obtained in Comparative Example 1 and the M3 3 strain obtained in the above step 4 The transformant culture solution was collected by centrifugation at 8,000 g for 10 minutes, and 80 mg of the obtained wet cells were resuspended in 2 ml of 5 OmM sodium phosphate buffer (pH 7). The bacteria were disrupted by using an ultrasonic breaker under cooling, and glycerin was added to the bacterial cell disrupted solution to 10% (weight/liquid amount), and each sample was subjected to a negative type using a 10% gel. 61) 1327596 Polyacrylamide gel electrophoresis for resolution. After electrophoresis, protein staining was performed using Coomassie Brilliant Blue 7 as a result of decolorization, and in the transform of M33 strain, except for the ribbon at the same position as the nitril hydrolase of the parent strain M33. In addition, at the lower electrophoretic position, there is also a band of the same position as the purified nitrilase of the Q6 strain, and it is confirmed that * each of them can achieve a rare amount of more than 30% of the total protein in the cell. ·* High performance. The ribbon at the same position as the purified nitrilase of the Q6 strain obtained from the sample of the transforming body was transferred onto a PVDF membrane (manufactured by MILLIPORE Co., Ltd.) using a transfer device (BIO-RAD) to perform CBB staining, and then The portion of the target ribbon that is adsorbed is cut from the PVDF film. Next, the fully automated protein-substructure analysis device PPSQ-23A (Shimadzu Corporation) was used to interpret the N-terminal amino acid sequence of the protein. As a result, it was found that the acid sequence described in the sequence number 23 of the sequence listing and the sequence described in the sequence number 24 of the sequence listing were mixed, and it was judged that the transformation system undoubtedly produced the nitrilase from the Q6 strain in a significant amount. . On the other side of the φ plane, in the sample of the M33 strain of the parent strain, of course, there is no ribbon from the nitrilase equivalent position of the Q6 ' strain. Industrial Applicability The microorganism of the present invention can be utilized in the field of converting a nitrile compound into a corresponding guanamine compound even when it is reacted at a high temperature and a high nitrile compound concentration or a high guanamine compound concentration. [Simple description of the diagram] • 65- (62) (62) 1327596 Figure 1 shows the genetic makeup and restriction enzyme maps of the nitrilase Θ subunit, α subunit and downstream gene group of Geobacillus thermoglucosidasius Q-6 strain. It also indicates the fragment obtained by colony hybridization (Hin2.3) and the position of the fragment introduced by Rhodococcus bacteria (yS αΐ). Fig. 2 is a diagram showing the restriction enzymes of the gene formation of the S subunit, the α subunit and the downstream gene group and the surrounding region of the Rhodococcus rhodochrous Μ33 strain nitrilase; The two ends of the restriction enzyme map correspond to the DN of the λ 3 1 1 obtained by spot hybridization and used for re-cloning of ΚρηΙ and SacI. The thick line is part of M33KSNH-pHSG298, and the broken part of the thick line indicates the inserter inserted. Fig. 3 is a graph showing the results of a acrylamide production reaction using Rhodococcus rhodochrous M3 3 strain and its transformant. -66-

Claims (1)

1327596 |~ Announcement Double-sided ten, patent application Fan Park No. 94 1 43462 Patent application Chinese patent application scope amendments revised December 30, 1998 of the Republic of China]. A microorganism of the genus Rhodococcus, characterized by the following (A) Or the transformation of any of the DNAs of (B), (A) the DN A line contains the amino acid sequence or the coding sequence sequence described in SEQ ID NO: 1 corresponding to the sequence listing of the nitrilase α subunit. In the amino acid sequence of SEQ ID NO: 1, the base sequence of the amino acid sequence having one or several amino acid substitutions, deletions or additions; and the coding corresponding to the nitril hydrolase yS subunit The amino acid sequence described in SEQ ID NO: 2 of the Sequence Listing, or the amino acid sequence described in SEQ ID NO: 2 of the Sequence Listing, wherein the amino acid having one or more amino acids substituted, deleted or added a base sequence of the sequence; at the same time, the DNA encodes a protein having nitrilase activity; (B) the DNA sequence contains the sequence number 3 of the sequence listing corresponding to the base sequence encoding the nitrilase α subunit 695 ~ 1312 a base sequence, or a base sequence r-column that hybridizes to the base sequence under stringent conditions; and a first sequence of sequence number 3 containing a sequence listing corresponding to a base sequence encoding a nitrilase/3 subunit The base sequence of the 681th, or the base sequence which hybridizes under the stringent conditions to the base sequence; and the DNA encodes a protein having a nitrilase activity. 