CN1648149A - Hydroxy alkanoic acid polymer and its producing method - Google Patents

Abstract

The present invention discloses one kind of hydroxy alkanoic acid polymer, that is, 3-hydroxy butyric acid-3-hydroxy caprylic acid polymer (PHBHO). The 3-hydroxy butyric acid-3-hydroxy caprylic acid polymer (PHBHO) is prepared with Sinorhizobium fredii as production strain, carbohydrate as carbon source and ammonium salt as nitrogen source, and through high density fermentation and metabolism regulation with lauric acid in optimal culture medium and under optimal culture condition. Corresponding fermentation management method, separation and purification method and quality detection method are established. The present invention can obtain PHBHO product up to 14.4-22.1 g/L, of molecular weight of 112000-185000 Da, and endotoxin content 4-6 EU/g, and the novel nanometer level material may be used in biomedicine and medical tissue engineering.

Description

A kind of hydroxy alkanoic acid polymer and production method thereof

Technical field

The present invention relates to a kind of macromolecular compound that makes by the reaction that on high polymer main chain, forms the carboxylic acid ester bond, specifically belong to a kind of hydroxy alkanoic acid polymer (hereinafter to be referred as PHA) and production method thereof.

Background technology

PHA be a class by biosynthetic high molecular polymer simple in structure, be prokaryotic organism main source of carbon and energy reserve substance.Prokaryotic organism just can utilize the synthetic PHA of remaining nutritive substance and are distributed in discretely in the cell with the particulate form under uneven metabolism condition, and its content reaches as high as 90% of dry cell weight, when growth needs, it is decomposed utilize again.Just because of this biological characteristics, biological recyclability of this class material and biodegradability have been given.

Studies show that PHA has and general-purpose plastics, as polypropylene, closely similar physico-chemical property can be made a lot of products by plastic working.Just because of its these physics, chemistry and biology characteristic, make it become the new bio thermoplastic materials that a class can substitute " environmentally friendly " of traditional petrochemical industry plastics.Discovered in recent years, PHA have character such as excellent biological compatibility, degraded product nontoxicity and surface modificability, and this has just greatly expanded its range of application, make it aspect medical treatment and the medical tissue engineering wide application prospect arranged.It not only can make operating sutures, wound overlay film and various medicine equipments etc., can also be as the timbering material of clone organ.The people such as WilliamsS.F of U.S. Metabolix company just be framework in 1998 with PHA vitro culture gone out the patient from the body blood vessel.2000, medical college of Harvard University used the PHA model successfully to clone heart valve again.Recently find also that the nano level PHA of molecular weight about 100,000 is the ideal material of making medicinal slow release agent.

Countries in the world mainly concentrate on the Development and Production bacterial strain, make up genetic engineering bacterium, explore pathways metabolism and Regulation Mechanism, establishment production method and technology, mensuration product structure and performance and widen aspect such as range of application for the research of PHA, and have obtained bigger progress.

So far found that more than 90 prokaryotic organism that belong to can accumulate PHA (Handbook of BiodegradablePlastic in vivo, Research Association of Biodegradable Plastic, NTS Co., Ltd., pp.178-197), however be used for the microorganism that PHA research and the microbial strains that produces mainly concentrate on a few genus such as Alcaligenes, Rhodopseudomonas, rhizobium.In addition, also made up the genetic engineering bacterium that many strains can be synthesized PHA by genetic engineering means.

Most of prokaryotic organism synthetic PHA kind is PHB, but also can synthesize homotype or the special-shaped PHA that is made up of other monomer by some microorganisms.Up to now, had been found that the hydroxy alkanoic acid monomer that kind more than 100 is different.These monomers can be saturated or unsaturated hydroxy alkanoic acids, can be the hydroxy alkanoic acids that has the aromatic series side chain, can also be to have halogen or the substituent hydroxy alkanoic acid of cyano group.The position of substitution of hydroxyl can be respectively on 2,3,4,5,6 carbon atoms of alkanoic acid, but wherein general with 3-hydroxy alkanoic acid monomer.The PHA that these different monomers constitute, the existing general character of its physics characteristic also has characteristic.Many research reports confirm that the monomer carbochain of PHA is long more, and its snappiness is just good more.In general, short chain PHA (ssc-PHA) is more harder than medium chain PHA (msc-PHA), and msc-PHA is more more pliable and tougher than ssc-PHA.Increasing some special side chain substituents on linear PHA chain usually can give PHA new nature and function.In addition, form PHA the number of monomers purpose what, i.e. the size of PHA molecular weight also can influence character and the purposes of PHA to a certain extent.In general, the molecular weight of PHA is between 50000～1000000Da, if its molecular weight is lower than 20000, just its thermoplastic property can be subjected to very big influence.

The PHA that biosynthesizing is different, closely related with factors such as microbe species, pathways metabolism, medium component, culture condition and control methods.It is reported, adopt really to support and have a liking for alkali bacterium H16 bacterial strain [Alcaligenes eutropus H16 (ATCCNo.17699)] and not only can produce PHB, and use its mutant strain can synthesize various ratios by different carbon sources 3-hydroxybutyric acid (3-HB) and 3-hydroxypentanoic acid (3-HV) polymer [P (HBHV)] (U.S.Pat.No.4,393,167 and 4,876,331).At U.S.Pat.No.5,200, in 332 patents, set up a kind of with first bacteria, secondary coccus, have a liking for alkali bacterium and pseudomonas (Methylo-bacterium sp.Paracoccus sp., Alcaligenes sp. and Pseudomonas sp.) for producing bacterial strain, with ethanol the method for the synthetic P (HBHV) of carbon source.

