CN109554406A - An a kind of step prepares ketone acid and the class of Non-natural chiral amino acid turns ammonia method - Google Patents
An a kind of step prepares ketone acid and the class of Non-natural chiral amino acid turns ammonia method Download PDFInfo
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Abstract
The class for preparing ketone acid and Non-natural chiral amino acid the invention discloses one step of one kind turns ammonia method, belongs to enzyme engineering and chiral medicinal intermediate preparation technical field.The class transamination reaction approach that the present invention constructs, it can change reaction balance, be directly realized by coenzyme in-situ regeneration, improve the advantages that conversion ratio, so that the conversion ratio of ketone acid is prepared ketone acid compared to single step oxidative deamination and improves 14 times or so, by being 0.01 μm of ol/L controlled at 30 DEG C, pH 9.0, concentration of substrate 100mmol/L, co-factor concentration, improve conversion ratio to 92%.The total turn over number TTN of coenzyme reaches 5.84 × 105.An effective way is provided efficiently to produce ketone acid and Non-natural chiral amino acid.
Description
Technical field
Prepare the class of ketone acid and Non-natural chiral amino acid the present invention relates to one step of one kind and turn ammonia method, belong to enzyme engineering and
Chiral medicinal intermediate preparation technical field.
Background technique
Chiral amino acid is widely used in pharmacy, food, cosmetics, agricultural and feed industry, the application of chiral amino acid
Impressive have stimulated the synthetic method largely innovated.In general, these methods are segmented into three kinds of different synthesis
Route, the amino acid including protein hydrolysis extraction method production, the synthesis of chemical method biocatalysis.Biocatalysis synthesis of chiral ammonia
There are four types of strategies for base acid: (1) the asymmetric reduction amination of ketone acid, and asymmetry transfer of (2) amino group to ketone acid, (3) are by ammonia
Enantioselectivity is added to α, and the amino acid converting acetal condensation of beta-unsaturated acid and (4) amino alcohol is aldehyde.
2-ketoacid is the compound of a kind of double carbonyls, more even more important than general compound and special since reaction center is more
Chemical property, be the important intermediate of organic synthesis, pharmaceutical synthesis and biosynthesis, medicine, chemical synthesis, cosmetics,
Feed and field of food have important application prospect.2-ketoacid industrial production mostly uses greatly double carboxylated methods, Hydrolyze method, oxidizing process
With the chemical synthesis such as glycolylurea method, raw materials technology is complicated, step is tediously long, environmental pollution is serious, utilizes environmental-friendly bioanalysis
2-ketoacid is prepared as development trend.Fermenting and producing method low relative to conversion ratio, by-product is more, at high cost, biotransformation method
The feature simple with its synthesis step, by-product is few becomes the first choice of biological green synthesis 2-ketoacid.It is excellent with price economy
The L-Leu of gesture is the ideal substrate of bioconversion, by transaminase (AT, EC2.6.2.X), amino acid oxidase (AAO,
EC1.4.3.2 and EC1.4.3.3) and the enzymes such as amino acid dehydrogenase (AADH, EC 1.4.1.X) can realize that amino group is de-
Ammonia effect prepares 2-ketoacid, is the preparation approach for most having industrial application value.The research of AAO and AT the preparation method is more at present, but AAO
Method by-product H2O2There is apparent toxicity and inhibiting effect to cell or enzyme, there are bottom products to inhibit for AT rule, conversion ratio compared with
Problem low, at high cost；And amino acid dehydrogenase can simultaneously reversible catalytic amino acid oxidase deamination and ketone acid reductive amination process,
But reaction selectivity is oriented in reduction amination direction, and equivalent coenzyme is needed to participate in, and approach realizes that difficulty is big, studies less.
In recent years, people deepen continuously to catalyst mechanism and the synthesis application research of amino acid dehydrogenase, and l-amino acid is de-
The synthesis application research of hydrogen enzyme is concentrated mainly on L-Leu dehydrogenase and PheDH, to the α -one of different structure
Acid is respectively provided with higher activity, therefore they are used to synthesize the amino acid of a series of branch and fragrance.Terleu
It is the key intermediate of synthetic drug, is just had been reported that using the dehydrogenase catalyzed synthesis Terleu of L-Leu from generation nineteen ninety,
The biotransformation method has been applied to industrialized production at present, becomes the allusion quotation using amino acid dehydrogenase synthesis unnatural amino acid
Model.The asymmetric reduction amination of some aliphatic amino-acid oxidase deamination and ketone acid of leucine dehydrogenase LeuDH catalyzed reversible
Reaction, be amino acid and ketone acid synthesis biological approach, but from the enzyme at present it has been reported that zymologic property and application study come
It sees, thermodynamically reaction balance generally tends to reduction direction, it is important amino acid and ketone acid preparation biocatalyst, and
And leucine dehydrogenase has extensive substrate to compose.The method and enzyme coupling method that leucine dehydrogenase is catalyzed single step oxidation ketone acid are all
Report, but enzyme coupling method has not been reported.PheDH normally tends to be applied to using fragrant 2-ketoacid as substrate
Synthesize L-phenylalanine and similar unnatural amino acid.
