CN1142536A - Bacillus-derived transglutaminase - Google Patents

Abstract

The present invention relates to a transglutaminase (TG) isolated from a Bacilli, with the TG or the fraction having TG activity to thereby form a protein, a non-proteinaceous amino acid polymer, or a peptide or derivatives thereof having a crosslinked structure. The present invention also relates to a DNA coding for a TG derived from a Bacilli, a vector comprising said DNA coding for said TG, a cell transformed with the vector, and a method for producing a Bacillus-derived transglutaminase by incubating the transformant. The crosslinked polymers produced using the Bacillus-derived TG of the present invention can be used in food.

Description

Bacillus-derived trans-glutaminases

The present invention relates to the trans-glutaminases (TG hereinafter referred to as) that (1) is produced by such bacillus such as resembling subtilis, (2) has the component of trans glutaminase active, (3) produce protein with crosslinking structure, the nonprotein amino acid polymer, the method of peptide or derivatives thereof, it is characterized in that by TG or the effect cross-linked proteins of the component of TG activity is arranged, the nonprotein amino acid polymkeric substance, Gln in peptide or the derivative and Lys residue, thus at protein, nonprotein amino acid polymkeric substance, peptide or derivatives thereof form the ε of intermolecular or intramolecular crosslinking-(γ-Glu)-Lys key.

The invention still further relates to (4) coding and derive from the DNA of the trans-glutaminases that resembles these genera bacilluss such as subtilis, relate to (5) recombinant DNA by carrier DNA and described DNA are linked to each other and obtain, relate to (6) and produce the method for bacillus-derived transglutamin-ase 9 by cultivating described transformant with described recombinant DNA cell transformed and (7).

Be referred to herein as crosslinked polymkeric substance by TG or the protein with crosslinking structure, nonprotein amino acid polymkeric substance, peptide and its derivative that have the effect of the component of TG activity to form.

TG is the enzyme of the transacylation of γ in the Gln residue that exists in a kind of catalysis peptide chain-Carboxylamide substrate.Epsilon-amino in peptide chain Lys residue is in the transacylation of acyl acceptor, between peptide molecule or the ε of interior formation intramolecularly or intermolecular cross-linking-(γ-Glu)-Lys key (hereinafter referred to as " GL key ").At water is in the transacylation of acyl acceptor, becomes the Glu residue by the Gln residue.And make the Gln residue in the peptide chain go amidation.

Utilize the bacillus-derived TG of the present invention can produce crosslinked polymkeric substance.The cross-linked polymer of producing thus is used for food, bean curd (Soybean curd) for example, pudding, sour milk, cheese, ground fish meat, the fish of boiling are stuck with paste, in sausage and other fishes and the interior product and in the makeup etc.

Up to now, known TG is present in many animal tissuess.For example, studied the TG that is present in the cavy liver (see Connelllan etal., Journal of Biological Chemistry, Vol 246, pp1093-1098,1971).Related microorganism deutero-TG, only reported that so far the TG that derives from actinomycetes (ray-fungus) or subtilis (sees M.V Ramanujam et al, FASEB J.Vol4, A2321) and the TG that derives from Acarasiales (mucous mould is arranged) (see J.D.Klein et al, J.Bacterial, Vol 174,2599-2605).Up to the present the TG that is produced by actinomycetes has dropped into practice and industrial application (seeing the open No.6-65280 of Japanese Patent, the open No.1-27471 of Japan's special permission).

Because underlying cause, particularly when producing cross-linked polymer, industrialized utilization derives from animal, as the TG of cavy obvious defects is arranged.

Be difficult to obtain the TG of a large amount of described animal derived with low cost.In addition, because TG has the characteristic of demand calcium ion, so its use is restricted.

Actinomycetes deutero-TG also has some unfavorable factors.Because unwrapping wire is grown slowly than common bacteria, so long incubation period of they needs, thereby the cost of producing TG increased.

People such as the Ramanujam of Mexico State University have reported and have had subtilis deutero-TG.Yet the TG by its report has following properties.

1) its suitable pH is 9.5 or higher.2), think that therefore the characteristic that TG has needs metal ion (3) is the Ca of 5mM or greater concn because its activity is subjected to the inhibition of sequestrant (EDTA) to a great extent ²⁺Suppress.4) suppressed by DTT.5) all can produce by vegetative cell and generation spore cell.

Because above-mentioned characteristic, particularly its suitable pH is high and be subjected to the influence of metal ion again, and therefore, the practical application of TG is restricted.

As mentioned above, problem is present in 1) when the TG of animal derived is used for industrial application, because itself needs the characteristic of calcium ion, and production cost is high and it is restricted, 2) when actinomycetes deutero-TG is used for industrial application, therefore the ratio bacterium of actinomycetic growth slow improved production cost, with 3) if the subtilis that will be reported by the investigator of Mexico State University is used for industrial application, then because it is 5mMCa ²⁺Suppress, so can not be used for food.

Therefore, the objective of the invention is, for example separate new TG in the subtilis etc., and the method for producing cross-linked polymer by using TG is provided from being used to produce the bacillus of food so far.

We inventor has carried out arduous research so that from being used for the microorganism of foodstuff production, for example in the subtilis etc., obtains a kind of new TG, found that bacillus, as subtilis etc. a kind of new TG is arranged.Be found to be the basis with this, we have finished the present invention.

Specifically, the present invention includes the bacillus-derived TG of following properties, with produce protein by the component of using TG or containing TG with crosslinking structure, the nonprotein amino acid polymkeric substance, the method of peptide or derivatives thereof, described method is characterised in that: make at protein by TG or the effect that contains the component of TG, the nonprotein amino acid polymkeric substance, Gln in the peptide or derivatives thereof and Lys residue are crosslinked, thus at protein, the nonprotein amino acid polymkeric substance, between the molecule of peptide or derivatives thereof or among form the ε of intermolecular or intramolecular crosslinking-(γ-Glu)-Lys key.

(characteristic of the present invention's bacillus-derived TG)

1) the suitable pH of TG is between about 7 and about 9.

2) its suitable temperature is between about 40 ℃ and about 65 ℃.

3) its about 60 ℃ or following be stable.

4) its activity does not rely on Ca ²⁺Be that TG does not rely on Ca ²⁺, and have 5mMCa ²⁺The time, have 50% or higher activity.

5) be NEM, cystamine and (NH ₄) ₂SO ₄In any material suppress.

6) be not subjected to EDTA, any material among DTT and the 2-ME suppresses.

7) its molecular wt (a) about 18,000 about 28,000 arrives about 30,000 (being measured by SDS-PAGE) to about 22,000 (being measured by gel infiltration) with (b).

8) transacylation of γ-Carboxylamide in the Gln residue in its catalysis peptide chain.

The present invention comprises that also coding is from bacillus, the DNA of the TG that obtains in for example typical subtilis, by with carrier DNA and the recombinant DNA that above-mentioned DNA links to each other and obtains, use the recombinant DNA cell transformed and by cultivating the method that transformant is produced bacillus-derived trans-glutaminases.

Known bacillus, high physics, chemistry and biology resistance have been arranged as the spore of subtilis etc.The inventor thinks that described resistance comes from the GL key in the bacillus spores.This is that they have strengthened the intensity of reticular tissue because the GL key extensively is present in the reticular tissue such as animal blood vessels, hair.

Therefore, the GL key extensively is present in the reticular tissue such as animal blood vessels, hair, in addition, recognizes that TG also is present in the tissue.Therefore, be enhanced by tissue intensity and the effect by TG of recognizing has formed the GL key.

Known case, the inventor utilizes solid phase NMR, and HPLC analyzes to wait and has analyzed bacillus, as the structure of spores such as subtilis, found that to have the GL key in the spore.Be found to be the basis with this, the inventor thinks the TG that also exists catalysis GL key to form in bacillus.

Based on above-mentioned idea, inventor's primary study be present in spore-bearing bacterium in the nature, particularly soil, typical as bacillus, and it is screened as subtilis, therefrom separated the bacterium of generation TG.

We have measured the TG activity at the product pityrosporion ovale in sporulation stage, have found that two have the active bacterial strain of strong TG.We are called AJ12866 and AJ1307 with these two bacterial strains.

We have carried out general evaluation by uncle Jie Shi systems biology classification manual to AJ12866 and AJ1307, show that these two bacterial strains are to belong to subtilis.February 2 nineteen ninety-five subtilis A.J12866 is deposited at the National Institute of Bioscience andHuman-Technology, the Agency of Industrial Scienceand Technology, the Ministry of International Tradeand Industry of Japan (hereinafter referred to as " NIBH "), preserving number is FERM P-14750.According to budapest treaty, subtilis AJ12866 is transferred to international preservation center December 4 nineteen ninety-five; International preserving number is FERM BP-5325.

Subtilis AJ1307 is deposited at NIBH August 22 nineteen ninety-five, and preserving number is FERMP-151123.According to budapest treaty, on January 18th, 1996 subtilis AJ1307 is transferred to international preservation center, preserving number is FERM BP-5367.

TG of the present invention extensively is present in the bacillus that spore is arranged.That is, TG does not exist only in the subtilis, and is present in the following bacillus.

Bacillus comprises Bacillus licheniformis, bacillus megaterium, bacstearothermophilus, bacillus brevis, Bacillus sphaericus bacillus polymyxa, Bacillus alcalo philus etc.

Then, the method for the described bacillus of cultivation and the method for purifying culture are described below, can from culture, obtain TG thus.

Arbitrary method by liquid culture or solid culture can be cultivated bacillus.Especially, deep ventilation and stir culture are industrial favourable.

As the nutrition source in the nutritional medium of cultivating bacillus, operable common carbon source, nitrogenous source, inorganic salt and other trace nutrient that has culturing micro-organisms to use always.Can use available all nutrition sources of bacillus.

As for ventilation, use aerobic conditions.Mention culture temperature, all can cultivate bacillus in its any temperature that can grow and produce TG.Therefore, needn't specifically limit culture temperature, but be generally 10 to 50 ℃, preferred 30 to 40 ℃.

Yet,, therefore can cultivate them in the temperature higher than above-mentioned scope because bacillus is a thermophile bacteria.

Incubation time is decided with culture temperature and other culture condition.Preferably, for example, cultivation bacillus in production maximum TG long-time.Usually, it was cultivated about 5 hours to 7 days preferred about 10 hours to 3 days.

