CN113528555A - Heterologous expression of extracellular neutral metalloprotease empr and application thereof - Google Patents

Abstract

The invention discloses an extracellular metalloprotease gene and application thereof, belonging to the technical field of biology. The full length of the empr gene is obtained by extracting the genomic DNA of bacillus LCB14 and adopting a PCR technology. The total length of the gene is 1566bp, and 521 amino acids are coded. The invention realizes the cloning, heterologous expression and purification of the empr gene in bacillus subtilis, characterization of the protease property and application thereof in sheep skin depilation. The protease empr shows excellent depilatory performance in the sheepskin depilatory process, and has wide prospects in leather depilatory applications.

Description

Heterologous expression of extracellular neutral metalloprotease empr and application thereof

Technical Field

The invention relates to the technical field of recombinant expression and application of neutral metalloprotease empr.

Background

The protease is an important industrial enzyme preparation as a substance for catalyzing the hydrolysis of protein into amino acid, and can be used in the industries of food processing, feed, tanning industry, medicine, environmental protection and the like.

The protease research in China also has the following problems: (1) the development of microbial resources secreting exoproteases is insufficient; (2) the types of proteases are few; (3) the enzyme preparation has single variety and high price; (4) the application range is not wide enough. The protease market is still in a state of short supply and short demand, and the current research focus is still on breeding high-yield protease strains, optimizing culture conditions and researching related properties of the high-yield protease strains, and improving the quality and yield of the protease in China.

With the increasing knowledge and research on proteases and the development of genome sequencing technology, the research on proteases is gradually shifted to high-efficiency expression in heterologous hosts, so as to be more beneficial to the development of industry. At present, proteases derived from bacillus, actinomycetes, molds and the like are available, and heterologous expression in hosts such as escherichia coli, bacillus subtilis, pichia pastoris and the like is realized. Wherein, the Escherichia coli is used as the first choice host, and successfully expresses a plurality of proteins. However, when Escherichia coli expresses a protease, the toxic effect of the protease on cells easily causes lysis or produces inclusion bodies having no protease activity. And the yeast heterologous expression cost is high, so that the application of the yeast heterologous expression is greatly limited. The bacillus subtilis is a highly efficient heterologous expression host, does not cause diseases, does not produce endotoxin, is certified as a safe host by the U.S. FDA, has relatively low cost and is suitable for large-scale production.

Disclosure of Invention

The invention firstly provides a gene empr for coding extracellular metalloprotease, which is characterized in that a high-yield protease strain LCB14 screened from saline-alkali soil of Xinjiang is taken as a starting strain, total genome DNA of the starting strain is extracted, a primer is designed and is combined with the whole genome sequence for analysis, an empr gene fragment (Genbank ID: MN 480449) is amplified through PCR, the total length of the gene is 1566bp, 521 amino acids are coded, wherein 1-27 amino acids are signal peptides, 28-221 amino acids are propeptides, and 222-521 amino acids are mature peptides of the protease.

The obtained gene is connected with a vector pUB10 to construct a recombinant expression vector pUB10-empr, and the recombinant expression vector pUB10-empr is transformed intoBacillus subtilisSCK6, and culturing the obtained recombinant bacillus subtilis for expression. Centrifuging to collect supernatant, purifying by cation ion exchange chromatography to obtain pure enzyme with protease activity.

The molecular weight of the obtained metalloproteinases is about 54kD, and the optimal temperature is 50^oAnd C, the pH is most suitable for 6.5, and the depilatory composition has excellent depilatory performance when being applied to the depilatory flow in the leather making process, and has good application prospect in the leather industry.

Drawings

FIG. 1 is an electrophoretogram of the empr gene; m: DNA marker; 1: recombinant plasmid pUB10-empr multimer; 2: an empr gene; 3: plasmid pUB 10.

FIG. 2 is a SDS-PAGE of recombinant Bacillus protease expression; m, standard protein; 1, protease empr.

