CN109295041A

CN109295041A - With active polypeptide of serrapeptase and preparation method thereof

Info

Publication number: CN109295041A
Application number: CN201811177143.4A
Authority: CN
Inventors: 田�健; 陈豪; 曹阳; 诸辉
Original assignee: Ningbo Xi Nuoya Marine Organisms Science And Technology Ltd
Current assignee: Ningbo Xi Nuoya Marine Organisms Science And Technology Ltd
Priority date: 2018-10-10
Filing date: 2018-10-10
Publication date: 2019-02-01

Abstract

The present invention provides a kind of with the active polypeptide of serrapeptase, gene order is as shown in SEQ ID NO:3, amino acid sequence is as shown in SEQ ID NO:4, using cloning serrapeptase gene from Serratieae, by technique for gene engineering by the gene cloning construction of expression vector into pHT43 carrier, the expression plasmid built is imported in different expression bacterial strains by converting, can be given expression to by culture and inducing expression, the bacterial strain with the active polypeptide of high serrapeptase.For the present invention by gene clone technology, realizing the expression in bacillus has the active polypeptide of serrapeptase.Strain culturing cycle time of the present invention, production cost reduce, and product purity is higher, and extraction process is more simple.The present invention has great application prospect and commercial value in biomedicine technical field.

Description

With active polypeptide of serrapeptase and preparation method thereof

Technical field

The present invention relates to biomedicine technical fields, specifically have the active polypeptide of serrapeptase, while being related to its system Preparation Method.

Background technique

Serrapeptase is a kind of protease synthesized by being called Serratia E15 (Serratieae E15) enterobacteria.It is this Bacterium can find in the enteron aisle of silkworm.There are many kinds of classes for Serratieae, but cause in addition to the most Serratieae of E15 bacterium has Characteristic of disease.Serrapeptase has the effects that very strong solution fibrin block, eliminates stickiness phlegm and purification inflammation foci face, clinical It is widely used in expectoration difficulties caused by respiratory disease.Serrapeptase has resolving sputum, mitigates inflammatory reaction, eliminates oedema or swelling Effect.Serrapeptase is all taken as alternative medicine in many countries (such as Japan) and uses, and has very long applicating history.Most Close research additionally provide it is more it in treatment pain and the effective evidence of aspect of inflammation.Including air flue chronic lung disease, Chronic ENT disease, carpal tunnel syndrome, lower jaw pain, osteoarthritis and oedema etc..

But the production of serrapeptase, should mainly using extracting from the fermentation liquid of Natural strains Serratieae E15 strain Since Serratieae research data of diving is limited, Natural strains ferment unstable, murder by poisoning of the serrapeptase for thallus itself technique Property, cause the technique to fail to obtain bigger breakthrough always.Finally, serrapeptase production cost is high, product marketing and Using limited.

Summary of the invention

In order to solve the above technical problems, the present invention provides, one kind is with short production cycle, have serrapeptase activity and activity is high Polypeptide.

The technical solution of the present invention is to provide one kind to have the active polypeptide of serrapeptase, and the gene order of the polypeptide is such as Shown in SEQ ID NO:3.

The present invention also provides express the above-mentioned expression bacterial strain with the active polypeptide of serrapeptase.

The present invention also provides the recombinant plasmid pHT43-SER for converting above-mentioned expression bacterial strain.

Further, above-mentioned serrapeptase gene SER derives from Serratieae.

Further, above-mentioned expression bacterial strain is bacillus.Bacillus can be selected from Bacillus sp WB600, gemma bar Bacterium WB800N, bacillus BS168, bacillus CMCC (B) 63501, one of bacillus SCK6.

It is a further object of the present invention to provide the preparation methods of aforementioned polypeptides, using the serrapeptase for deriving from Serratieae Gene SER, above-mentioned recombinant plasmid transformed expressed into bacillus, then sieved by construction recombination plasmid pHT43-SER High efficient expression transformant is selected, is induced through lactose, isopropylthiogalactoside (IPTG) or other lactose analogs and generates tool There is the active recombinant polypeptide of serrapeptase.

Further, it when above-mentioned screening high efficient expression transformant, chooses positive colony single colonie and is inoculated on LB culture medium and shake Bed culture, until being induced when OD600 ≈ 0.5-1 using lactose, isopropylthiogalactoside (IPTG) or other lactose analogs Generating has the active polypeptide of serrapeptase.

Further, the condition of shaking table culture is 28 DEG C -40 DEG C, 150-250r/min, when culture is to OD600=0.5-1, Using lactose, isopropylthiogalactoside (IPTG) and other lactose analog inducing expressions.

