WO2013027697A1 - Method for recovering activity of lipase - Google Patents
Method for recovering activity of lipase Download PDFInfo
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- WO2013027697A1 WO2013027697A1 PCT/JP2012/070983 JP2012070983W WO2013027697A1 WO 2013027697 A1 WO2013027697 A1 WO 2013027697A1 JP 2012070983 W JP2012070983 W JP 2012070983W WO 2013027697 A1 WO2013027697 A1 WO 2013027697A1
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- lipase
- containing composition
- activity
- mass
- moisture absorption
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
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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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
Definitions
- the present application is an application claiming priority based on Japanese Patent Application No. 2011-179720.
- the entire contents of Japanese Patent Application No. 2011-179720 are incorporated herein by reference.
- the present invention relates to a method for recovering lipase activity such as various esterification ability and transesterification ability in a composition containing a specific immobilized lipase or powdered lipase, and an immobilized lipase or powdered lipase having recovered activity.
- the present invention relates to an esterification reaction and a method of transesterification of fats and oils using a composition to be contained.
- Lipases are widely used in esterification reactions of various carboxylic acids such as fatty acids and alcohols such as monoalcohols and polyhydric alcohols, and transesterification reactions between multiple carboxylic acid esters.
- transesterification is an important technique as a method for producing esters of various fatty acids, sugar esters and steroid esters, including modification of animal and vegetable fats and oils.
- lipase which is an oil and fat hydrolyzing enzyme
- transesterification can be performed under mild conditions of about room temperature to about 90 ° C., and side reactions are suppressed compared to conventional chemical reactions. Not only energy costs are reduced, but also the safety is high because lipase as a catalyst is a natural product.
- the target substance can be efficiently produced by its substrate specificity and position specificity.
- Such lipases are immobilized on anion exchange resin (patent document 1), phenol adsorption resin (patent document 2), etc. for the purpose of maintaining activity, easiness of recovery, uniform dispersibility in raw materials, etc.
- Immobilized lipases, and powdered lipases (Patent Document 3) having predetermined pores and particle sizes have been used.
- the lipase which is an enzyme is expensive, it has been practiced that it is recovered after completion of the reaction and repeatedly used, and is discarded only when the lipase activity is considerably reduced. In addition, it has also been practiced to restore reduced lipase activity.
- Patent Document 4 For example, a method of recovering lipase activity by washing lipase with reduced activity with triacylglycerol (Patent Document 4), and a method of improving lipase activity in transesterification by adding water (Patent Document 5) ) has been proposed.
- the recovery method up to now, the recovery of the activity was insufficient or the activity of all the lipases could not be recovered uniformly.
- water when water is added, the lipase and water are brought into contact by spraying the water directly, but since the water is contacted unevenly with the lipase, recovery of the lipase activity becomes uneven.
- the present inventors examined a method that can restore the reduced lipase activity in order to achieve the above-mentioned task. As a result, it has been found out that contacting the lipase-containing composition with a wet gas under certain conditions can fully restore the reduced lipase activity, leading to the present invention. That is, the present invention may relate to the following aspects.
- the present invention relates to a method for recovering lipase activity, comprising a step of absorbing moisture in which a lipase-containing composition is brought into contact with a wet gas.
- the method according to [1] wherein the wet gas has a relative humidity of 50 to 90%.
- the moisture absorption step is performed until the amount of water contained in the lipase-containing composition is 8.5 to 20% by mass in the components excluding the oil component in the lipase-containing composition, [1] or [2]
- [5] The method according to any one of [1] to [4], wherein the moisture absorption step aerates the wet gas into the lipase-containing composition.
- the mass of the oil component contained in the lipase-containing composition is adjusted to 1.5 or less times the mass of the component excluding the oil component in the lipase-containing composition.
- the moisture absorption step reduces the amount of the oil component contained in the lipase-containing composition by aerating the moist gas into the lipase-containing composition, and absorbs moisture to the lipase-containing composition
- the present invention relates to a process for producing a lipase whose activity has been restored.
- the present invention as a result of being repeatedly used in the esterification method and the transesterification method, it is possible to provide a method of predominantly recovering the activity of the lipase whose activity has been reduced.
- the present invention as compared to the case where liquid water is directly sprayed onto the lipase as in the past, there is no spray unevenness, and the activity of the lipase can be surely improved.
- it is possible to provide a method for producing an ester compound by carrying out an esterification reaction or a transesterification reaction using the above-mentioned lipase whose activity has been restored.
- One aspect of the present invention is a method for recovering lipase activity, comprising a step of absorbing moisture in which the lipase-containing composition is contacted with a wet gas.
- This method will be described in detail below.
- Moisture absorption process The present invention includes a moisture absorption step for recovering the activity of the lipase whose activity has been reduced as a result of repeated use in the esterification method, transesterification method and the like.
- the moisture absorption step is carried out by contacting the lipase-containing composition with the moist gas while keeping the lipase-containing composition in the moist gas.
- Humid gas is intended to be a gas in which the relative humidity or the amount of water vapor is forcibly controlled rather than the atmosphere having a normal humidity.
- the gas used for the wet gas for example, air, nitrogen, carbon dioxide gas, argon and the like are preferable, among which air or nitrogen is preferable, and air is particularly preferable.
- a gas having a relative humidity of, for example, 50 to 90%, preferably 60 to 80%, more preferably 60 to 70% can be suitably used as the wetting gas. If the relative humidity is insufficient, steam or the like may be introduced into the system during the moisture absorption process to compensate for the relative humidity.
- the amount of water contained in the components excluding the oil component in the lipase-containing composition is, for example, 8.5 to 20% by mass, preferably 10 to 18.5% by mass, and more preferably 12 to 18%.
- the oily component means all oil-soluble components other than the aqueous component contained in the lipase-containing composition such as fat and oil, and can be measured from the oil of the standard fat and oil analysis test method described later.
- an oil-based component contained in the lipase containing composition of this invention a glyceride, a free fatty acid, fatty acid ester etc. are mentioned, for example.
- the moisture absorption step has a relative humidity of, for example, 50 to 90%, preferably 60 to 80%, more preferably 60 to 70%; for example, 30 to 60 ° C., preferably 40 to 60 ° C., more preferably 50 to 60 ° C.
- the activity of the lipase in the lipase-containing composition can be recovered or the decrease in activity can be suppressed.
- the degree of recovery of the lipase activity or the reduction of the activity can be evaluated by comparing the amounts of components in the middle stages of the esterification reaction or transesterification reaction. The amounts of the components can be compared among the components that greatly change depending on the reaction, for example, raw materials or main reaction products.
- POS 1-palmitoyl-2-oleoyl-3-stearoylglycerin
- SOS 1,3-distearoyl-2-oleoylglycerin
- the total mass% of POS and SOS in the triglyceride obtained after the reaction for 1 hour is 100.
- the total mass% of POS and SOS in triglyceride obtained after the lipase-containing composition before the moisture absorption step is changed to the lipase-containing composition after the moisture absorption step of the present invention and the same transesterification reaction is performed for 1 hour
- the lipase activity can be compared by determining the relative amount: POS + SOS value).
- the ratio (relative amount: POS + SOS value) of the total mass% of POS and SOS when using the lipase-containing composition after the moisture absorption step is preferably 100 or more or 100 or more, for example, 150 or more, preferably 200 or more And more preferably 200 to 250.
- ⁇ Oil component adjustment process> It is preferable to include an oil component adjustment step for reducing the oil component contained in the lipase-containing composition prior to the moisture absorption step.
- an oil component adjustment step for reducing the oil component contained in the lipase-containing composition prior to the moisture absorption step.
- the mass of the oil component fat and oil contained in the lipase containing composition is 1 based on the mass of the component excluding the oil component fat and oil in the lipase containing composition. It is preferable to adjust to .5 times or less.
- the mass of the oil component in the lipase-containing composition is equal to the mass of the component excluding the oil component in the lipase-containing composition), and most preferably 0.1 to 0. .7 times.
- the lipase-containing composition of the present invention comprises a lipase or a lipase and an auxiliary.
- lipases that can be used in the present invention include lipoprotein lipase, monoacylglycerolipase, diacylglycerolipase, triacylglycerolipase, galactolipase, phospholipase and the like. Of these, triacylglycerolipase is preferred.
- Microorganisms that produce these lipases include, but are not limited to, bacteria, yeast, filamentous fungi, actinomycetes, etc., but they are not particularly limited, but include Alcaligenes sp., Pseudomonas sp., And Arthrobacters. sp., Staphylococcus sp., Torulopsis sp., Escherichia sp., Mycotorula sp., Propionibacterum sp. Bacteria (Chromobacterum sp.), Xanthomonas sp.
- Xanthomonas sp. Lactobacillus (Lacto) bacillus sp.), Clostridium sp., Candida sp., Geotrichum sp., Sacchromycopsis sp., Nocardia sp. , Fusarium sp., Aspergillus sp., Rhizomucor sp., Mucor sp., Thermomyces sp. Rhizopus sp., Penicillium sp. (Penicillium sp.), Phycomyces sp., Puccinia sp.
- the genus Bacillus (Bacillus sp.), The genus Streptomyces (Streptmyces sp.), And the like.
- lipases derived from Alkaligenes, Pseudomonas, Rhizomucor, Mucor, Thermomyces, Rhizopus or Penicillium are preferable.
- Lipase from Rhizomucor miehei (Rhizomucor miehei), Lipase from Thermomyces lanchogenus (Thermomyces lanuginosus), Lipase from Rhizopus de lemar (Rhizopus delemar), Rhizopus lysops or More preferred is a lipase from Rhizopus oryzae). Particularly preferred are lipases from Rhizopus oryzae.
- the lipase used in the lipase-containing composition of the present invention may be one obtained by culturing and drying a lipase-containing aqueous liquid containing a medium component of the lipase, etc.
- the lipase consists essentially of the lipase itself.
- a lipase containing composition of this invention after culture
- the lipase used in the present invention may or may not have regiospecificity. When it has positional specificity, it is preferably 1,3-specific.
- a lipase culture solution used for culturing a lipase for example, an aqueous solution containing soybean powder, peptone, corn starch liquor, K 2 HPO 4 , (NH 4 ) 2 SO 4 , MgSO 4 ⁇ 7H 2 O, etc. is given.
- soy flour 0.1 to 20% by mass, preferably 1.0 to 10% by mass, peptone 0.1 to 30% by mass, preferably 0.5 to 10% by mass, corn starch and liquor
- the content is 0.1 to 30% by mass, preferably 0.5 to 10% by mass, and K 2 HPO 4 is 0.01 to 20% by mass, preferably 0.1 to 5% by mass.
- (NH 4 ) 2 SO 4 is 0.01 to 20% by mass, preferably 0.05 to 5% by mass
- MgSO 4 ⁇ 7H 2 O is 0.01 to 20% by mass, preferably 0.05 to 5%. It is mass%.
- the culture conditions are: culture temperature: 10 to 40 ° C., preferably 20 to 35 ° C., aeration amount: 0.1 to 2.0 VVM, preferably 0.1 to 1.5 VVM, stirring rotation speed: 100 to 800 rpm, preferably
- the pH is controlled to be in the range of 3.0 to 10.0, preferably 4.0 to 9.5, more preferably 7.0 to 8.0, using NaOH or ammonia.
- the cells are preferably separated by centrifugation, membrane filtration and the like.
- removal of low molecular components such as salts and sugars can be performed by UF membrane treatment.
- the procedure of UF membrane treatment, concentration of the lipase-containing aqueous solution to 1 ⁇ 2 volume, and addition of phosphate buffer of the same amount as the concentrate is repeated 1 to 5 times, A lipase-containing aqueous liquid from which low molecular weight components have been removed can be obtained.
- the stirring rotation number of the homogenizer is 500 to 30,000 rpm, preferably 1,000 to 15,000 rpm
- the rotation number of the Waring blender is 500 to 10,000 rpm, preferably 1,000 to 5,000 rpm.
- the stirring time may be 0.5 to 10 minutes, preferably 1 to 5 minutes.
- Ultrasonication may be performed under the conditions of 1 to 50 KHz, preferably 10 to 20 KHz.
- the ball mill should use small glass beads having a diameter of about 0.1 to 0.5 mm.
- the lipase-containing aqueous liquid one containing 5 to 30% by mass as solid content is preferably used.
- Drying and pulverization of the lipase-containing aqueous liquid are performed by, for example, spray drying, freeze drying (lyophilization), and a method of drying after solvent precipitation and the like.
- the spray drying may be performed using, for example, a spray dryer such as a nozzle countercurrent type, a disk countercurrent type, a nozzle cocurrent type, and a disk cocurrent type.
- the disk co-current system is more preferable, and the atomizer rotation speed is preferably 4,000 to 20,000 rpm.
- Spray drying is suitably performed by adjusting the temperature of the lipase-containing aqueous liquid to 10 to 60 ° C., preferably 20 to 40 ° C.
- the temperature of blowing (drying atmosphere) at the time of spray drying is an inlet temperature of 80 to 200 ° C., an outlet temperature of 30 to 100 ° C., preferably 40 ° C. to less than 70 ° C., more preferably 40 ° C. to 65 ° C., still more preferably 50 It is appropriate to set the temperature to 60 ° C. Lipase is weak to temperature, and lowering the temperature can reduce the decrease in enzyme activity. Freeze-drying (lyophilization) is preferably performed, for example, by a laboratory-sized small-scale lyophilizer or tray-type lyophilization. Furthermore, drying under reduced pressure may be performed. As a method of drying after solvent precipitation, for example, after solvent precipitation using ethanol, acetone or the like, a method of drying under reduced pressure may be mentioned.
- the pH of the lipase-containing aqueous liquid is 6 to 8.5 using NaOH or ammonia immediately before the drying / powdering step such as spray drying.
- the pH is preferably adjusted to 8.0 or less, and more preferably 7.5 to 8.0.
- the pH adjustment may be performed in any step prior to the drying / powdering step such as spray drying, and the pH of the lipase-containing aqueous liquid is previously adjusted so that the pH immediately before the drying / powdering step falls within the above range.
- the pH may be adjusted.
- alkali agents and acids can be used for pH adjustment, it is preferable to use alkali metal hydroxides, such as sodium hydroxide.
- the lipase-containing aqueous liquid may be concentrated in a process before the drying / powdering process.
- the concentration method is not particularly limited, but the evaporator, flash evaporator, UF membrane concentration, MF membrane concentration, salting out with inorganic salts, precipitation with a solvent, adsorption method with ion exchange cellulose etc., water absorption by a water absorbing gel Law etc. are mentioned.
- UF membrane concentration and an evaporator are preferable.
- a module for UF membrane concentration a flat membrane or hollow fiber membrane having a molecular weight cut off of 3,000 to 100,000, preferably 6,000 to 50,000, preferably made of polyacrylonitrile type or polysulfone type.
- the lipase of the present invention may be in the form of immobilized lipase or powdered lipase.
- the immobilized lipase is preferably one in which the above lipase is immobilized on a carrier such as silica, celite, diatomaceous earth, perlite, polyvinyl alcohol, anion exchange resin, phenol adsorption resin, hydrophobic carrier, hydrophobic resin, cation exchange resin, chelate resin, etc. .
- a carrier such as silica, celite, diatomaceous earth, perlite, polyvinyl alcohol, anion exchange resin, phenol adsorption resin, hydrophobic carrier, hydrophobic resin, cation exchange resin, chelate resin, etc.
- Such immobilized lipase can be obtained, for example, as Lipozyme TL-IM from Novozymes A / S.
- the immobilized lipase can be used as it is, or one obtained by crushing the immobilized lipase can be used.
- the pulverized product of immobilized lipase is an average particle size of 1 ⁇ m or more and less than 300 ⁇ m, preferably an average particle size of 1 to 200 ⁇ m, more preferably an average particle size of 1 to 100 ⁇ m, particularly preferably an average particle size using a usual pulverizer. It is preferable to grind so as to have a diameter of 20 to 100 ⁇ m.
- a grinding machine a mortar, a shear friction type grinding machine, a cutter type grinding machine, a stone mill (mycoroider, muscoloider), a coffee mill, a power mill, a pin mill, an impact type grinding machine (hammer mill, ball mill), roll type
- crushers pneumatic crushers, homogenizers, ultrasonic crushers and the like.
