CN109251867B - High-yield strain of acid protease and application thereof - Google Patents

Abstract

The invention relates to a Trichoderma reesei mutant strain with a preservation number of CCTCC NO of M2018390. The mutant strain is obtained by multiple rounds of ultraviolet mutagenesis screening, the yield of the acid protease can be greatly improved, after fermentation is carried out for 160 hours in a 20L tank, the enzyme activity of the acid protease in the fermentation supernatant of the mutant strain is as high as 51352u/ml, the enzyme activity is improved by 36% compared with that of the original strain, and unexpected technical effects are achieved. The Trichoderma reesei strain can be widely applied to the production of the acid protease, thereby being beneficial to reducing the production cost of the acid protease and promoting the popularization and application of the acid protease in the fields of alcohol processing, feed and the like.

Description

High-yield strain of acid protease and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to an acid protease high-yield strain and application thereof.

Background

The acidic protease is aspartic protease with the optimal action pH value of 2.5-5.0, the relative molecular mass is 30000-40000, the isoelectric point is 3.0-5.0, and the acidic protease can be widely applied to the fields of food, feed processing, medicines and the like. The acidic protease plays an important role in brewing white spirit. When the corn is used as the raw material to produce the white spirit, a certain amount of acid protease is added in the fermentation process to degrade the protein in the raw material and destroy the cell wall structure of the raw material, which is beneficial to the utilization of saccharifying enzyme; the sugar content in the raw materials is increased, which is beneficial to improving the wine yield. On the other hand, the organic nitrogen content in the raw materials is increased under the action of the acid protease, which is beneficial to the growth and the propagation of yeast, thereby being beneficial to the wine production capacity of the yeast, shortening the fermentation time and reducing the cost. In addition, the addition of the acid protease can also enhance the fragrance of the white spirit, the acid protease hydrolyzes protein in the raw materials into amino acid under the acidic environment of wine brewing, and the amino acid is further converted into substances such as alcohol, ester, phenol and the like under the action of enzyme, so that the white spirit has special white spirit fragrance.

In addition, the livestock and aquatic products are bred in large scale in industry, especially young breeding animals, because the digestive organs of the animals secrete insufficient enzyme systems and enzyme amounts (endogenous enzymes), the digestive ability of the animals to feed protein is poor, and diseases such as dyspepsia, diarrhea, emaciation and the like are easily caused. Researches show that the acidic protease added into the feed is favorable for improving the digestibility of the protein and degrading the protein into amino acid and polypeptide which are favorable for the digestion and absorption of young animals, thereby being favorable for the growth and development of the animals and shortening the fattening time.

At present, most of the acid proteases are derived from microorganisms, animals and plants. The acidic protease-producing microorganisms include Aspergillus niger, Aspergillus oryzae, Aspergillus saitoi, Aspergillus awamori, Aspergillus usamii, Mucor miehei, Penicillium, Rhizopus, and variants and mutants thereof. In addition, acid protease is secreted by Rhizopus zhonghua and terrestrial yeasts such as Saccharomyces cerevisiae, Candida albicans and Saccharomycopsis fibuligera. One of the remarkable features of microbial acid proteases is the diversity and complexity compared to animal and plant proteases, and usually one strain can secrete one or more acid proteases.

The research on the acid protease is relatively late in China. In 1970, the Shanghai Industrial microbiology research institute firstly screened out an acid protease-producing strain from Aspergillus niger, and cooperated with Shanghai alcohol plants to perform pilot-scale production, so that the blank of acidic protease preparations in China is filled. In recent years, domestic researches on acid protease are mainly dedicated to breeding strains with high enzyme production activity and good stress resistance, and the commercialized acid protease producing strains mainly comprise a few strains such as aspergillus niger, aspergillus usamii and aspergillus oryzae. The enzyme yield of the existing acid protease production strain is not high, so that the production cost of the enzyme is high, and the wide application of the acid protease is limited to a certain extent.

