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

The method of from the bacterium thalline, separating purification bacterium intracellular poly hydroxy fatty acid

The invention belongs to post-processing technology field, biotechnology downstream.

Polyhydroxyalkanoate (Poly-β-Hydroxyalkanoates is called for short PHA) is an inclusion in a kind of bacterium born of the same parents, has the characteristic of thermoplastics.Simultaneously because biodegradability and bio-compatible that it had make it that application potential be arranged in every respect.But owing to its form with inclusion is present in the bacterial body, complicated component makes its purification extremely difficult.Existing downstream aftertreatment purifying technique, perhaps utilize organic solvent (as chloroform, methylene dichloride etc.) to carry out extracting, the enzyme of perhaps taking heat to be used in combination the multiple pricing costliness decomposes non-polyhydroxyalkanoate composition in the cell to reach the purpose of purification.Complex manufacturing, the facility investment height, the raw materials cost costliness causes the polyhydroxyalkanoate product price too high, makes this coming biodegradable plastic be difficult to promote the use of.The present patent application people found through experiments, earlier with negatively charged ion alkalescence solution-treated bacterium thalline, separation and Extraction polyhydroxyalkanoate particle is further handled with proteolytic enzyme and is removed residual albumen impurity on the degranulation, can obtain highly purified polyhydroxyalkanoate product.Because this technological reaction mild condition does not have particular requirement to equipment, the raw material cheapness is fit to the requirement that large-scale industrialization is produced, and greatly reduces the production cost of polyhydroxyalkanoate.

The objective of the invention is to propose to use cheap anion surfactant and a spot of proteolytic enzyme, utilize the existing installation of general fermentation plant, from the tunning of bacterium, extract purifying polyhydroxyalkanoate product, product cost is greatly reduced.

The present invention proposes a kind of method of separating interior polyhydroxyalkanoate in the purification bacterium born of the same parents from the bacterium thalline, it is characterized in that may further comprise the steps: 1) earlier with anion surfactant basic solution agitator treating bacterium thalline; The result makes the bacterium broken wall and its cell wall is degraded to small shreds, discharges entocyte simultaneously.2) the poly-hydroxy fatty acid fat particle of separation and Extraction solid phase; Remove most of non-polyhydroxyalkanoate (PHA) composition that produces in the fermentation; 3) use the said particle of the further agitator treating of enzyme solution of basic protein again, remove residual albumen impurity, further improve its purity and reduce its albumen foreign matter content greatly, make it satisfy the further requirement of processing; 4) separation and Extraction purifying solid polycondensation hydroxy fatty acid fat particle; 5) drying obtains high purity powdered form shape poly-hydroxy fatty acid fat prod.The pH value of the said basic solution of the present invention is in the 9-13 scope.Wash temperature can be in 20-100 ℃ of scope.Said separating and extracting method can be the throw out in centrifugal or the filtering separation collection washings.The present invention also further comprises between said second, third step and washes said particle with water, further removes non-polyhydroxyalkanoate composition and adds tensio-active agent with institute, and separation and Extraction solid polycondensation hydroxy fatty acid fat particle.Between said the 4th, the 5th step, also can comprise washing said particle with water, and the solid polycondensation hydroxy fatty acid fat particle that is further purified of separation and Extraction.

The suitable process object of technology of the present invention is extensive, can handle the multiple bacterium of polyhydroxyalkanoate and the tunning of variant and genetic engineering recombination strain thereof of containing, and is not high to the polyhydroxyalkanoate content requirement of thalline.

The present invention can be according to the process object difference, in the anionic surfactant treatment process, select corresponding anion surfactant kind, suitable treatment condition (for example the concentration of the processing of solution, the amount ratio of tensio-active agent, the pH value condition of processing and the temperature of processing).The alkaline condition of anionic surfactant solution can obtain by adding various bases and alkaline salt.The present invention also can select suitable reaction conditions (as activity, pH value, temperature and time) according to the characteristic of use proteolytic enzyme, produces purity to improve, and reduces production costs.

The present invention compares with existing purifying technique, has following characteristics: (1) not with an organic solvent, facility investment is few, environmental pollution is little; (2) the anion surfactant low price of Shi Yonging; (3) proteolytic enzyme of Shi Yonging is more cheap relatively, and consumption seldom, and raw materials cost is low; (4) product purity height; (5) can adopt the existing installation of common fermentation factory to produce.

Polyhydroxyalkanoate product by explained hereafter of the present invention has the purity height, and protein content is little, the characteristics that molecular weight product is high.Being fit to further processing uses.

Embodiment one: extract polyhydroxyalkanoate from the thalline of vickers nitrogen-fixing bacteria (Azotobactor vinelandii).

Bacterial classification: vickers nitrogen-fixing bacteria UWD (Azotobactor vinelandii UWD)

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

Anion surfactant: sodium laurylsulfonate;

Anion surfactant washings concentration: 0.4-0.7% (w/v);

Anion surfactant washings pH value: 11;

Anion surfactant wash temperature: 40 ℃;

Anion surfactant washing time: 30 minutes;

The anion surfactant consumption becomes the ratio of branch: 1/10 (w/w) with non-polyhydroxyalkanoate in the dry cell weight;

Proteolytic enzyme: (it is about 50 that enzyme is lived, 000Unit/ml) for 2709 Sumizyme MP liquid;

Protease treatment pH value: 11;

Protease treatment temperature: 40 ℃;

The protease treatment time: 30 minutes;

The proteolytic enzyme consumption becomes the ratio of branch with non-polyhydroxyalkanoate in the initiating cell dry weight: 5, and 000Unit/g;

Reactive system alkaline conditioner: NaOH.

