CN1806047A

CN1806047A - Novel nitrile hydratase

Info

Publication number: CN1806047A
Application number: CNA2004800161703A
Authority: CN
Inventors: 古家加夫留; 玉木彰; 长泽伸一郎; 铃木绫乃
Original assignee: Asahi Kasei Kogyo KK
Current assignee: Asahi Kasei Corp
Priority date: 2003-06-10
Filing date: 2004-06-10
Publication date: 2006-07-19
Also published as: JPWO2004108942A1; KR20060016115A; AU2004245849B2; JP4108095B2; AU2004245849A1; WO2004108942A1; TW200504211A; KR100806991B1; TWI321151B

Abstract

A process for producing an amide compound with the use of a nitrile hydratase of high thermal stability capable of maintaining high activity even in the presence of high-concentration nitrile compound as a substrate and amide compound as a product. A recombinant microbe was produced by searching for a novel microbe capable of expression of the desired nitrile hydratase in nature, obtaining relevant gene from cells of the microbe or treatment product thereof, performing cloning of the gene and introducing the same in a different type of microbe for expression. With the use thereof, nitrile compounds can be efficiently converted to corresponding amide compounds even by the reaction conducted at high temperature and in the presence of high-concentration nitrile compounds and high-concentration amide compounds.

Description

Novel nitrile hydratase

Technical field

The present invention relates to by make the technology of amide compound by nitrile compound from the enzyme catalysis of the novel nitrile hydratase of the new isolated microorganism of nature.

Background technology

Carry out changing amide group into after the hydration at the itrile group to nitrile compound, make in the technology of corresponding amide compound, by the catalytic chemical process of copper, the enzyme of use microorganism was just becoming main flow as the method for catalyzer except in the past.Such enzyme is commonly referred to as Nitrile hydratase (Nitrile hydratase), since reported first, has found a lot of enzymes from various microorganisms again.For example, belong to (Agricultural andBiological Chemistry Vol.44p.2251-2252,1980) from Arthrobacter (Arthrobacter), Agrobacterium (Agrobacterium) belongs to (spy opens flat 05-103681), acinetobacter calcoaceticus (Acinetobacter) belongs to (spy opens clear 61-282089), Aeromonas (Aeromonas) belongs to (spy opens flat 05-030983), enterobacteria (Enterobacter) belongs to (spy opens flat 05-236975), Erwinia (Erwinia) belongs to (spy opens flat 05-161496), bacillus flavus (Xanthobacter) belongs to (spy opens flat 05-161495), klebsiella (Klebsiella) belongs to (spy opens flat 05-030982), (spy opens clear 54-129190 to rod bacillus (Corynebacterium) genus, distinguished afterwards and be Rhod), pseudomonas (Pseudomonas) belongs to (spy opens clear 58-86093), citric acid bacillus (Citrobacter) belongs to (spy opens flat 05-030984), streptomycete (Streptomyces) belongs to (spy opens flat 05-236976), (spy opens clear 51-86186 to genus bacillus (Bacillus) genus, Te Kaiping 7-255494), sickle mycete belongs to (spy opens flat 01-086889), (spy opens clear 63-137688 to Rhod (Rhodococcus), Te Kaiping 02-227069, the spy opens 2002-369697, Te Kaiping 2-470), rhizobium (Rhizobium) (spy opens flat 05-236977), the enzyme of false Nocardia bacteria Pseudomonas (Pseudonocardia) bacteriums such as (spy open flat 8-56684).These enzymes, are being studied according to various purposes so their physico-chemical property also is various owing to the diversity of their aminoacid sequence always.In physico-chemical property, as to relevant heat, or the example that the stability of amide compound and nitrile compound etc. is being illustrated, document (European Journal of Biochemistry Vol.196p.581-589 as relevant prunosus red coccus (Rhodococcus rhodochrous) J1 strain, 1991.Applied and Microbiology Biotechnology Vol.40p.189-195,1993.), (spy opens flat 8-187092 to the document of relevant thermophilic false Nocardia bacteria (Pseudonocardiathermophila) JCM3095 strain, Journal ofFermentation and Bioengineering Vol.83p.474-477,1997), relevant genus bacillus (Bacillus) belongs to document (the WO 99/55719.AppliedBiochemistry and Biotechnology Vol.77-79P.671-679 of BR449 strain, 1999.), (Enzyme and MicrobialTechnology Vol.26 p.368-373 for the document of relevant genus bacillus (Bacillus) genus RAPC8 strain, 2000.Extremophiles Vol.2p.347-357,1998), (Biochimica et Biophysica Acta Vol.1431 p.249-260 for the document of relevant bacillus pallidus (Bacillus palidus) DAC521 strain, 1999), the document of relevant Shi Shi genus bacillus (Bacillus Smithii) SC-J05-1 strain (Journal of Industrial Microbiology and Biotechnology Vol.20220-226,1998).

In addition, when using these unique Nitrile hydratases to make amide compound by the nitrile compound industrialization, making the shared cost of this enzyme in the manufacturing cost of amide compound is an important problem, wishes to use the host who establishes culture method already on industrial level to produce.Therefore, be purpose to make the Nitrile hydratase great expression by genetic engineering means, carrying out attempting the research of gene clone.For example, Rhodopseudomonas (spy opens flat 3-251184), Rhod (spy open flat 2-119778, spy open flat 4-211379, spy open that flat 09-00973, spy open flat 07-099980, the spy opens 2001-069978), rhizobium (spy opens flat 6-25296), Klebsiella (spy opens flat 6-303971), achromobacter (spy opens flat 08-266277), Pseudonocardia (spy opens flat 9-275978), bacillus (spy opens flat 09-248188) etc.

Summary of the invention

Even expectation obtains having the Nitrile hydratase that can keep thermostability and also keep this physico-chemical property of high reactivity in the presence of the high density of substrate nitrile compound or resultant amide compound, though the bibliographical information of this respect is arranged, but sometimes owing to the embodiment of the enzyme that in reaction, uses, the kind difference of nitrile compound, those physico-chemical properties and active absolute value all can change, and do not find to have both the enzyme of all characteristics as yet.In addition, the subject matter as using improvement physico-chemical properties such as evolution engineering from now on is expected to develop unprecedented plurality of enzymes.

When utilizing evolution engineering improvement physico-chemical property, it is self-evident needing clone and expressing gene, and the production cost related restriction of enzyme is considered from make with above-mentioned amide compound, wishes that the host who uses culture method to establish makes gene recombination bacterium.

Promptly the object of the present invention is to provide from nature and separate, the production method of this enzyme and use this enzyme to make the manufacture method of corresponding amide compound by nitrile compound heat or the high Nitrile hydratase of high concentrations of compounds stability.The aminoacid sequence and the gene order of this enzyme also are provided, contain this gene recombinant plasmid, contain this recombinant plasmid the conversion bacterial strain, use this conversion bacterial strain this enzyme production method and use this conversion bacterial strain to make the manufacture method of corresponding amide compound by nitrile compound.Proteinic aminoacid sequence and gene order with the further activatory effect of the Nitrile hydratase that makes this recombinant chou also are provided in addition.

The inventor has carried out wholwe-hearted research for solving above-mentioned problem, has found to have the active microorganism of Nitrile hydratase-thermophilic Polyglucosidase soil genus bacillus (Geobacillus thermoglucosidasius) near the soil of the result beautiful county of fine jade hot spring.And the microorganism of up to the present still not knowing the soil bacillus has Nitrile hydratase, can show the Nitrile hydratase activity, and 65 ℃ of these temperature of this microorganism common usefulness in cultivation have surpassed the common culture temperature (45 ℃～60 ℃) of the thermophile bacteria with Nitrile hydratase in the past.

By this microorganism purifying Nitrile hydratase, this Nitrile hydratase activity shows and has both heat and the nitrile compound of high density and the high stability of amide compound.In addition based on the aminoacid sequence of each subunit N-terminal of the enzyme behind the purifying, chromosomal DNA by this microorganism separates the Nitrile hydratase gene, according to the result that this aminoacid sequence and gene order are tentatively illustrated, can judge that the homology of itself and existing Nitrile hydratase is very low.Be in the gene order that is estimated as activation of protein in this gene downstream by expressing simultaneously in addition, but successfully obtain the recombinant bacterial strain of the gene of this enzyme of great expression, so far finish the present invention.

Promptly the invention provides following content:

(1) a kind of DNA is characterized by coded protein and has following physico-chemical property:

(a) has the Nitrile hydratase activity.

(b) substrate specificity: as substrate, show activity with vinyl cyanide, adiponitrile, acetonitrile, isopropyl cyanide, n-valeronitrile, n-butyronitrile, benzonitrile, own nitrile.

(c) molecular weight: by following 2 kinds of protein that subunit constitutes, the molecular weight that each subunit is determined through reduced form SDS-polyacrylamide gel electrophoresis is as follows as at least:

Subunit alpha molecule amount 25000 ± 2000

Subunit beta molecule amount 28000 ± 2000

(d) thermostability: when with the enzyme in the aqueous solution under 70 ℃ of temperature the heating 30 minutes after, remaining activity for the heating before active more than 35%.

(e) vinyl cyanide that promptly uses 6 weight % is compared during with the following concentration of substrate of 6 weight % as substrate, and its activity does not reduce yet.

(f), still have with the activity of vinyl cyanide as substrate even in the acrylamide solution of 35 weight %.

(2) following (A) or (B) in any DNA.

(A) a kind of DNA is characterized by by coding and contains the α subunit gene of aminoacid sequence shown in the sequence number 1 and the combination of beta subunit gene base sequence that coding contains aminoacid sequence shown in the sequence number 2 is constituted.

(B) a kind of DNA, coded protein is at the α subunit of aminoacid sequence shown in the sequence number 1 with contain among any or two of β subunit of aminoacid sequence shown in the sequence number 2 separately 1 or a plurality of amino acid displacement, disappearance, interpolation, the posttranslational modification carried out, contain the β subunit that sequence changes or do not have α subunit and the change that changes or do not have to change, and have the Nitrile hydratase activity.

(3) following (C) or (D) in any DNA.

(C) a kind of DNA is characterized by by DNA that contains the 695-1312 bit sequence with sequence number 3 and the combination of DNA with 1-681 bit sequence of sequence number 3 and is constituted.

(D) coding contain following two subunits and have an active protein DNA of Nitrile hydratase: wherein, one subunit is to comprise the coded α subunit of any DNA among the DNA of 695-1312 bit sequence of sequence number 3 or the DNA of hybridizing under stringent condition with this DNA, another subunit be comprise sequence number 3 1-681 bit sequence DNA or with this DNA under stringent condition, the β subunit that any DNA among the DNA of hybridizing is coded.Except the situation of but above-mentioned (C).

(4) each described DNA in (1)～(3), it is characterized in that recombinant contains the DNA of the base sequence of aminoacid sequence shown in the encoding sequence numbers 4, or coding characteristic in this aminoacid sequence 1 or a plurality of amino acid is replaced, is lacked, interpolation, posttranslational modification and coding be any DNA among the DNA with the activation proteins associated matter of Nitrile hydratase.

(5) each described DNA in (1)～(3) is characterized in that it is to activate any DNA among the DNA of proteins associated matter with Nitrile hydratase for what can hybridize under the condition of strictness with this DNA and encode that recombinant contains the DNA of 1325-1663 bit sequence of sequence number 3 or coding characteristic.

(6) contain the DNA of the α subunit gene of the Nitrile hydratase of aminoacid sequence shown in the encoding sequence number 1.

(7) contain the DNA of the beta subunit gene of the Nitrile hydratase of aminoacid sequence shown in the encoding sequence numbers 2.

(8) contain the DNA that is characterized as the coding gene relevant with the activation of the Nitrile hydratase of aminoacid sequence shown in the sequence number 4.

(9) each described DNA of (1)～(8), wherein, this DNA comes from soil genus bacillus (Geobacillus) and belongs to.

(10) each described DNA of (1)～(8), wherein, this DNA comes from thermophilic Polyglucosidase soil genus bacillus (Geobacillus thermoglucosidasius) and plants.

(11) each described DNA of (1)～(8), wherein, this DNA comes from thermophilic Polyglucosidase soil genus bacillus (Geobacillus thermoglucosidasius) Q-6 strain (FERM BP-08658).

(12) be integrated with (1)～recombinant vectors of (11) each described DNA.

(13) with each described DNA microorganism transformed of (1)～(11) or thermophilic Polyglucosidase soil genus bacillus (Geobacillus thermoglucosidasius) Q-6 strain (FERM BP-08658) with and varient in any microorganism.

(14) be this protein of feature or the manufacture method that contains this proteinic bacterial disposing thing so that each described DNA microorganism transformed of usefulness (1)～(11) is cultivated in substratum.

(15) a kind of protein or contain the manufacture method of this proteinic bacterial disposing thing, it is characterized by and in substratum, cultivate the microorganism that belongs to the soil bacillus, and this microorganism can produce the proteinic microorganism with following physico-chemical property, and said physico-chemical property comprises:

(a) has the Nitrile hydratase activity.

(b) substrate specificity: show activity as substrate with vinyl cyanide, adiponitrile, acetonitrile, isopropyl cyanide, n-valeronitrile, n-butyronitrile, benzonitrile, own nitrile.

Subunit alpha molecule amount 25000 ± 2000

Subunit beta molecule amount 28000 ± 2000

(16) from this protein of obtaining by (14) or (15) described any manufacture method cultured microorganism or contain this proteinic bacterial disposing thing.

(17) be characterized as protein with following physico-chemical property.

(a) has the Nitrile hydratase activity.

Subunit alpha molecule amount 25000 ± 2000

Subunit beta molecule amount 28000 ± 2000

(e) vinyl cyanide that promptly uses 6 weight % is compared during with the following concentration of substrate of 6 weight % as substrate, and activity does not reduce yet.

(18) following (A) or (B) in any protein.

(a) be characterized as the protein that contains α subunit and β subunit, wherein the α subunit contains aminoacid sequence shown in the sequence number 1, and the β subunit contains aminoacid sequence shown in the sequence number 2.

(b) contain the α subunit that contains aminoacid sequence shown in the sequence number 1 and any or two of β subunit of containing aminoacid sequence shown in the sequence number 2 separately to 1 or a plurality of amino acid is replaced, lacked, interpolation, posttranslational modification, contain sequence change or do not have reformed α subunit and a change or do not have reformed β subunit, and have the active protein of Nitrile hydratase.

(19) following (C) or (D) in any protein.

(c) be characterized as the protein that contains α subunit and β subunit, wherein, the α subunit is by the dna encoding of the 3695-1312 bit sequence with sequence number, and the β subunit is by the dna encoding of the 31-681 bit sequence with sequence number.

(d) be characterized as and contain α subunit and β subunit and have the active protein of Nitrile hydratase, wherein the α subunit is by the dna encoding of the 695-1312 bit sequence with sequence number 3, or any DNA among the DNA of hybridizing with this DNA under stringent condition is coded; And the β subunit is the DNA by the 1-681 bit sequence with sequence number 3, or any dna encoding among the DNA of under the condition of strictness, hybridizing with this DNA.Except the situation of but above-mentioned (C).

(20) have the protein of following feature: contain at least in the polypeptide of the polypeptide that comprises the α subunit or posttranslational modification any and comprise the polypeptide of β subunit or the polypeptide of posttranslational modification in any, or contain both, wherein, the α subunit is an aminoacid sequence shown in the sequence number 1, and the β subunit is an aminoacid sequence shown in the sequence numbering 2 in the sequence table.

(21) manufacture method of amide compound is characterized in that: will have the protein of following physico-chemical property or contain this proteinic bacterial disposing thing and act on nitrile compound, and obtain by this nitrile compound deutero-amide compound.

(a) has the Nitrile hydratase activity.

Subunit alpha molecule amount 25000 ± 2000

Subunit beta molecule amount 28000 ± 2000

(d) thermostability: when with the enzyme in the aqueous solution under 70 ℃ of temperature the heating 30 minutes after, residual activity for the heating before more than 35%.

