CN112662639B - Short-chain alcohol dehydrogenase and application thereof - Google Patents
Short-chain alcohol dehydrogenase and application thereof Download PDFInfo
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- CN112662639B CN112662639B CN202110079155.9A CN202110079155A CN112662639B CN 112662639 B CN112662639 B CN 112662639B CN 202110079155 A CN202110079155 A CN 202110079155A CN 112662639 B CN112662639 B CN 112662639B
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Abstract
The invention discloses a short-chain alcohol dehydrogenase and application thereof, belonging to the technical field of biological engineering. The invention obtains a short-chain alcohol dehydrogenase from the Pieris methystica (Tarenayahassleriana), and the amino acid sequence is shown as SEQ ID NO. 2. The short-chain alcohol dehydrogenase can reduce various carbonyl compounds and has excellent affinity to the carbonyl compounds, wherein the carbonyl compounds comprise propionaldehyde, valeraldehyde, caprylic aldehyde, 3-methylbutyraldehyde, benzaldehyde, 3-methylthiopropionaldehyde, 2, 5-dichloronicotinaldehyde, 2, 4-dimethyl-3-pentanone, 3-methylcyclohexanone, (-) -carvone, (+) -carvone, (-) -camphor, (-) -camphorquinone, ethyl 4-chloroacetoacetate and the like, can be suitable for various industrial production scenes, and has important industrial application value.
Description
Technical Field
The invention relates to a short-chain alcohol dehydrogenase and application thereof, belonging to the technical field of biological engineering.
Background
Catalytic hydrogenation of carbonyl compounds is an important class of reactions. In vivo, enzymes that catalyze the reduction of carbonyl compounds are collectively called carbonyl reductases (EC 1.1.1184), and are largely classified into the following categories: short-chain dehydrogenases/reductases family (SDR), aldone reductase family (AKR), Zinc-dependent alcohol dehydrogenases/reductases family (ADH). Among them, SDR family functions are relatively differentiated, and short-chain alcohol dehydrogenase is a relatively representative member in catalytic hydrogenation functions and has no metal dependence.
Short-chain alcohol dehydrogenases, are widely found in plants, animals and microorganisms. In the application of reducing carbonyl compounds, alcohol dehydrogenase derived from microorganisms is mostly studied. SDR is more representative in plants due to its diverse roles in primary and secondary metabolism (A multi-substrate reduction from plant major: structure-function in the short chain reduction enzyme superior. Sci Rep.2018; 8(1): 14796). Many SDRs have been characterized to date for elucidation of metabolic pathways, where most SDRs have a broader reduction of carbonyl compounds. For example, Rose Bengal reductase PAR reduces mainly several monoterpenes and aromatic carbonyl compounds, as well as reducing activity on acetaldehyde (Functional catalysis of rose Phenyl Aldehyde Reduction (PAR), an enzyme-absorbed in the biological synthesis of the scientific compound 2-phenyl ethyl alcohol. J Plant physiology.2011; 168(2): 88-95.). In addition, pea SAD short-chain dehydrogenases/reductases catalyze The reduction of a variety of quinone and aromatic carbonyl compounds, but are limited to these two classes of compounds (The pea SAD short-chain dehydrogenases/reductases: quinone reduction, tissue distribution, and biotechnology expression. plant physiology.2011; 155(4): 1839-50). However, there is no SDR that can simultaneously reduce aliphatic carbonyl compounds, aromatic compounds, quinones, and terpene ketones. Therefore, the method for mining the short-chain alcohol dehydrogenase with more excellent catalytic performance and wider substrate range in plants is an advantageous direction for developing a novel biocatalyst, and has important research significance and practical application value.
Disclosure of Invention
Aiming at the problems of weak catalytic performance, narrow substrate spectrum, small applicable range and the like of SDR discovered at present, the invention firstly characterizes a short-chain alcohol dehydrogenase from Indiana flower (Tarenayahassleiana). The enzyme has reduction activity on various carbonyl compounds, has strong catalytic performance and is suitable for production and application.
The invention provides application of short-chain alcohol dehydrogenase in carbonyl compound reduction, wherein the amino acid sequence of the short-chain alcohol dehydrogenase is shown as SEQ ID NO. 2.
In one embodiment, the nucleotide sequence of the short-chain alcohol dehydrogenase encoding gene is shown as SEQ ID No. 1.
In one embodiment, the carbonyl compound includes aldehydes, aliphatic ketones, terpenoids containing carbonyl groups, quinones, esters.
