CN100448911C - Method for extracting intracellular poly hydroxy fatty acid in microbe - Google Patents
Method for extracting intracellular poly hydroxy fatty acid in microbe Download PDFInfo
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Abstract
The present invention discloses a method for extracting intracellular poly hydroxy fatty acid from microbes. The method of the present invention has the steps that: 1, after microbe strains for producing poly hydroxy fatty acid ester are fermented, thallus cells in fermentation liquid are collected; 2, organic ester solvents with a similar structure to poly hydroxy fatty acid ester monomers are added into wet thalli or thallus powder (obtained through drying the wet thalli) collected in the first step, and obtained materials are uniformly mixed and are heated for 1 to 4 hours at 80 to 120 DEG C for extracting the poly hydroxy fatty acid ester; 3, thallus residue is separated and removed, and organic phases are reserved; 4, organic solvents which are intersoluble with ester extraction solvents used in the second step are used for settling the poly hydroxy fatty acid ester out of the organic phases obtained in the third step; 5, sediments are separated and washed; 6, after the sediments are dried, the poly hydroxy fatty acid ester is obtained. When used for the commercial process of high purity PHA, the method of the present invention has the advantages of short production time, low energy consumption, low production equipment investment, reduced environmental pollution in the production process, improved safety in the production process and wide application prospects.
Description
Technical field
The present invention relates to a kind of method of extracting intracellular poly hydroxy fatty acid in microbe.
Background technology
Polyhydroxyalkanoate (Poly-β-Hydroxyalkanoates is called for short PHA) is by polyester in a kind of born of the same parents of microorganism synthetic, exists with granular inclusion form in extracellular microbial.The physicals of PHA is similar to the plastics in petrochemical complex source, and has the biodegradable performance, is considered to a kind of " biodegradable plastic " of potentialization; At present, the PHA material that studies show that of field of tissue engineering technology has excellent biological compatibility, is very potential tissue engineering material.PHA is present in the microbe with the form of inclusion, and (general structure is seen formula 1, and R1, R2 can be different substituents groups according to the PHA monomer.The structure of typical R 1, R2 group is: CH
3, (CH
2) nCH
3(n=1-8)) difference of Zu Chenging, be divided into short chain PHA (monomeric carbon chain lengths 3-5, as poly 3-hydroxy butyrate PHB, 3-hydroxybutyric acid valeric acid copolyesters PHBV), middle long-chain PHA (the free carbon chain length is not less than 6), long-chain copolymerization PHA in the short chain (monomer of existing length 3-5 contains carbon chain lengths again and is not less than 6 monomer, as 3-hydroxybutyric acid caproic acid copolyesters PHBHHx).The physicals of these three kinds of PHA is widely different.Existing downstream aftertreatment PHA extraction process, extraction or broken wall reagent commonly used have chloroform, methylene dichloride, ethyl acetate etc., also have by carrying out extracting with organic solvent again behind the clorox processing lysing cell, also have by non-polyhydroxyalkanoate composition in the degraded microorganism to reach the purpose of purification.But all there is big defective in the said extracted method, as the rate of recovery is not high, product purity is low, extract back molecular weight product reduction etc., adds that many organic solvent toxicities of use are bigger, and boiling point is lower, and leaching process needs high pressure, brings potential safety hazard to production.For the PHA of middle long-chain, it is the very big material of viscosity that existing extracting method obtains the PHA precipitation usually, bring certain difficulty to extraction, and purity is lower difficult the separation and drying, has more impurity, has limited it in application on medical field.
Summary of the invention
The extracting method that the purpose of this invention is to provide a kind of easier, quick, intracellular poly hydroxy fatty acid in microbe that extraction yield is higher.
