CN107190029A - Use the preparation of the D type lactic acid of novel enzyme - Google Patents
Use the preparation of the D type lactic acid of novel enzyme Download PDFInfo
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- CN107190029A CN107190029A CN201610144852.7A CN201610144852A CN107190029A CN 107190029 A CN107190029 A CN 107190029A CN 201610144852 A CN201610144852 A CN 201610144852A CN 107190029 A CN107190029 A CN 107190029A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/56—Lactic acid
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
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- C12Y—ENZYMES
- C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
- C12Y305/05—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in nitriles (3.5.5)
- C12Y305/05001—Nitrilase (3.5.5.1)
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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Abstract
The present invention relates to a kind of preparation method of lactic acid, in more detail, it is related to a kind of method that D type lactic acid is produced by biochemical method.The feature of the production method of the present invention is in acetaldehyde and cyanide salt is reacted the reaction to prepare lactic acid, them to be reacted using enzyme.By using the preparation method of the lactic acid of the enzyme of the present invention, the dominant lactic acid of D types isomers can be prepared by simple course of reaction under the reaction condition easily realized.Preparation method by using the lactic acid of the enzyme of the present invention carrys out adjusting reaction time, so as to adjust the D types and the ratio of L-type isomers of prepared lactic acid.By the repeated using method of the enzyme of the present invention, for this reaction, enzyme can be reused minimum more than 5 times, so as to prepare lactic acid with low cost.
Description
Technical field
The present invention relates to a kind of preparation method of lactic acid, in more detail, it is related to one kind and D types breast is produced by biochemical method
The method of acid.
Background technology
Lactic acid is widely used as the raw material of food, field of medicaments, and is used as the multiple lactic acid of biological plastics material
Monomer using [1) C.Akerberg, K.Hofvendahl, G.Zac chi, B.Hahn-Hagerdal,
Appl.Microbiol.Biotechnol.1998,49,682-690；2) K.Hofvendahl, B.Hahn-Hagerdal,
Enzyme Microb.Technol.2000,26,87-107.].Recently, as the demand to biological plastics is sharply increased, environmental protection
Plastics get most of the attention, wherein, multiple lactic acid with biological degradability and as de- petroleum high-molecular because especially being attracted attention.It is logical
Cross and use multiple lactic acid as raw material, the biodegradable plastics such as automobile, household electrical appliances, house and disposable plastic container can be prepared
Material, and the processability and mechanical strength of plastics can be adjusted according to the ratio of lactic acid isomers.With multiple lactic acid
The increase of usability, the synthesis aspect to the lactic acid as monomer is also deploying positive research.
Lactic acid can be prepared with biochemical method chemically.Representative chemical method is to pass through lactonitrile
Hydrolysis prepare lactic acid [1) R.Datta, S.P.Tsai, P, Bo nsignore, S.H.Moon, J.R.Frank, FEMS
Microbiol.Rev.1995,16,221-231；2) Y.J.Wee, J.N.Kim, H.W.Ryu, Food
Technolo.Biotechnol.2006,44,163-172].Pass through the addition of hydrogen cyanide and acetaldehyde in the presence of base catalyst
Reaction, in 10 minutes, lactonitrile is obtained with more than 99% conversion ratio to synthesize.The lactonitrile of synthesis completes separation by distilling
After purification process, lactic acid is converted into by using the hydrolysis of sulfuric acid catalyst.Now, although conversion ratio be 99% with
On, but the lactic acid of generation is racemic lactic acid, the racemic lactic acid is mixed with same amount of following two kinds of isomers (D types
Lactic acid and L-type lactic acid).
