CN104762334A - Method for producing peroxyacetic acid through enzymatic catalytic reaction - Google Patents
Method for producing peroxyacetic acid through enzymatic catalytic reaction Download PDFInfo
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- CN104762334A CN104762334A CN201510176728.4A CN201510176728A CN104762334A CN 104762334 A CN104762334 A CN 104762334A CN 201510176728 A CN201510176728 A CN 201510176728A CN 104762334 A CN104762334 A CN 104762334A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01072—Acetylxylan esterase (3.1.1.72)
Abstract
The invention discloses a method for producing peroxyacetic acid through enzymatic catalytic reaction. Short-chain acetate and hydrogen peroxide are used as substrate, and peroxyacetic acid is generated through hydrolysis catalytic reaction of acetylxylan esterase.
Description
Technical field
The invention belongs to gene engineering technology field, being specifically related to a kind of acetyl xylan esterase AXE(Genbank:KC292495 from having hydrolytic activity) by acetic ester substrate production Peracetic Acid.
Background technology
Peracetic Acid (PAA), another name peroxyacetic acid, peracetic acid, acetyl hydrogen peroxide, be that in peroxide organic acid, structure is simple, synthesis is easy, the good high reactivity of relative stability, strong oxidizing property organic acid.The oxidation capacity of Peracetic Acid is higher than hydrogen peroxide, can be used as disinfection sanitizer, oxygenant, epoxidizing agent, it has unique effect, be a kind of wide spectrum, efficient, quick-acting, cheap disinfectant, sterilize and prophylactic disinfection with being widely used in the epidemic focus such as medical apparatus sterilizing, sterilizing and environment, thing table, air.(Han Shuanzhan, the synthesis of Peracetic Acid and application. Hebei chemical industry 2008 (6): 41-43.) due to the existence of active oxygen in its structure, except being used as disinfection sanitizer, Peracetic Acid is all widely used in papermaking weaving, chemosynthesis, environmental engineering, energy project, polymer industry and biomimetic chemistry field research, especially as the epoxidizing agent in organic synthesis and efficient germicide, have its outstanding premium properties, exploitation prospect is wide.(Zhang Tengyun, etc.; The synthesis of Peracetic Acid and industrial applied research progress. chemical industry progress 2007 (26): 194-197)
The synthetic method of Peracetic Acid has a variety of, but the method for current industrial employing mainly hydrogen peroxide method and acetaldehyde oxidation two kinds.Hydrogen peroxide method adopts acetic acid and hydroperoxidation, under the katalysis of strong acid, and obtained Peracetic Acid.Normal use sulfuric acid is as catalyzer, and reaction process is violent, and often needs high density acetic acid, and the Peracetic Acid obtained is strongly-acid.Acetaldehyde oxidation is on the basis that oxidation of acetaldehyde generates acetic acid, by changing the condition of oxidation of acetaldehyde, reducing temperature of reaction, obtaining Peracetic Acid.But this reaction is more complicated, obtain higher transformation efficiency and Peracetic Acid yield, need add suitable catalyzer.And there is strong oxidizing property and corrodibility due to Peracetic Acid, meet naked light, high heat can cause combustion explosion; Contact with reductive agent, meet metal powder wood and have combustion explosion dangerous, the packaging of Peracetic Acid and transport and store and have strict demand.(Liu Jiqi, the character of Peracetic Acid, Synthesis and applications. Henan preventive medicine magazine 2004 (03): 171-173)
Therefore, people start to try to explore biological process production Peracetic Acid.The advantage of enzyme process or microbial method production Peracetic Acid is to be swift in response, gentle, do not need high-strength hydrogen peroxide or substrate, the problems such as transport and storage can be solved.Esterase and lipase, it can catalyze ester hydrolysis, it is reported often there be the catalytic activity overlapping with non-heme halo superoxide, can catalysis peroxidation in the presence of hydrogen peroxide, produces peroxycarboxylic acid.React as follows:
Such as, O.Kirk etc. (Biocatalysis, 11:65-77 (1994)) have studied crossing of lytic enzyme (lipase, esterase and proteolytic enzyme) catalyzing acyl substrate and hydrogen peroxide and are hydrolyzed to form peroxycarboxylic acid; Report that several proteolytic enzyme and lipase combination original position produce the peracid (such as peracetic acid) (U.S. Patent application 11/413,246 and 11/588,523) that concentration is suitable for use as sterilizing agent and/or business SYNTHETIC OPTICAL WHITNER in the recent period.But most of known peroxyacetic acid concentration utilizing enzymes biocatalysis to produce is lower, does not possess effective using value.
Realize Biological preparation Peracetic Acid, the most important thing is to obtain a kind of enzyme being easy to acquisition, high reactivity, high expression level, just can reach that production efficiency is high, be swift in response sensitive requirement, and then realize scale operation.But this problem remains the key issue of restriction Biological preparation Peracetic Acid.
