CN104404098A - Biological preparation method of lipoic acid - Google Patents
Biological preparation method of lipoic acid Download PDFInfo
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- CN104404098A CN104404098A CN201410776657.7A CN201410776657A CN104404098A CN 104404098 A CN104404098 A CN 104404098A CN 201410776657 A CN201410776657 A CN 201410776657A CN 104404098 A CN104404098 A CN 104404098A
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- thioctic acid
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Abstract
The invention relates to a biological preparation method of lipoic acid. The biological preparation method comprises the following steps: in the existence of a biocatalyst, in a water phase system with a pH value of 8-10, and at the temperature of 30-50 DEG C, carrying out a hydrolysis reaction to lipoic acid ester to generate lipoic acid, wherein the biocatalyst is lipase or protease; the lipase is prepared from one of rhizo-mucor miehei, candida antarctica, pseudomonas cepacia, pseudomonas fluorescens, thermophilic fungi, bacillus subtilis and fusarium solani; the protease is prepared from one of bacillus subtilis and aspergillus niger.
Description
Technical field
The present invention relates to a kind of biological preparation method of Thioctic Acid.
Background technology
Thioctic Acid (lipoic acid, is often referred to alpha-lipoic acid) is water-fast white or light yellow crystal, is distributed widely in the biological tissues such as animals and plants.Thioctic Acid belongs to a compounds of vitamin B complex, is some microbial growth factors, plays cofactor effect in some multienzyme system, participates in pyruvate oxidation decarboxylation and forms acetyl-CoA, the oxidative decarboxylation reaction etc. of α-ketoglutaric acid.Thioctic Acid contains two sulphur five-membered ring structure, and electron density is very high, and have significant electrophilicity and the ability with free radical reaction, therefore it has oxidation-resistance.Thioctic Acid can multiple reactive oxygen free radical in purged body, and the resistance of oxidation of enhancing body, its oxidation-resistance is 400 times of vitamin-E, is the one that in the natural antioxidants known to the mankind, effect is the strongest.Thioctic Acid has concurrently fat-soluble and water-soluble, is widely used in the various diseases such as treatment and prevention of liver disease, heart trouble and diabetes.Thioctic Acid has two kinds of enantiomers, and wherein (R)-Thioctic Acid ratio (S)-Thioctic Acid is much effective.
Thioctic Acid is more and more applied in medicine, food and healthcare products.The annual requirement of global Thioctic Acid in 2008 is probably 400t, within 2010, reaches 500t, and with more than 10% speed increment, and its output is only about 200t, and huge supply breach makes Thioctic Acid market have important market position at home and abroad.
The principal synthetic route of Thioctic Acid has hexanodioic acid derivative synthesis method (J.Am.Chem.Soc, 1957,77,416-419), hexanodioic acid synthesis method (US 2792406), cyclohexanone derivative method and pimelinketone five-step approach (Chinese pharmaceutical chemistry, 1999,12 (9): 304-305) etc.In these methods, after completing the cyclization of disulfide linkage, need lipoate to be hydrolyzed to Thioctic Acid as the finished product, reaction equation is as follows:
The common method for hydrolysis of this committed step adds and the highly basic such as substrate equivalent or excessive sodium hydroxide, and the phase-transfer catalyst such as Tetrabutyl amonium bromide or anion surfactant are as catalyzer, as described in Chinese patent 201010605575.8 and 201310034941.2 etc.Because hydrolyzed waste water contains and substrate equivalent highly basic, directly can not discharge, need the strong acid neutralization adding equivalent.The tensio-active agent contained in waste water also exists can not the problem of natural degradation.The present invention, by using biological enzyme hydrolysis, with compared with open method, need not add the highly basic such as sodium hydroxide, and catalyzer can reuse and energy natural degradation, has a good application prospect, as follows:
Summary of the invention
The object of this invention is to provide a kind of biological preparation method of Thioctic Acid to solve the pollution problem existing for existing chemical method.
