Production method and application of special dietary protein

A construction method and technology of phenylalanine protein are applied in the production and application field of special dietary protein, which can solve the problems of limited yield, poor patient compliance with treatment, complicated production and purification process, etc.

Inactive Publication Date: 2020-02-04
湖北康肽药业有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are many disadvantages in the traditional phenylalanine-restricted diet therapy: (1) Since the protein of most foods contains phenylalanine, the food that can be selected is very limited, and you can only choose protein-free or very low-protein foods. (2) Using amino acid compound powder as a substitute for dietary protein, because many amino acids have a strong bitter taste and special unpleasant smell, resulting in amino acid compound powder The taste of the food is poor, and the patient's treatment compliance is poor; in addition, due to oral administration of a large amount of free amino acids, the osmotic pressure of the intestinal tract is disturbed, causing intestinal discomfort and intestinal metabolic disorders; thirdly, the food form that amino acid powder can be made into is very limited , long-term use leads to a decline in the quality of life of patients
(3) Continuous neurological and psychological problems due to poor quality of life; (4) Long-term dietary restrictions or single choices lead to nutritional deficiencies; (5) The cost of special foods and nutrients is relatively high, and the family burden is heavy
Because patients with phenylketonuria need to use it for a long time and consume at least 15-75g of protein per day, if the expression efficiency is low or the production and purification process is complicated, the production will be limited and the price will be expensive. Ordinary patients with phenylketonuria If it is unaffordable economically, such a product cannot meet the market demand

Method used

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  • Production method and application of special dietary protein
  • Production method and application of special dietary protein
  • Production method and application of special dietary protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1: Construction of Bacillus licheniformis strains expressing no / low phenylalanine content protein

[0069] The Bacillus licheniformis strain (B. licheniformis CICC 10266) used in the examples was purchased from China Industrial Microorganism Culture Collection and Management Center.

[0070] The pEBKan194-GFP plasmid used in the examples is pKGFP194ts in another patent application of the applicant (application number: 201610410811.8), and its construction information can be found in Example 1 of the specification of this application.

[0071] Unless otherwise specified, the reagents used in the experiment are all commercially available scientific research reagents or consumables. The ingredients and preparation methods of various reagents and media can refer to the operation in the routine experiment manual.

[0072] 1. Construction of phenylalanine-free protein expression strain BL01

[0073] 1. Knockout of the original gene in the expression strain

[0074] ...

Embodiment 2

[0122] Example 2: Fermentation production and purification preparation of no / low phenylalanine recombinant protein powder

[0123] This embodiment provides a production method of no / low phenylalanine recombinant protein powder, the specific steps are as follows:

[0124] 1) With the recombinant Bacillus licheniformis BL01 constructed in Example 1 as the starting strain, a 5L fermenter was used to carry out continuous feeding process fermentation, and the fermentation base medium was: 3% corn starch, 2% soybean protein isolate, and 0.5% yeast extract powder. %, K 2 HPO 4 0.45%, NaH 2 PO 4 0.7%, CaCl 2 0.04%, MgSO 4 ·7H 2 O 0.08%, MnSO 4 0.02%, defoamer 0.1%, α-high temperature amylase 0.01%, adjust pH to 7.5, base liquid volume is 50%, high temperature sterilization at 120°C for 20min; feeding medium: glucose, soybean hydrolyzed protein 10% , Yeast powder 10%, high temperature sterilization at 120°C. Fermentation inoculum amount is 5%, culture conditions: 37°C, aera...

Embodiment 3

[0127] Embodiment 3: the production of no / low phenylalanine recombinant protein powder

[0128] This embodiment provides a production method of no / low phenylalanine recombinant protein powder, the specific steps are as follows:

[0129] 1) Using the recombinant Bacillus licheniformis BL02 constructed in Example 1 as the starting strain, a 30L fermenter was used to carry out continuous feeding process fermentation, and the basic fermentation medium was: glucose 4%, soybean protein powder 3%, K 2 HPO 4 0.4%, NaH 2 PO 4 0.8%, CaCl 2 0.03%, MgSO 4 ·7H 2 O 0.06%, MnSO 4 0.03%, defoamer 0.2%, adjust the pH to 7.5, the basic liquid volume is 40%, 120 ℃ high-temperature sterilization for 20 minutes; feeding medium: glucose 40%, soybean hydrolyzed protein 15% + yeast powder 5%, Both were sterilized at high temperature at 120°C. Fermentation inoculum amount is 3%, culture conditions: 37°C, aeration rate 0.6-1.2vvm, DO control over 50% by adjusting the number of revolutions an...

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Abstract

The invention provides a production method and application of special dietary protein. A protease protein sequence from bacillus is used as a template, phenylalanine is knocked out and replaced, and part of amino acids is rearranged, so that it is ensured that phenylalanine-free protein has protease specific activity in the fermentation process, and other impure protein is degraded or growth of other impure protein is inhibited; a fed-batch fermentation process is adopted, so that the logarithmic growth period of a strain is prolonged, and the protein expression index is increased; besides, apost-treatment process is optimized, and the obtained protein powder is high in purity and good in mouth feel. The ratio of LNAA to Phe in the obtained recombinant phenylalanine-free protein exceeds 100 times, so that the situation that phenylalanine in blood enters the brain can be effectively reduced. According to the method, the production cost of the phenylalanine-free or low-phenylalanine protein powder can be reduced to reach the cost of a common protein product, and the protein powder has great practical application value and broad market prospects.

Description

technical field [0001] The present invention relates to the field of genetic engineering, in particular to a method for expression in Bacillus licheniformis and production of a recombinant protein suitable for patients with phenylketonuria, which includes using the protease protein sequence derived from Bacillus as a template when designing the recombinant protein, Knock out and replace phenylalanine, and rearrange some amino acids, so as to ensure that the newly designed phenylalanine-free protein has protease activity during the fermentation process, and can be simply processed during post-fermentation treatment It permanently loses its protease activity, thereby ensuring the safety of dietary protein. Background technique [0002] Phenylketonuria (PKU) is a rare genetic disease of amino acid metabolism. The incidence rate in Chinese newborns is about 1:11800, and the total number of PKU patients in China is about 120,000. PKU is autosomal recessive and is due to key enzy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/75C12N15/90C12N15/57C12N1/21C12N9/54A23L33/195A61K38/48A61P19/06C12R1/10
CPCA23V2002/00A61K38/48A23L33/195A61P19/06C12N9/54C12N15/75C12N15/902C12N2800/22A23V2200/30
Inventor 宋保平刘海峰李春艳陈火晴姚琦
Owner 湖北康肽药业有限公司
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