At Appl.Environ.Microbiol, 58 (2), it is sad as substrate synthesis of hydroxy butyric acid (HB), hydroxycaproic acid (HH), Hydroxyoctanoic acid (HO) and hydroxydecanoic acid (HD) polymer [P (HB-HH-HO-HD)] to have reported in 746 (1992) that Pseudomonas resinovorans (Pseudomonasresinovorans) can utilize, and wherein the ratio of monomer HB, HH, HO, HD is 1: 15: 75: 9; And with the capric acid during for substrate the monomer ratio of HB, HH, HO and the HD of synthetic P (HB-HH-HO-HD) be 8: 62: 23: 7.

At U.S.Pat.No.5, synthesized P (HBHH) with sweet oil and oleic acid as carbon source by Aeromonas caviae (Aeromonas caviae) in 292,860 and Japanese Patent Application Laid-Open No.7,265,065.It is that carbon source has also been synthesized P (HBHH) (food and biotechnology, Val, 21,76-79,2002) with soya-bean oil that water-based Aeromonas (Aeromonas hydrophila 4AK4) is had a liking in people such as Zhang Jin, Wu Qiong, Chen Guoqiang utilization.People such as Ou Yangshaoping, Wu Qiong, Chen Guoqiang have made up two strains and have had a liking for water-based Aeromonas (Aeromonas hydrophila WQ and Aeromonas hydrophila 4AK4) gene recombination bacterium, with Sunmorl N 60S and lauric acid is that carbon source has been synthesized P (HBHH), and carried out fermentation test (the biotechnology journal of 6L fermentor tank, Vol, 19,709-714,2003).

In sum, for its range of application of physicochemical property, processing characteristics and expansion of improving PHA, carried out many researchs at the aspects such as exploitation, transformation and production method thereof that produce bacterial classification, and obtained certain progress.Aspect the research of the akin hydroxy alkanoic acid polymer of the present invention, relevant achievement and characteristics thereof are as follows: (1) has developed the polymer P (HB-HH-HO-HD) that is made up of four kinds of hydroxy alkanoic acid monomers, dimer P (HBHV) and P (HBHH) by two kinds of hydroxy alkanoic acid monomers are formed do not report but see the dimeric exploitation of P (HBHO) as yet.(2) physico-chemical property of hydroxy alkanoic acid polymer, at first relevant with the polymeric monomeric species of composition.Compare the performance of P (HBHH) and P (HBHV), elasticity is strong than latter's fragility is low for the former, has good processing properties.Compare P (HBHO) and P (HBHH), elasticity and the processing characteristics of P (HBHO) are more excellent.Secondly, the character of hydroxy alkanoic acid polymer is relevant with polymeric monomer ratio.Monomeric species is more in P (HB-HH-HO-HD), and in general monomeric species is many more, and the various monomer ratio of control are just difficult more in fermentative production, and the metastable PHA product of production monomer ratio relative fixed and physicochemical property is just relatively more difficult.(3) in relevant hydroxy alkanoic acid polymer research, the microorganism strains of employing has pseudomonas, Aeromonas and relevant gene recombination bacterium.These bacterial strain common physiology characteristics are to synthesize the 3-hydroxy alkanoic acid polymer with lipid or lipid acid as sole carbon source, it utilizes ability relatively poor to the carbohydrate carbon source, particularly have a liking for the water-based Aeromonas, when glucose concn reaches 1%, will synthesize the formation restraining effect to thalli growth and PHA.Yet, for most microorganisms, carbohydrate is the optimum carbon source of its growth, and therefore research is matrix with the carbohydrate, be aided with metabolic regulation means synthesis of hydroxy alkanoic acid polymer, for utilizing microorganism exploitation hydroxy alkanoic acid polymer product to have more representativeness.In addition, in the production process of food and medical product, generally do not adopt gene recombination bacterium as producing bacterial strain, to avoid occurring the biological safety problem at present.(4) with the correlative study of the akin hydroxy alkanoic acid polymer of the present invention in, the main raw material of employing is respectively sweet oil and oleic acid, soya-bean oil and lauric acid, sodium gluconate and lauric acid etc., does not all report the downstream extraction and the course of processing.In general, producing PHA with oils as carbon source through fermentation can increase the difficulty that product extracts, and usually can influence the quality of PHA.And with sodium gluconate as fermentation raw material, can be higher because of its price, strengthen production cost, influence its range of application.

Summary of the invention

The present invention is intended to the good bacterial strain of the production traits, utilizes cheap raw material, and fermentation makes a kind of novel hydroxy alkanoic acid polymer.Good and the endotoxin content of these product performance meets the requirements, can be as the novel thermoplastic biomaterial in medical treatment, medical tissue engineering and other fields.

A kind of hydroxy alkanoic acid polymer provided by the invention is characterized in that it is 3-hydroxybutyric acid and 3-Hydroxyoctanoic acid polymer [P (3-HB-co-3-HO) is hereinafter to be referred as P (HBHO)].The production method of P (HBHO) is as follows:

Bacterial classification

The bacterial classification that the present invention adopts is Sinorhizobium fredii (Sinorhizobium fredii is hereinafter to be referred as S.fredii), and other people are deposited in Chinese typical culture collection center, and deposit number is CCTCC No.AB92049.

