CN101671716A - Method for manufacturing D-phenylalanine by bio-enzyme asymmetric transformation - Google Patents
Method for manufacturing D-phenylalanine by bio-enzyme asymmetric transformation Download PDFInfo
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Abstract
The invention provides a biological manufacturing method of D-phenylalanine, and belongs to the enzyme engineering technology in the bioengineering field, aiming at solving the technical problem of providing a manufacturing method of D-phenylalanine by bio-enzyme asymmetric transformation which has a simple manufacturing process and can lower the manufacturing cost. The technical key points are asfollows: taking DL-phenylalanine as a raw material to take part in enzyme catalysis reaction by being used as a substrate; taking L-phenylalanine ammonialyase as a catalyst to perform catalytic conversion on L-phenylalanine in the zymolyte DL-phenylalanine to generate trans-cinnamic acid and ammonia; extracting D-phenylalanine as a target which does not take part in reaction; adding zymolyte continuously to improve concentration of the target product D-phenylalanine, forming crystal in enzymatic reaction solution and separating directly; and dissolving D-phenylalanine in the enzymatic reaction solution, regulating pH value to be 1-5, separating and removing cinnamic acid by using the water-insoluble characteristic of cinnamic acid in acid environment, concentrating and crystallizing to obtain the product. The refining purpose can be realized by membrane filtration decoloration and recrystallization of the D-phenylalanine coarse product. The product in the invention has high chemical purity and optical purity and low manufacturing cost.
Description
Technical field:
The invention belongs to the enzyme engineering technology in the bioengineering field, the present invention relates to a kind of is raw material with the DL-phenylalanine, utilize the L-phenylalanine ammonia lyase as catalyzer, asymmetric conversion L-phenylalanine deamination wherein generates trans-cinnamic acid and ammonia, and the D-phenylalanine does not participate in reaction fully, extracts as product.
Technical background:
The amino acids biochemical products are the essential substance that is related to human life's health, and are of many uses.The D-phenylalanine is developed fully in the purposes aspect the food medicine in recent years, especially at field of medicaments, many in the world companies with the D-phenylalanine be development of raw materials a series of antitumour drugs (hundred scholars are glad, Sostatin etc.), diabetes B medicine (nateglinide), anti-AIDS drug (Indinavir) or the like.D-phenylalanine worldwide demand is also risen sharply.
The method of the known D-of producing phenylalanine mainly contains " L-Aminoacylase method ", " asymmetric chemical transfer method ", " using hydantoinase ".Wherein, " L-Aminoacylase method " is to produce D type amino acid ordinary method at present, also is widely used on other D type amino acid of production, but because L-Aminoacylase costs an arm and a leg, can only be used for small-scale production." asymmetric chemical conversion " reaction mechanism is too complicated, and technical process is long, and needs expensive homochiral reagent as resolving agent, the production cost height.Suitability for industrialized production D-phenylalanine generally adopts " using hydantoinase " both at home and abroad at present, and this method needs the participation of two kinds of enzyme catalysts, and concentration of substrate is low, and transformation time is long, and transformation efficiency is low, and product purity is not high, and cost is very high.The method of the suitability for industrialized production D-of enterprise phenylalanine all has both at home and abroad at present, small scale, and productive rate is low, shortcomings such as cost height.Can't tackle the world wide demand growing to the D-phenylalanine.
Countries in the world are in the input of the research and development of chiral drug, to the increase of the demand of D-phenylalanine, press for a kind of suitable large-scale industrial production of research and development, the technology that can significantly reduce production costs.According to the asymmetric degraded or the transforming principle of biological enzyme, research and produce the method for chiral amino acid, be an important direction, the technological innovation of this project just is based on this principle.
Summary of the invention:
The problem to be solved in the present invention is: provide a kind of raw material to be easy to get, production process is simple, mild condition, the method that the asymmetric conversion of the D-phenylalanine biological enzyme that can reduce production costs is significantly produced.
