CN103695323B - Stable and high-yield strain for alpha-transglucosidase - Google Patents
Stable and high-yield strain for alpha-transglucosidase Download PDFInfo
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Abstract
The invention provides a mutant strain aspergillus niger Anm2. The number of the mutant strain asperillus niger Anm2 is CGMCC No.8541. According to the mutant strain, alpha-transglucosidase can be efficiently recombined and expressed; the fermenting enzyme activity reaches 23,709 U/mL, and is improved by 54 percent in comparison with that of an original strain; the optimum temperature of the alpha-transglucosidase is 60 DEG C, and the optimum pH value of the alpha-transglucosidase is 5.0; the alpha-transglucosidase can be widely applied to the production of isomalto oligosacchadide syrup.
Description
Technical field
The invention belongs to microorganism mutagenesis screening technical field, be specifically related to a kind of aspergillus niger (Asperillus niger) bacterial strain and application thereof of stable, efficient heterogenous expression α-transfering grape glycosidase.
Background technology
α-transfering grape glycosidase (α-transglucosidase E.C.2.4.1.24) belongs to key specificity enzyme, it can cut α-1,4 glycosidic links of non-reducing end in saccharide substrates molecule in specific manner, discharge glucose, the glucosyl residue maybe dissociating is transferred on another saccharide substrates with α-1,6 glycosidic links, thus obtain the oligomeric isomaltose (be called for short IMO, mainly comprise the oligose of more than isomaltose, panose, Isomaltotriose and tetrose) of non-fermented, glycolipid or glycopeptide etc.In the production of oligomeric isomaltose, utilizing the hydrolytic action of α-transfering grape glycosidase and its to turn glycosides effect is the key of producing oligomeric isomaltose.Therefore the attention of α-transfering grape glycosidase foodstuffs industry circle extremely both at home and abroad.
α-transfering grape glycosidase is widely distributed at occurring in nature, of a great variety, distinct, is almost present in all organisms, in the sugar metabolism of the glycogen degradation of the mankind and animal, plant and microorganism, have important physiological function.α transfering grape glycosidase is mainly used in produces IMO, and IMO is the multiplicaiton factor of human intestine's profitable strain bifidus bacillus, not digested after absorption, also not easily utilize by the most putrefactive bacteriums in large intestine, but can be utilized as the carbon source of bifidus bacillus, have promote that intestinal beneficial flora rise in value, relax bowel, adjusting blood lipid, low sugariness, the unique effects such as low in calories, especially in the increment of promotion intestinal beneficial flora, effect is remarkable.As a kind of functional food ingredient, IMO has been widely used in the manufacture of various food as milk-product, candy class, bakery product etc., occupies first of various functional oligose.
Industrial at present had with starch or maltose for raw material, and by the technique of Production by Enzymes oligomeric isomaltose, but existing production technique transformation efficiency is not high.In addition, although China's isomaltose output is very high, the consumption of α transfering grape glycosidase is very large, and never realize suitability for industrialized production, this enzyme still dependence on import, these factors all greatly constrain the production of domestic oligomeric isomaltose.Therefore, this area is needed the α transfering grape glycosidase obtaining highly active α transfering grape glycosidase and corresponding high yield thereof badly and is produced bacterial strain, to adapt to the needs of oligomeric isomaltose production technique.
Summary of the invention
The object of this invention is to provide a kind of bacterial strain and application thereof of stable, high yield α-transfering grape glycosidase.The aspergillus niger engineering bacteria strain building the recombinant expressed α-transfering grape glycosidase obtained is carried out ultraviolet mutagenesis and screen mutation by the present invention, thus obtain a strain good stability, Aspergillus Niger Mutant that α-transfering grape glycosidase output is high, for the suitability for industrialized production of α-transfering grape glycosidase is laid a good foundation, thus make up the deficiencies in the prior art.
One aspect of the present invention provides a plant mutant bacterial strain, aspergillus niger Anm2(Aspergillus niger), be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) of No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on December 9th, 2013, bacterial strain deposit number is CGMCC No.8541.
