CN102952787A - Acetylxylan esterase and application thereof - Google Patents
Acetylxylan esterase and application thereof Download PDFInfo
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- CN102952787A CN102952787A CN2012104036662A CN201210403666A CN102952787A CN 102952787 A CN102952787 A CN 102952787A CN 2012104036662 A CN2012104036662 A CN 2012104036662A CN 201210403666 A CN201210403666 A CN 201210403666A CN 102952787 A CN102952787 A CN 102952787A
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Abstract
The invention discloses DNA (Deoxyribose Nucleic Acid) of a novel acetylxylan esterase, which has a nucleotide sequence shown as SEQ AXENO.1 and an amino acid sequence shown as SEQAXENO.2. Highest activity is obtained when a protein structure is a homotrimer. The invention also discloses a cloning vector containing the DNA of the novel acetylxylan esterase, a cloning technology and application of a recombinant esterase to hydrolysis of a short-chain fatty acid and acyl saccharides and application of the recombinant esterase to production of deacetylated 7-aminocephalosporanic acid and deacetylated cephalosporin C which are important intermediates of beta-lactam antibiotics; and the novel acetylxylan esterase has an important industrial application value.
Description
Technical field
The invention belongs to gene engineering technology field, be specifically related to a kind of from subtilis (
Bacillus subtilis) in the novel esterases that obtains and expression vector, Recombinant esterase and Recombinant esterase in the 7-amino-cephalosporanic acid of the important intermediate-removal of acylation of hydrolysis short chain fatty acid fat, removal of acylation reaction and β-lactam antibitics and the application among the deacylated cephaloporins C.
Background technology
Acetyl xylan esterase (acetyl xylan esterase; abbreviation AXE) be that a class can and discharge the esterase of acetic acid with the xylopyranose removal of acylation of the acylations in xylan or the xylo-oligosaccharide; classify in Procaine esterase family 7; can be hydrolyzed short chain fatty acid and various acylations compound, have widely substrate specificity.
Now, acetyl xylan esterase shows huge scientific research value and industrial application value, is mainly reflected in following two aspects.On the one hand, acetyl xylan esterase plays an important role in the degraded of hemicellulose xylan.This enzyme and inscribe β-1; 4-zytase, β-D-xylosidase, α-L furan type arabinofuranosidase/xylosidase and α-glucose aldehydic acid enzyme consist of xylanolytic enzyme system together; can be hydrolyzed the hemicellulose xylan with complex construction; so that the inexhaustible renewable resources of development and utilization occurring in nature becomes possibility, the structure and function that obtains in recent years the ethanoyl xylan esterase of high vigor and resolve this enzyme has become the focus of research.On the other hand, it is active that acetyl xylan esterase has good arylsterase, can catalysis 7-amino-cephalosporanic acid and the synthetic β-lactam antibitics parent nucleus of cephalosporin deacylated tRNA base, have huge industrial application value.Beta-lactam antibiotics is one of main type in the microbiotic, and whole world consumption is crossed ten thousand tons.There are the problems such as cost is high, three-waste pollution is serious in the chemical method that uses at present, and the field of much making more and more tends to use enzyme process synthetic.
The gemma quasi-microorganism of the production acetyl xylan esterase of present known report has
Bacillus pumilus, Bacillus subtilisATCC6633
, Bacillus subtilis168,
Bacillus haloduransC-125 etc.Wherein, remove
Bacillus subtilisThe acetyl xylan esterase of ATCC6633 is in the news and has outside the higher pNPA enzyme activity (1580 U/mg), vigor and the performance of the acetyl wood sugar esterase of other bacterial strains are still relatively poor, have limited its widespread use in hemicellulose degraded and the synthetic beta-lactam antibiotics of enzyme process.Therefore, need a kind of highly active acetyl xylan esterase of exploitation, enlarge its use range.
Summary of the invention
Technical purpose of the present invention provides a kind of encoding amino acid sequence and corresponding nucleotide sequence thereof of novel acetyl xylan esterase.
Second technical purpose of the present invention provides the protein structure of acetyl xylan esterase, and be wherein, the highest with the activity of homo-hexamer.
The 3rd technical purpose of the present invention provides the expression vector of the encoding gene that comprises acetyl xylan esterase.
The 4th technical purpose of the present invention provides the method for preparing the acetyl xylan esterase of recombinating.
The 5th technical purpose of the present invention provides the restructuring acetyl xylan esterase in hydrolysis short chain fatty acid fat, removal of acylation reaction, the 7-amino-cephalosporanic acid of producing removal of acylation and the application among the deacylated cephaloporins C.
The present invention is achieved through the following technical solutions:
The present invention utilizes round pcr to amplify the Cah gene from the subtilis genomic dna, and the Cah gene is connected in expression vector pET22b multiple clone site place, obtains recombinant plasmid pET-Cah, recombinant plasmid pET-Cah is transformed again
E. coliBL21 (DE3), the intestinal bacteria that obtain recombinating namely produce the genetic engineering bacterium of acetyl xylan esterase
E. coliBL21
-PET-Cah.
Use the IPTG abduction delivering after the strain culturing, produce the restructuring acetyl xylan esterase, the restructuring acetyl xylan esterase is separated getting final product.Embodiment 1 and embodiment 2 are seen in detailed operating process.
According to technical scheme of the present invention, can reach good technique effect:
One, acetyl xylan esterase sequence of the present invention and bacillus amyloliquefaciens FZB42(GenBank:YP 001419972.1) the sequence homology of acetyl xylan esterase be 99%, have 10 bases, 2 amino acid to be different from the ethanoyl xylan esterase gene of bacillus amyloliquefaciens FZB42: the 240th L-Ala (A) by the GCC coding of amino acid position replaces with the Threonine (T) of ACC or ACA or ACG coding; 289 amino acids are replaced with the glutamine (Q) of CAA or CAG coding by the L-glutamic acid (E) of GAA coding.In addition, acetyl xylan esterase of the present invention with characterized
Bacillus subtilis168(GenBank:NP_388200.1),
Bacillus subtilisATCC6633(GenBank:ZP_06875135.1),
Bacillus pumilusCect5072(GenBank:2XLC_A) acetyl xylan esterase homology is respectively 84%, 84%, 73%; With
Thermobacillus compostiAcetyl xylan esterase (GenBank:ZP_08919340.1) homology is 59%, with
Thermotoga maritimaMSB8 acetyl wood sugar esterase (GenBank:NP_227893.1) homology 41%, with
Bacillus haloduransCephalosporin desaturase (GenBank:BAB07045.1) homology 35% of C-125.
