阅读说明：本技术 一种同时制备d-脯氨酸和l-1-吡咯啉-5-羧酸的生物法 (Biological method for simultaneously preparing D-proline and L-1-pyrroline-5-carboxylic acid ) 是由 夏仕文 张凡凡 谢永芳 于 2020-03-30 设计创作，主要内容包括：本发明公开了一种同时制备D-脯氨酸和L-1-吡咯啉-5-羧酸的生物法。该方法以解木糖赖氨酸芽孢杆菌XX-40为发酵菌株,DL-脯氨酸为发酵前体。该菌株首先利用DL-脯氨酸中的部分L-脯氨酸生长并诱导产生L-脯氨酸脱氢酶,剩余L-脯氨酸在L-脯氨酸脱氢酶作用下转化为L-1-吡咯啉-5-羧酸,D-脯氨酸完全保留。发酵液中的细胞不经分离,直接或分批添加DL-脯氨酸,利用胞内L-脯氨酸脱氢酶将其中的L-脯氨酸转化为L-1-吡咯啉-5-羧酸。采用本发明中的发酵/转化级联方式,发酵液中D-脯氨酸浓度达到20g/L,ee>99％,发酵液中L-1-吡咯啉-5-羧酸浓度达到16g/L,产率达到40％。本发明提供的方法工艺简单,环境友好,适合D-脯氨酸和L-1-吡咯啉-5-羧酸的工业化生产。(The invention discloses a biological method for simultaneously preparing D-proline and L-1-pyrroline-5-carboxylic acid, which takes lysine bacillus xylolyticus XX-40 as a fermentation strain and D L-proline as a fermentation precursor.A strain firstly utilizes part of L1-proline in D L0-proline to grow and induce to generate L-proline dehydrogenase, the rest L-proline is converted into 855-1-pyrroline-5-carboxylic acid under the action of L-proline dehydrogenase, the D-proline is completely reserved, cells in fermentation liquor are not separated, D L-proline is directly or added in batches, intracellular L-proline dehydrogenase is utilized to convert L-proline in the D-proline into L-1-pyrroline-5-carboxylic acid, the fermentation/conversion cascade mode provided by the invention is adopted, the concentration of the D-proline in the fermentation liquor reaches 20 g/L, the concentration of the E48-1-pyrroline-5-carboxylic acid in the fermentation liquor reaches 16 g/L, the yield reaches 40%, the method is suitable for industrial production of D-proline, and the environment is friendly to produce D-1-pyrroline-5-carboxylic acid.)

1. A biological method for simultaneously preparing D-proline and L-1-pyrroline-5-carboxylic acid is characterized in that lysine bacillus xylolyticus XX-2 is cultured in a seed culture medium to prepare a seed solution, then the seed solution is inoculated into a fermentation culture medium, shaking tables are used for fermentation, and the fermentation culture medium takes D L-proline as a carbon-nitrogen source.

2. The biological method for simultaneously preparing D-proline and L-1-pyrroline-5-carboxylic acid according to claim 1, wherein L-proline in the D L-proline is used as a carbon and nitrogen source for growth of lysine bacillus xylolyticus XX-2, and simultaneously induces the lysine bacillus xylolyticus XX-2 to produce L-proline dehydrogenase.

3. The biological process for simultaneously preparing D-proline and L-1-pyrroline-5-carboxylic acid according to claim 1 or 2, wherein the seed culture medium comprises 3 g/L beef extract, 5 g/L yeast extract, 5 g/L sodium chloride, and has a pH of 6.0.

4. The biological method for simultaneously preparing D-proline and L-1-pyrroline-5-carboxylic acid as claimed in claim 3, wherein the fermentation medium comprises 3 g/L of beef extract, 5 g/L of yeast extract, 5 g/L of sodium chloride, 10-20 g/L of D L-proline, and the pH value is 6.0.

5. The biological method for simultaneously preparing D-proline and L-1-pyrroline-5-carboxylic acid according to claim 4, wherein the fermentation time is 48-72 hours.

