CN1070534C - Method for separating and refining polyhydroxy fatty acid ester in bacteria cell from bacteria - Google Patents

Abstract

The invention belongs to the technical field of bioengineering downstream post-processing. Including: (1) treating the bacterial cells obtained by fermentation with an anionic surfactant under alkaline conditions, and centrifuging to extract the polyhydroxyalkanoate particles contained in it; (2) treating the polyhydroxyalkanoate obtained in the previous step with protease Ester product, (3) collect and dry gained polyhydroxyalkanoate product. The invention utilizes relatively cheap raw materials, has mild reaction conditions, and requires less investment in production equipment, is suitable for industrialized large-scale production, and greatly reduces the production cost of polyhydroxyalkanoate.

Description

Method for Separating and Purifying Intracellular Polyhydroxyalkanoate from Bacteria

The invention belongs to the technical field of bioengineering downstream post-processing.

Poly-β-Hydroxyalkanoates (PHA for short) is a kind of bacterial intracellular content, which has the characteristics of thermoplastics. At the same time, because of its biodegradability and biocompatibility, it has potential applications in various aspects. However, because it exists in bacteria in the form of inclusions, its composition is complex, making its purification extremely difficult. Existing downstream post-processing purification processes, either use organic solvents (such as chloroform, methylene chloride, etc.) for extraction, or use high-temperature heating combined with the use of a variety of expensive enzymes to decompose non-polyhydroxyalkanoate components in cells for the purpose of purification. The complex production process, high equipment investment and expensive raw material cost lead to the high price of polyhydroxyalkanoate products, making it difficult to promote the use of this promising biodegradable plastic. The applicant of the present invention has found through experiments that the bacterial cells are first treated with an anionic alkaline solution, the polyhydroxyalkanoate particles are separated and extracted, and then the protein impurities remaining on the particles are further treated with protease to obtain high-purity polyhydroxyalkanoate ester products. Because the process has mild reaction conditions, no special requirements for equipment, and cheap raw materials, it is suitable for industrialized large-scale production and greatly reduces the production cost of polyhydroxyalkanoate.

The purpose of the present invention is to propose to use cheap anionic surfactant and a small amount of protease, utilize the existing equipment of general fermentation factory, extract and purify polyhydroxyalkanoate product from the fermentation product of bacteria, make product cost greatly reduce.

The present invention proposes a method for separating and purifying polyhydroxyalkanoate in bacterial cells from bacterial cells, which is characterized in that it comprises the following steps: 1) stirring and washing bacterial cells with an anionic surfactant alkaline solution; Bacteria break and degrade their cell walls into small fragments, releasing the cell contents. 2) separating and extracting the polyhydroxyalkanoate particles of the solid phase; removing most of the non-polyhydroxyalkanoate (PHA) components produced in the fermentation; 3) further stirring and washing the particles with an enzyme solution of alkaline protein to remove residual Protein impurities, further improve its purity and greatly reduce its protein impurity content, so that it meets the requirements of further processing; 4) Separation, extraction and purification of solid-phase polyhydroxy fatty acid lipid particles; 5) drying to obtain high-purity powdered polyhydroxy fatty acid lipid products . The pH value of said alkaline solution in the present invention is in the range of 9-13. The washing temperature may be in the range of 20-100°C. Said separation and extraction method can be centrifugation or filtration to separate and collect the precipitate in the washing liquid. The present invention further includes washing the particles with water between the second and third steps to further remove the non-polyhydroxyalkanoate components and the added surfactant, and separate and extract the solid-phase polyhydroxyalkanoate particles. Between the 4th and 5th steps, it may also include washing the particles with water, and separating and extracting further purified solid-phase polyhydroxyalkanoate particles.

The process of the invention is applicable to a wide range of processing objects, and can process fermentation products of various polyhydroxyalkanoate-containing bacteria and their mutant strains and genetically engineered recombinant strains, and does not have high requirements on the polyhydroxyalkanoate content of the bacteria.

The present invention can be different according to treatment object, selects corresponding anionic surfactant kind in the anionic surfactant treatment process, suitable treatment condition (for example the concentration of the treatment of solution, the consumption ratio of tensio-active agent, the pH value condition of treatment and processing temperature). The alkaline condition of the anionic surfactant solution can be obtained by adding various alkalis and alkaline salts. In the present invention, suitable reaction conditions (such as action concentration, pH value, temperature and time) can be selected according to the characteristics of the protease used, so as to improve the purity of the product and reduce the production cost.

