CN104805104A - Trehalose synthase and coding gene and application thereof - Google Patents
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Abstract
The invention relates to a trehalose synthase and a coding gene and application thereof. A nucleotide sequence of an expression gene of the trehalose synthase is shown in SEQ ID No. 1; the amino acid sequence of the trehalose synthase is shown in SEQ ID No. 2. According to the trehalose synthase and the coding gene and application thereof, the trehalose synthase is obtained from pseudomonas stutzeri for the first time, and a preparation method of the trehalose synthase is simple and convenient and is high in yield and high in purity; proved by experiments, the thermal stability of the trehalose synthase is good, the trehalose conversion ratio can reach 70% in 1-hour reaction, and the reaction time is greatly shortened compared with that of the existing trehalose synthase, so that the production cost of trehalose can be reduced, and a foundation is laid for the industrial production of trehalose.
Description
Technical field
The present invention relates to a kind of trehalose synthase and encoding gene thereof and application, belong to biological technical field.
Background technology
Trehalose (Trehalose) be a kind of by two pyranoid ring glucose molecules through α-1, the non-reducing disaccharide that 1-glycosidic link connects and composes, is extensively present in the organisms such as bacterium, yeast, filamentous fungus, plant, insect, invertebrates.Research shows, its stable in properties, has very important biological significance to organism.Be mainly manifested in the reserve that it is the organism energy and carbon source; stablizer in the severe environment such as dehydration, high temperature, oxyradical, low temperature of protein and biomembrane molecule and protective material; sensing mixture and growth regulatory factor; but also be one of component of some bacteria cell wall, the good reputation of " sugar of life " is therefore had at scientific circles' trehalose.Trehalose has magical provide protection to organism; because trehalose can form the protective membrane of uniqueness under the severe environmental conditions such as high temperature, high and cold, high osmotic pressure and dry dehydration at cell surface; protected protein matter molecule unchangeability inactivation effectively, thus the vital process of the body that sustains life and biological characteristic.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 (EC5.4.99.16, Trehalose synthase, TreS) be in a kind of molecule glucuronosyltransferases it only need single step reaction just the α of maltose-Isosorbide-5-Nitrae glycosidic link can be converted into α-1,1 glycosidic link generate 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 are had basic common ground: one is that substrate conversion efficiency is lower, and the highest only have about 80%, is generally 60%-70%; Two is that the thermostability of enzyme is poor, and optimal reactive temperature is about 25 DEG C; Three is that the reaction times is oversize, and be generally 48 hours, what have reaches 72h.
Summary of the invention
For the deficiencies in the prior art, the invention provides a kind of Heat stability is good, the trehalose synthase of what the reaction times shortened greatly derive from Pseudomonas stutzeri (Pseudomonas stutzeri Qlu3) and expressing gene thereof and application.
Technical solution of the present invention is as follows:
An expressing gene for trehalose synthase, nucleotide sequence is as shown in SEQ ID NO.1.
A kind of trehalose synthase, aminoacid sequence is as shown in SEQ ID NO.2.
Trehalose synthase of the present invention is by after intestinal bacteria High level prokaryotic expression, utilizes affinity chromatography, ion exchange chromatography, and a series of means of purification such as sieve chromatography, finally obtains.Pseudomonas stutzeri (Pseudomonas stutzeri CJ38) the trehalose synthase amino acid sequence homology of this trehalose synthase and bibliographical information is 98%, but its reaction times shortens and good thermal stability greatly.Above-mentioned recombinant protein reaction 1h can reach molecular balance point, and trehalose transformation efficiency is 70%.Above-mentioned recombinant protein places 30min trehalose transformation efficiency in 50 DEG C of metal baths still can reach 50%.Above-mentioned recombinant protein optimal reactive temperature is 35 DEG C.Above-mentioned recombinant protein optimal reaction pH is 8.0.
A kind of recombinant vectors inserting nucleotide sequence shown in above-mentioned SEQ ID No.1.
A kind of transgenic cell line, containing above-mentioned recombinant vectors.
The expressing gene of above-mentioned trehalose synthase, recombinant vectors or transgenic cell tie up to the application prepared in trehalose synthase.
Above-mentioned trehalose synthase is preparing the application in trehalose.
