Ethylenic bond reductase as well as mutant and application thereof

Abstract

The invention provides olefinic bond reductase as well as a mutant and application thereof, and belongs to the technical field of catalysis and transformation of functional enzyme molecules. The amino acid sequence of the olefinic bond reductase disclosed by the invention is as shown in SEQ ID NO: 1, and the amino acid sequence of the mutant of the olefinic bond reductase is as shown in SEQ ID NO: 3. The olefinic bond reductase and the mutant thereof provided by the invention are combined with glucose dehydrogenase for use, when the olefinic bond reductase and the mutant thereof are used for catalyzing citral, a coenzyme NADH does not need to be added into a reaction system, meanwhile, a byproduct gluconate can be produced, the economic benefit of the reaction is improved, the cost of the reaction is greatly reduced, and the olefinic bond reductase and the mutant thereof have more industrial production potential and value in preparation of (R)-citronellal.

Description

Technical Field

[0001] The invention belongs to the technical field of catalysis and modification of functional enzyme molecules, and specifically relates to an olefin reductase derived from Candida sake and a mutant thereof, a nucleic acid molecule encoding the olefin reductase or the mutant thereof, a recombinant vector and a recombinant cell comprising the nucleic acid molecule, a preparation method of the olefin reductase and the mutant thereof, and application of the olefin reductase and the mutant thereof, the nucleic acid molecule, the recombinant vector or the recombinant cell in preparing (R)-citronellal. Background Art

[0002] The molecular formula of citronellal is C 10 H 18 O, also known as 3,7-dimethyl-6-octenal, is an acyclic monoterpene aldehyde that occurs naturally in essential oils such as citronella oil and lemon eucalyptus oil, and under natural conditions mostly exists as a mixture of (R / S)-citronellal.

[0003] L-Menthol has a unique minty aroma and excellent cooling effect. It is widely used in fine chemicals, food, medical treatment, health and other fields. It is one of the top three fragrances in the world. (R)-Citronellal is an important chiral intermediate in the process of synthesizing L-menthol. It can also be used to synthesize important products such as natural vitamin E and male wax moth sex pheromones. Since the quality and yield of menthol extracted naturally from peppermint oil are greatly affected by natural environmental factors, and mainly uses (R)-configured citronellal, chemical synthesis is currently the mainstream method for industrial preparation of (R)-citronellal. However, when relying on chemical asymmetric hydrogenation to prepare products, the metal catalysts used may face a series of problems such as high cost, high requirements for the reaction environment, difficulty in catalyst recovery and use, and high reaction energy consumption.

[0004] The bio-enzyme method for catalyzing the synthesis of (R)-citronellal from citral can better solve the dilemma faced by chemical catalysis. However, the existing bio-enzyme method for catalyzing the synthesis of (R)-citronellal from citral has problems such as low conversion rate and low product ee value. Summary of the invention

[0005] In order to solve the above problems existing in the prior art, the present invention provides an olefin reductase and its mutants derived from Candida sake, which are used in combination with glucose dehydrogenase to obtain a catalytic reaction system for realizing coenzyme circulation, so that only NAD needs to be added to the reaction system. + , which can efficiently catalyze the conversion of citral into (R)-citronellal.

[0006] In a first aspect, the present invention provides an olefin reductase, the amino acid sequence of the olefin reductase is shown as SEQ ID NO:1.

[0007] Amino acid sequence of olefin reductase (SEQ ID NO: 1)

[0008] MSFVKDFKPQALGDTNLFKPIKIGNNELLHRAVIPPLTRRMRAQHPGNIPNRDWAVEYYAQRAQRPGTLIITEGTFPSPQSGGYDNAPGIWSEEQIKEWTK IFKAIHENKSFAWVQLWVLGWAAFPDTLARDGLRYDSASDNVYMNAEQEEKAKKANNPQHSITKDEIKQYVKEYVQAAKNSIAAGADGVEIHSANGYLLN QFLDPHSNNRTDEYGGSIENRARFTLEVVDAVVDAIGPEKVGLRLSPYGVFNSMSGGAETGIVAQYAYVLGELERRAKAGKRLAFVHLVEPRVTNPFLTE GEGEYNGGSNKFAYSIWKGPIIRAGNFALHPEVVREEVKDPRTLIGYGRFFISNPDLVDRLEKGLPLNKYDRDTFYKMSAEGYIDYPTYEEALKLGWDKK

[0009] The above-mentioned olefin bond reductase provided by the present invention can be a natural, recombinant or synthetic active polypeptide, and the active polypeptide can be a naturally purified product, a chemically synthesized product, or a product produced from a prokaryotic host (such as Escherichia coli) or a eukaryotic host (such as yeast, higher plants) using recombinant technology.

[0010] In some embodiments, the olefin reductase is obtained by introducing a recombinant vector containing its encoding gene into an expression host (eg, E. coli BL21 (DE3)) to obtain a recombinant genetic engineering strain, and then culturing the recombinant genetic engineering strain and inducing expression to obtain the olefin reductase.

[0011] In a second aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned olefin reductase, and the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:2.

[0012] Nucleotide sequence encoding olefin reductase (SEQ ID NO: 2)

[0013]

[0014] The nucleic acid molecules provided by the present invention can usually be obtained by PCR amplification or artificial synthesis.

[0015] In a third aspect, the present invention provides an olefin reductase mutant, the amino acid sequence of the olefin reductase mutant is shown in SEQ ID NO:3.

