CN106754820A - Albumen esterase E8 and its expression and purification, crystal structure and application - Google Patents

Albumen esterase E8 and its expression and purification, crystal structure and application Download PDF

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CN106754820A
CN106754820A CN201611154309.1A CN201611154309A CN106754820A CN 106754820 A CN106754820 A CN 106754820A CN 201611154309 A CN201611154309 A CN 201611154309A CN 106754820 A CN106754820 A CN 106754820A
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esterase
amino acid
spirals
acid section
crystal
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CN106754820B (en
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李继喜
郑军
匡思运
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Fudan University
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Abstract

The invention belongs to biological technical field, specially from deep-sea bacterium E4A9TAlbumen esterase E8 and its expression and purification, crystal structure and application.The esterase E8 has 241 amino acid, and molecular weight is 26.28kDa；Highly soluble expression, can obtain the esterase E8 of expression quantity high and enzyme activity high in bacterial strain is expressed；Substrate is p-nitrophenol butyrate（C4）When catalysis activity highest, esterase E8 still keeps greater activity in organic solvent.The crystal structure resolution ratio of esterase E8 reaches 1.5, and the structure is made up of 6 β-pleated sheets and 15 α spirals, wherein, the 77th serine, the disjunctor of the catalysis three composition zymetology activated centre that the 101st aspartic acid and the 222nd hyte propylhomoserin are constituted.The esterase the enzyme activity is high, it is with low cost, can be used for the field such as food processing and flavour of food products improvement, grease hydrolysis, the degreasing of leather silk spinning raw material, wastewater treatment, detergent industry, the three-dimensional structure of E8 can be with a wide range of applications in substrate transformation in industrial enzyme preparation and in terms of improving enzyme activity.

Description

Albumen esterase E8 and its expression and purification, crystal structure and application
Technical field
The invention belongs to biological technical field, and in particular to a kind of halmeic deposit albumen esterase E8, its vitro recombination table Up to purifying, crystal structure, and its application in industrial enzyme preparation.
Background technology
Esterase (esterase EC 3.1.1.1) is widely present in microorganism, can be catalyzed the hydrolysis of ester type compound And synthesis.EsterEnzyme has many excellent characteristics, such as with the chiral selection specificity of height, catalytic reaction do not need coenzyme and Co-factor, possesses wider substrate spectrum, high stability etc. is kept in organic solution so that esterase turns into weight in the industrial production The catalyst wanted.Esterase can be widely applied to chiral drug synthesis, food processing and flavour of food products improvement, grease hydrolysis, leather The fields such as silk spinning raw material degreasing, wastewater treatment, detergent industry.
The esterase of marine source generally has an advantageous property related to marine environment, for example temperature stability, salt tolerance, Alkali resistance, low temperature resistant and excellent chiral selectivity etc., therefore, the ester for showing unique characteristics is filtered out from marine microorganism Enzyme is just into an important directions of exploitation infant industry enzyme preparation.The present invention has obtained the egg of esterase E8 using the method for crystallization Bai Jingti has simultaneously parsed its three-dimensional structure.E8 crystal structures can have in substrate transformation in industrial enzyme preparation and in terms of improving enzyme activity It is widely applied prospect.
The content of the invention
Deep-sea bacterium E4A9 is derived from it is an object of the invention to provide one kindTEsterase E8, and gene work is carried out to it Journey is transformed, and the method for obtaining the high efficient expression, purifying and crystallization in Escherichia coli, and the offer esterase E8 the 1st is arrived The three-dimensional crystalline structure of the 241st amino acids, and its in the application of industrial aspect.
The present invention provides a kind of from deep-sea bacterium E4A9TEsterase E8, it has 241 amino acid, amino acid sequence As shown in SEQ.ID.NO.1, molecular weight is 26.28kDa.
The present invention will also be from deep-sea bacterium E4A9TEsterase E8 carry out genetic engineering transformation, by its full-length gene gram It is grand on the carrier of pET28b-sumo.Wherein, the sense primer of amplification esterase full genome is：
E8F (5 '-TCGCGGATCCATGGCTGTTTTTCTTCTG-3 ', BamHI), (SEQ.ID.NO.2)；
Anti-sense primer is：
E8R (5 '-TCCGCTCGAGTCATCCCACGATCTCCAGC-3 ', XhoI).(SEQ.ID.NO.3)；
Expression plasmid is pET28b-sumo, and method for transformation is CaCl2Conversion method, conversion bacterial strain is bacillus coli DH 5 alpha, table It is e. coli bl21 (DE3) plus up to bacterial strain.
