CN104946610B - High-temperature-resistant trehalose synthase and expression gene and application thereof - Google Patents
High-temperature-resistant trehalose synthase and expression gene and application thereof Download PDFInfo
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Abstract
The invention relates to a high-temperature-resistant trehalose synthase and an expression gene and application thereof. The nucleotide sequence of the modified expression gene of the high-temperature-resistant trehalose synthase is shown as SEQ ID NO. 1; the amino acid sequence of the modified high-temperature resistant trehalose synthase is shown as SEQ ID NO. 2. The invention firstly cuts off a flexible region in a structure based on a three-dimensional structure of Pseudomonas putida trehalose synthase (Pseudomonas putida KT2440), and retains a stable structural domain of an active center thereof, thereby obtaining more stable trehalose synthase. The trehalose synthase has simple preparation method, high yield and high purity; experiments prove that the modified trehalose synthase still keeps higher catalytic activity and greatly improves the thermal stability, thereby reducing the production cost of the trehalose and laying the foundation for the industrial production of the trehalose.
Description
Technical field
The present invention relates to a kind of high temperature resistant trehalose synthase and its expressing gene and application, more particularly to a kind of stench vacation list
High temperature resistant trehalose synthase and its expressing gene and application that born of the same parents' bacterium trehalose synthase is obtained after directional transformation, belong to biological
Technology technical field.
Background technology
Trehalose (Trehalose) is a kind of to be connected and composed by two pyranoid ring glucose molecule Jing α -1,1- glycosidic bonds
Non-reducing disaccharide, is widely present in the organisms such as antibacterial, yeast, filamentous fungis, plant, insecticide, invertebratess.Grind
Study carefully and show, its is stable in properties, there is very important biological significance to organism.Be mainly manifested in it be the organism energy and
The reserve of carbon source, is protein and biomembrane molecule stablizing in the adverse circumstances such as dehydration, high temperature, oxygen-derived free radicals, low temperature
Agent and protective agent, are sensing complex and growth regulatory factor, but also are one of components of some bacteria cell walls, because
This has the good reputation of " sugar of life " in scientific circles' trehalose.Trehalose has magical protective effect to organism, is because sea
Algae sugar can form the protection of uniqueness in high temperature, high and cold, hyperosmosises and under being dried the severe environmental conditions such as dehydration in cell surface
Film, effectively protected protein matter molecule invariance inactivation, so as to the life process and biological characteristic of the body that sustains life.This is unique
Functional characteristic so that trehalose except can as pharmaceutical grade protein, enzyme, vaccine and other biological product excellent activity protect
Beyond shield agent, cytoactive, the important component of moisturizing class cosmetics are also to maintain, more as preventing food from deteriorating, can keep eating
Product fresh flavor, the particular foodstuff dispensing for lifting food quality.Therefore trehalose can be widely applied to medicine, cosmetic industry with
And food service industry, with tempting development prospect and huge economic benefit.
In view of the extensive and important using value of trehalose, find that trehalose efficiently facilitates, Low-cost production method is ground
Study carefully and extensively paid attention to.At present the production method of trehalose mainly has yeast extraction method, fermentation method, Enzyme optrode.Wherein enzyme process life
Produce trehalose have higher specificity and it is quick gentle the features such as, have become the focus of research and development trehalose industrialized production simultaneously
One of feasible way that can be taken effect as short-term.
Trehalose synthase (EC5.4.99.16, Trehalose synthase, TreS) is a kind of intramolecular glucoside transfer
Enzyme it only need to single step reaction just can by the α -1,4 of maltose glycosidic bonds be converted into α -1,1 glycosidic bonds generate trehalose.The enzyme
Reaction process is short, easy-regulating, it is not necessary to consume anakinetomer, it is not necessary to which phosphate coexists, it is only necessary to which a kind of enzyme one-step reacts just
Trehalose can be obtained, therefore trehalose synthase conversion method is the method for suitable industrialized production trehalose, there is good application
Prospect, is widely paid close attention to.Up to the present, the microorganism that can produce trehalose synthase having been reported that both at home and abroad is alreadyd exceed
15 kinds.The catalytic efficiency of the trehalose synthase in different microorganisms source, zymologic property are had nothing in common with each other, but with basic common ground：
One is that substrate conversion efficiency is relatively low, and highest only has 80% or so, generally 60%-70%；Two be enzyme heat stability it is poor, it is most suitable
Reaction temperature is 25 DEG C or so；Three is that the response time is oversize, generally 48 hours, have up to 72h.
