CN104755623A

CN104755623A - Process for preparing polyhydroxyalkanoates (PHA) on oil substrate

Info

Publication number: CN104755623A
Application number: CN201380056284.XA
Authority: CN
Inventors: 伊凡娜·马罗瓦; 斯坦尼斯拉夫·奥布鲁恰; 拉德克·普里克里尔
Original assignee: VUT V BRNE
Current assignee: VUT V BRNE; Vysoke Uceni Technicke V Brne
Priority date: 2012-08-27
Filing date: 2013-08-23
Publication date: 2015-07-01
Also published as: CZ304183B6; CZ2012571A3; WO2014032633A1

Abstract

In a method of producing polyhydroxyalkanoates (PHAs) on an oil substrate according to the invention, whereby the oil substrate comprises vegetable oil and/or edible oil and/or waste edible oil, preferably frying oil, the bacterial strain Cupriavidus necator H16 is grown, converting oil into PHAs and at the same time producing its own extracellular lipolytic enzymes, which are at least partially isolated from the culture medium during the fermentation process before PHA production and isolation has completed. Lipolytic enzymes isolated from C.necator during the cultivation are used for the treatment of the oil substrate in such a manner that they are added to the production medium either before the beginning of the cultivation or at the beginning of the cultivation together with inoculum and in both cases also during the cultivation together with an additional dose of oil. In the isolation of PHAs produced by the method, after fermentation has completed, the content of the production reactor is warmed up to the temperature of 80 DEG C for at least 30 minutes, subsequently it is cooled to the temperature of about 60 DEG C and lytic agents containing a mixture of a detergent, such as SDS, and alkase (proteolytic enzyme) are added, by which means raw homogenate, or lysate, is obtained. Another option is isolation of PHAs by means of organic solvents - dimethyl sulfoxide or acetone.

Description

The method of polyhydroxyalkanoate (PHA) is prepared from oily substrate

Technical field

The present invention relates to the method preparing polyhydroxyalkanoate (PHA) on oily substrate, described oily substrate comprises vegetables oil and/or edible oil and/or waste edible oil, be preferably frying oil, described oily substrate make bacterial isolates hookworm covet copper bacterium H16 (Cupriavidus necator H16) growth, oil is changed into PHA, and produce extracellular lipolytic enzyme simultaneously, the preparation completing PHA be separated before, in substratum, be separated described extracellular lipolytic enzyme at least in part during the fermentation.

Background technology

A kind of most effective means manufacturing biomaterial utilizes the natural process occurred in microorganism.Some bacteriums create deposit (reserve) polymer P HA from the material be present in environment, and described material is used as food by them.Bacterium is for storing this polymkeric substance in the future, and then, in hungry period, they can it can be used as inner food to use.

Polyhydroxyalkanoate (PHAs) is the optically active polyester group of being synthesized as energy storage or deposit by some bacterial isolateses.The monomer of PHA is (R)-3-hydroxy alkanoic acid.Due to the stereospecificity of the enzyme of responsible synthesis PHA-PHA synthase, all monomeric building blocks are in (R) configuration.Only have in limited instances, find a small amount of (S) monomer in the polyester.Depend on microorganism and growth conditions, the molecular weight ranges of PHA is from 200,000Da to 3,000,000Da.PHA sees in cytoplasmic cell in the form of granules, and the size of described particle is from 0.2 μm to 0.5 μm different [1].

PHA can form the cell weight up to 90%.Unlike other biological polymer, such as polysaccharide, protein or DNA, PHA are thermoplastic.Due to their mechanics and processing performance, for their suitability, PHA polymkeric substance attracts people's attention very much, and it can be the substitute [2] from the synthesized plastics produced of oil.

According to the number of the atom in monomeric building blocks, we distinguish wherein every monomer have the former short chain of 3 to 5 carbon PHA (SCL) and wherein often monomer there is the PHA (MCL) of the medium chain length of 6 to 14 carbon atoms.The structure of PHA is shown in Figure 1, and wherein for the PHA of short chain, it uses R=CH ₃-C ₅h ₁₁, for the PHA of short chain, R=C ₆h ₁₃-C ₁₄h ₂₉.

When Lemoigne manages from bacillus megaterium bacteria distribution poly butyric ester (PHB), this is as far back as nineteen twenty-six.In the twentieth century later stage fifties, in many gram negative bacterium species, confirm PHB lays in form existence as energy and carbon.Except PHB, be also separated the multipolymer containing 3-hydroxyl valerate and 3-hydroxycaproic ester in 1974.From that time, having confirmed can many microorganism of synthesizing polyhydroxyalkanoateby, comprises industrial strain.These be all Gram-positive and Gram-negative bacteria (autotrophy, heterotrophism, phototrophy; Aerobic and anaerobism-with reference to patent WO2009156950A2), but they also comprise some bacterial strains of archeobacteria.In carbon source sufficient supplies and under lacking other nutraceutical conditions, Microbe synthesis PHA is as the deposit form of energy and carbon.After carbon source is used up, then utilize PHA as the energy and carbon source.PHA serves as the desirable deposit form of carbon source and energy simultaneously, and this is given by their low-solubility and high molecular.Due to these character, they do not participate in fact the osmotic pressure [1] in cell.

1976, Imperial Chemical Industries of UK corporation (ICI)-recognize butyric ester replaced the potentiality of the petrochemical polymer of the polymkeric substance of synthesis-mainly.Although it is expensive that the bacterium of PHB produces, it is cost-efficient for estimating that the price risen violently of oil can make the bacterium of PHB produce.Because the increase of the expection of oil price never occurred, polyhydroxyalkanoate finds that first its purposes is the material as special biodegradable and biocompatibility.But due to price high, the market penetration rate of known poly butyric ester under " Biopol " trade mark and P (HB-co-HV) (multipolymer of butyric ester and hydroxyl valerate) is quite not enough.Nineteen ninety, the bottle that German company Wella (Wella) uses Biopol to manufacture comes for new shampoo.1996, u s company Meng Shan bought biological polymer (Biopol) material from the Subsidiary Company ZENECA BioProducts of ICI.Even if in today, also submit to the patent by utilizing transgenic plant to produce Biopol next life, this will reduce production cost (such as US 2003/0017576 A1, US 2004/0101865 A1).

The biosynthetic biological chemistry aspect of PHB is widely studied.Utilize most of bacterium, such as alcaligenes eutrophus (Alcaligenes eutrophus) (Ralstonia eutropha (Ralstoniaeutropha) again; Present hookworm covets copper bacterium (Cupriavidus necator)) in three step reaction, synthesize PHB.First, in the reaction by 3-ketothiolase catalysis, two molecule acetyl-CoA couplings are to form acetoacetyl-CoA.In the reaction by the catalysis of NADPH dependency Acetoacetyl-CoA reductase, acetoacetyl-CoA subsequently and stereospecificity be reduced into (R)-3-hydroxybutyryl A.Finally, PHB [1] is synthesized by enzyme PHB synthase polymerization (R)-3-hydroxybutyryl A molecule.