In one embodiment, the aldehyde compounds include propionaldehyde, valeraldehyde, caprylic aldehyde, 3-methylbutyraldehyde, benzaldehyde, 3-methylthiopropionaldehyde, 2, 5-dichloronicotinaldehyde.
In one embodiment, the aliphatic ketones include 2, 4-dimethyl-3-pentanone, 3-methylcyclohexanone, (-) -carvone, (+) -carvone, (-) -camphor.
In one embodiment, the carbonyl-containing terpenoid includes (-) -camphor; the quinone compounds include (-) -camphorquinone; the ester compound comprises 4-chloroacetoacetic acid ethyl ester.
In one embodiment, the nucleotide sequence encoding the short-chain alcohol dehydrogenase is shown in SEQ ID NO. 1.
The invention provides a method for reducing carbonyl compounds, which is to catalyze the carbonyl compounds by using short-chain alcohol dehydrogenase with an amino acid sequence shown as SEQ ID NO. 2.
In one embodiment, the carbonyl compound comprises aldehydes, aliphatic ketones, terpenoids containing carbonyl groups, quinones, esters.
In one embodiment, the aldehyde compounds include propionaldehyde, valeraldehyde, caprylic aldehyde, 3-methylbutyraldehyde, benzaldehyde, 3-methylthiopropionaldehyde, 2, 5-dichloronicotinaldehyde.
In one embodiment, the aliphatic ketones include 2, 4-dimethyl-3-pentanone, 3-methylcyclohexanone, (-) -carvone, (+) -carvone, (-) -camphor.
In one embodiment, the carbonyl-containing terpenoid includes (-) -camphor; the quinone compounds include (-) -camphorquinone; the ester compound comprises 4-chloroacetoacetic acid ethyl ester.
In one embodiment, NADPH is present in the reaction system.
In one embodiment, the reaction is carried out at 45-50 deg.C and pH 4.5-5.5.
In one embodiment, the substrate concentration is 0.5 to 1.5mM, the NADPH concentration is 0.1 to 0.15mM, the pH of the reaction system is maintained by a phosphate buffer solution having a phosphate buffer salt concentration of 15 to 25 mM.
The beneficial effects that this hair demonstrates: the invention obtains a short-chain alcohol dehydrogenase from the Pieris methystica (Tarenayahasslerana), the enzyme can reduce a plurality of carbonyl compounds and has excellent affinity to the carbonyl compounds, the carbonyl compounds comprise propionaldehyde, valeraldehyde, caprylic aldehyde, 3-methylbutyraldehyde, benzaldehyde, 3-methylthiopropionaldehyde, 2, 5-dichloronicotinaldehyde, 2, 4-dimethyl-3-pentanone, 3-methylcyclohexanone, (-) -carvone, (+) -carvone, (-) -camphor, (-) -camphorquinone, ethyl 4-chloroacetoacetate and the like, and the invention can be suitable for various industrial production scenes and has important industrial application value.
Drawings
FIG. 1 is an SDS-PAGE image of the expression product, lane 1: a protein Marker; lane 2: crude enzyme solution; lane 3: pure enzyme liquid of short-chain alcohol dehydrogenase; lane 4: the supernatant was disrupted by recombinant null bacteria (E.coli BL 21/pRSFDuet-1).
Detailed Description
Analysis of enzymatic Properties of short-chain alcohol dehydrogenase:
3-methylcyclohexanone is used as a substrate to research the influence of pH on the enzymatic activity of the short-chain alcohol dehydrogenase;
3-methylcyclohexanone is used as a substrate to study the influence of temperature on the enzymatic activity of the short-chain alcohol dehydrogenase.
Substrate specificity analysis of short-chain alcohol dehydrogenase: the substrate used is formaldehyde, propionaldehyde, valeraldehyde, isovaleraldehyde, 2-ethylhexanal, octanal, 3-methylthiopropionaldehyde, 2, 4-dimethyl-3-pentanone, 3-methylcyclohexanone, 4-methylcyclohexanone, benzaldehyde, (+) -carvone, (-) -cuminone, (-) -menthone, (-) -camphor, (+) -camphor, tropinone, 3-quinuclidone, (-) -camphorquinone, 2, 5-dichloronicotinaldehyde, ethyl 4-chloroacetoacetate.