- the average particle size can be measured, for example, using a particle size distribution measuring apparatus (LA-500) manufactured by Horiba, Ltd.
- the powdered lipase is, for example, a powdered lipase in which 90% by mass or more of the total mass has an average particle diameter of 1 to 100 ⁇ m, preferably 10 to 80 ⁇ m, more preferably 20 to 50 ⁇ m.
- the particles of this powder are preferably spherical and, for example, 3,000 to 40,000 particles / mm 2 , preferably 3,000 to 20,000 pores with a diameter of 0.5 ⁇ m to 6 ⁇ m are formed on the surface of the particles. It is suitable to have / mm 2 , more preferably 3,000 to 10,000 / mm 2 .
- the number of pores on the particle surface can be easily measured using an electron microscope.
- Powdered lipase may be used in the form of a lipase powder formulation.
- the lipase powder preparation can be obtained, for example, by further drying and pulverizing the lipase-containing aqueous liquid obtained by dissolving and / or dispersing the lipase and the cereal powder and / or the saccharide powder in the aqueous liquid.
- Water is preferred as the aqueous liquid.
- the mass of water in the lipase-containing aqueous liquid is preferably 2.0 to 1,000 times, more preferably 2.0 to 500 times, the mass of water to the mass of the lipase, 3.0 It is most preferable to be 100 times.
- a lipase-containing aqueous liquid As a lipase-containing aqueous liquid, a lipase culture solution from which cells were removed, a purified culture solution, a solution obtained by dissolving and dispersing lipase powder obtained from these in water again, and a commercially available lipase powder were dissolved and dispersed in water again And commercially available liquid lipases and the like. Further, in order to further enhance the lipase activity, those from which low molecular components such as salts have been removed are more preferable, and those from which low molecular components such as sugars have been removed in order to improve powder properties are more preferable.
- the product of PICM Japan Co., Ltd .: Picantase R 8000, and the product of Amano Enzyme Co., Ltd .: Lipase F-AP15, etc. are preferably used.
- the most suitable powdered lipases include products of Amano Enzyme Co., Ltd. derived from Rhizopus oryzae: Lipase DF “Amano” 15-K (also referred to as Lipase D) and Lipase D “Amano” conc. In addition, about this lipase D, it was a description derived from Rhizopus delemar conventionally.
- cereal powder and / or saccharide powder examples include soybean powder such as whole fat soybean powder and defatted soybean powder, wheat flour, rice flour and dextrin.
- the grain powder and / or the sugar powder is suitably, for example, 250 to 1000% by mass, preferably 500 to 1000% by mass, more preferably 500 to 750% by mass, with respect to the lipase.
- auxiliary agents that can be used in the present invention, those that do not adversely affect the esterification reaction or transesterification reaction with lipase can be used.
- filter aids are preferred.
- the filter aid include inorganic filter aids such as celite, fibers such as cellulose, and organic filter aids such as ground products thereof.
- an organic filter aid particularly an organic polymer filter aid, is preferable, and in particular, cellulose powder is preferable.
- the filter aid is preferably in powder form.
- the average particle size of the powdery filter aid is, for example, 10 to 90 ⁇ m, more preferably 20 to 80 ⁇ m.
- the mass ratio of the immobilized lipase or powdered lipase to the filter aid is, for example, preferably 1/10 to 10/1, and particularly preferably 1/7 to 2/1.
- Another aspect of the present invention is to provide a method for producing a lipase whose activity has been restored.
- a lipase with reduced activity can be obtained by repeatedly performing esterification reaction and transesterification reaction.
- the mass of the lipase-containing composition is 0.05 to 10% by mass, preferably 0.1 to 5% by mass, more preferably 1 to 2% by mass, based on the starting substrate.
- the lipase-containing composition is added to the mixture and transesterification is performed for 10 to 50 hours while stirring at 20 to 80.degree.
- a lipase with reduced activity can be obtained.
- the good activity of the lipase is continuously maintained by alternately carrying out the esterification reaction or transesterification reaction of 1 to 10 times, preferably about 1 to 5 times, and the method for recovering lipase activity of the present invention. May be Moisture absorption process
- the lipase whose activity has been reduced recovers its activity by contacting the above-mentioned lipase-containing composition in a wet gas, and the activity can be recovered to produce a lipase whose activity has been restored.
- lipase is produced, for example, by placing the lipase with reduced activity in a container such as a tray and exposing it to a wet gas atmosphere as described above.
- the thickness of the lipase whose activity has been reduced in the container is, for example, 5 cm or less in thickness, preferably 3 cm or less, more preferably 0.1 to 1 cm.
- the moist gas used for this forced aeration can use the same gas as the moist gas used in the above-mentioned moisture absorption process.
- the forced aeration of the moist gas is suitably, for example, 10 minutes or less, preferably 7 minutes or less, more preferably 1 minute or more and 5 minutes or less.
- the mass of the oily component contained in the lipase-containing composition is not more than 1.5 times the mass of the component excluding the oily component in the lipase-containing composition before the moisture absorption step, ie, before contacting with the wet gas. It is preferable to More preferably, it is 1 times or less (ie, the mass of the oil component in the lipase-containing composition and the mass of the components excluding the oil component in the lipase-containing composition are equal) or less, most preferably 0.1 to 0.7. It is a double.
- the gas is passed through the lipase-containing composition in a column or a filter to reduce the amount of the oily component in the lipase-containing composition, and then the aforementioned wetting is performed.
- a gas may be bubbled into the lipase containing composition. Air, nitrogen, carbon dioxide gas, argon or the like can be used as the gas used for aeration for reducing the amount of the oily component, and dry nitrogen gas is particularly preferable.
- the aeration is suitably, for example, 10 minutes or less, preferably 7 minutes or less, more preferably 5 minutes or less.
- the amount of the oil component contained in the lipase-containing composition may be reduced, and moisture absorption may be performed on the lipase-containing composition.
- the moist gas used for this aeration can use the same gas as the moist gas used by the moisture absorption process mentioned above.
- the aeration of the wet gas is suitably, for example, 10 minutes or less, preferably 7 minutes or less, more preferably 1 minute to 5 minutes.
- the mass of the oil component contained in the lipase-containing composition is preferably reduced to 1.5 times or less the mass of the component excluding the oil component in the lipase-containing composition.
- the mass of the oil component in the lipase-containing composition and the mass of the components excluding the oil component in the lipase-containing composition are equal is 1 times or less (ie, the mass of the oil component in the lipase-containing composition and the mass of the components excluding the oil component in the lipase-containing composition are equal) or less, most preferably 0.1 to 0.7. It is a double.
- Another aspect of the present invention is a method for producing an ester compound (esterified product or transesterified product) obtained by carrying out an esterification reaction or a transesterification reaction using a lipase whose activity is restored.
- the esterification reaction carried out using the lipase having recovered activity of the present invention includes, for example, an esterification reaction of a partial ester of fatty acid with a fatty acid, or an esterification reaction of a monohydric or polyhydric alcohol with a fatty acid .
- the esterification reaction of glycerol and a fatty acid etc. are mentioned.
- restored of this invention is a transesterification reaction between 1 or more types chosen from fatty acid ester, a fatty acid, and alcohol, and fats and oils.
- preferable transesterification reactions include transesterification between fats and oils by a conventional method, transesterification between fatty acid esters and fats and oils, transesterification with alcoholysis and acidolysis.
- fatty acid esters and fats and oils are preferable as raw material substrates for transesterification.
- the fatty acid ester for example, a linear or branched, saturated or unsaturated fatty acid ester having 6 to 30, preferably 8 to 22 carbon atoms is suitable.
- examples of the fatty acid include octanoic acid, decanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, and behenic acid.
- Particularly preferred fatty acid esters include ethyl palmitate, methyl palmitate, ethyl stearate, methyl stearate, ethyl behenate and methyl behenate.
- the raw material substrate may contain water.
- the amount of water contained in the starting substrate is, for example, 10 to 1000 ppm, preferably 100 to 500 ppm, more preferably 200 to 400 ppm, based on the total mass of the starting substrate.
- transesterification using acidolysis with fats and oils and fatty acids it is possible to produce structural fats and oils using the 1,3-specific lipase possessed by lipases to a large extent. It leaves the specific fatty acid at the 2-position of the glycerin skeleton, replacing the fatty acid at the 1-3 position with the target fatty acid.
- the obtained product can be used for fats and oils used for chocolate etc., and can be used for fats and oils having specific nutritional effects.
- the conditions for the esterification reaction and the transesterification reaction using the lipase of which the activity is recovered according to the present invention are not particularly limited, and the reaction can be performed by a conventional method. Generally, it is carried out under normal pressure or reduced pressure while avoiding the inclusion of water which causes hydrolysis.
- As the reaction temperature although depending on the freezing point of each raw material substrate to be used, it is preferable to carry out at about 20 to 80 ° C., and it is more preferable to carry out at 40 to 60 ° C. if not limited by the freezing point.
- the addition amount of the lipase-containing composition containing lipase whose activity has been restored to the starting substrate is preferably 0.05 to 10% by mass with respect to the total mass of the lipase-containing composition and the starting substrate. -5% by mass is more preferable.
- the optimum amount is determined by the reaction temperature, the reaction time set, the activity of the lipase whose activity has been recovered, and the like. After completion of the reaction, the lipase is removed by filtration and / or centrifugation and the like, and is repeatedly used until it is reduced to an activity which can not be produced. The reduced activity lipase can be reactivated by the method of the present invention.
- esterified product or transesterified product is not particularly limited, but is also useful as a transesterified oil or esterified oil or fat used in the food field.
- lipase-containing composition As a powdery lipase, an enzyme solution (150,000 U / ml) of a product of Amano Enzyme Co., Ltd .: Lipase DF “Amano” 15-K (also referred to as Lipase D) was prepared. The amount of water in this enzyme solution was 15 times the mass of lipase D. Further, a 10% by mass aqueous solution of deodorized whole fat soybean powder (trade name: Alpha Plus HS-600, manufactured by Nisshin Oillio Group, Inc.) was prepared as grain powder.
- Alpha Plus HS-600 manufactured by Nisshin Oillio Group, Inc.
- the mass of the above-mentioned deodorized whole fat soybean powder was 500 mass% with respect to the mass of the above-mentioned lipase D.
- the above deodorized whole fat soybean powder was added to the above enzyme solution of Lipase D while the solution was being stirred.
- 1 ml of 0.5 N NaOH solution is added to the obtained mixed solution to adjust the whole mixed solution to pH 7.8, and then nozzle cocurrent spray drying (spray drying, temperature of the above mixed solution of 30 ° C., inlet temperature of air 100) C., outlet temperature 50.degree. C., Tokyo Scientific Instruments Co., Ltd., Model SD-1000) was carried out to obtain a lipase powder preparation.
- Cellulose powder (average particle diameter 30 ⁇ m) was added to the lipase powder preparation as a filter aid to obtain a lipase-containing composition.
- the average particle size was measured using a particle size distribution analyzer (LA-500) manufactured by Horiba, Ltd.
- the mass ratio of the powdered lipase to the filter aid contained in the obtained lipase-containing composition was 1/1.
- ethyl stearate (trade name: ethyl stearate, manufactured by Inoue Perfume Manufacturing Co., Ltd.) and high oleic sunflower oil (manufactured by Nisshin Oillio Group, Inc.) are mixed at a mass ratio of 6: 4, What adjusted so that the water
- the lipase-containing composition was added to the raw material substrate such that the composition containing lipase was 1% by mass, and the reaction was carried out for 16 hours while stirring at 50 ° C.
- the lipase-containing composition was recovered by filtration.
- the recovered lipase-containing composition was used again for the reaction.
- the above reaction procedure was repeated 30 times, and then the filtered and recovered lipase-containing composition (lipase filter cake A) was used in the following examples and comparative examples.
- POS + SOS value The degree of recovery or activity of lipase activity was evaluated by comparing the total mass of POS and SOS in triglyceride. Specifically, the total mass of POS and SOS in the triglyceride relative to the mass of triglyceride obtained after reacting for 1 hour in the transesterification using the untreated (before the moisture absorption step) lipase-containing composition as a comparison control % Relative to the total mass% of POS and SOS in the triglyceride obtained after reaction for 1 hour in the transesterification using the lipase-containing composition sample after treatment (after the moisture absorption step), where 100% is taken as 100 : (POS + SOS value) was determined.
- POS + SOS value (total mass% of POS and SOS in the triglyceride relative to mass of triglyceride after 1 hour of transesterification reaction of lipase-containing composition sample) / (transesterification reaction of comparative lipase-containing composition 1)
- the total mass% of POS and SOS in the triglyceride relative to the mass of triglyceride after time) x 100
- (POS + SOS value) is higher than 100, it can be evaluated that the lipase activity has recovered, and when it is lower than 100, it can be evaluated that the lipase activity has decreased.
- the relative mass (POS + SOS value) of the total mass% of POS and SOS in triglyceride was measured using gas chromatography. Specifically, 7.5 ⁇ l of the reaction solution was taken, diluted with 1.5 ml of hexane, and the filtrate obtained by filtering a solid containing a lipase powder preparation and the like was measured by gas chromatography (GC). As gas chromatography, Agilent Technologies Co., Ltd. product number 6890N and column 65 TG (manufactured by Shimadzu LC Co., Ltd.) were used. The conditions for gas chromatography were column temperature: 350 ° C., temperature rise: 1 ° C./min, and final temperature: 365 ° C.
- the lipase-containing composition was subjected to the Standard Methods for Analysis of Fats, oils and Related Materials, and the 1.4.1-1996 Moisture Moisture (heat-drying method Air oven) of the same test method (hereinafter referred to as a (a %) And 1.5-1996 oil content (hereinafter referred to as b (%)), and from the following formula, the ratio of water content to the amount of oil component contained in the lipase-containing composition Calculated.
- Example 1 10 g of lipase filter cake A (water content 8% by mass with respect to the mass of components excluding the oil component contained in the lipase-containing composition) is spread on a glass petri dish, and a temperature of 60 ° C. and a humidified gas (wet air) atmosphere of 70% relative humidity It kept under for 1 day.
- the water absorption capacity of the lipase filter cake after retention is 10% by mass (the water content is 18% by mass with respect to the mass of components excluding the oil component contained in the lipase-containing composition) with respect to the mass of lipase filter cake A There was no change in the amount of oil component in the lipase-containing composition.
- transesterification was performed once using the obtained lipase filter cake.
- transesterification was performed in the same manner as described above using lipase filter cake A before moisture absorption retention (trial 1-1 to trial 1-5) .
- Comparative Example 1 10 g of lipase filter cake A was spread on a glass petri dish and sprayed uniformly with water using a mister.
- the spray amount of water was 10% by mass (the amount of water relative to the mass of components excluding the oil component contained in the lipase-containing composition: 18% by mass) based on the mass of the lipase filter cake (lipase-containing composition).
- the lipase filter cake was thoroughly mixed to adjust the water, and then allowed to stand for 1 hour. Then, the above-mentioned transesterification was performed once using the obtained lipase filter cake.
- transesterification was carried out in the same manner as described above using lipase filter cake before moisture absorption retention (trial 1-6 to trial 1-10).
- lipase filter cake A before moisture absorption retention use lipase filter cake after moisture absorption retention when the total mass% of POS and SOS in triglyceride obtained after reaction for 1 hour is 100
- Table 2 shows the relative amounts (POS + SOS value) of the total mass% of POS and SOS in triglyceride obtained after reacting for 1 hour in the transesterification reaction.
- Example 2 10 g of lipase filter cake A is spread on a glass petri dish, and each temperature of 30 ° C., 40 ° C., 50 ° C. and 60 ° C .; 50 hours, 70% and 90% relative humidity in wet gas (wet air) atmosphere or 1 hour It was kept for 1 day.
- the water content (% by mass) based on the mass of the components excluding the oil-based component contained in the lipase-containing composition is shown in Table 3. Thereafter, the lipase filter cake was recovered and subjected to the above-described transesterification reaction once.
- ADVANTEC KST-142-JA pressure condition 0.1 MPa
- the amount of the oil component of the aerated filter cake is such that the mass of the oil component contained in the aerated filter cake (lipase-containing composition) is multiple times the mass of the component excluding the oil component in the filter cake It measured on the basis of. Moreover, the water content of this aerated filter cake was measured based on the mass (mass%) of the water with respect to the mass of the component except the oil-based component contained in a hygroscopic filter cake (lipase containing composition).