Above-mentioned restructuring acetyl xylan esterase is carried out abduction delivering, obtained the aminoacid sequence shown in SEQ ID No:2.In the present invention, the nucleotide sequence that this aminoacid sequence is corresponding is shown in SEQ ID No:1.But; according to those skilled in the art's common practise as can be known; the coding of Nucleotide codon of an amino-acid residue of can encoding may have at least a; perhaps two kinds and more than; therefore; the protection domain of acetyl xylan esterase of the present invention should at first be as the criterion based on the aminoacid sequence shown in the SEQ ID No:2; its extended protection scope is any nucleotide sequence of the aminoacid sequence shown in the SEQ ID No:2 of can encoding out, and is not limited to the sequence shown in the nucleotide sequence shown in the SEQ ID No:1.
Two, acetyl xylan esterase of the present invention (AXE), its protein structure is homo-hexamer, but depolymerization is homotrimer, and homotrimer has certain activity.
Restructuring acetyl xylan esterase monomer molecule amount of the present invention is 36 kDa, and the tripolymer molecular weight is 107 kDa, and six polymer molecular amounts are 216 kDa.Acetyl xylan esterase is homo-hexamer under the state of nature, under 1% SDS effect, can depolymerization be homotrimer, and when existing without SDS, the rapid autohemagglutination of tripolymer is combined into six aggressiveness, and recovers the high reactivity of six aggressiveness.The prior art report
Bacillus subtilis168 and bacillus pumilus acetyl xylan esterase state of nature under also be homo-hexamer, molecular weight is respectively 220 kDa and 190 kDa, but its tripolymer existence form has no report.（Multifunctional Xylooligosaccharide/Cephalosporin C Deacetylase Revealed by the Hexameric Structure of the Bacillus subtilis Enzyme at 1.9 A Resolution. Florence Vincent, Simon J et al. 2003. J. Mol. Biol. (2003) 330, 593-606;The crystal structure of the cephalosporin deacetylating enzyme acetyl xylan esterase bound to paraoxon explains the low sensitivity of this serine hydrolase to organophosphate inactivation. Silvia Montoro-Garcia, Fernando Gil-Ortiz et al. 2011.Biochem. J 436,321-330）。
Bacillus subtilisThe acetyl xylan esterase of ATCC6633 is single aggressiveness, and molecular weight is 37 kDa(YesT:A new rhamnogalacturonan acetyl esterase from
Bacillus subtilis.Irene Martinez-Martinez, Jose Navarro-Fernandez et al. Proteins 2008; 71:379 – 388.).
Bacillus haloduransThe cephalosporin desaturase of C-125 is single aggressiveness, and molecular weight is 45kDa(Characterization of a New C-125 Rhamnogalacturonan Acetyl Esterase from
Bacillus haloduransWith a New Putative Carbohydrate Binding Domain. Jose Navarro-Fernandez, Irene Martinez-Martinez et al. J. Bacteriol.2008,190 (4): 1375.).
The dna sequence dna efficient soluble expression restructuring acetyl xylan esterase in Escherichia coli system that derives from subtilis of the present invention has widely substrate spectrum, can be hydrolyzed short chain fatty acid fat and multiple acylations compound.Original pNPA enzyme activity is 383.3 U/mL behind the broken born of the same parents of restructuring acetyl xylan esterase of the present invention; PNPA reaches 2950U/mg than vigor behind the partial purification, and the vigor of cephalosporin reaches 640 U/mg, is the high reactivity of the known report of bacillus, sees Table 1.
Restructuring acetyl xylan esterase of the present invention has very strong chain length specificity to the fatty acid ester of C2, and activity had been reduced to 0.09% when chain length increased to C4, rarely seen report.With known report
Thermobifida fuscaAcetyl xylan esterase relatively see Table 2.
Table 2. restructuring acetyl xylan esterase is to the chain length specificity of fatty acid ester
| C-2 | C-4 | C-6 | C-8 | C-10 | C16 | |
| AXE of the present invention | 705.3 | 0.65 | 0 | 0 | 0 | |
| AXE from Thermobifida fusca a | 22 | 8 | -- | 4 | -- | 2 |
[a], Yang, C.-H., & Liu, W.-H. (2008). Purification and properties of an acetylxylan esterase from
Thermobifida fusca. Enzyme and Microbial Technology, 42(2), 181-186.
Three, the present invention's claimed expression vector that comprises the encoding gene of acetyl xylan esterase of the present invention also.According to those skilled in the art's common practise, described expression vector should include but not limited to recombinant vectors, recombinant bacterium, transgenic cell line, expression cassette or recombinant virus; In an embodiment of the present invention, described concrete expression vector is the recombinant vectors that obtains for the multiple clone site with the encoding gene insertion vector pET22b of acetyl xylan esterase of the present invention.
Four, a kind of method for preparing acetyl xylan esterase of the present invention is characterized in that: transform Host Strains with expression vector of the present invention, abduction delivering obtains the restructuring acetyl xylan esterase.
Five, acetyl xylan esterase of the present invention is in hydrolysis short chain fatty acid fat, removal of acylation reaction, the 7-amino-cephalosporanic acid of producing removal of acylation and the application among the deacylated cephaloporins C.
