CN111019108B - Method for extracting and purifying polyhydroxyalkanoate - Google Patents
Method for extracting and purifying polyhydroxyalkanoate Download PDFInfo
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- CN111019108B CN111019108B CN202010012479.6A CN202010012479A CN111019108B CN 111019108 B CN111019108 B CN 111019108B CN 202010012479 A CN202010012479 A CN 202010012479A CN 111019108 B CN111019108 B CN 111019108B
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
- C08G63/88—Post-polymerisation treatment
- C08G63/90—Purification; Drying
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
- C08G63/02—Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds
- C08G63/06—Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds derived from hydroxycarboxylic acids
- C08G63/08—Lactones or lactides
Abstract
The existing extraction and purification method of Polyhydroxyalkanoate (PHA) comprises organic solvent or surfactant and hydrolytic enzyme, and the process is complex and expensive. The invention completes the wall breaking and purification of bacterial cells by carrying out hot water treatment or superheated water treatment at more than 100 ℃ on the PHA-rich bacterial fermentation liquor, and can further improve the purity of PHA by combining the treatment of a surfactant, protease and/or a bleaching agent. The method has the advantages of low cost, simple operation and high product recovery rate, and solves the problems of complicated steps, high cost of adding chemical substances, difficult treatment of downstream sewage and the like of the traditional PHA extraction.
Description
Technical Field
The invention belongs to the field of downstream post-treatment of bioengineering, and particularly relates to a production and downstream extraction and purification process of Polyhydroxyalkanoate (PHA).
Background
Polyhydroxyalkanoates (PHA) is a degradable polyester that can be accumulated by various bacteria, and can be classified into short-chain/medium-long-chain PHAs, and also homopolymers and copolymers according to the monomer composition. PHA is an environment-friendly material, has wide application prospect, and can be used as an environment-friendly substitute for the traditional petroleum-based plastics. Among them, poly-3-hydroxybutyrate (PHB) is the most clearly studied among the current PHA family members, but its application is limited by its high crystallinity, low elasticity, poor flexibility, etc. At present, the method of biological fermentation is frequently used in industry7,8To obtain PHA-enriched bacteria, then to carry out complex purification work3,4,6。
PHA production is performed by Eubacterium rolfsii (Ralstonia eutropha), genetically modified Escherichia coli (E.coli), halophilic bacteria, and the like. Although the thallus in the fermentation liquor is rich in PHA, the extraction, purification and impurity removal processes of PHA are complicated, and some PHA are expensive due to the use of organic solvents. The properties of PHAs are very much related to their purity. The purity requirements for different applications vary. Among them, the purity is required to be very high in the fields of medical treatment and the like. However, PHA is a bacterial inclusion, and the intracellular components are quite complex and difficult to extract and purify.
The existing extraction processes include organic solvent extraction, mechanical disruption, surfactant method, and various enzyme combination methods. But all have their limitations: the organic solvent extraction method has the disadvantages of difficult solvent recovery, high cost and dangerous operation environment, and is not suitable for large-scale production;the mechanical crushing method mainly comprises ultrasonic wall breaking or high-pressure homogenate wall breaking, and has the advantages of high energy consumption, great product loss and low purity; the surfactant method has disadvantages that the surfactant seriously damages the molecular weight of PHA, the wastewater containing the surfactant is difficult to treat, and the surfactant remains2-5。
CN1070534C discloses a method for separating and extracting PHA from bacterial thallus, which comprises the following steps: 1) treating the cells with an alkaline solution containing a surfactant; 2) solid-liquid separation, most non-PHA components are separated; 3) treating the PHA with an alkaline protease; 4) separating and extracting PHA particles; 5) drying to obtain PHA product. The method has mild reaction conditions, but has large alkali consumption and large addition of the surfactant, additionally increases the cost and the sewage treatment cost, and has obvious effect in the industrial amplification process.