The advantages of the present invention: serrapeptase gene SER is cloned into pHT43 by technique for gene engineering and is carried Construction of expression vector in body is imported the expression vector built in different bacillus by method for transformation, by culture and Inducing expression, the recombinant bacterial strain can give expression to the active polypeptide of high serrapeptase.Strain culturing cycle time, is produced into This reduction, product purity is higher, and extraction process is more simple.The present invention is before biomedicine technical field has greatly application Scape and commercial value.

Detailed description of the invention

Fig. 1 is recombinant plasmid pHT43-SER schematic diagram.

Fig. 2 is the serrapeptase activity for the polypeptide that high activity bacterial strain is obtained by lactose inducing expression.

Fig. 3 is the house thunder peptide for the polypeptide that high activity bacterial strain is obtained by isopropylthiogalactoside (IPTG) inducing expression Enzymatic activity.

Fig. 4 is that the serrapeptase for the polypeptide that high activity bacterial strain is obtained through other lactose analog (ONPG) inducing expressions is living Property.

Fig. 5 is the SDS-PAGE electrophoresis with the active polypeptide of serrapeptase.

Specific embodiment

The invention will be further described With reference to embodiment.

Embodiment 1

By designing homology primer, carries out PCR amplification and obtain target gene.Wherein forward primer is SerF: Ccggaattc gccgcgacaacc (SEQ ID NO:1), reverse primer are as follows: tgctctaga ttacacgataaagtc (SEQ ID NO:2).PCR amplification is carried out as template using the genome extracted from serratia marcescens CICC 23703.PCR system With program according to following procedure

PCR reaction system:

PCR response procedures:

The DNA sequence dna that target gene has the active polypeptide of serrapeptase has been obtained by PCR amplification, such as SEQ ID NO: Shown in 3, according to the SEQ ID NO:3, translated according to central tenet, it is available with the active polypeptide of serrapeptase Amino acid sequence, as shown in SEQ ID NO:4.

The restriction endonuclease sites according to involved in primer: EcoR I (gaattc), Xba I (tctaga) is to purpose base Identical digestion is carried out because carrying out digestion, while to pHT43.After digestion products carry out after purification using T4DNA ligase to digestion PHT43 carrier and digestion after SER gene be attached.Connection product is electroporated into escherichia coli DH5a, picking single bacterium It falls in LB culture medium after cultivating 10-24 hours and extracts recombinant plasmid.The recombinant plasmid of extraction is electroporated into different gemma (bacillus spore bacillus WB600, bacillus WB800N, bacillus BS168, bacillus CMCC (B) in bacillus 63501, bacillus SCK6).The coating culture 12- on the LB culture plate of the ampicillin sodium salt containing 100ug/mL 24h。

Embodiment 2

After single colonie is grown, picking single colonie is inoculated into LB liquid medium, and 37 DEG C of condition of culture, culture shaking table turns Fast 150-250rpm/min.When cell concentration is grown between OD (600nm measurement) 0.5-1, IPTG is added and is induced, IPTG Induce final concentration 0.1-1mmol/L, induction time 72h.5 kinds of different bacillus, every kind has filtered out 5 high-activity expression bacterium Strain, as a result see the table below 1:

It can be seen that wherein WB800 series bacillus activity highest, is averagely higher than 2000SpU/g.It is selected from 5 groups of different strains The highest bacterial strain (WB6004, WB800N4, BS1682, CMCC02, SCK603) of activity is selected, SDS-PAGE electrophoresis is carried out, as a result sees Fig. 5, SDS-PAGE show that the molecular weight with serrapeptase active peptides is about 50kD.

Embodiment 3

The highest bacterial strain of activity is filtered out in every kind of bacillus, with lactose (final concentration 1%), isopropylthio gala Glucosides (final concentration 0.5mmol/L) or other lactose analogs (O- nitro galactoside (ONPG)) carry out inducing expression.Culture Condition and inducing expression condition are the same as embodiment 2.Experimental result is shown in Fig. 2, isopropylthiogalactosides for lactose (final concentration 1%) (final concentration 0.5mmol/L) experimental result is shown in Fig. 3, and experimental result is shown in Fig. 4 for O- nitro galactoside (ONPG).By comparative analysis It was found that after induction 72 hours, using the WB800N4 strain activity highest of IPTG induction.

By embodiment as it can be seen that can be obtained by genetic engineering using the present invention has the more of serrapeptase high activity Peptide, cultivation cycle is short, is conducive to production method popularization.