Application of the present invention shows as lives to the high enzyme of present 7-amino-cephalosporanic acid and especially cephalosporin.It is right that Montoro-Garc í a has attempted
B. pumilusBeing fixed of acetyl wood sugar esterase, catalyze and synthesize 7-ADCA and cephalosporin, transformation efficiency reaches 100%(A colorimetric assay for the determination of acetyl xylan esterase or cephalosporin C acetyl esterase activities using 7-amino phalosporanic acid behind the catalyzed reaction 100min, cephalosporin C, or acetylated xylan as substrate. Silvia Montoro-Garc í a, Fernando Gil-Ortiz et al. 2010. Bioresource Technology 101 331-336).And restructuring acetyl xylan esterase of the present invention is higher than far away the activity of 7-amino-cephalosporanic acid and cephalosporin
B. pumilusAcetyl xylan esterase, this demonstrates acetyl xylan esterase of the present invention and have huge application potential in the industry of beta-lactam antibiotics intermediate is synthetic.
Description of drawings
Fig. 1 is the Cah gene fragment that amplifies.Wherein, M:DNA Marker DL5000; 1,2:Cah.
Fig. 2 is Cah and pET22b connection result.Wherein, M:DNA Marker DL5000; 1:pDK7-Nox.
Fig. 3 is the SDS-PAGE gel (coomassie brilliant blue staining) of the acetyl xylan esterase of genetic engineering bacterium expression; Wherein, M: wide molecular weight maker (TaKaRa), the ethanoyl xylan esterase of 1-3 purifying.
Fig. 4 is bovine serum albumin (BSA) concentration-OD595 light absorption value typical curve.
Fig. 5 is p-NP (p-NP) concentration-OD405 light absorption value typical curve.
Fig. 6 is acetate concentration-OD210 light absorption value typical curve.
Fig. 7 is acetyl xylan esterase optimal reactive temperature, pH and the temperature pH stability experiment result of purifying.
Fig. 8 is the SDS-PAGE gel electrophoresis (coomassie brilliant blue staining) of the acetyl xylan esterase of non-depolymerization and depolymerization; Wherein, M: wide molecular weight maker (TaKaRa), 1,2 are respectively the ethanoyl xylan esterase of non-depolymerization and the depolymerization of purifying.
Fig. 9 is the typical curve of 4-methyl umbelliferone concentration-OD354.
Figure 10 is gel-filtration molecular weight standard curve.Wherein, Ve is the sample elution volume, and Vo is void volume.
Figure 11 is that SDS is on the impact of acetyl xylan esterase.Wherein, swimming lane 1 is for adding 0.5% SDS enzyme liquid, and swimming lane 2 is not for adding the enzyme liquid of SDS;
Figure 12 for determine trimerical activity and with the transformation relation of six aggressiveness.
Acetyl xylan esterase engineering strain of the present invention, its Classification And Nomenclature be intestinal bacteria (
Escherichia coli) BL21
-PET-Cah; Its preservation mechanism full name is Chinese Typical Representative culture collection center, is called for short CCTCC, and the address is China. Wuhan. and Wuhan University; Preservation date is on October 17th, 2012, and preserving number is numbered: CCTCC NO:M 2012408.
Embodiment
Bacterial classification used in the present invention source:
1, expresses bacterium
E. coliBL21 (DE3) buys from TaKaRa company.
2, subtilis (
Bacillus subtilis) the gene C ah source of acetyl xylan esterase of CICC 20034: Cah gene (GenBank:ZP_10041520.1) is as template among the bacillus amyloliquefaciens FZB42 that announces take Genebank, and the designed, designed primer obtains behind the pcr amplification.
3, related primer is designed, designed, and entrusts Nanjing Jin Sirui company synthetic.
Below in conjunction with accompanying drawing technical scheme of the present invention is described further, but should not be construed as limitation of the present invention:
The genetic engineering bacterium intestinal bacteria of present embodiment explanation product acetyl xylan esterase (
Escherichia coli) BL21
-The structure of pET-Cah.
The genetic engineering bacterium intestinal bacteria of the described product acetyl xylan esterase of present embodiment (
Escherichia coli) BL21
-PET-Cah, it be with subtilis (
Bacillus subtilis) the gene C ah of acetyl xylan esterase of CICC 20034 transforms by expression vector pET22b
E. coliThe genetic engineering bacterium that BL21 (DE3) obtains.
The construction process of said gene engineering bacteria is: utilize round pcr to amplify the Cah gene from plasmid vector subtilis genomic dna, the Cah gene is connected in expression vector pET22b multiple clone site place, obtain recombinant plasmid pET-Cah, again recombinant plasmid pET-Cah is transformed
E. coliBL21 (DE3), the intestinal bacteria that obtain recombinating namely produce the genetic engineering bacterium of acetyl xylan esterase
E. coliBL21
-PET-Cah.
Above-mentioned construction process specifically may further comprise the steps:
(1) clone of gene C ah:
According to Cah gene (GenBank:ZP_10041520.1) among the bacillus amyloliquefaciens FZB42 of Genebank announcement, sequences Design is closed primer:
P1：5’ TATA
CATATG（
NdeI）CAATTATACGACTTGC 3’（SEQ ID NO：3）。
P2：5’ G
GAATTC（
EcoRI）CTCAGCCTTTCAGATGCGCT 3’（SEQ ID NO：4）。
Restriction enzyme site is introduced respectively at the primer two ends
NdeI and
EcoRI extracts the explanation of test kit (OMEGA company) and extracts the subtilis genomic dna according to genome, finish the PCR reaction take the subtilis genomic dna as template;
The 8 tube reaction systems of PCR are: Buffer 22.5 μ L, ddH
2O 141.3 μ L, MgCl
213.5 μ L, dNTP-mix 18 μ L, primer P1 4.5 μ L, primer P2 4.5 μ L, Taq enzyme 2.7 μ L; After mixing, be divided into 8 pipes, add 2 μ L template DNAs to every pipe again; PCR reaction conditions: 94 ℃ of denaturation 4 min; Carry out 30 circulating reactions: 94 ℃ of sex change 45 s, 54.1 ℃ of annealing 30 s, 72 ℃ are extended 60 s; Last 72 ℃ of insulation 10 min, reaction finishes rear 4 ℃ of preservations, and behind the reaction terminating, 0.8 % agarose gel electrophoresis detects, and the result is as shown in Figure 1; Behind PCR purification kit (TaKaRa company) purifying, be connected with pMD-T Vector carrier, carry out sequencing,
NdeI and
EcoRThe I enzyme is cut restructuring pMD-T Vector carrier, reclaims 957 bp fragments,
NdeI and
EcoRThe I enzyme is cut the pET22b carrier, reclaims the large fragment product, the double digestion product is connected obtain recombinant plasmid pET-Cah under the effect of T4DNA ligase enzyme.
