CN108570486A - The bioconversion method of S-Leucine - Google Patents
The bioconversion method of S-Leucine Download PDFInfo
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Abstract
The invention discloses a kind of bioconversion methods of L Terleus, include the following steps：Using trimethyl Sodium Pyruvate as substrate, using coenzyme NAD+and the leucine dehydrogenase of immobilization, hydrogenlyase as biocatalyst, addition ammonium salt establishes transformation system and carries out bioconversion reaction, obtains the conversion fluid containing L Terleus.The present invention is biocatalyst using leucine dehydrogenase, the hydrogenlyase of immobilization, and capable of reusing 10 times, still there is enzyme activity, expensive coenzyme and enzyme can recycle, and save production cost；Using homemade trimethyl sodium pyruvate solution as substrate, preparation process is simplified, production cost is saved, convenient for purifying；The L Terleu crystal purities of preparation reach 98.5% or more, and optical purity is 99% or more.Technological process is simple, to equipment without particular/special requirement, is suitable for industrialized production.It is monitored using HPLC MS and HPLC, until substrate is fully utilized.
Description
Technical field
The present invention relates to the preparation methods of non-natural amino acid, in particular to a kind of bioconversion side of S-Leucine
Method.
Background technology
Non-natural amino acid and its derivative are important chemical intermediate, wherein S-Leucine is a kind of important
Non-natural amino acid can be used for synthesizing medicine intermediate, and the chiral induction agent as asymmetric syntheses and asymmetric fractionation
Resolution reagent is widely used in synthesising biological inhibitor, biologically active peptide, anticancer and the drugs such as antiviral.Current existing Shi Guibao,
The protease inhibitors class anti-HIV new medicament of the drugmakers such as Pfizer, Abbott Laboratories listing all uses S-Leucine as intermediate, such as anti-
HIV drugs atazanavir (Atazanavir), global marketing volume reaches 32.26 hundred million dollars within 2006, in addition have six kinds
The AntiHIV1 RT activity anti-cancer agent of clinical test also uses S-Leucine as intermediate.With Chinese society expanding economy and ring
The enhancing of border protective awareness, medication chemistry industry not only needs to improve the production capacity of S-Leucine, and needs to create green
The free of contamination production technology of color.Therefore, bioconversion production S-Leucine is carried out using enzyme process just more and more mutually shut down
The attention of structure.
In the prior art, the synthetic method of S-Leucine is very more, including Physical, chemical synthesis or Split Method, with
And biological enzymatic synthesis or Split Method.Physical is to detach N- acetyl-DL- Terleus using Simulated Moving Bed Chromatography isolation technics
Raceme, determination are to need professional equipment investment big, and stationary phase is expensive, and mobile phase consumption is big, and separation condition needs to optimize true
It is fixed.Chemical synthesis mainly has passes through multistep with chiral raw material such as (R)-tertiary butyl glycinol and (R) -4- phenyl -2- oxazolidones
Synthesis obtains S-Leucine, and the synthetic method chiral starting materials are expensive, and pollutant emission is more, and product yield is not high.It is another
Kind chemical synthesis process is asymmetric syntheses, using chiral induction agent such as (R)-phenyl glycine amide or chirality a- phenyl ethylamines point
Synthesis S-Leucine is not reacted with special valeral or tertiary butyl ketone acid, but asymmetric syntheses needs the catalyst price used high
It is expensive, seriously polluted and unsuitable large-scale production.Also the method for chemical resolution can be used to synthesize S-Leucine, the chirality of report is torn open
Agent is divided to have 10- camphorsulfonic acids, optical pure tartaric acid, optical pure mandel etc. to have the drawback that chiral resolving agent costliness, in vain
Enantiomer need to detach and racemization, and yield is not high, and consumption of organic solvent is big.
Due to biocatalysis technology have very strong Substratspezifitaet, higher catalytic efficiency, mild reaction condition with
And environmental pollution it is smaller the advantages that, have been widely used for preparing S-Leucine.S-Leucine is synthesized by living things catalysis
Include mainly two major classes method, i.e., split photoactive tert-leucine using enzyme is urged with the method and biology that prepare S-Leucine
Change the method for directly synthesizing S-Leucine.Such as：Application No. is 200610020854 Chinese invention patent applications to utilize rice
Aspergillus amino-acylase and application No. is 201010138158 Chinese invention patent applications to utilize a kind of dual-enzyme system (acylated ammonia
Base acid racemase and hydrolase) Terleu raceme can be split, prepare the S-Leucine (optical purity of high-optical-purity
Up to more than 99%ee values).Although the production cost of both methods is moderate, be convenient for industrialized production, entire production process also compared with
Simply, still, the theoretical yield of S-Leucine is less than 50%.S-Leucine volume side is directly synthesized using living things catalysis means
Method can improve the yield of S-Leucine.