2. The microorganism according to claim 1, which belongs to the genus Rhodococcus (1327596 Rhodococcus) which can be produced by transformation to produce a protein having the following physicochemical properties, (a) having nitrilase activity; (b) Matrix specificity: shows activity when acrylonitrile, adiponitrile, acetonitrile, isobutyronitrile, n-valeronitrile, n-butyronitrile, benzonitrile, hexanenitrile as matrix % (c) Molecular weight: a protein composed of at least the following two types of subunits, and the molecular weight obtained by electrophoresis of reduced SDS-polyacrylamide of each subunit is as follows, subunit α molecular weight: 25000 ± 2000Da, subunit / 3 molecular weight: 28000 ± 2000Da; (d) thermal stability: after the enzyme in the aqueous solution is heated at 7 (TC temperature for 30 minutes, the remaining activity is more than 35 % before heating; e) Even if 6% by weight of acrylonitrile is used as the substrate, the activity is not reduced as compared with the concentration of the substrate; (f) even when acrylonitrile is used as the matrix in 35% by weight of the aqueous solution of acrylamide 3. The microorganism according to claim 1, wherein the microorganism can produce a protein having the following physicochemical properties by transformation, (a) having nitrilase activity; (b) matrix specificity : exhibits activity (c) molecular weight when acrylonitrile, adiponitrile 'acetonitrile, isobutyronitrile, n-valeronitrile 'n-butyronitrile, benzonitrile, hexane nitrile as matrix; it is composed of at least the following two types of subunits Protein composed of each Reduced SDS_polyacrylamide electrophoresis • r -λ ·η LSJ -2- 1327596 The molecular weight obtained is as follows, subunit α molecular weight: 25000±2000Da, subunit Θ molecular weight: 28000±2000Da; , (d Thermal stability: after the enzyme in the aqueous solution is heated at 70 ° C for 30 minutes, the residual activity is more than 35% before heating; (e) even if 6% by weight of acrylonitrile is used as the matrix, The activity is not reduced as compared with the substrate concentration; (f) The activity in the case of acrylonitrile as a matrix even in a 35 wt% aqueous solution of acrylamide. 4. The scope of claims 1 to 3 The microorganism according to any one of the items, wherein the microorganism of the genus Rhodococcus is a strain of Rhodococcus rhodochrous M33 (VKM Ac-1515D or BCRC-9 10263) and/or a mutant thereof. The microorganism of any one of the items 1 to 3, which is a strain of Rhodococcus rhodococcus M33 (\ Rhodococcus rh〇d〇chrous M33) transformed with a nitrilase gene (VKM AC-1515D or BCRC) -9 1 0263 ) and / or its mutant strain. 6. A method for producing a microorganism according to any one of claims 1 to 5, characterized in that it comprises a genus Rhodococcus according to any of the following (A) or (B) DNA. The microorganism is transformed into a shape (A). The DNA system contains an amino acid sequence encoded by the sequence number 编码 corresponding to the sequence table of the nitril hydrolase unit, or the amino acid sequence described in SEQ ID NO: 1 of the coding sequence listing. a base sequence of an amino acid sequence having one or several amine-3- 1327596 base acids substituted, deleted or added: and a sequence number 2 comprising a sequence listing corresponding to a nitrilase/3 subunit a base sequence of an amino acid sequence described in the amino acid sequence described in SEQ ID NO: 2 in the coding sequence table, having an amino acid sequence in which one or more amino acids are substituted, deleted or added; Meanwhile, the DNA is encoded by a protein having a nitrilase activity; (B) the DNA contains a sequence number 3 of the sequence sequence corresponding to the base sequence of the nitril hydrolase subunit, 695 to 1 3 1 2 base sequences' or in strictness a base sequence for hybridizing the base sequence; and a first to 681th base sequence of SEQ ID NO: 3 in a sequence listing corresponding to a base sequence encoding a nitrilase/3 subunit, Or a base sequence which hybridizes to the base sequence under stringent conditions: at the same time, the DNA encodes a protein having nitrilase activity. A method for producing a nitrilase or a bacterium for treating a bacterium comprising the nitrilase, which comprises culturing a microorganism according to any one of items 1 to 5 of the patent application in a culture medium. 8. An enteric hydrolase or a bacterial cell treated product comprising the nitrilase, which is produced by the production method of claim 7 of the patent application. A method for producing a kinesin compound, which comprises subjecting a nitrilase according to item 8 of the patent application or a bacteriophage treatment containing the nitrilase to an eye compound and synthesizing hydrazine compound Amine compound 1327596 Patent application No. 94143462 Chinese map amendment page XI, Republic of China December 30, 1998 Amendment 7|&9649·—I. 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