Summary of the invention
Contriver finds, the acetyl xylan esterase AXE with independent intellectual property right (number of patent application CN201210403666.2) had hydrolytic activity, based on this, proposed the present invention program.Therefore, contriver requires the content of patent application CN201210403666.2 to be incorporated to the application in the lump, comprises the aminoacid sequence SEQ ID NO:2 of acetyl xylan esterase in application; The activated homotrimer of tool, six glycoprotein polyprotein precursor structures; And genetic engineering bacterium intestinal bacteria (
escherichia coli) building process of BL21-pET-Cah, and build the bacterial strain obtained.
Technical problem to be solved by this invention is, obtains a kind of enzyme being easy to acquisition, high reactivity, high expression level, carries out the Production by Enzymes of Peracetic Acid.
Technical purpose of the present invention is to provide this esterase crossing the application in hydrolysis reaction.Concrete, hydrolysis reaction is crossed in this enzyme catalysis available, and more specifically, under hydrogen peroxide exists situation, this enzyme catalysis short chain acetic ester available generates Peracetic Acid.
Concrete, technical scheme of the present invention comprises:
The acetyl xylan esterase with the aminoacid sequence that SEQ ID NO:1 represents is crossing the application in hydrolysis reaction.
Wherein, the consensus amino acid sequence represented in SEQ ID NO:2 described in the aminoacid sequence that in the application, SEQ ID NO:1 represents and Chinese patent application CN201210403666.2.First, contriver has found that this enzyme had hydrolytic activity, can generate peroxy acid by catalyzing acyl substrate when peroxygen source exists; Secondly, being construed as crossing hydrolysis reaction, having peroxygen source, during as hydrogen peroxide, the catalysis of acyl group substrate is generated peroxy acid by enzyme.
In preferred reaction, this enzyme can generate Peracetic Acid by catalysis acetyl substrate under hydrogen peroxide existence condition.
A method for enzyme catalysis production Peracetic Acid, with acetyl substrate and hydrogen peroxide for substrate, carried out hydrolysis reaction by the acetyl xylan esterase and substrate contact with the aminoacid sequence that SEQ ID NO:1 represents and generated Peracetic Acid.
Method of the present invention, wherein, with the carrier containing this esterase protein matter for enzyme catalyst, can carry out hydrolysis reaction with substrate contact and generated Peracetic Acid.
For with containing the carrier of this protein for enzyme catalyst, can be understood as, above-mentioned protein can be expressed in expression system purifying, immobilization afterwards; Also by after above-mentioned protein expression and the bacterial strain that will express is broken, thus crude enzyme liquid can be obtained, by crude enzyme liquid with substrate reactions to obtain target product, or will crude enzyme liquid or full substrate contacted with it after cell fixation and react acquisition target product.
Method of the present invention, described acetyl substrate is selected from the one in ethyl acetate, monoacetin, glyceryl diacetate, triacetin.
Method of the present invention, wherein, crosses in hydrolysis reaction system described, in milligram every milliliter, and described acetyl substrate: hydrogen peroxide: it is 1:0.5-8.5:0.015-0.075 that the quantitative proportion of enzyme closes.
Be interpreted as this proportionlity, having substrate kind to exist when meeting can realize the present invention, and ratio provided by the invention is preferred ratio.
Method of the present invention, wherein, the described reaction conditions crossing hydrolysis reaction is: pH value 6.5-9.5, is preferably pH 8.0.
Method of the present invention, wherein, the described reaction times crossing hydrolysis reaction is within 5 minutes to about 2 hours, within being preferably 5 minutes.
Method of the present invention, wherein, the described temperature crossing hydrolysis reaction is 20-37 DEG C, preferably 20 DEG C.
Understanding for the time be excessively hydrolyzed in the present invention, temperature, pH value is interpreted as, and having substrate kind to exist when meeting can realize the present invention, and condition provided by the invention is optimum condition.
Method of the present invention, wherein, by the coding that also comprises according to described protein or aminoacid sequence, obtains the process of crude enzyme liquid by protein expression.
Method of the present invention, wherein, in turn includes the following steps:
(1) with intestinal bacteria (
escherichia coli) BL21
/pET-CAH, for starting strain carries out seed culture; Wherein these intestinal bacteria are preserved in China typical culture collection center, and deposit number is: CCTCC NO:M2012408; Wherein, seed culture substratum is LB substratum; Culture condition is: 250 mL triangular flasks, liquid amount 50 mL, culture temperature 37 DEG C, and shaking speed 200 r/min, cultivates 12 h; In addition, through the crude enzyme liquid that broken wall obtains, catalyzed reaction can also be carried out further by immobilization.
Beneficial effect of the present invention is, provides a kind of brand-new zymin for biological process catalyzes and synthesizes Peracetic Acid.
In addition, the solution of the present invention is adopted can to obtain the higher Peracetic Acid final concentration obtained than existing biological process.
Accompanying drawing explanation
Fig. 1 is p-NP (p-NP) concentration-OD405 light absorption value typical curve.
Fig. 2 is bovine serum albumin (BSA) concentration-OD595 light absorption value typical curve.
Fig. 3 is Peracetic Acid (PAA) concentration-OD405 light absorption value typical curve.
