阅读说明：本技术 一种在生物反应器内利用半疏水晶胶基全细胞催化剂合成苯乳酸的方法 (Method for synthesizing phenyllactic acid by using semi-hydrophobic crystal gum base whole-cell catalyst in bioreactor ) 是由 张玮 李亦斌 楼小玲 贠军贤 于 2021-08-31 设计创作，主要内容包括：本发明公开了一种在生物反应器内利用半疏水晶胶基全细胞催化剂合成苯乳酸的方法,所述的方法是在搅拌式生物反应器内,以半疏水阴离子交换晶胶作为载体,通过将副干酪乳杆菌细胞固载在晶胶上形成高菌体浓度的全细胞催化剂,将晶胶投入生物反应器内进行悬浮培养,通过减轻底物抑制,提高转化过程的生物量,进而提高苯乳酸的产量。本发明提供的方法比常规游离细胞转化所得的苯乳酸的产量高,转化过程简单,操作方便,安全性好,放大容易,在苯乳酸制备领域具有广泛的应用前景。(The invention discloses a method for synthesizing phenyllactic acid by using a semi-hydrophobic crystal gum base whole-cell catalyst in a bioreactor, which is characterized in that semi-hydrophobic anion exchange crystal gum is used as a carrier in a stirring bioreactor, lactobacillus paracasei cells are immobilized on the crystal gum to form a whole-cell catalyst with high thallus concentration, the crystal gum is put into the bioreactor for suspension culture, and the biomass in the conversion process is improved by reducing substrate inhibition, so that the yield of the phenyllactic acid is improved. Compared with the conventional free cell transformation method, the method provided by the invention has the advantages of high yield of the phenyllactic acid, simple transformation process, convenience in operation, good safety, easiness in amplification and wide application prospect in the field of phenyllactic acid preparation.)

1. A method for synthesizing phenyllactic acid by using a semi-hydrophobic crystal gum base whole-cell catalyst in a bioreactor is characterized in that lactobacillus paracasei cells are immobilized on semi-hydrophobic anion exchange crystal gel to form a high-concentration whole-cell catalyst, and then the crystal gel immobilized with the lactobacillus paracasei cells is put into a stirring bioreactor containing a conversion solution for suspension culture to synthesize the phenyllactic acid through catalytic conversion.

2. A method for the synthesis of phenyllactic acid in a bioreactor using a semi-hydrophobic crystal gum based whole cell catalyst according to claim 1, characterized in that it comprises the following steps:

1) activating the lactobacillus paracasei strain in an MRS culture medium twice to obtain a seed solution, connecting the seed solution to the MRS culture medium, and performing standing culture;

2) putting the dried semi-hydrophobic anion exchange crystal gel into the MRS culture medium containing the seed liquid obtained in the step 1), and standing and culturing to obtain the seed crystal gel;

3) putting the seed crystal gel obtained in the step 2) into an MRS culture medium again, and continuing to perform static culture to obtain a high-concentration whole-cell catalyst, wherein the high-concentration whole-cell catalyst is the crystal gel immobilized with lactobacillus paracasei cells;

4) performing permeability treatment on the high-concentration whole-cell catalyst obtained in the step 3), putting the high-concentration whole-cell catalyst into a stirring bioreactor containing a conversion solution for suspension culture, and converting phenylalanine in the conversion solution into phenyllactic acid through whole-cell catalysis.

3. The method for synthesizing phenyllactic acid by using the semi-hydrophobic crystal gum base whole-cell catalyst in the bioreactor as claimed in claim 1, wherein the semi-hydrophobic anion exchange crystal gum is a poly hydroxyethyl methacrylate-butyl methacrylate based anion exchange spherical crystal gum carrier, the diameter of the crystal gum carrier is 2.0-5.0 mm, and the effective porosity is 74% -85%.

4. The method for synthesizing phenyllactic acid by using the semi-hydrophobic crystal gum base whole-cell catalyst in the bioreactor according to claim 2, wherein the seed solution of step 1) is inoculated into MRS culture medium, and the volume of the seed solution is 1-3%, preferably 2% of the volume of the MRS culture medium; in the step 1), the culture temperature is 33-37 ℃, preferably 35 ℃, and the standing culture time is 10-14 h, preferably 12 h.

