CN108841739A - A method of building efficient secretory expression bacterium heparin hydrolase recombinant bacterium - Google Patents

A method of building efficient secretory expression bacterium heparin hydrolase recombinant bacterium Download PDF

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CN108841739A
CN108841739A CN201810710021.0A CN201810710021A CN108841739A CN 108841739 A CN108841739 A CN 108841739A CN 201810710021 A CN201810710021 A CN 201810710021A CN 108841739 A CN108841739 A CN 108841739A
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heparin
hydrolase
recombinant bacterium
ala
heparin hydrolase
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康振
陈坚
原攀红
堵国成
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Jiangnan University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces

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Abstract

The invention discloses a kind of methods for constructing efficient secretory expression bacterium heparin hydrolase recombinant bacterium, belong to technical field of enzyme engineering.The present invention uses pichia yeast expression system, constructs a kind of method for expressing heparin hydrolase.And the secreting, expressing of heparin hydrolase is realized in pichia yeast expression system.It is a kind of method that fermentation produces heparin hydrolase, important impetus is played to the industrialized production of heparin hydrolase.

Description

A method of building efficient secretory expression bacterium heparin hydrolase recombinant bacterium
Technical field
The present invention relates to a kind of methods for constructing efficient secretory expression bacterium heparin hydrolase recombinant bacterium, belong to enzyme engineering skill Art field.
Background technique
Polysaccharide is widely present in animals and plants and microorganism as a kind of polymeric carbohydrate, has antitumor, disease-resistant The various biologicals activity such as malicious, anti-oxidant, immunological regulation.Polysaccharide is mainly made of disaccharide unit, is divided into Heparan sulfate, liver Element, hyaluronic acid, chondroitin sulfate, dermatan sulfate.Polysaceharide lyase mainly cracks heparin or sulfuric acid second in such a way that β is eliminated Acyl heparin, is mainly derived from Flavobacterium heparinum, therefrom isolates and purifies out three kinds of heparinases at present：Heparinase I, II, III, low Molecular heparin preparation and heparin class polysaccharide molecule structure elucidation field have highly important application prospect.For including heparinase Nearly all polysaceharide lyase inside, the unicity of catalytic way largely limit its corresponding application.However, for The unicity of this catalysis substrate, current understanding is only limitted to speculate, there is no direct experimental evidence, it is even more impossible to realize different knots The conversion that substrate is composed between structure polysaceharide lyase.
Heparin hydrolase is that inscribe-GRD beta-glucuronidase belongs to glycoside hydrolase Families 79, this enzyme hydrolysis sulfuric acid liver Element hydrolyzes β Isosorbide-5-Nitrae-glycosidic bond between glucuronic acid and Glucosamine.It is many institute's weeks that heparin hydrolase is overexpressed in cancer cell Know, related with angiogenesis, inflammation and increased metastatic potential, therefore, heparin hydrolase is a kind of important potential drug Target.Before 35 years, Hook and his colleagues first describe a kind of Heparan sulfate (HS) inscribe-β-D- glucuronic acid Glycosidase, hereafter, reporting this activity successively, there are various cell types and tissues.Heparanase is in nearly all analysis Human tumor in be all overexpressed, and observe correlation between the overexpression of tumour cell and metastatic potential.It seems It is also the key of mouse β cell survival and autoimmune diabetes.Therefore, heparin hydrolase is considered invading tumour cell It attacks and transfer is of great significance, become the ideal targets of anticancer drug discovery.
The method of production heparin hydrolase is BL21 at present, and yield do not report, there are the problem of be not implemented for intracellular expression Efficient secretory expression.
Summary of the invention
It is described the first purpose of the invention is to provide a kind of building efficient secretory expression bacterium heparin hydrolase recombinant bacterium Recombinant bacterium is with Pichia pastoris Pichia pastoris GS115 for host, and expression derives from Burkholderia The heparin hydrolase of pseudomallei.
In one embodiment of the invention, the nucleotide sequence of the heparin hydrolase such as SEQ ID NO:Shown in 1.
In one embodiment of the invention, the amino acid sequence of the heparin hydrolase gene such as SEQ ID NO:2 It is shown.