The main direction of studying of bioanalysis, which is focused on, at present is restored using L-Leu dehydrogenase from trimethyl pyruvic acid
For the route of S-Leucine, and hydrogenlyase in-situ regeneration coenzyme NAD H is utilized, ammonium formate is utilized to provide amino molecule and hydrogen
Atom.Degussa companies early in last century the nineties Germany are just combined using the route with ultrafiltration membrane reactor, and L- is produced
Terleu.The route has had several patent application publications, existing main problem to have：In presently disclosed patent, instead
It is 10-15% to answer initial substrate concentrations, and S-Leucine dehydrogenase can be pressed down when concentration of substrate is excessively high by significant substrate
System, reaction rate dramatic decrease, under conditions of high ammonia density, the product accumulation of excessive substrate and generation can all form by-product
Object illustrates that this method is difficult to repeat.In addition, enzyme inventory is excessive, expensive coenzyme NAD inventory reaches 1% (w/ of substrate
W), less economical, and without specific rear extraction process, still cannot be satisfied the needs of industrialized production.
Therefore, a kind of bioconversion method of efficient S-Leucine of research and development seems very necessary.
Invention content
Problem to be solved by this invention seeks to provide a kind of bioconversion method of S-Leucine, and this method is being protected
On the basis of demonstrate,proving highly-solid selectively, high conversion and high yield pulp1, concentration of substrate can be effectively improved, coenzyme input is reduced, simplifies
Production technology and reduction production cost.
In order to solve the above technical problems, the bioconversion method of S-Leucine provided by the present invention, which is characterized in that
Include the following steps：
1) leucine dehydrogenase, the hydrogenlyase of immobilization are prepared；
2) trimethyl sodium pyruvate solution is prepared by raw material of dichloro pinacolone；Its chemical equation is as follows：
Trimethyl sodium pyruvate solution is prepared to specifically comprise the following steps：
A. the water of the dichloro pinacolone and 2 parts by weight of 1 parts by weight is added into reaction kettle, 10mol/L is added dropwise while stirring
Sodium hydroxide solution, adjust pH to 10-11, drip off and be warming up to 75 degree, reaction is limpid transparent to solution, cooling；
B. the solution obtained by step a is heated to 40 DEG C, the potassium permanganate of 0.8 parts by weight is added, is increasing potassium manganate
In the process, temperature is warming up to 83 DEG C, reacts two hours, cooling, stands, and filtering, filter residue merges after fully being washed with water with filtrate
Obtain colourless transparent solution, as trimethyl sodium pyruvate solution.
3) using the trimethyl sodium pyruvate solution obtained by step 2) as substrate, with the fixation obtained by coenzyme NAD+and step 1)
Leucine dehydrogenase, the hydrogenlyase of change are biocatalyst, and addition ammonium salt establishes transformation system and carries out bioconversion reaction,
Obtain the conversion fluid containing S-Leucine.
Preferably, in step 1), leucine dehydrogenase, hydrogenlyase are fixed with carragheen or compound gel, consolidate
Surely leucine dehydrogenase, the hydrogenlyase changed.
Further, for compound gel by the mixture of alginate and polyethylene glycol, the alginate is calcium alginate, sea
The weight ratio of one or more of potassium alginate, sodium alginate, the alginate and polyethylene glycol is 1:4-9.
Further, the weight ratio of alginate and polyethylene glycol is 1:4, the alginate is by calcium alginate, sea
Potassium alginate, sodium alginate are 3 by weight:9:4 compositions.
Further, leucine dehydrogenase, hydrogenlyase are fixed with carragheen or compound gel, including following step
Suddenly：Carragheen or compound gel are dissolved by heating in distilled water, leucine dehydrogenase, hydrogenlyase, stirring are added after cooling
Uniformly, it then is cooled to its and solidifies leucine dehydrogenase, hydrogenlyase to get immobilization.
Further, the weight ratio of carragheen or compound gel and distilled water is 1:6-10 exists carragheen or compound gel
It is heated to 80-90 DEG C of dissolving in distilled water, leucine dehydrogenase, hydrogenlyase, the fixation are added after being cooled to 35-45 DEG C
In the leucine dehydrogenase of change, hydrogenlyase, leucine dehydrogenase, hydrogenlyase, carragheen mass ratio be 1:4-6:
7-12, leucine dehydrogenase, the hydrogenlyase of the immobilization are cut into 1 × 1cm3Cube it is spare.
Further, in step 3), the ammonium salt is ammonium formate；The molar ratio control of ammonium formate and trimethyl Sodium Pyruvate
It is made as 1.8-2.2:1, preferably 2:1；The leucine dehydrogenase of the immobilization, hydrogenlyase and trimethyl Sodium Pyruvate
Amount ratio is 8-12g:1mol, preferably 10g:The amount ratio of 1mol, coenzyme NAD+and trimethyl Sodium Pyruvate is 0.2-0.4g:
1mol, preferably 0.3g:1mol, final concentration control of the trimethyl Sodium Pyruvate in transformation system is 2mol/L, transformation system
PH value control be 5-10, preferably 6, control the transformation system temperature be 20-60 DEG C, preferably 55 DEG C；；Control conversion
Time is 12-36 hours, preferably 25 hours；The conversion reaction carries out in shaking table, and the rotating speed control of the shaking table is
150-250r/min, preferably 200r/min.