Another chemical method is to use the metal adsorption of iron or cobalt etc. in the catalyst on silica gel supporter, in acetaldehyde
And prepared in the presence of carbon monoxide, and water lactic acid method [S.K.Bhattacharyya, S.K.Palit, A.R.Das,
Ind.Eng.Chem.Prod.Res.Develo p.1970,1,92-95.].Using 13g catalyst, enter 5.5g acetaldehyde
After row reaction 3 hours, although generate lactic acid with 44% conversion ratio, but need in high-temperature and high-pressure conditions (230 DEG C, 350 gas
Pressure) under reacted, but also need to use expensive metallic catalyst as catalyst, therefore, deposited in economic aspect
In limitation.
Although chemical method has the advantages that reaction is simple, the time is shorter, but for the use of commerciality, in warp
It is restricted in terms of Ji, and the racemic breast for two isomers for being mixed with identical amount can only be prepared as product
Acid.
Biochemical method is the fermentation process of the carbohydrate of the microorganism by using bacterium or mould etc. to prepare
Lactic acid, wherein, the carbohydrate used is sucrose, lactose, starch and dextrin etc., very various.If by biochemistry side
Method prepares lactic acid, can respectively obtain pure D types lactic acid and L-type lactic acid according to the microorganism or carbohydrate used.[1)
Y.J.Wee, J.N.Kim, H.W.Ryu, Food Technol.Biotechnol.2006,44,163-172].
Generally, bacterial fermentation processes are mainly used when preparing lactic acid.In the prior art, disclose using by De Shi lactic acid
Bacillus LD 0025 (Lactobacillus delbrueckii) and LD0028 production amylase (α-amylase, β-
) and the hydrolysis of enzyme of amylopectase (pullulan ase) etc. sends out rice meal (rice powder) amylase
Ferment, thus produce the D type lactic acid of high-purity method [K.Fukushima, K.Sogo, S.Miura, Y.Kimur a,
Macromol.Biosci.2004,4,1021-1027].In addition, being also disclosed in L-type lactate dehydrogenase gene (L-lactate
Dehydrogenase gene) disappear Lactobacillus plantarum (L actobacillus plantarum) in import understand ox hammer
The plasmid of the α types amylase (α-amylase) of bacterium 148 (Streptococcus bov is 148), and optically come
Prepared from cornstarch pure D type lactic acid method [K.Okano, Q.Zhang, S.Shinkawa, S.Yoshida,
T.Tanaka, H.Fukuda, A.Kondo, Appl.Environ.Microbiol.2009,75,462-467].Also report virtuous
(the Lactobaci llus delbrueckii) bacterial strains of family name's Bacillus acidi lactici NBRC 3534 using the bagasse Jing Guo steam treatment as
Carbon source is used, thus prepare substantial amounts of D types lactic acid method [C.Sasaki, R.Okumura, A.Asakawa, C.Asada,
Y.Nakamura, J.Phys.Conf.Ser.2012,352-361].
L-type lactic acid can also prepare pure L-type lactic acid as D type lactic acid by using a variety of bacterial strains.Report has
Using blackstrap as substrate, the fermentation process by using enterococcus faecalis (Enterococ cus faecalis) has to prepare
Method [Y.J.Wee, J.N.Kim, J.S.Yun, H.W.Ryu, the Enzyme of the L-type lactic acid of high optical purity
Microb.Technol.2004,35,568-573.], also report has by using (the Streptococcus of bargen's streptococcus 148
Bovis 148) farinose fermentation process come prepare L-type lactic acid method [J.Narita, S.Nakahara, H.Fukuda,
A.Kondo, J.Biosci.Bioeng.2004,97,423-425.].In addition, propose to be not only to use bacterium, but also it is logical
Cross and use the Rhizopus oryzae (Rhizopus oryzae) in rhizopus (Rhizopus s pecies) as mould from glucose
The method for producing L-type lactic acid.Its effect that nutritional ingredient is brought to L-type production of lactic acid by inquiry, so that proposing is used for
Appropriate condition needed for a large amount of production L-type lactic acid.Also, the fibrous bioreactor of exploitation is also disclosed, and using in cotton
Piece upper fixed fungal hyphae come the method fermented, and by Rhizopus oryzae (Rhizopus oryzae) come from glucose and