The cellular form of S.fredii bacterial strain is shaft-like (see figure 3), and 0.5～0.9 μ m * 1.2～3.0 μ m does not produce gemma, and Gram-negative can effectively be given birth to knurl, symbiotic nitrogen fixation with leguminous plants.

For the research and the application of S.fredii bacterial strain, mainly concentrated on the symbiotic nitrogen fixation aspect in the past, other physiologic functions do not come into one's own as yet and are exploited.Discover through us, this bacterial strain has the excellent fermentation production traitss such as substrate wide ranges, growth velocity is fast, the production traits is stable, when this strain growth is on the substratum of carbohydrate such as glucose, just synthetic PHA in the cell growth, to the reduction of stationary phase, can utilize that remaining carbon source material accumulates PHA in a large number in the substratum along with nitrogen source concentration.Therefore, the fermented type of the synthetic PHA of this bacterial strain belongs to the part related type of growing.

Obviously, the carbohydrate metabolism of this bacterial strain is to generate acetyl-CoA by EMP Embden Meyerbof Parnas pathway.Wherein a part of acetyl-CoA enters TCA ring generate energy and reducing power, another part acetyl-CoA is then by ketothiolase catalysis two molecule acetylcoenzyme synthesis of acetyl acetylcoenzymes, the latter forms 3-maloyl group coenzyme under the effect of acetoacetyl coenzyme reductase enzyme, polysaccharase aggregates into PHB with 3-maloyl group coenzyme.

Yet, when having glucose and soap simultaneously in the substratum, can start the β-Yang Hua approach of lipid acid.When adding lauroleate, can synthesize P (3-HB-co-3-HO); And when adding octylate, hexanoate and butyrates and acetate, all only synthetic PHB.Hydroxy alkanoic acid monomer in the polymkeric substance is than few one the four carbon structure unit of matrix fatty acid chain.Though detailed mechanism waits further investigation, this bacterial strain obviously is through the β-Yang Hua approach, but not obtains synthesis of hydroxy alkanoic acid polymer precursor through the lipid acid route of synthesis.Be that to be different from bacterial strain such as pseudomonas be the metabolic way that forms hydroxy alkanoic acid polymer through the lipid acid route of synthesis in this.Just because of this metabolic characteristic, can use this bacterial strain, stably produce P (HBHO) product by the metabolic regulation of lauroleate substrate level.

Substratum

Research to S.fredii in the past mainly concentrates on the biological nitrogen fixation aspect, and therefore existing culture medium prescription and culture condition are all relevant with nitrogen fixation.In order to develop the function of the synthetic PHA of this bacterial strain, used as the production bacterial strain of PHA, the present invention uses orthogonal test, serves as the detection index with thalline biomass and PHA resultant quantity respectively, and the culture medium prescription and the culture condition of this bacterial strain carried out optimization Test.According to the result of variance analysis, make suitable culture based formulas and culture condition.

The component of substratum and content thereof (g/L): carbohydrate, 10～40; (NH ₄) ₂SO ₄, 1～5; KH ₂PO ₄12H ₂O, 0.61～1.22; K ₂HPO ₄, 0.39～0.78; CaCl ₂, 0～0.1; The bean sprouts, 100～300; H ₃BO ₃, 0.002; Na ₂MoO ₄, 0.002.Wherein carbohydrate is glucose, starch hydrolyzate or waste molasses, and concrete add-on is calculated with actual sugar degree.The bean sprouts, make bean sprouts juice, method for making: get the water infusion that the bean sprouts adds 3 times of weight, filter, filtrate is concentrated into 1/3.

(g/L) is as follows for the component of preferred substratum at different levels and content thereof:

(1) slant medium: glucose, 10; (NH ₄) ₂SO ₄, 1.0; KH ₂PO ₄.12H ₂O, 0.61; K ₂HPO ₄, 0.39; CaCl ₂, 0.1; The bean sprouts, 100; H ₃BO ₃, 0.002; Na ₂MoO ₄, 0.002; Agar, 20; PH7.0～7.2 (being used for the inclined-plane seed culture).

(2) shake-flask seed substratum: glucose, 10; (NH ₄) ₂SO ₄, 1.5; KH ₂PO ₄12H ₂O, 0.61; K ₂HPO ₄, 0.39; CaCl ₂, 0.1; The bean sprouts, 100; H ₃BO ₃, 0.002; Na ₂MoO ₄, 0.002; PH7.0～7.2 (being used for shake-flask seed cultivates).

(3) ferment-seeded substratum: glucose, 30; (NH ₄) ₂SO ₄, 3.0; KH ₂PO ₄12H ₂O, 0.61; K ₂HPO ₄, 0.39; CaCl ₂, 0.1; The bean sprouts, 200; H ₃BO ₃, 0.002; Na ₂MoO ₄, 0.002; PH7.0～7.2 (being used for seeding tank seed enlarged culturing).

