CN102168033A

CN102168033A - Acidophilic vulcanizing bacillus disulfurase gene and recombinant protein thereof

Info

Publication number: CN102168033A
Application number: CN2010105632762A
Authority: CN
Inventors: 陈新华; 刘倩
Original assignee: Third Institute of Oceanography SOA
Current assignee: Third Institute of Oceanography SOA
Priority date: 2010-11-26
Filing date: 2010-11-26
Publication date: 2011-08-31
Anticipated expiration: 2030-11-26
Also published as: CN102168033B

Abstract

The invention relates to a gene and a recombinant protein thereof, in particular to an acidophilic vulcanizing bacillus disulfurase gene and a recombinant protein thereof. The invention provides a gene sequence, a cloning method, a recombinant protein and a preparation method of acidophilic vulcanizing bacillus TPY disulfurase and application in the preparation of a growth accelerant for bacteria. The cloning method comprises the following steps of: extracting the genome of acidophilic vulcanizing bacillus TPY as a template, and amplifying a primer; recovering and connecting the PCR (Polymerase Chain Reaction) product of amplification to a pMD18T-Simple vector for sequencing; and recovering and connecting PET-His to construct an expression vector PET-His-sufs+or through double-enzyme digest for expressing. The acidophilic vulcanizing bacillus disulfurase can be used as a growth accelerant for bacteria, and the bacteria comprise Escherichia coli, acidophilic vulcanizing bacillus, thiobacillus ferrooxidans and the like.

Description

Have a liking for acid sulfuration genus bacillus disulfurase gene and recombinant protein thereof

Technical field

The present invention relates to a kind of gene and recombinant protein thereof, especially relate to a kind of function of having a liking for acid sulfuration genus bacillus (Sulfobacillus acidophilus) disulfurase gene order and recombinant protein thereof.

Background technology

It is the dimer enzyme of prothetic group with the 5-pyridoxal phosphate that disulfurase (Cysteine des ulfurase) is one; this enzyme can change L-type L-Ala into by catalysis L-type halfcystine; the sulphur atom of taking off generates sulfane; form the persulfide of halfcystine, thus the protection cysteine residues.([1] Zheng.L, White.R.H., Cash.V.L, Dean.D.R.Cysteine desulfurase activity indicates a role for NIFS in metallocluster biosynthesis.[j] Biochemistry.1994,33:4714-4720) many studies show that, this enzyme is at iron sulphur bunch and thiamines, sulfo-nucleosides among the tRNA, vitamin H, lipid acid, all play an important role in NAD etc. synthetic ([2] H.Mihara, N.Esaki.Bacterial cysteine desulfurases:their function and mechanisms.[j] Appl Microbiol Biotechnol.2002,60:12-23).This enzyme in born of the same parents iron balance and seleno-protein synthetic in ([3] Beinert.H that also plays an important role, Holm.R.H, Mu ¨ nck.E.Iron-sulfur clusters:Nature ' s modular, multipurpose structures.[j] Science, 1997,277:653-659).The mechanism of action of disulfurase in sulfo-is thanked is not clear at present, but it all plays an important role in the biosynthesizing that contains sulphur or seleno-protein.

