CN104711200A

CN104711200A - Neutral protease production bacterial strain and use thereof

Info

Publication number: CN104711200A
Application number: CN201310681169.3A
Authority: CN
Inventors: 李佩佩; 王华明; 黄亦钧
Original assignee: Qingdao Vland Biotech Group Co Ltd
Current assignee: Qingdao Vland Biotech Group Co Ltd
Priority date: 2013-12-12
Filing date: 2013-12-12
Publication date: 2015-06-17
Anticipated expiration: 2033-12-12
Also published as: CN104711200B

Abstract

The invention provides Trichoderma reesei engineering bacteria. The Trichoderma reesei engineering bacteria can realize high-efficiency recombinant expression of the neutral protease and fermented enzyme activity of 5000U/mL. The neutral protease obtained by engineering bacterium recombinant expression of the Trichoderma reesei engineering bacteria has optimal pH of 7.0, has 85% or more of enzyme activity at a pH value of 5.0-8.0, has optimal temperature of 65 DEG C and has 85% or more of enzyme activity at a temperature of 55-70 DEG C. The neutral protease can be widely used in the field of food and leather processing.

Description

A kind of neutral protease produces bacterial strain and application thereof

Technical field

The invention belongs to gene engineering technology field, be specifically related to a kind of neutral protease and produce bacterial strain and application thereof.

Background technology

Proteolytic enzyme is the general name of the class of enzymes of protein hydrolysate peptide bond.By the mode of its hydrolyzed peptide, endopeptidase and exopeptidase two class can be divided into.Endopeptidase is by protein molecule inner cut-out, and exopeptidase is from the free amine group of protein molecule or the end of carboxyl one by one by peptide bond hydrolysis, and the amino acid that dissociates, the former is aminopeptidase, and the latter is carboxypeptidase.By its active centre and optimum pH, proteolytic enzyme can be divided into serine protease, thiol proteinase, metalloprotease and aspartate protease again.By the optimum pH of its reaction, be divided into aspartic protease, neutral protease and Sumizyme MP.

Proteolytic enzyme is extensively present in animal and meets with, in plant stem-leaf, fruit and microorganism.Microbial protease, primarily of mould, bacterium, is secondly produced by yeast, actinomycetes.

The aspect such as proteolytic enzyme has been widely used in leather, fur, silk, medicine, food, brewage.The depilation of leather industry and softeningly utilize proteolytic enzyme in a large number, both saved time, improved labour health condition again.Proteolytic enzyme also can be used for natural silk degumming, tenderization, drinks clarification.Can do medicinal clinically, as used pepsin treatment maldigestion, by acidic protein enzyme treatment bronchitis, and with trypsinase, Quimotrase to the treatment of serous coat adhesion between the purification of the suppurative wound of surgery and thoracic cavity, also can be used for organized processing to become individual cells, carry out cell and tissue structrue.Enzymatic laundry powder is the product innovation in washing composition, containing Sumizyme MP, can remove the bloodstain on clothing and albumen dirt, but notes not contacting skin when using, in order to avoid the protein on injured skin surface, causes the allergic phenomena such as fash, eczema.

Research at present for neutral protease is also little, mainly produces senior seasonings and food enrichment relating to, and the manufacture field such as leather depilation, softening, wool silk scouring.

Summary of the invention

The object of this invention is to provide a kind of neutral protease and produce bacterial strain and application thereof.The present invention by by aspergillus oryzae ( aspergillus oryzae) neutral protease gene proceed to Trichodermareesei ( trichoderma reesei) in, build and obtain Trichodermareesei engineering strain, energy efficient secretory expression neutral protease, can be widely used in food and leather processing field.

One aspect of the present invention provides a kind of Trichodermareesei, and it carries the recombinant vectors of expressing neutral protease.

Described neutral protease is

A () aminoacid sequence is the enzyme of SEQ ID NO:1;

B () amino acid in (a) replaces, lacks or adds one or several amino acid obtains, there is the enzyme of neutral protease activity.

The coding nucleotide sequence of described neutral protease is SEQ ID NO:2.

Present invention also offers the application of above-mentioned Trichodermareesei.