Transaminase is highly selective with its, high conversion and mild reaction condition have won the favor of numerous researchers,
The transamination on 1 amino group donor (amino acid or simple amine) can be catalyzed to prochiral receptor ketone, obtain Chiral Amine
With by-product ketone 2-ketoacid, reaction process needs the participation of phosphopyridoxal pyridoxal phosphate (pyridoxal phosphate, PLP).Transaminase
Pass through the table tennis bi-directional mechanism of enzymatic catalysis transamination.Transaminase-catalyzed transamination reaction is a thermodynamical equilibrium process,
Mainly include two complementary reactions: the asymmetric syntheses of Chiral Amine and the transport power of racemic amines are split.Two above reaction
Process is influenced by factors such as reactant, product, reaction conditions, and the reverse movement of the balance that when reaction is occurred is in transamination reaction
Bottleneck problem.Transaminase amine substrate donor has specificity, generally very high to aromatic series donor activity, but to most of fat
Race's vigor is very low even without vibrant only for aliphatic carbon chain length C >=4 (butyrine and 6-aminocaprolc acid).It is right
In the specificity of amino acceptor, other than ketone acid, also there is very high reactivity for aliphatic and aromatic series aldehydes, usually shows
Good reactivity out.
From environment and economic viewpoint, transaminase is a kind of interesting substitute of chemical synthesis process.Although transaminase exists
It is concerned in terms of preparing drug and its intermediate, however is applied in industrialized production that there is also many problems conscientiously: 1.
The inhibition of substrate and product to enzymatic activity；2. enzyme is to the tolerance of high levels of organic solvents；3. reaction balance is unfavorable for product side
To etc..
Enzymatic coupling reaction generally comprises two types, and the coupling of the first substrate is with a kind of enzyme while to be catalyzed two kinds of bottoms
The reaction of object, it is desirable that two kinds of substrates convert while realizing substrate with auxiliary substrate to opposite the Direction of Reaction, and reaction has not
Invertibity, reducing power are strong, reduce auxiliary substrate surplus feature.Another enzyme coupling method is respectively present in a reaction system
Oxidation reaction enzyme system and reduction reaction enzyme system, and the two systems react simultaneously, and this method requires two enzyme systems
In substrate must be relatively independent, the activated centre for the same enzyme that avoids vying each other.Herein by substrate coupling method, obtain
The ketone acid and amino acid of higher yields.The present invention breaks original reaction balance by building enzyme coupling reaction, while realizing coenzyme
Highly efficient regeneration be to release LeuDH oxidative deamination to prepare the effective way of ketone acid.The class being coupled with leucine dehydrogenase catalyzing enzyme
Transamination reaction can make full use of the advantage of transaminase approach and prepare amino acid and ketone acid simultaneously.
Summary of the invention
In order to solve the problems, such as presently, there are ketone acid preparation, the balance of transaminase-catalyzed reaction inhibits, coenzyme circulatory problems.
The present invention provides one kind using ketone acid and unnatural amino acid as substrate, is catalyzed with leucine dehydrogenase, constructs a novel system
The method of standby ketone acid and the class transamination reaction of Non-natural chiral amino acid
It is described the first purpose of the invention is to provide the method that one step of one kind prepares ketone acid and Non-natural chiral amino acid
Method carries out oxygen using ketone acid and unnatural amino acid as substrate using leucine dehydrogenase or Phenylalanine dehydrogenase as catalyst
Change reduction reaction.
In one embodiment of the invention, in the reaction of leucine dehydrogenase catalysis, the substrate amino acid packet
Include but be not limited to valine, norvaline, leucine, isoleucine, C4H9NO2, Terleu, corresponding product ketone
Acid includes but is not limited to α-ketoisovaleric acid, oxopentanoic acid, α-ketoisocaproic acid, ketocaproic acid, 2- batanone acid, trimethyl pyruvic acid；It is described
Substrate ketone acid includes but is not limited to trimethyl pyruvic acid, α-ketoisocaproic acid, α-ketoisovaleric acid, 2- batanone acid；Corresponding product ammonia
Base acid includes but is not limited to Terleu, leucine, valine, C4H9NO2.