Iron-sulfur cluster albumen is that a class is at biological central ubiquitous albumen, and many important pathways metabolisms in the born of the same parents have been participated in, as: DNA repairs, transcriptional control, Nucleotide and amino acid are synthetic, and energy metabolism ([4] Zheng.L, White.R.H, Cash.V.L, Jack.R.F, Dean.D.R.Cysteine desulfurase activity indicates a role for NIFS in metallocluster biosynthesis.[j] Proc.Natl.Acad.Sci.USA, 1993,90:2754-2758).Although it is relevant Fe-S protein-actives that a lot of researchs are arranged, to the assembling of Fe-S in vivo and how to be inserted into the research of going in the albumen also few in number.In intestinal bacteria, two independent systems that participate in this process are respectively: by the ISC of IscSUA-hscBA-fdx gene cluster coding and ([5] the ToKumoto.U et of SUF system that is encoded by the SufABCDSE operon, al.A Suf operon requirement for Fe-S cluster assembly during iron starvation in Escherichia coli.[j] Proc.Natl.Acad.Sci.USA, 2004,136:199-201).IscSUA-hscBA-fdx participates in iron-sulfur cluster synthetic oligogene bunch ([6] Tokomoto.U et al, Transfer of Sulfur from IscS to IscU during Fe/S Cluster Assembly.[j] Biochem (ToKyo), 2001,131:63-65).This gene cluster is very conservative, 6 protein of codified, IscS wherein, IscU and IscA are iron-sulfur cluster synthetic main protein, HscA, HscB is a pair of heat shock cognate protein, relevant with the activity modifying of IscU ([7] Silberg.j.j et al, Crystal Structure of IscA, an Iron-sulfur Cluster Assembly Protein from Escherichia coli.[j] Biolchem, 2004,279:53924-53926); And Fdx is a kind of iron oxidation protein, in the assembling of iron-sulfur cluster, play a part electron donor ([8] Takag.I.S, Sasado.T, Tamiy.A.G, et al.An efficient expression vector for transgenic medaka construction.[J] Mol Mar Biol Bioteehnol, 1994,3:192-199).Studies show that, the Suf system may be relevant with the reparation of the damage of iron-sulfur cluster ([9] Kokata Y et al.Reconstitution of secretase activity.[j] Biochemistry, 2001,40:11007-11010).

Summary of the invention

One of purpose of the present invention is to provide the gene order of having a liking for acid sulfuration genus bacillus TPY disulfurase.

Two of purpose of the present invention is to provide the cloning process of having a liking for acid sulfuration genus bacillus TPY disulfurase.

Three of purpose of the present invention is to provide recombinant protein of having a liking for acid sulfuration genus bacillus TPY disulfurase and preparation method thereof.

Four of purpose of the present invention is to provide has a liking for the application of acid sulfuration genus bacillus TPY disulfurase in preparation bacterial growth promotor.

Of the present inventionly have a liking for acid sulfuration genus bacillus TPY (Sulfobacillus acidophilus TPY) and be preserved in Chinese typical culture collection center on 08 16th, 2010, deposit number is CCTCC NO:M2010203, the address at China typical culture collection center is a China, Wuhan, Wuhan University.

It is described that to have a liking for acid sulfuration genus bacillus TPY (Sulfobacillus acidophilus TPY) be the strain moderate thermophile acidophilic bacteria (referring to the applicant's Chinese invention patent application 200610135418.9) that separation and purification obtains from the hot fluid area sample of the Pacific Ocean.

The gene order of having a liking for acid sulfuration genus bacillus TPY disulfurase of the present invention is:

ATGACACGGACTTTAGTGGATATCCGGCAGGATTTTCCCATTTTGACGCGGGAAATTCACGGTCATCGACTCGTATATCTTGACAGTGCCGCCACCTCCC AAAAGCCGGAGTCGGTCATTCAAGCCATTGACCACTATTATCGACACTCGAATGCCAATGTGCTCCGGAGTGTGCATACGCTCGCGGAGGAGGCGACC A C GCTCTATGAAGACGCGCGGCGCAAGGTGGCCCGCTTCATTGGCGCCAAAACCTCGCAAGAAGTGATTTTTACTCGCGGGACCACCGAGAGTTTAAA CG TC GTCGCGCGAGGCTGGGGAGATAAATTTGTCCAGCCGGGCGACGAAATTGTCGTGTCACCGATGGAGCATCATTCGAATTTAATTCCTTGGCAGC AAT TGG CGCAACGCCGGCAAGCCGTTTTACGCTTTGTGGAATTAAACGGCGACGGCACGATTGCCTTGGACGAGGTCCGTCGGGTGATTAACGCCAAA ACCA AAAT CGTGGCCCTATCCCGCATTTCCAATGTTCTCGGGACCATTAACCCGATTGCGGAAATCAGTGCTTTAGCCCATCAACAGGGTGCGGTGAT GGTGG TGGAC GGCGCGCAGTCGGTGCCGCACGAACCGACGGACGTCAAACGTCTGGGAGCGGATTTTTTGGCCTTTTCCGGGCACAAAATGTTAGGGC CGACCG GCATCG GTGTCTTATGGGGACGTCGCGAGTTGTTAGACGCGATGGATCCCGTGCTTTTTGGCGGGGAAATGATTGCCTATGTCGATCGGGAA AGGGCGA CGTGGGC CGAATTGCCGGCCAAATTGGAGGGCGGCACGCCAAACATCGCCGGAGCCATTGGGTTAGGGGCGGCCGTCGATTATTTAACCGC CATTGGCA TGGACGTC GTCGCCGCGCACGGGCAAGCCTTAGGCGAATTGGCCTACCAACGGCTAAAAGCCGTCGGCGGCGTGACCGTTTATGGCCCCC CCGCACCTC GCGGTGCCC TGGTGGCATTTAACCTGGAAGGTATCCATCCCCATGACGTGGCCCAAGTGTTTGACTCCAATGGCGTGGCCATCCGAGCC GGGCATCATT GCGCACAACC GCTGTTGGCGTGGTTAGGCGTGGGATCTACCGCCCGTGCCAGTTTCTATGTCTATAATACCGAGGCTGACATCGATAC

The concrete steps of the cloning process of having a liking for acid sulfuration genus bacillus TPY disulfurase of the present invention are as follows:

1) extraction is had a liking for the sour genome that vulcanizes genus bacillus TPY as template, and amplimer is:

P1：5′-CA GAATTC ATGACACGGACTTTAGTGG-3′；

P2：5′-TT AAGCTT TCAGCGAAAGTACCTCTGC-3′；

2) the PCR product that comes out of amplification reclaims and pMD18T-Simple vector (can be provided by Japanese TaKaRa company) is provided checks order;

3) double digestion recovery connection PET-His construction of expression vector PET-His-sufs+ expresses.

The sequence of the recombinant protein of having a liking for acid sulfuration genus bacillus TPY disulfurase of the present invention is as follows:

MTRTLVDIRQDFPILTREIHGHRLVYLDSAATSQKPESVIQAIDHYYRHSNANVLRSVHTLAEEATTLYEDARRKVARFIGAKTSQEVIFTRGTTESLNVVARGWGDKFVQPGDEIVVSPMEHHSNLIPWQQLAQRRQAVLRFVELNGDGTIALDEVRRVINAKTKIVALSRISNVLGTINPIAEISALAHQQGAVMVVDGAQSVPHEPTDVKRLGADFLAFSGHKMLGPTGIGVLWGRRELLDAMDPVLFGGEMIAYVDRETATWAELPAKLEGGTPNIAGAIGLGAAVDYLTAIGMDVVAAHGQALGELAYQRLKAVGGVTVYGPPAPRGALVAFNLEGIHPHDVAQVFDSNGVAIRAGHHCAQPLLAWLGVGSTARASFYVYNTEADIDTLVEVIGETQRYFR*。

The preparation method of the recombinant protein of having a liking for acid sulfuration genus bacillus TPY disulfurase of the present invention is as follows:

With the expression vector PET-His-sufs that builds ⁺Changing expression strain BL21 (DE3) over to expresses.At first BL21 (DE3) bacterial strain is inserted the LB substratum, adding concentration in the LB substratum is the ammonia benzyl of 100 μ g/ml, puts into 37 ℃ of shaking tables again and cultivates, and cultivating the light absorption value OD600 of 600nm place is 0.5 o'clock, adds the IPTG of 0.1mM; After then putting into 37 ℃ of shaking tables and carrying out abduction delivering 6h, promptly get the recombinant protein of having a liking for acid sulfuration genus bacillus TPY disulfurase.