5. The method for synthesizing phenyllactic acid by using the semi-hydrophobic crystal gum base whole-cell catalyst in the bioreactor according to claim 2, wherein the semi-hydrophobic anion exchange crystal gum of step 2) is put into MRS culture medium, and the volume of the crystal gum is 4-8%, preferably 6% of the volume of the MRS culture medium; in the step 2), the temperature of the static culture is 33-37 ℃, preferably 35 ℃, and the time of the static culture is 14-18 h, preferably 16 h.

6. The method for synthesizing phenyllactic acid by using the semi-hydrophobic crystal gum base whole-cell catalyst in the bioreactor according to claim 2, wherein the seed crystal gum carrier in the step 3) is put into the MRS culture medium, and the standing culture time is 36-60 h, preferably 48 h.

7. The method for synthesizing phenyllactic acid in a bioreactor by using a semi-hydrophobic crystal gum base whole-cell catalyst according to claim 2, wherein the crystal gum immobilized with lactobacillus paracasei cells in step 4) is put into the transformation liquid, and the volume of the crystal gum is 1-5%, preferably 2% of the volume of the transformation liquid.

8. The method for synthesizing phenyllactic acid by using the semi-hydrophobic crystal gum base whole-cell catalyst in the bioreactor according to claim 2, wherein the suspension culture in the step 4) is performed by using a stirring bioreactor, the suspension culture is performed under stirring, the stirring speed is 60-140 rpm, preferably 80 rpm, the catalytic conversion temperature is 33-37 ℃, preferably 35 ℃, and the catalytic conversion time is 12-84 h.

9. The method for synthesizing phenyllactic acid by using the semi-borrelidin-based whole-cell catalyst in the bioreactor as claimed in claim 2, wherein the conversion solution in step 4) contains glucose, phenylalanine and lactobacillus paracasei thallus, wherein the glucose concentration is 1-3 mg/mL, the phenylalanine concentration is 0.8-1.5 mg/mL, and the lactobacillus paracasei thallus concentration is 80-120 mg/mL.

10. The method for synthesizing phenyllactic acid by using the semi-hydrophobic crystal gum base whole-cell catalyst in the bioreactor according to claim 2, wherein in the step 4), the process of the permeabilization treatment is as follows: freezing for 5-8 h at-18 to-25 ℃, and then thawing at normal temperature.

Technical Field

The invention belongs to the field of biochemical engineering, and particularly relates to a method for synthesizing phenyllactic acid by using a semi-hydrophobic crystal gum base whole-cell catalyst in a bioreactor.

Background

The phenyllactic acid is a novel high-value organic acid, has good antibacterial and bacteriostatic effects, is an important monomer for synthesizing a novel polyphenylactic acid material, and is widely applied to the fields of food, chemical industry, biology, pharmacy and the like.

The existing preparation method of phenyllactic acid has certain disadvantages, the chemical synthesis method has complex route, is easy to generate byproducts and has serious environmental pollution; the traditional biosynthesis method has high cost, low yield and lack of high-yield strains.

The method has the advantages that the ultra-large pore crystal glue carrier is used for immobilizing lactobacillus paracasei cells to form the biocatalyst, the byproducts of the phenyllactic acid synthesized by conversion in the bioreactor are less, the environmental pollution is less, the cost is lower, the catalytic efficiency is higher, and a new direction is provided for the synthesis of the phenyllactic acid.

Disclosure of Invention

In view of the above problems in the prior art, the present invention aims to provide a method for synthesizing phenyllactic acid in a bioreactor by using a semi-hydrophobic crystal gum base whole-cell catalyst.

The method for synthesizing the phenyllactic acid by using the semi-hydrophobic crystal gum base whole-cell catalyst in the bioreactor is characterized in that lactobacillus paracasei cells are immobilized on semi-hydrophobic anion exchange crystal gel to form a high-concentration whole-cell catalyst, and then the crystal gel immobilized with the lactobacillus paracasei cells is put into a stirring bioreactor containing a conversion solution for suspension culture to synthesize the phenyllactic acid through catalytic conversion.

