CN1313402A - Process for preparing D-lactone valerate by microbe enzyme method - Google Patents
Process for preparing D-lactone valerate by microbe enzyme method Download PDFInfo
- Publication number
- CN1313402A CN1313402A CN 01104070 CN01104070A CN1313402A CN 1313402 A CN1313402 A CN 1313402A CN 01104070 CN01104070 CN 01104070 CN 01104070 A CN01104070 A CN 01104070A CN 1313402 A CN1313402 A CN 1313402A
- Authority
- CN
- China
- Prior art keywords
- enzyme
- lactone
- pantoyl lactone
- substrate
- pantoyl
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Links
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
A process for preparing D-lactone valerate by microbe enzyme method features that the microbe strain Fusariummoniliforme SW-920 (CGMCC No.0536), which can generate D-lactone valerate hydrolase with high output, it used to ferment in optimized condition for generating enzyme. The fermented liquid has enzyme activity of 3-6V/L and the enzyme preparation of 1.5 V/g (dried). The transfer rate is 25-45% for enzyme and 70-90% for substrate. The output rate of product is 86.07%.
Description
The present invention relates to make substrate with chemosynthesis DL-pantoyl lactone, with the D-pantoic acid lactone hydrolase of microorganism strains beading Fusarium Fusarium moniliforme.SW-902 (being CGMCCNo.0536) fermentative production, carry out enzymic hydrolysis and split the method for making the D-pantoyl lactone.
D-pantoyl lactone (pantoyl laetone) is the important intermediate of producing pantothenic acid, panthenol and pantetheine, and it and Beta-alanine reaction promptly obtain pantothenic acid.Pantothenic acid, Pantothenic acid claims pantothenic acid, vitamins B again
5, or B
3, be a kind of important medicine, foodstuff additive and fodder additives.Its product form is generally its dextrorotatory form calcium salt D-calcium pantothenate.Pantothenic acid is the vitamin B complex material; it is the integral part of coenzyme A; participate in that body fat acid degradation, lipid acid are synthetic, the synthetic metabolism such as (promoting resistibility) of tricarboxylic acid cycle, choline acetylize (a kind of nerve impulse transmitter substance), antibody to pathogenic agent; because the physiological function of coenzyme A, pantothenic acid have absorption and a utilization that is beneficial to various nutritive substances.Pantothenic acid is used for the treatment of some diseases clinically, is used from extra-nutrition with other vitamin B group one, is used for Vitamin B deficiency disease, peripheral neuropathy, post operative ileus, streptomycin poisoning and similar rheumatism etc.Usually its calcium salt and other vitamin B group one are used from extra-nutrition.
Calcium pantothenate still is a kind of important fodder additives, the existing fodder industry that is used in a large number.Calcium pantothenate has important effect as vitamin feed additive in livestock and poultry cultivation, can make livestock and poultry the disorder of aspect physiological functions such as various metabolism, nerve, stomach occur when under-supply.Legal use D-of many countries or DL-calcium pantothenate, China only ratifies to use the D-type.It is the D-form with higher optical purity that the intermediate pantoyl lactone of production D-pantothenic acid then requires.
D-panthenol (pro-vitamin B5) is important makeup and medicinal raw material, and market is in short supply.The production of D-panthenol can not be adopted calcium salt induced crystallization method, and biological Split Method prepares the D-pantoyl lactone and more is of practical significance.