With the centrifugal receipts of culture 5000g bacterium behind the abduction delivering 6h, wash one time to remove residual LB substratum by phosphate buffered saline buffer (PBS); Then be resuspended in ultrasonication 10min in the phosphate buffered saline buffer, centrifugal supernatant liquor and the precipitation of getting of 8000g respectively got supernatant liquor and precipitation 100 μ l in the EP pipe, and each adds 2 * protein deposition damping fluid (giving birth to worker company available from Shanghai), 100 μ l, boil 10min, the SDS gel electrophoresis is analyzed.

Of the present inventionly have a liking for acid sulfuration genus bacillus TPY disulfurase and can be used as bacterial growth promotor, described bacterium such as intestinal bacteria, have a liking for acid sulfuration genus bacillus or thiobacillus ferrooxidant etc.

Outstanding advantage of the present invention and technique effect are as follows:

The described major function of having a liking for the recombinant protein of acid sulfuration genus bacillus TPY disulfurase is promote iron-sulfur cluster synthetic, and a lot of enzymes are all assembled by iron-sulfur cluster in the tricarboxylic acid cycle of bacterium.Owing to have a liking for the assembling that acid sulfuration genus bacillus TPY disulfurase can promote iron-sulfur cluster, can increase the activity of the tricarboxylic round-robin of participation bacterium, thereby promote tricarboxylic circulation by some enzymes of iron-sulfur cluster assembling, promote the growth of bacterium.In some rugged environments, bacterium can be resisted extraneous harsh conditions by the number that increases oneself.Such as intestinal bacteria under the very high situation of sulphur concentration is to be difficult to survival, and adds these enough albumen intestinal bacteria can grow (being confirmed in having tested, referring to the following drawings) in substratum.In the use of some engineering bacterias, usually can be slow because of bacterial growth speed, and can't utilize or increase cost, make the production cycle lengthening.Soak the ore deposit bacterium as some and can not be applied to so far in the middle of the production, one of them very main resistance is exactly that bacterial growth speed is slow, causes bacterial concentration low, makes that to soak the ore deposit cycle long, and ore-leaching speed is slow.The recombinant protein of having a liking for acid sulfuration genus bacillus TPY disulfurase can promote the growth of bacterium, can obviously shorten the bacterial leaching cycle, accelerates ore-leaching speed, thereby promotes that soaking the ore deposit bacterium leaches application in engineering at lean ore.

Description of drawings

Fig. 1 has a liking for the electrophorogram of the gene of acid sulfuration genus bacillus TPY disulfurase at the recombinant protein of intestinal bacteria E.coli BL21 (DE3) expression.In Fig. 1, swimming lane 1 is the molecular weight of albumen size, and swimming lane 2 is not for changing the whole bacterial protein of SufS gene over to, and swimming lane 3 is for changing the whole bacterial protein of SufS gene over to, and swimming lane 4 is for being the supernatant that changes the SufS gene over to, and swimming lane 5 is for changing the precipitation of SufS gene over to; The arrow place is a target stripe.

Fig. 2 crosses the growth curve of expressing flora and unloaded flora for having a liking for acid sulfuration genus bacillus TPY disulfurase in intestinal bacteria E.coli BL21 (DE3).In Fig. 2, X-coordinate is time time/min, and ordinate zou is 600nm light absorption value (OD600); ◆ be BL21/pet-his-sufs ⁺, ■ is BL21/pet-his.

Fig. 3 expresses bacterium and the bacterium colony size of unloaded bacterium on the LB flat board excessively for having a liking for acid sulfuration genus bacillus TPY disulfurase in intestinal bacteria E.coli BL21 (DE3).In Fig. 3, extent of dilution is 10 ^-6, incubation time is 24h; A is colony diameter 2～3mm, and b is colony diameter 1～1.5mm.