The method for synthesizing the phenyllactic acid by using the semi-hydrophobic crystal gum base whole-cell catalyst in the bioreactor is characterized by comprising the following steps of:

1) activating the lactobacillus paracasei strain in an MRS culture medium twice to obtain a seed solution, connecting the seed solution to the MRS culture medium, and performing standing culture;

2) putting the dried semi-hydrophobic anion exchange crystal gel into the MRS culture medium containing the seed liquid obtained in the step 1), and standing and culturing to obtain the seed crystal gel;

3) putting the seed crystal gel obtained in the step 2) into an MRS culture medium again, and continuing to perform static culture to obtain a high-concentration whole-cell catalyst, wherein the high-concentration whole-cell catalyst is the crystal gel immobilized with lactobacillus paracasei cells;

4) performing permeability treatment on the high-concentration whole-cell catalyst obtained in the step 3), putting the high-concentration whole-cell catalyst into a stirring bioreactor containing a conversion solution for suspension culture, and converting phenylalanine in the conversion solution into phenyllactic acid through whole-cell catalysis.

The method for synthesizing the phenyllactic acid by using the semi-hydrophobic crystal gum base whole-cell catalyst in the bioreactor is characterized in that the semi-hydrophobic anion exchange crystal gum is a poly (hydroxyethyl methacrylate) -butyl methacrylate based anion exchange spherical crystal gum carrier, the diameter of the crystal gum carrier is 2.0-5.0 mm, and the effective porosity is 74-85%.

The method for synthesizing the phenyllactic acid by using the semi-hydrophobic crystal gum base whole-cell catalyst in the bioreactor is characterized in that in the step 1), a seed solution is inoculated into an MRS culture medium, and the volume of the seed solution is 1-3% of that of the MRS culture medium, preferably 2%; in the step 1), the culture temperature is 33-37 ℃, preferably 35 ℃, and the standing culture time is 10-14 h, preferably 12 h.

The method for synthesizing the phenyllactic acid by using the semi-hydrophobic crystal gum base whole-cell catalyst in the bioreactor is characterized in that the semi-hydrophobic anion exchange crystal gum in the step 2) is put into an MRS culture medium, and the volume of the crystal gum is 4-8% of that of the MRS culture medium, preferably 6%; in the step 2), the temperature of the static culture is 33-37 ℃, preferably 35 ℃, and the time of the static culture is 14-18 h, preferably 16 h.

The method for synthesizing the phenyllactic acid by using the semi-hydrophobic crystal gum base whole-cell catalyst in the bioreactor is characterized in that the seed crystal gum carrier in the step 3) is put into an MRS culture medium, and the standing culture time is 36-60 hours, preferably 48 hours.

The method for synthesizing the phenyllactic acid by using the semi-hydrophobic crystal gum base whole-cell catalyst in the bioreactor is characterized in that the crystal gum immobilized with the lactobacillus paracasei cells in the step 4) is put into a conversion solution, and the volume of the crystal gum is 1-5% of that of the conversion solution, preferably 2%.

The method for synthesizing the phenyllactic acid by using the semi-hydrophobic crystal gum base whole-cell catalyst in the bioreactor is characterized in that step 4) is performed with suspension culture by using a stirring bioreactor, the suspension culture is performed under stirring, the stirring speed is 60-140 rpm, preferably 80 rpm, the catalytic conversion temperature is 33-37 ℃, preferably 35 ℃, and the catalytic conversion time is 12-84 hours.

The method for synthesizing the phenyllactic acid by using the semi-borrelidin crystal gum base whole-cell catalyst in the bioreactor is characterized in that the conversion solution in the step 4) contains glucose, phenylalanine and lactobacillus paracasei thallus components, wherein the glucose concentration is 1-3 mg/mL, the phenylalanine concentration is 0.8-1.5 mg/mL, and the lactobacillus paracasei thallus concentration is 80-120 mg/mL.

The method for synthesizing the phenyllactic acid by using the semi-hydrophobic crystal gum base whole-cell catalyst in the bioreactor is characterized in that in the step 4), the process of the permeability treatment is as follows: freezing for 5-8 h at-18 to-25 ℃, and then thawing at normal temperature.

By adopting the technology, the invention has the following beneficial effects:

1) according to the method provided by the invention, anion exchange crystal gel is used as a carrier, lactobacillus paracasei cells are immobilized on the crystal gel to form a high-concentration whole-cell catalyst, and a stirring bioreactor is used for suspension culture, so that the biomass in the conversion process is improved and the yield of phenyllactic acid is increased by reducing substrate inhibition. The yield of the phenyllactic acid synthesized by the method is higher than that of the phenyllactic acid synthesized by conventional free cell transformation, and the cell concentration in the transformation liquid is low, the viscosity is low and the phenyllactic acid is easy to separate because the cell is adsorbed on the crystal glue carrier. After the conversion is completed, the subsequent separation of the conversion solution can adopt the conventional separation methods, such as adsorption, crystallization, crystal gel chromatography, calcium salt precipitation and the like.