According to the present invention, bacillus shows the active stage of its TG and only limits to the sporulation stage.This is the most tangible place that TG of the present invention is different from the subtilis of being reported by Mexico State University substantially.

Beginning to cultivate bacilli-cell to forming spore, its TG activity increases gradually.Then in the sporulation Phase IV to about the VI, TG is active maximum, after this begins to reduce.In culture, only detect very little TG activity, but detected activity is much bigger in grown cell.

Under cold condition, fragmentation or dissolving be the cell of growth thus.The cell centrifugation that to handle thus in 20000 * g 10 minutes.Then supernatant liquor part and precipitation part are separated from each other, detect the TG activity of each several part.Use this method, confirm that the precipitation that contains spore partly has activity.

Hence one can see that, and TG is present in the surface of spore.

For the bacillus-derived TG of purifying, can directly handle the culture that contains the bacillus grown cell obtaining the TG of purifying, but the sporocyst of preferably at first broken or dissolving grown cell, handle the spore of gained then, to obtain the TG of purifying.

Through cultivating the sporocyst that bacillus obtains, can obtain the spore of bacillus by broken or dissolving.After fragmentation or dissolution process, the TG activity is collected in the insoluble part that contains spore.Therefore, can also concentrate insoluble part to obtain required enzyme preparation.

For the TG activity that will be collected in the insoluble part is returned to (promptly in order to dissolve the TG activity) in the soluble part, need carry out following operation.

As an example, with tensio-active agent, as Triton X-100, alkyl glucoside etc. are added in the insoluble part, thus the TG activity are recovered in the soluble part of gained.Can also use ealkaline buffer (for example, 20mMNaHCO ₃Damping fluid pH10) is handled the part that contains spore, thus the TG activity is recovered in the soluble part of gained, also the part that contains spore can be suspended from the damping fluid, heats the suspension of gained then, thus the TG activity is recovered in the soluble part of gained.For example, by adding hot suspension, the TG activity can be recovered in soluble part in 10 ℃ or higher temperature.

Available dissolved TG is as peptizing agent.Utilization can be used for any ordinary method of purifying enzyme, the example gel infiltration, and ion exchange chromatographies etc. can be further purified dissolved TG.As a result, can obtain having the TG of higher relative activity.The TG of purifying can be the peptizing agent that higher relative TG activity is arranged thus.

Use following method and measure the TG activity.With ¹⁴The putrescine of C-mark and dimethyl casein are as substrate, and the sample that will contain TG is added on these substrates, so that it reacts each other, with the dimethyl casein of 10%TCA precipitation putrescine bonding, then on the precipitated sorbent filter paper with gained.Because the radioactivity on the filter paper is directly proportional with the TG activity in the sample, therefore can quantitatively determine the TG activity in the sample.Can measure radioactivity with liquid scintillation counter.

Hereinafter subtilis AJ1307 deutero-TG is called TG-1, and subtilis AJ12866 deutero-TG is called TG-2.Based on the data of TG-1 and TG-2, the zymochemistry characteristic of TG of the present invention is described hereinafter.

Suitable pH scope:

The pH that TG of the present invention suits is about 7 and about about 9.

In order to determine that TG has the pH scope of suitable active, will carry out 30 minutes with the various enzyme reactions of TG in 37 ℃.

Suitable temperature range:

The optimal temperature of TG is at about 40 ℃ and about about 65 ℃.

In order to determine that TG has the temperature range of suitable active, at pH7.5, will carry out 30 minutes with the various enzyme reactions of TG.

Temperature stability:

TG about 60 ℃ or following be stable.

At pH7.5,, detect the temperature stability of TG then with TG pyroprocessing 10 minutes.Even TG stands 60 ℃ pyroprocessing, still keep about 80% TG activity.

The influence of inhibitor:

NEM (N-ethylomaleimide) and cystamine have largely suppressed the bacillus-derived TG of the present invention.In addition, (NH ₄) ₂SO ₄(ammonium sulfate) also has obvious restraining effect to it.

The influence of DTT and EDTA:

Exist under the condition of DTT (dithiothreitol (DTT)), can improve the activity of the bacillus-derived TG of the present invention.Yet the existence of EDTA (ethylenediamine tetraacetic acid (EDTA)) is to the almost not influence of TG activity.

TG of the present invention does not need Ca ²⁺Characteristic.That is, it is not rely on Ca ²⁺Enzyme.There is 5mMCa ²⁺Condition under, TG has still kept 50% or higher activity.

Molecular weight:

The molecular weight of TG is that (a) about 18,000 about 28,000 arrives about 30,000 (measuring through SDS-PAGE) to about 22,000 (being measured by gel infiltration) with (b).

Active:

There is the transacylation of γ-Carboxylamide on the Gln residue in the TG catalytic substrate in the peptide chain.Epsilon-amino is in the transacylation of acyl acceptor on peptide chain Lys residue, in peptide molecule or between form the ε of intramolecularly or intermolecular cross-linking-(γ-Glu)-Lys key.At water is in the transacylation of acyl acceptor, makes Gln residue in the peptide chain through going the acid amides effect by the Gln residue being become the Glu residue.

As indicated above, the characteristic that derives from the TG of the present invention of subtilis AJ12866 and subtilis AJ1307 obviously is different from the subtilis deutero-TG that is reported by research group of Mexico State University.

Then, the method for producing cross-linked polymer with TG of the present invention hereinafter will be described.

According to the present invention, the TG that is used to produce cross-linked polymer can be the insoluble enriched material that contains the spore part that (1) obtains by sporocyst broken or the dissolving culturing bacterium, (2) in all sorts of ways the insoluble part of dissolving and there is the purifying TG of high relative activity the have active part of TG and (3) that obtain.

Can also be by cultivating the bacillus-derived TG that the transformant cell that transforms with recombinant DNA obtain, link to each other with carrier DNA and make up described recombinant DNA with the bacillus-derived TG that will encode of method hereinafter described.

Except that above-mentioned situation, can also be with active all other parts of bacillus-derived TG are arranged.

As the substrate of TG or TG-active ingredient, use one or more protein, nonprotein amino acid polymkeric substance, peptide and its derivative.

Needn't limit especially proteinic source and characteristic, as long as used protein has Lys and Gln residue and is above-mentioned catalyst.For example, casein, gelatin, soybean proteins etc. can be used as substrate.Also can use through the partially digested protein of proteolytic enzyme in addition.

The nonprotein amino acid polymkeric substance comprises the aminoacid polymers of producing with chemical synthesis, as polylysine, etc.

Peptide can be obtain with chemical synthesis or with acid, degraded native protein such as alkali, proteolytic enzyme and obtain those.

The derivative of described material comprises the protein of glycoprotein, chemically modified etc.

Any other material is as long as it has Lys and Gln residue just can act on the substrate of TG or TG-active ingredient.

Add TG of the present invention or the active component of TG arranged, then with substrate in, contain concentration and be 0.1% or the more solution or the suspension reaction of high protein, thereby obtain crosslinked polymer product.According to its crosslinking degree, the cross-linked polymer that will obtain according to the present invention can be divided into gel product group, high sticking product group and the product group of polymkeric substance only.The present invention includes whole described cross-linked polymers.

Usually, the pH of reaction soln is about 4 to about 10, and temperature of reaction is about 5 ℃ to about 80 ℃, and the reaction times is about 10 seconds to about 24 hours.The result of reaction has obtained cross-linked polymer (gel product, high sticking product etc.).

The method of producing bacillus-derived TG according to the present invention with recombinant DNA technology will be described below.

Knownly manyly produce useful proteins matter such as enzyme, the example of the material etc. of physiologically active is arranged with recombinant DNA technology.With an advantage of recombinant DNA technology is the useful proteins matter that can mass production only exists on a small quantity at nature.

For with the bacillus-derived TG of described recombinant DNA technology production the present invention, just need the DNA of the bacillus-derived TG of coding.Described DNA is linked to each other with carrier DNA to make up required recombinant DNA.Use the recombinant DNA transformed host cell.In substratum, cultivate the transformant that can produce bacillus-derived TG that had transformed, in substratum or cell, make cells produce thus and accumulate bacillus-derived TG, collect TG thereafter.

The method of the DNA of the bacillus-derived TG that obtains encoding is described below.

At first, determine the aminoacid sequence of purifying TG.Can by the Edman method (see Edman, P, Acta chem.Scand, 4,227 (1950) determine required-aminoacid sequence.In addition, also can determine aminoacid sequence with the sequenator that AppliedBiosystems Co produces.

Determine that among the bacillus-derived TG of the present invention, from the aminoacid sequence of 35 residues of N-end to the, it is equivalent to the sequence of Sequence Num-ber1 in the sequence table.

Based on the aminoacid sequence of determining thus, can infer its DNA base sequence of coding.In order to infer the DNA base sequence, used is codon the most frequently used in universal code or the bacillus gene.

Based on the base sequence of inferring thus, the synthetic dna molecular that 30 to 50 left and right sides base pairs are arranged.At Tetrahedron Letters, the method for synthetic described dna molecular has been described in 22,1859 (1981).Also can synthesize described dna molecular with the synthesizer that Applied Biosystems Co produces.If separate the full length DNA of the bacillus-derived TG of coding from bacillus chromogene library, then available described dna molecular is made probe.In addition, if during with the DNA of the bacillus-derived TG of pcr amplification coding, also available its as probe.Yet,,, from bacillus chromogene library, separate the full length DNA of the bacillus-derived TG of coding so use DNA to make probe through pcr amplification owing to do not comprise the full length DNA of the TG that coding is bacillus-derived through the DNA of pcr amplification.

White has described the working method of PCR among the people such as T.J (in Trend Genet, 5,185 (1989) etc.).At Molecular Biologi-cal Methods for Bacillus (John Wiley ﹠amp; Sons, Ltd (1990), etc.) in the method for preparing the bacillus chromosomal DNA has been described.At Molecular Biologocal Methads for Bacillus, (JohnWiley ﹠amp; The method that makes up chromogene libraries such as bacillus has been described Sons, Ltd (1900) etc.).In Molecular Cloning (2ndEdition, Cold Spring Harbor Press (1989) etc.), described and made probe separates required dna molecular from gene library method with dna molecular.

In order to determine the TG that coding is bacillus-derived and the base sequence of separated DNA as stated above, used method is at A Practical Guideto Molecular Cloning, John Wiley ﹠amp; Sons, Inc is described in (1985).In order to carry out base order-checking, the dna sequencing instrument that also can use AppliedBiosystems Co. to produce.