Fig. 4 is the suitableeest Recombinant esterase AXE dosage optimization experimental result.
Fig. 5 is the suitableeest triacetin concentration optimization experimental result.
Fig. 6 is the suitableeest concentration of hydrogen peroxide optimization experiment result.
Fig. 7 is optimal pH optimization experiment result.
Embodiment
Below in conjunction with embodiment, the present invention will be further described.Listed embodiment is only used as to demonstrate, and shows that the spirit and scope of the present invention are not limited to details in this and amendment case thereof.
Bacterial classification source used in the present invention:
Genetic engineering bacterium (
escherichia coli) BL21/pET-CAH owns for this laboratory, and be preserved in China typical culture collection center, deposit number is: CCTCC NO:M2012408.The concrete building process of this bacterial classification, and the qualification process of enzyme is see patent CN 102952787 A.
embodiment 1the present embodiment illustrates the derivational expression method of esterase.
Utilize the concrete scheme of genetic engineering bacterium abduction delivering esterase 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, culture temperature 37 DEG C, shaking speed 200 r/min, cultivates 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), leavening temperature 37 DEG C, when OD reaches 0.6 ~ 0.8, add isopropyl-beta D-thio galactopyranoside (IPTG) induction, final concentration 1.0 mmol/L of IPTG, shaking speed 180 ~ 250 r/min, fermentation 2-10 h.
(4) acquisition of crude enzyme liquid:
Take out fermented liquid, the centrifugal 3min of 8000rpm, adds the Tris-HCl(pH 7.0 of 1/8 original fermented solution volume), concentrated 8 times; Ultrasonication 400W, 3s working hour, 7s off time, ultrasonic 4min (24 times); Centrifugal 6500 rpm, 10 min.Supernatant is crude enzyme liquid.
(5) mensuration of enzyme activity:
Use p-NP acetic ester to be substrate, 1 Ge Meihuo unit (U) is defined as: under certain reaction conditions, and per minute discharges 1 μ L
penzyme amount needed for-NP.
Standard curve making: the preparation of solution A: the phosphoric acid buffer (pH7.0) of 50 mmol/L, the Sudan Gum-arabic wherein containing 0.6% Triton X-100 and 0.1%; The preparation of B solution: accurately take 0.0139 g
pnP reference material, dissolve by solution A, volumetric flask is settled to 250 mL, is made into the B solution that reference material concentration is 1 μm of ol/mL.Adopt solution A to carry out gradient dilution to B solution, be made into different concns
pnP solution.In enzyme plate, add 10 μ L damping fluids, then add different concns successively
pthe each 240 μ L of NP standardized solution, add solution A in contrast.405 nm wavelength place light absorption values are measured by microplate reader.With
pthe concentration of NP, as X-coordinate, using light absorption value as ordinate zou, makes typical curve (Fig. 1).
Determination step is as follows: in reaction system, add 240 μ L substrate solutions (solution A mixes by 9:1 with B solution), the enzyme liquid that 10 μ L suitably dilute, be placed in water-bath, temperature is 40 DEG C, reaction times 10 min; Microplate reader is used to measure light absorption value at 405 nm places.Experiment establish three parallel, average, and use 10 μ L distilled water control group the most.
p-NP typical curve and equation are as Fig. 1.
Measurement result shows: the enzyme liquid of 100 times of dilutions, and under A405nm, light absorption value is respectively: 1.449,1.482,1.509, is calculated by typical curve (Fig. 1), and corresponding activity is 224.96U/mL.
(6) mensuration of protein content
Brandford method is used to measure protein content.
The making of albumen mark song: preparation Coomassie Brillant Blue solution: 100 mg Coomassie brilliant G-250 are dissolved in 50 mL 95 % ethanol, then add 100 mL 85 %(v/v) H
3pO
4, finally with distilled water diluting to 1 l, can use after filter paper filtering; Prepare 0.1 g/l BSA: take 0.01 g BSA, be dissolved in 10 mL distilled water, use front normal saline to become 0.01,0.02,0.03,0.04,0.06,0.08 mg/mL; Get 72 mL centrifuge tubes, the number of finishing (0,1,2,3,4,5,6), each concentration BSA solution 0.3 mL add above-mentioned dilution respectively in 1 ~ No. 6 centrifuge tube after, finally add 1.2 mL Coomassie Brillant Blue solution often propping up in centrifuge tube.1.2 mL Coomassie Brillant Blue solution and 0.3 mL physiological saline in No. 0 centrifuge tube.After after each pipe mixing, with No. 0 pipe for blank determination A
595.The typical curve recorded is as Fig. 2.
The detection concrete steps of albumen are as follows: first make typical curve equation, as Fig. 2 with the concentration of bovine serum albumin (BSA) standard specimen to OD595 light absorption value.Then remove the enzyme liquid 300 μ L of suitably dilution, add 1.2mL Xylene Brilliant Cyanine G working fluid, mixing, room temperature places 15min, measures light absorption value with under 595nm.Often group does two Duplicate Samples, take distilled water as blank.