In one embodiment of the invention, in the reaction of Phenylalanine dehydrogenase catalysis, the substrate amino acid
Including but not limited to methionine, leucine, isoleucine, valine, tryptophan, tyrosine, p- hydroxy phenylalanine, phenylpropyl alcohol
Propylhomoserin, corresponding product ketone acid include but is not limited to methyl mercapto batanone acid, ketocaproic acid, α-ketoisovaleric acid, α-ketoisocaproic acid, p- hydroxyl
Base phenylpyruvic acid, phenylpyruvic acid；The substrate ketone acid includes but is not limited to phenylpyruvic acid, p- hydroxyphenyl pyruvate, α -one dissident
Acid, corresponding product amino acid include but is not limited to phenylalanine, p- hydroxy phenylalanine, isoleucine.
In one embodiment of the invention, the method is by 2- batanone acid and leucine coupling preparation α -one dissident
Acid and α-ketoisocaproic acid.
In one embodiment of the invention, the method is by 2- batanone acid and valine coupling preparation L-2- amino
Butyric acid and α-ketoisovaleric acid.
In one embodiment of the invention, the method is by 2- batanone acid and norvaline coupling preparation L-2- ammonia
Base butyric acid and oxopentanoic acid.
In one embodiment of the invention, the method is by 2- batanone acid and leucine coupling preparation L-2- amino
Butyric acid and α-ketoisocaproic acid.
In one embodiment of the invention, the method is by 2- batanone acid and isoleucine coupling preparation L-2- ammonia
Base butyric acid and ketocaproic acid.
In one embodiment of the invention, the method is that trimethyl pyruvic acid and valine coupling preparation is tertiary bright
Propylhomoserin and α-ketoisovaleric acid.
In one embodiment of the invention, the method is by trimethyl pyruvic acid and norvaline coupling preparation uncle
Leucine and oxopentanoic acid.
In one embodiment of the invention, the method is that trimethyl pyruvic acid and leucine coupling preparation is tertiary bright
Propylhomoserin and α-ketoisocaproic acid.
In one embodiment of the invention, the method is by trimethyl pyruvic acid and isoleucine coupling preparation uncle
Leucine and ketocaproic acid.
In one embodiment of the invention, the method is by trimethyl pyruvic acid and C4H9NO2 coupling system
Standby Terleu and 2- batanone acid.
In one embodiment of the invention, the method is that α-ketoisovaleric acid and leucine coupling are prepared valine
And α-ketoisocaproic acid.
In one embodiment of the invention, the method is that α-ketoisovaleric acid and norvaline coupling are prepared figured silk fabrics ammonia
Acid and oxopentanoic acid.
In one embodiment of the invention, the method is that α-ketoisovaleric acid and leucine coupling are prepared valine
And α-ketoisocaproic acid.
In one embodiment of the invention, the method is that α-ketoisovaleric acid and isoleucine coupling are prepared figured silk fabrics ammonia
Acid and ketocaproic acid.
In one embodiment of the invention, the method is that α-ketoisovaleric acid and Terleu coupling are prepared figured silk fabrics ammonia
Acid and trimethyl pyruvic acid.
In one embodiment of the invention, the method is that α-ketoisocaproic acid and valine coupling are prepared the bright ammonia of figured silk fabrics
Acid and α-ketoisovaleric acid.
In one embodiment of the invention, the method is that α-ketoisocaproic acid and norvaline coupling are prepared bright ammonia
Acid and oxopentanoic acid.
In one embodiment of the invention, the method is that α-ketoisocaproic acid and leucine coupling are prepared leucine
And α-ketoisocaproic acid.
In one embodiment of the invention, the method is that α-ketoisocaproic acid and isoleucine coupling are prepared bright ammonia
Acid and ketocaproic acid.
In one embodiment of the invention, the method is that α-ketoisocaproic acid and Terleu coupling are prepared bright ammonia
Acid and trimethyl pyruvic acid.
In one embodiment of the invention, the method is by α-ketoisocaproic acid and Terleu coupling the third ammonia of preparation
Acid and α-ketoisocaproic acid.
In one embodiment of the invention, the method is that phenylpyruvic acid and Valine coupling are prepared alanine
And α-ketoisovaleric acid.