The method of extraction intracellular poly hydroxy fatty acid in microbe provided by the present invention may further comprise the steps:
1) microorganism strains of polyhydroxyalkanoate is produced in fermentation, collects the somatic cells in the fermented liquid;
2) add the ester class organic solvent similar to the polyhydroxyalkanoate monomer structure to wet thallus that step 1) is collected or in the thalline powder of making after being dried, mixing is at 80-120 ℃ of heating extraction in 1-4 hour polyhydroxyalkanoate down; The described ester class organic solvent similar to the polyhydroxyalkanoate monomer structure is one or more the mixed solvent in n-butyl acetate, isobutyl acetate, tert.-butyl acetate, pentyl acetate, methyl-butyrate, ethyl butyrate, propyl butyrate, the methyl caproate;
3) the thalline residue is removed in separation, keeps organic phase;
4) use and step 2) used ester class extracts the organic solvent that solvent dissolves each other polyhydroxyalkanoate is precipitated out from the organic phase that step 3) obtains; Describedly be used to precipitate the organic solvent of polyhydroxyalkanoate and the volume ratio of organic phase is 3-15: 1;
5) separation, washing precipitation;
6) will precipitate drying after, obtain polyhydroxyalkanoate.
In the said extracted method, the selection of producing the microorganism strains of polyhydroxyalkanoate in the step 1) is arbitrarily, as Aeromonas hydrophila 4AK4, E.coli JM109 (pBHR68), Wautersia eutropha H16 or Pseudomonas putida KT2442 etc., can it be fermented by the conventional culture condition of the microorganism strains that produces polyhydroxyalkanoate.
Step 2) by weight/volume of wet thallus and ester class organic solvent is 1 in: 1-5, and the by weight/volume of dry mycelium and ester class organic solvent is 1: 3-15; When directly extracting polyhydroxyalkanoate with wet thallus, for avoiding adding the wet thallus that extracts water behind the solvent and extract solvent and be difficult to mix, can be earlier with wet thallus with dehydrated alcohol or/and acetone mixes, add the extraction solvent again and extract; In addition, for obtaining higher extraction efficiency, the process of extracting polyhydroxyalkanoate from somatic cells is preferably under agitation condition carries out, and the speed of stirring can be 100-200 rev/min, is preferably 150 rev/mins.
The organic solvent that is used to precipitate polyhydroxyalkanoate in the step 4) can be 90-100% (V/V) ethanol, normal hexane or acetone.
Cleaning solvent in the step 5) can be dehydrated alcohol.
In the step 6) precipitation is carried out the exsiccant condition and can be, be preferably 50 ℃ of following heating under vacuum 4 hours at 45-55 ℃ of following heating under vacuum 3-5 hour.
In aforesaid method, step 3)-5) the sedimentary method of separate solid is centrifugal or filters from liquid-solid mixture.
The invention provides a kind of method of extracting intracellular poly hydroxy fatty acid in microbe.This method has the following advantages:
1) adopts the ester class organic solvent that has with PHA monomer analog structure to extract solvent,, have good solvability for PHA as n-butyl acetate, ethyl butyrate etc. as PHA; 2) used PHA extraction solvent is the industrial common agents with fruit aroma, and is not only cheap, and toxicity is very low, and boiling point is higher, is more than 100 ℃, thereby can realizes directly extracting PHA from wet thallus or exsiccant thalline under the normal pressure; 3) lower to the requirement of production unit; 4) for various types of other PHA good extraction effect is arranged all, extraction yield can reach 68-91%, and can obtain the higher PHA product of purity, and leaching process is to the not influence of molecular weight of product.Based on above-mentioned advantage, method of the present invention is applied to the suitability for industrialized production of high purity PHA, not only can shortens the production time, reduce the pollution of energy consumption, investment of production equipment and production process environment, and can improve security in the production process, have a extensive future.
Below in conjunction with specific embodiment the present invention is described in further details.