(22) manufacture method of amide compound, it is characterized in that: in substratum, cultivate with each described DNA microorganism transformed in the claim 1～11 or belong in the proteinic microorganism that producing of soil bacillus have following physico-chemical property any, the protein that obtains thus or contain this proteinic bacterial disposing thing.

(a) has the Nitrile hydratase activity.

Subunit alpha molecule amount 25000 ± 2000

Subunit beta molecule amount 28000 ± 2000

(d) thermostability: in the aqueous solution enzyme in heating under 70 ℃ of temperature after 30 minutes, residual activity before for heating more than 35%.

Description of drawings

Fig. 1 represents that Nitrile hydratase β subunit, α subunit and the downstream gene group's of thermophilic Polyglucosidase soil genus bacillus Q-6 (Geobacillusthermoglucosidasius Q-6) strain gene constitutes and restriction enzyme figure.Expressed the position of fragment (HiN2.3) that obtains by colony hybridization and the fragment of in colibacillary expression, using (β α, β α 1, β α 12).

Embodiment

At first Nitrile hydratase of the present invention is described.In the present invention, what is called have the Nitrile hydratase activity refer to have the nitrile compound of making addition water molecules after, change the activity of amide compound into, for example the N-propionitrile then changes the N-propionic acid amide into if vinyl cyanide then changes acrylamide into if acetonitrile then changes ethanamide into.The compound that generates uses gas-chromatography/mass analysis (GC/MS), infrared absorption spectrum (IR) and NMR (Nuclear Magnetic Resonance) spectrum (NMR) etc. to identify after the liquid chromatography classification is collected.

In the present invention when the Nitrile hydratase activity is measured, for example, the 1ml nitrile compound solution (0.05M phosphoric acid buffer pH7.7) to 0.1 weight % adds 10 μ l Nitrile hydratase solution, insulation is after 1 minute～60 minutes in 27 ℃～60 ℃ scopes of temperature of reaction, by adding the 1N HCl termination reaction of 0.1ml, the part of reaction solution is analyzed with liquid chromatography, can be identified the generation that has or not amide compound.

In the present invention as the nitrile compound of substrate, aliphatic nitrile compounds such as acetonitrile, N-propionitrile, N-butyronitrile, isopropyl cyanide, N-valeronitrile, the own nitrile of N-for example, 2-chloroethyl nitrile etc. contains the nitrile compound of halogen atom, vinyl cyanide, crotononitrile, methacrylonitrile etc. contain the aliphatic nitrile compound of unsaturated link(age), hydroxy nitrile compounds such as lactonitrile, hydroxybenzene acetonitrile, amino-nitrile compounds such as 2-phenylglycocoll base nitrile, aromatic nitrile compounds such as benzonitrile, cyanogen pyridine, dinitrile compounds such as propane dinitrile, succinonitrile, adiponitrile etc. and three nitrile compounds.

The substrate specificity of Nitrile hydratase of the present invention is measured and can be judged by changing various substrates under the said determination condition, corresponding to each substrate, whether has the Nitrile hydratase activity.If expansion substrate specificity, the kind of the amide compound of the correspondence that it can be made also increase and can enlarge preferably certainly, but this enzyme can use vinyl cyanide, adiponitrile, acetonitrile, isopropyl cyanide, n-valeronitrile, n-butyronitrile, benzonitrile, own nitrile as substrate at least.

Nitrile hydratase of the present invention is by reduced form SDS (sodium lauryl sulphate)-polyacrylamide gel electrophoresis, through coomassie brilliant blue staining, detect molecular weight 25000 ± 2000 and molecular weight 28000 ± 2,000 two subunit, the former is called the α subunit, the latter is called the β subunit.

Nitrile hydratase of the present invention before determination of activity, be in the aqueous solution that does not contain stablizers such as organic acid state in 70 ℃ carry out 30 minutes heat treated after, still can keep active 35% activity before the heating.

In addition, though but have the high density of reporting nitrile substrate chemical make enzyme deactivation, Nitrile hydratase of the present invention promptly use the vinyl cyanide of 6 weight % as substrate, do not observe above-mentioned phenomenon yet.

In addition, but the reaction product amide compound inhibited reaction that the high density reported is arranged, this has become big problem when obtaining the reaction product of high density, even but Nitrile hydratase of the present invention is added to the acrylamide of 35 weight % in the determination of activity solution, can find still that as the meaningful minimizing of the acrylonitrile concentration of substrate it is active that enzyme still keeps.

As the present invention, exemplified above-mentioned (18) described content.That is, as Nitrile hydratase of the present invention, preferred example is the enzyme that is made of α subunit of representing with 205 amino acid whose sequences shown in the sequence numbering in the sequence table 1 and the β subunit represented with 226 amino acid whose sequences shown in the sequence numbering in the sequence table 2.Except these two subunits, also can contain metal or other peptide etc.As metal, many especially is iron content or cobalt.In addition, also passable even contain the protein of any subunit in two subunits.And as the aminoacid sequence of each subunit, has the Nitrile hydratase activity so long as form complex body with other subunit, certainly can be contemplated to and in aminoacid sequence, can contain 1 or a plurality of amino acid whose displacement, disappearance or insert, and, may be modified after the translation owing to the host type difference.Particularly in the α of Nitrile hydratase subunit, often be modified to cysteine sulfinic acid or cysteine sulfenic acidcysteine sulfenate after the cysteine residues translation.Replaced, lacked, inserted or the aminoacid sequence of posttranslational modification as 1～30 amino acid in preferred example such as this aminoacid sequence, more preferably 1～10, more preferably 1～5, most preferably 1～3.Has such displacement and contain, disappearance, or the Nitrile hydratase enzyme of the aminoacid sequence that inserts also can be attempted by well-known rite-directed mutagenesis introductory technique, Molecular Cloning 2nd Edition for example, the described method of Cold Spring Harbor Laboratorypress (1989), use imports displacement at the position of base sequence correspondence, disappearance, or the DNA that inserts, as described below, import to and make this DNA expression back acquisition in the host microorganism, produce variant enzyme with character of expecting on thermostability or the industries such as organic solvent tolerance raising or substrate specificity variation.According to such technical level, those variant enzymes also can become the enzyme that comprises in the present invention when having the Nitrile hydratase activity.

Though the present invention has exemplified above-mentioned (19) described invention in addition, and this is had been described in detail in the explanation of DNA of the present invention.

Nitrile hydratase with above-mentioned physico-chemical property for example can belong to the microorganism acquisition of soil bacillus by cultivation.Spendable in the present invention microorganism is so long as belong to the soil bacillus, and has the active microorganism of hydration that makes nitrile compound change amide compound into, no matter any can.As the microorganism that belongs to the soil bacillus, as Geobacillus caldoxylosilyticus, thermophilic soil genus bacillus (Geobacilluskaustophilus), Geobacillus lituanicus, stearothermophilus soil genus bacillus (Geobacillus Stearothermophilus), Geobacillus Subterraneus, hot chain soil genus bacillus (Geobacillus thermocatenulatus), hot denitrification soil genus bacillus (Geobacillus thermodenitrificans), thermophilic Polyglucosidase soil genus bacillus (Geobacillus thermoglucosidasius), happiness heat is bitten oily soil genus bacillus (Geobacillus thermoleovorans), Geobacillustoebii, Geobacillus uzenensis.In addition, be not defined in microorganism especially, comprise Nitrile hydratase gene yet from other microbial strain from the soil bacillus.As such microbial strain, as belong to Agrobacterium (Agrobacterium) genus, colourless bar (Achromobacter) belongs to, acinetobacter calcoaceticus (Acinetobacter) belongs to, Aeromonas (Aeromonas) belongs to, enterobacteria (Enterobacter) belongs to, Erwinia (Erwinia) belongs to, bacillus flavus (Xanthobacter) belongs to, klebsiella (Klebsiella) belongs to, rod bacillus (Corynebacterium) belongs to, China root nodule bacterium (Sinorhizobium) belong to, pseudomonas (PSeudomonas) belongs to, streptomycete (Streptomyces) belongs to, Nocardia bacteria (Nocardia) belongs to, genus bacillus (Bacillus) belongs to, micrococci (Micrococcus) belongs to, rhodococcus (Rhodococcus) belongs to, rhodopseudomonas (RhodopSeudomonas) belongs to, root nodule bacterium (Rhizobium) belong to, the microorganism of false Nocardia bacteria bacterium (PSeudonocardia) genus etc.Specifically, screen by following gimmick in the present invention.The soil that at first takes a morsel and take from various place is added to and water or physiological saline is housed in vitro, and shaking culture is 2 days～14 days in 65 ℃ Shaking Incubators.Get this nutrient solution part, be added to widely usedly be used for substratum that microorganism growth grows, for example with the liquid nutrient medium as main component such as glycerine, peptone, yeast extract, under 65 ℃ culture temperature, cultivated about 1 day to 7 days.The part of the above-mentioned nutrient solution that obtains is layered on the Agar Plating that contains the above-mentioned medium component that is used for the microorganism growth growth, under 65 ℃, cultivates again, can form bacterium colony, separate microorganism by making it.The microorganism that obtains is like this used the test tube or the flask of the liquid nutrient medium that is added with amide compounds such as nitrile compounds such as in above-mentioned medium component, adding the N-valeronitrile again or Methacrylamide, by during suitable, for example about about 12 hours to 7 days time, under 65 ℃ culture temperature, carry out shaking culture and make microbial growth, according to the active ordinary method of said determination Nitrile hydratase, select the purpose microorganism.When the representative strain of such microorganism being identified according to 16SrRNA and following biochemical property, distinguish it is thermophilic Polyglucosidase soil genus bacillus (Geobacillusthermoglucosidasius), be referred to as numbering FERM P-19351 (accept day put down on May 16th, 15) with the name of thermophilic Polyglucosidase soil genus bacillus Q-6 (Geobacillus thermoglucosidasius Q-6) and be preserved in independent rows and lose legal person's industrial technology synthetic study institute (a kind of ground of 1 fourth order, east, ripple city, 1 central authorities the 6th are built in the Hitachinaka County, Japan), moved as FERM BP-08658 according to budapest treaty and guarantee Tibetans (accepting day puts down on March 11st, 16).Though retrieved multiple patent, document, any relevant microorganism that belongs to thermophilic Polyglucosidase soil genus bacillus of this discovery, relevantly had an active record of Nitrile hydratase.Confirm that thus thermophilic Polyglucosidase soil genus bacillus Q-6 strain is new bacterial strain.Use the mutant of this new bacterial strain, promptly can make amide compound by thermophilic Polyglucosidase soil genus bacillus Q-6 strain deutero-mutant strain, cytogamy strain and gene recombination strain.And the character of thermophilic Polyglucosidase soil genus bacillus Q-6 strain is as described below.

(a) morphologic character

Culture condition: (UK) substratum is 60 ℃ for Oxoid, England for nutrient agar medium

1. the shape of cell and size

Shape: bacillus

Size: 0.8 * 2.0～3.0 μ m

2. acellular polymorphism is arranged :-

3. have or not mobility :+

Flagellum give birth to state: peritricha

4. have or not sporozoite :-

The position of sporozoite: end is upright

(b) character of Pei Yanging

1. color: cream color

2. gloss :+

3. pigment production :-

Culture condition: (UK) substratum is 60 ℃ for Oxoid, England for nutrient broth

1. have or not the surface to grow :-

2. substratum has or not muddiness :+

Culture condition: 60 ℃ of gelatin stab culture

1. state grows :+

2. gelatine liquefication :+

Culture condition: 60 ℃ of litmus milks

1. solidify :-

2. liquefaction :-

(c) physiological property

1. gramstaining: indefinite

2. the reduction of nitrate :-

2. denitrification reaction :-

3.MR test :-

4.VP test :-

5. the generation of indoles :-

6. the generation of hydrogen sulfide :-

7. the hydrolysis of starch :-

8. the utilization of citric acid

Koser：-

Christensen：-

9. inorganic nitrogen-sourced utilization

Nitrate :-

Ammonium salt :+

10. the generation of pigment :-

11. urease activity :-

12. oxydase :+

13. catalase :+

14. the scope of growing

pH：5.5～8.0

Temperature: 45 ℃～72 ℃

15. attitude: general character anaerobism to oxygen

16.O-F test:-/-

(d) the product acid/aerogenesis of carbohydrate

1.L-pectinose-/-

2.D-wood sugar+/-

3.D-glucose+/-

4.D-seminose+/-

5.D-fructose+/-

6.D-semi-lactosi-/-

Maltose+/-

Sucrose+/-

Lactose-/-

Trehalose+/-

11.D-Sorbitol Powder-/-

12.D-maltose alcohol+/-

13. inositol-/-

14. glycerine-/-

(e) other character

1. betagalactosidase activity :-

2. arginine dihydro enzymic activity :-

3. lysine decarboxylase activity :-

4. tryptophane deaminase active :-

5. gelatinase activity :+

The method for culturing microbes of Shi Yonging can carry out according to general microbial culture method in the methods of the invention, and no matter solid culture or liquid culture can.Thermophilic Polyglucosidase soil genus bacillus (Geobacillus thermoglucosidasius) is owing to be general character anerobe, thus can with the same culture condition of common general character anerobe under cultivate.Culture temperature can be in microorganism growth be grown scope suitably change, for example, 40 ℃～75 ℃ scope.The pH of substratum can be for example 4～9.Particularly for thermophilic Polyglucosidase soil genus bacillus Q-6 (Geobacillus thermoglucosidasius Q-6), can under 45 ℃～72 ℃, the culture temperature of preferred 55 ℃～70 ℃ of scopes, cultivate under 5～8 the medium pH.Incubation time is because of the different differences to some extent of various conditions, usually, preferred about 1 day～about 7 days left and right sides time.As substratum, be used for the common various substratum that suitably contain carbon source, nitrogenous source, organic or inorganic salt etc. of general microorganism in addition.Can use glycerine, glucose, sucrose, molasses, organic acid, animals and plants wet goods as carbon source.As nitrogenous source, as yeast extract, peptone, malt extract, meat extract, urea, SODIUMNITRATE etc.As organic or inorganic salt, as using sodium-chlor, sal epsom, Repone K, ferrous sulfate, manganous sulfate, cobalt chloride, zinc sulfate, copper sulfate, sodium-acetate, lime carbonate, potassium primary phosphate, dipotassium hydrogen phosphate etc.In the methods of the invention, improve, preferably in substratum, add amide compounds such as nitrile compound, Methacrylamide such as N-valeronitrile, isovaleronitrile, crotononitrile for the Nitrile hydratase that makes microorganism used therefor is active.Addition, for example, for 1 liter of substratum, the amount of adding 0.01g～10g scope is for well.In addition, preferably there are above Fe ion of 0.1 μ g/ml or Co ion to exist.

In order to obtain the amino acid sequence information of enzyme of the present invention, behind the enzyme purification that this part can be invented, after by reduced form SDS-polyacrylamide gel electrophoresis each subunit being separated, from gel, cut out each band, measure the part of aminoacid sequence by protein sequencer.

The invention still further relates to the DNA of encoding nitrile hydratase in addition.Specifically, as contain sequence number 3 the 695-1312 position DNA and contain the DNA of the 1-681 position of sequence number 3, their are dna encoding α subunit and β subunit respectively, but is not limited thereto, so long as it is just passable to contain the DNA of this base sequence.Also can be the DNA that under the condition of strictness, can hybridize with the DNA that these sequence complementary base sequences constitute in addition,, be included among the present invention so long as have the Nitrile hydratase activity.Promptly use these DNA, can express Nitrile hydratase of the present invention.Refer under the strict condition and for example use ECL direct nucleic acid labelingand detection System (Amersham Pharmacia Biotech corporate system), the condition that handbook is recorded and narrated (Wash:42 ℃, contain the primary wash buffer of 0.5xSSC).As the DNA that under the condition of strictness, can hybridize, for example under the condition of above-mentioned strictness, at the DNA of the 695-1312 position of containing sequence number 3 or contain in the complementary base sequence of DNA of 1-681 position of sequence number 3 arbitrarily, at least 20 usually, preferred at least 50, particularly preferred at least 100 successive base sequences are as test sample, the DNA of hybridizing with this sample.

