WO2014032633A1 - Method of producing polyhydroxyalkanoates (pha) from oil substrate - Google Patents

Method of producing polyhydroxyalkanoates (pha) from oil substrate Download PDF

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WO2014032633A1
WO2014032633A1 PCT/CZ2013/000100 CZ2013000100W WO2014032633A1 WO 2014032633 A1 WO2014032633 A1 WO 2014032633A1 CZ 2013000100 W CZ2013000100 W CZ 2013000100W WO 2014032633 A1 WO2014032633 A1 WO 2014032633A1
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oil
pha
production
lipolytic enzymes
culture medium
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PCT/CZ2013/000100
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French (fr)
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Ivana MAROVA
Stanislav OBRUCA
Radek PRIKRYL
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Vysoke Uceni Technicke V Brne
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Priority to CN201380056284.XA priority Critical patent/CN104755623A/zh
Publication of WO2014032633A1 publication Critical patent/WO2014032633A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
    • C12P7/625—Polyesters of hydroxy carboxylic acids

Definitions

  • the invention relates to a method of producing polyhydroxyalkanoates (PHAs) on an oil substrate comprising vegetable oil and/or edible oil and/or waste edible oil, preferably frying oil, on which the bacterial strain Cupriavidus necator H16 is grown, converting oil into PHA and at the same time producing extracellular lipolytic enzymes, which are at least partially isolated from the culture medium during the fermentation process before finishing production and isolation of PHA.
  • PHAs polyhydroxyalkanoates
  • PHAs Polyhydroxyalkanoates
  • Monomers of PHA are (R)-3-hydroxyalkanoic acids. All monomeric building blocks are in (R) configuration due to stereo specificity of the enzyme, which is responsible for the synthesis PHA - PHA synthase. Only in a few cases a small amount of (S) monomer has been found in polyester.
  • the molecular weight of PHAs ranges from 200,000 Da to 3,000,000 Da depending upon the microorganism and growth conditions. PHA is found in the cell in cytoplasm in the form of granules which vary in size from 0.2 to 0.5 ⁇ [1].
  • microorganisms synthetize PHAs as a reserve form of energy and carbon under the condition of sufficient supply of carbon sources and lack of other nutrients. After exhaustion of the carbon source PHAs are then utilized as energy and carbon source. PHAs serve as an ideal reserve form of both carbon and energy, which is given by their low solubility and high molecular weight. Due to these properties they do not participate substantially in osmotic pressure in the cell [1].
  • PHB The biochemical aspect of PHB biosynthesis is widely studied.
  • Alcaligenes eutrophus also Ralstonia eutropha; now Cupriavidus necator
  • PHB is synthesized in a three-step reaction.
  • two molecules of acetyl-CoA are coupled to form acetoacetyl-CoA in a reaction catalysed by 3-ketothiolase.
  • the acetoacetyl-CoA is subsequently and stereospecifically reduced to (R)-3-hydroxybutyryl-CoA in a reaction catalysed by NADPH-dependent acetoacetyl-CoA reductase.
  • PHB is synthesized by polymerization of (R)-3-hydroxybutyryl-CoA molecules by the enzyme PHB synthase [1].
  • the diagram of the PHB biosynthesis is shown in Fig. 2.
  • PHB biosynthesis happens when a carbon souce is available in sufficient amounts and under limitations of for example nitrogen, iron, phosphorus, sulphur, potassium or oxygen.
  • PHB synthesis is regulated at the enzymatic level. From the point of view of the regulation of PHB synthesis, intracellular concentration of acetyl-CoA and of free HSCoA is essential. Under the balanced growth conditions acetyl-CoA is oxidized in the Krebs ' cycle. During the oxidation NADH, which is further used for biosynthetic purposes, is produced. After cessation of culture growth, the concentration of NADH increases, while the activities of citrate synthase and isocitrate dehydrogenase decrease.
  • Acetyl-CoA then cannot be oxidized in the Krebs ' cycle and enters the PHB biosynthetic pathway. 3-ketothiolase is inhibited by free HSCoA, which is generated by the oxidation of acetyl-CoA in the Krebs ' cycle under normal growth conditions [4].