- the aerated filter cakes adjusted in the amount of the oil component by the above method were each spread on a glass petri dish, and stored at 50 ° C. under an atmosphere of 70% relative humidity (wet air) for 1 day.
- Table 5 shows the relative amounts (POS + SOS value) of the total mass% of POS and SOS in triglycerides obtained after the reaction for 1 hour in the transesterification reaction.
- Table 5 * The amount of the oily component is based on the weight of the component excluding the oily component in the filter cake, the mass of the oily component contained in the filter cake (composition containing lipase) of comparative control and trial 3-1 to 3-6. A value indicating how many times The lower the amount of the oil component in the lipase powder-containing filter cake, the higher the degree of reactivation due to moisture absorption.
- ADVANTEC KST-90 pressure condition 0.1 MPa
- the reacted filter cake was aerated with moist air at 25 ° C. and 50% relative humidity for 10 minutes (trial 4-2), or wet air at 30 ° C. and relative humidity 70% for 10 minutes (try 4-3).
- the amount of the oil component of the absorbed filter cake is determined by multiplying the mass of the oil component contained in the absorbed filter cake (lipase-containing composition) with respect to the mass of the components excluding the oil component in the filter cake It measured on the basis of.
- the moisture content of this moisture absorbed filter cake was measured based on the mass (mass%) of moisture relative to the mass of the components excluding the oil component contained in the moisture absorbed filter cake (lipase-containing composition).
- Table 6 shows the relative amounts (POS + SOS value) of the total mass% of POS and SOS in triglyceride obtained after reacting for 1 hour in the transesterification reaction.
- Table 6 * The amount of the oily component is based on the weight of the component excluding the oily component in the filter cake, the mass of the oily component contained in the filter cake (composition containing lipase) of the comparative control and trials 4-1 to 4-3. A value indicating how many times The effect of moisture absorption by ventilating humidified air was seen in a shorter time than leaving, and the degree was further increased by raising the temperature and humidity.
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Abstract
Provided are: a method for recovering an activity of a lipase, comprising a moisture absorption step of bringing a composition containing the lipase into contact with a wet gas; a method for producing a lipase having a recovered activity, comprising a step of providing a lipase having a reduced activity and a step of recovering the activity of the lipase having a reduced activity by the aforementioned recovering method; and a method for producing an ester synthesis product, characterized by carrying out esterification or transesterification using the above-mentioned lipase having a recovered activity.
Description
本願は、日本国特許出願第2011-179720号に基づく優先権を主張する出願である。日本国特許出願第2011-179720号のすべての内容はここに参考として取り入れる。
本発明は、特定の固定化リパーゼ又は粉末リパーゼを含有する組成物における、各種エステル化能やエステル交換能などのリパーゼ活性を回復させる方法、及び活性を回復させた固定化リパーゼ又は粉体リパーゼを含有する組成物を用いるエステル化反応並びに油脂のエステル交換方法に関するものである。 The present application is an application claiming priority based on Japanese Patent Application No. 2011-179720. The entire contents of Japanese Patent Application No. 2011-179720 are incorporated herein by reference.
The present invention relates to a method for recovering lipase activity such as various esterification ability and transesterification ability in a composition containing a specific immobilized lipase or powdered lipase, and an immobilized lipase or powdered lipase having recovered activity. The present invention relates to an esterification reaction and a method of transesterification of fats and oils using a composition to be contained.
本発明は、特定の固定化リパーゼ又は粉末リパーゼを含有する組成物における、各種エステル化能やエステル交換能などのリパーゼ活性を回復させる方法、及び活性を回復させた固定化リパーゼ又は粉体リパーゼを含有する組成物を用いるエステル化反応並びに油脂のエステル交換方法に関するものである。 The present application is an application claiming priority based on Japanese Patent Application No. 2011-179720. The entire contents of Japanese Patent Application No. 2011-179720 are incorporated herein by reference.
The present invention relates to a method for recovering lipase activity such as various esterification ability and transesterification ability in a composition containing a specific immobilized lipase or powdered lipase, and an immobilized lipase or powdered lipase having recovered activity. The present invention relates to an esterification reaction and a method of transesterification of fats and oils using a composition to be contained.
リパーゼは、脂肪酸などの各種カルボン酸とモノアルコールや多価アルコールなどのアルコール類とのエステル化反応、複数のカルボン酸エステル間のエステル交換反応などに幅広く使用されている。このうち、エステル交換反応は、動植物油脂類の改質をはじめ、各種脂肪酸のエステル、糖エステルやステロイドエステルの製造法として重要な技術である。これらの反応の触媒として、油脂加水分解酵素であるリパーゼを用いると、室温~約90℃程度の温和な条件下でエステル交換反応を行うことができ、従来の化学反応に比べ、副反応の抑制やエネルギーコストが低減化されるだけでなく、触媒としてのリパーゼが天然物であることから安全性も高い。また、その基質特異性や位置特異性により目的物を効率良く生産することができる。
このようなリパーゼとしては、活性の維持、回収の容易性、原料への均一な分散性等を目的として、陰イオン交換樹脂(特許文献1)やフェノール吸着樹脂(特許文献2)等に固定化した固定化リパーゼ、及び、所定の細孔及び粒径を有する粉末リパーゼ(特許文献3)が使用されてきた。
しかし、酵素であるリパーゼは高価であるため、反応終了後回収して繰り返し使用し、リパーゼ活性がかなり低下した場合に初めて廃棄されることが行われてきた。さらに、低下したリパーゼ活性を回復させることも行われてきた。例えば、活性が低下したリパーゼをトリアシルグリセロールで洗浄してリパーゼ活性を回復する方法(特許文献4)、及び、水を添加することによりエステル交換反応におけるリパーゼ活性の向上を行う方法(特許文献5)が提案されている。しかしながら、これまでの回復方法では、活性の回復が不十分であったり、すべてのリパーゼの活性を均一に回復することができなかった。特に、水を添加する場合、直接に水を噴霧させることで、リパーゼと水とを接触させていたが、リパーゼに対して水が不均一に接触するため、リパーゼ活性の回復が不均一となるばかりか、リパーゼの一部に水が過剰に接触すると、当該一部分のリパーゼの活性が低下する現象もみられた。 Lipases are widely used in esterification reactions of various carboxylic acids such as fatty acids and alcohols such as monoalcohols and polyhydric alcohols, and transesterification reactions between multiple carboxylic acid esters. Among these, transesterification is an important technique as a method for producing esters of various fatty acids, sugar esters and steroid esters, including modification of animal and vegetable fats and oils. By using lipase, which is an oil and fat hydrolyzing enzyme, as a catalyst for these reactions, transesterification can be performed under mild conditions of about room temperature to about 90 ° C., and side reactions are suppressed compared to conventional chemical reactions. Not only energy costs are reduced, but also the safety is high because lipase as a catalyst is a natural product. In addition, the target substance can be efficiently produced by its substrate specificity and position specificity.
Such lipases are immobilized on anion exchange resin (patent document 1), phenol adsorption resin (patent document 2), etc. for the purpose of maintaining activity, easiness of recovery, uniform dispersibility in raw materials, etc. Immobilized lipases, and powdered lipases (Patent Document 3) having predetermined pores and particle sizes have been used.
However, since the lipase which is an enzyme is expensive, it has been practiced that it is recovered after completion of the reaction and repeatedly used, and is discarded only when the lipase activity is considerably reduced. In addition, it has also been practiced to restore reduced lipase activity. For example, a method of recovering lipase activity by washing lipase with reduced activity with triacylglycerol (Patent Document 4), and a method of improving lipase activity in transesterification by adding water (Patent Document 5) ) Has been proposed. However, with the recovery method up to now, the recovery of the activity was insufficient or the activity of all the lipases could not be recovered uniformly. In particular, when water is added, the lipase and water are brought into contact by spraying the water directly, but since the water is contacted unevenly with the lipase, recovery of the lipase activity becomes uneven. In addition, there was also a phenomenon that when part of the lipase was in excess of water, the activity of the part of the lipase was reduced.
このようなリパーゼとしては、活性の維持、回収の容易性、原料への均一な分散性等を目的として、陰イオン交換樹脂(特許文献1)やフェノール吸着樹脂(特許文献2)等に固定化した固定化リパーゼ、及び、所定の細孔及び粒径を有する粉末リパーゼ(特許文献3)が使用されてきた。
しかし、酵素であるリパーゼは高価であるため、反応終了後回収して繰り返し使用し、リパーゼ活性がかなり低下した場合に初めて廃棄されることが行われてきた。さらに、低下したリパーゼ活性を回復させることも行われてきた。例えば、活性が低下したリパーゼをトリアシルグリセロールで洗浄してリパーゼ活性を回復する方法(特許文献4)、及び、水を添加することによりエステル交換反応におけるリパーゼ活性の向上を行う方法(特許文献5)が提案されている。しかしながら、これまでの回復方法では、活性の回復が不十分であったり、すべてのリパーゼの活性を均一に回復することができなかった。特に、水を添加する場合、直接に水を噴霧させることで、リパーゼと水とを接触させていたが、リパーゼに対して水が不均一に接触するため、リパーゼ活性の回復が不均一となるばかりか、リパーゼの一部に水が過剰に接触すると、当該一部分のリパーゼの活性が低下する現象もみられた。 Lipases are widely used in esterification reactions of various carboxylic acids such as fatty acids and alcohols such as monoalcohols and polyhydric alcohols, and transesterification reactions between multiple carboxylic acid esters. Among these, transesterification is an important technique as a method for producing esters of various fatty acids, sugar esters and steroid esters, including modification of animal and vegetable fats and oils. By using lipase, which is an oil and fat hydrolyzing enzyme, as a catalyst for these reactions, transesterification can be performed under mild conditions of about room temperature to about 90 ° C., and side reactions are suppressed compared to conventional chemical reactions. Not only energy costs are reduced, but also the safety is high because lipase as a catalyst is a natural product. In addition, the target substance can be efficiently produced by its substrate specificity and position specificity.
Such lipases are immobilized on anion exchange resin (patent document 1), phenol adsorption resin (patent document 2), etc. for the purpose of maintaining activity, easiness of recovery, uniform dispersibility in raw materials, etc. Immobilized lipases, and powdered lipases (Patent Document 3) having predetermined pores and particle sizes have been used.
However, since the lipase which is an enzyme is expensive, it has been practiced that it is recovered after completion of the reaction and repeatedly used, and is discarded only when the lipase activity is considerably reduced. In addition, it has also been practiced to restore reduced lipase activity. For example, a method of recovering lipase activity by washing lipase with reduced activity with triacylglycerol (Patent Document 4), and a method of improving lipase activity in transesterification by adding water (Patent Document 5) ) Has been proposed. However, with the recovery method up to now, the recovery of the activity was insufficient or the activity of all the lipases could not be recovered uniformly. In particular, when water is added, the lipase and water are brought into contact by spraying the water directly, but since the water is contacted unevenly with the lipase, recovery of the lipase activity becomes uneven. In addition, there was also a phenomenon that when part of the lipase was in excess of water, the activity of the part of the lipase was reduced.
本発明は、低下したリパーゼ活性を回復させることができる方法並びに当該活性が回復したリパーゼの製造方法を提供することを目的とする。
本発明は、又、回復したリパーゼ活性を有する固定化リパーゼ又は粉末リパーゼを用いるエステル化方法及びエステル交換方法を提供することを目的とする。 An object of the present invention is to provide a method capable of restoring a reduced lipase activity as well as a method for producing a lipase with the activity restored.
Another object of the present invention is to provide an esterification method and a transesterification method using immobilized lipase or powdered lipase having recovered lipase activity.
本発明は、又、回復したリパーゼ活性を有する固定化リパーゼ又は粉末リパーゼを用いるエステル化方法及びエステル交換方法を提供することを目的とする。 An object of the present invention is to provide a method capable of restoring a reduced lipase activity as well as a method for producing a lipase with the activity restored.
Another object of the present invention is to provide an esterification method and a transesterification method using immobilized lipase or powdered lipase having recovered lipase activity.
本発明者らは、上記課題を達成するため、低下したリパーゼ活性を回復させることができる方法の検討を行った。その結果、リパーゼ含有組成物を一定条件下、湿潤気体に接触することにより、低下したリパーゼの活性を十分に回復することができることを見出し、本発明に至った。即ち、本発明は、以下の態様に関するものであり得る。
[1]リパーゼ含有組成物を湿潤気体に接触する吸湿工程を含む、リパーゼ活性の回復方法に関する。
[2]前記湿潤気体が、相対湿度50~90%である、[1]に記載の方法に関する。
[3]リパーゼ含有組成物中に含まれる水分量が、前記リパーゼ含有組成物中の油性成分を除く成分中8.5~20質量%となるまで前記吸湿工程を行う、[1]又は[2]に記載の方法に関する。
[4]前記吸湿工程が、相対湿度50~90%、温度30~60℃、保持時間0.5~36時間を満たす条件下で行われる、[1]~[3]のいずれかに記載の方法に関する。
[5]前記吸湿工程が、前記湿潤気体を前記リパーゼ含有組成物中に通気させることを特徴とする[1]~[4]のいずれかに記載の方法に関する。
[6]前記吸湿工程の前に、前記リパーゼ含有組成物中に含まれる油性成分の質量を、前記リパーゼ含有組成物中の油性成分を除く成分の質量に対して1.5倍以下に調節する油性成分調節工程を含む、[1]~[5]のいずれかに記載の方法に関する。
[7]前記吸湿工程が、前記湿潤気体を前記リパーゼ含有組成物に通気させることにより、前記リパーゼ含有組成物に含まれる油性成分量を低下させるとともに、前記リパーゼ含有組成物に対して吸湿を行なうことを特徴とする[1]~[5]のいずれかに記載の方法に関する。
[8]前記リパーゼ含有組成物が、粉末リパーゼ及びろ過助剤を含む、[1]~[7]のいずれかに記載の方法に関する。
[9]前記リパーゼが、リゾプス オリザエ(Rhizopus oryzae)由来のリパーゼである、[1]~[8]のいずれかに記載の方法に関する。
[10]前記ろ過助剤が、セルロースである、[8]に記載の方法に関する。
[11]活性が低下したリパーゼを準備する工程、及び
[1]~[10]のいずれかに記載の方法により前記活性が低下したリパーゼの活性を回復する工程、
を含む、活性が回復されたリパーゼの製造方法に関する。
[12][11]に記載の方法により製造された活性が回復されたリパーゼを用いてエステル化又はエステル交換することを特徴とする、エステル合成物の製造方法に関する。 The present inventors examined a method that can restore the reduced lipase activity in order to achieve the above-mentioned task. As a result, it has been found out that contacting the lipase-containing composition with a wet gas under certain conditions can fully restore the reduced lipase activity, leading to the present invention. That is, the present invention may relate to the following aspects.
[1] The present invention relates to a method for recovering lipase activity, comprising a step of absorbing moisture in which a lipase-containing composition is brought into contact with a wet gas.
[2] The method according to [1], wherein the wet gas has a relative humidity of 50 to 90%.
[3] The moisture absorption step is performed until the amount of water contained in the lipase-containing composition is 8.5 to 20% by mass in the components excluding the oil component in the lipase-containing composition, [1] or [2] Related to the method described in
[4] The method according to any one of [1] to [3], wherein the moisture absorption step is performed under the conditions of relative humidity 50 to 90%, temperature 30 to 60 ° C., and holding time 0.5 to 36 hours. On the way.
[5] The method according to any one of [1] to [4], wherein the moisture absorption step aerates the wet gas into the lipase-containing composition.
[6] Before the moisture absorption step, the mass of the oil component contained in the lipase-containing composition is adjusted to 1.5 or less times the mass of the component excluding the oil component in the lipase-containing composition The method according to any one of [1] to [5], which comprises an oil component adjustment step.
[7] The moisture absorption step reduces the amount of the oil component contained in the lipase-containing composition by aerating the moist gas into the lipase-containing composition, and absorbs moisture to the lipase-containing composition The method according to any one of [1] to [5], characterized in that
[8] The method according to any one of [1] to [7], wherein the lipase-containing composition comprises a powdered lipase and a filter aid.
[9] The method according to any one of [1] to [8], wherein the lipase is a lipase derived from Rhizopus oryzae.