The DNA of the TG that the code book invention is bacillus-derived is shown in the Sequence Number2 in the sequence table.From the chromosomal DNA of subtilis AJ1307, separate described DNA.The DNA of the TG that code book invention is bacillus-derived is not limited only to be shown in the sequence among the Sequence Num-ber2 in the sequence table.According to its bacillus specie of originating and bacterial strain, DNA should have different base sequences.

The DNA that encodes from the isolating TG of bacillus chromosomal DNA can be carried out artificial mutation to modify described DNA base sequence.A kind of common method of carrying out described artificial mutation is for example at Method in Enzymol, the locus specificities sudden change of describing in 154 (1987).As long as the DNA of any artificial mutation is the bacillus-derived TG of coding, then all in the present invention encodes the scope of DNA of bacillus-derived TG.

The DNA that contains the TG by the subtilis AJ1307 derivative of will encoding links to each other the E.coliAJ13172 cell of the recombinant DNA that makes up according to budapest treaty with carrier DNA, be deposited in the international preservation center of NIBH December 20 nineteen ninety-five, international preserving number is FERMBP-5346.

Link to each other by DNA and to make up recombinant DNA carrier DNA and the bacillus-derived TG of coding.Then with the recombinant DNA transformant obtaining the transformant cell, in substratum, cultivate the transformant cell so that cell produces and accumulate bacillus-derived TG in substratum and/or cell, collect TG then and can the bacillus-derived TG of mass production.

Produce under a large amount of proteinic many situations with recombinant DNA technology at needs, the protein of being produced is intracellular, and is relevant with the inclusion body in producing described proteinic transformant.The advantage of the expression method of this production protein is to make required protein to avoid by the protease digestion in the used cell, and by smudge cells, then the cell debris of the centrifugal gained required protein of purifying at an easy rate.

With the protein inclusion body that the protein denaturant dissolving obtains thus, remove denaturing agent then and recover molten activity of proteins basically, after this, institute's dissolved protein is become correct folding tool physiologically active protein matter.Known many examples relevant with this method, an example is exactly an activity (seeing the open No.61-257931 of Japan's special permission) of recovering human interleukin-12.

In order from the protein inclusion body, to obtain activatory protein, must need processes such as a series of dissolving, recovery activity, and described operating process is often proteinic more complicated than direct production activatory.Yet, as if production amounts of protein in cell, and, can in cell, accumulate protein with described non-activity inclusion body form if the growth of the protein pair cell of being produced is influential, lower proteinic influence thus.

Method as for mass production desired protein inclusion body, the known method that expression desired protein itself under strong promoter control is arranged, and express method with the desired protein of fusion rotein form, the another kind of protein fusion of wherein required protein and known great expression.

In addition, can earlier some limit protein enzyme enzyme recognition sites in position be inserted fusion rotein effectively, so that after it is expressed, required protein can be cut out from fusion rotein.

The proteinic host cell of mass production that is used for that transforms with recombinant DNA technology comprises bacterial cell, actinomycetes cell, yeast cell, fungal cell, vegetable cell, zooblast etc.Usually, intestinal bacteria, E.coli is preferred.This is owing to have many with the proteinic prior art of E.coli cell mass production.Method with the bacillus-derived TG of the E.coli cells produce the present invention who transforms is hereinafter described.

As for the promotor of the DNA that is used to express the bacillus-derived TG of coding, spendable is generally to be used to make E.coli to produce the promotor of exogenous protein.For example, spendable strong promoter such as T7 promotor, trp promotor, lac promotor, tac promotor, PL promotor etc.

In order to make the bacillus-derived TG of host cell production as the fused protein inclusion body, should be with the another kind of protein of coding, the gene of preferred hydrophilic peptide links the upstream of bacillus deutero-TG gene in each host cell or site, downstream so that construction of fusion protein gene therein.The another kind of proteinic gene of encoding can be anyly to have the ability to be increased in the desired protein of host cell accumulation and after fusion rotein sex change and renaturation, can improve the gene of fusion rotein stability.Candidate gene has T7 gene 10, beta-galactosidase gene, dihydrofolate reductase gene, interferon-gamma gene, interleukin-2 gene, prochymosin gene etc.

If any gene with the bacillus-derived TG that encodes in these genes is linked to each other, then the codon of the reading frame of these two kinds of genes should be identical.Can utilize the gene or any synthetic DNA that suitable sequence is arranged that are connected with each other in suitable restriction endonuclease site.

In order to improve the quantity of producing bacillus-derived TG by host cell, preferably will there be the terminator of transcription termination sequence to be connected in the site, downstream of antigen-4 fusion protein gene.Terminator comprises, T7 terminator for example, and fd phage terminator, the T4 terminator, the tetracycline resistance gene terminator, intestinal bacteria TrpA gene terminator, etc.

Be imported into the preferably so-called multi-copy vector of E.coli survey if contain the carrier of the gene of the fusion rotein that bacillus-derived TG of coding or coding be made up of bacillus-derived TG and another protein.Described carrier comprises: puc plasmid for example, pBR332 plasmid and its derivative.In order to screen the transformant of gained better, described carrier preferably contains such marks such as resembling ammonia benzyl penicillin resistant gene.For described plasmid, the various expression vectors that contain strong promoter all are (for example, the producing the pUC plasmid by TakaraShuzo Co, the pPROK plasmid of being produced by Clonetec Co., the pKK233-2 that is produced by Clonetec Co etc.) that can obtain from commercial channels.

The dna fragmentation that will contain promotor, the gene of the fusion rotein that encode bacillus-derived TG or coding are made up of bacillus-derived TG and another protein links to each other with such order with terminator, links to each other with carrier DNA then with the structure recombinant DNA.

With described recombinant DNA Transformed E .coli cell, the transformant cell of cultivating gained then is so that its expression and the fusion rotein producing bacillus-derived TG or be made up of bacillus-derived TG and another protein.

As for host transformed, can be with any bacterial strain that is suitable for expression alien gene.Particularly preferably be E.coli JM109 (DE3) bacterial strain and JM109 bacterial strain.(2nd Edition has described the method for method for transformation with screening gained transformant among the ColdSpring Harbor Press (1989) etc. at Molecular cloning.

If when having expressed fusion rotein, then can modify it, so that can cut TG with restricted proteolytic enzyme from fusion rotein, described limit protein enzyme contains not to be had in TG, as the sequence of recognition sequence, as blood Rh factor Xa, kallikrein etc.

As for producing substratum, can use any conventional substratum that is generally used for cultivating the E.coli cell, as the M9-Casamino substratum, LB substratum etc.Should be according to used carrier indicium, promotor, host cell type etc. are screening and culturing condition and induce the condition of production suitably.

For the fusion rotein that reclaims and collect bacillus-derived TG or form by bacillus-derived TG and another kind of protein, for example available following the whole bag of tricks.

If TG or fusion rotein are dissolved in the culturing cell, collecting cell then, broken then or dissolving, the liquid that contains cell debris that obtains thus can be used as crude enzyme liquid.If desired, liquid is further carried out routine precipitation, filtration, post layer bar etc., purifying TG or fusion rotein before use thus.

If desired, also can use method through its antibody purification TG or fusion rotein.

If form the protein inclusion body, then the available protein denaturing agent dissolves it.In this case, the protein inclusion body can be dissolved with the cell that contains it.Yet, consider purification step subsequently, preferably before dissolving, separate inclusion body earlier.All can from cell, separate inclusion body with any known method.For example, smudge cells, centrifugal then etc., reclaim inclusion body thus.

As for the protein denaturant that is used for the solubilising protein inclusion body, can use Guanidinium hydrochloride (for example, concentration is 6M, pH5-8), urea (for example concentration is 8M) etc.

Remove used protein denaturant through dialysis etc., obtain activated protein once more.The dialysis solution that is used for described dialysis can be the tris-Hcl damping fluid, phosphate buffered saline buffer etc.Its concentration can be from 20mM to 0.5M, and its pH can from 5 to 8.

Protein concn in the renaturation step is limited to and is not higher than about 500 μ g/ml.In order to prevent that the bacillus-derived TG of renaturation carries out self-crosslinking thus, just need make the infiltration temperature not be higher than 5 ℃, in order to reclaim protein denaturant, except that above-mentioned dialysis method, also available dilution method, ultrafiltration process etc.For the activity of recoverin matter, can use any in these methods.

If with the DNA of Sequence Number2 sequence in the tool sequence table DNA as the bacillus-derived TG of coding, the bacillus-derived TG of the aminoacid sequence shown in then can obtaining containing under Se-quence Number2 base sequence.In this DNA, the scope of open reading frame is from 118A to the 8692C residue.

Embodiment:

Utilize the following example to describe the present invention in more detail below.Yet the present invention is not limited only to the embodiment of these embodiment.

Embodiment 1, produces and purifying TG

Cultivate subtilis AJ1307 cell so that it has enough TG activity.Use Schaeffer ' s substratum, shake cultivation or liquid ventilation and stir culture in 37 ℃ through liquid and finish cultivation.The component of used Schaeffer ' s substratum has 8g/l Bacto nutritive medium, 1g/l KCl, 0.12g/lMgSO ₄7H ₂O, lmMCaCl ₂, 10 μ M MnCl ₂And 1 μ MFeSO ₄, pH7.0.

At first, the cell of AJ1307 bacterial strain was cultivated 24 hours in the 20ml substratum, carried out seed culture.The culture that again 5ml is contained seed cell is incubated in three batches of substratum of each 100ml.Cell in each substratum reaches logarithmic growth during the later stage, and each culture is forwarded in the another kind of substratum of 900ml.When the cultured continuously cell, three batches of substratum link to each other successively.Cell in each is cultivated is long again to arrive logarithmic growth during the later stage, and totally 3 liters of cultures forward in 27 liters of another kind of substratum, and then cultivate, and stirs with the 1/4vvm ventilation and with 350rpm simultaneously.

Reach logarithmic growth again during the later stage at cell, 30 liters of cultures are forwarded in 270 liters of another kind of substratum, further cultivate therein, stir with the 1/20vvm ventilation and with 200rpm simultaneously.This is last cultivation.After cell reaches stationary phase, continue again to cultivate 6 hours.After this, finish last cultivation.Last culture is cooled to rapidly below 20 ℃ or 20 ℃ with cold water.Use continuous centrifuge centrifugal then with collecting cell.As parent material, therefrom use following method purifying TG with the cell that obtains thus.