Disclosure of Invention

The invention provides a Trichoderma reesei (Trichoderma reesei) strain with high yield of acid protease for solving the problems of the prior art. The applicant firstly constructs and obtains a trichoderma reesei engineering strain for recombinant expression of the acid protease, and then screens and obtains a mutant strain with remarkably improved yield of the acid protease by an ultraviolet mutagenesis method, so that the mutant strain can be widely applied to production of the acid protease.

The invention provides a Trichoderma reesei engineering strain, wherein the strain carries a recombinant plasmid for expressing an acid protease gene.

The amino acid sequence of the acid protease is SEQ ID NO. 1, and the coding nucleotide sequence is SEQ ID NO. 2.

The invention provides a mutant strain Trichoderma reesei 4QA5(Trichoderma reesei 4QA5), which is preserved in the China center for type culture Collection of Wuhan university in Wuhan, China in 6 and 21 months in 2018, and the preservation number is CCTCC NO: M2018390.

The invention also provides application of the trichoderma reesei mutant strain in production of acid protease.

The invention expresses acid protease gene from Aspergillus niger in Trichoderma reesei host cell, constructs recombinant strain Trichoderma reesei 4Q-ASP, and after fermenting in 20L tank for 160h, the enzyme activity of acid protease in the fermented supernatant reaches 37691 u/ml. The applicant takes Trichoderma reesei 4Q-ASP as an original strain, and obtains a mutant strain Trichoderma reesei 4QA5 through multiple rounds of ultraviolet mutagenesis and final screening, the expression quantity of acid protease can be greatly improved, after fermentation is carried out for 160h in a 20L tank, the enzyme activity of the acid protease in fermentation supernatant reaches 51352u/ml, which is improved by 36% compared with the original strain, and unexpected technical effects are obtained. The Trichoderma reesei strain can be widely applied to the production of the acid protease, thereby being beneficial to reducing the production cost of the acid protease and promoting the popularization and application of the acid protease in the fields of alcohol processing, feed and the like.

Drawings

FIG. 1 is a map of plasmid pTG;

FIG. 2 is a 20L tank fermentation process curve;

FIG. 3 is an SDS-PAGE protein electrophoresis: wherein: m is a protein molecular weight Marker, and lanes 1 and 2 are respectively mutant strain trichoderma reesei 4QA5 and starting strain trichoderma reesei 4Q-ASP fermentation supernatant; the protein band at 42kDa as indicated by the arrow is the acid protease.

Detailed Description

The present invention uses conventional techniques and methods used IN the fields of genetic engineering and MOLECULAR BIOLOGY, such as the methods described IN MOLECULAR CLONING, A LABORATORY MANUAL,3nd Ed. (Sambrook,2001) and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, 2003). These general references provide definitions and methods known to those skilled in the art. However, those skilled in the art can adopt other conventional methods, experimental schemes and reagents in the field on the basis of the technical scheme described in the invention, and the invention is not limited to the specific embodiment of the invention.

The present invention will be described in detail with reference to specific embodiments.

EXAMPLE 1 cloning of acid protease Gene

An Aspergillus niger (Aspergillus niger) genome is taken as a template, and an acid protease gene fragment is amplified by using a primer 1 and a primer 2, wherein the nucleotide sequence of the acid protease gene fragment is SEQ ID NO:2, the encoded amino acid sequence is SEQ ID NO: 1.

the PCR primers and reaction conditions were as follows:

primer 1 (F): ATGGTCGTCTTCAGCAAAACC

Primer 2 (R): CTAAGCTTGAGCAGCGAAGCC

The reaction conditions are as follows: denaturation at 94 deg.C for 5 min; then denaturation at 94 ℃ for 30s, renaturation at 56 ℃ for 30s, extension at 72 ℃ for 80s, and after 30 cycles, heat preservation at 72 ℃ for 10 min. The agarose electrophoresis result shows that the size of the acid protease gene obtained by amplification is 1339 bp.

