CN104762276A - Esterase protein and encoding gene thereof as well as application of both esterase protein and encoding gene thereof - Google Patents
Esterase protein and encoding gene thereof as well as application of both esterase protein and encoding gene thereof Download PDFInfo
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Abstract
The invention discloses an esterase protein. The amino acid sequence of the esterase protein is shown in the SEQ ID No.2 (shown in the description). On the other hand, the invention further provides a gene for the encoding the esterase protein, the nucleotide sequence of the gene is a nucleotide sequence which can encode the amino acid sequence shown in the SEQ ID No.2. In addition, the invention further discloses application of both the esterase protein and the encoding gene thereof. The embodiment shows that the esterase protein provided by the invention has higher esterase activity and can effectively degrade nitrophenol ester.
Description
Technical field
The present invention relates to a kind of esterase protein, also relate to the encoding gene of this esterase protein, the recombinant vectors of the gene containing coding esterase protein and cell, the composition containing this esterase protein, and the application of described esterase protein and encoding gene thereof.
Background technology
Esterase (EsteraseE.C.3.1.1.1) is the common Ester hydrolysis enzyme of occurring in nature, and be prevalent in animal, plant and microorganism, wherein microbe-derived esterase kind is the abundantest.The esterase gene difference of different sources is large, and homology is not high, but on protein three-dimensional structure, have high similarity, i.e. α/β folding enzymes structure; Mechanism of catalytic reaction all follows the reaction mechanism of serine hydrolase, and namely catalyst structure domain comprises Serine-histidine-glutamic acid (or aspartic acid) triplet active sites and there is the feature conserved sequence G-X-S-X-G of serine hydrolase at serine proteinase activities location proximate.Esterase and lipase has high similarity in structure and catalytic mechanism, its key distinction: esterase is hydrolyzing short-chain soluble ester substrate mainly, follows typical Michaelis-Menton equation in catalytic process; And lipase can catalytic hydrolysis hydrophobicity longer chain fatty acid fat substrate, and show interfacial activity in catalytic process.
Microbe-derived esterase kind is the most various, according to their amino acid sequence similarity and Basic Biological Character, its system divides in 1999 was 8 large families by Arpigny; Wherein family one is lipase, and family two to eight is esterase.In the past few years along with the discovery of esterase gene new in a large number, 9 to 15 esterase families are proposed at present.
Esterase/lipase, due to advantages such as its protein molecular are stable, catalyzed reaction simply, does not rely on cofactor, substrate spectrum is wide, is widely used in food-processing, washing composition, and medicine, makeup, compound such as to prepare at the industry.The global industry enzyme output value reaches 2,000,000,000 2004 according to the preliminary statistics, and is increasing with the digit rate of annual 4-5%, and wherein the use of lipase accounts for 10% of total enzyme frequency.In recent years along with chipal compounds is applied widely in the industry such as pharmacy, agricultural chemicals, spices, liquid crystal, functional high-polymer, the characteristic that the chiral compound of esterase splits shows especially out more.Compared to lipase, esterase shows more significant effect in chiral separation, such as, utilize the chiral Naproxen Base of esterase NP to carry out fractionation and can obtain the corresponding body product that 99% optically pure transformation efficiency reaches 95%.
Summary of the invention
The present inventor, based on to the analysis from Glacier in Tibet soil, has found esterase protein and encoding gene thereof unexpectedly.In addition, present invention also offers recombinant vectors, the cell of the gene containing the described esterase protein of coding, and the composition containing described esterase protein, and their application.
First aspect, the invention provides a kind of esterase protein, and wherein, the aminoacid sequence of this esterase protein is as shown in SEQ ID No:2.
Second aspect, the invention provides a kind of gene of esterase protein of encoding, and wherein, the nucleotides sequence of this gene is classified as the nucleotide sequence of the aminoacid sequence shown in SEQ ID No:2 of can encoding.
The third aspect, the invention provides a kind of primer pair, and wherein, this primer pair comprises the upstream primer as shown in SEQ IDNo:3 and the downstream primer as shown in SEQ ID No:4.
Fourth aspect, the invention provides a kind of recombinant vectors, and wherein, this recombinant vectors contains gene as above.