The reacted OD595 light absorption value of enzyme liquid of measurement result display dilution 5 times is, 0.252,0.277.After calculating according to the typical curve of Fig. 2, protein concentration is: 0.16 mg/mL.Therefore, the ratio enzyme activity of the ethanoyl xylan esterase crude enzyme liquid of engineering bacterium fermentation liquid is 1406 U/mg.
embodiment 2the present embodiment illustrates that replacing genetic engineering bacterium expression vector is pET-28a, builds new gene engineering bacteria E. coli BL21pET28a-Cah and this esterase is carried out to the method for 5L tank scale abduction delivering.
(1) clone of gene C ah:
According to the Cah gene (Genbank:KC292495) announced in number of patent application CN201210403666.2, sequences Design closes primer:
P1：5’-CATG
CCATGGGCATGCAATTATACGACT-3’。
P2：5’-CC
CTCGAGGCCTTTCAGATGCGCTT-3’。
Restriction enzyme site is introduced respectively in primer two ends
ncoi and
xhoi, according to plasmid extraction kit (OMEGA company) explanation extract genetic engineering bacterium (
escherichia coli) BL21/pET-CAH plasmid, with genetic engineering bacterium (
escherichia coli) BL21/pET-CAH plasmid DNA be template complete PCR reaction;
The 8 tube reaction systems of PCR are: Buffer 22.5 μ L, ddH2O 141.3 μ L, MgCl2 13.5 μ L, dNTP-mix 18 μ LL, 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, then add 2 μ L template DNAs to every pipe; PCR reaction conditions: 94 DEG C of denaturation 4 min; Carry out 30 circulating reactions: 94 DEG C of sex change 45 s, 54.1 DEG C of annealing 30 s, 72 DEG C extend 60 s; Last 72 DEG C of insulation 10 min, reaction terminates rear 4 DEG C of preservations; After PCR purification kit (TaKaRa company) purifying,
ncoi and
xhoi enzyme is cut restructuring and is reclaimed product, reclaims 957 bp fragments,
ncoi and
xhoi enzyme cuts pET28a carrier, reclaims large fragment product, is connected by double digestion product and obtain recombinant plasmid pET28a-Cah under the effect of T4DNA ligase enzyme.
(2) acquisition of recombination engineering bacteria:
Recombinant plasmid pET28a-Cah is transformed
e. colibL21 (DE3), coating is dull and stereotyped containing the kantlex of 50 μ g/mL, picking positive transformant, and carries out bacterium colony PCR qualification, is recombinated
e.coli, called after
e.colibL21pET28a-Cah, extracts
e.colithe recombinant plasmid of BL21pET28a-Cah, carries out sequence verification.
(3) method of recombination engineering bacteria E. coli BL21pET28a-Cah 5L tank scale abduction delivering
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, culture temperature 37 DEG C, shaking speed 200 r/min, cultivates 12 h;
Fermentation culture:
Substratum forms: self-induction substratum: yeast powder 25g/L, Tryptones 15 g/L, sodium-chlor 10 g/L, glycerine (v/v) 0.1%, glucose (w/v) 0.2%, lactose (w/v) 0.2%;
Culture condition: 5L fermentor tank, liquid amount is 3L, inoculum size 3.3% (v/v), leavening temperature 30 DEG C, pH7.5, mixing speed 200 r/min, and air flow 1.5 vvm ferments 16 h.
(4) acquisition of crude enzyme liquid: with reference to the acquisition methods of crude enzyme liquid in example 1
(5) mensuration of enzyme activity: identify with reference to detection method in example 1.Result shows, express through 16 hours 5L tank self-inductions, can obtain esterase activity is 2.28 × 10
4u/ml, be pET-28a by replacing expression vector, and carry out 5L tank self-induction and express this esterase (replace expensive IPTG inductor), explanation can reduce further produces esterase culture medium cost, thus reduce the cost of Production by Enzymes Peracetic Acid, be more conducive to realizing suitability for industrialized production.
embodiment 3the present embodiment illustrates with different short chain acetic ester and hydrogen peroxide for substrate, the detection of Recombinant esterase catalytic production Peracetic Acid and Peracetic Acid.
Operation steps:
(1) enzyme liquid is obtained according to the method in embodiment 1;
(2) reaction system and the operating process of esterase catalyzed preparation Peracetic Acid is utilized: short chain acetic ester substrate (ethyl acetate, monoacetin, glyceryl diacetate, triacetin) 0.1g, hydrogen peroxide 100 μ L(1mol/l), esterase AXE 30 μ L (0.3g/ml), 0.1mol/l pH7.4 phosphate buffered, totally 1 mL reaction system.Wherein change enzyme liquid into equivalent damping fluid in blank;
(3) reaction under each condition do 3 parallel, react 5min, 10min, 15min time sample;
(4) process of product: get supernatant liquid after reaction terminates rear centrifugal 12000rpm, 2min, be reaction product;
(5) Peracetic Acid detection is carried out.The oxidation adopting 2,2-to join nitrogen-two (3-ethyl-benzothiazole-6-sulfonic acid) di-ammonium salts (ABTS) is detected the concentration of fluoroacetic acid.The acetic acid solution containing 0.03mg/ml potassiumiodide of reaction product and the 100 μ L 0.5g/l ABTS solution of 100 μ L after dilution and 50 μ L 1.5mol/l is mixed in 96 orifice plates, after room temperature reaction 10min, measures it in 405nm place absorbancy.