[0016] Amino acid sequence of olefin reductase mutant (SEQ ID NO: 3)

[0017] MSFVKDFKPQALGDTNLFKPIKIGNNELLHRAVIPLTRMRAQHPGNIPNRDWAVEYYAQRAQRPGTLIITEGTFPSPQSGGYDNAPGIWSEEQIKEWTKIFKAIHENKSFAWVQLWVLGWAAFPDTLARDGLRYDSASDNVYMNAEQEEKAK T ANNPQHSITKDEIKQYVKEYVQAAKNSIAAGADGVEIHSANGYLLNQFLDPHSNNRTDEYGGSIENRARFTLEVVDAVVDAIGPEKVGLRLSPYGVFNSMSGGAETGIVAQYAYVLGELERRAKAGKRLAF H HLVEPRVTNPFLTEGEGEYNGGSNKFAYSIWKGPIIRAGNFALHPEVVREEVKDPRTLIGYGRFFISNPDLVDRLEKGLPLNKYDRDTFYKMSAEGYIDYPTYEEALKLGWDKK

[0018] The olefin reductase mutant provided by the present invention is obtained by modifying the above-mentioned olefin reductase. Specifically, the present invention mainly uses a semi-rational design method to modify it. First, a molecular library of 96 enzyme mutations is obtained by error-prone PCR technology, and then after high-throughput reaction screening, an intermediate mutant of the olefin reductase with improved catalytic citral conversion is obtained, and the intermediate mutant has the following site mutations: the amino acid Lys at the 154th position is mutated to Asn, and the amino acid Val at the 286th position is mutated to Asp; the above-mentioned 154th and 286th sites are further subjected to site-directed saturation mutations, and after screening, a mutant of the olefin reductase with better catalytic citral conversion is obtained, and the mutant has the following site mutations: the amino acid Lys at the 154th position is mutated to Thr, and the amino acid Val at the 286th position is mutated to His. Compared with olefin reductase, the mutant has higher citral conversion activity.

[0019] The olefin reductase mutant provided by the present invention can be artificially synthesized, or its encoding gene can be synthesized first and then expressed biologically, such as by expressing it from a prokaryotic host (Escherichia coli) or a eukaryotic host (such as yeast, higher plants) using recombinant technology.

[0020] In some embodiments, the above-mentioned olefin reductase mutant is obtained by introducing a recombinant vector containing its encoding gene into an expression host (such as E. coli BL21 (DE3)) to obtain a recombinant genetic engineering strain, and then culturing the recombinant genetic engineering strain and inducing expression to obtain the olefin reductase mutant.

[0021] In a fourth aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned olefin reductase mutant, wherein the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:4.

[0022] Nucleotide sequence encoding an olefin reductase mutant (SEQ ID NO: 4)

[0023] atgtcatttgttaaggatttcaagccacaagctttgggtgacaccaacttattcaaaccaatcaaaattggtaacaatgaacttctacaccgtgctgtcattcctccattgact agaatgagagcccaacatccaggtaatattccaaacagagactgggccgttgaatactacgctcaacgtgctcaaagaccaggaaccttgattatcactgaaggtacctttccct ctccacaatctgggggttacgacaatgctccaggtatctggtccgaagaacaaattaaagaatggaccaagattttcaaggctattcatgagaataaatcgttcgcatgggtcca attatgggttctaggttgggctgctttcccagacacccttgctagggatggtttgcgttacgactccgcttctgacaacgtgtatatgaatgcagaacaagaagaaaaggctaag acggctaacaacccacaacacagtataacaaaggatgaaattaagcaatacgtcaaagaatacgtccaagctgccaaaaactccattgctgctggtgccga tggtgttgaaatccacagcgctaacggttacttgttgaaccagttcttggacccacactccaataacagaaccgatgagtatggtggatccatcgaaa acagagcccgtttcaccttggaagtggttgatgcagttgtcgatgctattggccctgaaaaagtcggtttgagattgtctccatatggtgtcttcaac agtatgtctggtggtgctgaaaccggtattgttgctcaatatgcttatgtcttaggtgaactagaaagaagagctaaagctggcaagcgtttggctttc cac catctagttgaacctcgtgtcaccaacccatttttaactgaaggtgaaggtgaatacaatggaggtagcaacaaatttgcttattctatctggaagggcccaattattagagctggtaactttgctctgcacccagaagttgtcagagaagaggtgaaggatcctagaaca ttgatcggttacggtagattttttatctctaatccagatttggttgatcgtttggaaaaagggttaccattaaacaaatatgacagagacactttctacaaaaatgtcagctgagggatacattgactaccctacgtacgaagaagctctaaaactcggctgggacaagaag

[0024] The nucleic acid molecules of the olefin reductase mutants provided by the present invention can usually be obtained by PCR amplification or artificial synthesis.

[0025] In a fifth aspect, the present invention provides a recombinant vector comprising any of the nucleic acid molecules described above.

[0026] The recombinant vector provided by the present invention includes a cloning vector and an expression vector. The cloning vector is used to replicate related sequences, and the expression vector is used to express related enzyme genes.

[0027] In some embodiments, the expression vector includes pET-28a(+), which is used for the expression of olefin reductase and mutant genes thereof.

[0028] In a sixth aspect, the present invention provides a recombinant cell comprising any of the recombinant vectors described above.

[0029] In some embodiments, the method for preparing the recombinant cell comprises the step of transforming the recombinant vector into an expression host cell.

[0030] In some preferred embodiments, the expression host cell is a prokaryotic cell or a eukaryotic cell, such as Escherichia coli, yeast, etc., and more preferably, the Escherichia coli expression host E. coli BL21 (DE3) is used.

[0031] More specifically, the recombinant cell construction method provided by the present invention comprises the following steps:

[0032] (i) obtaining olefin reductase genes and olefin reductase positive mutant genes;

[0033] (ii) constructing expression vectors for olefin reductase and olefin reductase mutant, respectively;

[0034] (iii) transforming the above recombinant expression vectors into host strains respectively;

[0035] (iv) Screening on plate resistance culture medium to obtain positive clone strains.

[0036] In a seventh aspect, the present invention provides a method for preparing an olefin reductase or a mutant thereof, comprising:

[0037] Cultivating any of the above-mentioned recombinant cells and inducing expression to obtain a culture;

[0038] A step of isolating the above olefin reductase or the above olefin reductase mutant from the culture.