(2) acquisition of recombination engineering bacteria:
Recombinant plasmid pET-Cah is transformed
E. coliBL21 (DE3), coating contains the paraxin flat board of 40 μ g/mL, the picking positive transformant, and carry out bacterium colony PCR and identify, obtain restructuring
E. coli, called after
E. coliBL21pET-Cah extracts
E. coliThe recombinant plasmid of BL21pET-Cah carries out
NdeI and
EcoRI double digestion checking (such as Fig. 2).
The derivational expression method of present embodiment explanation acetyl xylan esterase.
Utilize the concrete scheme of genetic engineering bacterium abduction delivering acetyl xylan esterase of embodiment 1 as follows:
(1) starting strain: intestinal bacteria (
Escherichia coli) BL21
-PET-Cah;
(2) seed culture:
Substratum: LB substratum: yeast powder 5g/L, peptone 10 g/L, sodium-chlor 10 g/L;
Culture condition: 250 mL triangular flasks, liquid amount 50 mL, 37 ℃ of culture temperature, shaking speed 200 r/min cultivate 12 h;
(3) fermentation culture:
Substratum forms: LB substratum: yeast powder 5g/L, peptone 10 g/L, sodium-chlor 10 g/L;
Culture condition: inoculum size 1.5% (v/v), 37 ℃ of leavening temperatures add sec.-propyl-β-D-sulfo-galactopyranoside (IPTG) and induce, final concentration 1.0 mmol/L of IPTG when OD reaches 0.6 ~ 0.8, shaking speed 180 ~ 250 r/min, fermentation 2-10 h.
(4) obtaining of crude enzyme liquid:
Take out fermented liquid, place the 50mL centrifuge tube, 4 ℃, 10000 leave heart 10min, and supernatant discarded adds the Tris-HCl damping fluid of 50mL pH7.4, and thalline is carried out ultrasonication, and broken condition is as follows: 3s work, 3s intermittently, 550w 15min.Broken rear 4 ℃, 10000 leave heart 30min.Supernatant is crude enzyme liquid.
(5) mensuration of enzyme activity:
Use pNPA to be substrate, 1 enzyme unit alive (U) is defined as: under certain reaction conditions, per minute discharges the required enzyme amount of 1 μ L p-NP.
Standard curve making: the preparation of A solution: the phosphoric acid buffer of 50 mmol/L (pH7.0), wherein contain the Sudan Gum-arabic of 0.6% Triton X-100 and 0.1%; The preparation of B solution: accurately take by weighing 0.0139 g pNP reference material, with the dissolving of A solution, volumetric flask is settled to 250 mL, and being made into reference material concentration is the B solution of 1 μ mol/mL.Adopt A solution that B solution is carried out gradient dilution, be made into the pNP solution of different concns.In enzyme plate, add 10 μ L damping fluids, then add successively each 240 μ L of pNP standardized solution of different concns, add A solution in the contrast.Measure 405 nm wavelength place light absorption values with microplate reader.As X-coordinate, as ordinate zou, make typical curve (Fig. 5) with light absorption value with the concentration of pNP.
Determination step is as follows: add 240 μ L substrate solutions (A solution mixes by 9:1 with B solution) in reaction system, 10 μ L are the enzyme liquid of dilution suitably, places water-bath, and temperature is 40 ℃, reaction times 10 min; Use microplate reader to measure light absorption value at 410 nm places.The experiment establish three parallel, average, and use the most control group of 10 μ L distilled water.P-NP typical curve and equation such as Fig. 5.
Measurement result shows: the enzyme liquid of 100 times of dilutions, and light absorption value is respectively under the A410nm: 1.449,1.482,1.509, to calculate by typical curve (Fig. 5), corresponding activity is 224.96U/mL.
(6) mensuration of protein content
Use the Brandford method to measure protein content.
The making of albumen mark song: preparation Xylene Brilliant Cyanine G solution: 100 mg Xylene Brilliant Cyanine G G-250 are dissolved in 50 mL, the 95 % ethanol, add 100 mL, 85 %(v/v again) H
3PO
4, with distilled water diluting to 1 l, can use behind the filter paper filtering at last; Prepare 0.1 g/l BSA: take by weighing 0.01 g BSA, be dissolved in the 10 mL distilled water, be mixed with 0.01,0.02,0.03,0.04,0.06,0.08 mg/mL with physiological saline before using; Get 72 mL centrifuge tubes, the number of finishing (0,1,2,3,4,5,6), in 1 ~ No. 6 centrifuge tube, add respectively above-mentioned dilution after each concentration BSA solution 0.3 mL, in every centrifuge tube, add at last 1.2 mL Xylene Brilliant Cyanine G solution.1.2 mL Xylene Brilliant Cyanine G solution and 0.3 mL physiological saline in No. 0 centrifuge tube.After after each pipe mixes, take No. 0 pipe as blank determination A
595The typical curve that records such as Fig. 4.
The detection concrete steps of albumen are as follows: at first with the concentration of bovine serum albumin (BSA) standard specimen the OD595 light absorption value is made the typical curve equation, such as Fig. 4.Then remove the suitably enzyme liquid 300 μ L of dilution, add 1.2mL Xylene Brilliant Cyanine G working fluid, mixing, room temperature is placed 15min, and measures light absorption value under the 595nm.Do two Duplicate Samples for every group, take distilled water as blank.