The making of Peracetic Acid mark song: the acetic acid solution containing 0.03mg/ml potassiumiodide of preparation 0.5g/l ABTS solution (lucifuge) and 1.5mol/l: add 0 μ L, 5 μ L, 10 μ L, 15 μ L, 20 μ L, 25 μ L, 30 μ L, 35 μ L, 40 μ L, 50 μ L ABTS solution successively in 96 orifice plate numberings 1 ~ 10,100 μ L, 95 μ L, 90 μ L, 85 μ L, 80 μ L, 75 μ L, 70 μ L, 65 μ L, 60 μ L, 50 μ L ddH
2o, adds the acetic acid solution containing 0.03mg/ml potassiumiodide of 50 μ L 1.5mol/l in numbering 1 ~ 10, and 100 μ L standard Peracetic Acid liquid, mixes rear room temperature reaction 10min, with No. 0 pipe for blank determination A
405.The typical curve recorded is as Fig. 3.
Experimental result is as follows: the peroxyacetic acid concentration obtained after reacting 5min at 25 DEG C is as shown in table 1, and the peroxyacetic acid concentration that triacetin produces as substrate is the highest, therefore selects triacetin as substrate.
the different acetic ester substrate of table 1. produces peroxyacetic acid concentration
embodiment 4the present embodiment illustrates with triacetin and hydrogen peroxide for substrate, the condition optimizing of Recombinant esterase catalytic production Peracetic Acid
1. the optimization step of Recombinant esterase AXE consumption:
(1) enzyme liquid is obtained according to the method in embodiment 1;
(2) reaction system and condition: Recombinant esterase AXE consumption is optimized, containing 250mmol/l triacetin in 1ml reaction system, 1mol/l hydrogen peroxide 25 DEG C, react 5min under pH:7.4 condition;
(3) obtain Peracetic Acid sample according to the method in embodiment 3 and detect.
Result shows: as shown in Figure 4, along with the increase of the amount of esterase, the amount of the Peracetic Acid of catalytic production is also in increase, when esterase is increased to 0.30mg/ml, the amount of the Peracetic Acid generated increases slowly, therefore from practical application and cost consideration, in this reaction system, the consumption optimum of Recombinant esterase is 0.30mg/ml, can produce 8259ppm Peracetic Acid.
2. the optimization of triacetin concentration.
Operation steps:
(1) enzyme liquid is obtained according to the method in embodiment 1;
(2) reaction system and condition: triacetin concentration is optimized, containing Recombinant esterase AXE 0.3mg/ml in 1ml reaction system, 1mol/l hydrogen peroxide 25 DEG C, react 5min under pH:7.4 condition;
(3) obtain Peracetic Acid sample according to the method in embodiment 3 and detect.
Result shows: as shown in Figure 5, along with the concentration of triacetin is increased to 300mmol/l from 100mmol/l, the amount of the Peracetic Acid of producing increases thereupon, but when triacetin concentration is higher than 300mmol/l, the amount of Peracetic Acid is not significantly increased, therefore in this reaction system, the concentration optimum of triacetin is 300mmol/l, can produce 9613ppm Peracetic Acid.
3. the optimization of concentration of hydrogen peroxide
Operation steps:
(1) enzyme liquid is obtained according to the method in embodiment 1;
(2) reaction system and condition: concentration of hydrogen peroxide is optimized, containing 300mmol/l triacetin in 1ml reaction system, Recombinant esterase AXE 0.3mg/ml 25 DEG C, react 5min under pH:7.4 condition;
(3) obtain Peracetic Acid sample according to the method in embodiment 3 and detect.
Result shows: as shown in Figure 6, and along with the increase of concentration of hydrogen peroxide, the amount of the Peracetic Acid of catalytic production first increases rear minimizing, and this may be because the hydrogen peroxide of high density causes the rapid deactivation of esterase to cause.In this reaction system, concentration of hydrogen peroxide optimum is 1mol/l, can produce 9613ppm Peracetic Acid.
4. the optimization of temperature
Operation steps:
(1) enzyme liquid is obtained according to the method in embodiment 1;
(2) reaction system and condition: under differing temps, containing 300mmol/l triacetin in 1ml reaction system, Recombinant esterase AXE 0.3mg/m, 1mol/l hydrogen peroxide reacts 5min under pH:7.4 condition;
(3) obtain Peracetic Acid sample according to the method in embodiment 3 and detect.