Compared with the existing purification process, the present invention has the following characteristics: (1) no organic solvent is used, less equipment investment and less environmental pollution; (2) the anionic surfactant used is cheap; (3) the protease used is relatively It is relatively cheap, and the dosage is small, and the cost of raw materials is low; (4) the product has high purity; (5) the existing equipment of a common fermentation factory can be used for production.

The polyhydroxyalkanoate product produced by the process of the invention has the characteristics of high purity, low protein content and high product molecular weight. Suitable for further processing.

Embodiment 1: Extracting polyhydroxyalkanoate from the thalline of Azotobactor vinelandii.

Strains: Azotobactor vinelandii UWD (Azotobactor vinelandii UWD)

Polyhydroxyalkanoate (PHA) content in dry cell weight: 50%;

Anionic surfactant: sodium dodecyl sulfate;

Anionic surfactant detergent concentration: 0.4-0.7% (w/v);

Anionic surfactant detergent pH value: 11;

Anionic surfactant washing temperature: 40°C;

Anionic surfactant washing time: 30 minutes;

The ratio of the amount of anionic surfactant to the non-polyhydroxyalkanoate component in the dry weight of the cells: 1/10 (w/w);

Protease: 2709 alkaline protease solution (enzyme activity about 50,000Unit/ml);

pH value of protease treatment: 11;

Protease treatment temperature: 40°C;

Protease treatment time: 30 minutes;

The ratio of the amount of protease to the non-polyhydroxyalkanoate components in the dry weight of the original cells: 5,000Unit/g;

Reaction system alkaline regulator: NaOH.

The production process of the present embodiment is as follows:

Azotobacter victorii UWD cells containing about 50% polyhydroxyalkanoate (PHA) were obtained by fermentation and centrifugation. Take about 1000g (equivalent dry weight) of bacteria, add 50g of sodium dodecylsulfonate, 10L of tap water, and adjust the pH value of the reaction system with NaOH solution to keep it at about 11. Keep the temperature above 40°C and wash with stirring for 30 minutes. Then add 5L of tap water and 50ml of alkaline protease solution to adjust the pH value of the reaction system to keep it at about 11. Keep the temperature at about 40°C and stir for 30 minutes, centrifuge to collect the precipitate, add tap water to stir and wash, and centrifuge again to collect the precipitate. Dry the precipitate to obtain a powdery high-purity polyhydroxyalkanoate (PHA) product.

The prepared polyhydroxyalkanoate (PHA) product has a purity greater than 96 percent and a protein content of less than 0.5 percent. Has good machinability. Example 2: Extract polyhydroxyl from the thalline of genetically engineered recombinant Escherichia coli (E.coli) with PHA synthesis ability

Fatty acid esters.

Bacteria: genetic engineering recombinant Escherichia coli (E.coli)

Polyhydroxyalkanoate (PHA) content in dry cell weight: 70%;

Anionic surfactant: sodium dodecyl sulfate;

Anionic surfactant washing concentration: 0.65-0.90% (w/v);

Anionic surfactant washing pH value: 11;

Anionic surfactant washing temperature: 60°C;

Anionic surfactant washing time: 30 minutes;

The ratio of the amount of anionic surfactant to the non-polyhydroxyalkanoate component in the dry weight of the cells: 1/8 (w/w);

Protease: 2709 alkaline protease solution (enzyme activity is about 50,000 Unit/ml, the optimum reaction conditions are temperature 40°C, pH value 11);

pH value of protease treatment: 11;

Protease treatment temperature: 40°C;

Protease treatment time: 1 hour;

The ratio of the amount of protease to the non-polyhydroxyalkanoate components in the dry weight of the original cells: 3,000Unit/g;

Reaction system alkaline regulator: Na ₂ CO ₃ .

The production process of the present embodiment is as follows:

By fermenting and producing strains (genetic engineered recombinant Escherichia coli), the cells whose polyhydroxyalkanoate (PHA) content accounts for about 70% of the dry weight of the cells are obtained, and the cells are collected by centrifugation. Take about 500 g (equivalent dry weight) of bacteria, add 18.75 g of sodium dodecylsulfonate, 2.5 L of tap water, and add Na ₂ CO ₃ to the solution to adjust the pH value of the reaction system to keep it at about 11. Keep the temperature above 60°C and wash with stirring for 30 minutes. Centrifuge to collect the precipitate, add tap water to stir and wash for 5 minutes, and centrifuge again to collect the precipitate. Add 9 ml of alkaline protease solution and 1 L of tap water to the precipitate, and add Na ₂ CO ₃ to adjust the pH value of the reaction system to keep it at about 11. Keep the temperature at about 40°C and stir and wash for 1 hour, centrifuge to collect the precipitate, add tap water to stir and wash for 5 minutes, and centrifuge again to collect the precipitate. Dry the precipitate to obtain a powdery high-purity polyhydroxyalkanoate (PHA) product.