Prioritizing selection of the present invention (but is not excluded for other expression systems using the procaryotic cell expression system of Escherichia coli System, is such as expressed in other bacteriums or other eukaryotic cells)；To the above albumen with the side of fusion protein Formula is expressed, and prioritizing selection His labels have obtained solubility expression and (but have been not excluded for other labels, such as MBP or GST is marked Label also have solubility expression).
The present invention is provided and derives from deep-sea bacterium E4A9TEsterase E8 expression and purification process, the method includes：Build The recombinant vector of the unmutated E8 full-length genes of fusion, converts Escherichia coli, so as to express the fusion with label with the carrier Albumen E8, destination protein is isolated by the albumen of above-mentioned acquisition by the specific method with the special tag recognition, is gone Except the albumen of the E8 for obtaining high-purity after label by the method for gel permeation chromatography again.
Wherein, level pad prioritizing selection the 50mM Tris-HCl, pH 8.0 of affinity chromatography；500mM NaCl；5- 10% glycerine；5-10mM imidazoles；2-5mMβ-Me；0.2mM PMSF, elution buffer prioritizing selection 50mM Tris-HCl, pH 8.0；500mM NaCl；5% glycerine；250mM imidazoles；2mMβ-Me.The buffer solution prioritizing selection 20mM of gel permeation chromatography Tris-HCl, pH7.4；100mM NaCl；2mM DTT.
The present invention also provides for coming from deep-sea bacterium E4A9TEsterase E8 be catalyzed ester-type hydrolysis in application.
Determined by esterase activity and shown, E8 esterases or the above-mentioned Host Strains that can express E8 esterases can be used for hydrolyzing short-chain fat Fat acid esters, such as C2-C10 short carbon chains fatty acid ester.Preferred SCFA fat is to nitro with C2-C10 short carbon chains Phenol ester, such as paranitrophenylacetic acid ester, p-nitrophenyl butyrate, p-nitrophenyl capronate, p-nitrophenyl caprylate and to nitre Catalysis activity highest when base benzene decylate etc., wherein substrate are p-nitrophenol butyrate (C4), enzyme activity reaches U/mg.
The esterase catalyzed temperature range for hydrolysis of E8 is 20~65 DEG C, preferably 45~55 DEG C；The pH value of the hydrolysis be 4.0~ 10.5, preferably 7.0~8.0.
Esterase E8 the enzyme activities are high, with low cost, can be used for food processing and flavour of food products improvement, grease hydrolysis, skin The fields such as the degreasing of leather silk spinning raw material, wastewater treatment, detergent industry.
The present invention provides the method for crystallising of above-mentioned esterase E8, and described method includes：Esterase E8 is concentrated into 10-40mg/ Ml, crystal growth condition is screened at 4-30 DEG C with vapor phase grafting, and prioritizing selection uses 15-20 DEG C.
The present invention provides the crystal of the good esterase E8 protein of diffraction property.
The present invention provides the three-dimensional structure of esterase E8 and its product, and the structure describes the secondary structure composition of esterase E8, Peptide chain trend, enterprise schema and three-dimensional molecular construction.Wherein, diffraction is carried out for esterase E8 albumen, obtains esterase E8 albumen Crystal diffraction data, with Indian medicinal plant Rauvolfia serpentina originate to poly- neuridine aldehydo-ester enzyme (PDB ID:2WFL) for model carries out structure elucidation, the three-dimensional structure of esterase E8 albumen is finally given.
In a particular embodiment, there is provided the crystal three-dimensional structure of esterase E8 (and its product p-nitrophenol), its Described in atom in crystal three-dimensional structure be at least one of atomic coordinates listed in subordinate list 1, it is or any with the seat The average root variance of the three-dimensional atomic coordinate of the main chain carbon skeleton of at least 40% amino acid residue is less than or equal in mark's Structure.
Present invention obtains the crystal three-dimensional structure of preferred esterase E8, resolution ratio isIts crystal structure space group is P21.Cell parameter is：α=γ=90 °, β=91.69 °.
