Method for preparing L-carnosine under catalysis of yeast whole-cell catalyst

Abstract

The invention discloses a method for catalytically preparing L-carnosine by using a yeast whole-cell catalyst, which comprises the following steps: adding the yeast whole-cell catalyst into a substrate solution containing 100mmol / L beta-alanine methyl ester and 200mmol / L L-histidine in a bioreactor, reacting for 60 minutes at 20 DEG C, maintaining the reaction pH to be 8.5 by using a 6mmol / L NaOH solution, and after the reaction is finished, separating and purifying to obtain the L-carnosine. The L-carnosine can be obtained; the yeast whole-cell catalyst is utilized to catalyze beta-alanine methyl ester and histidine to generate L-carnosine, and the whole-cell catalyst has the advantages of immobilized enzyme and can be repeatedly used, so that the problems of the existing biosynthesis method and chemical method are effectively solved, and the preparation process is simple and suitable for industrial production.

Description

Technical Field

[0001] The invention relates to the technical field of biocatalysis, and more specifically to a method for preparing L-carnosine by utilizing a yeast whole cell catalyst. Background Art

[0002] L-carnosine (β-alanyl-L-histidine) is an imidazole dipeptide composed of β-alanine and histamine, and is a natural active dipeptide widely found in the brain, muscles and other important tissues of mammals. Since the discovery of this active peptide over a hundred years ago, a large number of studies have found or proved that L-carnosine has significant antioxidant, intracellular free radical elimination, anti-aging and other activities, and is clinically used for auxiliary treatment of hypertension, heart disease, senile cataracts, ulcers, anti-tumor, promotion of wound healing, etc. Due to its strong antioxidant activity, low toxicity and side effects and multiple physiological activities, this active peptide and its derivatives are widely used in the fields of medicine, health care, hygiene, cosmetics, etc., and have broad market prospects.

[0003] At present, the synthesis methods of L-carnosine mainly include chemical synthesis and enzyme catalysis synthesis. The chemical synthesis method mainly uses phthalic anhydride and β-alanine to generate phthalyl-β-alanine to protect the amino group, and the carboxyl group reacts with thionyl chloride to generate phthalyl-β-alanyl chloride, and then forms a peptide bond with the protected L-histidine and removes the protecting group to obtain the product. This route is relatively complicated, with low yield, easy racemization during the peptide bond formation process, affecting the purity of the product, and high solvent consumption, which is easy to cause environmental pollution;

[0004] In the prior art, CN107217048A discloses an aminopeptidase for catalyzing the synthesis of carnosine and a preparation method thereof, which utilizes aminopeptidase to catalyze the synthesis of L-carnosine from β-alanine methyl ester and L-histidine in one step. However, since aminopeptidase has a very high exonuclease activity, it is impossible to accumulate high-concentration L-carnosine in the reaction system. In addition, the synthesis of L-carnosine using aminopeptidase has high requirements on the reaction system, and often requires an aqueous phase and an organic phase system to reduce the hydrolysis of the product. However, the organic phase has a certain degree of inhibition on the activity of the enzyme, and the solubility of the amino acid substrate in the organic phase is also relatively low. Various reasons all lead to a very low yield of L-carnosine synthesized based on aminopeptidase.

[0005] Therefore, how to provide a method for preparing L-carnosine using yeast whole cell catalyst to improve its yield is a problem that those skilled in the art need to solve urgently. Summary of the invention

[0006] In view of this, the present invention provides a method for preparing L-carnosine by using a yeast whole cell catalyst.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A method for preparing L-carnosine by using a yeast whole cell catalyst comprises: adding the yeast whole cell catalyst to a substrate solution containing 100 mmol / L beta-alanine methyl ester and 200 mmol / L L-histidine in a bioreactor, reacting for 60 minutes at 20° C., and maintaining the reaction pH at a constant level of 8.5 using a 6 mmol / L NaOH solution. After the reaction is completed, L-carnosine can be obtained.

[0009] Preferably, the yeast whole cell catalyst is obtained by amplifying the CocE / NonD family hydrolase gene AET of Sphingobacterium spp. after being treated with BamHI / XhoI restriction endonucleases, ligating it into a surface display expression vector pYD1 containing an a-lectin, and finally transferring the constructed yeast surface display expression vector pYD1-AET into flocculant bacteria.