In cornstarch produce L-type lactic acid method [1) Y.Zhou, J.M.Dominguez, N.Cao, J.Du, G.T.Tsao,
Appl.Biochem.Biotechnol.1999,78,401-407；.2) A.Tay, S.T.Yang,
Biotechnol.Bioeng.2002,80,1-12；3) Y.Kosakai, Y.S.Park, M.Okabe,
Biotechnol.Bioeng.1999,55,461-470].Recently, in order to reduce production cost, develop one kind and grasped by gene
The method that Pfansteihl is produced as bacterium, the genetic manipulation bacterium is as culture raw material using cheap glycerine
Bacterium.[S.Mazumdar, M.D.Blankschien, J.M.Clomburg, R.Gonzalez, Microb.C ell
Fact.2013,12,1-7].In addition, report has in the presence of lead ion (divalence) catalyst, exist at most in nature
Biomass-cellulose come synthesizing lactic acid method obtained a part of successfully content [Y.Wang, W.Deng, B.Wang,
Q.Zhang, X.Wan, Z.Tang, Y.Wang, C.Zhu, Z.Cao, G.Wang, H.Wan, Nature Commu n., 2013,7,
doi:10.1038/ncomms3141]。
Although biochemical method has the advantages that optically to prepare pure lactic acid, but course of reaction and change
Method compared to complex, and also exist the reaction time it is long the problem of.In addition, the bacterium or mould that use are anaerobic
When, because course of reaction is sensitive to oxygen, it is therefore desirable to note.Also, during the course of the reaction, due to the nutrient as bacterium
The yeast extract that must use or peptone it is expensive, therefore economic aspect exist limitation [A.W.Schepers,
J.Thibault, C.Lacroix, Enzyme Microb.Technol.2002,30,176-186.].
The content of the invention
The technical problem to be solved
The technical problem to be solved in the present invention is a kind of preparation section of D types lactic acid of offer, the reaction of the preparation section
Time is short, and reaction condition is simple, and reaction condition is easily realized, and lactic acid can be obtained with high yield, and can be obtained
The high lactic acid of optical purity.
The invention solves the problems that another technical problem for provide it is a kind of analyze D-ALPHA-Hydroxypropionic acid content method.
The invention solves the problems that another technical problem a kind of be used to prepare the enzyme of D type lactic acid to provide.
Technical scheme
In order to solve the above problems, feature of the invention is, in the reaction using acetaldehyde and cyanide salt to prepare lactic acid,
Them are reacted using enzyme.
In the present invention, the feature of the enzyme is, its can make acetaldehyde and cyanide salt reacted formed by lactonitrile
Itrile group is hydrolyzed, i.e. the enzyme utilized is the enzyme with itrile group hydrolysis ability.
Although not limiting in theory, the enzyme, which is played, makes acetaldehyde and cyanide salt be reacted and generated
The effect of the hydrolysis catalyst of hydrolysis occurs for lactonitrile, and the cyanide salt can be calcium, sodium or hydrogen.
In the present invention, the enzyme is the enzyme with itrile group hydrolysis ability, and passes through hydrolysis so that D-ALPHA-Hydroxypropionic acid
Growing amount is higher than Pfansteihl.
In the present invention, the enzyme cause the ratio of the D-ALPHA-Hydroxypropionic acid in whole lactic acid of production be preferably 60 moles of % with
On, more preferably 65 moles more than %, more preferably 70 moles more than %, most preferably 80 moles more than %.
In the present invention, the enzyme can be the enzyme from microorganism, plant or animal.Term enzyme not only represents enzyme sheet
Body, is also understood as that it includes cell of the enzyme through overexpression, the fragment of cell of the enzyme through overexpression or thus purifies and separates are obtained
Processed material on the enzyme arrived, and its science of heredity.Can be by recombinant DNA technology, in the place of Escherichia coli etc. as one
In master, the product on the science of heredity for producing enzyme is utilized.
