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High-purity extraction method of polyhydroxyalkanoate

A technology of polyhydroxyalkanoate and bacteria, which is applied in the field of bioengineering technology and biochemical industry, can solve the problems of difficult to meet the requirements of the application market, molecular weight loss, etc., and achieve the effect of industrial application of green production, high molecular weight and simple operation

Active Publication Date: 2022-04-22
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although the method is simple in extraction process, it still needs to add anionic surfactant
[0006] Patent document CN111019108B discloses a method for extracting and purifying polyhydroxyalkanoate, including using superheated water to break the wall, but due to degradation under extreme conditions, its molecular weight loss is serious and it is difficult to meet the requirements of the application market

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Determination of polyhydroxyalkanoate content (refer to the literature Unsterile and continuous production of polyhydroxybutyrate by Halomonas TD01, Tan D, et al, Bioresource Technology, 2011, 102(17): 8130-8136):

[0073] According to the different monomer components of the samples, PHB and butyrolactone were selected as the external standard, and benzoic acid was used as the internal standard. The standard sample and the sample form a monomer through acid hydrolysis, and esterify with methanol to generate the corresponding methyl ester, which is then detected by gas chromatography, such as the sum of the content of P34HB, 3HB and 4HB (see attached figure 1 ).

[0074] GPC method for determining the molecular weight of polyhydroxyalkanoate:

[0075] Polystyrenes with different molecular weights (20000Da~1800000Da) were used as standard products, and prepared into solutions with a concentration of 1mg / mL with chloroform to make a standard curve. The mobile phase was c...

Embodiment 2

[0078] The influence of embodiment 2 solid-liquid separation mode and number of times on PHA content

[0079]Take 200mL of fermentation liquid of halophilic bacteria in a tank, centrifuge at 8000rpm for 10min, wash with water for different times, and freeze-dry the obtained bacterial pellet for 48 hours to detect the PHA content. The results showed that the effect of breaking the wall was the best after centrifuging once to completely remove the supernatant before breaking the wall. Although excessive washing times can remove some soluble impurities and slightly improve product purity, it consumes a large amount of washing water, which is not conducive to industrial applications.

[0080] Further, by simulating an industrial disc centrifuge, take 200 mL of fermentation broth, centrifuge at 8000 rpm for 10 minutes, remove 65% of the supernatant, and mix the remaining part to form an industrial centrifugation. Add 0.5% of the purification aid sorbitol, pH9.5, and treat at 90°C ...

Embodiment 3

[0083] The influence of embodiment 3 wall breaking temperature and time on PHA content

[0084] Select the same batch of fermentation liquid in the tank to break the wall under the premise of other conditions unchanged (washing 3 times, 200g / L cell concentration, breaking pH 10.0, treatment time 120min, etc.), but design different broken walls Temperature, to study the effect of temperature on PHA content. The molecular weight test results show that the molecular weight remains unchanged when the temperature is lower than 40°C, and the molecular weight of the PHA finished product decreases rapidly as the wall breaking temperature continues to increase (see attached figure 2 ).

[0085] The GC test results of samples with different breaking temperatures show that when the temperature is too high, the PHA content of the finished product will decrease instead, indicating that too high a temperature will cause irreversible denaturation and precipitation of impurities such as pro...

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Abstract

The invention provides a method for extracting polyhydroxyalkanoate from bacteria. Specifically, the polyhydroxyalkanoate PHA derived from bacterial fermentation is optimized by optimizing the wall-breaking conditions (temperature, pH, concentration of bacterial solution, Wall-breaking aids and protective agents, etc.) for efficient wall-breaking, and enzymatic hydrolysis to achieve high recovery and high-purity extraction of PHA. The recovery rate of various PHA products obtained by the method reaches over 96%, the purity reaches over 95%, and the molecular weight reaches over 30% of the initial molecular weight.

Description

technical field [0001] The invention relates to the fields of bioengineering technology and biochemical technology, in particular to a high-purity extraction method of polyhydroxyalkanoate (PHA), including high-efficiency wall-breaking technology and high-purity enzymolysis extraction technology. Background technique [0002] As a biodegradable new biomaterial, polyhydroxyalkanoate has broad application prospects in the fields of medicine, chemical industry, agriculture, and daily use. It can be used to develop medical devices, medical microspheres, surgical sutures, patches, disposable packaging materials, textile fibers, etc. Due to its excellent biodegradability and biocompatibility, it is recognized as the most potential green environmental protection molecular material. [0003] Although polyhydroxyalkanoate has many advantages in application, and after metabolic engineering, bacteria can synthesize PHA accounting for more than 70% of dry cell weight, but the downstrea...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C08G63/78C08G63/06C12N1/06
CPCC08G63/78C08G63/06C12N1/06
Inventor 陈国强杨宏宇吴赴清
Owner TSINGHUA UNIV
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