Its technical scheme is: the asymmetric transformation production method of a kind of D-phenylalanine biological enzyme, and concrete steps and feature are as follows:
1. contain the making of enzyme cell suspension:
Bottle is shaken in employing or the fermentor tank ventilation is cultivated.Shaking bottle speed of rotation is 40-200 rev/min, 1000 milliliters of Erlenmeyer flask liquid amount 100-400 milliliters, incubation time 12～48 hours, 25 ℃-40 ℃ of culture temperature.Fermentation culture ventilating ratio 1: 0.05V-1: 0.4V (liquid volume: per minute bubbling air volume).
Substratum can use common carbohydrate as carbon source, as glucose, sucrose, starch, dextrin, wort, molasses etc.; Can be used alone or as a mixture various animals and plants peptones, soya-bean cake hydrolyzed solution, ammonium sulfate, yeast extract, corn steep liquor etc. as nitrogenous source; Inorganic salt can be used alone or as a mixture plurality of inorganic salt such as sodium-chlor, sal epsom, potassium primary phosphate, dipotassium hydrogen phosphate; Inductor is the L-phenylalanine that is used alone or as a mixture, DL-phenylalanine, L-tyrosine; Enzyme stabilizers is polyoxyethylene glycol, sorbyl alcohol, glutaraldehyde, glycerine, the L-glutamic acid that is used alone or as a mixture.Add soybean oil, various defoamer in addition in the substratum, avoid sterilizing or culturing process in the foaming phenomenon.PH value is regulated to 4.0-7.5 with acid or alkali in substratum preparation back.Then with 110-121 ℃ of sterilization of steam 10-30 minute.
Wherein carbon source concentration is 0-100g/L; Nitrogen concentration is 0.5-50g/L, and the concentration of inorganic salt is 0.1-10.0g/L.Inductor concentration is 0-1.0g/L, and the concentration of enzyme stabilizers is 0-1.0g/L.
What the present invention used is to produce L-phenylalanine ammonia lyase microorganism, mainly is Rhodotorula, and as rhodothece rubra, rhodotorula glutinis etc. also comprise streptomyces, Cladosporium, Aspergillus etc.By cultivating the microbial cell suspension that these microorganisms obtain containing enzyme, can directly add substrate reactions, also microorganism cells or the independent separation and Extraction of L-phenylalanine ammonia lyase can be come out use.Also separation can be obtained microorganism cells uses with mode immobilizations such as polymeric gel embeddings.
2. the asymmetric conversion reaction of enzyme
Cultured cell suspension (fermented liquid) is kept 25 ℃-55 ℃ of certain temperature, add substrate DL-phenylalanine in liquid, the ratio of two kinds of isomer of DL-phenylalanine of adding is any.The specific catalyzed conversion L-of L-phenylalanine ammonia lyase phenylalanine deamination in the reaction system generates trans-cinnamic acid and ammonia.D-phenylalanine is not wherein participated in reaction fully.Reaction process detects trans-cinnamic acid concentration, and the detection reaction system pH along with the carrying out reacting liquid pH value and can increase of reaction, can be kept optimum pH value at 4.0～10.0. by adding the pH value of sulfuric acid control reaction system
3. product extraction separation
Reaction process D-phenyl-alanine concentration increases, and runs up to a certain degree, and the D-phenylalanine crystallizes out from reaction system, just can obtain crude product by centrifugation.
Separate dissolved can be utilized styracin water-fast characteristic under sour environment by adding sour regulatory enzyme liquid pH 1.0 to 5.0 in enzyme liquid at the D-of reaction system phenylalanine, and precipitation is removed trans-cinnamic acid, and condensing crystal extracts then.
4.D-phenylalanine is refining
In distilled water, with the method decolouring of membrane filtration, the method for recrystallization is refining with dissolving crude product.Destainer reduces moisture with the method for vacuum concentration, thereby the D-phenylalanine is crystallized out.Centrifugal or suction filtration obtains finished product.
Its technique effect is: utilize the L-phenylalanine ammonia lyase, the L-phenylalanine in the asymmetric ground catalyzed conversion DL-phenylalanine generates trans-cinnamic acid and ammonia, and the D-phenylalanine is not participated in reaction fully.Be extracted out as target product.Optical purity of products up to 99.8%, chemical purity is up to 99.7%, production process is simple, mild condition, production cost is low.