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment, and wherein the extraction yield of intracellular poly hydroxy fatty acid in microbe (PHA) calculates according to following formula:
Embodiment 1, extraction Aeromonas hydrophila 4AK4 intracellular poly hydroxy fatty acid
Now use n-butyl acetate from Aeromonas hydrophila 4AK4 (Chen GQ, G.Zhang, S.J.Park, S.Y.Lee, Industrial scale Production of Poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) .Appl Microbiol Biotechnol, 57 (2001) 57:50-55) extract long-chain copolymerization PHA (PHBHHx) in the short chain in the dry mycelium, wherein the structural formula of the positive butyl ester of broken wall extraction solvent acetic acid is as follows:
Formula 2
Concrete leaching process may further comprise the steps:
1) weighing is through the Aeromonas hydrophila 4AK4 dry mycelium powder 20g of fermentation;
2) add the 100mL n-butyl acetate, mixing stirs down at 150 rev/mins, and 110 ℃ of following backflow heat treated 1 hour;
3) 5000g is centrifugal 10 minutes, removes the residue that degerms, and stays supernatant;
4) add 500mL 95% (v/v) alcohol in supernatant liquor, mix, left standstill 30 minutes, the cotton-shaped or Powdered precipitation of adularescent is separated out;
5) 5000g is centrifugal 2 minutes, and collecting precipitation washed 10 minutes centrifugal 5 minutes collecting precipitations of 5000g with the 50mL soaked in absolute ethyl alcohol to it;
6) will through washing be deposited in 50 ℃ of following vacuum-dryings 4 hours, obtain the PHA product.
After testing, the PHA product purity that obtains with aforesaid method is 96%, and extraction yield is 90%, and weight-average molecular weight is 480000 dalton, conformance with standard.
Embodiment 2, extraction Aeromonas hydrophila 4AK4 intracellular poly hydroxy fatty acid
Now use the mixed solvent of n-butyl acetate and isobutyl acetate to extract long-chain copolymerization PHA (PHBHHx) in the short chain from the dry mycelium of Aeromonas hydrophila 4AK4, wherein the structural formula of the isobutyl acetate in the broken wall extraction solvent is as follows:
Concrete leaching process may further comprise the steps:
1) weighing is through the Aeromonas hydrophila 4AK4 dry mycelium powder 20g of fermentation;
2) add 60mL n-butyl acetate and isobutyl acetate mixed solvent (volume ratio 1: 1), mixing, 200 rev/mins of stirrings down, and 110 ℃ of following backflow heat treated 1 hour;
3) 5000g is centrifugal 10 minutes, removes the residue that degerms, and stays supernatant;
4) add 900mL 95% (v/v) alcohol in supernatant liquor, mix, left standstill 30 minutes, the cotton-shaped or Powdered precipitation of adularescent is separated out;
5) 5000g is centrifugal 2 minutes, and collecting precipitation washed 10 minutes centrifugal 5 minutes collecting precipitations of 5000g with the 60mL soaked in absolute ethyl alcohol to it;
6) will through washing be deposited in 50 ℃ of following vacuum-dryings 4.5 hours, obtain the PHA product.
After testing, the PHA product purity that obtains with aforesaid method is 95%, and extraction yield is 75%, and weight-average molecular weight is 490000 dalton, conformance with standard.
Embodiment 3, extraction Aeromonas hydrophila 4AK4 intracellular poly hydroxy fatty acid
Now use n-butyl acetate to extract long-chain copolymerization PHA (PHBHHx) in the short chain from the dry mycelium of Aeromonas hydrophila 4AK4, concrete leaching process may further comprise the steps:
1) weighing is through the Aeromonas hydrophila 4AK4 dry mycelium powder 20g of fermentation;
2) add the 300mL n-butyl acetate, mixing stirs down at 130 rev/mins, and 120 ℃ of following backflow heat treated 1 hour;
3) 5000g is centrifugal 10 minutes, removes the residue that degerms, and stays supernatant;
4) add 900mL 95% (v/v) alcohol in supernatant liquor, mix, left standstill 30 minutes, the cotton-shaped or Powdered precipitation of adularescent is separated out;
5) 5000g is centrifugal 2 minutes, and collecting precipitation washed 10 minutes centrifugal 5 minutes collecting precipitations of 5000g with the 60mL soaked in absolute ethyl alcohol to it;
6) will through washing be deposited in 45 ℃ of following vacuum-dryings 5 hours, obtain the PHA product.