Result shows, as shown in table 2, and when 20 DEG C, the amount of the Peracetic Acid of production reaches and is up to 10481ppm, has significant using value.
the optimization of table 2. temperature of reaction
| Temperature of reaction | Peracetic Acid ppm |
| 20℃ | 10481.63 |
| 25℃ | 9818.63 |
| 30℃ | 9730.00 |
| 37℃ | 8407.54 |
The Optimizing operation step of 5.pH:
(1) enzyme liquid is obtained according to the method in embodiment 1;
(2) reaction system and condition: under condition of different pH, containing 300mmol/l triacetin in 1ml reaction system, Recombinant esterase AXE 0.3mg/ml, 1mol/l hydrogen peroxide reacts 5min under 20 DEG C of conditions;
(3) obtain Peracetic Acid sample according to the method in embodiment 3 and detect.
Result shows, as shown in Figure 7, during pH8.0, the amount of the Peracetic Acid of production reaches and is up to 11385ppm.
embodiment 5the present embodiment illustrates the step of immobilization AXE esterase production Peracetic Acid.
(1) enzyme liquid is obtained according to the method in embodiment 1;
(2) immobilization of Recombinant esterase AXE: select polyacrylamide resin, utilize Euplotes woodruffi to being fixed of Recombinant esterase, obtains immobilization AXE esterase, is: 17.5U/g to the vigor of paranitrophenylacetic acid ester
(3) reaction system and condition: containing 300mmol/l triacetin in 1ml reaction system, 1mol/l hydrogen peroxide, 0.5g immobilized enzyme, reacts 5min under pH 8.0,20 DEG C of conditions;
(4) obtain Peracetic Acid sample according to the method in embodiment 3 and detect.
Result shows: the Recombinant esterase AXE after 0.5g immobilization can produce the Peracetic Acid of 2678ppm.Compared to free esterase AXE, more convenient recyclable.The Peracetic Acid repeating to produce afterwards for 10 times is about 72% of first set reaction.
The invention provides following methods, the method acetyl xylan esterase AXE(unbound state or the immobilized state with hydrolytic activity), under hydrogen peroxide (concentration is 200mmol/l at least) exists, in acidity under neutral reaction condition, produce dense peroxide acetate aqueous solution from suitable short chain acetic ester (comprising glyceryl ester) original position.
Sequence table
<110> Nanjing University of Technology
The method of <120> enzymic catalytic reaction production Peracetic Acid
<130> xb15041502
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 957
<212> DNA
<213> Bacillus subtilis
<220>
<221> CDS
<222> (1)..(957)
<400> 1
atg caa tta tac gac ttg ccg ctc gac caa ctg caa acc tat aaa cca 48
Met Gln Leu Tyr Asp Leu Pro Leu Asp Gln Leu Gln Thr Tyr Lys Pro
1 5 10 15
aat aaa acc gct cct cat gac ttt tcg gat ttt tgg gca tca tca tta 96
Asn Lys Thr Ala Pro His Asp Phe Ser Asp Phe Trp Ala Ser Ser Leu
20 25 30
cat gag ctt gca aag gaa gag gca aag cct gaa ttg aag gct gaa agc 144
His Glu Leu Ala Lys Glu Glu Ala Lys Pro Glu Leu Lys Ala Glu Ser
35 40 45
tac ccg gct gac ggc gtg aaa gta ttc cgg ctg aca tac cgt agc ttc 192
Tyr Pro Ala Asp Gly Val Lys Val Phe Arg Leu Thr Tyr Arg Ser Phe
50 55 60
ggc aag gcg gaa att gag gga tgg tac gcc gtt ccc gac cgg caa gga 240
Gly Lys Ala Glu Ile Glu Gly Trp Tyr Ala Val Pro Asp Arg Gln Gly
65 70 75 80
ccg cat ccg gcc atc gtg aaa tat cac ggc tat aac gcg agc tat gac 288
Pro His Pro Ala Ile Val Lys Tyr His Gly Tyr Asn Ala Ser Tyr Asp
85 90 95
ggg gat ata cat gac atc gta aat tgg gcg ctt cac gga tat gcc gcg 336
Gly Asp Ile His Asp Ile Val Asn Trp Ala Leu His Gly Tyr Ala Ala
100 105 110