The DNA of code book invention Nitrile hydratase can obtain by the following method.In this manual, so long as do not specify, can adopt production technology, the analytical method of well-known gene recombination technology, recombinant protein in this field.

The DNA of code book invention Nitrile hydratase is according to measuring the sequence informations such as aminoacid sequence that obtain by above-mentioned purifying enzyme under the base sequence that provides in this specification sheets or aminoacid sequence and the different situations, can be from the microorganism that contains Nitrile hydratase of the present invention, for example obtain the thermophilic Polyglucosidase soil genus bacillus Q-6 strain.Use according to aminoacid sequence synthetic oligonucleotide as probe, the chromosomal DNA that will contain the microorganism of Nitrile hydratase imports in phage or the plasmid through the dna fragmentation that restriction enzyme digestion obtains, transform the host, from the library that obtains, also can obtain the DNA of code book invention Nitrile hydratase by plaque hybridization or colony hybridization etc.In addition not with oligonucleotide as probe, but according to the preparation primers such as N-terminal amino acid sequence information of measuring two subunits that obtain by above-mentioned purifying enzyme, the part of Nitrile hydratase gene is increased the product that obtains as probe, also can obtain this DNA by same process by polymerase chain reaction (PCR).The DNA that obtains inserts among plasmid vector, for example pUC118 and clones, by two deoxidation cessation method (Proceedings of the National Academyof Sciences.USA, 74:5463-5467,1977) etc. well-known method can be measured base sequence.Can survey work to the expression product in the escherichia coli host that uses this gene transformation by using the above-mentioned determination of activity ordinary method that provides, confirm that the gene of preparation is the DNA of encoding nitrile hydratase like this.

The invention provides recombinant vectors in addition, it is characterized in that above-mentioned DNA is connected on the carrier.

Recombinant vectors among so-called the present invention can be by connecting the 5 ' end side of the DNA that obtains with aforesaid method with the form that can play a role in the promoter region downstream that is suitable for host microorganism, insert transcription termination sequence in this fragment downstream as required, be incorporated into suitable expression with being prepared in the carrier.

As suitable expression,, be not particularly limited so long as can in host microorganism, duplicate propagation.In addition, so long as karyomit(e) can insert the host of gene, but do not need to contain the zone of self-replacation among this host.For example, as the host, if use intestinal bacteria, can select in any carrier that the pUC system of the promotor of PL or acetonic acid oxidase gene (No. the 2579506th, patent gazette) etc., pGEX system, pET system, pT7 system, pBluescript system, pKK system, pBS system, pBC system, pCAL system etc. use in intestinal bacteria usually from containing powerful promotor, for example lac, trp, tac, trc, T7.In addition, α subunit gene and beta subunit gene also can also can pass through the common promotor with polycistronic expression by each promotor as independently cistron expression.In addition, when being independent cistron, each subunit gene also can be on same carrier.

Be in the DNA of the gene in Nitrile hydratase gene of the present invention downstream in addition by integrative gene expression in the recombinant vectors of above-mentioned Nitrile hydratase, find that the active of Nitrile hydratase further rises, so the present invention also provides such carrier.Specifically, with above-mentioned same, use contains the plasmid vector of expressing essential promotor or transcription termination factor etc., coding can also can be regulated and control the zone with polycistronic expression by common with each independently cistron expression with the gene of the activation proteins associated matter of Nitrile hydratase, the α subunit gene and the beta subunit gene of Nitrile hydratase.Same each gene also can be on same carrier.

Therefore, the invention provides the DNA of the activation proteins associated matter of coding and Nitrile hydratase.Specifically,, but be not limited to this as the base sequence of 1325-1663 position in the sequence numbering in the sequence table 3, can so long as contain the DNA of this base sequence.Can be the DNA that under the condition of strictness, can hybridize by the DNA that constitutes with these sequence complementary base sequences in addition, so long as be included in the present invention relevant with the activation of Nitrile hydratase.That is, use these DNA that Nitrile hydratase of the present invention is further activated.Refer under the strict condition and for example use ECL direct nucleic acid labeling and detection system (Amersham Pharmacia Biotech corporate system), the condition that handbook provides (wash:42 ℃, contain the primary wash buffer of 0.5xSSC).As the DNA that under the condition of strictness, can hybridize, for example, under above-mentioned stringent condition, to contain in the complementary base sequence among the DNA of base sequence of sequence number 31325-1663 position arbitrarily, at least 20 usually, preferably at least 50, especially preferably at least 100 successive base sequences are as test sample, can with the DNA of this sample hybridization.In addition, activating proteins associated matter with Nitrile hydratase of the present invention can be 112 protein that amino acid whose sequence is represented of sequence number 4, so long as have the activatory ability that participates in Nitrile hydratase, certainly also can give and expecting in aminoacid sequence, also can contain displacement, disappearance or insert 1 or a plurality of amino acid whose aminoacid sequence, and, may be modified after the translation owing to the host type difference.Replaced, lacked, inserted or the aminoacid sequence ideal example of posttranslational modification as 1～25 amino acid in this aminoacid sequence, as preferred 1～10, more preferably 1～5, most preferably 1～3.

In addition, the invention provides transformant, it is characterized in that above-mentioned DNA is imported into host cell, transforms the host.

Can obtain transformant by using the expression vector transformed host cell that makes by aforesaid method.As host cell, comprise microorganism, cells of mamma animals and vegetable cell etc., preferably utilize microorganism.As the example of microorganism, as the intestinal bacteria among the following embodiment, there is no particular limitation to this, as bacillus, Rhodopseudomonas, Corynebacterium, Brevibactcrium, streptococcus, Rhod, actinomycetes or yeast etc.

As introductory technique, can for example pass through: transform, transduce, merge transmission or electroporation etc. in well-known in the art ordinary method arbitrarily, import among the preferred host to the host microorganism quiding gene.

In addition as Nitrile hydratase of the present invention or contain the manufacture method of the bacterial disposing thing of this enzyme, as mentioned above, well-known purification process that can be by appropriate combination from the microorganism that can produce this Nitrile hydratase, for example, obtain this enzyme the culture of the microorganism of soil bacillus, particularly thermophilic Polyglucosidase soil genus bacillus Q-6 strain, in addition as mentioned above, also can from the transformant of the gene transformation of using Nitrile hydratase, obtain this enzyme.

When obtaining this enzyme, the method for culturing microbes of soil bacillus is aforesaid method, preferably but above-mentioned transformant is cultivated in the substratum of the nutrition source that contains usually these microorganism digestibility and utilizations, for example, can cultivate with the usual method of producing enzyme or antibiotic substance etc.Usually no matter cultivation is that liquid culture or solid culture can.For example, can use the suitable carbohydrate such as glucose, sucrose that mixed; Alcohols such as Sorbitol Powder, glycerine; Organic acids such as citric acid, jealous woman acid; The mixture of soybean wet goods carbon source or these materials; Nitrogenous inorganic organic nitrogen sources such as yeast extract, meat extract, sulphur ammonium, ammonium; Inorganic nutrients sources such as phosphoric acid salt, magnesium, iron, cobalt, manganese, potassium; And the substratum of vitamins such as vitamin H, VitB1.Even more ideal is to have above Fe ion of 0.1 μ g/ml or Co ion in above-mentioned medium component.Culture condition preferably carries out under aerobic condition usually.Culture temperature is not particularly limited so long as the temperature that host microorganism can grow gets final product, for example usually 5 ℃～80 ℃, preferred 20～70 ℃, more preferably carry out under 25～42 ℃.And the pH in the culturing process is not particularly limited so long as the pH that host microorganism can grow gets final product, for example usually in pH3～9, preferred pH5～8, more preferably time carry out in pH6～7.

In addition, in the present invention, use enzyme of the present invention can make nitrile compound.In this invention, when using above-mentioned enzyme, so long as do not hinder the effect of enzyme of the present invention, its degree of purification etc. is not particularly limited, except the enzyme of the present invention of purifying, also can contain thing, can use the microorganism that produces this enzyme maybe the gene of this enzyme to be imported the back transformant that transforms etc. in addition with other enzyme.When using microorganism or transformant etc., can utilize thalline, can use the viable bacteria body or use as thalline and implement acetone or the toluene equal solvent is handled or processing such as lyophilize, increased the thalline of the saturating property of compound.Also can make bacterial cell disruption thing or thalline extract etc. because of the situation difference and contain the enzyme thing.Preparation method as the bacterial disposing thing that contains this enzyme, as at first culture being carried out solid-liquid separation, the wet thallus that obtains is suspended in the damping fluids such as phosphoric acid buffer or Tris hydrochloride buffer, appropriate combination utilization ultrasonic treatment, French press are handled or are used the pulverization process of granulated glass sphere or handle with bacterial cell disruptions such as Lysozyme processing such as N,O-Diacetylmuramidase or proteolytic enzyme then, in thalline, extract this enzyme, can obtain rough Nitrile hydratase and contain liquid.As required, can be further purified this rough enzyme and contain liquid by using separation, means of purification such as well-known protein, enzyme.For example, contain organic solvents such as adding acetone, ethanol in the liquid, make its fractionation precipitation, or add sulphur ammonium etc. it is saltoutd, from the aqueous solution, make and contain the fractional precipitation of Nitrile hydratase level, the method that reclaims to rough enzyme.The affinity chromatography of appropriate combination appropriate combination anionresin again, cationic exchange, gel-filtration, use antibody or sequestrant etc. is carried out purifying.Certainly, can be by well-known method, enzyme or thalline, the bacterial disposing thing etc. that contains enzyme are filled in the pillar, also can be fixed on the carrier, particularly for thalline, also can be embedded in the polymers such as polyacrylamide gel.Thalline or bacterial disposing thing are suspended in the water-based aqueous solution of damping fluids such as water or phosphoric acid buffer etc.,, reaction are carried out by in this solution, adding nitrile compound.The thalline that uses or the concentration of bacterial disposing thing are 0.01 weight %～20 weight %, preferred 0.1 weight %～10 weight %.Preferred 90 ℃, more preferably 85 ℃, preferably preferred 70 ℃ of the upper limits of temperature of reaction, for example 1 ℃, preferred 4 ℃, more preferably 10 ℃ of the lower limits of temperature of reaction, reaction pH for example 5～10, preferred 6～8, for example 1 minute～72 hours reaction times.Also can lead to nitrile compound is dripped slowly, make it to generate high density amide compound and savings.In order from reaction solution, to reclaim amide compound, can adopt and filter or after centrifugation etc. removes thalline or bacterial disposing thing etc., utilize method that gimmick such as partial crystallization takes out etc.

Embodiment

The present invention is described in detail below to provide embodiment, and these embodiment do not limit this

Scope of invention.

Embodiment 1: thalline separates

Near the soil (about 1g) that takes a morsel and take the beautiful county of fine jade hot spring joins and 5ml physiological saline is housed in vitro, and shaking culture is 3 in 65 ℃ Shaking Incubators.Get a part this nutrient solution (0.5ml), be added in the substratum of forming by glucose 1.0 weight %, peptone 0.5 weight %, yeast extract 0.3 weight % (pH7.0), under 65 ℃, carry out reciprocating vibration on the 2nd and cultivate.By being coated on the Agar Plating that contains above-mentioned medium component through the part (0.1ml) of the nutrient solution that shaking culture obtains, under 65 ℃, cultivated again 2 days, make it form bacterium colony, microorganism is separated.By with isolating microbial inoculant to the substratum of above-mentioned same composition in added the liquid nutrient medium of N-valeronitrile of 0.1 weight % after, cultivated 24 hours down in 65 ℃, obtain to contain the nutrient solution of the high microorganism of nitrile usability.This nutrient solution 1ml is added to the acrylonitrile solution (0.05M phosphoric acid buffer pH7.7) of the 1.1 weight % of 9ml, under 27 ℃ of temperature of reaction, begins reaction.After 10 minutes, by adding the 1N HCl termination reaction of 1ml.Utilize liquid chromatography (HPLC) to analyze the part of reaction solution, have or not acrylamide to generate by detection, screening has the active microorganism of Nitrile hydratase.Obtain making that nitrile compound changes amide compound into has the thermophilic Polyglucosidase soil of the active microorganism of a hydration genus bacillus Q-6 strain by such operation.

(liquid-phase chromatographic analysis condition)

Body: HITACHI D-7000 (Hitachi, Ltd's system)

Pillar; InertSil ODS-3 (GLscience corporate system)

Length; 200mm

Column temperature; 35 ℃

Flow; 1ml/min

The sample injection rate; 10 μ l

Solution: 0.1wt% phosphate aqueous solution

Embodiment 2: the ceiling temperature that grows of yeast culture

The thermophilic Polyglucosidase soil genus bacillus Q-6 strain that embodiment 1 is obtained is coated on the Agar Plating of the medium component that contains embodiment 1 use, cultivates the growth and development state of research thalline under a plurality of differing tempss.Its result is as shown in table 1.Thermophilic Polyglucosidase soil genus bacillus Q-6 strain all shows common multiplication characteristic until 70 ℃, even also can grow under 72 ℃.

Table 1

Metewand :-do not breed ,+propagation, ++ propagation is good

Culture temperature (℃)	The propagation degree of thermophilic Polyglucosidase soil genus bacillus Q-6
Culture temperature (℃)		20	-
30	-	20	-
30	-	40	-
50	++	40	-
50	++	60	++
65	++	60	++
65	++	70	++
72	+	70	++
72	+	75	-

Embodiment 3: the active mensuration of Nitrile hydratase in the thermophilic Polyglucosidase soil genus bacillus Q-6 strain thalline and to dependence on temperature

To contain glycerine 0.2 weight %, citrate trisodium dihydrate 0.2 weight %, potassium primary phosphate 0.1 weight %, dipotassium hydrogen phosphate 0.1 weight %, peptone 0.1 weight %, yeast extract 0.1 weight %, sodium-chlor 0.1 weight %, N-valeronitrile 0.1 weight %, magnesium sulfate 7 hydrate 0.02 weight %, ferric sulfate (II) heptahydrate 0.003 weight %, substratum (pH7.0) 100ml of the bacterium of going out of cobalt chloride hexahydrate 0.0002% joins in the 500ml Erlenmeyer flask, inoculates 1ml then in advance at the thermophilic Polyglucosidase soil genus bacillus Q-6 strain nutrient solution of same culture medium culturing.Postvaccinal nutrient solution was cultivated 1 day with the 200stroke/min rotational oscillation under 65 ℃, obtained bacterial culture fluid.From the bacterial culture fluid of this thermophilic Polyglucosidase soil genus bacillus Q-6 strain of 300ml, collect thalline by centrifugation (10000 * g, 15 minutes), after 0.05M phosphoric acid buffer (pH7.5) washing, be suspended in the same damping fluid of 50ml.For the thalline suspension liquid of such preparation, with aforesaid method reaction 5 minutes, mensuration made nitrile compound change the hydration activity of amide compound into.If unit of enzyme activity (unit) is defined as the activity that made 1 μ mol vinyl cyanide change acrylamide in 1 minute be 1 unit (following, be designated as U), the Nitrile hydratase activity (U/Mg) of 27 ℃ of following per unit wet thallus weight is 9.37U/mg so.The thalline suspension liquid (5U/ml) that preparation contains 0.5 weight % vinyl cyanide under 10 ℃ uses this thalline suspension liquid again, under 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃ condition, obtains the Nitrile hydratase activity equally, and is active as shown in table 2.This result shows that in reaction optimum temperuture when using thalline near 60 ℃, particularly shows very high activity in the high-temperature zone.