Again further, the conversion fluid concentration containing S-Leucine is placed in 0-4 DEG C and places 8-16h, crystallized,
Obtain S-Leucine coarse crystal；S-Leucine coarse crystal is rinsed with methanol, is filtered, is crystallized again, obtain the tertiary bright ammonia of L-
Acid crystal.
As another embodiment, the conversion fluid containing S-Leucine is first exchanged with 732 type highly acidic cations
Resin adsorption, then adsorbed with 201 type strong-base anion-exchange resins, obtain S-Leucine crystal.In this way, L- can be improved
The yield and purity of Terleu.
Leucine dehydrogenase is the leucine dehydrogenation from bacillus cereus (Bacillus cereus) of purchase
Enzyme.
Hydrogenlyase is the hydrogenlyase from Candida boidinii (Candida boidinii) of purchase.
Advantages of the present invention is mainly reflected in following several respects：
First, the present invention fixes leucine dehydrogenase, hydrogenlyase using carragheen or compound gel, and after fixed, bright ammonia
Acidohydrogenase, hydrogenlyase are reacted after reusing 10 times still with enzyme activity, and expensive coenzyme and enzyme can be recycled, be saved
Save production cost；
Second, the present invention is with trimethyl pyruvic acid using homemade trimethyl sodium pyruvate solution as substrate, with traditional
Substrate is compared, on the one hand, is simplified preparation process, is reduced the acidization of trimethyl Sodium Pyruvate, save production cost；
On the other hand, the reduction that hydrochloric acid feeds intake, so as to get the conversion fluid containing S-Leucine in salt content reduce, contain convenient for the later stage
There is the purifying of the conversion fluid of S-Leucine；
Third, S-Leucine crystal purity prepared by the present invention reaches 98.5% or more, optical purity is 99% or more；
Fourth, present invention process flow is simple, to equipment without particular/special requirement, it is suitable for industrialized production；
Fifth, the present invention is monitored in biotransformation using HPLC-MS and HPLC, until substrate is completely sharp
With.
Description of the drawings
Fig. 1 is the mass spectrogram after S-Leucine prepared by the embodiment of the present invention 1 derives；
Fig. 2 is the liquid phase figure of S-Leucine standard items；
Fig. 3 is the liquid phase figure of S-Leucine prepared by the embodiment of the present invention 1；
Fig. 4 is the Rate activity datagram that 1 leucine dehydrogenase of the embodiment of the present invention reuses 10 times；
Fig. 5 is the Rate activity datagram that 1 hydrogenlyase of the embodiment of the present invention reuses 10 times；
Fig. 6 is the Rate activity datagram that 2 leucine dehydrogenase of the embodiment of the present invention reuses 10 times；
Fig. 7 is the Rate activity datagram that 2 hydrogenlyase of the embodiment of the present invention reuses 10 times.
Specific implementation mode
Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail, but the embodiment should not manage
Solution is limitation of the present invention.
The bioconversion method of S-Leucine provided by the present invention, includes the following steps：
1) leucine dehydrogenase, the hydrogenlyase of immobilization are prepared；
2) trimethyl sodium pyruvate solution is prepared by raw material of dichloro pinacolone；Its chemical equation is as follows：
Trimethyl sodium pyruvate solution is prepared to specifically comprise the following steps：
A. the water of the dichloro pinacolone and 2 parts by weight of 1 parts by weight is added into reaction kettle, 10mol/L is added dropwise while stirring
Sodium hydroxide solution, adjust pH to 10-11, drip off and be warming up to 75 degree, reaction is limpid transparent to solution, cooling；
B. the solution obtained by step a is heated to 40 DEG C, the potassium permanganate of 0.8 parts by weight is added, is increasing potassium manganate
In the process, temperature is warming up to 83 DEG C, reacts two hours, cooling, stands, and filtering, filter residue merges after fully being washed with water with filtrate
Obtain colourless transparent solution, as trimethyl sodium pyruvate solution.
3) using the trimethyl sodium pyruvate solution obtained by step 2) as substrate, with the fixation obtained by coenzyme NAD+and step 1)
Leucine dehydrogenase, the hydrogenlyase of change are biocatalyst, and addition ammonium salt establishes transformation system and carries out bioconversion reaction,
Obtain the conversion fluid containing S-Leucine.
Preferably, in step 1), leucine dehydrogenase, hydrogenlyase are fixed with carragheen or compound gel, consolidate
Surely leucine dehydrogenase, the hydrogenlyase changed.
Further, for compound gel by the mixture of alginate and polyethylene glycol, the alginate is calcium alginate, sea
The weight ratio of one or more of potassium alginate, sodium alginate, the alginate and polyethylene glycol is 1:4-9.
Further, the weight ratio of alginate and polyethylene glycol is 1:4, the alginate is by calcium alginate, sea
Potassium alginate, sodium alginate are 3 by weight:9:4 compositions.
Further, leucine dehydrogenase, hydrogenlyase are fixed with carragheen or compound gel, including following step
Suddenly：Carragheen or compound gel are dissolved by heating in distilled water, leucine dehydrogenase, hydrogenlyase, stirring are added after cooling
Uniformly, it then is cooled to its and solidifies leucine dehydrogenase, hydrogenlyase to get immobilization.
