CN104531629A - Cyclodextrin glucosyltransferase mutant for improving AA-2G conversion rate - Google Patents

Cyclodextrin glucosyltransferase mutant for improving AA-2G conversion rate Download PDF

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CN104531629A
CN104531629A CN201410779194.XA CN201410779194A CN104531629A CN 104531629 A CN104531629 A CN 104531629A CN 201410779194 A CN201410779194 A CN 201410779194A CN 104531629 A CN104531629 A CN 104531629A
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mutant
sudden change
cyclomaltodextrin glucanotransferase
aspartic acid
aminoacid sequence
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CN104531629B (en
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吴敬
宿玲恰
陈晟
熊艳军
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Jiangnan University
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C12N9/1074—Cyclomaltodextrin glucanotransferase (2.4.1.19)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01019—Cyclomaltodextrin glucanotransferase (2.4.1.19)

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Abstract

The invention discloses a cyclodextrin glucosyltransferase mutant for improving AA-2G conversion rate and belongs to the field of genetic engineering and enzyme engineering. According to the cyclodextrin glucosyltransferase mutant, the 228th lysine near the active center of the cyclodextrin glucosyltransferase of thermophilic fat bacillus (Bacillus stearotherm opilus NO2) is mutated into arginine to obtain a mutant K228R, the 367th aspartic acid is mutated into serine to obtain a mutant D367S and amphimutation is carried out on the basis to obtain a mutant D367S/K228R. The mutant can be used for realizing the improvement of the AA-2G conversion rate and has relatively high industrial value.