Table 2

Temperature of reaction (℃)	Nitrile hydratase activity (U/ml)
Temperature of reaction (℃)	Nitrile hydratase activity (U/ml)	10	5.0
30	19.2	10	5.0
30	19.2	40	39.4
50	49.2	40	39.4
50	49.2	60	50.6
70	42.4	60	50.6

Embodiment 4: the thermostability of the Nitrile hydratase in the thermophilic Polyglucosidase soil genus bacillus Q-6 strain thalline

In order to study the active thermostability of Nitrile hydratase in the thermophilic Polyglucosidase soil genus bacillus Q-6 strain thalline, to be suspended in the distilled water according to 10U/ml by the thalline that embodiment 3 culture methods obtain, both carrying out insulation processing in 30 fens under the fixed temperature, measure remaining activity.Back thalline liquid is handled in the insulation that adds 0.5ml in the 1 weight % acrylonitrile solution (0.05M potassium phosphate buffer, pH7.5) of 0.5ml, and in 27 ℃ of following stirrings, the limit begins reaction.After 5 minutes, by adding the 1 equivalent hydrochloric acid termination reaction of 100 μ L.Calculate the activity of handling after preceding active preservation is handled with respect to preserving, the activity of preserving before handling is as shown in table 3 as the scaled value of benchmark (100).Even can think that by this result the enzymic activity of the Nitrile hydratase in the thermophilic Polyglucosidase soil genus bacillus Q-6 strain thalline at high temperature also still can stablize maintenance, under 70 ℃ high temperature, also can keep the activity more than 80%, under 80 ℃ high temperature, also can keep the activity more than 30%.

Table 3

Treatment temp (℃)	Active (U/ml)	Remaining activity (%)
Treatment temp (℃)	Active (U/ml)	Remaining activity (%)	30	5	100
40	4.8	96	30	5	100
40	4.8	96	50	4.5	90
60	4.4	88	50	4.5	90
60	4.4	88	70	4.2	84
80	1.6	32	70	4.2	84

Embodiment 5: with the reaction of various nitrile compounds as substrate

Study at the Nitrile hydratase activity that makes the various nitrile compounds that provide in the following table 4 change corresponding amide compound into.In the 1.1% nitrile solution (0.05M potassium phosphate buffer, pH7.5) of 9ml, add 1ml thalline suspension liquid, under 30 ℃ of temperature of reaction, begin reaction.After 10 minutes by adding the 1N HCl termination reaction of 1ml.The analysis condition of used HPLC is identical with embodiment 1, solution is made the distilled water that contains the 10wt% acetonitrile.Its result shows that all nitrile compounds that table 4 is provided as substrate, all have the Nitrile hydratase activity.

Table 4

For the experiment nitrile compound
For the experiment nitrile compound		Adiponitrile acetonitrile isopropyl cyanide n-valeronitrile	The own nitrile benzene of n-butyronitrile nitrile

With being summarized as follows of embodiment 6 the following stated until illustrating from the aminoacid sequence of Nitrile hydratase α subunit (ORF2), β subunit (ORF1) and the Nitrile hydratase sensitization factor (ORF3) of thermophilic Polyglucosidase soil genus bacillus Q-6 strain and the process of the present invention of base sequence.

After will cultivating the bacterial cell disruption that obtains to thermophilic Polyglucosidase soil genus bacillus Q-6 strain, carry out ammonium sulfate precipitation, anion exchange chromatography, DEAE column chromatography, hydroxyapatite column chromatography, gel permeation chromatography, dialysis, the Nitrile hydratase enzyme is carried out purifying.

The aminoacid sequence of the α subunit of the Nitrile hydratase of mensuration purifying and about 30 residues of N-terminal of β subunit, the amino acid whose codon of considering the affiliated Pseudomonas of this bacterium uses, preparation is used for the oligonucleotide degenerated primer of gene amplification, with the chromosomal DNA that extracts from this thalline as template, carry out degenerate pcr, obtain amplification of DNA fragments.The dna fragmentation that is amplified is cloned, measure and insert segmental base sequence.Compare to the aminoacid sequence of inferring out with from the Nitrile hydratase α subunit of thermophilic Polyglucosidase soil genus bacillus Q-6 strain purifying and the N-terminal aminoacid sequence of β subunit, confirm the sequence encoding Nitrile hydratase of being cloned from this base sequence.

The result shows that the Nitrile hydratase gene that is begun by 5 ' end side upstream exists according to β subunit, α subunit order adjacency in thermophilic Polyglucosidase soil genus bacillus Q-6 strain.

The oligonucleotide degenerated primer that is used for gene amplification according to the sequence preparation that homology is high in the downstream gene of well-known various Nitrile hydratase α subunits, carry out degenerate pcr with the chromosomal DNA that extracts from this thalline as template, obtain amplification of DNA fragments.Amplification of DNA fragments to the α subunit part of this bacterium of obtaining is cloned, and measures base sequence.

The Nitrile hydratase α subunit and the β subunit of the above thermophilic Polyglucosidase soil genus bacillus Q-6 strain that obtains are imported suitable expression.Use the expression plasmid that makes up, transform suitable host bacterium.As the host, as Rhod, Corynebacterium, intestinal bacteria etc.The host who does not preferably have Ntn hydrolase.Make in addition and immerse oneself in the resulting thalline of transformant and in aqueous solvent, contact, confirm the generation of acrylamide, this formation efficiency and Nitrile hydratase activity are compared with this with vinyl cyanide.

Then with the above-mentioned dna fragmentation that obtains as probe, carry out colony hybridization, the peripheral gene of the downstream gene of the Nitrile hydratase α subunit that comprises thermophilic Polyglucosidase soil genus bacillus Q-6 strain and β subunit is cloned.

Make downstream gene and Nitrile hydratase α subunit and β subunit coexpression, relatively the Nitrile hydratase activity.Found that the relevant gene of activation that downstream gene is and the Nitrile hydratase activity is significantly risen.

Embodiment 6: from the purifying of the Nitrile hydratase of thermophilic Polyglucosidase soil genus bacillus Q-6 strain

Strain is cultivated to thermophilic Polyglucosidase soil genus bacillus Q-6, by various column chromatographies the Nitrile hydratase active fraction is carried out purifying.

The measuring method of the Nitrile hydratase active fraction in the chromatography is following to carry out.In with each fraction elutriant of HEPES damping fluid (100mM, pH7.2) dilution, add the vinyl cyanide of 1 weight %, reacted 1 minute down in 27 ℃.Make reaction terminating by add 10 liquid measure %1N HCl to reaction solution, the acrylamide concentration of utilizing embodiment 1 described HPLC assay to generate.

For the Nitrile hydratase enzyme from thermophilic Polyglucosidase soil genus bacillus Q-6 strain is carried out purifying, at first thermophilic Polyglucosidase soil genus bacillus Q-6 strain is seeded in V/F substratum (the glycerine 0.2 weight % of the N-valeronitrile that contains 0.1 weight %, citrate trisodium dihydrate 0.2 weight %, potassium primary phosphate 0.1 weight %, dipotassium hydrogen phosphate 0.1 weight %, peptone 0.1 weight %, yeast extract 0.1 weight %, sodium-chlor 0.1 weight %, N-valeronitrile 0.1 weight %, magnesium sulfate 7 hydrate 0.02 weight %, ferric sulfate (II) heptahydrate 0.003 weight %, cobalt chloride hexahydrate 0.0002 weight %), cultivated 24 hours in 65 ℃.In cultivation, use the dark base plate (COSTAR company) of 96 hole 2ml.Cultivate to finish the back, by 8000g, 10 minutes centrifugation collection bacterium, will obtain 3g wet thallus resuspending in the HEPES of 20ml damping fluid (100mM, pH7.2).Under cooling, thalline is carried out fragmentation with the broken instrument of ultrasound, in bacterial cell disruption liquid, add ammonium sulfate (30% saturation concentration), under 4 ℃, stirred lentamente 30 minutes, under 20000g, carry out 10 minutes centrifugation, obtain supernatant.In centrifuged supernatant, add ammonium sulfate (70% saturation concentration), after stirring 30 minutes lentamente under 4 ℃, by in 20000g centrifugation in following 10 minutes, the throw out that obtains is dissolved in again the HEPES damping fluid (100mm, pH7.2) of 9ml, in the same solution of 1L, dialysed 24 hours down in 4 ℃, carry out anion-exchange chromatography (Amersham Bioscience company; HiTrap DEAE FF (column volume 5ml * 5 piece)).Developping solution uses HEPES damping fluid (100mm, pH7.2), increases until the 0.5M straight line from 0.0M by making potassium chloride concentration, carries out the classification wash-out, obtains containing the active fraction of Nitrile hydratase.This fraction is carried out phosphatic rock column chromatography (BIO-RAD corporate system; CHT2-I (column volume 2ml)).As developping solution, carry out the classification wash-out from 0.01M until the increase of 0.3M straight line with the 0.01M potassiumphosphate aqueous solution (pH7.2), obtain containing the active fraction of Nitrile hydratase by making potassiumphosphate.This fraction is carried out with the 0.05M sodium phosphate aqueous solution (pH7.2) that contains 0.15M NaCl gel permeation chromatography (the Amersham Bioscience company as developping solution; Superdex 200HR 10/30), obtain the Nitrile hydratase active fraction.Use the Nitrile hydratase active fraction of the gel permeation chromatography that obtains like this to carry out following embodiment.

Embodiment 7: by the temperature of reaction dependency of the Nitrile hydratase in the Nitrile hydratase active fraction of thermophilic Polyglucosidase soil genus bacillus Q-6 strain purifying

For Nitrile hydratase active fraction solution (3.2mg/mL, 0.05M phosphoric acid buffer (pH7.5)) from thermophilic Polyglucosidase soil genus bacillus Q-6 strain, under temperature of reaction shown in the table 5 to the Nitrile hydratase activity that nitrile compound changes amide compound into is measured.0.5 weight % acrylonitrile solution (0.05M potassium phosphate buffer, pH7.5) to 1mL adds Nitrile hydratase active fraction solution, is beginning reaction at each temperature while stirring.After 2 minutes by adding the 1N hydrochloric acid termination reaction of 100 μ L.It is 1 unit (following, be designated as U) that unit of enzyme activity (unit) is defined as in 1 minute activity that vinyl cyanide with 1 μ mol changes acrylamide into, and table 5 has provided the hydration activity (U/mg) of per unit enzyme weight.The activity that this result shows the Nitrile hydratase from the Nitrile hydratase active fraction of thermophilic Polyglucosidase soil genus bacillus Q-6 strain purifying is under the high temperature of 60 degree, is accompanied by the rising of temperature of reaction and rises.Same when using thalline in optimum temperuture and the reaction, think to be near 60 ℃, even under the high temperature of 70 degree, also show very high Nitrile hydratase activity.

Table 5

Temperature of reaction (℃)	Active (U/mg)
Temperature of reaction (℃)	Active (U/mg)	20	210.4
27	550.7	20	210.4
27	550.7	40	1135.3
50	2228.8	40	1135.3
50	2228.8	60	2823.3
70	2781.1	60	2823.3

Embodiment 8: from the thermostability of the Nitrile hydratase of thermophilic Polyglucosidase soil genus bacillus Q-6 strain purifying

In order to study from the active thermostability of the Nitrile hydratase of thermophilic Polyglucosidase soil genus bacillus Q-6 strain purifying, Nitrile hydratase active fraction solution (3.2mg/ml, 0.05M phosphoric acid buffer (pH7.5)) both carrying out 30 minutes insulation processing under the fixed temperature, is measured remaining activity.In the 0.5 weight % acrylonitrile solution (0.05M potassium phosphate buffer, pH7.5) of 1mL, add the Nitrile hydratase solution 5 μ l after insulation is handled, under 27 ℃, begin reaction while stirring.After 2 minutes by adding the 1N HCl termination reaction of 100 μ l.Calculate with respect to preserving and handle preceding active preservation processing back active (remaining activity), preceding active as shown in table 6 to preserve processing as the scaled value of benchmark (100).Even can think that by this result the enzymic activity of Nitrile hydratase at high temperature also keeps stable in the aqueous solution from the Nitrile hydratase active fraction of thermophilic Polyglucosidase soil genus bacillus Q-6 strain purifying, under 60 ℃ high temperature, also can keep the activity more than 60%, under 70 ℃ high temperature, also can also keep the activity more than 35%.

Table 6

Treatment temp (℃)	Remaining activity (%)
Treatment temp (℃)	Remaining activity (%)	20	89.2
27	85.6	20	89.2
27	85.6	40	82.3
50	78.9	40	82.3
50	78.9	60	66.9
70	38.8	60	66.9

Embodiment 9: from the Nitrile hydratase of thermophilic Polyglucosidase soil genus bacillus Q-6 strain purifying dependency and the concentration tolerance to acrylonitrile concentration

For Nitrile hydratase from thermophilic Polyglucosidase soil genus bacillus Q-6 strain purifying, in order to study to dependency and tolerance as the acrylonitrile concentration of substrate, the Nitrile hydratase active fraction solution (3.2mg/mL, 0.05M phosphoric acid buffer (pH7.5)) of 4 μ l is added to 5ml contains in the acrylonitrile solution (0.05M potassium phosphate buffer, pH7.5) of various weight %, under 27 ℃, begin reaction while stirring.After 5 minutes, 10 minutes, 20 minutes, 40 minutes, respectively get 1ml, by adding the 1N HCl termination reaction of 100 μ L,, as shown in table 7 by the concentration of the quantitative acrylamide that generates of HPLC.Even can think that by this result the Nitrile hydratase enzymic activity from the aqueous solution of the Nitrile hydratase active fraction of thermophilic Polyglucosidase soil genus bacillus Q-6 strain purifying also can keep stable under high acrylonitrile concentration.Even reaction is 40 minutes in 6% high density acrylonitrile solution, compare with lower acrylonitrile concentration situation, do not observe active decline, be accompanied by the rising of concentration of substrate on the contrary, activity also rises.

Table 7

Acrylonitrile concentration (weight %) during the reaction beginning	Acrylamide concentration after 5 minutes (weight %)	Acrylamide concentration after 10 minutes (weight %)	Acrylamide concentration after 20 minutes (weight %)	Acrylamide concentration after 40 minutes (weight %)
	Acrylamide concentration after 5 minutes (weight %)	Acrylamide concentration after 10 minutes (weight %)	Acrylamide concentration after 20 minutes (weight %)	Acrylamide concentration after 40 minutes (weight %)	0.5％	0.018	0.032	0.052	0.087
2.0％	0.023	0.042	0.070	0.107	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	4.0％	0.026	0.046	0.077	0.137

Embodiment 10: from the acrylamide concentration tolerance of the Nitrile hydratase of thermophilic Polyglucosidase soil genus bacillus Q-6 strain purifying

For Nitrile hydratase from thermophilic Polyglucosidase soil genus bacillus Q-6 strain purifying, suppress in order to study the enzyme that causes by the resultant acrylamide, the Nitrile hydratase active fraction solution (3.2mg/ml, 0.05M phosphoric acid buffer (pH7.5)) of 10 μ l is added in the 1ml solution (0.05M potassium phosphate buffer, pH7.5) that contains 0.5 weight % vinyl cyanide and 35 weight % acrylamides, under 27 ℃, carry out reaction in 10 minutes while stirring, by the acrylonitrile concentration in the solution after the HPLC quantitative reaction, find that all vinyl cyanide all change acrylamide into.Even can think that by this result the enzymic activity of the Nitrile hydratase from the aqueous solution of the Nitrile hydratase active fraction of thermophilic Polyglucosidase soil genus bacillus Q-6 strain purifying also still keeps active under 35% high acrylamide concentration.

Embodiment 11: from the clone of the gene of the Nitrile hydratase β subunit of thermophilic Polyglucosidase soil genus bacillus Q-6 strain and α subunit part

(1) from the affirmation and the N-terminal determined amino acid sequence of the Nitrile hydratase enzyme of thermophilic Polyglucosidase soil genus bacillus Q-6 strain

To under reductive condition, carry out reduced form sds polyacrylamide electrophoresis by the Nitrile hydratase active fraction elutriant that embodiment 6 obtains through gel permeation chromatography.Carry out protein staining, decolouring with Xylene Brilliant Cyanine G (CBB) behind the electrophoresis, 2 main bands that the result has about 25K dalton and about 28K Dalton molecular weight are identified.Use blotter (BIO-RAD company) that these 2 main protein purifications are transferred on the pvdf membrane (MILLIPORE corporate system), carry out CBB dyeing, the part that is adsorbed with the band of 2 clauses and subclauses is scaled off from pvdf membrane.Use full-automatic prlmary structure of protein analyser PPSQ-23A (Shimadzu Seisakusho Ltd.) to measure 2 kinds of protein N terminal amino acid sequences then.The result shows that the daltonian proteinic N of molecular weight 25K end aminoacid sequence is the sequence of sequence number 23 expressions, and the daltonian proteinic N of molecular weight 28K end aminoacid sequence is the sequence of sequence number 24 expressions.