For solving above technical problem, the present invention takes following technical scheme:
A kind of biological preparation method of Thioctic Acid, it is by making lipoate under biological catalyst exists, in aqueous phase system in pH8 ~ 10 and temperature 30 ~ 50 DEG C issues raw hydrolysis reaction and generates Thioctic Acid, described biological catalyst is lipase or proteolytic enzyme, wherein:
Lipase is selected from and derives from a meter black root hair enzyme (Rhizomucor miehei), antarctic candida (Candida Antarctica), pseudomonas cepacia (Pseudomonas cepacia), Pseudomonas fluorescens (Pseudomonas fluorescens), thermophilic fungus (Thermomyces lanuginose), Bacillus subtilus (Bacillus subtilis), Fusarium solani (Fusarium solani pisi), one in the lipase of Fusarinm solani (Fusarium solani pisi),
Proteolytic enzyme is selected from the one in the proteolytic enzyme deriving from Bacillus subtilus (Bacillus subtilis), aspergillus niger (Aspergillus niger).
According to the present invention, the form of described lipase, proteolytic enzyme can be resolvase or immobilized enzyme, is not particularly limited.
Preferably, biological catalyst is be selected from the Novozym 435 deriving from antarctic candida, derive from the IMMOZYME AUL1 of Bacillus subtilus, derive from the Lipolase of thermophilic fungus, derive from the IMMOZYME IMMABC of pseudomonas cepacia, derive from the one in the IMMOZYME APF of Pseudomonas fluorescens.。
Particularly, described lipoate is Thioctic Acid methyl esters or Thioctic Acid ethyl ester or the combination of the two.
Further, described biological catalyst is preferably 0.1 ~ 1:1 with the charged material weight ratio of lipoate, is more preferably 0.1 ~ 0.5:1.
Further, the pH of described aqueous phase system is preferably 9 ~ 10, is more preferably 9.5 ~ 10.
Preferably, described hydrolysis reaction carries out at temperature 38 ~ 42 DEG C.
Preferably, described method also comprises makes hydrolysis reaction carry out under solubility promoter exists, and solubility promoter is specially acetonitrile, DMSO or the combination of the two.
Preferably, the volume by volume concentration of solubility promoter is 10% ~ 15%.
According to the present invention, lipoate is specifically as follows Thioctic Acid ethyl ester, Thioctic Acid methyl esters etc., also can be other alkyl esters of Thioctic Acid.
According to of the present invention one specifically and preferred embodiment, biological catalyst is be selected from the Novozym 435 deriving from antarctic candida, derive from the IMMOZYME AUL1 of Bacillus subtilus, derive from the Lipolase of thermophilic fungus, derive from the IMMOZYME IMMABC of pseudomonas cepacia, derive from the one in the IMMOZYME APF of Pseudomonas fluorescens.; Biological catalyst is 0.1 ~ 0.5:1 with the charged material weight ratio of lipoate, and the pH of aqueous phase system is 9.5 ~ 10, and the temperature of hydrolysis reaction is 38 ~ 42 DEG C, and hydrolysis reaction also carries out under the existence of DMSO, and the volume by volume concentration of DMSO is 10% ~ 15%.According to this embodiment, after 6h is carried out in reaction, namely transformation efficiency reaches more than 90%.
According to a preferred forms of the present invention, biological catalyst is 0.1 ~ 0.5:1 with the charged material weight ratio of lipoate, biological catalyst is be selected from the Novozym 435 deriving from antarctic candida or the IMMOZYME AUL1 deriving from Bacillus subtilus, the pH of aqueous phase system is 10, the temperature of hydrolysis reaction is 39 ~ 41 DEG C, hydrolysis reaction carries out under the existence of DMSO, and the volume by volume concentration of DMSO is 15%.According to this embodiment, after 6h is carried out in reaction, namely transformation efficiency reaches more than 99%.