In a specific embodiment, the three-dimensional structure of described esterase E8 has in a crystallography asymmetry unit Two protein moleculars.The structure is made up of 6 β-pleated sheets and 15 α spirals altogether, wherein, 6 described β-pleated sheets are located at center, institute 15 α for stating are spiraled about around β-pleated sheet.Wherein β-pleated sheet 1, i.e., the amino acid section containing Ala2-Met7, α spirals 1, i.e., Amino acid section containing Gly13-Trp17, α spirals 2, i.e., the amino acid section containing Lys18-Lys27, β-pleated sheet 2 contains The amino acid section of Thr30-Phe32, α spirals 3, i.e., the amino acid section containing Phe39-Ile46, α spirals 4, i.e., containing Gly51- The amino acid section of Glu66, β-pleated sheet 3, i.e., the amino acid section containing Val71-Ser77, α spirals 5, i.e., containing Tyr78-Val82's Amino acid section, α spirals 6, i.e., the amino acid section containing Ala83-Gly91, β-pleated sheet 4, the i.e. amino acid containing Arg93-Gly101 Section, α spirals 7, i.e., the amino acid section containing Arg110-Met115, α spirals 8, i.e., the amino acid area containing Ser119-Gly132 Section, α spirals 9, i.e., the amino acid section containing Gly140-His152, α spirals 10, i.e., the amino acid area containing Asp157-Leu168 Section, α spirals 11, i.e., the amino acid section containing Met173-Cys178, α spirals 12, i.e., the amino acid area containing Gly183-Ile187 Section, β-pleated sheet 5, i.e., the amino acid section containing Pro190-His198, α spirals 13, i.e., the amino acid area containing Met201-Leu211 Section, β-pleated sheet 6, i.e., the amino acid section containing Thr214-His222, α spirals 14, i.e., the amino acid area containing Ala223-Asp228 Section, α spirals 15, i.e., the amino acid section containing Pro229-Val240.
The present invention provides the mechanism of action that esterase E8 plays esterase hydrolyzed function.The crystal structure of described esterase E8, the 77 serines (Ser77), the catalysis that the 101st aspartic acid (Asp101) is constituted with the 222nd hyte propylhomoserin (His222) Three disjunctors constitute activated centre.
The present invention provides the amino acid residue that esterase E8 is located at activated centre, including the 77th serine, the 101st asparagus fern Propylhomoserin and the 222nd hyte propylhomoserin and by hydrogen bond action Stable conformation.
The three-dimensional structure of esterase E8 can have a wide range of applications in substrate transformation in industrial enzyme preparation and in terms of improving enzyme activity Prospect.
Brief description of the drawings
For the three-dimensional structure ribbon schematic diagram of esterase E8, (α spirals are represented Fig. 1 with purple；β-pleated sheet is represented with yellow；Randomly Then curling is represented with green；Structure includes 15 α spirals and 6 β-pleated sheets).Wherein, A：E8 monomers；B：E8 isomerization units (two Molecule).
Fig. 2 is the protein purification figure of esterase E8.Wherein, A：E8 Polyacrylamide Gel Electrophoresis figures；B：Gel filtration Chromatographic analysis figure (Superdex200 16/600, GE Healthcare).
Fig. 3 is esterase E8 substrate specificity figures.C2：P-nitrophenol acetic acid esters；C4：P-nitrophenol butyrate, C6：It is right Nitrophenol capronate, C8：P-nitrophenol caprylate, C10：P-nitrophenol decylate, C12：P-nitrophenol lauric acid/dodecanoic acid Ester, C14：P-nitrophenol myristinate.
Fig. 4 schemes for esterase E8 optimal reactions pH.
Fig. 5 is esterase E8 optimal reactive temperature figures.
Fig. 6 is esterase E8 heat endurance figures.
Fig. 7 is bivalent cation to esterase E8 activity influence figures.
Fig. 8 is organic solvent and detergent to esterase E8 activity influence figures.
Fig. 9 is the enzyme activity site schematic diagram of esterase E8.Enzyme activity site includes 77 serines, 101 aspartic acids and 222 Hyte propylhomoserin.
Specific embodiment
Present disclosure is further illustrated using specific embodiment below.
Herein below is to combine specific preferred embodiment further description made for the present invention, it is impossible to assert Specific implementation of the invention is confined to these explanations.For general technical staff of the technical field of the invention, On the premise of not departing from present inventive concept, some simple deduction or replace can also be made, should be all considered as belonging to of the invention Protection domain.
