CN106190998A - A kind of method improving Q-enzyrne vigor - Google Patents

A kind of method improving Q-enzyrne vigor Download PDF

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CN106190998A
CN106190998A CN201610725645.0A CN201610725645A CN106190998A CN 106190998 A CN106190998 A CN 106190998A CN 201610725645 A CN201610725645 A CN 201610725645A CN 106190998 A CN106190998 A CN 106190998A
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enzyrne
gene
mutant
enzyme
vigor
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CN106190998B (en
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李兆丰
顾正彪
刘艺婷
李才明
程力
洪雁
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Jiangnan University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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)
    • C12N9/107—1,4-Alpha-glucan branching enzyme (2.4.1.18)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
    • 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)
    • C12Y204/01018—1,4-Alpha-glucan branching enzyme (2.4.1.18), i.e. glucan branching enzyme

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Abstract

The invention discloses a kind of method improving Q-enzyrne vigor, belong to genetic engineering and enzyme engineering field.The present invention uses the method for rite-directed mutagenesis that the 349th methionine deriving from the Q-enzyrne of Geobacillus thermoglucosidasius (Geobacillus thermoglueosidan) is mutated into threonine (Thr) or serine (Ser) respectively, gained mutant is compared to wild starch branching enzyme, vigor significantly improves, beneficially the industrialized production application of Q-enzyrne.