(4) fermentation basic medium: carbohydrate (glucose, starch hydrolyzate or waste molasses), 30; (NH ₄) ₂SO ₄, 3.0; KH ₂PO ₄12H ₂O, 1.22; K ₂HPO ₄, 0.78; CaCl ₂, 0.1; The bean sprouts, 200; H ₃BO ₃, 0.002; Na ₂MoO ₄, 0.002; PH7.0～7.2 (basic medium that is used for batch fermentation or fed-batch fermentation).

Starch hydrolyzate preparation method: get starch 20g, add water 80ml, make starch milk, regulate pH6.5, after the heating gelatinization, the α-Dian Fenmei 0.05ml that adds 20000U/ml, the 2h that liquefies in 95 ℃ of water-baths adds the saccharifying enzyme 0.02g of 50000U/g again, transfer pH to 4.5, hydrolysis 6h under 60 ℃ of water bath condition filters, and filtrate decompression is concentrated into 30ml.With 3,5-dinitrosalicylic acid method is measured reducing sugar content 0.5g/ml.

Waste molasses is from sugar refinery, Datong, Shanxi Province, and measuring reducing sugar content through anthrone method is 27.5%.

Production stage

The production process of P among the present invention (HBHO) can be divided into inclined-plane and shake-flask seed cultivation, seeding tank seed culture, the high density fermentation of cell and four operating units of separation and purification of lauroleate metabolic regulation and product P (HBHO).Concrete production method comprises the steps:

(1) inclined-plane and shake-flask seed are cultivated

The inclined-plane seed culture: inoculation S.fredii preservation seed is cultivated in 28 ℃～32 ℃ incubators to slant medium.Through twice switching activation,, standby as the inclined-plane seed.

Shake-flask seed is cultivated: with shake-flask seed substratum the shaking in the bottle of 300mL of packing into, loading amount is 100mL, shake bottle with inclined-plane seed inoculation one-level, at 28 ℃～32 ℃, cultivate 14h～18h under 150r/min (eccentricity is 30mm) the shaking table condition, shake bottle with one-level shake-flask seed liquid inoculation secondary again, inoculum size is 10%.When cell grows into logarithmic phase mid-term, cell concn is about 1.53g stem cell/L～2.85g stem cell/L (OD ₆₀₀Be 3.0～5.5) between, standby as the seeding tank seed liquor.

(2) seeding tank seed culture

Adopting the 10L automatically controlled fermentor is seeding tank, use the ferment-seeded substratum, coefficient 0.7 inserts seed liquor, inoculum size 8%～10%, temperature is controlled at 28 ℃～32 ℃, the pH value is constant in 7.0～7.2, and air flow 1: 1 (v/vmin) maintains more than 20% the dissolved oxygen saturation ratio by regulating stirring velocity, when cell grows into logarithmic phase mid-term, cell concn (is OD about 10.2g stem cell/L～12.2g stem cell/L ₆₀₀Be 20～24) between, as the seed liquor of fermentor tank.

In seeding tank seed culture process, the concentration of cell and main nutrient matter in the detection nutrient solution, the microscopy cellular form is by the quality of control incubation time assurance seed liquor.Require the cell concn of seed liquor suitable, nutritive ingredients such as sugar, nitrogen, phosphorus are at more than 30% of original bulk, the neat homogeneous of cellular form, and the PHA particle is less, guarantees that seed liquor both had been in logarithmic phase, has higher cell concn.

(3) high density fermentation of cell and lauroleate metabolic regulation

The high density fermentation of cell

Adopt 100L to control fermentor tank automatically, use the fermentation basic medium, coefficient 0.7, inoculum size 8%-10%, temperature is controlled at 28 ℃～32 ℃, and the pH value is constant in 7.0, air flow 1: 1 (v/vmin) maintains more than 10% the dissolved oxygen saturation ratio by regulating stirring velocity.

In the fermentation culture process, every 4h sampling and measuring cell concn, sugared concentration, ammonium concentration and phosphorus acid ion concentration.After cell grows into logarithmic phase, changing conditions according to cell, sugar, ammonium ion and phosphorus acid ion concentration in the fermented liquid, by add nutritive substances such as carbohydrate and ammonium sulfate in good time, simultaneously temperature, pH value, dissolved oxygen etc. are controlled under the optimum condition, impel the cell ramp to high-density state.

The lauroleate metabolic regulation

When cell concn reaches 27.5g stem cell/L (is OD ₆₀₀Be 55) when above, sugared content remains on more than 30% of original bulk in the control fermented liquid, simultaneously limit nitrogen content original bulk 10%～30% between.Adopt two step of lauroleate addition method to carry out the metabolic regulation of lauroleate.The first step, the lauroleate that adds 3mmol/L～4mmol/L forms certain pressure of coercing as basic salt, starts the β-Yang Hua approach.Second step added lauroleate solution with certain speed stream, made that total dosage of lauroleate reaches 6mmol/L～10.1mmol/L in the system.Add behind the lauroleate in the control fermented liquid ammonium sulfate content and about 0.5g/L, add liquid glucose simultaneously its concentration is remained on about 10g/L, regulate pH value, promote P (HBHO) to synthesize 7.5.Regulation and control 2～5h when P (HBHO) reaches certain content and the ratio of HO monomer in P (HBHO) is in the desired extent in cell, can be put jar, the cell of gathering.

