CN105524936A - Mutant trehalose synthase as well as expression gene and application thereof - Google Patents
Mutant trehalose synthase as well as expression gene and application thereof Download PDFInfo
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Abstract
The invention relates to mutant trehalose synthase as well as an expression gene and application thereof. The nucleotide sequence of the expression gene of the mutant trehalose synthase is as shown in SEQ ID NO. 1. The amino acid sequence of the mutant trehalose synthase is as shown in SEQ ID NO. 2. On the basis of a three-dimensional structure predicted by pseudomonas stutzeri Qlu3, the invention conducts key amino acid site-specific mutagenesis on the active center for the first time, so that the mutant protein of the trehalose synthase is obtained; the mutant trehalose synthase is simple and convenient in preparation method, high in yield, high in purity and good in thermal stability; and compared with wild trehalose synthase, the trehalose conversion rate of the mutant is improved by 4.5% and the generation amount of glucose, as a byproduct, is reduced by 69.4%.
Description
Technical field
The present invention relates to
a kind of trehalose synthase of sudden change and expressing gene thereof and application, belong to gene engineering technology field.
Background technology
Trehalose is a kind of fool proof reliable irreducibility disaccharide, and the expense thunder Derek using NMR technology that takes the lead in is probed into its chemical structure, has 2 glucopyranose monomers by 1, the disaccharide that 1 glycosidic link links.The 19th-century first half is extracted trehalose first time by Wei Gesi and is obtained from the ergot of a kind of rye, and has confirmed that trehalose has non-specific provide protection to various bioactivators.Again because characteristics such as its drought-resistant, anti-low temperature, anti-severe cold, be " sugar of life " by a lot of person.Trehalose can make organism stand high temperature, high and cold, the rugged environment condition such as high osmotic pressure and dry dehydration, reason is that it can produce a kind of film and is attached to cell surface, maintain protein molecular preferably and sex change does not occur, and then animals and plants life characteristics is existed.The functional performance of this uniqueness; make trehalose except can as except the excellent activity protective material of pharmaceutical grade protein, enzyme, vaccine and other biological goods; still the important component of cytoactive, moisturizing class makeup is kept, the particular foodstuff batching of the Food Quality that more can be used as and prevent foodstuff deteriorate, keeps food fresh flavor, promotes.Therefore trehalose can be applied to medicine, cosmetic industry and food service industry widely, has tempting development prospect and huge economic benefit.
In view of the using value that trehalose is extensive and important, find trehalose research that is efficiently convenient, Low-cost production method and extensively paid attention to.The production method of current trehalose mainly contains yeast extraction method, fermentation method, Enzyme optrode.Wherein Production by Enzymes trehalose has higher specificity and the quick feature such as gentle, has become the focus of research and development trehalose suitability for industrialized production and one of feasible way that can take effect as short-term.
Trehalose synthase (Trehalosesynthase, TreS) is glucuronosyltransferases in a kind of molecule, and it only needs single step reaction just the α of maltose-Isosorbide-5-Nitrae glycosidic link can be converted into α-1,1 glycosidic link generation trehalose.This enzyme reaction flow process is short, easy-regulating, does not need to consume anakinetomer, do not need phosphoric acid salt to coexist, only need a kind of enzyme one-step to react and just can obtain trehalose, therefore trehalose synthase conversion method is the method that suitability for industrialized produces trehalose, there is good application prospect, paid close attention to widely.Up to the present, the microorganism that can produce trehalose synthase of report is had both at home and abroad more than 15 kinds.Catalytic efficiency, the zymologic property of the trehalose synthase in different microorganisms source are had nothing in common with each other, but its generation all with by product in reaction process, have up to 20%, be generally about 5%-10%, this brings difficulty to the separation and purification of downstream product, improves the production cost of trehalose simultaneously.
Summary of the invention
For the deficiencies in the prior art, the invention provides
a kind of trehalose synthase of sudden change and expressing gene thereof and application.The growing amount of the trehalose synthase by product glucose of this sudden change reduces, and the transformation efficiency of trehalose improves.The trehalose synthase that this trehalose synthase origin comes from Pseudomonas stutzeri (PseudomonasstutzeriQlu3) obtains through rite-directed mutagenesis A309E.
Technical solution of the present invention is as follows:
An expressing gene for the trehalose synthase of sudden change, nucleotide sequence is as shown in SEQIDN0.1.
A trehalose synthase for sudden change, aminoacid sequence is as shown in SEQIDNO.2.