Fig. 4 is the growth curve of intestinal bacteria E.coli BL21 (DE3) in the LB substratum.In Fig. 4, X-coordinate is time time/min, and ordinate zou is 600nm light absorption value (OD600); ◆ the growth curve of E.coli BL21 (DE3) in the LB substratum, the growth curve of ■ E.coli BL21 (DE3) in the proteic LB substratum of the actin that is added with 520ng/ml, the growth curve of ▲ E.coli BL21 (DE3) in the proteic LB substratum of the SufS that is added with 520ng/ml.

The H that the absorption liquid of the different amounts of Fig. 5 and reference liquid produce ₂The typical curve that the concentration of S is drawn at the light absorption value at 670nm place.In Fig. 5, X-coordinate is H ₂The concentration μ g of S, ordinate zou are 670nm light absorption value (OD670).

Embodiment

Following examples will the present invention is further illustrated in conjunction with the accompanying drawings.

1. have a liking for the genomic extraction of acid sulfuration genus bacillus TPY.

1) get the bacteria culture medium that is cultured to stationary phase (this moment precipitate minimum) of 100ml, in 10, centrifugal 10min collects bacterium under the 000rpm;

2) with the deionized water wash bacterium of pH=2,, remove salt ion in the substratum until not precipitating;

3) resuspended with the TE (pH8.0) of 500 μ L, add the N,O-Diacetylmuramidase (50 μ g/ μ L) (because this bacterium be that the gram-positive microorganism broken wall is difficult) of 30 μ L, mixing, 37 ℃ of water-bath 10min;

4) SDS solution and 10 μ l RNase (the 20 μ g/ μ L) mixing of adding 30 μ L 10%, 55 ℃ of water-bath 10min add 3 μ L Proteinase Ks (20 μ g/ μ L) again, and 55 ℃ of water-bath 10min become clarification until bacterium liquid;

5) add the NaCl of 150 μ L 5mol/L, abundant mixing adds the CTAB/NaCl solution of 80 μ L again, and mixing is in 65 ℃ of water-bath 10min;

6) add isopyknic phenol/chloroform/primary isoamyl alcohol (25: 24: 1), mixing, 12, the centrifugal 10min of 000rpm, supernatant liquor are transferred in the new pipe; If can many extracting several times than higher to the DNA specification of quality;

7) Virahol of 0.6 times of volume of adding, mixing is placed 2～3h for-20 ℃ gently, precipitate up to DNA, 12, the centrifugal 10min of 000rpm;

8) abandon supernatant, wash twice, dry with 70% ethanol;

9) resolution of precipitate is in 30～60 μ L water, and-20 ℃ store for future use.

2.PCR reactive system comprises:

Dna profiling 80～100ng

Each 1 μ M of forward primer and reverse primer

10 * PCR damping fluid, 2.5 μ L

25mM MgCl ₂ 2μL

2.5mM dNTP 2μL

Taq archaeal dna polymerase 1U

Sterilized water is mended to 25 μ L.

The PCR program:

In the PCR reactive system, add required component and carry out pcr amplification by following program.

94℃ 5min

94℃ 1min

67℃ 1min

72 ℃ of 1.5min circulate 30 times

72℃ 7min

4 ℃ stop.

The PCR product recovery that amplification is come out is connected to pMD18T-Simple vector (TaKaRa) and checks order.Utilize intestinal bacteria E.coli BL21 (DE3) host as an alternative, the plasmid PET-SufS+ that will contain the SufS gene changes among the E.coli BL21 (DE3) and makes it cross expression.

3. have a liking for the recombinant expressed of acid sulfuration genus bacillus disulfurase

1) (the E.coli BL21 (DE3) of Pignus pignoris grain PET-SufS+ picking list bacterium colony access 5ml respectively contains in the LB substratum of penbritin (100 μ g/ml) 37 ℃ of shaking table overnight incubation to contrast bacterium (promptly only changeing the E.coli BL21 (DE3) of PET-His carrier) and reorganization bacterium.