The content of the invention
For the deficiencies in the prior art, there is provided a kind of high temperature resistant trehalose synthase and its expressing gene and application, high temperature resistant
Trehalose synthase be by pseudomonas putida trehalose synthase is oriented transformation after obtain.
Technical solution of the present invention is as follows：
A kind of expressing gene of high temperature resistant trehalose synthase, nucleotide sequence is as shown in SEQ ID NO.1.
A kind of high temperature resistant trehalose synthase, aminoacid sequence is as shown in SEQ ID NO.2.
The high temperature resistant trehalose synthase is tied on the basis of existing pseudomonas putida trehalose synthase according to simulation three-dimensional
Structure, is intercepted N-terminal and C-terminal protein flexibility region by molecular biology method, retaining protein rock-steady structure domain
N42-C680 is obtained.Improved trehalose synthase after escherichia coli High level prokaryotic expression, using affinity chromatograph, hand over by ion
A series of means of purification such as chromatography, sieve chromatography are changed, it is final to obtain.The trehalose synthase and original trehalose synthase
(KT2440) compare its expression to increase, heat stability is also significantly improved.
A kind of recombinant vector, inserts nucleotide sequence shown in above-mentioned SEQ ID No.1 in plasmid.
According to currently preferred, the plasmid is pET-15b vector plasmids.
Conversion in a kind of transgenic cell line, host cell has above-mentioned recombinant vector.
According to currently preferred, the host cell is e. coli bl21 DE (3).
The expressing gene of trehalose synthase, recombinant vector or transgenic cell are tied up to and prepare trehalose synthase after above-mentioned transformation
In application.
Application of the above-mentioned trehalose synthase in trehalose is prepared.
Trehalose synthase of the present invention, by pseudomonas putida trehalose synthase (Pseudomonas putida
KT2440) obtain through molecular modification.Concrete grammar is：According to pseudomonas putida trehalose synthase three dimensional structure simulation, profit
With the method for PCR, design primer and intercepted the flex region in its three dimensional structure, obtain trehalose synthase rock-steady structure domain
Gene.The gene is connected on pET-15b carriers and is built into plasmid, and be transformed in e. coli bl21 DE (3) and carry out original
Nuclear expression.Then by affinity chromatography, Source-Q ion-exchange chromatographies, the method for Superdex-200 sieve chromatographies
Separate and obtain highly purified trehalose synthase.
Beneficial effect
Pseudomonas putida trehalose synthase is carried out molecular modification by the present invention first, is retaining its catalysis activity region
The flex region in its structure has been cut out simultaneously, so as to obtain a kind of new trehalose synthase.It is verified by experiments Sargassum after transformation
Synthase heat stability can improve at the same temperature 20% than original trehalose synthase, and expression also increases.This is
The industrialized production of trehalose is laid a good foundation.
Description of the drawings
Fig. 1 is pseudomonas putida trehalose synthase three dimensional structure simulation figure；
In figure：The darker regions of N-terminal and C-terminal are the flexible region at albumen two ends, and remaining light areas is the steady of the albumen
Constant domain part.
Fig. 2 is PCR amplifications and agarose gel electrophoresiies result photo；
In figure：M, Marker, 1, the purpose band of original trehalose synthase, 2, improved trehalose synthase (tres
Purpose band 42-680).
Fig. 3 is the standard specimen crest result figure of the glucose, maltose and trehalose that are determined by efficient liquid phase.
Fig. 4 is the curve chart that the trehalose synthase response time affects on conversion ratio after molecular modification.
Fig. 5 is trehalose synthase optimal reaction pH curves and pH stability curve figure after molecular modification；
Wherein, Fig. 5 A are trehalose synthase optimal reaction pH curves after transformation；Fig. 5 B are steady for trehalose synthase pH after transformation
Qualitative curve figure.
Fig. 6 is optimal reactive temperature curve and enzyme activity temperature stability curve chart before and after trehalose synthase transformation；
Fig. 7 is expressing quantity comparison diagram before and after trehalose synthase transformation；
In figure, M, Marker；1,2 is supernatant protein electrophoretogram after cell breakage before transformation；3,4 are cell breakage after transformation
Supernatant protein electrophoretogram afterwards；What arrow was indicated is destination protein.