Described aspergillus niger Anm2 is producing the application in α-transfering grape glycosidase.
Present invention also offers the application of above-mentioned α-transfering grape glycosidase in food processing field.
High efficiency recombinant expressed α-the transfering grape glycosidase of mutant strain aspergillus niger Anm2 energy provided by the invention, its fermenting enzyme work is up to 23709U/mL, and than setting out, bacterium improves 54%.The optimum temperature of described α-transfering grape glycosidase is 60 DEG C, and Optimun pH is 5.0.α-transfering grape glycosidase of producing can be widely used in the production of isomaltooligosaccharide syrup.
Accompanying drawing explanation
The genetic map of Fig. 1: pGm-An recombinant plasmid;
Fig. 2: mutant strain aspergillus niger Anm2 fermented supernatant fluid SDS-PAGE electrophoresis detection figure, wherein swimming lane M is depicted as protein standard molecular weight marker, is from top to bottom 116.0kDa, 66.2kDa, 45.0kDa, 35.0kDa, 25.0kDa and 18.4kDa; Swimming lane 1 is depicted as protein expression situation in starting strain aspergillus niger An1 fermented supernatant fluid; Swimming lane 2 is depicted as protein expression situation in mutant strain aspergillus niger Anm2 fermented supernatant fluid, and the protein band at arrow indication 109kDa place is recombinant expressed α-transfering grape glycosidase;
Action pH-enzyme the graphic representation alive relatively of Fig. 3: α-transfering grape glycosidase;
Operative temperature-enzyme the graphic representation alive relatively of Fig. 4: α-transfering grape glycosidase.
Embodiment
The present invention has used the routine techniques and method that use in genetic engineering and biology field.Such as MOLECULAR CLONING:A LABORATORY MANUAL, 3nd Ed. (Sambrook, 2001) and technology described in the book of reference such as CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, 2003).But this does not also mean that and limits the invention to described any concrete grammar, experimental program and reagent, and those of ordinary skill in the art can select published technology to implement the scheme recorded in the embodiment of the present invention.
In the present invention, 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.
The oligomeric isomaltose recorded in specification sheets of the present invention and dextrinosan are used interchangeably, and all refer to one or more sugar being selected from lower group: isomaltose, Isomaltotriose, Isomaltotetraose or panose.In addition, should be understood that described term also comprises the mixture of above-mentioned sugar.
α transfering grape glycosidase described in the present invention, be also called alpha-D-glucose glycosides lytic enzyme, α-1 in maltose and malto-oligosaccharide molecular structure can be cut, 4 glycosidic links, and the α-1 that a free glucosyl residue out can be transferred in another glucose molecule or maltose or trisaccharide maltose equimolecular, on 6, its transglycosylation can by the α-1 in oligose, 4 glycosidic links change into α-1,6 glycosidic links or other forms of link, thus the oligomeric isomaltose or sugar ester, the glycopeptide etc. that obtain non-fermented.
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).
Nucleic acid comprises DNA, RNA, strand or double-strand, and their chemical modification object; Nucleic acid and polynucleotide can exchange use in this manual.
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 coding for alpha transfering grape glycosidase 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.