[10] The method according to [8], wherein the filter aid is cellulose.
[11] A step of preparing a lipase with reduced activity, and a step of recovering the activity of the lipase whose activity has been reduced by the method described in any of [1] to [10],
The present invention relates to a process for producing a lipase whose activity has been restored.
[12] A method for producing an ester compound, which comprises esterification or transesterification using the lipase with recovered activity produced by the method according to [11].
[1]リパーゼ含有組成物を湿潤気体に接触する吸湿工程を含む、リパーゼ活性の回復方法に関する。
[2]前記湿潤気体が、相対湿度50~90%である、[1]に記載の方法に関する。
[3]リパーゼ含有組成物中に含まれる水分量が、前記リパーゼ含有組成物中の油性成分を除く成分中8.5~20質量%となるまで前記吸湿工程を行う、[1]又は[2]に記載の方法に関する。
[4]前記吸湿工程が、相対湿度50~90%、温度30~60℃、保持時間0.5~36時間を満たす条件下で行われる、[1]~[3]のいずれかに記載の方法に関する。
[5]前記吸湿工程が、前記湿潤気体を前記リパーゼ含有組成物中に通気させることを特徴とする[1]~[4]のいずれかに記載の方法に関する。
[6]前記吸湿工程の前に、前記リパーゼ含有組成物中に含まれる油性成分の質量を、前記リパーゼ含有組成物中の油性成分を除く成分の質量に対して1.5倍以下に調節する油性成分調節工程を含む、[1]~[5]のいずれかに記載の方法に関する。
[7]前記吸湿工程が、前記湿潤気体を前記リパーゼ含有組成物に通気させることにより、前記リパーゼ含有組成物に含まれる油性成分量を低下させるとともに、前記リパーゼ含有組成物に対して吸湿を行なうことを特徴とする[1]~[5]のいずれかに記載の方法に関する。
[8]前記リパーゼ含有組成物が、粉末リパーゼ及びろ過助剤を含む、[1]~[7]のいずれかに記載の方法に関する。
[9]前記リパーゼが、リゾプス オリザエ(Rhizopus oryzae)由来のリパーゼである、[1]~[8]のいずれかに記載の方法に関する。
[10]前記ろ過助剤が、セルロースである、[8]に記載の方法に関する。
[11]活性が低下したリパーゼを準備する工程、及び
[1]~[10]のいずれかに記載の方法により前記活性が低下したリパーゼの活性を回復する工程、
を含む、活性が回復されたリパーゼの製造方法に関する。
[12][11]に記載の方法により製造された活性が回復されたリパーゼを用いてエステル化又はエステル交換することを特徴とする、エステル合成物の製造方法に関する。 The present inventors examined a method that can restore the reduced lipase activity in order to achieve the above-mentioned task. As a result, it has been found out that contacting the lipase-containing composition with a wet gas under certain conditions can fully restore the reduced lipase activity, leading to the present invention. That is, the present invention may relate to the following aspects.
[1] The present invention relates to a method for recovering lipase activity, comprising a step of absorbing moisture in which a lipase-containing composition is brought into contact with a wet gas.
[2] The method according to [1], wherein the wet gas has a relative humidity of 50 to 90%.
[3] The moisture absorption step is performed until the amount of water contained in the lipase-containing composition is 8.5 to 20% by mass in the components excluding the oil component in the lipase-containing composition, [1] or [2] Related to the method described in
[4] The method according to any one of [1] to [3], wherein the moisture absorption step is performed under the conditions of relative humidity 50 to 90%, temperature 30 to 60 ° C., and holding time 0.5 to 36 hours. On the way.
[5] The method according to any one of [1] to [4], wherein the moisture absorption step aerates the wet gas into the lipase-containing composition.
[6] Before the moisture absorption step, the mass of the oil component contained in the lipase-containing composition is adjusted to 1.5 or less times the mass of the component excluding the oil component in the lipase-containing composition The method according to any one of [1] to [5], which comprises an oil component adjustment step.
[7] The moisture absorption step reduces the amount of the oil component contained in the lipase-containing composition by aerating the moist gas into the lipase-containing composition, and absorbs moisture to the lipase-containing composition The method according to any one of [1] to [5], characterized in that
[8] The method according to any one of [1] to [7], wherein the lipase-containing composition comprises a powdered lipase and a filter aid.
[9] The method according to any one of [1] to [8], wherein the lipase is a lipase derived from Rhizopus oryzae.
[10] The method according to [8], wherein the filter aid is cellulose.
[11] A step of preparing a lipase with reduced activity, and a step of recovering the activity of the lipase whose activity has been reduced by the method described in any of [1] to [10],
The present invention relates to a process for producing a lipase whose activity has been restored.
[12] A method for producing an ester compound, which comprises esterification or transesterification using the lipase with recovered activity produced by the method according to [11].
本発明によれば、エステル化方法及びエステル交換方法で繰り返し使用された結果、活性が低下したリパーゼの活性を優位に回復する方法を提供することができる。
特に、本発明によれば、これまでのような、液体の水をリパーゼに直接噴霧する場合と比べ、噴霧ムラがなく、確実にリパーゼの活性を向上することができる。
また、本発明によれば、活性が回復されたリパーゼの製造方法を提供することができる。
さらに、本発明によれば、活性が回復された上記リパーゼを用いてエステル化反応又はエステル交換反応を行うことによりエステル合成物を製造する方法を提供することができる。 According to the present invention, as a result of being repeatedly used in the esterification method and the transesterification method, it is possible to provide a method of predominantly recovering the activity of the lipase whose activity has been reduced.
In particular, according to the present invention, as compared to the case where liquid water is directly sprayed onto the lipase as in the past, there is no spray unevenness, and the activity of the lipase can be surely improved.
Moreover, according to the present invention, it is possible to provide a method for producing a lipase whose activity has been restored.
Furthermore, according to the present invention, it is possible to provide a method for producing an ester compound by carrying out an esterification reaction or a transesterification reaction using the above-mentioned lipase whose activity has been restored.
特に、本発明によれば、これまでのような、液体の水をリパーゼに直接噴霧する場合と比べ、噴霧ムラがなく、確実にリパーゼの活性を向上することができる。
また、本発明によれば、活性が回復されたリパーゼの製造方法を提供することができる。
さらに、本発明によれば、活性が回復された上記リパーゼを用いてエステル化反応又はエステル交換反応を行うことによりエステル合成物を製造する方法を提供することができる。 According to the present invention, as a result of being repeatedly used in the esterification method and the transesterification method, it is possible to provide a method of predominantly recovering the activity of the lipase whose activity has been reduced.
In particular, according to the present invention, as compared to the case where liquid water is directly sprayed onto the lipase as in the past, there is no spray unevenness, and the activity of the lipase can be surely improved.
Moreover, according to the present invention, it is possible to provide a method for producing a lipase whose activity has been restored.
Furthermore, according to the present invention, it is possible to provide a method for producing an ester compound by carrying out an esterification reaction or a transesterification reaction using the above-mentioned lipase whose activity has been restored.
本発明の一つの態様は、リパーゼ含有組成物を湿潤気体に接触する吸湿工程を含む、リパーゼ活性の回復方法である。この方法について、以下詳細に説明する。
<吸湿工程>
本発明は、エステル化方法及びエステル交換方法等で繰り返し使用された結果、活性が低下したリパーゼの活性を回復するための、吸湿工程を含む。吸湿工程は、リパーゼ含有組成物を湿潤気体中に保持等して、リパーゼ含有組成物を湿潤気体に接触することによって行われる。
湿潤気体は、通常の湿度を有する大気と言うよりはむしろ相対湿度若しくは水蒸気量を強制的にコントロールした気体を意図したものである。湿潤気体に用いる気体としては、例えば、空気、窒素、炭酸ガス、アルゴン等が好ましく、中でも空気もしくは窒素が好ましく、特に空気が好ましい。これらの気体でも、相対湿度が、例えば、50~90%、好ましくは60~80%、より好ましくは60~70%の気体が湿潤気体として好適に用いることができる。相対湿度が不足する場合は、吸湿工程の途中で水蒸気等を系内に導入して相対湿度を補ってもよい。
吸湿工程は、リパーゼ含有組成物中の油性成分を除く成分に含まれる水分量が、例えば、8.5~20質量%、好ましくは10~18.5質量%、より好ましくは、12~18質量%、さらに好ましくは13~18質量%となるまで行われることが適当である。ここで、油性成分とは、油脂等のリパーゼ含有組成物中に含まれる水性成分以外の油溶解性成分全般を意味し、後述の基準油脂分析試験法の油分から測定することができる。本発明のリパーゼ含有組成物に含まれる油性成分としては、例えば、グリセリド、遊離の脂肪酸、脂肪酸エステル等が挙げられる。
吸湿工程は、例えば、50~90%、好ましくは60~80%、より好ましくは60~70%の相対湿度;例えば、30~60℃、好ましくは40~60℃、より好ましくは50~60℃、及び、例えば、0.5~36時間、好ましくは1~24時間を満たす条件下で行われることが適当である。好ましい吸湿工程の条件の組み合わせは、(a)70~90%の相対湿度、30~60℃に1日、又は(b)50~90%の相対湿度、40~50℃に1日保持した場合のいずれかである。
このような本発明の吸湿工程を経ることにより、リパーゼ含有組成物中のリパーゼの活性は優位に回復もしくは活性の低下を抑えることができる。リパーゼ活性の回復もしくは活性の低下の程度は、エステル化反応又はエステル交換反応の反応途中段階における成分量を比較することで評価できる。当該成分量は、反応によって、大きく変化する成分で比較することができ、例えば、原料もしくは主反応生成物である。例えばOOO(トリオレイン)とパルミチン酸エチル/ステアリン酸エチルのエステル交換反応の場合、POS(1-ステアロイル-2-オレオイル-3-パルミトイルグリセリン又は1-パルミトイル-2-オレオイル-3-ステアロイルグリセリン)及びSOS(1,3-ジステアロイル-2-オレオイルグリセリン)が生成するが、トリグリセリド中のPOSとSOSの合計質量を比較することでリパーゼ活性の回復もしくは活性の低下の程度を求めることができる。具体的には、本発明の場合、吸湿工程前のリパーゼ含有組成物を使用したエステル交換反応において、1時間反応させた後に得られたトリグリセリド中のPOS及びSOSの合計質量%を100とした場合、前記吸湿工程前のリパーゼ含有組成物を本発明の吸湿工程後のリパーゼ含有組成物に変えて同様にエステル交換反応を1時間行った後に得られたトリグリセリド中のPOS及びSOSの合計質量%(相対量:POS+SOS値)を求めることで、リパーゼ活性を比較することができる。吸湿工程後のリパーゼ含有組成物を使用した場合のPOS及びSOSの合計質量%の比(相対量:POS+SOS値)が100以上あるいは100超であることが好ましく、例えば、150以上、好ましくは200以上、より好ましくは200~250であることが好適である。 One aspect of the present invention is a method for recovering lipase activity, comprising a step of absorbing moisture in which the lipase-containing composition is contacted with a wet gas. This method will be described in detail below.
Moisture absorption process
The present invention includes a moisture absorption step for recovering the activity of the lipase whose activity has been reduced as a result of repeated use in the esterification method, transesterification method and the like. The moisture absorption step is carried out by contacting the lipase-containing composition with the moist gas while keeping the lipase-containing composition in the moist gas.
Humid gas is intended to be a gas in which the relative humidity or the amount of water vapor is forcibly controlled rather than the atmosphere having a normal humidity. As the gas used for the wet gas, for example, air, nitrogen, carbon dioxide gas, argon and the like are preferable, among which air or nitrogen is preferable, and air is particularly preferable. Even in these gases, a gas having a relative humidity of, for example, 50 to 90%, preferably 60 to 80%, more preferably 60 to 70% can be suitably used as the wetting gas. If the relative humidity is insufficient, steam or the like may be introduced into the system during the moisture absorption process to compensate for the relative humidity.
In the moisture absorption step, the amount of water contained in the components excluding the oil component in the lipase-containing composition is, for example, 8.5 to 20% by mass, preferably 10 to 18.5% by mass, and more preferably 12 to 18%. It is suitably carried out to a%, more preferably 13 to 18% by mass. Here, the oily component means all oil-soluble components other than the aqueous component contained in the lipase-containing composition such as fat and oil, and can be measured from the oil of the standard fat and oil analysis test method described later. As an oil-based component contained in the lipase containing composition of this invention, a glyceride, a free fatty acid, fatty acid ester etc. are mentioned, for example.
The moisture absorption step has a relative humidity of, for example, 50 to 90%, preferably 60 to 80%, more preferably 60 to 70%; for example, 30 to 60 ° C., preferably 40 to 60 ° C., more preferably 50 to 60 ° C. And, for example, it is suitable to be carried out under the conditions that satisfy 0.5 to 36 hours, preferably 1 to 24 hours. (A) 70 to 90% relative humidity, one day at 30 to 60 ° C., or (b) 50 to 90% relative humidity, one day at 40 to 50 ° C. It is either.
By passing through the moisture absorption step of the present invention, the activity of the lipase in the lipase-containing composition can be recovered or the decrease in activity can be suppressed. The degree of recovery of the lipase activity or the reduction of the activity can be evaluated by comparing the amounts of components in the middle stages of the esterification reaction or transesterification reaction. The amounts of the components can be compared among the components that greatly change depending on the reaction, for example, raw materials or main reaction products. For example, in the case of transesterification of OO (triolein) with ethyl palmitate / ethyl stearate, POS (1-stearoyl-2-oleoyl-3-palmitoylglycerin or 1-palmitoyl-2-oleoyl-3-stearoylglycerin ) And SOS (1,3-distearoyl-2-oleoylglycerin), but comparing the total mass of POS and SOS in triglyceride to determine the degree of recovery or decrease of lipase activity it can. Specifically, in the case of the present invention, in the transesterification using the lipase-containing composition before the moisture absorption step, the total mass% of POS and SOS in the triglyceride obtained after the reaction for 1 hour is 100. The total mass% of POS and SOS in triglyceride obtained after the lipase-containing composition before the moisture absorption step is changed to the lipase-containing composition after the moisture absorption step of the present invention and the same transesterification reaction is performed for 1 hour The lipase activity can be compared by determining the relative amount: POS + SOS value). The ratio (relative amount: POS + SOS value) of the total mass% of POS and SOS when using the lipase-containing composition after the moisture absorption step is preferably 100 or more or 100 or more, for example, 150 or more, preferably 200 or more And more preferably 200 to 250.