After testing, the PHA product purity that obtains with aforesaid method is 94%, and extraction yield is 78%, and weight-average molecular weight is 455000 dalton, conformance with standard.
Embodiment 4, extraction Aeromonas hydrophila 4AK4 intracellular poly hydroxy fatty acid
Now use n-butyl acetate to extract long-chain copolymerization PHA (PHBHHx) in the short chain from the dry mycelium of Aeromonas hydrophila 4AK4, concrete leaching process may further comprise the steps:
1) weighing is through the Aeromonas hydrophila 4AK4 dry mycelium powder 20g of fermentation;
2) add the 300mL n-butyl acetate, mixing stirs down at 150 rev/mins, and 80 ℃ of following backflow heat treated 4 hours;
3) 5000g is centrifugal 10 minutes, removes the residue that degerms, and stays supernatant;
4) add 500mL 95% (v/v) alcohol in supernatant liquor, mix, left standstill 30 minutes, the cotton-shaped or Powdered precipitation of adularescent is separated out;
5) 5000g is centrifugal 2 minutes, and collecting precipitation washed 10 minutes centrifugal 5 minutes collecting precipitations of 5000g with the 20mL soaked in absolute ethyl alcohol to it;
6) will through washing be deposited in 55 ℃ of following vacuum-dryings 3 hours, obtain the PHA product.
After testing, the PHA product purity that obtains with aforesaid method is 96%, and extraction yield is 72%, and weight-average molecular weight is 415000 dalton, conformance with standard.
Embodiment 5, extraction Pseudomonas putida KT2442 intracellular poly hydroxy fatty acid
Now use n-butyl acetate from Pseudomonas putida KT2442 (Kellerhals MB et al, Closed-loop control of bacterial high-cell-density fed-batch cultures:Production of mcl-PHAs by Pseudomonas putida KT2442 under single-substrateand cofeeding conditions.BIOTECHNOLOGY AND BIOENGINEERING, 65 (3): 306-315NOV 5 1999) dry mycelium in extract in long-chain PHA, concrete leaching process may further comprise the steps:
1) weighing is through the Pseudomonas putida KT2442 dry mycelium powder 20g of fermentation;
2) add the 120mL n-butyl acetate, mixing stirs down at 150 rev/mins, and 100 ℃ of following backflow heat treated 1 hour;
3) 5000g is centrifugal 10 minutes, removes the residue that degerms, and stays supernatant;
4) add 600mL 95% (v/v) alcohol in supernatant liquor, mix, left standstill 30 minutes, the cotton-shaped or Powdered precipitation of adularescent is separated out;
5) 5000g is centrifugal 2 minutes, and collecting precipitation washed 10 minutes centrifugal 5 minutes collecting precipitations of 5000g with the 20mL soaked in absolute ethyl alcohol to it;
6) will through washing be deposited in 50 ℃ of following vacuum-dryings 4 hours, obtain the PHA product.
After testing, the PHA product purity that obtains with aforesaid method is 95%, and extraction yield is 89%, and weight-average molecular weight is 385000 dalton, conformance with standard.
Embodiment 6, extraction Aeromonas hydrophila 4AK4 intracellular poly hydroxy fatty acid
Now use ethyl butyrate to extract long-chain copolymerization PHA (PHBHHx) in the short chain from the wet thallus of Aeromonas hydrophila 4AK4, wherein the structural formula of broken wall extraction solvent ethyl butyrate is as follows:
Concrete leaching process may further comprise the steps:
1) weighing is through the Aeromonas hydrophila 4AK4 wet thallus 60g of fermentation;
2) add 20mL ethanol, mix, add the 150mL ethyl butyrate again, mixing stirs down at 150 rev/mins, and 100 ℃ of following backflow heat treated 2 hours;
3) 5000g is centrifugal 10 minutes, removes the residue that degerms, and stays supernatant;
4) add 600mL 95% (v/v) alcohol in supernatant liquor, mix, left standstill 30 minutes, the cotton-shaped or Powdered precipitation of adularescent is separated out;
5) 5000g is centrifugal 2 minutes, and collecting precipitation washed 10 minutes centrifugal 5 minutes collecting precipitations of 5000g with the 50mL soaked in absolute ethyl alcohol to it;
6) will through washing be deposited in 50 ℃ of following vacuum-dryings 4 hours, obtain the PHA product.