5th aspect, the invention provides a kind of transgenic cell, and wherein, this transgenic cell contains gene as above.
6th aspect, the invention provides a kind of composition, and wherein, said composition contains esterase protein as above.
7th aspect, the invention provides esterase protein as above, gene as above, recombinant vectors as above, transgenic cell as above and composition as above in degrading nitrobenzene phenolic ester and/or the application in food, articles for washing, medicine, makeup.
As can be seen from embodiment, the esterase protein provided of the present invention has higher esterase activity, can degrading nitrobenzene phenolic ester effectively.And there is the characteristic of pH value tolerance, cold tolerance and salt tolerant widely.
Other features and advantages of the present invention are described in detail in embodiment part subsequently.
Accompanying drawing explanation
Accompanying drawing is used to provide a further understanding of the present invention, and forms a part for specification sheets, is used from explanation the present invention, but is not construed as limiting the invention with embodiment one below.In the accompanying drawings:
The SDS-PAGE electrophorogram of recombinant protein solution in each stage that Fig. 1 is expression and purification; M: albumen Marker; 1: contrast enzyme liquid; 2: recombinant bacterium crude enzyme liquid; The recombinant protein solution obtained after 3:Ni-NDA His-Bind Column first time purifying; The recombinant protein solution obtained after 4:Ni-NDA His-Bind Column second time purifying.
Fig. 2 shows the specific activity of recombinant protein to different substrate.
Fig. 3 shows recombinant protein specific activity at different temperatures.
Fig. 4 shows the specific activity of recombinant protein under different pH.
Fig. 5 shows the specific activity of recombinant protein under different N aCl concentration.
Embodiment
Below the specific embodiment of the present invention is described in detail.Should be understood that, embodiment described herein, only for instruction and explanation of the present invention, is not limited to the present invention.
First aspect, the invention provides a kind of esterase protein, and wherein, the aminoacid sequence of this esterase protein is as shown in SEQ ID No:2.
According to the present invention, place esterase protein can be obtained by synthetic, also can obtain its encoding gene by the aminoacid sequence of this esterase protein, and then carries out corresponding biological expression acquisition.
Based on this, second aspect, the invention provides a kind of gene of the as above esterase protein of encoding.
As well known to those skilled in the art, genetic codon has degeneracy, therefore, when understanding the aminoacid sequence of as above esterase protein, those skilled in the art according to the technique means of routine can obtain nucleotide sequence different and as above esterase protein encoding gene of can encoding.In preferred situation, the nucleotide sequence of described encoding gene is as shown in SEQ ID No:1.
The third aspect, the invention provides a kind of primer pair, and wherein, this primer pair comprises the upstream primer (5'-GCG as shown in SEQ ID No:3
gGATCCaTGACTGTGAATCCTC-3') downstream primer (5'-GCG and as shown in SEQ IDNo:4
gAGCTCgGACTATTTTTGTCCCAG-3').
Above-mentioned primer pair provided by the invention can the gene shown in specific amplification SEQ ID No:1.Described specific amplification refers to that described primer pair can increase the nucleic acid shown in SEQ ID No.1 produce the target stripe of specific size, and can not increase nucleic acid beyond the nucleic acid shown in SEQ ID No.1 or the amplification of described primer pair can not produce the target stripe of specific size.
In addition, according to the present invention, the gene fragment increased for the ease of this primer pair can be connected with carrier effectively, described primer pair is also designed with restriction enzyme site, upstream primer underscore part as shown in SEQ ID No:3 is the restriction enzyme site of BamHI, and the downstream primer underscore part shown in SEQ ID No:4 is the restriction enzyme site of SacI.
The present invention it should be noted that, restriction enzyme site in described primer provided by the invention can not as the restriction to primer of the present invention, those skilled in the art, according to the restriction enzyme site on the predetermined carrier be connected with the gene of described primer pair amplifies, can design the primer pair with different restriction enzyme site accordingly.
Fourth aspect, present invention also offers a kind of recombinant vectors, and wherein, this recombinant vectors contains gene as above.