Filamentous fungus refers to that the Eumycotina biology of all filamentous form is (see INTRODUCTORYMYCOLOGY, 4th Ed. (Alexopoulos, 2007) and AINSWORTH AND BISBYDICTIONARY OF THE FUNGI, 10th Ed. (Kirk et al., 2008)).The feature of these fungies is the vegetative myceliums with the cell walls be made up of chitin, Mierocrystalline cellulose and other complicated polysaccharide.Filamentous fungus of the present invention is different from yeast on morphology, physiology and genetics.Nourishing and growing of filamentous fungus has been come by the extension of mycelia, and carbon metablism is obligate aerobic.In the present invention, filamentous fungal parent cell can make, but is not limited to, Aspergillus certain (Aspergillus sp.) (such as excellent aspergillus (A.clavatus), Aspergillus fumigatus (A.fumigatus), Aspergillus awamori (A.awamori), flavus (A.flavus), terreus (A.terreus) and aspergillus oryzae (A.oryzae)), Penicillium certain (Penicillium sp.) (such as Penicllium chrysogenum (P.chrysogenum)), Xin Satuo Pseudomonas certain (Neosartorya sp.) (such as Fei Xixinsatuo bacterium (N.fischeri)), gliocladium germ belongs to certain (Gliocladium sp.) (such as Gliocladium roseum (G.roseum)), Trichoderma certain (Trichoderma sp.) (such as Trichodermareesei (T.reesei), viride (T.viride), healthy and free from worry wood mould (T.koningii), trichoderma harziarum (T.harzianum)), Humicola certain (Humicola sp.) (such as Humicola insolens (H.insolens) and grey humicola lanuginosa (H.grisea)), the gold mould genus of spore certain (Chrysosporium sp.), Fusarium certain (Fusarium sp.), Neurospora sp belongs to certain (Neurospora sp.), the cell of Hypocrea certain (Hypocrea sp.) and Emericella certain (Emericella sp.).
Aspergillus involved in the present invention or Aspergillus certain referred to before or be classified as any fungi of Aspergillus at present.
Analytical procedure and the enzyme activity determination method of expression of enzymes described in the present invention are as follows:
In order to evaluate the expression of α transfering grape glycosidase, can analyze at protein level or nucleic acid level.The in situ hybridization that adaptable analytical procedure comprises Northern trace, Dot blot (DNA or RNA analysis), Southern trace, radioautograph, RT-PCR (ThermoScript II polymerase chain reaction) and carries out containing the probe (based on nucleic acid coding sequence) suitably marked.In addition, genetic expression can pass through immunological method, the such as immunohistochemical staining of cell, tissue slice or the immunity test of tissue culture medium (TCM).Such as assessed by western blot or ELISA.Such immunity test may be used for qualitatively and evaluates the expression of α transfering grape glycosidase (such as Anm2) quantitatively.The details of these class methods is well known by persons skilled in the art, and can business obtain for the many reagent implementing these class methods.In some embodiments, the expression of α transfering grape glycosidase (such as Anm2) is analyzed by SDS-PAGE.
Specific embodiment as herein described, should be understood that these embodiments are only not used in for illustration of the present invention and limits the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition is as people such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989) condition described in, or according to the condition that manufacturer advises.In the present invention, per-cent is weight percentage, unless stated otherwise.
The structure of embodiment 1 starting strain aspergillus niger An1
According to the specification sheets of manufacturers, use fungal genomic DNA to extract test kit (Omega) and extract genomic dna from aspergillus niger (Asperillus niger) overnight culture.According to α-transfering grape glycosidase gene order design PCR primer NCBI being numbered XM_001402016.Be 5 '-AATCTAGAATCATGGTGAAGTTGACGCA for cloning the forward primer An1-F sequence of the An1 gene in aspergillus niger, reverse primer An1-R sequence is 5 '-GCTCTAGAGTGAAACAAAGGTGGAAGT, increases out from aspergillus niger genomic dna by this gene Phusion archaeal dna polymerase (Thermo scientific).
Use gel purification kit (Fermentas) by above-mentioned PCR primer purifying.Carry out enzyme with restriction enzyme XbaI (Fermentas) to sublimed PCR primer to cut; Meanwhile, carry out enzyme with restriction enzyme XbaI to plasmid pGm to cut.Use gel purification kit by digestion products purifying, and with T4DNA ligase enzyme (Fermentas), above-mentioned two digestion products are connected.Connection product conversion is entered Trans5 α intestinal bacteria (Transgen), select with penbritin.For guaranteeing accurately, to check order (Invitrogen) to some clones.Sequencing result shows, and the nucleotides sequence of the α that pcr amplification of the present invention obtains-transfering grape glycosidase gene fragment is classified as SEQ ID NO:2, and encoding amino acid sequence is SEQ ID NO:1.
Use amount in plasmid to prepare test kit (Axygen) plasmid purification from the correct escherichia coli cloning of sequencing result, gained plasmid called after pGm-An(plasmid map is shown in Fig. 1).