Embodiment 1, the expression of esterase E8, purifying
From deep-sea bacterium E4A9TEsterase E8, its amino acid sequence is as follows：
MAVFLLMHGGGMGGWTWKFMRDALESKGHTVFTPTFTGFGEREHLIGRDVGNAVHVTDIVNVLKYEDLSDVILVAHS YAGTVAPGVLAQAGDRVRRVIYLDAIVPHAGERIASMMGFASEEEAAGLDAMLDGGEGPIGSGVHEQQRAMAKDHPH MMDRNREKWLLDHLSDQPMRATSCVIPVGAESITHPVDYIAAEHTIMGMMHERAEKLGWTIHKHPGDHAFNVGDPEG LADMMLEIVG(SEQ.ID.NO.1)。
The full-length gene of esterase E8 is cloned on the carrier of pET28b-sumo by molecule clone technology, for expressing 6 fusion proteins of His of amino-terminal fusion, recombinant vector is transformed into e. coli bl21 (DE3) plus, 37 DEG C of vibrations Culture adds when reaching 0.5-0.8 to OD600 the IPTG of final concentration of 0.4-1.0mM to carry out induced expression, and expression condition is： 20-25 DEG C of shaken cultivation 8-16h.
The recombinant expression carrier that will be built is transformed into e. coli bl21 (DE3) plus the solubility for carrying out esterase E8 Expression.To be selection pressure with 50 μ g/ml kanamycins and 34 μ g/ml chloramphenicol, switching recombinant strains are in containing above-mentioned In the LB fluid nutrient mediums of selection pressure, 37 DEG C of shaken cultivations to OD600The IPTG of final concentration of 0.5mM is added to enter when reaching 0.8 Row induced expression, expression condition is：25 DEG C of shaken cultivation 8h.To OD600The IPTG of final concentration of 0.5mM is added to enter when reaching 0.8 Row induced expression.25 DEG C, 200rpm shaken cultivations, by the way that bacterium is collected by centrifugation after 8h to 10 hours, for purifying.
The expression bacterium that will be collected by centrifugation is with appropriate amount of buffer solution (50mM Tris, pH 8.0；500mM NaCl；5% glycerine； 10mM imidazoles；2mMβ-Me；0.2mM PMSF) outstanding bacterium, using low-temperature ultrahigh-pressure cell crushing instrument, (Guangzhou cumulative biotechnology has Limit company) cracking thalline, with 18000rpm high speed centrifugations 1h removals precipitation and other particulate contaminations.By centrifuged supernatant with After Ni-NTA affinity medias are combined, 50mM Tris, pH 8.0 are used；500mM NaCl；5% glycerine；50mM imidazoles；2mM β-Me's Wash buffer medium, removes foreign protein.Finally use 50mM Tris, pH 8.0；500mM NaCl；5% glycerine；250mM miaows Azoles；The eluent of 2mM β-Me elutes destination protein from affinity media, is gone with the digestions of Ulp I of albumen quality 1/500 Sumo-His labels, use 50mM Tris, pH 8.0；500mM NaCl；5% glycerine；10mM imidazoles；The buffer solution of 2mM β-Me is saturating Analysis reduces imidazole concentration, and after being combined with Ni-NTA affinity medias, collection is flowed through, and flowing through the concentration tube retained with 10kDa will elute Liquid is concentrated.Protein solution after concentration is further purified with the method for gel permeation chromatography (superdex200,16/600), The buffer solution for using is 20mM Tris, pH7.4；100mM NaCl；2mM DTT.The destination protein that will be eluted is concentrated It is 15-30mg/ml to concentration, in -80 DEG C of preservations, for crystallization experiment.
Embodiment 2, the analysis of esterase E8 substrate specificities
The substrate specificity analysis of esterase E8, using system：100mM phosphate buffers (pH 7.5), 1mM substrates, plus Enter 3 μ g pure enzyme proteins, the METHOD FOR CONTINUOUS DETERMINATION light absorption value A at 40 DEG C4052min.The substrate that uses of measure for：P-nitrophenol acetic acid Ester (C2), p-nitrophenol butyrate (C4), p-nitrophenol capronate (C6), p-nitrophenol caprylate (C8), to nitro Phenol decylate (C10), p-nitrophenol dodecanoate (C12), p-nitrophenol myristinate (C14).Show after measured, ester P-nitrophenyl phenolic ester (C2, C4, C6, C8, C10 and C12) enzyme E8 shorter to acyl group carbochain has catalysis activity, and wherein substrate is Catalysis activity highest during p-nitrophenol butyrate (C4), it is more difficult to hydrolyze p-nitrophenol myristinate (C14) (Fig. 2).As a result Show, esterase E8 has catalysis activity to the shorter lipid material of acyl group carbochain, the hydrolysis vigor for short-chain lipid is better than long-chain Lipid.