Accompanying drawing explanation
Fig. 1 is the liquid chromatogram of embodiment 3 products therefrom.
Embodiment
Below in conjunction with specific embodiment, the present invention will be further described in detail, but the invention is not restricted to following examples.
The transformation efficiency that Thioctic Acid is prepared in the different lipase of embodiment 1 or the hydrolysis of proteases catalyze Thioctic Acid ethyl ester compares
In 2ml 96 orifice plate, add different hydrolase 10 mg, substrate Thioctic Acid ethyl ester 10mg, the phosphate buffer soln of 1ml pH 7.0 100mM, shakes reaction 8 hours after sealing at 30 DEG C, and sampling detects transformation efficiency, in table 1.
The different enzyme of table 1 is to the katalysis of hydrolysis reaction
| Title | Type | Source species | Supplier | Transformation efficiency |
| Lipase AK | Lipase | Pseudomonas fluorecens | AMANO | 51.6％ |
| Lipase AS | Lipase | Aspergillus niger | AMANO | 5.0％ |
| Lipase AY | Lipase | Candida rugosa | AMANO | 67.9％ |
| Lipase PS | Lipase | Burkholderia cepacia | AMANO | 84.5％ |
| Amano M4 | Lipase | Mucor javanicus | AMANO | 93.8％ |
| Amano F-AP154 | Lipase | Rhizopus oryzae | AMANO | 25.3％ |
| CALA | Lipase | Candida antarctica | Novozymes | 54.2％ |
| Novozym 435 | Lipase | Candida antarctica | Novozymes | >99％ |
| Lipolase | Lipase | Thermomyces lanuginosa | Novozymes | 94.9％ |
| Cutinase | Lipase | Fusarium solani pisi | Novozymes | 92.1％ |
| RML | Lipase | Rhizomucor miehei | sigma-aldrich | 90.8％ |
[0030]
| IMMOZYME IMMABC | Immobilized lipase | Pseudomonas cepacia | chiral vision | 97.9％ |
| IMMOZYME APF | Immobilized lipase | Pseudomonas fluorescens | chiral vision | 98.6％ |
| IMMOZYME AULI | Immobilized lipase | Bacillus subtilis | chiral vision | >99％ |
| IMMOZYME CALB | Immobilized lipase | Candida antarctica | chiral vision | 72.1％ |
| IMMCRL | Immobilized lipase | Candida rugosa | chiral vision | 89.6％ |
| Lipase OF | Lipase | Candida cylindracea sp. | Meito | 3.1％ |
| Papain | Proteolytic enzyme | Carica papaya | sigma-aldrich | 12.4％ |
| Alkaline protease | Proteolytic enzyme | Bacillus subtilis | AMANO | 89.6％ |
| Neutral protease | Proteolytic enzyme | Bacillus subtilis | AMANO | 18.6％ |
| Acid protease | Proteolytic enzyme | Aspergillus niger | AMANO | 46.7％ |
| Alcalase | Proteolytic enzyme | Bacillus subtilis | Novozymes | 72.0％ |
As seen from Table 1, derive from a meter black root hair enzyme (Rhizomucor miehei), antarctic candida (Candida Antarctica), pseudomonas cepacia (Pseudomonas cepacia), Pseudomonas fluorescens (Pseudomonas fluorescens), thermophilic fungus (Thermomyces lanuginose), Bacillus subtilus (Bacillus subtilis), Fusarium solani (Fusarium solani pisi), the lipase of Fusarinm solani (Fusarium solani pisi), derive from Bacillus subtilus (Bacillus subtilis), the proteolytic enzyme of aspergillus niger (Aspergillus niger) all has katalysis in various degree to the hydrolysis reaction preparing Thioctic Acid.Wherein, adopt the Novozym 435 deriving from antarctic candida, derive from the IMMOZYME AUL1 of Bacillus subtilus, derive from the Lipolase of thermophilic fungus, derive from the IMMOZYME IMMABC of pseudomonas cepacia, the transformation efficiency deriving from the lipase of the IMMOZYME APF of Pseudomonas fluorescens reaches more than 95%.