CN1211489C discloses a method for separating and extracting PHA from bacterial thallus, which comprises using one or more of organic solvents of five and/or four carbon alcohol to destroy cell wall and extract PHA.
CN109575264A relates to a method for extracting PHA with an organic solvent valerolactone as a solvent.
CN109504715A discloses a method for preparing PHA, which comprises the step of adding anionic surfactant (SDS) to perform cell wall breaking and cracking of thalli.
All the existing methods relate to the use of organic solvents and/or surfactants, and have the problems of complicated extraction steps, high cost of adding chemical substances, difficult treatment of downstream sewage and the like.
Therefore, the development of a simple and effective downstream extraction and purification process of PHA is crucial to reduce the production cost of PHA.
Disclosure of Invention
The invention aims to provide a method for preparing Polyhydroxyalkanoate (PHA), which is a process method for extracting PHA with high efficiency and low cost from PHA production bacteria fermentation liquor, and has the advantages of mild process, low cost, low requirement on equipment and realization of large-scale industrial production.
The inventor surprisingly finds that a microbial thallus wall-broken lysate can be obtained by treating microbial thallus for producing Polyhydroxyalkanoate (PHA) with hot water, particularly superheated water, so that the use of an organic solvent and/or a surfactant is avoided, and the problems of complicated traditional PHA extraction steps, high cost of added chemical substances, difficulty in downstream sewage treatment and the like are solved.
Accordingly, the present invention provides a method for extracting Polyhydroxyalkanoate (PHA) from PHA-producing microbial cells, comprising the steps of: and treating the microbial thallus with hot water or overheated water to obtain thallus wall-broken lysate. Preferably, the method does not use an organic solvent and/or a surfactant (e.g., an anionic surfactant) to treat the microbial cells while breaking the cell walls. The cell wall-broken lysate is rich in PHA granules, and can be further concentrated and dried to obtain PHA powder. The purity of PHA obtained after treatment is improved by 5-15 wt% relative to the PHA content in the microbial cells before treatment.
Unless otherwise indicated, the microbial cells described in the present invention are PHA-producing bacteria, and may be any microorganism (wild-type bacterial strain or genetically engineered bacterial strain) capable of producing PHA, which may be gram-positive or gram-negative bacteria, including but not limited to, Pseudomonas rhodinii (Ralstonia eutropha), genetically engineered escherichia coli (e.g., colibacillus), halophilic bacteria (e.g., Halomonas), and the like, such as Pseudomonas spp.
PHA as referred to herein means polyhydroxyalkanoate, which can be classified into homopolymers and copolymers according to monomer composition. Depending on the number of carbon atoms of the monomer, the PHA of the present invention may be a short chain PHA (i.e., a hydroxy fatty acid monomer C3-C5) or a medium-long chain PHA (i.e., a hydroxy fatty acid monomer C6-C18), but is not limited thereto. In some embodiments of the invention, the PHA may be a homopolymer, including but not limited to polyhydroxypropionate, Polyhydroxybutyrate (PHB), polyhydroxyvalerate, and the like, for example, poly-3-hydroxybutyrate (P3HB), poly-4-hydroxybutyrate (P4HB), poly-3-hydroxypropionate (P3HP), or poly-3-hydroxyvalerate (P3HV), and the like.
In one embodiment of the process of the invention, the hot or superheated water has a temperature of from 60 ℃ to 200 ℃, preferably from 80 ℃ to 160 ℃, further preferably from 100 ℃ to 140 ℃, more preferably from 100 ℃ to 130 ℃, most preferably greater than 100 ℃ to 125 ℃ (e.g., 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125 ℃).
In a preferred embodiment of the method of the invention, the microbial cells are treated with superheated water to obtain a cell wall broken lysate. According to the invention, the superheated water is obtained by heating water under pressure to a temperature above 100 ℃.