[0039] In the present invention, there are no special requirements for the culture method and culture conditions, as long as the normal growth of the recombinant cells is ensured. And the methods for isolating the above-mentioned olefin reductase or the above-mentioned mutant from the culture are all conventional methods in the art.

[0040] In some embodiments, the culture medium used in the method for preparing the olefin reductase or a mutant thereof is a culture medium that can express proteins in the art, preferably TB culture medium.

[0041] In some embodiments, the induction temperature is 16-25°C, for example, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C or a value or range between any two of these values, preferably the induction temperature is 22°C; the final concentration of the inducer IPTG is preferably 0.5mM.

[0042] In some embodiments, the separation method is as follows: 8000rpm, 4°C, 10min, centrifugation to collect bacteria, adding 5-10 times the mass volume of pH 8.5 PBS solution to wash and resuspend the bacteria, using ultrasonic disruption or high-pressure homogenizer to disrupt the resuspended bacteria, 10000rpm, 4°C, 20min, centrifugation to collect the supernatant, purifying the olefin reductase or mutant through a Ni ion column, using an ultrafiltration tube to concentrate the purified protein and exchange the solution to pH 8.5 PBS buffer and freeze it at -80°C for use.

[0043] In an eighth aspect, the present invention provides the use of the above-mentioned olefin reductase, the above-mentioned nucleic acid molecule, the above-mentioned olefin reductase mutant, the above-mentioned recombinant vector, the above-mentioned recombinant cell or the olefin reductase or its mutant obtained by the above-mentioned preparation method in the preparation of (R)-citronellal.

[0044] In the ninth aspect, the present invention provides a method for preparing (R)-citronellal, comprising the following steps: using the above-mentioned olefin reductase, the above-mentioned olefin reductase mutant, the above-mentioned recombinant cell or the olefin reductase or its mutant obtained by the above-mentioned preparation method as a catalyst to catalyze the reaction of citral to obtain (R)-citronellal.

[0045] In some preferred embodiments, the catalyst further comprises glucose dehydrogenase, and the glucose dehydrogenase is preferably derived from Cedecea neteri; in some more preferred embodiments, the amino acid sequence of the glucose dehydrogenase is shown in SEQ ID NO:5.

[0046] Amino acid sequence of glucose dehydrogenase (SEQ ID NO: 5)

[0047] VNTNKTGSQLFVTLSALFAVLCGLYLLFGGIWLVAIGGSWYYPIAGLVMLGVAWLLWKSKASALWLYAALLLCTMAWGVWEVDFWALTPRCHVLVFFGVWLLLPFVSRRLVIPASGAAASLVVSLLITAVVLVWAGFNDPQEINGTLQLDNTAAAAGSSIPDADWPAYGRNQEGQRFSPLKQITTQNVGQLKEAWRF QTGDVKRATDPGEITNEVTPIKIRDMLYLCTAHQQLFALDASTGKEKWKFDPQLNTDPSFQHVTCRGVTYHEATADNASADVVADCPRRIILPVNDGRLFALNADTGKLCESFANKGILNLQTNQPVTTPGMYEPTSPPIVTDKVIVIAGAVTDNFSTREPSGVIRGFDVNTGKLLWAFDPGAKDPNAIPDDGHHYTLN SPNSWAPAAYDAKLDLVYLPMGVTTPDIWGGNRTAEQERYASSIVALNATTGKLAWSYQTVHHDLWDMDMPAQPTLADITDKSGNKVPVVYAPAKTGNIFVLDRRDGKLVVPAPEQPVPQGAAKGDHVSPTQPFSELTFRPKKDLSGADMWGATMFDQLVCRVMFHSMRYEGIFTPPSEKGTLVFPGNLGMFEWGGISV DPNNQVAMTNPMARPFVSKLIPRGPGNPMEPPKDAKGSGTESGVQPQYGVPYGVTLNPFLSPLGLPCKQPAWGYISGVDLKTNEIVWKKRIGTVEDSMP FPTGIPMKFRMGMPMLGGPISTAGNVMFIGATADNYLRAFDMRDGKQLWEARLPAGGQATPMTYEVNGKQYVVISAGGHGSFGTKMGDYIVAYALPDDAK

[0048] The glucose dehydrogenase provided by the present invention can be a natural, recombinant or synthetic active polypeptide, and the active polypeptide can be a naturally purified product, a chemically synthesized product, or a product produced from a prokaryotic host (such as Escherichia coli) or a eukaryotic host (such as yeast, higher plants) using recombinant technology.

[0049] In some embodiments, the glucose dehydrogenase is obtained by introducing a recombinant vector containing its encoding gene into an expression host (eg, E. coli BL21 (DE3)) to obtain a recombinant genetic engineering strain, and then culturing the recombinant genetic engineering strain and inducing expression to obtain glucose dehydrogenase.

[0050] In some embodiments, the present invention provides a nucleic acid molecule encoding the above-mentioned glucose dehydrogenase, and the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:6.

[0051] Nucleotide sequence encoding glucose dehydrogenase (SEQ ID NO:6)

[0052]

[0053] The nucleic acid molecules provided by the present invention can usually be obtained by PCR amplification or artificial synthesis.

[0054] In some embodiments, the present invention provides a recombinant vector comprising a nucleic acid molecule encoding the above-mentioned glucose dehydrogenase. The recombinant vector provided by the present invention includes a cloning vector and an expression vector, wherein the expression vector includes pET-21a(+), and the pET-21a(+) is used for the expression of the glucose dehydrogenase gene.

[0055] In some embodiments, the present invention provides a recombinant cell, comprising a recombinant vector encoding the above-mentioned glucose dehydrogenase nucleic acid molecule. The preparation method of the recombinant cell comprises the step of transforming the recombinant vector into an expression host cell.