EXAMPLE 2 construction of recombinant vectors

The acid protease gene is amplified by PCR, and XbaI sites are introduced at two ends of the primer. The primer sequences are as follows:

primer 3 (F): GCTCTAGA ATGGTCGTCTTCAGCAAAACC

Primer 4 (R): GCTCTAGA CTAAGCTTGAGCAGCGAAGCC

The PCR reaction conditions are as follows: denaturation at 94 deg.C for 5 min; then denaturation at 94 ℃ for 30s, renaturation at 56 ℃ for 30s, extension at 72 ℃ for 80s, and after 30 cycles, heat preservation at 72 ℃ for 10 min. The result of agarose gel electrophoresis showed that the acid protease gene was a 1339bp fragment.

And (3) performing single restriction enzyme digestion on the obtained acid protease gene fragment and an expression vector pTG by using a restriction enzyme XbaI under the following digestion conditions:

carrying out enzyme digestion treatment for 2h in water bath at 37 ℃, respectively recovering two target fragments after electrophoresis, and dissolving in 20ul ddH₂And O. Ligation was performed with T4DNA ligase in the following system:

connecting for 1h at 22 ℃, transforming escherichia coli DH5a competence, coating an LB + AAP plate, culturing overnight at 37 ℃, growing a single colony, verifying the correctly connected transformant by colony PCR, extracting plasmid, sequencing, and obtaining the recombinant vector pTG-ASP containing the acid protease gene after sequencing is correct.

Example 3 construction of recombinant strains of Trichoderma reesei

1. Preparing protoplasts:

inoculating host cell Trichoderma reesei (Trichoderma reesei)4Q to PDA + U (potato 200g/L, boiling for 20-30min, filtering to remove residue, glucose 2%, Uridine 1%, agar powder 1.5%) plate, and culturing at 30 deg.C for 5-7 d; cutting 2cm × 2 cm-sized fungus block, inoculating into 100ml liquid PDA + U (potato 200g/L, boiling for 20-30min, filtering to remove residue, glucose 2%, and Uridine 1%) culture medium, and culturing at 30 deg.C for 16 hr to grow mycelium for transformation; after the grown mycelia were filtered, it was resuspended in 20ml of 1.2M magnesium sulfate solution; adding 0.2g of lysozyme, culturing at 30 ℃ and 100rpm for 2-3 h; filtering the cracked mycelium with 2 layers of mirror paper, centrifuging at 3000rpm for 10min to obtain protoplast; filtering the cracked mycelium with a piece of lens wiping paper, and centrifuging to obtain a protoplast; then, the mixture is resuspended by using a proper amount of sorbitol solution.

2. And (3) transformation:

washing the obtained Trichoderma reesei 4Q protoplast with 1.2M sorbitol solution for 2 times, and re-suspending with appropriate amount of sorbitol solution to make the protoplast concentration reach 10⁸Per ml; adding 10ul of the prepared recombinant vector pTG-ASP into 200ul of protoplast, adding 50ul of 25% PEG6000, ice-cooling for 20min, adding 2ml of 25% PEG6000, and standing at room temperature for 5 min; adding 4ml sorbitol solution, mixing, pouring 50ml conversion upper layer culture medium, pouring into 4 conversion lower layer flat plates, solidifying the upper layer culture medium, and culturing in 30 deg.C incubator for 5 d.

3. And (3) transformant screening:

after 5 days of culture, the grown colonies are picked up, spotted on a transformation lower layer plate for re-screening, and cultured for 3 days at 30 ℃. The transformants which grew normally were inoculated into fresh PDA plates, respectively, and cultured at 30 ℃ for 5-7 days. Each transformant was harvested into 2cm × 2 cm-sized clumps, inoculated into 50ml of liquid shake flask medium (1% glucose, 2% lactose, 1.5% corn steep liquor, 0.9% ammonium sulfate, 0.15% magnesium sulfate, 0.073% citric acid, 0.1125% calcium chloride, 0.1% trace elements) respectively, fermented at 28 ℃ for 5 days. After culturing for 5 days, centrifuging the thalli to obtain supernatant fluid which is crude enzyme liquid, and carrying out SDS-PAGE protein electrophoresis detection and acid protease enzyme activity detection.