Specific embodiment
Technical scheme is further elaborated with reference to embodiment, but institute's protection domain of the present invention is not limited to
This.
Embodiment 1：Clone obtains pseudomonas putida trehalose synthase (Pseudomonas putida KT2440) N42-
The trehalose synthase gene of C680 fragments
According to disclosed pseudomonas putida trehalose synthase (Pseudomonas putida KT2440) total length on NCBI
Aminoacid sequence carry out three dimensional structure simulation, analog result is as shown in figure 1, find trehalose synthase full-length proteins two ends tool
There are obvious flex region, i.e. N-terminal to the 41st glutamic acid and 680 isoleucine to C-terminal.Design primer gets rid of two ends
Flex region, primer sequence is as follows：
Forward primer：5’-atc gga tcc ATG GCCCAGCCCCGC-3’；
Downstream primer：5’-tca ctc gag tta CCGCAGGCACAG-3’；
With pseudomonas putida trehalose synthase (Pseudomonas putida KT2440) full-length gene as template, profit
Enter performing PCR amplification with above-mentioned primer, PCR reaction systems are as follows：
Above-mentioned PCR reactions are carried out according to following program：
95 DEG C of denaturations 5min；94 DEG C of degeneration 30s, 54 DEG C of annealing 30s, 72 DEG C of extension 4min, 28 circulations；72 DEG C of ends prolong
Stretch 10min.
PCR passes through 1% agarose gel electrophoresiies analytic plate segment length after terminating is short, and according to clip size purpose bar is cut
Band, carries out recovery and cuts glue product using the DNA purification kits of vast Tyke.
Embodiment 2：Trehalose synthase gene after transformation is transformed in expressive host, positive expression bacterial strain is obtained.
The double digestion reaction of PCR primer and plasmid vector
The enzyme action system of PCR primer：
Reaction condition：37 DEG C of 2～3h of reaction.
The enzyme action system of plasmid vector：
Reaction condition：37 DEG C of 6～8h of reaction.
The agarose gel electrophoresiies of product Jing 1% after PCR primer and carrier double digestion, and using DNA gel reclaim reagent
Box carries out purification recovery.
Coupled reaction system：
The several seconds is centrifuged after fully mixing, tube wall drop is received into ttom of pipe, 16 DEG C of connections overnight, are obtained connection product.
The conversion of recombiant plasmid
(1) preparation of competent cell
1. during picking BL21 single bacterium colonies (or picking preservation strain) are seeded to 10ml LB liquid mediums, 37 DEG C, 210rpm
Incubated overnight；
2. take 5ml bacterium solutions to be inoculated in 500ml LB culture medium, 37 DEG C, 210rpm shakes 70～80min to OD600Reach
0.375；
3. bacterium solution is positioned over into 10min on mixture of ice and water, while pre-cooling 50ml centrifuge tubes；
4. bacterium solution is transferred in centrifuge tube, 4 DEG C, 3700rpm, 10min collects thalline abandons supernatant；
5. activation buffer (the 0.1M CaCl of about 10ml ice pre-coolings are added in each centrifuge tube2), with sterilized
5ml rifle points break up precipitation, then again to the activation buffer of each Guan Zhongjia about 30ml ice pre-coolings, overturn and mix, quiet on ice
Put 20min；
6. 4 DEG C, 3700rpm is centrifuged 10min；Supernatant is abandoned, residual liquid evacuation is stored by the pre-cooling of 500ml bacterium solution 12ml ice
buffer(0.1M CaCl2, 15% glycerol) amount, precipitation is broken up, (shift by several times, then pressure-vaccum is broken up).
7. competence is dispensed in the sterilizing EP of ice pre-cooling, often 100 microlitres of pipe, is placed in and (prepares a basin frozen water to mix on ice
Compound).
8. -80 DEG C of competence is frozen, and competent cell is obtained.
Note：Whole process allows cell in low temperature, rifle point used as far as possible, and centrifuge tube, EP pipes and buffer etc. are intended to
Sterilizing, whole process operates all in super-clean bench, and competent cell wants its efficiency of test and whether microbiological contamination after finishing.
(2) connection product conversion
During 1. 15 μ L connection products to be added the competent cell BL21DE (3) of the fresh preparations of 100 μ L, gently mix, ice
Bath 30min.
2. 42 DEG C of heat shock 90s, are then immediately placed in ice bath and cool down 3min.