Determine the hereinafter TG activity of gained enzyme liquid by following enzymic activity detection method.50 μ l reagent solutions (100mMTris-HCl, pH7.5,6.3mg/ml dimethyl casein, the 10nM that will contain 10 μ l enzyme liquid to be checked ¹⁴The C-putrescine, 1.2 μ Ci) in 37 ℃ of reactions 30 minutes, the liquid adsorption that 40 μ l are reacted was thus fixed with 10%TCA to filter paper then.Then use 5%TCA solution washing 3 times, count its radioactivity with liquid scintillation counter.The TG activity that the value of counting thus is called enzyme liquid.

1. washed cell:

Be suspended among the 50mM Tris-HCl (pH7.5) cultivating the cell of collecting the back, with 20, centrifugal 30 minutes of 000xg is the cell of collecting precipitation portion-form once more.This suspension and centrifugation step are for washed cell.This cell washing process is repeated 2 times altogether.

2. dissolved cell:

In the wet cell that washs thus of 1 part of weight, add 9 parts of weight and used ice-cooled Buffer 1 (100mM Tris-HCl (pH7.5); 0.5mg/ml N,O-Diacetylmuramidase, 20 μ g/ml DNase I, 1mM EDTA; 2mM phenylmethane alkylsulfonyl is fluoridized thing (PMSF), and cell is suspended from the damping fluid.The suspension of gained being stirred 1 to 3 hour, make cytolysis thus on ice.

3. preparation spore:

After the dissolving, in 4 ℃, with 20,000g was with gained solution centrifugal 30 minutes.Measure each centrifuged supernatant and centrifugal precipitation is suspended from Buffer 2 (100mM Tris-HCl (pH7.5), 1mM EDTA) and the TG activity of the suspension of preparation.As a result, in precipitation suspension, detect the TG activity.Precipitate suspension with microscopic examination, the result shows that suspension contains microbial spores and dissolved cell debris residue.

To detect the active precipitation suspension of TG and stir 30 minutes, use ice-cooledly simultaneously, and then, with its centrifugal 30 minutes, measure each centrifugal supernatant liquor then and the centrifugal precipitation is suspended among the Buffer2 and the TG activity of the suspension of preparation with 20,000 * g.As a result, in precipitation suspension, detect the TG activity.

Carrying out this suspension, stirring and centrifugation step with Buffer2 is in order to wash spore.Described spore washing is repeated 4 times altogether.In this process, can detect the TG activity in the precipitation part.After spore washed 4 times altogether, detected the last suspension of the precipitation part of TG activity with microscopic examination, the result shows that suspension contains the cell debris residue hardly, and only contains microbial spores.Microscopic examination also shows the spore that does not have sprouting.

4. dissolve TG:

After the washing, the centrifugal and collecting precipitation part with spore is suspended from it Buffer3 (0.1M Na that is heated to 37 ℃ in advance then ₂CO ₃, 1mM EDTA, 50mM DTT, pH10.0) in.The pH of gained suspension is adjusted to 10.0, in 37 ℃ suspension was stirred 30 minutes then.Then with it with 20, centrifugal 30 minutes of 000xg detects each centrifuged supernatant and centrifugation is suspended from Buffer3 and the TG activity of the suspension for preparing.

As a result, detect the TG activity at the centrifugal supernatant liquor.The result confirms to have dissolved TG.The solution that contains TG is called thick TG solution.

5. precipitation and remove the protein deposited of companion under acidic conditions

By the thick TG solution of filter paper filtering, then acetate is added in the filter thing of gained so that the pH of filter thing reaches 5.8.Then will filter thing at 5 ℃ stirred 1 hour.Consequently form white precipitate, think the isoelectric protein precipitation.Then with 20,000xg is its centrifugal 30 minutes, thus precipitation separation from centrifuged supernatant.Precipitation is dissolved in Buffer3.Detect the TG activity of the solution of centrifuged supernatant and precipitation respectively.As a result, in centrifuged supernatant, detect the TG activity.

6. with (NH ₄) ₂SO ₄Precipitation TG

The 1M Tris-HCl (pH7.5) that in having detected the active centrifuged supernatant of its TG, adds 1/20 volume (for supernatant liquor).Then add (NH ₄) ₂SO ₄, be dissolved in wherein, make final concentration reach 50% saturation ratio.Adding NaOH makes the pH of gained solution reach 7.5.Follow on ice it being stirred 2 hours, then with 20, centrifugal 30 minutes of 000xg.To Buffer4 (25mM Tris-HCl (pH7.5), 5mM folds sodium hydride) dialysis centrifugal supernatant liquor thus, simultaneously the centrifugal precipitation is dissolved in Buffer4, then to the Buffer4 dialysis.By dialysis, removed (NH fully ₄) ₂SO ₄, detect supernatant liquor part and precipitation TG activity partly respectively.As a result, in centrifugal precipitation part, or in other words, there is the saturated (NH of 50%- ₄) ₂SO ₄The time detect the TG activity in the precipitation part that forms.

7. hydrophobic chromatography:

To Buffer5 (50mM Tris-HCl, 0.75M MgSO ₄, 0.02% (w/v) sodiumazide, pH9.0) dialysis has detected the active solution of its TG.With 20,000xg with centrifugal 30 minutes of gained dialysis thing with clear liquid analytically.The supernatant liquor that obtains thus is added to uses damping fluid equilibrated hydrophobic chromatography post in advance, phenyl Sepharose HP (producing) by pharmaciaCo.By this step, TG is adsorbed on the gel.

Wash the protein (not Xi Fu protein) that is not adsorbed onto on the gel off with Buffer5.Then with the protein of the damping fluid that contains ethylene glycol as the absorption of elutriant wash-out.In wash-out, MgSO ₄Be linear change with the concentration of ethylene glycol in damping fluid.That is, make MgSO in the used damping fluid ₄Concentration from the 0.75M linear change to 0M; And wherein glycol concentration from 0% (v/v) linear change to 10% (v/v), so that finish wash-out.

Detect the TG activity of the eluate part that obtains through wash-out respectively, the result is the MgSO about 150 to 200mM ₄Observe the TG activity in the eluate of the glycol concentration wash-out of concentration and about 7 to 8% (v/v).

8. gel infiltration:

With ultra-filtration equipment (centriprep is produced by Amicon Co) is concentrated the active part of TG is arranged, then to Buffer6 (25mMTris-HCl, 150mM NaCl, 1% (v/v) ethylene glycol, 0.02% (2/v) sodiumazide, pH8.0) dialysis.With 20,000xg concentrates 10 minutes to collect the supernatant liquor of gained with the dialysis thing of gained.The supernatant liquor of gained thus is used for using in advance Buffer6 equilibrated gel permeation column, Sepharacryl S200HR (producing) by pharmacia Co.Detect the TG activity of each wash-out part.As a result, be about 18,000 to detect the TG activity in about part of about 22,000 at molecular weight

9. anion-exchange chromatography:

Use ultra-filtration membrane to concentrate the TG part of gained thus, then to Buf-fer7 (25mM piperazine, 1% (v/v) ethylene glycol, 0.02% sodiumazide, pH10.5) dialysis.With 20, the dialysis thing of the centrifugal gained of 000xg 10 minutes is to collect supernatant liquor.The supernatant liquor of gained thus is used for by Buffer7 equilibrated anion-exchange chromatography post Mono-Q (producing) by Pharmacia Co..Thus, TG is adsorbed onto on the gel.

Wash the not protein of absorption off with Buffer7.Then with the protein of the damping fluid that contains NaCl as the absorption of elutriant wash-out.Make NaCl concentration in the used damping fluid from the 0mM linear change to 500mM, finish wash-out with this.The TG activity of each eluate part that detection is obtained by wash-out, the result shows, observes the TG activity at about 50mM in the eluate of the NaCl concentration wash-out of 150mM.

The active part of gained thus is used for SDS-PAGE, dyes with Coomassie brilliant blue then.The TG that confirms purifying thus has a band, and the molecular weight of estimation TG may be for about 28,000 to about 30,000 (see figure 1)s.

The relative activity of determining purification part increases to some extent.Specifically, measure the TG activity of the active part of the TG activity of above-mentioned thick TG solution and purifying.The result shows: so bring up to 600 times of thick solution owing to passed through the heavy relative TG activity of serial purifying per unit purification part protein.In the active method of measurement used herein, estimate that the relative TG activity of purification part is about 2.5 * 10 ⁴Dpm/mg/30 minute (37 ℃, pH7.5).

10. determine TGN-end aminoacid sequence on every side.By following definite with the aminoacid sequence around the TGN-end of aforesaid method purifying.

Exist under the situation of SDS, the TG of about 10 μ g purifying part (with proteinometer) through polyacrylamide gel electrophoresis, is forwarded to the TG in the gel on the film filter paper then.Thus, begin to analyze the aminoacid sequence of TG from its N-end with protein sequencer.

Specifically, (see Protein Structure Analysis by the half-dried system that uses Milliblot (producing) by Millipore Co., write by H.Hirano, Tokyo Kagaku Dojin publishes), with required enzyme from through electrophoresis and gel transcribe on polyvinylidene difluoride (PVDF) (PVDF) film.Use protein sequencer (Model476A is produced by ABI Co) to measure the aminoacid sequence of transcribing the required enzyme on the pvdf membrane thus then.

Therefore, determine to begin to contain the aminoacid sequence of the TG of 35 residues from its N-end.Aminoacid sequence around the N-end of the trans-glutaminases of Que Dinging is shown in the Sequence Number1 in the sequence table thus.

Embodiment 2: pH and the temperature range of determining TG

Measure the suitable pH scope of the definite thus TG of variation that the TG enzymic activity relies on pH with following method.

As for the damping fluid that is used for enzymatic reaction, can use sodium formiate damping fluid (pH:2.0,3.0,3.5,4.0), sodium acetate buffer (pH:4.5,5.0,5.5,6.0), Tris-HCl damping fluid (pH:7.0,7.5,8.0,8.5,9.0) and Na ₂CO ₃Damping fluid (pH:9.0,9.5,10.5,12.0).

In order to measure the TG activity, available use ¹⁴The putrescine of C-mark and dimethyl casein are the aforesaid method of substrate.Respectively the concentration of damping fluid with 50mM is added in the reactive system.As for the enzyme source, can use previously prepared concentration is the pure TG part of 2 μ g/ml.Being reflected at 37 ℃ carried out 30 minutes.