[0056] In some embodiments, the present invention provides a method for preparing glucose dehydrogenase, comprising culturing a recombinant cell containing a recombinant vector containing a nucleic acid molecule encoding the above-mentioned glucose dehydrogenase, and inducing the expression of glucose dehydrogenase to obtain a culture; and isolating the above-mentioned glucose dehydrogenase from the culture.

[0057] In the present invention, there are no special requirements for the culture method and culture conditions, as long as the normal growth of the recombinant cells is ensured. And the methods for isolating the above-mentioned glucose dehydrogenase from the culture are all conventional methods in the art.

[0058] In some embodiments, the culture medium used in the method for preparing glucose dehydrogenase is a culture medium that can express proteins in the art, preferably TB culture medium.

[0059] In some embodiments, the induction temperature is 16-25°C, for example, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C or a value or range between any two of these values, preferably the induction temperature is 18°C; the final concentration of the inducer IPTG is preferably 0.5mM.

[0060] In some embodiments, the separation method is as follows: 8000rpm, 4°C, 10min, centrifugation to collect bacteria, adding 5-10 times the mass volume of pH 8.5 PBS solution to wash and resuspend the bacteria, using ultrasonic disruption or high-pressure homogenizer to disrupt the resuspended bacteria, 10000rpm, 4°C, 20min, centrifugation to collect the supernatant, purifying glucose dehydrogenase through a Ni ion column, using an ultrafiltration tube to concentrate the purified protein and exchange the solution to pH 8.5 PBS buffer and freeze it at -80°C for use.

[0061] The olefin reductase or its mutant and glucose dehydrogenase provided by the present invention can be added to the reaction system in the form of whole cells, or can be unpurified crude enzyme solution, or can be partially purified or completely purified semi-pure enzyme or pure enzyme form. The olefin reductase or its mutant and glucose dehydrogenase described in the present invention can also be prepared as a catalyst in the form of immobilized enzyme or immobilized cells by using conventional immobilization means in the art and added to the reaction system.

[0062] In some preferred embodiments, the olefin reductase or its mutant and glucose dehydrogenase in the present invention are added to the reaction system in the form of whole cells. Whole cells are more conducive to maintaining enzyme activity and have a lower cost advantage.

[0063] In some preferred embodiments, the olefin reductase or its mutant and glucose dehydrogenase in the present invention are expressed separately in different cells and then added to the reaction system in the form of whole cells to avoid the inability to reconcile the ratios of the two enzymes in the catalytic cascade reaction due to the mismatch in expression rhythms caused by co-expression of the enzymes in a single cell, thereby causing problems such as insufficient reaction, poor reaction effect, and difficulty in separating the resulting products.

[0064] In some embodiments, the wet cells obtained by centrifugation and washing of the induced cultured cells (cells containing olefin reductase or its mutants and cells containing glucose dehydrogenase) are used as catalysts, and the wet cell catalyst concentration of the olefin reductase or its mutants is 40-110 g / L, for example, 40 g / L, 50 g / L, 60 g / L, 70 g / L, 80 g / L, 90 g / L, 100 g / L, 110 g / L or a value or range between any two of these values, preferably 80 g / L; the wet cell concentration of glucose dehydrogenase is 64-176 g / L, for example The wet cell mass ratio of the olefin reductase or its mutant to the wet cell mass ratio of the glucose dehydrogenase is 1:0.6-1:2.0, for example, 1:0.6, 1:0.8, 1:1.0, 1:1.2, 1:1.4, 1:1.6, 1:1.8, 1:2.0 or a ratio or a ratio range between any two of these ratios, preferably 1:1.6.

[0065] In some embodiments, the recombinant cells containing olefin reductase or its mutant and the recombinant cells containing glucose dehydrogenase can be lyophilized into bacterial powder. And the method for preparing lyophilized bacterial powder can be a conventional method in the art, for example, prepared by the following method: 8000rpm, 4°C, 10min, centrifugal collection of bacterial cells, 5-10 times the mass volume of PBS solution with pH 8.5 to wash the bacterial cells, centrifugal collection, freezing at -20°C overnight, running a vacuum freeze dryer, -20°C, vacuum degree 15Pa for 48h, and obtaining lyophilized bacterial powder.

[0066] In some preferred embodiments, the concentration of the freeze-dried powder catalyst of the olefin reductase or its mutant is 12-32 g / L, for example, 12 g / L, 14 g / L, 16 g / L, 18 g / L, 20 g / L, 22 g / L, 24 g / L, 26 g / L, 28 g / L, 30 g / L, 32 g / L or a value or range between any two of these values, preferably 24 g / L; the concentration of the freeze-dried powder of glucose dehydrogenase is 24-64 g / L, for example 24g / L, 34g / L, 48g / L, 54g / L, 64g / L or a value or range between any two of these values, preferably 48g / L; the mass ratio of the freeze-dried powder of the olefin reductase or its mutant to the freeze-dried powder of the glucose dehydrogenase is 1:1.6-1:2.4, for example, 1:1.6, 1:1.8, 1:2.0, 1:2.2, 1:2.4 or a ratio or ratio range between any two of these ratios, preferably 1:2.0.

[0067] In some embodiments, as a reaction involving oxidoreductase known in the art, it is understood that DMSO, glucose and NAD can also be added to the reaction system for catalyzing citral to prepare (R)-citronellal. + .

[0068] Wherein, the DMSO is used as a cosolvent, and its volume concentration is 15-35%, for example, it can be 15%, 17%, 19%, 21%, 23%, 25%, 27%, 29%, 31%, 33%, 35% or a value or range between any two of these values, preferably 25%; the concentration of the glucose is 100-800mM, for example, 100mM, 200mM, 300mM, 400mM, 500mM, 600mM, 700mM, 800mM or a value or range between any two of these values, preferably 700mM; the NAD +The concentration is 0.05-0.2 mM, for example, 0.05 mM, 0.07 mM, 0.09 mM, 0.11 mM, 0.13 mM, 0.15 mM, 0.17 mM, 0.19 mM, 0.20 mM or a value or range between any two of these values, preferably 0.15 mM.