The biosynthetic schematic diagram of PHB is shown in Fig. 2.

When carbon source is enough obtaining, and under such as nitrogen, iron, phosphorus, sulphur, potassium or oxygen are limited, PHB biosynthesizing occurs.In enzyme level regulation and control PHB synthesis.From the angle of the regulation and control of PHB synthesis, the IC of acetyl-CoA and free HSCoA is vital.Under the condition of balanced growth, acetyl-CoA is oxidized in Krebs cycle.In the process of oxidation, create the NADH being further used for biosynthetic object.After stopping culture (culture) growth, the concentration of NADH increases, and the activity of Oxalacetic transacetase and isocitric enzyme reduces simultaneously.Then acetyl-CoA can not be oxidized in Krebs cycle, and enter PHB biosynthetic pathway.3-ketothiolase is suppressed by HSCoA freely, and described HSCoA is freely generated [4] by the oxidation of the acetyl-CoA under normal growing conditions, in Krebs cycle.

Some bacterial strains of bacterium produce various copolymer p HA.Copolymer p HB and PHV can such as by growing on the substrate comprising glucose and propionic acid time alcaligenes eutrophus or other type microorganism and synthesize, or directly to be synthesized by 3-hydroxyl valerate (3HV) precursor.If use propionic acid, described synthesis is similar to the synthesis of PHB, and difference is that acetyl-CoA and propionyl coenzyme A condensation form 3-ketone group valeryl coenzyme A (3-ketovaleryl-CoA), and this causes 3-hydroxyl valerate to be incorporated in polymer architecture.If use the acid with odd number carbon atom as carbon source, 3-hydroxyl valerate directly produces from the beta-oxidation of lipid acid.When alcaligenes eutrophus, the low dissolved axygen level also just in substratum (medium) adds 3-hydroxyl valerate being incorporated to [1] in polymer architecture.The schematic diagram of the structure of PHB and P (common-HV of HB-) is shown in Fig. 3.

Some microorganisms (some mutant of such as alcaligenes eutrophus), when growing in the substratum of precursor not comprising 3-hydroxyl valerate, can synthesize P (common-HV of HB-).The multipolymer of 3-hydroxybutyrate ester and 4 hydroxybutyric acid ester can be synthesized by 4 hydroxybutyric acid, BDO, butyrolactone, 4-neoprene acid esters [1] in alcaligenes eutrophus.

PHA is in the form of granules at intracellular storage (deposited).Number and the size of particle depend on growth conditions, also change in different bacterial culturess.The density range of PHB particle is about 1.18g.cm ^-3to 1.24g.cm ^-3, the density of MCL PHA particle is about 1.05g.cm ^-3.This particle comprises polyester, protein and lipid.Described particle has polyester core, surface is formed by phospholipid monolayer, fulfils different functional proteins and is incorporated in described phospholipid monolayer.Usually, PHA has hydrophobic property, and therefore, phosphatide and protein form the interface [1] between PHA and surrounding environment.

One in the protein of PHA particle is PHA synthase.Likely there is the PHA synthase of three types, they are different on their substrate specificity and primary structure.Their common trait is the avtive spot containing halfcystine.The synthesis (short chain) of the alcohol acid catalysis SCL PHA of the free 3-5 of the first type carbon atom formation of PHA synthase.Long chain allcyl acid (6-14 carbon atom) is incorporated in the structure of PHA polymkeric substance (MCL PHA) by the second type.The structure of the third type is different from first two type.Type before PHA synthase is made up of only a kind of subunit of the size with 60-70kDa, but the third type of PHA synthase is made up of Liang Zhong subunit: C subunit (~ 40kDa) and E subunit (~ 40kDa).For the PHA synthesis undertaken by such enzyme, Liang Zhong subunit is all required.Substrate specificity is not regulation strict as when PHA synthase above, but generally speaking, the preferably synthesis [5] of SCL PHA.

The schematic diagram of PHA grain pattern is shown in Fig. 4.

The another kind of protein that can find in PHA particle is intracellular PHA depolymerizing enzyme.It is responsible for, when limited from the carbon source of environment, PHA being used as energy source and carbon source.Up to now, research shows, approximately slower than their synthesis 10 times of the process of the PHA degraded undertaken by intracellular depolymerizing enzyme.But the regulation and control of intracellular depolymerizing enzyme are not yet fully explained.The protein of on-catalytic effect is also had, i.e. so-called phasins in the structure of PHA particle.In the cytoplasmic aqueous environments of cell, they should be the stabilizations [6] participating in hydrophobicity PHA.

PHB homopolymer is that wherein all unsymmetrical carbons are in the polyester of (R) configuration.It is that relatively highly crystallization (crystalline) (about 50% to 80%) this makes it hard and crisp.Second-order transition temperature is 5 DEG C to 9 DEG C, and melt temperature is between 173 DEG C to 180 DEG C.In the temperature of 200 DEG C close to fusing point, PHB decomposes.In chloroformic solution, it creates right-hand(ed)screw sigmoid curve.Mechanical property (such as elastic Young's modulus (3.5GPa), the elasticity of elongation (40MPa)) and the polypropylene of PHB are similar.But ductility is only about 3%, this is than in little many [1] of polyacrylic situation.

The properties of PHA derivative is listed in the table below 1.

If be incorporated in polymer architecture by 3-hydroxyl valerate, then mechanical property significantly improves.Elastic Young's modulus is reduced to lower than 0.7GPa, and the elasticity of elongation is reduced to 30MPa.Material ductility increases along with the ratio of the growth of 3-hydroxyl valerate.The fusing point of multipolymer drops to about 130 DEG C, but decomposition temperature is also in a slight decrease.Due to these character, multipolymer can melt when not decomposing.As mentioned above, poly-(common-HV of HB-) that comprise the 3-hydroxyl valerate of 0 to 30% carries out commercially available [1] under trade mark Biopol.

The multipolymer of 3-hydroxybutyrate ester and 4 hydroxybutyric acid ester does not form crystalline texture.Along with the growth (0-100%) of the 4 hydroxybutyric acid ester content in polymkeric substance, second-order transition temperature drops to-50 DEG C from 5 DEG C, and melt temperature also drops to 54 DEG C from 180 DEG C.For for 94% 4 hydroxybutyric acid ester, elastic Young's modulus is about 55MPa, and the elasticity of elongation is 39MPa, and ductility is 500% [1].