Construction method a second object of the present invention is to provide the recombinant bacterium described in one kind is：Nucleotides sequence is classified as The heparin hydrolase gene of SEQ ID NO.1 is connected on Expression vector pPIC9K, is then transformed into Pichia pastoris Pichia In pastoris GS115, correct transformant is screened to get recombinant yeast pichia pastoris Pichia pastoris GS115/ pPIC9K-BpHEP。
In one embodiment of the invention, the construction method of recombinant bacterium addition group on heparin hydrolase gene His tag.
Third object of the present invention is to provide a kind of method of recombinant bacterium fermenting and producing heparin hydrolase, the sides Method in the fermenter, using methanol induction strategy fermenting and producing heparin hydrolase.
In one embodiment of the invention, fermentation process the specific steps are：
The YPD seed liquor of shaking flask culture is inoculated in 3L automatic fermenter (LiFlus GM with 10% inoculum concentration BioTRON, Korea) in, temperature is controlled at 25-30 DEG C, and adjusting pH using 25% ammonium hydroxide is 5-6, and initial speed of agitator is 500- 1000r·min-1, ventilatory capacity 2-4Lmin-1, dissolved oxygen maintains 30%-35% or more.Cultivate about 20-40h, OD600About 70-80 waits for glycerol depletion, exponential fed-batch 30-50% (WV-1) glycerite (and contain 12mLL-1PTM1), speed of agitator with it is molten Oxygen is associated, controls revolving speed in 500-1000rmin-1Feed supplement 12h stops feed supplement and waits for that glycerol exhausts again, OD600About 250 is (dry Weigh about 60.0gL-1), revolving speed is in 600-1000rmin-1Cocurrent adds 100% methanol (containing 12mLL-1PTM1), methanol concentration Control is in 18.0gL-1, 30 DEG C, 25 DEG C, 22 DEG C and the temperature-induced strategy of stage control is respectively adopted in inducing temperature.Of the invention 4th purpose is to provide a kind of method of heparin hydrolysis enzyme purification, and specific step is as follows：It is centrifuged fermentation liquid, is collected Supernatant, loading；Using Ni column, 10 column volume of pillar is balanced with phosphate buffer；It is eluted using imidazole gradient, imidazoles is used Phosphate buffer preparation is stated, according to 10mmolL-1, 30mmolL-1, 200mmolL-1Gradient elution.
Beneficial effects of the present invention：
(1) it is expressed in Pichia pastoris using the genetic engineering bacterium that method of the invention constructs, is a kind of fast fast-growing The method for producing heparin hydrolase, is purified, method is simple using nickel column.
(2) Pichia pastoris has strong promoter AOX, and expression is high, recombinant bacterial strain high stability, heterologous egg White gene and expression plasmid are integrated on P.pastoris genome by homologous recombination, with chromosome replication heredity and are not easy It loses.With posttranslational modifications machining functions such as glycosylation, protein phosphorylation, fatty phthaleins.Adaptable, nutritional requirement is low. It is fewer to be secreted into extracellular protein classes, and is free of protein in culture medium, is easy to the isolation and purification of downstream product.It is highly dense It is more mature to spend zymotechnique, easily amplifies culture.
(3) the heparin hydrolase expression quantity of Pichia pastoris secreting, expressing is high, zymologic property is stable, resistance to pH, wide temperature range.
Detailed description of the invention
Fig. 1 is the plasmid map that heparin hydrolase is constructed in Pichia pastoris；
Fig. 2 is the plasmid map that heparin hydrolase is constructed in Escherichia coli；
Fig. 3 is the enzyme activity that heparin hydrolase is detected in Pichia pastoris；
Fig. 4 is the SDS-PAGE electrophoresis result that heparin hydrolase is constructed in Pichia pastoris.
Specific embodiment
BMGY culture medium：Yeast powder 10g L–1, peptone 20g L–1, glycerine 10mL L–1, 100mM potassium phosphate buffering Liquid, 0.34% (mass percent) is without amino yeast nitrogen (YNB), 1% (NH4)2SO4, 4x 10–5% biotin, pH=6.0.
Heparin hydrolase enzyme activity determination method：800 μ L substrate heparin (HP), chondroitin sulfate A (CSA) (CSA), sulfuric acid are taken respectively Chondroitin C (CSC) (2mgmL-1) 5-10 μ L fermentation liquid, residue 50mmolL is added-15.5 citrate buffer solution (lemon of pH Lemon acid+disodium hydrogen phosphate) supply 1mL.2mLDNS is added to mix, reaction 10min is boiled in water-bath.It is cold to go to room temperature, it adds ultrapure Water measures the A540 value of each number pipe to 10mL system at 540nm.