Description

A kind of method improving Q-enzyrne vigor
Technical field
The present invention relates to a kind of method improving Q-enzyrne vigor, belong to genetic engineering and enzyme engineering field.
Background technology
Q-enzyrne (1,4-α-glucan branching enzyme；GBE；EC 2.4.1.18) it is that one belongs to sugar The glycosyl transferase of glycosides hydrolase family 13, is the key enzyme of glycogen biosynthesis and amylopectin.Through the shallow lake that Q-enzyrne is modified Powder has the feature of highly-branched, therefore has broad application prospects in the industry such as food, medicine.
At present, the vigor of wild starch branching enzyme is the most relatively low, and thermostability is poor, is unfavorable for industrialized production.Microorganism The Q-enzyrne great majority in source achieve intracellular expression in escherichia coli, it is still necessary to this production stage of breaking cellular wall so that Its commercial production is relatively costly.
With deriving from the hot glucosidase of heat-resisting bacterial strain bacillus (Geobacillus used in the present invention Thermoglueosidan) vigor of the Q-enzyrne of STB02 is about 384U/mg, improves the cyclisation vigor of this enzyme further, Industrialized production in Q-enzyrne be would be more advantageous.
Summary of the invention
First technical problem that the invention solves the problems that is to provide the Q-enzyrne mutant that a kind of vigor improves, described Mutant is by aminoacid sequence methionine (Met) of the 349th of the Q-enzyrne of sequence point as shown in SEQ ID NO.2 It is not mutated into threonine (Thr) or serine (Ser), the most named M349T of gained single mutant, M349S.
Encode the nucleotide sequence of described Q-enzyrne as shown in SEQ ID NO.1.
Second technical problem that the invention solves the problems that is to provide a kind of side obtaining described mutant M349T, M349S Method.According to the gene order shown in SEQ ID NO.1, separately design and synthesize rite-directed mutagenesis primer, gene being carried out fixed point prominent Become, it is thus achieved that the gene of coding M349T, M349S mutant, and converted and carry out table to host E.coli BL21 (DE3) Reach.
Said method comprising the steps of:
(1) utilize fast PCR technology, with containing wild starch branching enzyme gene expression vector pET-20b (+)/gbe is as mould Plate suddenlys change；
(2) expression and purification of mutant
Picking contains expressive host E.coli BL21 (DE3) of mutant plasmid, carries out fermentation culture and uses IPTG to induce The expression of enzyme, collects fermented supernatant fluid, uses nickel post affinity column, the isolated and purified enzyme preparation obtaining mutant M349T, M349S.
Beneficial effects of the present invention:
The present invention, by building mutant M349T and M349S, all achieves the raising of Q-enzyrne enzyme activity, with open country Raw type Q-enzyrne is compared, and the vigor of mutant M349T, M349S all improves 60%；Mutated body M349T and M349S changes Amylopectin α-1,6 glycosidic bond relative amount after property is respectively increased 27.9% and 22.1%.The mutant ratio that the present invention obtains Wild type starch branching enzyme is more suitable for the industrialized production of highly-branched starch.
Accompanying drawing explanation
The pure amylopectin of Fig. 1 Rhizoma Solani tuber osi is after wild starch branching enzyme and mutant modification 4h thereof1HNMR measures spectrogram.A: Wild type starch branching enzyme, B: mutant M349T, C: mutant M349S.
Detailed description of the invention
The preparation of embodiment 1 mutant M349T, M349S
(1) rite-directed mutagenesis
According to the sequence of the wild starch branching enzyme gene gbe shown in SEQ ID NO.1, separately design and synthesize introducing The primer of M349T, M349S codon mutation.
Utilize fast PCR technology, with containing wild starch branching enzyme gene expression vector pET-20b (+)/gbe is as template Suddenly change, the primer respectively:
The primer introducing Met349Thr sudden change is:
Forward primer:
5’-GGGCATATTGACGATTGCCGAAGATTCGACGGAATGGCCGCTCGTCACTGC-3 ', underscore is prominent Become base,
Reverse primer:
5’-CGAATCTTCGGCAATCGTCAATATGCCCGGGTCATGCGCAAATACGGTCT-3 ', underscore is sudden change Base；
The primer introducing Met349Ser sudden change is:
Forward primer: 5 '-ACCCGGGCATATTGAGCATTGCCGAAGATTCGACGGAATGGCCGC-3 ', underscore is Mutating alkali yl,
Reverse primer: 5 '-AATCTTCGGCAATGCTCAATATGCCCGGGTCATGCGCAAATACGG-3 ', underscore is Mutating alkali yl.
PCR reaction system is: 5 × PrimeSTAR Buffer (Mg2+Plus) 20 μ L, dNTPs (each 2.5mM) 8 μ L, just To primer 1 μ L, reverse primer 1 μ L, template DNA 1 μ L, PrimeSTAR HS DNA Polymerase (2.5U/ μ L) 1 μ L, add Enter distilled water 68 μ L.
