CN1113953A - No.118 Position mutation of bacillus subtilis alkaline protease and its thermal stable enzyme - Google Patents

Abstract

The present invention provided the mutation on the 118 th position of bacillus subtilis basic proteinase the 118th asparagine is substituted by serine: Asn (118) ser, after mutation the gene expressing product is a kind of proteinase, which is different from the natural bacillus substilis basic proteinase, the thermal stability of the former is much higher than the latter, thus overcoming the inactivation while heating shortage of natural basic proteinase which is used as one component of detergent for tannery and the industry for cleaning the protein dirt.

Description

No.118 Position mutation of bacillus subtilis alkaline protease and its thermal stable enzyme

The present invention relates to bacillus alkaline protease, particularly sudden change and the high proteolytic enzyme of consequent thermostability on the 118th of the Ki2 bacillus alkaline protease.

Bacillus alkaline protease belongs to serinase, and is less to the specificity of amino acid kind during its protein hydrolysate, has broad spectrum.As one of component of washing composition, be used for tanning industry and cleaned the spot of protein one class.But natural Sumizyme MP heats easy inactivation, can not coexist with SYNTHETIC OPTICAL WHITNER, influenced its use value, abroad the Sumizyme MP to multiple different sources has carried out big quantity research with genetically engineered and protein engineering means, obtained multiple good mutant, wherein have oxidation resistantly, heat-stable, the surface charge that also changed is to change the mutant of improveing washing effect with the interaction of substrate.Except having done on the known site some rite-directed mutagenesis, also there is not the mutant proteinase kind of on unknown site, not suddenling change and obtaining improved properties at home according to external existing being reported in.

The patented technology WO8906279 and the WO9100345 of Denmark Nuo Wo company have described respectively by methods such as deletion, replacement or insertions, on the single or multiple sites of subtilisin, or carry out aminoacid replacement, or change its surface charge and obtain the mutant of different loci, these mutational sites have the 6th, 9,11-12,19,25,36-38,53-59,67,71,89,111,115,120,121-122,124,128,131,140,153,154 etc.Chinese patent application number provides the 123rd of subtilisin and 274 site mutation bodies for 90108892.7, but foregoing invention does not illustrate the specific performance of these mutant.

The object of the invention is to provide the sudden change on the 118th site of bacillus alkaline protease, on the 118th site, 118 amino-succinamic acids are replaced by Serine, be Asn(118) Ser, gene expression product after this sudden change is a kind of different with neutral protease, the proteolytic enzyme that thermotolerance is much better than neutral protease, thus solved on the natural Sumizyme MP industrial application as one of detergent components, in process hides and the defective of cleaning the easy inactivation of heating in the protein one class spot industry.

The present invention carries out random mutation with PCR method to the bacillus alkaline protease gene, screen a kind of mutant Asn(118) Ser, it has much better thermotolerance than natural Sumizyme MP, crystalline structure with the neutral protease that has recorded carries out computer simulation, finds to play stabilization at 118 Ser by water molecules and the amino acid interaction partners protease molecule structure of being close to.

Below in conjunction with accompanying drawing,, describe the sudden change of the 118th of bacillus alkaline protease and the process that makes of sudden change back thermophilic enzyme thereof in detail to select for use Ki2 bacillus alkaline protease gene as embodiment:

The present invention selects Ki2 bacillus alkaline protease gene for use, comprises that upstream regulatory region signal peptide and the gene fragment of leading peptide moiety are 1.9Kb, and the fragment of 1.9Kb is inserted the recombinant plasmid PY that plasmid PUC-19 constitutes.Protein engineering work of the present invention is from the PY plasmid, from the PY plasmid, the 1.9Kb fragment is cut out, be inserted into PBE-2 shuttle plasmid (biotechnology journal 7(3) .224-229.1991. Guo Xinghua etc.) in, thereby make alkaline protease gene in Bacillus subtilus, obtain expressing.

1. accompanying drawing 1 is for containing the recombinant plasmid PBY of subtilisin, single line partly is PBE-2(biotechnology journal 7(3 among its figure) the .224-229.1991. Guo Xinghua etc.) carrier, two-wire partly is the proteinase gene fragment, arrow is represented the proteinase gene direction, accompanying drawing 2 is that plasmid PUB or PUA make up synoptic diagram, accompanying drawing 3 prepares the gene fragment synoptic diagram that contains sudden change for PCR, and accompanying drawing 4 inserts for the gene fragment that will contain sudden change and contains without synoptic diagram in the upstream region of gene carrier partly of sudden change.

Know by Fig. 2, with the Hind III enzymolysis of the PBY shown in Fig. 1, obtain (1), (2) two fragments, fragment (1) is the fragment that comprises the proteolytic enzyme structure gene about 0.9Kb in downstream part, fragment (2) is to comprise whole carriers and the about 7Kb fragment of proteinase gene upstream portion, is that the 0.9Kb fragment is inserted the PUC-18 carrier with fragment (1), obtains plasmid PUA or PUB owing to direction of insertion is different.Fragment (2) is that the big fragment of 7Kb is called PBY ', and step is used after giving over to, and promptly is connected with the complete genome of recoverin with Hind III fragment that PCR obtains and constitutes expression plasmid PBM;