According to the present invention, described recombinant vectors is specifically as follows recombinant expression vector.The recombinant expression vector of described gene can be contained with existing expression vector establishment.In order to strengthen transcribing of goal gene, when using described gene constructed recombinant expression vector, can add enhancement type promotor or the constitutive promoter of any one this area routine before its translation initiation codon, they can be used alone or are combined with other promotor.In addition, in order to strengthen transcribing and/or translating of goal gene further, when using gene constructed recombinant expression vector of the present invention, also transcriptional enhancer and/or translational enhancer can be used.
Those skilled in the art, but must be identical with the reading frame of encoding sequence it is understood that these enhanser regions can be ATG initiator codon or neighboring region initiator codon etc., to ensure the correct translation of whole sequence.
According to the present invention, above-mentioned promotor and enhanser can be promotor and the enhanser of this area routine, and in this not go into detail.
According to the present invention, described " carrier " can select various carrier known in the art, as commercially available various plasmids, clay, phage and retrovirus etc.Preferably, described carrier is pET28a plasmid.
According to the present invention, for the ease of the isolation and purification of target protein, when building described carrier, the nucleotide sequence of expressing label as shown in table 1 can also be added, in target protein, the label shown in table 1 is connected to N-terminal or the C-terminal of target protein.
The sequence of table 1 label
| Label | Residue | Sequence |
| Poly-Arg | 5-6(is generally 5) | RRRRR |
| Poly-His | 2-10(is generally 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
5th aspect, the invention provides a kind of transgenic cell, and wherein, this transgenic cell contains gene as above.
Described transgenic cell is preferably prokaryotic cell prokaryocyte, is more preferably intestinal bacteria.
According to the present invention, transgenic cell provided by the invention may be used for the preparation of described esterase protein, particularly, described transgenic cell can be intestinal bacteria, can cultivate under the condition of 35-40 DEG C, when OD value reaches 0.7-0.8, add inductor, under the condition of 28-32 DEG C, then induce the expression of goal gene.Described inductor is generally isopropylthiogalactoside (IPTG), and its final concentration can be such as 0.4-0.6mM.
6th aspect, the invention provides a kind of composition, and wherein, said composition contains esterase protein as above.
According to the present invention, described in described composition, the concentration of esterase protein has no particular limits, and can carry out concrete selection according to specific circumstances, in this not go into detail.
In addition, different according to predetermined purposes, composition provided by the invention can be prepared as different formulations, and is added with the compositions such as corresponding vehicle.Concrete being chosen as is conventionally known to one of skill in the art, and in this not go into detail.
7th aspect, the invention provides esterase protein as above, gene as above, recombinant vectors as above, transgenic cell as above and composition as above in degrading nitrobenzene phenolic ester and/or the application in food, articles for washing, medicine, makeup.
According to the present invention, described oil of mirbane phenolic ester is p-NP acetic ester, pnitrophenyl propionate, p-NP butyric ester, one or more in p-NP capronate, p-NP octanoate and p-NP decylate.
According to the present invention, esterase provided by the invention can any can not make the condition of its inactivation under use, such as, temperature can be 10-80 DEG C, be preferably 30-50 DEG C; PH value can be 4-9.5, is preferably 7-9; The highest tolerance can be not less than 2.5M, is preferably the sodium chloride concentration of 0.5-1.5M.
Further describe the present invention by the following examples.
The test method of unreceipted actual conditions in the following example, conveniently condition is carried out, such as the condition described in " molecular cloning: laboratory manual ", or according to the condition that the manufacturer of corresponding biological reagent advises.
Embodiment 1
The present embodiment is for illustration of the clone of coding esterase protein nucleotide sequence
(1) acquisition of goal gene
According to DNA recovery from soils of diverse composition(Zhou etc., ApplEnviron Microbiol62:316-322) the middle method recorded, draw the soil sample of glacier from Kano, Tibet and extract STb gene, purifying is carried out afterwards by pulsed field gel electrophoresis PFGE method, obtain the STb gene solution of purifying, the ultraviolet spectrophotometer measurement result of STb gene solution is: A260/A280=1.947, A260/A230=2.15.