The plasmid pGm-An obtained by above-mentioned purifying is proceeded in aspergillus niger Host Strains G1 by protoplast transformation, obtain 23 aspergillus niger transformants (respectively called after An1, An2, An3 ..., An23).The spore suspension of described 23 transformants is inoculated in respectively in 20mL TSB fermention medium, at 30 DEG C, under the condition of 200rpm, cultivates 5d; By gained fermented liquid 8 layers of filtered through gauze; Filtrate is centrifugal 10min under 14000g condition, collects supernatant liquor; By supernatant liquor in concentration be 12% SDS-PAGE glue on carry out electrophoresis, result shows the present invention and builds transformant An1, An3, An5, An6, An9, An10, An13, An14, An15, An18, An19, An20, An21, An23 of obtaining and have obvious protein band at 109kD place, consistent with α of the present invention-transfering grape glycosidase theoretical molecular, thus above-mentioned positive transformant all recombinant expressed α-transfering grape glycosidases of energy are described; Select positive transformant An1, An6, An13, An14, An18, An21 that wherein α-transfering grape glycosidase expressing quantity is higher, the enzyme measuring its fermented supernatant fluid is respectively lived, result shows, and the fermented supernatant fluid enzyme of aspergillus niger An1 is lived the highest, reaches 15210U/mL.
α-transfering grape glycosidase enzyme activity determination method
Adopt People's Republic of China (PRC) light industry standard QB2525-2001, α transfering grape glycosidase is acted on substrate Alpha-Methyl-D-Glucose glycosides and generate glucose, the glucose generated carries out color reaction with the 4-AA (4-Aminoantipyrin) containing glucose oxidase, peroxidase and phenol reagent and carrys out quantitative assay.
Concrete measuring method comprises: draw 2% Alpha-Methyl-D-Glucose glycosides substrate solution 1ml and 0.02mol/L acetic acid-sodium acetate buffer solution (pH5.0) 1ml and add in test tube (15mm × 150mm), in the constant water bath box of (60 ± 0.5) DEG C, be incubated 10min.Add sample enzyme liquid 0.5ml, mixing, in the constant water bath box of (60 ± 0.5) DEG C accurately after insulation 60min, test tube is transferred in boiling water bath and heats 5min, then cool fast with flowing water.After cooling, inhale this solution 0.1ml in test tube, and add 4-AA-phenol developer 3ml, mixing.The water bath with thermostatic control of this test tube being put into (40 ± 0.5) DEG C is incubated 20min, measure the absorbancy at 500nm place, the enzyme activity (U/ml) of α transfering grape glycosidase is calculated according to absorbance, namely under this test conditions, in reaction mixture 2.5ml, the 60min enzyme amount produced needed for 1 μ g glucose is defined as a α transfering grape glycosidase unit of activity.
TSB fermention medium: 12g NaNO
3, 0.5g KCl, 1.5g KH
2pO
4, 2.05g MgSO
47H
2o, 3.5g NaH
2pO
4h
2o, 45g Tryptose soy meat soup, 70g Trisodium Citrate, 1g tween 80,1mL trace elements (see below), adds dlH
2o, to final volume 700mL, adds 40% maltose that the 300mL filtering with microporous membrane of 0.22 μm is degerming after autoclaving.
Trace elements: at 250mL dlH
21g FeSO is added in O
47H
2o, 8.8g ZnSO
4.
7h
2o, 0.4g CuSO
45H
2o, 0.15g MnSO
44H
2o, 0.1g Na
2b
4o
710H
2o, 50mg (NH
4)
6mo
7o
244H
2the dense HCl of O, 0.2mL, uses dlH after dissolving completely
2o is settled to 1L, degerming with the filtering with microporous membrane of 0.22 μm.
