CN102337254A - Mutant of beta-galactosidase and preparation method and application thereof - Google Patents
Mutant of beta-galactosidase and preparation method and application thereof Download PDFInfo
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- CN102337254A CN102337254A CN2011102473383A CN201110247338A CN102337254A CN 102337254 A CN102337254 A CN 102337254A CN 2011102473383 A CN2011102473383 A CN 2011102473383A CN 201110247338 A CN201110247338 A CN 201110247338A CN 102337254 A CN102337254 A CN 102337254A
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Abstract
The invention discloses a mutant of beta-galactosidase and a preparation method and application thereof. In the invention, phenyl alanine close to the active center of beta-galactosidase of sulfolobussolfataricus is mutated into tyrosine to obtain a mutant enzyme F441Y. Under the optimized enzymatic conversion condition of the mutant enzyme, the yield of galactooligosaccharide produced by the mutant enzyme F441Y is up to 61 percent, which is about 10 percent higher than that of a wild enzyme. According to the invention, increase in the yield of galactooligosaccharide is realized, and a higher industrial value is achieved.
Description
Technical field
The present invention relates to a kind of beta-galactosidase enzymes two mutants and preparation method thereof, after amino acid is undergone mutation near the site, active site, obtain the active beta-galactosidase enzymes that improves, belong to genetically engineered and enzyme engineering field.
Technical background
Oligomeric galactose (GOs) is a kind of new type functional oligose, has been applied in the industries such as milk formula, bakery, beverage at present.According to statistics, the Japan at present YO of oligomeric galactose reaches 6500~7000 tons, only is lower than oligomeric isomaltose, but annual sales amount is first of all functions property oligose.The America and Europe, people are also growing to the demand of oligomeric galactose.In China, oligomeric galactose still is an emerging industry, does not form fairly large as yet.After " 95 ", at Yucheng, Shandong, ground such as Guangdong Jiangmen have just begun the production of oligomeric galactose successively, but output is limited.
Beta-galactosidase enzymes is the main enzyme of using of industrial Production by Enzymes oligomeric galactose.Beta-galactosidase enzymes acts on β (1 → 4) glycosidic link of lactose, forms galactosyl-enzyme complex, and under the commentaries on classics glycosides vigor effect of this enzyme, mixture further combines with semi-lactosi, disaccharides or trisaccharide, generates oligomeric galactose.The beta-galactosidase enzymes in different microorganisms source is owing to different in kind own, and synthetic GOs ability also there are differences, as derives from
E. coli,
A. nigerThe hydrolytic activity of beta-galactosidase enzymes stronger, and derive from
B. circulans,
A. oryzae,
Kluyveromyces lactisBeta-galactosidase enzymes then have stronger commentaries on classics glycosides active.At present, domestic a lot, but be mostly the research of its hydrolysis aspect to beta-galactosidase enzymes research, change glycosides active aspect the research relative deficiency,, and industrialization promotion is few, has limited the raising of oligomeric galactose production capacity.
Used in the present invention deriving from
Sulfolobus solfataricusThough the beta-galactosidase enzymes of P2 can be realized the High-efficient Production of oligomeric galactose in conversion process, the back extraction process is still indispensable.How to improve substrate conversion efficiency, practice thrift cost, simplify the research direction that the back extraction process will become beta-galactosidase enzymes.
Summary of the invention
The invention provides a kind of beta-galactosidase enzymes two mutants, this two mutants be with sulphur ore deposit sulfolobus solfataricus (
Sulfolobus solfataricus) beta-galactosidase enzymes (NCBI numbering: near AAK43121) amino acid whose replacement the active site, compare with its parental generation beta-galactosidase enzymes, it has higher transformation efficiency to lactose.
Said beta-galactosidase enzymes two mutants is that 441 amino acids phenylalanine(Phe)s sport tyrosine, called after F441Y.
The present invention is applied to the production of oligomeric galactose, and optimum temperuture is 70 ℃, and ph optimum is 6.5.
The present invention has optimized the enzymatic conversion condition of mutant enzyme on the basis of the mutant strain that makes up, realized the raising of oligomeric galactose output, has higher commercial value.Use this two mutants to produce oligomeric galactose, under optimum enzymatic conversion condition, the productive rate of the oligomeric galactose that mutant enzyme F441Y produces reaches 61%, and wilder enzyme is high by about 10%.