PCR reaction amplification condition is: 98 DEG C of denaturations 4min；98 DEG C of degeneration 10s subsequently, 60 DEG C of annealing 15s, 72 DEG C are prolonged Stretching 5min, carry out 23 circulations, each cycle annealing temperature arranges fall 0.2 DEG C；Last 72 DEG C of insulation 10min.
By PCR primer after DpnI digestion 2h, proceed to big escherichia coli (Escherichia coli) JM109 competence thin In born of the same parents, being applied to overnight incubation in the LB solid medium containing agar, picking list bacterium colony was cultivated in LB fluid medium Extract plasmid after night and carry out sequence verification.Proceed to express place by the expression vector of the gene containing coding Q-enzyrne mutant In main E.coli BL21 (DE3) competent cell.Above-mentioned each culture medium is all added the ampicillin of 100 μ g/mL.
(2) expression and purification of mutant
Picking contains the monoclonal of expressive host E.coli BL21 (DE3) of mutant plasmid in blue or green containing 100 μ g/mL ammonia benzyls In the LB culture medium of mycin, 37 DEG C, cultivate 8～12h under 200r/min, be inoculated into containing 100 μ with the inoculum concentration of volume ratio 2% In the TB culture medium of g/mL ampicillin, start cultivation temperature be 37 DEG C, shaking speed be 200r/min, when cell concentration is trained Support to OD600During to 0.6, go to rapidly after adding the IPTG of 0.01mM 30 DEG C, continue induction 48h under 200r/min, will fermentation Liquid in 4 DEG C, 10000rpm be centrifuged 15min to remove thalline, collect supernatant.
Fermented supernatant fluid is used affinity chromatography His Trap HP post, respectively purification obtain mutant M349T, The enzyme preparation of M349S.
Embodiment 2 enzyme activity determination analysis
(1) mensuration of enzyme activity
0.25% (w/v) amylopectin potato or straight is prepared with the 50mmol/L phosphate buffer (pH 7.5) of 0.9mL Chain starch standard solution, adds 0.1mL enzyme liquid, reacts 15min at 50 DEG C.React and terminate rear boiling water bath enzyme denaturing 10min, and with 10000r/min is centrifuged 2min and treats that colour developing is used.Color development system is 0.3mL reaction supernatant and 5.0mL nitrite ion (0.05% (w/v) KI, 0.005% (w/v) I2, pH 7.5) mix in 7mL centrifuge tube, measure at 530nm or 660nm after colour developing 15min and inhale Light value.Enzyme is lived and is defined: at 530nm or 660nm, and the enzyme amount needed for light absorption value reduction per minute 1% is enzyme unit alive.
(2) enzyme activity compares
Experimental result is listed in table 1, it was found that compared with wild type starch branching enzyme, the work of mutant M349T, M349S Power all improves 60%.After finding to be mutated into threonine (Thr) and serine (Ser) by crystal structure sunykatuib analysis, strengthen With other amino acid whose hydrogen bond actions, beneficially stable space structure.
Table 1
Embodiment 3 utilizes NMR to analyze α-1,6 glycosidic bond relative amount of modified starch
Using the pure amylopectin solution of Rhizoma Solani tuber osi of 5mg/mL of preparation as substrate, weigh the pure amylopectin of 0.5g Rhizoma Solani tuber osi It is dissolved in 90mL sodium phosphate buffer (pH7.5), is settled to 100mL, gelatinizing 30min in boiling water.It is separately added into certain The wild starch branching enzyme of amount, mutant M349T, M349S are placed at 50 DEG C reaction 4h, carry out lyophilization after boiling enzyme denaturing.
α-1,6 glycosidic bond relative amount of modified starch measures and uses proton nmr spectra (NMR) to measure.Weigh 20mg to treat Test sample product, are dissolved in 0.5mLD2In O, must not there is bubble.
Experimental result is as shown in Fig. 1 and Biao 2, when with the 5% pure amylopectin of Rhizoma Solani tuber osi for substrate, forms sediment compared to wild type Powder branching enzyme, mutant M349T, M349S have higher vigor.After 4h enzyme reaction, compared with wild type, mutant Amylopectin α-1,6 glycosidic bond relative amount modified for M349T and M349S is respectively increased 27.9% and 22.1%.To sum up may be used To find out, mutant M349T and M349S is more suitable for the industrialized production application of highly-branched starch.
Table 2
Although the present invention is open the most as above with preferred embodiment, but it is not limited to the present invention, any is familiar with this skill The people of art, without departing from the spirit and scope of the present invention, can do various changes and modification, therefore the protection model of the present invention Enclosing should be with being as the criterion that claims are defined.