In one embodiment of the invention, the method is that α-ketoisocaproic acid and phenylalanine coupling are prepared bright ammonia
Acid and phenylpyruvic acid.
In one embodiment of the invention, the pH value control of the conversion reaction is 7-11.
In one embodiment of the invention, the concentration of substrate is 5-500mmol/L.
In one embodiment of the invention, the co-factor concentration is 0.01-10 μm of ol/L.
In one embodiment of the invention, the NH4+Concentration is 2-10mmol/L.
In one embodiment of the invention, the transformation time 0.5h-8h.
A second object of the present invention is to provide the method answering in terms of preparing the product containing amino acid and/or ketone acid
With.
Beneficial effects of the present invention: the present invention is had and was prepared using the catalytic oxidation-reduction reaction simultaneously of single dehydrogenase
Journey is simple, high catalytic efficiency feature, and coenzyme circulation is constituted in system, realizes the efficient circulation regeneration of coenzyme, and simultaneously
Realize that one-step method prepares ketone acid and unnatural amino acid.The class transamination reaction approach that the present invention constructs, thus it is possible to vary reaction balance,
It is directly realized by coenzyme in-situ regeneration, improves the advantages that conversion ratio, the conversion ratio of ketone acid is made to prepare ketone compared to single step oxidative deamination
Acid improves 14 times or so, by being controlled at 30 DEG C, pH 9.0, concentration of substrate 100mmol/L, co-factor concentration
0.01 μm of ol/L, improves conversion ratio to 92%.The total turn over number of coenzyme (TTN) reaches 5.84 × 105.For efficiently produce ketone acid and
Non-natural chiral amino acid provides an effective way.
Detailed description of the invention
Fig. 1 is conversion reaction schematic diagram of the invention.
Specific embodiment
Ketone acid detection: parallax detector；60 DEG C of column temperature；Mobile phase is 5mmol/L dilute sulfuric acid；Flow velocity 0.6mL/min；Sample introduction
Measure 10 μ L.
Amino acid detection: On-chip derivatization is carried out first with OPA, then is detected.Chromatographic condition: UV detector wavelength
338nm；5 μm of C of chromatographic column Diamonsil18(250*4.6mm)；40 DEG C of column temperature；Flow velocity 1mL/min；10 μ L of sample volume；Flowing
Phase A:20mmol/L sodium acetate, with 1% acetic acid diluted tune pH value to 7.2；Mobile phase B is that 20mmol/L sodium acetate/methanol/acetonitrile is molten
Liquid (2:4:4, V/V/V, pH 7.2)；Pump program: B component is risen to by 7% in 27min rises to 80% again in 50%, 4min, keep
Then 3min is down to 7% in 1min, keep 3min (total time 38min).
Single step oxidation reaction system: with the NAD of various concentration+To originate coenzyme, reaction condition: 10mmol/L leucine,
The pure enzyme solution of 2U/mL leucine dehydrogenase, 10mmol/L Tris-HCL (pH=8.5).
Single step reduction reaction system: being starting coenzyme, reaction condition: 10mmol/L 2- butanone with the NADH of various concentration
Acid, the pure enzyme solution of 2U/mL leucine dehydrogenase, 1mol/L NH4 +, 10mmol/L Tris-HCL (pH=9.5).
Class transamination reaction system: the concentration for adding substrate amino acid is 100mmol/L, and the concentration of substrate ketone acid is
100mmol/L, coenzyme NAD+Concentration is 0.2 μm of ol/L NAD+, the pure enzyme solution/Phenylalanine dehydrogenase of 2U/mL leucine dehydrogenase
The pure enzyme solution of leucine dehydrogenase, reaction temperature are 30 DEG C, control pH 9.0, reaction time 6h with ammonium hydroxide.
Embodiment 1
E.coli BL21/pET 28a-LDH is inoculated into 5mL LB (50 μ g/mL kanamycins) fluid nutrient medium, in
37 DEG C, cultivate 8h under the conditions of 200r/min, be then transferred in 50mL LB (50 μ g/mL kanamycins) fluid nutrient medium, in 37
DEG C, under the conditions of 200r/min culture to OD600Between 0.6-0.8, it is added IPTG (0.1mmol/L), in 17 DEG C, 200r/min
Under the conditions of inducing expression 17h.Thallus is collected, 14mL 0.1molL is added in the wet thallus after weighing 10g induction-1Tris-HCL
Cell is resuspended in (pH 7.2) buffer, carries out ice-bath ultrasonic broken (power 600W, broken 1s, interval 3s, broken time
20min).Liquid is crushed in 12000rmin-1Refrigerated centrifuge 30min collects supernatant, and it is stand-by to obtain purification of samples.Utilize GE public affairs
The HisTrap HP affinity column of department purifies crude enzyme liquid.Enzyme solution a part after purification measures enzyme activity and moves
Mechanics parameter, another part catalyzed coupling reaction.