After testing, the PHA product purity that obtains with aforesaid method is 92%, and extraction yield is 79%, and weight-average molecular weight is 375000 dalton, conformance with standard.
Embodiment 7, extraction Pseudomonas putida KT2442 intracellular poly hydroxy fatty acid
Now use n-butyl acetate from the wet thallus of Pseudomonas putida KT2442, extract in long-chain PHA, concrete leaching process may further comprise the steps:
1) weighing is through the Pseudomonas putida KT2442 wet thallus 50g of fermentation;
2) add 20mL acetone, mix, add the 150mL n-butyl acetate again, mixing stirs down at 150 rev/mins, and 90 ℃ of following backflow heat treated 1 hour;
3) 5000g is centrifugal 10 minutes, removes the residue that degerms, and stays supernatant;
4) add 500mL 95% (v/v) alcohol in supernatant liquor, mix, left standstill 30 minutes, the cotton-shaped or Powdered precipitation of adularescent is separated out;
5) 5000g is centrifugal 2 minutes, and collecting precipitation washed 10 minutes centrifugal 5 minutes collecting precipitations of 5000g with the 50mL soaked in absolute ethyl alcohol to it;
6) will through washing be deposited in 50 ℃ of following vacuum-dryings 4 hours, obtain the PHA product.
After testing, the PHA product purity that obtains with aforesaid method is 96%, and extraction yield is 87%, and weight-average molecular weight is 390000 dalton, conformance with standard.
Embodiment 8, extraction Pseudomonas putida KT2442 intracellular poly hydroxy fatty acid
Now use methyl caproate from the wet thallus of Pseudomonas putida KT2442, extract in long-chain PHA, it is as follows that wherein broken wall extracts the structural formula of solvent methyl caproate:
Concrete leaching process may further comprise the steps:
1) weighing is through the Pseudomonas putida KT2442 wet thallus 50g of fermentation;
2) add 20mL acetone, mix, add the 200mL methyl caproate again, mixing stirs down at 150 rev/mins, and 90 ℃ of following backflow heat treated 4 hours;
3) 5000g is centrifugal 10 minutes, removes the residue that degerms, and stays supernatant;
4) add 600mL 95% (v/v) alcohol in supernatant liquor, mix, left standstill 30 minutes, the cotton-shaped or Powdered precipitation of adularescent is separated out;
5) 5000g is centrifugal 2 minutes, and collecting precipitation washed 10 minutes centrifugal 5 minutes collecting precipitations of 5000g with the 50mL soaked in absolute ethyl alcohol to it;
6) will through washing be deposited in 50 ℃ of following vacuum-dryings 4 hours, obtain the PHA product.
After testing, the PHA product purity that obtains with aforesaid method is 94%, and extraction yield is 78%, and weight-average molecular weight is 370000 dalton, conformance with standard.
Embodiment 9, extraction Wautersia eutropha H16 intracellular poly hydroxy fatty acid
Now use n-butyl acetate from Wautersia eutropha H16 (Loo CY et al, Biosynthesis andcharacterization of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) from palmoil products in a Wautersia eutropha mutant.BIOTECHNOLOGY LETTERS 27 (18): extract short chain PHA (PHB) in dry mycelium 1405-1410 SEP 2005), concrete leaching process may further comprise the steps:
1) weighing is through the Wautersia eutropha H16 dry mycelium powder 20g of fermentation;
2) add the 100mL n-butyl acetate, mixing stirs down at 150 rev/mins, and 100 ℃ of following backflow heat treated 1 hour;
3) 5000g is centrifugal 10 minutes, removes the residue that degerms, and stays supernatant;
4) add 500mL 95% (v/v) alcohol in supernatant liquor, mix, left standstill 30 minutes, the cotton-shaped or Powdered precipitation of adularescent is separated out;
5) 5000g is centrifugal 2 minutes, and collecting precipitation washed 10 minutes centrifugal 5 minutes collecting precipitations of 5000g with the 50mL soaked in absolute ethyl alcohol to it;
6) will through washing be deposited in 50 ℃ of following vacuum-dryings 4 hours, obtain the PHA product.
