CN117844773A - Phenylalanine dehydrogenase mutant and application thereof in synthesis of L-unnatural amino acid - Google Patents
Phenylalanine dehydrogenase mutant and application thereof in synthesis of L-unnatural amino acid Download PDFInfo
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- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a phenylalanine dehydrogenase mutant and application thereof in synthesis of L-unnatural amino acid, belonging to the technical fields of enzyme engineering and microbial engineering. The phenylalanine dehydrogenase mutant provided by the invention can asymmetrically and reductively aminate alpha-keto acid substrate 2- (naphthalene-1-yl) -2-oxoacetic acid, 2- (naphthalene-2-yl) -2-oxoacetic acid, 3- (naphthalene-1-yl) -2-oxopropionic acid and 3- (naphthalene-2-yl) -2-oxopropionic acid, wherein four mutants V329G/S176G/L326M/T144I can efficiently synthesize (S) -2-amino-2- (naphthalene-1-yl) acetic acid, the conversion rate can reach 99.84% in the reaction time of 10 hours, the reaction condition is mild, the environment is friendly, the operation is simple and convenient, and the industrial amplification is easy. Therefore, the phenylalanine dehydrogenase mutant has good industrial application and development prospects.
Description
Technical Field
The invention relates to the technical fields of enzyme engineering and microbial engineering, in particular to a phenylalanine dehydrogenase mutant and application thereof in synthesizing L-unnatural amino acid.
Background
Amino acid and amino acid derivatives are very important medical intermediates, and have very wide application prospects in medical research. It is estimated that about 40% of chiral drugs on the market at present contain structural blocks of chiral amines, such as anti-inflammatory drugs, anti-alzheimer drugs, antidepressants, anti-aids drugs, etc., which are important in the medical field. Because of the importance and great value of chiral amine in the synthesis of medicine molecules, the research and development of synthetic technology has become a research hotspot in the field of new medicine development.
Although synthesis of chiral amines has received increasing attention over the last decades, currently, achieving an easy and efficient synthesis of specific chiral amines remains a major challenge. Classical chemical methods for synthesizing chiral amines include racemate resolution and asymmetric transformations of latent chiral compounds. The theoretical yield of the asymmetric resolution method is only 50%, and the problems of harsh reaction conditions, use of toxic and harmful chemical reagents such as transition metal and the like, complicated reaction operation process, low enantioselectivity and the like exist in the ways of asymmetric hydrogenation, enantioselective C-H bond amination, asymmetric reductive amination, asymmetric nucleophilic addition of C=N double bond, asymmetric hydroamination and the like of imine or enamine related to the asymmetric resolution method. Compared with chemical methods, the biocatalysis method has the advantages of mild reaction conditions, high selectivity, environmental friendliness and the like, and is widely applied to the industrial production of medical intermediates, agrochemicals, fine chemicals and other products. Currently, a variety of biocatalysts have been successfully used in the synthesis of chiral amines, including hydrolases, amino acid dehydrogenases, transaminases, amine oxidases, imine reductases, reductive amination enzymes, ammonia lyase, amine dehydrogenases, and the like.
The amino acid dehydrogenase (Amino acid dehydrogenase, AADH) can utilize inorganic ammonium or organic amine as an amino donor, and utilize NADH as reducing power to synthesize chiral amino acid and chiral amine through asymmetric reductive amination, and the byproduct is only water, so that the product has high selectivity and single configuration, has high atom utilization rate, accords with the aim of green chemistry, and has high development and application values. Among the numerous amino acid dehydrogenases, phenylalanine dehydrogenase (phenylalanine dehydrogenase, pheDH) is a class of amino acid dehydrogenases having phenylpyruvate (aminated) and phenylalanine (deaminated) as natural substrates and NADH (aminated)/nad+ (deaminated) as a coenzyme. Phenylalanine dehydrogenase has a wide range of applications in the synthesis of phenylalanine and aromatic amino acids, amino acid derivatives, such as the synthesis of important unnatural amino acids such as L-phenylglycine, L-homophenylalanine, 2- (3-hydroxy-1-adamantyl) - (2S) -aminoglycolic acid (see, in particular, references "Cheng, et al, RSC adv, 2016,6,80557-80563", "Wu, et al, biotechnol Biofuels,2021,14,207", "r.l., et al, adv. Synth. Catalyst.2007, 349, 1369-1378").