Embodiment 3, the analysis of esterase E8 optimum reaction conditionses
Esterase E8 optimal reactive temperatures are determined in the range of 25~55 DEG C.Concrete operations are：100mM phosphate buffers (pH 7.5), 1mM p-nitrophenol butyrates add 3 μ g pure enzyme proteins, respectively at 25,30,35,40,45,50,55 and 60 DEG C Under the conditions of METHOD FOR CONTINUOUS DETERMINATION light absorption value A4052min.Measurement result shows that the range of reaction temperature of E8 is 25~60 DEG C, optimal reaction Temperature is 25~55 DEG C (Fig. 3).
Esterase E8 optimal reactions pH is determined in the range of 3.0~10.0.Concrete operations are：Added in different pH buffer solutions 1mM p-nitrophenols butyrate and 3 μ g pure enzyme proteins, the METHOD FOR CONTINUOUS DETERMINATION light absorption value A at 50 DEG C4052min.It is slow that measure is used Fliud flushing is：100mM citric acid-sodium citrate buffer solutions (pH 3.0~6.0), 100mM potassium dihydrogen phosphates-sodium hydroxide buffer Liquid (pH6.0~8.0), 100mM Tris- hydrochloride buffers (pH 7.5~8.5) and 50mM 2- cyclohexylaminos ethyl sulfonic acid-hydrogen-oxygen Change sodium buffer solution (pH 8.5~10.0).Measurement result shows that esterase E8 optimal reactions pH is pH 7.5, in pH 5.0~9.5 In the range of active (Fig. 4).
Embodiment 4, esterase E8 zymetology stability analyses
Measure concrete operations of the bivalent cation to esterase E8 activity influences are：10mM is separately added into reaction system Co2+、Cu2+、Ca2+、Mg2+、Zn2+、Sr2+、Mn2+、Ni2+、Ba2+With ethylenediamine tetra-acetic acid (EDTA), enzymatic activity is determined.Survey enzyme activity System is：100mM Tris- hydrochloride buffers (pH 7.5), 1mM p-nitrophenol butyrate, 3 μ g pure enzyme proteins, at 50 DEG C METHOD FOR CONTINUOUS DETERMINATION light absorption value A4052min.Measurement result shows that esterase E8 activity can be by Ni2+And Cu2+Complete inhibition, in Sr2+With Remain to keep more strongly active (Fig. 5) in the presence of EDTA.
The measure concrete operations of organic solvent and detergent to esterase E8 activity influences are：It is separately added into reaction system 15% (v/v) organic solvent (isopropanol, acetonitrile, ethanol, methyl alcohol, acetone, dimethyl sulfoxide (DMSO) and dimethylformamide) and 1% goes Dirty agent (w/v or v/v) (SDS, polysorbas20, Tween 80 and Triton X-100) and then the activity of measure enzyme.Live body system is： 100mM phosphate buffers (pH 7.5), 1mM p-nitrophenol butyrates, 3 μ g pure enzyme proteins are inhaled in METHOD FOR CONTINUOUS DETERMINATION at 50 DEG C Light value A4052min.Measurement result shows that esterase E8 activity can be by SDS, DMF, acetonitrile, dimethyl sulfoxide (DMSO), methyl alcohol, Triton X-100, polysorbas20 and 80 suppress (Fig. 6).
The method for crystallising of embodiment 5, esterase E8
As above the good esterase E8 of method expression and purification is concentrated into concentration about 20-30mg/ml, crystalline reagents box is used (Crystal Screen Kit I/II, Index, Salt, PEG/Ion from companies such as Hampton Research etc.) makees It is the primary dcreening operation condition of crystal growth, is crystallized using hanging drop air plllutant method.The present inventor is in multiple different crystalline reagents Under the conditions of obtain primary crystalline.By optimizing and revising for later stage, preferably 0.1M imidazoles, pH7.0-8.0；0.1-0.2M Ca (Ac)2；Used as crystal growth condition, be collected into a set of resolution ratio is the buffer solution of 15%-25%PEG1000X-ray Diffraction data.