The prepared polyhydroxyalkanoate (PHA) product has a purity greater than 97 percent and a protein content of less than 0.2 percent. Has good machinability. Example 3: Extract polyhydroxyalkanoate from the thalline of Alcaligenes latus.

Strain: Alcaligenes latus DSM (Alcaligenes latus DSM)

Polyhydroxyalkanoate (PHA) content in dry cell weight: 60%;

Anionic surfactant: sodium dodecylbenzenesulfonate;

Anionic surfactant washing concentration: 0.5% (w/v);

Anionic surfactant washing pH value: 12;

Anionic surfactant washing temperature: 80°C;

Anionic surfactant washing time: 1 hour;

The ratio of the amount of anionic surfactant to the non-polyhydroxyalkanoate component in the dry weight of the cells: 1/5 (w/w);

Protease: high temperature alkaline protease solution (enzyme activity is about 100,000Unit/ml, the optimum reaction conditions are temperature 60℃, pH value 10);

pH value of protease treatment: 10;

Protease treatment temperature: 60°C; protease treatment time: 1 hour;

The ratio of the amount of protease to the non-polyhydroxyalkanoate components in the dry weight of the original cells: 4,000Unit/g;

Reaction system alkaline regulator: NaOH.

The production process of the present embodiment is as follows:

The bacterial cells whose polyhydroxyalkanoate (PHA) content accounts for about 60% of the dry weight of cells are obtained by fermenting Alcaligenes megaterium DSM, and the bacterial cells are collected by centrifugation. Take about 500 g (equivalent dry weight) of bacteria, add 40 g of sodium dodecylbenzenesulfonate, 8 L of tap water, and adjust the pH value of the reaction system with NaOH solution to keep it at about 12. Keep the temperature above 80°C and wash with stirring for 1 hour. Centrifuge to collect the precipitate, add tap water to stir and wash for 10 minutes, and centrifuge again to collect the precipitate. Add 8ml of high-temperature alkaline protease solution and 4L of tap water to the precipitate, and adjust the pH value of the reaction system with NaOH solution to keep it at about 10. Keep the temperature at about 60°C for 1 hour with stirring and washing, centrifuge to collect the precipitate, add tap water to stir and wash for 10 minutes, and centrifuge again to collect the precipitate. Dry the precipitate to obtain a powdery high-purity polyhydroxyalkanoate (PHA) product.

The prepared polyhydroxyalkanoate (PHA) product has a purity greater than 94 percent and a protein content of less than 0.6 percent. Has good machinability.

Claims (5)

1. A method for isolating and purifying polyhydroxyalkanoate in bacterial cells from bacterial cells, characterized in that it comprises the following steps: 1) stirring and washing bacterial cells with an anionic surfactant alkaline solution, the washing temperature is 20- 100°C; 2) separating and extracting the polyhydroxyalkanoate particles in the solid phase; 3) further stirring and washing the particles with an alkaline protease solution with a pH value of 9-13 to remove residual protein impurities; 4) separating, extracting and purifying the Solid-phase polyhydroxyalkanoate ester particles; 5) drying to obtain powdered polyhydroxyalkanoate ester products. 2. A method for separating and purifying intracellular polyhydroxyalkanoate of bacteria from bacterial cells according to claim 1, characterized in that said separation and extraction method is centrifugation or filtration to separate and collect the precipitate in the washing liquid. 3. A method for separating and purifying polyhydroxyalkanoate in bacterial cells as claimed in claim 1 or 2, further comprising washing with water between said second and third steps said particles, and separating and extracting the solid-phase polyhydroxyalkanoate particles. 4. A method for isolating and purifying intracellular polyhydroxyalkanoate from bacterial cells as claimed in claim 1 or 2, characterized in that between said 4th and 5th steps, including washing said Particles, and the further purified solid-phase polyhydroxyalkanoate particles were separated and extracted. 5. A method for isolating and purifying intracellular polyhydroxyalkanoate from bacterial cells as claimed in claim 3, characterized in that between said 4th and 5th steps, washing said Particles, and the further purified solid-phase polyhydroxyalkanoate particles were separated and extracted.