CN102443578B - Glucose isomerase mutant and application thereof - Google Patents
Glucose isomerase mutant and application thereof Download PDFInfo
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- CN102443578B CN102443578B CN2011104064117A CN201110406411A CN102443578B CN 102443578 B CN102443578 B CN 102443578B CN 2011104064117 A CN2011104064117 A CN 2011104064117A CN 201110406411 A CN201110406411 A CN 201110406411A CN 102443578 B CN102443578 B CN 102443578B
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Abstract
The invention discloses a glucose isomerase mutant and application thereof, belonging to the technical field of enzyme genetic engineering and enzyme engineering. According to the invention, a high temperature glucose isomerase gene (NCBI coding: CP000088) is obtained from total DNA of thermobifida fusca, and after site-directed mutagenesis, high efficiency expression of the glucose isomerase gene with a high conversion rate is realized, with the plasmid pT7-7 or a vector that can express glucose isomerase as an expression vector and E. coli BL21 (DE3) or a bacterial strain that can express glucose isomerase as an expression host; the glucose isomerase gene has altogether 1158 nucleotide and encodes 385 amino acids; expression plasmid is constructed in the invention, glucose isomerase is expressed through conversion of bacteria or yeast, and an obtained recombinase mutant has activity of glucose isomerase and has a conversion rate of 60% at a temperature of 70 DEG C, 7% higher than the conversion rate of a parent; optimum temperature of the recombinant glucose isomerase is 80 DEG C, an optimum pH is 10, and the half life of the recombinant glucose isomerase at a temperature of 70 DEG C is no less than 30 h. The recombinant glucose isomerase is particularly applicable to production of F55 high-fructose syrup in the industry of foodstuffs.
Description
Technical field
The present invention relates to a kind of glucose isomerase, relate in particular to a kind of high conversion glucose isomerase mutant and production thereof and application.
Technical background
Glucose isomerase (glucose isomerase, GI) claims again xylose isomerase (xylose isomerase, XI) (EC5.3.1.5), can the catalysis D-Glucose etc. aldose to the isomerization reaction of the corresponding ketoses such as D-Fructose.Therefore be one of extensive key enzyme for preparing high fructose syrup with starch on the industrial production.
High fructose syrup also claims high fructose syrup or isomery syrup, and it is that wherein a part of glucose isomerase becomes fructose with the saccharified liquid of the enzyme process starch saccharification gained isomerization through glucose isomerase, a kind of mixing sugar syrup that is comprised of glucose and fructose.High fructose syrup is that sugariness and sucrose are suitable, local flavor is pure, nutritious, and have synergy, cold sweet and tasty mouthful, carious tooth low in calories, unlikely: the characteristics such as preservative effect is good, good water absorption, leavening property are good are bestly to substitute that sucrose uses or the novel sweetener of substituting saccharose use.In addition, do not need the auxiliary of Regular Insulin in the fructose metabolic process, the glycogen growing amount of metabolic conversion is three times of glucose in vivo, has hepatoprotective effect; It can also suppress the consumption of body internal protein, is conducive to sportsmen and physical labourer's nutrient.Because high fructose syrup has than glucose, sucrose obviously superior health care and nutritive effect and good food processing properties, be applied to more and more widely in the food-processings such as beverage, cold foods, candy, baking goods, fruit can and preserved fruit, jam at present, its demand will constantly increase.
High fructose syrup is a kind of mixture that is comprised of fructose and glucose, difference according to its fructose content, mainly contain in the market F42 (fructose accounts for 42%) and two kinds of F55 (fructose accounts for 55%), the F55 syrup is with respect to the major advantage of F42 syrup: the a.F42 syrup can not store at low temperatures, easy crystallization glucose, the F55 syrup then can store in 26～30 ℃ scope; B. under the prerequisite of equal carbohydrate, its sugariness of F42 syrup only has common sucrose sugariness 75%, and F55 syrup sugariness and sucrose sweetness are suitable; C is because F55 syrup fructose content is higher, and its health care is worth higher.And according to present domestic concrete production, technical qualification and cost requirement, overwhelming majority manufacturing enterprise can only produce first the F42 product, concentrate out F90 syrup (fructose accounts for 90%) through chromatographic fractionation system again, blend out the F55 syrup with the F42 syrup again, if can direct production go out the F55 syrup, then investment of production will reduce greatly.
