CN1094519C

CN1094519C - Process for extracting poly-beta-hydroxyalkanates from alkaligenes

Info

Publication number: CN1094519C
Application number: CN97106977A
Authority: CN
Inventors: 陈坚; 陈银广; 伦世仪
Original assignee: WUXI LIGHT INDUSTRY UNIV
Current assignee: WUXI LIGHT INDUSTRY UNIV
Priority date: 1997-05-22
Filing date: 1997-05-22
Publication date: 2002-11-20
Anticipated expiration: 2017-05-22
Also published as: CN1171410A

Abstract

The present invention relates to a novel method for extracting poly-beta-hydroxy naphthenic acid ester from cultivated alkaligenous bacilli, which is characterized in that poly-beta-hydroxy naphthenic acid ester (shortly named as 'PHA') is extracted from the cultivated alkaligenous bacilli with a water solution of a surface active agent and a complexing agent by one step. With the advantages of less environmental pollution and easy operation and the characteristics of high quality of products, low cost of reagents and low operation cost, the present invention is particularly suitable for commercial production.

Description

From alcaligenes eutrophus, extract the method for poly-beta-hydroxyalkanates

The present invention relates to a kind of method of from microbe, directly extracting poly-beta-hydroxyalkanates (poly-β-hydroxyalkanoate, abbreviation PHA) with tensio-active agent.

PHA is that microorganism is stored in intracellular a series of high molecular polymer under the unbalanced growth condition, poly-beta-hydroxy-butanoic acid ester (poly-β-hydroxybutyrate wherein, abbreviation PHB), poly-beta-hydroxy valerate (PHV) and both multipolymers (PHBV) not only have and the similar character of chemosynthesis plastics polypropylene, but also has the unexistent property of chemosynthesis plastics, as: biological degradability, biocompatibility, optical activity etc., and can utilize renewable resource to carry out biosynthesizing.Therefore closely during the last ten years, along with the chemosynthesis plastics cause seriousness to " white pollution " of environment, the research of the synthetic PHA of microorganism is subjected to people's extensive attention.Britain ICI company takes the lead in adopting alcaligenes eutrophus (A.eutrophus) to produce the biodegradable plastic of commodity " Biopol " by name in nineteen ninety through the effort in 15 years.

Being used for extracting the most frequently used method of PHA from this microorganism of alcaligenes eutrophus is organic solvent method, usually the organic solvent of usefulness comprises: chloroform is (as European patent 46,017), ethylene dichloride (European patent 58,480), 1,1,2-trichloroethane (United States Patent (USP) 4,310,684), diacetyl oxide (Deutsches Wirtschafts Patent 223,428), NSC 11801 and propylene carbonate (United States Patent (USP) 4,101,533) etc.The disadvantage that this method exists is: (1) uses a large amount of organic solvents, although the recyclable usefulness again of solvent, but still cause huge raw material consumption; (2) even, still have a certain amount of PHA to be present in the thalline, thereby extraction yield is difficult to reach very high through repeatedly extracting repeatedly; (3) when containing the PHA that surpasses 5% (w/v) in the solvent, solution becomes gets very sticking, and it is just very difficult to carry out lock out operation; (4) use of poisonous in a large number, explosive, solvent flashing causes severe contamination to environment, makes troubles to operation.

The another kind of method of extracting PHA is that sodium-hypochlorite process (is seen Biotechnology Techniques, 1989; 3 (4): 227), because used clorox has very strong oxidisability, thereby it has serious Degradation to the PHA molecule, make extract the PHA molecular weight that obtains reduced near half.

The method of the extraction PHA that people also know is a chloroform---sodium-hypochlorite process (is seen Biotechnology andBioengineering, 1994; 44:256), it comes down to a kind of method that above-mentioned two kinds of methods are combined.Owing to still need to use a large amount of organic solvents, and still used clorox, so it can't overcome the common shortcoming that exists of above-mentioned two kinds of methods.