Trehalose synthase of the present invention is L-glutamic acid by the alanine mutation of method to key amino acid 309 of Quickchange rite-directed mutagenesis, then proceed to after intestinal bacteria carry out high expression, utilize affinity chromatography, ion exchange chromatography, a series of means of purification such as molecular sieve, the highly purified trehalose synthase mutant protein of final acquisition.Above-mentioned recombinant protein reaction 2h can reach maximum conversion rate, and trehalose transformation efficiency can reach 74.7%, and by product is only 1.1%.Above-mentioned recombinant protein places 30min trehalose transformation efficiency in 50 DEG C of metal baths still can reach 51.2%.Above-mentioned recombinant protein optimal reactive temperature is 35 DEG C.Above-mentioned recombinant protein optimal reaction pH is 7.0-8.0.
A kind of recombinant vectors inserting the expressing gene of the trehalose synthase of said mutation.
A kind of transgenic cell line, containing above-mentioned recombinant vectors.
The expressing gene of the trehalose synthase of said mutation, recombinant vectors or transgenic cell tie up to the application prepared in trehalose synthase.
The trehalose synthase of said mutation is preparing the application in trehalose.
Trehalose synthase of the present invention, obtain by Pseudomonas stutzeri (Pseudomonasstutzeri) Qlu3, concrete grammar is: transform the multiple clone site of commercial pET-15b carrier, delete unnecessary restriction enzyme site, retain BamHI, XhoI, EcolI, HindIII restriction enzyme site, and add Prescission restriction enzyme site wherein, obtain pGLO1 carrier.This gene is connected on pGLO1 carrier and is built into plasmid, then utilize the L-Ala (A) of the method for Quickchange rite-directed mutagenesis to key amino acid 309 to suddenly change L-glutamic acid (E), and be transformed in e. coli bl21 DE (3) and carry out prokaryotic expression.Then by affinity chromatography, Source-Q ion exchange chromatography, the method for Superdex-200 sieve chromatography is separated and obtains highly purified mutant trehalose synthase.
Beneficial effect
Based on the three-dimensional structure of the present invention first according to (Pseudomonasstutzeri) Qlu3 prediction, its active centre is carried out to the rite-directed mutagenesis of key amino acid, obtain the mutant protein of trehalose synthase, the trehalose synthase preparation method of this sudden change is easy, output is large, purity is high, Heat stability is good, and trehalose transformation efficiency brings up to 74.7% by 71.5% of wild-type, by product is reduced to 1.1% by 3.6% of wild-type, comparatively wild-type trehalose synthase, the trehalose transformation efficiency of mutant improves 4.5%, the growing amount of by product glucose reduces 69.4%.The separation costs of trehalose and glucose can be reduced, for the suitability for industrialized production of trehalose is laid a good foundation; Also possible ways is provided for other similar proteins improve transformation efficiency.
Accompanying drawing explanation
fig. 1pcr amplification and agarose gel electrophoresis result photo;
in figure: M, Marker, T is the object band of TreS-pET-15b;
fig. 2for trehalose synthase SDS-PAGE electrophoresis result picture after ni-sepharose purification;
in figure, ce: full bacterium liquid after broken, s: supernatant after high speed centrifugation, elu: albumen after nickel post wash-out;
fig. 3for the standard specimen crest result of glucose, maltose and trehalose measured by high performance liquid phase
figure;
fig. 4for being terminated the crest result of rear glucose, trehalose and maltose by high performance liquid phase assaying reaction
figure;
fig. 5 Afor the optimal reactive temperature curve alive of trehalose synthase enzyme after purifying
figure;
fig. 5 Btrehalose synthase enzyme temperature-stable curve alive after purifying
figure;
fig. 6 Afor trehalose synthase optimal reaction pH curve after purifying
figure;
fig. 6 Bthe trehalose synthase pH curve of stability after purifying
figure;
fig. 7 Afor the curve that trehalose synthase after purifying and mutant reaction times affect trehalose transformation efficiency
figure;
in figure: TreS is wild-type trehalose synthase, and TreSA309E is the trehalose synthase of sudden change;
fig. 7 Bfor the curve that trehalose synthase after purifying and mutant reaction times affect inversion rate of glucose
figure;
in figure: TreS is wild-type trehalose synthase, and TreSA309E is the trehalose synthase of sudden change.
Embodiment
Below in conjunction with embodiment, technical scheme of the present invention is further elaborated, but institute of the present invention protection domain is not limited thereto.
Embodiment 1: clone obtains the trehalose synthase gene suddenlyd change.