The present invention purchases the high efficiency recombinant expressed neutral protease of Trichodermareesei engineering bacteria energy built, and fermenting enzyme is lived and reached 5000U/mL.The suitableeest action pH of neutral protease of the present invention is 7.0, and can keep the enzymic activity of more than 60% when pH50-8.0; Optimum temperature is 65 DEG C, and can keep the enzymic activity of more than 85% when 55-70 DEG C.Described neutral protease can be widely used in food and leather processing field.Wherein, utilize the collagen protein in described neutral proteinase hydrolysis pigskin, percent hydrolysis is up to 68%, and efficiency is high, and pollute few, effect is good.

Accompanying drawing explanation

Fig. 1 is neutral protease action pH of the present invention-enzyme curve alive relatively.

Fig. 2 is neutral protease operative temperature of the present invention-enzyme curve alive relatively.

Embodiment

The present invention has used routine techniques and the method for genetic engineering and biology field use, such as MOLECULAR CLONING:A LABORATORY MANUAL, 3nd Ed. (Sambrook, 2001) method and described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, 2003).These general reference provide definition well known by persons skilled in the art and method.But this does not also mean that and limits the invention to described any concrete grammar, experimental program and reagent, because they can change.

Unless be separately construed as limiting in this article, whole technical term used herein and scientific terminology have usual the understood identical meanings of common counting personnel in field belonging to the present invention.DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 3nd Ed. (Singleton et al., 2006) and COLLINS DICTIONARY BIOLOGY (Hale et al., 2003) for technician provide the many terms used in the present invention generality explain.

Unless otherwise mentioned, nucleic acid writes from left to right by 5 ' to 3 ' direction; Amino acid writes from left to right by the direction of amino to carboxyl.

As used herein, term " restructuring ", when being used to refer to cell, nucleic acid, albumen or carrier, represents this cell, nucleic acid, albumen or carrier by importing heterologous nucleic acids or albumen or being modified by change natural acid or albumen.Therefore, such as, reconstitution cell expresses the gene never found in this cell of natural (non-recombinant) form, or express natural gene.

Term " protein " and " polypeptide " can exchange use in this article.Use traditional single-letter or the three-letter codes of amino-acid residue herein.

As used herein, term " gene " refers to the DNA fragmentation participating in producing polypeptide, comprises the region before and after coding region, and the insertion sequence (intron) between each encode fragment (exon).

Term " nucleic acid " comprises DNA, RNA, strand or double-strand, and their chemical modification object.

Term " nucleic acid " and " polynucleotide " can exchange use in this article.

Term " carrier " refers to the polynucleotide sequence being designed to nucleic acid be imported one or more cell types.Carrier comprises cloning vector, expression vector, shuttle vectors, plasmid, phagemid, sequence box and analogue.

Term " expression vector " represents the DNA construction comprising DNA sequence dna, and described DNA sequence dna is connected to the suitable control sequence that can affect this DNA and express in suitable host by steerable.This type of control sequence can comprise the sequence of the termination that the promotor of transcribing, the sequence optionally controlling ribosome bind site suitable on the operon sequence of transcribing, the mRNA that encodes, enhanser and control are transcribed and translated.

Term " promotor " represent participate in conjunction with RNA polymerase with promotor gene transcribe regulating and controlling sequence.Promotor can be inducible promoter or constitutive promoter.

Have the polynucleotide of the sequence iden of a certain per-cent with another sequence or polypeptide refers to, when comparing this two sequences, the base of described per-cent or amino-acid residue are identical.

Because genetic code is degeneracy, so more than one codon can be used to specific amino acid of encoding, the present invention includes the polynucleotide of specific aminoacid sequence of encoding.

Term " host strain " or " host cell " refer to the suitable host of expression vector or DNA construction, and described expression vector or DNA construction comprise the polynucleotide of encoding lipase of the present invention.Specifically, host strain is preferably filamentous fungal cells.This host cell can be wild-type filamentous fungal host cells or genetically modified host cell.Term " host strain " or " host cell " refer to the nucleus protoplastis produced by filamentous fungal strains cell.