Further, the weight ratio of carragheen or compound gel and distilled water is 1:6-10 exists carragheen or compound gel
It is heated to 80-90 DEG C of dissolving in distilled water, leucine dehydrogenase, hydrogenlyase, the fixation are added after being cooled to 35-45 DEG C
In the leucine dehydrogenase of change, hydrogenlyase, leucine dehydrogenase, hydrogenlyase, carragheen mass ratio be 1:4-6:
7-12, leucine dehydrogenase, the hydrogenlyase of the immobilization are cut into 1 × 1cm3Cube it is spare.
Further, in step 3), the ammonium salt is ammonium formate；The molar ratio control of ammonium formate and trimethyl Sodium Pyruvate
It is made as 1.8-2.2:1, preferably 2:1；The leucine dehydrogenase of the immobilization, hydrogenlyase and trimethyl Sodium Pyruvate
Amount ratio is 8-12g:1mol, preferably 10g:The amount ratio of 1mol, coenzyme NAD+and trimethyl Sodium Pyruvate is 0.2-0.4g:
1mol, preferably 0.3g:1mol, final concentration control of the trimethyl Sodium Pyruvate in transformation system is 2mol/L, transformation system
PH value control be 5-10, preferably 6, control the transformation system temperature be 20-60 DEG C, preferably 55 DEG C；；Control conversion
Time is 12-36 hours, preferably 25 hours；The conversion reaction carries out in shaking table, and the rotating speed control of the shaking table is
150-250r/min, preferably 200r/min.
Again further, the conversion fluid concentration containing S-Leucine is placed in 0-4 DEG C and places 8-16h, crystallized,
Obtain S-Leucine coarse crystal；S-Leucine coarse crystal is rinsed with methanol, is filtered, is crystallized again, obtain the tertiary bright ammonia of L-
Acid crystal.
As another embodiment, the conversion fluid containing S-Leucine is first exchanged with 732 type highly acidic cations
Resin adsorption, then adsorbed with 201 type strong-base anion-exchange resins, obtain S-Leucine crystal.
Embodiment 1
The preparation process of S-Leucine crystal, includes the following steps：
1) 8 parts by weight compound gels are heated to 80 DEG C of dissolvings in 50 parts by weight distilled water, compound gel includes 0.3 parts by weight
Calcium alginate, 0.9 parts by weight potassium alginate, 0.4 parts by weight sodium alginate and 6.4 parts by weight polyethylene glycol, after being cooled to 40 DEG C
1 parts by weight leucine dehydrogenase, 5.5 parts by weight hydrogenlyases are added, stir evenly, then be placed in refrigerator at 4 DEG C and preserve,
It is allowed to cool leucine dehydrogenase, hydrogenlyase of the solidification to get immobilization.Again by the leucine dehydrogenase of immobilization, first
Acidohydrogenase is cut into 1 × 1cm3Cube, it is spare.
2) trimethyl sodium pyruvate solution is prepared by raw material of dichloro pinacolone；
Trimethyl sodium pyruvate solution is prepared to specifically comprise the following steps：
A. the water of the dichloro pinacolone and 2 parts by weight of 1 parts by weight is added into reaction kettle, 10mol/L is added dropwise while stirring
Sodium hydroxide solution, adjust pH to 10-11, drip off and be warming up to 75 degree, reaction is limpid transparent to solution, cooling；
B. the solution obtained by step a is heated to 40 DEG C, the potassium permanganate of 0.8 parts by weight is added, is increasing potassium manganate
In the process, temperature is warming up to 83 DEG C, reacts two hours, cooling, stands, and filtering, filter residue merges after fully being washed with water with filtrate
Obtain colourless transparent solution, as trimethyl sodium pyruvate solution.
3) de- with coenzyme NAD+and the leucine dehydrogenase of immobilization, formic acid using trimethyl sodium pyruvate solution as substrate
Hydrogen enzyme is biocatalyst, and addition ammonium formate establishes transformation system and carries out bioconversion reaction, obtains containing S-Leucine
Conversion fluid.Wherein, the molar ratio control of ammonium formate and trimethyl Sodium Pyruvate is 2:1；The leucine dehydrogenase of the immobilization,
The amount ratio of hydrogenlyase and trimethyl Sodium Pyruvate is 10g:1mol, the amount ratio of coenzyme NAD+and trimethyl Sodium Pyruvate
For 0.3g:1mol, final concentration control of the trimethyl Sodium Pyruvate in transformation system is 2mol/L, and the pH value of transformation system controls
It is 6.The temperature control of the transformation system is 55 DEG C；Transformation time control is 25 hours；The conversion reaction in shaking table into
The rotating speed control of row, the shaking table is 200r/min.
4) the conversion fluid concentration containing S-Leucine is placed in 4 DEG C and places 12h, crystallized, obtain S-Leucine
Coarse crystal；S-Leucine coarse crystal is rinsed with methanol, is filtered, is crystallized again, obtain S-Leucine crystal.
Prepared S-Leucine crystal purity reaches 99.4%, optical purity 99.7%.