Description of drawings
Fig. 1 utilizes wild enzymatic conversion lactose to generate the optimum temperuture of oligomeric galactose;
Fig. 2 utilizes mutant enzyme to transform the optimum temperuture that lactose generates oligomeric galactose;
Fig. 3 utilizes wild enzymatic conversion lactose to generate the righttest Ph of oligomeric galactose;
Fig. 4 utilizes mutant enzyme to transform the ph optimum that lactose generates oligomeric galactose.
Embodiment
embodiment 1: the preparation of the wild beta-galactosidase enzymes of this example explanation.
, beta-galactosidase enzymes the clone
(available from ATCC, ATCC numbers: 35095), and extract its total DNA to cultivate sulphur ore deposit sulfolobus solfataricus P2.
Gene according to the beta-glycosidase of sulphur ore deposit sulfolobus solfataricus
LacSGene (NCBI numbering: AE006641) design primer:
Forward primer P1:GTCTG
CATATGTACTCATTTCCAAATAGC (underscore is a restriction enzyme site)
Reverse primer P2:GAAT
CTCGAGTTAGTGCCTTAATGGCTTTAC (underscore is a restriction enzyme site)
Utilizing above-mentioned primer, is template with total DNA of sulphur ore deposit sulfolobus solfataricus P2, and the pcr amplification beta-galactosidase gene is reflected in the 50 μ L systems and carries out.Reaction conditions is: 94 ℃ of preparatory sex change 4 min; Carry out 30 circulations (94 ℃ of 10 s, 60 ℃ of 5 s, 72 ℃ of 1min50s) subsequently; 72 ℃ are extended 10 min; Last 4 ℃ of insulations.The PCR fragment that amplification obtains after glue reclaims, is connected with pMD18-T simple carrier; And will connect product and be converted into e. coli jm109, it is dull and stereotyped that converted product is coated the LB that contains the 100mg/L penbritin, through 37 ℃ of overnight cultures; Choose 5 bacterium colonies on the flat board; Insert the LB liquid nutrient medium, extracting plasmid and order-checking behind the 8h, the result is correct.
, beta-galactosidase enzymes expression and preparation:
With above-mentioned plasmid that obtains and pT7-7 carrier, carry out NdeI and XhoI 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,, select transformant and in the LB liquid nutrient medium that contains the 100mg/L penbritin, cultivate through 37 ℃ of overnight cultures; Extracting plasmid behind the 8h obtains the lacS/pT7-7 plasmid of enrichment.
With plasmid lacS/pT7-7 transformed into escherichia coli BL21 (DE3) cell; Selecting transformant 37 ℃ of liquid culture in LB substratum (containing 100 μ g/mL penbritins) spends the night; The back is inserted and is induced with 4 mg/L IPTG (isopropylthio-β-D galactoside) after 37 ℃ of TB fermentation broth (containing 100 μ g/mL penbritins) are cultivated 2 h, is cooled to 25 ℃ of constant temperature culture 20 h.
Fermented liquid is in 4 ℃, and centrifugal 10 min of 8000 rpm collect thalline, with the potassium phosphate buffer redissolution of pH 6.5 50mmol/L of equal volume.Water-bath 30min in 80 ℃ of shaking baths, the centrifugal 20min of back 10000rpm removes deposition, collects supernatant, obtains purer wild enzyme.
embodiment 2: the preparation of this example explanation mutant enzyme F441Y.
, rite-directed mutagenesis
Utilize the fast PCR technology, the rite-directed mutagenesis of single mutation F441Y: expression vector
LacS/ pT7-7 is a template, and F441Y rite-directed mutagenesis primer is:
Forward primer P3:5 '-GATTCTCTATGAGG
TATGGTCTGTTAAAGGTCG-3 ' (underscore is a mutating alkali yl)
Reverse primer P4:5 '-CGACCTTTAACAGACC
ATACCTCATAGAGAATC-3 ' (underscore is a mutating alkali yl)
The PCR reaction system is: 2 * PrimeSTAR
TMGC Buffer (contains Mg
2+) 25 μ L, dNTPs (each 2.5 mmol/L) 4 μ L, forward primer P3 (10 μ M) 1 μ L, reverse primer P4 (10 μ M) 1 μ L, template DNA 1 μ L, PrimeSTAR
TMHS DNA Polymerase (2.5 U/ μ l) 0.5 μ L adds distilled water to 50 μ L.