Although amino acid dehydrogenases have shown great potential, there are still problems of low catalytic activity on unnatural substrates, narrow substrate spectrum, etc. In addition, the enzyme has poor stability under the slightly alkaline condition, and the reaction process for preparing chiral amine by utilizing amino acid dehydrogenase is still to be further developed and optimized.
Disclosure of Invention
In order to solve the technical problems, the invention provides a phenylalanine dehydrogenase mutant with good heat stability and good stability under a alkalescent condition, which is obtained by mutating valine 329, serine 176, threonine 144 and leucine 326 of phenylalanine dehydrogenase taking SEQ ID NO.1 as a starting sequence, and the obtained phenylalanine mutant can be used for asymmetrically reductively aminating alpha-keto acid substrates and can be applied to the preparation of L-unnatural amino acid.
The first object of the invention is to provide a phenylalanine dehydrogenase mutant which is modified by modifying a starting sequence with an amino acid sequence shown as SEQ ID NO. 1:
(1) Valine at position 329 is mutated to glycine;
(2) Valine 329 is mutated to glycine, serine 176 is mutated to glycine or alanine;
(3) Valine 329 is mutated to glycine, serine 176 is mutated to glycine, threonine 144 is mutated to valine or leucine or isoleucine or methionine;
(4) Valine 329 is mutated to glycine, serine 176 is mutated to glycine, leucine 326 is mutated to valine or serine or cysteine or methionine;
(5) Valine 329 is mutated to glycine, serine 176 is mutated to glycine, leucine 326 is mutated to methionine, threonine 144 is mutated to methionine or isoleucine;
(6) Valine 329 is mutated to glycine, serine 176 is mutated to glycine, leucine 326 is mutated to cysteine, and threonine 144 is mutated to methionine or isoleucine.
A second object of the present invention is to provide a gene encoding the above phenylalanine dehydrogenase mutant.
A third object of the present invention is to provide a recombinant expression vector comprising the above gene.
Further, the expression vector is pET-28a (+) plasmid, pET-28b (+) plasmid or pET-20b (+) plasmid.
It is a fourth object of the present invention to provide a host cell comprising the phenylalanine dehydrogenase mutant described above.
It is a fifth object of the present invention to provide the use of the above phenylalanine dehydrogenase mutant, the above gene, the above recombinant expression vector or the above host cell for producing an L-unnatural amino acid.
It is a sixth object of the present invention to provide a method for producing an L-unnatural amino acid, comprising the steps of: the phenylalanine dehydrogenase mutant or an expression system containing the mutant is added to a reaction system containing a substrate to perform a reaction.
Further, the reaction system contains a coenzyme and a coenzyme circulation system.
Further, the coenzyme circulation system is any one of D-glucose and glucose dehydrogenase, phosphite and phosphite dehydrogenase, formate and formate dehydrogenase, lactate and lactate dehydrogenase, glycerol and glycerol dehydrogenase.
Further, the reaction system contains D-glucose and glucose dehydrogenase.
Further, the substrate comprises one or more of 2- (naphthalen-1-yl) -2-oxoacetic acid, 2- (naphthalen-2-yl) -2-oxoacetic acid, 3- (naphthalen-1-yl) -2-oxopropionic acid, and 3- (naphthalen-2-yl) -2-oxopropionic acid.
Compared with the prior art, the technical scheme of the invention has the following technical effects:
the phenylalanine dehydrogenase mutant provided by the invention can efficiently and asymmetrically aminate alpha-keto acid substrates to synthesize L-unnatural amino acid, compared with a wild type, the amination specific activity is greatly improved, the ee value of the product can reach 99.9%, the conversion rate of (S) -2-amino-2- (naphthalene-1-yl) acetic acid synthesized by four mutants V329G/S176G/L326M/T144I reaches 99.84% in the reaction time of 10 hours, and the three mutants V329G/S176G/T144V and V329G/S176G/T144M can catalyze four different types of alpha-keto acid substrates, so that the catalytic substrate spectrum of phenylalanine dehydrogenase is greatly widened, the phenylalanine dehydrogenase mutant has high heat stability and good stability in a partial alkali environment, is applied to the field of unnatural amino acid synthesis, has mild reaction conditions, is environment-friendly, is easy to operate, and is easy to be industrially amplified, and has good industrial application development prospects.