Embodiment 1：Produce the building of heparin hydrolase recombinant yeast pichia pastoris
A kind of building for the genetic engineering bacterium producing heparin hydrolase：
Using pPIC9K as carrier, the heparin hydrolase gene (nucleotide sequence such as SEQ ID NO is cloned:Shown in 1), base Because adding 6-his label after ATG.As shown in Figure 1, upstream primer introduces EcoR I restriction enzyme site；Downstream primer introduces Not I restriction enzyme site；Gene Hepa, plasmid pPIC9K through restriction enzyme EcoR I, Not I digestion after purification, 16 DEG C of connections overnight are transferred to Escherichia coli JM109 and are expanded, and select the correct plasmid of sequencing and are transferred to Pichia Pastoris GS115 is expressed, and the recombinant yeast pichia pastoris Pichia containing recombination heparin hydrolase gene is obtained pastoris GS115/pPIC9K-BpHEP。
The upstream and downstream primer is：
F:GAATTCATGCCGCAGCAGCCGCCG(SEQ ID NO:3)
R:GCGGCCGCTTACAATTGAATAGATT(SEQ ID NO:4)
Embodiment 2：Produce the building of the Escherichia coli recombinant strain of heparin hydrolase
A kind of building for the genetic engineering bacterium producing heparin hydrolase：
Using pET20 as carrier, the heparin hydrolase gene is cloned.As shown in Fig. 2, upstream primer introduces EcoR I limitation Property restriction enzyme site；Downstream primer introduces Not I restriction enzyme site；Gene Hepa, plasmid pET20 are through restriction enzyme After purification, 16 DEG C of connections overnight are transferred to Escherichia coli JM109 and are expanded for EcoR I, Not I digestion, select survey The correct plasmid of sequence is transferred to BL21 and is expressed, and obtains the recombination bacillus coli BL21DE3/ containing recombination heparin hydrolase gene PET20-BpHEP。
The upstream and downstream primer is：
F:GAATTCATGCCGCAGCAGCCGCCG(SEQ ID NO:3)
R:GCGGCCGCTTACAATTGAATAGATT(SEQ ID NO:4)
Embodiment 3：The horizontal fermenting and producing heparin hydrolase of shaking flask
It is production bacterial strain with the recombination bacillus coli BL21DE3/PET20-BpHEP containing recombination heparin hydrolase gene, Single colonie is inoculated in LB culture medium, and 37 DEG C, overnight incubation under the conditions of 220rmp obtains seed culture fluid；By seed culture fluid 1% inoculum concentration is inoculated into the TB culture medium of 25mL by mass percentage, to OD6000.6 is grown to, 0.1M is added in the medium IPTG, 28 DEG C of inducing temperature, inducing expression 28h.After fermentation, measurement heparin enzyme activity is 10U/mL.
With the recombinant yeast pichia pastoris Pichia pastoris GS115/pPIC9K- containing recombination heparin hydrolase gene BpHEP is production bacterial strain, and single colonie is inoculated in YPD culture medium, and 28 DEG C, overnight incubation under the conditions of 200rmp obtains seed training Nutrient solution；By seed culture fluid by mass percentage 2% inoculum concentration be inoculated into the BMGY culture medium of 25mL, be added in the medium 1% methanol is induced, and 28 DEG C of inducing temperature, inducing expression 70h.After fermentation, as shown in figure 3, measurement heparin enzyme activity For 89U/mL, chondroitin sulfate A (CSA) enzyme activity 39U/mL, chondroitin sulfate C enzyme activity 47U/mL.
Therefore, the yield of recombinant yeast pichia pastoris Pichia pastoris GS115/pPIC9K-BpHEP is higher, and recombination is big The low output of enterobacteria.