Measurement result shows that the reacted OD595 light absorption value of enzyme liquid of 5 times of dilutions is 0.252,0.277.After the typical curve calculating according to Fig. 4, protein concentration is: 0.16 mg/ μ L.Therefore, the ratio enzyme activity of the ethanoyl xylan esterase crude enzyme liquid of engineering bacterium fermentation liquid is 1406 U/mg.
Purifying, molecular weight and the polymer form thereof of present embodiment explanation acetyl xylan esterase.
1. the purifying of acetyl xylan esterase:
Passing through 50%-80%(w/v) ammonium sulfate precipitation and ultrafiltration acetyl xylan esterase concentrate and purifying.Purification step is as follows:
(1) 50%-80% ammonium sulfate precipitation: get broken good solution, 12000g, 30min is centrifugal; Transferase 45 0mL supernatant slowly adds the SAS solution (saturated ammonium sulphate solution) of 50mL while stirring to the 100mL beaker, with solution be placed on the magnetic stirring apparatus stir 6 hours after, be positioned over 4 degree refrigerator overnight and fully precipitate.The centrifugal 30min of protein soln 10000g.The 50mL supernatant is transferred to new beaker, slowly adds while stirring the SAS solution of 75mL, making final concentration is 80% ammoniumsulphate soln, with solution be placed on stir 6 hours on the magnetic stirring apparatus after, be positioned over 4 degree refrigerator overnight and fully precipitate.Behind the centrifugal 30min of protein soln 10000g, abandon filtrate, with the phosphate buffer solution dissolving of precipitation with 5mL pH7.0, again after the solution after the dissolving is transferred to 12h in the dialysis tubing (4 ℃), place the dialysis buffer of 500mL, every 3h changes dialysis buffer liquid once, thoroughly to remove ammonium sulfate.Protein content and enzyme activity behind the mensuration purifying.
(2) gel-filtration: adopt gel-filtration that the acetyl xylan esterase after concentrated is further purified, use HiLoad 16/60 Superdex, 200 gel chromatographic columnses (Healthcare Bio-Sciences company), 50 mM Tris-HCl (comprise 100 mM KCl, pH 7.5) as moving phase, applied sample amount 100 μ L, flow velocity 0.1 mL/min, 280nm detects.Use automatic fraction collector to collect elutriant, the elutriant that the uv-absorbing peak value is arranged is carried out enzyme activity determination, great-hearted part is merged, behind the ultrafiltration and concentration, be pure enzyme liquid.Protein content and enzyme activity behind the mensuration purifying.
Purification effect is as shown in table 3, and the acetyl xylan esterase behind the purifying demonstrates single band, such as Fig. 3.Ethanoyl wood sugar esterase after purified obtains single protein band through the SDS electrophoresis.This shows, the coded polypeptide of the described nucleotide sequence of SEQ ID NO.1 is intestinal bacteria
E. coliEfficiently expressed among the BL21 (DE3), and all recombinant proteins all are soluble, form without inclusion body.
2. acetyl xylan esterase molecular weight and polymer form thereof
The molecular weight under the acetyl xylan esterase state of nature, the purifying flow process of operating process such as gel-filtration are determined in the employing gel-filtration.Gel-filtration protein standard (available from SIGMA, article No.: MWGF 1000), the making of mark song is marked Qu Jianbiao Figure 10 according to the protein standard specification sheets.The gel-filtration result shows, V
0Be 43.49 mL, Ve is that 61.76 mL(Ve are the mobile phase volume that ultraviolet absorption peak uses when occurring), do not detect other ultraviolet absorption peaks.The molecular weight that calculates under the acetyl xylan esterase state of nature according to the mark song is 216 kDa.Adopt the molecular weight of SDS-PAGE electrophoresis (12%) acetyl xylan esterase list subunit, preparation such as the table 4 of SDS-PAGE gel use the Bio-Rad electrophoresis apparatus, and deposition condition is: 80V 45 min; 120V 45min.The electrophoresis sample boils after SDS-PAGE loading buffered, the thorough depolymerization of protein protomer.The result shows that its molecular weight is 36 kDa, such as Fig. 8, and swimming lane 2.This shows under the acetyl xylan esterase native state to be six aggressiveness forms.
3. the tripolymer of acetyl xylan esterase
Adopt SDS-PAGE electrophoresis (12%) that the acetyl xylan esterase (enzyme liquid 40 ℃ of temperature after SDS-PAGE loading buffered are bathed 5 min, and protein molecular is depolymerization not) of not depolymerization is analyzed other operation stepss such as above-mentioned SDS-PAGE electrophoresis.The result shows that the acetyl xylan esterase molecular weight is 107 kDa(such as Fig. 8, swimming lane 1).This exists with trimeric form after showing the acetyl xylan esterase electrophoresis, and it is tripolymer that certain material in the possible electrophoresis process in some conditioned disjunction electrophoresis solution makes six aggressiveness depolymerization.
