CN104513839A - Biocatalysis preparation method of D-tert-leucine - Google Patents

Abstract

The invention discloses a new method for synthesizing optically pure D-tert-leucine by reductive ammoniation using a mutant biocatalyst derived from Symbiobacterium thermophilum meso-diaminopimelic acid dehydrogenase (StDAPDH). It is characterized in that the 121st position of the StDAPDH protein sequence or the tryptophan (W) corresponding to the 121st position in the homology comparison is replaced by the leucine (L), and the 146th position or the benzene at the 146th position is equivalent to the homology comparison. Alanine (F) is replaced with leucine (L), and histidine (H) at position 227 or equivalent to position 227 in homology comparison is replaced with phenylalanine (F), and these three positions Point mutations were combined into triple mutants. The three-mutant pure enzyme is used to establish a catalytic reaction system, and the coenzyme NADPH cycle system is used to synthesize optically pure D-tert-leucine through reductive amination, and the ee value of the synthesized D-tert-leucine product is greater than 99%.

Description

A kind of biocatalytic preparation method of D-tert-leucine

technical field

The invention belongs to the field of biocatalysis, and relates to a variant of biocatalyst meso-diaminopimelic acid dehydrogenase, which is used to synthesize optically pure D-tert-leucine by reductive amination of α-keto acid as a substrate acid.

Background technique

Amino acids refer to organic compounds containing carboxylic acids containing amino groups. The difference between different amino acids lies in the difference in their side chain R groups. There are more than 300 kinds of amino acids found in nature so far. According to whether they are the basic components of natural proteins Composition, amino acids can be divided into natural amino acids and unnatural amino acids, unnatural amino acids refer to various synthetic amino acids. Except for glycine, amino acids have asymmetric carbon atoms and are optically active. According to different spatial arrangements, they can be divided into D-amino acids and L-amino acids. Different optically active amino acids play different physiological roles in organisms. Tert-leucine is a kind of unnatural amino acid, its side chain is large sterically hindered hydrophobic tert-butyl group, and it is very close to amino and carboxyl groups in space, the peptide bond containing tert-leucine is difficult to be degraded, thus increasing The enzymatic stability of related compounds and the large steric hindrance can well control the molecular conformation. These characteristics make tert-leucine an important pharmaceutical intermediate and a chiral induction template and catalyst for asymmetric synthesis [Bommarius, AS, et. al (1995). Tetrahedron: Asymmetry 6(12): 2851-2888].