Embodiment 4：Ferment tank produces heparin hydrolase
The YPD seed liquor of shaking flask culture is inoculated in 3L automatic fermenter (LiFlus GM with 10% inoculum concentration BioTRON, Korea) in, temperature is controlled at 30 DEG C, and adjusting pH using 25% ammonium hydroxide is 5.5, and initial speed of agitator is 500r min-1, ventilatory capacity 2.5Lmin-1, dissolved oxygen maintains 30% or more.Cultivate about 28h, OD600About 70-80 waits for that glycerol consumes To the greatest extent, 50% (WV of exponential fed-batch-1) glycerite (and contain 12mLL-1PTM1), speed of agitator is associated with dissolved oxygen, controls revolving speed In 500-1000rmin-1Feed supplement 12h stops feed supplement and waits for that glycerol exhausts again, OD600About 250 (dry weight about 60.0gL-1), Revolving speed is in 1000rmin-1Cocurrent adds 100% methanol (containing 12mLL-1PTM1), methanol concentration control is in 18.0gL-1, lure It leads temperature and 30 DEG C, 25 DEG C, 22 DEG C and the temperature-induced strategy of stage control is respectively adopted.96h heparin enzyme activity is up to 340U/ mL。
Embodiment 5：Recombinate the purifying preparation of heparin hydrolase
Purifying recombination heparin hydrolase, specific steps：Using the Ni column of 5mL, with phosphate buffer (50mmolL- 1pH7.0) balance 10 column volume of pillar.4 DEG C are collected by centrifugation fermented supernatant fluid, loading 20mL.It is eluted using imidazole gradient, imidazoles It is prepared with above-mentioned phosphate buffer, according to 10mmolL-1, 30mmolL-1, 200mmolL-1Gradient elution finally elutes What is got off is destination protein.The destination protein eluted carries out desalination and takes off imidazoles, 4 DEG C of preservations.Purified enzyme activity： 130U/mL.As shown in figure 4, swimming lane 1 indicates that heparin hydrolase in fermented liquid supernatant, swimming lane 2 indicate purifying shown in SDS-PAGE The band of postheparin hydrolase.
Embodiment 6：Enzymatic property measurement
A series of solution of pH value (4.0-11.0) is used to measure the optimal reaction pH of heparin hydrolase, is sodium acetate respectively Buffer (20mM, pH 4.0-5.0), phosphate buffer (20mM, pH 5.0-7.0), Tris-HCl buffer (20mM, pH 7.0-9.0) with Gly-NaOH buffer (20mM, pH 9.0-11.0), optimal pH 4.5 is measured.And different temperatures hydrolyzes heparin The influence measurement of enzymatic activity mainly uses 20 DEG C -70 DEG C of temperature range, and measuring optimum temperature is 40 DEG C.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention Enclosing subject to the definition of the claims.
SEQUENCE LISTING
<110>Southern Yangtze University
<120>A method of building efficient secretory expression bacterium heparin hydrolase recombinant bacterium
<130> 1
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 1344
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<213>It is artificial synthesized
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atgccgcagc agccgccgcc cgcggggccg agctcgagcg cgaacgtcgc gatgacgctg 60
ccggccgacg cgccgcgcat cgctcgcgat ttcgcggggc tcagcatcga aaaggcggcg 120
ttgagctatc cgctgctgag cggcgagaac ggcaacatgg tcggcctgtt caaccggctc 180
ggcgccggcg tgttgcgcat cggcggcaat agcagcgacg cgtccggctg gcagcgcacc 240
ggtccggacg agacgtcggg cgtcatcacg cccgccgccg tggaccggct cgcgagcttc 300
gttcaggctt gccgctggcg cgtgatctac gggctcaatt tcgtcggcaa cgaccccgcg 360
acgatcgccg acgaagccgc gtatgcggcc caagcgctgg gcgtccagct cgcgggcttc 420
gagatcggca acgagcccga tctctacgcg cagcatggcc tcgcgccgaa cgcgaacacc 480
tatccgggct tcgtgagccg ctggaccaca ttcgcgaatg cgatccgggc ggcggtgccc 540