Using 2- batanone acid and leucine as substrate, by class transamination reaction, C4H9NO2 and α-ketoisocaproic acid are prepared.
Reaction system is 2mL, and system includes Tris-HCL buffer (0.1mol/L), L-Leu (100mmol/L), 2- batanone acid
(100mmol/L), the pure enzyme solution of 2U/mL leucine dehydrogenase, NAD+(0.2mmol/L) reacts under the conditions of 30 DEG C, 200r/min
6h。
Leucine dehydrogenase aoxidizes L-Leu and generates α-ketoisocaproic acid yield 4% or so, and restores the yield of ketone acid
Reach 72.73%, be 18 times of oxidation reaction yield, at the same research also indicate that it is simple by improve oxidisability coenzyme concentration for
Improving oxidation reaction yield does not have obvious positive acting.Therefore, by constructing coupling reaction, break original reaction balance, simultaneously
The highly efficient regeneration for realizing coenzyme is the effective way for releasing LeuDH oxidative deamination and preparing ketone acid.
Embodiment 2
Under the coupling reaction system of leucine dehydrogenase catalysis, respectively with valine, norvaline, leucine, different bright
Propylhomoserin, C4H9NO2, methionine, Terleu, phenylalanine are oxidation substrates, and with 3- methyl -2- butyric acid, α -one
Isocaproic acid, trimethyl pyruvic acid, 2- batanone acid, pyruvic acid, phenylpyruvic acid, ketoglutaric acid are that reduction substrate progress combination of two is anti-
It answers, class transamination reaction carries out in Tris-HCL buffer (0.1mol/L), and the concentration for adding substrate amino acid is 100mmol/
L, the concentration of substrate ketone acid are 100mmol/L, coenzyme NAD+Concentration is 0.2 μm of ol/L, and the pure enzyme solution of leucine dehydrogenase is 2U/
ML, reaction temperature are 30 DEG C, control pH 9.0, reaction time 6h with ammonium hydroxide.As a result as shown in 1~table of table 2.
The yield of 1 reaction product ketone acid of table
The yield of 2 reaction product amino acid of table
3 2- batanone acid of embodiment and leucine coupling preparation α-ketoisocaproic acid and C4H9NO2
Coupling reaction system be 2mL, system include 0.1mol/L Tris-HCL buffer, 100mmol/L L-Leu,
100mmol/L 2- batanone acid, the pure enzyme solution of 2U/mL leucine dehydrogenase, 0.2 μm of ol/L NAD+, with ammonium hydroxide control pH 9.0, in
30 DEG C, react 6h under the conditions of 200r/min.The yield for obtaining α-ketoisocaproic acid is 56.3%, and the yield of C4H9NO2 is
55.2%.
4 α-ketoisovaleric acid of embodiment and Terleu coupling prepare trimethyl pyruvic acid and L-Leu
Coupling reaction system is 2mL, and system includes 0.1mol/L Tris-HCL buffer, the tertiary bright ammonia of 100mmol/L L-
Acid, 100mmol/L α-ketoisovaleric acid, the pure enzyme solution of 2U/mL leucine dehydrogenase, 0.2 μm of ol/L NAD+, with ammonium hydroxide control pH
9.0,6h is reacted under the conditions of 30 DEG C, 200r/min.The yield for obtaining trimethyl pyruvic acid is 77.8%, the yield of L-Leu
It is 75.3%.
5 2- batanone acid of embodiment and Terleu coupling prepare trimethyl pyruvic acid and C4H9NO2
Coupling reaction system is 2mL, and system includes 0.1mol/L Tris-HCL buffer, the tertiary bright ammonia of 100mmol/L L-
Acid, 100mmol/L 2- batanone acid, the pure enzyme solution of 2U/mL leucine dehydrogenase, 0.2 μm of ol/L NAD+, with ammonium hydroxide control pH
9.0,6h is reacted under the conditions of 30 DEG C, 200r/min.The yield for obtaining trimethyl pyruvic acid is 76.7%, C4H9NO2
Yield is 77.4%.