After testing, the PHA product purity that obtains with aforesaid method is 94%, and extraction yield is 91%, and weight-average molecular weight is 890000 dalton, conformance with standard.
Embodiment 10, extraction Wautersia eutropha H16 intracellular poly hydroxy fatty acid
Now use n-butyl acetate to extract short chain PHA (PHBV) from the dry mycelium of Wautersia eutropha H16, concrete leaching process may further comprise the steps:
1) weighing is through the Wautersia eutropha H16 dry mycelium powder 20g of fermentation;
2) add the 100mL n-butyl acetate, mixing stirs down at 150 rev/mins, and 110 ℃ of following backflow heat treated 2 hours;
3) 5000g is centrifugal 10 minutes, removes the residue that degerms, and stays supernatant;
4) add 500mL 95% (v/v) alcohol in supernatant liquor, mix, left standstill 30 minutes, the cotton-shaped or Powdered precipitation of adularescent is separated out;
5) 5000g is centrifugal 2 minutes, and collecting precipitation washed 10 minutes centrifugal 5 minutes collecting precipitations of 5000g with the 50mL soaked in absolute ethyl alcohol to it;
6) will through washing be deposited in 50 ℃ of following vacuum-dryings 4 hours, obtain the PHA product.
After testing, the PHA product purity that obtains with aforesaid method is 95%, and extraction yield is 88%, and weight-average molecular weight is 810000 dalton, conformance with standard.
Embodiment 11, extraction JM109 (pBHR68) intracellular poly hydroxy fatty acid
Now use n-butyl acetate from JM109 (pBHR68) (Patricia Spiekermann et al, A sensitive, viable-colony staining method using Nile red for direct screening of bacteriathat accumulate polyhydroxyalkanoic acids and other lipid storage compounds.Arch Microbiol, 171:73-80,1999) extract short chain PHA (PHB) in the dry mycelium, concrete leaching process may further comprise the steps:
1) weighing is through JM109 (pBHR68) the dry mycelium powder 20g of fermentation;
2) add the 60mL n-butyl acetate, mixing stirs down at 150 rev/mins, and 120 ℃ of following backflow heat treated 3 hours;
3) 5000g is centrifugal 10 minutes, removes the residue that degerms, and stays supernatant;
4) add 300mL 95% (v/v) alcohol in supernatant liquor, mix, left standstill 30 minutes, the cotton-shaped or Powdered precipitation of adularescent is separated out;
5) 5000g is centrifugal 2 minutes, and collecting precipitation washed 10 minutes centrifugal 5 minutes collecting precipitations of 5000g with the 50mL soaked in absolute ethyl alcohol to it;
6) will through washing be deposited in 50 ℃ of following vacuum-dryings 4 hours, obtain the PHA product.
After testing, the PHA product purity that obtains with aforesaid method is 95%, and extraction yield is 87%, and weight-average molecular weight is 850000 dalton, conformance with standard.