FIG. 3 shows the pH optimum and pH stability of the purified protease empr.

FIG. 4 shows the optimum temperature and temperature stability of the purified protease empr.

Figure 5 epilation effect.

Detailed Description

The first embodiment is as follows: obtaining the empr Gene

Extracting genome DNA of the bacillus LCB14, sequencing the whole genome sequence (completed by Meiji organisms), combining the obtained ORF reading frames, consulting related documents and comparing databases, and selecting an extracellular protease gene empr.

Example two: expression of the empr Gene

Primers were designed to amplify the empr gene, the forward primer (5'-CTCGGGGAATTTATCTTGTAGCCATAACAGTTCTTGATCTGTCCGTCCTTCCTTTTTTC-3'), the reverse primer (5'-GTTGCTAGTAACATCTGACCGAGATTTTTTTGAGCAACTTCAGTTTTCATTTGGAATGG-3'). And primers were designed to linearize plasmid pUB10, the forward primer (5'-CAAGAACTGTTATGGCTACAAG-3'), the reverse primer (5'-AGTTGCTCAAAAAAATCTCGGTC-3'). Recovering the amplified linearized plasmid pUB10 and empr gel, performing overlap extension PCR to obtain pUB10-empr nucleic acid polymer, transformingBacillus subtilisSCK6, positive clones were picked to LB liquid Medium, 37^oC, culturing the cells in a medium of 220rpm for 48 hours, and analyzing the expression amount of the protease by SDS-PAGE.

Example three: purification of protease empr

Recombinant bacterium channel 37^oC, culturing the culture medium at 220rpm for 48h, and centrifuging at 10000rpm to collect supernatant. Concentration by ultrafiltration, purification on a Hitrap SP FF cation exchange column, equilibration of the column with (20 mM Tris-HCl, pH 6.5), elution of the protein of interest on a linear gradient containing 1M NaCl and SDS-PAGE identification.

Example four: method for measuring protease activity

100 mul of purified protease was added to 700 mul of 50mM Tris-HCl buffer solution, 200 mul of casein solution (2%), 50%^oC, reacting for 20min in a water bath kettle, adding 200 mul of trichloroacetic acid (6.56 percent) to terminate the reaction, and centrifuging for 10min at 10000 rpm. After centrifugation, 400 mul of supernatant is taken, 2ml of sodium carbonate (4.24%) and 400 mul of furin phenol reagent are added, and the absorbance at 660nm is measured. Blank control was performed by adding trichloroacetic acid and then adding an equal volume of enzyme solution.

Example five: study of the enzymatic Properties of protease empr

Respectively measuring the activity of protease under different pH conditions (HAC-NaAC pH 4-6.5, Tris-HCl pH 6-9, Gly-NaOH pH 9-11); incubating for one hour at different pH (HAC-NaAC pH 4-6.5, Tris-HCl pH 6-9, Gly-NaOH pH 9-11) to determine the pH stability of the protease; respectively at different temperatures (20)^oC，25^oC，30^oC，35^oC，40^oC，45^oC，50^oC，55^oC，60^oC，65^oC，70^oC，75^oC) Measuring protease activity; at different temperatures (20)^oC，25^oC，30^oC，35^oC，40^oC，45^oC，50^oC，55^oC，60^oC，65^oC，70^oC，75^oC) The following incubation was performed for one hour and the thermostability of the protease was determined.

Example six: study of depilatory Properties with protease empr

Soaking sheepskin in water, removing meat and defatting, weighing, adding into rotary drum, adding water to liquid ratio of 1, and temperature of 37^oAnd C, adding protease empr according to 200IU/g of sheepskin, rotating the rotary drum for 30min, and stopping for 30min until the wool is completely removed.

