CN113481175B - Active and stereoselectivity improved olefinic bond reductase mutant and encoding gene and application thereof - Google Patents
Active and stereoselectivity improved olefinic bond reductase mutant and encoding gene and application thereof Download PDFInfo
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Abstract
The invention discloses an activity and stereoselectivity improved olefinic bond reductase mutant, a coding gene and application thereof, wherein the olefinic bond reductase mutant comprises olefinic bond reductase mutants Y84A, Y84V, Y84L, Y84I, Y84T and Y84C which are formed by taking a wild olefinic bond reductase OYE2p shown in SEQ ID NO.1 as a template and mutating an 84 th amino acid residue, and the amino acid sequences are shown in SEQ ID NO. 3-8. The mutant of the olefinic bond reductase provided by the invention can catalyze citral to prepare (R) -citronellal with high activity and high stereoselectivity, has the characteristics of economy, environmental protection and high chiral selectivity, and provides a potential biocatalyst for industrial production of (R) -citronellal.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to an olefinic bond reductase mutant with improved activity and stereoselectivity, and a coding gene and application thereof.
Background
The chemical formula of the (R) -Citronellal is shown as (3R) -3, 7-dimethyl-6-octenal, which is an important spice and medical intermediate, can be used for flavoring foods, daily necessities and the like, and can also be used as a raw material for synthesizing L-menthol and vitamin E. At present, (R) -citronellal is mainly obtained by extracting natural essential oil, and can also be prepared by catalytic hydrogenation of citral by a metal catalyst.
Compared with other methods, the method for synthesizing (R) -citronellal by catalyzing the asymmetric reduction of citral by using the olefinic bond reductase has the advantages of environmental friendliness, mild reaction conditions, high stereoselectivity of products and the like. The reported olefinic bond reductase capable of catalyzing citral to generate (R) -citronellal is few, and the catalytic activity of the common tablet is low. Stewart et al obtained a strain of olefinic reductase OYE2.6 from Pichia stipitis that catalyzes the production of 150mM E-citral to (R) -citronellal in 5.75 hours, with product ee values up to 98% [ Chemical Communications,2010,46 (45): 8558-8560 ]; however, the substrate E-citral is obtained by separation from citral (mixture of E, Z-citral). Hauer et al engineered the olefinic reductase NCR from Zymomonas mobilis by protein engineering means and finally the mutant W66A was inverted from 99% (S) enantioselectivity to E-citral to 46% (R), but the enantioselectivity to Z-citral was still only 88% (S), thus making it difficult to meet the need for producing (R) -citronellal. Therefore, the development of the olefinic bond reductase capable of catalyzing citral to generate (R) -citronellal with high activity and high selectivity has important industrial application value.
Disclosure of Invention
The invention aims to provide an olefinic bond reductase mutant with improved activity and stereoselectivity, and a coding gene and application thereof, so as to solve the problems of low stereospecific activity and low stereoselectivity in the existing technology for synthesizing (R) -citronellal by using olefinic bond reductase.
In order to solve the technical problems, the invention adopts the following technical scheme:
according to a first aspect of the present invention, there is provided an olefinic reductase mutant having improved activity and stereoselectivity, comprising an olefinic reductase mutant having an amino acid sequence shown in SEQ ID NO.1 and a nucleotide sequence shown in SEQ ID NO.2, wherein the olefinic reductase mutant has an olefinic reductase mutant OYE2p as a template, and the 84 th amino acid has the following mutation: an olefinic reductase mutant Y84A (SEQ ID NO. 3) in which tyrosine Y at position 84 is mutated to alanine A; an olefinic reductase mutant Y84V (SEQ ID NO. 4) in which tyrosine Y at position 84 is mutated to valine V; an olefinic reductase mutant Y84L (SEQ ID NO. 5) in which tyrosine Y at position 84 is mutated to leucine L; an olefinic reductase mutant Y84I (SEQ ID NO. 6) in which tyrosine Y at position 84 is mutated to isoleucine I; an olefinic reductase mutant Y84T (SEQ ID NO. 7) in which tyrosine Y at position 84 is mutated to threonine T; tyrosine Y at position 84 is mutated to cysteine C, an olefinic reductase mutant Y84C (SEQ ID NO. 8).
The invention takes the preserved olefinic bond reductase OYE2p (from Saccharomyces cerevisiae YJM1341, see document Bioresource.Bioprocess (2018) 5:9) constructed in the earlier stage of a laboratory as a template, the amino acid sequence is shown as SEQ ID NO.1, the nucleotide sequence is shown as SEQ ID NO.2, and site-directed mutagenesis is carried out on the olefinic bond reductase, so that the olefinic bond reductase mutant with remarkable improvement in catalytic activity, stereoselectivity and the like is obtained.
According to a second aspect of the present invention, there is provided a gene encoding an olefinic reductase mutant having improved enzymatic activity and stereoselectivity as described above, the gene encoding an amino acid sequence shown in the olefinic reductase mutant as described above.
Particularly preferred, the invention provides an olefinic bond reductase mutant Y84V with improved enzyme activity and stereoselectivity, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 9.
According to a third aspect of the present invention, there is also provided a recombinant genetically engineered bacterium comprising a gene encoding an olefinic reductase mutant having improved enzymatic activity and stereoselectivity as described above. The host cell may be any of a variety of conventional host cells in the art, preferably the host cell is E.coli BL21 (DE 3).
According to a fourth aspect of the invention there is also provided the use of said olefinic reductase mutant in the catalysis of citral to prepare (R) -citronellal.