2) get 50 μ l overnight culture and be linked into 5ml and contain in the LB substratum of penbritin (100 μ g/ml), 37 ℃ of shaking tables are cultured to logarithmic phase (OD600 is 0.5).

3) IPTG of adding 0.1mM in substratum continues to cultivate 6h in 37 ℃ of shaking tables.

4) get culture centrifugal 10min of 5000g in the EP pipe of 1ml, precipitation is resuspended in the 100 μ l phosphate buffered saline buffers (PBS), adds 2 * sds gel sample loading buffer (giving birth to worker company available from Shanghai), 100 μ l, and 100 ℃ are boiled 5min.

5) above-mentioned sample is splined on the 12%SDS polyacrylamide gel.

6) coomassie brilliant blue staining (referring to Fig. 1).

4, have a liking for the purifying of acid sulfuration genus bacillus disulfurase

This recombinant protein is in the middle of supernatant, so we are according to supernatant His-tag fusion rotein this albumen of purification process purifying at supernatant.

1) 100ml induces bacterium liquid, and centrifugal (5000g 10min) gets precipitation, with 10ml bufferA washing 1 time.

2) with 10ml bufferA resuspended ultrasonication 10min (broken power 60%, 5s, 5s).

3) the bacterium liquid of ultrasonication is at 18000g, 4 ℃ of centrifugal 20min.

4) Ni Sepharose 4Fast Flow (invitrogen) medium is removed alcohol twice with distilled water wash, adds BufferA balance 10min.

5) bacterium lysate upper prop is at 4 ℃ of jog 4h.

6) collect effluent liquid and repeating upper prop 2 times.

7) with buffer B washing medium, totally 4～5 times (making dielectric suspension) at every turn.

8) with buffer C washing medium, totally 4～5 times (making dielectric suspension) at every turn.

9) the buffer D of adding 1ml soaks 15min, collects.Collect 5 pipes altogether.

5, the mensuration of growth curve

At first obtain containing PET-SufS ⁺The BL21 of plasmid and the bacterium liquid of BL21 same concentrations that contains the PET-His plasmid are as seed.The Erlenmeyer flask of every group of 3 bottles of 250ml is equipped with 100ml LB substratum, in 1: 100 ratio inoculum density identical BL21 that contains the PET-SufS+ plasmid and the BL21 bacterium liquid that contains the PET-His plasmid.Put into 37 ℃, 180 change shaking table cultivates, and surveys OD in different time period samplings with nucleic acid-protein determinator (model smartspe).Survey and average for 3 times, do X-coordinate with the time, OD is that ordinate zou is drawn growth curve (referring to Fig. 2 and 4).

Same BL21 that contains the PET-SufS+ plasmid with identical OD that obtains and the identical extent of dilution of the BL21 bacterium liquid that contains PET-His plasmid dilution used, each extent of dilution is coated with 3 identical plates (referring to Fig. 3).

6, have a liking for the enzyme mensuration alive of acid sulfuration genus bacillus disulfurase

Because but the desulfurization of this enzyme catalysis L-halfcystine forms L-Ala, the sulfonium ion of taking off generates methylene blue with P-aminodimethylaniline under the situation that iron trichloride exists under acidic conditions, at the 670nm place absorption peak is arranged, the proportional colorimetric assay that carries out of the content of its shade and sulfonium ion.

That 1) we at first will survey before the survey enzyme is lived is H ₂The typical curve of S is according to the concentration of typical curve calculating sulfonium ion.Required reagent has:

Absorption liquid: get 5g zinc sulfate and be dissolved in the 500ml water, other gets 6g sodium hydroxide and is dissolved in the 300ml water, and two kinds of solution are mixed, and gets 70g ammonium sulfate again and adds in the solution, adds 50g glycerine after the stirring and dissolving, and thin up is to 1L.