The Data Collection and structure elucidation of embodiment 6, esterase E8
After the crystal that will be prepared is using 25% glycerine as antifreezing agent treatment, freeze in liquid nitrogen.Crystal X-Ray Diffraction Data Collection is carried out in SSRF (SSRF) biological macromolecule crystal light beam line station (BL17U).Data processing is used HKL2000, structure elucidation with Indian medicinal plant Rauvolfia serpentina originate to poly- neuridine aldehydo-ester enzyme (PDB ID:It is 2WFL) model, using molecular replacement technique, using Refmac programs and is aided with Coot softwares and does further amendment, last solution Analysis obtains the crystal structure of esterase E8 albumen.One of ordinary skill in the art understands, in wherein described crystal three-dimensional structure Atom have in table 1 listed at least 40% atomic coordinates, or at least 40% ammonia in the crystal three-dimensional structure of 3D albumen The average root variance of the coordinate in atomic structure coordinate and the table 1 of the main chain carbon skeleton of base acid is less than or equal toAtom sit Mark, can be considered as having identical structure with 3D albumen.
Specifically, the crystal three-dimensional structure of described esterase E8, there is two albumen in a crystallography asymmetry unit Molecule.It is made up of 6 β-pleated sheets and 15 α spirals, wherein, 6 described β-pleated sheets are located at center, and 15 described α spirals enclose It is wound on around β-pleated sheet.Wherein β-pleated sheet 1, i.e., the amino acid section containing Ala2-Met7, α spirals 1, i.e., containing Gly13-Trp17's Amino acid section, α spirals 2, i.e., the amino acid section containing Lys18-Lys27, β-pleated sheet 2, the i.e. amino acid containing Thr30-Phe32 Section, α spirals 3, i.e., the amino acid section containing Phe39-Ile46, α spirals 4, i.e., the amino acid section containing Gly51-Glu66, β Folding 3, i.e., the amino acid section containing Val71-Ser77, α spirals 5, i.e., the amino acid section containing Tyr78-Val82, α spirals 6, Contain the amino acid section of Ala83-Gly91, β-pleated sheet 4, i.e., the amino acid section containing Arg93-Gly101, α spirals 7 contain The amino acid section of Arg110-Met115, α spirals 8, i.e., the amino acid section containing Ser119-Gly132, α spirals 9 contain The amino acid section of Gly140-His152, α spirals 10, i.e., the amino acid section containing Asp157-Leu168, α spirals 11 contain The amino acid section of Met173-Cys178, α spirals 12, i.e., the amino acid section containing Gly183-Ile187, β-pleated sheet 5 contains The amino acid section of Pro190-His198, α spirals 13, i.e., the amino acid section containing Met201-Leu211, β-pleated sheet 6 contains The amino acid section of Thr214-His222, α spirals 14, i.e., the amino acid section containing Ala223-Asp228, α spirals 15 contain The amino acid section of Pro229-Val240.
The atomic coordinates group of the albumen esterase E8 crystal of subordinate list 1 is as follows：
Crystal space group：P21。
Cell parameter is：α=γ=90 °, β=91.69 °.
I/σ：14.9(2.29)
Rwork/Rfree (%) is respectively：14.41 and 16.94
Resolving range50-1.50
Data integrity (%)：97.9
SEQUENCE LISTING
<110>Fudan University
<120>Albumen esterase E8 and its expression and purification, crystal structure and application
<130> 001
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 241
<212> PRT
<213>
<400> 1
Met Ala Val Phe Leu Leu Met His Gly Gly Gly Met Gly Gly Trp Thr
1 5 10 15
Trp Lys Phe Met Arg Asp Ala Leu Glu Ser Lys Gly His Thr Val Phe
20 25 30
Thr Pro Thr Phe Thr Gly Phe Gly Glu Arg Glu His Leu Ile Gly Arg
35 40 45
Asp Val Gly Asn Ala Val His Val Thr Asp Ile Val Asn Val Leu Lys
50 55 60
Tyr Glu Asp Leu Ser Asp Val Ile Leu Val Ala His Ser Tyr Ala Gly
65 70 75 80
Thr Val Ala Pro Gly Val Leu Ala Gln Ala Gly Asp Arg Val Arg Arg
85 90 95
Val Ile Tyr Leu Asp Ala Ile Val Pro His Ala Gly Glu Arg Ile Ala
100 105 110
Ser Met Met Gly Phe Ala Ser Glu Glu Glu Ala Ala Gly Leu Asp Ala
115 120 125
Met Leu Asp Gly Gly Glu Gly Pro Ile Gly Ser Gly Val His Glu Gln
130 135 140
Gln Arg Ala Met Ala Lys Asp His Pro His Met Met Asp Arg Asn Arg
145 150 155 160
Glu Lys Trp Leu Leu Asp His Leu Ser Asp Gln Pro Met Arg Ala Thr
165 170 175
Ser Cys Val Ile Pro Val Gly Ala Glu Ser Ile Thr His Pro Val Asp
180 185 190
Tyr Ile Ala Ala Glu His Thr Ile Met Gly Met Met His Glu Arg Ala
195 200 205
Glu Lys Leu Gly Trp Thr Ile His Lys His Pro Gly Asp His Ala Phe
210 215 220
Asn Val Gly Asp Pro Glu Gly Leu Ala Asp Met Met Leu Glu Ile Val
225 230 235 240
Gly
<210> 2
<211> 28
<212> DNA
<213>
<400> 2
tcgcggatcc atggctgttt ttcttctg 28
<210> 3
<211> 29
<212> DNA
<213>
<400> 3
tccgctcgag tcatcccacg atctccagc 29

Claims (10)

1. it is a kind of to derive from deep-sea bacterium E4A9TEsterase E8, it is characterised in that have 241 amino acid, amino acid sequence is such as SEQ. shown in ID. NO.1, molecular weight is 26.28kDa.