Above-mentioned STb gene solution syringe abstracting method is cut off DNA and reclaims the DNA fragmentation of 36-48kb, with CopyControl Fosmid library kits (purchased from Epicentre) and according to its specification sheets, the DNA fragmentation of acquisition is connected on CopyControl carrier and forms Fosmid clone library, and Transformed E .coli, coat on the LB flat board of the paraxin containing 12.5 μ g ml-1 afterwards, cultivate 14-16h for 37 DEG C.Obtain nearly 10000 clones.Clone passes through the LB plate screening of the tributyrin containing 1%, obtains positive colony that has esterase activity, checks order and sequential analysis, obtain sequence shown in SEQ ID NO.1, called after estH9 through order-checking company to its Insert Fragment.
The aminoacid sequence of the albumen that sequence shown in SEQ ID NO.1 is expressed is as shown in SEQ ID NO.2, called after H9Est albumen, with the homology from the supposition albumen in Acinetobacter sp genome sequencing with 65-99%, and with identifying the esterase from Acinetobacter lowffii of protein characteristic, there is highest homology, identity is 67%.This enzyme can be attributed to esterase/lipase family IV according to esterase H9Est protein sequence.
(2) clone of goal gene
According to the nucleotide sequence design Auele Specific Primer pair of the esterase gene obtained in step (1), wherein, upstream primer is (5'-GCG as shown in SEQ ID NO.3
gGATCCaTGACTGTGAATCCTC-3', underscore part is the restriction enzyme site of BamHI), downstream primer is (5'-GCG as shown in SEQ ID NO.4
gAGCTCgGACTATTTTTGTCCCAG-3', underscore part is SacI restriction enzyme site).
With the above-mentioned recombinant vectors DNA with positive colony of esterase activity for template, use described Auele Specific Primer, and amplification kit (precious biological purchased from Dalian, article No. is D332C) is to carrying out pcr amplification.Amplification system is as follows:
50 μ l are supplemented to sterilized water.
PCR reaction conditions: 94 DEG C of denaturation 4min; 94 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C extend 30 seconds, 30 circulations; 72 DEG C extend 10min.
PCR primer 1% agarose gel electrophoresis detects output and specificity, and with DNA purification kit (purchased from sky root biochemistry) purifying.Checked order by purified product, sequence, as shown in SEQ ID NO.1, proves that primer pair of the present invention can specific amplification goal gene fragment.
Embodiment 2
The present embodiment is for illustration of the acquisition of esterase protein provided by the invention.
(1) structure of recombinant plasmid pETEst
PCR primer after the purifying obtained by BamHI and SacI double digestion embodiment 1, reaction system is: PCR primer (about containing the DNA of 30 μ g) each 5 μ l of 50 μ l, BamHI and SacI, reaction buffer (product carries) 10 μ l and deionized water 30 μ l, 37 DEG C of incubation 4h, agarose electrophoresis reclaims digestion products.Plasmid pET28a carries out double digestion under identical condition and electrophoresis reclaims.
Use T4DNA ligase enzyme (purchased from TAKARA, D2011A), and according to the record of its specification sheets, the PCR primer after reclaimed double digestion is connected with plasmid, is built into pETEst recombinant expression vector.By electroporated for this recombinant vectors e. coli bl21 (DE3).Then, recombinant bacterium is coated on the LB flat board containing 50 μ g/mL kantlex, cultivate 14-16h, obtain the conversion bacterial strain pETEst/E.coliBL21(DE3 of the recombinant expression vector containing SEQ ID NO.1 for 37 DEG C).
(2) expression and purification of esterase protein
1. by recombinant bacterium pETEst/E.coliBL21(DE3) join in 200ml LB liquid nutrient medium (containing 50 μ g/ml kantlex), 37 DEG C of shaking tables are cultivated;
2., when OD600 reaches about 0.8, adding inductor IPTG(final concentration is 0.5mM), 30 DEG C of overnight induction;
3. centrifugal 10 minutes of 6000rpm, collects thalline, by 1:6(1mg:6mL) add 50mMTris-HCl(pH8.0) damping fluid, ultrasonic disruption 10 minutes (60W, ultrasonic 2, stopping 2s);
4. the centrifugal 10min of 15000rpm, removes cell debris, obtains crude enzyme liquid.