SDS is a kind of sequestrant, can cause protein denaturation, may cause the depolymerization of six aggressiveness.Therefore, for whether checking is tripolymer because the SDS in the electrophoresis liquid makes natural six aggressiveness depolymerization, adopt native polyacrylamide gel electrophoresis and esterase dyeing to detect.Implementation process is as follows:
The preparation of solution: the preparation of gel is not added SDS such as table 4(); Tris – glycine electrophoretic buffer: 25 mmol/L Tris; 250 mmol/L glycine (pH 8.3); The loading buffering of sample buffer (0.1 mol/L Tris-Hcl, pH 6.8) 2*: add 1 mL glycerine and 0. 0001 g tetrabromophenol sulfonphthaleins in the Tris buffering (PH 6.8) of 0.5 mL, 1 mol/L, the distilled water of sterilization is diluted to 10 mL.After processing sample, add SDS, making its final concentration is 0.5 %, carries out electrophoresis after 40 ℃ of temperature are bathed 2 h, take the enzyme liquid sample that do not add SDS as blank.Use Bio-Rad vertical electrophoresis apparatus 80 V electrophoresis 45 min, 120 V electrophoresis, 45 min; Carry out afterwards esterase activity dyeing.The concrete steps of active coloring are as follows: accurately take by weighing 100 mg α-naphthylacetates and 100 mg β-naphthylacetates, fully dissolving adds 100 mg fast blues in 2 mL acetone, mixes.Be settled to 100 mL with 0.1 mol/L pH, 7.0 phosphoric acid buffers; Gel is put in the staining fluid, placed on the shaking table and slowly shake, 37 ℃, 55 turn, and 2 min dye.The result as shown in figure 11, the blank sample of processing without SDS is an esterase band, and the acetyl xylan esterase that adds SDS an other esterase band occurs below normal esterase band.This explanation is that SDS makes acetyl xylan esterase become tripolymer by six natural aggressiveness depolymerization, and 0.5% SDS is not enough so that the complete depolymerization of six aggressiveness.The concentration that strengthens SDS is 1%, and treat enzyme liquid 2 h carry out polyacrylamide gel electrophoresis and esterase dyeing, and it is tripolymer (such as Figure 12, swimming lane 1) that result such as Figure 12,1% SDS effect 2 h can make the basic fully depolymerization of six aggressiveness.
For determine trimerical activity and with the transformation relation of six aggressiveness, in esterase dyeing liquid, add 1% SDS and dye, guaranteeing that acetyl xylan esterase exists with tripolymer all the time in the esterase dyeing process, thereby determine trimerical esterase activity.The result shows that the tripolymer of acetyl xylan esterase has certain activity, but is lower than six aggressiveness (such as Figure 12, swimming lane 2).Gel is transferred in the staining fluid that does not contain SDS dyes, the result shows that (1 min) reverts to high reactivity in the acetyl xylan esterase short period of time, such as Figure 12, and swimming lane 3.This shows that SDS can make the acetyl xylan esterase depolymerization temporarily be tripolymer, and behind the flush away SDS, acetyl wood sugar esterase at short notice rapid autohemagglutination is combined into six aggressiveness.
The preparation of table 4. Native polyacrylamide gel electrophoresis gel
| Solution | Separation gel | 12% (10 mL) | |
| H 2O | 3.2 | 2.78 | |
| 30 % acrylamides | 4.0 | 0.66 | |
| 1.5 mol/L Tris-Hcl(PH8.8) | 2.6 | --- | |
| 1.0 mol/L Tris-Hcl(PH6.8) | --- | 0.5 | |
| 10% | 0.1 | 0.06 | |
| 10 % ammonium persulphates | 0.1 | 0.04 | |
| TEMED | 0.01 | 0.01 |
The basic zymologic property research of present embodiment explanation restructuring acetyl xylan esterase.
1. the optimal reactive temperature of acetyl xylan esterase, optimal pH:
Take 4-methyl umbrella shape ethyl ester as substrate, detect optimal reactive temperature, the optimal pH of acetyl xylan esterase.1 enzyme unit alive (U) is defined as: under certain reaction conditions, per minute discharges the required enzyme amount of 1 μ L 4-methyl umbelliferone.
Concrete steps are as follows:
(1) making of 4-methyl umbrella shape ethyl ester enzyme activity mark song: the 4-methyl umbelliferone of preparation 0.1,0.15,0.2,0.25,0.3,0.35,0.4 mM, measure respectively its light absorption value under 354 nm.Result such as Fig. 9.
(2) mensuration of peak optimization reaction temperature: mix the phosphoric acid buffer (0.1 mol/L) and 280 μ L distilled water of 100 μ L pH 7.0,40 ℃ of preheating 10 min; Add 10 μ L enzyme liquid; Add 10 μ L, 100 mol/L 4-methyl umbrella shape ethyl esters (being dissolved in DMSO); Behind 30,40,45,50,55,60,70 ℃ of lower reaction 10 min, add 600 μ L, 50 mmol/L citric acids, survey the light absorption value under 354 nm.Typical curve according to Fig. 9 is scaled enzyme activity.The result is shown in Fig. 7 (a), and the peak optimization reaction temperature is 50 ℃.
(3) mensuration of peak optimization reaction pH: mix 100 μ L pH and be respectively 3.0,4.0,5.0,6.0,7.0,7.5,8.0,8.5,9,10,11,12 phosphoric acid buffer (0.1 mol/L) and 280 μ L distilled water, 40 ℃ of preheating 10 min; Other steps are described in (2).Typical curve according to Fig. 9 is scaled enzyme activity.The result is shown in Fig. 7 (c), and peak optimization reaction pH is 8.0.
2. the temperature of acetyl xylan esterase, pH stability:
Use pNPA to measure temperature, the pH stability of acetyl xylan esterase as substrate.
(1) temperature stability determination step: prepare 62 mL centrifuge tubes, add respectively the enzyme liquid of 2 mL purifying; Place 25-70 ℃ of water-bath, survey remaining pNPA enzyme activity at 0.5h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 9 h, 12 h, 24 h respectively, and calculate relative activity.The result is shown in Fig. 7 (b), and the acetyl xylan esterase of restructuring remains at enzyme activity below 55 ℃ and reaches 5 days more than 80 %, only can stablize 4 h when being higher than 60 ℃.
(2) pH Stability Determination step: prepare 10 2 mL centrifuge tubes, put into the damping fluid of 1.9 mL pH 3-12, add respectively 100 μ L enzyme liquid.Survey remnant enzyme activity behind 40 ℃ of water-bath 1 h, and computational is to vigor.The result is shown in Fig. 7 (b), and the acetyl xylan esterase of restructuring is very stable in pH 7.0-11.0 scope, and is unstable under the acidic conditions.
Present embodiment explanation acetyl xylan esterase is to the hydrolytic activity of short chain fatty acid fat and acyl compounds.