In embodiments of the present invention, the enzyme can be centrifuged by the nutrient solution to thalline and obtain thin
After cell space, cell body is crushed, supernatant is then obtained by centrifuging, then it is freeze-dried and obtained.
In another embodiment of the present invention, the enzyme can be obtained by the following method：Utilize RP chromatography
Enzyme by purification is further purified, so that it is determined that the amino acid sequence of protein, and based on the amino acid sequence come
Probe is prepared, the DNA of the active main body of coding itrile group hydrolase is then cloned from the DNA fragmentation of thalline.Then, by poly-
Synthase chain reaction (PCR) is expanded to it carrys out Prepare restructuring plasmid, and is inserted in Escherichia coli, then to the Escherichia coli
Deng being cultivated, it is hereby achieved that required enzyme.
In the present invention, the enzyme is extracted from the YC6258 (KCCM 43015), and using the enzyme extracted, carried out as
The hydrolysis of lactonitrile (lactonitrile) shown in following reaction equations 1, wherein, the YC6258 is from halophytes rough leaf
The bacterial strain separated in sedge.In one day, the ratio of D type isomers high lactic acid can be prepared with 75% separation yield
([α]26=-1.14 (c=1.0 in water), reference value：L-type lactic acid, [α]21-22=+2. 60 (c=2.5 in water)).
In the present invention, from the molten bacillus of capsicum (Lysobacter capsici) YC5194T(KC TC 22007T=DSM
19286T)、Martelella endophyticaYC6887T(KCC M 43011T=NBRC 109149T) or Hoeflea
suaedaeYC6898T(KACC 14911T=NBRC 107700T) middle extraction other enzymes.
In embodiments of the present invention, reaction temperature can be 25 DEG C and 40 DEG C, it is preferable that be carried out at 25 DEG C anti-
Should.
In embodiments of the present invention, novel enzyme maintains high activity in pH6,7,8 times, it is preferable that be conducive to during pH8
The preparation of lactic acid.
In the present invention, there is the structure of chemical formula (1) as the D types lactic acid and L-type lactic acid of product, can be by this
Reaction method adjusts the ratio of D types lactic acid and L-type lactic acid, so as to prepare product.
In embodiments of the present invention, by using dialysis membrane, the enzyme may be reused more than 5 times.
On the one hand, the present invention provides a kind of analysis method, and the analysis method is by comprising D types lactic acid and L-type lactic acid
Lactic mixt be converted into benzyl lactate (benzyl lactate), and divided using high performance liquid chromatograph (HPLC)
Analysis.
In the present invention, the synthesis of the benzyl lactate is implemented according to such as following reaction equations (2).
In the present invention, D types benzyl lactate and L-type benzyl lactate have the structure shown in chemical formula (2), and by making
Calculate the area ratio of D types benzyl lactate and L-type benzyl lactate respectively with the high performance liquid chromatograph (HPLC) of chiral column, and by
This calculates the optical purity of prepared lactic acid.
Beneficial effect
By using the preparation method of the lactic acid of the enzyme of the present invention, letter can be passed through under the reaction condition easily realized
Single course of reaction is come to prepare D types isomers be dominant lactic acid.
The preparation method of lactic acid by using the enzyme of the present invention carrys out adjusting reaction time, prepared so as to adjust
The D types isomers of lactic acid and the ratio of L-type isomers.
By the repeated using method of the enzyme of the present invention, in the present reaction, enzyme can be reused minimum more than 5 times,
So as to prepare lactic acid with low cost.
Brief description of the drawings
Fig. 1 is to prepare the flow chart of lactic acid in the embodiment of the present invention 2.