Below in conjunction with specific embodiment, the present invention is described in detail.

the clone of embodiment 1 proteinase gene

With aspergillus oryzae ( aspergillus oryzae) genome DNA is template, utilizes upstream and downstream primer to increase.Pcr amplification condition is 95 DEG C of 4min; 94 DEG C of 30S; 55 DEG C of 40S, 72 DEG C of 1min 30 circulations; 72 DEG C of 7min.Gel reclaims test kit and reclaims pcr amplification product, is sent by amplified production Beijing Hua Da gene to carry out sequencing analysis.Result shows, and the nucleotides sequence reclaiming the amplified production obtained is classified as SEQ ID NO:2, and the aminoacid sequence of its coding is SEQ ID NO:1.Above-mentioned sequence found by NCBI Blast compare of analysis, the protease amino acid sequence similarity of SEQ ID NO:1 and aspergillus oryzae is the highest, is 83%, is a new allelotrope.

embodiment 2 Trichodermareesei ( trichoderma reesei) structure of engineering strain

The amplified production reclaimed in embodiment 1 is connected to pMD18-T carrier, obtains cloning vector pT-Pro plasmid, then carry out double digestion with NcoI and KpnI, reclaim TR fragment; Get 2 μ l recovery products and be connected with NcoI and KpnI double digestion pKDN-5 carrier the importing bacillus coli DH 5 alpha that spends the night, obtain recombinant expression plasmid pKDN-Pro.

Trichodermareesei mycelium is inoculated in PDA flat board, grows 4 days; The bacterium colony cutting diameter about 3 cm is placed in about YEG(0.5% yeast powder, 1% glucose) liquid nutrient medium, 30 DEG C, 200 rpm shaking culture spend the night; Multilayer filtered through gauze collects mycelia; Mycelia is placed in and fills 10-20 ml lyase liquid (Sigma L1412) enzymolysis 2-3 hour; Take out enzymolysis solution, add 0.7 M NaCl solution, jiggle, fall in three layers of sterilizing lens wiping paper and filter, collect filtrate, 3000 rpm, centrifugal 10 min; Abandon supernatant, add 10-20 ml STC liquid (20% sucrose, 50mM Tris-Cl, 50mM CaCl ₂) suspend, 3000 rpm, centrifugal 10 min; Add appropriate STC suspension packing (150 μ l/ manage, 10 ⁸individual/ml).

Get 2 μ g pKDN-Pro DNA to join in 150 μ l protoplastiss, then add 500 μ l 25%PEG and mix gently, room temperature leaves standstill 25 min; Then divide 2-3 time and add 1ml 25%PEG again, mix gently, room temperature leaves standstill 25min, is cooled to the upper strata semisolid medium (0.1%MgSO of 45-55 DEG C after protoplastis is added to about 50 ml fusing ₄, 1%KH ₂pO4,0.6% (NH ₄) ₂sO ₄, 1% glucose, 18.3% sorbyl alcohol, 0.35% agarose), pour into containing 100 μ g/ml Totomycin subfoundation culture medium flat plate (2% glucose, 0.5% (NH4) after mixing gently ₂sO ₄, 1.5%KH ₂pO ₄, 0.06%MgSO ₄, 0.06%CaCl ₂, 1.5% agar), 28 DEG C of dark culturing a couple of days grow to transformant.Will wherein a strain positive transformant called after Trichodermareesei ( trichoderma reesei) Pro-1.

embodiment 3 is fermented and zymologic property measures

Trichodermareesei Pro-1 is inoculated in MM fermention medium (1.5% glucose, 1.7% lactose, 2.5% corn steep liquor, 0.44% (NH ₄) ₂sO ₄, 0.09%MgSO ₄, 2%KH ₂pO ₄, 0.04%CaCl ₂, 0.018% tween-80,0.018% trace element, 0.018% polypropylene glycol-2000) cultivate, cultivate 48 hours for 28 DEG C, then cultivate 48 hours for 25 DEG C; Gained fermented liquid 8 layers of filtered through gauze, filtrate is centrifugal 10 min under 14000 g conditions, collect supernatant liquor; By supernatant liquor in concentration be 12% SDS-PAGE glue on carry out electrophoresis detection, locate appearance protein band at about 38kDa, molecular weight with prediction consistent, be recombinant expressed proteolytic enzyme.

proteinase activity measuring method is as follows:

Use folin's methods to measure the vigor of proteolytic enzyme, the solution used comprises: forint uses solution (a commercially available folin solution mixes with two parts of water, shakes up), sodium carbonate solution (42.4 g/L), trichoroacetic acid(TCA) (65.4 g/L), gradient pH value damping fluid, casein solution (10.0 g/L).Reaction process is as follows: add 1ml enzyme liquid in test tube, and 40 DEG C of temperature bath 2min, add casein solution 1ml, shakes up rear 40 DEG C of temperature bath 10min, adds 2ml solution of trichloroacetic acid, shake up (blank first adds trichoroacetic acid(TCA), then adds casein solution).Take out static 10min, qualitative filter paper filters at a slow speed.Get 1ml filtrate, add sodium carbonate solution 5ml, add Folin reagent and use solution 1ml, 40 DEG C of colour developing 20min, in 680nm wavelength, measure absorbancy with 10mm cuvette.

