CN108103048B - Low-temperature matrix metalloproteinase and coding gene and application thereof - Google Patents
Low-temperature matrix metalloproteinase and coding gene and application thereof Download PDFInfo
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- CN108103048B CN108103048B CN201711172228.9A CN201711172228A CN108103048B CN 108103048 B CN108103048 B CN 108103048B CN 201711172228 A CN201711172228 A CN 201711172228A CN 108103048 B CN108103048 B CN 108103048B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6489—Metalloendopeptidases (3.4.24)
- C12N9/6491—Matrix metalloproteases [MMP's], e.g. interstitial collagenase (3.4.24.7); Stromelysins (3.4.24.17; 3.2.1.22); Matrilysin (3.4.24.23)
Abstract
The invention discloses a low-temperature matrix metalloproteinase and a coding gene and application thereof. The invention firstly discloses a low-temperature matrix metalloproteinase, which is a protein shown in the following (a) or (b): (a) a protein consisting of an amino acid sequence shown in a sequence table SEQ ID NO. 1; (b) the protein with the protein degradation activity under the low temperature condition is obtained by substituting and/or deleting and/or inserting one or more amino acid residues in an amino acid sequence shown in a sequence table SEQ ID NO. 1. The invention further discloses application of the low-temperature matrix metalloproteinase in degrading proteins under a low-temperature condition. The low-temperature matrix metalloproteinase still has high-efficiency protein degradation activity at the low temperature of 10-20 ℃, and has a low heat inactivation temperature.
Description
Technical Field
The present invention relates to the field of enzyme engineering. More particularly, relates to a low-temperature matrix metalloproteinase, a coding gene and application thereof.
Background
Matrix metalloproteinases, a large group requiring Ca2+、Zn2+The metal ions are used as accessory factors and can degrade protease of various extracellular matrix components (collagen, gelatin, adhesive protein, fibronectin, proteoglycan and the like). The matrix metalloproteinase with collagen degradation activity can be widely applied to industries such as light industry production, medical treatment and health.
Collagenase can be used as a dyeing auxiliary agent of leather, and has certain catalytic activity on a collagen fiber matrix, so that the dyeing performance of the leather can be improved. Leather is a material made of collagen, which undergoes a change in structure after tanning with various cross-linking agents (e.g., chromium) and chemical cross-linking due to the reaction of the collagen with the tanning agent. The enzyme has high specificity to the combination of the substrate, so that the leather is tanned by chrome and uses collagenase, collagen can not generate comprehensive hydrolysis, but the reticular structure of the leather fiber can be more stretched, thereby promoting the dye to permeate into the interior of the grass fiber structure, simultaneously increasing the exposed area of the leather contacted with the dye, and further improving the dyeing effect of the leather.
In medicine, a disclysis method developed by collagenase is used for treating diseases such as lumbar disc herniation. Under the guidance of an imaging device, collagenase is accurately injected into and around the herniated intervertebral disc, and the collagen tissue is dissolved by the pharmacological action of the collagenase for decomposing the collagen fibers, so that the herniation is reduced or eliminated, the compression of the collagenase on the nerve tissue is relieved or eliminated, and the effect similar to that of the operation for removing the herniated intervertebral disc is achieved. Collagenase is an enzyme which mainly dissolves collagen, can effectively dissolve type I and type II collagen in nucleus pulposus and annulus fibrosus, does not damage tissue cells and nerve cells due to collagenase solution with the same osmotic pressure as human tissues, and has no damage to proteins such as hemoglobin, milk casein, keratan sulfate and the like.
At present, collagenase which is applied in a large scale and commercialized mainly comes from microorganisms (Clostridium histolyticum) and terrestrial mammals (cattle, pigs and the like), is normal temperature protease, and the optimal catalytic temperature is 30-40 ℃. Matrix metalloproteases with low-temperature collagen hydrolysis activity are rarely reported, and low-temperature catalysis has unique technical advantages compared with normal-temperature catalysis, and is represented by the following steps:
(1) the low-temperature catalysis reduces the enzyme reaction temperature, and can effectively realize the energy conservation and consumption reduction of the process. Taking leather dyeing pretreatment as an example, if middle-low temperature collagenase pretreatment can be realized, energy consumption investment for heating (usually 40 ℃) in the cylinder body is saved, and equipment investment for maintaining constant temperature and heat transfer in the cylinder body is saved;
(2) the low reaction temperature, and correspondingly the reduction of the inactivation temperature of the enzyme, can obviously reduce the side reaction or the nonspecific degradation of products and reduce the damage to the processed sample. Taking collagenase as an example for tissue cell digestion in the early stage of mammalian cell culture, the digestion process is carried out at 37 ℃, animal cells are in an active state, and effective oxygen supply is lacked in the reaction process, so that the cells can carry out anaerobic respiration, and metabolic byproducts such as lactic acid and the like are greatly generated and accumulated, thereby damaging the activity of the subsequent cell culture process. If the low-temperature collagenase can be introduced to replace the traditional normal-temperature collagenase, the reduction of the reaction temperature can greatly relieve the accumulation of byproducts in the process, thereby improving the activity of cell culture.