(3) the Ni-NDA HisBind Column purifying of esterase protein
Crude enzyme liquid is flow through the NDA of the HisBind Column(band Ni that Novagen company produces, 1.25mL prepacked column).After crude enzyme liquid flows through chromatography column completely, rinse Ni post by 5ml solution A (imidazoles of the NaCl of the Tris-Cl of 20mM pH7.9,0.5M, 10mM) and remove unconjugated foreign protein.Use 10ml solution B (imidazoles of the NaCl of the Tris-Cl of 20mM pH7.9,0.5M, 60mM) rinsing Ni post to remove again and combine more weak foreign protein.Finally use the 5ml solution C (Tris-Cl of 20mM pH7.9, the NaCl of 0.5M, the imidazoles of 500mM) wash-out, collect elutriant 1, elutriant 1 is repeated purify once with Ni-NDA HisBind Column, obtains elutriant 2, then by elutriant 2 FPLC(fast protein liquid chromatography) desalination, obtain the sample after desalination, the esterase protein solution namely after purifying.
In each stage of expression and purification, the SDS-PAGE electrophoresis result of esterase protein solution is shown in Fig. 1, and wherein, M is albumen Marker; 1: the contrast crude enzyme liquid of contrast enzyme liquid (the only conversion carrying out processing according to embodiment 2 step (2) has the E.coliBL21(DE3 of pET28a)); 2: recombinant bacterium crude enzyme liquid; The recombinant protein solution obtained after 3:Ni-NDA His-Bind Column first time purifying; The recombinant protein solution obtained after 4:Ni-NDA His-Bind Column second time purifying.
Test case 1
Temperature is on the impact of the activity of esterase of the present invention.
Using nitrophenols butyric ester as esterase hydrolyzed substrate, reaction system is 1ml, buffer system is 50mM Tris-HCl(pH8.0), the final concentration of substrate is 0.2mM, esterase protein solution after the purifying that 10 μ l embodiments 2 obtain or contrast enzyme liquid, measure the change curve of the absorbance value of 405nm in 1 minute with DU800 ultraviolet-visible pectrophotometer (Beckman) under differing temps, 1M sodium chloride concentration.The results are shown in Figure 2.
Result shows, the optimum temperuture of esterase protein is 40 DEG C, and contrast enzyme liquid does not all have activity.Live as 100% with this highest enzyme, the specific activity of described esterase protein at other several temperature as shown in Figure 2.
Test case 2
PH is on the impact of the activity of esterase of the present invention.
Using p-NP butyric ester as esterase hydrolyzed substrate, reaction system is 1ml, buffer system is the one in A-C, the final concentration of substrate is 0.2mM, esterase protein solution after the purifying that 10 μ l embodiments 2 obtain or contrast enzyme liquid, 40 DEG C, measure the change curve of the absorbance value of 405nm in 1 minute under 1M sodium chloride concentration with DU800 ultraviolet-visible pectrophotometer (Beckman).The results are shown in Figure 3.
A: buffer system is sodium radio-phosphate,P-32 solution (pH6.0-8.0);
B: buffer system is Tris-HCl solution (pH8.0-9.0);
C: buffer system is Glycine-NaOH solution (pH9.0-10.0);
Result shows, esterase protein optimal pH is 8.0, and under pH7-9, this enzyme can play good catalytic activity.Contrast enzyme liquid does not all have activity.Live as 100% with this highest enzyme, the specific activity of described esterase protein under other several pH as shown in Figure 3.
Test case 3
Salt concn is on the impact of enzymic activity.
Using nitrophenols butyric ester as esterase hydrolyzed substrate, reaction system is 1ml, buffer system is 50mM Tris-HCl(pH8.0), the final concentration of substrate is 0.2mM, esterase protein solution after the purifying that 10 μ l embodiments 2 obtain or contrast enzyme liquid, 40 DEG C, measure the change curve of the absorbance value of 405nm in 1 minute under different salinity (NaCl of 0-2.5M) with DU800 ultraviolet-visible pectrophotometer (Beckman).The results are shown in Figure 4.
Result shows, the optimal salinity of esterase protein is 1M, and contrast enzyme liquid does not all have activity.Live as 100% with this highest enzyme, the specific activity of described esterase protein under other several salt concn as shown in Figure 4.