Embodiment
The method that embodiment 1. prepares the enzyme liquid of YC6258 bacterial strains and other bacterial strains
Mass propgation with fluid nutrient medium (1g sucrose, 2g yeast extract, 5g casein, 4g MgSO4、
2g/L MgCl2) in, at 28 DEG C, under conditions of 120rpm, shaken cultivation novel strain YC6258 72 hours.With 8000rpm
Nutrient solution is centrifuged 20 minutes, so as to obtain cell body, then hanged with pH6.5 50mM phosphate buffers
It is floating.Suspension is placed in ice, and carries out ultrasonication in this condition, so that by after clasmatosis, under 12000rpm
It is centrifuged 30 minutes, so as to obtain supernatant.Freeze-drying process is carried out to supernatant, and used in each reaction.
Prepare YC5194T、YC6887T、YC6898TEnzyme liquid method with the above-mentioned method for preparing YC6258.Difference is, makes
Standby YC5194T、YC6887T、YC6898TWhen culture medium used respectively 50% soybean broth (Trytic soy broth),
M10broth (10g yeast extract, 4g MgSO4, 2g MgCl2, 5g NaCl, 1g K2HPO 4, 10g/L sucrose)
And sea gives birth to bacterial context soup (Marine broth) to be cultivated.
Embodiment 2. has used the preparation of the lactic acid of a variety of D types/L-type ratios of novel enzyme
2.0M cyaniding aqueous solutions of potassium (1.0 equivalents, 5 0mL, pH8) is added in 100mL round-bottomed flask, and is added new
After type enzyme YC6258 (200-500mg), acetaldehyde (100mmol) is added, and stir at normal temperatures.Add 1 centinormal 1 chlorination
Hydrogen solution (2.5 equivalent) and make solution be acidified after, continuously extracted with isopropyl ether after 24 hours, remove organic solvent, so as to obtain
Liquid product.[reaction equation 3] according to Fig. 1 is prepared for lactic acid.
In order to confirm the optical purity of the lactic acid prepared, the synthetic reaction of the benzyl lactate shown in [reaction equation 2] has been carried out.
Obtained lactic acid (1.0mmol) and methanol (0.5mL) are added in 4mL vial, and 1,8- diazabicyclos 11 are added dropwise
Carbon -7- alkene (1,8-diazabicycl oundec-7-ene；DBU) after (1.0 equivalent), stir 30 minutes at normal temperatures.Afterwards
Methanol is removed, and adds dimethylformamide (dimethylformamide；DMF) (0.5mL), benzyl chloride is then added dropwise, and (1 works as
Amount) after, stir 24 hours at normal temperatures.Organic solvent is removed, after then being extracted and concentrated with ethyl acetate, ethyl acetate is utilized
With hexane (1:4 volume ratios), and obtain by column chromatography liquid product (134mg, 74% yield).Use efficient liquid phase
Chromatograph (HPLC) determines the optical purity (analysis condition of the benzyl lactate of synthesis；Post：Chiralcel-OD, developing solvent：
Hexane/isopropyl alcohol=98/2, flow velocity=1.0mL/ minutes, UV=217nm, holdup time：16.2 minutes (L-type benzyl lactate),
17.5 minutes (D types benzyl lactate)).As a result as shown in table 1 below.
Table 1
Lactic acid conversion ratio in the present invention is by the lactonitrile generated in the reactant that is determined by nuclear magnetic resonance (NMR)
With the area of lactic acid than calculating what is obtained.Separation yield calculates obtain in the following manner.Determine by continuously extracting
Come the weight of the lactic acid obtained by after purifying, and counted according to the theoretic acquisition amount of the product of the amount of used acetaldehyde
Calculate.
The ratio of D types/L-type lactic acid of obtained lactic acid is calculated in the following manner to be obtained.Lactic acid is converted into breast
After acid benzyl ester, the area ratio of D types benzyl lactate and L-type benzyl lactate is determined using high performance liquid chromatograph, and by considering
Lactic acid conversion ratio obtains to calculate.The optical purity of lactic acid is by mathematical expression ((D type benzyl lactate areas ratio-L-type lactic acid benzyl
Ester area ratio)/(D type benzyl lactate areas ratio+L-type benzyl lactate area ratio) × 100) calculate what is obtained.