  • Copolymer PHB and PHV may be synthesized for example by the strain Alcaligenes eutrophus or by other types of microorganisms, namely when grown on substrates comprising glucose and propionic acid, or directly by 3- hydroxyvalerate (3HV) precursors. If propionic acid is used, the synthesis is similar to PHB synthesis, except that acetyl-CoA condensates with propionyl- CoA to form 3-ketovaleryl-CoA, which leads to the incorporation of 3- hydroxyvalerate into the polymer structure.
  • Some microorganisms are capable of synthesizing P(HB-co-HV) when grown on a medium which does not include precursors of 3-hydroxyvalerate, such as some mutants of Alcaligenes eutrophus.
  • the copolymer of 3- hydroxybutyrate and 4-hydroxybutyrate may be synthetized in Alcaligenes eutrophus from 4-hydroxybutyric acid, 1 ,4-butanediol, butyrolactone a 4-chloro butyrate [1].
  • PHAs are deposited intracellular ⁇ in the form of granules. The number and size of the granules depend on growth conditions, also varying within different bacterial cultures.
  • the density of PHB granules ranges from about 1.18 to 1.24 g.cm "3 , the density of MCL PHA granules is approximately 1.05g.cm "3 .
  • the granules contain polyesters, proteins and lipids.
  • the granules have a polyester core, the surface being formed by a phospholipid monolayer, into which proteins fulfilling different functions are incorporated.
  • PHAs have a hydrophobic character, therefore phospholipids and proteins constitute the interface between PHAs and the surrounding environment [1].
  • PHA synthase One of the proteins of PHA granules is the PHA synthase. There are probably three types of the PHA synthase, which differ in their substrate specificity and their primary structure. Their common feature is the active site containing cysteine. The first type of the PHA synthase catalyzes the SCL PHA synthesis (short-chain-length) from hydroxy acids consisting of 3-5 carbon atoms. The second type incorporates long-chain hydroxy acids (6-14 carbon atoms) into the structure of PHA polymers (MCL PHA). The third type differs from the first two types by its structure.
  • the third type of PHA synthases consists of two subunits: C-subunit ( ⁇ 40 kDa) and E- subunit ( ⁇ 40 kDa).
  • C-subunit ⁇ 40 kDa
  • E- subunit ⁇ 40 kDa
  • the substrate specificity is not as strict a stipulation as in the case of the preceding PHA synthases, but, on the whole, it is the SCL PHA synthesis that is preferred [5].
  • PHA depolymerase Another protein that can be found in the PHA granule is an intracellular PHA depolymerase. It is responsible for utilization PHA as a source of energy and carbon in case of limitation of carbon source from the environment. So far, research suggests that the process of PHA degradation by intracellular depolymerases is approximately 10 times slower than their synthesis. However, regulation of intracellular depolymerases has not been fully explained yet. In the structure of PHA granules there are also non-catalytic proteins, the so-called phasins. They are supposed to participate in the stabilization of hydrophobic PHAs in the aqueous environment of the cellular cytoplasm [6].
  • the PHB homopolymer is a polyester with all asymmetrical carbon atoms in the (R) configuration. It is relatively highly crystalline (approximately 50 to 80%), which makes it hard and brittle. The glass transition temperature is 5 to 9°C, the melting temperature is between 173 and 180°C. PHB decomposes at the temperature of 200°C, which is close to the melting point. In a chloroform solution it creates a dextrorotatory helical curve.
  • the mechanical properties of PHB for example Young ' s modulus of flexibility (3.5 GPa), elasticity of elongation (40 MPa) is similar to that of polypropylene. Ductility, however, is only around 3%, which is considerably less than in the case of polypropylene [1].
  • Copolymer of 3-hydroxybutyrate and 4-hydroxybutyrate does not form crystalline structures.
  • the glass transition temperature drops from 5 to - 50°C, the melting temperature also decreases from 180 to 54°C with the growing content of 4-hydroxybutyrate (0-100%) in the polymer.
  • Young ' s modulus of flexibility is approximately 55 MPa, elasticity of elongation 39 MPa and ductility 500 % [1].