Description

A kind of cyclomaltodextrin glucanotransferase mutant improving AA-2G transformation efficiency
Technical field
The present invention relates to a kind of cyclomaltodextrin glucanotransferase mutant improving AA-2G transformation efficiency, belong to genetically engineered and enzyme engineering field.
Background technology
Vitamins C (VC) is the water-soluble vitamins that a kind of human body self can not synthesize, participate in physiological activities a lot of in body, as promoted, cholesterol changes bile acide into, promote the synthesis of adrenocortical hormone, participate in the metabolism of aromatic amino acid, promote the absorption of iron and participate in multiple redox reaction in body, maintaining and in promoting health, play important role.In addition, vitamins C can also promote the synthesis of collagen protein, reduces established melanocyte, has certain effect to maintenance skin elasticity, whitening, smoothing wrinkle.Be widely used in the fields such as food, medicine, makeup.But the hydroxyl extremely unstable of VC bis-carbon, is very easy to oxidative degradation and limits its application.2-O-α-D-glucopyranosyl xitix (AA-2G) is a kind of ascorbic carbohydrate derivative, is the most stable, that performance is best VC substitute, is applied in the high-end makeup of multiple brand in recent years mainly as a kind of whitening additive.
At present, the AA-2G that the whole world is applied in makeup provides by one, Japanese Lin Yuan company, and it commercially occupies the status of monopolization.The price of AA-2G is about 3000 yuan/kg, the chances are ascorbic more than 100 times, its marketable value as seen.China is production of vitamin C big country, and domestic VC manufacturing enterprise has and exceedes global 80% production capacity, but China VC product 3/4ths is for outlet, and inner pin portion is mainly used in medicine, and kind is more dull.The research of China to AA-2G also has report, but only belongs to the preliminary exploratory stage, not yet reaches industrialized level.Therefore excavate the glycosyltransferase optimizing the applicable AA-2G of production the industrialization tool promoting China AA-2G is of great significance.
Although the Bacillus of deriving from stearothermophilus NO2 cyclomaltodextrin glucanotransferase CGTase used in the present invention can realize the production of AA-2G in conversion process, extraction process is still indispensable thereafter.How improving substrate conversion efficiency, cost-saving, after simplifying, extraction process will become the research direction of cyclomaltodextrin glucanotransferase.
Summary of the invention
A technical problem to be solved by this invention is to provide a kind of mutant of cyclomaltodextrin glucanotransferase, comprise containing one or two replacement relative to Bacillus stearothermophilus NO2 cyclomaltodextrin glucanotransferase reactive amino acid residues, have higher AA-2G transformation efficiency compared with parent.
The parental gene of described cyclomaltodextrin glucanotransferase and the Bacillus stearothermophilusNO2 cyclomaltodextrin glucanotransferase consistent (accession number X59042.1) in ncbi database.
The aminoacid sequence of the maturation protein of the parental gene coding of described cyclomaltodextrin glucanotransferase is as described in SEQ ID NO.1.
Described mutant relative to aminoacid sequence for the cyclomaltodextrin glucanotransferase shown in SEQ ID NO.1 there occurs the sudden change of one or two amino acid sites.
Described sudden change is relevant to the transformation efficiency that CGTase produces AA-2G.
Described amino acid sites of undergoing mutation is the Methionin of the 228th or the aspartic acid of the 367th.
Described mutant is that the Methionin (Lys) of the 228th in the aminoacid sequence shown in SEQ ID NO.1 is sported arginine (Arg), called after K228R, and its aminoacid sequence is as shown in SEQ ID NO.2.
Described mutant is that the aspartic acid (Asp) of the 367th in the aminoacid sequence shown in SEQ ID NO.1 is mutated into Serine (Ser), shown in called after D367S.
Described mutant is that the Methionin (Lys) of the 228th of the aminoacid sequence shown in SEQ ID NO.1 is sported arginine (Arg), the aspartic acid (Asp) of the 367th is mutated into Serine (Ser), the mutant called after K228R/D367S obtained simultaneously.
Another technical problem to be solved by this invention is to provide a kind of preparation method of mutant of cyclomaltodextrin glucanotransferase, comprises the steps:
(1) on the basis of Bacillus stearothermophilus NO2 cyclomaltodextrin glucanotransferase aminoacid sequence, mutational site is determined; The mutant primer of design rite-directed mutagenesis, with the carrier carrying cyclodextrin glucosyl transferase gene for template carries out rite-directed mutagenesis; Build the plasmid vector containing mutant;
(2) mutant plasmid is transformed into host cell;
(3) select positive colony and carry out fermentation culture, and purifying cyclomaltodextrin glucanotransferase mutant K228R, D367S, D367S/K228R.
Described plasmid vector is pUC series, pET series, or any one in pGEX.
Described host cell is bacterium and fungal cell, and it is also protection scope of the present invention.
Described bacterium is Gram-negative bacteria or gram-positive microorganism.
The production of the present invention's application and AA-2G, optimum temperuture is 35 DEG C, and optimal pH is 5.0.Present invention achieves the raising of AA-2G output, under the enzymatic conversion condition of optimum, the productive rate that mutant enzyme K228R, D367S, D367S/K228R produce AA-2G reaches 42%, 40%, 45% respectively, is 1.17,1.11,1.25 times that wild enzyme produces AA-2G transformation efficiency.
Embodiment
Embodiment 1: recombinant bacterium builds
According to the cgt gene order (NCBI sequence number X59042.1) that NCBI logs in, adopt chemical synthesis synthesis cyclodextrin glucosyl transferase gene sequence cgt.PET20b (+) for building colibacillary plasmid, with T7 promotor.PET20b (+) plasmid and the plasmid containing cgt gene are carried out Nco I and Hind III double digestion respectively, after digestion products rubber tapping is reclaimed, connect with T4 ligase enzyme again, connect product conversion E.coli JM109 competent cell, 8h is cultivated through 37 DEG C, choose transformant and shake cultivation in the LB substratum containing 100mg/L ampicillin liquid, extract plasmid, digestion verification obtains expression plasmid cgt/pET20b (+).
By plasmid cgt/pET20b (+) Transformed E .coli BL21 (DE3) Host Strains, coating is dull and stereotyped containing the LB of penbritin (100mg/L), cultivate 8h, called after cgt/pET20b (+)/E.coli BL21 (DE3) for 37 DEG C.Choose single bacterium colony to containing in 100mg/L ampicillin liquid LB substratum, 37 DEG C of overnight incubation, preserve glycerine pipe.
Embodiment 2: the preparation of mutant
(1) single mutation
Derive from two kinds of mutant enzymes K228R, D367S of the cyclomaltodextrin glucanotransferase of B.stearothermophilus NO2:
According to the gene order (NCBI sequence number X59042.1) of B.stearothermophilus NO2 cyclomaltodextrin glucanotransferase, design and synthesize the primer introducing K228R, D367S sudden change respectively, rite-directed mutagenesis is carried out to cyclodextrin glucosyl transferase gene, measure DNA encoding sequence, the Lys codon identifying the 228th respectively becomes Arg codon, and the Asp codon of the 367th becomes Ser codon.Mutant gene is placed in suitable expression vector and imports subtilis, intestinal bacteria or bacillus pumilus and express, obtain single mutation cyclomaltodextrin glucanotransferase.The rite-directed mutagenesis of single mutation K228R, D367S: utilize fast PCR technology, with expression vector cgt/pET20b (+) for template,
The rite-directed mutagenesis primer of the K228R sudden change of calling sequence as shown in SEQ ID NO.3, SEQ ID NO.4 is:
Forward primer: 5 '-TGGATGCTGTT cGCcACATG-3 ' (underscore is mutating alkali yl)
Reverse primer: 5 '-ACGGCATGTG gCGaACAGC-3 ' (underscore is mutating alkali yl)
The rite-directed mutagenesis primer of calling sequence D367S sudden change as shown in SEQ ID NO.5, SEQ ID NO.6 is:
Forward primer: 5 '-CGGTAACGGC tCTcCGAAC-3 ' (underscore is mutating alkali yl)
Reverse primer: 5 '-GGTTGTTCGG aGAgCCGTTA-3 ' (underscore is mutating alkali yl)
PCR reaction system is: 5 × PS buffer 10 μ L, dNTPs Mix (2.5mM) 4 μ L, forward primer (10 μMs) 1 μ L, reverse primer (10 μMs) 1 μ L, template DNA 1 μ L, PrimerStar HS (5U/ μ L) 0.5 μ L, adds distilled water to 50 μ L.
Pcr amplification condition is: 94 DEG C of denaturation 4min; 30 circulations (98 DEG C of 10s, 55 DEG C of 5s, 72 DEG C of 8min) subsequently; 72 DEG C are continued to extend 10min.
PCR primer digests through Dpn I, transformation of E. coli JM109 competence, competent cell is after LB solid medium (containing 100 μ g/mL penbritins) overnight incubation, choose to be cloned in LB liquid nutrient medium (containing 100 μ g/mL penbritins) and extract plasmid after cultivation, transformed by mutant plasmid and express host e. coli BL21 (DE3) competent cell, all mutant plasmids all check order correctly.