The optical purity of the lactic acid of generation is determined by changing the amount of novel enzyme used and reaction time.Its result
For lactic acid conversion ratio is lower, and the optical purity of the lactic acid of the generation shown is higher., can be with 41% point by this method
D type lactic acid of the highest with 84% optical purity is generated from yield, and can be with the separation yield next life of highest 75%
Into the D type lactic acid with 40% optical purity.
When using Cymag to replace potassium cyanide, under identical reaction conditions, also generated with 73% separation yield
There is the D-ALPHA-Hydroxypropionic acid of 39% optical purity.
Embodiment 3. has used the preparation of the lactic acid of various new enzyme
2.0M cyaniding aqueous solutions of potassium (1.0 equivalents, 50mL, pH8) is added in 100mL round-bottomed flask, and is added new
After type enzyme (500mg), acetaldehyde (100mmol) is added, and stir 3 hours at normal temperatures.Add 1 centinormal 1 hydrogen chloride molten
Liquid (2.5 equivalent) and make solution be acidified after, continuously extracted with isopropyl ether after 24 hours, remove organic solvent, so as to obtain liquid
Product.As a result as shown in table 2 below.
Table 2
The result reacted using the novel enzyme extracted from a variety of bacterial strains is that various new enzyme is to the anti-of this reaction
Answering property is similar.By carrying out the reaction of 3 hours, lactic acid, and the Plasma lactate to being generated are generated with 51-57% conversion ratio
The result of optical purity is to show 64-68%, and generates the dominant lactic acid of D types isomers on the whole.
Embodiment 4. has used the reuse of the novel enzyme of dialysis membrane
After novel enzyme (500mg) is dissolved in distilled water (5mL), molecular cut off (molecular is added it to
Weight cut-off) to be made in 10,000 dialysis membrane after " enzyme membrane ", in the beaker for putting into 100mL, and add 2.0M
Cyaniding aqueous solutions of potassium (1.0 equivalents, 45mL, pH8) and acetaldehyde (100mmol), and at normal temperatures stir 24 hours.1 is added to work as
Measure the hydrogen chloride solution (2.5 equivalent) of concentration and make after solution acidifying, continuously to be extracted with isopropyl ether after 24 hours, remove organic molten
Agent, so as to obtain liquid product.As a result as shown in Table 3 below.
Table 3
The result that enzyme membrane is prepared with novel enzyme and is reused is, it is thus identified that minimum reusable more than 5 times of enzyme.And
And confirm, the conversion ratio and yield of the lactic acid of reaction are also definitely maintained, and also maintain the light of generated lactic acid
Learn purity.
The present invention is not limited to above-mentioned specific embodiment, those skilled in the art without departing from
In the range of the purport of the present invention, the embodiment of various deformation can be carried out to the present invention.Therefore, the scope of the present invention is not limited to
In above-described embodiment, and not only determined by claims described later, and Ying Youyu claim guarantors
The equivalent content of shield scope is determined.
Claims (17)
1. a kind of make acetaldehyde and cyanide salt be reacted the method to prepare lactic acid, it is characterised in that makes acetaldehyde and cyanide salt
Reacted in the reaction to prepare lactic acid, them are reacted using enzyme.
2. according to the method described in claim 1, it is characterised in that the enzyme is the enzyme with itrile group hydrolysis ability.
3. according to the method described in claim 1, it is characterised in that in the lactic acid, the content of D type lactic acid is higher than L-type lactic acid
Content.
4. according to the method described in claim 1, it is characterised in that the cyanide salt is selected from cyanogas, Cymag and hydrogen cyanide
One or more of.