Drawings
In order that the invention may be more readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings, in which
FIG. 1 is a PCR amplification electropherogram of the recombinant plasmid of example 1 of the present invention, wherein M is Marker, and the different lanes correspond to mutants M1-M3.6 of Table 1, respectively;
FIG. 2 is an electropherogram of PCR extension of the recombinant plasmid of example 1 of the present invention, wherein M is Marker, and the different lanes correspond to mutants M3.7-M4.4 of Table 1, respectively;
FIG. 3 is a SDS-PAGE analysis of an expression product obtained by shake flask induction fermentation of recombinant E.coli in example 1 of the present invention; wherein the lanes are Marker, flow-through, 50mM, 100mM, 150mM, 200mM, 250mM, 300mM imidazole collection solution in sequence;
FIG. 4 is a chiral chromatogram of the product (S) -2-amino-2- (naphthalen-1-yl) acetic acid/(R) -2-amino-2- (naphthalen-1-yl) acetic acid in example 3 of the present invention.
FIG. 5 is a graph showing the progress of conversion of the product obtained by asymmetric reductive amination of α -keto acid 2- (naphthalen-1-yl) -2-oxoacetic acid by mutant M4.2 in example 3 according to the invention;
FIG. 6 is a schematic representation of asymmetric reduction of phenylalanine dehydrogenase mutants.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.
Coli e.coli BL21 (DE 3) as referred to in the examples was purchased from north nanoorganisms; the pET-28a (+) plasmid and NADH referred to in the examples below were purchased from Novagen; glucose Dehydrogenase (GDH), lactate Dehydrogenase (LDH) referred to in the examples below were purchased from Novain; the D-glucose referred to in the examples below was purchased from Jieli corporation; the alpha-keto acid substrates referred to in the examples below were purchased from Shanghai Biotechnology (E.coli BL21 (DE 3) of the above strain is commercially available and does not require preservation for the patent procedure).
The media referred to in the examples are as follows:
LB liquid medium: yeast powder 5.0g/L, tryptone 10.0g/L, naCl 10.0.0 g/L, kanamycin 100mg/L.
LB solid medium: yeast powder 5.0g/L, tryptone 10.0g/L, naCl 10.0.0 g/L, agar powder 15g/L, kanamycin 50mg/L.
The detection method of phenylalanine dehydrogenase enzyme activity is as follows:
200. Mu.L of NH4Cl/NH4OH buffer (2M, pH 9.0) containing 4mM NADH and 2.0mM alpha-keto acid substrate was incubated at 30℃for 2 minutes, 10. Mu.L of pure enzyme solution was added to the above buffer to carry out a reaction at 30℃for 30 minutes, and 20. Mu.L of 6M concentrated hydrochloric acid was added to terminate the reaction, and the amount of the produced product was measured by high performance liquid chromatography, based on which the enzyme activity was calculated.
The calculation formula of the enzyme activity is as follows:
enzyme activity (U/mg) = (c1×v1) ×103/(t×c2×v2);
wherein, the concentration of the C1 generated product is expressed in mol/L; v1 is the volume of the reaction solution, and the unit is mL; c2 is the protein concentration of the enzyme in mg/ml; v2 is the volume of enzyme added in the reaction system, and the unit is mu L; t is the reaction time in min;
definition of enzyme activity: the amount of enzyme required to catalytically oxidize l. Mu. Mol of substrate per minute to produce l. Mu. Mol of product under this condition is one enzyme activity unit (1U).