Embodiment 2
The preparation process of S-Leucine crystal, includes the following steps：
1) 7 parts by weight carragheens are heated to 85 DEG C of dissolvings in 42 parts by weight distilled water, 1 weight is added after being cooled to 35 DEG C
Part leucine dehydrogenase, 6 parts by weight hydrogenlyases are measured, stirs evenly, then be placed in refrigerator at 4 DEG C and preserve, is allowed to cool
Solidify leucine dehydrogenase, the hydrogenlyase to get immobilization.Again by the leucine dehydrogenase of immobilization, hydrogenlyase
It is cut into 1 × 1cm3Cube, it is spare.
2) trimethyl sodium pyruvate solution is prepared by raw material of dichloro pinacolone；Preparation method such as embodiment 1.
3) de- with coenzyme NAD+and the leucine dehydrogenase of immobilization, formic acid using trimethyl sodium pyruvate solution as substrate
Hydrogen enzyme is biocatalyst, and addition ammonium formate establishes transformation system and carries out bioconversion reaction, obtains containing S-Leucine
Conversion fluid.Wherein, the molar ratio control of ammonium formate and trimethyl Sodium Pyruvate is 1.8:1；The leucine dehydrogenation of the immobilization
The amount ratio of enzyme, hydrogenlyase and trimethyl Sodium Pyruvate is 9g:1mol, the dosage of coenzyme NAD+and trimethyl Sodium Pyruvate
Than for 0.2g:1mol, final concentration control of the trimethyl Sodium Pyruvate in transformation system is 2mol/L, the pH value control of transformation system
It is made as 5.The temperature control of the transformation system is 60 DEG C；Transformation time control is 30 hours；The conversion reaction is in shaking table
It carries out, the rotating speed control of the shaking table is 150r/min.
4) the conversion fluid concentration containing S-Leucine is placed in 3 DEG C and places 8h, crystallized, it is thick to obtain S-Leucine
Crystal；S-Leucine coarse crystal is rinsed with methanol, is filtered, is crystallized again, obtain S-Leucine crystal.
Prepared S-Leucine crystal purity reaches 99.2%, optical purity 99.6%.
Embodiment 3
The preparation process of S-Leucine crystal, includes the following steps：
1) 9 parts by weight compound gels are heated to 88 DEG C of dissolvings in 72 parts by weight distilled water, compound gel includes 0.4 parts by weight
Calcium alginate, 0.3 parts by weight potassium alginate, 0.2 parts by weight sodium alginate and 8.1 parts by weight polyethylene glycol, after being cooled to 45 DEG C
1 parts by weight leucine dehydrogenase, 4 parts by weight hydrogenlyases are added, stirs evenly, then be placed in refrigerator at 4 DEG C and preserve, allows
Leucine dehydrogenase, hydrogenlyase of its cooled and solidified to get immobilization.Again by the leucine dehydrogenase of immobilization, formic acid
Dehydrogenase is cut into 1 × 1cm3Cube, it is spare.
2) trimethyl sodium pyruvate solution is prepared by raw material of dichloro pinacolone；Preparation method such as embodiment 1.
3) de- with coenzyme NAD+and the leucine dehydrogenase of immobilization, formic acid using trimethyl sodium pyruvate solution as substrate
Hydrogen enzyme is biocatalyst, and addition ammonium formate establishes transformation system and carries out bioconversion reaction, obtains containing S-Leucine
Conversion fluid.Wherein, the molar ratio control of ammonium formate and trimethyl Sodium Pyruvate is 2.2:1；The leucine dehydrogenation of the immobilization
The amount ratio of enzyme, hydrogenlyase and trimethyl Sodium Pyruvate is 8g:1mol, the dosage of coenzyme NAD+and trimethyl Sodium Pyruvate
Than for 0.4g:1mol, final concentration control of the trimethyl Sodium Pyruvate in transformation system is 2mol/L, the pH value control of transformation system
It is made as 10.The temperature control of the transformation system is 50 DEG C；Transformation time control is 36 hours；The conversion reaction is in shaking table
It carries out, the rotating speed control of the shaking table is 250r/min.
4) the conversion fluid concentration containing S-Leucine is placed in 0 DEG C and places 16h, crystallized, obtain S-Leucine
Coarse crystal；S-Leucine coarse crystal is rinsed with methanol, is filtered, is crystallized again, obtain S-Leucine crystal.
Prepared S-Leucine crystal purity reaches 98.7%, optical purity 99.4%.
Embodiment 4
The preparation process of S-Leucine crystal, includes the following steps：
1) 12 parts by weight compound gels are heated to 82 DEG C to dissolve in 80 parts by weight distilled water, in compound gel, compound gel packet
1 parts by weight potassium alginate, 1 parts by weight sodium alginate and 10 parts by weight polyethylene glycol are included, then 1 parts by weight are added after being cooled to 38 DEG C
Leucine dehydrogenase, 5 parts by weight hydrogenlyases, stir evenly, then be placed in refrigerator at 4 DEG C and preserve, are allowed to cool solidification,
Leucine dehydrogenase, hydrogenlyase up to immobilization.The leucine dehydrogenase of immobilization, hydrogenlyase are cut into 1 again
×1cm3Cube, it is spare.