P-aminodimethylaniline solution: get 25ml sulfuric acid, add in the 15ml water, get 6g P-aminodimethylaniline hydrochloride after the cooling, be dissolved in the above-mentioned solution, store in the refrigerator.During use, reach the 2.5ml stock solution, use the 1+1 Dilution of sulphuric acid to 100ml.Ferric chloride Solution: take by weighing 50g iron trichloride (FeCl ₃6H ₂O) soluble in water, be diluted to 50ml.If there is precipitation to filter.Standardized solution: take by weighing 0.71g sodium sulfide crystal (Na2S9H2O) and be dissolved in and newly boil in the cooled water, be diluted to 100ml.Demarcate the back and be diluted to the standard use liquid that every ml contains 5ug with newly boiling cooled water.

Scaling method: the standardized solution of getting 20.00ml 10.0100N places the 200ml iodine flask, accurately adds in the 10.00ml sodium sulphite standardized solution, adds and newly boils cooled water 80ml, adds the 5ml Glacial acetic acid immediately, in the dark places 2-3min behind the mixing; Extremely faint yellow with the titration of 0.0100N sodium thiosulfate standard solution, 0.5% starch that adds the new configuration of 1ml is blue, with less water washing bottle inwall, just disappearing in continuation titration to blueness is terminal point (have this moment sulphur to generate and make the little muddiness of solution, pay special attention to the sudden change of terminal colour).Write down the volume V (ml) of used sodium thiosulfate standard solution, simultaneously, other gets 10ml water, does blank titration, and its operation steps is the same.The volume V of the used sodium thiosulfate standard solution of record blank titration ₀Ml.

The concentration of hydrogen sulfide is calculated as follows:

Hydrogen sulfide (H ₂S, mg/ml)=(V-V ₀) N17/10

In the formula:

V ₀---when titration is blank, consume the ml number of Sulfothiorine;

V---during titration hydrogen sulfide stock solution, consume the ml number of hypo solution;

The equivalent concentration of N---sodium thiosulfate standard solution;

17---the equivalent of hydrogen sulfide.

The data of the concentration standard curve (referring to Fig. 5) of drafting hydrogen sulfide are referring to table 1.

Table 1

The method for expressing that enzyme is lived is 1 enzyme unit alive for the sulfonium ion that produces 1nmol.The data that record OD670 are as shown in table 2.

Table 2

The enzyme of the disulfurase of having a liking for sour genus bacillus of the calculating by typical curve is lived and is 95u/ μ L, and negative control is 0.84u/ μ L.

More than each pipe add the 1ml P-aminodimethylaniline respectively and use liquid, add a cover mixing (not jolting), place 1min, add 1～2 liquor ferri trichloridi again, shake up, get rid of Fe after placing 30min ³⁺Color.Using the 2cm cuvette, in wavelength 670nm place, is reference with water, measures absorbancy drawing standard curve.

2) enzyme activity determination step:

(1) under anaerobic gets 1ml and contain 50mmol/l Tris-HCl (pH=8.0), 10mmol/l MgCl ₂, 1.0mmol/lL-Cysteine, 5mmol/l DTT, 10um/l pyridoxal 5 '-5phosphate.The enzyme that adds 2 μ l.

(2) behind the reaction 20min, add 100 μ l and be dissolved in 30mmol/l FeCl in the hydrochloric acid of 1.2mol/l ₃With termination reaction.

(3) add 100 μ l and be dissolved in 20mmol/l N in the hydrochloric acid of 7.2mol/l, N-dimethy-p-phenediamine dihydrochloride reacts 30min to produce Methylene blue under dark condition.

(4) 670nm surveys OD down.

(5) repeat 3 times.