  • bacterial strains capable of producing PHAs only a few of them can be used in industrial applications. Applicability of a bacterial strain is influenced by a number of factors. First of all, it is stability and safety, growth and accumulation capabilities, attainable amount of biomass and the amount of PHA. Next, it is the extractability rate of PHA, the molecular weight of PHA, the amount of usable substrates as well as financial demands on individual components of the medium [1].
  • the company ZENECA Bioproducts employed mutant strains of Alcaligenes eutrophus for the production of PHB and P(HB-co-HV). The process was realized as a two-phase fed-batch system.
  • biomass was cultivated in a mineral medium comprising glucose as a source of carbon and energy and a precisely determined amount of phosphate. After the culture growth the phosphate was exhausted and in the second step phosphorus limitation occurred, which resulted in PHA accumulation.
  • glucose was supplied to the culture, until a required amount of PHA was produced by biosynthesis. Each phase took approximately 48 hours and the final concentration of biomass dry weight was approximately 100 g/l.
  • the copolymer P(HB-co-HV) was synthesized by adding a mixture of glucose and propionic acid in the second phase of the cultivation. The 3-hydroxy valerate content in the polymer was controlled by the ratio of glucose to propionic acid [7].
  • Production costs could be reduced if for example methanol was used as a substrate, one of the cheapest carbon sources.
  • the strain Methylobacterium extorquens produced PHB at discontinuous fed-batch cultivation on methanol.
  • the optimum concentration of methanol was 1.7 g/l. 9-10 g/l of biomass concentration was achieved and the amount of PHB reached 30-33% of biomass. Nevertheless, even using the cheapest carbon source will not reduce operation costs, since a small amount of produced PHB makes the subsequent separation process more costly and more difficult.
  • Lipolytic enzymes belong to ester hydrolases, catalyzing in a two-phase system water - lipid, decomposition of mono-, di- a triacylglycerols to higher fatty acids, alcohol and glycerol through a complex mechanism dependent on many factors.
  • Lipolytic enzymes are defined as carboxylesterases, which hydrolyze acylglycerols.
  • Lipolytic enzymes which hydrolyze acylglycerols with fatty acids with short chain lengths up to 10 carbon atoms, are considered to be esterases or carboxylases (EC 3.1.1.1).
  • Esterases or carboxylases are active in aqueous solutions, whereas contextgenuine" lipases are more active at the water/lipid interface than in the water phase [20].
  • Lipolytic enzymes are divided into three groups: • . The first group is non-specific. Lipolytic: enzymes of this group release fatty acids from all three positions of acylglycerol and completely hydrolyze triacylglycerols into fatty acids and glycerol.
  • the second group of lipolytic enzymes is 1 ,3-specific. They release fatty acids from the outer positions of the triacylglycerol molecules to form 1 ,2- diacylglycerol, 2,3-diacylglycerol a 2-monoacylglycerol, releasing fatty acids. Long incubation of triacylglycerol with 1 ,3-specific lipases generally leads to complete hydrolysis of triacylglycerols into fatty acids and glycerol.
  • the third group includes lipolytic enzymes which prefer only some fatty acids.
  • lipases belong to extracellular enzymes, which are released to the environment during late exponential and early stationary phases of growth.
  • the production of lipases is influenced by a number of different factors, such as temperature, pH, source of nitrogen, carbon and lipids, stress, and concentrations of dissolved oxygen and inorganic salts.
  • the optimum pH for lipase activity is usually in the range of 6-9.
  • lipases from A. niger and Rhizopus sp. are active even under acidic conditions at pH 4.
  • an alkaline lipase which is active at pH 11 , has been isolated from P. nitroreducens.
  • optimum temperature and thermal stability vary.
  • Lipolytic enzymes belong to the so-called serine hydrolases. Three- dimensional 3D structure of these enzymes displays typical ⁇ / ⁇ -folding patterns - a-helices and ⁇ -sheets. Catalytic triad consists of three amino acid residues, namely serine, asparagine and histidine; in some lipases glutamine is found instead of asparagine. Lipolytic reaction takes place only at the lipid - water interface, therefore reaction rate is directly influenced by the substrate concentration at the phase interface. Thus in one phase there may be mojecules of the substrate in different states without directly influencing the reaction rate [22].
  • Esterase activity is a function of substrate concentration and undergoes Michaelis-Menten kinetics, the maximum reaction rate being achieved at a substrate concentration many times lower than saturation concentration.