2) trimethyl sodium pyruvate solution is prepared by raw material of dichloro pinacolone；Preparation method such as embodiment 1.
3) de- with coenzyme NAD+and the leucine dehydrogenase of immobilization, formic acid using trimethyl sodium pyruvate solution as substrate
Hydrogen enzyme is biocatalyst, and addition ammonium formate establishes transformation system and carries out bioconversion reaction, obtains containing S-Leucine
Conversion fluid.Wherein, the molar ratio control of ammonium formate and trimethyl Sodium Pyruvate is 1.9:1；The leucine dehydrogenation of the immobilization
The amount ratio of enzyme, hydrogenlyase and trimethyl Sodium Pyruvate is 11g:1mol, the use of coenzyme NAD+and trimethyl Sodium Pyruvate
Amount is than being 0.25g:1mol, final concentration control of the trimethyl Sodium Pyruvate in transformation system is 2mol/L, the pH of transformation system
Value control is 7.The temperature control of the transformation system is 30 DEG C；Transformation time control is 12 hours；The conversion reaction is being shaken
It is carried out in bed, the rotating speed control of the shaking table is 180r/min.
4) conversion fluid containing S-Leucine is first adsorbed with 732 type storng-acid cation exchange resins, then with 201 types
Strong-base anion-exchange resin adsorbs, and obtains S-Leucine coarse crystal.
Prepared S-Leucine crystal purity reaches 99.1%, optical purity 99.3%.
Embodiment 5
The preparation process of S-Leucine crystal, includes the following steps：
1) 10 parts by weight compound gels are heated to 90 DEG C of dissolvings in 100 parts by weight distilled water, compound gel includes 1.25 weights
Part potassium alginate and 8.75 parts by weight polyethylene glycol are measured, then 1 parts by weight leucine dehydrogenase, 4.5 weights are added after being cooled to 43 DEG C
Part hydrogenlyase is measured, is stirred evenly, then be placed in refrigerator at 4 DEG C and preserve, is allowed to cool solidification to get the bright ammonia of immobilization
Acidohydrogenase, hydrogenlyase.The leucine dehydrogenase of immobilization, hydrogenlyase are cut into 1 × 1cm again3Cube, it is standby
With.
2) trimethyl sodium pyruvate solution is prepared by raw material of dichloro pinacolone；Preparation method such as embodiment 1.
3) de- with coenzyme NAD+and the leucine dehydrogenase of immobilization, formic acid using trimethyl sodium pyruvate solution as substrate
Hydrogen enzyme is biocatalyst, and addition ammonium formate establishes transformation system and carries out bioconversion reaction, obtains containing S-Leucine
Conversion fluid.Wherein, the molar ratio control of ammonium formate and trimethyl Sodium Pyruvate is 2.1:1；The leucine dehydrogenation of the immobilization
The amount ratio of enzyme, hydrogenlyase and trimethyl Sodium Pyruvate is 12g:1mol, the use of coenzyme NAD+and trimethyl Sodium Pyruvate
Amount is than being 0.35g:1mol, final concentration control of the trimethyl Sodium Pyruvate in transformation system is 2mol/L, the pH of transformation system
Value control is 8.The temperature control of the transformation system is 40 DEG C；Transformation time control is 24 hours；The conversion reaction is being shaken
It is carried out in bed, the rotating speed control of the shaking table is 220r/min.
4) conversion fluid containing S-Leucine is first adsorbed with 732 type storng-acid cation exchange resins, then with 201 types
Strong-base anion-exchange resin adsorbs, and obtains S-Leucine coarse crystal.
Prepared S-Leucine crystal purity reaches 98.9%, optical purity 99.2%.
Comparative example 1
The preparation process of S-Leucine crystal, includes the following steps：
1) 8 parts by weight compound gels are heated to 80 DEG C of dissolvings in 50 parts by weight distilled water, compound gel includes 0.3 parts by weight
Potassium alginate, 0.9 parts by weight sodium alginate, 0.4 parts by weight calcium alginate and 6.4 parts by weight polyethylene glycol, after being cooled to 40 DEG C
1 parts by weight leucine dehydrogenase, 5.5 parts by weight hydrogenlyases are added, stir evenly, then be placed in refrigerator at 4 DEG C and preserve,
It is allowed to cool leucine dehydrogenase, hydrogenlyase of the solidification to get immobilization.Again by the leucine dehydrogenase of immobilization, first
Acidohydrogenase is cut into 1 × 1cm3Cube, it is spare.
2) using trimethyl Sodium Pyruvate as substrate, with leucine dehydrogenase, the hydrogenlyase of coenzyme NAD+and immobilization
For biocatalyst, addition ammonium formate establishes transformation system and carries out bioconversion reaction, obtains the conversion containing S-Leucine
Liquid.Wherein, the molar ratio control of ammonium formate and trimethyl Sodium Pyruvate is 2:1；Leucine dehydrogenase, the formic acid of the immobilization
The amount ratio of dehydrogenase and trimethyl Sodium Pyruvate is 6g:1mol, coenzyme NAD+with the amount ratio of trimethyl Sodium Pyruvate are
0.5g:1mol, final concentration control of the trimethyl Sodium Pyruvate in transformation system are 2mol/L, and the pH value control of transformation system is
6.The temperature control of the transformation system is 75 DEG C；Transformation time control is 8 hours；The conversion reaction carries out in shaking table,
The rotating speed control of the shaking table is 200r/min.