[0069] In the present invention, the reaction system for catalyzing citral to prepare (R)-citronellal can be catalyzed to convert citral into (R)-citronellal without adding coenzyme NADH. Meanwhile, gluconate is a by-product of the reaction, which can be separated and purified to generate additional economic benefits.

[0070] In some embodiments, the concentration of citral is 20-850mM, for example, 20mM, 80mM, 140mM, 200mM, 260mM, 320mM, 380mM, 460mM, 520mM, 580mM, 640mM, 650mM, 700mM, 760mM, 820mM, 850mM or a value or range between any two of these values, preferably 650mM; the temperature of the reaction is 20-45°C, for example, 20°C, 23°C, 26°C, 29°C, 32°C, 35°C, 38°C, 41°C, 44°C, 45°C or a value or range between any two of these values, preferably 650mM. The reaction temperature is 35 ℃; the reaction temperature is 7.5-9.0, for example, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0 or a value or range between any two of these values, preferably 8.5; in some preferred embodiments, 2M sodium hydroxide can be used to adjust the pH in the reaction system; the reaction time is 22-40h, for example, 22h, 24h, 26h, 28h, 30h, 32h, 34h, 36h, 38h, 40h or a value or range between any two of these values, preferably 28h.

[0071] Compared with the prior art, the present invention has the following beneficial effects:

[0072] The invention provides an olefin reductase derived from Candida sake, and further mutates the olefin reductase to obtain an olefin reductase mutant. Compared with the wild-type olefin reductase, the mutant has a 28h conversion rate of 99.9% when catalyzing citral to convert into (R)-citronellal, an ee value of 98% for the product (R)-citronellal, a yield of 92%, a time-space yield of 3.29g / (L*h), and a conversion rate of 2.64 times that of the original wild-type olefin reductase. In addition, by using in combination with glucose dehydrogenase, the catalytic efficiency of the olefin reductase mutant is further improved, and there is no need to add coenzyme NADH to the reaction system, while producing gluconate as a byproduct, the economic benefit of the reaction is improved, the cost of the reaction is greatly reduced, and the potential and value of industrial production are more in the preparation of (R)-citronellal. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 This is a schematic diagram of the reaction of synthesizing (R)-citronellal catalyzed by olefin reductase and glucose dehydrogenase of the present invention;

[0074] Figure 2 is a gas chromatogram of the citral standard in Example 5 of the present invention;

[0075] Figure 3 is a gas chromatogram of the (R)-citronellal standard substance in Example 5 of the present invention;

[0076] Figure 4 is a gas chromatogram of the conversion of citral into (R)-citronellal in Example 5 of the present invention;

[0077] Figure 5 This is a chromatogram showing the ee value of the reaction product (R)-citronellal in Example 5 of the present invention. DETAILED DESCRIPTION

[0078] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0079] The experimental methods without specific conditions in the examples are usually carried out according to conventional conditions and conditions described in the manual, or according to conditions recommended by the manufacturer. The general equipment, materials, reagents, etc. used are all available from commercial channels unless otherwise specified.

[0080] Candida sake has been disclosed in the literature (Gueguen, Y. "Purification and characterization of an intracellular beta-glucosidase from a candida sake strain isolated from fruit juices." Applied Biochemistry & Biotechnology, 95(3), 151.) and is publicly available from Wanhua Chemical Group Co., Ltd.

[0081] Cedecea neteri has been disclosed in the literature (Chan K G. Complete Genome Sequence of Cedecea neteri Strain SSMD04, a Bacterium Isolated from Pickled Mackerel Sashimi.". American Society for Microbiology, 2014(6).01339-14) and can be obtained by the public from Wanhua Chemical Group Co., Ltd.

[0082] In the present invention, the conversion rate of citral is measured by gas chromatography, and the specific method is as follows:

[0083] Take about 1 mL of the sample to be tested, extract it with twice the volume of ethyl acetate, take the supernatant after centrifugation and add an appropriate amount of anhydrous sodium sulfate to remove water, and take the supernatant after centrifugation for gas chromatography detection.

[0084] The conditions for gas chromatography detection include: using an SH-RTX-5 capillary column, nitrogen as the carrier gas, FID detector temperature: 300°C, injection port temperature of 280°C, column oven temperature of 100°C, injection volume of 1 μL, column flow rate of 0.86 mL / min, and area normalization to calculate the citral conversion rate.

[0085] Example 1 Acquisition of olefin reductase gene

[0086] 1) Extracting genomic DNA of Candida sake;

[0087] 2) Using the genomic DNA obtained in step 1) as a template, PCR amplification is performed using upstream primer 1 and downstream primer 2 to obtain a DNA fragment containing a wild-type olefin reductase gene.

[0088] The primer sequences are as follows:

[0089] Upstream primer 1: 5'-cgcggatccatgtcatttgttaagg-3' (SEQ ID NO: 7)

[0090] Downstream primer 2: 5'-ctcgaattcttacttcttgtcccagcc-3' (SEQ ID NO: 8)

[0091] The nucleotide sequence of the olefin reductase is shown in SEQ ID NO:2; and the amino acid sequence of the olefin reductase is shown in SEQ ID NO:1.

[0092] Example 2 Establishment of an olefin reductase gene mutant library using error-prone PCR technology

[0093] Using the DNA fragment of the wild-type olefin reductase gene obtained in Example 1 as a template, upstream primer 1 and downstream primer 2 as primers, an error-prone PCR reaction was performed according to the following reaction system to obtain 96 olefin reductase gene mutants.