(4) separation and purification of P (HBHO)

We take the solvent extraction technology route to carry out the separation and purification of product, and basic operation unit is followed successively by: the liquid-solid separation of fermented liquid, wet cell drying, cell mix with extraction agent, extraction liquid separates, extraction liquid concentrates, ethanol sedimentation, separate and drying, P (HBHO) product.

Pass through orthogonal experiment, yield and purity with P (HBHO) serve as to detect index, kind, consumption, extraction temperature, extraction time, the extraction mode of extraction agent have been carried out selecting test, determined that chloroform is an optimum extractant, and optimized extraction conditions and extraction mode.Simultaneously pair cell and extraction liquid liquid-solid separates and carried out centrifugally also studying with operational conditions such as filtration, extraction liquid cycles of concentration, precipitation agent alcoholic acid dosages and optimizing, set up the method for a whole set of isolation and purification P (HBHO), concrete operation method is as follows:

After fermentation ends, at first through tubular-bowl centrifuge, be under the condition of 15700 * g in centrifugal factor, the control flow velocity carries out continuous liquid-solid separation, collects wet cell, and again through secondary water washing and eccentric cell, 80 ℃ of oven dry down get stem cell.At 30 ℃～60 ℃ following oscillation extraction 2h～4h, suction filtration is collected extraction liquid to stem cell with chloroform, and normal pressure concentrates; Add cold ethanol precipitation, get P (HBHO) crude product, through 2～3 chloroforms dissolvings and ethanol sedimentation, get the pure product of P (HBHO) again.

Detection method

1. dry cell weight: be the finding speed of accelerating cell concn in the fermenting process, nutritive substance based on no graininess in the nutrient solution, we adopt spectrophotometry, measure the light absorption value of cell suspension at 600nm wavelength place, and then are converted to cell concn according to light absorption value and dry cell weight curve.

2. reducing sugar test: 3,5-dinitrosalicylic acid method, anthrone method.

3. ammonium ion is measured: improvement indophenol blue colorimetry.

4. the mensuration of inorganic phosphorus: ammonium molybdate colorimetry.

5. PHA assay: take by weighing 15mg stem cell or 5mg P (HBHO) product, adding 1ml contains in the 15% vitriolic methanol solution and dissolves, add 1ml chloroform-benzoic acid solution (phenylformic acid is as interior mark) again and carry out esterification in 100 ℃ of water bath heat preservation 140min, then reaction solution cooling back is added 1ml deionized water vibration mixing, the centrifugal 15min of 3500 * g, collect organic phase, add anhydrous Na ₂SO ₄After the dehydration, use gas chromatographic analysis.

6. the mensuration of PHA structure: extract and refining the pure product of PHA through the chloroform method, use the 75MHz-nmr determination ¹³C NMR collection of illustrative plates, the PHA structure is determined in interpretation of result according to collection of illustrative plates.

7. the mensuration of molecular weight: Ubbelohde viscometer method.

8. measuring endotoxin: tachypleus amebocyte lysate box method.

Advantage and effect

The present invention adopts the good Sinorhizobium fredii of proterties for producing bacterial strain, use cheapness and the raw materials for production of wide material sources, use distinctive fermentation and control methods, obtained a kind of novel PHA product, through 75MHz-nmr determination and atlas analysis, determine that its structure is 3-hydroxybutyric acid and 3-Hydroxyoctanoic acid polymer [P (3-HB-co-3-HO)] (seeing accompanying drawing 1).And created from bacterial classification, upstream and fermented to a whole set of P (HBHO) new process of production of downstream extraction.By P (HBHO) product of this explained hereafter, its molecular weight is moderate, endotoxin content is low, and excellent property is of many uses, especially can be used as the Performances of Novel Nano-Porous meter level matrix material of biological medicine and medical tissue engineering.

We select the production bacterial strain of S.fredii as P (HBHO) for use.This bacterial strain has the substrate wide ranges, growth velocity is fast, the production traits is stable, the good production traits such as chromogenesis not, realizes high density fermentation production easily, and can the synthesis of hydroxy alkanoic acid polymer through the regulation and control of soap substrate level.S.fredii is a Gram-negative bacteria, is beneficial to the isolation and purification that carries out P (HBHO).In addition, this bacterial classification is natural vinelandii, even flow into the problem that does not also have biological safety in the environment in process of production accidentally.

In the present invention, we have studied culture medium prescription and the culture condition of application S.fredii bacterial strain production P (HBHO) in great detail.The result shows that glucose, starch hydrolyzate, waste molasses all can be used as carbon source, and ammonium sulfate, ammonium chloride, ammonium nitrate etc. all can be used as nitrogenous source, and yeast extract paste or bean sprouts juice all can be used as its somatomedin, so main production raw material is cheap and the source abundant.As adopting the most cheap raw material to produce, raw materials cost can reduce by 1/2～3/4.In addition, because this strain growth speed is fast, culture condition such as temperature, dissolved oxygen are moderate, and the fermentation overhead charges are lower, and these all help reducing the cost of P (HBHO).

In the present invention, according to the fermented type of the growth power mathematic(al) parameter of S.fredii bacterial strain and the synthetic P (HBHO) of related type that partly grows, set up the fermentation management novel process of the high density fermentation and the lauroleate metabolic regulation of cell.In the fs of fermentation, fermentation management focus on the needs aspect that satisfies the cell growth, adopt the mode of fed-batch fermentation to make somatic cells grow into the high-density level fast, make that cell concn reaches more than 27.5g stem cell/L in the fermented liquid.In subordinate phase, the synthetic aspect that focuses on adjusting P (HBHO) product of fermentation management adds lauroleate through two step method and regulates and control, and has synthesized P (HBHO) hydroxy alkanoic acid polymer, and its output reaches between 14.4g/L～22.1g/L.