Protease activity unit of force is defined as 1g solid enzyme powder (or 1 ml liquid enzymes), and under certain temperature and pH value condition, 1min caseinhydrolysate produces 1 μ g tyrosine, is 1 enzyme activity unit.

Adopt above-mentioned detection method to carry out Enzyme activity assay to Trichodermareesei engineering bacterium fermentation liquid supernatant of the present invention, result shows, and fermentation broth enzyme work, up to 5000U/mL, illustrates the proteolytic enzyme of the engineering bacteria energy efficient secretory expression aspergillus oryzae that the present invention builds.

(1) optimal pH analysis

The damping fluid of 3.0,4.0,5.0,6.0,7.0,8.0,9.0,10.0,11.0 is respectively by pH value, under temperature 50 C condition, measure enzyme live, live as 100% with the highest enzyme, calculate relative enzyme to live, do the relative enzyme of pH-curve alive, as shown in Figure 1, the optimal pH of proteolytic enzyme of the present invention is 7.0 to result, be a neutral protease, and the enzymic activity of more than 60% can be kept when pH50-8.0.

(2) optimum temperuture analysis

Respectively at 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C, 75 DEG C, 80 DEG C, 85 DEG C, 90 DEG C, measure enzyme under the condition of pH 7.0 and live, live as 100% with the highest enzyme, calculate relative enzyme and live, do temperature-enzyme curve alive relatively.As shown in Figure 2, the optimum temperuture of neutral protease of the present invention is 65 DEG C to result, can keep the enzymic activity of more than 85% when 55-70 DEG C.

the application in collagen polypeptide prepared by embodiment 4 neutral protease

The present invention utilizes above-mentioned neutral protein ferment treatment pigskin, and prepare collagen polypeptide, output is high, pollutes little.

Specific embodiment is as follows:

1) pre-treatment of pigskin

Cleaning fresh porcine skin, strikes off the grease of pig skin surfaces after removing dirt; Add appropriate water, be heated to 100 DEG C, keep 5min; Take out the pigskin boiled, the fat on its surface scrapes off by slightly cool rear cutter, pulls out pig hair; Then the pigskin block degreasing of 10mm × 10mm size is cut into;

2) saponification method degreasing

Grease-removing agent is Na ₂cO ₃, temperature 40 DEG C, time 40 min, refrigerator freezing saves backup;

3) enzyme-squash techniqued collagen polypeptide technique

The pigskin of pre-treatment is added deionized water, and adjust pH is 7.0-7.5, adds 1%-2% neutral protease of the present invention, and steady temperature hydrolysis 1h, boiling water bath 10 min go out enzyme, centrifugally discards throw out.

The percent hydrolysis adopting formol titration to measure above-mentioned Collagen Protein From Pig Skin is about 68%, and visible neutral protease of the present invention can be widely used in the production of collagen polypeptide.

SEQUENCE LISTING

<110> Qingdao Weilan Biology Group Co., Ltd.