[0010] More preferably, the preparation of the flocculant comprises the following steps:

[0011] (1) The promoter PGK and the flocculation gene FLO1 were amplified respectively, treated with XhoI / HindIII and BamHI / NotI restriction enzymes respectively, and then inserted into the pRS425 vector in sequence to obtain the yeast flocculation expression vector pRS425-PGK-FLO1;

[0012] (2) The yeast flocculation expression vector pRS425-PGK-FLO1 was transferred into Escherichia coli DH5α for cultivation, the plasmid was extracted from the mixed bacteria, and a mutant library with random loss of the FLO1 flocculation gene repeat sequence was prepared;

[0013] (3) The library of mutants with random loss of flocculation gene repeat sequences was transferred to Saccharomyces cerevisiae EBY100 for screening and verification, and a strain with flocculation gene was screened out.

[0014] Preferably, the method for preparing the yeast whole cell catalyst comprises the following steps:

[0015] (1) Inoculate the yeast whole cell catalyst into 100 mL of seed culture medium and culture at 30°C and 200 rpm until the OD 620 is 2.0, and bacterial cell A is obtained;

[0016] (2) Cell A was transferred to 200 mL of induction medium and cultured at 25°C and 200 rpm for 48 h to obtain yeast whole-cell catalyst.

[0017] More preferably, in step (1), the preparation of the seed culture medium comprises: weighing 1.34 g of YNB, adding 80 mL of ddH2O, sterilizing at 121°C for 15 min; after sterilization, adding 10 mL of 10× amino acid mixture sterilized by membrane and 10 mL of 40% glucose.

[0018] More preferably, in step (2), the preparation of the induction medium comprises: weighing 2.68 g of YNB, adding 160 mL of ddH2O, and sterilizing at 121°C for 15 min; after sterilization, adding 20 mL of 10× amino acid mixture sterilized by membrane and 20 mL of 40% galactose.

[0019] More preferably, the 10× amino acid mixture comprises: 0.2 g / L arginine, 1.0 g / L aspartic acid, 1.0 g / L glutamic acid, 0.3 g / L isoleucine, 0.3 g / L lysine, 1.5 g / L valine, 0.2 g / L methionine, 0.5 g / L phenylalanine, 3.75 g / L serine, 0.3 g / L tyrosine and 0.4 g / L adenine.

[0020] It can be seen from the above technical solution that, compared with the prior art, the present invention discloses a method for preparing L-carnosine using yeast whole cell catalyst, which has the following beneficial effects:

[0021] (1) The present invention obtains a whole-cell catalyst for the green synthesis of L-carnosine by screening a mutant strain containing a flocculation gene, connecting a CocE / NonD family hydrolase gene to a surface display expression vector, and then transferring the vector into a mutant strain containing a flocculation gene. The prepared cell catalyst is used to catalyze the preparation of L-carnosine, thereby obtaining a high-yield and high-concentration L-carnosine, which effectively solves the problem that the current chemical method and biosynthesis method cannot obtain a high-yield L-carnosine, and lays a foundation for the industrial whole-cell catalytic production of L-carnosine.

[0022] (2) The yeast whole cell catalyst prepared by the present invention has the advantages of immobilized enzyme, good stability, reusability, simple preparation, saving enzyme immobilization equipment and reducing production costs. DETAILED DESCRIPTION

[0023] The following will be described clearly and completely in conjunction with the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] Example 1 Screening and preparation of flocculants

[0025] The promoter PGK (SEQ ID NO.1) and the flocculation gene FLO1 (SEQ ID NO.2) were amplified by PCR, treated with XhoI / HindIII and BamHI / NotI restriction enzymes, and then inserted into the pRS425 vector in sequence. The constructed yeast flocculation expression vector pRS425-PGK-FLO1 was transferred into Escherichia coli DH5α for culture, and the mixed bacteria were used to extract plasmids to prepare a mutant library with random loss of the repeat sequence of the FLO1 flocculation gene; the mutant library with random loss of the repeat sequence of the flocculation gene was transferred to Saccharomyces cerevisiae EBY100 for screening and verification, and the positive transformant EP-FLO1 was evaluated. m The flocculation characteristics were sequenced.

[0026] The nucleotide sequence of the promoter PGK is shown in SEQ ID NO.1.