6 α-ketoisovaleric acid of embodiment and leucine coupling preparation α-ketoisocaproic acid and Valine
Coupling reaction system be 2mL, system include 0.1mol/L Tris-HCL buffer, 100mmol/L L-Leu,
100mmol/L α-ketoisovaleric acid, the pure enzyme solution of 2U/mL leucine dehydrogenase, 0.2 μm of ol/L NAD+, with ammonium hydroxide control pH 9.0,
6h is reacted under the conditions of 30 DEG C, 200r/min.The yield for obtaining α-ketoisocaproic acid is 62.2%, and the yield of Valine is
65.2%.
7 α-ketoisovaleric acid of embodiment and isoleucine coupling preparation α-ketoisocaproic acid and Valine
Coupling reaction system is 2mL, and system includes 0.1mol/L Tris-HCL buffer, the different bright ammonia of 100mmol/L L-
Acid, 100mmol/L α-ketoisovaleric acid, the pure enzyme solution of 2U/mL leucine dehydrogenase, 0.2 μm of ol/L NAD+, with ammonium hydroxide control pH
9.0,6h is reacted under the conditions of 30 DEG C, 200r/min.The yield for obtaining α-ketoisocaproic acid is 58.9%, and the yield of Valine is
57.7%.
The coenzyme NAD of 7 various concentration of embodiment+Influence to yield and TTN
On the basis of embodiment 1, adjustment coenzyme concentration is 0.01 μm of ol/L, investigates the coenzyme NAD of various concentration+To production
The influence of rate and TTN, the results showed that, when coenzyme concentration is 0.01 μm of ol/L, coupling reaction has highest TTN, can reach
5.84×105, industrialized level is had reached, illustrates coenzyme NAD+There is good power of regeneration, cost can be substantially reduced.And
α-ketoisocaproic acid and the corresponding yield of C4H9NO2 are 58.3% and 65.3%.As coenzyme concentration is mentioned from 0.01 μm of ol/L
Height is to 10 μm of ol/L, and the concentration of product α-ketoisocaproic acid and C4H9NO2 is basically unchanged, but the TTN of coupling reaction is but shown
Writing reduces, and illustrates that the additional amount of coenzyme is smaller to the yield impact of reaction, to establish being successfully established for coupling reaction mode.
The coenzyme NAD of 8 various concentration of embodiment+Influence to yield and TTN
On the basis of embodiment 8, adjustment coenzyme concentration is 1 μm of ol/L, investigates the coenzyme NAD of various concentration+To yield
With the influence of TTN, the results showed that, when coenzyme concentration is 1 μm of ol/L, coupling reaction has highest TTN, it can reach 5.84 ×
103, industrialized level is had reached, illustrates coenzyme NAD+There is good power of regeneration, cost can be substantially reduced.And α -one is different
Caproic acid and the corresponding yield of C4H9NO2 are 58.2% and 64.0%.As coenzyme concentration is increased to 1 μ from 0.01 μm of ol/L
The concentration of mol/L, product α-ketoisocaproic acid and C4H9NO2 is basically unchanged, but the TTN of coupling reaction is but significantly reduced,
Illustrate that the additional amount of coenzyme is smaller to the yield impact of reaction, to establish being successfully established for coupling reaction mode.
The coenzyme NAD of 9 various concentration of embodiment+Influence to yield and TTN
On the basis of embodiment 9, adjustment coenzyme concentration is 10 μm of ol/L, investigates the coenzyme NAD of various concentration+To yield
With the influence of TTN, the results showed that, when coenzyme concentration is 10 μm of ol/L, coupling reaction TTN is 584, and regenerating coenzyme ability is not
It is good.α-ketoisocaproic acid and the corresponding yield of C4H9NO2 are 59.2% and 66.0% at this time.As coenzyme concentration is from 1 μ
Mol/L is increased to 10 μm of ol/L, and the concentration of product α-ketoisocaproic acid and C4H9NO2 is basically unchanged, but coupling reaction
TTN is but significantly reduced, and illustrates that the additional amount of coenzyme is smaller to the yield impact of reaction, to establish the success of coupling reaction mode
It establishes.
10 NH of embodiment4 +Influence of the concentration to class transamination reaction
On the basis of embodiment 1, NH4 is adjusted+Concentration is 2mmol/L, investigates NH4 +Shadow of the concentration to class transamination reaction
It rings, needs NH during leucine dehydrogenase (LeuDH) reduction amination 2- batanone acid4 +, therefore NH4 +Concentration is to reduction reaction
Generate certain influence, NH4 +When concentration is 2mmol/L, α-ketoisocaproic acid and the corresponding yield of C4H9NO2 are 57.0% He
62.9%.