The method for detecting the conversion efficiency and stereoselectivity of phenylalanine dehydrogenase and asymmetric reductive amination of alpha-keto acid 2- (naphthalen-1-yl) -2-oxoacetic acid and 2- (naphthalen-2-yl) -2-oxoacetic acid is as follows:
mu.L of NH containing 4mM NADH, 2.0mM alpha-keto acid substrate 4 Cl/NH 4 After the OH buffer solution (2M, pH 9.0) is kept at 30 ℃ for 2min, 10 mu L of pure enzyme solution is added into the buffer solution to react for 30min at 30 ℃, and 6M of concentrated hydrochloric acid is added to terminate the reaction, so that the conversion efficiency and ee value are analyzed by chiral liquid chromatography;
the conversion efficiency chromatographic conditions are specifically as follows: ZORBAX SB-Aq (5 μm,250 mM. Times.4.6 mM) liquid chromatography column with acetonitrile as mobile phase, 20mM potassium dihydrogen phosphate (20:80, v/v), flow rate of 1mL/min, column temperature of 35deg.C, UV detection wavelength of 280nm, sample injection amount of 5 μl, and retention time of substrate 2- (naphthalen-1-yl) -2-oxoacetic acid and product (S) -2-amino-2- (naphthalen-1-yl) acetic acid of 4.12min and 10.71min, respectively; the retention times of the substrate 2- (naphthalen-2-yl) -2-oxoacetic acid and the product (S) -2-amino-2- (naphthalen-2-yl) acetic acid were 4.02min and 10.51min, respectively;
the stereoselective chromatography conditions are specifically as follows: astec CHIROBIOTICTM T (15 cm. Times.4.6mm, 5 μm) liquid chromatography column with methanol/water (60:40, v/v) as mobile phase, flow rate of 0.6mL/min, column temperature of 35 ℃, UV detection wavelength of 280nm, sample injection amount of 5. Mu.L, retention time of (S) -2-amino-2- (naphthalen-1-yl) acetic acid and (R) -2-amino-2- (naphthalen-1-yl) acetic acid of 3.82min and 7.01min respectively; the retention times of (S) -2-amino-2- (naphthalen-2-yl) acetic acid and (R) -2-amino-2- (naphthalen-1-yl) acetic acid were 4.22min and 8.91min, respectively.
The conversion efficiency was calculated as follows:
the ee value is calculated as follows:
as is defined As: the molar concentration of (S) -2-amino-2- (naphthalen-1-yl) acetic acid in the reaction solution; a is that R The definition is as follows: the molar concentration of (R) -2-amino-2- (naphthalen-1-yl) acetic acid in the reaction solution; a is that sub The definition is as follows: molar concentration of unreacted 2- (naphthalen-1-yl) -2-oxoacetic acid in the reaction solution.
Example 1: preparation, expression and purification of phenylalanine dehydrogenase mutant
Chemically synthesizing a gene encoding phenylalanine dehydrogenase with an amino acid sequence shown as SEQ ID NO.1 (the nucleotide sequence of the gene is shown as SEQ ID NO. 2), connecting the obtained gene with pET-28a (+) plasmid after double digestion (Nde I and Xho I), converting the connection product into escherichia coli E.coli BL21 (DE 3), coating the conversion product on an LB solid culture medium, culturing at 37 ℃ for 8-10h, picking up 5 transformants on the LB solid culture medium, inoculating into an LB liquid culture medium for culturing at 37 ℃ for 10h, extracting plasmids, carrying out enzyme digestion verification and sequencing verification on the extracted plasmids, and obtaining recombinant plasmids pET28 a-QtDH containing the gene encoding the wild phenylalanine dehydrogenase and recombinant bacteria E.coli BL21/pET28a-QtPheDH containing the gene encoding the wild phenylalanine dehydrogenase correctly.
Site-directed mutagenesis was performed using the obtained recombinant plasmid pET28a-QtPheDH as a template by using the whole plasmid PCR technique, and a recombinant plasmid containing the mutant of phenylalanine dehydrogenase V329G (mutation of valine at position 329 to glycine), S176G (mutation of serine at position 176 to glycine), S176A (mutation of serine at position 176 to alanine), L326S (mutation of leucine at position 326 to serine), L326V (mutation of leucine at position 326 to valine), L326C (mutation of leucine at position 326 to cysteine), L326M (mutation of leucine at position 326 to methionine), T144I (mutation of threonine at position 144 to isoleucine), T144M (mutation of threonine at position 144 to methionine), and T144V (mutation of threonine at position 144 to valine) was obtained. The mutation sites and numbers of the mutants are shown in the following table.