[0094] Error-prone PCR reaction system: 10× amplification buffer 5 μl, 4 μl of 2.5 mM dNTP mixture, 1.5 μl each of upstream primer 1 and downstream primer 2, 1.5 μl template DNA, 1 μl Taq DNA polymerase, Mn 2+ 5mM, add double distilled water to 50μl.

[0095] PCR reaction procedure: (1) Pre-denaturation: 94°C, 3 min; (2) Denaturation: 94°C, 30 s; Annealing: 60°C, 30 s; Extension: 72°C, 3 min, 30 cycles; (3) Post-extension: 72°C, 10 min; (4) Incubation at 4°C.

[0096] Example 3 High-throughput screening to obtain potential modification sites of olefin reductase and site-directed saturation mutagenesis

[0097] 1) The DNA fragments of the 96 olefin reductase gene mutants obtained in Example 2 were double-digested with BamH I and EcoR I to obtain gene fragments, the pET-28a(+) vector was double-digested with BamH I and EcoR I to obtain vector fragments, the gene fragments and the vector fragments were connected to obtain recombinant expression plasmids, and after sequencing, the recombinant expression plasmids were respectively transferred into the expression host E. coli BL21 (DE3) by heat shock, and the positive clone strains obtained by screening were cultured in 8 ml TB medium for 4-6 h, and IPTG was added at a final concentration of 0.5 mM, and low-temperature induction was performed at 22° C. for 16 h, 6 mL of the bacterial solution was centrifuged and resuspended with 0.2 mL of pH 8.5 PBS, and 110 μL was transferred to a 96-well plate, and olefin reductase was used to catalyze citral to generate (R)-citronellal and NAD in the presence of NADH. + , which will cause a change in the absorbance value at a wavelength of 340nm to determine the catalytic activity of the olefin reductase.

[0098] 2) Continue to add 90 μL substrate solution (0.5 mM NADH; 56.5 μL 2.3 M citral DMSO solution; 19 μL DMSO solution, 14.5 μL pH 8.5 PBS solution; can be prepared as a mother solution), incubate at 35°C and 120 rpm for 4 h, and scan at 340 nm to measure the absorbance.

[0099] 3) Extract the plasmid of the strain corresponding to the reaction solution with the lower absorbance value, and sequence it to confirm the mutation site: the amino acid Lys at position 154 mutated to Asn, and the amino acid Val at position 286 mutated to Asp. For the amino acids at these two sites, design a single-point site-directed saturation mutation of the codon using NNK, and obtain 48 mutants. Continue screening according to the screening method in steps 1) and 2), and obtain 2 positive single mutants with good catalytic effect: positive single mutant 1: the amino acid Lys at position 154 mutated to Thr, positive single mutant 2: the amino acid Val at position 286 mutated to His. According to the mutation information of the above two positive single mutants, design primers to perform site-directed saturation mutagenesis.

[0100] The primer sequences for site-directed saturation mutagenesis are as follows:

[0101] 154 upstream primer 3: 5'-gaacaagaagaaaaggctaagnnkgctaac-3' (SEQ ID NO: 9)

[0102] 154 downstream primer 4: 5'-tgtgggttgttagcnnkcttagcc-3' (SEQ ID NO: 10)

[0103] 286 upstream primer 5: 5'-cgtttggctttcnnkcatctagtt-3' (SEQ ID NO: 11)

[0104] 286 downstream primer 6: 5'-cacgaggttcaactagatgnnkgaaa-3' (SEQ ID NO: 12)

[0105] Site-directed saturation mutagenesis full plasmid PCR reaction system: 10 μl of DNA polymerase mixture, 1 μl of primers and template (the plasmid of the strain corresponding to the reaction solution with the lower absorbance value mentioned above), add double distilled water to 20 μl.

[0106] PCR reaction procedure: (1) Pre-denaturation: 96°C, 3 min (2) Denaturation: 96°C, 30 s; Annealing: 60°C, 30 s; Extension: 72°C, 2.0 min, 30 cycles; (3) Extension: 72°C, 5 min; (4) Continuous incubation at 4°C.

[0107] Among them, upstream primer 3 at position 154, downstream primer 4 at position 154, upstream primer 5 at position 286 and downstream primer 6 at position 286 were used to obtain a combined mutant in which the amino acid Lys at position 154 was mutated to Thr and the amino acid Val at position 286 was mutated to His.

[0108] Example 4 Comparison of catalytic efficiency of combined mutants and single mutations

[0109] 1) The DNA fragment of the combined mutant (or positive single mutant 1, positive single mutant 2) obtained in Example 3 was double-digested with BamH I and EcoR I to obtain a gene fragment, the pET-28a (+) vector was double-digested with BamH I and EcoR I to obtain a vector fragment, the gene fragment and the vector fragment were connected to obtain a recombinant expression plasmid, and the plasmid with correct sequencing was transferred into the expression host E. coli BL21 (DE3) by heat shock, and a single clone colony was picked by plate culture, inoculated into 5 ml LB medium for activation, and then transferred to 100 ml TB medium at a 1% inoculum, and cultured in a shake flask at 37° C. for 4-6 h, IPTG was added at a final concentration of 0.5 mM, and low-temperature induction was performed at 22° C. for 12-16 h, 40 ml of the bacterial solution was centrifuged, and the bacteria were washed with pH 8.5 PBS, the wet bacteria concentration was adjusted to 80 g / L, and 0.2 mM NADH, 100 mM citral, and 25% DMSO (volume fraction) were reacted at 35° C. for 1 h, and the citral conversion rate was measured by gas chromatography.

[0110] The results showed that the conversion rate of the positive single mutant 1 (K154T) was 43.18%, the conversion rate of the positive single mutant 2 (V286H) was 40.86%, and the conversion rate of the combined mutants (K154T and V286H) was 57%. It can be determined that the combined mutant has the best reaction effect, and therefore, an olefin reductase mutant with higher citral conversion activity is obtained.