<吸湿工程>
本発明は、エステル化方法及びエステル交換方法等で繰り返し使用された結果、活性が低下したリパーゼの活性を回復するための、吸湿工程を含む。吸湿工程は、リパーゼ含有組成物を湿潤気体中に保持等して、リパーゼ含有組成物を湿潤気体に接触することによって行われる。
湿潤気体は、通常の湿度を有する大気と言うよりはむしろ相対湿度若しくは水蒸気量を強制的にコントロールした気体を意図したものである。湿潤気体に用いる気体としては、例えば、空気、窒素、炭酸ガス、アルゴン等が好ましく、中でも空気もしくは窒素が好ましく、特に空気が好ましい。これらの気体でも、相対湿度が、例えば、50~90%、好ましくは60~80%、より好ましくは60~70%の気体が湿潤気体として好適に用いることができる。相対湿度が不足する場合は、吸湿工程の途中で水蒸気等を系内に導入して相対湿度を補ってもよい。
吸湿工程は、リパーゼ含有組成物中の油性成分を除く成分に含まれる水分量が、例えば、8.5~20質量%、好ましくは10~18.5質量%、より好ましくは、12~18質量%、さらに好ましくは13~18質量%となるまで行われることが適当である。ここで、油性成分とは、油脂等のリパーゼ含有組成物中に含まれる水性成分以外の油溶解性成分全般を意味し、後述の基準油脂分析試験法の油分から測定することができる。本発明のリパーゼ含有組成物に含まれる油性成分としては、例えば、グリセリド、遊離の脂肪酸、脂肪酸エステル等が挙げられる。
吸湿工程は、例えば、50~90%、好ましくは60~80%、より好ましくは60~70%の相対湿度;例えば、30~60℃、好ましくは40~60℃、より好ましくは50~60℃、及び、例えば、0.5~36時間、好ましくは1~24時間を満たす条件下で行われることが適当である。好ましい吸湿工程の条件の組み合わせは、(a)70~90%の相対湿度、30~60℃に1日、又は(b)50~90%の相対湿度、40~50℃に1日保持した場合のいずれかである。
このような本発明の吸湿工程を経ることにより、リパーゼ含有組成物中のリパーゼの活性は優位に回復もしくは活性の低下を抑えることができる。リパーゼ活性の回復もしくは活性の低下の程度は、エステル化反応又はエステル交換反応の反応途中段階における成分量を比較することで評価できる。当該成分量は、反応によって、大きく変化する成分で比較することができ、例えば、原料もしくは主反応生成物である。例えばOOO(トリオレイン)とパルミチン酸エチル/ステアリン酸エチルのエステル交換反応の場合、POS(1-ステアロイル-2-オレオイル-3-パルミトイルグリセリン又は1-パルミトイル-2-オレオイル-3-ステアロイルグリセリン)及びSOS(1,3-ジステアロイル-2-オレオイルグリセリン)が生成するが、トリグリセリド中のPOSとSOSの合計質量を比較することでリパーゼ活性の回復もしくは活性の低下の程度を求めることができる。具体的には、本発明の場合、吸湿工程前のリパーゼ含有組成物を使用したエステル交換反応において、1時間反応させた後に得られたトリグリセリド中のPOS及びSOSの合計質量%を100とした場合、前記吸湿工程前のリパーゼ含有組成物を本発明の吸湿工程後のリパーゼ含有組成物に変えて同様にエステル交換反応を1時間行った後に得られたトリグリセリド中のPOS及びSOSの合計質量%(相対量:POS+SOS値)を求めることで、リパーゼ活性を比較することができる。吸湿工程後のリパーゼ含有組成物を使用した場合のPOS及びSOSの合計質量%の比(相対量:POS+SOS値)が100以上あるいは100超であることが好ましく、例えば、150以上、好ましくは200以上、より好ましくは200~250であることが好適である。 One aspect of the present invention is a method for recovering lipase activity, comprising a step of absorbing moisture in which the lipase-containing composition is contacted with a wet gas. This method will be described in detail below.
Moisture absorption process
The present invention includes a moisture absorption step for recovering the activity of the lipase whose activity has been reduced as a result of repeated use in the esterification method, transesterification method and the like. The moisture absorption step is carried out by contacting the lipase-containing composition with the moist gas while keeping the lipase-containing composition in the moist gas.
Humid gas is intended to be a gas in which the relative humidity or the amount of water vapor is forcibly controlled rather than the atmosphere having a normal humidity. As the gas used for the wet gas, for example, air, nitrogen, carbon dioxide gas, argon and the like are preferable, among which air or nitrogen is preferable, and air is particularly preferable. Even in these gases, a gas having a relative humidity of, for example, 50 to 90%, preferably 60 to 80%, more preferably 60 to 70% can be suitably used as the wetting gas. If the relative humidity is insufficient, steam or the like may be introduced into the system during the moisture absorption process to compensate for the relative humidity.
In the moisture absorption step, the amount of water contained in the components excluding the oil component in the lipase-containing composition is, for example, 8.5 to 20% by mass, preferably 10 to 18.5% by mass, and more preferably 12 to 18%. It is suitably carried out to a%, more preferably 13 to 18% by mass. Here, the oily component means all oil-soluble components other than the aqueous component contained in the lipase-containing composition such as fat and oil, and can be measured from the oil of the standard fat and oil analysis test method described later. As an oil-based component contained in the lipase containing composition of this invention, a glyceride, a free fatty acid, fatty acid ester etc. are mentioned, for example.
The moisture absorption step has a relative humidity of, for example, 50 to 90%, preferably 60 to 80%, more preferably 60 to 70%; for example, 30 to 60 ° C., preferably 40 to 60 ° C., more preferably 50 to 60 ° C. And, for example, it is suitable to be carried out under the conditions that satisfy 0.5 to 36 hours, preferably 1 to 24 hours. (A) 70 to 90% relative humidity, one day at 30 to 60 ° C., or (b) 50 to 90% relative humidity, one day at 40 to 50 ° C. It is either.
By passing through the moisture absorption step of the present invention, the activity of the lipase in the lipase-containing composition can be recovered or the decrease in activity can be suppressed. The degree of recovery of the lipase activity or the reduction of the activity can be evaluated by comparing the amounts of components in the middle stages of the esterification reaction or transesterification reaction. The amounts of the components can be compared among the components that greatly change depending on the reaction, for example, raw materials or main reaction products. For example, in the case of transesterification of OO (triolein) with ethyl palmitate / ethyl stearate, POS (1-stearoyl-2-oleoyl-3-palmitoylglycerin or 1-palmitoyl-2-oleoyl-3-stearoylglycerin ) And SOS (1,3-distearoyl-2-oleoylglycerin), but comparing the total mass of POS and SOS in triglyceride to determine the degree of recovery or decrease of lipase activity it can. Specifically, in the case of the present invention, in the transesterification using the lipase-containing composition before the moisture absorption step, the total mass% of POS and SOS in the triglyceride obtained after the reaction for 1 hour is 100. The total mass% of POS and SOS in triglyceride obtained after the lipase-containing composition before the moisture absorption step is changed to the lipase-containing composition after the moisture absorption step of the present invention and the same transesterification reaction is performed for 1 hour The lipase activity can be compared by determining the relative amount: POS + SOS value). The ratio (relative amount: POS + SOS value) of the total mass% of POS and SOS when using the lipase-containing composition after the moisture absorption step is preferably 100 or more or 100 or more, for example, 150 or more, preferably 200 or more And more preferably 200 to 250.
<油性成分調節工程>
上記吸湿工程の前に、リパーゼ含有組成物に含まれる上記油性成分を低減するための油性成分調節工程を含むことが好ましい。吸湿工程に先立ってリパーゼ含有組成物中の油性成分を取り除くことにより、リパーゼの吸湿効果の向上が期待できる。具体的には、吸湿工程前、すなわち湿潤気体に接触する前に、リパーゼ含有組成物中に含まれる油性成分油脂の質量をリパーゼ含有組成物中の油性成分油脂を除く成分の質量に対して1.5倍以下に調節することが好ましい。より好ましくは1倍(即ち、リパーゼ含有組成物中の油性成分油脂の質量とリパーゼ含有組成物中の油性成分油脂を除く成分の質量とが等しい)以下であり、最も好ましくは0.1~0.7倍である。 <Oil component adjustment process>
It is preferable to include an oil component adjustment step for reducing the oil component contained in the lipase-containing composition prior to the moisture absorption step. By removing the oil component in the lipase-containing composition prior to the moisture absorption step, improvement in the moisture absorption effect of the lipase can be expected. Specifically, before the moisture absorption step, that is, before contacting with the wet gas, the mass of the oil component fat and oil contained in the lipase containing composition is 1 based on the mass of the component excluding the oil component fat and oil in the lipase containing composition. It is preferable to adjust to .5 times or less. More preferably, it is 1 times or less (that is, the mass of the oil component in the lipase-containing composition is equal to the mass of the component excluding the oil component in the lipase-containing composition), and most preferably 0.1 to 0. .7 times.
上記吸湿工程の前に、リパーゼ含有組成物に含まれる上記油性成分を低減するための油性成分調節工程を含むことが好ましい。吸湿工程に先立ってリパーゼ含有組成物中の油性成分を取り除くことにより、リパーゼの吸湿効果の向上が期待できる。具体的には、吸湿工程前、すなわち湿潤気体に接触する前に、リパーゼ含有組成物中に含まれる油性成分油脂の質量をリパーゼ含有組成物中の油性成分油脂を除く成分の質量に対して1.5倍以下に調節することが好ましい。より好ましくは1倍(即ち、リパーゼ含有組成物中の油性成分油脂の質量とリパーゼ含有組成物中の油性成分油脂を除く成分の質量とが等しい)以下であり、最も好ましくは0.1~0.7倍である。 <Oil component adjustment process>
It is preferable to include an oil component adjustment step for reducing the oil component contained in the lipase-containing composition prior to the moisture absorption step. By removing the oil component in the lipase-containing composition prior to the moisture absorption step, improvement in the moisture absorption effect of the lipase can be expected. Specifically, before the moisture absorption step, that is, before contacting with the wet gas, the mass of the oil component fat and oil contained in the lipase containing composition is 1 based on the mass of the component excluding the oil component fat and oil in the lipase containing composition. It is preferable to adjust to .5 times or less. More preferably, it is 1 times or less (that is, the mass of the oil component in the lipase-containing composition is equal to the mass of the component excluding the oil component in the lipase-containing composition), and most preferably 0.1 to 0. .7 times.
<リパーゼ>
本発明のリパーゼ含有組成物は、リパーゼ又はリパーゼと助剤を含む。
本発明において使用できるリパーゼとしては、リポプロテインリパーゼ、モノアシルグリセロリパーゼ、ジアシルグリセロリパーゼ、トリアシルグリセロリパーゼ、ガラクトリパーゼ、フォスフォリパーゼ等が挙げられる。これらのうち、トリアシルグリセロリパーゼが好ましい。
これらのリパーゼを産生する微生物としては、細菌、酵母、糸状菌、放線菌等特に限定されるものではないが、アルカリゲネス属(Alcaligenes sp.)、シュードモナス属(Pseudomonas sp.)、アスロバクター属(Arthrobacter sp.)、スタフィロコッカス属(Staphylococcus sp.)、トルロプシス属(Torulopsis sp.)、エスチエリシア属(Escherichia sp.)、マイコトルラ属(Mycotorula sp.)、プロピオニバクテリウム属(Propionibacterum sp.)、クロモバクテリウム属(Chromobacterum sp.)、キサントモナス属(Xanthomonas sp.)、ラクトバチルス属(Lactobacillus sp.)、クロストリデイウム属(Clostridium sp.)、キャンデイダ属(Candida sp.)、ジオトリカム属(Geotrichum sp.)、サッカロマイコプシス属(Sacchromycopsis sp.)、ノカルデイア属(Nocardia sp.)、フザリウム属(Fuzarium sp.)、アスペルギルス属(Aspergillus sp.)、リゾムコール属(Rhizomucor sp.)、ムコール属(Mucor sp.)、サーモマイセス属(Thermomyces sp.)リゾプス属(Rhizopus sp.)、ペニシリウム属(Penicillium sp.)、フィコマイセス属(Phycomyces sp.)、プチニア属(Puccinia sp.)、バチルス属(Bacillus sp.)、ストレプトマイセス属(Streptmyces sp.)、などが挙げられる。
本発明では、これらのうち、アルカリゲネス属、シュードモナス属、リゾムコール属、ムコール属、サーモマイセス属、リゾプス属又はペニシリウム属由来のリパーゼが好ましい。中でも、アルカリゲネス属のAlcaligenes sp.由来のリパーゼ、リゾムコール属のリゾムコール ミーヘイ(Rhizomucor miehei)由来のリパーゼ、サーモマイセス属のサーモマイセス ラヌゲノウス(Thermomyces lanuginosus)由来のリパーゼ、リゾプス属のリゾプス デレマー(Rhizopus delemar)由来のリパーゼ、及びリゾプス属のリゾプス オリザエ(Rhizopus oryzae)由来のリパーゼがより好ましい。リゾプス オリザエ(Rhizopus oryzae)由来のリパーゼが特に好ましい。
本発明のリパーゼ含有組成物で使用するリパーゼとしては、培養し、リパーゼの培地成分等を含有したリパーゼ含有水性液体を乾燥して得られたものでもよいが、これらを含有していないもの、つまり実質的にリパーゼ自体から構成されるものが好ましい。本発明のリパーゼ含有組成物としては、リパーゼの培養後、菌体を除去し、固定化したもの、もしくはさらに粉末化したものがより好ましい。
本発明で用いるリパーゼは、位置特異性を有していても有していなくてもよい。位置特異性を有している場合、1,3-特異性であるのが好ましい。 <Lipase>
The lipase-containing composition of the present invention comprises a lipase or a lipase and an auxiliary.
Examples of lipases that can be used in the present invention include lipoprotein lipase, monoacylglycerolipase, diacylglycerolipase, triacylglycerolipase, galactolipase, phospholipase and the like. Of these, triacylglycerolipase is preferred.
Microorganisms that produce these lipases include, but are not limited to, bacteria, yeast, filamentous fungi, actinomycetes, etc., but they are not particularly limited, but include Alcaligenes sp., Pseudomonas sp., And Arthrobacters. sp., Staphylococcus sp., Torulopsis sp., Escherichia sp., Mycotorula sp., Propionibacterum sp. Bacteria (Chromobacterum sp.), Xanthomonas sp. (Xanthomonas sp.), Lactobacillus (Lacto) bacillus sp.), Clostridium sp., Candida sp., Geotrichum sp., Sacchromycopsis sp., Nocardia sp. , Fusarium sp., Aspergillus sp., Rhizomucor sp., Mucor sp., Thermomyces sp. Rhizopus sp., Penicillium sp. (Penicillium sp.), Phycomyces sp., Puccinia sp. , The genus Bacillus (Bacillus sp.), The genus Streptomyces (Streptmyces sp.), And the like.
In the present invention, among these, lipases derived from Alkaligenes, Pseudomonas, Rhizomucor, Mucor, Thermomyces, Rhizopus or Penicillium are preferable. Among them, Alcaligenes sp. Lipase from Rhizomucor miehei (Rhizomucor miehei), Lipase from Thermomyces lanchogenus (Thermomyces lanuginosus), Lipase from Rhizopus de lemar (Rhizopus delemar), Rhizopus lysops or More preferred is a lipase from Rhizopus oryzae). Particularly preferred are lipases from Rhizopus oryzae.
The lipase used in the lipase-containing composition of the present invention may be one obtained by culturing and drying a lipase-containing aqueous liquid containing a medium component of the lipase, etc. It is preferred that the lipase consists essentially of the lipase itself. As a lipase containing composition of this invention, after culture | cultivation of lipase, what removed the microbial cell and fix | immobilized, or what was further powdered was more preferable.
The lipase used in the present invention may or may not have regiospecificity. When it has positional specificity, it is preferably 1,3-specific.
本発明のリパーゼ含有組成物は、リパーゼ又はリパーゼと助剤を含む。
本発明において使用できるリパーゼとしては、リポプロテインリパーゼ、モノアシルグリセロリパーゼ、ジアシルグリセロリパーゼ、トリアシルグリセロリパーゼ、ガラクトリパーゼ、フォスフォリパーゼ等が挙げられる。これらのうち、トリアシルグリセロリパーゼが好ましい。
これらのリパーゼを産生する微生物としては、細菌、酵母、糸状菌、放線菌等特に限定されるものではないが、アルカリゲネス属(Alcaligenes sp.)、シュードモナス属(Pseudomonas sp.)、アスロバクター属(Arthrobacter sp.)、スタフィロコッカス属(Staphylococcus sp.)、トルロプシス属(Torulopsis sp.)、エスチエリシア属(Escherichia sp.)、マイコトルラ属(Mycotorula sp.)、プロピオニバクテリウム属(Propionibacterum sp.)、クロモバクテリウム属(Chromobacterum sp.)、キサントモナス属(Xanthomonas sp.)、ラクトバチルス属(Lactobacillus sp.)、クロストリデイウム属(Clostridium sp.)、キャンデイダ属(Candida sp.)、ジオトリカム属(Geotrichum sp.)、サッカロマイコプシス属(Sacchromycopsis sp.)、ノカルデイア属(Nocardia sp.)、フザリウム属(Fuzarium sp.)、アスペルギルス属(Aspergillus sp.)、リゾムコール属(Rhizomucor sp.)、ムコール属(Mucor sp.)、サーモマイセス属(Thermomyces sp.)リゾプス属(Rhizopus sp.)、ペニシリウム属(Penicillium sp.)、フィコマイセス属(Phycomyces sp.)、プチニア属(Puccinia sp.)、バチルス属(Bacillus sp.)、ストレプトマイセス属(Streptmyces sp.)、などが挙げられる。
本発明では、これらのうち、アルカリゲネス属、シュードモナス属、リゾムコール属、ムコール属、サーモマイセス属、リゾプス属又はペニシリウム属由来のリパーゼが好ましい。中でも、アルカリゲネス属のAlcaligenes sp.由来のリパーゼ、リゾムコール属のリゾムコール ミーヘイ(Rhizomucor miehei)由来のリパーゼ、サーモマイセス属のサーモマイセス ラヌゲノウス(Thermomyces lanuginosus)由来のリパーゼ、リゾプス属のリゾプス デレマー(Rhizopus delemar)由来のリパーゼ、及びリゾプス属のリゾプス オリザエ(Rhizopus oryzae)由来のリパーゼがより好ましい。リゾプス オリザエ(Rhizopus oryzae)由来のリパーゼが特に好ましい。
本発明のリパーゼ含有組成物で使用するリパーゼとしては、培養し、リパーゼの培地成分等を含有したリパーゼ含有水性液体を乾燥して得られたものでもよいが、これらを含有していないもの、つまり実質的にリパーゼ自体から構成されるものが好ましい。本発明のリパーゼ含有組成物としては、リパーゼの培養後、菌体を除去し、固定化したもの、もしくはさらに粉末化したものがより好ましい。
本発明で用いるリパーゼは、位置特異性を有していても有していなくてもよい。位置特異性を有している場合、1,3-特異性であるのが好ましい。 <Lipase>
The lipase-containing composition of the present invention comprises a lipase or a lipase and an auxiliary.
