CN104513839A - Biocatalysis preparation method of D-tert-leucine - Google Patents
Biocatalysis preparation method of D-tert-leucine Download PDFInfo
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Abstract
The present invention discloses a new method for reduce ammonification synthesis of optically pure D-tert-leucine by using a biocatalyst derived from a Symbiobacterium thermophilum internal compensation-diaminopimelate dehydrogenase (StDAPDH) mutant. The method is characterized in that the site 121 on the StDAPDH protein sequence or the tryptophane (W) with the homology comparison equivalent to the site 121 is replaced by leucine (L), the site 146 on the StDAPDH protein sequence or the phenylalanine (F) with the homology comparison equivalent to the site 146 is replaced by leucine (L), the site 227 on the StDAPDH protein sequence or the histidine (H) with the homology comparison equivalent to the site 227 is replaced by phenylalanine (F), the mutations on the three sites are combined to form the three-mutant, the three-mutant pure enzyme is adopted to establish the catalysis reaction system, the coenzyme NADPH circulation system is matched, and the optically pure D-tert-leucine is subjected to reduce ammonification synthesis, wherein the ee value of the synthesized D-tert-leucine product is more than 99%.
Description
Technical field
The invention belongs to biocatalysis field, relate to a kind of biological catalyst meso-diaminopimelate dehydrogenase variant, utilize this biological catalyst to take alpha-ketoacid as the pure D-Terleu of substrate reduction amination synthesizing optical.
Background technology
Amino acid refers to the organic compound both containing amino carboxylic acid, between different aminoacids, difference is the difference of their side chain R group, up to the present occurring in nature finds that there is 300 multiple amino acids altogether, according to being whether the ultimate constituent forming natural protein, amino acid can be divided into natural amino acid and alpha-non-natural amino acid, and alpha-non-natural amino acid refers to each seed amino acid of synthetic.Except glycine, amino acid has unsymmetrical carbon, and in opticity, different according to steric position, can be divided into D-amino acid and two kinds, L-amino acid, different optically-active amino acid plays different physiological actions in vivo.Terleu is a class alpha-non-natural amino acid, its side chain is the large steric hindrance hydrophobicity tertiary butyl, and spatially very close to amino and carboxyl, peptide bond containing Terleu is difficult to be degraded, thus add related compound enzymolysis stability, because large steric hindrance well can control molecular conformation, these features make Terleu become chiral induction template and the catalyzer [Bommarius of important medicine intermediate and asymmetric synthesis, A.S., et.al (1995).
tetrahedron:Asymmetry6 (12): 2851-2888].
The method of synthesizing optical pure amino acid mainly contains: the chemistry of racemate resolution method, chemical synthesis and biological process and biological synthesis process.The feature that biological process in recent years, especially enzymatic conversion method are pollution-free with it, cost is low, product optical purity is high has demonstrated wide market outlook.In Terleu enzymatic clarification; the synthesis of S-Leucine mainly splits [continuous enzymatic method for producing L-tert-leucine, the patent No.: 201010622182] by L-Aminoacylase, and L-Leu desaturase reduction amination carries out [Hummel; W., et.al (2003).
org Lett5 (20): 3649-3650; Menzel, A., et.al (2004).
engineering in Life Sciences4 (6): 573-576].And be applied to production [a kind of method preparing S-Leucine, the patent No.: 201110202325; A production method for S-Leucine, number of patent application: 2012105080840].D-Terleu can be synthesized by some enzyme process, as penicillin acylase [Liu, S.L., et.al (2006).
prep Biochem biotechnol36 (3): 235-241], D-hydantoin enzyme [Turner, R.J., et.al (2004).
engineering in Life Sciences4 (6): 517-520], Nitrile hydratase/D-Ntn hydrolase [Marion Ansorge, et.al (2003) .EP patent application1,318,193), proteolytic enzyme [Laumen, K., et.al (2001).
biosci Biotechnol Biochem65 (9): 1977-1980] etc., but these synthetic methods split raceme, and the highest theoretical yield is 50%.Amino acid dehydrogenase (EC1.4.1.X) can at coenzyme NAD (P)
+existence under, catalytic amino acid reversible oxidation deamination/reduction amination [Ohshima, T.et.al (1990).