Extract the method for PHA (as United States Patent (USP) 4 with enzyme process, 910,145) be adopt for the shortcoming that overcomes above-mentioned several method a kind of new extracting method, but it is not high to use enzyme process to extract the purity of the PHA that obtains merely, often to for example handle (United States Patent (USP) 4,101 with tensio-active agent again in conjunction with other method, 533), just can obtain the PHA of higher degree.The thermal treatment, enzyme that this method generally includes cell is handled and with steps such as anionic surfactant treatment, is operated very complicated.Although it has realized extracting PHB in aqueous phase system, but because the impurity component more complicated of cell, particularly the condition of enzyme effect is relatively harsher, and highly purified product needed multistep is handled operation, thereby the complicated operation of process, so the aspects such as extraction cost, process amplification that are applied in of enzyme process still are very limited.

In order to overcome the deficiency of the many and enzyme action condition harshness of operation steps that Enzymatic Extraction PHA exists, simultaneously with an organic solvent extract PHB with aqueous phase system in order to avoid as far as possible, people have studied and have used tensio-active agent---and clorox extracts method (Biotechnology Techniques, 1990 of PHA; 4 (4): 221).The weak point of this method is to use relatively large tensio-active agent (sodium lauryl sulphate or TX-100), and the use of clorox caused the degraded of PHA inevitably, and this just must limit its industrial application.

The method that the purpose of this invention is to provide the extraction poly-beta-hydroxyalkanates that a kind of environmental pollution is little, easy and simple to handle, expense is low, finished product purity is high.

Concrete operations step of the present invention is as follows:

(1) cryodesiccated thalline is joined in the aqueous solution that contains tensio-active agent and complexing agent, under certain temperature and pH, stir;

(2) centrifugal or filtration said mixture;

(3) with the solids that obtains with deionized water wash and centrifugal or filtering separation;

(4) use washing with acetone again;

(5) solids centrifugal or that filtration obtains is dried in 60 ℃ baking oven.

The used tensio-active agent of the present invention is a kind of empgen BB type amphoterics, its intramolecularly with quaternary ammonium salt base as cationic moiety, with carboxyl as negatively charged ion, structural formula is as follows:

It has stronger avidity to lyophobic dust, can in conjunction with and make cellular material solubilising such as phosphatide or be dissolved in the aqueous solution.

Here, the consumption of tensio-active agent is 1～80% of thalline (being an alcaligenes eutrophus) amount, and optimum amount is 12%.

Because alcaligenes eutrophus is a Gram-negative bacteria, the outer membrane structure is usually by divalent cation Ca ²⁺Or Mg ²⁺Keep in conjunction with lipopolysaccharides and protein.If add complexing agent EDTA with Ca ²⁺Or Mg ²⁺Chelating, a large amount of lipopolysaccharide molecules will come off, and the cave appears in outer membrane, and these zones are filled up by the phosphatide of inner layer film, thereby make this regional permeability increase, and help the release of intracellular organic matter PHA.

According to the present invention, the consumption of EDTA is 0～50% of a biomass, obtain highly purified product, and optimum amount is 8%.

The concentration of thalline in the aqueous solution is 1～100g/l, and optimum amount is 20g/l.

In order to help extracting, it is very important controlling suitable pH and keeping suitable temperature.Here, the pH value of aqueous solution that contains tensio-active agent (or EDTA) is 5～14, and the best is 13; Temperature is 5～120 ℃, and the best is 50 ℃.

The above-mentioned time with tensio-active agent processing thalline is 2～120 minutes, and the best use of time is 10 minutes.

In lock out operation subsequently, because the consumption of tensio-active agent is less, and the temperature that contains the mixed solution of PHA is not very low, so the viscosity of mixed solution is little; Because PHA is water insoluble, therefore can obtain crude product PHA again by centrifugal or filtering separation.

In the superincumbent filter operation, add an amount of diatomite, will help filtering as flocculating aids.

With proper amount of deionized water washing crude product PHA, can remove the water-soluble substances and the ion that are bonded on the PHA.Still the cell impurity substances that exists to remove some with acetone or methanol wash just can obtain more highly purified PHA again; And because acetone or methyl alcohol can form azeotrope with water, its boiling point reduces greatly than the boiling point of water, thereby also helps drying.

PHA after the washing was dried 24 hours in 60 ℃ thermostatic drying chamber, can obtain the product of white.

The quantitative analysis of PHA vapor-phase chromatography (Journal of Chromatography.1988,445:285); The mensuration of molecular weight is used viscosimetry (Macromolecules.1976,9 (5): 774).