Two step of lauroleate addition method, hydroxy alkanoic acid polymer synthetic regulation and control needs have not only been satisfied, and eliminated because of disposable adding soap and produced foamy phenomenon, overcome the soap excessive concentration and suppressed the synthetic active disadvantage of P (HBHO) of cell, improved the utilization ratio of soap, keep the coefficient of fermentor tank, guaranteed higher fermentation productivity.

The present invention is the separation purification method that a cover P (HBHO) has been set up on the basis with the extraction process route, and it is simple that this method has technology, advantages such as easy handling.By P (HBHO) product that this method obtains, yield reaches more than 95%, and purity reaches more than 98%.This method can not reduce the molecular weight of product and influence the character of P (HBHO).In addition, use this separation purification method, can reduce endotoxic content in P (HBHO) product, its endotoxic content can be controlled between 4～6EU/g, be lower than the standard of the minimum 20EU/g of U.S. FDA regulation.

P (HBHO) hydroxy alkanoic acid dimer product by the present invention obtains is a kind of novel PHA product.Still there is not the patent report of producing this product both at home and abroad, the P (HBHO) that we produce, its molecular weight is 11.2 * 10 ⁴About Da, can be used as the nano level matrix material, can be used on fields such as medical treatment and medical tissue engineering.

On the basis of shaking bottle regulation and control experiment, carry out the repeatedly expansion fermentation test and the small-sized production-scale separation and purification test of 100L fermentor tank among the present invention, obtained the correlation technique parameter that expansion is produced, can realize suitability for industrialized production.

Description of drawings

Fig. 1, the 75MHz-of P (3-HB-co-3-HO) ¹³C NMR collection of illustrative plates

Fig. 2 is the gas chromatogram of carbon source synthetic P (3-HB-co-3-HO) with glucose,

Fig. 3 is the gas chromatogram of carbon source synthetic P (3-HB-co-3-HO) with the starch hydrolyzate

Fig. 4 is the gas chromatogram of carbon source synthetic P (3-HB-co-3-HO) with the waste molasses

1 is the methyl alcohol characteristic peak among Fig. 2,3,4, and 2 is the chloroform characteristic peak, and 3 is 3-HB monomer peak, and 4 is interior mark peak, and 5 is 3-HO monomer peak.

Fig. 5, Sinorhizobium fredii cellular form figure, dark in the somatic cells among the figure is the PHA particle partly.

Embodiment

The same specification sheets of the component concentration of substratum in following examples, used fermentation equipment are 10L, the 100L automatically controlled fermentor support equipment that east, Zhenjiang biotechnology equipment and technology company produces.

Example 1 is the fermentative production of the synthetic P (HBHO) of carbon source with glucose

(1) inclined-plane and shake-flask seed are cultivated

With the S.fredii bacterial classification inoculation of preservation on slant medium, in 30 ℃ incubator, activate twice, switching goes into to be equipped with the reactivate twice in the bottle that shakes of shake-flask seed substratum, treats back one-level shake-flask culture 14h, when somatic cells concentration reaches 1.53g stem cell/L (OD ₆₀₀Value is 3.0) time, the cell growth still is in logarithmic phase, and is standby as the seed liquor of seeding tank.

(2) seeding tank seed culture

With 10L automatic control jar is seeding tank, adopt the ferment-seeded substratum, coefficient 0.7, insert seed liquor, inoculum size is 8%, 30 ℃ of cultivations, regulate control pH7.0～7.2 by the NaOH solution of 8mol, keeping the dissolved oxygen saturation ratio more than 20%, through the seed culture of 20h, (is OD when cell concn reaches 10.2g stem cell/L ₆₀₀Be 20), as the seed liquor of 100L fermentor tank.

(3) high density fermentation of cell and lauroleate metabolic regulation

Adopt 100L to control fermentor tank automatically and ferment, use the fermentation basic medium, coefficient 0.7, inoculum size 8%.During the fermentation, controlled temperature is at 30 ℃, and the pH value is constant in 7.0, and air flow 1: 1 (v/vmin) is kept the dissolved oxygen saturation ratio more than 10%.Every 4h sampling and measuring glucose concn, ammonium concentration, phosphorus acid ion concentration and cell concn.During the fermentation by add glucose and ammonium sulfate etc. in good time, make its concentration in fermented liquid maintain 10g/L respectively and more than the 1g/L, satisfy the growth demand of thalline, prolong its logarithmic phase.When cell concn reaches 40.8g stem cell/L (is OD ₆₀₀Be 80) time, adopt two step of lauroleate addition method to carry out the lauroleate metabolic regulation.The first step adds a certain amount of 0.2mol/L lauroleate solution as basic salt, makes its dosage reach 3mmol/L, forms to coerce pressure, starts the β-Yang Hua approach.Second step added lauroleate solution with certain speed stream, made that total dosage of lauroleate reaches 8mmol/L in the system.Supply glucose simultaneously and keep its concentration about 10g/L about 0.5g/L with keeping in the fermented liquid ammonium sulfate content after the salt, keep pH value, promote the accumulation of P (HBHO) 7.5, continue cultivation 3h after, put jar.