ttc ggc atg ctt gtc cgc ggc cag cac agc agc aca gat aca agc gtc 384
Phe Gly Met Leu Val Arg Gly Gln His Ser Ser Thr Asp Thr Ser Val
115 120 125
tca ccg cac gga cac gtt ccc ggc tgg atg acg aaa ggg att tta gat 432
Ser Pro His Gly His Val Pro Gly Trp Met Thr Lys Gly Ile Leu Asp
130 135 140
aaa gat aca tat tat tac cgc ggc gtg tac tta gac gcc gtc cgg gct 480
Lys Asp Thr Tyr Tyr Tyr Arg Gly Val Tyr Leu Asp Ala Val Arg Ala
145 150 155 160
ttg gaa gtc atc agc ggc ttt gat gaa gtg gat gaa acg agg atc gcc 528
Leu Glu Val Ile Ser Gly Phe Asp Glu Val Asp Glu Thr Arg Ile Ala
165 170 175
gtc atc ggc ggg agc cag gga ggc gga ctt tca atc gct gcg gcg gca 576
Val Ile Gly Gly Ser Gln Gly Gly Gly Leu Ser Ile Ala Ala Ala Ala
180 185 190
cta tct gat att ccg aga gcg gtt gcg gcc gat tat cct tat tta agc 624
Leu Ser Asp Ile Pro Arg Ala Val Ala Ala Asp Tyr Pro Tyr Leu Ser
195 200 205
aat ttt gag cgg gcc att gat gtt gct ctt gat gag ccg tat ctt gag 672
Asn Phe Glu Arg Ala Ile Asp Val Ala Leu Asp Glu Pro Tyr Leu Glu
210 215 220
att aac tcc ttt ttc aga aag aac ggc agc cct gaa acg gag aag acc 720
Ile Asn Ser Phe Phe Arg Lys Asn Gly Ser Pro Glu Thr Glu Lys Thr
225 230 235 240
gcg atg aat aca ctg gcc tat ttt gat atc atg aat ctg gcg gac cgc 768
Ala Met Asn Thr Leu Ala Tyr Phe Asp Ile Met Asn Leu Ala Asp Arg
245 250 255
gta aaa gtt ccg gtg ctc atg tcc atc ggg ctg att gac cgc gtg act 816
Val Lys Val Pro Val Leu Met Ser Ile Gly Leu Ile Asp Arg Val Thr
260 265 270
ccg ccg tcg acg gtg ttt gcc gct tac aat cat ctg gaa acg gaa aaa 864
Pro Pro Ser Thr Val Phe Ala Ala Tyr Asn His Leu Glu Thr Glu Lys
275 280 285
caa ttg aag gtt tac cgg tat ttc gga cat gaa tac ata cct tct ttc 912
Gln Leu Lys Val Tyr Arg Tyr Phe Gly His Glu Tyr Ile Pro Ser Phe
290 295 300
cat acg gaa aag ctg gcc ttt ctg aaa gcg cat ctg aaa ggc tga 957
His Thr Glu Lys Leu Ala Phe Leu Lys Ala His Leu Lys Gly
305 310 315
<210> 2
<211> 318
<212> PRT
<213> Bacillus subtilis
<400> 2
Met Gln Leu Tyr Asp Leu Pro Leu Asp Gln Leu Gln Thr Tyr Lys Pro
1 5 10 15
Asn Lys Thr Ala Pro His Asp Phe Ser Asp Phe Trp Ala Ser Ser Leu
20 25 30
His Glu Leu Ala Lys Glu Glu Ala Lys Pro Glu Leu Lys Ala Glu Ser
35 40 45
Tyr Pro Ala Asp Gly Val Lys Val Phe Arg Leu Thr Tyr Arg Ser Phe
50 55 60
Gly Lys Ala Glu Ile Glu Gly Trp Tyr Ala Val Pro Asp Arg Gln Gly
65 70 75 80
Pro His Pro Ala Ile Val Lys Tyr His Gly Tyr Asn Ala Ser Tyr Asp
85 90 95
Gly Asp Ile His Asp Ile Val Asn Trp Ala Leu His Gly Tyr Ala Ala
100 105 110
Phe Gly Met Leu Val Arg Gly Gln His Ser Ser Thr Asp Thr Ser Val
115 120 125
Ser Pro His Gly His Val Pro Gly Trp Met Thr Lys Gly Ile Leu Asp
130 135 140
Lys Asp Thr Tyr Tyr Tyr Arg Gly Val Tyr Leu Asp Ala Val Arg Ala
145 150 155 160
Leu Glu Val Ile Ser Gly Phe Asp Glu Val Asp Glu Thr Arg Ile Ala
165 170 175
Val Ile Gly Gly Ser Gln Gly Gly Gly Leu Ser Ile Ala Ala Ala Ala
180 185 190
Leu Ser Asp Ile Pro Arg Ala Val Ala Ala Asp Tyr Pro Tyr Leu Ser
195 200 205
Asn Phe Glu Arg Ala Ile Asp Val Ala Leu Asp Glu Pro Tyr Leu Glu
210 215 220
Ile Asn Ser Phe Phe Arg Lys Asn Gly Ser Pro Glu Thr Glu Lys Thr
225 230 235 240
Ala Met Asn Thr Leu Ala Tyr Phe Asp Ile Met Asn Leu Ala Asp Arg
245 250 255
Val Lys Val Pro Val Leu Met Ser Ile Gly Leu Ile Asp Arg Val Thr
260 265 270
Pro Pro Ser Thr Val Phe Ala Ala Tyr Asn His Leu Glu Thr Glu Lys
275 280 285
Gln Leu Lys Val Tyr Arg Tyr Phe Gly His Glu Tyr Ile Pro Ser Phe
290 295 300
His Thr Glu Lys Leu Ala Phe Leu Lys Ala His Leu Lys Gly
305 310 315
Claims (10)
1. the acetyl xylan esterase with the aminoacid sequence that SEQ ID NO:1 represents is crossing the application in hydrolysis reaction.