The preparation method of D-pantoyl lactone has following several:
1, chemical process splits, and as splitting pantoyl lactone with resolving agents such as quinine, brucine or Chiral Amine, the shortcoming of chemical resolution method is that resolving agent is too expensive, the cost height, and separation difficulty also has environmental pollution and toxicity problem;
2, physical method splits, and produces the D-calcium pantothenate as directly split the DL-calcium pantothenate with the induced crystallization method, and condition control requires high, and yield is low, and optical purity is poor, and the cost height can only be used for the production of D-calcium pantothenate, and is not suitable for the production of other derivatives such as D-panthenol;
3, biological process splits and mainly contains enzyme process, microbial method.The research of having reported has the multiple technologies route.As:
1. with thoroughly degrade L-pantoyl lactone in the DL-pantoyl lactone of microorganism, obtain undecomposed D-form pantoyl lactone.(JPA?47-19745)。The shortcoming of this method is to lose half DL-pantoyl lactone raw material at least;
2. become the ketone group pantoyl lactone with the L-configuration pantoyl lactone in the microbiological oxidation DL-pantoyl lactone, asymmetric reduction is converted into the D-pantoyl lactone again.(JPA?61-293386、JPA?47-19745、JPB?61-14797)。Because substrate concn is low, speed of reaction is low, does not almost have practical significance;
3. use the L-pantoyl lactone in the microbial selective ground asymmetric hydrolysis DL-pantoyl lactone, and obtain unhydrolysed D-pantoyl lactone.(JPA?57-152895)。Also because substrate concn is low, speed of reaction is low, does not almost have practical significance, and the interior esterolytic D-pantoyl lactone that very thoroughly just can obtain high-optical-purity that has only the L-configuration;
4. use the D-pantoyl lactone in the microbial selective ground asymmetric hydrolysis DL-pantoyl lactone, obtain the D-pantoic acid, with the D-pantoic acid of the high-optical-purity generation D-pantoyl lactone that lactonizes, (USPatent 5,275, and 949,1994; US Patent 5,372,940,1994), this method is the United States Patent (USP) of Japanese FuJi company (the invention people is the Sakamoto Keiji of Kyoto Univ Japan etc.) application, is used for industrial production in Japanese first drugmaker.But the shortcoming of this method is that the bacterial enzyme cultivation and fermentation time is long, needs 7 days, and the thalline of cultivation only carries out enzymatic conversion reaction one time, and the thalline utilization ratio is low, enzyme cost height.
The report that also has some other enzyme process, microbial method to split, as: lipase or acyl ammonia enzymic hydrolysis pantothenate or pantothenic acid acyl ammonia (JP-A 1-228487, JP-A 1-228488), with microorganism D-pantoic acid in the DL-pantoic acid and Beta-alanine are optionally synthesized the asymmetric cyanohydrination route (Adam that generates D-pantothenic acid (JP-A 5-23191), uses the aldehyde alcohol of cyanohydrin lytic enzyme (oxynitrilase), G., and Seebach, D., Synthesis, 373,1988) etc.All low, the low Practical significance that lacks of product optical purity owing to yield.
In addition, 1996, Japan Takede Chemical Industries Ltd has applied for the patent (application number: 96194779 of " producing the method for D-calcium pantothenate " in China, notification number: 1187854), Japan and United States Patent (USP) (JP-A 6-261772, U S Patent 5,932 are arranged again recently successively, 457,1999; US Patent 6,013,492,2000), by microbial fermentation processes direct production D-pantothenic acid.They have screened and can generate the D-pantoic acid by direct fermentation glucose, add Beta-alanine in fermented liquid, just can directly obtain D-pantothenic acid.But, be difficult to use from methods such as friendship, crystallizations and remove because fermented liquid component complexity particularly has about 10% monose and oligosaccharides, concentrate, during crystallization because sticking, operational difficulty, solvent consumption is big, product colour is dark, and extract yield is low, and scale production amplification is had any problem.
The present invention adopts microorganism asymmetric hydrolysis DL-pantoyl lactone method to obtain the D-pantoic acid, generates the method for D-pantoyl lactone again through lactonization reaction.Obtain not hydrolysis D-pantoyl lactone method with asymmetric hydrolysis L-pantoyl lactone and compare, meet very much the practicality requirement.
If with the hydrolysis of L-stereospecificity lactone hydrolase, the L-pantoyl lactone is hydrolyzed to the L-pantoic acid, can directly obtains unhydrolysed D-pantoyl lactone.But owing to require enzymic hydrolysis thoroughly can guarantee the optical purity of product.Like this, very high to enzyme and enzymatic hydrolysis condition requirement thereof, the reaction times is also long, also may bring chemical hydrolysis to cause other impurity because of long-time reaction.
The present invention does not require that with D-stereospecificity lactone hydrolase hydrolysis D-pantoyl lactone hydrolysis is thorough, and the reaction times is short, and hydrolyzable moiety can not use repeatedly through racemization.The stereospecificity of the microbial enzyme that obtains that screens good (i.e. not hydrolysis L-pantoyl lactone) can obtain the very D-pantoyl lactone of high-optical-purity.