TABLE 1 mutation sites and numbering
Wherein, the primers used for the mutation V329G, S176G, S176A, L326C, L326M, T144I, T M are as follows:
V329G-F：5’-GGTTTGATCCAGggtGCGGACGAA-3’(SEQ ID NO.3)
V329G-R：5’-CAGTTCGTCCGCaccCTGGATCAA-3’(SEQ ID NO.4)
S176G-F：5’-GGAGGCGGAGACggtTCCGTTCCG-3’(SEQ ID NO.5)
S176G-R：5’-GGTCGGAACGGAaccGTCTCCGCC-3’(SEQ ID NO.6)
S176A-F：5’-GGAGGCGGAGACgcaTCCGTTCCG-3’(SEQ ID NO.7)
S176A-R：5’-GGTCGGAACGGAtgcGTCTCCGCC-3’(SEQ ID NO.8)
L326C-F：5’-AACGCGGGGGGTtgtATCCAGGGT-3’(SEQ ID NO.9)
L326C-R：5’-CGCACCCTGGATacaACCCCCCGC-3’(SEQ ID NO.10)
L326M-F：5’-AACGCGGGGGGTatgATCCAGGGT-3’(SEQ ID NO.11)
L326M-R：5’-CGCACCCTGGATcatACCCCCCGC-3’(SEQ ID NO.12)
T144I-F：5’-TTCTATACAGGAattGATATGGGC-3’(SEQ ID NO.13)
T144I-R：5’-TGTGCCCATATCaatTCCTGTATA-3’(SEQ ID NO.14)
T144M-F：5’-TTCTATACAGGAatgGATATGGGC-3’(SEQ ID NO.15)
T144M-R：5’-TGTGCCCATATCcatTCCTGTATA-3’(SEQ ID NO.16)
the PCR reaction system (50. Mu.L) was: KOD enzyme (2.5U/mL) l.0. Mu.L, template (5-50 ng) l.0. Mu.L, dNTP 4.0. Mu.L, 10 Xreaction buffer 5.0. Mu.L, 1.0. Mu.L each of the upstream and downstream primers, ddH 2 O was made up to 50. Mu.L.
The PCR product amplification conditions were: (1) denaturation at 94℃for 3min, (2) denaturation at 94℃for 30sec, (3) annealing at 54℃for 30sec, (4) extension at 72℃for 150sec, repeating steps (2) - (4) for 10-15 cycles, and finally extension at 72℃for 10min, and preserving the PCR amplification product at 4 ℃.
Detecting the PCR amplified product by using 1% agarose gel electrophoresis, adding 0.5 mu L of methylation template digestive enzyme (Dpn I) into 10 mu L of amplified product after detection, blowing and sucking the mixture at a gun head for uniform mixing, reacting for 1h at 37 ℃, converting the amplified product treated by Dpn I into E.coli BL21 (DE 3), coating the converted product on LB solid culture medium, culturing for 8-10h at 37 ℃, picking up 5 transformants on LB solid culture medium, inoculating LB liquid culture medium for culturing for 10h, extracting plasmids after culturing for 37 ℃, carrying out enzyme digestion verification (the verification result is shown in figures 1 and 2) and sequencing verification, and obtaining recombinant plasmids pET28a-QtPheDH-1 to recombinant plasmids pET28a-QtPheDH-4.4 respectively containing phenylalanine dehydrogenase mutants M1-M4.4 and recombinant bacteria E.Phei QDH 21/QPhe 2-Phe 4.E/pET 28a-QtPhe 4.4 respectively.
The obtained recombinant bacteria E.coli BL21/pET28a-QtPheDH and recombinant bacteria E.coli BL21/pET28 a-QtPheDH-1-recombinant bacteria E.coli BL21/pET28a-QtPheDH-4.4 are respectively coated on LB solid medium and cultured for 8-10h at 37 ℃ to obtain single colony; selecting single bacterial colony, inoculating into LB liquid culture medium, culturing at 37deg.C for 12-14 hr to obtain seed liquid; mixing the seed solutionInoculating LB liquid culture medium according to 2% (v/v), culturing at 37deg.C and 200rpm to OD 600 After reaching 0.8, adding IPTG with the final concentration of 0.2mM into the fermentation broth, and continuously performing induction culture at 25 ℃ for 8 hours to obtain the fermentation broth; centrifuging the fermentation broth at 4deg.C and 8000rpm for 10min, and collecting cells; the collected cells were suspended in potassium phosphate buffer (100 mmol/L, pH 7.0) and subjected to ultrasonication, and cell disruption supernatants each containing wild-type phenylalanine dehydrogenase and phenylalanine dehydrogenase mutants M1 to M4.4 were collected.