[0111] The nucleotide sequence of the mutant is shown in SEQ ID NO:4; the amino acid sequence of the mutant is shown in SEQ ID NO:3.

[0112] Example 5 Determination of the efficiency of catalyzing the conversion of citral to (R)-citronellal using a combination of olefin reductase or its mutant and glucose dehydrogenase

[0113] 1) Extracting genomic DNA of Cedecea neteri;

[0114] 2) Using the genomic DNA obtained in step 1) as a template, PCR amplification is performed using upstream primer 1 and downstream primer 2 to obtain a DNA fragment containing the glucose dehydrogenase gene.

[0115] The primer sequences are as follows:

[0116] Upstream primer 7: 5'-atccgaattcgtgaatacaaataaaaccgg-3' (SEQ ID NO: 13)

[0117] Downstream primer 8: 5'-gcaagcttttacttcgcgtcgtc-3' (SEQ ID NO: 14)

[0118] The nucleotide sequence of the glucose dehydrogenase is shown in SEQ ID NO:6; the amino acid sequence of the glucose dehydrogenase is shown in SEQ ID NO:5:

[0119] 3) The DNA fragment of the glucose dehydrogenase gene obtained in step 2) was double-digested with EcoRI and Hind III, and the DNA fragment of the wild-type olefin reductase gene obtained in Example 1 was double-digested with BamH I and EcoR I to obtain a first gene fragment and a second gene fragment, respectively; the pET-21a(+) vector was double-digested with EcoR I and Hind III, and the pET-28a(+) vector was double-digested with BamH I and EcoR I to obtain a first vector fragment and a second vector fragment, respectively; the first gene fragment and the first vector fragment were connected to obtain a first recombinant expression plasmid; the second gene fragment and the second vector fragment were connected to obtain a second recombinant expression plasmid; after sequencing, the first recombinant expression plasmid and the second recombinant expression plasmid were respectively transferred into the expression host E. coli BL21(DE3) by heat shock, and a positive clone strain containing glucose dehydrogenase and a positive clone strain containing olefin reductase were screened. Using TB medium, the strain containing the olefin reductase mutant obtained in Example 4, the above-mentioned strain containing glucose dehydrogenase, and the above-mentioned strain containing olefin reductase were respectively subjected to shake flask fermentation, and after induction culture (the induction conditions for the strain containing the olefin reductase mutant and the strain containing the olefin reductase were as follows: adding IPTG with a final concentration of 0.5 mM, and inducing at a low temperature of 22°C for 16 h; the induction conditions for the strain containing glucose dehydrogenase were as follows: adding IPTG with a final concentration of 0.5 mM, and inducing at a low temperature of 18°C ​​for 16 h), the wet cells after the induced culture were respectively collected by centrifugation, and the cells were washed with a PBS solution with a pH of 8.5 and then centrifuged and collected for use.

[0120] 4) A 100 mL three-necked reaction bottle was selected for catalytic reaction, the reaction system was fixed to 20 mL, and two system reactions were performed respectively: wet cells were added to system 1, specifically as follows: the final concentration of the wet cells of the olefin reductase mutant was 80 g / L, and the concentration of the wet cells of glucose dehydrogenase was 128 g / L; the wet cells of the olefin reductase mutant and the wet cells of glucose dehydrogenase were added to different batches of reaction systems at a mass ratio of 1:0.6, 1:0.8, 1:1.0, 1:1.2, 1:1.4, 1:1.6, 1:1.8, and 1:2.0, respectively, and the proportion of cells added was screened. The reaction system also included a 25% volume concentration of DMSO solution, a final concentration of 700 mM glucose solution, 650 mM citral, and a final concentration of 0.15 mM NAD + , the pH of the reaction system was controlled to 8.5 using 2M sodium hydroxide solution, the reaction was continued for 28 hours, and the conversion rate of citral was determined using gas chromatography. The results are shown in Table 1 below:

[0121] Table 1 Citral conversion results

[0122]

[0123] As can be seen from Table 1, when the mass ratio of the wet cells of the olefin reductase mutant to the wet cells of the glucose dehydrogenase is 1:1.6, the conversion rate of citral is the highest.

[0124] Freeze-dried bacterial powder was added to system 2, specifically as follows: the freeze-dried bacterial powder concentration of the olefin reductase mutant was 24 g / L, and the freeze-dried bacterial powder concentration of the glucose dehydrogenase was 48 g / L; the freeze-dried bacterial powder of the olefin reductase mutant and the freeze-dried bacterial powder of the glucose dehydrogenase were added to the reaction systems of different batches at a mass ratio of 1:1.6, 1:1.8, 1:2.0, 1:2.2, and 1:2.4, respectively, and the proportion of bacterial addition was screened. The reaction system also included a 25% volume concentration of DMSO solution, a final concentration of 700 mM glucose solution, 650 mM citral, and a final concentration of 0.15 mM NAD+. The pH of the reaction system was controlled to 8.5 using a 2M sodium hydroxide solution. If the volume was insufficient, a PBS solution with a pH of 8.5 was used to make up for it. The reaction was continued for 28 hours, and the conversion rate of citral was determined using gas chromatography. The results are shown in Table 2 below:

[0125] Table 2 Citral conversion results

[0126]

[0127] It can be seen from Table 2 that when the mass ratio of the freeze-dried bacterial powder of the olefin reductase mutant to the freeze-dried bacterial powder of glucose dehydrogenase is 1:2.0, the conversion rate of citral is the highest.

[0128] In the present invention, Figure 1 Schematic diagram of the reaction of synthesizing (R)-citronellal catalyzed by olefin reductase and glucose dehydrogenase.

[0129] Furthermore, the ee value and yield of the reaction product of the freeze-dried bacterial powder of the olefin reductase mutant and the freeze-dried bacterial powder of glucose dehydrogenase in system 2, in a mass ratio of 1:2.0, were determined by gas chromatography. At the same time, the freeze-dried bacterial powder of the olefin reductase mutant was replaced by the freeze-dried bacterial powder of the olefin reductase, and the reaction was carried out, and the conversion rate of citral was determined by gas chromatography.