The microorganism strains that produces the D-pantoic acid lactone hydrolase mainly contains Fusarium Fusarium, gibberella Gibberella, and Gliocladium Gliocladium, aspergillus niger belongs to Aspergillus, and Cylindrocarpon.Volutella etc.The present invention's screening and mutagenesis have obtained energy high yield stereospecificity D-pantoic acid lactone hydrolase and have not utilized the microorganism strains beading Fusarium Fusariummoniliforme.SW-902 of do not degrade pantoyl lactone or pantoic acid.
The operational path that the present invention adopts (contrasts document: USPatent 5,275,949,1994 with the United States Patent (USP) of Japanese FuJi company application; US Patent 5,372,940,1994) identical, but used bacterial strain is beading fusarium (it is mould that rice the is disliked seedling) Fusarium.moniliforme (SW-902) in the Fusarium, belong to red mould group [Lisola], this bacterium can be fermented and be produced plant growth hormone gibberellin, does not cause withered corruption of plant or the listless disease of withering plant excessive growth such as (but easily cause) paddy rice.And the contrast document is used is fusarium Fusariumoxysporum in the Fusarium, belong to beautiful fusarium group [Elegans], this bacterial classification can cause all generally that leaf is withered, root-rot or the listless disease of withering, be a kind of plant pathogen, can produce harm (the contrast document is undeclared) to farm crop after its discharging, the diffusion.The bacterial classification that the present invention screened is the industrial production bacterial classification that fermentation produces plant growth hormone gibberellin, and the products production that it is used for this invention does not then have these misgivings.The level that the present invention reaches at present is: produce 2～3 days enzymic fermentation time; Enzymic disposable transformation efficiency 25～45%; To substrate total conversion rate 7～90%; The enzymatic conversion time short (5～30 hours), enzyme can be recycled repeatedly (more than 6 times); After carrying out immobilization with carrageenin, enzymatic conversion can reach more than 10 times in batches repeatedly; To the product extract yield more than 60%; The main quality index specific rotation of D-pantoyl lactone [α]
D 20Be-48～51 °, optical purity is 99 (%e.e).Part index number slightly is better than contrasting level (5～7 days enzymic fermentation time of product that document is reported; Enzymic disposable transformation efficiency 25～30%; 2～3 days enzymatic conversion time, enzyme utilizes 1 time; The main quality index specific rotation of D-pantoyl lactone [α]
D 20Be-45.6 °, optical purity is 91～98%e.e).
Purpose of the present invention provides a kind of microorganism of high-yield D-pantolactone hydrolysis new bacterial strain, provides a kind of new microbial enzyme method to split the method for preparing the D-pantoyl lactone.The optical purity of the D-pantoyl lactone that the D-pantoic acid extracts after lactonizing in the resulting enzymic hydrolysate reaches more than the 99%e.e., satisfies the specification of quality that D-calcium pantothenate and D-panthenol are produced intermediate.
The present invention realizes by following scheme:
(1) the microorganism strains beading Fusarium Fusarium moniliforme.SW-902-CGMCCNo.0536 (this bacterial strain has been kept at Zhong Guan-cun, BeiJing, China China Committee for Culture Collection of Microorganisms common micro-organisms preservation center February 12 calendar year 2001) of the energy high-yield D-pantolactone hydrolytic enzyme of seed selection of inventor laboratory and preservation is as bacterium producing multi enzyme preparation;
(2) bacterial strain is through slant activation, and enzymatic production is a zymin with its wet thallus or its immobilized cell, as the enzyme source;
(3) with the isobutyric aldehyde be raw material, adopt formaldehyde method, the sodium cyanide route is synthetic, produces the DL-pantoyl lactone, and refining back is as the enzymatic conversion substrate;
(4) zymin with (2) adds in the substrate of (3), the D-pantoic acid that enzymatic conversion generates, and through lactonizing, the D-pantoyl lactone is made in solvent extraction, decolouring, concentrated, crystallization.
Bacterial classification and mutagenesis thereof:
From fermentation industry is produced, collect and the microorganism strains of inventor laboratory preservation in, through primary dcreening operation, multiple sieve and separation and purification, obtain a strain and produce the Fusarium Fusariumsp.SW-9 of D-pantoic acid lactone hydrolase, this strain enzyme-producing is stable, stereoselectivity is good, does the enzyme source, the D-pantoic acid that hydrolysis D-pantoyl lactone obtains at the mycelium that produces shake flask fermentation on the enzyme substratum, after lactonizing, extract the D-pantoyl lactone optical purity height that obtains, [α]
D 20=-47 °.Present method with this bacterium as bacterium producing multi enzyme preparation.