Trehalose synthase of the present invention, obtain by Pseudomonas stutzeri (Pseudomonas stutzeri Qlu3), concrete grammar is: utilize degenerated primer to increase from Pseudomonas stutzeri (Pseudomonas stutzeri Qlu3) genome and obtain the full-length gene of trehalose synthase.This gene is connected on pET-15b carrier and is built into plasmid, 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 trehalose synthase.
Beneficial effect
The present invention obtains trehalose synthase by Pseudomonas stutzeri (Pseudomonas stutzeri Qlu3) first, and preparation method is easy for this trehalose synthase, and output is large, and purity is high; Experiment confirms its Heat stability is good, and reaction 1h trehalose transformation efficiency can reach 70%, and the more existing trehalose synthase reaction times shortens greatly, can reduce the production cost of trehalose, for the suitability for industrialized production of trehalose is laid a good foundation.
Accompanying drawing explanation
Fig. 1 is pcr amplification and agarose gel electrophoresis result photo;
In figure: M, Marker, T is the object band of trehalose synthase.
Fig. 2 is trehalose synthase SDS-PAGE electrophorogram result photo after molecular sieve purification;
In figure: M is Marker; B11, B12, C1, C2, C3, C4, C5, C6, C7 are the sample that after molecular sieve purification, different collection tube is collected;
Fig. 3 is the standard specimen crest result figure of glucose, maltose and the trehalose measured by high performance liquid phase;
Fig. 4 is the crest result figure being terminated rear glucose, trehalose and maltose by high performance liquid phase assaying reaction;
Fig. 5 is the graphic representation that affects transformation efficiency of trehalose synthase reaction times after purifying;
Fig. 6 is the graphic representation affected transformation efficiency in the trehalose synthase reaction times of article report;
Fig. 7 is trehalose synthase enzyme optimal reactive temperature curve alive and enzyme temperature-stable graphic representation alive after purifying;
Wherein, Fig. 7 A is the optimal reactive temperature curve that after purifying, trehalose synthase enzyme is lived;
Fig. 7 B is trehalose synthase enzyme temperature-stable curve alive after purifying;
Fig. 8 is trehalose synthase optimal reaction pH curve and pH curve of stability figure after purifying;
Wherein, Fig. 8 A is trehalose synthase enzyme optimal reaction pH alive curve after purifying;
Fig. 8 B is the trehalose synthase enzyme pH curve of stability alive after purifying.
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 Pseudomonas stutzeri (Pseudomonas stutzeri Qlu3) trehalose synthase gene
According to Pseudomonas stutzeri (Pseudomonas stutzeri) trehalose synthase full length nucleotide sequences Design degenerated primer disclosed on NCBI, primer sequence is as follows:
Upstream primer: 5 '-atc gga tcc atg agc ahn cca gac aah anc tat atc-3 ';
Downstream primer: 5 '-tca ctc gag tta ran cac cgg hgr-3 ';
With the genome of Pseudomonas stutzeri (Pseudomonas stutzeri Qlu3) 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:
95 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 54 DEG C of annealing 30s, 72 DEG C extend 4min, 28 circulations; 72 DEG C of ends extend 10min.
Agarose gel electrophoresis analytic plate segment length by 1% after PCR terminates is short, cuts object band according to clip size, uses the DNA purification kit of vast Tyke to carry out recovery and cuts glue product.
Embodiment 2: be transformed into by trehalose synthase gene in expressive host, obtains positive expression bacterial strain.
The double digestion reaction of PCR primer and plasmid vector
The enzyme of PCR primer cuts system:
Reaction conditions: 37 DEG C of reaction 2 ~ 3h.
The enzyme of plasmid vector cuts system:
Reaction conditions: 37 DEG C of reaction 6 ~ 8h.
Product after PCR primer and carrier double digestion through 1% agarose gel electrophoresis, and uses DNA gel to reclaim test kit to carry out purifying recovery.
Ligation system:
The centrifugal several seconds after abundant mixing, receive at the bottom of pipe by tube wall drop, 16 DEG C of connections are spent the night, obtained connection product.