This study tour the application of acetyl xylan esterase in hydrolysis short chain fatty acid fat and removal of acylation class reaction (p-NP acetic ester, p-NP butyric ester, five acyl glucoses, 4-methyl umbrella shape ethyl ester, α-naphthylacetate, β-naphthylacetate).Specific embodiments is as follows:
1. the enzyme activity determination of p-NP acetic ester, p-NP butyric ester: as implementing the mensuration of enzyme activity in the case 2.The results are shown in Table 5.
2. to the enzyme activity determination of 4-methyl umbrella shape ethyl ester: as implementing the experimental procedure in the case 4.The results are shown in Table 5.
3. to the enzyme activity determination of five acyl glucoses: 1 enzyme unit (U) that lives is defined as: under certain reaction conditions, per minute discharges the required enzyme amount of 1 μ L acetic acid.The concentration of acetic acid is detected by high performance liquid chromatography (Japanese to Shimadzu company, the C18 post).Testing conditions is as follows: flow velocity 0.6 mL/min, and the 0.1 % phosphoric acid of 93.5 %, the acetonitrile of 3.5 %, the ultraviolet method detects.
(1) making of acetate concentration typical curve: the acetum of preparation 0.1,0.2,0.4,0.6 g/L, the light absorption value under Liquid Detection 210 nm.The result as shown in Figure 6.
(2) to the enzyme activity determination of five acyl glucoses: five acyl glucose solution (being dissolved in the Tris-HCl damping fluid of pH 8.0), 10 min of 50 ℃ of preheating 490 μ L 2 mmol/L; Add 10 μ L enzyme liquid, blank group is distilled water, 50 ℃ of water-bath 10 min behind the mixing; Add 100 μ L, 0.33 mol/L sulphuric acid soln, termination reaction; The high-efficient liquid phase chromatogram technique analysis acetic acid content.By the vigor of standard curve determination acetyl xylan esterase shown in Figure 6 to five acyl glucoses.
The result is: the peak area of solution acetic acid under 210 nm detect is 0.504,0.468 mAU*min after the acetyl xylan esterase reaction of two 50 times of parallel dilutions; 0.486 mAU*min averages; calculating acetyl xylan esterase according to typical curve shown in 6 is 328.5 U/mL to the vigor of five acyl glucoses, is 896U/mg than vigor.
4. to the enzyme activity determination of α-naphthylacetate, β-naphthylacetate
Concrete steps to the enzyme activity determination of α-naphthylacetate, β-naphthylacetate are as follows:
(1) preparation of each solution: A substrate solution: 2 mmol/ L α-naphthylacetates, take by weighing and add 0. 1 mol/ L pH, 7.4 phosphate buffered saline buffers, 49 mL after 20 mg α-naphthylacetates (chemical pure) dissolve with 1 mL Virahol, jolting hydrotropy firmly, 4 ℃ keep in Dark Place available 1 week; 0. 1 mol/ L pH, 7.4 phosphate buffered saline buffers: 1.36 g KH
2PO
4Be dissolved in the 80 mL distilled waters, transfer pH to 7.4 with 1 mol/L NaOH, be settled to 100 mL; B stop buffer: 1. 75 mol/L acetic acid (11 mL Glacial acetic acid are diluted to 100mL); The C nitrite ion: the solid blue B salt of 1 mmol/ L, take by weighing the solid blue B (analytical pure) of 50 mg and add 0. 8% tween 20,100 mL, 4 ℃ keep in Dark Place available 2 weeks; The D reference liquid: 2 mmol/ L α-naphthols, water complements to 100 mL after taking by weighing 28. 8 mg α-naphthols (analytical pure) and adding dehydrated alcohol 0. 5 mL dissolving, and 4 ℃ keep in Dark Place.
(2) operation steps: get 3 1.5 mL centrifuge tubes, indicate respectively mensuration, standard, blank.In centrifuge tube, add 0.5 mL substrate solution, 50 μ L testing samples or reference liquid or distilled water, 40 ℃ of water-bath 20 min respectively; Take out respectively the reacted solution of 250 μ L, add 0.5 mL stop buffer and 0.5 mL nitrite ion, mixing, 40 ℃ of water-bath 20 min;
With the blank tube zeroing, read absorbancy under 520 nm.
(3) calculating of enzyme activity: measure pipe A/standard pipe A*100=α/β-naphthyl acetate esterase enzyme activity (U/L); The enzyme activity definition: it is 1 unit of enzyme activity (U/ L) that every liter of enzyme liquid produces 1 mol α/β-naphthols at 40 ℃ of enzyme reaction 1 min.
The vigor of the α/β that records-naphthylacetate sees Table 5.
Table 5. acetyl xylan esterase is to the hydrolytic activity of short chain fatty acid fat and acyl compounds
| Substrate | Enzyme activity (U/mL) | Than vigor (U/mg) |
| The p-NP acetic ester | 705.4 | 2949.4 |
| The p-NP butyric ester | 0.65 | 1.8 |
| 4-methyl umbrella shape ethyl ester | 286.4 | 781.1 |
| α-naphthylacetate | 195.3 | 532.7 |
| β-naphthylacetate | 253.7 | 691.9 |
| Five acyl glucoses | 328.5 | 896 |
| Cephalosporin | 127.6 | 348 |
| 7-amino-cephalosporanic acid | 135.6 | 638.7 |
Present embodiment explanation restructuring acetyl xylan esterase is in the important intermediate of producing β-lactam antibitics---the application in the 7-amino-cephalosporanic acid of removal of acylation.
Acetyl xylan esterase can utilize 7-amino-cephalosporanic acid to be raw material, is converted into important intermediate---the 7-amino-cephalosporanic acid of β-lactam antibitics through removal of acylation.The present invention has tentatively probed into the removal of acylation enzyme activity of this novel acetyl xylan esterase to 7-amino-cephalosporanic acid.1 enzyme unit alive (U) is defined as: under certain reaction conditions, per minute discharges the required enzyme amount of 1 μ L acetic acid.