Claims (10)

1. a Q-enzyrne, it is characterised in that its aminoacid sequence is as shown in (a) or (b) or (c):
A the methionine of the 349th is mutated into threonine or serine in sequence basis shown in SEQ ID NO.2 by () respectively；
B () aminoacid sequence in (a) is through replacing, lacking or add one or several aminoacid and have Q-enzyrne The protein derivative by (a) of activity.
2. a gene, it is characterised in that Q-enzyrne described in coding claim 1.
3. a carrier, it is characterised in that containing gene described in claim 2.
4. a cell, it is characterised in that containing gene described in claim 2.
5. the method obtaining Q-enzyrne described in claim 1, it is characterised in that according to the base of coding Q-enzyrne The sequence of cause, separately designs and synthesizes rite-directed mutagenesis primer, gene is carried out rite-directed mutagenesis, it is thus achieved that coding M349T, M349S are prominent The gene of variant, and converted and express to host E.coli BL21 (DE3).
Method the most according to claim 5, it is characterised in that picking contains the expressive host E.coli of mutant gene BL21 (DE3), carries out fermentation culture and uses the expression of IPTG inducible enzyme, collects fermented supernatant fluid, uses nickel affinity column, separates Purification obtains the enzyme preparation of mutant M349T, M349S.
Method the most according to claim 6, it is characterised in that picking contains the expressive host E.coli of mutant gene The monoclonal of BL21 (DE3) in the LB culture medium containing 100 μ g/mL ampicillin, 37 DEG C, cultivate under 200r/min 8～ 12h, is inoculated in the TB culture medium containing 100 μ g/mL ampicillin with the inoculum concentration of volume ratio 2%, starts cultivation temperature and is 37 DEG C, shaking speed be 200r/min, when cell concentration is cultivated to OD600When being 0.6, turn rapidly after adding the IPTG of 0.01mM To 30 DEG C, continue induction 48h under 200r/min；By fermentation liquid in 4 DEG C, 10000rpm be centrifuged 15min to remove thalline, collect Supernatant purification, respectively obtain mutant M349T and M349S.
8. the method improving Q-enzyrne vigor, it is characterised in that be by aminoacid sequence as shown in SEQ ID NO.2 The 349th methionine of Q-enzyrne be mutated into threonine or serine respectively.
9. the application in glycogen biosynthesis and amylopectin of the Q-enzyrne described in claim 1.
10. the application in producing Q-enzyrne of the gene described in claim 2.
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108660121A (en) * 2018-05-29 2018-10-16 江南大学 A kind of Q-enzyrne mutant that thermal stability improves
CN108841801A (en) * 2018-05-29 2018-11-20 江南大学 A kind of method of amino acid residue relevant to enzyme activity in screening enzyme
WO2019174137A1 (en) * 2018-03-16 2019-09-19 江南大学 Method for improving transparency of starch liquefied product
CN110684751A (en) * 2019-10-23 2020-01-14 江南大学 Starch branching enzyme mutant with improved catalytic capability
CN111172221A (en) * 2020-01-16 2020-05-19 齐鲁工业大学 Preparation method and application of modified starch
CN112391365A (en) * 2020-11-30 2021-02-23 江南大学 Starch branching enzyme mutant with improved catalytic activity and application thereof
CN113881648A (en) * 2021-10-20 2022-01-04 江南大学 Method for improving catalytic activity of starch branching enzyme

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AU5632099A (en) * 1998-09-25 2000-04-17 Roquette Freres Method for preparing a mixture of starch branching enzymes extracted from algae
CN1875105A (en) * 2003-06-30 2006-12-06 联邦科技产业研究组织 Wheat with altered branching enzyme activity and starch and starch containing products derived therefrom
CN102816778A (en) * 2012-07-30 2012-12-12 上海市农业科学院 Mutant gene of rice starch branching enzyme SBE3 gene and application of mutant gene

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AU5632099A (en) * 1998-09-25 2000-04-17 Roquette Freres Method for preparing a mixture of starch branching enzymes extracted from algae
CN1875105A (en) * 2003-06-30 2006-12-06 联邦科技产业研究组织 Wheat with altered branching enzyme activity and starch and starch containing products derived therefrom
CN102816778A (en) * 2012-07-30 2012-12-12 上海市农业科学院 Mutant gene of rice starch branching enzyme SBE3 gene and application of mutant gene

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019174137A1 (en) * 2018-03-16 2019-09-19 江南大学 Method for improving transparency of starch liquefied product
US11124816B2 (en) 2018-03-16 2021-09-21 Jiangnan University Method for improving the transparency of starch liquefaction
CN108660121A (en) * 2018-05-29 2018-10-16 江南大学 A kind of Q-enzyrne mutant that thermal stability improves
CN108841801A (en) * 2018-05-29 2018-11-20 江南大学 A kind of method of amino acid residue relevant to enzyme activity in screening enzyme
CN110684751A (en) * 2019-10-23 2020-01-14 江南大学 Starch branching enzyme mutant with improved catalytic capability
CN110684751B (en) * 2019-10-23 2021-06-25 江南大学 Starch branching enzyme mutant with improved catalytic capability
CN111172221A (en) * 2020-01-16 2020-05-19 齐鲁工业大学 Preparation method and application of modified starch
CN112391365A (en) * 2020-11-30 2021-02-23 江南大学 Starch branching enzyme mutant with improved catalytic activity and application thereof
CN113881648A (en) * 2021-10-20 2022-01-04 江南大学 Method for improving catalytic activity of starch branching enzyme
CN113881648B (en) * 2021-10-20 2022-12-13 江南大学 Method for improving catalytic activity of starch branching enzyme

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