The application uses thalli, thalli immobilized cells, enzymes extracted after ultrasonic disruption of thalli or pure enzymes obtained by fermenting and culturing engineering bacteria containing an ethylenic reductase mutant encoding gene as a catalyst, uses citral as a substrate, uses buffer solution with pH of 7-9 as a reaction medium, reacts at 20-40 ℃ under the condition of 150-300rpm, and separates and purifies reaction liquid after the reaction is finished to obtain (R) -citronellal.
It will be appreciated that the olefinic reductase mutants of the present invention may be used in whole cells of the engineering bacterium, as crude enzyme without purification, as partially or fully purified enzyme. The olefinic reductase mutants of the present invention can also be made into immobilized enzymes or biocatalysts in immobilized cell form using immobilization techniques known in the art.
Preferably, the substrate concentration is 50-200mM, the glucose concentration is 55-220mM, the amount of the crude enzyme powder of the olefinic reductase is 2U/mL in terms of enzyme activity, and the amount of the crude enzyme powder of the glucose dehydrogenase is 3U/mL in terms of enzyme activity.
Preferably, the reaction medium is PBS buffer solution with pH value of 8.5, and the catalytic reaction temperature is 30 ℃.
Preferably, the crude enzyme powder of the olefinic bond reductase is an olefinic bond reductase mutant Y84V after freeze-drying.
The medium used for recombinant expression of the transformant may be a medium which is known in the art for growing the transformant and producing the olefinic reductase of the present invention, preferably an LB medium: 10g/L peptone, 10g/L sodium chloride, 5g/L yeast extract and pH 7.0.
The culture method and culture conditions are not particularly limited as long as the transformant is capable of growing and expressing the olefinic reductase. The method specifically comprises the following steps:
(1) Plate culture: streaking the recombinant engineering bacteria related to the invention onto LB solid plate culture medium containing screening antibiotics, and culturing overnight at 37 ℃;
(2) Seed culture: picking single colonies on the flat plate obtained in the step (1) on an ultra-clean bench, inoculating the single colonies into LB liquid medium containing screening antibiotics, and culturing at 37 ℃ for 8 hours;
(3) Induction culture: inoculating the seed culture solution obtained in the step (2) into LB liquid culture medium containing screening antibiotics at the ultra-clean bench, culturing at 37 ℃ until the bacterial liquid OD 600 The value reaches 0.6, IPTG with the final concentration of 0.2mM is added, and induction culture is carried out for 12 hours at 20 ℃;
(4) And (3) collecting thalli: centrifuging the bacterial liquid obtained by the induction culture obtained in the step (3) for 10min under the condition that the rotating speed is 8000rpm, and separating to obtain bacterial precipitate; washing the obtained thalli with physiological saline for 2 times;
(5) Protein purification: and (3) dissolving the thalli obtained in the step (4) in 20mM PBS buffer solution (pH 7.4) to obtain cell suspension, carrying out ultrasonic crushing, centrifuging for 30min under the condition of rotating speed of 10000rpm, separating to obtain supernatant crude enzyme solution, and purifying by a nickel column to obtain a completely purified olefinic bond reductase mutant, namely the catalyst.
Preferably, the concentration of the cell suspension in step (5) is 50g/L.
Preferably, the ultrasonic crushing power in the step (5) is 200-300W, the ultrasonic is performed for 5 seconds, the ultrasonic is stopped for 5 seconds, and the ultrasonic is accumulated for 99 times.
According to the present invention, it was found that the 84 th site of the wild-type ethylenic reductase plays a key role in both activity and stereoselectivity enhancement. Particularly preferably, the olefinic bond reductase mutant Y84V provided by the invention can catalyze citral to prepare (R) -citronellal with high activity and high stereoselectivity, has the characteristics of economy, environmental protection and high chiral selectivity, and provides a potential biocatalyst for industrial production of (R) -citronellal.
Compared with the prior art, the invention has the following beneficial effects:
(1) The olefinic bond reductase mutant Y84V provided by the invention can catalyze high-concentration citral (200 mM) to generate (R) -citronellal, the conversion rate is more than 95%, and the ee value of the product (R) -citronellal is more than 95%;
(2) The invention improves the activity and the stereoselectivity of the olefinic bond reductase through protein engineering, and has good application prospect in the industrial production of (R) -citronellal.
Drawings
FIG. 1 is an SDS-PAGE protein gel of wild-type olefinic reductase OYE2P and its mutant Y84V, wherein M is the standard molecular weight of the protein, T is the whole cell fraction, S is the supernatant of the cell disruption solution, P is the precipitate of the cell disruption solution, and E is the pure enzyme;
FIG. 2 is a graph showing the reaction process of the wild-type olefinic reductase OYE2p catalyzed citral;
FIG. 3 is a reaction profile of the catalytic citral reaction of the olefinic reductase OYE2p mutant Y84V.
Detailed Description
The invention will be further illustrated with reference to specific examples. It should be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The technical means used in the examples are conventional in the art as specifically described.
The parent olefinic bond reductase adopted in the specific example is the olefinic bond reductase OYE2p constructed in advance in the subject group, the amino acid sequence of the olefinic bond reductase is shown as SEQ ID NO.1, and the nucleotide sequence of the olefinic bond reductase is shown as SEQ ID NO. 2.
EXAMPLE 1 construction of recombinant E.coli containing mutants
In order to carry out site-directed mutagenesis on tyrosine (Tyr) at position 84 in the amino acid sequence of the parent olefinic bond reductase, mutation sites Y84A, Y84V, Y L, Y84I, Y T and Y84C are designed on primers, recombinant plasmids pET21a-OYE2p containing a target gene fragment are used as templates, and plasmids with mutated base sequences are amplified by PCR, and the sequences of the primers are shown in Table 1.