2. it is a kind of to derive from deep-sea bacterium E4A9 as claimed in claim 1TEsterase E8 expression and purification process, its feature It is, including：The recombinant vector of the unmutated E8 full-length genes of fusion is built, Escherichia coli is converted with the carrier, so as to express Fusion protein E8 with label, the albumen of above-mentioned acquisition is separated by the specific method with the special tag recognition Go out destination protein, obtain the albumen of the E8 of high-purity after removal label by the method for gel permeation chromatography again.
3. it is a kind of to derive from deep-sea bacterium E4A9 as claimed in claim 1TEsterase E8 method for crystallising, it is characterised in that bag Include：Esterase E8 is concentrated into 20-30mg/ml, using crystalline reagents box as crystal growth primary dcreening operation condition, it is meteorological using hanging drop Diffusion method is crystallized, and is collected into the X ray diffracting data that resolution ratio is 1.5.
4. described in a kind of claim 1 from deep-sea bacterium E4A9TEsterase E8 crystal three-dimensional structures, it is characterised in that should Crystal three-dimensional structure is obtained by the following method：Diffraction is carried out for esterase E8 albumen, the crystal diffraction number of esterase E8 albumen is obtained According to Indian medicinal plantRauvolfia serpentinaSource to poly- neuridine aldehydo-ester enzyme for model carries out structure solution Analysis, finally gives the three-dimensional structure of esterase E8 albumen；Atom therein has in subordinate list 1 listed at least 40% atomic coordinates, or The average root variance of the coordinate in the atomic structure coordinate of the main chain carbon skeleton of person's at least 40% amino acid and table 1 is less than or equal to 1.5Å。
5. the crystal three-dimensional structure of esterase E8 according to claim 4, it is characterised in that resolution ratio is 1.5；Wherein, institute The crystal structure space group for stating esterase E8 is P21, an asymmetry unit has two molecules；Cell parameter is：A=48.84, B=69.74, c=70.60, α=γ=90 °, β=91.69 °.
6. the crystal three-dimensional structure of esterase E8 according to claim 4, it is characterised in that in an asymmetric list of crystallography There are two protein moleculars in position；The structure is made up of 6 β-pleated sheets and 15 α spirals altogether, wherein, 6 described β-pleated sheets are located at Center, 15 described α are spiraled about around β-pleated sheet；Wherein, β-pleated sheet 1, i.e., the amino acid section containing Ala2-Met7, α Spiral 1, i.e., the amino acid section containing Gly13-Trp17, α spirals 2, i.e., the amino acid section containing Lys18-Lys27, β-pleated sheet 2, Contain the amino acid section of Thr30-Phe32, α spirals 3, i.e., the amino acid section containing Phe39-Ile46, α spirals 4 contain The amino acid section of Gly51-Glu66, β-pleated sheet 3, i.e., the amino acid section containing Val71-Ser77, α spirals 5, i.e., containing Tyr78- The amino acid section of Val82, α spirals 6, i.e., the amino acid section containing Ala83-Gly91, β-pleated sheet 4, i.e., containing Arg93-Gly101 Amino acid section, α spirals 7, i.e., the amino acid section containing Arg110-Met115, α spirals 8, i.e., containing Ser119-Gly132's Amino acid section, α spirals 9, i.e., the amino acid section containing Gly140-His152, α spirals 10, the i.e. ammonia containing Asp157-Leu168 Base acid section, α spirals 11, i.e., the amino acid section containing Met173-Cys178, α spirals 12, the i.e. ammonia containing Gly183-Ile187 Base acid section, β-pleated sheet 5, i.e., the amino acid section containing Pro190-His198, α spirals 13, the i.e. amino containing Met201-Leu211 Sour section, β-pleated sheet 6, i.e., the amino acid section containing Thr214-His222, α spirals 14, the i.e. amino acid containing Ala223-Asp228 Section, α spirals 15, i.e., the amino acid section containing Pro229-Val240；
Also, the disjunctor of the catalysis three composition activity that the 77th serine, the 101st aspartic acid and the 222nd hyte propylhomoserin are constituted Center.