Although there are many facts that can produce the bacterial isolates of PHA, in them, only there is minority can use in industrial application.The suitability of bacterial isolates is by being permitted multifactorial impact.First, it is stability and security, growth and the ability of accumulation, the amount of accessible biomass (biomass) and the amount of PHA.Secondly, it is the credit requirement [1] of each component in the extraction yield of PHA, the molecular weight of PHA, the amount of spendable substrate and substratum.

ZENECA Bioproducts company adopts the mutant strain of alcaligenes eutrophus to come for the production of PHB and P (common-HV of HB-).This process quilt is embodied as two benches batch feeding system.In a first step, biomass are cultivated comprising in as carbon source and the glucose of energy source and the phosphatic ineral media of accurately determined amount.After culture growth, phosphoric acid salt is depleted, and the restriction of phosphorus occurs in the second step, and this causes the accumulation of PHA.In the second step, provide glucose to culture, until the PHA of amount needed for being produced by biosynthesizing.Each stage needs about 48 hours, and the ultimate density of biomass dry weight is about 100g/l.By adding the mixture of glucose and propionic acid and synthetic copolymer P (common-HV of HB-) in the subordinate phase of cultivating.The content of the 3-hydroxyl valerate in polymkeric substance is controlled [7] by the ratio of glucose and propionic acid.

Another kind of technique is developed by Austrian Company Biotechnologische Forschungsgesellschaft, the said firm uses bacterial strain extensive Alcaligenes (Alcaligenes latus) DSM1124 to come for the production of PHB homopolymer, because this bacterial strain can produce PHB with the amount up to 80% dry cell weight.This process quilt is embodied as a step batch feeding, and uses sucrose as carbon source [8].

If use the methyl alcohol of one of such as the most cheap carbon source as substrate, then production cost can be reduced.Bacterial strain is turned round demethylation bacillus (Methylobacterium extorquens) and producing PHB during discontinuous Fed batch fementation on methyl alcohol.The optimum concn of methyl alcohol is 1.7g/l.Reach the biomass concentration of 9g/l-10g/l, and the amount of PHB reaches the 30%-33% of biomass.But, even if use the most cheap carbon source also can not reduce running cost, because a small amount of PHB produced makes subsequent separation process more expensive and more difficult.

Can produce great intracellular amount (80%-90% up to dry weight) PHA genetic modification (genetically modified) bacteria Escherichia coli in have very large potentiality.In addition, by engineered method, the bacterial strain producing copolymer p (common-HV of HB-) or other multipolymer can be developed.Although the synthesis by means of the PHA of recombination bacillus coli does not need restriction, this depends on the amount of available acetyl-CoA.Advantage comprises fast growth, the ability utilizing cheap carbon source, the high-cell density of culture grown and easy purified product (such as patent KR20030070790 (A)-2003-09-02, KR20030070789 (A)-2003-09-02, KR20070097883 (A)-2007-10-05, KR20070097884 (A)-2007-10-05, US 2003/0004299 A1 etc.).

Compared with the petrochemical polymer of synthesis, the main drawback of PHA is temporarily the problem of high production cost.For this reason, bacteriogenic PHA ratio is as polypropylene or the expensive about 5-10 of polyethylene times.Our object finds the effective ways that control PHA produces in bacterium, produces polymkeric substance as much as possible to make bacterium.The final price of polymkeric substance particularly introduced for the cost of raw material cultivated and the impact by isolating polymer arduously from cell.Our strategy is considered by using suitable waste material to reduce input cost as the food (namely based on waste oil) of bacterium.We also imagine optimized purification technique, and use the by product-extracellular lipase of fermentation-for the substrate in substratum pretreated object or may as the independent co-product (co-product) with a large amount of industrial application.

Lipolytic enzyme belongs to ester hydrolase, and it is in two-phase system water-lipid, by depending on the mechanism of multifactorial complexity perhaps, is higher fatty acid, alcohol and glycerine by monoacylglycerol, two acylglycerol and triacylglycerol catalytic decomposition.Lipolytic enzyme is defined as Procaine esterase, its acylglycerols.Hydrolysis has the lipolytic enzyme that short chain can reach the acylglycerol of the lipid acid of nearly 10 carbon atoms, is considered to esterase or carboxylase (EC3.1.1.1).Hydrolysis comprises and has >=the enzyme of the acylglycerol of the lipid acid of the carbonatoms of 10, is called as lipase or is also called as triacylglycerol Acyl-hydrolase (triacylglycerol acylhydrolases) (EC3.1.1.3).Esterase or carboxylase are activated in aqueous solution, and " real (genuine) " lipase has more activity [20] at water/lipid interface than in aqueous phase.

Lipolytic enzyme is divided into three groups:

● first group is nonspecific.The lipolytic enzyme of this group is sour from three whole position release fats of acylglycerol, and completely triacylglycerol is hydrolyzed to lipid acid and glycerine.

● second group of lipolytic enzyme is that 1,3-is specific.They are from the external position release fat acid of triacylglycerol molecule, to form 1,2-DG, 2,3-DGs and 2-monoacylglycerol, and release fat acid.Hatch triacylglycerol for a long time with 1,3-specific lipase usually to cause triacylglycerol complete hydrolysis to become lipid acid and glycerine.

● the 3rd group comprises the lipolytic enzyme being more prone to only some lipid acid.

Most of lipase belongs to extracellular enzyme, is released in environment when its exponential phase of growth in the later stage and early stage stationary growth phase.The generation of lipase by the impact of many different factors, as the concentration of temperature, pH value, nitrogenous source, carbon source, lipid source, pressure and dissolved oxygen and inorganic salt.The Optimal pH of lipase activity is usually in the scope of 6-9.But, from aspergillus niger (A.niger) even and if the lipase of Rhizopus be also activated at the acidic conditions of pH 4.Form contrast, be separated from Pseudomonas nitroreducens (P.nitroreducens) at the activated alkaline lipase of pH 11.Optimum temperuture and thermostability are also different.Addicted in warm microorganism, maximum lipase activity is the temperature at 30 DEG C-35 DEG C.In thermophilic MO, it is mainly at 60 DEG C.Addicted in cold MO, produce lipase at the low temperature of about 5 DEG C.Most of microbe also can produce more than a type, there is not homospecific extracellular lipase [21].

The mechanism of lipase activity is shown in Fig. 5.

Lipolytic enzyme belongs to so-called serine hydrolase.The three-dimensional 3D structure of these enzymes shows typical α/β folding mode-α spiral and β-pleated sheet structure.Catalytic triads (triad) is made up of three amino-acid residues (i.e. Serine, l-asparagine and Histidine); In some lipase, glutamine instead of l-asparagine are found.Lipolysis in vitro occurs over just lipid-water interface, and therefore speed of reaction is directly received in the impact of the concentration of substrate of phase interface.Therefore, the substrate molecule of different states can be had in a phase, and directly do not affect speed of reaction [22].