Therefore, the matrix metalloproteinase with collagen degradation activity under low temperature has important application value.
Disclosure of Invention
An object of the present invention is to provide a low-temperature matrix metalloproteinase and a gene encoding the same, which can have a protein degradation activity under low-temperature conditions.
Another object of the present invention is to provide the use of the above low temperature matrix metalloprotease.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a low-temperature matrix metalloproteinase, which is named nrMMP14 and is derived from Antarctic Luo fish (Notothenia rossii); the low-temperature matrix metalloproteinase is protein shown in the following (a) or (b):
(a) a protein consisting of an amino acid sequence shown in a sequence table SEQ ID NO. 1;
(b) the protein with the protein degradation activity under the low temperature condition is obtained by substituting and/or deleting and/or inserting one or more amino acid residues in an amino acid sequence shown in a sequence table SEQ ID NO. 1.
Wherein, the amino acid sequence of the sequence table SEQ ID NO.1 consists of 491 amino acid residues.
The gene encoding the low-temperature matrix metalloproteinase is also within the scope of the present invention, and the encoding gene is represented by (a) or (b):
(a) a nucleotide sequence shown as SED ID NO.2 in a sequence table;
(b) a nucleotide sequence of an amino acid sequence shown in a sequence table SED ID NO. 1.
Wherein, the nucleotide sequence 1473 bases shown in the sequence table SEQ ID NO.2, the coding sequence is the bases from the 1 st to the 1473 rd position of the 5' end, and the protein of the amino acid sequence shown in the sequence table SED ID NO.1 is coded.
It should be noted that the expression vector, cell line, engineering bacterium and host bacterium containing the above coding gene of the present invention all fall into the protection scope of the present invention.
The invention also provides a method for expressing the low-temperature matrix metalloproteinase, which is to introduce a recombinant expression vector containing the encoding gene of the low-temperature matrix metalloproteinase into a host cell and express the recombinant expression vector to obtain the low-temperature matrix metalloproteinase.
Wherein the host can be Escherichia coli, yeast, mammal, insect, Bacillus subtilis, Bacillus or Lactobacillus, preferably yeast.
The yeast is preferably Pichia pastoris (Pichia pastoris), such as Pichia pastoris X33.
The starting vector for constructing the recombinant escherichia coli expression vector and the recombinant yeast expression vector can be an expression vector for expressing a foreign gene in the host, such as a pEB vector capable of being expressed in escherichia coli, and pPIC9K, pPIC9, pGAPza and the like expressed in Pichia pastoris (Pichia pastoris).
The recombinant expression vectors can be constructed by conventional methods.
The invention also provides application of the low-temperature matrix metalloproteinase in degrading proteins under the low-temperature condition.
The invention further provides application of the coding gene of the low-temperature matrix metalloproteinase in degrading protein under the low-temperature condition.
Further, the proteins include, but are not limited to, casein, soy protein, collagen, gelatin, wheat gluten, and the like.
The invention has the following beneficial effects:
the low-temperature matrix metalloproteinase has high-efficiency protein degradation activity at the low temperature of 10-20 ℃, and has a low heat inactivation temperature (40 ℃).
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 shows a SDS-PAGE pattern of the expression product of the recombinant expression vector pGAPZa-nrMMP 14.
FIG. 2 shows a graph of the determination of the degradation ability of matrix metalloproteinases at low temperature.
Detailed Description
In order to more clearly illustrate the invention, the invention is further described below with reference to preferred embodiments and the accompanying drawings. Similar parts in the figures are denoted by the same reference numerals. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.
