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Method for producing hyaluronic acids with different molecular weights through pichia pastoris gene-regulation fermentation

A technology of Pichia pastoris and gene regulation, applied in the field of genetic engineering and biological fermentation, to achieve the effect of solving the difficulty of molecular weight grading

Inactive Publication Date: 2013-02-06
INST OF BIOPHARM OF SHANDONG PROVINCE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The enzymatic degradation method has mild reaction conditions and fast degradation speed, which is more suitable for the preparation of HA with different molecular weights, but it needs to prepare the enzyme solution first, and then use the enzyme solution to degrade HA

Method used

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  • Method for producing hyaluronic acids with different molecular weights through pichia pastoris gene-regulation fermentation
  • Method for producing hyaluronic acids with different molecular weights through pichia pastoris gene-regulation fermentation
  • Method for producing hyaluronic acids with different molecular weights through pichia pastoris gene-regulation fermentation

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Experimental program
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Effect test

Embodiment 1

[0022] Example 1: Construction of recombinant plasmids pGAPZαA-szHasA and pPICZαA-ph20 and transformation of Pichia pastoris

[0023] 1 Construction of recombinant plasmid pGAPZαA-szHasA

[0024] 1 The whole gene synthesis sequence of the hyaluronic acid synthase gene szHasA of Streptococcus zooepidemicus in Table 1 was cloned and amplified by PCR. The PCR primer sequences are as follows:

[0025] Forward: 5'-CCGCTCGAGGCAGAACATTAAAAAACCTCA-3'

[0026] Reverse: 5'-CATTTCTAGATTATAATAATTTTTTACGTGTT-3'

[0027] PCR reaction conditions:

[0028] Pre-denaturation: 94°C, 5min

[0029] Denaturation: 94°C, 1min

[0030] Refolding: 56°C, 1min

[0031] Extension: 72°C, 2min

[0032] Cycles: 30

[0033] Extension: 72°C, 10min

[0034] 2 Construction of recombinant plasmids

[0035] Detected by 0.7% agarose electrophoresis, a PCR product with a size of about 1.2 kb was recovered for cloning and expression. The PCR recovered product and the pPICZαA vector were digested with restri...

Embodiment 2

[0065] Embodiment 2: the method for producing multiple different molecular weight HA by fermentation of yeast engineering bacteria HAS-PH20

[0066] In a 1-liter fermenter, the engineered yeast strain HAS-PH20 can directly produce HA with different molecular weights. First, under the condition of 30°C, activate and culture the recombinant yeast HAS-PH20 in YPD medium for 24 hours; inoculate the yeast cultivated overnight in the YPD seed medium at a ratio of 0.5% to 2%, and ferment in the tank as follows :

[0067] (1) Cell culture stage: the fermentation temperature is maintained at 28-30°C, the pH value of the fermentation medium is adjusted to 5.0-6.0 with 28% ammonia water, and then the seed medium is inoculated into the Add 4.37mL PTM1 salt solution to the Basal Salts upper tank medium at the same time, and aerate and stir for 18 to 24 hours;

[0068] (2) Glycerol feeding stage, that is, the stage of constitutively expressing the hyaluronic acid synthase of Streptococcus...

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Abstract

The invention relates to a novel method for producing hyaluronic acids (HA) with different molecular weights through pichia pastoris gene-regulation fermentation. The method comprises the steps that: recombinant plasmid pGAPZalphaA-szHasA constitutively expressing streptococcus zooepidemicus hyaluronic acid synthase and recombinant plasmid pPICZalphaA-ph20 inducibly expressing recombinant human hyaluronidase are used for converting yeast cells, such that a target strain HAS-PH20 is obtained. Through different gene regulation and induction methods, the yeast engineering strain HAS-PH20 directly produces various hyaluronic acids with different molecular weights through fermentation processes. With the method, the coupling of hyaluronic acid molecular weights and gene regulation methods is realized, and hyaluronic acids with specific molecular weights can be prepared according to requirements.

Description

technical field [0001] The invention relates to the fields of genetic engineering and biological fermentation, in particular to a method for producing hyaluronic acid with different molecular weights by using Pichia pastoris gene regulation and fermentation. Background technique [0002] Hyaluronic acid (HA) is a linear polysaccharide structure composed of repeating structures of N-hexanoylglucosamine and D-glucuronic acid. HA plays a significant role in lubricating joints, regulating the permeability of blood vessel walls, and promoting wound healing. More importantly, it has a special water-retaining effect and is one of the best moisturizing substances found in nature. [0003] However, due to the large molecular weight of HA, it will not be absorbed by the dermis when it is applied to the epidermis, so that its effect is greatly reduced. And low molecular weight HA oligosaccharides (1×10 5 ~4×10 5 ) can penetrate into the dermis, regulate skin metabolism, promote blo...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12P19/04C12R1/84
Inventor 朱希强刘飞张莉刘霞钊倩倩王绍花陈勉侯重文袁丹丹张林军陈晓燕
Owner INST OF BIOPHARM OF SHANDONG PROVINCE
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