Enzymatic production
(2) two sudden change
The double-mutant enzyme D367S/K228R of B.stearothermophilus NO2 cyclomaltodextrin glucanotransferase: the aspartic acid (Asp) of the 367th in single-mutant enzyme K228R gene is mutated into Serine (Ser), called after D367S/K228R.The preparation method of double-mutant enzyme, with single mutant K228R encoding gene for template, design and synthesize the primer introducing D367S sudden change respectively, rite-directed mutagenesis is carried out to single mutant K228R encoding gene, measure sequence, the Asp codon identifying 367 becomes Ser codon, mutant gene is placed in suitable expression vector and imports subtilis, intestinal bacteria or bacillus pumilus to express, and obtains two sudden change cyclomaltodextrin glucanotransferase mutant.
The rite-directed mutagenesis of two sudden change D367S/K228R: utilize fast PCR technology, with expression vector K228R/pET20b (+) for template,
The rite-directed mutagenesis primer introducing D367S sudden change is:
Forward primer: 5 '-CGGTAACGGC tCTcCGAAC-3 ' (underscore is mutating alkali yl)
Reverse primer: 5 '-GGTTGTTCGG aGAgCCGTTA-3 ' (underscore is mutating alkali yl)
The sequence measurement of PCR reaction system, reaction conditions and mutator gene is with the method for single mutant.
(3) fermentation of mutant enzyme and purifying
The positive colony that picking proceeds to expressive host e. coli bl21 (DE3) grows 8 ~ 10h in LB liquid nutrient medium (containing 100 μ g/mL penbritins), by 5% inoculum size, seed fermentation liquid is received in TB substratum (containing 100 μ g/mL penbritins), cultivate 48h in 25 DEG C of shaking tables after, by fermented liquid in 4 DEG C, the centrifugal 10min of 8000rpm except thalline, collect centrifuged supernatant.The ferment supernatant liquor that obtains of the cgt/pET20b (+) obtained with embodiment 1/E.coli BL21 (DE3) is the contrast of wild enzyme.
Embodiment 3: concentrating of mutant crude enzyme liquid
It is ammonium sulfate relative to enzyme liquid massfraction 26% that the enzyme liquid obtained in embodiment 2 is slowly added concentration while stirring, is stirred to ammonium sulfate and dissolves, under 4 DEG C of conditions, leave standstill 8 ~ 10 hours protein precipitations.Mixture through centrifugal (8000rpm, 10min) collecting precipitation, then uses the 50mM KH of minimum volume 2pO 4-Na 2hPO 4damping fluid (pH 6.0) redissolves, and through recentrifuge removing solid substance after redissolution, obtains concentrated enzyme liquid after collecting supernatant dialysis.
Embodiment 4:HPLC detects the output of 2-oxygen-α-D-glucopyranosyl xitix
Add the L-AA of 50g/L in the reactor, the beta-cyclodextrin of 50g/L is as substrate, aqueous sodium hydroxide solution with 20% by pH regulator to 5.0, add the concentrated enzyme liquid of wild enzyme and the mutant obtained in a certain amount of enzyme the same example 3 alive, at 37 DEG C, react 24 hours in the shaking bath of 150rpm, 60U glucoamylase is added after reaction terminates, at 60 DEG C, in the shaking bath of 150rpm, reaction samples after 24 hours and adds the solution of trichloroacetic acid (10% of same volume, v/v) termination reaction protein precipitation, precipitate centrifugal for sample 12000rpm 10min after 4 hours, get the rear 0.45 μm of ultrafiltration membrance filter of supernatant liquor appropriateness dilution, and carry out HPLC analysis.Chromatographic condition is as follows: Agilent 1200HPLC chromatographic instrument, Agilent automatic sampler, Agilent SB-Aq 5 μm (4.6mm × 250mm), LC-9A UV-detector; Moving phase is the dilute phosphoric acid of 20mM, flow velocity 0.8mL min -1; Column temperature 35 DEG C.
The wild enzyme of table 1 and mutant produce the transformation efficiency of AA-2G
Enzyme Produce the transformation efficiency (%) of AA-2G
Wild enzyme 36
K228R 42
D367S 40
D367S/K228R 45
The results are shown in Table 1, mutant expresses the mutant enzyme of acquisition compared with wild enzyme, can find, mutant achieves the raising of AA-2G transformation efficiency, wherein the transformation efficiency of single mutation K228R, D367S synthesis AA-2G improves 6%, 4% respectively compared with wild enzyme, and the transformation efficiency of two sudden change D367S/K228R synthesis AA-2G and wild enzyme improve 9%.
Although the present invention with preferred embodiment openly as above; but it is also not used to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, what therefore protection scope of the present invention should define with claims is as the criterion.