Sequence listing

<110> Sichuan university

<120> heterologous expression of extracellular neutral metalloprotease empr and application thereof

<141> 2020-04-16

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1566

<212> DNA

<213> Bacillus sp. LCB14(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 1

gtgggtttag gtaagaaatt gtctgttgct gtcgccgctt cctttatgag tttaaccatc 60

agtcttccgg gtgttcaggc cgctgagaat cctcagctta aagaaaacct gacgaacttt 120

gtgccgaagc attctttggt gcaatctgaa ttgccttcag tcagtgacaa agcaatcaag 180

caatacttga aacaaaacgg caaagtcttc aaaggcaacc cttctgagag actgaagcta 240

attgaccaca cgaccgatga tctcggctac aagcacttcc gttatgtgcc tgtcgttaac 300

ggtgtgcctg tgaaagactc gcaagtcatt attcacgtcg ataaatccaa caatgtctat 360

gcgattaacg gagaattaaa caacgatgct tctgccaaaa cggcaaacag caaaaaatta 420

tctgcaaatc aagcgctgga tcatgctttt aaagcaatcg gcaaatcacc tgaagccgtc 480

tctaacggca acgttgcaaa caaaaacaaa gccgagctga aagcagcggc cacaaaagac 540

ggtaaatacc gactcgccta tgatgtaacc atccgctaca tcgaaccgga accagctaac 600

tgggaagtaa ccgttgatgc ggaaacaggg aaagtcctga aaaagcaaaa caaagtggag 660

catgccgctg caaccggaac aggtacgact cttaaaggaa aaacggtctc attaaatatt 720

tcttctgaaa gcggcaaata tgtaatgcgt gatctttcta aacctaccgg aacgcaaatt 780

attacgtacg atctgcaaaa ccgacaatat aacctgccgg gcacgctcgt atcaagcact 840

acaaaccagt tcacaacttc ttctcagcgc gctgccgttg atgcgcatta caatctcggc 900

aaagtgtacg attatttcta tcagacgttt aaacgcaaca gctacgacaa taaaggcggc 960

aaaatcgtat cttccgttca ttacggcagc aaatacaaca acgcggcctg gatcggcgac 1020

caaatgattt acggtgacgg tgacggctca ttcttctcgc ctctttccgg ttcaatggac 1080

gtaacggccc atgaaatgac acacggtgtt acacaggaaa cagccaacct gaactatgaa 1140

aatcaaccgg gtgctttaaa cgaatccttc tctgatgtat tcggatactt caatgatact 1200

gaggactggg atatcggtga agatattacg gtcagccagc cggctctccg cagtttatcc 1260

aatccgacaa aatacggaca gcccgaccat tacaaaaatt atcgaaacct tccgaatact 1320

gatgccggcg actacggcgg cgtgcataca aacagcggaa ttccgaacaa agccgcttac 1380

aacacgatta caaaaatcgg cgtgaaaaaa gcggagcaga tttactaccg tgcactgacg 1440

gtatatctca ctccgtcatc aagctttaaa gatgcaaaag cagctttgat tcaatcagcg 1500

cgggaccttt acggctctca agacgctgca agcgtagaag cggcctggaa tgcggtcggc 1560

ttgtaa 1566

<210> 2

<211> 521

<212> PRT

<213> Bacillus sp. LCB14(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 2

Val Gly Leu Gly Lys Lys Leu Ser Val Ala Val Ala Ala Ser Phe Met

1 5 10 15

Ser Leu Thr Ile Ser Leu Pro Gly Val Gln Ala Ala Glu Asn Pro Gln

20 25 30

Leu Lys Glu Asn Leu Thr Asn Phe Val Pro Lys His Ser Leu Val Gln

35 40 45

Ser Glu Leu Pro Ser Val Ser Asp Lys Ala Ile Lys Gln Tyr Leu Lys

50 55 60

Gln Asn Gly Lys Val Phe Lys Gly Asn Pro Ser Glu Arg Leu Lys Leu

65 70 75 80

Ile Asp His Thr Thr Asp Asp Leu Gly Tyr Lys His Phe Arg Tyr Val

85 90 95

Pro Val Val Asn Gly Val Pro Val Lys Asp Ser Gln Val Ile Ile His