3. 200 μ L LB culture medium are added, 37 DEG C, 180rpm/min shaken cultivation 60min makes antibacterial restore normal growth
State, and the antibiotics resistance gene of expression plasmid coding；
4. the μ L of above-mentioned bacterium solution 200 are taken, is coated with resistant LB solid mediums (ampicillin 100mg/L).
5. after bacterium solution is blotted, it is inverted flat board and cultivates 12～16h in 37 DEG C.
The identification of positive colony
(1) bacterium colony PCR identifications
Picking single bacterium colony, 37 DEG C of 6～8h of shaken cultivation draw 1 μ L bacterium solutions, according to 15 μ L PCR reaction systems, enter performing PCR
Identification.If positive colony, by agarose gel electrophoresiies a purpose band is can detect that.
(2) protein expression and solubility are identified
Bacterium colony PCR is identified the IPTG (isopropylthiogalactoside) that final concentration of 0.6mM is added in remaining bacterium solution, is lured
Expression 1h, 12000rpm/min are led, 1min is centrifuged, abandon supernatant, collects thalline.2 times of sample-loading buffers are added (to give birth to work purchased from Shanghai
Albumen sample-loading buffer), hang precipitation, 90 DEG C of degeneration 10min with rifle point.It is detectable by SDS-PAGE if positive colony
To there is protein expression.
(3) DNA sequencing
By two methods of the positive colony after being identified with the above, Jing sequencings, the nucleoside inserted in the positive colony for obtaining
Nucleotide sequence of the acid sequence as shown in SEQ ID NO.1.
Embodiment 3：Fermentation culture positive expression bacterial strain, isolates and purifies trehalose synthase recombiant protein after transformation
Seed culture：In conventional manner picking positive colony is placed in the LB liquid containing 100mg/L ampicillin of 5mL
In culture medium, in 37 DEG C of shaken cultivation 5-6h；
Ultrasonication somatic cells：Ultrasonic 3s, is spaced 6s, 400W, works 60 times.
Ultracentrifugation：Cell breakage liquid after ultrasound is centrifuged 45min in 14000rpm under the conditions of 4 DEG C, collect supernatant
Liquid, carries out isolating and purifying for next step.
Ni-NTA affinity chromatographs：The supernatant fluid containing soluble protein collected is poured into the Ni-NTA posts for having regenerated
In；After supernatant stream is net, with wash buffer (25mM Tris-HCl, pH8.0,100mMNaCl, 15mM imidazoles) 10 are rinsed
Column volume, removes the albumen of non-specific adsorption；Finally using elution buffer (25mM Tris-HCl, pH8.0,
100mM NaCl, 250mM imidazoles) destination protein is eluted, collected with clean pre-cooling beaker；Examined using SDS-PAGE electrophoresis
Survey albumen it is whether solvable, whether solvable albumen can be combined with Ni-NTA, if can be eluted and albumen concentration.
Anion-exchange chromatography purification (Source-Q)：The soluble protein solution that Ni-NTA affinity chromatographs system is taken off
A (25mM Tris-HCl, pH8.0) dilutes 3～4 times, is then loaded to the ion exchange column for balancing using solution A
On SourceQ, using solution A and solution B (25mM Tris-HCl, pH8.0,1M NaCl) linear gradient elution is carried out.Observation
Light absorption value (A280) situation of change of 280nm, collects each collecting pipe gone out near peak position, and carries out SDS-PAGE electrophoresis,
To obtain target protein.
Molecular sieve purification：According to the shape of ion exchange column protein peak, whether there is " shoulder " and whether it is symmetrical, sharply sentencing
Character of the disconnected protein on ion exchange column.The protein good for character is concentrated by ultrafiltration to 2mL, is loaded to and has been used
On the gel permeation chromatography post Superdex-200 that solution C (25mM Tris-HCl, pH8.0,100mM NaCl) has been balanced, stream
Speed is 0.4mL/min.Collect protein peak and carry out SDS-PAGE electrophoresis, detect the purity and character of protein.Jing is sequenced, amino
Acid sequence is as shown in SEQ ID NO.2.