5. method according to any one of claim 1 to 4, it is characterised in that the enzyme source is in bacterial strain YC6258
(KCCM 43015), the molten bacillus of capsicum (Lysobacter capsici) YC5194T(KCTC 22007T=DSM 19286T)、
Martelella endophytica YC6887T(KCCM 43011T=NBRC 109149T) or Hoeflea suaedae
YC6898T(KACC 14911T=NBRC 107700T)。
6. method according to claim 5, it is characterised in that the enzyme is to carry out centrifugation point by the nutrient solution to thalline
From and obtain after cell body, cell body is crushed, supernatant is then obtained by centrifuging, freezing is then carried out to it dry
Obtained from dry.
7. method according to claim 1 or 2, it is characterised in that the enzyme is used with enzyme membrane form.
8. method according to claim 1 or 2, it is characterised in that the enzyme is the enzyme expressed by recombination.
9. method according to claim 1 or 2, it is characterised in that be further used as substrate comprising aldehyde.
10. method according to claim 1 or 2, it is characterised in that the reaction is under 25 DEG C or 40 DEG C of reaction temperature
Implement.
11. method according to claim 1 or 2, it is characterised in that the reaction is produced under the scope of pH6~8.
12. method according to claim 1 or 2, it is characterised in that the reaction is accomplished below at 24 hours.
13. a kind of enzyme, it is characterised in that it is to be reacted acetaldehyde and cyanide salt to prepare the enzyme of lactic acid, and the enzyme is included
From selected from YC6258 (KCCM 43015), the molten bacillus of capsicum (Lysobacter capsici) YC5194T(KCTC 22007T
=DSM 19286T)、Martelella endophytica YC6887T(KCCM 43011T=NBRC 109149T) or
Hoeflea suaedae YC6898T(KACC 14911T=NBRC 107700T) one or more of bacterial strain enzyme.
14. enzyme according to claim 13, it is characterised in that the enzyme is included, and the nutrient solution of thalline is centrifuged
And obtain after cell body, supernatant obtained from being centrifuged after cell body is crushed.
15. a kind of analysis method, it is characterised in that the polylactides comprising D types lactic acid and L-type lactic acid are converted into lactic acid benzyl
Ester, and analyzed using high performance liquid chromatograph.
16. analysis method according to claim 15, it is characterised in that the high performance liquid chromatograph is with chiral column
High performance liquid chromatograph.
17. analysis method according to claim 15, it is characterised in that the lactic mixt is to make acetaldehyde and cyanide salt
Mixture obtained from being reacted.
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| CN201610144852.7A Pending CN107190029A (en) | 2016-03-14 | 2016-03-14 | Use the preparation of the D type lactic acid of novel enzyme |
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| CN (1) | CN107190029A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0473328A2 (en) * | 1990-08-16 | 1992-03-04 | Nitto Chemical Industry Co., Ltd. | Biological process for preparing optically active lactic acid |
| CN101724592A (en) * | 2009-12-18 | 2010-06-09 | 南京第一农药集团有限公司 | Bacillus subtilis and application thereof in biocatalytic production of L-lactic acid |
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2016
- 2016-03-14 CN CN201610144852.7A patent/CN107190029A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0473328A2 (en) * | 1990-08-16 | 1992-03-04 | Nitto Chemical Industry Co., Ltd. | Biological process for preparing optically active lactic acid |
| CN101724592A (en) * | 2009-12-18 | 2010-06-09 | 南京第一农药集团有限公司 | Bacillus subtilis and application thereof in biocatalytic production of L-lactic acid |
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| Title |
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| 余剑霞: "气相色谱法测定白酒中的乳酸", 《酿酒科技》 * |
| 申世刚等: "生物酶催化氰化钠制备光学活性乳酸的研究", 《第十届全国催化学术会议》 * |
| 肖婧等: "腈水解酶的特性与应用进展研究", 《安徽农业科学》 * |
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