Claims (10)

1. a cyclomaltodextrin glucanotransferase mutant, is characterized in that, relative to aminoacid sequence for the cyclomaltodextrin glucanotransferase shown in SEQ ID NO.1 there occurs the sudden change of one or two amino acid sites; The transformation efficiency that described sudden change and cyclomaltodextrin glucanotransferase produce 2-O-α-D-glucopyranosyl xitix is relevant.
2. mutant according to claim 1, is characterized in that, described in the amino acid sites of undergoing mutation be the Methionin of the 228th or the aspartic acid of the 367th.
3. mutant according to claim 1, is characterized in that, described sudden change is arginine by the lysine mutation of the 228th, and the aminoacid sequence of mutant is the sequence shown in SEQ ID NO.2.
4. mutant according to claim 1, is characterized in that, described sudden change is that the Aspartic acid mutations of the 367th is become Serine.
5. mutant according to claim 1, is characterized in that, described sudden change is arginine by the lysine mutation of the 228th, the Aspartic acid mutations of the 367th become Serine, the mutant called after K228R/D367S obtained simultaneously.
6. express the genetic engineering bacterium of the arbitrary described mutant of claim 1-5.
7. a preparation method for mutant described in claim 1, comprises the steps:
(1) on the basis of Bacillus stearothermophilus NO2 cyclomaltodextrin glucanotransferase aminoacid sequence, mutational site is determined;
(2) design the mutant primer of rite-directed mutagenesis, carry out rite-directed mutagenesis with the carrier carrying cyclodextrin glucosyl transferase gene for template and also build the plasmid vector containing mutant;
(3) mutant plasmid is transformed into host cell; Select positive colony and carry out fermentation culture, and purifying cyclomaltodextrin glucanotransferase mutant.
8. preparation method according to claim 7, is characterized in that, described plasmid vector is pUC series, pET series, or any one in pGEX.
9. preparation method according to claim 7, is characterized in that, described host cell is bacterium or fungal cell; Described bacterium is Gram-negative bacteria or gram-positive microorganism.
10. the arbitrary described application of mutant in AA-2G production of claim 1-5.
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CN108018268A (en) * 2018-01-15 2018-05-11 江南大学 A kind of yclodextrin glycosyltransferase mutant of raising AA-2G yield
CN108034645A (en) * 2018-01-15 2018-05-15 江南大学 The preparation and its application of a kind of yclodextrin glycosyltransferase mutant
CN108350474A (en) * 2015-09-25 2018-07-31 奥地利工业生物技术中心有限公司 The glycosylated method of small molecule
CN109486786A (en) * 2018-12-07 2019-03-19 江南大学 A kind of yclodextrin glycosyltransferase mutant
CN111534498A (en) * 2020-05-28 2020-08-14 江南大学 Cyclodextrin glucosyltransferase mutant with improved disproportionation specific activity and AA-2G yield
CN112210544A (en) * 2020-10-15 2021-01-12 江南大学 Cyclodextrin glucosyltransferase mutant and application thereof
CN112301011A (en) * 2019-07-23 2021-02-02 波顿（上海）生物技术有限公司 Glycosyltransferase variants and uses thereof
CN112301012A (en) * 2020-10-15 2021-02-02 江南大学 Cyclodextrin glucosyltransferase mutant and construction method thereof

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CN109486786A (en) * 2018-12-07 2019-03-19 江南大学 A kind of yclodextrin glycosyltransferase mutant
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CN112301011B (en) * 2019-07-23 2023-12-08 波顿（上海）生物技术有限公司 Glycosyltransferase variants and uses thereof
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CN111534498A (en) * 2020-05-28 2020-08-14 江南大学 Cyclodextrin glucosyltransferase mutant with improved disproportionation specific activity and AA-2G yield
CN111534498B (en) * 2020-05-28 2022-03-25 江南大学 Cyclodextrin glucosyltransferase mutant with improved disproportionation specific activity and AA-2G yield
CN112301012A (en) * 2020-10-15 2021-02-02 江南大学 Cyclodextrin glucosyltransferase mutant and construction method thereof
CN112210544A (en) * 2020-10-15 2021-01-12 江南大学 Cyclodextrin glucosyltransferase mutant and application thereof

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