Embodiment 4：Trehalose synthase enzyme activity determination method
20% maltose, 20mM Na are added in reaction system2HPO4-NaH2PO4Buffer solution pH7.0,1 μM of Amur vacation
Zymomonas mobiliss (Pseudomonas stutzeri Qlu3) trehalose synthase, at 37 DEG C 2h is reacted.By the method for high-pressure liquid phase
The conversion ratio of trehalose is determined, nh 2 column is adopted in continuous mode；Column temperature is that 40 DEG C of mobile phases are molten with the mixing of water using acetonitrile
Liquid, the two volume ratio is 3:1；Flow velocity is 1mL/min；Detector is Composition distribution；Detection time is 25min.Standard product examine
Survey result and see that Fig. 3 calculates conversion ratio according to equation below：
According to maltose peak area in efficient liquid phase result, trehalose peak area and glucose peaks area are fitted using software
Obtain curve, calculate the quality of three, wherein m3 is the quality for being converted into trehalose, m2 is the quality for being converted into glucose, m1
For the quality of remaining maltose.
Embodiment 6：Trehalose synthase optimum reacting time is determined after transformation
Reaction system for 20wt% maltose, 20mM Na2HPO4-NaH2PO4, pH7.2,1 μM of trehalose synthase restructuring
Albumen, reacts at 37 DEG C, and in 2h, 3h, 4h, 5h, 6h, 7h, 8h, 10h, 12h different time is sampled, then using HPLC
Detected, detection method is shown in embodiment 4.By being calculated conversion ratio.As a result show, reaction carries out 8h and reaches balance, turn
Rate can reach 60% (as shown in Figure 4).
Embodiment 5：Trehalose synthase optimum pH and pH Stability Determinations after transformation
The measure of optimal reaction pH value：
Use pH5.0,5.5,6.0,6.5,7.0,7.5,8.0,8.5,9.0,9.5,10,0.10.5,11.0.A series of phosphorus
Phthalate buffer dilutes trehalose synthase of recombinating after transformation.It is subsequently adding 20% maltose and is placed in 37 DEG C of reaction 8h, determines enzyme activity.
As a result as shown in Figure 5A.The optimal reaction pH of trehalose synthase is 8.0 after transformation.
The measure of pH stability：
Enzyme liquid is dilute with pH5.0,5.5,6.0,6.5,7.0,7.5,8.0,8.5,9.0,9.5,10,0.10.5,11.0
After releasing certain multiple, in 37 DEG C, after water-bath 30min, add 20% maltose to be placed in 37 DEG C of reaction 8h, determine enzyme activity.As a result such as
Shown in Fig. 5 B.Trehalose synthase is stable in pH7.0-8.5 enzyme activity.
Comparative example
Remove the flex region in structure due to improved trehalose synthase so that protein is more stable, its temperature
Stability increases.Trehalose synthase enzyme liquid is diluted after suitable multiple before and after transforming, in 20 DEG C, 25 DEG C, and 30 DEG C, 35
DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 30min is incubated in 60 DEG C, determine trehalose conversion ratio.As a result it is as shown in Figure 6.As a result table
Albumen increases with original trehalose synthase in same reaction temperature stability inferior after bright transformation described herein.Original sea
Algae sugar synthase conversion ratio at 50 DEG C only has 50%, and trehalose synthase still keeps 80% in 50 DEG C of conversion ratios after transforming.
The escherichia coli of trehalose synthase before and after expression transformation are cultivated at identical conditions and induced, ultrasound is broken
After broken, supernatant is carried out into protein electrophorese, as a result show that expression of the improved trehalose synthase in escherichia coli is obvious
Improve.As a result it is as shown in Figure 7.
Claims (8)
1. a kind of expressing gene of high temperature resistant trehalose synthase, nucleotide sequence is as shown in SEQ ID NO.1.
2. a kind of high temperature resistant trehalose synthase, aminoacid sequence is as shown in SEQ ID NO.2.
3. a kind of recombinant vector, inserts the expressing gene of high temperature resistant trehalose synthase described in claim 1 in plasmid.
4. recombinant vector as claimed in claim 3, it is characterised in that the plasmid is pET-15b vector plasmids.
5. a kind of transgenic cell line, converts recombinant vector described in requirement 3 of having the right in host cell.
6. transgenic cell line as claimed in claim 5, it is characterised in that the host cell is e. coli bl21 DE (3).
7. described in recombinant vector described in the expressing gene of trehalose synthase described in claim 1, claim 3 or claim 5
Transgenic cell ties up to the application prepared in trehalose synthase.
8. application of the trehalose synthase described in claim 2 in trehalose is prepared.
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