7. deep-sea bacterium E4A9 is derived from as claimed in claim 1TEsterase E8 be catalyzed ester-type hydrolysis in application.
8. application according to claim 7, it is characterised in that described esters are short-chain aliphatic ester.
9. application according to claim 8, it is characterised in that the short-chain aliphatic ester is C2-C10 short carbon chains.
10. application according to claim 9, it is characterised in that described C2-C10 short-chain aliphatic esters are p-nitrophenyl Acetic acid esters, p-nitrophenyl butyrate, p-nitrophenyl capronate, p-nitrophenyl caprylate or p-nitrophenyl decylate.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109837259A (en) * 2019-02-24 2019-06-04 复旦大学 From the thermophilic esterase crystal three-dimensional structure and expression and purification method of marine bacteria
CN110256540A (en) * 2019-03-22 2019-09-20 复旦大学 Expression and purification, crystal structure and the application of mycobacterium tuberculosis ribosomal protein S7
CN111647577A (en) * 2020-05-22 2020-09-11 复旦大学 Three-dimensional structure of RNA helicase and application thereof
CN114736887A (en) * 2022-03-25 2022-07-12 上海威高医疗技术发展有限公司 Use of carboxylesterase

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105132392A (en) * 2015-09-12 2015-12-09 复旦大学 Abyssal sediment albumen esterase expression and purification, abyssal sediment albumen esterase crystal structure and application thereof
CN105132393A (en) * 2015-09-12 2015-12-09 复旦大学 Abyssal salt-tolerant alkali-resisting albumen esterase expression and purification, abyssal salt-tolerant alkali-resisting albumen esterase crystal structure and application thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105132392A (en) * 2015-09-12 2015-12-09 复旦大学 Abyssal sediment albumen esterase expression and purification, abyssal sediment albumen esterase crystal structure and application thereof
CN105132393A (en) * 2015-09-12 2015-12-09 复旦大学 Abyssal salt-tolerant alkali-resisting albumen esterase expression and purification, abyssal salt-tolerant alkali-resisting albumen esterase crystal structure and application thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JUN ZHENG ET AL.: "Enzymatic characterization and crystal structure of a novel carboxylesterase E8", 《中国生物化学与分子生物学会2016年》 *
XUE-WEI XU ET AL.: "Croceicoccus marinus gen. nov., sp. nov., a yellow-pigmented bacterium from deep-sea sediment, and emended description of the family Erythrobacteraceae", 《INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY》 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109837259A (en) * 2019-02-24 2019-06-04 复旦大学 From the thermophilic esterase crystal three-dimensional structure and expression and purification method of marine bacteria
CN109837259B (en) * 2019-02-24 2022-10-11 复旦大学 Thermophilic esterase crystal three-dimensional structure derived from marine bacteria and expression purification method
CN110256540A (en) * 2019-03-22 2019-09-20 复旦大学 Expression and purification, crystal structure and the application of mycobacterium tuberculosis ribosomal protein S7
CN110256540B (en) * 2019-03-22 2022-10-11 复旦大学 Expression and purification of mycobacterium tuberculosis ribosomal protein S7, crystal structure and application
CN111647577A (en) * 2020-05-22 2020-09-11 复旦大学 Three-dimensional structure of RNA helicase and application thereof
CN111647577B (en) * 2020-05-22 2023-02-10 复旦大学 Three-dimensional structure of RNA helicase and application thereof
CN114736887A (en) * 2022-03-25 2022-07-12 上海威高医疗技术发展有限公司 Use of carboxylesterase

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