CN104513839B - A kind of biocatalysis preparation method of D Terleus - Google Patents

Abstract

The invention discloses it is a kind of it is new using from Symbiobacterium thermophilum mesos diaminopimelate dehydrogenase (StDAPDH) mutant biocatalysts come the method for the pure D Terleus of reduction amination synthesizing optical.It is characterized in that replace with leucine (L) by 121 of StDAPDH protein sequences or in tryptophan (W) of the tetraploid rice equivalent to 121,146 or replace with leucine (L) in phenylalanine (F) of the tetraploid rice equivalent to 146,227 or phenylalanine (F) is replaced with histidine (H) of the tetraploid rice equivalent to 227, and these three site mutations are combined into Trimutant.Catalystic converter system is established using the pure enzyme of Trimutant, coordinates coenzyme NADP 11 circulating system, the pure D Terleus of reduction amination synthesizing optical, the ee values of synthesized D Terleus product are more than 99%.

Description

A kind of biocatalysis preparation method of D- Terleus

Technical field

The invention belongs to biocatalysis field, is related to a kind of biocatalyst meso-diaminopimelate dehydrogenase and becomes Body, using the biocatalyst using 2-ketoacid as the pure D- Terleus of substrate reduction amination synthesizing optical.

Background technology

Amino acid refers to the organic compound of both carboxylic acids containing amino, is distinguished between different aminoacids and is their sides The difference of chain R group, up to the present find there are 300 several amino acids altogether in nature, according to whether being to form native protein The ultimate constituent of matter, amino acid can be divided into natural amino acid and alpha-non-natural amino acid, and alpha-non-natural amino acid refers to manually close Into various amino acid.In addition to glycine, amino acid has asymmetric carbon atom, in optical activity, according to steric position not Together, two kinds of D- amino acid and l-amino acid can be divided into, different optically-active amino acid play different physiology and made in vivo With.Terleu is a kind of alpha-non-natural amino acid, and its side chain is the big steric hindrance hydrophobicity tert-butyl group, and spatially very close to ammonia Base and carboxyl, the peptide bond containing Terleu are difficult to be degraded, thus add related compound enzymolysis stability, due to big position Resistance can control molecular conformation well, and these features cause Terleu to turn into important medicine intermediate and asymmetric syntheses Chiral induction template and catalyst【Bommarius, A.S., et.al (1995)Tetrahedron：Asymmetry6(12)： 2851-2888】。

The method of synthesizing optical pure amino acid mainly has：The chemistry of racemate resolution method, chemical synthesis and bioanalysis And biological synthesis process.Bioanalysis in recent years, especially enzymatic conversion method are with its spy pollution-free, that cost is low, product optical purity is high Point has shown that wide market prospects.In terms of Terleu enzymatic clarification, the synthesis of S-Leucine mainly passes through Amino-acylase is split【Continuous enzymatic method for producing L-tert-leucine, the patent No.：201010622182】, and L-Leu dehydrogenation Enzyme reduction amination is carried out【Hummel, W., et.al (2003)Org Lett5(20)：3649-3650；Menzel, A., et.al (2004).Engineering in Life Sciences4(6)：573-576】.And it is applied to production【One kind prepares L- The method of Terleu, the patent No.：201110202325；A kind of production method of S-Leucine, number of patent application： 2012105080840】.D- Terleus can be synthesized by some enzyme process, such as PA ase【Liu, S.L., et.al(2006).Prep BiochemBiotechnol36(3)：235-241】, D- hydantoin enzymes【Turner, R.J., et.al (2004).Engineering in Life Sciences4(6)：517-520】, nitrile hydratase/D- amidases【Marion Ansorge, et.al (2003) .EP patent application1,318,193), protease【Laumen, K., et.al (2001).Biosci Biotechnol Biochem65(9)：1977-1980】Deng, but these synthetic methods are that raceme is entered Row is split, and highest theoretical yield is 50%.Amino acid dehydrogenase (EC1.4.1.X) can be at coenzyme NAD (P)⁺In the presence of, catalysis 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 NH₃As amino group donor synthesizing amino acid, its Accessory substance is water, and product enantiomer reason theoretical yield is 100%, should from the aspect of financial cost and effect on environment Method is the method for the green economy of synthesizing amino acid【Zhu, D.et.al (2009)Biotechnol J4(10)：1420- 1431】.The wild-type amino acid dehydrogenase reported is mostly L- selectivity【Yonaha, K.et.al (1986) .AdvBiochem Eng Biotechnol33：95-130】, many enzymes are employed successfully in commercial scale 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】.It can be used for reduction amination generation D- amino acid currently without the D- amino acid dehydrogenases of wild type.Meso-diamino Base pimelic acid dehydrogenase, (DAPDH, EC1.4.1.16) it is reversible catalysis diaminopimelic acid D-form amino oxidative deamination/also Former ammonification, the mutant of the enzyme are already used to using ketone acid as 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】, still Enzyme in these reports can not synthesize D- Terleus, also synthesize uncle D- without using amino acid dehydrogenase reduction amination so far The pertinent literature report of leucine.