Test case 4
This test case is for illustration of the degraded of esterase protein p-nitrophenyl phenolic ester provided by the invention.
Respectively with p-NP acetic ester (C2), pnitrophenyl propionate (C3), p-NP butyric ester (C4), p-NP octanoate (C8), p-NP decylate (C10), p-NP cetylate (C16) is substrate.
Reaction system is 1ml, buffer system is 50mM Tris-HCl(pH8.0), the final concentration of substrate is 0.2mM, esterase protein solution after the purifying that 10 μ l embodiments 2 obtain or contrast enzyme liquid, 40 DEG C, under 1M sodium chloride concentration, measure the change curve of the absorbance value in 1 minute under 405nm with DU800 ultraviolet-visible pectrophotometer (Beckman).The results are shown in Figure 5.
Wherein, esterase protein p-NP butyric ester (short chain substrate) has the highest enzyme and lives, and contrast enzyme liquid does not all have activity.And live as 100% with this highest enzyme, described esterase protein is to the specific activity of other several substrate as shown in Figure 5.
More than describe the preferred embodiment of the present invention in detail; but the present invention is not limited to the detail in above-mentioned embodiment, within the scope of technical conceive of the present invention; can carry out multiple simple variant to technical scheme of the present invention, these simple variant all belong to protection scope of the present invention.
It should be noted that in addition, each the concrete technical characteristic described in above-mentioned embodiment, in reconcilable situation, can be combined by any suitable mode.In order to avoid unnecessary repetition, the present invention illustrates no longer separately to various possible array mode.
In addition, also can carry out arbitrary combination between various different embodiment of the present invention, as long as it is without prejudice to thought of the present invention, it should be considered as content disclosed in this invention equally.
Claims (10)
1. an esterase protein, is characterized in that, the aminoacid sequence of this esterase protein is as shown in SEQ ID No:2.
2. encode the gene of esterase protein, it is characterized in that, the nucleotides sequence of this gene is classified as the nucleotide sequence of the aminoacid sequence shown in SEQ ID No:2 of can encoding.
3. gene according to claim 2, wherein, the nucleotide sequence of this gene is as shown in SEQ IDNo:1.
4. a primer pair, is characterized in that, this primer pair comprises the upstream primer as shown in SEQ ID No:3 and the downstream primer as shown in SEQ ID No:4.
5. a recombinant vectors, is characterized in that, this recombinant vectors contains gene according to claim 2.
6. a transgenic cell, is characterized in that, this transgenic cell contains gene according to claim 2.
7. transgenic cell according to claim 6, wherein, described transgenic cell is prokaryotic cell prokaryocyte.
8. a composition, is characterized in that, said composition contains esterase protein according to claim 1.
9. esterase protein according to claim 1, gene according to claim 2, recombinant vectors according to claim 5, transgenic cell according to claim 6 and composition according to claim 8 are in degrading nitrobenzene phenolic ester and/or the application in food, articles for washing, medicine, makeup.
10. application according to claim 9, wherein, described oil of mirbane phenolic ester is p-NP acetic ester, pnitrophenyl propionate, p-NP butyric ester, one or more in p-NP capronate, p-NP octanoate and p-NP decylate.
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| CN105176943A (en) * | 2015-10-13 | 2015-12-23 | 福州大学 | Salt-tolerant and organic solvent-tolerant low-temperature alkaline esterase EstSL3 and gene and application thereof |
| CN105176943B (en) * | 2015-10-13 | 2018-09-18 | 福州大学 | The low-temperature alkali esterase EstSL3 and its gene of a kind of salt tolerant organic solvent-resistant and application |
| CN105368802A (en) * | 2015-12-09 | 2016-03-02 | 广东轻工职业技术学院 | Salt-tolerant esterase, coding gene of salt-tolerant esterase and application of salt-tolerant esterase |
| CN105368802B (en) * | 2015-12-09 | 2018-09-04 | 广东轻工职业技术学院 | A kind of salt tolerant esterase and its encoding gene and application |
| CN106834250A (en) * | 2016-10-08 | 2017-06-13 | 中国海洋大学 | A kind of esterase EstK1 albumen and its application |
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