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Method for preparing product of active lactic acid galactococcus

A Lactococcus lactis, active technology, applied in the field of preparation of Lactococcus lactis products, can solve the problems of low yield of Lactococcus lactis, low yield of expression products, limited practical application, etc., achieves good preservation effect, and prolongs logarithmic growth time. , the effect of reducing the cost of fermentation

Active Publication Date: 2009-11-25
BEIJING TRI PRIME GENE PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The obvious disadvantage of this technology is that the yield of expression products is very low, and the yield of Lactococcus lactis is also low, so the cost is extremely high, which limits its practical application

Method used

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  • Method for preparing product of active lactic acid galactococcus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Preparation of genetically engineered Lactococcus lactis highly expressing phenylalanine deaminase

[0022] 1. Using the PAL gene fragment in the plasmid pET23b-PAL as a template, all the codons on the parsley PAL were changed to the preferred codons of Lactococcus lactis, and artificially synthesized to obtain PAL art , the primers used were:

[0023] T1: 5'-GAGAACGGTAACGGTGCAACTAC-3',

[0024] T2: 5'-GCTCTAGAGCATGTCAGTTAAC-3'.

[0025] T2 contains Xba I restriction site;

[0026] 2. PAL art After being treated with T4 DNA polymerase, it was digested with Xba I to obtain a cDNA fragment with preferred codons of Lactococcus lactis;

[0027] 3. Digest the expression vector pNZ8149 with Nco I, and connect it with the cDNA fragment with the preferred codon of Lactococcus lactis;

[0028] 4. Transforming competent Lactococcus lactis with the above-mentioned recombinant plasmid;

[0029] 5. Screen high-expression strains in transformed Lactococcus lactis;

...

Embodiment 2

[0036] Embodiment 2: Determination of PAL enzyme activity expressed by Lactococcus lactis in the present invention

[0037] The bacteria collected in Example 1 were prepared into a 1 mg / ml suspension with 0.1 mol / L borate buffer (containing 10 mM phenylalanine) at pH 8.8, reacted at 30° C. for 1 hour, and reacted at 0, 15, and 30 °C respectively. , 45, and 60 minutes to collect the reaction supernatant, use spectrophotometry to measure the amount of conversion product cinnamic acid, and calculate the specific activity of PAL enzyme in the bacteria.

Embodiment 3

[0038] Embodiment 3: Pharmacodynamic identification of the present invention for treating phenylketonuria

[0039] Feed the bacterium obtained in Example 1 of the present invention to the gene-deficient phenylketonuria model mice, and the usage is a single oral administration per day, 0.5g / only, for 7 consecutive days. The results show that the phenylketonuria in the blood of the treated mice The amino acid levels were significantly lower than those in the control mice, and there was a significant difference between the two groups (P<0.01).

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Abstract

The invention discloses a method for preparing the product of active lactic acid galactococcus, which comprises the technical processes of constructing strains (constructing, connecting, transforming and selecting plasmids), preparing a fermentation culture medium, activating the strains, inoculating, fermentaing and drying. The strains with high expression and an optimized medium formulation are used in the method, the methods of pH control and the like are adopted for prolonging the logarithmic phase of the thalli, the density of the thalli in a fermenting system is increased by 100 times, the yield of living thalli is larger than 10 CFU / ml, and the expressing quantity of a PAL protein is larger than 12 percent. The obtained thalli have the ability for efficiently transforming phenyl alanine into cinnamylate, and the specific enzyme activity of the PAL is larger than 50U / g. The stability of the obtained thalli powder is good and can be kept for six months under 4 DEG C, the number of the living thalli can reach more then 10 CFU / g, and the specific enzyme activity of the PAL is not obviously reduced. The fermenting cost is reduced by 2 / 3. The PAL with low cost can be conveniently and largely provided for PKU treatment by using the invention.

Description

technical field [0001] The invention relates to a preparation method of a Lactococcus lactis product, in particular to a preparation method of an active Lactococcus lactis product expressing phenylalanine ammonia-lyase with high efficiency and low cost. Background technique [0002] Phenylalanine (phenylalanlne, phe) is one of the essential amino acids for the human body. Except for a small part of phenylalanine that normal people ingest from the diet and recycled in the body, most of it is in the phenylalanine in the liver. It is converted into tyrosine by the action of phenylalanine hydroxylase (PAH) and then utilized. Patients with autosomal recessive genetic disease - phenylketonuria (PKU), due to phenylalanine hydroxylase gene defect, lead to reduced or absent liver PAH enzyme activity, unable to phenylalanine It is converted into tyrosine, causing phenylalanine to be converted into phenylpyruvate, phenylacetic acid, phenyllactic acid, etc. through other pathways. The...

Claims

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Application Information

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IPC IPC(8): C12N15/60C12N15/74C12N1/21C12R1/225
Inventor 牛春刘金毅高天星孙俭波程永庆
Owner BEIJING TRI PRIME GENE PHARMA CO LTD
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