(4) separation and purification of P (HBHO):

Adopting tubular-bowl centrifuge, is under the condition of 15700 * g in centrifugal factor, and the control flow velocity carries out continuous liquid-solid separation, collects wet cell, and washing is also centrifugal twice, 80 ℃ of oven dry.Stem cell adds 10 times of volumes (v/w) extraction agent chloroform, and fully mixing stirs 4h down at 50 ℃, and suction filtration is collected extraction liquid.With extraction liquid under 78 ℃ of water-baths, distillation is concentrated into 1/5 of original volume, gets concentrated extract, and reclaims chloroform, the cold ethanol that then concentrated extract is added 5 times of volumes, and slowly stir precipitation, and filter, abandon filtrate, collect P (HBHO) precipitation, get P (HBHO) crude product, again through 2～3 chloroforms dissolvings and ethanol sedimentation, the dry pure product of P (HBHO) that get under the room temperature.

(5) by P (HBHO) content and product purity in the GC gas Chromatographic Determination stem cell, P (HBHO) content is 18g/L, (wherein HB accounts for 79.8%, and HO accounts for 20.2%, sees Fig. 2).

(6) viscosimetry is measured molecular weight product, and molecular weight is 11.2 * 10 ⁴Da.

(7) the tachypleus amebocyte lysate box method is measured product P (HBHO) endotoxin content, endotoxin content 4.0EU/g.

Embodiment 2 is with the fermentative production of starch hydrolyzate as the synthetic P (HBHO) of carbon source

(1) inclined-plane and shake-flask seed are cultivated

With the Sfredii bacterial classification inoculation of preservation on slant medium, in 28 ℃ incubator, activate twice, switching goes into to be equipped with the reactivate twice in the bottle that shakes of shake-flask seed substratum, treats back one-level shake-flask culture 16h, when somatic cells concentration reaches 2.3g stem cell/L (OD ₆₀₀Value is 4.5) time, the cell growth still is in logarithmic phase, and is standby as the seed liquor of seeding tank.

(2) seeding tank seed culture

With 10L automatic control jar is seeding tank, adopt the ferment-seeded substratum, coefficient 0.7, insert seed liquor, inoculum size is 8%, 28 ℃ of cultivations, regulate control pH7.0～7.2 by the NaOH solution of 8mol, keeping the dissolved oxygen saturation ratio more than 20%, through the seed culture of 21h, (is OD when cell concn reaches 11.3g stem cell/L ₆₀₀Be 22), as the seed liquor of 100L fermentor tank.

(3) high density fermentation of cell and lauroleate metabolic regulation

Adopt 100L to control fermentor tank automatically and ferment, use the fermentation basic medium, coefficient 0.7, inoculum size 10%.During the fermentation, controlled temperature is at 28 ℃, and the pH value is constant in 7.0, and air flow 1: 1 (v/vmin) is kept the dissolved oxygen saturation ratio more than 10%.Every 4h sampling and measuring sugar concentration, ammonium concentration, phosphorus acid ion concentration and cell concn.During the fermentation by add starch hydrolysis concentrated solution and ammonium sulfate etc. in good time, make in the fermented liquid sugared concentration and ammonium sulfate concentrations maintains 10g/L and more than the 1g/L, satisfy the growth demand of thalline, prolong its logarithmic phase.When cell concn reaches 43.7g stem cell/L (is OD ₆₀₀Be 86) time, adopt two step of lauroleate addition method to carry out the lauroleate metabolic regulation.The first step adds a certain amount of 0.2mol/L lauroleate solution as basic salt, makes its concentration reach 4mmol/L, forms to coerce pressure, starts the β-Yang Hua approach.Second step added lauroleate solution with certain speed stream, made that total dosage of lauroleate reaches 10.1mmol/L in the system.About 0.5g/L, add starch hydrolysis concentrated solution with keeping in the fermented liquid ammonium sulfate content after the salt simultaneously, keep sugared concentration about 10g/L, keep pH value, promote the accumulation of P (HBHO), continue to put jar behind the cultivation 3h 7.5.

(4) separation and purification of P (HBHO):

The stem cell that obtains adds 12 times of volumes (v/w) extraction agent chloroform, stirs 2h down at 60 ℃, and other steps are with embodiment 1.

Resulting P (HBHO) content is 22.1g/L in the present embodiment, (wherein HB accounts for 65.1%, and HO accounts for 34.9%, sees Fig. 3), and molecular weight is 18.5 * 10 ⁴Da, endotoxin content 5.0EU/g.

Embodiment 3, with the fermentative production of waste molasses as the synthetic P (HBHO) of carbon source

(1) inclined-plane and shake-flask seed are cultivated

With the S.fredii bacterial classification inoculation of preservation on slant medium, in 32 ℃ incubator, activate twice, switching goes into to be equipped with the reactivate twice in the bottle that shakes of shake-flask seed substratum, treats back one-level shake-flask culture 18h, when somatic cells concentration reaches 2.85g stem cell/L (OD ₆₀₀Value is 5.5) time, the cell growth still is in logarithmic phase, and is standby as the seed liquor of seeding tank.