Table 1: primer design table
The PCR amplification system was (50. Mu.L): template DNA 1-5ng,PrimeSTAR Max Premix (2X) 25. Mu.L, 1.5. Mu.L upstream and downstream of the mutation primer each, was supplemented with sterile distilled water to 50. Mu.L.
PCR reaction parameters: (1) pre-denaturation at 98℃for 2 min; (2) denaturation at 95℃for 10 sec; (3) annealing at 55 ℃ for 5 seconds; (4) Extending at 72 ℃ for 2 minutes, and cycling the steps (2) - (4) for 30 times; (5) extension thoroughly at 72℃for 5min, preservation at 16 ℃.
After target bands of the PCR products are observed through 1.0% agarose gel electrophoresis, the residual PCR products are purified to obtain purified linearization plasmids, and 50ng of the purified linearization plasmids are taken for seamless cloning.
The seamless cloning system was (20 μl): linearized plasmid 50ng,Seamless Cloning Mix (2×) 5 μl was supplemented with sterilized distilled water to 10 μl.
The seamless cloning reaction was placed in a 50℃water bath for 15 minutes.
5. Mu.L of the seamless cloning product was transformed into E.coli BL21 (DE 3) competent cells by heat shock, and after resuscitation, the cells were plated on LB plates containing ampicillin and cultured overnight.
Then 5-10 clones are selected to LB culture medium, after culturing for 8h at 37 ℃, bacterial solution is taken for sequencing verification, so as to obtain the recombinant engineering bacteria E.coli BL21 (DE 3)/pET 21a-OYE2p/Y84A, E.coli BL21 (DE 3)/pET 21a-OYE2p/Y84V, E.coli BL21 (DE 3)/pET 21a-OYE2p/Y84L, E.coli BL21 (DE 3)/pET 21a-OYE2p/Y84I, E.coli BL21 (DE 3)/pET 21a-OYE2p/Y84T and E.coli BL21 (DE 3)/pET 21a-OYE2p/Y84C of the ethylene reductase mutant, and the amino acid sequences of the ethylene reductase mutants are shown as SEQ ID NO. 3-8.
EXAMPLE 2 expression and purification of wild type OYE2p and mutants
Step 1: the recombinant engineering bacteria of the olefin bond reductase OYE2p and the mutant obtained in the example 1 are streaked on LB solid plate culture medium containing screening antibiotics, cultured overnight at 37 ℃, single colonies on a single plate are selected, inoculated on LB liquid culture medium containing the screening antibiotics, and cultured for 8 hours at 37 ℃. Inoculating the seed culture solution into 200mL LB liquid culture medium containing screening antibiotics, culturing at 37 ℃, adding IPTG with final concentration of 0.2mM when the OD600 value of the bacterial solution reaches 0.6, and performing induction culture at 20 ℃ for 12h.
Step 2: purifying wild OYE2p and 6 mutants thereof by immobilized metal ion affinity method, wherein the nickel column for purification is HisTrap of GE company TM HP (5 mL). The obtained bacterial liquid is centrifugated for 10min under the condition of 8000rpm, bacterial precipitate is obtained by separation, the obtained bacterial precipitate is dissolved in 20mM PBS buffer (pH 7.4) to obtain cell suspension, the cell suspension is centrifugated for 30min under the condition of 10000rpm by ultrasonic crushing, and the supernatant is taken to pass through a 0.22 mu m water-based filter membrane, thus obtaining crude enzyme liquid by separation.
Step 3: and (3) after the nickel column is balanced by the balancing buffer solution, sampling the crude enzyme solution obtained in the step (2), removing the impurity protein by using the low-concentration eluting buffer solution, eluting and collecting the target protein by using the high-concentration eluting buffer solution, and dialyzing to obtain the pure enzyme. Protein concentration was quantified using Bradford protein concentration detection kit. The purity of the protein is detected by SDS-PAGE, and the detection result is shown in figure 1, so that the wild type and the mutant can obtain a single protein band after purification.
Example 3 Activity and stereoselectivity of wild type OYE2p and mutants on citral
And (3) freeze-drying the OYE2p and crude enzyme liquid of each mutant expression strain obtained in the step (2) of the example to obtain freeze-dried crude enzyme powder.
Under the condition of unified enzyme dosage, the wild type OYE2p and each mutant are utilized to catalyze the reduction of the citral so as to determine the activity of the citral and the ee value of the product.
The enzyme catalytic reduction reaction system comprises: 100mM phosphate buffer (pH 8.5), 15g/L lyophilized wild type OYE2p, crude enzyme powder of each mutant, 5g/L lyophilized crude enzyme powder of glucose dehydrogenase, 20mM citral, 0.2mM NAD + And 100mM glucose. Sequentially adding all the substances into a centrifuge tube, placing the centrifuge tube at 200rpm and 30 ℃ for shake reaction for 6 hours, adding an equal volume of ethyl acetate for extraction twice after the reaction is finished, and combining the organic phases for gas phase detection.
The results showed that the catalytic activities of mutants Y84A, Y84V, Y84L, Y84I, Y T and Y84C were improved by 25.2%, 40.0%, 25.5%, 34.5%, 24.7% and 33.75% respectively compared to the wild type. And the products of the wild type OYE2p and the mutant catalytic citral are mainly (R) -citronellal, wherein the stereoselectivity of the mutant Y84V, Y84L, Y I and Y84T is respectively improved to 98.0%, 92.1%, 95.5% and 90.1%.
Therefore, the catalytic performance of the mutant obtained by the invention is greatly improved compared with that of the wild-type olefinic bond reductase OYE2p, wherein the mutant Y84V has the optimal catalytic activity and the highest stereoselectivity.