Selecting Fusarium sp.SW-9 is bacterial strain, carries out ultraviolet ray, C according to a conventional method
o-60 mutagenic treatment.Uviolizing is 15W, apart from 27cm, and 2～3 minutes time: C
o-60 irradiations are dosage 2～80,000 roentgens, and the time is 20～30 minutes.Treated mycelium culture transferring is on the minimum medium and perfect medium that contain DL-pantoyl lactone and DL-pantoic acid, cultivated 3～7 days for 20～40 ℃, detect at the minimum medium that contains DL-pantoyl lactone and DL-pantoic acid not long, the bacterium colony of on perfect medium, growing.Meanwhile, the mycelium culture transferring of above-mentioned mutagenesis and screening on the screening culture medium that contains D-pantoyl lactone and indicator, was cultivated 3～7 days, and detected the big growth bacterium colony of variable color circle for 20～40 ℃.
The minimum medium that contains DL-pantoyl lactone and DL-pantoic acid comprises Na
2SO
4, Na
2HPO
4, KH
2PO
4, MgSO
4, CaCl
2, VITAMIN, trace element solution and all components of agar.
Be called Fusarium sp.SW-902 through mutagenic obtained bacterial strain, i.e. CGMCC No.0536.This mutagenic fungi is compared with parental plant, the mode of appearance unanimity, but on the minimum medium that contains DL-pantoyl lactone and DL-pantoic acid, do not grow, do not decompose DL-pantoyl lactone and DL-pantoic acid, and its product enzymic fermentation time shortened to 2～3 days from 7 days, produce enzyme activity and improved 25%, the required time of enzymatic conversion also shortened to 5～6 hours from 24～48 hours.This bacterial strain is kept at Zhong Guan-cun, BeiJing, China China Committee for Culture Collection of Microorganisms common micro-organisms preservation center with preservation CGMCC No.0536.
Bacterial classification preliminary evaluation result:
Appearance, the growthhabit of this bacterium, the internal structure of having observed it have been observed.On potato or rice medium, produce a large amount of gas mycelia and be flocculence, mycelia chromogenesis not in substratum is not seen ascus, sexual spore is not quite clear.Do not see the large-scale spore of sickleshaped.Thalline is a filament in the liquid medium within, and barrier film is arranged, and more small-sized spore is arranged, and most spores are unicellular, circle, and Dan Sheng does not see and concatenates.This bacterium can be fermented and be produced plant growth hormone gibberellin, does not cause withered corruption of plant or the listless disease of withering.Wo Lunweibai and Lai Yinqin (Wollenweber are pressed in contrast " fungi identification handbook " (the super posthumous work of Wei Jing, Shanghai science tech publishing house, 1979, Shanghai p609～638); Reinking) categorizing system, we think that tentatively the rice that should belong in the Fusarium dislikes the mould Fusarium.moniliforme of seedling, belong to red mould group [Lisola], intend called after beading Fusarium Fusarium moniliforme.SW-902.
The cultivation of bacterium producing multi enzyme preparation and fermentation:
Slant culture: preparation contains glucose 1～10%, and potato is soaked juice 5～30%, agar 1～2.5%, and the substratum of pH6～9, each components contents is percent weight in volume, and promptly g/100ml is together following.100～120 ℃ of sterilizations, 20～50 minutes, sterilization postcooling, bevel, inoculation were cultivated 3～7 days for 20～40 ℃.The product enzyme is cultivated: preparation contains glycerol 1～10%, peptone 0.5～5%, yeast extract paste 0.5～5%, corn steep liquor 0.5～5%, the substratum of pH6～9, liquid amount 20～200ml/500ml triangular flask, 100～120 ℃ of sterilizations, 20～50 minutes, sterilization postcooling, inoculation, inoculum size 5～10%, is shaken bottle rotating speed a 100～220r/min by 20～40 ℃.Carry out the shake flask fermentation test under these conditions, cultivated 3-7 days, produce enzyme and can reach more than the 4U/l (1.0U/g dry mycelium).