In one embodiment of the process of the invention, the pressure is preferably set at a sufficiently high level that the water is in a liquid state and does not boil. Preferably, the treatment of the microbial cells with hot or superheated water is carried out at the following pressure: 0.1 to 30MPa (e.g.0.12 to 0.15MPa), preferably 0.5 to 20MPa, more preferably 1 to 10MPa (e.g.1, 2, 3, 4, 5, 6, 7, 8, 9, 10MPa), most preferably 1.5 to 5 MPa.
In one embodiment of the method of the present invention, wherein the microbial cells are treated with hot water or superheated water at a pH of 8-11.
In one embodiment of the method of the present invention, the treatment of the microbial cells with superheated water according to the present invention is performed in an autoclave (e.g. autoclave). In one embodiment of the process of the present invention, the autoclave (e.g., autoclave) may be warmed to 100 ℃ or in a range of greater than 100 ℃ to 200 ℃ and the pressure may be 2MP or less. Preferably, the treatment is carried out at a constant temperature and pressure. In a preferred embodiment of the present invention, the microbial cells are treated to effect wall-breaking lysis by raising the temperature of an aqueous solution (e.g., fermentation broth or cell suspension) in which the microbial cells are suspended in an autoclave (e.g., autoclave) so that the water in the aqueous solution becomes superheated water.
In one embodiment of the method of the present invention, the treatment of the microbial cells with hot water or superheated water is performed for 0.2 to 4 hours, preferably 0.5 to 1 hour.
In one embodiment of the method of the present invention, 0.01 to 5 v/v% (e.g., 0.05 v/v%, 0.1 v/v%, 0.5 v/v%, 1 v/v%, 1.5 v/v%, 2 v/v%, 3 v/v%, 4 v/v%, 5 v/v%, preferably 0.1 to 2 v/v%) of glycerol (based on the volume of hot water or superheated water) may be added to the hot water or superheated water to increase the efficiency of the hot water or superheated water in destroying microbial cells (cell wall disruption). It is believed that the hot or superheated water and glycerol have a synergistic effect in the method of extracting PHA in the present invention.
In one embodiment of the method of the present invention, the method further comprises treating the disrupted cell lysate with a protease to further increase the purity of the PHA.
In one embodiment of the method of the present invention, wherein the protease is selected from the group consisting of: proteinase K, alkaline protease, acid protease, papain, SUMO protease, or combinations thereof. The above-mentioned proteases are commercially available unless otherwise specified. The amount of protease added is preferably 0.0001-1% (w/v), more preferably 0.0001-0.1% (w/v), as compared with the cell wall disruption lysate.
In one embodiment of the method of the present invention, it further comprises treating the solution obtained after treating the cell wall-broken lysate with a protease with a bleaching agent. Bleaching agents are used to oxidatively remove impurities, again increasing PHA purity. The bleaching agent is preferably added in an amount of 0.0001 to 2 wt.%, more preferably 0.001 to 0.1 wt.%, based on the weight of the solution to be treated.
In one embodiment of the method of the present invention, wherein said bleaching agent is selected from the group consisting of: hydrogen peroxide, sodium hypochlorite, sodium sulfite, thiourea dioxide, chlorine dioxide, or combinations thereof.
In one embodiment of the method of the present invention, further comprising washing, filtering, centrifuging and/or drying steps to obtain a purified Polyhydroxyalkanoate (PHA).
In one embodiment of the method of the present invention, wherein said Polyhydroxyalkanoate (PHA) is selected from the group consisting of short-chain poly-3-hydroxybutyrate (PHB), 3-hydroxybutyrate and 3-hydroxyvalerate copolyester (PHBV), 3-hydroxybutyrate and 4-hydroxybutyrate copolyester (P3HB4HB), medium-and long-chain PHAs (the monomer is a hydroxy ester of C6-C18), and short-and medium-and long-chain PHA copolymers.