Claims

1. have a liking for the gene order of acid sulfuration genus bacillus TPY disulfurase, it is characterized in that describedly having a liking for acid sulfuration genus bacillus TPY (Sulfobacillus acidophilus TPY) and being preserved in Chinese typical culture collection center on 08 16th, 2010, deposit number is CCTCC NO:M2010203;

The described gene order of having a liking for acid sulfuration genus bacillus TPY disulfurase is:

ATGACACGGACTTTAGTGGATATCCGGCAGGATTTTCCCATTTTGACGCGGGAAATTCACGGTCATCGACTCGTATATCTTGACAGTGCCGCCACCTCCCAAAAGCCGGAGTCGGTCATTCAAGCCATTGACCACTATTATCGACACTCGAATGCCAATGTGCTCCGGAGTGTGCATACGCTCGCGGAGGAGGCGACCACGCTCTATGAAGACGCGCGGCGCAAGGTGGCCCGCTTCATTGGCGCCAAAACCTCGCAAGAAGTGATTTTTACTCGCGGGACCACCGAGAGTTTAAACGTCGTCGCGCGAGGCTGGGGAGATAAATTTGTCCAGCCGGGCGACGAAATTGTCGTGTCACCGATGGAGCATCATTCGAATTTAATTCCTTGGCAGCAATTGGCGCAACGCCGGCAAGCCGTTTTACGCTTTGTGGAATTAAACGGCGACGGCACGATTGCCTTGGACGAGGTCCGTCGGGTGATTAACGCCAAAACCAAAATCGTGGCCCTATCCCGCATTTCCAATGTTCTCGGGACCATTAACCCGATTGCGGAAATCAGTGCTTTAGCCCATCAACAGGGTGCGGTGATGGTGGTGGACGGCGCGCAGTCGGTGCCGCACGAACCGACGGACGTCAAACGTCTGGGAGCGGATTTTTTGGCCTTTTCCGGGCACAAAATGTTAGGGCCGACCGGCATCGGTGTCTTATGGGGACGTCGCGAGTTGTTAGACGCGATGGATCCCGTGCTTTTTGGCGGGGAAATGATTGCCTATGTCGATCGGGAAACGGCGACGTGGGCCGAATTGCCGGCCAAATTGGAGGGCGGCACGCCAAACATCGCCGGAGCCATTGGGTTAGGGGCGGCCGTCGATTATTTAACCGCCATTGGCATGGACGTCGTCGCCGCGCACGGGCAAGCCTTAGGCGAATTGGCCTACCAACGGCTAAAAGCCGTCGGCGGCGTGACCGTTTATGGCCCCCCCGCACCTCGCGGTGCCCTGGTGGCATTTAACCTGGAAGGTATCCATCCCCATGACGTGGCCCAAGTGTTTGACTCCAATGGCGTGGCCATCCGAGCCGGGCATCATTGCGCACAACCGCTGTTGGCGTGGTTAGGCGTGGGATCTACCGCCCGTGCCAGTTTCTATGTCTATAATACCGAGGCTGACATCGATACCTTGGTGGAAGTCATCGGAGAAACGCAGAGGTACTTTCGCTGA。

2. have a liking for the cloning process of acid sulfuration genus bacillus TPY disulfurase, it is characterized in that concrete steps are as follows:

P1：5′-CA GAATTC ATGACACGGACTTTAGTGG-3′；

P2：5′-TT AAGCTT TCAGCGAAAGTACCTCTGC-3′；

3) double digestion reclaims and connects PET-His construction of expression vector PET-His-sufs ⁺Express.

3. have a liking for the recombinant protein of acid sulfuration genus bacillus TPY disulfurase, it is characterized in that its sequence is as follows:

4. the preparation method who has a liking for the recombinant protein of acid sulfuration genus bacillus TPY disulfurase as claimed in claim 3 is characterized in that concrete steps are as follows:

5. have a liking for the application of acid sulfuration genus bacillus TPY disulfurase in preparation bacterial growth promotor.

6. application as claimed in claim 5 is characterized in that described bacterium is intestinal bacteria, has a liking for acid sulfuration genus bacillus or thiobacillus ferrooxidant.