Embodiment:
Below, the present invention will be described with embodiment, but the present invention is not subjected to the restriction of these embodiment.The raw material that uses among the embodiment is that specific rotation is zero DL-phenylalanine
Embodiment 1:
Prepare the following substratum 1000ml of each constituent mass mark.
Glucose 0.5%, peptone 1%, yeast extract paste 1%, NaCl0.5%, K
2HPO
40.1%, L-tyrosine 0.1%, bubble enemy 0.01%, DL-phenylalanine 0.1%, L-glutamic acid 0.1%.
With sulphur acid for adjusting pH value to 5.5.
This 1000ml substratum branch is packed in the 1000ml Erlenmeyer flask into every bottled liquid measure 250ml.With 121 ℃ of autoclavings of above-mentioned substratum 15 minutes.Be cooled to 30 ℃, will be kept on the inclined-plane rhodotorula glutinis and go in the above-mentioned substratum with ф 1mm transfering loop picking one articulating.Use gyrate shaker, under 30 ℃, 110rpm, cultivated 24 hours.
Merge 1 liter of above-mentioned cell culture fluid, regulate pH to 8.5, add DL-phenylalanine-3,4-quinone 0g, 45 ℃ of controlled temperature react and begin stream after 5～10 hours and add the DL-phenylalanine, reaction was carried out 40 hours continuously, accumulative total adds DL-phenylalanine 250g, stops to feed in raw material, and continues reaction 1 hour, be cooled to 15 ℃, the D-phenylalanine crude product 90g that goes out with the method for pumping filtration fractional crystallization.Add sulfuric acid in the suction filtration liquid, regulate its pH to 2, the styracin precipitation is separated out, and suction filtration is removed the styracin precipitation once more, and the suction filtration liquid vacuum concentration that obtains obtains D-phenylalanine 25g, merges crude product 115g dissolving crude product in 4L distilled water, membrane filtration and decolorization, suction filtration.Use the rotary film evaporator Concentrated and crystallized in vacuum, get D-phenylalanine highly finished product 100g.Optical purity of products 99.5%, chemical purity 99.6%.
Embodiment 2:
Prepare the following substratum 1000ml of each constituent mass mark.
Glucose 0.5%, peptone 1%, yeast extract paste 1%, NaCl0.5%, K
2HPO
40.1%, L-tyrosine 0.1%, bubble enemy 0.01%, DL-phenylalanine 0.1%, L-glutamic acid 0.1%.
With sulphur acid for adjusting pH value to 5.5.
This 1000ml substratum branch is packed in the 1000ml Erlenmeyer flask into every bottled liquid measure 250ml.With 121 ℃ of autoclavings of above-mentioned substratum 15 minutes.Be cooled to 30 ℃, will be kept on the inclined-plane rhodotorula glutinis and go in the above-mentioned substratum with ф 1mm transfering loop picking one articulating.Use the rotation oscillator, under 30 ℃, 110rpm, cultivated 24 hours.
Centrifugal above-mentioned cell culture fluid is got the centrifugal gained thalline of 6g and is added the DL-phenylalanine solution 200ml (concentration 3%) for preparing in advance, 45 ℃, 110rpm, sustained reaction 10 hours.Add sulfuric acid and regulate pH to 2 in this reaction solution, styracin is separated out, and suction filtration is removed styracin, and gained suction filtration liquid vacuum concentration obtains D-phenylalanine crude product 2.5g.The 2.5g dissolving crude product in 100ml distilled water, is used membrane filtration, the refining D-phenylalanine highly finished product 2.0g that obtains of crystalline method.Optical purity of products 99.8%, chemical purity 99.7%.
Embodiment 3:
Preparation contains peptone 20g/L, yeast extract paste 20g/L, glucose 10g/L, sodium-chlor 5g/L, Dried Corn Steep Liquor Powder 18g/L, sal epsom 0.2g/L, 150 milliliters of the one-level substratum of potassium primary phosphate 1g/L, adjust pH value to 5.5 with ammoniacal liquor or sulfuric acid, divide in the Erlenmeyer flask of the 500ml that packs into every bottled liquid measure 150ml.With 121 ℃ of autoclavings of above-mentioned substratum 15 minutes.Be cooled to 30 ℃, choose an articulating with ф 1mm transfering loop and go in the above-mentioned substratum being kept at rhodotorula glutinis on the inclined-plane.Use the constant temperature shaking table, at 30 ℃, 110rpm cultivated 18 hours down, obtained 400 milliliters of first order seed cell suspensions.