Esterase activity is the function of concentration of substrate, and experiences Michaelis (Michaelis-Menten) kinetics, reaches maximum reaction velocity at the concentration of substrate of more much lower than saturation concentration times.Form contrast, as long as substrate (lipid) is the state of individual molecule in water, lipase does not show any activity.When concentration of substrate exceedes solubleness point, emulsion starts to be formed, and speed of reaction enlarges markedly.Then lipase activity directly depends on the existence of phase interface.By explaining that the space structure of lipase has confirmed that enzyme active center is protected by polypeptide chain, described polypeptide chain blocks enzyme molecule itself and the combination of enzyme and the formation subsequently of activated complex.But, if lipase directly interacts mutually with lipid, initial conformational change transfer (shift) described protective polypeptide chain, thus for lipid, the active centre of enzyme becomes accessible.Due to the result of the hydrophobic interaction with lipid phase, enzyme-substrate combines and becomes stronger.The phenomenon of this facts explain lipase activation under the existence of phase interface.If the enzyme acting on triacylglycerol does not show such activation, then it should be regarded as esterase.Unlike esterase; the kinetics of lipolysis in vitro does not follow Michaelis-Menton equation; be presumably two step mechanism; described two steps mechanism comprise the surface (itself and the activation of enzyme and the transfer of described protective polypeptide simultaneously occur) of lipase physical adsorption to lipid phase; with the formation subsequently of enzyme-substrate complex, after described enzyme-substrate complex, be hydrolyzed into product and regeneration enzyme [20-22].

The hydrolysis mechanism principle that ester combines is the same for lipase and esterase, and comprises four steps as follows in Fig. 6, Fig. 6 shows the schematic diagram of microbiological deterioration lipid.According to the comparison of the aminoacid sequence of 3D lipase and esterase structure, show to distinguish them on the basis of pH value; In the scope of the pH relevant to maximum lipase activity (typically at pH8.0), the avtive spot of lipase is in (disposes of) negative potential, and the avtive spot of esterase shows similar behavior at pH6.0, it is associated with active usually lower pH optimum condition.

The known microorganism of major part can produce lipase, but only has some kinds industrially to use.Such reason is production of enzyme deficiency, the physical-chemical property of undesirable lipase, the limited possibility etc. from substratum separation.The modal mould of commercial use comprises species Aspergillus, Penicillium, Mucor and head mold.The main producers of commercial fatty enzyme is aspergillus niger, Humicola lanuginosa (Humicola lanuginosa), rice black wool mould (Mucor miehei), rhizopus arrhizus (Rhizopus arrhizus), R.delemar (Dai Shi head mold), Japanese head mold (R.japonicus), Rhizopus niveus (R.niveus) and Rhizopus oryzae (R.oryzae) [20].

Lipolytic enzyme, due to its huge biotechnological potential, causes sizable concern at present.They constitute most important group of the biological catalyst for biotechnology applications, it is successfully used to the synthesis of biological polymer, bio oil, for the production of agrochemicals and aromatics.Here it is why for industrial enzyme, and the demand of particularly microbe-derived industrial enzyme is also improving constantly.Enzyme is just being used to industry-by-industry (industry such as such as food, medicine, weaving and makeup), and for washing composition.Lipase is used for brewageing (brewing) and wine making, cheese-making and dietary supplements.They play an important role in the transesterify and hydrolysis reaction of pharmacy industry, and are absolutely necessary for the production of special lipid.They are very important for the modification (modifications) of monoglyceride, then the monoglyceride of described modification are used as emulsifying agent.By also being produced by lipase from some industrial important chemical of chemical technology manufacture of fat and oil, the specificity levels of described lipase is large many and good many.In addition, lipase is used to produce the substitute of theobroma oil, and the production of ester class for using in cosmetic industry.The hydrolysis that lipase comes for butterfat is used in dairy industry.The current application adding other products to as raw material contributes to strengthening the fragrance of cheese, accelerate cheese produce in weathering process, cheese-making.Lipase from aspergillus oryzae is used to washing composition.As makeup tenderizer, and as the commercial catalysts [21] for the preparation of prostaglandin(PG), steroid, carboxyl nucleoside analog and pharmaceutically important polyphenolic compound, lipase is widely used in the processing of fat and oil.

PHA may be used for many fields equally.By inference, main purposes will be in the scope of packaging industry, especially for production nursing-bottle and feeding bottle, for the plastics of juvenile product and ecological product (such as toy), for the packaging of cosmetic industry and the so-called intelligent packaging of food.Other interesting application comprises the container (such as cup) that production designs for the process after use (such as fast food chain dining room), and this can provide again waste oil as substrate conversely, for the production of biological plastics.

Polymer P HA also may be used for other application: can prepare nanofiber and nano particle by it, for targeted drug delivery system, and the implant producing biocompatibility can be used it for, the implant of described biocompatibility can be used as fiber in medical science, blood vessel alternative (vascular substitutions) etc.

Their cost it should be noted that the production of current biological plastics is lower, because temporarily, than the height of synthetic plastics, which prevent demand.But, in the near future, it is expected to the regulation stricter in fact of the use for ecological plastics, this expansion that biological plastics may be caused to produce.

Nowadays it is well-known for producing PHA by bacterium from oily substrate, but with traditional compared with the crude production plastics, its cost is too high, so be difficult to promote it.

The object of the invention is the method proposing to prepare PHA from oily substrate, described method will be economical, and can produce a large amount of PHA.

inventive principle

Object of the present invention is realized by the method preparing polyhydroxyalkanoate (PHA) on oily substrate, described oily substrate comprises vegetables oil and/or edible oil and/or waste edible oil, be preferably frying oil, described oily substrate makes bacterial isolates hookworm covet copper bacterium H16 to grow, oil is changed into PHA, and produce extracellular lipolytic enzyme simultaneously, the generation completing PHA be separated before, described extracellular lipolytic enzyme is separated at least in part during the fermentation from substratum, thus, principle of the present invention is before starting to cultivate, described oily substrate is joined by being coveted the extracellular lipolytic enzyme that copper bacterium H16 produces by hookworm, thus accelerate the growth of bacterial cultures.

During PHA and extracellular lipolytic enzyme produce simultaneously, before cultivation starts, described oily substrate is joined by being coveted the extracellular lipolytic enzyme that copper bacterium H16 produces by hookworm, this represent the inventive process that PHA produces, thus by the lipolytic enzyme existed outside inducing cell of oily substrate, and bacterium hookworm is coveted copper bacterium H16 and produces effective molecular form, described effective molecular form can effectively close to the oily substrate utilized.Enzyme is extracellular products, and PHA is thin intracellular product, and this is very large advantage, and this is conducive to the separation of two kinds of products.