2) The method provided by the invention has the advantages that the crystal gel carrier has high porosity and large aperture, the mass transfer and diffusion of the substrate and the product are rapid, and the crystal gel is used as the carrier of the somatic cells, so that the stability of the enzyme system on the cell surface can be improved, and the substrate inhibition is reduced.

3) The method provided by the invention needs to be carried out in a stirring bioreactor through suspension culture. The method has the advantages of simple conversion process, convenient operation, good safety, easy amplification and wide application prospect in the field of biochemical synthesis.

Detailed Description

The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention.

Example 1

A method for synthesizing phenyllactic acid in a bioreactor by using a semi-hydrophobic crystal gum base whole-cell catalyst, comprising the following steps:

1) preparing MRS culture medium, and sterilizing at 121 deg.C for 20 min. Inoculating the preserved lactobacillus paracasei strain into 10 mL of MRS liquid culture medium, performing static culture at 35 ℃ for 12 h, then inoculating the strain into the liquid MRS culture medium in an inoculation amount of 2%, performing static culture at 35 ℃ for 12 h, and performing secondary activation to obtain seed liquid. Inoculating the seed solution into 500 mL of MRS liquid culture medium according to the inoculation amount of 6% of volume fraction, and performing static culture at 35 ℃ for 12 h to obtain the MRS culture medium containing the seed solution;

2) measuring a crystal gel carrier according to 6% of the volume fraction of the culture medium, wherein the crystal gel carrier is a poly (hydroxyethyl methacrylate) -butyl methacrylate based anion exchange crystal gel microsphere carrier, the diameter of the crystal gel microsphere carrier is 2.6-4.3 mm, the effective porosity is 76%, and the absolute dry porosity is 88%. Putting a crystal gel carrier into the MRS culture medium containing the seed liquid obtained in the step 1), and standing and culturing for 16 h at 35 ℃ to obtain seed crystal gel; then putting the seed crystal gel into an MRS culture medium again, and performing static culture at 35 ℃ for 48 hours to prepare a whole-cell catalyst; freezing at-20 deg.C for 6 hr, thawing at room temperature, and adding into a stirred bioreactor containing 5L of transformation solution containing Lactobacillus paracasei thallus concentration of 100 mg/mL, glucose concentration of 2 mg/mL, and phenylalanine concentration of 1 mg/mL. Setting the conversion temperature to 35 ℃, the stirring speed to 60 rpm and the conversion time to 84 h, centrifuging, taking the supernatant, detecting the content of the phenyllactic acid in the conversion solution to be 0.11 mg/mL, and calculating the result to be that the conversion rate of the phenyllactic acid is 11%.

Example 2

Experimental procedures the example 1 was repeated except that "in the step 2), the stirring speed in the stirring bioreactor was changed to 80 rpm", and the other steps were repeated the example 1, after the reaction was completed, the supernatant was centrifuged, the content of phenyllactic acid in the conversion solution was measured, and the conversion rate of phenyllactic acid was calculated to be 18%.

Example 3

Experimental procedures the example 1 was repeated except that in the step 2), the stirring speed in the stirring bioreactor was changed to 100 rpm, and the other steps were repeated in the example 1, after the reaction was completed, the supernatant was centrifuged, and the content of phenyllactic acid in the conversion solution was measured, with the result that the conversion rate of phenyllactic acid was 15%.

Example 4

Experimental procedures example 1 was repeated except that "step 2) was performed in which the stirring speed in the stirred bioreactor was changed to 120 rpm", and the rest of procedures example 1 was repeated, after the reaction was completed, the supernatant was centrifuged, and the content of phenyllactic acid in the conversion solution was measured, with the result that the conversion rate of phenyllactic acid was 14%.

Example 5

Experimental procedures example 1 was repeated except that "step 2) was performed in which the stirring speed in the stirred bioreactor was changed to 140 rpm", and the rest of procedures example 1 was repeated, after the reaction was completed, the supernatant was centrifuged, and the content of phenyllactic acid in the conversion solution was measured, with the result that the conversion rate of phenyllactic acid was 14%.

The statements in this specification merely set forth a list of implementations of the inventive concept and the scope of the present invention should not be construed as limited to the particular forms set forth in the examples.