adv Biochem Eng Biotechnol42:187-209], can be used to from keto acid substrate, utilize NH
3as amino group donor synthesizing amino acid, its by product is water, and product enantiomorph reason theoretical yield is 100%, from the viewpoint of Financial cost with to environmental influence, the method be the green economy of synthesizing amino acid method [Zhu, D.et.al (2009).
biotechnol J4 (10): 1420-1431].The wild-type amino acid desaturase reported mostly be L-optionally [Yonaha, K.et.al (1986).
adv biochem Eng Biotechnol33:95-130], many enzymes are employed successfully in industrial-scale production L-amino acid [Ohshima dt.al (1990)
bioprocesses and Applied Enzymology, Springer Berlin/Heidelberg.42:187-209; Galkin, et.al (1997).
appl Environ Microbiol63 (12): 4651-4656].Do not have the D-amino acid dehydrogenase of wild-type can be used for reduction amination at present and generate D-amino acid.Meso-diaminopimelate dehydrogenase, (DAPDH, EC1.4.1.16) oxidative deamination/reduction amination of reversible catalysis diaminopimelic acid D-form amino, the mutant of this enzyme has been used to ketone acid be substrate, selectivity synthesis D-amino acid [Vedha-Peters, et.al (2006).
j Am Chem Soc128 (33): 10923-10929; Akita, H., et.al (2012).
biotechnol Lett34 (9): 1693-1699; Akita, H., et.al (2013).
appl Microbiol biotechnol.1-9], but the enzyme in these reports all can not synthesize D-Terleu, does not also utilize amino acid dehydrogenase reduction amination to synthesize the pertinent literature report of D-Terleu so far.
We previously obtained energy reduction amination synthesis D type amino acid as D-alanine, D-Val, D-Leu meso-diaminopimelate dehydrogenase [Gao, X., et.al (2012).
appl Environ Microbiol78 (24): 8595-8600; Gao, X., et.al (2013).
appl Environ Microbiol, and apply for a patent " synthesis D-amino acid whose a kind of novel method " [number of patent application: 201210334554.6] 79 (16): 5078-5081].By carrying out protein engineering transformation to this meso-diaminopimelate dehydrogenase, obtain the enzyme mutant that can carry out reduction amination synthesis D-Terleu, this enzyme mutant can be used as biological catalyst and is used for the pure D-Terleu of synthesizing optical.
Summary of the invention:
The invention provides a kind of meso-diaminopimelate dehydrogenase (StDAPDH) variant biological catalyst deriving from Symbiobacterium thermophihum of transformation, this catalyzer can be used to the D-Terleu of reduction amination synthesis ee value > 99%.
The obtaining step of mutant enzyme albumen is as follows:
1. with pET
32-Dapdh plasmid is template, introduces W121L, F146L, H227F tri-Sites Combination sudden change, and carry out sequence verification to mutant plasmid by Quick Change Mutagenesis Kit mutagenesis kit;
2. by mutant plasmid with e. coli bl21 (DE3) for Host Strains is built into engineering bacteria;
3. the engineering bacteria pair built is cultivated, abduction delivering, and mutant protein is present in born of the same parents with soluble form;
4. by mutant protein through Ni-NTA affinitive layer purification to SDS-PAGE single slice;
5. by the muton albumen desalination and concentration after purifying, for setting up catalystic converter system.
D-Terleu synthetic method is:
Under the existence of glucose/Hexose phosphate dehydrogenase coenzyme NADP 11 cyclic regeneration system, substrate 3 in reaction system, 3-dimethyl-ALPHA-ketobutyric acid and ammonium chloride, after adding biological catalyst StDAPDH mutant, in 30 DEG C of reactions 24 hours, in every milliliter of reaction system, the consumption of biological catalyst was about 0.5U.
Reaction product configuration detection method is:
Add perchloric acid/heat denatured protein in reaction product after, centrifuging and taking supernatant, carries out FDAA to the product in supernatant and derives, L, D-Terleu standard specimen is utilized to contrast, analyze through high performance liquid chromatography, according to the retention time of product, determine product configuration and ee value.