gatgcggtgt tcacgggccc ggcgaccgcg tggaactatc agcgttacac cgtgccgttc 600
gcaagcgatg cggcgggctt ggtgtcgttg ctgacgcagc accactaccg caaccccgat 660
agcgcgacga tcgaggcgat gctgagcccc gatccgagcc tcgcgccgat gctgcaagcg 720
ttgcagggtg cggcgagcgc gcgcggcatc ggctttcgtc tcgcggagac gaacagctat 780
tggggcggcg gcaagccggg cgtgagcgat gcgcacgcat ccgcgctctg ggtgatcaac 840
ttcctgttcg ccgtggcgca agggggcgct tcgggcgtga acctgcatac cggcggcgga 900
gcgtcgtatt cggcgatcaa gacgaacaag accgccggga cggtcgcggc gatcgggccg 960
gagtactacg gcatctatct gttcaaccag gccgcgggcg ggcgactgat gcaaacccgc 1020
gtcgattcgg cgggtaccac gctgttcgcg catgcggtcg cggccgacgg cggcggcgtg 1080
cgcctcatcc tcgtgaatac cgatgcgaac agcggctatg acgtcgccgt cgattgcagc 1140
agcgtgccga acgcgcgcgc cggcatcgtc acgacgctcg gcgggccgtc gctcggcagc 1200
ctgacgggca cgcagatcga cggcgcgacg tttgcgctcg acgggagcgg ggcgccgcag 1260
ggcggccggc cggtcgcttg cgtgaacggc gtgctcggcg tgcatgtcgc gtccgcgagc 1320
gcgttgctgg tcgacttcgc gtga 1344
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Met Pro Gln Gln Pro Pro Pro Ala Gly Pro Ser Ser Ser Ala Asn Val
1 5 10 15
Ala Met Thr Leu Pro Ala Asp Ala Pro Arg Ile Ala Arg Asp Phe Ala
20 25 30
Gly Leu Ser Ile Glu Lys Ala Ala Leu Ser Tyr Pro Leu Leu Ser Gly
35 40 45
Glu Asn Gly Asn Met Val Gly Leu Phe Asn Arg Leu Gly Ala Gly Val
50 55 60
Leu Arg Ile Gly Gly Asn Ser Ser Asp Ala Ser Gly Trp Gln Arg Thr
65 70 75 80
Gly Pro Asp Glu Thr Ser Gly Val Ile Thr Pro Ala Ala Val Asp Arg
85 90 95
Leu Ala Ser Phe Val Gln Ala Cys Arg Trp Arg Val Ile Tyr Gly Leu
100 105 110
Asn Phe Val Gly Asn Asp Pro Ala Thr Ile Ala Asp Glu Ala Ala Tyr
115 120 125
Ala Ala Gln Ala Leu Gly Val Gln Leu Ala Gly Phe Glu Ile Gly Asn
130 135 140
Glu Pro Asp Leu Tyr Ala Gln His Gly Leu Ala Pro Asn Ala Asn Thr
145 150 155 160
Tyr Pro Gly Phe Val Ser Arg Trp Thr Thr Phe Ala Asn Ala Ile Arg
165 170 175
Ala Ala Val Pro Asp Ala Val Phe Thr Gly Pro Ala Thr Ala Trp Asn
180 185 190
Tyr Gln Arg Tyr Thr Val Pro Phe Ala Ser Asp Ala Ala Gly Leu Val
195 200 205
Ser Leu Leu Thr Gln His His Tyr Arg Asn Pro Asp Ser Ala Thr Ile
210 215 220
Glu Ala Met Leu Ser Pro Asp Pro Ser Leu Ala Pro Met Leu Gln Ala
225 230 235 240
Leu Gln Gly Ala Ala Ser Ala Arg Gly Ile Gly Phe Arg Leu Ala Glu
245 250 255
Thr Asn Ser Tyr Trp Gly Gly Gly Lys Pro Gly Val Ser Asp Ala His
260 265 270
Ala Ser Ala Leu Trp Val Ile Asn Phe Leu Phe Ala Val Ala Gln Gly
275 280 285
Gly Ala Ser Gly Val Asn Leu His Thr Gly Gly Gly Ala Ser Tyr Ser
290 295 300
Ala Ile Lys Thr Asn Lys Thr Ala Gly Thr Val Ala Ala Ile Gly Pro
305 310 315 320
Glu Tyr Tyr Gly Ile Tyr Leu Phe Asn Gln Ala Ala Gly Gly Arg Leu
325 330 335
Met Gln Thr Arg Val Asp Ser Ala Gly Thr Thr Leu Phe Ala His Ala
340 345 350
Val Ala Ala Asp Gly Gly Gly Val Arg Leu Ile Leu Val Asn Thr Asp
355 360 365
Ala Asn Ser Gly Tyr Asp Val Ala Val Asp Cys Ser Ser Val Pro Asn
370 375 380
Ala Arg Ala Gly Ile Val Thr Thr Leu Gly Gly Pro Ser Leu Gly Ser
385 390 395 400
Leu Thr Gly Thr Gln Ile Asp Gly Ala Thr Phe Ala Leu Asp Gly Ser
405 410 415
Gly Ala Pro Gln Gly Gly Arg Pro Val Ala Cys Val Asn Gly Val Leu
420 425 430
Gly Val His Val Ala Ser Ala Ser Ala Leu Leu Val Asp Phe Ala
435 440 445
<210> 3
<211> 24
<212> DNA
<213>It is artificial synthesized
<400> 3
gaattcatgc cgcagcagcc gccg 24
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<212> DNA
<213>It is artificial synthesized
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gcggccgctt acaattgaat agatt 25

Claims (8)

1. a kind of expression bacterium heparin hydrolase recombinant bacterium, feature are being that the recombinant bacterium is with Pichia pastoris Pichia Pastoris GS115 is host, and expression derives from the heparin hydrolase of Burkholderia pseudomallei.