  • lipases do not display any activity, as long as the substrate (lipid) is in the state of individual molecules in water. When the substrate concentration exceeds the solubility point, emulsion begins to form, the reaction rate increasing considerably. Lipase activity then depends directly on the presence of the phase interface. It has been confirmed by explaining the spatial structure of lipases that the active centre of an enzyme is protected by polypeptide chain, which blocks binding of the enzyme molecule itself to the enzyme and subsequent formation of the active complex.
  • the mechanism for the hydrolysis of the ester binding is in principle identical both for lipase and for esterase and consists of four steps, as follows from Fig.6, which shows the diagram of microbial degradation of lipids.
  • Fig.6 shows the diagram of microbial degradation of lipids.
  • the active site of lipases disposes of negative potential in the range of pH connected with the maximum lipase activity (typically at pH 8.0), whereas the active site of esterase displays similar behaviour at pH 6.0, which is connected to the usual lower pH optimum of activity.
  • the most well-known microorganisms are capable of producing lipases, but only some kinds are used industrially. The reason for this is insufficient enzyme production, undesirable physical-chemical properties of lipases, limited possibilities of isolation form the cultivation medium, etc.
  • the most common moulds that are commercially used include the species Aspergillus, Penicillium, Mucor and Rhizopus.
  • the main producers of commercial lipases are Aspergillus niger, Humicola lanuginosa, Mucor miehei, Rhizopus arrhizus, R. delemar, R. japonicus, R. niveus and R. oryzae [20].
  • Lipolytic enzymes are currently attracting considerable attention because of their tremendous biotechnological potential. They constitute the most important group of biocatalysts for biotechnological applications, which are successfully used for the synthesis of biopolymers, bio-oil, for the production of agrochemicals and aromatic compounds. That is why the demand for industrial enzymes, particularly of microbial origin, is ever increasing. Enzymes are being exploited in various industries such as food, pharmaceutical, textile, and cosmetic industries, as well as in detergents. Lipases are used in brewing and wine making, cheese making and dietary supplements. They play an important role in pharmacy in transesterification and hydrolysis reactions and are essential for the production of special lipids.
  • Lipases are of great importance for modifications of monoglycerides, which are then used as emulsifiers.
  • Some industrially important chemicals manufactured through a chemical process from fats and oils may be also produced by lipases whose specificity level is a lot greater and better.
  • lipases are used for the production of substitute for cocoa butter and for the production of esters which are employed in cosmetics industry. Lipases are used in the dairy industry for the hydrolysis of milk fat. Current applications contribute to intensifying the flavour of cheese, speeding up the aging process in the production of cheese, cheese making, as ingredients added to other products. Lipase from Aspergillus oryzae is exploited in detergents. Lipases are widely used in the processing of fats and oils, as cosmetic softeners, as well as industrial catalysts for the preparation of prostaglandins, steroids, carboxylic nucleoside analogues and pharmaceutically important polyphenolic compounds [21].
  • PHAs can be used in many fields as well. It is presumed that the main use will be in the sphere of packaging industry, particularly for the production of feeding bottles and baby bottles, plastics for children ' s products and ecological products (e.g. toys), packaging for cosmetics industry and the so-called intelligent packaging of food.
  • Other interesting applications include production of containers (e.g. cups) designed for disposal after use, for instance for fast food restaurant chains, which in turn can provide waste oil as a substrate for the production of bioplastics.
  • Polymer PHA can be also used in other applications: nanofibres and nanoparticles can be prepared from it for targeting drug delivery system, and it is possible to use it for the production of biocompatible implants that can be employed in medicine as fibres, vascular substitutions, etc.
  • biocompatible implants that can be employed in medicine as fibres, vascular substitutions, etc.
  • bioplastics are relatively low, because their cost is for the time being higher than that of synthetic plastics, which discourages demand.
  • substantially stricter regulations for use of ecological plastics can be expected, which will presumably bring the expansion of bioplastic production.
  • Today the production of PHA from oil substrates by means of bacteria is well-known, but compared to classical production of plastics from crude oil it is too expensive, and so it is difficult to promote it.
  • the aim of the invention is to propose a method of PHA production from oil substrates which would be economical and would produce large quantities of PHA.