High temperature is conducive to the raising of glucose isomerase transformation efficiency, at present, people have isolated the thermotolerance glucose isomerase many from some natural thermophile bacteria, such as Bacills thermoantarcti.CUS, Thermus aquaticus HB8, Thermotoga neapolitana etc.People have carried out purifying, qualitative to the heat-resisting glucose isomerase of its generation, have affirmed its great potential in the industry of the high candy slurry of preparation.But because a large amount of generations that the culture condition of thermophile bacteria harshness, low cell yield and the isomery invert point more than 90 ℃ cause by product and pigment, it produces enzyme and also is not suitable for suitability for industrialized production.So can be under 60-70 ℃ really be high fructose syrup industrial production first-class problem to be solved with regard to the glucose isomerase that the transformation efficiency more than 55% is arranged!
Summary of the invention
Technical problem to be solved by this invention provides a kind of glucose isomerase mutant, its parent has NCBI coding: the aminoacid sequence shown in the YP_289661, described glucose isomerase mutant are that the 243rd or 244 or 284 or 286 s' amino acid mutation is for sporting proline(Pro) (Pro) or Serine (Ser) or L-Ala (Ala) or Isoleucine (Ile) or Threonine (Thr) or α-amino-isovaleric acid (Val).
Described glucose isomerase mutant is that the 244th hyte propylhomoserin, the 243rd phenylalanine sport respectively a kind of in proline(Pro), Serine, L-Ala, Isoleucine, Threonine, the α-amino-isovaleric acid.
Described glucose isomerase mutant is that the 243rd phenylalanine, the 244th hyte propylhomoserin, 284 proline(Pro), 286 hyte propylhomoserins all sport a kind of in proline(Pro), Serine, L-Ala, Isoleucine, Threonine, the α-amino-isovaleric acid.
Described glucose isomerase mutant is to be that 286 hyte propylhomoserins sport a kind of in proline(Pro), Serine, L-Ala, Isoleucine, Threonine, the α-amino-isovaleric acid.
The gene order of glucose isomerase mutant of the present invention of encoding also belongs to the scope of protection of present invention.
Another technical problem that the present invention will solve provides a kind of method of producing glucose isomerase mutant, concrete technical scheme is as follows: encode according to thermophilic ascomycete (Thermobifida fusca) colt Glucose Isomerase Gene NCBI: CP000088, the mutant primer of design rite-directed mutagenesis carries out rite-directed mutagenesis take the cloning vector that carries colt Glucose Isomerase Gene as template and makes up mutant; The carrier that maybe can express this enzyme take plasmid pT7-7 is as expression vector, and recombinant plasmid transformed e. coli bl21 (DE3) cell maybe can be expressed the host cell of this enzyme, and the positive monoclonal of selecting after the checking carries out fermentation culture.
The present invention is applied to the production of high fructose syrup, and application of temperature is 60-70 ℃, and application pH is 5.0-8.5, and the application phosphate concn is that 50-100mM, magnesium ion concentration are 5-8mM.
Use this mutant to produce high fructose syrup, under above-mentioned conversion condition, the transformation efficiency that mutant enzyme is produced high fructose syrup reaches 55%-60%, about the high 3%-8% of wilder enzyme, this enzyme mutant has solved the industrial technical barrier of long-term puzzlement high fructose syrup, has outstanding technical progress.
Description of drawings
The separation and purification SDS-PAGE collection of illustrative plates of Fig. 1 glucose isomerase mutant
M: standard protein Marker, 1 broken wall supernatant liquor, the enzyme liquid behind the 2 thermal treatment purifying, the enzyme liquid behind 3 ion-exchanges and the gel exclusion purifying,
The optimum temperuture of Fig. 2 glucose isomerase mutant
The optimal pH of Fig. 3 glucose isomerase mutant
NaAc-HAc damping fluid (pH 4.0-5.0), Na-K-PO
4Damping fluid (pH 5.0-9.0) and Gly-NaOH damping fluid (pH9.0-12.0)
Fig. 4 glucose isomerase mutant is at 70 ℃ of stability studies
Fig. 5 glucose isomerase mutant is studied at 70 ℃ of transformation efficiencys
pH7.5(○)，pH5.0(□)
Embodiment
The structure of embodiment 1, glucose isomerase unit point mutant
Number according to thermophilic ascomycete (Thermobifida fusca) colt Glucose Isomerase Gene NCBI: CP000088, adopt chemical complete synthesizing process to synthesize parental array, be cloned into pMD18-T, carrier construction xylA/pMD18-T.Mutant primer according to parental array design rite-directed mutagenesis utilizes the fast PCR technology, and take carrier xylA/pMD18-T as template, single mutation P284S primer is:
Primer B-ST-1:5 '-AGTGGCTACGACGGA
TCGCGCCACTTCGACTTCAAGACC-3 ' (underscore is mutating alkali yl)
Primer B-ST-2:5 '-GGTCTTGAAGTCGAAGTGGCG
CGATCCGTCGTAGCCACT-3 ' (underscore is mutating alkali yl)
The PCR reaction system is: 2 * PrimeSTARTM GC Buffer (containing Mg2+), 25 μ L, dNTPs (each 2.5mmol/L) 4 μ L, forward primer (10 μ M) 1 μ L, reverse primer (10 μ M) 1 μ L, template DNA 1 μ L, PrimeSTARTM HS DNA Polymerase (2.5U/ μ l) 0.5 μ L adds distilled water to 50 μ L.