With the aminoacid sequence of known Nitrile hydratase enzyme relatively, although the result shows that the daltonian polypeptide chain of 25K and Nitrile hydratase α subunit, the daltonian polypeptide chain of 28K and Nitrile hydratase β subunit homology are low, all show homology, hint this protein of encoding.

(2) synthetic corresponding to the Oligonucleolide primers of N-terminal aminoacid sequence

By 2 kinds of proteinic N-terminal aminoacid sequences of said determination, according to the synthetic following 12 kinds of degeneracy oligonucleotide primers that are used for PCR of codon use of this Pseudomonas.Primer 1 shown in the sequence number 5 (α F1), primer 2 shown in the sequence number 6 (α F2), primer 3 shown in the sequence number 7 (α F3), primer 4 shown in the sequence number 8 (α R1), primer 5 shown in the sequence numbering 9 in the sequence table (α R2), primer 6 shown in the sequence number 10 (α R3), primer 7 shown in the sequence numbering 11 in the sequence table (β F1), primer 8 shown in the sequence number 12 (β F2), primer 9 shown in the sequence number 13 (β F3), primer 10 shown in the sequence number 14 (β R1), primer 11 shown in the sequence number 15 (β R2), primer 12 shown in the sequence number 16 (β R3).Wherein y represents C or T, and R represents a or g, and m represents a or c, and k represents g or t, and s represents c or g, and w represents a or t, and d represents a, g or t, and n represents a, c, g or t.In addition, consider the position of gene on karyomit(e) of coding for alpha subunit and β subunit, synthetic primer.

(3) extraction and the degenerate pcr of thermophilic Polyglucosidase soil genus bacillus Q-6 strain chromosomal DNA

Thermophilic Polyglucosidase soil genus bacillus Q-6 strain is cultivated, reclaimed by method similarly to Example 6, use Genomic-tip system (500/G) test kit of QIAGEN company from thalline, to extract chromosomal DNA.The chromosomal DNA that is dissolved in the thermophilic Polyglucosidase soil genus bacillus Q-6 strain of TE solution with 0.1 μ g carries out degenerate pcr as template.Primer shown in the sequence number 5 to 10 1 to 6 and the primer 7 to 12 shown in the sequence number 11 to 16 are carried out degenerate pcr according to 36 kinds of combinations.Use contains each 2 kinds of the primers of 100pmol, and the reaction solution of the ExTaq archaeal dna polymerase of the TA kara company of 5U and the total amount 100 μ l of damping fluid carries out degenerate pcr, attempts amplification of DNA fragments.Reaction conditions is as follows.After 96 ℃, 3 minutes thermally denatures, carry out 35 circulations of 96 ℃, 30 seconds thermally denatures, annealing in 42 ℃, 30 seconds, 72 ℃, 1 minute 30 seconds extension after, carry out 5 minutes extensions in 72 ℃, remain under 4 ℃.Each PCR product is carried out 1 weight % agarose gel electrophoresis, when confirming whether DNA increases, the primer 8 (β F2) that the primer 5 (α R2) that has only the combination of the primer 7 (β F1) that the primer 5 (α R2) that provides with the sequence numbering in the sequence table 9 and sequence numbering 11 in the sequence table provide and provide with the sequence numbering in the sequence table 9 and the sequence numbering 12 in the sequence table provide is when being combined into performing PCR, and the dna fragmentation of the 700bp that can confirm to have an appointment increases.

(4) base sequence of the clone of degenerate pcr product and amplification of DNA fragments is measured

From gel, the dna fragmentation that is amplified is cut out, extract, this fragment is connected with pGEM-T carrier (Promega company) with T4DNA ligase enzyme (TAkara company) with QIAquick GE1 Extraction Kit (QIAGEN company).Utilized use ExTaq to carry out PCR, the result can be in the character of 3 ' terminal additional 1 base A.After the ligation, transformed into escherichia coli JM109 strain, in LB nutrient agar (50 μ g/ml penbritins, 0.5 weight % bacterium are used agar (pH7.5) with yeast extract, 1 weight % bacterium with trypsinase peptone, 0.5 weight %NaCl, 2.0 weight % bacteriums),, utilize penbritin to select transformant in 37 ℃ of following overnight incubation.From with extracting plasmid DNA the transformant of LB culture medium culturing that contains penbritin, the sequence of using the SP6 that is on the carrier and T7 promotor is measured the base sequence of about 700bp insertion sequence as primer according to ordinary method.

The result confirms the open reading frame (being called ORF1 later on) of 3681bp in amplification of DNA fragments.Between the translation initiation codon ATG of the translation stop codon of ORF1 and next open reading frame (being called ORF2 later on), 13bp is arranged.25 aminoacid sequences of N-terminal side of the daltonian polypeptide chain of 28K of being inferred 25 aminoacid sequences of N-terminal side that and above-mentioned purifying by the base sequence of ORF1 are in full accord, are equivalent to the 1st to the 25th sequence of aminoacid sequence shown in the sequence number 2.Although homology is low between the aminoacid sequence of the aminoacid sequence of ORF1 and known Nitrile hydratase β subunit, also show homology, this protein of hint coding.

The Nitrile hydratase β subunit of thermophilic Polyglucosidase soil genus bacillus Q-6 strain 226 amino acid of encoding, with the consistent degree of the proteinic aminoacid sequence that in existing database, has homology according to the rank order that begins by height, be 43% with the Nitrile hydratase β subunit of Klebsiella MC12609 strain, be 42% with the Nitrile hydratase β subunit of Agrobacterium, with Rhodopseudomonas CGA009 strain Nitrile hydratase β subunit be 40%, very low.In addition, with from the proteinic amino acid whose consistent degree of the near genus bacillus of soil bacillus sibship also according to ordering from high to low, be 35.0% with the Nitrile hydratase β subunit of thermophile bacteria Bacillus BR449 strain, with the Nitrile hydratase β subunit of thermophile bacteria Shi Shi genus bacillus SC-J05-1 strain be 34.5%, very low.On the other hand, the Nitrile hydratase β subunit of thermophile bacteria Bacillus BR449 strain and the Nitrile hydratase β subunit of thermophile bacteria Shi Shi genus bacillus SC-J05-1 strain have 85.6% very high consistent degree.The aminoacid sequence of the daltonian polypeptide chain N-terminal of 25K of the aminoacid sequence of the N-terminal of being inferred by the base sequence of ORF2 and above-mentioned purifying is in full accord in addition.

Distinguish in thermophilic Polyglucosidase soil genus bacillus Q-6 strain by above result, the gene of the gene of the coding 28K dalton Nitrile hydratase β subunit that 5 ' end side upstream begins, coding 25K dalton Nitrile hydratase α subunit according to such order in abutting connection with existing.

(5) clone of the gene of thermophilic Polyglucosidase soil genus bacillus Q-6 strain Nitrile hydratase α subunit part

For the peripheral gene of the gene of the Nitrile hydratase β subunit of the above-mentioned thermophilic Polyglucosidase soil genus bacillus Q-6 strain that obtains part and the gene of α subunit part are cloned, carry out degenerate pcr.Gene with reference to being positioned at the downstream of known Nitrile hydratase α subunit prepares following 2 kinds of Oligonucleolide primers that are used for degenerate pcr.Be the primer 13 (pR1) that provides of sequence number 17, the primer 14 (pR2) that sequence number 18 provides.

In addition, the following 2 kinds of Oligonucleolide primers that are used for pcr amplification of the interior preparation of Nitrile hydratase β subunit of the thermophilic Polyglucosidase soil genus bacillus Q-6 strain of base sequence have formerly been measured.Be the primer 15 (Q6AposF) that provides of sequence number 19, the primer 16 (Q6abF1) that sequence number 20 provides.Carry out degenerate pcr with the thermophilic Polyglucosidase soil of 0.1 μ g genus bacillus Q-6 strain chromosomal DNA as template.Degenerate pcr is under annealing temperature 50 degree, and 4 kinds of combinations of the primer 15,16 that primer 13,14 that provides according to sequence number 17,18 and sequence number 18,19 provide are carried out.Can confirm to have the DNA amplification product of about 0.8kb among the PCR that the combination of the primer 15 (Q6AposF) that the primer 13 (pR1) that provides with sequence number 17 and sequence number 18 provide carries out by this result.And being combined in the performing PCR of the primer 16 (Q6abF1) that the primer 13 (pR1) that provides with sequence number 16 and sequence number 19 provide confirms to have the DNA amplification product of 1.5k.When being combined into performing PCR, can not confirm the existence of DNA amplification product by other.

The dna fragmentation of the 0.8kb of the degenerate pcr amplification that the combination of the primer 15 (Q6AposF) that will provide by the primer 13 (pR1) that provides with sequence number 16 and sequence number 19 is carried out cuts out from sepharose, extract DNA according to ordinary method, be incorporated in the pGEM-T carrier (Promega company), transformed into escherichia coli JM109 strain is selected recombinant chou by the penbritin of 50 μ g/ml.With the LB culture medium culturing transformant that contains penbritin, utilize ordinary method to extract plasmid DNA, measured the base sequence of the insertion portion of about 0.8kb.This result can confirm the open reading frame (being called ORF2 later on) of 618bp.29 aminoacid sequence of the aminoacid sequence that the N-terminal side of being inferred by the base sequence of ORF2 is 29 and the daltonian polypeptide chain N-terminal of the 25K side of above-mentioned purifying is in full accord, is equivalent to the 1st to the 29th sequence of aminoacid sequence shown in the sequence number 1.Although the aminoacid sequence homology of the aminoacid sequence of ORF2 and known Nitrile hydratase α subunit is low, show homology, this protein of hint coding.

205 amino acid of Nitrile hydratase α subunit coding of thermophilic Polyglucosidase soil genus bacillus Q-6 strain, with the consistent degree of the proteinic aminoacid sequence that has homology in the data with existing storehouse according to ordering from high to low, be 66.3% with the Nitrile hydratase β subunit of thermophile bacteria Bacillus BR449 strain, be low to moderate 63.9% with the Nitrile hydratase β subunit of thermophile bacteria Shi Shi genus bacillus SC-J05-1 strain.And the Nitrile hydratase β subunit consistence of the Nitrile hydratase β subunit of thermophile bacteria Bacillus BR449 strain and thermophile bacteria Shi Shi genus bacillus SC-J05-1 strain reaches 88.8%, has very high homology.

Embodiment 12: the Nitrile hydratase α subunit of thermophilic Polyglucosidase soil genus bacillus Q-6 strain and β subunit part are to expression vector integration and the expression in intestinal bacteria

(1) the Nitrile hydratase α subunit of thermophilic Polyglucosidase soil genus bacillus Q-6 strain and β subunit part is to the integration of expression vector

As the basis, preparation is used for following 2 kinds of Oligonucleolide primers of Nitrile hydratase α subunit and β subunit partly being increased by PCR with the base sequence of said determination.Be the primer 17 (Q6ab-F1-T) that provides of sequence number 21, the primer 18 (Q6ABall-R1-BglII-T) that sequence number 22 provides.The primer 17 that sequence number 21 provides has designed the translation initiation codon of the Nitrile hydratase β subunit of thermophilic Polyglucosidase soil genus bacillus Q-6 strain among the NdeEI of restriction enzyme position.The primer 18 that provides for sequence number 22 is importing restriction enzyme BglII site below translation stop codon of the Nitrile hydratase α subunit of thermophilic Polyglucosidase soil genus bacillus Q-6 strain.As template, primer 17,18 each 100pmol that use sequence number 21,22 to provide carry out PCR with the chromosomal DNA of thermophilic Polyglucosidase soil genus bacillus Q-6 strain.Use the ExTaq archaeal dna polymerase, in total amount 100 μ l after carrying out 30 round-robin PCR under the condition under 96 ℃, after 3 minutes thermally denatures, according to 96 ℃ of 30 seconds thermally denatures, 60 ℃ of annealing in 30 seconds, 72 ℃ of 1 fen 30 seconds extensions, under 72 ℃, carry out 5 minutes extensions, then 4 ℃ of coolings down.When the solution behind the PCR being carried out 1.5 weight % agarose gel electrophoresis, can confirm to amplify the dna fragmentation of about 1.3kb.This DNA amplification product is extracted with ordinary method from sepharose, be connected on the pGEM-Teasy carrier of Promega company transformed into escherichia coli JM109 strain then.Extract plasmid DNA from transformant, measure the base sequence of insertion portion, confirming does not have mistake in the pcr amplification.

Next with this plasmid NdeI, 1.5 weight % agarose gel electrophoresis are carried out in the digestion of EcoRI restriction enzyme, and the insertion DNA of about 1.3Kb is cut out from sepharose, utilize the ordinary method extraction.PET-26b (+) carrier of use NOvagene company and pET-28a (+) carrier are as expression vector.With these 2 kinds of carrier DNA NdeI, 1 weight % agarose gel electrophoresis is carried out in the digestion of EcoRI restriction enzyme, utilizes ordinary method to extract the dna fragmentation of about 5.3Kb.According to conventional methods these insertion fragments and carrier are carried out ligation, transformed into escherichia coli JM109 strain is extracted plasmid DNA from the transformant that utilizes kalamycin resistance to select, and selects to have imported and inserts segmental plasmid.Can obtain the Nitrile hydratase α subunit of thermophilic Polyglucosidase soil genus bacillus Q-6 strain and the expression plasmid that β subunit part imports as the insertion fragment by above.Below the plasmid that will finally prepare is referred to as pET-26b (+)-β α and pET-28a (+)-β α.

(2) utilize the Nitrile hydratase activity of the thermophilic Polyglucosidase soil genus bacillus Q-6 strain of escherichia coli expression

Transform e. coli bl21 (DE3) the LysE strain of Novagene company with expression plasmid pET-26b (+)-β α and pET-28A (+)-β α, make the protein of the Nitrile hydratase β subunit of thermophilic Polyglucosidase soil genus bacillus Q-6 strain and α subunit as the polycistron coexpression by the T7 promotor.

Transform the competent cell of e. coli bl21 (DE3) the LysE strain of Novagene corporate system with each expression plasmid, with the bacterium liquid after the conversion processing be coated in the LB nutrient agar that contains 30 μ g/ml kantlex (0.5 weight % bacterium with yeast extract, 1 weight % bacterium with trypsinase peptone, 0.5 weight %NaCl, 2.0 weight % agar; PH7.5) on,, utilize kantlex to select in 30 ℃ of following overnight incubation.This transformant is inoculated into 2ml contains in the LB substratum of 30 μ g/ml kantlex, under 30 ℃, spend the night with the 200rpm shaking culture.Above-mentioned nutrient solution is inoculated into according to 2% weight contains 20 μ g/ml cobalt chloride sky hydrate (CoCl ₂6H ₂O) and in the 10ml LB substratum of 30 μ g/ml kantlex, under 30 ℃ about 3 hours with the 200rpm shaking culture, until OD600=0.5, add the IPTG of 0.1mM, induce the expression that begins from the T7 promotor after, 200rpm shaking culture 4 hours reclaims thalline.

Use this thalline, mensuration makes nitrile compound change the Nitrile hydratase hydration activity of amide compound under 27 ℃.

To the coli somatic of expressing from the Nitrile hydratase of thermophilic Polyglucosidase soil genus bacillus Q-6 strain be dissolved in the TBS damping fluid (pH7.5) of the NaCl that contains 20mM Tris-HCl and 15mM again, be diluted to OD=0.2, make the reaction solution of final concentration 0.2 weight % acrylonitrile solution.Under 27 ℃ while stirring the reaction, after 30 minutes by adding the 1 equivalent hydrochloric acid termination reaction of 100 μ L.It is 1 unit (following, be designated as U) that unit of enzyme activity (unit) is defined as the activity that the 1mol vinyl cyanide is changed into acrylamide in 1 minute, and table 8 has provided the hydration activity (U/mg) of per unit wet thallus weight.