In the present invention, by the reduction amination effect of StDAPDH muton, the catalyzed reaction of 3,3-dimethyl-ALPHA-ketobutyric acid and ammonium chloride can obtain the D-Terleu of the enantioselectivity (ee) being greater than 99%.
Tool of the present invention has the following advantages:
The inventive method utilizes the StDAPDH mutant enzyme through transformation to make catalyzer, utilizes free NH
4 +as amino group donor, under the existence of glucose/gluconate dehydrogenase circulating system, by D-Terleu pure for 3,3-dimethyl-ALPHA-ketobutyric acid reduction amination synthesizing optical.
Accompanying drawing illustrates:
Shown in Fig. 1 is the SDS-PAGE electrophoretogram of StDAPDH muton protein purification;
Shown in Fig. 2 is the HPLC collection of illustrative plates utilizing StDAPDH muton proteins carry reaction product to detect.
Embodiment
Content that by the following specific examples further illustrate the invention, but these embodiments are not construed as limiting the invention.Embodiment 1: the acquisition of mutant
No. Genbank, StDapdh wild type gene is AP006840.1, is first connected to pET32 carrier obtains and expresses the plasmid of goal gene by this total gene synthesis: pET
32-StDapdh, and in e. coli bl21 (DE3), soluble-expression is carried out to wild type gene.The N-end band of the albumen given expression to has 6*his label, and this will be conducive to utilizing Ni-NTA to carry out purifying to target protein.The site suddenlyd change as required, illustrates with reference to Quick Change Mutagenesis Kit test kit, PCR mutant primer used in the lower list 1 of synthesis, PCR primer amplification, Dpn1 digestion and follow-up nucleic acid reclaim and all undertaken by test kit operation instruction.
Table 1: sudden change the primer
Primer sequence number | Primer | Primer direction 5 '-3 ' |
1 | W121L5 | GTTATCTCTGCGGGTctgGACCCGGGCACT |
2 | W121L3 | AGTGCCCGGGTCcagACCCGCAGAGATAAC |
3 | F146L5 | CACCTACACCAACctgGGTCCGGGTATGTCT |
4 | F146L3 | AGACATACCCGGACCcagGTTGGTGTAGGTG |
5 | H227F5 | ATGGACGTTGGTtttGGTGTTGTTATGGAAC |
6 | H227F3 | GTTCCATAACAACACCaaaACCAACGTCCAT |
With pET
32-StDapdh plasmid is template, uses primer 1 and 2 on plasmid, introduce W121L sudden change, and is converted into intestinal bacteria TOP10 competence, extracts plasmid and check order to confirm to obtain single mutation plasmid pET
32-StDapdh W121L.With the single mutation plasmid obtained for template, use primer 3 and 4 to continue to introduce F146L sudden change on this muton, obtain two mutant plasmid: pET
32-StDapdh W121L/F146L; Again with two mutant plasmid for template, use primer 5 and 6 introduce H227F sudden change, obtain three mutant plasmids: pET
32three mutant plasmids finally obtained are transformed in e. coli bl21 (DE3) Efficiency Competent, and extract plasmid through sequence verification by-StDapdh W121L/F146L/H227F.
Embodiment 2: the expression of mutant enzyme, purifying
Cultivated in 2LLB liquid nutrient medium by e. coli bl21 (DE3) containing three mutant plasmids, 37 DEG C are cultured to OD
600after about 0.8, adding final concentration is wherein that the isopropyl-beta D-thio galactopyranoside (IPTG) of 0.5mM carries out abduction delivering, and inducing temperature is 25 DEG C, induces 20 hours.After abduction delivering terminates, within centrifugal 5 minutes, collect thalline in 5000 × rpm, utilize buffer A (20mM Tris-Cl pH8.0,500mM sodium-chlor, 5% glycerine) resuspended and wash thalline.Follow-up all purification experiment all carry out at 4 DEG C, and all damping fluids are all first chilled to 4 DEG C in advance.First use the resuspended thalline of buffer A, high-pressure homogenization is broken, and 14000 × rpm removes broken precipitation for centrifugal 30 minutes, the Ni-NTA chromatography column (GE health care) that on supernatant, pre-buffer A is equilibrated, and with buffer B (20mM Tris-Cl pH8.0,50mM imidazoles, 500mM sodium-chlor, 5% glycerine) remove foreign protein, with damping fluid C (20mM Tris-Cl pH8.0,250mM imidazoles, 500mM sodium-chlor, 5% glycerine) elute target protein.Target protein is dialysed to damping fluid D (20mM Tris-Cl pH8.0,50mM sodium-chlor, 5% glycerine), to remove high density imidazoles and sodium-chlor.Accompanying drawing 1 is the sub-protein electrophoresis collection of illustrative plates of purified mutant, and in figure, swimming lane M is protein molecular weight Marker, and swimming lane 1 is the muton enzyme after purifying.As we know from the figure, the molecular size range of the muton enzyme be purified into is correct, and purity > 95%.