The pcr amplification condition is: 94 ℃ of denaturation 4min; Carry out subsequently 30 circulations (94 ℃ of 10s, 60 ℃ of 5s, 72 ℃ of 4min30s); 72 ℃ are extended 10min; Last 4 ℃ of insulations.
The PCR product transforms escherichia coli jm109 competent cell, competent cell is after LB solid medium (containing 100 μ g/mL penbritins) overnight incubation, choosing mono-clonal cultivates in LB liquid nutrient medium (containing 100 μ g/mL penbritins), rear extraction plasmid, the mutant plasmid order-checking is correct, the mutant called after xylA-S of structure.
The structure of embodiment 2, glucose isomerase dibit Point mutont
Number according to thermophilic ascomycete (Thermobifida fusca) colt Glucose Isomerase Gene NCBI: CP000088, adopt chemical complete synthesizing process to synthesize parental array, be cloned into pMD18-T, carrier construction xylA/pMD18-T.Mutant primer according to parental array design rite-directed mutagenesis utilizes the fast PCR technology, take carrier xylA/pMD18-T as template, and two sudden change F243I, H244T, the rite-directed mutagenesis primer is:
Primer A-IT-1:5 '-TGGCACGGCAAACTG
ATCACCATCGACCTCAACGGC-3 ' (underscore is mutating alkali yl)
Primer A-IT-2:5 '-GCCGTTGAGG TCGATGGTGA TCAGTTTGCC GTGCCA-3 ' (underscore is mutating alkali yl)
The PCR reaction system is: 2 * PrimeSTARTM GC Buffer (containing Mg2+), 25 μ L, dNTPs (each 2.5mmol/L) 4 μ L, forward primer (10 μ M) 1 μ L, reverse primer (10 μ M) 1 μ L, template DNA 1 μ L, PrimeSTARTM HS DNA Polymerase (2.5U/ μ l) 0.5 μ L adds distilled water to 50 μ L.
The pcr amplification condition is: 94 ℃ of denaturation 4min; Carry out subsequently 30 circulations (94 ℃ of 10s, 60 ℃ of 5s, 72 ℃ of 4min30s); 72 ℃ are extended 10min; Last 4 ℃ of insulations.
The PCR product transforms escherichia coli jm109 competent cell, competent cell is after LB solid medium (containing 100 μ g/mL penbritins) overnight incubation, choosing mono-clonal cultivates in LB liquid nutrient medium (containing 100 μ g/mL penbritins), rear extraction plasmid, the mutant plasmid order-checking is correct, the mutant called after xylA-IT of structure.
The structure of embodiment 3, glucose isomerase four site mutation bodies
Number according to thermophilic ascomycete (Thermobifida fusca) colt Glucose Isomerase Gene NCBI: CP000088, adopt chemical complete synthesizing process to synthesize parental array, be cloned into pMD18-T, carrier construction xylA/pMD18-T.Mutant primer according to parental array design rite-directed mutagenesis utilizes the fast PCR technology, take carrier xylA/pMD18-T as template, and two sudden change F243I/H244T, the rite-directed mutagenesis primer is:
Primer A-IT-1:5 '-TGGCACGGCAAACTG
ATCACCATCGACCTCAACGGC-3 ' (underscore is mutating alkali yl)
Primer A-IT-2:5 '-GCCGTTGAGG TCGAT
GGTGA TCAGTTTGCC GTGCCA-3 ' (underscore is mutating alkali yl)
Again take carrier xylA (F243I/H244T)/pT7-7 as template, two sudden change P284S/H286T, the rite-directed mutagenesis primer is:
Primer B-ST-1:5 '-AGTGGCTACGACGGA
TCGCGC
ACCTTCGACTTCAAGACC-3 ' (underscore is mutating alkali yl)
Primer B-ST-2:5 '-GGTCTTGAAGTCGAA
GGTGCG
CGATCCGTCGTAGCCACT-3 ' (underscore is mutating alkali yl)
The PCR reaction system is: 2 * PrimeSTARTM GC Buffer (containing Mg2+), 25 μ L, dNTPs (each 2.5mmol/L) 4 μ L, forward primer (10 μ M) 1 μ L, reverse primer (10 μ M) 1 μ L, template DNA 1 μ L, PrimeSTARTM HS DNA Polymerase (2.5U/ μ l) 0.5 μ L adds distilled water to 50 μ L.