The present invention not only can be in laboratory implementation, and very easily uses in industrial production.When handling thalline with water phase surfactant mixture, the most frequently used stirring tank of used device just.During solid-liquid separation, centrifugally operated only needed under 8000rpm centrifugal 5 minutes or 4000rpm under centrifugal 15 minutes; If use filter operation, then can under normal pressure or decompression (1 normal atmosphere), carry out.All these shows that the present invention is easy to realize from the laboratory to industrial amplification.

Among all embodiment below, used thalline all is after cultivate finishing, through centrifugal (2500rpm, 15min), deionized water wash, obtain through lyophilize again; The viscosity-average molecular weight of PHB is 402,000 before extracting; Used water all is deionized water; The content of PHB all is 60% of dry cell weight in the thalline; All take off data all are three mean values of measuring, and per-cent all is in mass.

Can understand the present invention better by following embodiment, they have narrated from Ralstonia eutropha the specific operation process of extracting PHA, and wherein major part is an example to extract PHB, but the present invention not merely is applicable to extracts PHB from Ralstonia eutropha.

Embodiment 1

4 gram 30% alkali solution of beet and 0.4 gram EDTA are dissolved in 200 milliliters the deionized water, transfer pH to 14 with NaOH.To wherein adding 4 gram thalline, under 50 ℃ temperature, stirred 10 minutes.Mixture after centrifugal 5 minutes, is used 100 ml waters and 40 milliliters of washing with acetones respectively under 8000rpm, the centrifugal throw out that obtains was dried by the fire 24 hours under 60 ℃ temperature.

The result is as follows:

The purity 99.9% of PHB

Extraction yield 56.7%

Viscosity-average molecular weight 8.9 * 10 ⁴

Obviously, though high pH extracts the product that can obtain extreme high purity, molecular weight generation severely degrade.

Embodiment 2

The pH that changes when extracting is 13, and other is operated with example 1.The gained result is as follows:

The purity 96.3% of PHB

Extraction yield 90.5%

Viscosity-average molecular weight 3.16 * 10 ⁵

Embodiment 3

The temperature that changes when extracting is 85 ℃, and other is operated with example 2.The gained result is as follows:

The purity 99.7% of PHB

Extraction yield 97.2%

Viscosity-average molecular weight 1.62 * 10 ⁵

Obviously, though high-temperature extracts the product that can obtain extreme high purity and fine extraction yield, the part degraded takes place in molecular weight.

Embodiment 4

The consumption that changes tensio-active agent is 12% of a biomass, and the consumption of EDTA is 8% of a biomass, and the pH during extraction is 13, and other is operated with example 1.The gained result is as follows:

The purity 98.7% of PHB

Extraction yield 93.3%

Viscosity-average molecular weight 3.16 * 10 ⁵

Claims

1, a kind of method of extracting poly-beta-hydroxyalkanates from alcaligenes eutrophus is characterized in that:

(1) cryodesiccated thalline is joined in the aqueous solution that contains tensio-active agent and complexing agent, under certain temperature and PH, stirs,

The concentration of thalline in the aqueous solution is 1～100 grams per liter;

Dosage of surfactant is 1～80% of a dry cell weight amount;

The complexing agent consumption is a dry cell weight amount 0～50%;

Extracting temperature is 5～120 ℃;

PH value is 5～14 during extraction;

Extraction time is 2～120 minutes;

(2) centrifugal or filtration said mixture;

(3) solids that obtains is spent ion-cleaning and centrifugal or filtering separation;

(4) use washing with acetone again;

(5) with solids oven dry centrifugal or that filtration obtains.

2, by the described method of claim 1, it is characterized in that described tensio-active agent is an empgen BB type amphoterics.

3, by the described method of claim 1, it is characterized in that described complexing agent is a disodium ethylene diamine tetraacetate.

4, by the described method of claim 1, it is characterized in that the concentration of described thalline in the aqueous solution is 20 grams per liters.

5, by the described method of claim 1, it is characterized in that described dosage of surfactant is 12% of a dry cell weight amount.

6, by the described method of claim 1, it is characterized in that described complexing agent consumption is a dry cell weight amount 8%.

7, by the described method of claim 1, it is characterized in that described extraction temperature is 50 ℃.

8, by the described method of claim 1, pH value is 13 when it is characterized in that extracting.

9, by the described method of claim 1, it is characterized in that described extraction time is 10 minutes.