Example 1 obtaining of Low temperature matrix Metalloprotease Gene and expression of Low temperature matrix Metalloprotease 1, establishment of transcription library of Low temperature matrix Metalloprotease Gene
Adult Luo's Antarctic fish (Notothenia rossii) is dissected from living body, oral cavity sample is taken, liquid nitrogen is frozen quickly, and the sample is preserved at-80 ℃ for standby. After the tissue samples were ground with liquid nitrogen, total RNA was extracted with RNAiso Plus (Takara, large ligation) kit as reference. RNA samples are digested by RNase-free DNase I to remove genomic DNA pollution, agarose gel electrophoresis and an ultraviolet spectrophotometer are used for detection, and the concentration is adjusted to 500 ng/mu L. The RNA sample is used as a template, and is reversely transcribed into cDNA by an AMV reverse transcription kit (Promega), and the cDNA is preserved at the temperature of-20 ℃.
2. Obtaining of Low temperature matrix metalloproteinases Gene
And (3) carrying out PCR reaction under the guide of a primer 1 and a primer 2 by taking the cDNA sequence obtained in the step 1 as a template, and amplifying the sequence of the low-temperature matrix metalloproteinase (nrMMP14) gene.
Primer 1: 5' -CTCGAGGCGACGGAGCGACAAGT-3' (the nucleotide sequence is shown in sequence table SED ID NO.3, and the base of the underlined part is Xho I site);
primer 2: 5' -GCGGCCGCTTAGCCTCCATCAGTCTTGG-3' (the nucleotide sequence is shown in sequence table SED ID No.4, the base of the underlined part is Not I recognition site)
In the PCR reaction, the PCR reaction conditions are as follows: at 94 ℃ for 5 minutes, followed by 30 cycles with the following temperature program: heating to 94 ℃, keeping for 1 minute, cooling to 54 ℃, keeping for 1 minute, heating to 68 ℃, and keeping for 2 minutes; the amplification reaction was then terminated by holding at 68 ℃ for 10 minutes and finally at 4 ℃ for 10 minutes. A single band of about 1.5kb was obtained by agarose electrophoresis analysis, and PCR products were purified by a PCR product purification kit after detection, and the concentration was measured. The PCR product is subjected to TA cloning, is connected with a pMD18-T vector, is transformed into Escherichia coli DH5 alpha, and is subjected to sequencing identification of the recombinant plasmid pMD18-nrMMP 14. The DNA sequence of the low-temperature matrix metalloproteinase gene is shown as a sequence table SEQ ID NO.2, and the corresponding amino acid sequence is shown as a sequence table SEQ ID NO. 1.
3. Construction of recombinant expression vector of low-temperature matrix metalloproteinase encoding gene sequence
The obtained PCR product had Xho I and Not I restriction enzyme sites at both ends, and the PCR product and the plasmid pGAPZA alpha were subjected to double digestion simultaneously with the Xho I and Not I restriction enzymes. The enzyme digestion system is 50 μ L: 20 μ L, 10 XBuffer 5 μ L, Xho I2 μ L, Not I2 μ L, ddH of the fragment or plasmid of interest2O21. mu.L, and the digestion conditions are 37 ℃ for 3 h. And (4) carrying out sequencing verification on the enzyme digestion product after column recovery. The target fragment and the vector fragment are ligated in vitro with T4DNA ligase. Ligation reaction 10. mu.L: mu.L of the target fragment, 2. mu.L of pGAPZA. alpha. vector, 1. mu.L of 10 XT 4DNA ligation buffer, 1. mu.L of T4DNA ligase (350U/. mu.L), ddH2O1. mu.L, 16 ℃ overnight. And transforming the connecting product into escherichia coli JM109, screening kanamycin resistance, selecting colonies, carrying out shake culture at 37 ℃ for 6-8h, and respectively carrying out PCR identification and restriction enzyme digestion identification of recombinant plasmids. The obtained recombinant expression vector was named post-pGAPZA alpha-nrMMP 14. Sequencing pPIC9K-Dasag proves that the DNA sequence of the cloning connection is the same as the sequence shown in the sequence table SEQ ID NO.2, and the recombinant expression vector pGAPZA alpha-nrMMP 14 containing the gene sequence of the low-temperature matrix metalloproteinase is constructed correctly.