And detecting the activity of the acid protease in the fermentation supernatant of the positive transformant, screening the positive transformant with the highest enzyme activity, and naming the positive transformant as Trichoderma reesei 4Q-ASP (Trichoderma reesei 4Q-ASP), wherein the enzyme activity of the acid protease in the shake flask fermentation supernatant reaches 3145 u/ml.

Example 4 mutagenesis screening

The mutation caused by ultraviolet mutagenesis has strong randomness, and the effect generated by mutation is random and difficult to predict. Therefore, in order to obtain effective positive mutations, technicians usually need to perform multiple rounds of ultraviolet mutagenesis, the screening workload is large, and the possibility that effective positive mutations cannot be obtained exists. However, ultraviolet mutagenesis requires simple equipment and low cost, and can obtain a large number of mutants in a short time, so that it is still a common mutagenesis breeding method.

The applicant takes Trichoderma reesei 4Q-ASP as an original strain, and carries out genetic modification on the original strain by an ultraviolet mutagenesis method, thereby further improving the yield of the acid protease.

1. Determination of the lethality rate:

inoculating original strain Trichoderma reesei 4Q-ASP on PDA plate, and culturing at 30 deg.C for 5-7 d. When a large amount of spores are generated on the surface of the colony, 5ml of sterile water is absorbed for elution to obtain a spore liquid, the spore liquid is resuspended by the sterile water after centrifugation, and a blood counting chamber is used for counting. A90 mm petri dish was taken and 5ml of diluted spore suspension (concentration 1X 10) was added⁷) Adding a rotor and stirring on a magnetic stirrer to make the spore liquid in a uniform state. Irradiating with ultraviolet lamp with power of 9w at a vertical distance of 20cm in a sterile ultra-clean bench for 30s, 45s, 60s, 75s, 90s, 105s and 120s, diluting the irradiated spore solution for 10, 100 and 1000 times, coating 100ul PDA plate, culturing at 30 deg.C for 2-3d, counting, and calculating lethality with unirradiated spore solution as control. Wherein the lethality is 95% when the irradiation time is 90s, and the irradiation time is selected for subsequent mutagenesis experiments.

2. First round mutagenesis screening:

a90 mm petri dish was taken and 5ml of diluted spore suspension (concentration 1X 10) was added⁷) Adding a rotor and stirring on a magnetic stirrer to make the spore liquid in a uniform state. Irradiating with ultraviolet lamp with power of 9w in sterile ultra-clean bench at vertical distance of 20cm for 90s, diluting 1000 times, coating 100ul PDA plate, and culturing at 30 deg.C for 2-3 d.

Totally coating 200 PDA plates, culturing at 30 ℃ for 2-3d, growing 30-50 colonies on each plate, and screening short-branched mutants through colony morphology. The applicant selects 90 mutant bacteria with small colony morphology, compact hyphae and short villus around the colony, and the mutant bacteria are respectively inoculated to a PDA plate and cultured for 5-7 days at 30 ℃. Each mutant strain was harvested into 2cm × 2cm blocks, inoculated into 50ml liquid shake flask culture medium (glucose 1%, lactose 2%, corn steep liquor 1.5%, ammonium sulfate 0.9%, magnesium sulfate 0.15%, citric acid 0.073%, calcium chloride 0.1125%, trace elements 0.1%) for fermentation, and cultured at 28 deg.C for 5 days. After culturing for 5 days, centrifuging the thallus to obtain supernatant, namely crude enzyme liquid, and respectively carrying out protein electrophoresis detection and acid protease enzyme activity detection.