Example 4 kinetic parameters of wild type OYE2p and mutant Y84V thereof
The kinetic parameters of the olefinic reductase OYE2p and its mutants on citral were determined by fixing the concentration of NADH at 0.2mM under standard conditions, and varying the concentration of E-citral or Z-citral between 0.01 and 10 mM. The standard detection method is as follows: the total reaction volume was 200. Mu.L, 2-20. Mu.g of the pure enzyme obtained in example 2, 10mM of the substrate, 100mM of PBS buffer (pH 7.4) were added to make up to 196. Mu.L, and after shaking and heat-preserving at 30℃for 5min, 4. Mu.L of NADH solution (10 mM) was added, and the mixture was immediately placed in an microplate reader to determine the change in absorbance at 340 nm. To ensure the accuracy of the experiment, the assay was repeated three times for each sample.
The results show that for E-citral, the wild type and Y84V mutants are k cat /K m The values are 1.78S respectively -1 /mM -1 And 3.05S -1 /mM -1 . For Z-citral, wild type and Y84V mutant k cat /K m The values are respectively 0.42S -1 /mM -1 And 0.50S -1 /mM -1 。
Catalytic efficiency of mutant Y84V on E-citral and Z-citral (k cat /K m Values) increased by about 71% and 19% respectively over the wild type, i.e.when the amino acid residue at this position was mutated, the mutant strain increased to a different extent in E-citral and Z-citral, indicating that this amino acid residue has an important role in the catalytic activity of the olefinic reductase.
Example 5 application of wild type OYE2p and mutant Y84V thereof in catalyzing production of (R) -citronellal
And (3) freeze-drying the OYE2p and crude enzyme liquid of each mutant expression strain obtained in the step (2) of the example to obtain freeze-dried crude enzyme powder. The reaction system of the citral catalyzed by the wild type OYE2p and the mutant Y84V thereof is as follows: 200mM citral (dissolved in DMSO), 0.2mM NAD + 250mM glucose, 100mM phosphate buffer (pH 8.5), 0.15g of lyophilized OYE2p or mutant Y84V crude enzyme powder thereof, and 0.05g of lyophilized Glucose Dehydrogenase (GDH) crude enzyme powder in a total volume of 5mL. The reaction was carried out in 25mL Erlenmeyer flasks at 30℃and 200rpm shaking. By adding Na during the reaction 2 CO 3 The pH value of the solution regulating system is maintained at 8.5.
As shown in FIG. 2, after the wild type OYE2p catalyzes 200mM citral for 10 hours, the conversion rate reached 91.6%, but the ee value of the resulting product (R) -citronellal was only 87.6%. As shown in FIG. 3, the conversion rate of the mutant Y84V obtained by the invention reaches 100% after catalyzing 200mM citral for 10 hours, and the ee value of the product is more than 95%. Therefore, the catalytic performance of the mutant obtained by the invention is greatly improved compared with that of the wild type olefinic bond reductase OYE2 p.
The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, and various modifications can be made to the above-described embodiment of the present invention. All simple, equivalent changes and modifications made in accordance with the claims and the specification of the present application fall within the scope of the claims of the present invention. The present invention is not described in detail in the conventional art.
SEQUENCE LISTING
<110> university of Industy of Huadong
<120> an activity and stereoselectivity-enhanced olefinic reductase mutant, and coding gene and application thereof
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<170> PatentIn version 3.5
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Leu Ala Arg Asp Gly Leu Arg Tyr Asp Ser Ala Ser Asp Asn Val Tyr
130 135 140
Met Asn Ala Glu Gln Glu Glu Lys Ala Lys Lys Ala Asn Asn Pro Gln
145 150 155 160
His Ser Ile Thr Lys Asp Glu Ile Lys Gln Tyr Val Lys Glu Tyr Val