D-pantoic acid lactone hydrolase enzyme activity determination method: get the 10ml fermented liquid, the leaching mycelium adds 2% pantoyl lactone 2.5ml, CaCl in thalline
250mmol and pH7.5, the 0.2mol/LTris-HCl damping fluid, 28 ℃, 150r/min shaking table reaction 30min, suction filtration is removed thalline, and reaction solution is measured the D-pantoic acid with high pressure liquid chromatographic analysis (HPLC).The enzyme activity unit definition: under these conditions, per minute hydrolysis D-pantoyl lactone becomes the enzyme amount of the μ mol amount of D-pantoic acid, is defined as 1 enzyme activity unit (U).
The HPLC of pantoyl lactone and pantoic acid measures: column type is ZORBAX SB C
185 μ M, 250 * 4.6mm, moving phase is acetonitrile: KH
2PO
4(0.02mol/L)=1: 9, transferring pH with HCl is 3, and flow velocity is 1ml/min, and the ultraviolet detection wavelength is 215nm.
Enzymatic conversion reaction:
Mycelium after the cultivation is substrate as the enzyme source with the DL-pantoyl lactone, and concentration of substrate is 1～70%, and enzyme concentration is 0.1～5U/g substrate, and enzyme reaction temperature is 20～40 ℃, and enzymolysis time is 3～30 hours.The DL-pantoyl lactone enzymolysis solution of gained carries out high pressure liquid chromatographic analysis and specific rotation and measures and show that the main ingredient of enzymolysis is the D-pantoic acid with this understanding.
Perhaps, the fermentation produce wet mycelium, make the mycelium suspension with physiological saline, with 4～6%, 1～5 times the amount carrageenan solutions at 35 ℃～60 ℃ following mixings, add the KCl solution hardening after the cooling and make immobilized cell.Carry out enzymic hydrolysis with this as the enzyme source.Except carrageenin as the fixation support embedding, can also use the method for carrier embeddings such as alginate calcium, gelatin, chitin.Immobilized cell is reuse repeatedly, carries out repeatedly enzymatic hydrolysis reaction.
The extraction of D-pantoyl lactone and refining:
The enzymolysis solution clear liquid of gained after the enzymic hydrolysis, main component is L-pantoyl lactone and D-pantoic acid, also has unconverted D-pantoyl lactone, also contain some inorganic salt impurity and darker color, can remove L-pantoyl lactone and unconverted D-pantoyl lactone with the appropriate solvent extraction earlier, isolate the D-pantoic acid that has transformed, after carrying out lactonization reaction, extract with the appropriate solvent extraction again, and carry out desalination and decolour handling, or recrystallization method made with extra care, thus the high purity of obtaining, high-quality D-pantoyl lactone product.
Process flow diagram of the present invention is seen accompanying drawing 1.
The present invention has overcome that induced crystallization method resolution DL-calcium pantothenate yield is low, and optical purity is poor, and cost is high, can only be used for the limitation that D-VB5 calcium is produced, and it is expensive to have overcome the chemical resolution method resolving agent, and cost is high, and separation difficulty has and pollutes and the shortcoming such as toxicity problem. To the shortcoming of other enzyme process or fermentation method,, reaction rate low, product optical purity low etc. low such as substrate concentration makes moderate progress. One strain that advantage of the present invention has been seed selection can high yield stereocpecificity D-pantoyl lactone hydrolase, do not utilize the microbial strains Fusorium moniliforme Sheldon mould Fusarium moniliforme.SW-902-CGMCC No 0536 of do not degrade pantoic acid lactone or pantoic acid, and, produce the enzyme fermentation time short (3 days), the enzymatic conversion time short (5～10 hours), enzyme can be recycled repeatedly (more than 6 times). Provide the simple microbial enzyme method of a kind of technique to split the method for preparing D-pantoyl lactone. Resulting enzymic hydrolysates Pantothenic acid extracts the D-pantoyl lactone that obtains after lactonizing optical purity reaches more than the 99%e.e., can satisfy the quality requirement that D-VB5 calcium or D-pantothenyl aleohol are produced intermediate.
The following examples elaborate to the present invention.
Embodiment 1
Slant culture: substratum is that potato is soaked juice (20%) 100ml, glucose 2g, agar 2g, pH6.5 adds water to 1000ml, sterilizes 20 minutes for 121 ℃, the inoculation of sterilization postcooling, bacterial classification is beading Fusarium Fusarium moniliforme SW-902-CGMCC No.0536, cultivates 5 days for 25 ℃, as the slant activation seed.