The enzyme activity determination method comprises the following steps: the amount of NADPH consumed at 340nm was measured by a spectrophotometer to determine the activity of the enzyme. The substrate screening system was 3mL in volume and contained 0.13mM NADPH, 1mM substrate and 20mM phosphate buffer (pH 7.2), and these mixtures were incubated at 37 ℃ for 10min to prime the reaction by adding 50. mu.L of purified enzyme solutionMeasurement of OD by spectrophotometry340A change in (c). The blank analysis contained the same mixture without substrate. All measurements were repeated three times.
The specific activity of the enzyme is calculated by the formula:
Δ a is the difference in decrease in absorbance
TVTotal reaction system
mass of added enzyme
L-cell optical path
Epsilon is the molar extinction coefficient, epsilon340＝6220L·moL-1·cm-1
Example 1: construction of engineering bacterium of colibacillus for expressing short-chain alcohol dehydrogenase
1. Acquisition of nucleic acid sequence of short-chain alcohol dehydrogenase Gene
According to the amino acid sequence of the short-chain alcohol dehydrogenase, after the codon optimization of cDNA and mRNA is carried out by Visual Gene Developer, the Gene synthesis is carried out to obtain the target Gene segment (the nucleotide sequence is shown as SEQ ID NO. 1), and a recombinant plasmid is constructed.
2. Competent preparation of E.coli
(1) Coli DH 5. alpha. and BL21(DE3) were each cultured overnight at 37 ℃ and 200rpm/min in a small tube containing 3mL of LB medium.
(2) Inoculating into 50mL LB medium at 1% inoculum size, and culturing at 37 deg.C to OD600About 0.6 (about 2-3 h).
(3) Transferring the bacterial liquid into a 50mL precooled centrifuge tube, placing the centrifuge tube on ice for 30min, and centrifuging the centrifuge tube at 8000rpm/min and 4 ℃ for 5 min.
(4) The supernatant was discarded and 5mL of pre-cooled 0.1mol/L CaCl were added2The solution (containing 15% glycerol) was suspended in the bacterial cells, and the suspension was centrifuged at 8000rpm/min at 4 ℃ for 5min on ice for 20 min. Repeat 2 times.
(5) The supernatant was discarded and 2mL of pre-cooled 0.1mol/L CaCl was added2The cells were gently suspended in the solution, and then added to each centrifuge tube (1.5mL)Subpackaging with 100 μ L of bacterial solution, and preserving in a refrigerator at-80 deg.C for use.
3. Preparation of recombinant Escherichia coli
(1) Mu.l of recombinant plasmid was added to 100. mu.l of DH 5. alpha./BL 21 competent bacteria, gently mixed, and ice-cooled for 30 min.
(2) Placing into preheated 42 deg.C water bath, and standing for 90s for heat shock treatment.
(3) Immediately ice-bath for 2 min.
(4) 1ml of LB medium containing no antibiotic was added and cultured at 37 ℃ for 1 hour to resuscitate the cells.
(5) The cells were spread evenly on kanamycin-resistant LB plates.
(6) The growth vigor is good after 24h of culture. And selecting a single colony for colony PCR, carrying out sequencing verification after nucleic acid electrophoresis verification, and storing the positive transformant for later use, wherein the positive transformant is determined as the correct transformant.
Example 2: induced expression, separation and purification of short-chain alcohol dehydrogenase
1. Adding 500. mu.l of the recombinant bacterial solution prepared in example 1 to 50ml of LB medium, culturing at 37 ℃ for 2.5 hours, and adjusting the Optical Density (OD) at 600nm600) When the concentration reaches 0.6, 40 mul of 0.5M IPTG is added, and the mixture is subjected to cold induction culture at 15 ℃ for 24 hours to obtain fermentation liquor. Centrifuging the fermentation liquid (8000rmp/min, 10min), collecting precipitate to obtain thallus, re-dissolving thallus with disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution (20mmol/L, pH 7.0), crushing thallus with ultrasonicator, centrifuging (8000rmp/min, 10min), and collecting supernatant to obtain crude enzyme solution.
2. By using
and (2) performing nickel column affinity purification on the crude enzyme solution obtained in the step (1) by using an avant 150 protein purification system, wherein an elution method comprises the following steps: the four pipes A1, A2, B1 and B2 were placed in water, and the system flow was set at 20ml/min for pump purge. Then setting system flow 1ml/min, flow path (column position 3), delta pressure 0.3, pre-pressure 0.5, Gradient 0, insert A1, connecting nickel column after water drops flow out uniformly, placing A1 into the binding liquid after balancing for ten minutes, placing B1 into the eluent, and then proceedingThe air was discharged by one pump, the binding solution was equilibrated for twenty minutes, and then the crude enzyme solution was loaded, and the target protein was linearly eluted with 500mM of a high concentration imidazole buffer solution (B1-based solution), and the protein adsorbed on the nickel column was eluted to obtain a purified enzyme (see FIG. 1). The purified enzyme is freeze-dried for later use.