In a preferred embodiment of the present invention, there is provided a method for purifying Polyhydroxyalkanoate (PHA) using water, the method comprising the steps of:
(1) treating PHA-rich thallus with hot water or superheated water (105 deg.C), optionally adding a certain amount of glycerol to break cell wall and obtain bacterial wall-broken mixed liquor, wherein no anionic surfactant is used, and PHA purity is increased by 5-15%;
(2) adding a certain amount of protease into the PHA wall breaking liquid obtained in the step (1) for treatment, so that the purity can be further improved;
(3) adding bleaching agent into the protease treatment wall-breaking liquid in the step (2) for treatment, and oxidizing to remove impurities so as to improve the PHA purity again; and
(4) removing non-PHA components in the mixed solution obtained in the step (3), and collecting and drying the concentrated solution containing PHA.
In a preferred embodiment, the above-mentioned cell wall breaking and lysis of the bacterium comprises the steps of:
(1) adjusting the temperature of the bacterial liquid to the optimal wall breaking temperature (60-160 ℃, preferably 80-140 ℃, more preferably 101-130 ℃);
(2) adjusting the pH value of the bacterial liquid to the optimum pH value, and controlling the pH value to be 8-11;
(3) maintaining the optimum treatment time (such as 0.2-2 hr) at constant temperature and pressure, and controlling the pressure at 0.12-0.15 MPa.
In a particularly preferred embodiment, the cell wall breaking only needs to be carried out by superheated water treatment, specifically, the required temperature is 60-160 ℃, preferably 80-140 ℃, and the required time for cell wall breaking is 0.2-4 hours, preferably 0.5-1 hour.
In a preferred embodiment, after treatment with hot water/superheated water, glycerol, protease treatment, bleaching agent, water is added to the resulting PHA concentrate, washing is stirred well, then the washing liquid is removed by centrifugation, optionally repeating this process 1-6 times, obtaining purified PHA concentrate, drying or obtaining powdered PHA. In a particularly preferred embodiment, the process comprises adding water to the PHA concentrate after wall breaking and bleaching, washing with thorough agitation, and then centrifuging to remove the wash, repeating this process several times until the PHA meets the purification requirements. Preferably, the amount of water added during the washing process of the PHA concentrate with water is 1-10 times, more preferably 1-5 times, the volume of the concentrate.
Compared with the existing PHA purification process, the method of the invention has the following advantages:
(1) the whole process of the process does not use any organic solvent and/or surfactant, and the production environment is simple and safe;
(2) the extraction equipment is simple and can be realized only by high-pressure heating equipment; and
(3) and any chemical harmful substance (optionally adding trace protease) for assisting in breaking the wall is not added, so that the extraction cost is greatly reduced, and the difficulty in treating downstream production wastewater is reduced.
Detailed Description
The invention is further illustrated in the following examples. These examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Unless otherwise indicated, terms used herein have the ordinary technical meaning as understood by those skilled in the art.
The chemicals used in the following reactions are all commercially available products unless otherwise indicated.
The product purity, such as P3HB4HB content (wt%), referred to herein is the mass of P34HB as a percentage of the mass of the ice-dried cells participating in the esterification, where the mass of P34HB is the total mass of 34HB obtained after esterification.
The product recovery rate mentioned here is the ratio of the quality of the freeze-dried thallus after wall breaking, purification and collection to the thallus after the direct freeze-drying of the fermentation product.
Example 1: extracting and purifying PHA from fermentation liquor containing PHA thallus by using superheated water method
The extraction method comprises the following steps:
(1) centrifugation and heavy suspension desalination of the cells
Collecting Halomonas containing 60% PHA (P3HB, P34 HB)1250mL of fermentation liquor of the thallus is symmetrically placed in a high-speed centrifuge after being balanced, and the centrifugation conditions are as follows: the rotation speed is 6000rpm, and the time is 20 min. After the centrifugation is finished, carefully removing the supernatant, taking care to avoid losing the precipitate as much as possible, then adding water to restore the volume, stirring uniformly, and continuing the centrifugation, wherein the centrifugation conditions are as follows: 6000rpm, time 20 min. And after the centrifugation is finished, discarding the supernatant, recovering the volume of the thalli to 250mL, uniformly stirring, pouring the washed bacterial liquid into a 500mL beaker, and preparing for breaking the walls.