Due to several hours before the generation of PHA reaches its maximum value, the generation of extracellular lipolytic enzyme reaches the fact of its maximum value, in the process that PHA produces, design the new process of the lipolytic enzyme outside isolated cell by means of aseptic or non-sterile ultrafiltration (ultrafiltration).

By adding NaOH or H ₂sO ₄the pH value of maintain base is about 7 (changing in the interval of +/-0.3), and forms the top condition of bacterial growth, and during the fermentation, the oxygen concn of dissolving maintains the scope of 10%-50% by controlling aeration.The content of the 3-hydroxyl valerate in the growth of culture, the generation of PHA and multipolymer is regulated by aeration.

In order to be supported in the growth of the bacterium on oily substrate, by the extracellular lipolytic enzyme after separation, together with the oil/oily substrate of additional dose, be added back to substratum at least in part.

Accompanying drawing explanation

In the accompanying drawings, Fig. 1 represents the structure of PHA; Fig. 2 is the schematic diagram of biosynthesizing PHB; Fig. 3 is the schematic diagram of PHB and P (common-HV of HB-) structure; Fig. 4 is the schematic diagram of PHA grain pattern; Fig. 5 is the mechanism of lipase activity; Fig. 6 is the schematic diagram of microbiological deterioration lipid; Fig. 7 is the comparison of the induction of extracellular lipase activity on different carbon substrates; Fig. 8 is the productive rate of the metabolite in the process of exemplary fermentation technique; Fig. 9 is the sign of the exploitation of the centrifugation of polymer product by analysis centrifugal partition method (4000rpm, 2 hours, 5 DEG C); Figure 10 makes hookworm covet the impact of the process that copper bacterium H16 grows to use oil as carbon source adding of lipase; Figure 11 is the example of the GC-FID color atlas of PHA; Figure 12 is the example of the GPC color atlas of PHA; Figure 13 is that the TGA of PHB analyzes; Figure 14 is the dsc analysis of PHB; Figure 15 is the pH optimum condition of extracellular lipase; Figure 16 is the impact of ionic strength on the figure of lipase activity; Figure 17 is the record of the protein separation by PAGE-SDS-silver dye (silver dying).

Embodiment

production bacterial strain

In order to produce extracellular lipolytic enzyme and PHA simultaneously, bacterial isolates hookworm is used to covet copper bacterium H16; Czech Collection institute (CCM 3726).This bacterial strain is embodied in microbial preservation institute, and is approved for the PHA producing and be used for contacting with foodstuff products, and described bacterial strain is not carried out genetic modification.

bacterial growth on waste oil

In numerous patents, all use discarded substrate to produce PHA.Process may be that one of the most conventional method of the most polytype waste is disclosed in patent US2009/0317879 A1, but wherein, waste is processed into low-grade carboxylic acid's (propionic acid, acetic acid) and methane primarily of methane-oxidizing bacteria (methanotrophic bacteria), makes waste be come-at-able for production bacterial strain by the method.Another patent (US 2010/0190221 A1) even describes that use may to the substrate of microorganism or environmental toxic.By enzyme methane-monooxygenase, organic compound is converted to available substrate.

Various PHA production on waste oil itself is also included in many different patents.The waste edible oil (JP2004254668 (A)-2004-09-16) that authors propose to use the waste from refuse landfill (CN101255227 (A)-2008-09-03) such as containing oil, produced for PHA by hydrogen bacterium, dissimilar vegetables oil in general (WO 2009/156950A2-namely sunflower seed oil, rapeseed oil, hempseed oil and other oils do not indicated further; Don't you mention sintering frying oil (fritting frying? oil)).Another kind of possible substrate be by the bacterial species of Rhodopseudomonas use from the oil (WO2010/0441180A1) of Aesop than sub-mustard (Brassica carinata).Some are also had to relate to the publication [9-19] carrying out PHA production from oil and related substrates.Available resource is not had to mention the possibility utilizing discarded sintering frying oil, the enzyme that described sintering frying oil is induced by the production bacterial strain itself in the process of growth of production bacterial strain and being hydrolyzed in advance.

Produce while two kinds of industrial important metabolites (PHA and extracellular lipolytic enzyme), not yet be described in the situation that PHA in technical literature or patent documentation produces, one (born of the same parents' external fat lytic enzyme) is wherein by having specific chemical composition (composition) (vegetables oil), uniquely the substrate of (sole) type induced, and in addition, described substrate is preferably discarded and specific (not having the sintering frying oil of other purposes), and can be degraded by the latter's product (lipolytic enzyme), and the maximum output of the metabolite of the latter (yields) (having realized the biomass of maximum output on oil-up to 96% in production bacterial strain) is provided simultaneously.

In some patents (such as WO02/070659 A2), describe by product, but, in most of the cases, problematic process is that (it is oil to substrate under exceptional circumstances, but it must be utilized by aerobic) process of anaerobism at least in part that processes, and problematic product is the product (acetic acid, propionic acid etc.) of the fermenting process of small molecules-usually mostly, it can be used further.A disclosed patent describe thin intracellular PHA and and extracellular polysaccharide while produce, but, use the bacterial strain (WO 95/33838) of genetic modification.

The activity of extracellular lipolytic enzyme to dissimilar carbon substrate is given in table 2.

And the comparison of the activity of extracellular lipolytic enzyme having illustrated in Fig. 7 on dissimilar carbon substrate, induced.

culture media composition: inoculation; Produce; Add precursor

Up to the present, the modal type of the PHA accumulated in the bacterium of production bacterial strain is homopolymer PHB.Due to low-crystallinity and snappiness, develop the technique for the production of copolymer p (common-3HHx of 3HB-) gradually, wherein, the transgenic strain of the transgenic strain usually utilizing Ralstonia eutropha or the gene with certain pseudomonas, especially creates the multipolymer (Japanese Patent Publication 57-150393,59-220192) with 3-hydroxypentanoic acid.Nearest patent application (patent open 5-93049,7-93049) for being produced the P (common-3-HHx of 3HB-) containing 3-hydroxycaproic acid by soil bacteria Aeromonas caviae is disclosed (deposited).Due to the molar fraction of 3-HH, achieve the high-flexibility of polymkeric substance.Other patents submitted to comprise the file concentrating on the multipolymer producing the 3-hydroxy fatty acid (3-hydroxyalkaonic acid) with extra methyl, and it the most often uses recombinant bacterial strain (such as US2011/0081692 A1).The patent that another part proposes relate to produces PHA by the species selected by Rhodopseudomonas, wherein the controlled composition (composition) of multipolymer regulates (US2011/0166318 A1) via culture media composition (C-source-lipid acid) and the suitable precursor of interpolation, or has proposed the patent (WO0006762 A1) for being prepared segmented copolymer by control enzymic activity and nutrient media components.