According to the pH value scope of each reactive system, determine the relative value of enzymic activity.Be 8.2 at pH when (is that 8.5 Tris-HCl is when being damping fluid with pH), reactive system has the highest TG activity, with it as standard: 100.The results are shown in Fig. 2.

The suitable pH scope of having found TG of the present invention is about 7 to about 9, is to about 8.8 (see figure 2)s strictly speaking from about 7.7.On the other hand, the appropriate pH value scope by the subtilis deutero-TG of group of Mexico State University report is 9.5 or higher.Therefore, have significantly different by they the bacillus-derived TG of TG and the present invention of report.

The temperature dependency of measuring TG enzymic activity aspect as follows changes, and determines the temperature range that TG is suitable thus.

In order to measure the TG activity, can use above-mentioned introduction to use ¹⁴The putrescine of C-mark and dimethyl casein are the method for substrate.Specifically, with 0.1MTris-HCl the pH of reactive system being adjusted to 7.5, as for the enzyme source, is that the previously prepared pure TG of 2 μ g/ml partly is added in the reactive system with concentration.In being 25 ℃ to 80 ℃ water-bath, temperature makes reactive system reaction 30 minutes.

Relative value according to the measure of the change enzymic activity of temperature of reaction.At 60 ℃, reactive system has maximum TG activity, and this is decided to be 100.The results are shown in Fig. 3.

The optimal temperature scope of having found TG of the present invention more strictly speaking, arrives about 62 ℃ of (see figure 3)s at about 50 ℃ at about 40 ℃ to about 65 ℃.

Embodiment 3: the temperature stability of determining TG

Determine the temperature stability of TG of the present invention as follows.

In 10 ℃ to 80 ℃ transformation temperature water-bath, make previously prepared pure TG partial reaction 10 minutes.After this, by with embodiment 2 in identical method measure the TG activity of each system.Determine the relative value of enzymic activity according to the variation of temperature of reaction.The highest TG activity of reactive system is decided to be 100.The results are shown in Fig. 4.

Having found TG of the present invention in not being higher than about 60 ℃ temperature, all is stable (see figure 4)s.

Embodiment 4: use the TG cross-linked proteins

Determine protein---the crosslinking activity of TG of the present invention as follows.

It is the 1mg/ml alpha-casein that preparation contains final concentration, 0.1MTris-HCl (pH7.5), the reactive system of 5mM DTT and 5mM sodiumazide.

Alpha-casein used herein is the commercially available prod that is produced by Sigma Co..Adding final concentration to this system is the previously prepared pure TG parts of 440 μ g/ml.

37 ℃ of water-baths, with this reactive system reaction 18 hours.Make thus the system of reaction carry out SDS-PAGE, the result also has a band in high polymers one side more except that the band of substrate alpha-casein.This has shown the caseic crosslinking polymerization of α.The results are shown in Fig. 5.

These results have confirmed protein-crosslinking activity (see figure 5) of TG of the present invention

When bovine serum albumin (BSA) being reacted, also can obtain result same as described above with TG of the present invention.That is it is crosslinked that, TG of the present invention also can make substrate protein BSA.

Embodiment 5: all ingredients is to the active influence of TG

Detect, if having, all ingredients is to the active influence of TG.The reagent that the present invention detects is N-ethyl maleinamide (NEM), and cystamine, phenylmethane alkylsulfonyl are fluoridized thing (PMSF), (NH ₄) ₂SO ₄, Na ₂SO ₄, EDTA, EGTA, CaCl ₂, DTT and 2 mercapto ethanol (2-ME).All reagent are all available from Nacalai Tesque, Inc.Co..

As for the enzyme source, be the previously prepared pure TG parts of 2 μ g/ml with concentration.Measure protein concn with protein detection box (producing) by Bio Rad Co..

In order to detect the TG activity, can use above-mentioned with ¹⁴The putrescine of C-mark and dimethyl casein are the method for substrate (pH7.5,37 ℃).As for the enzyme source, be the pure TG parts of 2 μ g/ml ready-formed with concentration.In 37 ℃ of reactions 30 minutes.

Detect the TG activity with following method.With above-mentioned pure TG part and various modulated reagent mix, put on ice then 30 minutes to suitable concentration.To transfer to concentration with the enzyme source solution of each reagent react is 2 μ g/ml, then with substrate reactions, detects the TG activity (pH7.5,37 ℃) that still has in the gained reactive system.

Being not 100 (contrasts), be designated as the relative value of contrast with the TG activity that keeps in the reactive system of each reagent react with the TG activity of the TG of any reagent react part.The results are shown in hereinafter table 1.

The TG activity of TG of the present invention is subjected to the inhibition of NEM, and is not subjected to the inhibition of reductive agent DTT and 2-ME, can improve its activity on some degree on the contrary.These presentation of results Cys residue may participate in the active expression of TG.

TG of the present invention is not subjected to the inhibition of DTT, and the known subtilis deutero-TG that is reported by group of Mexico State University can be subjected to the inhibition of DTT.Promptly two kinds of TG have visibly different characteristic.

In addition, TG of the present invention is not Na ₂SO ₄Suppress, but be subjected to (NH ₄) ₂SO ₄Inhibition with cystamine.Therefore, illustrate that TG of the present invention is characterised in that in containing the reactive system of some amine, its activity is suppressed.

In addition, sequestrant EGTA and EDTA do not suppress TG of the present invention.From its this characteristic, TG of the present invention is different from the TG by group of Mexico State University report.In other words, should not need metal ion such as Ca by TG of the present invention ²⁺, the characteristic that waits.This paper is used to detect the active reactive system that contains TG of the present invention of TG and does not contain Ca ²⁺, and TG of the present invention does not have Ca at these ²⁺Reactive system, in the TG activity is arranged.From these results, can reach a conclusion: TG of the present invention does not rely on Ca ²⁺.

In addition, be lower than 5mMCa in existence ²⁺Condition under, TG of the present invention still has and is no less than 50% TG activity.Therefore, TG of the present invention is different from the TG by group of Mexico State University report, and the promptly known latter can be the Ca of 5mM or greater concn to a great extent ²⁺Suppress (seeing Table 1).

Embodiment 6:

Purifying is from the TG of bacillus subtilis AJ12866 bacterial strain, and detects its characteristic.

In 37 ℃ of cells of in the Schaeffer substratum, cultivating subtilis AJ12866 in 16 hours by shaking culture.With the 3ml culture be added to 30ml in addition-the Schaeffer substratum in, and cultivated again 12 hours in 37 ℃.Used Schaeffer substratum contains the Bacto nutritive medium of 8g/l, 1g/l KCl, 0.12g/l MgSO ₄7H ₂O), 1mM CaCl ₂, 10 μ M MnCl ₂With 1 μ M FeSO ₄PH7.0.

With 10, centrifugal 20 minutes of 000xg is with precipitation separation and supernatant liquor with culture.

Grind precipitation with granulated glass sphere.Detect culture supernatants, culture precipitation and ground throw out liquid (cell debris liquid) TG activity respectively.The following is the method that this paper is used to detect enzymic activity.50 μ l reactive systems (100mM Tris-HCl, pH7.5,6.3mg/ml dimethyl casein, the 10nM that will contain 10 μ l enzyme sample to be measured ¹⁴The C-putrescine, 1.2 μ Ci) kept 30 minutes at 37 ℃, make substrate reactions thus.After the reaction, 40 μ l reaction mixtures are adsorbed onto on the filter paper.In reaction mixture, because the wherein katalysis of TG, putrescine will react with the dimethyl casein.Putrescine and dimethyl casein bonded reaction product are adsorbed onto on the filter paper.By to wherein adding 10%TCA, the bonded reaction product is fixed on the filter paper.With 5%TCA solution filter paper is washed 3 times then, being fixed on the filter paper with the liquid scintillation counter counting again ¹⁴The C radioactivity.The value of Ce Lianging is equivalent to the relative TG activity of enzyme sample thus.Gained the results are shown in hereinafter table 2.

These results show as the ground throw out liquid that contains the microbial spores part to have the TG activity that is higher than other samples.

Embodiment 7: the stage of determining to induce bacillus-derived TG

By the method identical, cultivate the cell of subtilis AJ12866, with the TG activity of the cell debris suspension of given interval measurement sampling with embodiment 6.In order to prepare the cell debris suspension and to measure the TG activity, relate to the method for embodiment 1 and embodiment 6.Represent the extent of growth of the subtilis AJ12866 cell of being cultivated with the turbidity of culture.In order to determine the turbidity of culture, be that the ray of 610nm is used for culture with wavelength, measure the absorption value of ray then.Gained the results are shown in Fig. 6.From these results understand be listed in the cell growth and reach steady stage and cell and begin to form its spore (beginning to cultivate the back about 4 hours) after, the TG activity of culture begins to increase.

Embodiment 8: the TG that purifying is bacillus-derived

Use the method cultured cells of pressing among the embodiment 7, finish following experiment.

The cell of subtilis AJ12866 bacterial strain is suspended from reactive system (0.5mg/ml N,O-Diacetylmuramidase, 20 μ g/ml DNase I, 0.1M Tris-HCl (pH7.5), 2mM DTT, 1mM EDTA, 2mMPMSF) in, react 2 hours then on ice so that cytolysis.With 20,000xg is centrifugal 20 minutes of reaction mixture, and the precipitation of gained partly is suspended from washing system (0.1M Tris-HCl, (pH7.5), 1mMEDTA, 2mM PMSF).The suspension of gained is centrifugal, the collecting precipitation thing.This operating process is repeated 2 times altogether.

Gained precipitation part is suspended from damping fluid (0.1M Na ₂CO ₃(pH10), 1mM EDTA, 2mM PMSF) in, put 30 minutes at 37 ℃.The part material that is present in the throw out part simultaneously is dissolved in the damping fluid, and the TG activity is transferred in the soluble part.It is centrifugal, and the supernatant liquor of gained has the TG activity.Add acetate, the pH of supernatant liquor is transferred to 6.0.Be referred to as crude enzyme liquid.The centrifugal crude enzyme liquid of ultrafiltration, then to damping fluid (50mM Tris-HCl (pH7.5), 0.1M NaCl) dialysis, thus exchange buffering liquid.The gained crude enzyme liquid is carried out gel infiltration, collect the TG active part of wash-out.Determine the enzymatic property of this part.

Presentation of results the following fact, in order to determine these characteristics, relate to the method for embodiment 1 to 5.