Preparation contains soya-bean cake hydrolyzed solution 20g/L (giving money as a gift), malt meal 20g/L, and glucose 5g/L, sodium-chlor 5g/L, Dried Corn Steep Liquor Powder 5g/L, sal epsom 02g/L, 10 liters of the fermented liquids of potassium primary phosphate 1g/L, L-phenylalanine 5g/L are regulated pH value to 5.5.Above-mentioned substratum is added in the fermentor tank of 10 liters of useful volumes, 115 ℃ autoclaving 15-20 minute.Be cooled to 30 ℃, insert 400 milliliters of above-mentioned first order seed cell suspensions.Per minute feeds 2 liters of sterile airs, cultivates 24 hours.The centrifugation thalline is standby.
(1) with the embodiment of free cell reaction: get wet thallus 3kg, add the enzyme reaction jar, compound concentration 3% in advance in the enzyme reaction jar, the substrate solution 100L of the DL-phenylalanine of pH9.5.45 ℃ of sustained reactions 10 hours.Add sulfuric acid, conditioned reaction liquid pH to 2.0 removes by filter styracin.Membrane filtration and decolorization, vacuum concentration enzyme reaction solution obtain D-phenylalanine 1150g, mother liquor 20L.Optical purity of products 998%, chemical purity 99.7%.
(2) embodiment of the cell response of immobilized thallus: get wet thallus 3kg, use carrageenin 0.5kg, in 45 ℃ of mixing of temperature, cooling preparation immobilized cell.With the immobilized cell synthetic glass immobilized cell reaction column of packing into.Compound concentration 3%, the DL-phenylalanine solution 100L of pH9.5,40 ℃ of controlled temperature feed reaction column with DL-phenylalanine solution from bottom cycle, and the control flow velocity makes L-phenylalanine transformation efficiency transformation efficiency be not less than 98.5%.Collect effluent liquid 95L, reaction finishes with 3 washing reaction posts of 10L distillation moisture.Merge and obtain reaction solution 105L.With sulphur acid for adjusting pH value to 2.0, remove by filter styracin, by membrane filtration and decolorization, Concentrated and crystallized in vacuum obtains D-phenylalanine 1200g, mother liquor 20L.Optical purity of products 99.8%, chemical purity 99.7%.
Claims (6)
1. the asymmetric conversion of the biological enzyme of the D-phenylalanine method of producing is characterized in that:
1) the D-phenylalanine production method that the present invention relates to is to be raw material with the DL phenylalanine, utilize the L-phenylalanine ammonia lyase as catalyzer, asymmetric conversion L-phenylalanine wherein, make its deamination generate trans-cinnamic acid and ammonia, and the D-phenylalanine does not participate in reaction fully, extracts as product.
2) making of the used enzyme reaction solution of the asymmetric conversion reaction of biological enzyme
Carbon source, nitrogenous source, inorganic salt, water and inductor and enzyme stabilizers are prepared substratum in proportion, its pH value is adjusted to 4.0-8.0 with acid or alkali; Insert the microorganism that produces the L-phenylalanine ammonia lyase then, ventilate with gyrate shaker or fermentor tank and cultivated 12-48 hour, culture temperature is 25 ℃-40 ℃.
3) the asymmetric conversion reaction of biological enzyme.
Continuous current adding substrate DL-phenylalanine in cultured cell suspension liquid, make the specific catalyzed conversion L-of the L-phenylalanine ammonia lyase phenylalanine in the reaction system, deamination generates trans-cinnamic acid and ammonia, by strengthening the pH value that means such as ventilation or adding acid reduce solution, make the pH value of solution maintain 4.0-10.0,25 ℃-55 ℃ of temperature of reaction.