According to the primer at Pseudomonas stutzeri (Pseudomonasstutzeri) trehalose synthase full length nucleotide sequences Design catastrophe point place disclosed on NCBI, primer sequence is as follows:
Upstream primer: GTGGCCCTCCGACCAttcGGTGCC
Downstream primer: CGTGCCGAGGGCACCgaaTGGTCG
With the tres-pGLO1 plasmid built for template, utilize above-mentioned primer to carry out pcr amplification, PCR reaction system is as follows:
Above-mentioned PCR reaction is carried out according to following program:
98 DEG C of denaturation 3min; 98 DEG C of sex change 10s, 58 DEG C of annealing 30s, 72 DEG C extend 4min, 25 circulations; 72 DEG C of ends extend 10min.
Agarose gel electrophoresis analytic plate segment length by 1wt% after PCR terminates is short.
Embodiment 2: be transformed in expressive host by the trehalose synthase gene of sudden change, obtains positive expression bacterial strain.
PCR primer is through the reaction of DpnI endonuclease digestion, and endonuclease reaction system is as follows:
The enzyme of PCR primer cuts system:
The centrifugal several seconds after abundant mixing, tube wall drop is received at the bottom of pipe, 37 DEG C of ligation 30min.
The conversion of recombinant plasmid
(1) preparation of competent cell
1. picking e. coli bl21 (DE3) (purchased from Shanghai past bio tech ltd) single bacterium colony (or picking preservation bacterial classification) is seeded in 10ml LB liquid medium, 37 DEG C, 210rpm incubated overnight;
2. getting 5ml bacterium liquid is inoculated in 500mlLB substratum, and 37 DEG C, 210rpm, shakes 70-80min to OD
600reach 0.375;
3. bacterium liquid is positioned over 10min on mixture of ice and water, the 50ml of precooling simultaneously centrifuge tube;
4. transfer in centrifuge tube by bacterium liquid, 4 DEG C, 3700rpm, 10min collect thalline, abandon supernatant;
5. the activation damping fluid (0.1MCaCl of about 10ml ice precooling is added in each centrifuge tube
2), break up precipitation with sterilized 5ml rifle point, and then add the activation damping fluid of about 30ml ice precooling in each pipe, put upside down mixing, leave standstill 20min on ice;
6. 4 DEG C, 3700rpm, centrifugal 10min; Abandon supernatant, by raffinate evacuation, store buffer (0.1MCaCl by the precooling of 500ml bacterium liquid 12ml ice
2, volume percent 15% glycerine) amount, precipitation is broken up, (gradation shift, then pressure-vaccum is broken up).
7. competent cell is dispensed in the sterilizing EP of ice precooling, often pipe 100 microlitre, is placed on ice (0 DEG C) 30min.
8. competence-80 DEG C is frozen, obtained competent cell BL21 (DE3).
Attention: whole process allows cell be in low temperature as far as possible, rifle point used, centrifuge tube, EP pipe and buffer etc. all want sterilizing, and whole process all operates in super clean bench, and competent cell wants its efficiency of test and whether microbiological contamination after finishing.
(2) product conversion is connected
1. 15 μ L are connected product to add in the competent cell BL21DE (3) of the fresh preparation of 100 μ L, mix gently, ice bath 30min.
2. 42 DEG C of heat shock 90s, are then placed in rapidly ice bath and cool 3min.
3. add 200 μ LLB liquid nutrient mediums, 37 DEG C, 180rpm/min shaking culture 60min, bacterium is restore normal growth state, and the antibiotics resistance gene of expression plasmid coding;
4. get above-mentioned bacterium liquid 200 μ L, coat the LB solid medium (penbritin 100mg/L) with resistance.
5., after bacterium liquid is blotted, be inverted dull and stereotyped in 37 DEG C of cultivation 12 ~ 16h.
LB liquid nutrient medium component is as follows:
5g yeast powder, 10g peptone, 10gNaCl, adds water to 1L, and 121 DEG C of autoclaving 20min are for subsequent use.
LB solid medium component is as follows:
5g yeast powder, 10g peptone, 10gNaCl, 2% agar powder, adds water to 1L, and 121 DEG C of autoclaving 20min are for subsequent use.
The qualification of positive colony
(1) bacterium colony PCR identifies
Picking list bacterium colony, 37 DEG C of shaking culture 6 ~ 8h, draw 1 μ L bacterium liquid, according to 15 μ LPCR reaction systems, carry out PCR qualification.If positive colony, the band of an entry can be detected by agarose gel electrophoresis,
as Fig. 1shown in.