(1) the suitable pH scope of TG is about 7 to about 9 (see figure 7)s.(2) the suitable temperature range of TG be about 40 ℃ to about 65 ℃ of (see figure 8)s.(3) relevant its temperature stability, TG about 60 ℃ or following be stable (see figure 9).(4) cystamine, NEM and (NH ₄) ₂SO ₄Suppress TG (seeing Figure 10 and table 3) to a great extent.(5) DTT does not suppress the TG activity, and on the contrary, when having 1mM DTT, it is active to improve 1.5 times or highlyer (see Figure 11.(6) EDTA is to the almost not influence (seeing Figure 12) of TG activity.(7) Ca of 5mM or greater concn ²⁺Do not suppress the TG activity.The active expression of TG does not need to exist Ca ²⁺In other words, TG does not rely on Ca ²⁺(seeing Figure 13).(8) molecular weight of TG is that (a) about 18,000 about 28,000 arrives about 30,000 (being measured by SDS-PAGE) to about 22,000 (being measured by gel infiltration) with (b).

In addition, determined near the aminoacid sequence the TG N-terminal, that is, from the terminal beginning of its N-35 the aminoacid sequence of residue.The result shows that the TG of this paper gained and the subtilis AJ1307 deutero-TG of former gained have homology highly.Specifically, the aminoacid sequence of two kinds of TG is only different at the 22nd amino-acid residue each other.AJ1307 deutero-TG is Asn at the 22nd amino acids residue, and AJ12866 deutero-TG is Asp at the 22nd times amino-acid residue.

Above-mentioned result of experiment shows that the TG and the above-mentioned subtilis AJ1307 deutero-TG that derive from subtilis AJ12866 have identical characteristics.

The gel of embodiment 9. protein and TG of the present invention

With (25mM Tris-HCl (pH7.5) is 5mMDTT) with 1: 9 mixed, then in 37 ℃ of reactions 24 hours through the TG of gel infiltration gained active ingredient and 10% tyrosine solution among the embodiment 8.After the reaction, make solution gelatinizing.

The pure TG of gained among the embodiment 1 is added in 7% gelatin solution with the proteinic concentration of 2 units/g, in 35 ℃ of reactions 2 hours.After the reaction, make the gelatin protein solution gelatinizing.

Embodiment 10. separates bacillus deutero-TG gene

(1) purifying TG and definite its-terminal amino acid sequence

Detect determined bacillus-derived TG partial amino-acid series (corresponding to the Sequence Number 1 in the sequence table) among the embodiment 1 whether with any amino acid sequence homologous of known peptide.Yet, find and GenBank (LASL-GDB) that any aminoacid sequence of registration does not all have homology among SWISS-PROT and the NBRF (PIR).

The aminoacid sequence of TG reverses on ordinary password subbase plinth and translates, and therefrom infers the base sequence of encoding amino acid sequence.Therefore, detect described base sequence whether with the base sequence homology of any known nucleic acid.

The result shows the former and is registered in a kind of base sequence of GeneBank (LASL-GDB) high homology.The base sequence homologous base sequence registration number with TG of the present invention that this paper finds is L29189, and its source is D.W.Hanlon ﹠amp; G.W.Ordal, (J.Biol., Chem, Vol269, PP.14038-14046 (1994)).This reference discloses the gene base sequence of the coding subtilis transmembrane receptor that is positioned at the lateral areas, upstream, and is wherein disclosed, and this paper finds and the base sequence homologous base sequence of this paper TG.Specifically, the base sequence of finding TG of the present invention on the 1st to the 68th base residue with disclosed base sequence homology.Back a kind of base sequence of being made up of 68 base pairs is positioned at 5 ' upstream site of subtilis mcpB gene, and its transcriptional orientation and mcpB gene is opposite.The function of the peptide of the sequence encoding of forming by 68 base pairs with by D.W.Hanlon ﹠amp; (J.Biol Chem Vol 269, pp14038-14046 (1994) is disclosed irrelevant for G.W.Ordal.

The inventor infers that the sequence of being made up of 68 base pairs may be the part of the gene of the bacillus-derived TG of code book invention, separates full-length gene then.

(2) collecting cell:

Cultivate the cell of bacillus subtilis strain AJ1307 under the following conditions.Use the Schaeffer substratum, finish shaking culture in 37 ℃.At first, the cell of incubated overnight AJ1307 bacterial strain is cultivated the 5ml culture that contains inoculating cell at last to carry out inoculation culture in another Schaeffer substratum of 100ml in 20ml Schaeffer substratum.

(3) from cell, separate chromosomal DNA.

Under these conditions, cell reaches logarithmic growth after the later stage, with 100ml culture centrifugal (12,000xg, 4 ℃, 15 minutes), collecting cell.Cell is suspended among 10ml 50: the 20TE (50mM Tris-HCl, pH8.0,20mM EDTA) washing, centrifugal collecting cell again.And then cell is suspended from 10ml 50: among the 20TE.Lysozyme soln and the 1ml 10%SDS solution of 0.5ml 20mg/ml are added in the gained suspension, wherein in 55 ℃ with cell cultures 20 minutes.After the cultivation, will be added to the phenol of 10 of suspension equal volume: 1TE in the suspension, make the suspension deproteinize thus.To be added to the 2-propyl alcohol of water layer equal volume in the water layer of gained, with this deposit D NA and collection.Gained DNA throw out is dissolved in 0.5ml 50: 20TE, adds 5 μ l 10mg/ml RNase and 5 μ l10mg/ml Proteinase Ks then, in 55 ℃ of reactions 2 hours.After the reaction, will be added in the solution with the saturated phenol of 10 of solution equal volume: 1TE, thereby make the solution deproteinize.To isolating water layer add 24: the 1 chloroform/primary isoamyl alcohol identical with this layer volume, water layer is then collected in stirring then.This process is repeated 2 times altogether.Adding final concentration in the water layer of last gained is 3M sodium acetate solution (pH5.2) and 2 times of this layer volume of ethanol of 0.4M.The DNA of collecting precipitation uses 70% washing with alcohol, and drying is dissolved in 1ml10: among the 1TE then.

(4) prepare dna fragmentation through PCR:

In order to separate and the dna molecular of the gene that contains the bacillus-derived TG of coding of increasing, can use TakaRa LA PCR body outer clone box (Takara shuzo Co production).Hereinafter unless stated otherwise, will finish following experiment by the explanation of the appended specification sheets of this box.

With 5 prepared μ g chromosomal DNAs of restriction enzyme Hind III digestion preceding step (3).Then collect the dna fragmentation that links to each other with Hind III box by ethanol sedimentation.Make it through ethanol sedimentation again, make the DNA of collection carry out the PCR first time with primer C1 and primer S1 then.The base sequence of the primer C1 is corresponding to the sequence Number 3 in the sequence table, primer S1 correspondence Sequence Number4 wherein.Contain primer C1 in used TaKaRa LAPCR body outer clone box, its base sequence is in the base sequence of Hind III box.The 566th G in the base sequence of the gene of the base sequence of primer S1 and above-mentioned coding subtilis transmembrane receptor is to the regional complementarity of 600A.

Carry out 30 round-robin PCR reactions under the following conditions altogether with Gene Amp PCR System 9600 (producing) by Perkin ElmerCo..

A circulation:

98 ℃ 20 seconds,

68 ℃ 3 minutes

Then reaction mixture is diluted to 1/100,, carries out PCR the 2nd time to wherein adding primer C2 and primer S2.The condition of the 2nd PCR is identical with the 1st PCR's.Sequence Number5 and Sequeuce Num-ber6 in the corresponding sequence table of base sequence difference of primer C2 and S2.Contain primer C2 in used TaKaRa LA PCR body outer clone box, its base sequence is in the base sequence of Hind III box.34T in the gene base sequence of the base sequence of primer S2 and above-mentioned coding subtilis transmembrane receptor is to the regional complementarity of 68T.

After the reaction, make 3 μ l reaction mixtures, the dna fragmentation of about 2kb that confirms to have increased through 0.8% agarose gel electrophoresis.

5) dna fragmentation that increases with the puc18 clone PCR:

The dna fragmentation of about 2kb pcr amplification is cloned by linking to each other with puc18.Finish the clone with Sure Clone Ligation box (producing) by Pharmacia Co..Hereinafter unless stated otherwise, finish following experiment by the explanation of the appended specification sheets of this box.Dna fragmentation to the amplification of the about 2kb of 400ng is processed so that two end becomes flush end, then with its phosphorylation.After the phosphorylation, purifying DNA fragment links to each other with the pUC18 that digests with SmaI again.The reaction solution of the dna fragmentation that connects with containing is with DNA Transformed E .coli cell.

The JM109 transformant that screening transforms as required with the pUC18 that contains about 2kb dna fragmentation from the transformant of gained.As for screening, relate to the screening method of in Molecular Cloning (2nd Edition, Cold Springg HarborPress (1989)), describing.

(6) determine the dna sequence dna of this gene:

Press the method preparation of describing among the Molecular Cloning (2nol Edition, Cold SpringHarbor Press (1989)) and screen the plasmid that transforms, determine the base sequence of the dna fragmentation of wherein about 2kb amplification thus.Check order with Dye Terminator Cycle Sequencing box (by ABICo. production) and the method that illustrates by the appended specification sheets of this box.Carry out electrophoresis with DNA Sequencer373 (producing) by ABI CO..

The result of order-checking shows that the dna fragmentation of pcr amplification contains in the ordered list Sequencer Number2 118A to the base sequence zone of 1042T.As for the base sequence of Sequence Number2, the zone from 118A to 852C is an open reading frame, and the amino acid sequence of polypeptide of ORF coding can be inferred on the basis of ordinary password.The aminoacid sequence of being studied is shown in hereinafter with the base sequence of Sequence Number2.

For aminoacid sequence, the aminoacid sequence of being made up of 35 amino-acid residues that relates to from the zone of 35 amino-acid residues of its N-end to the and preceding step (1) is identical.Can reach a conclusion thus: the dna fragmentation of pcr amplification is required bacillus-derived TG gene.

Can reach a conclusion: Sequence Number2 base sequence and D.W.Hanlon ﹠amp; (J.Biol Chem., the difference between the disclosed base sequence of Vol.269pp14038-14046 (1994) should be owing to the difference between their bacterial strain uses therefors for G.W.Ordal.

The plasmid that will contain bacillus-derived TG gene is called pBSTG75-11, and wherein said TG gene inserts with the direction that pUC18 deutero-lac promotor acts on this gene.