165 170 175
Gln Ala Ala Lys Asn Ser Ile Ala Ala Gly Ala Asp Gly Val Glu Ile
180 185 190
His Ser Ala Asn Gly Tyr Leu Leu Asn Gln Phe Leu Asp Pro His Ser
195 200 205
Asn Asn Arg Thr Asp Glu Tyr Gly Gly Ser Ile Glu Asn Arg Ala Arg
210 215 220
Phe Thr Leu Glu Val Val Asp Ala Val Val Asp Ala Ile Gly Pro Glu
225 230 235 240
Lys Val Gly Leu Arg Leu Ser Pro Tyr Gly Val Phe Asn Ser Met Ser
245 250 255
Gly Gly Ala Glu Thr Gly Ile Val Ala Gln Tyr Ala Tyr Val Leu Gly
260 265 270
Glu Leu Glu Arg Arg Ala Lys Ala Gly Lys Arg Leu Ala Phe Val His
275 280 285
Leu Ile Glu Pro Arg Val Thr Asn Pro Phe Leu Thr Glu Gly Glu Gly
290 295 300
Glu Tyr Asn Gly Gly Ser Asn Glu Phe Ala Tyr Ser Ile Trp Lys Gly
305 310 315 320
Pro Ile Ile Arg Ala Gly Asn Phe Ala Leu His Pro Glu Val Val Arg
325 330 335
Glu Glu Val Lys Asp Pro Arg Thr Leu Ile Gly Tyr Gly Arg Phe Phe
340 345 350
Ile Ser Asn Pro Asp Leu Val Asp Arg Leu Glu Lys Gly Leu Pro Leu
355 360 365
Asn Lys Tyr Asp Arg Asp Thr Phe Tyr Lys Met Ser Ala Glu Gly Tyr
370 375 380
Ile Asp Tyr Pro Thr Tyr Glu Glu Ala Leu Lys Leu Gly Trp Asp Lys
385 390 395 400
Asn
<210> 4
<211> 401
<212> PRT
<213> artificial sequence
<400> 4
Met Val Pro Phe Val Lys Asp Phe Lys Pro Gln Ala Leu Gly Asp Thr
1 5 10 15
Asn Leu Phe Lys Pro Ile Lys Ile Gly Asn Asn Glu Leu Leu His Arg
20 25 30
Ala Val Ile Pro Pro Leu Thr Arg Met Arg Ala Gln His Pro Gly Asn
35 40 45
Ile Pro Asn Arg Asp Trp Ala Val Glu Tyr Tyr Ala Gln Arg Ala Gln
50 55 60
Arg Pro Gly Thr Leu Ile Ile Thr Glu Gly Thr Phe Pro Ser Pro Gln
65 70 75 80
Ser Gly Gly Val Asp Asn Ala Pro Gly Ile Trp Ser Glu Glu Gln Ile
85 90 95
Lys Glu Trp Thr Lys Ile Phe Lys Ala Ile His Glu Lys Lys Ser Phe
100 105 110
Ala Trp Val Gln Leu Trp Val Leu Gly Trp Ala Ala Phe Pro Asp Thr
115 120 125
Leu Ala Arg Asp Gly Leu Arg Tyr Asp Ser Ala Ser Asp Asn Val Tyr
130 135 140
Met Asn Ala Glu Gln Glu Glu Lys Ala Lys Lys Ala Asn Asn Pro Gln
145 150 155 160
His Ser Ile Thr Lys Asp Glu Ile Lys Gln Tyr Val Lys Glu Tyr Val
165 170 175
Gln Ala Ala Lys Asn Ser Ile Ala Ala Gly Ala Asp Gly Val Glu Ile
180 185 190
His Ser Ala Asn Gly Tyr Leu Leu Asn Gln Phe Leu Asp Pro His Ser
195 200 205
Asn Asn Arg Thr Asp Glu Tyr Gly Gly Ser Ile Glu Asn Arg Ala Arg
210 215 220
Phe Thr Leu Glu Val Val Asp Ala Val Val Asp Ala Ile Gly Pro Glu
225 230 235 240
Lys Val Gly Leu Arg Leu Ser Pro Tyr Gly Val Phe Asn Ser Met Ser
245 250 255
Gly Gly Ala Glu Thr Gly Ile Val Ala Gln Tyr Ala Tyr Val Leu Gly
260 265 270
Glu Leu Glu Arg Arg Ala Lys Ala Gly Lys Arg Leu Ala Phe Val His
275 280 285
Leu Ile Glu Pro Arg Val Thr Asn Pro Phe Leu Thr Glu Gly Glu Gly
290 295 300
Glu Tyr Asn Gly Gly Ser Asn Glu Phe Ala Tyr Ser Ile Trp Lys Gly
305 310 315 320
Pro Ile Ile Arg Ala Gly Asn Phe Ala Leu His Pro Glu Val Val Arg
325 330 335
Glu Glu Val Lys Asp Pro Arg Thr Leu Ile Gly Tyr Gly Arg Phe Phe
340 345 350
Ile Ser Asn Pro Asp Leu Val Asp Arg Leu Glu Lys Gly Leu Pro Leu
355 360 365
Asn Lys Tyr Asp Arg Asp Thr Phe Tyr Lys Met Ser Ala Glu Gly Tyr
370 375 380
Ile Asp Tyr Pro Thr Tyr Glu Glu Ala Leu Lys Leu Gly Trp Asp Lys
385 390 395 400
Asn
<210> 5
<211> 401
<212> PRT
<213> artificial sequence
<400> 5
Met Val Pro Phe Val Lys Asp Phe Lys Pro Gln Ala Leu Gly Asp Thr
1 5 10 15
Asn Leu Phe Lys Pro Ile Lys Ile Gly Asn Asn Glu Leu Leu His Arg
20 25 30
Ala Val Ile Pro Pro Leu Thr Arg Met Arg Ala Gln His Pro Gly Asn
35 40 45
Ile Pro Asn Arg Asp Trp Ala Val Glu Tyr Tyr Ala Gln Arg Ala Gln
50 55 60
Arg Pro Gly Thr Leu Ile Ile Thr Glu Gly Thr Phe Pro Ser Pro Gln
65 70 75 80
Ser Gly Gly Leu Asp Asn Ala Pro Gly Ile Trp Ser Glu Glu Gln Ile
85 90 95
Lys Glu Trp Thr Lys Ile Phe Lys Ala Ile His Glu Lys Lys Ser Phe
100 105 110
Ala Trp Val Gln Leu Trp Val Leu Gly Trp Ala Ala Phe Pro Asp Thr
115 120 125
Leu Ala Arg Asp Gly Leu Arg Tyr Asp Ser Ala Ser Asp Asn Val Tyr
130 135 140