The methods for synthesizing optically pure amino acids mainly include: racemate resolution, chemical synthesis, and biological chemical and biological synthesis. In recent years, biological methods, especially enzymatic conversion, have shown broad market prospects due to their characteristics of no pollution, low cost, and high optical purity of products. In terms of enzymatic synthesis of tert-leucine, the synthesis of L-tert-leucine is mainly split by aminoacylase [continuous enzymatic production of L-tert-leucine, patent number: 201010622182], and L-leucine [Hummel, W., et.al(2003). Org Lett 5(20): 3649-3650; Menzel, A., et.al(2004). Engineering in Life Sciences 4 (6): 573-576]. And it has been applied to the production [a method of preparing L-tert-leucine, patent number: 201110202325; a production method of L-tert-leucine, patent application number: 2012105080840]. D-tert-leucine can be synthesized by some enzymatic methods, such as penicillin acylase [Liu, SL, et.al(2006). Prep Biochem Biotechnol 36(3): 235-241], D-hydantoinase [ Turner, RJ, et.al(2004). Engineering in Life Sciences 4(6):517-520], nitrile hydratase/D-amidase [Marion Ansorge, et.al(2003).EP patent application1, 318, 193), protease [Laumen, K., et.al (2001). Biosci Biotechnol Biochem 65 (9): 1977-1980], etc., but these synthesis methods are to resolve the racemate, the highest theoretical yield is 50% . Amino acid dehydrogenase (EC1.4.1.X) can catalyze the reversible oxidative deamination/reductive amination reaction of amino acids in the presence of the coenzyme NAD(P) ⁺ [Ohshima, T.et.al(1990). Adv Biochem Eng Biotechnol 42:187-209], can be used to synthesize amino acids from ketoacid substrates, using NH ₃ as amino donors, the by-product is water, and the enantiomeric theoretical yield of the product is 100%. Considering the impact on the environment, this method is a green and economical method for the synthesis of amino acids [Zhu, D.et.al(2009). Biotechnol J 4(10):1420-1431]. Most of the reported wild-type amino acid dehydrogenases are L-selective [Yonaha, K.et.al(1986). Adv Biochem Eng Biotechnol 33:95-130], and many enzymes have been successfully used in industrial scale production of L- Amino acids [Ohshima dt.al(1990) Bioprocesses and Applied Enzymology , Springer Berlin/Heidelberg. 42:187-209; Galkin, et.al(1997). Appl Environ Microbiol 63(12):4651-4656]. There is currently no wild-type D-amino acid dehydrogenase that can be used for reductive amination to D-amino acids. Meso-diaminopimelate dehydrogenase, (DAPDH, EC1.4.1.16) reversibly catalyzes the oxidative deamination/reductive amination of the D-configuration amino group of diaminopimelate, and mutants of this enzyme have been Used for the selective synthesis of D-amino acids with ketoacids as substrates [Vedha-Peters, et.al(2006). J Am Chem Soc 128(33):10923-10929; Akita, H., et.al(2012 ). Biotechnol Lett 34(9): 1693-1699; Akita, H., et.al(2013). Appl Microbiol Biotechnol .1-9], but none of the enzymes in these reports can synthesize D-tert-leucine, So far, there is no relevant literature report on the synthesis of D-tert-leucine by reductive amination of amino acid dehydrogenase.

We previously obtained a meso-diaminopimelate dehydrogenase capable of reductive amination to synthesize D-type amino acids such as D-alanine, D-valine, and D-leucine [Gao, X., et.al(2012). Appl Environ Microbiol 78(24): 8595-8600; Gao, X., et.al(2013). Appl Environ Microbiol 79(16): 5078-5081], and applied for a patent "Synthetic D - A new method for amino acids” [patent application number: 201210334554.6]. Through protein engineering of this meso-diaminopimelate dehydrogenase, an enzyme mutant capable of reductive amination to synthesize D-tert-leucine was obtained, which can be used as a biocatalyst for synthesis Optically pure D-tert-leucine.

Invention content:

The present invention provides a modified meso-diaminopimelic acid dehydrogenase (StDAPDH) variant biocatalyst derived from Symbiobacterium thermophihum, which can be used to reduce ammonification to synthesize D with an ee value > 99%. - tertiary leucine.

The steps for obtaining the mutant enzyme protein are as follows:

1. Using the pET ₃₂ -Dapdh plasmid as a template, introduce the W121L, F146L, and H227F three-site combination mutation through the Quick Change Mutagenesis Kit mutation kit, and perform sequencing verification on the mutant plasmid;

2. Construct the mutant plasmid into an engineering bacterium with Escherichia coli BL21 (DE3) as the host bacterium;

3. Cultivate and induce expression of the constructed engineering bacteria, and the mutant protein exists in the cell in a soluble form;

4. Purify the mutant protein by Ni-NTA affinity chromatography to a single band on SDS-PAGE;

5. Desalting and concentrating the purified mutant protein for establishing a catalytic reaction system.

D-tertiary leucine synthetic method is:

In the presence of the glucose/glucose dehydrogenase coenzyme NADPH cycle regeneration system, the substrate 3,3-dimethyl-2-carbonylbutyric acid and ammonium chloride in the reaction system, after adding the biocatalyst StDAPDH mutant, in After reacting at 30°C for 24 hours, the amount of biocatalyst used in each milliliter of the reaction system is about 0.5U.

The reaction product configuration detection method is:

After adding perchloric acid/heat denatured protein to the reaction product, centrifuge to take the supernatant, derivatize the product in the supernatant with FDAA, use L, D-tert-leucine standard sample as a control, and analyze it by high performance liquid chromatography , according to the retention time of the product, determine the product configuration and ee value.