The cell disruption supernatant obtained was purified using an affinity column HisTrap FF credit (nickel column) as follows: the nickel column was equilibrated with buffer A (20 mmol/L sodium phosphate, 500mmol/L NaCl,20mmol/L imidazole, pH 7.4), and the cell disruption supernatant obtained in example 1 was passed through the nickel column, and after the breakthrough peak was completed, the proteins not bound to the nickel column were eluted continuously with buffer A, and after the breakthrough peak was completed, gradient elution was performed from buffer A to buffer B (20 mmol/L sodium phosphate, 500mmol/L NaCl,500mmol/L imidazole, pH 7.4), and the recombinant proteins bound to the nickel column were eluted to obtain pure enzyme solutions of wild-type phenylalanine dehydrogenase and phenylalanine dehydrogenase mutants M1 to M4.4.
The purified enzyme solution of the phenylalanine dehydrogenase mutant M1 was subjected to SDS-PAGE analysis, and the analysis results are shown in FIG. 3. Lanes were in turn Marker, flow-through, 50mM, 100mM, 150mM, 200mM, 250mM, 300mM imidazole collections. As is clear from FIG. 3, the purified enzyme solution of the phenylalanine dehydrogenase mutant M1 showed a single band at about 45kDa, and the amount of the impurity protein was small, indicating that the purification effect by the nickel column was good. Other mutants were protein purified and analyzed in the same manner.
Example 2: determination of kinetic parameters of phenylalanine dehydrogenase mutants and relative activities of substrates containing naphthyl alpha-keto acids
The wild-type phenylalanine dehydrogenase and phenylalanine dehydrogenase enzyme mutants M1 to M4.4 obtained in example 1 were selected, and the reductive amination activities of the wild-type phenylalanine dehydrogenase and phenylalanine dehydrogenase enzyme mutants M1 to M4.4 obtained in example 1 were measured using 2- (naphthalen-1-yl) -2-oxoacetic acid as a substrate at a concentration of 0.1 to 50mM, respectively, usingFitting the data by a nonlinear regression method in Graph Pad Prism 7.0 software to obtain K of Mies (Michaelis-Menten) equation respectively m The value is recalculated to obtain K cat And K cat /K m The values and calculation results are shown in Table 2.K (K) cat The calculation formula of the value is: k (K) cat ＝V max M/1; wherein M is the molecular mass of the enzyme in kDa.
TABLE 2 kinetic parameters of wild-type phenylalanine dehydrogenase and phenylalanine dehydrogenase mutants M1-M4.4
As can be seen from Table 2, the wild-type phenylalanine dehydrogenase and the single mutation (M1) had no catalytic activity on the substrate 2- (naphthalen-1-yl) -2-oxoacetic acid, and an asymmetric amination activity was generated starting from mutant M2.1. The double mutations M2.1 and M2.2 have similar catalytic efficiency. In the three mutations, the catalytic efficiency of the mutant M3.4 is improved by about 4 times compared with that of the double mutation, and the catalytic efficiencies of M3.1, M3.3, M3.5 and M3.6 are similar, and are improved by about 2 times compared with that of the double mutation. In the four mutations, M4.1, M4.3 and M4.4 have similar catalytic efficiency, which is improved by about 4 times compared with the double mutation catalytic efficiency. Mutant M4.2 exhibited the highest catalytic efficiency, which was about 8 times that of the double mutation.
The wild-type phenylalanine dehydrogenase enzyme obtained in example 1 and phenylalanine dehydrogenase mutant M1-M4.4 were examined for the relative activities of α -keto acid 2- (naphthalen-1-yl) -2-oxoacetic acid, 2- (naphthalen-2-yl) -2-oxoacetic acid, 3- (naphthalen-1-yl) -2-oxopropionic acid and 3- (naphthalen-2-yl) -2-oxopropionic acid to unnatural amino acids by asymmetric reductive amination, and the results of the examination are shown in Table 3.