We had previously obtained one can reduction amination synthesis D types amino acid such 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 Microbiol79(16)：5078-5081】, and apply special Profit " a kind of new method of synthesis D- amino acid "【Number of patent application：201210334554.6】.By to this meso-diaminourea Pimelic acid dehydrogenase carries out protein engineering transformation, obtains the enzyme mutant that can carry out reduction amination synthesis D- Terleus, This enzyme mutant can be used for the pure D- Terleus of synthesizing optical as biocatalyst.

The content of the invention：

The invention provides a kind of meso-diamino from Symbiobacterium thermophihum of transformation Base pimelic acid dehydrogenase (StDAPDH) variant biocatalyst, the catalyst can be used to reduction amination synthesis ee values ＞ 99% D- Terleus.

The obtaining step for being mutated zymoprotein is as follows：

1. with pET₃₂- Dapdh plasmids are template, are drawn by Quick Change Mutagenesis Kit mutagenesis kits Enter the mutation of the Sites Combination of W121L, F146L, H227F tri-, and sequence verification is carried out to mutant plasmid；

2. mutant plasmid is built into engineering bacteria with e. coli bl21 (DE3) for Host Strains；

3. the engineering bacteria of pair structure is cultivated, induced expression, mutant protein is present in intracellular with soluble form；

4. by mutant protein through Ni-NTA affinitive layer purifications to SDS-PAGE single slice；

5. by muton albumen desalination and concentration after purification, for establishing catalystic converter system.

D- Terleu synthetic methods are：

In the presence of glucose/glucose dehydrogenase coenzyme NADP 11 circular regeneration system, the substrate 3 in reaction system, 3- dimethyl-ALPHA-ketobutyric acid and ammonium chloride, after biocatalyst StDAPDH mutant is added, reacted 24 hours in 30 DEG C, The dosage of biocatalyst is about 0.5U in every milliliter of reaction system.

Reaction product configuration detection method is：

After adding perchloric acid/heat denatured protein into reaction product, centrifuging and taking supernatant, the product in supernatant is carried out FDAA derives, and is compareed using L, D- Terleu standard specimen, is analyzed through high performance liquid chromatography, during according to the reservation of product Between, determine product configuration and ee values.

In the present invention, acted on by the reduction amination of StDAPDH mutons, 3,3- dimethyl-ALPHA-ketobutyric acid and chlorination The catalytic reaction of ammonium can obtain the D- Terleus of the enantioselectivity (ee) more than 99%.

The invention has the advantages that：

The inventive method makees catalyst using engineered StDAPDH mutant enzymes, utilizes free NH₄ ⁺Supplied as amino Body, in the presence of glucose/gluconate dehydrogenase circulating system, 3,3- dimethyl-ALPHA-ketobutyric acid reduction amination is closed Into optically pure D- Terleus.