4) product extraction separation
Utilizing the low characteristic of phenylalanine solubleness in water, by constantly add the DL-phenylalanine in transformation system, increase the concentration of D-phenylalanine, is that the D-phenylalanine crystallizes out from reaction system, obtains crude product by centrifugation
Reaction finishes to be dissolved in the D-phenylalanine in the enzyme liquid, by adding sour regulatory enzyme liquid pH 1.0 to 5.0 in enzyme liquid, utilizes styracin water-fast characteristic under sour environment, and precipitation is removed trans-cinnamic acid, and condensing crystal extracts then.
5) the D-phenylalanine is refining
Dissolving crude product is in distilled water, and with the method decolouring of membrane filtration, condensing crystal promptly obtains D-phenylalanine finished product then.
2. the method that the asymmetric conversion of the biological enzyme of a kind of D-phenylalanine according to claim 1 is produced is characterized in that carbon source in the described substratum is glucose, sucrose, starch, dextrin, wort and the molasses in the carbohydrate that is used alone or as a mixture; Nitrogenous source is various animal/vegetable protein peptones, soya-bean cake hydrolyzed solution, ammonium sulfate, yeast extract and the corn steep liquor that is used alone or as a mixture; Inorganic salt are sodium-chlor, sal epsom, potassium primary phosphate, the dipotassium hydrogen phosphate that is used alone or as a mixture; Described inductor is the L-phenylalanine that is used alone or as a mixture, DL-phenylalanine, L-tyrosine; Enzyme stabilizers is polyoxyethylene glycol, sorbyl alcohol, glutaraldehyde, glycerine, the L-glutamic acid that is used alone or as a mixture.
3. according to claim 1 and the asymmetric transformation production method of 2 described a kind of D-phenylalanine biological enzymes, it is characterized in that: described carbon source concentration is 0-100g/L; The concentration of nitrogenous source is 0.5-50g/L; The concentration of inorganic salt is 0.1-10.0g/L, and inductor concentration is 0-10.0g/L, and enzyme stabilizers concentration is 0-10.0g/L.
4. the method that the asymmetric conversion of the biological enzyme of a kind of D-phenylalanine according to claim 1 is produced is characterized in that: the microorganism that produces the L-phenylalanine ammonia lyase is a Rhodotorula, as rhodothece rubra, rhodotorula glutinis etc. also comprise streptomyces, Cladosporium, Aspergillus etc.
5. the method that the asymmetric conversion of the biological enzyme of a kind of D-phenylalanine according to claim 1 is produced, it is characterized in that: the ratio as two kinds of optically active isomers in the raw materials for production DL-phenylalanine can be arbitrarily.
6. the method that the asymmetric conversion of the biological enzyme of a kind of D-phenylalanine according to claim 1 is produced, it is characterized in that: can directly add substrate DL-phenylalanine in the cultured microorganism cells suspension, also can isolate microorganism cells in the cell culture fluid, react with substrate solution mixing manufacture reaction system by centrifugal means.Equally also can from microorganism cells, extract the L-phenylalanine ammonia lyase, participate in reaction with the form of resolvase.The microorganism cells that also separation can be obtained uses with method immobilizations such as polymeric gel embeddings.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102234241A (en) * | 2010-05-07 | 2011-11-09 | 北京金源化学集团有限公司 | Chemical synthesis method of chiral D-phenylalanine |
| CN104140987A (en) * | 2013-05-07 | 2014-11-12 | 南京中医药大学 | A constant-pH feeding fermentation method for production of high-purity D-alanine |
| CN106434827A (en) * | 2016-11-26 | 2017-02-22 | 熊廷珍 | Production of phenylalanine through co-fermentation of immobilized mixed bacteria |
-
2009
- 2009-10-15 CN CN200910184831A patent/CN101671716A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102234241A (en) * | 2010-05-07 | 2011-11-09 | 北京金源化学集团有限公司 | Chemical synthesis method of chiral D-phenylalanine |
| CN104140987A (en) * | 2013-05-07 | 2014-11-12 | 南京中医药大学 | A constant-pH feeding fermentation method for production of high-purity D-alanine |
| CN106434827A (en) * | 2016-11-26 | 2017-02-22 | 熊廷珍 | Production of phenylalanine through co-fermentation of immobilized mixed bacteria |
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Application publication date: 20100317 |