In the present invention, through the reductive amination of the StDAPDH mutant, the catalyzed reaction of 3,3-dimethyl-2-carbonylbutanoic acid and ammonium chloride can obtain the D- tertiary leucine.

The present invention has the following advantages:

The method of the present invention utilizes the transformed StDAPDH mutant enzyme as a catalyst, utilizes free NH ₄ ⁺ as an amino donor, and in the presence of a glucose/gluconate dehydrogenase cycle system, 3,3-dimethyl-2-carbonyl Synthesis of optically pure D-tert-leucine by reductive amination of butyric acid.

Description of drawings:

What Fig. 1 shows is the SDS-PAGE electrophoresis pattern of StDAPDH mutant protein purification;

Figure 2 shows the HPLC spectrum for detection of reaction products catalyzed by the StDAPDH mutant protein.

Detailed ways

The content of the present invention is further described below through specific examples, but these examples do not constitute a limitation to the present invention. Embodiment 1: the acquisition of mutant

The StDapdh wild-type gene Genbank number is AP006840.1. First, the gene is fully synthesized and connected to the pET32 vector to obtain a plasmid expressing the target gene: pET ₃₂ -StDapdh, and the wild-type gene can be identified in Escherichia coli BL21 (DE3). dissolved expression. The N-terminus of the expressed protein has a 6*his tag, which will facilitate the purification of the target protein using Ni-NTA. According to the site to be mutated, refer to the instructions of the Quick Change Mutagenesis Kit kit to synthesize the PCR mutation primers used in Table 1 below. PCR product amplification, Dpn1 digestion and subsequent nucleic acid recovery were all carried out according to the kit instructions.

Table 1: Primers used for mutation

Primer number Primer name Primer direction 5'-3' 1 W121L5 GTTATCTCTGCGGGTctgGACCCGGGCACT 2 W121L3 AGTGCCCGGGTCcagACCCGCAGAGATAAC 3 F146L5 CACCTACACCCAACctgGGTCCGGGTATGTCT 4 F146L3 AGACATACCCGGACCcagGTTGGTGTAGGTG 5 H227F5 ATGGACGTTGGTtttGGTGTTGTTATGGAAC 6 H227F3 GTTCCATAACAACACCaaaACCAACGTCCAT

Using the pET ₃₂ -StDapdh plasmid as a template, the W121L mutation was introduced into the plasmid using primers 1 and 2, and transformed into Escherichia coli TOP10 competent. The plasmid was extracted and sequenced to confirm that the single mutant plasmid pET ₃₂ -StDapdh W121L was obtained. The obtained single mutant plasmid was used as a template, and primers 3 and 4 were used to continue to introduce the F146L mutation on the mutant to obtain a double mutant plasmid: pET ₃₂ -StDapdh W121L/F146L; then, using the double mutant plasmid as a template, primers 5 and 6 were used The H227F mutation was introduced to obtain a three-mutant plasmid: pET ₃₂ -StDapdh W121L/F146L/H227F, and the finally obtained three-mutant plasmid was transformed into Escherichia coli BL21 (DE3) highly competent, and the plasmid was extracted and verified by sequencing.