<120> neutral protease produces bacterial strain and application thereof

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 353

<212> PRT

<213> protease enzyme sequence

<400> 1

Met Arg Phe Ile Pro Val Ser Phe Leu Leu Leu Pro Leu Ala Pro Ala

1 5 10 15

Leu Lys Pro Leu Pro Val Glu Val Ala Gly Ser Pro Glu Gly Leu Asp

20 25 30

Val Thr Val Arg Lys Val Gly Asn Pro Arg Ile Lys Ala Val Val Lys

35 40 45

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

50 55 60

Asp Ala Ala Pro Val Pro Lys Val Phe Leu Phe Arg Asn Ala Thr Glu

65 70 75 80

Val Gln Phe Gln Gly Leu Lys Gln Arg Leu Ile Ser Lys Gly Phe Ser

85 90 95

Asp Asp Pro Phe Arg Thr Leu Ala Pro Gly Ala Thr Ile Glu Asp Glu

100 105 110

Leu Glu Thr Ala Ser Thr Ser Glu Leu Ser Glu Gly Gly Thr Ile Thr

115 120 125

Thr Lys Ser Asn Gly Leu Val Pro Ile Thr Thr Asp Asn Lys Val Thr

130 135 140

Gly Tyr Val Pro Phe Ser Ser Asn Glu Leu Ser Val Asp Val Asp Glu

145 150 155 160

Ala Glu Ala Ala Ser Val Thr Gln Ala Val Lys Ile Leu Glu Leu Arg

165 170 175

Thr Lys Val Thr Ser Cys Ser Gly Ser Arg Leu Ser Ala Leu Gln Thr

180 185 190

Ala Leu Arg Asn Thr Val Ser Leu Ala Arg Ala Ala Ala Thr Ala Ala

195 200 205

Gln Ser Gly Ser Ser Ser Arg Phe Gln Glu Tyr Phe Lys Thr Thr Ser

210 215 220

Ser Pro Thr Arg Ser Thr Val Val Pro Arg Leu Asn Ala Val Ala Lys

225 230 235 240

Glu Ala Ala Ser Thr Ser Ser Gly Ser Thr Thr Tyr Tyr Cys Ser Asp

245 250 255

Val Tyr Gly Tyr Cys Ser Ser Asn Val Leu Ala Tyr Thr Leu Pro Ser

260 265 270

Tyr Asn Ile Ile Ala Asn Cys Asp Leu Asn Tyr Ser Tyr Leu Ser Asp

275 280 285

Leu Thr Ser Thr Cys His Ala Gln Glu Lys Ala Ser Thr Thr Leu His

290 295 300

Glu Phe Pro His Pro Pro Gly Val Ser Thr Pro Gly Thr Asp Asp Phe

305 310 315 320

Gly Ser Gly Tyr Ser Ala Ala Thr Ser Phe Arg Ala Ser Gln Ala Leu

325 330 335

Leu Asn Ala Glu Thr Ser Pro Leu Phe Ala Asn Ala Val Lys Leu Lys

340 345 350

Cys

<210> 2

<211> 1062

<212> DNA

<213> protease gene sequence

<400> 2

atgcgtttca ttcctgtctc ctttcttctt ttgccccttg caccggctct caaacccctt

60

cctgtagagg ttgccggtag tcccgaaggt cttgatgtga ctgttaggaa ggtgggaaat

120

cctcggatca aggccgtggt aaagaacact ggcagcgagg atgtcacctt tgtgcacctc

180

aaattgttga aagatgccgc tccggtgccg aaagtttttc tgttccgcaa tgcgaccgag

240

gttcaattcc agggactcaa gcagcgtctt atctccaaag gtttttccga tgatcctttc

300

agaactcttg cccctggtgc tactatcgag gacgagctcg aaaccgcaag tactagtgaa

360

ctgtccgagg gtggtaccat cacgaccaaa agcaacggtt tagtacctat taccaccgat

420

aacaaggtca ctggatacgt tccattctcc tcgaacgagc tctccgttga tgtagatgaa

480

gctgaggccg cgagtgttac tcaagcagtt aagatcctgg agctccgcac caaggtcact

540

tcctgctctg gcagcagatt gtcggccctt cagactgctc tgagaaacac agtctctttg

600

gcacgtgcag ctgctactgc cgcgcagtcg ggatcttcct cccgtttcca ggagtatttc

660

aagacgacat ccagccccac ccgtagcacc gttgttcctc gcctgaacgc cgttgctaag

720

gaggccgcgt cgacctcttc gggaagtacc acgtactact gcagcgacgt gtatggatac

780

tgcagctcca acgtgcttgc gtataccctt ccgtcttata acatcatcgc caactgcgac

840

ctcaactatt cctatctttc ggacctgact agcacctgcc atgctcaaga aaaggcctcc

900

accaccctgc atgagttccc tcaccccccc ggtgtgtcca cccctggcac tgacgacttt

960

ggatctggat actcggctgc cacctccttc agggccagtc aggctctgct gaatgccgaa

1020

acctctccct tgtttgccaa cgctgtcaag ctcaagtgtt ag

1062

Claims

1. a Trichodermareesei, it carries the recombinant vectors of expressing neutral protease.

2. Trichodermareesei as claimed in claim 1, it is characterized in that, described neutral protease is

A () aminoacid sequence is the enzyme of SEQ ID NO:1;

3. Trichodermareesei as claimed in claim 2, it is characterized in that, the coding nucleotide sequence of described neutral protease is SEQ ID NO:2.

4. the application of Trichodermareesei described in claim 1.