The pcr amplification condition is: 94 ℃ of preparatory sex change 4 min; Carry out 30 circulations (94 ℃ of 10 s, 60 ℃ of 5 s, 72 ℃ of 4 min30s) subsequently; 72 ℃ are extended 10 min; Last 4 ℃ of insulations.
PCR product warp
DpnI (available from Canadian Fermentas company) digestion; Transformed into escherichia coli JM109 competent cell; Competent cell is after LB solid medium (containing 100 μ g/mL penbritins) overnight cultures; Choose mono-clonal and in LB liquid nutrient medium (containing 100 μ g/mL penbritins), cultivate, plasmid is extracted in the back, and the mutant plasmid order-checking is correct.
, mutant enzyme expression and preparation:
With mutant plasmid F441Y/pT7-7 transformed into escherichia coli BL21 (DE3) cell; Selecting transformant 37 ℃ of liquid culture in LB substratum (containing 100 μ g/mL penbritins) spends the night; The back is inserted and is induced with 4 mg/L IPTG (isopropylthio-β-D galactoside) after 37 ℃ of TB fermentation broth (containing 100 μ g/mL penbritins) are cultivated 2 h, is cooled to 25 ℃ of constant temperature culture 20 h.
Fermented liquid is in 4 ℃, and centrifugal 10 min of 8000 rpm collect thalline, with the potassium phosphate buffer redissolution of pH 6.5 50mmol/L of equal volume.Water-bath 30min in 80 ℃ of shaking baths, the centrifugal 20min of back 10000rpm removes deposition, collects supernatant, obtains purer mutant enzyme F441Y.
embodiment 3: the present embodiment explanation utilizes mutant enzyme to transform the optimum temperuture that lactose generates oligomeric galactose.
Can know that by Fig. 1 the righttest enzymatic conversion temperature of wild-type beta-galactosidase enzymes is 75 ℃, can know that the optimum temperuture of mutant enzyme F441Y is 70 ℃ by Fig. 2.
embodiment 4: the present embodiment explanation utilizes mutant enzyme to transform the ph optimum that lactose generates oligomeric galactose.
Can know that by Fig. 3 the righttest enzymatic conversion pH of wild-type beta-galactosidase enzymes is 6.0, can know that the pH of mutant enzyme F441Y is 6.5 by Fig. 4.
embodiment 5: the productive rate of present embodiment explanation HPLC methods analyst oligomeric galactose.
In the reaction solution, the tetrose in the oligomeric galactose, trisaccharide and the disaccharides amount in product adopts HPLC to confirm.The chromatographic condition of measuring trisaccharide and tetrose is: Agilent 1200 HPLC chromatographic instruments, Agilent automatic sampler, chromatographic column Bio-rad Aminex HPX-87H (7.8 mm * 300 mm), Agilent 2410 differential detectors; Moving phase is the 5mmol/L dilution heat of sulfuric acid, flow velocity 0.8 mL min
-150 ℃ of column temperatures.
The chromatographic condition of measuring disaccharides is: Agilent 1200 HPLC chromatographic instruments, Agilent automatic sampler, chromatographic column Thermo Aps-2 HYPERSIL (4.6 mm * 250 mm), Agilent 2410 differential detectors; Moving phase (V/V) is 70% acetonitrile solution, flow velocity 0.8 mL min
-130 ℃ of column temperatures.
The mutant enzyme that the said mutation body surface is reached acquisition is compared with wild enzyme, can find, two mutants F441Y has realized the raising of oligomeric galactose trisaccharide and tetrose content, and whole output reaches 61%.
Sequence table
< 110>Southern Yangtze University
< 120>a kind of two mutants of beta-galactosidase enzymes
<160>?4
<170>?PatentIn?version?3.3
<210>?1
<211>?29
<212>?DNA
< 213>artificial synthesized sequence
<220>
< 223>according to the gene order design, be used for gene amplification.