(2) protein expression and solubility qualification
Identified by bacterium colony PCR in residue bacterium liquid and add the IPTG (isopropylthiogalactoside) that final concentration is 0.2mM, abduction delivering 1h, 12000rpm/min, centrifugal 1min, abandons supernatant, collects thalline.Add 2 times of sample-loading buffers, hang precipitation, 95 DEG C of sex change 10min with rifle point.If positive colony, protein overexpression can be detected by SDS-PAGE.
(3) DNA sequencing
By with the positive colony after above two kinds of methods qualification, correct through sequencing sequence, the nucleotide sequence inserted in the positive colony obtained is as shown in SEQIDNO.1.
Embodiment 3: fermentation culture positive expression bacterial strain, the trehalose synthase recombinant protein of separation and purification sudden change
Seed culture: the positive colony that picking embodiment 2 is obtained is in conventional manner placed in the LB liquid nutrient medium containing 100 μ g/L penbritins of 5mL, at 37 DEG C of shaking culture 5-6h;
Thalline enlarged culturing: seed is accessed 1L and contain in the liquid nutrient medium of 100mg penbritin, 37 DEG C of shaking culture are to the dense OD of bacterium
600when being 1.0, be cooled to 16 DEG C; Add the isopropylthiogalactoside (IPTG) that final concentration is 0.2mM after 1 hour, spend the night abduction delivering.
Collect thalline: 4200rpm, 4 DEG C of centrifugal 15min, supernatant discarded, results thalline; Add resuspended solution (25mMTris-HCl, pH8.0,200mMNaCl), vibration precipitation somatic cells; Adding proteinase inhibitor PMSF (Phenylmethylsulfonylfluoride, phenylformic acid sulfonic acid fluoride) is 2mM to final concentration.
Ultrasonication somatic cells: ultrasonic 3s, interval 6s, 500W, work 60 times.
Ultracentrifugation: the cytoclasis liquid after ultrasonic is at 14000rpm, and centrifugal 45min under 4 DEG C of conditions, collects supernatant liquor, carry out next step separation and purification.
Ni-NTA affinity chromatography: the supernatant fluid containing soluble proteins collected is poured in the nickel post regenerated; After supernatant liquor stream is clean, rinse 10 column volumes with washbuffer (25mMTris-HCl, pH8.0,100mMNaCl, 15mM imidazoles), the albumen of removing non-specific adsorption; Finally use elutionbuffer (25mMTris-HCl, pH8.0,100mMNaCl, 250mM imidazoles) to be eluted by target protein, collect with clean precooling beaker; Whether use SDS-PAGE electrophoresis detection albumen solvable, whether whether solvable albumen can be combined with Ni-NTA, can be eluted, and the concentration of albumen, result
as Fig. 2shown in.
Anion-exchange chromatography purifying (Source-Q): the soluble proteins solution A (25mMTris-HCl that Ni-NTA affinity chromatography system is taken off, pH8.0) 3 ~ 4 times are diluted, then be loaded to used solution A to balance ion exchange column SourceQ on, use solution A and solution B (25mMTris-HCl, pH8.0,1MNaCl) carry out linear gradient elution.Observe light absorption value (A280) changing conditions of 280nm, collect each and go out collection tube near peak position, and carry out SDS-PAGE electrophoresis, to obtain target protein matter.
Molecular sieve purification: according to the shape of ion exchange column protein peak, with or without " shoulder " and whether symmetrical, sharply judge the proterties of protein on ion exchange column.Ultrafiltration and concentration is carried out to 2mL for the protein that proterties is good, be loaded to use solution C (25mMTris-HCl, pH8.0,100mMNaCl) to balance gel permeation chromatography post Superdex-200 on, flow velocity is 0.4mL/min.Collect protein peak and carry out SDS-PAGE electrophoresis, detecting purity and the proterties of protein.Through order-checking, aminoacid sequence, as shown in SEQIDNO.2, is the trehalose synthase of sudden change.
Embodiment 4: the enzyme activity determination method of the trehalose synthase of sudden change
150mM maltose is added, 20mMNa2HPO in reaction system
4-NaH
2pO
4buffered soln pH7.0, the trehalose of the sudden change that 1 μM of embodiment 3 is obtained closes, at 37 DEG C, react 2h.Measured the transformation efficiency of trehalose by the method for high-pressure liquid phase, in mensuration process, adopt nh 2 column; Column temperature is the mixing solutions that 40 DEG C of moving phases adopt acetonitrile and water, and the two volume ratio is 3:1; Flow velocity is 1mL/min; Detector is Composition distribution; Detection time is 25min.Standard substance detected result is shown in
fig. 3according to following formulae discovery transformation efficiency:
According to
fig. 4maltose peak area in shown high performance liquid phase result, trehalose peak area and glucose peaks area utilize software matching to obtain curve, and calculate the quality of three, wherein m3 is the quality being converted into trehalose, m2 is the quality being converted into glucose, and m1 is the quality of residue maltose.