Embodiment 11. is from the bacillus-derived TG gene of chromosomal dna library clone

(1) formation in karyomit(e) NA library

1 μ g chromosomal DNA with preparation among the Hind III complete digestion embodiment 10.Reclaim DNA through ethanol sedimentation, be dissolved in 10 μ l 10 then: among the 1TE.5 μ l gained solution are mixed with the 1ng pUC118 that has digested with Hind III (being produced by Takara Shuzo Co.), use the BAP dephosphorylation, use DNA Ligation.Kit Ver 2 (producing) that described DNA is linked to each other with pUC118 again by Takara Shuzo.The reaction mixture that the competent cell of 100 μ l E.coli JM109 bacterial strains (being produced by Takara Shuzo Co) is connected with 3 μ l is with described DNA Transformed E coli JM 109 bacterial strains.The solid medium that bag is suited by one deck on the transformant cell is with the preparation chromosomal dna library.

(2) formation of probe:

As for probe, used is the total length TG gene that obtains among the embodiment 1.With pBSTG75-11 is template, carries out PCR with primer S2 and primer S3.Ver.2 carries out PCR with TaKaRa LA PCR box.

Prepare 100 μ l and contain 10ng template pBSTG75-11, the reactive system of 20pmol primer S2 and 20pmol primer S3, reaction then.Primer S3 is and 35 monomers of TG gene (Sequence Number2) the 818th base to the regional complementarity of 852 bases that its base sequence is corresponding to the Sequence Number7 in the sequence table.Under following condition, carry out 30 round-robin PCR altogether.

A circulation:

94 ℃ 30 seconds

55 ℃ 30 seconds

72 ℃ 1 minute

With the dna fragmentation of 1% sepharose (Seaplaque GTG is produced by FMC CO) electrophoretic separation with above-mentioned reaction amplification.Cut out required band, use therefrom purify DNA of Easy Prep System (producing) and PCR Products Prep box (producing) by Pharnacia Co. by Phamacia z Co..Therefore obtain 200 μ l DNA (4ng/ μ l) solution at last.

Use the 32P labeled dna fragment, then as probe.With RandomPrimer DNA Labeling Kit Ver.2 (Takara Shuzo Co. production), by the method for the appended specification sheets of this box explanation with (α- ³²P) dCTP (3000Ci/mmol) (Amersham Co. production) label probe.

(3) colony hybridization

An example that relates to the colony hybridization of in Molecular Cloning (2nd EditiOn, Cold Spring Harbor Press (1989)), describing hereinafter.

With the colony lift of chromosomal dna library on nylon leaching film or (Hybond-N, Amersham Co. produces), alkaline denaturation then, neutralization is also fixing.Finish hybridization with Rapid-Hyb Buffer (Amersham Co. production).Specifically, filter membrane is immersed in the damping fluid, 65 ℃ of prehybridizations 4 hours.After this, the label probe for preparing in the above-mentioned steps (2) is added in the damping fluid, in 65 ℃ of hybridization 2 hours.Then, filter membrane was washed 20 minutes in room temperature with the 2 * SSC that contains 0.1%SDS.In addition, 0.1 * SSC with containing 0.1%SDS washes 2 times through 15 minutes at 65 ℃.

The result of this step shows: produced 5 bacterium colonies with probe hybridization.

(4) determine the dna sequence dna of TG gene

By with embodiment 5 in identical method, determine to have inserted the base sequence of the dna fragmentation among the pUC118.The result of order-checking shows: the base sequence of Sequence Number2 in this DNA ordered list.

Embodiment 12: at the bacillus-derived TG gene of expression in escherichia coli:

(1) contain inducing of reorganization TG expression of gene in the cultivation of E.coli cell of reorganization TG gene and the cell:

The plasmid pBSTG75-11 that obtains in embodiment 10 contains DNA and the coding lacZ protein DNA of the bacillus-derived TG of coding, and preceding kind of DNA is connected in the site, downstream of a kind of DNA in back.From its base sequence, infer designed plasmid energy expressed fusion protein, described fusion rotein contains the peptide with aminoacid sequence of Sequence Number8 in the sequence table, and this peptide is made up of 11 amino acid, and be added among the bacillus-derived TG, be positioned at before the Met residue of TG sequence beginning.

In this test, the control cells of the experimental cell of the E.coli JM109 that used is transforms with pBSTG75-11 and the E.coliJM109 that transforms with pUC18 is carried out shaking culture respectively at 37 ℃ with experimental cell and control cells in the LB substratum that contains the blue or green toxin of 100mg/ml ammonia benzyl.Specifically, cell is implanted in the 30ml substratum, shaking culture is spent the night with preparation inoculation culture thing.On the other hand, 4 flasks that respectively contain the 30ml fresh culture of preparation.Cell concn with 5%, the inoculation culture thing of the E.coli JM109 experimental cell that will transform with pBSTG75-11 is implanted in 2 flasks.Two flasks are called experimental group 1 and experimental group 2.On the other hand, the cell concn with 5%, the inoculation culture thing of the E.coliJM109 control cells that will transform with pUC18 is implanted two flasks in addition.Two flasks are called experimental group 3 and experimental group 4.Cultivate and respectively organize cell.After the absorption value of 610nm reaches about 0.7, only in experimental group 1 and experimental group 3, add IPTG with the final concentration of 1mM.After 4 hours, stop respectively organizing the cultivation of cell.

(2) determine to induce and expressed protein:

After stopping cultivating, with each cells in culture of microscopic examination.The result shows: have only with pBSTG25-11 to transform, and contained the protein inclusion body to the JM109 cell that has wherein added IPTG.

After stop cultivating, with each 10ml culture centrifugal (with 12,000xg, 15 minutes) with collecting cell.Cell is suspended among the 2ml 10mMTris-HCl (pH7.5), washing, and then centrifugal with collecting cell.Cell is suspended from the identical damping fluid of 1ml, by with 0.1mm Zirconia pearl, gained suspension was vibrated 3 minutes and fragmentation with Mini-beadBeater (Nakenyaku Co production).With broken suspension carry out SDS-PAGE, dye with CBB then.The result shows: have an appointment 29,000 to about 30,000 band of the suspension of experimental group 1 (transform with pBSTG75-11 and by IPTG inductive JM109 cell).From the molecular weight of being identified, infer experimental group 1 cell expressing the fusion rotein of expection.

(3) determine the TG activity

Measure expressed proteinic TG activity.Specifically, the ready-formed suspension that 10 μ l is contained smudge cells be added to contain the dimethyl casein and ¹⁴In the reactive system of the putrescine of C-mark, reaction then.After the reaction, reaction mixture is adsorbed onto on the filter paper, with dimethyl casein bonded putrescine amount on the liquid scintillation counter measurement filter paper.The results are shown in hereinafter table 4.That these results confirm to transform with pBSTG75-11 and by in the IPTG inductive JM109 cell experiment group 1) the TG activity arranged.

The E.coli KM109 bacterial strain that transforms with pBSTG75-11 is called E.coli AJ13172, according to budapest treaty, has been deposited in the international preservation center of Bioengineering laboratory December 20 nineteen ninety-five.International preservation is FERM BP-5446.

The dna encoding that has now proved Sequence Number2 base sequence in its ordered list has the active enzyme of TG.That is, clear, this DNA has bacillus-derived TG gene.In addition, proof also, even other DNA of the different peptides of will encode are added among this DNA, gained also can express bacillus-derived TG gene in conjunction with DNA, and the TG activity is also arranged by fusion rotein in conjunction with DNA expression.In addition, required transformant that clearly can E.coli, at the fusion rotein of cell inner expression protein inclusion body form, and described protein expression can be by suitable promotor control.

Advantage of the present invention:

According to the present invention, can be from food industry available microorganism, bar Bacterium obtains before this unknown new TG such as bacillus subtilis etc.

The advantage of the new TG of the present invention is (1) and any other animal derived The TG difference, it does not need calcium, so its use is unrestricted, and can To compare unwrapping wire with the bacillus of low-cost production and (2) production TG of the present invention Bacteria growing is fast, therefore can give birth to by low cost with the TG that derives than actinomyces Produce bacillus-derived TG.

TG of the present invention is different from the TG by Mexico State University's report, is that (1) the former activity is not subjected to 5mM or higher concentration Ca²⁺Press down The system, (2) the former appropriate pH scope in neutrality between the alkalescent, (3) the former can't help DTT and suppresses, and the former is not subjected to chelating agent such as EGTA (4) Deng inhibition, the former is only produced (5) by the bacillus in the sporogenesis stage.

Since can produce crosslinked polymer with TG of the present invention, therefore can With the various food of its industrial production.

In addition, because TG of the present invention derives from the bar that is used in particular for producing food Therefore bacterium has very high real value in food industry.

Sequence table

Sequence Number：1

Sequence length: 35

Sequence type: amino acid

Topological structure: linearity

Sequence type: peptide

The source

Biological name: bacillus subtilis

Strain name: AJ1307

Sequence: Met Ile Ile Val Ser Gly Gln Leu Leu Arg Pro Gln Asp Ile Glu Asn 15 10 15 Trp Gln Ile Asp Gln Asn Leu Asn Pro Leu Leu Lys Glu Met Ile Glu

        20                   25                30 Thr Pro Val

35 Sequence Number:2 sequence length: 1042 sequence types: nucleic acid chains number: 2 (two strands) topological structures: linear sequence type: genome DNA source: biological name: bacillus subtilis strain title: AJ1307 sequence: CTGCTTAAAA AGTTTTAAAA TAAAAAATGG AAGAAGTTCT TTTTGGCAGT CTTCTGTCTT 60 TTTAGCTTTC ATTGCCCAAG CTCTTTGCAT ATCTTATATA AACAAGGGGG GCTAAAC 117 ATG ATT ATT GTG TCA GGA CAA TTG CTC CGT CCC CAG GAT ATT GAA AAT 153 Met Ile Ile Val Ser Gly Gln Leu Leu Arg Pro Gln Asp Ile Glu Asn 15 10 15 TGG CAG ATT GAT CAA AAT CTG AAT CCG CTG TTA AAA GAG ATG ATT GAG 213 Trp Gln Ile Asp Gln Asn Leu Asn Pro Leu Leu Lys Glu Met Ile Glu

         20                  25                  30 ACG CCT GTT CAG TTT GAT TAT CAT TCA ATT GCT GAA CTG ATG TTT GAG     261 Thr Pro Val Gln Phe Asp Tyr His Ser Ile Ala Glu Leu Met Phe Glu