<400>?1
gtctgcatat?gtactcattt?ccaaatagc 29
?
<210>?2
<211>?31
<212>?DNA
< 213>artificial synthesized sequence
<220>
< 223>according to the gene order design, be used for gene amplification.
<400>?2
?
gaatctcgag?ttagtgcctt?aatggcttta?c 31
<210>?3
<211>?33
<212>?DNA
< 213>artificial synthesized sequence
<220>
< 223>according to the gene order design, be used for gene amplification.
<400>?3
gattctctat?gaggtatggt?ctgttaaagg?tcg 33
<210>?4
<211>?33
<212>?DNA
< 213>artificial synthesized sequence
<220>
< 223>according to the gene order design, be used for gene amplification.
<400>?4
cgacctttaa?cagaccatac?ctcatagaga?atc 33
Claims (5)
1. the two mutants of a beta-galactosidase enzymes, it is characterized in that with sulphur ore deposit sulfolobus solfataricus (
Sulfolobus solfataricus) the betagalactosidase activity center near phenylalanine(Phe) sport tyrosine, the gene of the beta-glycosidase of sulphur ore deposit sulfolobus solfataricus in the aminoacid sequence of the beta-galactosidase enzymes of said sulphur ore deposit sulfolobus solfataricus and the ncbi database
LacSThe aminoacid sequence of genes encoding is identical,
LacSGene is numbered AE006641.
2. the described two mutants of claim 1 is characterized in that the 441st amino acids phenylalanine(Phe) sports tyrosine, called after F441Y.
3. the preparation method of the described two mutants of claim 1 is characterized in that on the basis of sulphur ore deposit sulfolobus solfataricus beta-galactosidase enzymes crystalline structure, confirming the mutational site; The mutant primer of design rite-directed mutagenesis is that template is carried out rite-directed mutagenesis structure mutant plasmid with the carrier that carries beta-galactosidase gene; With mutant plasmid transformed into escherichia coli BL21 (DE3) cell, the positive monoclonal of selecting after the checking carries out fermentation culture, 25 ℃ of constant temperature culture to 24 h; Collecting cell, suspend with damping fluid after; 80 ℃ of water-bath thermal treatment 30min, the centrifuging and taking supernatant obtains two mutants F441Y.
4. method according to claim 3 is characterized in that the said carrier that carries beta-galactosidase enzymes is a pUC series, pET series, pT7-7, or any among the pGEX.
5. the application of the said two mutants of claim 1 is characterized in that optimum temperuture is 70 ℃, and ph optimum is 6.5, and oligomeric galactose output reaches 61%.
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Cited By (10)
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CN102586312A (en) * | 2012-02-27 | 2012-07-18 | 江南大学 | Method for expressing intracellular protein matrix and application thereof |
CN103275953A (en) * | 2013-06-25 | 2013-09-04 | 山东大学 | Beta-galactosidase with broadened nucleoside substrate specificity |
CN103805581A (en) * | 2012-11-15 | 2014-05-21 | 中国科学院微生物研究所 | Beta-glycosidase mutant and coding gene thereof, and application thereof in producing ginsenoside CK |
CN103881994A (en) * | 2014-04-14 | 2014-06-25 | 中国农业科学院生物技术研究所 | Beta-galactosidase mutant with high transglycosylation activity and preparation method and application thereof |
CN105132446A (en) * | 2015-09-23 | 2015-12-09 | 昆明理工大学 | Promoter screening system |
CN105754969A (en) * | 2016-04-01 | 2016-07-13 | 北京农学院 | Beta-galactosidase mutant and related biological material and application thereof |
CN105765067A (en) * | 2013-09-30 | 2016-07-13 | 天野酶株式会社 | Modified beta-galactosidase |
CN107937365A (en) * | 2018-01-15 | 2018-04-20 | 江南大学 | A kind of mutant of beta galactosidase and its preparation method and application |
CN113980936A (en) * | 2021-10-20 | 2022-01-28 | 江南大学 | Beta-galactosidase mutant and application thereof in preparation of long-chain GOS |