Embodiment 2: ultraviolet mutagenesis and screening
2.1 ultraviolet mutagenesis methods
(1) embodiment 1 is built the aspergillus niger An1 that obtains as starting strain, be inoculated on CMA inclined-plane and activate, at 37 DEG C, cultivate 4d;
(2) wash the fresh inclined-plane of An1 with the 0.1%Tween-80 that 3ml is aseptic and obtain spore suspension, absorption 10ul is placed on blood counting chamber and counts, and is about 106/about ml according to count results dilution spore suspension to spore number;
(3) spore suspension drawn after 5ml dilution is placed in 9cm culture dish, under the condition of ultraviolet lamp 30w, irradiation distance 22cm, irradiation time 4min, carry out ultraviolet mutagenesis;
(4) after uv irradiating, the spore suspension after mutagenesis is diluted 100 times, draw the spore suspension coating CMA after dilution dull and stereotyped, each CMA flat board is coated with 100ul, and be about coated with 100 dull and stereotyped left and right, at 37 DEG C, lucifuge cultivates 40h;
(5), after cultivating 40h, the individual morphology that picking flat board grows is little, mycelia is dense and the sudden change bacterium colony of consolidation, and on another CMA flat board, carry out line purifying, streak plate cultivates 40h at 37 DEG C;
(5) choose single bacterium colony that about 100 streak plates grow, be seeded to CMA inclined-plane, cultivate more than 4 days at 37 DEG C;
CMA is dull and stereotyped: 20g glucose, 20g Fructus Hordei Germinatus extract, 1g peptone, 15g agar, adds dlH
2o to final volume 1000mL, autoclaving.
The screening of 2.2 mutant strains
1, primary dcreening operation
According to the growing state of mutagenic strain on CMA inclined-plane, select the close mutagenic strain of growing way and carry out fermentation expression in batches, compare with starting strain An1 simultaneously.Fermentation employing one step shake flask culture: be inoculated in respectively by the spore suspension of aspergillus niger mutagenic strain in 20mL TSB fermention medium, each bacterial strain connects 1 bottle, at 30 DEG C, cultivates 5d under the condition of 200rpm; By gained fermented liquid 8 layers of filtered through gauze, filtrate is centrifugal 10min under 14000 × g condition, collects supernatant liquor; By supernatant liquor in concentration be 12% SDS-PAGE glue on carry out electrophoresis, by the expression amount of observation and comparison α transfering grape glycosidase recombinant protein band, pick out the mutant strain that 13 strains are better than starting strain An1 expression effect.
2, multiple sieve
The 13 plant mutant bacterial strains picked out are carried out fermentation expression again, compares with starting strain An1 simultaneously.Fermentation employing two step shake flask culture: the spore suspension of aspergillus niger mutagenic strain is inoculated in 20ml CLS fermention medium, at 30 DEG C, cultivates 48h under the condition of 200rpm; Then by 10% inoculum size, draw 2mlCSL nutrient solution thalline and be inoculated in 20mL TSB fermention medium, at 30 DEG C, under the condition of 200rpm, cultivate 5d; By gained fermented liquid 8 layers of filtered through gauze, filtrate is centrifugal 10min under 14000 × g condition, collects supernatant liquor; Use Econo-Pac10DG Columns(BOI-RAD) desalination is carried out except sugar process to supernatant liquor; Then carried out 12%SDS-PAGE detection analyze and measure enzyme work; Finally pick out the mutant strain that a strain α transfering grape glycosidase expression amount is the highest, called after aspergillus niger Anm2(Aspergillus nigerAnm2), be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) of No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on December 9th, 2013, bacterial strain deposit number is CGMCC No.8541.
CSL fermention medium: 100g corn steep liquor, 1g NaH
2pO
4h
2o, 0.5g MgSO
4, 100g maltose, 10g glucose, 50g fructose, adds dlH
2o to 900mL, is settled to 1L, autoclaving after adjusting pH5.8 with Solid NaOH pellets.