Embodiment 12, extraction Aeromonas hydrophila 4AK4 intracellular poly hydroxy fatty acid
Now use the mixed solvent of tert.-butyl acetate and pentyl acetate from the dry mycelium of Aeromonas hydrophila 4AK4, to extract long-chain copolymerization PHA (PHBHHx) in the short chain, wherein the structural formula of the tert.-butyl acetate in the broken wall extraction solvent is seen formula 6, and the structural formula of pentyl acetate is seen formula 7:
Concrete leaching process may further comprise the steps:
1) weighing is through the Aeromonas hydrophila 4AK4 dry mycelium powder 20g of fermentation;
2) add the mixed solvent mixed solvent (volume ratio 1: 1) of 60mL tert.-butyl acetate and pentyl acetate, mixing, 150 rev/mins of stirrings down, and 110 ℃ of following backflow heat treated 2 hours;
3) 5000g is centrifugal 10 minutes, removes the residue that degerms, and stays supernatant;
4) add 300mL 95% (v/v) alcohol in supernatant liquor, mix, left standstill 30 minutes, the cotton-shaped or Powdered precipitation of adularescent is separated out;
5) 5000g is centrifugal 2 minutes, and collecting precipitation washed 10 minutes centrifugal 5 minutes collecting precipitations of 5000g with the 20mL soaked in absolute ethyl alcohol to it;
6) will through washing be deposited in 50 ℃ of following vacuum-dryings 4 hours, obtain the PHA product.
After testing, the PHA product purity that obtains with aforesaid method is 90%, and extraction yield is 68%, and weight-average molecular weight is 470000 dalton, conformance with standard.
Embodiment 13, extraction Aeromonas hydrophila 4AK4 intracellular poly hydroxy fatty acid
Now use the mixed solvent of methyl-butyrate and propyl butyrate to extract long-chain copolymerization PHA (PHBHHx) in the short chain from the dry mycelium of Aeromonas hydrophila 4AK4, wherein the structural formula of the methyl-butyrate in the broken wall extraction solvent is as follows:
Concrete leaching process may further comprise the steps:
1) weighing is through the Aeromonas hydrophila 4AK4 dry mycelium powder 20g of fermentation;
2) add the mixed solvent mixed solvent (volume ratio 1: 1) of 80mL methyl-butyrate and propyl butyrate, mixing, 150 rev/mins of stirrings down, and 110 ℃ of following backflow heat treated 1 hour;
3) 5000g is centrifugal 10 minutes, removes the residue that degerms, and stays supernatant;
4) add the 400mL normal hexane in supernatant liquor, mix, left standstill 30 minutes, the cotton-shaped or Powdered precipitation of adularescent is separated out;
5) 5000g is centrifugal 2 minutes, and collecting precipitation washed 10 minutes centrifugal 5 minutes collecting precipitations of 5000g with the 20mL soaked in absolute ethyl alcohol to it;
6) will through washing be deposited in 50 ℃ of following vacuum-dryings 4 hours, obtain the PHA product.
After testing, the PHA product purity that obtains with aforesaid method is 91%, and extraction yield is 70%, and weight-average molecular weight is 465000 dalton, conformance with standard.
Embodiment 14, extraction Aeromonas hydrophila 4AK4 intracellular poly hydroxy fatty acid
Now use the mixed solvent (volume ratio 1: 1: 1) of n-butyl acetate, isobutyl acetate and ethyl butyrate to extract long-chain copolymerization PHA (PHBHHx) in the short chain from the dry mycelium of Aeromonas hydrophila 4AK4, concrete leaching process may further comprise the steps:
1) weighing is through the Aeromonas hydrophila 4AK4 dry mycelium powder 20g of fermentation;
2) add the mixed solvent (volume ratio 1: 1: 1) of 90mL n-butyl acetate, isobutyl acetate and ethyl butyrate, mixing, 150 rev/mins of stirrings down, and 110 ℃ of following backflow heat treated 1 hour;
3) 5000g is centrifugal 10 minutes, removes the residue that degerms, and stays supernatant;
4) add 360mL acetone in supernatant liquor, mix, left standstill 30 minutes, the cotton-shaped or Powdered precipitation of adularescent is separated out;
5) 5000g is centrifugal 2 minutes, and collecting precipitation washed 10 minutes centrifugal 5 minutes collecting precipitations of 5000g with the 20mL soaked in absolute ethyl alcohol to it;
6) will through washing be deposited in 50 ℃ of following vacuum-dryings 4 hours, obtain the PHA product.
After testing, the PHA product purity that obtains with aforesaid method is 90%, and extraction yield is 75%, and weight-average molecular weight is 445000 dalton, conformance with standard.