Embodiment 2 differential responses condition is for the impact of hydrolysis reaction
Example 2.1 adds lipase Novozym 435 5mg in 2ml 96 orifice plate, substrate Thioctic Acid ethyl ester 10mg, the phosphate buffer soln of 1ml pH 7.0 100mM, shakes reaction 24 hours at sealing is placed on 30 DEG C, and it is 77% that sampling detects transformation efficiency.
Example 2.2 adds lipase Novozym 435 5mg in 2ml 96 orifice plate, substrate Thioctic Acid ethyl ester 10mg, the phosphate buffer soln of 1ml pH 7.0 100mM, shakes reaction 24 hours after sealing at being placed in 30 DEG C, 40 DEG C and 50 DEG C respectively, it is 97% and 72% that sampling detects transformation efficiency.
Example 2.3 adds lipase Novozym 435 5mg in 2ml 96 orifice plate, substrate Thioctic Acid ethyl ester 10mg, add the phosphate buffer soln of 1ml pH 8.0 100mM, at 40 DEG C, reaction 6 hours is shaken after sealing, it is 82% that sampling detects transformation efficiency, concussion reaction 22 hours, it is 98% that sampling detects transformation efficiency.
Example 2.4 is substantially identical with example 2.3, unlike, the pH of the phosphate buffer soln added is respectively 9.0,10.0,11.0,12.0, the transformation efficiency detected for 6 hours is followed successively by 92%, 99%, 87% and 73%, and the transformation efficiency detected for 22 hours is followed successively by 98%, 99%, 98% and 78%.
Example 2.5 adds lipase Novozym 435 5mg in 2ml 96 orifice plate, substrate Thioctic Acid ethyl ester 10 mg, add phosphate buffer soln and the 0.1ml DMSO of 0.9ml pH 10.0 100mM, shake reaction 6 hours after sealing at 40 DEG C, it is 88% that sampling detects transformation efficiency.
Example 2.6 is substantially identical with example 2.5, unlike, substitute DMSO with toluene, acetonitrile, octane-iso respectively, the transformation efficiency detected for 6 hours is followed successively by 44%, 76%, 68%.
Example 2.7 is substantially identical with example 2.5, unlike, change the volume by volume concentration of DMSO, namely to 10%, 15%, 20%, 25%, 30%, the transformation efficiency detected for 6 hours is followed successively by 68%, 86%, 44%, 46%, 42%.
Embodiment 3
In 40 DEG C of water-bath devices, add 5g Thioctic Acid ethyl ester, add deionized water 102ml and DMSO 18ml, regulate between pH to 9.0 ~ 9.5, control magnetic agitation 800rpm, stir, add 2.5g Novozym 435 immobilized lipase, start reaction, with 8%NaOH aqueous solution control ph 10.0, HPLC monitoring reaction to 6 hours, substrate conversion efficiency 99.7%, product purity 97.9%, product liquid chromatogram is shown in Fig. 1.
Embodiment 4
In 15ml chromatography column, add Novozym 435 immobilized lipase 1.2g, deionized water 10ml.In the container for storing liquid of 40 DEG C of water-bath preservations, add 5g Thioctic Acid ethyl ester, then add deionized water 100mL, regulate pH 10.0, magnetic agitation is about 800rpm, inputs in chromatography column with peristaltic pump, start reaction, HPLC monitoring was reacted to 12 hours, substrate conversion efficiency 98.5%.
Above-described embodiment, only for technical conceive of the present invention and feature are described, its object is to person skilled in the art can be understood content of the present invention and implement according to this, can not limit the scope of the invention with this.All equivalences done according to spirit of the present invention change or modify, and all should be encompassed within protection scope of the present invention.