Wherein, the activity test method of serrapeptase with reference to Japanese Pharmacopoeia (JP XVI) detection method (serrapeptase, Serrapeptase), as follows

(i) it sample solution: by the serrapeptase vitriolization ammonium salt solution (1/20) of lucky 0.100g, accurately dissolves 100mL.The 1mL solution is drawn, borate-hydrochloric acid buffer solution (dissolution 19 grams of boraxs (borax), 900 milliliters of water) are added.Use 1N Salt acid for adjusting pH value is to 9.Volume is settled to 1 liter with water), accurate quantitative analysis to 200mL, and use the solution molten as sample Liquid.

(ii) 0.160g tyrosine accurately tyrosine standard solution: is dissolved in 0.2mol/L hydrochloric acid TS (in advance at 105 DEG C Lower drying 3 hours), precise volume setting to 1000mL.10 milliliters of solution are drawn, and add the accurate constant volume 100 of 0.2mol/L hydrochloric acid Milliliter.Use preceding preparation.

(iii) substrate solution: (60 DEG C, decompression (being no more than 0.67 kPa) is 3 hours dry) are pre-dried in casein.Accurately It weighs based on the calculated 1.20g casein of drying loss, adds 160mL dobell's solution (19g is dissolved in 1000ml purified water), And it dissolves by heating in a water bath.After cooling, pH is adjusted to 9.0 with 1mol/L hydrochloric acid, it is slow that borate-hydrochloric acid is then added Rush solution (dissolution 19 grams of boraxs (borax), 900 milliliters of water), accurate constant volume to 200mL.It is used after being warming up to 37 ± 0.59C.Make With preceding preparation.

(iv) precipitation reagent: 15 grams of anhydrous vinegar are added in 150mL deionized water in 9 grams of trichloroacetic acids (CCl3COOH) of dissolution Sour sodium (anhydrous CH3COONa) and 10 milliliters of glacial acetic acid (CH3COOH) are completely dissolved.Volume is settled to 500 millis with deionized water It rises.It is used after being warming up to 37 ± 0.59C.

(v) step: drawing 1mL sample solution, be put into glass stopper pipe (15 × 130mm), stands 5 points at 37 ± 0.5 DEG C Clock, accurate that 5mL substrate solution is added, mixing is at once all right.It is placed 20 minutes at 37 ± 0.5 DEG C, it is accurate that tri- chloroethene of 5mL is added Acid, mixing are placed 30 minutes at 37 ± 0.5 DEG C, and are filtered with dry filter paper.2mL filtrate is drawn, accurate addition 5mL is anhydrous Sodium carbonate liquor (30 grams of natrium carbonicum calcinatums (anhydrous Na2CO3) of dissolution are in 500 ml deionized waters), 1mL is accurately added in mixing Diluted Folin's solution (1/3 dilution), is sufficiently mixed, and be allowed to rest in 37 ± 0.59C30 minutes.

According to spectrophotometry measuring method, use water as blank, the extinction of the solution is measured at 660nm Spend A1.

Individually absorption 1mL sample solution, the accurate 5mL trichloroacetic acid TS that is added is mixed as charait pepsin, accurate to add Enter 5mL substrate solution, placed at 37 ± 0.5 DEG C 30 minutes, then carries out determining absorbance as described above in an identical manner A2。

2mL tyrosine standard solution is individually drawn, it is accurate that 5mL Carbon Dioxide sodium solution (30 grams of Carbon Dioxides of dissolution are added Sodium (anhydrous Na2CO3) is in 500 ml deionized waters), mixing is accurate that the diluted Folin's solution of 1mL (1/3 dilution) is added, It is sufficiently mixed, then carries out mode same as described above and determine absorbance A 3.

2mL 0.2mol/L hydrochloric acid solution is individually drawn, carries out the measurement of absorbance A 4 according to the above method.

Enzyme activity definition: a charait pepsin unit corresponds to per minute from 5mL substrate solution under the above conditions Generate the amount of the charait pepsin of 1mg tyrosine.

Enzyme activity calculates:

The charait pepsin unit of every milligram of Serrapeptase=(A1-A2)/(A3-A4) × 1/20 × 200 × 176

20: the reaction time (minute)

200: dilution rate

176: conversion ratio (amount of enzyme reaction solution total amount/take tyrosine in amount of filtrate × tyrosine standard solution 2mL)

The present embodiments relate to the material arrived, reagent and experimental facilities, are to meet biological medicine unless otherwise instructed The commercial product of technical field.

The above is merely a preferred embodiment of the present invention, it is noted that for those skilled in the art For, under the premise of not departing from core of the invention technology, improvements and modifications can also be made, these improvements and modifications are also answered Belong to scope of patent protection of the invention.With any change in the comparable meaning and scope of claims of the present invention, all It is considered as being included within the scope of the claims.