4. Expression of low temperature matrix metalloproteinase in pichia pastoris
After the recombinant expression vector pGAPZA alpha-nrMMP 14 is cut by BspHI restriction enzyme for linearization, the linearized vector pGAPZA alpha-nrMMP 14 is introduced into Pichia pastoris X33 by an electric shock mode, and the high expression strain resisting bleomycin is screened after the selective culture medium is cultured. A single colony grown on the selective medium was selected and inoculated into 5ml of YPD liquid medium (10 g/L yeast extract, 20g/L peptone, 20g/L glucose) and cultured at 28 ℃ for 12 to 24 hours, and then transferred into 500ml of BMGY medium (1% yeast extract, 2% peptone, 1.34% yeast nitrogen source (YNB), 100mM phosphate buffer pH6.0, 4X 10-5Biotin, 1% glycerol) until the OD600 of the bacterial solution becomes 2-3, and the culture temperature is adjustedAnd (3) adding glucose for induction culture at 15 ℃, adding glucose every 24 hours till the final concentration is 1%, and stopping culture till 120 hours. The supernatant was collected by centrifugation, and 15. mu.L of the supernatant was examined by SDS-PAGE. The results are shown in FIG. 1: lane 1 is protein standard molecular weight marker; lanes 2, 3 and 4 are expression products and arrows indicate the bands of interest, indicating that the recombinant strain expresses proteins with a molecular weight of about 56kDa under the low temperature induction of glucose, which is consistent with the theoretical molecular weight (56kd) deduced from amino acids.
The low-temperature matrix metalloproteinase expressed in pichia pastoris can be directly secreted into the supernatant of the culture solution, and the protein component in the supernatant is single and can be directly used for enzyme activity determination.
Example 2 Activity assay of Low temperature matrix Metalloprotease (hydroxyproline assay)
2.1 principle of the test
The matrix metalloproteinase catalyzes the solid insoluble collagen to be decomposed into soluble peptide which enters the solution, unreacted protein is filtered after the reaction is ended, and the filtrate is heated and acidolyzed into free amino acid. Hydroxyproline is specific amino acid of collagen, and the mass of free hydroxyproline in acidolysis solution is measured by hydroxyproline colorimetry, so that the mass of hydrolyzed collagen is indirectly represented. The ratio of this value to the total hydroxyproline content of the substrate collagen is called the collagen degradation rate and may reflect the collagen hydrolysis activity of the matrix metalloproteinase.
2.2 Experimental methods
Adding Tris-HCl buffer (pH 7.4, 5mmol/ml CaCl)20.5mmol of acetic acid-4-aminophenylmercury) was added to the reaction system, a substrate of bovine achilles tendon collagen (Sigma C9879) was added to 2mg/ml, 0.1mg/ml of an enzyme was added, the reaction was stirred at 15 ℃ for 30 hours, and a sample of the supernatant was periodically collected. Filtering the supernatant, adding an equal volume of concentrated hydrochloric acid solution into an acidolysis tube, heating in an oven at 110 ℃ overnight, and determining the content of hydroxyproline by a colorimetric method (561 nm).
The enzyme added in the experimental group was the low temperature matrix metalloproteinase (nrMMP14) obtained in example 1; the enzyme added to the control group was collagenase type I (MMP _ from _ Ch) derived from Clostridium histolyticum (Clostridium histolyticum). The results show that the low-temperature matrix metalloproteinase nrMMP14 of the invention can effectively hydrolyze 80% of substrate collagen within 12 hours. Meanwhile, the average specific activity of nrMMP14 collagenase in the whole reaction period under the condition of low temperature (15 ℃) is 1.98 times of that of the control group (Clostridium histolyticum type I collagenase MMP _ from _ Ch) (see figure 2).
The thermal stability of the low-temperature matrix metalloproteinase nrMMP14 is verified through a thermal inactivation experiment, and the experimental conditions are as follows: preparing 0.5mg/ml enzyme stock solution, bathing for 30 minutes at 40 ℃, and then determining the activity of the inactivated enzyme according to the activity determination method. The results showed that collagenase activity of nrMMP14 was completely inactivated by treatment in water bath at 40 ℃ for 30 minutes (see fig. 2, nrMMP _ inactivated).
It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention, and it will be obvious to those skilled in the art that other variations or modifications may be made on the basis of the above description, and all embodiments may not be exhaustive, and all obvious variations or modifications may be included within the scope of the present invention.