6. The biological process for simultaneously preparing D-proline and L-1-pyrroline-5-carboxylic acid according to any one of claims 1 to 5, wherein when L-proline in the fermentation medium is completely consumed, D L-proline is added again for microbial conversion.

7. The biological method for simultaneously preparing D-proline and L-1-pyrroline-5-carboxylic acid as claimed in claim 6, wherein D L-proline is directly added without separating cells in the fermentation broth so that the concentration of D L-proline is 10-20 g/L.

8. The biological process for simultaneously preparing D-proline and L-1-pyrroline-5-carboxylic acid according to claim 7, wherein the D L-proline is added in a one-time or fed-batch manner.

9. The biological method for simultaneously preparing D-proline and L-1-pyrroline-5-carboxylic acid according to claim 6, wherein the conversion time is 48-96 h.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a biological method for simultaneously preparing D-proline and L-1-pyrroline-5-carboxylic acid.

Background

As an important five-membered ring non-proteinogenic amino acid, D-Proline (D-Proline) is a key chiral intermediate in the synthesis of various optically active pyrrolidine derivatives such as serum amyloid P component (SAP) depleting agents (CN 108368095a), Eletriptan (Eletriptan), a migraine treating drug.

The preparation method of the D-proline mainly comprises a chemical method and a biological method. Chemical methods include asymmetric synthesis and asymmetric transformation.

An asymmetric synthesis method (CN 107827802A) uses pyrrolidine-2-formaldehyde as a raw material, asymmetrically reduces the raw material into D-pyrrolidine-2-methanol, and then oxidizes the D-pyrrolidine-2-methanol into D-proline, the method adopts expensive raw materials and chiral metal catalysts, the ee value of the D-proline is not high, an asymmetric conversion method uses L-proline as a raw material and n-butyl aldehyde as a catalyst, the D-proline-tartaric acid salt is prepared by reacting with L-tartaric acid in an n-butyl acid solvent, and then D-proline is obtained by treating the methanol with ammonia water.

A biological method is mainly adopted, a biological degradation method is adopted, Japanese synergistic fermentation (JP 95-289275) takes D L-proline as a raw material, 50 g/L Candida PRD-234 strains utilize and assimilate L-proline, the concentration of the D-proline in fermentation liquor reaches 50 g/L, ee is 99.5%, and the yield is 47%.

Disclosure of Invention

The invention provides a biological method for simultaneously preparing D-proline and L-1-pyrroline-5-carboxylic acid to solve the problems in the prior art.

The technical route of the invention is as follows:

the lysine bacillus xylolyticus XX-2 is preserved in China center for type culture Collection with the preservation number of CCTCC No. M2015520.

In view of the above, the technical scheme adopted by the invention is that the biological method for simultaneously preparing D-proline and L-1-pyrroline-5-carboxylic acid comprises the steps of culturing lysine bacillus xylolyticus XX-2 in a seed culture medium to prepare a seed solution, then inoculating the seed solution into a fermentation culture medium, oscillating a shaking table to ferment, wherein the fermentation culture medium takes D L-proline as a carbon-nitrogen source, L-proline in D L-proline is taken as a carbon-nitrogen source for growth of lysine bacillus xylolyticus XX-2, and simultaneously induces the lysine bacillus xylolyticus XX-2 to generate L-proline dehydrogenase.

When L-proline in the fermentation medium is completely consumed, D L-proline is added again for microbial conversion.

Therefore, the method comprises the following specific steps:

(1) the method comprises the following steps of fermenting lysine bacillus xylolyticus XX-2, culturing the lysine bacillus xylolyticus XX-2 in a seed culture medium at 30 ℃ for 48 hours in a shaking table vibration (180rpm) mode to prepare a seed solution, wherein the seed culture medium comprises 3 g/L beef extract, 5 g/L yeast extract, 5 g/L sodium chloride, the pH value is 6.0, inoculating the seed solution into a fermentation culture medium at 10% of inoculation amount, and fermenting at 30 ℃ for 48-72 hours in a shaking table vibration (180rpm) mode, and the fermentation culture medium comprises 3 g/L beef extract, 5 g/L yeast extract, 5 g/L sodium chloride, 10-20 g/L D L-proline, and the pH value is 6.0.