Due to range of application widely, and the structure of copolymer p (3-HB-HV) meet European Parliament about in food service industry use (namely potential application), we are absorbed in the production of the copolymer p (3-HB-HV) of the 3HV content with 4%-10%.

Culture media composition

for the substratum preserved and inoculum I

substratum and production substratum for other inoculation steps:

the cultivation of bacterium, the production of biomass and control thereof

Between the seed stage of two steps of bacterial cultures, prepare inoculum I, described inoculum I has the cumulative volume (usually in Erlenmeyer flask) of 2000ml, afterwards, the second inoculum II is prepared in the fermentor tank with 25l volume, in cultivation after 23 hours, the substratum of 200l is seeded in together with the second inoculum and produces in tank (tank).

Cultivation in production substratum carries out 32 hours-38 hours in order.Between 8th hour and the 12nd hour, add precursor (Sodium Propionate), the 3g/l (NH of 10g/l ₄) ₂sO ₄with the oil (C-source additive) of 20g/l.During 8-21.5 hour, usually the maximum activity of extracellular lipolytic enzyme is reached, therefore the substratum (10%-50%) of part is taken out, isolate biomass by ultrafiltration, and turn back to this process by aseptic for biomass (I) or joined in reactor under non-sterile condition before the separation of PHA subsequently.By ultrafiltration concentrated penetrant (Permeate) containing extracellular lipolytic enzyme (MW exclusion limit, i.e. " retaining " of so-called 10kDa) further.Between the 20th hour and the 25th hour, add the oil (20g/l) of other dosage.

The productive rate of the metabolite in typical fermenting process is shown in Fig. 8, and is described in table 3:

Yield coefficient Y _pHA/S=0.7 (oil and precursor all in the calculation involved).

In culturing process, by adding NaOH or the 0.5M H of 1M ₂sO ₄the pH of substratum is maintained the value of 7 ± 0.3, regulate aeration, to make the scope of concentration at 10%-50% of dissolved oxygen during the fermentation.The content of the 3-hydroxyl valerate in the growth of aerating regulation culture, the generation of PHA and multipolymer.

after completing cultivation, immediately directly from the ecological polymerized thing of cellular segregation in industrial reactor one-step method

First, usually by centrifugal or filtration, cell is separated from substratum.Then, in order to release polymers must make cell rupture.The most frequently used method extracts polymkeric substance by suitable solvent (chloroform, methylene dichloride, propylene carbonate, Ethylene Dichloride (dichlorethylen)).But this process is very harsh for the consumption of solvent, and true due to this, and this process is expensive.Another kind method uses clorox (sodium hypochorite), but this causes the Partial digestion of PHB and reduces its molecular weight.Disclosed many patents, these patents disclose use almost various available organic solvent-comprise PHA unprovability be wherein dissolve those solvents.Separation method by means of the polymer P HA of organic solvent can find in many publications and patent (such as RU 2,199 587 C2, US 2002/0081646 A1 etc.).

On the other hand, describe (normally broadly) can be used as solvent extraction replacement scheme and for the technique commercially produced of PHB and P (common-HV of HB-).A kind of selection scheme is lyophilization (lyophilize; WO 2006103699 A1, WO 2010/082810 A1), in addition, also describe the method for the mixture using enzyme and washing composition, and do not need the composition (such as WO2010116681 (A1)-2010-10-14) of specifying cleavage mixture.

In proposed application, there is no first isolated cell, after cultivation completes (usual 32-38 hour), direct isolating polymer in fermentation reactor immediately.When substratum is heated to the temperature of 80 DEG C (30min), first cell in substratum is heated, and after being cooled to envrionment temperature subsequently, add such as, containing proteolytic ferment (i.e. the enzyme of protein hydrolysate, Sumizyme MP) and there is optimization concentration (the SDS/1g CDW of 0.04g; Alcojet-neutral industrial detergent) the mixture of washing composition (such as sodium lauryl sulphate).Most cells component is hydrolyzed by the effect of these two kinds of reagent, and polymkeric substance remains unchanged.After by membrane ultrafiltration (the fractional membrane ultrafiltration) isolating polymer of classification, wash polymkeric substance with water and carry out drying by lyophilization.

Centrifugation, as the common technology for isolated cell in substratum or the rest part isolating polymer from cell, proves unworkable.First, the centrifugation of product is quite difficult, because the centrifugation sample of quite most of (about 1/4-1/3) is carried into surface by residual oil, this represents considerable damage, following from shown in Fig. 9, Fig. 9 shows the sign of the process being separated the centrifugation of the polymer product of the method for (4000rpm, 2 hours, 5 DEG C) by analysis centrifugal.

For this reason, the patent application proposed proposes to use the filter cartridge (filtration cassettes) of " retaining " exclusion limit had in the scope of 200-400kDa to optimize the membrane ultrafiltration of classification.

Also the finished product can be washed, to increase its cleanliness factor-purity.Following table (table 5) shows the list of possible purification condition and the impact on the cleanliness factor purity of PHA product thereof.

Table 5

the generation of extracellular lipolytic enzyme, separation and utilization

If bacterium grows on oil (and only on oil), then their produce and contribute to decomposing described oil and utilize the extracellular lipolytic enzyme of described oil, and described oil can be vegetables oil and/or edible oil and/or waste edible oil, is preferably frying oil.Extracellular lipolytic enzyme is industrial important enzyme, and in this culturing process, they are by a large amount of generations, and this is attractive economically.Therefore, we propose to comprise PHA (intracellular polymkeric substance, productive rate 93%-96%) and lipase (extracellular enzyme; Active is about 100U/ml) while total technical scheme of preparing.In the process prepared at the same time, be necessary to perform by following formed step: before final cytolysis is separated with PHA, the lipolytic enzyme in substratum outside isolated cell.

We have proposed the effective technology scheme of the lipolytic enzyme outside isolated cell by the method for the membrane filtration of classification during the fermentation.The maximum production reaching extracellular lipolytic enzyme than reach maximum PHA output in advance several little time, this means in PHA production process, substratum or be completely removed in aseptic mode, or or be that partial medium is removed (it depends on biotechnological device) together with cell in non-sterile mode.And subsequently, have the method for two kinds of feasible, outside isolated cell lipolytic enzymes technically:

A) in the suitable time period (after normally cultivating 24 hours), carry out the sterile ultrafiltration of substratum from cell, in the cell of growth, add the new substratum with substrate and precursor.By precursor, join in substratum together with the oil (additive) of additional dose and extracellular lipolytic enzyme.