Examples of lipases that can be used in the present invention include lipoprotein lipase, monoacylglycerolipase, diacylglycerolipase, triacylglycerolipase, galactolipase, phospholipase and the like. Of these, triacylglycerolipase is preferred.
Microorganisms that produce these lipases include, but are not limited to, bacteria, yeast, filamentous fungi, actinomycetes, etc., but they are not particularly limited, but include Alcaligenes sp., Pseudomonas sp., And Arthrobacters. sp., Staphylococcus sp., Torulopsis sp., Escherichia sp., Mycotorula sp., Propionibacterum sp. Bacteria (Chromobacterum sp.), Xanthomonas sp. (Xanthomonas sp.), Lactobacillus (Lacto) bacillus sp.), Clostridium sp., Candida sp., Geotrichum sp., Sacchromycopsis sp., Nocardia sp. , Fusarium sp., Aspergillus sp., Rhizomucor sp., Mucor sp., Thermomyces sp. Rhizopus sp., Penicillium sp. (Penicillium sp.), Phycomyces sp., Puccinia sp. , The genus Bacillus (Bacillus sp.), The genus Streptomyces (Streptmyces sp.), And the like.
In the present invention, among these, lipases derived from Alkaligenes, Pseudomonas, Rhizomucor, Mucor, Thermomyces, Rhizopus or Penicillium are preferable. Among them, Alcaligenes sp. Lipase from Rhizomucor miehei (Rhizomucor miehei), Lipase from Thermomyces lanchogenus (Thermomyces lanuginosus), Lipase from Rhizopus de lemar (Rhizopus delemar), Rhizopus lysops or More preferred is a lipase from Rhizopus oryzae). Particularly preferred are lipases from Rhizopus oryzae.
The lipase used in the lipase-containing composition of the present invention may be one obtained by culturing and drying a lipase-containing aqueous liquid containing a medium component of the lipase, etc. It is preferred that the lipase consists essentially of the lipase itself. As a lipase containing composition of this invention, after culture | cultivation of lipase, what removed the microbial cell and fix | immobilized, or what was further powdered was more preferable.
The lipase used in the present invention may or may not have regiospecificity. When it has positional specificity, it is preferably 1,3-specific.
リパーゼの培養に用いられるリパーゼ培養液としては、例えば、大豆粉、ペプトン、コーン・ステープ・リカー、K2HPO4、(NH4)2SO4、MgSO4・7H2O等含有する水溶液があげられる。これらの濃度としては、大豆粉0.1~20質量%、好ましくは1.0~10質量%、ペプトン0.1~30質量%、好ましくは0.5~10質量%、コーン・ステープ・リカー0.1~30質量%、好ましくは0.5~10質量%、K2HPO4 0.01~20質量%、好ましくは0.1~5質量%である。又、(NH4)2SO4は0.01~20質量%、好ましくは0.05~5質量%、MgSO4・7H2Oは0.01~20質量%、好ましくは0.05~5質量%である。培養条件は、培養温度は10~40℃、好ましくは20~35℃、通気量は0.1~2.0VVM、好ましくは0.1~1.5VVM、攪拌回転数は100~800rpm、好ましくは200~400rpm、pHはNaOHやアンモニアを用いて、3.0~10.0、好ましくは4.0~9.5、より好ましくは7.0~8.0に制御するのがよい。
As a lipase culture solution used for culturing a lipase, for example, an aqueous solution containing soybean powder, peptone, corn starch liquor, K 2 HPO 4 , (NH 4 ) 2 SO 4 , MgSO 4 · 7H 2 O, etc. is given. Be As these concentrations, soy flour 0.1 to 20% by mass, preferably 1.0 to 10% by mass, peptone 0.1 to 30% by mass, preferably 0.5 to 10% by mass, corn starch and liquor The content is 0.1 to 30% by mass, preferably 0.5 to 10% by mass, and K 2 HPO 4 is 0.01 to 20% by mass, preferably 0.1 to 5% by mass. Also, (NH 4 ) 2 SO 4 is 0.01 to 20% by mass, preferably 0.05 to 5% by mass, and MgSO 4 · 7H 2 O is 0.01 to 20% by mass, preferably 0.05 to 5%. It is mass%. The culture conditions are: culture temperature: 10 to 40 ° C., preferably 20 to 35 ° C., aeration amount: 0.1 to 2.0 VVM, preferably 0.1 to 1.5 VVM, stirring rotation speed: 100 to 800 rpm, preferably The pH is controlled to be in the range of 3.0 to 10.0, preferably 4.0 to 9.5, more preferably 7.0 to 8.0, using NaOH or ammonia.
菌体の分離は、遠心分離、膜濾過などで行うのが好ましい。また、塩類や糖等の低分子成分の除去は、UF膜処理により行うことができる。具体的には、UF膜処理を行い、リパーゼを含有する水溶液を1/2量の体積に濃縮後、濃縮液と同量のリン酸バッファーを添加するという操作を1~5回繰り返すことにより、低分子成分を除去したリパーゼ含有水性液体を得ることができる。
遠心分離は200~20,000×g、膜濾過はMF膜、フィルタープレスなどで圧力を3.0kg/m2以下にコントロールするのが好ましい。菌体内酵素の場合は、ホモジナイザー、ワーリングブレンダー、超音波破砕、フレンチプレス、ボールミル等で細胞破砕し、遠心分離、膜濾過などで細胞残さを除去することが好ましい。ホモジナイザーの攪拌回転数は500~30,000rpm、好ましくは1,000~15,000rpm、ワーリングブレンダーの回転数は500~10,000rpm、好ましくは1,000~5,000rpmである。攪拌時間は0.5~10分、好ましくは1~5分がよい。超音波破砕は1~50KHz、好ましくは10~20KHzの条件で行うのが良い。ボールミルは直径0.1~0.5mm程度のガラス製小球を用いるのがよい。
本発明では、リパーゼ含有水性液体としては、固形分として5~30質量%含むものを用いるのが好ましい。 The cells are preferably separated by centrifugation, membrane filtration and the like. In addition, removal of low molecular components such as salts and sugars can be performed by UF membrane treatment. Specifically, the procedure of UF membrane treatment, concentration of the lipase-containing aqueous solution to 1⁄2 volume, and addition of phosphate buffer of the same amount as the concentrate is repeated 1 to 5 times, A lipase-containing aqueous liquid from which low molecular weight components have been removed can be obtained.
It is preferable to control the pressure to 3.0 kg / m 2 or less by centrifugation at 200 to 20,000 × g, and membrane filtration using an MF membrane, a filter press or the like. In the case of intracellular enzymes, it is preferable to disrupt cells with a homogenizer, Waring blender, sonication, French press, ball mill or the like, and remove cell residue by centrifugation, membrane filtration or the like. The stirring rotation number of the homogenizer is 500 to 30,000 rpm, preferably 1,000 to 15,000 rpm, and the rotation number of the Waring blender is 500 to 10,000 rpm, preferably 1,000 to 5,000 rpm. The stirring time may be 0.5 to 10 minutes, preferably 1 to 5 minutes. Ultrasonication may be performed under the conditions of 1 to 50 KHz, preferably 10 to 20 KHz. The ball mill should use small glass beads having a diameter of about 0.1 to 0.5 mm.
In the present invention, as the lipase-containing aqueous liquid, one containing 5 to 30% by mass as solid content is preferably used.
遠心分離は200~20,000×g、膜濾過はMF膜、フィルタープレスなどで圧力を3.0kg/m2以下にコントロールするのが好ましい。菌体内酵素の場合は、ホモジナイザー、ワーリングブレンダー、超音波破砕、フレンチプレス、ボールミル等で細胞破砕し、遠心分離、膜濾過などで細胞残さを除去することが好ましい。ホモジナイザーの攪拌回転数は500~30,000rpm、好ましくは1,000~15,000rpm、ワーリングブレンダーの回転数は500~10,000rpm、好ましくは1,000~5,000rpmである。攪拌時間は0.5~10分、好ましくは1~5分がよい。超音波破砕は1~50KHz、好ましくは10~20KHzの条件で行うのが良い。ボールミルは直径0.1~0.5mm程度のガラス製小球を用いるのがよい。
本発明では、リパーゼ含有水性液体としては、固形分として5~30質量%含むものを用いるのが好ましい。 The cells are preferably separated by centrifugation, membrane filtration and the like. In addition, removal of low molecular components such as salts and sugars can be performed by UF membrane treatment. Specifically, the procedure of UF membrane treatment, concentration of the lipase-containing aqueous solution to 1⁄2 volume, and addition of phosphate buffer of the same amount as the concentrate is repeated 1 to 5 times, A lipase-containing aqueous liquid from which low molecular weight components have been removed can be obtained.
It is preferable to control the pressure to 3.0 kg / m 2 or less by centrifugation at 200 to 20,000 × g, and membrane filtration using an MF membrane, a filter press or the like. In the case of intracellular enzymes, it is preferable to disrupt cells with a homogenizer, Waring blender, sonication, French press, ball mill or the like, and remove cell residue by centrifugation, membrane filtration or the like. The stirring rotation number of the homogenizer is 500 to 30,000 rpm, preferably 1,000 to 15,000 rpm, and the rotation number of the Waring blender is 500 to 10,000 rpm, preferably 1,000 to 5,000 rpm. The stirring time may be 0.5 to 10 minutes, preferably 1 to 5 minutes. Ultrasonication may be performed under the conditions of 1 to 50 KHz, preferably 10 to 20 KHz. The ball mill should use small glass beads having a diameter of about 0.1 to 0.5 mm.
In the present invention, as the lipase-containing aqueous liquid, one containing 5 to 30% by mass as solid content is preferably used.
リパーゼ含有水性液体の乾燥及び粉末化は、例えば、スプレードライ、フリーズドライ(凍結乾燥)、及び溶剤沈殿後乾燥する方法等によって行われる。
スプレードライは、例えば、ノズル向流式、ディスク向流式、ノズル並流式、ディスク並流式等の噴霧乾燥機を用いて行うのがよい。ディスク並流式がより好ましく、アトマイザー回転数は4,000~20,000rpmとすることが好ましい。スプレードライは、リパーゼ含有水性液体の温度を10~60℃、好ましくは20~40℃に調整して行うことが適当である。スプレードライの際の送風(乾燥雰囲気)の温度は、入口温度80~200℃、出口温度30~100℃、好ましくは40℃以上70℃未満、より好ましくは40℃~65℃、さらに好ましくは50℃~60℃とすることが適当である。リパーゼは温度に弱く、低温にすることで酵素活性の低下を抑えられる。
フリーズドライ(凍結乾燥)は、例えば、ラボサイズの少量用凍結乾燥機、棚段式凍結乾燥により行うのが好ましい。さらに、減圧乾燥を行ってもよい。
溶剤沈殿後乾燥する方法としては、例えば、エタノール、アセトン等を使用して溶剤沈殿させた後、減圧乾燥する方法が挙げられる。 Drying and pulverization of the lipase-containing aqueous liquid are performed by, for example, spray drying, freeze drying (lyophilization), and a method of drying after solvent precipitation and the like.
The spray drying may be performed using, for example, a spray dryer such as a nozzle countercurrent type, a disk countercurrent type, a nozzle cocurrent type, and a disk cocurrent type. The disk co-current system is more preferable, and the atomizer rotation speed is preferably 4,000 to 20,000 rpm. Spray drying is suitably performed by adjusting the temperature of the lipase-containing aqueous liquid to 10 to 60 ° C., preferably 20 to 40 ° C. The temperature of blowing (drying atmosphere) at the time of spray drying is an inlet temperature of 80 to 200 ° C., an outlet temperature of 30 to 100 ° C., preferably 40 ° C. to less than 70 ° C., more preferably 40 ° C. to 65 ° C., still more preferably 50 It is appropriate to set the temperature to 60 ° C. Lipase is weak to temperature, and lowering the temperature can reduce the decrease in enzyme activity.
Freeze-drying (lyophilization) is preferably performed, for example, by a laboratory-sized small-scale lyophilizer or tray-type lyophilization. Furthermore, drying under reduced pressure may be performed.
As a method of drying after solvent precipitation, for example, after solvent precipitation using ethanol, acetone or the like, a method of drying under reduced pressure may be mentioned.
スプレードライは、例えば、ノズル向流式、ディスク向流式、ノズル並流式、ディスク並流式等の噴霧乾燥機を用いて行うのがよい。ディスク並流式がより好ましく、アトマイザー回転数は4,000~20,000rpmとすることが好ましい。スプレードライは、リパーゼ含有水性液体の温度を10~60℃、好ましくは20~40℃に調整して行うことが適当である。スプレードライの際の送風(乾燥雰囲気)の温度は、入口温度80~200℃、出口温度30~100℃、好ましくは40℃以上70℃未満、より好ましくは40℃~65℃、さらに好ましくは50℃~60℃とすることが適当である。リパーゼは温度に弱く、低温にすることで酵素活性の低下を抑えられる。
フリーズドライ(凍結乾燥)は、例えば、ラボサイズの少量用凍結乾燥機、棚段式凍結乾燥により行うのが好ましい。さらに、減圧乾燥を行ってもよい。
溶剤沈殿後乾燥する方法としては、例えば、エタノール、アセトン等を使用して溶剤沈殿させた後、減圧乾燥する方法が挙げられる。 Drying and pulverization of the lipase-containing aqueous liquid are performed by, for example, spray drying, freeze drying (lyophilization), and a method of drying after solvent precipitation and the like.
The spray drying may be performed using, for example, a spray dryer such as a nozzle countercurrent type, a disk countercurrent type, a nozzle cocurrent type, and a disk cocurrent type. The disk co-current system is more preferable, and the atomizer rotation speed is preferably 4,000 to 20,000 rpm. Spray drying is suitably performed by adjusting the temperature of the lipase-containing aqueous liquid to 10 to 60 ° C., preferably 20 to 40 ° C. The temperature of blowing (drying atmosphere) at the time of spray drying is an inlet temperature of 80 to 200 ° C., an outlet temperature of 30 to 100 ° C., preferably 40 ° C. to less than 70 ° C., more preferably 40 ° C. to 65 ° C., still more preferably 50 It is appropriate to set the temperature to 60 ° C. Lipase is weak to temperature, and lowering the temperature can reduce the decrease in enzyme activity.
Freeze-drying (lyophilization) is preferably performed, for example, by a laboratory-sized small-scale lyophilizer or tray-type lyophilization. Furthermore, drying under reduced pressure may be performed.
As a method of drying after solvent precipitation, for example, after solvent precipitation using ethanol, acetone or the like, a method of drying under reduced pressure may be mentioned.