Met Asn Ala Glu Gln Glu Glu Lys Ala Lys Lys Ala Asn Asn Pro Gln
145 150 155 160
His Ser Ile Thr Lys Asp Glu Ile Lys Gln Tyr Val Lys Glu Tyr Val
165 170 175
Gln Ala Ala Lys Asn Ser Ile Ala Ala Gly Ala Asp Gly Val Glu Ile
180 185 190
His Ser Ala Asn Gly Tyr Leu Leu Asn Gln Phe Leu Asp Pro His Ser
195 200 205
Asn Asn Arg Thr Asp Glu Tyr Gly Gly Ser Ile Glu Asn Arg Ala Arg
210 215 220
Phe Thr Leu Glu Val Val Asp Ala Val Val Asp Ala Ile Gly Pro Glu
225 230 235 240
Lys Val Gly Leu Arg Leu Ser Pro Tyr Gly Val Phe Asn Ser Met Ser
245 250 255
Gly Gly Ala Glu Thr Gly Ile Val Ala Gln Tyr Ala Tyr Val Leu Gly
260 265 270
Glu Leu Glu Arg Arg Ala Lys Ala Gly Lys Arg Leu Ala Phe Val His
275 280 285
Leu Ile Glu Pro Arg Val Thr Asn Pro Phe Leu Thr Glu Gly Glu Gly
290 295 300
Glu Tyr Asn Gly Gly Ser Asn Glu Phe Ala Tyr Ser Ile Trp Lys Gly
305 310 315 320
Pro Ile Ile Arg Ala Gly Asn Phe Ala Leu His Pro Glu Val Val Arg
325 330 335
Glu Glu Val Lys Asp Pro Arg Thr Leu Ile Gly Tyr Gly Arg Phe Phe
340 345 350
Ile Ser Asn Pro Asp Leu Val Asp Arg Leu Glu Lys Gly Leu Pro Leu
355 360 365
Asn Lys Tyr Asp Arg Asp Thr Phe Tyr Lys Met Ser Ala Glu Gly Tyr
370 375 380
Ile Asp Tyr Pro Thr Tyr Glu Glu Ala Leu Lys Leu Gly Trp Asp Lys
385 390 395 400
Asn
<210> 6
<211> 401
<212> PRT
<213> artificial sequence
<400> 6
Met Val Pro Phe Val Lys Asp Phe Lys Pro Gln Ala Leu Gly Asp Thr
1 5 10 15
Asn Leu Phe Lys Pro Ile Lys Ile Gly Asn Asn Glu Leu Leu His Arg
20 25 30
Ala Val Ile Pro Pro Leu Thr Arg Met Arg Ala Gln His Pro Gly Asn
35 40 45
Ile Pro Asn Arg Asp Trp Ala Val Glu Tyr Tyr Ala Gln Arg Ala Gln
50 55 60
Arg Pro Gly Thr Leu Ile Ile Thr Glu Gly Thr Phe Pro Ser Pro Gln
65 70 75 80
Ser Gly Gly Ile Asp Asn Ala Pro Gly Ile Trp Ser Glu Glu Gln Ile
85 90 95
Lys Glu Trp Thr Lys Ile Phe Lys Ala Ile His Glu Lys Lys Ser Phe
100 105 110
Ala Trp Val Gln Leu Trp Val Leu Gly Trp Ala Ala Phe Pro Asp Thr
115 120 125
Leu Ala Arg Asp Gly Leu Arg Tyr Asp Ser Ala Ser Asp Asn Val Tyr
130 135 140
Met Asn Ala Glu Gln Glu Glu Lys Ala Lys Lys Ala Asn Asn Pro Gln
145 150 155 160
His Ser Ile Thr Lys Asp Glu Ile Lys Gln Tyr Val Lys Glu Tyr Val
165 170 175
Gln Ala Ala Lys Asn Ser Ile Ala Ala Gly Ala Asp Gly Val Glu Ile
180 185 190
His Ser Ala Asn Gly Tyr Leu Leu Asn Gln Phe Leu Asp Pro His Ser
195 200 205
Asn Asn Arg Thr Asp Glu Tyr Gly Gly Ser Ile Glu Asn Arg Ala Arg
210 215 220
Phe Thr Leu Glu Val Val Asp Ala Val Val Asp Ala Ile Gly Pro Glu
225 230 235 240
Lys Val Gly Leu Arg Leu Ser Pro Tyr Gly Val Phe Asn Ser Met Ser
245 250 255
Gly Gly Ala Glu Thr Gly Ile Val Ala Gln Tyr Ala Tyr Val Leu Gly
260 265 270
Glu Leu Glu Arg Arg Ala Lys Ala Gly Lys Arg Leu Ala Phe Val His
275 280 285
Leu Ile Glu Pro Arg Val Thr Asn Pro Phe Leu Thr Glu Gly Glu Gly
290 295 300
Glu Tyr Asn Gly Gly Ser Asn Glu Phe Ala Tyr Ser Ile Trp Lys Gly
305 310 315 320
Pro Ile Ile Arg Ala Gly Asn Phe Ala Leu His Pro Glu Val Val Arg
325 330 335
Glu Glu Val Lys Asp Pro Arg Thr Leu Ile Gly Tyr Gly Arg Phe Phe
340 345 350
Ile Ser Asn Pro Asp Leu Val Asp Arg Leu Glu Lys Gly Leu Pro Leu
355 360 365
Asn Lys Tyr Asp Arg Asp Thr Phe Tyr Lys Met Ser Ala Glu Gly Tyr
370 375 380
Ile Asp Tyr Pro Thr Tyr Glu Glu Ala Leu Lys Leu Gly Trp Asp Lys
385 390 395 400
Asn
<210> 7
<211> 401
<212> PRT
<213> artificial sequence
<400> 7
Met Val Pro Phe Val Lys Asp Phe Lys Pro Gln Ala Leu Gly Asp Thr
1 5 10 15
Asn Leu Phe Lys Pro Ile Lys Ile Gly Asn Asn Glu Leu Leu His Arg
20 25 30
Ala Val Ile Pro Pro Leu Thr Arg Met Arg Ala Gln His Pro Gly Asn
35 40 45
Ile Pro Asn Arg Asp Trp Ala Val Glu Tyr Tyr Ala Gln Arg Ala Gln
50 55 60
Arg Pro Gly Thr Leu Ile Ile Thr Glu Gly Thr Phe Pro Ser Pro Gln
65 70 75 80
Ser Gly Gly Thr Asp Asn Ala Pro Gly Ile Trp Ser Glu Glu Gln Ile
85 90 95
Lys Glu Trp Thr Lys Ile Phe Lys Ala Ile His Glu Lys Lys Ser Phe
100 105 110
Ala Trp Val Gln Leu Trp Val Leu Gly Trp Ala Ala Phe Pro Asp Thr
115 120 125