Example 3: optimum temperature of short-chain alcohol dehydrogenase
The reaction system is 3ml (1mM substrate, 0.13mM NADPH, 20mM phosphate buffer solution, pH 5.0), 3-methylcyclohexanone and phosphate buffer solution with pH5.0 are bathed for 10min at 25-65 ℃ and under the condition of 5 ℃ as gradient, 50 mu l of NADPH is added, 50 mu l of enzyme solution is added and shaken uniformly to carry out colorimetric determination, and the enzyme activity of the short-chain alcohol dehydrogenase is calculated, as shown in Table 1, under the condition of 45-50 ℃, the enzyme activity of the short-chain alcohol dehydrogenase can be kept above 90%.
TABLE 1 residual enzyme Activity of short-chain alcohol dehydrogenase at different temperatures
Example 4: optimum pH of short-chain alcohol dehydrogenase
The reaction system is 3ml (1mM substrate, 0.13mM NADPH, 20mM phosphate buffer solution, temperature 50 ℃), 3-methylcyclohexanone is respectively dissolved in the phosphate buffer solution with pH 4.0-7.0, water bath is carried out for 10min at 50 ℃, 50 mul NADPH is added, 50 mul enzyme solution is added to be shaken up and then colorimetric determination is carried out, the highest enzyme activity is 100%, relative enzyme activities under different pH values are calculated, and the results are shown in Table 2, the activity of the short-chain alcohol dehydrogenase can be kept above 89% under the condition of pH 5.5, and the activity of the short-chain alcohol dehydrogenase can be kept better under the conditions of pH4.5 and pH 5.0.
TABLE 2 enzymatic Activity of short-chain alcohol dehydrogenases at different pH
Example 5: reaction characteristics of short-chain alcohol dehydrogenases with different substrates
The substrate concentration was 1mM, and the poorly water-soluble substrate was dissolved in 5% methanol. The specific enzyme activity was calculated, and the highest value was defined as 100%. The results are shown in Table 1, where LMA (low measured activity) means that the enzyme has catalytic activity on the substrate but low assay activity.
TABLE 3 Activity of short-chain alcohol dehydrogenases on different substrates
Example 6: catalytic ability of alcohol dehydrogenases with different substrates
Selecting a substrate to set a reaction system with different concentrations, adding a proper amount of enzyme, measuring the specific activity of the enzyme, and calculating the enzymatic kinetic parameters of the short-chain dehydrogenase to each substrate according to the nonlinear fitting of the substrate concentration and the specific enzyme activity. The results are shown in Table 4. It can be seen that the short-chain alcohol dehydrogenase catalyzes camphorquinone most efficiently, and has outstanding affinity and catalytic efficiency for the important prodrug COBE. The enzyme has good industrial application prospect.