Embodiment 4: the shake flask fermentation checking of mutant strain aspergillus niger Anm2
By mutant strain aspergillus niger Anm2(CGMCC No.8541) and starting strain aspergillus niger An1 Simultaneous vaccination to CMA flat board, cultivate 4-5d, get its separately spore suspension be inoculated in 20ml CLS fermention medium respectively, at 30 DEG C, under the condition of 200rpm, cultivate 48h; Then by 10% inoculum size, draw 2ml CSL nutrient solution thalline and be inoculated in 20mL TSB fermention medium respectively, at 30 DEG C, under the condition of 200rpm, cultivate 5d, collect the fermented supernatant fluid of aspergillus niger Anm2 and aspergillus niger An1 respectively; With Econo-Pac10DGColumns(BOI-RAD) desalination is carried out except sugar process to supernatant liquor; Then carried out the analysis of 12%SDS-PAGE electrophoresis detection, as shown in Figure 2, two strain bacterium all have obvious protein band at 109kDa place to result, illustrate that mutant strain aspergillus niger Anm2 that the present invention obtains also can recombinant expressed α transfering grape glycosidase; Enzyme activity determination result shows, and the fermenting enzyme of the bacterium aspergillus niger An1 that sets out is lived as 15351U/mL, and the fermenting enzyme work of mutant strain aspergillus niger Anm2 is up to 23709U/mL, and than setting out, bacterium improves 54%.
Embodiment 4: characterization analysis
With pH value be 2.0,3.0,4.0,5.0,6.0,7.0,8.0,9.0,10.0, the fermented supernatant fluid of set out described in the damping fluid of 11.0 dilution embodiment 3 bacterium and mutant bacteria, measure its enzyme respectively to live, live as 100% with the highest enzyme, calculate relative enzyme and live, do the relative enzyme of effect pH-curve alive.As shown in Figure 3, α-transfering grape glycosidase that mutant strain aspergillus niger Anm2 of the present invention expresses is compared with the bacterium that sets out, and action pH-relative enzyme curve alive does not change, and Optimun pH is 5.0 for result.
Respectively at 30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C, 70 DEG C, 80 DEG C, 90 DEG C, measure to set out described in embodiment 3 bacterium and mutant bacteria fermented supernatant fluid enzyme under pH5.0 condition and live, live as 100% with the highest enzyme, calculate relative enzyme and live, do the enzyme of temperature-relatively and to live curve.As shown in Figure 4, α-transfering grape glycosidase that mutant strain aspergillus niger Anm2 of the present invention expresses is compared with the bacterium that sets out, and operative temperature-relative enzyme curve alive does not change, and optimum temperature is 60 DEG C for result.
The above results shows, α-transfering grape glycosidase zymologic property generation Inactivating mutations that the sudden change of mutant strain aspergillus niger Anm2 that the present invention obtains does not cause it to express.
Embodiment 5 α-transfering grape glycosidase is producing the application in isomaltooligosaccharide syrup
Industrial production isomaltooligosaccharide syrup, take starch as raw material, add water and make 30% powder slurry, pH value be 6.0, under temperature is 90 ~ 120 DEG C of conditions, through thermotolerance α-amylase post liquefaction, be 5.0 in pH value, under temperature is the condition of 60 DEG C, acts on β-amylase, Pullulanase and Fungal Alpha amylase simultaneously, change into maltose; Then α-the transfering grape glycosidase adding mutant strain aspergillus niger Anm2 of the present invention expression carries out turning glycosides reaction, generates IMO and panose that isomaltose, Isomaltotriose, tetrose and pentasaccharides etc. contain α-1,6 key.
By above-mentioned turn glycosides reaction product through activated carbon decolorizing, ion exchange resin desalination, being concentrated to solid quality volume ratio is 75%, namely obtain common iso-maltose syrup, wherein the mass volume ratio of dextrinosan is 46% ~ 57%, and the mass volume ratio of glucose is 51%; Again by the glucose yeast fermentation in iso-maltose syrup or membrane filtration removing, the mass volume ratio that just can obtain containing dextrinosan is the product of 92%.
Claims (2)
1. a strain aspergillus niger, is characterized in that, the bacterial strain deposit number of described aspergillus niger is CGMCCNo.8541.
2. aspergillus niger according to claim 1 is producing the application in α-transfering grape glycosidase.
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