Brief description of the drawings：

Shown in Fig. 1 is the SDS-PAGE spectrum of StDAPDH muton protein purifications；

Shown in Fig. 2 is the HPLC collection of illustrative plates using the detection of StDAPDH muton proteins carries reaction product.

Embodiment

Content that by the following specific examples further illustrate the invention, but these embodiments are not formed to the present invention Limitation.Embodiment 1：The acquisition of mutant

StDapdh wild type genes Genbank are AP006840.1, first by the total gene synthesis and are connected to The plasmid of expression target gene is obtained on pET32 carriers：pET₃₂- StDapdh, and to wild in e. coli bl21 (DE3) Type gene carries out soluble-expression.The N- ends of the albumen given expression to carry 6*his labels, and this is beneficial to utilize destination protein Ni-NTA is purified.The site being mutated as needed, illustrate with reference to Quick Change Mutagenesis Kit kits, PCR mutant primers used in following table 1 are synthesized, PCR primer amplification, Dpn1 digestion and follow-up nucleic acid recovery are made by kit Carried out with explanation.

Table 1：It is mutated 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 pET₃₂- StDapdh plasmids are template, introduce W121L mutation on plasmid using primer 1 and 2, and convert to big Enterobacteria TOP10 competence, extract plasmid and confirmation acquisition single mutation plasmid pET is sequenced₃₂-StDapdh W121L.With acquisition Single mutation plasmid is template, continues to introduce F146L mutation on the muton using primer 3 and 4, obtains double mutant plasmids： pET₃₂-StDapdh W121L/F146L；Again using double mutant plasmids as template, H227F mutation are introduced using primer 5 and 6, are obtained Three mutant plasmids：pET₃₂- StDapdh W121L/F146L/H227F, three mutant plasmids finally obtained are transformed into large intestine bar In bacterium BL21 (DE3) Efficiency Competent, and plasmid is extracted through sequence verification.

Embodiment 2：Expression, the purifying of mutant enzyme

E. coli bl21 (DE3) containing three mutant plasmids is cultivated in 2LLB fluid nutrient mediums, 37 DEG C of trainings Support to OD₆₀₀After about 0.8, the isopropyl-beta D-thio galactopyranoside (IPTG) for adding final concentration of 0.5mM thereto enters Row induced expression, inducing temperature are 25 DEG C, are induced 20 hours.After induced expression terminates, centrifuge 5 minutes and collect in 5000 × rpm Thalline, simultaneously washing thalline is resuspended using buffer A (20mM Tris-Cl pH8.0,500mM sodium chloride, 5% glycerine).Follow-up institute There is purification experiment to be first cooled to 4 DEG C in advance in 4 DEG C of progress, all buffer solutions.Thalline first is resuspended with buffer A, high-pressure homogenization is broken Broken, 14000 × rpm, which is centrifuged, removes broken precipitation for 30 minutes, the pre- Ni-NTA chromatographic column (GE equilibrated with buffer A on supernatant Health care), and removed with buffer B (20mM Tris-Cl pH8.0,50mM imidazoles, 500mM sodium chloride, 5% glycerine) Foreign protein, purpose is eluted with buffer solution C (20mM Tris-Cl pH8.0,250mM imidazoles, 500mM sodium chloride, 5% glycerine) Albumen.Destination protein is dialysed to buffer solution D (20mM Tris-Cl pH8.0,50mM sodium chloride, 5% glycerine), to go Except high concentration imidazoles and sodium chloride.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, swimming lane 1 are muton enzyme after purification.It can be seen that the molecular size range for the muton enzyme being purified into is correct, and And purity ＞ 95%.