The technological process of production of present embodiment is as follows:

By fermentation and the centrifugal vickers nitrogen-fixing bacteria UWD thalline that obtains containing polyhydroxyalkanoate (PHA) about 50%.Get about 1000g (equivalent dry weight) thalline, add sodium laurylsulfonate 50g, tap water 10L makes it to remain on about 11 with NaOH solution conditioned reaction system pH.Keep temperature agitator treating 30 minutes more than 40 ℃.Add 5L tap water and 50ml Sumizyme MP liquid again, the conditioned reaction system pH makes it to remain on about 11.Keep temperature to stir 30 minutes for about 40 ℃, centrifugal collecting precipitation adds the tap water agitator treating, the recentrifuge collecting precipitation.The oven dry precipitation promptly gets Powdered high purity polyhydroxyalkanoate (PHA) product.

The polyhydroxyalkanoate that makes (PHA) product purity is greater than 96%, and protein content is less than 0.5%.Has good workability.Embodiment two: extract poly-hydroxyl from the thalline of genetically engineered recombinant escherichia coli (E.coli) with PHA synthesis capability

Fatty acid ester.

Bacterial classification: genetically engineered recombinant escherichia coli (E.coli)

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

Anion surfactant: sodium laurylsulfonate;

Anion surfactant wash concentration: 0.65-0.90% (w/v);

Anion surfactant washing pH value: 11;

Anion surfactant wash temperature: 60 ℃;

Anion surfactant washing time: 30 minutes;

The anion surfactant consumption becomes the ratio of branch: 1/8 (w/w) with non-polyhydroxyalkanoate in the dry cell weight;

Proteolytic enzyme: 2709 Sumizyme MP liquid (it is about 50 that enzyme is lived, and 000Unit/ml, optimum reaction conditions are 40 ℃ of temperature, pH value 11);

Protease treatment pH value: 11;

Protease treatment temperature: 40 ℃;

The protease treatment time: 1 hour;

The proteolytic enzyme consumption becomes the ratio of branch with non-polyhydroxyalkanoate in the initiating cell dry weight: 3, and 000Unit/g;

Reactive system alkaline conditioner: Na ₂CO ₃

The technological process of production of present embodiment is as follows:

Obtain the thalline that polyhydroxyalkanoate (PHA) content accounts for dry cell weight 70%, centrifugal collection thalline by fermentative production bacterial classification (genetically engineered recombinant escherichia coli).Get about 500g (equivalent dry weight) thalline, add sodium laurylsulfonate 18.75g, tap water 2.5L adds Na in solution ₂CO ₃The conditioned reaction system pH makes it to remain on about 11.Keep temperature agitator treating 30 minutes more than 60 ℃.Centrifugal collecting precipitation added the tap water agitator treating 5 minutes, the recentrifuge collecting precipitation.Add Sumizyme MP liquid 9ml in precipitation, tap water 1L adds Na ₂CO ₃The conditioned reaction system pH makes it to remain on about 11.Kept 40 ℃ of left and right sides agitator treatings of temperature 1 hour, centrifugal collecting precipitation added the tap water agitator treating 5 minutes, the recentrifuge collecting precipitation.The oven dry precipitation promptly gets Powdered high purity polyhydroxyalkanoate (PHA) product.

The polyhydroxyalkanoate that makes (PHA) product purity is greater than 97%, and protein content is less than 0.2%.Has good workability.Embodiment three: extract polyhydroxyalkanoate from the thalline of huge Alcaligenes (Alcaligenes latus).

Bacterial classification: huge Alcaligenes DSM (Alcaligenes latus DSM)

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

Anion surfactant: Sodium dodecylbenzene sulfonate;

Anion surfactant wash concentration: 0.5% (w/v);

Anion surfactant washing pH value: 12;

Anion surfactant wash temperature: 80 ℃;

Anion surfactant washing time: 1 hour;

The anion surfactant consumption becomes the ratio of branch: 1/5 (w/w) with non-polyhydroxyalkanoate in the dry cell weight;

Proteolytic enzyme: high-temperature alkaline liquid of protease (it is about 100 that enzyme is lived, and 000Unit/ml, optimum reaction conditions are 60 ℃ of temperature, pH value 10);

Protease treatment pH value: 10;

Protease treatment temperature: 60 ℃; The protease treatment time: 1 hour;

The proteolytic enzyme consumption becomes the ratio of branch with non-polyhydroxyalkanoate in the initiating cell dry weight: 4, and 000Unit/g;

Reactive system alkaline conditioner: NaOH.

The technological process of production of present embodiment is as follows:

Obtain the thalline that polyhydroxyalkanoate (PHA) content accounts for dry cell weight 60%, centrifugal collection thalline by the huge Alcaligenes DSM that ferments.Get about 500g (equivalent dry weight) thalline, add Sodium dodecylbenzene sulfonate 40g, tap water 8L makes it to remain on about 12 with NaOH solution conditioned reaction system pH.Keep temperature agitator treating 1 hour more than 80 ℃.Centrifugal collecting precipitation added the tap water agitator treating 10 minutes, the recentrifuge collecting precipitation.Add high-temperature alkaline liquid of protease 8ml in precipitation, tap water 4L makes it to remain on about 10 with NaOH solution conditioned reaction system pH.Kept 60 ℃ of left and right sides agitator treatings of temperature 1 hour, centrifugal collecting precipitation added the tap water agitator treating 10 minutes, the recentrifuge collecting precipitation.The oven dry precipitation promptly gets Powdered high purity polyhydroxyalkanoate (PHA) product.

The polyhydroxyalkanoate that makes (PHA) product purity is greater than 94%, and protein content is less than 0.6%.Has good workability.

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.