  • the goal of the invention was achieved by a method of production of polyhydroxyalkanoates (PHAs) on an oil substrate comprising vegetable oil and/or edible oil and/or waste edible oil, preferably frying oil, on which the bacterial strain Cupriavidus necator H16 is grown, converting oil into PHA and at the same time producing extracellular lipolytic enzymes, which are at least partially isolated from the culture medium during the fermentation process before finishing production and isolation of PHA, whereby the principle of the invention consists in that before starting the cultivation, extracellular lipolytic enzymes produced by Cupriavidus necator H16 are added to the oil substrate, thus accelerating the growth of the bacterial culture.
  • PHAs polyhydroxyalkanoates
  • Concurrent production of PHA and extracellular lipolytic enzymes, during which, before the beginning of the cultivation, extracellular lipolytic enzymes produced by Cupriavidus necator H16 are added to the oil substrate, represents an innovative method of PHA production, whereby extracellular lipolytic enzymes are induced by the presence of the oil substrate and bacteria Cupriavidus necator H16 produce an effective molecular form capable of effective accessing the oil substrate for utilization. It is of great advantage that enzymes are an extracellular product, whereas PHA is an intracellular product, which facilitates the separation of both products.
  • the extracellular lipolytic enzymes after being isolated, are at least partially added back to the culture medium together with an additional dose of oil/oil substrate.
  • Fig. 1 represents the structure of PHA
  • Fig. 2 the diagram of biosynthesis PHB
  • Fig. 3 the diagram of PHB and P(HB-co-HV) structure
  • Fig. 4 the diagram of.PHA granule structure
  • Fig. 5 the mechanism of lipase activity
  • Fig. 6 the diagram of microbial degradation of lipids
  • Fig. 7 comparison of induction of extracellular lipase activity on different carbon substrates
  • Fig. 8 the yield of metabolites during a typical fermentation process
  • Fig. 9 characterization of the development of the centrifugation of a polymer product by the method of analytical centrifugation (4000 rpm, 2 hours, 5°C), Fig.
  • Fig. 10 the effect of lipase addition on the process of growth of Cupriavidus necator H16 using oil as a carbon source
  • Fig. 11 an example of GC-FID chromatogram of PHA
  • Fig. 12 an example of GPC chromatogram of PHA
  • Fig. 13 TGA analysis of PHB
  • Fig. 14 DSC analysis of PHB
  • Fig. 15 pH optimum of extracellular lipase
  • Fig. 16 the effect of ionic strength on lipase activity
  • Fig. 17 record of protein separation by PAGE-SDS - silver dying.
  • Waste substrates are used for PHA production in a number of patents.
  • One of the most general method of processing probably most types of waste is disclosed in the patent US 2009/0317879 A1, where, however, waste is mostly processed by methanotrophic bacteria to lower carboxylic acids (propionic, acetic) and to methane, by which means the waste is made accessible to the production strain.
  • Another patent (US 2010/0190221 A1) even describes using substrates that may be toxic for microorganisms or the environment. By means of the enzyme methane-monooxygenase organic compounds are converted into utilizable substrates. PHA production itself on waste oil of various kinds is also included in a number of various patents.
  • PHA and extracellular lipolytic enzymes Concurrent production of two industrially important metabolites (PHA and extracellular lipolytic enzymes), one of which (extracellular lipolytic enzyme) is induced by a sole type of substrate with a specific chemical composition (vegetable oil), and, furthermore, the substrate is preferably waste and specific (fritting oil frying?, which has no other uses), is degradable by the latter product (lipolytic enzymes) and at the same time provides the highest yields of the latter metabolite (on oil the highest yields have been achieved in the production strain - up to 96 % of biomass), has not yet been described in the case of PHA production either in technical literature or patent literature.
  • Palmitate 54.3 ⁇ 3.5 18.67 ⁇ 1.20 37.92+2.44 and comparison of induced activity of extracellular lipolytic enzymes on different types of carbon substrate is shown in Fig. 7.
  • Another presented patent deals with the PHA production by selected species of the genus Pseudomonas with controlled composition of copolymer regulated by means of the medium composition (C-source - fatty acids) and addition of suitable precursors (US 2011/0166318 A1), or a patent for the preparation of block copolymers by means of controlling enzymatic activities and medium composition (WO 0006762 A1) has been presented.