TABLE 4 kinetic parameters of short-chain alcohol dehydrogenases on different substrates
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> short-chain alcohol dehydrogenase and application thereof
<130> BAA201690A
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 810
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<213> Artificial sequence
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gcactggtga ccggcggcac aaaaggttta ggtgaagcag tggtggaaga attagcgggt 120
tttggagcgc gtatacacac gtgcgcccgc gacggtgacc atctcaataa gtgtctccgc 180
gaatggcagg ccaaaggttt tcaggtaacc ggttcggtgt gcgacgtctc cctgcgaacc 240
gaacgcgaaa aattgatgga aacagttagt tctctgttca acggagaact gaatatactc 300
atcaataacg tgggcaccaa tatgacgaag cccacgacag aatatacggc agaagatttc 360
tccttcctta tggccacaaa tttcgaatct tcatatcacc tttgtcaatt gtcgcatcct 420
ttactgaaag cttctggcag cggtagcatc gtctttatgt cctctgtttg tggggtggta 480
tcgatcaatg ttggctcgat ttacggtgca acaaagggcg ctatgaatca gttaacccgt 540
aatcttgcat gtgagtgggc cagtgataat attcgagcaa atagtgtttg cccgtggttc 600
atttctaccc cgttggcgta tcgctatctg gaggatgaaa aatttaaaga agcggtggtg 660
tcccgtaatc ctatccgtcg tgtgggagaa ccggaagaag tgtcttcgct ggttaccttt 720
ctgtgcttgc cggcggcgtc atacattacc ggccaaacta tctgtgtcga tgggggcatg 780
acagttaacg gctttagctt cagtgcttaa 810
<210> 2
<211> 269
<212> PRT
<213> Tarenaya hassleriana
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Met Ala Lys Ala Glu Glu Ser Ile Arg Gln Asn Ser Arg Trp Ser Leu
1 5 10 15
Gln Gly Lys Thr Ala Leu Val Thr Gly Gly Thr Lys Gly Leu Gly Glu
20 25 30
Ala Val Val Glu Glu Leu Ala Gly Phe Gly Ala Arg Ile His Thr Cys
35 40 45
Ala Arg Asp Gly Asp His Leu Asn Lys Cys Leu Arg Glu Trp Gln Ala
50 55 60
Lys Gly Phe Gln Val Thr Gly Ser Val Cys Asp Val Ser Leu Arg Thr
65 70 75 80
Glu Arg Glu Lys Leu Met Glu Thr Val Ser Ser Leu Phe Asn Gly Glu
85 90 95
Leu Asn Ile Leu Ile Asn Asn Val Gly Thr Asn Met Thr Lys Pro Thr
100 105 110
Thr Glu Tyr Thr Ala Glu Asp Phe Ser Phe Leu Met Ala Thr Asn Phe
115 120 125
Glu Ser Ser Tyr His Leu Cys Gln Leu Ser His Pro Leu Leu Lys Ala
130 135 140
Ser Gly Ser Gly Ser Ile Val Phe Met Ser Ser Val Cys Gly Val Val
145 150 155 160
Ser Ile Asn Val Gly Ser Ile Tyr Gly Ala Thr Lys Gly Ala Met Asn
165 170 175
Gln Leu Thr Arg Asn Leu Ala Cys Glu Trp Ala Ser Asp Asn Ile Arg
180 185 190
Ala Asn Ser Val Cys Pro Trp Phe Ile Ser Thr Pro Leu Ala Tyr Arg
195 200 205
Tyr Leu Glu Asp Glu Lys Phe Lys Glu Ala Val Val Ser Arg Asn Pro
210 215 220
Ile Arg Arg Val Gly Glu Pro Glu Glu Val Ser Ser Leu Val Thr Phe
225 230 235 240
Leu Cys Leu Pro Ala Ala Ser Tyr Ile Thr Gly Gln Thr Ile Cys Val
245 250 255
Asp Gly Gly Met Thr Val Asn Gly Phe Ser Phe Ser Ala
260 265
Claims (5)
1. The application of the short-chain alcohol dehydrogenase in reducing carbonyl compounds is characterized in that the amino acid sequence of the short-chain alcohol dehydrogenase is shown in SEQ ID NO.2, and the carbonyl compounds are 3-methylthiopropanal, 3-methylcyclohexanone, 4-methylcyclohexanone, (-) -camphorquinone and 4-chloroacetoacetic acid ethyl ester.
2. The use according to claim 1, wherein the nucleotide sequence encoding the short-chain alcohol dehydrogenase is as shown in SEQ ID No. 1.
3. A method for reducing carbonyl compounds is characterized in that short-chain alcohol dehydrogenase with an amino acid sequence shown as SEQ ID No.2 is used for catalyzing the carbonyl compounds, wherein the carbonyl compounds are 3-methylmercapto propionaldehyde, 3-methylcyclohexanone, 4-methylcyclohexanone, (-) -camphorquinone and 4-ethyl chloroacetoacetate.
4. The method according to claim 3, wherein NADPH is contained in the reaction system.
5. The method according to claim 4, wherein the reaction is carried out at 45 to 50 ℃ and at a pH of 4.5 to 5.5.
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Non-Patent Citations (3)
| Title |
|---|
| A multisubstrate reductase from Plantago major: structure-function in the short chain reductase superfamily;Rachel Fellows，et al;《Sci Rep》;20181004;第8卷;第1-13页 * |
| XP_010521893.1;NCBI;《NCBI Reference Sequence》;20161118;第1页 * |
| 基于 RNA-Seq 的油茶种仁萜类功能组分代谢调控研究;曾艳玲等;《经济林研究》;20161231;第34卷(第4期);第1-6页 * |
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