2. a method for enzyme catalysis production Peracetic Acid, with acetyl substrate and hydrogen peroxide for substrate, is characterized in that, with acetyl xylan esterase according to claim 1 for enzyme catalyst, carried out hydrolysis reaction generate Peracetic Acid with substrate contact.
3. method according to claim 2, is characterized in that, with the carrier containing the acetyl xylan esterase described in claim 1 for enzyme catalyst, carried out hydrolysis reaction generate Peracetic Acid with substrate contact.
4. according to the method in claim 2 or 3, it is characterized in that, described acetyl substrate is selected from the one in ethyl acetate, monoacetin, glyceryl diacetate, triacetin.
5. method according to claim 4, is characterized in that, crosses in hydrolysis reaction system described, in milligram every milliliter, and described acetyl substrate: hydrogen peroxide: it is 1:0.5-8.5:1.5-75 that the quantitative proportion of enzyme closes.
6. according to the method in claim 2 or 3, it is characterized in that, the described reaction conditions crossing hydrolysis reaction is: pH value 6.5-9.5, is preferably pH 8.0.
7. according to the method in claim 2 or 3, it is characterized in that, the described temperature crossing hydrolysis reaction is 20-37 DEG C, preferably 20 DEG C.
8. according to the method in claim 2 or 3, it is characterized in that, the described reaction times crossing hydrolysis reaction is within 5 minutes, arrive about within an hour, within being preferably 5 minutes.
9. method according to claim 2, is characterized in that, by the coding that also comprises according to described protein or aminoacid sequence, is expressed by described acetyl xylan esterase and obtains the process of crude enzyme liquid.
10. method according to claim 9, is characterized in that, in turn includes the following steps:
(1) with intestinal bacteria (
escherichia coli) BL21
/pET-CAH, for starting strain carries out seed culture; Wherein these intestinal bacteria are preserved in China typical culture collection center, and deposit number is: CCTCC NO:M2012408; Wherein, seed culture substratum is LB substratum; Culture condition is: 250 mL triangular flasks, liquid amount 50 mL, culture temperature 37 DEG C, and shaking speed 200 r/min, cultivates 12 h;
(2) fermentation culture, fermention medium consists of LB liquid medium; Culture condition is, according to volume ratio be 1.5% inoculum size inoculation seed culture fluid, leavening temperature 37 DEG C, when OD reaches 0.6 ~ 0.8, add isopropyl-beta D-thio galactopyranoside (IPTG) induction, final concentration 1.0 mmol/L of IPTG, shaking speed 180 ~ 250 r/min, fermentation 2-10 h;
(3) acquisition of crude enzyme liquid, take out fermented liquid, the centrifugal 3min of 8000rpm, adds the Tris-HCl(pH 7.0 of 1/8 original fermented solution volume), concentrated 8 times; Ultrasonication 400W, 3s working hour, 7s off time, ultrasonic 4min; Centrifugal 6500 rpm, 10 min, supernatant is crude enzyme liquid.
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| CN201510176728.4A CN104762334A (en) | 2015-04-15 | 2015-04-15 | Method for producing peroxyacetic acid through enzymatic catalytic reaction |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201510176728.4A CN104762334A (en) | 2015-04-15 | 2015-04-15 | Method for producing peroxyacetic acid through enzymatic catalytic reaction |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101970674A (en) * | 2007-11-21 | 2011-02-09 | 纳幕尔杜邦公司 | Production of peracids using an enzyme having perhydrolysis activity |
| CN102782143A (en) * | 2009-10-01 | 2012-11-14 | 纳幕尔杜邦公司 | Perhydrolase for enzymatic peracid production |
| CN102952787A (en) * | 2012-10-22 | 2013-03-06 | 南京工业大学 | Acetylxylan esterase and application thereof |
| CN103275955A (en) * | 2013-05-26 | 2013-09-04 | 山东隆科特酶制剂有限公司 | Xylosidase Xyl43B with high xylose tolerance, and gene and application thereof |
-
2015
- 2015-04-15 CN CN201510176728.4A patent/CN104762334A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101970674A (en) * | 2007-11-21 | 2011-02-09 | 纳幕尔杜邦公司 | Production of peracids using an enzyme having perhydrolysis activity |
| CN102782143A (en) * | 2009-10-01 | 2012-11-14 | 纳幕尔杜邦公司 | Perhydrolase for enzymatic peracid production |
| CN102952787A (en) * | 2012-10-22 | 2013-03-06 | 南京工业大学 | Acetylxylan esterase and application thereof |
| CN103275955A (en) * | 2013-05-26 | 2013-09-04 | 山东隆科特酶制剂有限公司 | Xylosidase Xyl43B with high xylose tolerance, and gene and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| SEUNG-MOON PARK: "Acetyl xylan esterase of Aspergillus ficcum catalyzed the synthesis of peracetic acid from ethyl acetate and hydrogen peroxide", 《JOURNAL OF BIOSCIENCE AND BIOENGINEERING》 * |
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