The result shows that the enzyme activity of the acid protease in the fermentation supernatant enzyme of no mutant strain is higher than that of the starting strain in 90 mutant strains obtained by the first round of ultraviolet mutagenesis screening; wherein, the enzyme activity of 53 mutant strains is basically equivalent to that of the original strain, and the enzyme activity of the rest 37 mutant strains is even reduced by 9-23 percent compared with that of the original strain.

The applicant carries out 6 rounds of mutagenesis screening according to the method, and finally obtains 5 mutant strains with the yield of the acid protease which is improved by more than 30 percent compared with the yield of the original strain, wherein the mutant strains are named as Trichoderma reesei 4QA1, 4QA2, 4QA3, 4QA4 and 4QA5 respectively. The enzyme activity of the acid protease in the shake-flask fermentation supernatant of the trichoderma reesei 4QA5 is the highest and reaches 5214u/ml, which is improved by 65 percent compared with that of the original strain.

(1) Definition of the enzyme Activity Unit of acid proteases

The amount of enzyme that hydrolyzes casein at 40 ℃ and pH3.0 per minute to produce 1. mu.g of tyrosine is defined as one enzyme activity unit U.

(2) Enzyme activity measuring method

Casein solution (10 g/L): weighing 1.000g of casein, wetting with a small amount of lactic acid, adding a proper amount of sodium lactate buffer solution with pH of 3.0 to about 80ml, heating in a boiling water bath while stirring, boiling for 30min until the solution is completely dissolved, cooling, and transferring into a 100ml volumetric flask for constant volume.

Enzyme solution: diluting with pH3.0 sodium lactate buffer solution to proper amount, and controlling absorbance to 0.25-0.4.

Drawing an L-tyrosine standard curve: 0, 1, 2, 3, 4 and 5ml of prepared 100ug/ml L-tyrosine standard solution respectively, and water is used for fixing the volume to 10ml to be used as standard point solution. And (3) taking 1ml of each standard point solution, adding 5ml of 0.4mol/L sodium carbonate solution and 1ml of the formalin reagent use solution, uniformly oscillating, developing in a water bath at 40 ℃ for 20min, taking out, using a spectrophotometer to measure the absorbance of the solution at the wavelength of 680nm and taking a tube 0 without tyrosine as a blank. And drawing a standard curve by taking the absorbance A as a vertical coordinate and the concentration C of the tyrosine as a horizontal coordinate. From the plot, the amount of tyrosine (ug) at an absorbance of 1 was calculated, which is the absorbance constant K, which should be in the range of 95-100.

And (3) determination: placing casein solution in 40 deg.C constant temperature water bath, and preheating for 5 min; adding 1ml of diluted enzyme solution into a test tube, balancing for 2min at 40 ℃, adding 1ml of casein solution, uniformly mixing, reacting for 10min at 40 ℃, adding 2ml of trichloroacetic acid, uniformly mixing, taking out, standing for 10min, and filtering; taking 1ml of filtrate, adding 5ml of 0.4mol/L sodium carbonate solution, adding 1ml of the welan reagent use solution, developing in a water bath at 40 ℃ for 20min, taking out, using a spectrophotometer at the wavelength of 680nm, taking a tube 0 without tyrosine as a blank, and respectively measuring the absorbance.