100 105 110

Val Asp Lys Ser Asn Asn Val Tyr Ala Ile Asn Gly Glu Leu Asn Asn

115 120 125

Asp Ala Ser Ala Lys Thr Ala Asn Ser Lys Lys Leu Ser Ala Asn Gln

130 135 140

Ala Leu Asp His Ala Phe Lys Ala Ile Gly Lys Ser Pro Glu Ala Val

145 150 155 160

Ser Asn Gly Asn Val Ala Asn Lys Asn Lys Ala Glu Leu Lys Ala Ala

165 170 175

Ala Thr Lys Asp Gly Lys Tyr Arg Leu Ala Tyr Asp Val Thr Ile Arg

180 185 190

Tyr Ile Glu Pro Glu Pro Ala Asn Trp Glu Val Thr Val Asp Ala Glu

195 200 205

Thr Gly Lys Val Leu Lys Lys Gln Asn Lys Val Glu His Ala Ala Ala

210 215 220

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

225 230 235 240

Ser Ser Glu Ser Gly Lys Tyr Val Met Arg Asp Leu Ser Lys Pro Thr

245 250 255

Gly Thr Gln Ile Ile Thr Tyr Asp Leu Gln Asn Arg Gln Tyr Asn Leu

260 265 270

Pro Gly Thr Leu Val Ser Ser Thr Thr Asn Gln Phe Thr Thr Ser Ser

275 280 285

Gln Arg Ala Ala Val Asp Ala His Tyr Asn Leu Gly Lys Val Tyr Asp

290 295 300

Tyr Phe Tyr Gln Thr Phe Lys Arg Asn Ser Tyr Asp Asn Lys Gly Gly

305 310 315 320

Lys Ile Val Ser Ser Val His Tyr Gly Ser Lys Tyr Asn Asn Ala Ala

325 330 335

Trp Ile Gly Asp Gln Met Ile Tyr Gly Asp Gly Asp Gly Ser Phe Phe

340 345 350

Ser Pro Leu Ser Gly Ser Met Asp Val Thr Ala His Glu Met Thr His

355 360 365

Gly Val Thr Gln Glu Thr Ala Asn Leu Asn Tyr Glu Asn Gln Pro Gly

370 375 380

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

385 390 395 400

Glu Asp Trp Asp Ile Gly Glu Asp Ile Thr Val Ser Gln Pro Ala Leu

405 410 415

Arg Ser Leu Ser Asn Pro Thr Lys Tyr Gly Gln Pro Asp His Tyr Lys

420 425 430

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

435 440 445

His Thr Asn Ser Gly Ile Pro Asn Lys Ala Ala Tyr Asn Thr Ile Thr

450 455 460

Lys Ile Gly Val Lys Lys Ala Glu Gln Ile Tyr Tyr Arg Ala Leu Thr

465 470 475 480

Val Tyr Leu Thr Pro Ser Ser Ser Phe Lys Asp Ala Lys Ala Ala Leu

485 490 495

Ile Gln Ser Ala Arg Asp Leu Tyr Gly Ser Gln Asp Ala Ala Ser Val

500 505 510

Glu Ala Ala Trp Asn Ala Val Gly Leu

515 520

Claims (4)

1. The gene sequence of extracellular metalloprotease empr, its heterogenous expression in bacillus and its application in depilation.

2. The extracellular metalloprotease empr according to claim 1, wherein said protease gene consists of 1566 nucleotides encoding 521 amino acids, and wherein amino acids 1-27 are signal peptides.

3. The empr gene is connected into a pUB110 vectorBacillus subtilisHigh-efficiency expression is realized in SCK6, and excellent unhairing performance is shown when the SCK6 is applied to leather unhairing experiments.

4. The purified protease is characterized in that: optimum temperature 50^oC, the optimum pH value is 6.5, and the method has good application prospect in the leather unhairing industry.

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