Table 8

Expression plasmid	Active (U/mg)
Expression plasmid	Active (U/mg)	PET-26b (+) carrier	0
pET-26b(+)-βα	0.200	PET-26b (+) carrier	0
pET-26b(+)-βα	0.200	PET-28a (+) carrier	0
pET-28a(+)-βα	0.212	PET-28a (+) carrier	0

Distinguish that by this result it has the Nitrile hydratase activity that vinyl cyanide is converted to acrylamide when the Nitrile hydratase α subunit that makes thermophilic Polyglucosidase soil genus bacillus Q-6 strain and β subunit during at expression in escherichia coli.

Embodiment 13: obtain from the peripheral gene of the Nitrile hydratase of thermophilic Polyglucosidase soil genus bacillus Q-6 strain by colony hybridization

(1) preparation of fluorescent mark DIG probe

The DNA of the Nitrile hydratase α subunit part of the thermophilic Polyglucosidase soil genus bacillus Q-6 strain that provides with sequence number 25 uses the DIG-DNA labelling kit of Roche Holding Ag's system to prepare fluorescence labeling probe as template.The preparation method is according to the DIG handbook of Roche Holding Ag.

(2) chromosomal DNA hybridization

The chromosomal DNA of the thermophilic Polyglucosidase soil genus bacillus Q-6 strain of preparation among the embodiment 11 is digested with various restriction enzymes, carry out 1 weight % agarose gel electrophoresis.DNA in the sepharose is transferred to nylon membrane Hybond)-N+ (Amersham company) after, use the fluorescent mark DIG probe of previous preparation, carry out chromosomal DNA hybridization.With the film that shifts, fixed DNA according to the ratio of every 10ml be immersed in hybridization buffer (contain 1 weight % skim-milk, 0.1 weight %N-Sarkosyl L ester, 0.02 weight %SDS, 50 weight % methane amides 5 * SSC) in, under 42 ℃, carry out 2 hours prehybridizations.With above-mentioned same, the fluorescence labeling probe of 100ng preparation is made its thermally denature by boiling in 10 minutes to handle with chilling under 95 ℃, add in the prehybridization damping fluid, in 42 ℃ down hybridization spend the night.Film after the hybridization is washed 2 times under room temperature in the 2 * SSC that contains 0.1 weight %SDS of 150ml.In being heated to 65 ℃ the 1 * SSC that contains 0.1 weight %SDS of 150ml, carry out 2 times 5 minutes washing then.Next toxilic acid damping fluid (being transferred to pH7.5 with 0.1M toxilic acid, 0.15M NaCl, the NaOH) washing of using 100ml is after 5 minutes, at lock solution (the 0.1M toxilic acid damping fluid that contains 0.3 weight %Tween20,0.15M NaCl and 1 weight % skimming milk of 50ml; PH7.5) in, under room temperature, carry out 30 minutes sealing treatment.Lock solution with 20ml will resist digoxigenin-AP to be diluted to 75mU/ml, after carrying out 30 minutes antibody response under the room temperature, at lavation buffer solution (the 0.1M toxilic acid damping fluid that contains 0.3 weight %Tween20,0.15M NaCl of 100ml; PH7.5) in film is washed 5 times, will do not had bonded antibody to wash away.After in the detection damping fluid (0.1M Tris-HCl, 0.1M NaCl, pH9.5) of 20ml, carrying out 5 minutes Balance Treatment, be formed in NBT (nitroblue tetrazolium(NBT)) solution that contains the 100mg/ml of 34 μ l in the detection damping fluid of 10ml, chromophoric substrate solution NBT/BCIP (5-bromo-4-chloro-3-indolylphosphate) after the dilution of the BCIP solution of the 50mg/ml of 35 μ l, film is immersed in this solution fully lucifuge incubation 1 minute to 16 hours.Dish is moved on to, do not shake yet, carry out color development and confirm.The result shows in the about 2.3kb gene fragment with restriction enzyme HiIII digestion, contains the downstream gene of Nitrile hydratase α subunit part.

(3) obtain the purpose clone by colony hybridization

1. be used for the making of the plasmid library of colony hybridization

Use same fluorescent mark DIG probe, carry out colony hybridization.The chromosomal DNA of the thermophilic Polyglucosidase soil of 10 μ g genus bacillus Q-6 strain is carried out 1 weight % agarose gel electrophoresis, the part that will contain the dna fragmentation of the 2.0kb to 2.6kb that has an appointment from sepharose cuts out, and uses with above-mentioned same method dna fragmentation is extracted and purifying.The dna fragmentation that obtains is imported the interior Hind III restriction enzyme position of multiple clone site that is in pUC118 plasmid vector (Takara corporate system) with dna ligation kit (Takara corporate system).The pUC118 plasmid vector DNA that uses in the connection is the DNA through following processing: after using restriction enzyme Hind III digestion, carry out purifying by phenol/chloroform processing and ethanol sedimentation, after the dephosphorylation of using alkaline phosphatase (Takara corporate system) to carry out 5 ' end is then handled, carrying out phenol/chloroform once again handles and ethanol sedimentation, carry out agarose gel electrophoresis, carry out repurity by from sepharose, extracting.

Use has connected the solution transformed into escherichia coli JM109 strain of chromosomal DNA of the thermophilic Polyglucosidase soil genus bacillus Q-6 strain of about 2.0kB to 2.6kB fragmentation at Hind III restriction enzyme position in the pUC118 plasmid vector, be seeded in the penbritin that contains 50 μ g/ml, (0.5 weight % bacterium is used yeast extract to the LB nutrient agar of the IPTG of 1mM (isopropyl-) and 2 weight %X-GAl (5-bromo-4-chloro-3-indoles-β-D-galactopyranoside), 1 weight % bacterium is used the trypsinase peptone, 0.5 weight %NaCl, 2.0 weight % agar; PH7.5) on, in 37 ℃ of overnight incubation.The result can obtain many pieces every piece plate that occurs 50 to 500 white colonies.

Plasmid library for these chromosomal DNAs uses the previous fluorescent mark DIG probe for preparing, and carries out colony hybridization, and screening contains the clone of purpose Nitrile hydratase α subunit downstream gene.

2. obtain the purpose clone by colony hybridization

The white bacterium colony that at first will occur about 1000 clones rule on new LB nutrient agar with the toothpick of sterilizing.At this moment, line equally on the LB nutrient agar of L B nutrient agar that makes film hybridization and preservation usefulness is in 30 ℃ of following overnight incubation.

Then, on the plate of growth bacterium colony, put the nylon membrane Hybond-N+ of Amersham corporate system gently, from the limit, take out at leisure after 1 minute with tweezers.With the film that peels off according to adhere to thalline towards last in denaturing soln (the 0.5M NaOH aqueous solution that contains 1.5M NaCl), soak 7 minutes like that after, at neutralization solution (the 0.5M Tris aqueous hydrochloric acid that contains 1.5M NaCl and 1mM EDTA2Na; PH7.2) soaked 3 minutes in, and then in new neutralization solution, soaked 3 minutes.Next after using 2 * SSC solution (containing NaCl 8.76g, Trisodium Citrate 4.41g among 1 liter of 1 * SSC) to wash 1 time, on the filter paper of doing that film is air-dry.Again by carrying out 120mJ/cm ²UV irradiation, DNA is fixed on the film.

3. utilize DIG antibody to detect and separating that purpose is cloned

With fixing of above-mentioned processing DNA film according to the ratio of 1 film 10ml be dipped into 10ml hybridization buffer (contain 1 weight % skimming milk, 0.1 weight %N-Sarkosyl L ester, 0.02 weight %SDS, 50 weight % methane amides 5 * SSC) in, under 42 ℃, carry out 2 hours prehybridizations.With above-mentioned same, the 100ng fluorescence labeling probe of preparation is made its thermally denature by boiling in 10 minutes to handle with chilling under 95 ℃, add in the prehybridization damping fluid, in 42 ℃ down hybridization spend the night.Film after the hybridization is washed under room temperature 2 times in the 2 * SSC that contains 0.1 weight %SDS of 150ml.In being heated to 65 ℃ the 1 * SSC that contains 0.1 weight %SDS of 150ml, carry out 2 times 5 minutes washing then.Next toxilic acid damping fluid (being transferred to pH7.5 with 0.1M toxilic acid, 0.15M NaCl, the NaOH) washing of using 100ml is after 5 minutes, at lock solution (the 0.1M toxilic acid damping fluid that contains 0.3 weight %Tween20,0.15M NaCl and 1 weight % skimming milk of 50ml; PH7.5) in, under room temperature, carry out 30 minutes sealing treatment.Lock solution with 20ml will resist digoxigenin-AP to be diluted to 75mU/ml, after carrying out 30 minutes antibody response under the room temperature, at lavation buffer solution (the 0.1M toxilic acid damping fluid that contains 0.3 weight %Tween20,0.15M NaCl of 100ml; PH7.5) in film is washed 5 times, will do not had bonded antibody to wash away.After in the detection damping fluid (0.1MTris-HCl, 0.1M NaCl, pH9.5) of 20ml, carrying out 5 minutes Balance Treatment, be formed in NBT (nitroblue tetrazolium(NBT)) solution that contains the 100mg/ml of 34 μ l in the detection damping fluid of 10ml, the chromophoric substrate solution NBT/BCIP (5-bromo-4-chloro-3-indolylphosphate) of the dilution of the BCIP solution of the 50mg/ml of 35 μ l, film is immersed in this solution fully lucifuge incubation 1 minute to 16 hours.Dish is moved on to, do not shake yet, carry out color development and confirm.Among 1000 clones of result on this film, find 4 place's positive signal, on original plate, confirmed positive colony with this location overlap.

(4) positive colony that contains the Nitrile hydratase α subunit downstream gene partly of thermophilic Polyglucosidase soil genus bacillus Q-6 strain is resolved

With confirmed positive colony from plating to the LB liquid nutrient medium that contains penbritin, under 37 ℃, spend the night with the 250rpm shaking culture, by centrifugal recovery thalline, utilize ordinary method to extract plasmid DNA.After plasmid DNA usefulness restriction enzyme Hind III digestion, carry out 1.5 weight % agarose gel electrophoresis, confirm to insert segmental size and be about 2.3kB.In addition, can confirm to insert the Nitrile hydratase α subunit part that contains thermophilic Polyglucosidase soil genus bacillus Q-6 strain in the fragment by PCR polymorphism and restriction enzyme digestion pattern.

With the plasmid called after pUC118-Q6Hin2.3 of above acquisition, measure and insert segmental whole base sequences.The Nitrile hydratase of thermophilic Polyglucosidase soil genus bacillus Q-6 strain and downstream gene group's restriction enzyme figure and gene constitute as shown in Figure 1.

The result can confirm that in inserting fragment the open reading frame that the base sequence by 339bp constitutes (following, be called ORF3) is present in the same direction in 5 ' end side downstream of Nitrile hydratase α subunit (ORF2) thus.Sequence is 12bp between the translation initiation codon of the translation stop codon of ORF2 and ORF3, and the translation stop codon of ORF3 sequence sub and between the translation initiation codon of the ORF in downstream more is 145bp.ORF3 112 amino acid of encoding, though have homology with following protein in the data with existing storehouse, very low.With the amino acid whose concordance rate of the sequence that homology is high in the data with existing storehouse be: be 31% with the P12K of bacillus BR449 strain, be 31%, be 21% with the Nh1E of prunosus red coccus J1 strain with the NhhG of prunosus red coccus J1 strain, with the P16 of thermophilic false Nocardia bacteria JCM3095 strain be 23%.

Embodiment 14: the structure of the Nitrile hydratase α subunit of thermophilic Polyglucosidase soil genus bacillus Q-6 strain and the expression plasmid of β subunit and downstream gene ORF3

PET-26b (+)-β α and pET-28a (+)-β α plasmid are digested with restriction enzyme Hind III, carry out the dephosphorylation reaction, handle by carry out dephosphorylation with the phenol chloroform extraction.This digestion product is carried out 1 weight % agarose gel electrophoresis, extract the dna fragmentation of about 6.1Kb by ordinary method.In this dna fragmentation, contain the Nitrile hydratase β subunit of pET carrier and thermophilic Polyglucosidase soil genus bacillus Q-6 strain and until the Hind III of the 60th amino acids that is positioned at α subunit restriction enzyme position.

Next, the pUC118-Q6Hin2.3 plasmid with restriction enzyme Hind III digestion, is carried out 1 weight % agarose gel electrophoresis, extract the insertion sheet segment DNA of about 2.3kB.This is inserted fragment be connected with the previous fragment of extracting, transformed into escherichia coli JM109 strain is selected from the transformant of selecting by kalamycin resistance and is contained the plasmid that inserts DNA.Confirm direction of insertion by PCR, obtain in pET-26b (+) and pET-28a (+) carrier, to contain the Nitrile hydratase α subunit of thermophilic Polyglucosidase soil genus bacillus Q-6 strain and the plasmid of β subunit and downstream gene ORF3 and catchment thereof.Below will be called pET-26b (+)-β α 12 and pET-28a (+)-β α 12 by the plasmid that aforesaid operations prepares.

Then, import thermophilic Polyglucosidase soil genus bacillus Q-6 strain Nitrile hydratase α subunit, β subunit and until translation stop codon of downstream gene ORF3, structure can make the expression plasmid of 3 kinds of protein coexpressions.With plasmid pET-26B (+)-β α 12 usefulness restriction enzyme NdeI and the Bg1II digestion that had before made up, carry out 1.5 weight % agarose gel electrophoresis, extract by ordinary method and contain α subunit, β subunit and until the 1.7kB gene fragment of translation stop codon of downstream gene ORF3.Simultaneously expression vector pET-26b (+) and pET-28a (+) are digested with NdeI and BamHI, carry out 1 weight % agarose gel electrophoresis, extract the gene fragment of the carrier part of 5.3kB by ordinary method.With these carriers with as inserting containing α subunit, β subunit and carrying out ligation by ordinary method of segmental previous extraction until the 1.7kB gene fragment of translation stop codon of downstream gene ORF3.When carrying out this reaction, the end that digests with the BglII restriction enzyme is connected with the end that digests with the BamHI restriction enzyme.Solution transformed into escherichia coli JM109 strain after the use ligation is extracted plasmid DNA from the transformant of selecting with kalamycin resistance, select to have imported and insert segmental plasmid.By above operation, can obtain the Nitrile hydratase α subunit of thermophilic Polyglucosidase soil genus bacillus Q-6 strain, β subunit part and downstream gene ORF3 as inserting fragment expression plasmid that be imported into, that can make 3 kinds of protein coexpressions.Below these purpose plasmids are referred to as pET-26b (+)-β α 1 and pET-28a (+)-β α 1.

Embodiment 15: in intestinal bacteria with the Nitrile hydratase activity of the thermophilic Polyglucosidase soil genus bacillus Q-6 strain of downstream gene coexpression

Intestinal bacteria BL 21 (DE3) LysE strain with expression plasmid pET-26b (+)-β α and pET-28a (+)-β α conversion NOvagene company makes the Nitrile hydratase β subunit of thermophilic Polyglucosidase soil genus bacillus Q-6 strain and the protein of α subunit pass through T7 promotor coexpression.Same pET-26b (+)-β α 1 of use and pET-28A (+)-β α 1 transforms e. coli bl21 (DE3) the Lyse strain of NOvagene company, makes the protein of Nitrile hydratase β subunit, α subunit and the downstream gene ORF3 of thermophilic Polyglucosidase soil genus bacillus Q-6 strain pass through T7 promotor coexpression.In contrast, use the transformant of e. coli bl21 (DE3) the Lyse strain of the NOvagene company that transforms with expression vector pET-26b (+) and pET-28a (+).