11 NH of embodiment4 +Influence of the concentration to class transamination reaction
On the basis of embodiment 11, NH4 is adjusted+Concentration is 10mmol/L, investigates NH4 +Shadow of the concentration to class transamination reaction
It rings, needs NH during leucine dehydrogenase (LeuDH) reduction amination 2- batanone acid4 +, therefore NH4 +Concentration is to reduction reaction
Generate certain influence NH4 +When concentration is 10mmol/L, α-ketoisocaproic acid and the corresponding yield of C4H9NO2 are 60.8% He
65.1%.NH4 +Concentration is improved from 2mmol/L to 10mmol/L, and the yield of product has almost no change, and illustrates NH4 +It is several to yield
It does not influence, NH4 +It can be recycled in coupling reaction, oxidative deamination can generate NH4 +, further it is proved to be successful
Establish coupling reaction mode.
Influence of 12 pH value of embodiment to class transamination reaction
On the basis of embodiment 1, adjustment pH value is 7.0, influence of the pH value to class transamination reaction is investigated, in biocatalysis
In reaction, the pH value of reaction system can have an impact the ionic condition of substrate and enzyme, especially to the polarity of enzyme active center, pH
The change of value not only will affect the activity of enzyme, but also can have an impact to regenerating coenzyme ability, and when pH value is 7.0, α -one is different
Caproic acid and the corresponding yield of C4H9NO2 are 60.0% and 57.2%.
Influence of 13 pH value of embodiment to class transamination reaction
On the basis of embodiment 12, adjustment pH value is 11.0, influence of the pH to class transamination reaction is investigated, in biocatalysis
In reaction, the pH value of reaction system can have an impact the ionic condition of substrate and enzyme, especially to the polarity of enzyme active center, pH
The change of value not only will affect the activity of enzyme, but also can have an impact to regenerating coenzyme ability, and when pH value is 11.0, α -one is different
Caproic acid and the corresponding yield of C4H9NO2 are 59.8% and 57.5%.
Influence of 14 concentration of substrate of embodiment to class transamination reaction
On the basis of embodiment 1, adjustment concentration of substrate is 1mmol/L, investigates concentration of substrate to the shadow of class transamination reaction
Ring, concentration of substrate increases in 0-100mmol/L, yield with the increase of concentration of substrate, concentration of substrate in 1mmol/L, this
When α-ketoisocaproic acid and the corresponding yield of C4H9NO2 be 51.4% and 49.2%.
Influence of 15 concentration of substrate of embodiment to class transamination reaction
On the basis of embodiment 14, adjustment concentration of substrate is 100mmol/L, investigates concentration of substrate to class transamination reaction
It influencing, within 100mmol/L, yield increases concentration of substrate with the increase of concentration of substrate, but when concentration of substrate is greater than
When 100mmol/L, with the increase of concentration of substrate, yield is gradually reduced, and this phenomenon is anti-other redox enzymatics
Answer in system there is also.In 100mmol/L, α-ketoisocaproic acid and the corresponding yield of C4H9NO2 are concentration of substrate at this time
83.4% and 81.6%.
Influence of 16 concentration of substrate of embodiment to class transamination reaction
On the basis of embodiment 14, adjustment concentration of substrate is 500mmol/L, investigates concentration of substrate to class transamination reaction
It influencing, within 100mmol/L, yield increases concentration of substrate with the increase of concentration of substrate, but when concentration of substrate is greater than
When 500mmol/L, with the increase of concentration of substrate, yield is gradually reduced, and this phenomenon is anti-other redox enzymatics
Answer in system there is also.In 500mmol/L, α-ketoisocaproic acid and the corresponding yield of C4H9NO2 are concentration of substrate at this time
53.4% and 51.6%.
Influence of 17 transformation time of embodiment to class transamination reaction
On the basis of embodiment 1, adjustment transformation time is respectively 0h, 0.5h, 1h, 2h, 4h, 6h, 8h, convert anti-
It answers, when transformation time is from 0h to 2h, the yield of α-ketoisocaproic acid and C4H9NO2 is rapidly increased to 75.3% He respectively
76.4%, but when transformation time is from 1h to 6h, the yield of α-ketoisocaproic acid and C4H9NO2 rises to respectively
83.2% and 85.4%.After 6h, yield is almost unchanged.Thus it can determine that transformation time is that 6h is best.
18 phenylpyruvic acid of embodiment and L-Leu coupling prepare phenylalanine and α-ketoisocaproic acid
Using phenylpyruvic acid and L-Leu as substrate, by class transamination reaction, phenylalanine and α-ketoisocaproic acid are prepared.?