(2) Cell wall breaking and PHA release of bacterial liquid
5M NaOH was added to a 500mL beaker containing 250mL of the bacterial solution to adjust the pH of the bacterial solution to 9.5, and the 500mL beaker containing the bacterial solution was placed in a 1000mL beaker containing water, with the 500mL beaker not being tilted, and the lower half of the beaker was immersed in water. Placing the two in an autoclave, and reacting at 80-121 deg.C for 30-60 min.
(3) Purification and Collection of PHA
After the wall breaking reaction is finished, centrifuging to remove non-PHA impurities, wherein the centrifugation conditions are as follows: 4500rpm, 10 min. After the centrifugation is finished, the supernatant is carefully removed, water is added to restore the volume, the centrifugation is continued after the uniform stirring, and the washing and centrifugation operations are repeated for 2 times. The washed and centrifuged PHA is tightly adhered to a centrifuge tube, so that the supernatant can be completely discarded, and PHA solid matters can not be lost.
(4) PHA parameter measurement
Freezing the centrifuge tube containing PHA precipitate in-80 deg.C ultra-low temperature refrigerator for 2 hr, freeze-drying in vacuum freeze-drying machine, grinding PHA, mixing, and measuring PHA quality and content by gas chromatography2,5. Three replicates were set for each set of experiments and the results were averaged. The results are shown in Table 1.
Table 1: effect of treatment temperature and wall breaking time on PHA purity and recovery
| Experimental number | Temperature of wall breaking | Time of wall breaking | pH of broken wall | Purity of raw material | Purity of the product | Recovery rate |
| 1-1 | 80℃ | 30min | 10 | 60％ | 87.98％ | 90.17％ |
| 1-2 | 80℃ | 60min | 10 | 60％ | 88.16％ | 86.83％ |
| 1-3 | 100℃ | 30min | 10 | 60％ | 88.97％ | 88.41％ |
| 1-4 | 100℃ | 60min | 10 | 60％ | 89.71％ | 86.76％ |
| 1-5 | 125℃ | 30min | 10 | 60％ | 89.05％ | 85.57％ |
| 1-6 | 125℃ | 60min | 10 | 60％ | 91.06％ | 83.91％ |
Example 2: extracting and purifying PHA from fermentation liquor containing PHA thallus by using superheated aqueous protease method
The extraction method comprises the following steps:
(1) centrifugation and heavy suspension desalination of the cells
Collecting Halomonas containing 60% PHA (P3HB, P34 HB)1250mL of fermentation liquor of the thallus is symmetrically placed in a high-speed centrifuge after being balanced, and the centrifugation conditions are as follows: the rotation speed is 6000rpm, and the time is 20 min. After centrifugation, the supernatant was carefully removed, taking care not to lose the precipitate as much as possible, then water was added to restore the volume, and stirring was carried outAnd (3) continuing to centrifuge after uniformly stirring, wherein the centrifugation conditions are as follows: 6000rpm, time 20 min. And after the centrifugation is finished, discarding the supernatant, recovering the volume of the thalli to 250mL, uniformly stirring, pouring the washed bacterial liquid into a 500mL beaker, and preparing for breaking the walls.