Embodiment 5: the biochemical property of the trehalose synthase of sudden change measures
The mensuration of optimal reactive temperature:
After enzyme liquid is diluted suitable multiple, in 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, in 60 DEG C, react 2h, utilize the method for high-pressure liquid phase to measure the transformation efficiency of trehalose synthase.Result
as Fig. 5 Ashown in.The optimal reactive temperature of trehalose synthase recombinant protein is 35 DEG C.
The mensuration of temperature stability:
After enzyme liquid is diluted certain multiple, in 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, be incubated 30min in 60 DEG C, add substrate maltose, then react 2h, measure trehalose transformation efficiency.The results are shown in
as Fig. 5 Bshown in.Trehalose synthase is placed 30min and is still kept the enzyme of 70% to live in 50 DEG C of metal baths.
The mensuration of optimal reaction pH value:
With pH5.0,5.5,6.0,6.5,7.0,7.5,8.0,8.5,9.0,9.5,10,0.10.5,11.A series of phosphate buffered saline buffer dilution restructuring trehalose synthase mutant.Then be placed in 35 DEG C of reaction 2h, measure enzyme and live.The results are shown in
as Fig. 6 Ashown in.
The mensuration of pH stability:
After enzyme liquid is diluted certain multiple, with pH5.0,5.5,6.0,6.5,7.0,7.5,8.0,8.5,9.0,9.5,10,0.10.5,11 a series of phosphate buffered saline buffer dilution restructuring trehalose synthase mutant.Be incubated 30min in 35 DEG C, add substrate maltose, then react 2h, measure trehalose transformation efficiency.The results are shown in
as Fig. 6 Bshown in.The maintenance more than 95% alive of trehalose synthase mutant enzyme during pH7.0-8.0.
Embodiment 6: the optimum reacting time of the trehalose synthase of sudden change measures
Reaction system is 150mM maltose, 20mMNa
2hPO
4-NaH
2pO
4pH7.2,1 μM of trehalose synthase mutant recombinant protein, reacts, samples at 5min, 20min, 40min, 1h, 2h, 3h, 4h different time at 37 DEG C, and then use HPLC to detect, detection method is with embodiment 4.By calculating transformation efficiency.The reaction of trehalose synthase mutant is carried out trehalose transformation efficiency and can be reached 74.7%, and inversion rate of glucose is 1.1%.
as Fig. 7shown in.
Contrast experiment
Because trehalose synthase mutant makes the transformation efficiency of trehalose synthase improve, by product reduces.After trehalose synthase enzyme liquid before and after sudden change is diluted suitable multiple, react, sample at 5min, 20min, 40min, 1h, 2h, 3h, 4h different time at 37 DEG C, then use HPLC to detect, detection method is with embodiment 4.By calculating transformation efficiency.This trehalose synthase protein preparation method is easy, and output is large, and purity is high; Its Heat stability is good of experimental verification, and trehalose transformation efficiency brings up to 74.7%, result by 71.5% of wild-type
as Fig. 7 Ashown in; And by product is reduced to 1.1% by the inversion rate of glucose 3.6% of wild-type,
as Fig. 7 Bshown in.Comparatively trehalose synthase before sudden change, the trehalose transformation efficiency of mutant improves 4.5%, and the output of by product glucose reduces 69.4%.The separation costs of trehalose and glucose can be reduced, for the suitability for industrialized production of trehalose is laid a good foundation; Also possible ways is provided for other similar proteins improve transformation efficiency.
Claims (7)
1. an expressing gene for the trehalose synthase of sudden change, nucleotide sequence is as shown in SEQIDNO.1.
2. a trehalose synthase for sudden change, aminoacid sequence is as shown in SEQIDNO.2.
3. a recombinant vectors, inserts expressing gene according to claim 1 in expression vector.
4. recombinant vectors as claimed in claim 3, it is characterized in that, described expression vector is pET-15b.
5. a transgenic cell line, containing recombinant vectors according to claim 3.
6. the expressing gene of the trehalose synthase suddenlyd change described in claim 1, recombinant vectors according to claim 3 or transgenic cell according to claim 5 tie up to the application prepared in trehalose synthase.
7. the trehalose synthase of sudden change according to claim 2 is preparing the application in trehalose.
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