Embodiment 3: the vitality test of mutant
The vigor of muton to 3,3-dimethyl-ALPHA-ketobutyric acid utilizes SPECTRAMAXM2e (MD, USA) microplate reader, uses 96 orifice plates to measure.Survey live body system as follows: the final concentration of each composition is respectively: 20mM substrate 3,3-dimethyl-ALPHA-ketobutyric acid, 200mM substrate ammonium chloride, 1mM coenzyme NADP 11, the pure enzyme of appropriate StDAPDH muton, surveying damping fluid of living is 100mM sodium carbonate/bicarbonate buffered soln pH9.0, final volume 200 μ L.Vitality test substrate used and protein sample all first to add in 96 orifice plates in 30 DEG C of balances 10 minutes, then add appropriate coenzyme NADP 11 initial action wherein, by measuring OD
340the minimizing locating NADPH measures enzyme activity, and (NADPH is 6.22mM at 340nm molar extinction coefficient
-1cm
-1), when enzyme activity unit is defined as catalyzed reaction, per minute consumes the enzyme amount needed for 1 μm of ol coenzyme NADP 11.
Embodiment 4: catalystic converter system is set up
In 1mL sodium carbonate/bicarbonate buffered soln (100mM, pH9.0), add final concentration 25mM substrate 3,3-dimethyl-ALPHA-ketobutyric acid, 20mg glucose, 1mg Hexose phosphate dehydrogenase GDH, 1mM coenzyme NAD P
+, 250mM ammonium chloride, the pure enzyme 0.5U of StDAPDH muton, and adjust final system pH to 9.0.Reaction system carries out reaction 24 hours with 200*rpm rotating speed in 30 DEG C.By adding 10 μ l perchloric acid or heating termination reaction in catalystic converter system, metaprotein is by removing after high speed centrifugation, and supernatant utilizes HPLC to analyze after crossing 0.22 μm of film.
Embodiment 5: catalytic reaction products ee pH-value determination pH
Product ee value measures after catalyzed reaction supernatant FDAA is derivative.Deriving method carries out with reference to FDAA derivative reagent operational manual, Eclipse XDB-C18 post (4.6 × 150mm) is used to measure, moving phase is: phosphoric acid triethylamine (50mM, pH3.0)/acetonitrile ratio is: 65/35 (v/v), flow velocity is 0.6mL/min, and determined wavelength is 340nm.The retention time of two kinds of corresponding bodies of Terleu is respectively: t
r(D-Terleu)=22.4min, t
r(S-Leucine)=12.7min.The enantiomeric excess (ee) of catalysate is > 99%.Accompanying drawing 2 is that the ee value of catalytic reaction products in embodiment 4 detects liquid phase spectrogram.In figure, upper width is the liquid phase result after L, D Terleu mixed sample derives; Middle width is the liquid phase result after contrast catalystic converter system derives; Lower width is the liquid phase result after example 4 catalytic reaction products derives.Contrast three figure known, the product of catalysis in example 4 is D-Terleu, its ee value > 99%.
Claims (10)
1. one kind utilizes meso-diaminopimelate dehydrogenase mutant to carry out the method for synthesizing optical purity > 99%D-Terleu as biological catalyst, comprise: combinatorial mutagenesis is carried out to meso-diaminopimelate dehydrogenase StDAPDH, with the combination mutant obtained for biological catalyst, with 3, the reaction system that 3-dimethyl-ALPHA-ketobutyric acid and ammonium chloride are formed as substrate, add coenzyme circulating system and carry out catalytic reduction amination reaction, the D-Terleu of obtained optical purity > 99%.