Sequence listing
<110> research institute of physical and chemical technology of Chinese academy of sciences
<120> low-temperature matrix metalloproteinase, coding gene and application thereof
<130> JLC17I0835E
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 491
<212> PRT
<213> Notothenia rossii
<400> 1
Ala Thr Glu Arg Gln Val Asn Pro Gln Thr Trp Leu Gln Gln Tyr Gly
1 5 10 15
Tyr Leu Pro Pro Gly Asp Met Arg Ala His Ser Leu Arg Ser Pro His
20 25 30
Ser Val Thr Ser Ala Ile Ser Ala Met Gln Lys Phe Tyr Gly Leu Thr
35 40 45
Val Thr Gly Thr Phe Asp Pro Asn Thr Ile Glu Ala Met Lys Arg Pro
50 55 60
Arg Cys Gly Val Pro Asp Lys Phe Gly Ala Glu Leu Lys Ser Asn Leu
65 70 75 80
Arg Lys Lys Arg Tyr Ala Ile Gln Gly Leu Lys Trp Gly Lys Asn Glu
85 90 95
Ile Thr Phe Ser Ile Gln Asn Tyr Thr Pro Lys Ile Gly Glu Tyr Asn
100 105 110
Ser Tyr Glu Ala Ile Arg Arg Ala Phe Lys Val Trp Gln Gln Val Thr
115 120 125
Pro Leu Thr Phe Asp Glu Ile Pro Tyr Gln Glu Ile Lys Tyr Gly Arg
130 135 140
Arg Lys Glu Pro Asp Ile Met Ile Phe Phe Ala Ser Gly Phe His Gly
145 150 155 160
Asp Ser Ser Pro Phe Asp Gly Glu Gly Gly Phe Leu Ala His Ala Tyr
165 170 175
Phe Pro Gly Pro Gly Met Gly Gly Asp Thr His Phe Asp Ser Asp Glu
180 185 190
Pro Trp Thr Ile Gly Asn His Asn Val Gln Gly Asn Asp Leu Phe Leu
195 200 205
Val Ala Val His Glu Leu Gly His Ala Leu Gly Leu Glu His Ser Asn
210 215 220
Asn Pro Leu Ala Ile Met Ala Pro Phe Tyr Gln Trp Met Glu Thr Glu
225 230 235 240
Asn Phe Gln Leu Pro Asp Asp Asp Leu Arg Gly Ile Gln Gln Ile Tyr
245 250 255
Gly Ser Gly Ser Gly Pro Gln Pro Pro Pro Val Thr Thr Arg Thr Pro
260 265 270
Asn His Asp Pro Asp Val Glu Tyr Ser Pro Asp Lys Pro His Phe Gly
275 280 285
Pro Asn Ile Cys Asp Gly His Phe Asp Thr Ile Ala Ile Leu Arg Gly
290 295 300
Glu Met Phe Val Phe Lys Asp Lys Trp Phe Trp Arg Val Arg Asn Asn
305 310 315 320
His Val Leu Asp Gly Tyr Pro Met Pro Ile Gly His Phe Trp Arg Gly
325 330 335
Leu Pro Thr His Val Thr Ala Ala Phe Glu Arg Glu Asp Gly Lys Phe
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Val Phe Phe Lys Gly Asp Lys Tyr Trp Val Phe Thr Glu Ser Leu Leu
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Asp Pro Gly Phe Pro Lys Asn Ile Lys Glu Met Gly Thr Gly Leu Pro
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Lys Asp Arg Ile Asp Ala Gly Leu Phe Tyr Thr Pro Thr Gly Gln Thr
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Phe Tyr Phe Arg Ala Asn Lys Tyr Tyr Arg Phe Asn Glu Asp Met Arg
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Ser Val Asp Glu Gly Tyr Pro Lys Ala Val Ser Val Trp Gln Gly Val
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Pro Asp Asn Ile Lys Ala Ala Phe Met Ser Lys Asp Gln Glu His Thr
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Tyr Phe Tyr Lys Ala Asn Lys Tyr Trp Lys Phe Asn Asn Gln Val Met
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Arg Val Glu Pro Gly Tyr Pro Lys Ser Ala Leu Arg Asp Trp Met Gly
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Cys Pro Asn Glu Asp Pro Lys Thr Asp Gly Gly
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<210> 2
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<212> DNA
<213> Notothenia rossii
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gcgacggagc gacaagtcaa tccacagaca tggctgcagc agtatggtta cctgcccccg 60
ggggacatgc gagcccactc cctccgctcc cctcactccg tcacctcggc tatcagcgcc 120
atgcagaagt tctacggcct caccgtcact ggcaccttcg accccaacac catcgaggcc 180
atgaagcgac cgcgctgtgg agtgccagat aagtttggtg ctgagttgaa aagcaacctg 240
aggaagaagc gatacgccat ccagggcctg aagtggggca agaatgaaat cactttcagt 300