Concrete operation step is as follows:
(1) making of acetate concentration typical curve: as implementing the making of acetate concentration typical curve in the case 5.
(2) mensuration of 7-amino-cephalosporanic acid enzyme activity: 7-amino-cephalosporanic acid solution (being dissolved in the Tris-HCl damping fluid of pH 8.0) 10 min of 50 ℃ of preheating 490 μ L 2 mmol/L; Add 10 μ L enzyme liquid, blank group is distilled water, 50 ℃ of water-bath 10 min behind the mixing; Add 100 μ L, 0.33 mol/L sulphuric acid soln, termination reaction; The high-efficient liquid phase chromatogram technique analysis acetic acid content.Press standard curve determination acetyl xylan esterase shown in Figure 6 to five acyl glucose enzyme activities.
The result is: the peak area of solution acetic acid under 210 nm detect is 1.556,1.506 mAU*min after the acetyl xylan esterase reaction of two 10 times of parallel dilutions; 1.531 mAU*min average; calculating acetyl xylan esterase according to typical curve shown in 6 is 135.6 U/mL to five acyl glucose enzyme activities, is 638.7U/mg than vigor.
Present embodiment explanation Recombinant esterase is in the important intermediate of producing β-lactam antibitics---the application among the deacylated cephaloporins C.
Acetyl xylan esterase can utilize cephalosporin to be raw material, is converted into the cephalosporin of the important intermediate-removal of acylation of β-lactam antibitics through removal of acylation.The present invention has probed into the removal of acylation enzyme activity of this enzyme to cephalosporin.1 enzyme unit alive (U) is defined as: under certain reaction conditions, per minute discharges the required enzyme amount of 1 μ L acetic acid.
Concrete steps are with case study on implementation 7, the result is: the peak area of solution acetic acid under 210 nm detect is 1.417,1.406 mAU*min after the acetyl xylan esterase reaction of two 10 times of parallel dilutions, 1.4115 mAU*min average, calculating acetyl xylan esterase according to typical curve shown in 6 is 127.6 U/mL to the vigor of 7-amino-cephalosporanic acid, is 348 U/mg than vigor.
In sum; the dna sequence dna efficient soluble expression restructuring acetyl xylan esterase in Escherichia coli system that derives from subtilis of the present invention has widely substrate spectrum; can be hydrolyzed short chain fatty acid fat and multiple acylations compound; and the cephalosporin of the synthetic 7-amino-cephalosporanic acid of highly active catalytic and removal of acylation has huge application potential in the application facet of xylan degrading and β-lactam antibitics production.
Above-described embodiment is the better embodiment of the present invention; but embodiments of the present invention are not limited by above-described embodiment; other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify the substitute mode that all should be equivalence, is included in protection scope of the present invention.
Sequence table
＜110〉Nanjing University of Technology
＜120〉a kind of acetyl xylan esterase and application thereof
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Claims (5)
1. an acetyl xylan esterase is characterized in that it has the aminoacid sequence shown in the SEQ ID NO:2.
2. acetyl xylan esterase according to claim 1 is characterized in that its protein structure is homo-hexamer or homotrimer.
3. the expression vector that comprises the encoding gene of the described acetyl xylan esterase of claim 1.
4. a method for preparing acetyl xylan esterase claimed in claim 1 is characterized in that: transform Host Strains with expression vector claimed in claim 3, abduction delivering acquisition restructuring acetyl xylan esterase.
5. acetyl xylan esterase claimed in claim 1 is in hydrolysis short chain fatty acid fat, removal of acylation reaction, the 7-amino-cephalosporanic acid of producing removal of acylation and the application among the deacylated cephaloporins C.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104762334A (en) * | 2015-04-15 | 2015-07-08 | 南京工业大学 | Method for producing peroxyacetic acid through enzymatic catalytic reaction |
| CN106422963A (en) * | 2016-09-09 | 2017-02-22 | 南京工业大学 | Application of acetyl xylan esterase (AXE) as emulsifying agent |
| CN111254152A (en) * | 2020-03-27 | 2020-06-09 | 广东药科大学 | Acetyl xylan esterase gene, its coding product and preparation method |
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| WO1999038982A1 (en) * | 1998-01-28 | 1999-08-05 | Biochemie Gesellschaft Mbh | Nucleic acid molecule encoding a cephalosporin acetylesterase |
| JP2001510037A (en) * | 1997-07-15 | 2001-07-31 | ジェネンコア インターナショナル インコーポレーテッド | Proteases from Gram-positive microorganisms |
| EP2121951A1 (en) * | 2006-12-12 | 2009-11-25 | E.I. Du Pont De Nemours And Company | Production of peracids using an enzyme having perhydrolysis activity |
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| JP2001510037A (en) * | 1997-07-15 | 2001-07-31 | ジェネンコア インターナショナル インコーポレーテッド | Proteases from Gram-positive microorganisms |
| WO1999038982A1 (en) * | 1998-01-28 | 1999-08-05 | Biochemie Gesellschaft Mbh | Nucleic acid molecule encoding a cephalosporin acetylesterase |
| EP2121951A1 (en) * | 2006-12-12 | 2009-11-25 | E.I. Du Pont De Nemours And Company | Production of peracids using an enzyme having perhydrolysis activity |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104762334A (en) * | 2015-04-15 | 2015-07-08 | 南京工业大学 | Method for producing peroxyacetic acid through enzymatic catalytic reaction |
| CN106422963A (en) * | 2016-09-09 | 2017-02-22 | 南京工业大学 | Application of acetyl xylan esterase (AXE) as emulsifying agent |
| CN111254152A (en) * | 2020-03-27 | 2020-06-09 | 广东药科大学 | Acetyl xylan esterase gene, its coding product and preparation method |
| CN111254152B (en) * | 2020-03-27 | 2023-06-23 | 广东药科大学 | Acetylxylan esterase gene, coded product and preparation method thereof |
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