スプレードライ等の乾燥・粉末化工程の直前に、NaOHやアンモニアを用いて、リパーゼ含有水性液体のpHを6~8.5に調整するのが好ましい。特にpHを8.0以下に、さらにpHを7.5~8.0の範囲となるように調整するのが好ましい。pH調整は、スプレードライなどの乾燥・粉末化工程の前のいずれかの工程において行ってもよく、乾燥・粉末化工程の直前のpHが上記範囲内となるように、予めリパーゼ含有水性液体のpHを調整しておいてもよい。pH調整には、各種アルカリ剤や酸を用いることができるが、水酸化ナトリウムなどのアルカリ金属水酸化物を用いるのが好ましい。
又、乾燥・粉末化工程前の途中の工程において、リパーゼ含有水性液体を濃縮してもよい。濃縮方法は、特に限定されるものではないが、エバポレーター、フラッシュエバポレーター、UF膜濃縮、MF膜濃縮、無機塩類による塩析、溶剤による沈殿法、イオン交換セルロース等による吸着法、吸水性ゲルによる吸水法等が挙げられる。中でも、UF膜濃縮、エバポレーターが好ましい。UF膜濃縮用モジュールとしては、分画分子量3,000~100,000、好ましくは6,000~50,000の平膜または中空糸膜、材質はポリアクリルニトリル系、ポリスルフォン系などが好ましい。 It is preferable to adjust the pH of the lipase-containing aqueous liquid to 6 to 8.5 using NaOH or ammonia immediately before the drying / powdering step such as spray drying. In particular, the pH is preferably adjusted to 8.0 or less, and more preferably 7.5 to 8.0. The pH adjustment may be performed in any step prior to the drying / powdering step such as spray drying, and the pH of the lipase-containing aqueous liquid is previously adjusted so that the pH immediately before the drying / powdering step falls within the above range. The pH may be adjusted. Although various alkali agents and acids can be used for pH adjustment, it is preferable to use alkali metal hydroxides, such as sodium hydroxide.
In addition, the lipase-containing aqueous liquid may be concentrated in a process before the drying / powdering process. The concentration method is not particularly limited, but the evaporator, flash evaporator, UF membrane concentration, MF membrane concentration, salting out with inorganic salts, precipitation with a solvent, adsorption method with ion exchange cellulose etc., water absorption by a water absorbing gel Law etc. are mentioned. Among them, UF membrane concentration and an evaporator are preferable. As a module for UF membrane concentration, a flat membrane or hollow fiber membrane having a molecular weight cut off of 3,000 to 100,000, preferably 6,000 to 50,000, preferably made of polyacrylonitrile type or polysulfone type.
又、乾燥・粉末化工程前の途中の工程において、リパーゼ含有水性液体を濃縮してもよい。濃縮方法は、特に限定されるものではないが、エバポレーター、フラッシュエバポレーター、UF膜濃縮、MF膜濃縮、無機塩類による塩析、溶剤による沈殿法、イオン交換セルロース等による吸着法、吸水性ゲルによる吸水法等が挙げられる。中でも、UF膜濃縮、エバポレーターが好ましい。UF膜濃縮用モジュールとしては、分画分子量3,000~100,000、好ましくは6,000~50,000の平膜または中空糸膜、材質はポリアクリルニトリル系、ポリスルフォン系などが好ましい。 It is preferable to adjust the pH of the lipase-containing aqueous liquid to 6 to 8.5 using NaOH or ammonia immediately before the drying / powdering step such as spray drying. In particular, the pH is preferably adjusted to 8.0 or less, and more preferably 7.5 to 8.0. The pH adjustment may be performed in any step prior to the drying / powdering step such as spray drying, and the pH of the lipase-containing aqueous liquid is previously adjusted so that the pH immediately before the drying / powdering step falls within the above range. The pH may be adjusted. Although various alkali agents and acids can be used for pH adjustment, it is preferable to use alkali metal hydroxides, such as sodium hydroxide.
In addition, the lipase-containing aqueous liquid may be concentrated in a process before the drying / powdering process. The concentration method is not particularly limited, but the evaporator, flash evaporator, UF membrane concentration, MF membrane concentration, salting out with inorganic salts, precipitation with a solvent, adsorption method with ion exchange cellulose etc., water absorption by a water absorbing gel Law etc. are mentioned. Among them, UF membrane concentration and an evaporator are preferable. As a module for UF membrane concentration, a flat membrane or hollow fiber membrane having a molecular weight cut off of 3,000 to 100,000, preferably 6,000 to 50,000, preferably made of polyacrylonitrile type or polysulfone type.
<リパーゼの形態>
本発明のリパーゼは、固定化リパーゼ又は粉末リパーゼの形態であり得る。
固定化リパーゼは、上記リパーゼをシリカ、セライト、珪藻土、パーライト、ポリビニールアルコール、陰イオン交換樹脂、フェノール吸着樹脂、疎水性担体、陽イオン交換樹脂、キレート樹脂等の担体に固定化したものが好ましい。このような固定化リパーゼは、例えば、ノボザイムズA/S社からリポザイムTL-IMとして入手することができる。
固定化リパーゼは、そのまま使用するか、又は該固定化リパーゼを粉砕したものを使用することができる。固定化リパーゼの粉砕品は、通常の粉砕機を用いて、1μm以上で300μm未満の平均粒子径、好ましくは平均粒子径1~200μm、より好ましくは平均粒子径1~100μm、特に好ましくは平均粒子径20~100μmとなるように粉砕するのがよい。ここで、粉砕機としては、乳鉢、せん断摩擦式粉砕機、カッター式粉砕機、石臼(マイコロイダー、マスコロイダー)、コーヒーミル、パワーミル、ピンミル、衝撃式粉砕機(ハンマーミル、ボールミル)、ロール式粉砕機及び気流式粉砕機、ホモジナイザー、超音波破砕機などがあげられる。なお、平均粒子径は、例えば、株式会社堀場製作所の粒度分布測定装置(LA-500)を用いて測定することができる。 <Form of lipase>
The lipase of the present invention may be in the form of immobilized lipase or powdered lipase.
The immobilized lipase is preferably one in which the above lipase is immobilized on a carrier such as silica, celite, diatomaceous earth, perlite, polyvinyl alcohol, anion exchange resin, phenol adsorption resin, hydrophobic carrier, hydrophobic resin, cation exchange resin, chelate resin, etc. . Such immobilized lipase can be obtained, for example, as Lipozyme TL-IM from Novozymes A / S.
The immobilized lipase can be used as it is, or one obtained by crushing the immobilized lipase can be used. The pulverized product of immobilized lipase is an average particle size of 1 μm or more and less than 300 μm, preferably an average particle size of 1 to 200 μm, more preferably an average particle size of 1 to 100 μm, particularly preferably an average particle size using a usual pulverizer. It is preferable to grind so as to have a diameter of 20 to 100 μm. Here, as a grinding machine, a mortar, a shear friction type grinding machine, a cutter type grinding machine, a stone mill (mycoroider, muscoloider), a coffee mill, a power mill, a pin mill, an impact type grinding machine (hammer mill, ball mill), roll type There are crushers, pneumatic crushers, homogenizers, ultrasonic crushers and the like. The average particle size can be measured, for example, using a particle size distribution measuring apparatus (LA-500) manufactured by Horiba, Ltd.
本発明のリパーゼは、固定化リパーゼ又は粉末リパーゼの形態であり得る。
固定化リパーゼは、上記リパーゼをシリカ、セライト、珪藻土、パーライト、ポリビニールアルコール、陰イオン交換樹脂、フェノール吸着樹脂、疎水性担体、陽イオン交換樹脂、キレート樹脂等の担体に固定化したものが好ましい。このような固定化リパーゼは、例えば、ノボザイムズA/S社からリポザイムTL-IMとして入手することができる。
固定化リパーゼは、そのまま使用するか、又は該固定化リパーゼを粉砕したものを使用することができる。固定化リパーゼの粉砕品は、通常の粉砕機を用いて、1μm以上で300μm未満の平均粒子径、好ましくは平均粒子径1~200μm、より好ましくは平均粒子径1~100μm、特に好ましくは平均粒子径20~100μmとなるように粉砕するのがよい。ここで、粉砕機としては、乳鉢、せん断摩擦式粉砕機、カッター式粉砕機、石臼(マイコロイダー、マスコロイダー)、コーヒーミル、パワーミル、ピンミル、衝撃式粉砕機(ハンマーミル、ボールミル)、ロール式粉砕機及び気流式粉砕機、ホモジナイザー、超音波破砕機などがあげられる。なお、平均粒子径は、例えば、株式会社堀場製作所の粒度分布測定装置(LA-500)を用いて測定することができる。 <Form of lipase>
The lipase of the present invention may be in the form of immobilized lipase or powdered lipase.
The immobilized lipase is preferably one in which the above lipase is immobilized on a carrier such as silica, celite, diatomaceous earth, perlite, polyvinyl alcohol, anion exchange resin, phenol adsorption resin, hydrophobic carrier, hydrophobic resin, cation exchange resin, chelate resin, etc. . Such immobilized lipase can be obtained, for example, as Lipozyme TL-IM from Novozymes A / S.
The immobilized lipase can be used as it is, or one obtained by crushing the immobilized lipase can be used. The pulverized product of immobilized lipase is an average particle size of 1 μm or more and less than 300 μm, preferably an average particle size of 1 to 200 μm, more preferably an average particle size of 1 to 100 μm, particularly preferably an average particle size using a usual pulverizer. It is preferable to grind so as to have a diameter of 20 to 100 μm. Here, as a grinding machine, a mortar, a shear friction type grinding machine, a cutter type grinding machine, a stone mill (mycoroider, muscoloider), a coffee mill, a power mill, a pin mill, an impact type grinding machine (hammer mill, ball mill), roll type There are crushers, pneumatic crushers, homogenizers, ultrasonic crushers and the like. The average particle size can be measured, for example, using a particle size distribution measuring apparatus (LA-500) manufactured by Horiba, Ltd.
粉末リパーゼは、例えば全質量の90質量%以上が平均粒子径1~100μm、好ましくは10~80μm、より好ましくは20~50μmである粉末状のリパーゼである。この粉末の粒子は、好ましくは球形であり、粒子の表面に、例えば直径0.5μm~6μmの細孔を3,000~40,000個/mm2、好ましくは3,000~20,000個/mm2、より好ましくは3,000~10,000個/mm2有するものが適当である。
粒子表面の細孔の数は、電子顕微鏡を用いて容易に測定することができる。
粉末リパーゼは、リパーゼ粉末製剤の形で用いてもよい。
リパーゼ粉末製剤は、例えば、リパーゼと、穀物粉末及び/又は糖類粉末とを水性液体に溶解及び/又は分散させて得たリパーゼ含有水性液体を、さらに乾燥して粉末化することによって得られる。
水性液体としては、水が好ましい。リパーゼ含有水性液体中の水の量は、リパーゼの質量に対する水の質量が、2.0~1,000倍であるのが好ましく、2.0~500倍であるのがより好ましく、3.0~100倍が最も好ましい。
リパーゼ含有水性液体としては、菌体を除去したリパーゼ培養液、精製培養液、これらから得たリパーゼ粉末を再度水に溶解・分散させたもの、市販のリパーゼ粉末を再度水に溶解・分散させたもの、市販の液状リパーゼ等が挙げられる。さらに、リパーゼ活性をより高めるために塩類等の低分子成分を除去したものがより好ましく、また、粉末性状をより高めるために糖等の低分子成分を除去したものがより好ましい。
リパーゼとしては、上記列挙したリパーゼの中でも、特にディー・エス・エムジャパン株式会社の商品:ピカンターゼR8000や、天野エンザイム株式会社の商品:リパーゼF-AP15等が好適に用いられる。最も適した粉末リパーゼとしては、Rhizopus oryzae由来の、天野エンザイム株式会社の商品:リパーゼDF“Amano”15-K(リパーゼDともいう)やリパーゼD “アマノ”コンクが挙げられる。なお、このリパーゼDについては、従来はRhizopus delemar由来の表記であった。
穀物粉末及び/又は糖類粉末としては、例えば、全脂大豆粉末、脱脂大豆粉末等の大豆粉末、小麦粉、米粉、デキストリンが挙げられる。穀物粉末及び/又は糖類粉末は、リパーゼに対して、例えば250~1000質量%、好ましくは、500~1000質量%、より好ましくは、500~750質量%であることが適当である。 The powdered lipase is, for example, a powdered lipase in which 90% by mass or more of the total mass has an average particle diameter of 1 to 100 μm, preferably 10 to 80 μm, more preferably 20 to 50 μm. The particles of this powder are preferably spherical and, for example, 3,000 to 40,000 particles / mm 2 , preferably 3,000 to 20,000 pores with a diameter of 0.5 μm to 6 μm are formed on the surface of the particles. It is suitable to have / mm 2 , more preferably 3,000 to 10,000 / mm 2 .
The number of pores on the particle surface can be easily measured using an electron microscope.
Powdered lipase may be used in the form of a lipase powder formulation.
The lipase powder preparation can be obtained, for example, by further drying and pulverizing the lipase-containing aqueous liquid obtained by dissolving and / or dispersing the lipase and the cereal powder and / or the saccharide powder in the aqueous liquid.
Water is preferred as the aqueous liquid. The mass of water in the lipase-containing aqueous liquid is preferably 2.0 to 1,000 times, more preferably 2.0 to 500 times, the mass of water to the mass of the lipase, 3.0 It is most preferable to be 100 times.
As a lipase-containing aqueous liquid, a lipase culture solution from which cells were removed, a purified culture solution, a solution obtained by dissolving and dispersing lipase powder obtained from these in water again, and a commercially available lipase powder were dissolved and dispersed in water again And commercially available liquid lipases and the like. Further, in order to further enhance the lipase activity, those from which low molecular components such as salts have been removed are more preferable, and those from which low molecular components such as sugars have been removed in order to improve powder properties are more preferable.
Among the lipases listed above, among the lipases listed above, the product of PICM Japan Co., Ltd .: Picantase R 8000, and the product of Amano Enzyme Co., Ltd .: Lipase F-AP15, etc. are preferably used. The most suitable powdered lipases include products of Amano Enzyme Co., Ltd. derived from Rhizopus oryzae: Lipase DF “Amano” 15-K (also referred to as Lipase D) and Lipase D “Amano” conc. In addition, about this lipase D, it was a description derived from Rhizopus delemar conventionally.
Examples of cereal powder and / or saccharide powder include soybean powder such as whole fat soybean powder and defatted soybean powder, wheat flour, rice flour and dextrin. The grain powder and / or the sugar powder is suitably, for example, 250 to 1000% by mass, preferably 500 to 1000% by mass, more preferably 500 to 750% by mass, with respect to the lipase.
粒子表面の細孔の数は、電子顕微鏡を用いて容易に測定することができる。
粉末リパーゼは、リパーゼ粉末製剤の形で用いてもよい。
リパーゼ粉末製剤は、例えば、リパーゼと、穀物粉末及び/又は糖類粉末とを水性液体に溶解及び/又は分散させて得たリパーゼ含有水性液体を、さらに乾燥して粉末化することによって得られる。
水性液体としては、水が好ましい。リパーゼ含有水性液体中の水の量は、リパーゼの質量に対する水の質量が、2.0~1,000倍であるのが好ましく、2.0~500倍であるのがより好ましく、3.0~100倍が最も好ましい。
リパーゼ含有水性液体としては、菌体を除去したリパーゼ培養液、精製培養液、これらから得たリパーゼ粉末を再度水に溶解・分散させたもの、市販のリパーゼ粉末を再度水に溶解・分散させたもの、市販の液状リパーゼ等が挙げられる。さらに、リパーゼ活性をより高めるために塩類等の低分子成分を除去したものがより好ましく、また、粉末性状をより高めるために糖等の低分子成分を除去したものがより好ましい。
リパーゼとしては、上記列挙したリパーゼの中でも、特にディー・エス・エムジャパン株式会社の商品:ピカンターゼR8000や、天野エンザイム株式会社の商品:リパーゼF-AP15等が好適に用いられる。最も適した粉末リパーゼとしては、Rhizopus oryzae由来の、天野エンザイム株式会社の商品:リパーゼDF“Amano”15-K(リパーゼDともいう)やリパーゼD “アマノ”コンクが挙げられる。なお、このリパーゼDについては、従来はRhizopus delemar由来の表記であった。
穀物粉末及び/又は糖類粉末としては、例えば、全脂大豆粉末、脱脂大豆粉末等の大豆粉末、小麦粉、米粉、デキストリンが挙げられる。穀物粉末及び/又は糖類粉末は、リパーゼに対して、例えば250~1000質量%、好ましくは、500~1000質量%、より好ましくは、500~750質量%であることが適当である。 The powdered lipase is, for example, a powdered lipase in which 90% by mass or more of the total mass has an average particle diameter of 1 to 100 μm, preferably 10 to 80 μm, more preferably 20 to 50 μm. The particles of this powder are preferably spherical and, for example, 3,000 to 40,000 particles / mm 2 , preferably 3,000 to 20,000 pores with a diameter of 0.5 μm to 6 μm are formed on the surface of the particles. It is suitable to have / mm 2 , more preferably 3,000 to 10,000 / mm 2 .