Leu Ala Arg Asp Gly Leu Arg Tyr Asp Ser Ala Ser Asp Asn Val Tyr
130 135 140
Met Asn Ala Glu Gln Glu Glu Lys Ala Lys Lys Ala Asn Asn Pro Gln
145 150 155 160
His Ser Ile Thr Lys Asp Glu Ile Lys Gln Tyr Val Lys Glu Tyr Val
165 170 175
Gln Ala Ala Lys Asn Ser Ile Ala Ala Gly Ala Asp Gly Val Glu Ile
180 185 190
His Ser Ala Asn Gly Tyr Leu Leu Asn Gln Phe Leu Asp Pro His Ser
195 200 205
Asn Asn Arg Thr Asp Glu Tyr Gly Gly Ser Ile Glu Asn Arg Ala Arg
210 215 220
Phe Thr Leu Glu Val Val Asp Ala Val Val Asp Ala Ile Gly Pro Glu
225 230 235 240
Lys Val Gly Leu Arg Leu Ser Pro Tyr Gly Val Phe Asn Ser Met Ser
245 250 255
Gly Gly Ala Glu Thr Gly Ile Val Ala Gln Tyr Ala Tyr Val Leu Gly
260 265 270
Glu Leu Glu Arg Arg Ala Lys Ala Gly Lys Arg Leu Ala Phe Val His
275 280 285
Leu Ile Glu Pro Arg Val Thr Asn Pro Phe Leu Thr Glu Gly Glu Gly
290 295 300
Glu Tyr Asn Gly Gly Ser Asn Glu Phe Ala Tyr Ser Ile Trp Lys Gly
305 310 315 320
Pro Ile Ile Arg Ala Gly Asn Phe Ala Leu His Pro Glu Val Val Arg
325 330 335
Glu Glu Val Lys Asp Pro Arg Thr Leu Ile Gly Tyr Gly Arg Phe Phe
340 345 350
Ile Ser Asn Pro Asp Leu Val Asp Arg Leu Glu Lys Gly Leu Pro Leu
355 360 365
Asn Lys Tyr Asp Arg Asp Thr Phe Tyr Lys Met Ser Ala Glu Gly Tyr
370 375 380
Ile Asp Tyr Pro Thr Tyr Glu Glu Ala Leu Lys Leu Gly Trp Asp Lys
385 390 395 400
Asn
<210> 8
<211> 401
<212> PRT
<213> artificial sequence
<400> 8
Met Val Pro Phe Val Lys Asp Phe Lys Pro Gln Ala Leu Gly Asp Thr
1 5 10 15
Asn Leu Phe Lys Pro Ile Lys Ile Gly Asn Asn Glu Leu Leu His Arg
20 25 30
Ala Val Ile Pro Pro Leu Thr Arg Met Arg Ala Gln His Pro Gly Asn
35 40 45
Ile Pro Asn Arg Asp Trp Ala Val Glu Tyr Tyr Ala Gln Arg Ala Gln
50 55 60
Arg Pro Gly Thr Leu Ile Ile Thr Glu Gly Thr Phe Pro Ser Pro Gln
65 70 75 80
Ser Gly Gly Cys Asp Asn Ala Pro Gly Ile Trp Ser Glu Glu Gln Ile
85 90 95
Lys Glu Trp Thr Lys Ile Phe Lys Ala Ile His Glu Lys Lys Ser Phe
100 105 110
Ala Trp Val Gln Leu Trp Val Leu Gly Trp Ala Ala Phe Pro Asp Thr
115 120 125
Leu Ala Arg Asp Gly Leu Arg Tyr Asp Ser Ala Ser Asp Asn Val Tyr
130 135 140
Met Asn Ala Glu Gln Glu Glu Lys Ala Lys Lys Ala Asn Asn Pro Gln
145 150 155 160
His Ser Ile Thr Lys Asp Glu Ile Lys Gln Tyr Val Lys Glu Tyr Val
165 170 175
Gln Ala Ala Lys Asn Ser Ile Ala Ala Gly Ala Asp Gly Val Glu Ile
180 185 190
His Ser Ala Asn Gly Tyr Leu Leu Asn Gln Phe Leu Asp Pro His Ser
195 200 205
Asn Asn Arg Thr Asp Glu Tyr Gly Gly Ser Ile Glu Asn Arg Ala Arg
210 215 220
Phe Thr Leu Glu Val Val Asp Ala Val Val Asp Ala Ile Gly Pro Glu
225 230 235 240
Lys Val Gly Leu Arg Leu Ser Pro Tyr Gly Val Phe Asn Ser Met Ser
245 250 255
Gly Gly Ala Glu Thr Gly Ile Val Ala Gln Tyr Ala Tyr Val Leu Gly
260 265 270
Glu Leu Glu Arg Arg Ala Lys Ala Gly Lys Arg Leu Ala Phe Val His
275 280 285
Leu Ile Glu Pro Arg Val Thr Asn Pro Phe Leu Thr Glu Gly Glu Gly
290 295 300
Glu Tyr Asn Gly Gly Ser Asn Glu Phe Ala Tyr Ser Ile Trp Lys Gly
305 310 315 320
Pro Ile Ile Arg Ala Gly Asn Phe Ala Leu His Pro Glu Val Val Arg
325 330 335
Glu Glu Val Lys Asp Pro Arg Thr Leu Ile Gly Tyr Gly Arg Phe Phe
340 345 350
Ile Ser Asn Pro Asp Leu Val Asp Arg Leu Glu Lys Gly Leu Pro Leu
355 360 365
Asn Lys Tyr Asp Arg Asp Thr Phe Tyr Lys Met Ser Ala Glu Gly Tyr
370 375 380
Ile Asp Tyr Pro Thr Tyr Glu Glu Ala Leu Lys Leu Gly Trp Asp Lys
385 390 395 400
Asn
<210> 9
<211> 1203
<212> DNA
<213> artificial sequence
<400> 9
atggttccat ttgttaagga ctttaagcca caagctttgg gtgacaccaa cttattcaaa 60
ccaatcaaaa ttggtaacaa tgaacttcta caccgtgctg tcattcctcc attgactaga 120
atgagagccc aacatccagg taatattcca aacagagact gggccgttga atactacgct 180
caacgtgctc aaagaccagg aaccttgatt atcactgaag gtacctttcc ctctccacaa 240
tctgggggtg ttgacaatgc tccaggtatc tggtccgaag aacaaattaa agaatggacc 300
aagattttca aggctattca tgagaagaaa tcgttcgcat gggtccaatt atgggttcta 360
ggttgggctg ctttcccaga cacccttgct agggatggtt tgcgttacga ctccgcttct 420