(2) Cell wall breaking and PHA release of bacterial liquid
5M NaOH was added to a 500mL beaker containing 250mL of a fermentation broth containing PHA cells to adjust the pH of the broth to 9.5, and the 500mL beaker containing the broth was placed in a 1000mL beaker containing water, taking care that the 500mL beaker was not tilted, and the lower half was immersed in water. Placing the two in autoclave, and reacting at 121 deg.C for 90min
(3) Protease treatment of wall-broken liquid
And (3) placing a 500mL beaker filled with 250mL of fermentation liquor containing PHA thallus in a heating device with magnetic stirring, after the temperature of the bacterial liquid is recovered to 60 ℃, adjusting the pH to 9.5 by using concentrated hydrochloric acid again, and adding 0.1% (v/v) of selected protease. Maintaining 60 deg.C enzyme treatment for 30-60min
(4) Purification and Collection of PHA
After the wall breaking reaction is finished, centrifuging to remove non-PHA impurities, wherein the centrifugation conditions are as follows: 4500rpm, 10 min. After the centrifugation is finished, the supernatant is carefully removed, water is added to restore the volume, the centrifugation is continued after the uniform stirring, and the washing and centrifugation operations are repeated for 2 times. The washed and centrifuged PHA is tightly adhered to a centrifuge tube, so that the supernatant can be completely discarded, and PHA solid matters can not be lost.
(5) PHA purification parameter measurement
Freezing the centrifuge tube containing PHA precipitate in-80 deg.C ultra-low temperature refrigerator for 2 hr, freeze-drying in vacuum freeze-drying machine, grinding PHA, mixing, and measuring PHA quality and content by gas chromatography2,5. Three replicates were set for each set of experiments and the results were averaged. The results are shown in Table 2.
Table 2: influence of superheated aqueous protease method on PHA purity and recovery rate
Protease from MACKLIN/Michalin: proteinase K cat # P6157, papain cat # P815680, alkaline proteinase cat # P824138, acid proteinase cat # P832411. SUMO protease was purchased from a leaf organism of origin under accession number S25841.
Example 3: extracting and purifying PHA from fermentation liquor containing PHA thallus by using superheated water, protease and bleaching agent method
The extraction method comprises the following steps:
(1) centrifugation and heavy suspension desalination of the cells
Collecting Halomonas containing 60% PHA (P3HB, P34 HB)1250mL of fermentation liquor of the thallus is symmetrically placed in a high-speed centrifuge after being balanced, and the centrifugation conditions are as follows: the rotation speed is 6000rpm, and the time is 20 min. After the centrifugation is finished, carefully removing the supernatant, taking care to avoid losing the precipitate as much as possible, then adding water to restore the volume, stirring uniformly, and continuing the centrifugation, wherein the centrifugation conditions are as follows: 6000rpm, time 20 min. And after the centrifugation is finished, discarding the supernatant, recovering the volume of the thalli to 250mL, uniformly stirring, and pouring the washed bacterial liquid into a 500mL beaker to prepare for breaking the walls.
(2) Cell wall breaking and PHA release of bacterial liquid
5M NaOH was added to a 500mL beaker containing 250mL of a fermentation broth containing PHA cells to adjust the pH of the broth to 9.5, and the 500mL beaker containing the broth was placed in a 1000mL beaker containing water, taking care that the 500mL beaker was not tilted, and the lower half was immersed in water. The two were reacted together in an autoclave at 121 ℃ for 90 min.
(3) Protease treatment of wall-broken liquid
Placing 500mL beaker containing 250mL fermentation liquid containing PHA thallus in a heating device with magnetic stirring, after the temperature of the bacterial liquid is recovered to 60 ℃, adjusting pH to 9.5 again by concentrated hydrochloric acid, and adding 0.1% (v/v) of selected protease (alkaline protease). Maintaining 60 deg.C enzyme treatment for 30-60min
(4) Bleaching of liquid after enzyme treatment
Placing 500mL beaker containing 250mL fermentation liquid containing PHA thallus in a heating device with magnetic stirring, after the temperature of the bacterial liquid is returned to room temperature, adjusting the pH value to about 6 with concentrated hydrochloric acid again, and adding 0.01% (v/v) of bleaching agent which is all purchased from ASONE. Maintaining the room temperature for 30-60 min. And (3) adjusting the bleached liquid to be neutral by using a sodium hydroxide solution, and adding hydrogen peroxide for neutralization dechlorination and desulfurization.