2. recombinant bacterium according to claim 1, feature is being, the nucleotide sequence such as SEQ of the heparin hydrolase ID NO:Shown in 1.
3. recombinant bacterium according to claim 1, feature is being, the amino acid sequence of the heparin hydrolase gene is such as SEQ ID NO:Shown in 2.
4. recombinant bacterium according to claim 1, feature is being, the construction method of the recombinant bacterium is：By nucleosides Acid sequence is that the heparin hydrolase gene of SEQ ID NO.1 is connected on Expression vector pPIC9K, is then transformed into Pichia pastoris In Pichia pastoris GS115, correct transformant is screened to get recombinant yeast pichia pastoris Pichia pastoris GS115/pPIC9K-BpHEP。
5. recombinant bacterium according to claim 4, feature is being, the construction method of the recombinant bacterium is in heparin hydrolase Histidine tag is added on gene.
6. a kind of method of fermenting and producing heparin hydrolase, feature are being, the method is any using claim 1-5 The recombinant bacterium carries out fermenting and producing.
7. according to the method described in claim 6, it is characterized in that, the method is in the fermenter using methanol induction strategy Fermenting and producing heparin hydrolase.
8. according to the method described in claim 6, it is characterized in that, fermentation process the specific steps are：
The YPD seed liquor of shaking flask culture is inoculated in 3L automatic fermenter (LiFlus GM with 10% inoculum concentration BioTRON, Korea) in, temperature is controlled at 25-30 DEG C, and adjusting pH using 25% ammonium hydroxide is 5-6, and initial speed of agitator is 500- 1000r·min-1, ventilatory capacity 2-4Lmin-1, dissolved oxygen maintains 30%-35% or more.Cultivate about 20-40h, OD600About 70-80 waits for glycerol depletion, exponential fed-batch 30-50% (WV-1) glycerite (and contain 12mLL-1PTM1), speed of agitator with it is molten Oxygen is associated, controls revolving speed in 500-1000rmin-1Feed supplement 12h stops feed supplement and waits for that glycerol exhausts again, OD600About 250 is (dry Weigh about 60.0gL-1), revolving speed is in 600-1000rmin-1Cocurrent adds 100% methanol (containing 12mLL-1PTM1), methanol concentration Control is in 18.0gL-1, 30 DEG C, 25 DEG C, 22 DEG C and the temperature-induced strategy of stage control is respectively adopted in inducing temperature.Of the invention 4th purpose is to provide a kind of method of heparin hydrolysis enzyme purification, and specific step is as follows：It is centrifuged fermentation liquid, is collected Supernatant, loading；Using Ni column, 10 column volume of pillar is balanced with phosphate buffer；It is eluted using imidazole gradient, imidazoles is used Phosphate buffer preparation is stated, according to 10mmolL-1, 30mmolL-1, 200mmolL-1Gradient elution.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1948461A (en) * 2006-03-15 2007-04-18 中国人民解放军军事医学科学院野战输血研究所 Expression method of human heparinase and its special engineering bacteria
CN102477417A (en) * 2010-11-24 2012-05-30 中国农业大学 Beta-glucosaccharase and encoding gene and application thereof
CN104212779A (en) * 2014-08-29 2014-12-17 宁波美成生物科技有限公司 Novel ribonuclease A and purification production process thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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Application publication date: 20181120