[0028] The nucleotide sequence of the flocculation gene FLO1 is shown in SEQ ID NO.2.

[0029] According to the sequencing results, the number of repeat sequences in the flocculation gene is positively correlated with the size of the flocculation particles. The three flocculation gene mutants EP-FLO1-a10 (flocculation gene nucleotide sequence is SEQ ID NO.3), EP-FLO1-a14 (flocculation gene nucleotide sequence is SEQ ID NO.4) and EP-FLO1-a16 (flocculation gene nucleotide sequence is SEQ ID NO.5) with 10, 14 and 16 repeat sequences lost intuitively showed significant differences in the size of flocculation particles. Among them, EP-FLO1-a10 and EP-FLO1-a14 can still maintain millimeter-level flocculation particles, but EP-FLO1-a16, which has lost 16 repeat sequences, has almost lost its flocculation characteristics.

[0030] The nucleotide sequence of EP-FLO1-a10 is shown in SEQ ID NO.3.

[0032] The nucleotide sequence of EP-FLO1-a14 is shown in SEQ ID NO.4.

[0033] The nucleotide sequence of EP-FLO1-a16 is shown in SEQ ID NO.5.

[0034] Example 2

[0035] A method for preparing L-carnosine by catalysis using a yeast whole cell catalyst comprises the following steps:

[0036] (1) Preparation of yeast whole-cell catalyst: The CocE / Non D family hydrolase gene AET (SEQ ID NO.6, SEQ ID NO.7) of Sphingobacterium was amplified by PCR, treated with BamHI / XhoI restriction endonucleases, and then ligated into the surface display expression vector pYD1 containing a-lectin. Finally, the constructed yeast surface display expression vector pYD1-AET was transferred into the flocculant EP-FLO1-a10 to obtain the yeast whole-cell catalyst EPA-FLO1-a10;

[0037] (2) adding the yeast whole cell catalyst in step (1) to a substrate solution containing 100 mmol / L β-alanine methyl ester (β-Ala-OMe) and 200 mmol / L L-histidine (L-His) in a bioreactor, reacting at 20° C. for 60 min, and maintaining the reaction pH at a constant level of 8.5 with 6 mmol / L NaOH solution. After the reaction is completed, L-carnosine can be obtained;

[0038] (3) Finally, the concentration of L-carnosine was detected by high performance liquid chromatography (HPLC). It was calculated that 72.8 mmol / L of L-carnosine could be generated, with a conversion rate of up to 73%.

[0039] The nucleotide sequence of AET is shown in SEQ ID NO.6.

[0040] The amino acid sequence of AET is shown in SEQ ID NO.7.

[0041] Example 3

[0042] A method for preparing L-carnosine by catalysis using a yeast whole cell catalyst comprises the following steps:

[0043] (1) Preparation of yeast whole-cell catalyst: The CocE / Non D family hydrolase gene AET of Sphingobacterium was amplified by PCR and treated with BamHI / XhoI restriction endonucleases, and then ligated into the surface display expression vector pYD1 containing a-lectin. Finally, the constructed yeast surface display expression vector p YD1-AET was transferred into the flocculant EP-FLO1-a14 to obtain the yeast whole-cell catalyst EPA-FLO1-a14;

[0044] (2) adding the yeast whole cell catalyst in step (1) to a substrate solution containing 100 mmol / L β-alanine methyl ester (β-Ala-OMe) and 200 mmol / L L-histidine (L-His) in a bioreactor, reacting at 20° C. for 60 min, and maintaining the reaction pH at a constant level of 8.5 with 6 mmol / L NaOH solution. After the reaction is completed, L-carnosine can be obtained;

[0045] (3) Finally, the concentration of L-carnosine was detected by high performance liquid chromatography (HPLC). It was calculated that 70.8 mmol / L of L-carnosine could be generated, and the conversion rate was as high as 71.2%.