Sequence table

<110>Ningbo Xi Nuoya marine organisms Science and Technology Ltd.

<120>there is active polypeptide of serrapeptase and preparation method thereof

<130> 2018

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<170> SIPOSequenceListing 1.0

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<212> DNA

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ccggaattcg ccgcgacaac c 21

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<212> DNA

<213>reverse primer (artificial sequence)

<400> 2

tgctctagat tacacgataa agtc 24

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<211> 1416

<212> DNA

<213>with the DNA sequence dna (artificial sequence) of the active polypeptide of serrapeptase

<400> 3

gccgcgacaa ccggttacga tgctgtagac gacctgctgc attatcatga gcggggtaac 60

gggattcaga ttaatggcaa ggattcattt tctaacgagc aagctgggct gtttattacc 120

cgtgagaacc aaacctggaa cggttacaag gtatttggcc agccggtcaa attaaccttc 180

tcgttcccgg actataagtt ctcttccacc aacgtcgccg gcgacaccgg gctgagcaag 240

ttcagcgcgg aacagcagca gcaggctaag ctgtcgctgc agtcctgggc cgacgtcgcc 300

aatatcacct tcaccgaagt ggcggccggt caaaaggcca atatcacctt cggcaattac 360

agccaggatc gtcccggcca ctatgattac ggcacccagg cctacgcctt cctgccgaac 420

accatttggc agggccagga tttgggcggc cagacctggt acaacgtcaa ccaatccaac 480

gtgaagcatc cggcgaccga agactacggc cgccagacgt tcacccatga gattggccat 540

gcgctgggcc tgagccaccc gggcgactac aacgccggtg agggcaaccc gacctataga 600

gatgtcacct atgcggaaga tacccgccag ttcagcctga tgagctactg gagtgaaacc 660

aataccggtg gcgacaacgg cggtcactat gccgcggctc cgctgctgga tgacattgcc 720

gccattcagc atctgtatgg cgccaacctg tcgacccgca ccggcgacac cgtgtacggc 780

tttaactcca ataccggtcg tgacttcctc agcaccacca gcaactcgca gaaagtgatc 840

tttgcggcct gggatgcggg cggcaacgat accttcgact tctccggtta caccgctaac 900

cagcgcatca acctgaacga gaaatcgttc tccgacgtgg gcggcctgaa gggcaacgtc 960

tcgatcgccg ccggtgtgac cattgagaac gccattggcg gttccggcaa cgacgtgatc 1020

gtcggcaacg cggccaacaa cgtgctgaaa ggcggcgcgg gtaacgacgt gctgttcggc 1080

ggcggcgggg cggatgaatt gtggggcggt gccggcaaag acatcttcgt gttctctgcc 1140

gccagcgatt ccgcaccggg cgcttcagac tggatccgcg acttccagaa ggggatcgac 1200

aagatcgacc tgtcgttctt caataaagaa gcgcagagca gcgatttcat tcacttcgtc 1260

gatcacttca gcggcacggc cggtgaggcg ctgctgagct acaacgcgtc cagcaacgtg 1320

acagatttgt cggtgaacat cggtgggcat caggcgccgg acttcctggt gaaaatcgtc 1380

ggccaggtag acgtcgccac tgactttatc gtgtaa 1416

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<213>with the amino acid sequence (artificial sequence) of the active polypeptide of serrapeptase

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Ala Ala Thr Thr Gly Tyr Asp Ala Val Asp Asp Leu Leu His Tyr His