(5) Purification and Collection of PHA
After the wall breaking reaction is finished, centrifuging to remove non-PHA impurities, wherein the centrifugation conditions are as follows: 4500rpm, 10 min. After the centrifugation is finished, the supernatant is carefully removed, water is added to restore the volume, the centrifugation is continued after the uniform stirring, and the washing and centrifugation operations are repeated for 2 times. The washed and centrifuged PHA is tightly adhered to a centrifuge tube, so that the supernatant can be completely discarded, and PHA solid matters can not be lost.
(6) PHA purification parameter measurement
Freezing the centrifuge tube containing PHA precipitate in-80 deg.C ultra-low temperature refrigerator for 2 hr, freeze-drying in vacuum freeze-drying machine, grinding PHA, mixing, and measuring PHA quality and content by gas chromatography2,5. Finally, the purity of the product can reach 95.29 percent, and the recovery rate can reach more than 90 percent. Three replicates were set for each set of experiments and the results were averaged. The results are shown in Table 3.
Table 3: influence of superheated water, protease and bleaching agent method on PHA purity and recovery
Example 4: extracting and purifying PHA from fermentation liquor containing PHA thallus by superheated water and glycerol method
The extraction method comprises the following steps:
(1) centrifugation and heavy suspension desalination of the cells
Collecting Halomonas containing 60% PHA (P3HB, P34 HB)1250mL of fermentation liquor of the thallus is symmetrically placed in a high-speed centrifuge after being balanced, and the centrifugation conditions are as follows: the rotation speed is 6000rpm, and the time is 20 min. After centrifugation, the supernatant was carefully removed, taking care not to lose the pellet as much as possible, and then water was added to restore the volumeAnd after stirring uniformly, continuing centrifuging under the centrifugation conditions: 6000rpm, time 20 min. And after the centrifugation is finished, discarding the supernatant, recovering the volume of the thalli to 250mL, uniformly stirring, and pouring the washed bacterial liquid into a 500mL beaker to prepare for breaking the walls.
(2) Cell wall breaking and PHA release of bacterial liquid
5M NaOH was added to a 500mL beaker containing 250mL of a fermentation broth containing PHA cells to adjust the pH of the broth to 9.5, and 0.01-1% (v/v) glycerol was added, and the 500mL beaker containing the broth was placed in a 1000mL beaker containing water, taking care that the 500mL beaker did not topple over, and the lower half was immersed in water. The two were reacted together in an autoclave at 121 ℃ for 90 min.
(3) Purification and Collection of PHA
After the wall breaking reaction is finished, centrifuging to remove non-PHA impurities, wherein the centrifugation conditions are as follows: 4500rpm, 10 min. After the centrifugation is finished, the supernatant is carefully removed, water is added to restore the volume, the centrifugation is continued after the uniform stirring, and the washing and centrifugation operations are repeated for 2 times. The washed and centrifuged PHA is tightly adhered to a centrifuge tube, so that the supernatant can be completely discarded, and PHA solid matters can not be lost.
(4) PHA purification parameter measurement
Freezing the centrifuge tube containing PHA precipitate in-80 deg.C ultra-low temperature refrigerator for 2 hr, freeze-drying in vacuum freeze-drying machine, grinding PHA, mixing, and measuring PHA quality and content by gas chromatography2,5. Three replicates were set for each set of experiments and the results were averaged. The results are shown in Table 3.
Table 4: influence of superheated water + Glycerol method on PHA purity and recovery
Reference documents:
1. chengkongqiang, Xue Yuansheng, Tan Dan, Wu Qiong, a strain of Halomonas and its application, CN102120973B.