Preferably, the concentration of D L-proline in the fermentation medium is 20 g/L and the fermentation time is 72 h.

(2) And (3) microbial transformation, adding 10-20 g/L D L-proline when L-proline in the fermentation liquor is completely consumed, and continuing to transform for 48-96 h until L-proline is completely consumed.

Preferably, the concentration of the added D L-proline is 20 g/L, and the D L-proline is added in two batches, 10 g/L in each addition, and the conversion time is 96 hours.

The concentrations of D-proline, L-proline and L-1-pyrroline-5-carboxylic acid in the fermentation and conversion processes are detected by using pre-column chiral derivatization-high performance liquid chromatography.

The conditions of pre-column chiral derivatization-high performance liquid chromatography are that 4 g/L triethylamine/acetonitrile solution and 2 g/L2, 3,4, 6-tetra-O-acetyl- β -D-Glucopyranose Isothiocyanate (GITC)/acetonitrile solution are added into supernatant after fermentation liquor is centrifuged, water bath heating is carried out for 20min at 30 ℃, centrifugation is carried out, HP L C is adopted to separate derivatives of D-proline and L-proline and L-1-pyrroline-5-carboxylic acid in the supernatant, concentration is calculated according to a correction curve, a chromatographic column is a C18 column, a mobile phase is 0.1% trifluoroacetic acid water solution/methanol (51:49, v/v), pH is 2.5, flow rate is 1.0ml/min, detection wavelength is 254nm, and column temperature is 25 ℃.

Compared with the existing biological preparation method of D-proline, the biological method provided by the invention has the advantages that 1) the process is simple, L-proline in D L-proline is used as a carbon-nitrogen source for cell growth and induces cells to produce L-proline dehydrogenase, L-proline is converted into L-1-pyrroline-5-carboxylic acid under the action of L-proline dehydrogenase, L-proline is converted into L-1-pyrroline-5-carboxylic acid under the action of L-proline dehydrogenase, D-proline is completely reserved in the fermentation or conversion stage, the fermentation and conversion cascade is enabled not to separate cells generated in the fermentation stage, the fermentation and conversion are carried out in the same system, 2) the cost is low, the D-proline and L-1-pyrroline-5-carboxylic acid can be simultaneously prepared, L-1-pyrroline-5-carboxylic acid can be reduced into L-proline through chemical catalysis, then the racemic D-proline-L-proline-5-carboxylic acid is obtained through chemical or biological racemization, the conversion yield of the D-proline-5-pyrroline-5-carboxylic acid reaches more than 638 g, and the fermentation broth reaches more than 638-9/3-L% by fermentation.

Drawings

FIG. 1 shows the pre-column chiral derivative-HP L C patterns of D-proline and L-proline derivatives and L-1-pyrroline-5-carboxylic acid in example 3, the retention times of D-proline derivative (D-Pro) and L-proline derivative (L-Pro) are 17.8min and 15.3min, respectively, and the retention time of L-1-pyrroline-5-carboxylic acid (P5C) is 12.7 min.

FIG. 2 shows the spectrum of procollagen derivative-HP L C of D-proline and L-proline derivatives and L-1-pyrroline-5-carboxylic acid in example 5, the retention times of D-proline derivative (D-Pro) and L-proline derivative (L-Pro) are 17.9min and 15.4min, respectively, and the retention time of L-1-pyrroline-5-carboxylic acid is 12.8 min.

Detailed Description

The following examples are only basic illustrations of the concept of the present invention, and any equivalent changes made according to the technical solutions of the present invention are within the protection scope of the present invention.