The product enzyme is cultivated: substratum is a glycerol 3%, peptone 0.5%, yeast extract paste 0.5%, corn steep liquor 0.5%, pH6.0, liquid amount are the bottled liquid 100ml of 500ml triangle, sterilize 20 minutes for 121 ℃, sterilization postcooling inoculation inclined-plane seed was cultivated 3-7 days for 28 ℃, as fermenting enzyme liquid.
Shake bottle and produce enzyme test result following (table 1).By table 1 as seen, incubation time 3 days, enzyme work reaches maximum, and enzyme work in later several days is kept constant substantially.
Table 1 shakes bottle and produces the enzyme test result
| Incubation time d | Thalline weight in wet base g/L | Dry cell weight g/L | Enzyme U/g (dry mycelium) alive |
| ????1 ????2 ????3 ????4 ????5 ????6 | ????10.385 ????86.125 ????174.41 ???105.135 ????77.28 ????97.225 | ????0.46 ????6.005 ????9.795 ????11.85 ????13.92 ????14.785 | ??????0 ????0.44 ????1.62 ????1.22 ????1.20 ????1.27 |
Average thalline weight in wet base/dry cell weight=9.69
Embodiment 2
By the DL-pantoyl lactone of chemosynthesis preparation, with containing 50mmolCaCl
2The aqueous solution to be mixed with 30% concentration be substrate; Add wet thallus (D-pantoic acid lactone hydrolase) by the preparation of example 1 method, enzyme concentration is the 0.3U/g pantoyl lactone, puts 28 ℃ of shaking tables, the 150r/min reaction, every 20min sampling carrying out high pressure liquid chromatographic analysis and specific rotation are measured, and are 7.0 with the pH value of strong aqua conditioned reaction liquid.Enzymolysis time is 5～10 hours.With the mycelium that leaches after the reaction end, add new reaction substrate and continue reaction, so repeatedly, reuse 6 times.Obtain enzymic hydrolysis and the results are shown in Table 2.Table 2 is the result show, after the enzymic hydrolysis in 5～30 hours, the enzymatic conversion rate that obtains is about 38%, and the specific rotatory power of the product D-pantoyl lactone of extraction is [α] after lactonizing
D 20=-47 °.After thalline used 6 times, the enzymatic conversion rate did not still significantly descend.The result is as shown in table 2:
Table 2 is enzymatic conversion result in batches repeatedly
| Transform number of times | ??1 | ??2 | ??3 | ??4 | ??5 | ??6 |
| Transformation efficiency (%) | ?30.5 | ?31.1 | ?29.3 | ?28.6 | ?29.3 | ?29.6 |
Embodiment 3
Press example 1 method, enzymatic production.Get wet mycelium 100g, add 100ml physiological saline and make the mycelium suspension, 30 ℃ of insulations; Other takes by weighing the 20g carrageenin, adds 400ml physiological saline, after the heating for dissolving, is cooled to 55 ℃ of insulations.Both pour plate at mixing below 50 ℃, the cooling, add KCl solution, put the 3hr that hardens in the refrigerator, weigh about 500g immobilized cell.With this immobilized cell, press example 2 method enzymic hydrolysiss.Enzymic hydrolysis the results are shown in Table 3 10 times in batches repeatedly.
Table 3 immobilized cell is enzymatic conversion result in batches repeatedly
| Transform number of times | ??1 | ??2 | ??3 | ??4 | ??5 | ??6 | ??7 | ??8 | ??9 | ??10 |
| Transformation efficiency (%) | ?28.5 | ?30.1 | ?32.2 | ?30.5 | ?29.3 | ?29.6 | ?30.5 | ?31.2 | ?28.6 | ?31.5 |
Embodiment 4
Press the method for embodiment 2, compound concentration is 30% DL-pantoyl lactone substrate, adds the wet thallus (D-pantoic acid lactone hydrolase) by the preparation of example 1 method, the method enzymic hydrolysis of pressing embodiment 2.The enzymolysis solution 100ml of gained filters the clear liquid of gained, is 1: 1～2 dichloromethane extraction 3 times with volume ratio, isolates water, wherein mainly contains the D-pantoic acid that has transformed.Water with HCl transfer pH be 1 carry out lactonization reaction after, extract with dichloromethane extraction again, organic phase is evaporated the recovery solvent, obtains D-pantoyl lactone crude product 12.5g, crude product yield 41.67% (to DL-pantoyl lactone meter), measuring specific rotation is [α]
D 20=-48 °.Crude product gets the pure product 10.5g of D-pantoyl lactone with acetone/isopropyl ether recrystallization, recrystallization yield 84%, and to DL-pantoyl lactone yield 35%, the product specific rotation is [α]
D 20=-51 °, it is 99%e.e. that HPLC measures D-pantoyl lactone optical purity.