Transform the competent cell of e. coli bl21 (DE3) the Lyse strain of NOvagene corporate system with each expression plasmid, the bacterium liquid after the conversion processing is layered on the LB nutrient agar of the kantlex that contains 30 μ g/ml, and (0.5 weight % bacterium is cultivated tryptone, 0.5 weight %NACl, 2.0 weight % agar with yeast extract, 1 weight %; PH7.5) on,, carry out kalamycin resistance and select in 30 ℃ of overnight incubation.This transformant is seeded in the LB substratum that 2ml contains 30 μ g/ml kantlex, under 30 ℃, spends the night with the 200rpm shaking culture.Be seeded in preceding nutrient solution in the 10ml LB substratum that contains 20 μ g/ml cobalt chloride hexahydrates (COCl26H20) and 30 μ g/ml kantlex according to 2 weight %, under 30 ℃ about 3 hours with the 200rpm shaking culture, till OD600=0.5, add the IPTG of 0.1MM, induce the expression that begins from the T7 promotor, carry out about 4 hours shaking culture with 200rpm again, reclaim thalline.

The coli somatic resuspending that expressing of obtaining like this expressed from the Nitrile hydratase of thermophilic Polyglucosidase soil genus bacillus Q-6 strain is in the TBS damping fluid (pH7.5) that contains 20mM Tris-HCl and 15mM NaCl, be diluted to OD=0.2, make the reaction solution of final concentration 0.2 weight % acrylonitrile solution.Under 27 ℃, react while stirring, after 10 minutes, after 30 minutes by adding the 1 equivalent hydrochloric acid termination reaction of 100 μ L.It is 1 unit (following, be designated as U) that unit of enzyme activity (unit) is defined as the activity that in 1 minute 1 μ mol vinyl cyanide is changed into acrylamide, and the hydration activity (U/mg) of per unit wet thallus weight is as shown in table 9.

Table 9

Expression plasmid	Active (U/mg)
Expression plasmid	Active (U/mg)	PET-26b (+) carrier	0
pET-26b(+)-βα	0.20	PET-26b (+) carrier	0
pET-26b(+)-βα	0.20	pET-26b(+)-βα1	2.74
PET-28a (+) carrier	0	pET-26b(+)-βα1	2.74
PET-28a (+) carrier	0	pET-28a(+)-βα	0.21
pET-28a(+)-βα1	4.22	pET-28a(+)-βα	0.21

By this result to the situation of the Nitrile hydratase α subunit that only makes thermophilic Polyglucosidase soil genus bacillus Q-6 strain and β subunit expression with the situation of downstream gene ORF3 coexpression is compared, because the existence of downstream gene ORF3 is arranged, the Nitrile hydratase activity can significantly increase as can be known.The function that the enzymic activity that obvious ORF3 has the Nitrile hydratase that makes thermophilic Polyglucosidase soil genus bacillus Q-6 strain significantly improves.

Embodiment 16: in the thermostability of the thermophilic Polyglucosidase soil genus bacillus Q-6 strain Nitrile hydratase of expression in escherichia coli

In order to study active thermostability, to have expression vector pET26b (+)-β α, and make the intestinal bacteria liquid that thermophilic Polyglucosidase soil genus bacillus Q-6 strain Nitrile hydratase expresses (the TBS damping fluid (pH7.5) that contains the NaCl of the Tris-HCl of 20mM and 15mM) carry out insulation processing in 30 minutes down in 30,65,70 degree.After insulation is handled, in cooled on ice, be incubated after making temperature certain under 27 degree mensuration Nitrile hydratase activity under temperature of reaction 27 degree.Express the intestinal bacteria of thermophilic Polyglucosidase soil genus bacillus Q-6 strain Nitrile hydratase according to the turbidity suspension of OD=0.2, make the reaction solution of final concentration 0.5 weight % acrylonitrile solution, under 27 ℃, begin while stirring the reaction, after 30 minutes by adding the 1 equivalent hydrochloric acid termination reaction of 10 liquid measure %.Activity when handling to be incubated down in 30 degree is as shown in table 10 as the scaled value of benchmark (100%).

Table 10

Treatment temp (℃)	Remaining activity (%)
Treatment temp (℃)	Remaining activity (%)	30	100.0
65	102.5	30	100.0
65	102.5	70	83.9

Can find out by this result, even the Nitrile hydratase enzymic activity in the intestinal bacteria of the Nitrile hydratase of expressing thermophilic Polyglucosidase soil genus bacillus Q-6 strain still can keep the activity more than 80% under the high temperature of 70 degree, we can say under intestinal bacteria make the situation of its expression also have high heat resistance, is industrial very useful enzyme.

Utilizability on the industry

Constituent of the present invention shows very high stability for nitrile, the amide compound of heat or high concentration, has the effect that can effectively nitrile compound be changed into corresponding amide compound. Even constituent of the present invention is fit to be applied to also nitrile compound to be changed in the field of corresponding amide compound in the reaction under high temperature and high nitrile compound concentration or high amide compound substrate concentration.

Sequence table

＜110〉Asahi Kasei Corporation

＜120〉novel nitrile hydratase

<130>A41354A

<160>25

<210>1

<211>205

<212>PRT

＜213〉thermophilic Polyglucosidase soil genus bacillus (Geobacillus thermoglucosidasius)

＜223〉Nitrile hydratase α-subunit

<400>1

Met Ser Val Gln Lys Val His His Asn Val Leu Pro Glu Lys Pro Ala

1 5 10 15

Gln Thr Arg Thr Lys Ala Leu Glu Ser Leu Leu Ile Glu Ser Gly Leu

20 25 30

Val Ser Thr Asp Ala Leu Asp Ala Ile Ile Glu Ala Tyr Glu Asn Asp

35 40 45

Ile Gly Pro Met Asn Gly Ala Lys Val Val Ala Lys Ala Trp Val Asp

50 55 60

Pro Asp Tyr Lys Glu Arg Leu Leu Arg Asp Gly Thr Ser Ala Ile Ala

65 70 75 80

Glu Leu Gly Phe Leu Gly Leu Gln Gly Glu His Met Val Val Val Glu

85 90 95

Asn Thr Pro Lys Val His Asn Val Val Val Cys Thr Leu Cys Ser Cys

100 105 110

Tyr Pro Trp Pro Val Leu Gly Leu Pro Pro Ser Trp Tyr Lys Ser Ala

115 120 125

Ser Tyr Arg Ala Arg Ile Val Ser Glu Pro Arg Thr Val Leu Lys Glu

130 135 140

Phe Gly Leu Glu Leu Asp Asp Asp Val Glu Ile Arg Val Trp Asp Ser

145 150 155 160

Ser Ala Glu Ile Arg Tyr Leu Val Leu Pro Glu Arg Pro Ala Gly Thr

165 170 175

Glu Gly Trp Ser Glu Glu Glu Leu Ala Lys Leu Val Thr Arg Asp Ser

180 185 190

Met Ile Gly Val Ala Lys Ile Lys Ser Pro Val Lys Lys

195 200 205

<210>2

<211>226

<212>PRT

＜213〉thermophilic Polyglucosidase soil genus bacillus

＜223〉Nitrile hydratase β-subunit

<400>2

Met Asn Gly Pro His Asp Leu Gly Gly Lys Arg Asp Phe Gly Pro

1 5 10 15

Ile Ile Lys His Asp Gln Glu Pro Leu Phe His Glu Glu Trp Glu

20 25 30

Ala Lys Val Leu Ala Met His Phe Ala Leu Leu Gly Gln Gly Val

35 40 45

Ile Asn Trp Asp Glu Phe Arg His Gly Ile Glu Arg Met Gly Tyr

50 55 60

Val Tyr Tyr Leu Thr Ser Ser Tyr Tyr Glu His Trp Leu Ala Ser

65 70 75

Leu Glu Thr Val Leu Ala Glu Lys Asn Ile Ile Asn Ser Glu Gln

80 85 90

Tyr Arg Lys Arg Ile Arg Glu Ile Glu Tyr Gly Met Ser Val Pro

95 100 105

Val Ser Glu Lys Pro Glu Leu Lys Glu Ser Leu Leu Ser Glu Val

110 115 120

Ile Tyr Gly Thr Lys Ile Ser Ser Glu Arg Arg Glu Ser Thr Val

125 130 135

Ser Pro Arg Phe Arg Pro Gly Asp Arg Val Arg Val Lys His Phe

140 145 150

Tyr Thr Asn Lys His Thr Arg Cys Pro Gln Tyr Val Met Gly Lys

155 160 165

Val Gly Val Val Glu Leu Leu His Gly Asn His Val Phe Pro Asp

170 175 180

Ser Asn Ala His Gly Asp Gly Glu Ala Pro Gln Pro Leu Tyr Asn

185 190 195

Val Arg Phe Glu Ala Arg Glu Leu Trp Gly Gly Glu Ala His Glu

200 205 210

Lys Asp Ser Leu Asn Leu Asp Leu Trp Asp Ser Tyr Leu Thr His

215 220 220

Ala

226

<210>3

<211>1663

<212>DNA

＜213〉thermophilic Polyglucosidase soil genus bacillus

＜223〉Nitrile hydratase β-subunit, α-subunit, orf3

<221>CDS

<222>1..681

＜223〉Nitrile hydratase β-subunit

<221>CDS

<222>695..1312

＜223〉Nitrile hydratase α-subunit

<221>CDS

<222>1325..1663

<223>orf3

<400>3

atg aac ggc ccg cac gat tta ggt gga aaa cgt gat ttt ggc cca 45

Met Asn Gly Pro His Asp Leu Gly Gly Lys Arg Asp Phe Gly Pro

1 5 10 15

atc att aaa cat gat caa gaa cct ctt ttt cat gaa gaa tgg gaa 90

Ile Ile Lys His Asp Gln Glu Pro Leu Phe His Glu Glu Trp Glu

20 25 30

gca aaa gta ctg gcg atg cat ttt gct tta ctt gga caa gga gta 135

Ala Lys Val Leu Ala Met His Phe Ala Leu Leu Gly Gln Gly Val

35 40 45

atc aac tgg gat gaa ttt agg cat ggt ata gaa cgg atg gga tat 180

Ile Asn Trp Asp Glu Phe Arg His Gly Ile Glu Arg Met Gly Tyr

50 55 60

gtt tat tac ctt act tca agc tat tat gaa cat tgg ctt gct tca 225

Val Tyr Tyr Leu Thr Ser Ser Tyr Tyr Glu His Trp Leu Ala Ser

65 70 75

cta gaa acc gta ttg gcc gag aaa aat atc att aac agt gaa cag 270

Leu Glu Thr Val Leu Ala Glu Lys Asn Ile Ile Asn Ser Glu Gln

80 85 90

tat aga aag aga att agg gaa ata gaa tat ggc atg agt gta cct 315

Tyr Arg Lys Arg Ile Arg Glu Ile Glu Tyr Gly Met Ser Val Pro

95 100 105

gtc agc gaa aag cct gag tta aaa gag tct ttg tta tcc gaa gtg 360

Val Ser Glu Lys Pro Glu Leu Lys Glu Ser Leu Leu Ser Glu Val

110 115 120

atc tat ggc acg aaa ata tca tcc gaa cgg aga gaa agc act gta 405

Ile Tyr Gly Thr Lys Ile Ser Ser Glu Arg Arg Glu Ser Thr Val

125 130 135

tct ccg cgg ttt cgt cct gga gat aga gtg agg gta aaa cac ttt 450

Ser Pro Arg Phe Arg Pro Gly Asp Arg Val Arg Val Lys His Phe

140 145 150

tat aca aac aag cat act aga tgt cct caa tat gtc atg ggg aaa 495

Tyr Thr Asn Lys His Thr Arg Cys Pro Gln Tyr Val Met Gly Lys

155 160 165

gta gga gtt gta gaa ctt ctt cat ggg aat cat gtt ttc cca gac 540

Val Gly Val Val Glu Leu Leu His Gly Asn His Val Phe Pro Asp

170 175 180

tct aac gct cat ggt gat ggc gag gct ccg caa ccg ctt tac aat 595

Ser Asn Ala His Gly Asp Gly Glu Ala Pro Gln Pro Leu Tyr Asn

185 190 195

gtg cgc ttt gaa gca aga gaa ctg tgg gga ggc gag gct cac gaa 630

Val Arg Phe Glu Ala Arg Glu Leu Trp Gly Gly Glu Ala His Glu

200 205 210

aaa gat agt cta aat ctc gac tta tgg gat agc tat cta act cac 675

Lys Asp Ser Leu Asn Leu Asp Leu Trp Asp Ser Tyr Leu Thr His

215 220 225

gcg taa aggaggaaaa atc 694

Ala

226

atg agt gta caa aaa gtt cat cac aac gtt ctg cct gaa aag cct 739

Met Ser Val Gln Lys Val His His Asn Val Leu Pro Glu Lys Pro

1 5 10 15

gct caa act cgg aca aag gct ttg gaa tcg ctg ttg atc gaa tct 784

Ala Gln Thr Arg Thr Lys Ala Leu Glu Ser Leu Leu Ile Glu Ser

20 25 30

gga ttg gtc tcc act gat gcc ctt gat gcg att att gaa gcc tat 829

Gly Leu Val Ser Thr Asp Ala Leu Asp Ala Ile Ile Glu Ala Tyr

35 40 45

gaa aat gat att ggg cct atg aat ggg gca aaa gtt gtt gca aaa 874

Glu Asn Asp Ile Gly Pro Met Asn Gly Ala Lys Val Val Ala Lys

50 55 60

gct tgg gtt gat cct gat tac aaa gaa aga ttg ctt cgg gat ggg 910

Ala Trp Val Asp Pro Asp Tyr Lys Glu Arg Leu Leu Arg Asp Gly

65 70 75

act tcg gct att gca gag ctt ggc ttt tta ggg ttg cag ggg gag 964

Thr Ser Ala Ile Ala Glu Leu Gly Phe Leu Gly Leu Gln Gly Glu

80 85 90

cac atg gtt gtt gtc gaa aat acg cct aaa gtt cat aat gta gta 1009

His Met Val Val Val Glu Asn Thr Pro Lys Val His Asn Val Val

95 100 105

gtt tgt acg cta tgt tcc tgc tat ccg tgg cct gtc cta ggc ttg 1054

Val Cys Thr Leu Cys Ser Cys Tyr Pro Trp Pro Val Leu Gly Leu

110 115 120

cct cct tca tgg tat aaa agt gct tca tac agg gct cga att gtt 1099

Pro Pro Ser Trp Tyr Lys Ser Ala Ser Tyr Arg Ala Arg Ile Val

125 130 135

tca gag cca aga act gta ctt aaa gag ttt ggg ctt gaa ctg gat 1144

Ser Glu Pro Arg Thr Val Leu Lys Glu Phe Gly Leu Glu Leu Asp

140 145 150

gat gat gtt gaa att agg gtt tgg gac agc agt gct gaa att cga 1189

Asp Asp Val Glu Ile Arg Val Trp Asp Ser Ser Ala Glu Ile Arg

155 160 165

tat tta gtt ctt cca gaa aga cct gca ggt act gaa ggg tgg tcg 1234

Tyr Leu Val Leu Pro Glu Arg Pro Ala Gly Thr Glu Gly Trp Ser

170 175 180

gaa gag gaa ctg gct aaa ctt gta acg cgt gac tct atg atc ggt 1279

Glu Glu Glu Leu Ala Lys Leu Val Thr Arg Asp Ser Met Ile Gly

185 190 195

gtg gcc aag ata aag tcg cct gtt aaa aaa taa ggggggacaa aa 1324

Val Ala Lys Ile Lys Ser Pro Val Lys Lys

200 205

atg gtt caa tca aat ctt caa ata aaa ccg gat gag att cta cct 1369

Met Val Gln Ser Asn Leu Gln Ile Lys Pro Asp Glu Ile Leu Pro

1 5 10 15

gaa cct agg aga aca gaa aat gag ccg gtt ttt aat tcc ccg tgg 1414

Glu Pro Arg Arg Thr Glu Asn Glu Pro Val Phe Asn Ser Pro Trp

20 25 30

gaa gcc cgg att ttt gct atg aca atc aat ttg tat gac aaa aaa 1459

Glu Ala Arg Ile Phe Ala Met Thr Ile Asn Leu Tyr Asp Lys Lys

35 40 45

ttc ttt gat tgg gag gac ttt cga caa gga tta ata gct gaa att 1504

Phe Phe Asp Trp Glu Asp Phe Arg Gln Gly Leu Ile Ala Glu Ile

50 55 60

gca gtt gcg gac agc ctt cct gag aat gaa cga cca acc tac tac 1549

Ala Val Ala Asp Ser Leu Pro Glu Asn Glu Arg Pro Thr Tyr Tyr

65 70 75

gaa agt tgg ctg gcc gct ttg gaa aag ttg tta atc aag gat ggt 1594

Glu Ser Trp Leu Ala Ala Leu Glu Lys Leu Leu Ile Lys Asp Gly

80 85 90

ata tta aca aaa gaa caa ata gat gaa cgc act aaa gaa ttg aaa 1639

Ile Leu Thr Lys Glu Gln Ile Asp Glu Arg Thr Lys Glu Leu Lys

95 100 105

gaa ggt ata aga aaa agt tgc tag 1663

Glu Gly Ile Arg Lys Ser Cys

110

<210>4

<211>112

<212>PRT

＜213〉thermophilic Polyglucosidase soil genus bacillus

<223>ORF3

<400>4

Met Val Gln Ser Asn Leu Gln Ile Lys Pro Asp Glu Ile Leu Pro Glu

1 5 10 15

Pro Arg Arg Thr Glu Asn Glu Pro Val Phe Asn Ser Pro Trp Glu Ala

20 25 30

Arg Ile Phe Ala Met Thr Ile Asn Leu Tyr Asp Lys Lys Phe Phe Asp

35 40 45

Trp Glu Asp Phe Arg Gln Gly Leu IIe Ala Glu Ile Ala Val Ala Asp

50 55 60

Ser Leu Pro Glu Asn Glu Arg Pro Thr Tyr Tyr Glu Ser Trp Leu Ala

65 70 75 80

Ala Leu Glu Lys Leu Leu Ile Lys Asp Gly Ile Leu Thr Lys Glu Gln

85 90 95

Ile Asp Glu Arg Thr Lys Glu Leu Lys Glu Gly Ile Arg Lys Ser Cys

100 105 110

<210>5

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be used to the to increase degenerate pcr primer of part dna fragmentation of the thermophilic Polyglucosidase soil of part genus bacillus Q6