gacaacgtgt atatgaatgc agaacaagaa gaaaaggcta agaaggctaa caacccacaa 480
cacagtataa caaaggatga aattaagcaa tacgtcaaag aatacgtcca agctgccaaa 540
aactccattg ctgctggtgc cgatggtgtt gaaatccaca gcgctaacgg ttacttgttg 600
aaccagttct tggacccaca ctccaataac agaaccgatg agtatggtgg atccatcgaa 660
aacagagccc gtttcacctt ggaagtggtt gatgcagttg tcgatgctat tggccctgaa 720
aaagtcggtt tgagattgtc tccatatggt gtcttcaaca gtatgtctgg tggtgctgaa 780
accggtattg ttgctcaata tgcttatgtc ttaggtgaac tagaaagaag agctaaagct 840
ggcaagcgtt tggctttcgt ccatctaatt gaacctcgtg tcaccaaccc atttttaact 900
gaaggtgaag gtgaatacaa tggaggtagc aacgaatttg cttattctat ctggaagggc 960
ccaattatta gagctggtaa ctttgctctg cacccagaag ttgtcagaga agaggtgaag 1020
gatcctagaa cattgatcgg ttacggtaga ttttttatct ctaatccaga tttggttgat 1080
cgtttggaaa aagggttacc attaaacaaa tatgacagag acactttcta caaaatgtca 1140
gctgagggat acattgacta ccctacgtac gaagaagctc taaaactcgg ttgggacaaa 1200
aat 1203
<210> 10
<211> 20
<212> DNA
<213> artificial sequence
<400> 10
acccccagat tgtggagagg 20
<210> 11
<211> 43
<212> DNA
<213> artificial sequence
<400> 11
cctctccaca atctgggggt gccgacaatg ctccaggtat ctg 43
<210> 12
<211> 43
<212> DNA
<213> artificial sequence
<400> 12
cctctccaca atctgggggt gttgacaatg ctccaggtat ctg 43
<210> 13
<211> 43
<212> DNA
<213> artificial sequence
<400> 13
cctctccaca atctgggggt ttggacaatg ctccaggtat ctg 43
<210> 14
<211> 43
<212> DNA
<213> artificial sequence
<400> 14
cctctccaca atctgggggt attgacaatg ctccaggtat ctg 43
<210> 15
<211> 43
<212> DNA
<213> artificial sequence
<400> 15
cctctccaca atctgggggt accgacaatg ctccaggtat ctg 43
<210> 16
<211> 43
<212> DNA
<213> artificial sequence
<400> 16
cctctccaca atctgggggt tgtgacaatg ctccaggtat ctg 43
Claims (4)
1. An olefinic bond reductase mutant with improved activity and stereoselectivity in catalyzing citral preparationR) The application of citronellal is characterized in that the mutant of the vinyl reductase takes a wild vinyl reductase OYE2p shown in SEQ ID NO.1 as a template,
an olefinic bond reductase mutant Y84V with tyrosine Y at 84 th position mutated into valine V, and the amino acid sequence is shown in SEQ ID NO. 4;
an olefinic bond reductase mutant Y84L with tyrosine Y at 84 th position mutated into leucine L, and the amino acid sequence is shown in SEQ ID NO. 5;
the 84 th tyrosine Y is mutated into an isoleucine I olefinic bond reductase mutant Y84I, and the amino acid sequence is shown in SEQ ID NO. 6.
2. The application of claim 1, wherein the application comprises: fermenting recombinant genetically engineered bacteria containing an olefinic bond reductase mutant Y84V coding gene, culturing, centrifuging to obtain thalli, thalli immobilized cells, ultrasonically crushing thalli, extracting enzyme, immobilized enzyme or pure enzyme as a catalyst, taking citral as a substrate, taking buffer solution with pH of 7-9 as a reaction medium, reacting at 20-40 ℃ under 150-300r/min, separating and purifying the reaction solution after the reaction is finished to obtain the productR) Citronellal.
3. The use according to claim 2, wherein the substrate concentration in the reaction system is 20-200mM, the glucose concentration is 30-220mM, the amount of the olefinic reductase is 1-5U/mL in terms of enzyme activity, and the amount of the glucose dehydrogenase is 1.5-7.5U/mL in terms of enzyme activity.
4. The use according to claim 3, wherein the reaction is carried out by adding Na according to pH changes 2 CO 3 The solution was adjusted to pH 8.5.
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| SG172783A1 (en) * | 2008-12-25 | 2011-08-29 | Codexis Inc | Enone reductases |
| EP3601582A4 (en) * | 2017-03-29 | 2021-01-27 | University of Florida Research Foundation, Inc. | Materials and methods for alkene reduction of levoglucosenone by an alkene reductase |
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