2. Chengqiang, wuqiong, research on production technology of biodegradable plastic-Polyhydroxyalkanoate (PHA) [ J ] fine and specialty chemicals, 2001 (18): 22-25.
3. Chenzhong, plum blossom, Chenjinchun, etc. the method for separating and purifying intracellular polyhydroxyalkanoate from bacterial thallus is CN1070534C [ P ].
4. Chen Guo Qiang, Ganzhixiong, a method for separating and extracting polyhydroxyalkanoate from bacterial thallus, CN1211489C [ P ].
5. The identification and detection method of polyhydroxyalkanoate has advanced the research progress [ J ] Yunnan chemical industry, 2007(4):86-89.
6. Kangshimin, Yanminli, Xiuqiu, etc. a method for extracting polyhydroxyalkanoate by using valerolactone as solvent, CN109575264A [ P ].
7. Jiang leaf, Wandong Sheng, Chenfeng, a method for preparing Polyhydroxyalkanoate (PHA) CN109504715A [ P ].
8. Zhang Mengyao, Li Yahui, Zhanyuelong, et al research progress of halophilic bacteria biosynthesis Polyhydroxyalkanoates (PHAs) [ J ] biotechnological report, 2019 (6): 172-177.
It will be appreciated by persons skilled in the art that although the invention has been described with reference to specific embodiments thereof, the invention is not limited to these specific embodiments. Based on the teaching of the present invention and the technical solutions, those skilled in the art can make appropriate modifications or improvements without departing from the spirit of the present invention, and thus the resulting equivalent embodiments are within the scope of the present invention.
Claims (10)
1. A method for extracting PHA from microbial cells producing polyhydroxyalkanoate PHA, comprising the steps of: treating the microbial thallus with superheated water separately under the pH value of 7-11 to obtain thallus wall-broken lysate, wherein the temperature of the superheated water is more than 100 ℃ and less than or equal to 200 ℃, the treatment time is 0.2-4 hours, and the method does not use a surfactant,
the method further comprises treating the cell wall-broken lysate with a protease, wherein the protease is selected from the group consisting of: proteinase K, alkaline protease, acid protease, papain, SUMO protease, or a combination thereof; and is
The method further comprises treating a solution obtained after treating the cell wall-broken lysate with a protease with a bleaching agent, wherein the bleaching agent is selected from the group consisting of: hydrogen peroxide, sodium hypochlorite, sodium sulfite, thiourea dioxide, chlorine dioxide, or combinations thereof.
2. The method of claim 1 wherein the superheated water has a temperature of greater than 100 ℃ and less than or equal to 125 ℃.
3. The method according to claim 1, wherein the treatment of the microbial cells with superheated water is performed at a pressure of 0.1-30 MPa.
4. The method according to claim 3, wherein the treatment of the microbial cells with superheated water is performed at a pressure of 1.5-5 MPa.
5. The method according to claim 3, wherein the treatment of the microbial cells with superheated water is performed at a pressure of 0.12-0.15 MPa.
6. The method of claim 1, wherein 0.1-2 v/v% glycerol is added to the superheated water.
7. The method according to claim 1, further comprising washing, filtering, centrifuging and/or drying steps to obtain purified polyhydroxyalkanoate PHA.
8. The method of any one of claims 1-7, wherein the polyhydroxyalkanoate PHA is selected from the group consisting of short chain PHA with hydroxy fatty acids monomeric from C3-C5, medium long chain PHA with hydroxy fatty acids monomeric from C6-C18, and short and medium long chain PHA copolymers.
9. The method of claim 8, wherein the short chain PHA is polyhydroxypropionate, polyhydroxybutyrate PHB, or polyhydroxyvalerate.
10. The method according to any one of claims 1 to 7, wherein the polyhydroxyalkanoate PHA is a copolyester of 3-hydroxybutyrate and 3-hydroxyvalerate or a copolyester of 3-hydroxybutyrate and-4-hydroxybutyrate.
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