[0130] The results showed that when the obtained freeze-dried bacterial powder of the olefin reductase mutant and the freeze-dried bacterial powder of glucose dehydrogenase were catalyzed in a mass ratio of 1:2.0, a higher conversion rate of citral could be obtained: the conversion rate was 99.9% in 28 hours, and the ee value of the product (R)-citronellal was 98%, the yield was 92%, and the space-time yield was 3.29 g / (L*h), which was 2.64 times the conversion rate of the freeze-dried bacterial powder of the wild olefin reductase and the freeze-dried bacterial powder of glucose dehydrogenase (37.8%).

[0131] Among them, the calculation formula of the spatiotemporal yield of (R)-citronellal is as follows:

[0132] (R)-citronellal space-time yield = molar concentration of citral × (R)-citronellal yield × (R)-citronellal molar mass × 10 -3 / Reaction time

[0133] Figure 2 This is a gas chromatogram of a citral standard; the citral standard contains two standards, namely, neral with a peak time of 7.557 min and geranial with a peak time of 7.907 min; Figure 3 This is the gas chromatogram of (R)-citronellal standard; wherein, the peak time of (R)-citronellal is 5.964min; Figure 4 The gas chromatogram of the sample in which citral is converted into (R)-citronellal by olefin reductase and glucose dehydrogenase; Figure 5 This is the chromatogram for determining the ee value of the reaction product (R)-citronellal.

[0134] Comparative Example

[0135] The reaction was carried out using system 2 in Example 5, except that glucose dehydrogenase was replaced by commercial glucose dehydrogenase from Pseudomonas (purchased from Aladdin Biochemical Technology, product number G139687), and the citral conversion reaction was carried out at a mass ratio of lyophilized bacterial powder of the olefin reductase mutant to lyophilized bacterial powder of glucose dehydrogenase of 1:2.0. The conversion rate of citral was determined by gas chromatography, and the conversion rate was 72.1%.

[0136] The results show that the olefin reductase mutant of the present invention further improves the conversion rate of citral by using glucose dehydrogenase that matches its specificity.

[0137] The above embodiments are only specific implementation methods of the present invention, which are used to illustrate the technical solutions of the present invention rather than to limit them. The protection scope of the present invention is not limited thereto. Although the present invention has been described in detail with reference to the aforementioned embodiments, ordinary technicians in the field should understand that any technician familiar with the technical field can still modify the technical solutions recorded in the aforementioned embodiments within the technical scope disclosed by the present invention, or can easily think of changes, or make equivalent replacements for some of the technical features therein; and these modifications, changes or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be covered within the protection scope of the present invention.

Claims

1. An olefin reductase, characterized in that The amino acid sequence of the olefin reductase is shown in SEQ ID NO:

1.

2. A nucleic acid molecule encoding the olefin reductase according to claim 1, characterized in that The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

2.

3. An olefin reductase mutant, characterized in that The amino acid sequence of the olefin reductase mutant is shown in SEQ ID NO:

3.

4. A nucleic acid molecule encoding the olefin reductase mutant according to claim 3, characterized in that: The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

4.

5. A recombinant vector, characterized in that: Comprising the nucleic acid molecule of claim 2 or claim 4.

6. A recombinant cell, characterized in that Comprising the recombinant vector according to claim 5.

7. A method for preparing an olefin reductase or a mutant thereof, characterized in that: include: Cultivating the recombinant cell according to claim 6, inducing expression, and obtaining a culture; The olefin reductase according to claim 1 or the olefin reductase mutant according to claim 3 is isolated from the culture.

8. Use of the olefin reductase according to claim 1, the nucleic acid molecule according to claim 2, the olefin reductase mutant according to claim 3, the nucleic acid molecule according to claim 4, the recombinant vector according to claim 5, the recombinant cell according to claim 6 or the olefin reductase or its mutant obtained by the preparation method according to claim 7 in the preparation of (R)-citronellal.

9. A method for preparing (R)-citronellal, characterized in that: The method comprises the following steps: using the olefin reductase according to claim 1, the mutant according to claim 3, the recombinant cell according to claim 6 or the olefin reductase or the mutant thereof prepared by the preparation method according to claim 7 as a catalyst to catalyze the reaction of citral to obtain (R)-citronellal; Preferably, the catalyst further comprises glucose dehydrogenase; More preferably, the amino acid sequence of the glucose dehydrogenase is shown in SEQ ID NO: 5; Further preferably, when the olefin reductase or its mutant is in the form of wet bacteria, its concentration is 40-110 g / L, preferably 80 g / L; when the glucose dehydrogenase is in the form of wet bacteria, its concentration is 64-176 g / L, preferably 128 g / L; the mass ratio of the wet bacteria of the olefin reductase or its mutant to the wet bacteria of the glucose dehydrogenase is 1:0.6-1:2.0, preferably 1:1.6; when the olefin reductase or its mutant is in the form of freeze-dried powder, its concentration is 12-32 g / L, preferably 24 g / L; when the glucose dehydrogenase is in the form of freeze-dried powder, its concentration is 24-64 g / L, preferably 48 g / L; the mass ratio of the freeze-dried powder of the olefin reductase or its mutant to the freeze-dried powder of the glucose dehydrogenase is 1:1.6-1:2.4, preferably 1:2.

0.

10. The preparation method according to claim 9, characterized in that: NAD was also added to the reaction + ; The NAD + The concentration is 0.05-0.2mM, preferably 0.15mM; The reaction temperature is 20-45°C, preferably 35°C; The pH of the reaction is 7.5-9.0, preferably 8.5; The reaction time is 22-40h, preferably 28h; The concentration of citral is 20-850 mM, preferably 650 mM.

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