[0046] Example 4

[0047] A method for preparing L-carnosine by catalysis using a yeast whole cell catalyst comprises the following steps:

[0048] (1) Preparation of yeast whole-cell catalyst: The CocE / Non D family hydrolase gene AET of Sphingobacterium was amplified by PCR and treated with BamHI / XhoI restriction endonucleases, and then ligated into the surface display expression vector pYD1 containing a-lectin. Finally, the constructed yeast surface display expression vector p YD1-AET was transferred into the flocculant EP-FLO1-a16 to obtain the yeast whole-cell catalyst EPA-FLO1-a16;

[0049] (2) adding the yeast whole cell catalyst in step (1) to a substrate solution containing 100 mmol / L β-alanine methyl ester (β-Ala-OMe) and 200 mmol / L L-histidine (L-His) in a bioreactor, reacting at 20° C. for 60 min, and maintaining the reaction pH at a constant level of 8.5 with 6 mmol / L NaOH solution. After the reaction is completed, L-carnosine can be obtained;

[0050] (3) Finally, the concentration of L-carnosine was detected by high performance liquid chromatography (HPLC). It was calculated that 69.7 mmol / L of L-carnosine could be generated, and the conversion rate was as high as 70.5%.

[0051] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0052] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing L-carnosine using a yeast whole cell catalyst, characterized in that: include: In a bioreactor, add yeast whole cell catalyst to a substrate solution containing 100 mmol / L β-alanine methyl ester and 200 mmol / L L-histidine, react at 20°C for 60 minutes, and use 6 mmol / L NaOH solution to maintain the reaction pH constant at 8.

5. After the reaction is completed, L-carnosine can be obtained.

2. A method for preparing L-carnosine using a yeast whole cell catalyst according to claim 1, characterized in that: The yeast whole cell catalyst is obtained by amplifying the CocE / NonD family hydrolase gene AET of Sphingobacterium sphingosineum, treating it with BamHI / XhoI restriction endonucleases, connecting it to a surface display expression vector pYD1 containing a-lectin, and finally transferring the constructed yeast surface display expression vector pYD1-AET into flocculants.

3. A method for preparing L-carnosine using a yeast whole cell catalyst according to claim 2, characterized in that: The preparation of the flocculant comprises the following steps: (1) The promoter PGK and the flocculation gene FLO1 were amplified respectively, treated with XhoI / HindIII and BamHI / NotI restriction enzymes respectively, and then inserted into the pRS425 vector in sequence to obtain the yeast flocculation expression vector pRS425-PGK-FLO1; (2) The yeast flocculation expression vector pRS425-PGK-FLO1 was transferred into Escherichia coli DH5α for cultivation, and the plasmid was extracted by mixing the bacteria to prepare a mutant library with random loss of the FLO1 flocculation gene repeat sequence; (3) The library of mutants with random loss of flocculation gene repeat sequences was transferred to Saccharomyces cerevisiae EBY100 for screening and verification, and a strain with flocculation gene was screened out.

4. A method for preparing L-carnosine using a yeast whole cell catalyst according to claim 1, characterized in that: The method for preparing the yeast whole cell catalyst comprises the following steps: (1) Inoculate the yeast whole cell catalyst into 100 mL of seed culture medium and culture at 30°C and 200 rpm until the OD 620 is 2.0, and bacterial cell A is obtained; (2) Cell A was transferred to 200 mL of induction medium and cultured at 25°C and 200 rpm for 48 h to obtain yeast whole-cell catalyst.

5. A method for preparing L-carnosine using a yeast whole cell catalyst according to claim 4, characterized in that: In step (1), the preparation of the seed culture medium comprises: weighing 1.34 g of YNB, adding 80 mL of ddH2O, and sterilizing at 121°C for 15 min; after sterilization, adding 10 mL of 10× amino acid mixture sterilized by membrane and 10 mL of 40% glucose.

6. A method for preparing L-carnosine using a yeast whole cell catalyst according to claim 4, characterized in that: In step (2), the preparation of the induction medium includes: weighing 2.68 g of YNB, adding 160 mL of ddH2O, and sterilizing at 121°C for 15 min; after sterilization, adding 20 mL of 10× amino acid mixture sterilized by membrane and 20 mL of 40% galactose.

7. A method for preparing L-carnosine using a yeast whole cell catalyst according to any one of claims 5-6, characterized in that: The 10× amino acid mixture comprises: 0.2 g / L arginine, 1.0 g / L aspartic acid, 1.0 g / L glutamic acid, 0.3 g / L isoleucine, 0.3 g / L lysine, 1.5 g / L valine, 0.2 g / L methionine, 0.5 g / L phenylalanine, 3.75 g / L serine, 0.3 g / L tyrosine and 0.4 g / L adenine.