  • C-source - fatty acids C-source - fatty acids
  • suitable precursors US 2011/0166318 A1
  • WO 0006762 A1 a patent for the preparation of block copolymers by means of controlling enzymatic activities and medium composition
  • copolymer P(3-HB-HV) structure With regard to a wide range of uses and compliance of the copolymer P(3-HB-HV) structure with the European Congress concerning uses in food industry (i.e. for potential applications), we focused on the production of copolymer P(3-HB-HV) with the 3HV content in the range 4-10%.
  • Metabolite yields during a typical fermentation process are represented in
  • cells are not separated first, polymer is isolated directly in the fermentation reactor immediately after the cultivation has completed (usually 32-38 hours).
  • the cells in the culture medium are first exposed to warming-up, when the culture medium is warmed up to the temperature of 80°C (30 min) and, after subsequent cooling to the environment temperature, a mixture containing proteolytic enzyme is added (i.e. an enzyme hydrolyzing proteins, e.g. alcalase) and a detergent (e.g. sodium dodecyl sulfate) with optimized concentration (0,04 g SDS/1 g CDW; Alcojet - neutral industrial detergent).
  • proteolytic enzyme i.e. an enzyme hydrolyzing proteins, e.g. alcalase
  • a detergent e.g. sodium dodecyl sulfate
  • Most cell components are hydrolyzed by the acting of these two agents, whereas polymer remains untouched. After that polymer is separated by fractional membrane ultrafiltration, is washed with water and dried by lyophilization.
  • Centrifugation as a common technique for separating cells from the medium, or polymer from the rest of the cells proved to be difficult to use. Above all, centrifugation of the product is rather difficult, since the residual oil carries a relatively large part (about 1/4-1/3) of the centrifuged sample to the surface, which represents a considerable loss, as follows from Fig. 9, illustrating the characterization of the process of centrifugation of the polymer product by a method of analytical centrifugation (4000 rpm, 2 hours, 5°C).
  • the final product can be also washed in order to increase its cleanness - purity?.
  • Table 5 shows a list of possible purification conditions and their influence on the cleanness purity? of the PHA product.
  • the bacteria grows on oil (and solely on oil), which may be vegetable oil and/or edible oil and/or waste edible oil, preferably frying oil, they produce extracellular lipolytic enzymes, which help to decompose the oil and utilize it.
  • Extracellular lipolytic enzymes are industrially important enzymes and during this cultivation process they are produced in large quantities, which is economically attractive. Therefore we propose an overall technological solution including the concurrent production of PHA (intracellular polymer; yield 93-96 %) and lipase (extracellular enzyme; activity approximately 100 U/ml)).
  • PHA intracellular polymer
  • lipase extracellular enzyme; activity approximately 100 U/ml
  • the precursor is added to the culture medium together with the additional dose of oil (addition) and with extracellular lipolytic enzymes.
  • b) non-sterile withdrawal of part of the culture e.g. 1/3 of the cells in the culture medium
  • Extracellular lipolytic enzymes can be further utilized in the subsequent cultivation as a factor enhancing biomass production and consequently accelerating the whole process. If extracellular lipolytic enzymes, isolated according to the procedure described above in the amount of 0,5 - 3 U per ml of growth medium (i.e. about 2%) are added to a medium containing oil and subsequently the medium is inoculated by the culture Cupriavidus necator, the bacterial culture growth increases by approximately 20-30%. Extracellular lipolytic enzymes produced by Cupriavidus necator seem to be more suitable for this purpose than for example commercially available lipase produced by the microorganism Rhizopus oryzae. The effect of the added extracellular lipolytic enzymes on the process of cultivation of Cupriavidus necator H16 using oil as carbon source is shown in Fig. 10.
  • DMSO dimethyl sulfoxide
  • the concentration and structure of PHA is most often determined by a method of gas chromatography (GC) with detection by FID (flame ionization detector).
  • GC gas chromatography
  • FID flame ionization detector
  • Fig. 11 shows a typical chromatogram of polymer composed of 3-hydroxybutyrate and 3- hydroxyvalerate.