Embodiment 3：The vitality test of mutant

Muton utilizes SPECTRAMAXM2e (MD, USA) ELIASA to the vigor of 3,3- dimethyl-ALPHA-ketobutyric acid, makes It is measured with 96 orifice plates.Live body system is as follows：The final concentration of each composition is respectively：20mM substrates 3,3- dimethyl -2- carbonyls Butyric acid, 200mM substrate ammonium chlorides, 1mM coenzyme NADP 11s, the appropriate pure enzyme of StDAPDH mutons, it is 100mM carbonic acid to survey buffer solution living Sodium/sodium bicarbonate buffer solution pH9.0, the μ L of final volume 200.Substrate used in vitality test and protein sample first add 96 holes Balanced 10 minutes in 30 DEG C in plate, then add appropriate coenzyme NADP 11 initial action thereto, by measuring OD₃₄₀Locate NADPH's To determine enzyme activity, (NADPH is 6.22mM in 340nm molar extinction coefficients for reduction^-1·cm^-1), enzyme activity unit is defined as urging Change the enzyme amount needed for 1 μm of ol coenzyme NADP 11 of consumption per minute during reaction.

Embodiment 4：Catalystic converter system is established

In 1mL sodium carbonate/bicarbonates cushioning liquid (100mM, pH9.0), final concentration 25mM substrates 3,3- bis- are added Methyl-ALPHA-ketobutyric acid, 20mg glucose, 1mg glucose dehydrogenases GDH, 1mM coenzyme NAD P⁺, 250mM ammonium chlorides, The pure enzyme 0.5U of StDAPDH mutons, and final system pH is adjusted to 9.0.Reaction system is with 200*rpm rotating speeds in 30 DEG C of progress Reaction 24 hours.Reacted by adding 10 μ l perchloric acid or heating termination into catalystic converter system, albuminate passes through at a high speed Removed after centrifugation, supernatant is analyzed after crossing 0.22 μm of film using HPLC.

Embodiment 5：Catalytic reaction products ee values determine

Product ee values after catalytic reaction supernatant FDAA is derived by being measured.Deriving method is with reference to FDAA derivative reagents Operation manual is carried out, and is measured using Eclipse XDB-C18 posts (4.6 × 150mm), mobile phase is：Phosphoric acid triethylamine (50mM, pH3.0)/acetonitrile ratio is：65/35 (v/v), flow velocity 0.6mL/min, Detection wavelength 340nm.Two kinds of tertiary bright ammonia The retention time of the corresponding body of acid is respectively：t_r(D- Terleus)=22.4min, t_r(S-Leucine)=12.7min.Catalysis The enantiomeric excess (ee) of product is ＞ 99%.Accompanying drawing 2 is that the ee values of catalytic reaction products in embodiment 4 detect liquid phase spectrogram. In figure, upper width is the liquid phase result after L, D Terleu mixed sample derive；After middle width derives for control catalystic converter system Liquid phase result；Lower width is the liquid phase result after the catalytic reaction products of example 4 derive.Contrast three figures to understand, the production being catalyzed in example 4 Thing is D- Terleus, its ee value ＞ 99%.

Claims (2)

1. one kind is by the use of meso-diaminopimelate dehydrogenase mutant as biocatalyst come synthesizing optical purity ＞ The method of 99%D- Terleus, including：To from Sybiobacterium thermopilum mesos-diaminourea heptan two Acidohydrogenase StDAPDH is combined mutation, and StDAPDH protein sequences are characterised by 121 of sequence or in tetraploid rice Tryptophan (W) equivalent to 121 replaces with leucine (L)；146 or in phenylpropyl alcohol ammonia of the tetraploid rice equivalent to 146 Sour (F) replaces with leucine (L)；227 or replace with phenylalanine in histidine (H) of the tetraploid rice equivalent to 227 (F)；The mutant combines for above-mentioned three site mutation；Using the combination mutant of acquisition as biocatalyst, with 3,3- diformazans The reaction system that base-ALPHA-ketobutyric acid is formed with ammonium chloride as substrate, add coenzyme circulating system and carry out catalysis reduction amination Reaction, optical purity ＞ 99% D- Terleus are made；Coenzyme circulating system includes nicotinamide-adenine dinucleotide phosphate (NADPH or NADP⁺), glucose, glucose dehydrogenase (GDH).

2. the method as described in claim 1, from Sybiobacterium thermopilum meso-diaminourea heptan No. GenBank of two acidohydrogenases is AP006840.1.