The conversion of recombinant plasmid
(1) preparation of competent cell
1. the mono-bacterium colony of picking BL21 (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 500ml LB 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. activation damping fluid (the 0.1M CaCl 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 (0.1M CaCl by the precooling of 500ml bacterium liquid 12ml ice
2, 15% glycerine) amount, precipitation is broken up, (gradation shift, then pressure-vaccum is broken up).
7. competence is dispensed in the sterilizing EP of ice precooling, often pipe 100 microlitre, is placed on ice (preparing a basin mixture of ice and water).
8. competence-80 DEG C is frozen, obtained competent cell.
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 L LB substratum, 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.
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.
(2) protein expression and solubility qualification
Identified by bacterium colony PCR in residue bacterium liquid and add the IPTG (isopropylthiogalactoside) that final concentration is 0.6mM, abduction delivering 1h, 12000rpm/min, centrifugal 1min, abandons supernatant, collects thalline.Add 2 times of sample-loading buffers (purchased from Shanghai raw work albumen sample-loading buffer), hang precipitation, 90 DEG C of sex change 10min with rifle point.If positive colony, protein expression can be detected by SDS-PAGE.
(3) DNA sequencing
By with the positive colony after above two kinds of methods qualification, through order-checking order-checking, the nucleotide sequence of the nucleotide sequence inserted in the positive colony obtained as shown in SEQ ID NO.1.
Embodiment 3: fermentation culture positive expression bacterial strain, separation and purification trehalose synthase recombinant protein
Seed culture: picking positive colony is placed in the LB liquid nutrient medium containing 100mg/L penbritin of 5mL in conventional manner, 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/L penbritin, when 37 DEG C of shaking culture are 1.0 to the dense OD600 of bacterium, is cooled to 15 DEG C; Add the IPTG that final concentration is 0.6mM 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, pH8.0,100mM NaCl), vibration precipitation somatic cells; Adding proteinase inhibitor PMSF (Phenylmethylsulfonyl fluoride, phenylformic acid sulfonic acid fluoride) is 2mM to final concentration.
Ultrasonication somatic cells: ultrasonic 3s, interval 6s, 400W, 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 Ni-NTA post regenerated; After supernatant liquor stream is clean, rinse 10 column volumes with wash buffer (25mM Tris-HCl, pH8.0,100mMNaCl, 15mM imidazoles), the albumen of removing non-specific adsorption; Finally use elution buffer (25mM Tris-HCl, pH8.0,100mM NaCl, 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.
Anion-exchange chromatography purifying (Source-Q): soluble proteins solution A (the 25mM Tris-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 (25mM Tris-HCl, pH8.0,1M NaCl) 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 (25mM Tris-HCl, pH8.0,100mM NaCl) 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 is as shown in SEQ ID NO.2.
Embodiment 4: trehalose synthase enzyme activity determination method
20% maltose is added, 20mM Na in reaction system
2hPO
4-NaH
2pO
4buffered soln pH7.0,1 μM of Pseudomonas stutzeri (Pseudomonas stutzeri Qlu3) trehalose synthase, reacts 2h at 37 DEG C.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 that figure (3) is according to following formulae discovery transformation efficiency:
According to maltose peak area in high performance liquid phase result (as Fig. 4), trehalose peak area and glucose peaks area utilize software matching to obtain curve, 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: trehalose synthase recombinant protein biochemical property 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.Result as shown in Figure 7 A.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, 50 DEG C, 55 DEG C, in 60 DEG C, be incubated 30min, measure trehalose transformation efficiency.Result as shown in Figure 4.Trehalose synthase is placed 30min and is still kept 64% enzyme to live in 50 DEG C of metal baths.
The mensuration of optimal reaction pH value:
With pH5.0,5.5,6.0,6.5,7.0,7.5,8.0,8.5,9.0,9.5,10,0.10.5,11.0.A series of phosphate buffered saline buffer dilution restructuring trehalose synthase.Then add 20% maltose and be placed in 37 DEG C of reaction 2h, measure enzyme and live.Result as shown in Figure 8 A.The optimal reaction pH of trehalose synthase is 8.0.
The mensuration of pH stability:
By enzyme liquid 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, after 11.0 dilution certain multiple, in 37 DEG C, after water-bath 30min, add 20% maltose and be placed in 37 DEG C of reaction 2h, measure enzyme and live.Result as shown in Figure 8 B.Trehalose synthase is lived stable at pH7.0-9.0 enzyme.