The enzyme activity calculation formula is as follows:

in the formula:

x is the enzyme activity of the sample, and the unit is U/ml;

a is the difference of blank light absorption values of the sample;

k-absorption constant;

4-total volume of reaction reagents (ml);

n is dilution multiple;

10-reaction time, 10 min;

example 4 fermentation Scale-Up

The applicant further ferments the original strain Trichoderma reesei 4Q-ASP and 5 mutant strains (Trichoderma reesei 4QA1, 4QA2, 4QA3, 4QA4 and 4QA5) in 20L tanks respectively; and when the fermentation is finished for 160h, respectively measuring the enzyme activity of the acid protease in the fermentation supernatant. The result shows that the enzyme activity of the acid protease in the supernatant fermented by the original strain trichoderma reesei 4Q-ASP reaches 37691u/ml, the fermentation enzyme activity of the mutant strain is generally improved by 16-36% compared with the original strain, wherein the enzyme activity of the acid protease in the supernatant fermented by the mutant strain trichoderma reesei 4QA5 is the highest and reaches 51352u/ml, and is improved by 36% compared with the original strain.

The fermentation process curves of the developed trichoderma reesei 4Q-ASP and the mutant trichoderma reesei 4QA5 are shown in figure 2, and after fermentation is carried out for 40 hours, the fermentation enzyme activity of the mutant trichoderma reesei 4QA5 is obviously higher than that of the developed trichoderma reesei; when the fermentation is finished for 160h, the enzyme activity of the acid protease in the supernatant obtained by fermenting the original strain trichoderma reesei 4Q-ASP reaches 37691u/ml, and the enzyme activity of the acid protease in the supernatant obtained by fermenting the mutant strain trichoderma reesei 4QA5 reaches 51352 u/ml.

Meanwhile, the applicant carries out SDS-PAGE electrophoresis detection on the fermentation supernatant of the original strain Trichoderma reesei 4Q-ASP and the mutant strain Trichoderma reesei 4QA 5. As a result, as shown in FIG. 3, the protein band at 42kDa indicated by the arrow is the acidic protease, and the content of the acidic protease in the supernatant obtained by fermenting Trichoderma reesei 4QA5 as the mutant strain in Lane 1 is significantly higher than that in the supernatant obtained by fermenting Trichoderma reesei 4Q-ASP as the starting strain in Lane 2, thereby achieving unexpected technical effects.

The applicant has deposited the mutant strain Trichoderma reesei 4QA5(Trichoderma reesei 4QA5) in 21.6.2018 in China center for type culture Collection in Wuhan university, Wuhan, China with the preservation number of CCTCC NO: M2018390.

Sequence listing

<110> Islands blue biological group Co Ltd

<120> high-yield strain of acid protease and application thereof

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<170> SIPOSequenceListing 1.0

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<211> 394

<212> PRT

<213> Aspergillus niger (Aspergillus niger)

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

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Gln Ile Ala Arg Pro Ala Asn Lys Thr Arg Thr Ile Asn Leu Pro Gly

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Met Tyr Ala Arg Ser Leu Ala Lys Phe Gly Gly Ala Val Pro Gln Ser

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Val Lys Glu Ala Ala Ser Lys Gly Ser Ala Val Thr Thr Pro Gln Asn

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Asn Asp Glu Glu Tyr Leu Thr Pro Val Thr Val Gly Lys Ser Thr Leu

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His Leu Asp Phe Asp Thr Gly Ser Ala Asp Leu Trp Val Phe Ser Asp

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Glu Leu Pro Ser Ser Glu Arg Thr Gly His Asp Val Tyr Thr Pro Ser

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Ser Ser Ala Thr Lys Leu Ser Gly Tyr Ser Trp Asp Ile Ser Tyr Gly

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Asp Gly Ser Ser Ala Ser Gly Asp Val Tyr Arg Asp Thr Val Thr Val

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

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

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Ala Phe Ser Ser Ile Asn Thr Val Gln Pro Lys Ala Gln Thr Thr Phe

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

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<210> 2

<211> 1185

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<213> Aspergillus niger (Aspergillus niger)

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ttcaactctg agggtcctaa gctgggcttc gctgctcaag cttag 1185

Claims (2)

1. The Trichoderma reesei (Trichoderma reesei) mutant strain is 4QA5, and the preservation number is CCTCC NO: M2018390.

2. Use of the mutant strain of trichoderma reesei of claim 1 for the production of an acid protease.

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