2. biological catalyst as claimed in claim 1, its wild type gene be derive from Symbiobacterium thermophilum meso-diaminopimelate dehydrogenase (No. Genbank is AP006840.1) mutant or be not less than the protein of 80% with its amino acid sequence similarity.
3. biological catalyst meso as claimed in claim 2-diaminopimelate dehydrogenase variant, its protein sequence is characterized in that 121 of sequence or replace with leucine (L) at the tryptophane (W) that tetraploid rice is equivalent to 121; 146 or replace with leucine (L) at the phenylalanine (F) that tetraploid rice is equivalent to 146; 227 or replace with phenylalanine (F) at the Histidine (H) that tetraploid rice is equivalent to 227; Described mutant is above-mentioned three site mutation combinations.
4. the method for biological catalyst synthesizing optical purity > 99%D-Terleu as described in claim 1 and 2, it is characterized in that, described substrate is 3,3-dimethyl-ALPHA-ketobutyric acid.
5. the method for biological catalyst synthesizing optical purity > 99%D-Terleu as described in claim 1 and 2, it is characterized in that, in reaction system, the concentration of substrate is 20 ~ 250mmol/L.
6. the method for biological catalyst synthesizing optical purity > 99%D-Terleu as described in claim 1 and 2, it is characterized in that, described circulating reaction system comprises Triphosphopyridine nucleotide, reduced (NADPH or NADP
+), glucose, Hexose phosphate dehydrogenase (GDH).
7. the method for biological catalyst synthesizing optical purity > 99%D-Terleu as described in claim 1 and 2, it is characterized in that, in reaction system, the consumption of biological catalyst is about 0.5U/mL.
8. the method for biological catalyst synthesizing optical purity > 99%D-Terleu as described in claim 1 and 2, it is characterized in that, temperature of reaction is 20 DEG C ~ 50 DEG C, and optimal reactive temperature is 30 DEG C.
9. the method for biological catalyst synthesizing optical purity > 99%D-Terleu as described in claim 1 and 2, it is characterized in that, described pH value in reaction is 7 ~ 11, and the suitableeest initial ph value is 90.
10. the method for biological catalyst synthesizing optical purity > 99%D-Terleu as described in claim 1 and 2, the aimnosubstrate in described reaction can be ammonium salt also can be ammoniacal liquor.
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Cited By (4)
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CN105821014A (en) * | 2015-01-07 | 2016-08-03 | 中国科学院天津工业生物技术研究所 | Symbiobacterium thermophilum meso-diaminopimelate dehydrogenase mutants |
CN106191150A (en) * | 2015-05-06 | 2016-12-07 | 中国科学院天津工业生物技术研究所 | A kind of method utilizing co-immobilization enzymatic synthesis D-alanine |
CN113817785A (en) * | 2021-08-13 | 2021-12-21 | 山东大学 | Method for synthesizing (S) -6-nitro norleucine and catalytic enzyme thereof |
CN115786296A (en) * | 2022-09-27 | 2023-03-14 | 山东理工大学 | Meso-diaminopimelate dehydrogenase mutant and production method thereof |
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Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
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CN105821014A (en) * | 2015-01-07 | 2016-08-03 | 中国科学院天津工业生物技术研究所 | Symbiobacterium thermophilum meso-diaminopimelate dehydrogenase mutants |
CN106191150A (en) * | 2015-05-06 | 2016-12-07 | 中国科学院天津工业生物技术研究所 | A kind of method utilizing co-immobilization enzymatic synthesis D-alanine |
CN113817785A (en) * | 2021-08-13 | 2021-12-21 | 山东大学 | Method for synthesizing (S) -6-nitro norleucine and catalytic enzyme thereof |
CN113817785B (en) * | 2021-08-13 | 2023-08-18 | 山东大学 | Method for synthesizing (S) -6-nitronorleucine and catalytic enzyme thereof |
CN115786296A (en) * | 2022-09-27 | 2023-03-14 | 山东理工大学 | Meso-diaminopimelate dehydrogenase mutant and production method thereof |
CN115786296B (en) * | 2022-09-27 | 2024-01-30 | 山东理工大学 | Meso-diaminopimelate dehydrogenase mutant and production method thereof |
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