The pcr amplification condition is: 94 ℃ of denaturation 4min; Carry out subsequently 30 circulations (94 ℃ of 10s, 60 ℃ of 5s, 72 ℃ of 4min30s); 72 ℃ are extended 10min; Last 4 ℃ of insulations.
The PCR product transforms escherichia coli jm109 competent cell, competent cell is after LB solid medium (containing 100 μ g/mL penbritins) overnight incubation, choosing mono-clonal cultivates in LB liquid nutrient medium (containing 100 μ g/mL penbritins), rear extraction plasmid, the mutant plasmid order-checking is correct, the mutant called after xylA-ITST of structure.
Example 4, expression wild or the sudden change recombinant glucose isomerase prepare with fermentation
With plasmid obtained above and pT7-7 carrier (commercialization carrier), carry out NdeI and HindIII double digestion, enzyme is cut product after glue reclaims, spend the night with 16 ℃ of connections of T4 ligase enzyme, connect product and be converted into e. coli jm109, through 37 ℃ of overnight incubation, select transformant and in the LB liquid nutrient medium that contains the 100mg/L penbritin, cultivate, extracting plasmid behind the 8h obtains parent's glucose isomerase plasmid and the glucose isomerase mutant plasmid of enrichment.
They are transformed respectively behind e. coli bl21 (DE3) cell in the dull and stereotyped upper 37 ℃ of cultivations of LB/Amp (containing 100 μ g/mL penbritins), selecting transformant 37 ℃ of liquid culture in LB substratum (containing 100 μ g/mL penbritins) behind the 8h spends the night, behind 37 ℃ of cultivations of rear access TB fermentation broth (containing 100 μ g/mL penbritins) 2h, after being cooled to 25 ℃, with 4mg/LIPTG (isopropylthio-β-D galactoside) inducing culture 30h.
Example 5, wild or the sudden change recombinant glucose isomerase purifying
Get the thalline 10 after 100ml cultivates, 000rpm, 4 ℃ of centrifugal 10min remove most supernatant, add 10mL PBS damping fluid (pH 7.5 for 50mM KPO4,10mM MgSO4), abundant mixing, ON 10s, OFF 10s, ultrasonication.4 ℃, 13,000rpm, centrifugal 30min, supernatant is crude enzyme liquid, crude enzyme liquid at 70 ℃ of water-bath 10min, 4 ℃, 13,000rpm, centrifugal 30min gets supernatant and carries out the ion column purifying.Adopt the ARTK protein purification system, adopt Sepharose Fast Flow chromatography column, DEAE is its anionic exchange medium group, elutriant is: A liquid (pH 7.5 for 50mM KPO4,5mMMgSO4), B liquid (0mM KPO4,5mMMgSO4,1M NaClpH 7.5).Carry out enzyme activity determination to going out peak albumen, choose the absorption peak that suitable elutriant ratio wash-out contains target protein.Carry out target protein secondarily purified with molecular-exclusion chromatography again, carry out enzyme activity determination to going out peak albumen, choose suitable elution time and reclaim the absorption peak that contains target protein, target protein is carried out electrophoresis with the SDS-PAGE protein electrophoresis to the stage product of crude enzyme liquid and different purifying with molecule, relatively purification effect (seeing Fig. 1).
The zymologic property of multi-mutant site, two mutational site and single mutation site glucose isomerase is basic identical, and hereinafter the multi-mutant site glucose isomerase of preparation describes as example in the example 1.
The characteristic of example 6, recombinant glucose isomerase.
When take glucose as substrate, the optimum temperuture of glucose isomerase is 80 ℃ (Fig. 2), and optimal pH 10 (Fig. 3) has very high stability at 70 ℃, and the transformation period is greater than 30h (Fig. 4).
The transformation efficiency of example 7, HPLC methods analyst glucose isomerase.