atccaaaact acactcccaa gattggagag tataactcat acgaagccat ccgtcgggcg 360
tttaaagtct ggcagcaggt gaccccattg acctttgacg aaatccccta ccaggagatc 420
aaatatggac gccgcaagga gcccgacatt atgatctttt tcgcttcggg tttccatgga 480
gacagctctc cttttgatgg ggagggaggc ttcctagctc atgcctactt ccctggacct 540
ggaatgggcg gggacacaca ctttgactcc gatgaaccat ggaccatagg aaaccataac 600
gtacaaggta acgacctctt cctggttgca gtccatgagc tgggccacgc ccttggttta 660
gagcactcca acaacccact tgccatcatg gctccctttt accagtggat ggagacggag 720
aatttccaac tgccagatga cgacctgcga ggcatccagc agatctatgg ttctggatca 780
ggccctcagc cccctcctgt aacaactcgt acgccgaacc atgaccctga cgtcgaatat 840
tcaccagaca agcctcactt tggccccaac atctgtgatg gacacttcga cacaatcgcc 900
atcctcagag gagaaatgtt tgtgtttaag gataaatggt tctggagagt acggaacaac 960
catgttctgg atggatatcc aatgccgatt ggtcactttt ggaggggact gcccacacat 1020
gtgactgctg cttttgaaag ggaagatggg aaatttgttt tctttaaagg ggacaagtat 1080
tgggtgttta ccgaatcact tttagatcct ggtttcccga agaatataaa ggaaatgggc 1140
actggacttc ccaaggaccg aatagatgct ggtctcttct acacccccac tggacaaacg 1200
ttttacttca gagcaaacaa atattaccgt ttcaacgaag acatgcgaag cgttgatgag 1260
ggatatccaa aagctgtcag tgtgtggcaa ggagtgcctg acaatatcaa ggcagctttc 1320
atgagcaaag atcaagagca cacctacttc tacaaagcca acaagtattg gaagtttaac 1380
aaccaggtga tgcgtgtgga gcctggatat cctaaatcag cccttcgtga ctggatgggt 1440
tgtcccaacg aagaccccaa gactgatgga ggc 1473
<210> 3
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ctcgaggcga cggagcgaca agt 23
<210> 4
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
gcggccgctt agcctccatc agtcttgg 28
Claims (10)
1. The low-temperature matrix metalloproteinase is characterized in that the low-temperature matrix metalloproteinase is a protein consisting of an amino acid sequence shown in a sequence table SEQ ID NO. 1.
2. The gene encoding the low temperature matrix metalloproteinase of claim 1.
3. The gene encoding the low-temperature matrix metalloproteinase of claim 2, wherein the gene encoding the low-temperature matrix metalloproteinase is represented by (a) or (b):
(a) a nucleotide sequence shown as SED ID NO.2 in a sequence table;
(b) a nucleotide sequence of an amino acid sequence shown in a sequence table SED ID NO. 1.
4. An expression vector, cell line, engineering bacterium or host bacterium comprising the coding gene of claim 2 or 3.
5. A method for expressing the cold-temperature matrix metalloproteinase of claim 1, wherein the cold-temperature matrix metalloproteinase is obtained by introducing a recombinant expression vector containing the gene encoding the cold-temperature matrix metalloproteinase of claim 2 or 3 into a host cell and expressing the recombinant expression vector.
6. The method of claim 5, wherein the starting vectors used for constructing the recombinant expression vector are pEB, pPIC9K, pPIC9, pGAPza vectors.
7. The method of claim 5, wherein the host is Escherichia coli, yeast, mammal, insect, Bacillus subtilis, Bacillus or Lactobacillus.
8. The use of the low-temperature matrix metalloproteinase of claim 1 for degrading proteins at low temperature, wherein the low-temperature matrix metalloproteinase is subjected to protein degradation at a low temperature of 10-20 ℃.
9. The use according to claim 8, wherein the low temperature matrix metalloproteinase is heat inactivated at 40 ℃ for 30 minutes.
10. Use according to claim 8, wherein the protein is casein, soy protein, collagen, gelatin or wheat gluten.
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