  • 3HB (mg-mr 1 ) 3HB area (-) 3HV (mg-mr 1 ) 3HV are (-)
  • the molecular weight of PHA is a very important parameter, especially with respect to its applications. Its value is important not only for processing of PHA for example to bioplastics, nanofibres or particles, but also for numerous pharmaceutical applications, particularly in connection with biodegradability of the used materials. Therefore it is essential to characterize the produced microbial polymer in the greatest detail possible, or generalize the conditions for the production of polymer having a defined molecular weight.
  • the molecular weight of the produced PHA was determined by a method of gel permeation chromatography (GPC). An example of GPC chromatogram of PHA is shown in Fig. 12.
  • the molecular weight of the produced polymer achieved values in the range 1.85-2.41 E+05, whereby the values achieved on a saccharide substrate were more than twice as high.
  • Biomer PHB (commercially available material) 5 5
  • the thermal properties of PHA depend on the content of individual monomers (see Tab. 1). Specific parameters, such as the temperature of material degradation, the melting point etc. are routinely determined by means of TGA (thermogravimetric analysis), which is represented in Fig. 13, and DSC (differential scanning calorimetry), which is shown in Fig. 14.
  • Biomer PHB (commercially available material) 264 168.52
  • substrate oil precursor sodium propionate 256 166.40
  • PHA is not soluble in polar implicit solvents or in non-polar implicit solvents. Solvents from the centre of an eluotropic series (medium Rf values) have the ability to partly dissolve a polymer or create gel. Solubility of PHA in selected organic solvents is shown in Table 9:
  • Basic molecular characteristics include pH optimum, whose values are summarized in Fig. 15 and shown in Table 10, which indicates changes of extracellular lipolytic enzyme activity at varying pH assessed in % compared to the maximum value of 100 %.
  • the culture medium contains, apart from extracellular lipolytic enzymes, also a number of other proteins and other substances. Furthermore, all these substances are considerably attenuated, including the targeted enzyme.
  • condensation and more detailed characterization of extracellular enzymes were carried out in order to verify cheap and effective isolation and concurrent enzyme purification.
  • Extracellular fraction of C.necator H16 comprises 5 main protein fractions having molecular weights of 15.95 kDa; 18.80 kDa; 24.34 kDa; 47.54 kDa and 64.52 kDa. The first four of them were visible even after the dialysis. After the application of ultrafiltration (membrane filter with 10 kDa extensivelycut-off" exclusion limit) the majority fractions were proteins with membrane filter 18.80 kDa and 24.34 kDa, the fraction having 15.95 kDa was only slightly visible.
  • Hasan F., Shah A.A., Hameed A. Industrial applications of microbial lipases. Enzyme and Microbial Technology 2006, vol. 39, 35-251 p. Joseph, B., Ramteke P. W., Thomas, G., Schvastava, N.: Standard Review Cold-active microbial Lipases: a versatile tool for industrial applications. Biotechnology and Molecular Biology Rewiew, June 2007, vol. 2, pp. 39-48. ISSN 1538/2273.

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WO2017076374A1 (en) 2015-11-06 2017-05-11 Vysoké Učení Technické V Brně Polymer-made fibre preparation method
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US11279957B2 (en) 2017-08-29 2022-03-22 Mitsubishi Gas Chemical Company, Inc. Method for producing polyester
EP3560479A1 (en) 2018-04-24 2019-10-30 NAFIGATE Corporation, a.s. A uv filter based on polyhydroxybutyrate and a method of its preparation
JP2020054334A (ja) * 2018-10-02 2020-04-09 オブシェストヴォ ス オグラニチェンノイ オトヴェトストヴェンノスチュ ギプロビオシンテズＯｂｓｈｃｈｅｓｔｖｏ Ｓ Ｏｇｒａｎｉｃｈｅｎｎｏｙ Ｏｔｖｅｔｓｔｖｅｎｎｏｓｔｙｕ Ｇｉｐｒｏｂｉｏｓｉｎｔｅｚ 微生物タンパク質を得るための共棲菌 従属栄養細菌Ｃｕｐｒｉａｖｉｄｕｓ ｇｉｌａｒｄｉｉ ＧＢＳ−１５−１菌株
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