B) take out the culture (such as, the cell of 1/3 in substratum) of part, isolated cell non-sterilely, and subsequently by the extracellular lipolytic enzyme of ultrafiltration purification; Afterwards, just before finally will being separated PHA, the cell of the polymer content with about 70% is returned again and is added to reactor.

Extracellular lipolytic enzyme can be improved yield of biomass by being utilized as further and the factor therefore accelerating whole process in follow-up cultivation.If the extracellular lipolytic enzyme be separated according to method described above is joined in the substratum containing oil with the amount of the growth medium of 0.5U/ml – 3U/ml (namely about 2%), and described substratum cultured object hookworm is coveted copper bacterium and inoculates subsequently, then bacterial culture growth improves about 20%-30%.Covet by hookworm the extracellular lipolytic enzyme that copper bacterium produces to seem to be more suitable for this object than the such as commercially available lipase produced by microorganism Rhizopus oryzae.The extracellular lipolytic enzyme added is shown in Figure 10 to the impact that the hookworm using oil as carbon source covets the culturing process of copper bacterium H16.

The digestion of biomass and utilize the extraction of PHA of organic solvent

In principle, when being separated PHB in bacterium living beings matter, two kinds of methods-a kind of can be adopted to be the logical overchlorinated organic solvent (such as chloroform etc.) of solvent by dissolving PHB-be the most often and to extract PHB from bacterium living beings matter.Second method is to break also the biomass (protein, cell walls, lipid etc.) of " dissolving " (digestion) except PHB, for this purpose, following chemical reagent can be used: such as sodium hydroxide (NaOH), washing composition, clorox, lytic enzyme or depend on the circumstances as their combination.[Jacquel N.et al.,2008]。

For the digestion of the biomass except PHB, under being used in the condition of separation, significantly do not dissolve organic solvent-DMSO and the acetone of PHB.The cleanliness factor of product is brought up to the value of 92.0% (acetone) and 91.1% (DMSO) by this strategy being applied to the biomass of the PHB content with 79.2%.As far as we know, this strategy is not also announced by anyone.Be separated from biomass in unique publication of PHB at use acetone, employ the first strategy, when very high temperature and pressure, PHB is dissolved in [Koller M.et al., 2013] in acetone.The technology that we propose now provides under so inviolent (dramatic) condition (energy helpfulness (energeticprofitability)), method when PHB does not dissolve.In addition, in our case, there is no need to be separated PHB from the biomass of drying.Cell does not need the dried fact to be that on energy, (energetically) is favourable before separation yet, and is that in logistics, (logistically) is useful during the course yet.

Step (Procedure): after cultivation completes, by centrifugation from substratum separating biomass, subsequently the organic solvent from dimethyl sulfoxide (DMSO) (DMSO) or acetone group is applied to wet biomass with the amount of the wet biomass corresponding to every 1 liter of solvent 20g-80g.Temperature within the scope of 30 DEG C-80 DEG C, hatching process needs 1 hour-5 hours.By such as centrifugation, organic solvent is removed subsequently, and utilize the either method or arbitrary known method mentioned in patent application, can be further purified containing the solid fraction (fraction) of PHB had higher than the cleanliness factor of 90%.Used organic solvent regeneration can be made by such as distilling.

the sign of PHA polymkeric substance

The determination of the content and structure of of the PHA produced

The concentration of PHA and structure are the most often determined by the method for the gas-chromatography (GC) of the detection utilizing FID (flame ionization detector).Before actual analysis, polymkeric substance is made to stand acid hydrolysis and methylating, to form the volatile methyl ester of 3-alcohol acid subsequently.Separated and detected by GC after these esters.Figure 11 shows the typical color spectrogram of the polymkeric substance be made up of 3-hydroxybutyrate ester and 3-hydroxyl valerate.The content of each component can be determined by peak anomalous integral by calibration curve (table 6), and represent their ratio, in other words, the content of the 3-HV namely in copolymer p HB-HV.

Ratio between the concentration of table 6 peak area and 3HB (3HV)

Produce the determination of the molecular weight of PHA

The molecular weight of PHA is very important parameter, particularly with regard to its application.Its value is not only important for PHA being processed into biological example plastics, nanofiber or particle, and for many medicinal applications, particularly relevant to the biodegradable of used material medicinal application is also important.Therefore, the microbial polymer that produces is characterized as far as possible in detail or the condition summarized for generation of having a polymkeric substance limiting molecular weight is necessary.

The molecular weight of the PHA produced is determined by gel permeation chromatography (GPC) method.The example of the GPC chromatogram of PHA is shown in Figure 12.In many test experiments processes, discovery can pass through the molecular weight only changing growth conditions (such as Nutrient Stress (nutrition stress)) and affected final polymkeric substance by Selective agar medium.For this reason, some inventors are with zymin (enzymepreparative), chemical substance or radiation (see such as U.S. US 2006/0183205A1).

When us, the molecular weight of the polymkeric substance produced reaches the value within the scope of 1.85-2.41E+05, the value wherein reached on sugared substrate up to twice more than.

The molecular weight of selected sample is shown in table 7

Thermostability

The thermal properties of PHA depends on the content (see table 1) of each monomer.The parameter that the degradation temperature, fusing point etc. of such as material are concrete carries out conventional determining by TGA (thermogravimetric analysis) (being shown in Figure 13) and DSC (dsc) (being shown in Figure 14).

The fusing point of selected PHA sample and degraded point temperature are provided in table 8.

The solvability of PHA

PHA is insoluble to polarity implicit expression solvent or nonpolar implicit expression solvent.There is from the solvent of (medium Rf value) in the middle part of eluotropic series the ability of partly dissolve polymer or generation gel.The solvability of the PHA in selected organic solvent is shown in table 9:

R – high soluble; R – is solvable; Mr – is sl. sol.; Nr – is insoluble; Gel – produces gel

Extracellular lipolytic enzyme characterize

Basic characterization of molecules comprises the optimum condition (optimum) of pH, its value is summarized in Figure 15 and is shown in Table 10, and table 10 indicates under different pH, carries out the change of the activity of the extracellular lipolytic enzyme assessed with the percentage ratio of the maximum value relative to 100% (%).

The exploitation of the optimum condition of pH shows two maximum values, and it corresponds to two kinds of molecular form of extracellular lipolytic enzyme.In microorganism, the secretion of the similar type of lipolytic enzyme is common; With regard to the value of pH optimum condition, can suppose, two kinds of forms all decompose acylglycerol, and they may be the enzymes ester generic key in triacylglycerol to different preferences.