3) the conversion fluid concentration containing S-Leucine is placed in 4 DEG C and places 12h, crystallized, obtain S-Leucine
Coarse crystal；S-Leucine coarse crystal is rinsed with methanol, is filtered, is crystallized again, obtain S-Leucine crystal.
Prepared S-Leucine crystal purity reaches 85.6%, optical purity 71.5%.
Comparative example 2
The preparation process of S-Leucine crystal, includes the following steps：
1) 9 parts by weight compound gels are heated to 88 DEG C of dissolvings in 72 parts by weight distilled water, compound gel includes 0.4 parts by weight
Potassium alginate, 0.3 parts by weight sodium alginate, 0.2 parts by weight calcium alginate and 8.1 parts by weight polyethylene glycol, after being cooled to 45 DEG C
1 parts by weight leucine dehydrogenase, 4 parts by weight hydrogenlyases are added, stirs evenly, then be placed in refrigerator at 4 DEG C and preserve, allows
Leucine dehydrogenase, hydrogenlyase of its cooled and solidified to get immobilization.Again by the leucine dehydrogenase of immobilization, formic acid
Dehydrogenase is cut into 1 × 1cm3Cube, it is spare.
2) using trimethyl Sodium Pyruvate as substrate, with leucine dehydrogenase, the hydrogenlyase of coenzyme NAD+and immobilization
For biocatalyst, addition ammonium formate establishes transformation system and carries out bioconversion reaction, obtains the conversion containing S-Leucine
Liquid.Wherein, the molar ratio control of ammonium formate and trimethyl Sodium Pyruvate is 2.4:1；The leucine dehydrogenase of the immobilization, first
The amount ratio of acidohydrogenase and trimethyl Sodium Pyruvate is 3g:1mol, coenzyme NAD+with the amount ratio of trimethyl Sodium Pyruvate are
0.5g:1mol, final concentration control of the trimethyl Sodium Pyruvate in transformation system are 2mol/L, and the pH value control of transformation system is
10.The temperature control of the transformation system is 50 DEG C；Transformation time control is 36 hours；The conversion reaction in shaking table into
The rotating speed control of row, the shaking table is 250r/min.
3) the conversion fluid concentration containing S-Leucine is placed in 0 DEG C and places 16h, crystallized, obtain S-Leucine
Coarse crystal；S-Leucine coarse crystal is rinsed with methanol, is filtered, is crystallized again, obtain S-Leucine crystal.
Prepared S-Leucine crystal purity reaches 79.3%, optical purity 82.4%.
Embodiment 1 compares mole it is found that ammonium formate and trimethyl Sodium Pyruvate with comparative example 1,2 comparative example 2 of embodiment
It is 1.8-2.2 than control:1；The amount ratio of the leucine dehydrogenase of immobilization, hydrogenlyase and trimethyl Sodium Pyruvate is 8-
12g:The amount ratio of 1mol, coenzyme NAD+and trimethyl Sodium Pyruvate is 0.2-0.4g:When 1mol, prepared S-Leucine
Crystal purity and optical purity are high.
Fig. 1 is the mass spectrogram of S-Leucine prepared by the method for the present invention；It is theoretical after Terleu derives by OPA
Molecular weight is 308.1, and actual measurement molecular weight is 308.1.
Fig. 2 is the liquid phase figure of S-Leucine standard items；As shown, there is absorption peak at 10.283min；
Fig. 3 is the liquid phase figure of S-Leucine prepared by the embodiment of the present invention 1；As shown, there is suction at 10.459min
Receive peak；
Fig. 4 is the Rate activity datagram that 1 leucine dehydrogenase of the embodiment of the present invention reuses 10 times；Fig. 6 is the present invention
2 leucine dehydrogenase of embodiment reuses 10 Rate activity datagrams；After fixation, leucine dehydrogenase reuses 10 times
After react still have enzyme activity, and at the 10th time in use, the Rate activity of its enzyme be more than 20U/mg.In this way, expensive leucine
Dehydrogenase can recycle, and greatly save production cost.
Fig. 5 is the Rate activity datagram that 1 hydrogenlyase of the embodiment of the present invention reuses 10 times；Fig. 7 is that the present invention is real
The Rate activity datagram that 2 hydrogenlyase of example reuses 10 times is applied, after fixed, hydrogenlyase reacts after reusing 10 times
Still there is enzyme activity, and at the 10th time in use, the Rate activity of its enzyme is more than 15U/mg.In this way, expensive hydrogenlyase can
To recycle, production cost is greatly saved.
Enzyme activity defines:Under conditions of pH7.0,37 DEG C of temperature, the enzyme activity of 1 μm of olNADH of catalysis reduction per minute is 1
A enzyme-activity unit.The calculation formula of enzyme activity is:(in formula, EW is absorbance at 340nm in 1min to enzyme activity (U)=EWV/6.22
Variation；V is the volume (ml) of reaction solution；6.22 being molar extinction coefficient.