Claims (10)
1, a kind of method of extracting middle long-chain polyhydroxyalkanoate in the extracellular microbial may further comprise the steps:
1) after the microorganism strains of polyhydroxyalkanoate is produced in fermentation, collects the somatic cells in the fermented liquid;
2) add the ester class organic solvent similar to the polyhydroxyalkanoate monomer structure to wet thallus that step 1) is collected or in the thalline powder of making after being dried, mixing is at 80-120 ℃ of heating extraction in 1-4 hour polyhydroxyalkanoate down; The described ester class organic solvent similar to the polyhydroxyalkanoate monomer structure is methyl caproate;
3) the thalline residue is removed in separation, keeps organic phase;
4) use and step 2) used ester class extracts the organic solvent that solvent dissolves each other polyhydroxyalkanoate is precipitated out from the organic phase that step 3) obtains; Describedly be used to precipitate the organic solvent of polyhydroxyalkanoate and the volume ratio of organic phase is 3-15: 1;
5) separation, washing precipitation;
6) will precipitate drying after, obtain polyhydroxyalkanoate;
The free carbon chain length of long-chain polyhydroxyalkanoate is not less than 6 in described.
2, method according to claim 1 is characterized in that: the microorganism strains that produces polyhydroxyalkanoate in the described step 1) is Pseudomonas putida KT2442.
3, method according to claim 1 is characterized in that: the by weight/volume of wet thallus and ester class organic solvent is 1 described step 2): 1-5, the by weight/volume of dry mycelium and ester class organic solvent is 1: 3-15.
4, method according to claim 1 is characterized in that: when directly extracting polyhydroxyalkanoate described step 2) with wet thallus, earlier with wet thallus with dehydrated alcohol or/and acetone mixes, add the extraction solvent again and extract.
5, according to claim 1 or 2 or 3 or 4 described methods, it is characterized in that: the process of described step 2) extracting polyhydroxyalkanoate from somatic cells is carried out under agitation condition.
6, method according to claim 5 is characterized in that: the speed of described stirring is 100-200 rev/min.
7, according to claim 1 or 2 or 3 or 4 described methods, it is characterized in that: the organic solvent that is used to precipitate polyhydroxyalkanoate in the described step 4) is 90-100% ethanol, normal hexane or acetone; Described alcoholic acid percentage composition is a volumn concentration.
8, according to claim 1 or 2 or 3 or 4 described methods, it is characterized in that: the cleaning solvent in the described step 5) is a dehydrated alcohol.
9, according to claim 1 or 2 or 3 or 4 described methods, it is characterized in that: it is at 45-55 ℃ of following heating under vacuum 3-5 hour that described step 6) is carried out the exsiccant condition to precipitation.
10, according to claim 1 or 2 or 3 or 4 described methods, it is characterized in that: described step 3)-5) the sedimentary method of separate solid is centrifugal or filters from liquid-solid mixture.
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| CN101591575A (en) | 2008-05-30 | 2009-12-02 | 汕头大学 | The hydroxy fatty acid derivative Application of Additives that acts as a fuel |
| CN103589503B (en) * | 2012-08-13 | 2015-09-30 | 丰益(上海)生物技术研发中心有限公司 | A kind of method of efficient extraction of microbial oil |
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| CN109575264B (en) * | 2018-10-26 | 2021-09-28 | 亿科优生物降解材料研究院(广东)有限公司 | Method for extracting polyhydroxyalkanoate by taking valerolactone as solvent |
| CN109517156A (en) * | 2019-01-02 | 2019-03-26 | 清华大学 | A kind of purification process of polyhydroxyalkanoate |
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| CN113121807B (en) * | 2021-04-07 | 2022-05-06 | 珠海麦得发生物科技股份有限公司 | Method for removing endotoxin in PHAs by fermentation method |
| CN115807044B (en) * | 2022-11-09 | 2023-10-13 | 华南理工大学 | Method for efficiently extracting and purifying high-purity polyhydroxyalkanoate |
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