Collect L-pantoyl lactone and unconverted D-pantoyl lactone, after evaporation concentration reclaims solvent,, carry out racemization reaction with 130 ℃ of heating of NaOH solution 2 hours.Recovery obtains DL-pantoyl lactone 17.8g, is directly used in next enzyme reaction.Calculate by this, the pure product D-pantoyl lactone that obtains is 86.07% to the yield that consumes DL-pantoyl lactone substrate (the D-pantoyl lactone of enzymic hydrolysis).
Embodiment 5
Press example 1 method, produce enzyme with the 15L ferment tank.Press the method for embodiment 2, compound concentration is 30% DL-pantoyl lactone substrate, adds the wet thallus (D-pantoic acid lactone hydrolase) of jar fermentative preparation, the method for pressing embodiment 2, carry out enzymic hydrolysis with the 15L retort, adding strong aqua adjusting pH value automatically is 6.9～7.0.Carry out product extraction and unconverted substrate recovery by example 3 methods.Enzyme is recycled 5 times.The result is: fermentation time 2 days, enzyme work reach 1.02 U/g (dry mycelium), thalline weight in wet base 42g/L, and the average enzymatic conversion rate of 6 enzymatic conversions is 30.2%, the specific rotatory power of crude product D-pantoyl lactone is [α]
D 20=-47 °, crude product is to the extraction yield 90.5% of the D-pantoyl lactone of enzymic hydrolysis.
Claims (4)
1. the microorganism strains of a high-yield D-pantolactone hydrolytic enzyme, it is beading Fusarium (Fusarium moniliforme) SW-902 CGMCC No:0536.
2. make the method for D-pantoyl lactone, comprising:
(1) with the described bacterial strain SW-902 of claim 1, carry out routine and cultivate, fermentation, obtain wet thallus or with carrier immobilized backs such as carrageenins as zymin;
(2) with the isobutyric aldehyde be raw material, adopt formaldehyde method, the sodium cyanide route is produced the general ester lactone of separating of DL-, and refining back is as the enzymatic conversion substrate;
(3) with the wet thallus of (1) or join the substrate reactions of (2) after carrier immobilized with carrageenin etc., generate the D-pantoic acid, through lactonizing, make the D-pantoyl lactone.
3. method according to claim 2 is characterized in that: concentration of substrate is 1-70% in the step (3), and enzyme concentration is the 0.1-5U/g substrate, and enzyme reaction temperature is 20-50 ℃, and enzymolysis time is 5-30 hour.
4. method according to claim 2 is characterized in that the substratum that produces the enzymic fermentation cultivation is: glycerol 1-10%, and peptone 0.5-5%, yeast extract paste 0.5-5%, corn steep liquor 0.5-5%, pH6-9, cultivated 2-7 days by temperature 20-40 ℃.
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| WO2008046328A1 (en) * | 2006-10-19 | 2008-04-24 | East China University Of Science And Technology | A levorotatory lactonohydrolase producing strain and its use for producing chiral oxyacid |
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| CN108624513A (en) * | 2018-06-15 | 2018-10-09 | 成都本则生科技有限公司 | A kind of method of High Density Cultivation D-pantoyl lactone hydrolase producing strains and application |
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2001
- 2001-02-21 CN CN 01104070 patent/CN1111604C/en not_active Expired - Lifetime
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| US6720302B2 (en) * | 1999-07-22 | 2004-04-13 | Firmenich Sa | Method for preparing a γ-unsaturated β-lactone and use thereof as an aromatic and flavouring ingredient |
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| CN110845453A (en) * | 2019-11-28 | 2020-02-28 | 安徽泰格生物科技有限公司 | Racemization method of L-pantoic acid lactone |
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| CN112481345A (en) * | 2020-12-18 | 2021-03-12 | 合肥工业大学 | Method for preparing D-pantoic acid by continuous catalysis |
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