(2) seeding tank seed culture

With 10L automatic control jar is seeding tank, adopt the ferment-seeded substratum, coefficient 0.7, insert seed liquor, inoculum size is 10%, 32 ℃ of cultivations, regulate control pH7.0～7.2 by the NaOH solution of 8mol, keeping the dissolved oxygen saturation ratio more than 20%, through the seed culture of 22h, (is OD when cell concn reaches 12g stem cell/L ₆₀₀Be 24), as the seed liquor of 100L fermentor tank.

(3) high density fermentation of cell and lauroleate metabolic regulation

Adopt 100L to control fermentor tank automatically and ferment, use the fermentation basic medium, coefficient 0.7, inoculum size 8%.During the fermentation, controlled temperature is at 32 ℃, and the pH value is constant in 7.0, and air flow 1: 1 (v/vmin) is kept the dissolved oxygen saturation ratio more than 10%.Every 4h sampling and measuring sugar concentration, ammonium concentration, phosphorus acid ion concentration and cell concn.During the fermentation by add waste molasses and ammonium sulfate etc. in good time, make in the fermented liquid sugared concentration and ammonium sulfate concentrations maintains 10g/L and more than the 1g/L, satisfy the growth demand of thalline, prolong its logarithmic phase.When cell concn reaches 27.5g stem cell/L (is OD ₆₀₀Be 55) time, adopt two step of lauroleate addition method to carry out the lauroleate metabolic regulation.The first step adds a certain amount of 0.2mol/L lauroleate solution as basic salt, makes its concentration reach 3mmol/L, forms to coerce pressure, starts the β-Yang Hua approach.Second step added lauroleate solution with certain speed stream, made that total dosage of lauroleate reaches 6mmol/L in the system.About 0.5g/L, add waste molasses with keeping in the fermented liquid ammonium sulfate content after the salt simultaneously, keep sugared concentration about 10g/L, keep pH value, promote the accumulation of P (HBHO), continue to put jar behind the cultivation 3h 7.5.

(4) separation and purification of P (HBHO):

The stem cell that obtains adds 8 times of volumes (v/w) extraction agent chloroform, stirs 4h down at 30 ℃, and other steps are same as embodiment 1.

The resulting P of this example (HBHO) content is 14.4g/L, (wherein HB accounts for 71.4%, and HO accounts for 28.6%, sees Fig. 4), and molecular weight is 18.3 * 10 ⁴Da, endotoxin content are 6EU/g.

Claims (5)

1. a hydroxy alkanoic acid polymer is characterized in that it is 3-hydroxybutyric acid and 3-Hydroxyoctanoic acid polymer [P (3-HB-co-3-HO)].

2. method for producing hydroxy alkanoic acid polymer according to claim 1, the bacterial classification that adopts in it is characterized in that producing is a Sinorhizobium fredii, preserving number is CCTCC No.AB92049.

3. method for producing hydroxy alkanoic acid polymer according to claim 2 is characterized in that production method comprises the steps:

(1) cultivation of inclined-plane and shake-flask seed: bacterial classification is activated on substratum, and 28 ℃～32 ℃ of controlled temperature are when somatic cells grows to the mid-term of logarithmic phase, as the seed liquor of seeding tank;

(2) cultivation of seeding tank seed: above-mentioned seed liquor is inserted in the seeding tank, substratum coefficient 0.7, inoculum size is 8%～10%, 28 ℃～32 ℃ of controlled temperature, pH7.0～7.2, keep the dissolved oxygen saturation ratio more than 20%, when somatic cells grows to the mid-term of logarithmic phase, as the fermentor tank seed liquor;

(3) high density fermentation of cell and lauroleate metabolic regulation: the fermentor tank seed liquor is inserted in the fermentor tank, substratum coefficient 0.7, inoculum size is 8%～10%, 28 ℃～32 ℃ of culture temperature, control pH7.0 keeps the dissolved oxygen saturation ratio more than 10%, carries out fed-batch fermentation; When cell concentration reaches 28.1g stem cell/L when above, add the lauroleate of 3mmol/L～4mmol/L earlier as basic salt, stream adds lauroleate solution then, make that total dosage of lauroleate reaches 6mmol/L～10.1mmol/L in the fermented liquid, limitting this moment nitrogen to mend sugar cultivates, regulate pH7.5, regulation and control 2～5h is put jar;

(4) product separation purifying: solid-liquid separation, collect wet cell, oven dry; At 30 ℃～60 ℃ following oscillation extraction 2h～4h, suction filtration is collected extraction liquid to stem cell with chloroform, and normal pressure concentrates; Add cold ethanol precipitation, get crude product, through 2～3 chloroforms dissolvings and ethanol sedimentation, get pure product again.

4. method for producing hydroxy alkanoic acid polymer according to claim 3 is characterized in that the component and the content (g/L) thereof of described substratum: carbohydrate, 10～40; (NH ₄) ₂SO ₄, 1～5; KH ₂PO ₄.12H ₂O, 0.61～1.22; K ₂HPO ₄, 0.39～0.78; CaCl ₂, 0～0.1; The bean sprouts, 100～300; H ₃BO ₃, 0.002; Na ₂MoO ₄, 0.002.

5. method for producing hydroxy alkanoic acid polymer according to claim 4 is characterized in that described carbohydrate is glucose, starch hydrolyzate or waste molasses.

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