1 5 10 15

Glu Arg Gly Asn Gly Ile Gln Ile Asn Gly Lys Asp Ser Phe Ser Asn

20 25 30

Glu Gln Ala Gly Leu Phe Ile Thr Arg Glu Asn Gln Thr Trp Asn Gly

35 40 45

Tyr Lys Val Phe Gly Gln Pro Val Lys Leu Thr Phe Ser Phe Pro Asp

50 55 60

Tyr Lys Phe Ser Ser Thr Asn Val Ala Gly Asp Thr Gly Leu Ser Lys

65 70 75 80

Phe Ser Ala Glu Gln Gln Gln Gln Ala Lys Leu Ser Leu Gln Ser Trp

85 90 95

Ala Asp Val Ala Asn Ile Thr Phe Thr Glu Val Ala Ala Gly Gln Lys

100 105 110

Ala Asn Ile Thr Phe Gly Asn Tyr Ser Gln Asp Arg Pro Gly His Tyr

115 120 125

Asp Tyr Gly Thr Gln Ala Tyr Ala Phe Leu Pro Asn Thr Ile Trp Gln

130 135 140

Gly Gln Asp Leu Gly Gly Gln Thr Trp Tyr Asn Val Asn Gln Ser Asn

145 150 155 160

Val Lys His Pro Ala Thr Glu Asp Tyr Gly Arg Gln Thr Phe Thr His

165 170 175

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

180 185 190

Gly Glu Gly Asn Pro Thr Tyr Arg Asp Val Thr Tyr Ala Glu Asp Thr

195 200 205

Arg Gln Phe Ser Leu Met Ser Tyr Trp Ser Glu Thr Asn Thr Gly Gly

210 215 220

Asp Asn Gly Gly His Tyr Ala Ala Ala Pro Leu Leu Asp Asp Ile Ala

225 230 235 240

Ala Ile Gln His Leu Tyr Gly Ala Asn Leu Ser Thr Arg Thr Gly Asp

245 250 255

Thr Val Tyr Gly Phe Asn Ser Asn Thr Gly Arg Asp Phe Leu Ser Thr

260 265 270

Thr Ser Asn Ser Gln Lys Val Ile Phe Ala Ala Trp Asp Ala Gly Gly

275 280 285

Asn Asp Thr Phe Asp Phe Ser Gly Tyr Thr Ala Asn Gln Arg Ile Asn

290 295 300

Leu Asn Glu Lys Ser Phe Ser Asp Val Gly Gly Leu Lys Gly Asn Val

305 310 315 320

Ser Ile Ala Ala Gly Val Thr Ile Glu Asn Ala Ile Gly Gly Ser Gly

325 330 335

Asn Asp Val Ile Val Gly Asn Ala Ala Asn Asn Val Leu Lys Gly Gly

340 345 350

Ala Gly Asn Asp Val Leu Phe Gly Gly Gly Gly Ala Asp Glu Leu Trp

355 360 365

Gly Gly Ala Gly Lys Asp Ile Phe Val Phe Ser Ala Ala Ser Asp Ser

370 375 380

Ala Pro Gly Ala Ser Asp Trp Ile Arg Asp Phe Gln Lys Gly Ile Asp

385 390 395 400

Lys Ile Asp Leu Ser Phe Phe Asn Lys Glu Ala Gln Ser Ser Asp Phe

405 410 415

Ile His Phe Val Asp His Phe Ser Gly Thr Ala Gly Glu Ala Leu Leu

420 425 430

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

435 440 445

Gly His Gln Ala Pro Asp Phe Leu Val Lys Ile Val Gly Gln Val Asp

450 455 460

Val Ala Thr Asp Phe Ile Val

465 470

Claims

1. have the active polypeptide of serrapeptase, which is characterized in that the gene order of the polypeptide as shown in SEQ ID NO:3, Amino acid sequence is as shown in SEQ ID NO:4.

2. expressing the expression bacterial strain described in claim 1 with the active polypeptide of serrapeptase, which is characterized in that the expression Bacterial strain is bacillus.

3. the recombinant plasmid pHT43-SER for converting expression bacterial strain as claimed in claim 2.

4. having the active polypeptide of serrapeptase as described in claim 1, which is characterized in that the gene order template comes from Serratieae.

5. expression bacterial strain as claimed in claim 2, which is characterized in that the bacillus is selected from Bacillus sp WB600, gemma Bacillus WB800N, bacillus BS168, bacillus CMCC (B) 63501, one of bacillus SCK6.

6. the preparation method described in claim 1 with the active polypeptide of serrapeptase, which is characterized in that using from sand The gene SER of the serrapeptase of thunder Salmonella, construction recombination plasmid pHT43-SER, by the recombinant plasmid transformed to bacillus In expressed, then filter out high efficient expression transformant, and induce generation have the active polypeptide of serrapeptase.

7. preparation method as claimed in claim 6, which is characterized in that when the screening high efficient expression transformant, choose positive gram Grand single colonie is inoculated in shaking table culture on LB culture medium, until when OD600 ≈ 0.5-1, using lactose, isopropylthiogalactoside Or the induction of other lactose analogs is generated with the active polypeptide of serrapeptase.

8. preparation method as claimed in claim 7, which is characterized in that the condition of the shaking table culture is 25 DEG C -40 DEG C, 150- 250r/min。

9. preparation method as claimed in claim 6, which is characterized in that when construction recombination plasmid pHT43-SER, restriction enzyme site is EcoR I and Xba I.

10. preparation method as claimed in claim 6, which is characterized in that when construction recombination plasmid pHT43-SER, PCR amplification Forward primer sequence is as shown in SEQ ID NO:1, and reverse primer is as shown in SEQ ID NO:2.