Investigate the conversion capability of recombinant glucose isomerase under two kinds of pH conditions, the conversion of glucose system is 5mL, comprise 50mM Na-K-PO4 damping fluid (pH 7.5 or 5.0), 42% (W/V) D-Glucose aqueous solution, the 5mM MgSO4 aqueous solution, the enzyme liquid of 100U, 70 ℃ of water-baths, interval certain hour sampling, sample heats 5min and makes enzyme deactivation sex change in the sample in boiling water after, dilution is the centrifugal 25min of 1/100,12000r/min, and supernatant is got 500 μ L and analyzed behind 0.45 μ m ultrafiltration membrance filter.The condition that HPLC analyzes is: Agilent1200 HPLC chromatographic instrument, and the Agilent automatic sampler, chromatographic column Aminex HPX-87H lon Exclusion Cloumn (7.8mm * 300mm), Agilent1200 differential detector; Moving phase is 0.0005M H2SO4 solution, flow velocity 0.6mL/min; 50 ℃ of column temperatures; 30 ℃ of Chi Wen (detector); Sample size 10 μ L.
Experimental result such as Fig. 5, the mutant enzyme that the said mutation body surface is reached acquisition is compared with wild enzyme, can find that mutant has been realized the raising of inversion rate of glucose, and whole output reaches 60%.
Claims (5)
1. a glucose isomerase mutant is characterized in that carrying out the replacement of an amino acid sites in the aminoacid sequence shown in the NCBI number of the logging in YP_289661, and the 284th proline(Pro) sported Serine.
2. the described mutant of claim 1 is in glucose application in the high fructose syrup to the conversion of fructose or preparation.
3. the described application of claim 2 is characterized in that glucose is 70 ℃ to the treatment process control temperature of the conversion of fructose, and pH is 7.5.
4. the nucleotide sequence of coding claim 1 described glucose isomerase mutant.
5. method of producing the described glucose isomerase mutant of claim 1, it is characterized in that obtaining colt Glucose Isomerase Gene by the total DNA of thermophilic ascomycete (Thermobifida fusca), NCBI is encoded to CP000088, behind rite-directed mutagenesis, mutator gene is take plasmid pT7-7 as expression vector, the bacterial strain that maybe can express this enzyme take E.coli BL21 (DE3) is realized efficiently expressing of colt Glucose Isomerase Gene as expressive host.
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| CN102994490B (en) * | 2012-12-27 | 2014-10-22 | 江南大学 | Glucose isomerase immobilization method |
| CL2013003356A1 (en) | 2013-11-22 | 2014-08-08 | Univ Pontificia Catolica Chile | Mutants of the gene coding for the phenylacetone monooxygenase (pamo) enzyme isolated from fusca or thermononospora fusca thermofibide with substitutions at positions 93.94, and 440 (n, dyf, respectively) and specific combinations of positions 441,442,443 and / or 444 (god , poe, t, v, iowyq respectively) with high performance as catalysts in the conversion of cyclohexanone to epailon-caprolactone and high thermal stability, DNA conditioning and encoded amino acidic sequences. |
| CN104745563A (en) * | 2015-03-05 | 2015-07-01 | 浙江工业大学 | Glucose isomerase and gene, mutant, engineering bacteria and application thereof |
| MX2022002712A (en) * | 2019-09-13 | 2022-08-10 | Danisco Us Inc | Thermostable glucose isomerase variants. |
| CN116064456B (en) * | 2022-11-28 | 2023-08-08 | 江南大学 | Oligosaccharide debranching enzyme mutant and application thereof in glucose mother liquor |
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| GB8815902D0 (en) * | 1988-07-04 | 1988-08-10 | Imperial College | Xylose isomerase mutants |
| CN1693473A (en) * | 2004-05-08 | 2005-11-09 | 张骏 | Method of prepairing peltoglucose syrup and high temperature resistant glucose isomerase mutant |
| CN1743454A (en) * | 2004-09-02 | 2006-03-08 | 百瑞全球有限公司 | Glucose isomerase mutant and its use |
| WO2007028811A1 (en) * | 2005-09-06 | 2007-03-15 | Cargill, Incorporated | Thermostable xylose isomerase enzymes |
| CN1966679B (en) * | 2005-11-18 | 2011-08-17 | 百瑞全球有限公司 | Glucose isomerase mutant, its application and DNA encoding same |
| CN101323858B (en) * | 2008-07-24 | 2010-06-02 | 河南天冠企业集团有限公司 | Xylose isomerase, and encoding gene and use thereof |
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