The content not being described in detail in this specification belongs to the prior art well known to those skilled in the art.
Claims (10)
1. a kind of bioconversion method of S-Leucine, which is characterized in that include the following steps：
1) leucine dehydrogenase, the hydrogenlyase of immobilization are prepared；
2) trimethyl sodium pyruvate solution is prepared by raw material of dichloro pinacolone；
3) using the trimethyl sodium pyruvate solution obtained by step 2) as substrate, with the immobilization obtained by coenzyme NAD+and step 1)
Leucine dehydrogenase, hydrogenlyase are biocatalyst, and addition ammonium salt establishes transformation system and carries out bioconversion reaction, obtains
Conversion fluid containing S-Leucine.
2. the bioconversion method of S-Leucine according to claim 1, which is characterized in that in the step 1), use
Carragheen or compound gel fix leucine dehydrogenase, hydrogenlyase, obtain leucine dehydrogenase, the formate dehydrogenase of immobilization
Enzyme.
3. the bioconversion method of S-Leucine according to claim 2, which is characterized in that the compound gel is by seaweed
The mixture of hydrochlorate and polyethylene glycol, the alginate are calcium alginate, potassium alginate, one kind in sodium alginate or several
Kind, the weight ratio of the alginate and polyethylene glycol is 1:4-9.
4. the bioconversion method of S-Leucine according to claim 3, which is characterized in that the alginate with it is poly-
The weight ratio of ethylene glycol is 1:4, the alginate is 3 by weight by calcium alginate, potassium alginate, sodium alginate:9:4 groups
At.
5. the bioconversion method of S-Leucine according to claim 2, which is characterized in that use carragheen or compound gel
Leucine dehydrogenase, hydrogenlyase are fixed, included the following steps：Carragheen or compound gel are heated in distilled water molten
Solution is added leucine dehydrogenase, hydrogenlyase, stirs evenly after cooling, then is cooled to its solidification to get the bright ammonia of immobilization
Acidohydrogenase, hydrogenlyase.
6. the bioconversion method of S-Leucine according to claim 5, which is characterized in that the carragheen or compounding
The weight ratio of glue and distilled water is 1:Carragheen or compound gel in distilled water are heated to 80-90 DEG C of dissolving, are cooled to by 6-10
It is added leucine dehydrogenase, hydrogenlyase after 35-45 DEG C, it is bright in the leucine dehydrogenase of the immobilization, hydrogenlyase
Propylhomoserin dehydrogenase, hydrogenlyase, carragheen mass ratio be 1:4-6:7-12, the leucine dehydrogenase of the immobilization, first
Acidohydrogenase is cut into 1 × 1cm3Cube it is spare.
7. the bioconversion method of S-Leucine according to claim 1, which is characterized in that in the step 3), institute
It is ammonium formate to state ammonium salt；The molar ratio control of ammonium formate and trimethyl Sodium Pyruvate is 1.8-2.2:1；The bright ammonia of the immobilization
The amount ratio of acidohydrogenase, hydrogenlyase and trimethyl Sodium Pyruvate is 8-12g:1mol, coenzyme NAD+and front three benzylacetone
The amount ratio of sour sodium is 0.2-0.4g:1mol, final concentration control of the trimethyl Sodium Pyruvate in transformation system is 2mol/L, is turned
The pH value control of change system is 5-10, and the temperature for controlling the transformation system is 20-60 DEG C；It is that 12-36 is small to control transformation time
When；The conversion reaction carries out in shaking table, and the rotating speed control of the shaking table is 150-250r/min.
8. the bioconversion method of S-Leucine according to claim 7, which is characterized in that ammonium formate and trimethyl third
The molar ratio control of ketone acid sodium is 2:1；The leucine dehydrogenase of the immobilization, hydrogenlyase and trimethyl Sodium Pyruvate
Amount ratio is 10g:The amount ratio of 1mol, coenzyme NAD+and trimethyl Sodium Pyruvate is 0.3g:1mol, trimethyl Sodium Pyruvate exist
Final concentration control in transformation system is 2mol/L, and the pH value control of transformation system is 6, and the temperature for controlling the transformation system is
55℃；It is 25 hours to control transformation time；The conversion reaction carries out in shaking table, and the rotating speed control of the shaking table is 200r/
min。
9. the bioconversion method of S-Leucine according to claim 1, which is characterized in that containing S-Leucine
Conversion fluid purified, include the following steps：Conversion fluid concentration containing S-Leucine is placed in 0-4 DEG C and places 8-
16h, crystallization, obtains S-Leucine coarse crystal；S-Leucine coarse crystal is rinsed with methanol, is filtered, is crystallized, obtain again
S-Leucine crystal.
10. the bioconversion method of S-Leucine according to claim 1, which is characterized in that containing the tertiary bright ammonia of L-
The conversion fluid of acid is purified, and is included the following steps：By the conversion fluid containing S-Leucine first with 732 type highly acidic cations
Exchanger resin adsorbs, then is adsorbed with 201 type strong-base anion-exchange resins, obtains S-Leucine crystal.
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