Under reaction system, system includes Tris-HCL buffer (0.1mol/L), L-Leu (100mmol/L), phenylpyruvic acid
(100mmol/L), the pure enzyme solution of 2U/mL Phenylalanine dehydrogenase, NAD+(0.2mmol/L), it is anti-under the conditions of 30 DEG C, 200r/min
Answer 6h.Phenylalanine and α-ketoisocaproic acid yield are respectively 62.5.1% and 56%.
19 phenylpyruvic acid of embodiment and Valine coupling prepare alanine and α-ketoisovaleric acid
Using phenylpyruvic acid and Valine as substrate, by class transamination reaction, phenylalanine and α-ketoisovaleric acid are prepared.?
Under reaction system, system includes Tris-HCL buffer (0.1mol/L), L-Leu (100mmol/L), phenylpyruvic acid
(100mmol/L), the pure enzyme solution of 2U/mL Phenylalanine dehydrogenase, NAD+(0.2mmol/L), it is anti-under the conditions of 30 DEG C, 200r/min
Answer 6h.Phenylalanine and α-ketoisovaleric acid yield are respectively 43.3% and 40.3%.
20 α-ketoisocaproic acid of embodiment and phenylalanine coupling prepare leucine and phenylpyruvic acid
Using α-ketoisocaproic acid and phenylalanine as substrate, by class transamination reaction, leucine and phenylpyruvic acid are prepared.Anti-
It answers under system, system includes Tris-HCL buffer (0.1mol/L), L-Leu (100mmol/L), phenylpyruvic acid
(100mmol/L), the pure enzyme solution of 2U/mL Phenylalanine dehydrogenase, NAD+(0.2mmol/L), it is anti-under the conditions of 30 DEG C, 200r/min
Answer 6h.Leucine and phenylpyruvic acid yield are respectively 31.2% and 28.7%.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Claims (9)
1. the method that a step prepares ketone acid and Non-natural chiral amino acid, which is characterized in that the method is with leucine dehydrogenase
Or Phenylalanine dehydrogenase carries out redox reaction using ketone acid and unnatural amino acid as substrate as catalyst.
2. the method according to claim 1, wherein leucine dehydrogenase catalysis reaction in, the substrate ammonia
Base acid includes but is not limited to valine, norvaline, leucine, isoleucine, C4H9NO2, Terleu, corresponding
Product ketone acid includes but is not limited to α-ketoisovaleric acid, oxopentanoic acid, α-ketoisocaproic acid, ketocaproic acid, 2- batanone acid, front three benzylacetone
Acid；The substrate ketone acid includes but is not limited to trimethyl pyruvic acid, α-ketoisocaproic acid, α-ketoisovaleric acid, 2- batanone acid；It is corresponding
Product amino acid includes but is not limited to Terleu, leucine, valine, C4H9NO2.
3. the method according to claim 1, wherein Phenylalanine dehydrogenase catalysis reaction in, the substrate
Amino acid includes but is not limited to methionine, leucine, isoleucine, valine, tryptophan, tyrosine, the third ammonia of p- hydroxy benzenes
Acid, phenylalanine, corresponding product ketone acid includes but is not limited to methyl mercapto batanone acid, ketocaproic acid, α-ketoisovaleric acid, α -one dissident
Acid, p- hydroxyphenyl pyruvate, phenylpyruvic acid；The substrate ketone acid includes but is not limited to phenylpyruvic acid, p- hydroxyphenyl pyruvate, α-
Ketoisocaproate, corresponding product amino acid include but is not limited to phenylalanine, p- hydroxy phenylalanine, isoleucine.
4. any method according to claim 1~3, which is characterized in that the pH value control of the conversion reaction is 7-11.
5. any method according to claim 1~3, which is characterized in that oxidation/restores concentration of substrate as 5-
500mmol/L。
6. any method according to claim 1~3, which is characterized in that co-factor concentration is 0.01-10 μm of ol/L.
7. any method according to claim 1~3, which is characterized in that transformation time 0.5h-8h.
8. any the method for claim 1~7 is in the application of food, biology, chemical field.
9. application of any the method for claim 1~7 in terms of preparing the product containing amino acid and/or ketone acid.
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| CN1380408A (en) * | 2001-04-10 | 2002-11-20 | 上海博德基因开发有限公司 | A polypeptide-Glu/Leu/Phe/Val dehydrogenase-11.55 and polynucleotide for coding this polypeptide |
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