CN114214304A (en) * | 2021-10-20 | 2022-03-22 | 江南大学 | Beta-galactosidase mutant with improved GOS conversion rate and application thereof |
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JP2003274991A (en) * | 2002-03-20 | 2003-09-30 | Nippon Origo Kk | Method for producing galactooligosaccharide |
CN101597614A (en) * | 2008-11-24 | 2009-12-09 | 中国农业科学院生物技术研究所 | Coding beta-galactosidase gene and expression thereof and application |
CN102115718A (en) * | 2010-12-16 | 2011-07-06 | 南京工业大学 | Recombinant strain for expressing beta-galactosidase and construction method and application thereof |
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AU1368300A (en) * | 1999-07-09 | 2001-01-30 | Universite De Liege | Cold-active beta galactosidase, the process for its preparation and the use thereof |
JP2003274991A (en) * | 2002-03-20 | 2003-09-30 | Nippon Origo Kk | Method for producing galactooligosaccharide |
CN101597614A (en) * | 2008-11-24 | 2009-12-09 | 中国农业科学院生物技术研究所 | Coding beta-galactosidase gene and expression thereof and application |
CN102115718A (en) * | 2010-12-16 | 2011-07-06 | 南京工业大学 | Recombinant strain for expressing beta-galactosidase and construction method and application thereof |
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CN102586312B (en) * | 2012-02-27 | 2014-07-09 | 江南大学 | Method for expressing intracellular protein matrix and application thereof |
CN103805581A (en) * | 2012-11-15 | 2014-05-21 | 中国科学院微生物研究所 | Beta-glycosidase mutant and coding gene thereof, and application thereof in producing ginsenoside CK |
CN103805581B (en) * | 2012-11-15 | 2015-06-17 | 中国科学院微生物研究所 | Beta-glycosidase mutant and coding gene thereof, and application thereof in producing ginsenoside CK |
CN103275953A (en) * | 2013-06-25 | 2013-09-04 | 山东大学 | Beta-galactosidase with broadened nucleoside substrate specificity |
CN103275953B (en) * | 2013-06-25 | 2014-06-18 | 山东大学 | Beta-galactosidase with broadened nucleoside substrate specificity |
CN105765067A (en) * | 2013-09-30 | 2016-07-13 | 天野酶株式会社 | Modified beta-galactosidase |
US10053682B2 (en) | 2014-04-14 | 2018-08-21 | Biotechnology Research Institute, Chinese Academy Of Agricultural Sciences | β-galactosidase mutant with high transglycosidase activity, and preparation method thereof and uses thereof |
WO2015158213A1 (en) * | 2014-04-14 | 2015-10-22 | 中国农业科学院生物技术研究所 | Β-galactosidase mutant with high transfer-glycoside activity, and preparation method therefor and uses thereof |
CN103881994A (en) * | 2014-04-14 | 2014-06-25 | 中国农业科学院生物技术研究所 | Beta-galactosidase mutant with high transglycosylation activity and preparation method and application thereof |
CN105132446A (en) * | 2015-09-23 | 2015-12-09 | 昆明理工大学 | Promoter screening system |
CN105754969A (en) * | 2016-04-01 | 2016-07-13 | 北京农学院 | Beta-galactosidase mutant and related biological material and application thereof |
CN107937365A (en) * | 2018-01-15 | 2018-04-20 | 江南大学 | A kind of mutant of beta galactosidase and its preparation method and application |
CN107937365B (en) * | 2018-01-15 | 2020-03-24 | 江南大学 | β -galactosidase mutant and preparation method and application thereof |
CN113980936A (en) * | 2021-10-20 | 2022-01-28 | 江南大学 | Beta-galactosidase mutant and application thereof in preparation of long-chain GOS |
CN114214304A (en) * | 2021-10-20 | 2022-03-22 | 江南大学 | Beta-galactosidase mutant with improved GOS conversion rate and application thereof |
CN114214304B (en) * | 2021-10-20 | 2023-07-18 | 江南大学 | Beta-galactosidase mutant with improved GOS conversion rate and application thereof |
CN113980936B (en) * | 2021-10-20 | 2023-07-25 | 江南大学 | Beta-galactosidase mutant and application thereof in preparation of long-chain GOS |
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