Embodiment 6: trehalose synthase optimum reacting time measures
Detect according to the condition that document (Cloning and expression of a trehalose synthase from Pseudomonas stutzeri CJ38in Escherichia coli for the production of trehalose) is reported, reaction system is the maltose of 20wt%, 20mM Na
2hPO
4-NaH
2pO
4, pH7.2,1 μM of trehalose synthase recombinant protein, reacts, samples at 5min, 20min, 40min, 1h, 2h, 3h different time at 37 DEG C, and then use HPLC to detect, detection method is shown in embodiment 4.By calculating transformation efficiency.Result shows, reaction is carried out 1h reaction and reached balance, and transformation efficiency can reach for 70% (as shown in Figure 5).And the homologous protein of bibliographical information reacts under the same conditions and carries out 19h and reach balance (see Fig. 6).
Comparative example
According to the reaction times measured under the condition that document (Cloning and expression of a trehalose synthase from Pseudomonas stutzeri CJ38in Escherichia coli for the production of trehalose) is reported, result show with the albumen homology described in the application be 98% homologous protein the reaction times is 19h (see Fig. 6) under the same conditions.
Claims (6)
1. an expressing gene for trehalose synthase, nucleotide sequence is as shown in SEQ ID NO.1.
2. a trehalose synthase, aminoacid sequence is as shown in SEQ ID NO.2.
3. one kind is inserted the recombinant vectors of nucleotide sequence shown in SEQ ID No.1 in claim 1.
4. a transgenic cell line, containing recombinant vectors according to claim 3.
5. transgenic cell described in recombinant vectors described in the expressing gene of trehalose synthase described in claim 1, claim 3 or claim 4 ties up to the application prepared in trehalose synthase.
6. trehalose synthase described in claim 2 is preparing the application in trehalose.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105524936A (en) * | 2016-02-02 | 2016-04-27 | 齐鲁工业大学 | Mutant trehalose synthase as well as expression gene and application thereof |
| CN106290606A (en) * | 2016-07-25 | 2017-01-04 | 河南省科学院生物研究所有限责任公司 | A kind of method of Quantitative detection trehalose synthase enzyme activity in antibacterial TreS approach trehalose producing strains screens |
| CN106636170A (en) * | 2016-09-21 | 2017-05-10 | 齐鲁工业大学 | Construction method and application of double-promoter self-induction type recombinant bacillus subtilis |
| CN106754486A (en) * | 2016-11-30 | 2017-05-31 | 山东隆科特酶制剂有限公司 | One plant height produces pseudomonad and its enzymatic production method of trehalose synthase |
| CN108048439A (en) * | 2017-11-20 | 2018-05-18 | 齐鲁工业大学 | A kind of preparation method and application of saltant type trehalose synthase |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105524936A (en) * | 2016-02-02 | 2016-04-27 | 齐鲁工业大学 | Mutant trehalose synthase as well as expression gene and application thereof |
| CN105524936B (en) * | 2016-02-02 | 2018-11-06 | 齐鲁工业大学 | The trehalose synthase and its expressing gene of a kind of mutation and application |
| CN106290606A (en) * | 2016-07-25 | 2017-01-04 | 河南省科学院生物研究所有限责任公司 | A kind of method of Quantitative detection trehalose synthase enzyme activity in antibacterial TreS approach trehalose producing strains screens |
| CN106636170A (en) * | 2016-09-21 | 2017-05-10 | 齐鲁工业大学 | Construction method and application of double-promoter self-induction type recombinant bacillus subtilis |
| CN106636170B (en) * | 2016-09-21 | 2019-10-15 | 齐鲁工业大学 | The construction method of double-promoter self-induction type recombined bacillus subtilis and application |
| CN106754486A (en) * | 2016-11-30 | 2017-05-31 | 山东隆科特酶制剂有限公司 | One plant height produces pseudomonad and its enzymatic production method of trehalose synthase |
| CN108048439A (en) * | 2017-11-20 | 2018-05-18 | 齐鲁工业大学 | A kind of preparation method and application of saltant type trehalose synthase |
| CN108048439B (en) * | 2017-11-20 | 2021-02-26 | 齐鲁工业大学 | Preparation method and application of mutant trehalose synthase |
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