Claims (10)
1. the biological preparation method of a Thioctic Acid, it is characterized in that: described method by make lipoate biological catalyst exist under, in the aqueous phase system of pH 8 ~ 10 and temperature 30 ~ 50 DEG C issue raw hydrolysis reaction generate Thioctic Acid, described biological catalyst is lipase or proteolytic enzyme, wherein:
Described lipase is selected from and derives from a meter black root hair enzyme (Rhizomucor miehei), antarctic candida (Candida Antarctica), pseudomonas cepacia (Pseudomonas cepacia), Pseudomonas fluorescens (Pseudomonas fluorescens), thermophilic fungus (Thermomyces lanuginose), Bacillus subtilus (Bacillus subtilis), Fusarium solani (Fusarium solani pisi), one in the lipase of Fusarinm solani (Fusarium solani pisi),
Described proteolytic enzyme is selected from the one in the proteolytic enzyme deriving from Bacillus subtilus (Bacillus subtilis), aspergillus niger (Aspergillus niger).
2. the biological preparation method of Thioctic Acid according to claim 1, is characterized in that: the form of described lipase, proteolytic enzyme is resolvase or immobilized enzyme.
3. the biological preparation method of Thioctic Acid according to claim 1, it is characterized in that: described biological catalyst is be selected from the Novozym 435 deriving from antarctic candida, derive from the IMMOZYME AUL1 of Bacillus subtilus, derive from the Lipolase of thermophilic fungus, derive from the IMMOZYME IMMABC of pseudomonas cepacia, derive from the one in the IMMOZYME APF of Pseudomonas fluorescens.
4. the biological preparation method of Thioctic Acid according to claim 1, is characterized in that: described lipoate is Thioctic Acid methyl esters or Thioctic Acid ethyl ester or the combination of the two, and described biological catalyst is 0.1 ~ 1:1 with the charged material weight ratio of lipoate.
5. the biological preparation method of Thioctic Acid according to claim 4, is characterized in that: described biological catalyst is 0.1 ~ 0.5:1 with the charged material weight ratio of lipoate.
6. the biological preparation method of Thioctic Acid according to claim 1, is characterized in that: the pH of described aqueous phase system is 9 ~ 10.
7. the biological preparation method of Thioctic Acid according to claim 6, is characterized in that: the pH of described aqueous phase system is 9.5 ~ 10.
8. the biological preparation method of Thioctic Acid according to claim 1, is characterized in that: described hydrolysis reaction carries out at temperature 38 ~ 42 DEG C.
9. the biological preparation method of Thioctic Acid according to claim 1, is characterized in that: described method also comprises makes described hydrolysis reaction carry out under solubility promoter exists, and described solubility promoter is acetonitrile, DMSO or the combination of the two.
10. the biological preparation method of Thioctic Acid according to claim 9, is characterized in that: the volume by volume concentration of described solubility promoter is 10% ~ 15%.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1553959A (en) * | 2001-09-25 | 2004-12-08 | - | Enzymatic process for the preparation of substituted 2-amino-3-(2-amino-phenylsulfanyl)-propionic acid |
| CN101652483A (en) * | 2007-01-22 | 2010-02-17 | Zach系统股份公司 | Method for the chemo-selective enzymatic hydrolysis if a diester compound for preparing a monoester monoacid compound |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1553959A (en) * | 2001-09-25 | 2004-12-08 | - | Enzymatic process for the preparation of substituted 2-amino-3-(2-amino-phenylsulfanyl)-propionic acid |
| CN101652483A (en) * | 2007-01-22 | 2010-02-17 | Zach系统股份公司 | Method for the chemo-selective enzymatic hydrolysis if a diester compound for preparing a monoester monoacid compound |
Non-Patent Citations (1)
| Title |
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| 韩丽等: "《脂肪酶催化拆分外消旋6-羟基-8-氯辛酸乙酯》", 《精细化工》 * |
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Application publication date: 20150311 |