TABLE 3 relative specific Activity of wild-type phenylalanine dehydrogenase and phenylalanine dehydrogenase mutants M1-M4.4 reductive amination of substrates containing naphthyl α -keto acid
As can be seen from Table 3, the wild-type phenylalanine dehydrogenase had no catalytic activity for 2- (naphthalen-1-yl) -2-oxoacetic acid and very low catalytic activity for 2- (naphthalen-2-yl) -2-oxoacetic acid. For the substrate 2- (naphthalen-1-yl) -2-oxoacetic acid, all phenylalanine dehydrogenase mutants showed an increase in catalytic specific activity compared to the wild type, starting from double mutation, the catalytic activity was generated starting from about 2 times the relative activity of the double mutation, four mutation M4.2 showed the highest catalytic amination activity, and the catalytic activity of the other four mutants was about 3 times that of the double mutant. For the substrate 2- (naphthalene-2-yl) -2-oxoacetic acid, all phenylalanine dehydrogenase mutants show about 4 times of catalytic amination activity improvement compared with the wild type, the catalytic amination activity improvement of double mutation M2.1 is about 6 times, the superposition of three mutations M3.2 and M3.4 can improve the catalytic amination activity by about 9 times, and the four mutations M4.1 show the highest catalytic amination activity, which is different from 2- (naphthalene-1-yl) -2-oxoacetic acid.
Phenylalanine dehydrogenase mutants M1-M4.4 are more biased towards the highly sterically hindered, highly rigid, short-chain alpha-keto acid substrates 2- (naphthalen-1-yl) -2-oxoacetic acid, 2- (naphthalen-2-yl) -2-oxoacetic acid, whereas for long-chain 3- (naphthalen-1-yl) -2-oxopropionic acid, 3- (naphthalen-2-yl) -2-oxopropionic acid substrates are detrimental to their reductive amination. Among all phenylalanine mutants, the three mutants M3.1 and M3.4 can catalyze the four different types of substrates, greatly broaden the catalytic substrate spectrum of phenylalanine dehydrogenase, and have high application value and research value.
Example 3: conversion efficiency of phenylalanine dehydrogenase mutant to asymmetrically aminated 2- (naphthalen-1-yl) -2-oxoacetic acid
The cyclohexanone monooxygenase mutant M4.2 obtained in example 1 was selected and added to 2M NH containing 25mM, 50mM, 75mM, 100mM of the alpha-keto acid substrate 2- (naphthalen-1-yl) -2-oxoacetic acid in an amount of 5g/L 4 Cl/NH 4 In an OH buffer solution (pH 9.0), reacting for 10 hours at 30 ℃ and pH 9.0 and 200rpm to obtain a reaction solution; NH in addition to the alpha-keto acid substrate 2- (naphthalen-1-yl) -2-oxoacetic acid 4 Cl/NH 4 The OH buffer also contains coenzyme NAD at a concentration of 0.5mM + Glucose at a concentration of 120mM, glucose dehydrogenase GDH at a concentration of 1.5mM and methanol at a concentration of 5% (v/v). The conversion rate of 2- (naphthalene-1-yl) -2-oxoacetic acid, which is an alpha-keto acid substrate, was asymmetrically reduced and aminated by the phenylalanine dehydrogenase mutant M4.2 at different times of the reaction was measured, and the measurement results are shown in Table 4.