     35                  40                  45 CTT AAA CTG CGG ATG AAT ATT GTA GCA GCG GCA AAG ACG CTG CAC AAA     309 CTT AAA CTG CGG ATG AAT ATT GTA GCA GCG GCA AAG ACG CTG CAC AAA     309 Leu Lys Leu Arg Met Asn Ile Val Ala Ala Ala Lys Thr Leu His Lys

 50                  55                  60 AGC GGG GCG AAG TTT GCC ACT TTT TTA AAA ACA TAC GGG AAT ACA ACG     357 Ser Gly Ala Lys Phe Ala Thr Phe Leu Lys Thr Tyr Gly Asn Thr Thr  65                  70                  75                  80 TAT TGG AGG GTT TCA CCG GAG GGC GCC TTG GAG CTG AAA TAC AGA ATG    405 Tyr Trp Arg Val Ser Pro Glu Gly Ala Leu Glu Leu Lys Tyr Arg Met

             85                  90                  95 CCG CCT TCA AAA GCG ATT CGG GAC ATT GCA GAG AAC GGC CCG TTT TAT    453 Pro Pro Ser Lys Ala Ile Arg Asp Ile Ala Glu Asn Gly Pro Phe Tyr

        100                 105                 110 GCG TTT GAA TGC GCA ACC GCA ATC GTT ATC ATT TAT TAC TTG GCC TTA    501 Ala Phe Glu Cys Ala Thr Ala Ile Val Ile Ile Tyr Tyr Leu Ala Leu

      115                 120                 125 ATC GAT ACA ATC GGT GAA GAT AAA TTC AAT GCC AGC TTT GAC AGA ATT    549 Ile Asp Thr Ile Gly Glu Asp Lys Phe Asn Ala Ser Phe Asp Arg Ile

130                 135                 140 ATT TTA TAT GAC TGG CAT TAT GAG AAA TTG CCG ATC TAT ACG GAA ACA    597 Ile Leu Tyr Asp Trp His Tyr Glu Lys Leu Pro Ile Tyr Thr Glu Thr 145               150                   155                 160 GGA CAC CAC TTT TTC CTT GGA GAT TGT TTG TAT TTT AAG AAT CCT GAA    645 Gly His His Phe Phe Leu Gly Asp Cys Leu Tyr Phe Lys Asn Pro Glu

            165                 170                 175 TTT GAT CCG CAA AAG GCG CAA TGG AGA GGC GAA AAT GTG ATT TTA CTG    693 Phe Asp Pro Gln Lys Ala Gln Trp Arg Gly Glu Asn Val Ile Leu Leu

        180                 185                 190 GGG GAA GAT AAA TAT TTT GCC CAT GGT CTT GGA ATC TTA AAC GGA AAG    741 Gly Glu Asp Lys Tyr Phe Ala His Gly Leu Gly Ile Leu Asn Gly Lys

    195                 200                 205 CAA ATT ATA GAT AAG CTG AAT TCT TTT AGG AAA AAA GGA GCC TTA CAG     789 Gln Ile Ile Asp Lys Leu Asn Ser Phe Arg Lys Lys Gly Ala Leu Gln

210                 215                 220 TCA GCC TAC CTT CTG TCT CAG GCG ACC AGA CTG GAT GTT CCG TCT CTT     837 Ser Ala Tyr Leu Leu Ser Gln Ala Thr Arg Leu Asp Val Pro Ser Leu 225                 230                 235                 240 TTC CGC ATC GTC CGC TAAAAAGCCC CATCGCCTAT TTTCGGGACG ATGGGGTTTC     892 Phe Arg Ile Val Arg

245 AAATGCCTTT CGTTTTCGAT AGAAGGGGGC TGTGCCGAAA TATTGGTTCG CAGCCCACTC, 952 CATTTTTTCA AGGTCATTTC TTGTCACGAT TGGATCCTGG CTGCTCCATT TGATAAAGCG, 1012 GACAAAATAG TAGCCTTTGA TAGGAACCAT, 1042 Sequence Number:3 sequence lengths: 35 sequence types: nucleic acid chains number: (strand) topological structure: linear order type: synthetic DNA

The primer C1 sequence of PCR: GTACATATTG TCGTTAGAAC GCGTAATACG ACTCA (35) Sequence Number:4 sequence length: 35 sequence types: nucleic acid chains number: 1 (strand) topological structure: linear order type: synthetic DNA

Primer S1 sequence TGGCGCTTGT ACATAAGTGC CGTTATCTGC GCCCC (35) the Sequence Number:5 sequence length of PCR: 35 sequence types: nucleic acid chain: 1 (strand) topological structure: linear order type: synthetic DNA

PCR primer C2 sequence: CGTTAGAACG CGTAATACGA CTCACTATAG GGAGA (35) Sequence Number:6 sequence length: 35 sequence types: nucleic acid chains number: 1 (strand) topological structure: sequence type: synthetic DNA

Primer S2 sequence A TGATTATTG TATCAGGACA ATTGCTCCGT CCCCA (35) the Sequence Number:7 sequence length of PCR: 35 sequence types: nucleic acid chain: 1 (strand) topological structure: sequence type: synthetic DNA

PCR primer s3 sequence: GCGGACGATG CGGAAAAGAG ACGGAACATC CAGTC (35) Sequence Number:8 sequence length: 11 sequence types: amino acid chain number: 1 (strand) topological structure: linear order type: peptide source biological name: E.Coli sequence Met (1) Thr Met Ile Thr (5) Asn Ser Ser Ser Val (10) Pro

The SDS-PAGE result of the pure TG-1 of Brief Description Of Drawings [Fig. 1] expression.[Fig. 2] expression and the active relevant pH curve of TG-1.[Fig. 3] expression and the active relevant temperature curve of TG-1.The temperature stability of [Fig. 4] expression TG-1.[Fig. 5] expression and the crosslinked alpha-casein of TG-1.Relation between the growth of [Fig. 6] expression TG-production cell and the TG-2 activity of production.[Fig. 7] expression and the active relevant pH curve of TG-2.[Fig. 8] expression and the active relevant temperature curve of TG-2.The temperature stability of [Fig. 9] expression TG-2.[Figure 10] expression suppresses the influence to TG-2.[Figure 11] expression DTT is to the influence of TG-2.[Figure 12] expression EDTA is to the influence of TG-2.[Figure 13] represents Ca ²⁺Influence to TG-2.

Each reagent of table 1. is to the impact of TG activity

Reagent	Concentration	Remaining activity (relative value)
			-	-	1.0
NEM	1mM	17
			Cystamine	2mM		0
PMSF	5mM	89
			(NH 4) 2SO 4	10mM	1
Na 2SO 4	10mM	87
			EDTA	10mM	107
EGTA	10mM	98
			CaCl 2	5mM	65
DTT	10mM	119
			2-ME	10mM	111

Table 2

Relative activity
	Culture supernatants 280
Culture precipitation 510
	The precipitated liquid 10200 that grinds

Each reagent of table 3. is to the impact of TG activity

Reagent	Concentration	Remaining active relative value
			-	-	100
PMSF	5mM	95
			(NH 4) 2SO 4	10mM	3
Na 2SO 4	10mM	85
			EGTA	10mM	94
2-ME	10mM	122

The TG activity of each transformant of table 4.

Contained plasmid	Induce with IPTG	TG activity (dpm)
			PBSTG75-11 experimental group 1	+	496
PBSTG75-11 experimental group 2	-	0
			PUC18 experimental group 3	+	0
PUC18 experimental group 4	-	0

Claims (13)

1. bacillus-derived trans-glutaminases is characterized in that

(1) its suitable pH is between about 7 and 9;

(2) its suitable temperature is between about 40 ℃ and 65 ℃;

(3) its about be stable below 60 ℃ or 60 ℃,

(4) it does not rely on Ca ²⁺, and in 5mMCa ²⁺Under the condition that exists, have 50% or more active,

5) it can be by NEM, cystamine and (NH ₄) ₂SO ₄In any material suppress,

6) EDTA, any material among DTT and the 2ME does not all have inhibition to it,

7) its molecular weight be (a) about 18,000 to about 22,000 (measuring) through gel infiltration and (b) about 28,000 to about 30,000 (through the SDS-PAGE measurement) and

8) transacylation of γ-Carboxylamide on the glutamine residue in its catalysis peptide chain.

2. according to the bacillus-deutero-trans-glutaminases of claim 1, it has the aminoacid sequence of Sequence Number1 in the sequence table.

3. isolated bacillus-derived trans-glutaminases component from the spore that obtains by bacillus spores capsule broken or that dissolving is cultivated.

4. according to the trans-glutaminases of claim 1, wherein said bacillus is a subtilis.

5. according to the trans-glutaminases component of claim 3, wherein said bacillus is a subtilis.

6. produce protein with crosslinking structure, the nonprotein amino acid polymkeric substance, the method of peptide or derivatives thereof, it is characterized in that the effect of the trans-glutaminases by claim 1 and crosslinked at protein, the nonprotein amino acid polymkeric substance, Gln in the peptide or derivatives thereof and Lys residue, thus at protein, between nonprotein amino acid polymkeric substance, the peptide or derivatives thereof molecule or within form the ε of intermolecular or intramolecular crosslinking-(γ-Glu)-Lys key.

7. produce protein with crosslinking structure, the nonprotein amino acid polymkeric substance, the method of peptide or derivatives thereof, it is characterized in that the trans-glutaminases component by claim 3 effect and will be at protein, Gln and Lys residue in nonprotein amino acid polymkeric substance, the peptide or derivatives thereof are crosslinked, thus at protein, between nonprotein amino acid polymkeric substance, the peptide or derivatives thereof molecule or within form between the molecule or the ε of intramolecular crosslinking-(γ-Glu)-Lys key.

8. the DNA of coding claim 1 trans-glutaminases.

9. DNA according to Claim 8 has the 118th base in the base sequence of listed Sequence Number2 in the sequence table at least to 852 base sequences.

10. the recombinant DNA by carrier DNA and the DNA of claim 8 are linked to each other and obtain.

11. recombinant DNA cell transformed with claim 10.

12. produce the method for bacillus-derived trans-glutaminases, it is characterized in that the cell of cultivation claim 11 in substratum produces and accumulate bacillus-derived trans-glutaminases with this in substratum and/or cell, collect trans-glutaminases then.

13. press the bacillus-derived trans-glutaminases that the method for claim 12 is produced.

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