Ionic strength (ionic stregth) (NaCl, the 0-5mol/l) impact on the activity of extracellular lipolytic enzyme is shown in Figure 16.From dependent process, clearly, the activity of extracellular lipolytic enzyme reduces along with the ionic strength increased, and this is important for culture media composition.

The partial purification of extracellular lipolytic enzyme

After the separation of cell, substratum except containing except extracellular lipolytic enzyme, also containing many other protein and other materials.In addition, all these materials (comprising target enzyme) all significantly reduce.In order to the object of the more detailed sign of concentrated (condensation) and extracellular enzyme, the experiment below having carried out, with verify cheap effective be separated and while enzyme purification.

As concentrated by acetone as a result, lose the enzymic activity of essence, productive rate shows as about 1%.In the concentration range of 60% to 80%, when by ammonium sulfate concentration, saturated (saturation) is more successful, but in fact causes activity to completely lose in dialysis subsequently, and productive rate reaches about 3%.Another kind of method for the purifying of born of the same parents' lipolytic enzyme is classification ultrafiltration.In the first phase, cell is removed (on the strainer of cutoff value with 1 [mil.] [A1] kDa), afterwards, the lipolytic enzyme on the ultra-fine filter of " retaining " exclusion limit with 10kDa outside isolated cell.Therefore, concentration adds 8 times, and productive rate is the culture of about 91U/ml, see table 11.Therefore, the prime of this technology is proposed as in ultrafiltration.

Table 11

The preparation purity of the extracellular lipolytic enzyme measured by PAGE/SDS, the molecular weight of extracellular protein

In order to the purity of the concentrate formulation of the sign and extracellular lipolytic enzyme therefore that realize protein composition, be used in polyacrylamide gel electrophoresis (PAGE) method under the existence of sodium lauryl sulphate (SDS), wherein according to the isolated protein according to the mobility in unidirectional electric field.Mobility is determined by the ratio of molecular weight and particle charge.Because protein all under these conditions has identical electric charge and shape, be separated on the basis of different molecular weight in principle.It is the record being separated (silver dye) in Figure 17.The indicative explaination of Figure 17 is in following table 12 and table 13, and table 13 describes the protein fractions (protein fractions) of electrophoresis.

Table 12

Table 13

The extracellular part that hookworm covets copper bacterium H16 comprises 5 kinds of main protein fractions, and described 5 kinds of main protein fractions have 15.95kDa; 18.80kDa; 24.34kDa; The molecular weight of 47.54kDa and 64.52kDa.Even if after dialysis, first four kinds in them is also visible.After application ultrafiltration (having the film filter of " retaining " exclusion limit of 10kDa), most of fraction is the protein of the film filter with 18.80kDa and 24.34kDa, and the fraction with 15.95kDa is only slight visible.In addition, although confirmed that by PAGE/SDS method the preparation of the extracellular lipolytic enzyme obtained by classification ultrafiltration has relatively low concentration, but it contains minimum impurity, and may be used for direct application-no matter be add substratum to for the industrial production of PHA or the pre-treatment for oily substrate, or for other industrial uses.

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Claims

1. on oily substrate, prepare the method for polyhydroxyalkanoate (PHA), described oily substrate comprises vegetables oil and/or edible oil and/or waste edible oil, be preferably frying oil, described oily substrate makes bacterial isolates hookworm covet copper bacterium H16 to grow, oil is changed into PHA, and produce extracellular lipolytic enzyme simultaneously, the generation completing PHA be separated before, during the fermentation, described extracellular lipolytic enzyme is separated at least in part in substratum, it is characterized in that, before starting to cultivate, join in described oily substrate by being coveted the extracellular lipolytic enzyme that copper bacterium H16 produces by hookworm, thus accelerate the growth of bacterial cultures.

2. the method preparing PHA according to claim 1, it is characterized in that, the lipolytic enzyme in described substratum outside isolated cell by the sterile ultrafiltration of described substratum, wherein, bacterium hookworm is coveted copper bacterium H16 cellular segregation and return be added in production equipment.

3. the method preparing PHA according to claim 1, it is characterized in that, by the non-sterile lipolytic enzyme in described substratum outside isolated cell through the growth medium of fermentation taking out part, thus bacterial cell is separated in the taking-up part of described growth medium, before PHA is separated, described bacterial cell is returned and is added in described substratum, and the lipolytic enzyme in remaining substratum outside isolated cell.

4. the method preparing PHA according to claim 1 and 2, it is characterized in that, after lipolytic enzyme outside isolated cell, these at least part of extracellular lipolytic enzymes are returned together with the oily substrate of additional dose and is added to described substratum, to support that bacterium hookworm covets the growth of copper bacterium H16.

5. according to the method preparing PHA in any one of the preceding claims wherein, it is characterized in that, in culturing process, by adding NaOH nebo H ₂sO ₄and the pH value of described substratum is maintained the scope of 7+/-0.3, and during the fermentation, by controlled aeration, make the concentration of dissolved oxygen maintain the scope of 10%-50%, regulated the content of the 3-hydroxyl valerate in the growth of culture, the generation of PHA and multipolymer thus by aeration.

6. according to the method preparing PHA in any one of the preceding claims wherein, it is characterized in that, described oily substrate contains the additional substances of the group from Sodium Propionate, n-propyl alcohol, levulinic acid.

7. according to the method preparing PHA in any one of the preceding claims wherein, it is characterized in that, after completing fermentation, the content of production reactor is heated to the temperature of 80 DEG C, and this heating temperatures at least 30 minutes, be cooled to the temperature of 20 DEG C to 60 DEG C subsequently, afterwards, add the solvating agent containing the washing composition of such as SDS and the mixture of proteolytic ferment, obtain original homogenate or lysate thus.

8. the method preparing PHA according to claim 7, it is characterized in that, filtered by gradocol membrane and purify described original homogenate or lysate, and washing at the temperature water of 20 DEG C to 60 DEG C, thus based on the gained PHA productive rate of initial cellular biomass up to 96%.

9. the method preparing PHA according to Claims 2 or 3, is characterized in that, is concentrated extracellular lipolytic enzyme by ultrafiltration, and this makes their cleanliness factor scope be 100U/mg to 200U/mg.

10. the method preparing PHA according to any one of claim 1-6, it is characterized in that, after the fermentation was completed, by centrifugation separating biomass from described substratum, and the direct nontoxic organic solvent using the group from dimethyl sulfoxide (DMSO) (DMSO) or acetone in described undried biomass with the amount of the wet biomass 1L solvent of every 20g-80g subsequently, and described nontoxic organic solvent is stayed thereon, with at the temperature action 1-5 hour of 30 DEG C to 80 DEG C, remove described organic solvent subsequently.