TABLE 4 conversion of phenylalanine dehydrogenase mutants M4.2 asymmetric reductive amination of alpha-keto acid substrates 2- (naphthalen-1-yl) -2-oxoacetic acid at different concentrations
The cyclohexanone monooxygenase mutant M4.2 obtained in example 1 was added to 2M NH containing 100mM alpha-keto acid substrate 2- (naphthalen-1-yl) -2-oxoacetic acid in addition amounts of 5g/L, 10g/L, 15g/L, 20g/L, respectively 4 Cl/NH 4 In an OH buffer solution (pH 9.0), reacting for 10 hours at 30 ℃ and pH 9.0 and 200rpm to obtain a reaction solution; NH in addition to the alpha-keto acid substrate 2- (naphthalen-1-yl) -2-oxoacetic acid 4 Cl/NH 4 The OH buffer also contains coenzyme NAD at a concentration of 0.5mM + Glucose at a concentration of 120mM, glucose dehydrogenase GDH at a concentration of 1.5mM and methanol at a concentration of 5% (v/v). The conversion rate of 2- (naphthalene-1-yl) -2-oxoacetic acid, which is an alpha-keto acid substrate, was asymmetrically reduced and aminated by the phenylalanine dehydrogenase mutant M4.2 at different times of the reaction was measured, and the measurement results are shown in Table 5.
TABLE 5 conversion of the asymmetric reductive amination of the alpha-keto acid substrate 2- (naphthalen-1-yl) -2-oxoacetic acid with different phenylalanine dehydrogenase mutants M4.2 enzyme additions
As is clear from Table 5, the 25mM and 50mM substrates were able to be completely converted at 10 hours under the condition that the enzyme addition amount of phenylalanine dehydrogenase mutation M4.2 was 5g/L, whereas the reaction tended to stagnate after 1 hour from the beginning with an increase in the substrate concentration, and the conversion was maintained at about 5% at the final 75mM substrate concentration and at about 1.5% at the 100mM substrate concentration. As is clear from Table 5, at a substrate addition of 100mM, the enzyme addition of 5g/L, 15g/L, 20g/L was not effective in converting 2- (naphthalen-1-yl) -2-oxoacetic acid, and only when the enzyme addition was increased to 25g/L, 100mM of the substrate could be rapidly converted.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.
Claims (10)
1. The phenylalanine dehydrogenase mutant is characterized in that the phenylalanine dehydrogenase mutant is modified by any one of the following starting sequences with the amino acid sequence shown as SEQ ID NO. 1:
(1) Valine at position 329 is mutated to glycine;
(2) Valine 329 is mutated to glycine, serine 176 is mutated to glycine or alanine;
(3) Valine 329 is mutated to glycine, serine 176 is mutated to glycine, threonine 144 is mutated to valine or leucine or isoleucine or methionine;
(4) Valine 329 is mutated to glycine, serine 176 is mutated to glycine, leucine 326 is mutated to valine or serine or cysteine or methionine;
(5) Valine 329 is mutated to glycine, serine 176 is mutated to glycine, leucine 326 is mutated to methionine, threonine 144 is mutated to methionine or isoleucine;
(6) Valine 329 is mutated to glycine, serine 176 is mutated to glycine, leucine 326 is mutated to cysteine, and threonine 144 is mutated to methionine or isoleucine.
2. A gene encoding the phenylalanine dehydrogenase mutant according to claim 1.
3. An expression vector comprising the gene of claim 2.
4. The expression vector of claim 3, wherein the expression vector is a pET-28a (+) plasmid, a pET-28b (+) plasmid, or a pET-20b (+) plasmid.
5. A host cell comprising the phenylalanine dehydrogenase mutant according to claim 1.
6. Use of the phenylalanine dehydrogenase mutant according to claim 1, the gene according to claim 2, the expression vector according to claim 3 or the host cell according to claim 5 for the production of L-unnatural amino acids.
7. A method for producing an L-unnatural amino acid comprising the steps of: the phenylalanine dehydrogenase mutant or the expression system containing the mutant according to claim 1 is added to a reaction system containing a substrate to perform a reaction.
8. The method according to claim 7, wherein: the reaction system comprises coenzyme and a coenzyme circulation system.
9. The method according to claim 8, wherein: the coenzyme circulatory system is any one of D-glucose and glucose dehydrogenase, phosphite and phosphite dehydrogenase, formate and formate dehydrogenase, lactic acid and lactic acid dehydrogenase, glycerol and glycerol dehydrogenase.
10. The method according to claim 7, wherein: the substrate is one or more of 2- (naphthalene-1-yl) -2-oxoacetic acid, 2- (naphthalene-2-yl) -2-oxoacetic acid, 3- (naphthalene-1-yl) -2-oxopropionic acid and 3- (naphthalene-2-yl) -2-oxopropionic acid.
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