2. known by Fig. 3 that PUA or PUB are as the template of PCR, (3) are the universal sequencing primer thing of MB, forward of two primers, one is oppositely, passes through PCR method, through 25 circulations, each cycling condition be 93 ℃ 1 minute, 52 ℃ of 1 minute seconds, 70 ℃ 1 minute 10 seconds, the PCR fragment 4 that obtains, use Hind III enzymolysis again, remove the primer part at two, and obtain the Hind III fragment (5) of pcr amplification through the gel electrophoresis purifying;

3. know by Fig. 4, Hind III fragment (2) with the above-mentioned pcr amplification that obtains, (5) turning back to fragment (2) in the step 2 is that the big fragment of 7Kb also claims among the PBY ', obtain recombinant expression plasmid PBM, because PCR has 1/400 wrong probability, so this recombinant expression plasmid PBM is a mixture, sudden change is arranged, also have normal, there is direction of insertion identical, also there is direction of insertion opposite, the gene that makes that direction of insertion is opposite loses function, and the mutant that has also can make gene lose function, has only the identical recombinant plasmid of those direction of insertion can express normal Sumizyme MP, the activity that has in these normal proteolytic enzyme has improved, the reduction that has, what have has a thermotolerance, and what have is then the same with natural enzyme.Recombinant plasmid mixture PBM is transformed the DB104 recipient bacterium, at the dull and stereotyped enterprising row filter of milk, do not have transparent circle be direction of insertion opposite make the transformant of the recombinant plasmid of gene inactivation with sudden change.Though those have transparent circle promptly be contain normal protein enzyme gene or contain sudden change but do not make the transformant of the recombinant plasmid of gene inactivation.These transformants that contain transparent circle are cultivated one by one again, measured the relative reactivity and the thermostability of their excretory Sumizyme MPs one by one.Therefrom select the mutant Asn(118 that significantly increases of a thermostability) Ser.

4. the mutant of choosing is determined the nucleotide position of undergoing mutation through determined dna sequence, thereby know that sudden change has taken place which amino acid, accompanying drawing 5 is mutant and near the dna sequence dna of wild type gene saltation zone, wild-type is AAT, and mutant then is AGT, and AAT is the coding of Asn, AGT is the coding of Ser, known that by Fig. 5 the difference of a Nucleotide is only arranged in the dna sequence dna of mutant and wild-type, A becomes G.This sudden change just in time is positioned at the 118th Asn(amino-succinamic acid) coding AAT intermediary A, the AGT after the sudden change has become the Ser(Serine) coding.So alkaline protease gene that the present invention has obtained suddenling change on 118, gene expression product after this sudden change is the different proteolytic enzyme of a kind of and natural Sumizyme MP, it has much better thermotolerance than natural enzyme, and it will improve the application of natural enzyme in industry and orthobiosis.Accompanying drawing 6 is that bacillus alkaline protease and Bacillus subtilus 168 Sumizyme MP dna sequence dnas compare synoptic diagram, both different places of expression, band point place among the figure.Accompanying drawing 7 is the comparison synoptic diagram of the enzyme amino acid sequence of Ki2 bacillus alkaline protease and Bacillus subtilus 168 alkaline eggs, both different places of expression, band point place among the figure, accompanying drawing 10 is the Ki2 bacillus alkaline protease dna sequence dna after suddenling change, and accompanying drawing 11 is the aminoacid sequence of the Bacillus subtilus alkalescence thermostable protease of the 118th sudden change; Accompanying drawing 8 is the Asn(118 after wild-type protease and 118 sudden changes) Ser protease activity synoptic diagram relatively, curve A represents that the wild-type protease activity with temperature changes and the curve of variation among the figure, curve B is represented mutant Asn(118) the Ser protease activity varies with temperature and the curve that changes, by drawing mutant Asn(118 among the figure) the Ser protease heat stability is more much better than wild-type protease, and 60 ℃ of inactivation transformation period are longer 3 times than natural enzyme.

Accompanying drawing 9 is three-dimensional right for the proteolytic enzyme local structure after suddenling change, " " expression water molecules is known by Fig. 9 among the figure, Ser(118) makes up three pairs of hydrogen bonds by water molecules and two other amino acid, molecular structure is tended towards stability, thereby the thermostability of its proteolytic enzyme is improved.

The present invention has disclosed the sudden change of bacillus alkaline protease the 118th, and the expression product after the sudden change is a kind of thermostability proteolytic enzyme more much better than natural Sumizyme MP, this kind of enzyme is a kind of thermophilic enzyme, and the present invention is applicable to the rite-directed mutagenesis of the 118th of bacillus alkaline protease.

Claims (3)

1, the sudden change of the 118th of bacillus alkaline protease is characterized in that amino-succinamic acid is replaced by Serine on the 118th site of bacillus alkaline protease: Asn (118) Ser;

2, by the sudden change of the 118th of the described bacillus alkaline protease of claim 1, the back dna sequence dna that it is characterized in that suddenling change is:

3, the Bacillus subtilus of the 118th sudden change alkalescence thermostable protease is characterized in that its aminoacid sequence is:

AQSVPYGISQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLNVRGGRSF

VPSETNPYQDGSSHGTHVAGTIAALNNSIGVLGVRPSASLYAVKVLDSTG

SGQYSWIINGIEWAISNSMDVINMSLGGPTGSTALKTVVDKAVSSGIVVA

AAAGNEGSSGSTSTVGYPAKYPSTIAVGAVNSSNQRASFSSAGSELDVMA

PGVSIQSTLPGGTYGAYNGTSMATPHVAGAAALILSKHPTWTNAQVRDRL

ESTATYLGNSFYYGKGLINVQAAAQ

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