Example 2: Expression and purification of mutant enzymes

Escherichia coli BL21(DE3) containing the triple mutant plasmid was cultured in 2LLB liquid medium, and after culturing at 37°C to an _OD600 of about 0.8, wasopropyl-β-D-thiol was added to it at a final concentration of 0.5mM The expression of galactopyranoside (IPTG) was induced, and the induction temperature was 25° C. for 20 hours. After induction of expression, the cells were collected by centrifugation at 5000×rpm for 5 minutes, resuspended and washed with buffer A (20 mM Tris-Cl pH 8.0, 500 mM sodium chloride, 5% glycerol). All subsequent purification experiments were performed at 4°C, and all buffers were pre-cooled to 4°C. First resuspend the bacteria with buffer A, high-pressure homogenate, and centrifuge at 14000×rpm for 30 minutes to remove the broken precipitate. The supernatant is pre-balanced with buffer A Ni-NTA chromatography column (GE health care), and buffered Solution B (20mM Tris-Cl pH8.0, 50mM imidazole, 500mM NaCl, 5% glycerol) to remove impurities, and buffer C (20mM Tris-Cl pH8.0, 250mM imidazole, 500mM NaCl, 5% glycerol) to elute the target protein. The target protein was dialyzed against buffer D (20 mM Tris-Cl pH8.0, 50 mM sodium chloride, 5% glycerol) to remove high concentration of imidazole and sodium chloride. Accompanying drawing 1 is the electrophoresis pattern of the purified mutant protein. In the figure, lane M is the protein molecular weight marker, and lane 1 is the purified mutant enzyme. It can be seen from the figure that the molecular weight of the purified mutant enzyme is correct, and the purity is more than 95%.

Example 3: Viability assay of mutants

The activity of the mutants against 3,3-dimethyl-2-oxobutanoic acid was measured using a SPECTRAMAXM2e (MD, USA) microplate reader using a 96-well plate. The activity measurement system is as follows: the final concentrations of each component are: 20mM substrate 3,3-dimethyl-2-carbonylbutanoic acid, 200mM substrate ammonium chloride, 1mM coenzyme NADPH, appropriate amount of StDAPDH mutant pure enzyme, activity measurement The buffer is 100 mM sodium carbonate/sodium bicarbonate buffer solution pH 9.0, the final volume is 200 μL. The substrate and protein samples used for activity determination were first added to a 96-well plate and equilibrated at 30°C for 10 minutes, then an appropriate amount of coenzyme NADPH was added to it to initiate the reaction, and the enzyme activity was determined by measuring the reduction of NADPH at OD ₃₄₀ (NADPH at 340nm molar The extinction coefficient is 6.22mM ^-1 ·cm ^-1 ), and the enzyme activity unit is defined as the amount of enzyme required to consume 1 μmol of coenzyme NADPH per minute when catalyzing the reaction.

Embodiment 4: catalytic reaction system is established

In 1 mL of sodium carbonate/sodium bicarbonate buffer solution (100 mM, pH 9.0), add a final concentration of 25 mM substrate 3,3-dimethyl-2-carbonylbutanoic acid, 20 mg of glucose, 1 mg of glucose dehydrogenase GDH, 1 mM Coenzyme NADP ⁺ , 250mM ammonium chloride, StDAPDH mutant pure enzyme 0.5U, and adjust the pH value of the final system to 9.0. The reaction system was reacted at 200*rpm at 30° C. for 24 hours. The reaction was terminated by adding 10 μl perchloric acid to the catalytic reaction system or heating, the denatured protein was removed by high-speed centrifugation, and the supernatant was analyzed by HPLC after passing through a 0.22 μm membrane.

Embodiment 5: Determination of ee value of catalytic reaction product

The ee value of the product was determined by derivatizing the supernatant FDAA of the catalytic reaction. The derivatization method was carried out according to the FDAA derivatization reagent operation manual, and the Eclipse XDB-C18 column (4.6×150mm) was used for determination, and the mobile phase was: triethylamine phosphate (50mM, pH3.0)/acetonitrile ratio: 65/35 (v/ v), the flow rate is 0.6mL/min, and the detection wavelength is 340nm. The retention times of the two tert-leucine counterparts are respectively: t _r (D-tert-leucine)=22.4min, t _r (L-tert-leucine)=12.7min. The enantiomeric excess (ee) of the catalytic product was >99%. Accompanying drawing 2 is the ee value detection liquid chromatogram of catalytic reaction product among the embodiment 4. In the figure, the upper panel is the liquid phase result derived from the mixed standard sample of L and D tert-leucine; the middle panel is the liquid phase result derived from the control catalytic reaction system; the lower panel is the liquid phase result derived from the catalytic reaction product of Example 4 result. Comparing the three figures, it can be seen that the product catalyzed in Example 4 is D-tert-leucine, and its ee value is >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.