<400>5

gtncaraarg tncaycayaa yg 22

<210>6

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<400>6

ccncaraarc cngcncarac 20

<210>7

<211>17

<212>DNA

＜213〉artificial sequence

<220>

<400>7

caycancanc ayytncc 17

<210>8

<211>23

<212>DNA

＜213〉artificial sequence

<220>

<400>8

acrttrtgrt gnacyttytg nac 23

<210>9

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<400>9

gtytgngcng gyttytgngg 20

<210>10

<211>17

<212>DNA

＜213〉artificial sequence

<220>

<400>10

ggnarrtgnt gntgrtg 17

<210>11

<211>17

<212>DNA

＜213〉artificial sequence

<220>

<400>11

atgaayggnc cncayga 17

<210>12

<211>17

<212>DNA

＜213〉artificial sequence

<220>

<400>12

aarmgngayt tyggncc 17

<210>13

<211>23

<212>DNA

＜213〉artificial sequence

<220>

<400>13

athathaarc aygaycarga rcc 23

<210>14

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<400>14

arrtcrtgng gnccrttcat 20

<210>15

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<400>15

thrtcrtgyt tdatdatngg 20

<210>16

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<400>16

tcytcytcra araanarngg 20

<210>17

<211>15

<212>DNA

＜213〉artificial sequence

<220>

<400>17

gcgraartcy yccca 15

<210>18

<211>17

<212>DNA

＜213〉artificial sequence

<220>

<400>18

ccartgytcr tarttcc 17

<210>19

<211>25

<212>DNA

＜213〉artificial sequence

<220>

<400>19

agatagtcta aatctcgact tatgg 25

<210>20

<211>26

<212>DNA

＜213〉artificial sequence

<220>

<400>20

atgaacggcc cgcacgattt aggtgg 26

<210>21

<211>29

<212>DNA

＜213〉artificial sequence

<220>

<400>21

catatgaacg gcccgcacga tttaggtgg 29

<210>22

<211>37

<212>DNA

＜213〉artificial sequence

<220>

<400>22

cagatcttat tttttaacag gcgactttat cttggcg 37

<210>23

<211>29

<212>PRT

＜213〉thermophilic Polyglucosidase soil genus bacillus

＜223〉the-terminal amino acid sequence of Nitrile hydratase α-subunit

<400>23

Met Ser Val Gln Lys Val His His Asn Val Leu Pro Glu Lys Pro

1 5 10 15

Ala Gln Thr Arg Thr Lys Ala Leu Glu Ser Leu Leu Ile Glu

20 25

<210>24

<211>25

<212>PRT

＜213〉thermophilic Polyglucosidase soil genus bacillus

＜223〉the-terminal amino acid sequence of Nitrile hydratase β-subunit

<400>24

Met Asn Gly Pro His Asp Leu Gly Gly Lys Arg Asp Phe Gly Pro

1 5 10 15

Ile Ile Lys His Asp Gln Glu Pro Leu Phe

20 25

<210>25

<211>618

<212>DNA

＜213〉thermophilic Polyglucosidase soil genus bacillus

＜223〉be used for the probe (Nitrile hydratase α-subunit) of colony hybridization

<400>25

atgagtgtac aaaaagttca tcacaacgtt ctgcctgaaa agcctgctca aactcggaca 60

aaggctttgg aatcgctgtt gatcgaatct ggattggtct ccactgatgc ccttgatgcg 120

attattgaag cctatgaaaa tgatattggg cctatgaatg gggcaaaagt tgttgcaaaa 180

gcttgggttg atcctgatta caaagaaaga ttgcttcggg atgggacttc ggctattgca 240

gagcttggct ttttagggtt gcagggggag cacatggttg ttgtcgaaaa tacgcctaaa 300

gttcataatg tagtagtttg tacgctatgt tcctgctatc cgtggcctgt cctaggcttg 360

cctccttcat ggtataaaag tgcttcatac agggctcgaa ttgtttcaga gccaagaact 420

gtacttaaag agtttgggct tgaactggat gatgatgttg aaattagggt ttgggacagc 480

agtgctgaaa ttcgatattt agttcttcca gaaagacctg caggtactga agggtggtcg 540

gaagaggaac tggctaaact tgtaacgcgt gactctatga tcggtgtggc caagataaag 600

tcgcctgtta aaaaataa 618

Claims

(according to the modification of the 19th of treaty)

1. DNA is characterized by coded protein and has following physico-chemical property:

(a) has the Nitrile hydratase activity;

(b) substrate specificity: as substrate, show activity with vinyl cyanide, adiponitrile, acetonitrile, isopropyl cyanide, n-valeronitrile, n-butyronitrile, benzonitrile, own nitrile;

Subunit alpha molecule amount 25000 ± 2000

Subunit beta molecule amount 28000 ± 2000

(d) thermostability: when with the enzyme in the aqueous solution under 70 ℃ of temperature the heating 30 minutes after, remaining activity for the heating before active more than 35%;

(e) vinyl cyanide that promptly uses 6 weight % is compared during with the following concentration of substrate of 6 weight % as substrate, and activity does not reduce yet;

2. following (A) or (B) in any DNA:

(A) the following DNA of feature: the α subunit gene that is contained aminoacid sequence shown in the sequence number 1 by coding is constituted with the combination that coding contains the base sequence of the beta subunit gene of aminoacid sequence shown in the sequence number 2;

(B) a kind of DNA, coded protein is at the α subunit that contains aminoacid sequence shown in the sequence number 1 with contain among any or two of β subunit of aminoacid sequence shown in the sequence number 2 separately 1 or a plurality of amino acid is replaced, lacked, interpolation, posttranslational modification, contain that change or that do not have reformed α subunit and change or do not have reformed β subunit, and this protein has the Nitrile hydratase activity.

3. following (C) or (D) in any DNA:

(C) the following DNA of feature: constituted by the DNA of the 695-1312 bit sequence that contains sequence number 3 and the combination of the DNA of the 1-681 bit sequence that contains sequence number 3;

(D) a kind of DNA, coded protein contains α subunit and β subunit, and have the Nitrile hydratase activity, wherein, the α subunit be by the DNA of the 695-1312 bit sequence that contains sequence number 3 or under the condition of strictness with the DNA of this DNA hybridization in any DNA coded; The β subunit be by the DNA of the 1-681 bit sequence that contains sequence number 3 or under the condition of strictness with the DNA of this DNA hybridization in any DNA coded; But except the situation of above-mentioned (C).

4. each described DNA in the claim 1～3, it is characterized by the DNA that recombinant contains the base sequence of aminoacid sequence shown in the encoding sequence numbers 4, or coding characteristic for 1 in this aminoacid sequence or a plurality of amino acid are replaced, lack, interpolation, posttranslational modification and coding be any DNA among the DNA with the activation proteins associated matter of Nitrile hydratase.

5. each described DNA in the claim 1～3 is characterized in that it is to activate any DNA among the DNA of proteins associated matter with Nitrile hydratase for what can hybridize under the condition of strictness with this DNA and encode that recombinant contains the DNA of 1325-1663 bit sequence of sequence number 3 or coding characteristic.

6. a DNA contains the gene that coding has the Nitrile hydratase α subunit of aminoacid sequence shown in the sequence number 1.

7. a DNA contains the gene that coding has the Nitrile hydratase β subunit of aminoacid sequence shown in the sequence number 2.

8. DNA, it contains and is characterized as and the relevant gene of activation of encoding and having the Nitrile hydratase of aminoacid sequence shown in the sequence number 4.

9. each described DNA of claim 1～8, wherein, this DNA comes from soil genus bacillus (Geobacillus) and belongs to.

10. each described DNA of claim 1～8, wherein, this DNA comes from thermophilic Polyglucosidase soil genus bacillus (Geobacillus thermoglucosidasius) and plants.

11. each described DNA of claim 1～8, wherein, this DNA comes from thermophilic Polyglucosidase soil genus bacillus (Geobacillus thermoglucosidasius) Q-6 strain (FERM BP-08658).

12. be integrated with the recombinant vectors of each described DNA of claim 1～11.

13. a microorganism, its for each described DNA microorganism transformed of claim 1～11 or thermophilic Polyglucosidase soil genus bacillus (Geobacillusthermoglucosidasius) Q-6 strain (FERM BP-08658) with and varient in any microorganism.

14. a protein or contain the manufacture method of this proteinic bacterial disposing thing is characterized by and cultivates in substratum with each described DNA microorganism transformed of claim 1～11.

15. a protein or contain the manufacture method of this proteinic bacterial disposing thing it is characterized by and cultivate the microorganism that belongs to the soil bacillus in substratum, and this microorganism can be produced the protein with following physico-chemical property:

(a) has the Nitrile hydratase activity;

(b) substrate specificity: show activity as substrate with vinyl cyanide, adiponitrile, acetonitrile, isopropyl cyanide, n-valeronitrile, n-butyronitrile, benzonitrile, own nitrile;

Subunit alpha molecule amount 25000 ± 2000

Subunit beta molecule amount 28000 ± 2000

16. a protein or contain this proteinic bacterial disposing thing, it is from by obtaining claim 14 or described any manufacture method cultured microorganism of claim 15.

17. a protein is characterized by and has following physico-chemical property:

(a) has the Nitrile hydratase activity;

Subunit alpha molecule amount 25000 ± 2000

Subunit beta molecule amount 28000 ± 2000

Following 18. (A) or (B) in any protein:

(A) be characterized as the protein that contains α subunit and β subunit, wherein, the α subunit contains aminoacid sequence shown in the sequence number 1, and the β subunit contains aminoacid sequence shown in the sequence number 2;

(B) at the α subunit of aminoacid sequence shown in the sequence number 1 with contain among any or two in the β subunit of aminoacid sequence shown in the sequence number 2 separately 1 or a plurality of amino acid is replaced, lacked, interpolation, posttranslational modification, contain by through that change or that do not have reformed α subunit and change or do not have reformed β subunit, and have the active protein of Nitrile hydratase.

Following 19. (C) or (D) in any protein:

(C) be characterized as the protein that contains α subunit and β subunit, wherein, the α subunit is by the dna encoding of the 695-1312 bit sequence with sequence number 3, and the β subunit is by the dna encoding of the 1-681 bit sequence with sequence number 3;

(D) contain α subunit and β subunit and have the active protein of Nitrile hydratase, wherein the α subunit is by the dna encoding of the 695-1312 bit sequence with sequence number 3; The β subunit is the DNA by the 1-681 bit sequence with sequence number 3, or any dna encoding among the DNA of under the condition of strictness, hybridizing with this DNA; But except the situation of above-mentioned (C).

20. have the protein of following feature: comprise at least after the polypeptide that contains the α subunit or its translation by modifier and contain the polypeptide of β subunit or its translation after by in the modifier any, or both, wherein, the α subunit is an aminoacid sequence shown in the sequence number 1, and the β subunit is an aminoacid sequence shown in the sequence number 2 in the sequence table.

21. the manufacture method of amide compound is characterized in that: will have the protein of following physico-chemical property or contain this proteinic bacterial disposing thing and act on nitrile compound, and obtain by this nitrile compound deutero-amide compound, said physico-chemical property comprises:

(a) has the Nitrile hydratase activity;

Subunit alpha molecule amount 25000 ± 2000

Subunit beta molecule amount 28000 ± 2000

22. the manufacture method of amide compound, it is characterized in that: in substratum, cultivate with each described DNA microorganism transformed in the claim 1～11 or belong in the proteinic microorganism that producing of soil bacillus have following physico-chemical property any, make resulting protein or contain this proteinic bacterial disposing thing and act on nitrile compound, acquisition is by this nitrile compound deutero-amide compound, and wherein said physico-chemical property comprises:

(a) has the Nitrile hydratase activity;

Subunit alpha molecule amount 25000 ± 2000

Subunit beta molecule amount 28000 ± 2000

Claims

(a) has the Nitrile hydratase activity;

Subunit alpha molecule amount 25000 ± 2000

Subunit beta molecule amount 28000 ± 2000

2. following (A) or (B) in any DNA:

3. following (C) or (D) in any DNA:

(a) has the Nitrile hydratase activity;

Subunit alpha molecule amount 25000 ± 2000

Subunit beta molecule amount 28000 ± 2000

17. a protein is characterized by and has following physico-chemical property:

(a) has the Nitrile hydratase activity;

Subunit alpha molecule amount 25000 ± 2000

Subunit beta molecule amount 28000 ± 2000

Following 18. (A) or (B) in any protein:

(B) at the α subunit of aminoacid sequence shown in the sequence number 1 with contain among any or two in the β subunit of aminoacid sequence shown in the sequence number 2 separately 1 or a plurality of amino acid is replaced, lacked, interpolation, posttranslational modification, contain by through change or do not have reformed α subunit and a change or do not have reformed β subunit, and have the active protein of Nitrile hydratase.

Following 19. (C) or (D) in any protein:

(a) has the Nitrile hydratase activity;

Subunit alpha molecule amount 25000 ± 2000

Subunit beta molecule amount 28000 ± 2000

(d) thermostability: when with the enzyme in the aqueous solution under 70 ℃ of temperature the heating 30 minutes after, remaining activity for the heating before more than 35%;

22. the manufacture method of amide compound, it is characterized in that: cultivate with each described DNA microorganism transformed in the claim 1～11 in substratum or belong to any the resulting protein in the proteinic microorganism that producing of soil bacillus have following physico-chemical property or contain this proteinic bacterial disposing thing, said physico-chemical property comprises:

(a) has the Nitrile hydratase activity;

Subunit alpha molecule amount 25000 ± 2000

Subunit beta molecule amount 28000 ± 2000

(d) thermostability: in the aqueous solution enzyme in heating under 70 ℃ of temperature after 30 minutes, remaining activity before for heating more than 35%;