The number of pores on the particle surface can be easily measured using an electron microscope.
Powdered lipase may be used in the form of a lipase powder formulation.
The lipase powder preparation can be obtained, for example, by further drying and pulverizing the lipase-containing aqueous liquid obtained by dissolving and / or dispersing the lipase and the cereal powder and / or the saccharide powder in the aqueous liquid.
Water is preferred as the aqueous liquid. The mass of water in the lipase-containing aqueous liquid is preferably 2.0 to 1,000 times, more preferably 2.0 to 500 times, the mass of water to the mass of the lipase, 3.0 It is most preferable to be 100 times.
As a lipase-containing aqueous liquid, a lipase culture solution from which cells were removed, a purified culture solution, a solution obtained by dissolving and dispersing lipase powder obtained from these in water again, and a commercially available lipase powder were dissolved and dispersed in water again And commercially available liquid lipases and the like. Further, in order to further enhance the lipase activity, those from which low molecular components such as salts have been removed are more preferable, and those from which low molecular components such as sugars have been removed in order to improve powder properties are more preferable.
Among the lipases listed above, among the lipases listed above, the product of PICM Japan Co., Ltd .: Picantase R 8000, and the product of Amano Enzyme Co., Ltd .: Lipase F-AP15, etc. are preferably used. The most suitable powdered lipases include products of Amano Enzyme Co., Ltd. derived from Rhizopus oryzae: Lipase DF “Amano” 15-K (also referred to as Lipase D) and Lipase D “Amano” conc. In addition, about this lipase D, it was a description derived from Rhizopus delemar conventionally.
Examples of cereal powder and / or saccharide powder include soybean powder such as whole fat soybean powder and defatted soybean powder, wheat flour, rice flour and dextrin. The grain powder and / or the sugar powder is suitably, for example, 250 to 1000% by mass, preferably 500 to 1000% by mass, more preferably 500 to 750% by mass, with respect to the lipase.
<助剤>
本発明で使用し得る助剤としては、リパーゼによるエステル化反応若しくはエステル交換反応に悪影響を与えないものを用いることができる。特に、ろ過助剤が好ましい。ろ過助剤としては、例えば、セライトなどの無機ろ過助剤及びセルロースなどの繊維やその粉砕物などの有機ろ過助剤が挙げられる。ろ過助剤としては、有機ろ過助剤、特に有機高分子ろ過助剤が好ましく、なかでもセルロースパウダーなどが好ましい。ろ過助剤は、粉状であるのが好ましい。粉状のろ過助剤の平均粒子径は、例えば、10~90μm、より好ましくは20~80μmであることが適当である。
上記固定化リパーゼ又は粉末リパーゼとろ過助剤との質量比は、例えば、1/10~10/1であることが好ましく、特に1/7~2/1であることが好ましい。 <Auxiliaries>
As auxiliary agents that can be used in the present invention, those that do not adversely affect the esterification reaction or transesterification reaction with lipase can be used. In particular, filter aids are preferred. Examples of the filter aid include inorganic filter aids such as celite, fibers such as cellulose, and organic filter aids such as ground products thereof. As the filter aid, an organic filter aid, particularly an organic polymer filter aid, is preferable, and in particular, cellulose powder is preferable. The filter aid is preferably in powder form. The average particle size of the powdery filter aid is, for example, 10 to 90 μm, more preferably 20 to 80 μm.
The mass ratio of the immobilized lipase or powdered lipase to the filter aid is, for example, preferably 1/10 to 10/1, and particularly preferably 1/7 to 2/1.
本発明で使用し得る助剤としては、リパーゼによるエステル化反応若しくはエステル交換反応に悪影響を与えないものを用いることができる。特に、ろ過助剤が好ましい。ろ過助剤としては、例えば、セライトなどの無機ろ過助剤及びセルロースなどの繊維やその粉砕物などの有機ろ過助剤が挙げられる。ろ過助剤としては、有機ろ過助剤、特に有機高分子ろ過助剤が好ましく、なかでもセルロースパウダーなどが好ましい。ろ過助剤は、粉状であるのが好ましい。粉状のろ過助剤の平均粒子径は、例えば、10~90μm、より好ましくは20~80μmであることが適当である。
上記固定化リパーゼ又は粉末リパーゼとろ過助剤との質量比は、例えば、1/10~10/1であることが好ましく、特に1/7~2/1であることが好ましい。 <Auxiliaries>
As auxiliary agents that can be used in the present invention, those that do not adversely affect the esterification reaction or transesterification reaction with lipase can be used. In particular, filter aids are preferred. Examples of the filter aid include inorganic filter aids such as celite, fibers such as cellulose, and organic filter aids such as ground products thereof. As the filter aid, an organic filter aid, particularly an organic polymer filter aid, is preferable, and in particular, cellulose powder is preferable. The filter aid is preferably in powder form. The average particle size of the powdery filter aid is, for example, 10 to 90 μm, more preferably 20 to 80 μm.
The mass ratio of the immobilized lipase or powdered lipase to the filter aid is, for example, preferably 1/10 to 10/1, and particularly preferably 1/7 to 2/1.
<活性が回復されたリパーゼの製造方法>
本発明の他の一つの態様は、活性が回復されたリパーゼの製造方法を提供することにある。
<活性が低下したリパーゼの準備>
まず、活性が低下したリパーゼを準備する。活性が低下したリパーゼは、エステル化反応やエステル交換反応を繰り返し行うことによって得られる。例えば、エステル交換反応の場合、原料基質に対してリパーゼ含有組成物の質量が0.05~10質量%、好ましくは、0.1~5質量%、より好ましくは1~2質量%となるようにリパーゼ含有組成物を加え、20~80℃で撹拌しながら10~50時間エステル交換反応を行う。この反応を10~50回、好ましくは20~50回、より好ましくは、30~50回繰り返すことによって、活性が低下したリパーゼを得ることができる。また、1~10回、好ましくは1~5回程度のエステル化反応又はエステル交換反応と、本発明のリパーゼ活性の回復方法とを交互に行って、リパーゼの良好な活性を継続的に維持してもよい。
<吸湿工程>
当該活性が低下したリパーゼは、上述したリパーゼ含有組成物を湿潤気体中に接触する吸湿工程を経ることによって活性を回復し、活性が優位に回復したリパーゼを製造することができる。具体的には、リパーゼの製造は、例えば、活性が低下したリパーゼをトレイなどの容器に静置し、上記のごとく湿潤気体雰囲気に曝すことによって行われる。このとき、容器に置かれた活性が低下したリパーゼの厚さは、例えば、厚さ5cm以下、好ましくは、3cm以下、より好ましくは0.1~1cmであることが適当である。
また、湿潤気体をリパーゼ含有組成物中に通気させることが、効率的に活性を回復できるので好ましい。例えば、カラムやろ過器にリパーゼ含有組成物を保持し、湿潤気体をリパーゼ含有組成物中に強制的に通気させることが好ましい。この強制的通気に使用する湿潤気体は、上述した吸湿工程で使用する湿潤気体と同じ気体を使用することができる。湿潤気体の強制的通気は、例えば、10分以下、好ましくは7分以下、より好ましくは1分以上5分以下とすることが適当である。
<油性成分調節工程>
リパーゼの吸湿効果は、リパーゼ含有組成物中の油性成分量に影響を受けるため、リパーゼ含有組成物中に含まれる油性成分量を低減することが好ましい。その場合、吸湿工程前、すなわち湿潤気体に接触する前に、リパーゼ含有組成物中に含まれる油性成分の質量をリパーゼ含有組成物中の油性成分を除く成分の質量に対して1.5倍以下にすることが好ましい。より好ましくは1倍(即ち、リパーゼ含有組成物中の油性成分の質量とリパーゼ含有組成物中の油性成分を除く成分の質量とが等しい)以下であり、最も好ましくは0.1~0.7倍である。リパーゼ含有組成物中の油性成分量を低減させるためには、カラムやろ過器中で気体をリパーゼ含有組成物に通気させてリパーゼ含有組成物中の油性成分量を低下させた後、上述した湿潤気体をリパーゼ含有組成物中に通気させてもよい。油性成分量の低減のための通気に使用する気体は、空気、窒素、炭酸ガス、アルゴン等を使用することができるが、特に乾燥窒素ガスが好ましい。通気は、例えば、10分以下、好ましくは7分以下、より好ましくは5分以下とすることが適当である。
さらに、湿潤気体をリパーゼ含有組成物に通気させることにより、リパーゼ含有組成物に含まれる油性成分量を低下させるとともに、リパーゼ含有組成物に対して吸湿を行ってもよい。この通気に使用する湿潤気体は、上述した吸湿工程で使用する湿潤気体と同じ気体を使用することができる。湿潤気体の通気は、例えば、10分以下、好ましくは7分以下、より好ましくは1分以上5分以下とすることが適当である。なお、リパーゼ含有組成物中に含まれる油性成分の質量は、リパーゼ含有組成物中の油性成分を除く成分の質量に対して1.5倍以下になるまで低下させることが好ましい。より好ましくは1倍(即ち、リパーゼ含有組成物中の油性成分の質量とリパーゼ含有組成物中の油性成分を除く成分の質量とが等しい)以下であり、最も好ましくは0.1~0.7倍である。 <Method for producing lipase whose activity has been restored>
Another aspect of the present invention is to provide a method for producing a lipase whose activity has been restored.
<Preparation of lipase with reduced activity>
First, prepare a lipase with reduced activity. The lipase with reduced activity can be obtained by repeatedly performing esterification reaction and transesterification reaction. For example, in the case of transesterification, the mass of the lipase-containing composition is 0.05 to 10% by mass, preferably 0.1 to 5% by mass, more preferably 1 to 2% by mass, based on the starting substrate. The lipase-containing composition is added to the mixture and transesterification is performed for 10 to 50 hours while stirring at 20 to 80.degree. By repeating this reaction 10 to 50 times, preferably 20 to 50 times, more preferably 30 to 50 times, a lipase with reduced activity can be obtained. In addition, the good activity of the lipase is continuously maintained by alternately carrying out the esterification reaction or transesterification reaction of 1 to 10 times, preferably about 1 to 5 times, and the method for recovering lipase activity of the present invention. May be
Moisture absorption process
The lipase whose activity has been reduced recovers its activity by contacting the above-mentioned lipase-containing composition in a wet gas, and the activity can be recovered to produce a lipase whose activity has been restored. Specifically, production of lipase is carried out, for example, by placing the lipase with reduced activity in a container such as a tray and exposing it to a wet gas atmosphere as described above. At this time, the thickness of the lipase whose activity has been reduced in the container is, for example, 5 cm or less in thickness, preferably 3 cm or less, more preferably 0.1 to 1 cm.
In addition, it is preferable to aerate the moist gas into the lipase-containing composition because the activity can be recovered efficiently. For example, it is preferable to hold the lipase-containing composition in a column or a filter and to force a wet gas into the lipase-containing composition. The moist gas used for this forced aeration can use the same gas as the moist gas used in the above-mentioned moisture absorption process. The forced aeration of the moist gas is suitably, for example, 10 minutes or less, preferably 7 minutes or less, more preferably 1 minute or more and 5 minutes or less.
<Oil component adjustment process>
Since the hygroscopic effect of the lipase is affected by the amount of the oil component in the lipase-containing composition, it is preferable to reduce the amount of the oil component contained in the lipase-containing composition. In that case, the mass of the oily component contained in the lipase-containing composition is not more than 1.5 times the mass of the component excluding the oily component in the lipase-containing composition before the moisture absorption step, ie, before contacting with the wet gas. It is preferable to More preferably, it is 1 times or less (ie, the mass of the oil component in the lipase-containing composition and the mass of the components excluding the oil component in the lipase-containing composition are equal) or less, most preferably 0.1 to 0.7. It is a double. In order to reduce the amount of the oily component in the lipase-containing composition, the gas is passed through the lipase-containing composition in a column or a filter to reduce the amount of the oily component in the lipase-containing composition, and then the aforementioned wetting is performed. A gas may be bubbled into the lipase containing composition. Air, nitrogen, carbon dioxide gas, argon or the like can be used as the gas used for aeration for reducing the amount of the oily component, and dry nitrogen gas is particularly preferable. The aeration is suitably, for example, 10 minutes or less, preferably 7 minutes or less, more preferably 5 minutes or less.
Furthermore, by allowing the lipase-containing composition to aerate the moist gas, the amount of the oil component contained in the lipase-containing composition may be reduced, and moisture absorption may be performed on the lipase-containing composition. The moist gas used for this aeration can use the same gas as the moist gas used by the moisture absorption process mentioned above. The aeration of the wet gas is suitably, for example, 10 minutes or less, preferably 7 minutes or less, more preferably 1 minute to 5 minutes. The mass of the oil component contained in the lipase-containing composition is preferably reduced to 1.5 times or less the mass of the component excluding the oil component in the lipase-containing composition. More preferably, it is 1 times or less (ie, the mass of the oil component in the lipase-containing composition and the mass of the components excluding the oil component in the lipase-containing composition are equal) or less, most preferably 0.1 to 0.7. It is a double.
本発明の他の一つの態様は、活性が回復されたリパーゼの製造方法を提供することにある。
<活性が低下したリパーゼの準備>
まず、活性が低下したリパーゼを準備する。活性が低下したリパーゼは、エステル化反応やエステル交換反応を繰り返し行うことによって得られる。例えば、エステル交換反応の場合、原料基質に対してリパーゼ含有組成物の質量が0.05~10質量%、好ましくは、0.1~5質量%、より好ましくは1~2質量%となるようにリパーゼ含有組成物を加え、20~80℃で撹拌しながら10~50時間エステル交換反応を行う。この反応を10~50回、好ましくは20~50回、より好ましくは、30~50回繰り返すことによって、活性が低下したリパーゼを得ることができる。また、1~10回、好ましくは1~5回程度のエステル化反応又はエステル交換反応と、本発明のリパーゼ活性の回復方法とを交互に行って、リパーゼの良好な活性を継続的に維持してもよい。
<吸湿工程>
当該活性が低下したリパーゼは、上述したリパーゼ含有組成物を湿潤気体中に接触する吸湿工程を経ることによって活性を回復し、活性が優位に回復したリパーゼを製造することができる。具体的には、リパーゼの製造は、例えば、活性が低下したリパーゼをトレイなどの容器に静置し、上記のごとく湿潤気体雰囲気に曝すことによって行われる。このとき、容器に置かれた活性が低下したリパーゼの厚さは、例えば、厚さ5cm以下、好ましくは、3cm以下、より好ましくは0.1~1cmであることが適当である。
また、湿潤気体をリパーゼ含有組成物中に通気させることが、効率的に活性を回復できるので好ましい。例えば、カラムやろ過器にリパーゼ含有組成物を保持し、湿潤気体をリパーゼ含有組成物中に強制的に通気させることが好ましい。この強制的通気に使用する湿潤気体は、上述した吸湿工程で使用する湿潤気体と同じ気体を使用することができる。湿潤気体の強制的通気は、例えば、10分以下、好ましくは7分以下、より好ましくは1分以上5分以下とすることが適当である。
<油性成分調節工程>
リパーゼの吸湿効果は、リパーゼ含有組成物中の油性成分量に影響を受けるため、リパーゼ含有組成物中に含まれる油性成分量を低減することが好ましい。その場合、吸湿工程前、すなわち湿潤気体に接触する前に、リパーゼ含有組成物中に含まれる油性成分の質量をリパーゼ含有組成物中の油性成分を除く成分の質量に対して1.5倍以下にすることが好ましい。より好ましくは1倍(即ち、リパーゼ含有組成物中の油性成分の質量とリパーゼ含有組成物中の油性成分を除く成分の質量とが等しい)以下であり、最も好ましくは0.1~0.7倍である。リパーゼ含有組成物中の油性成分量を低減させるためには、カラムやろ過器中で気体をリパーゼ含有組成物に通気させてリパーゼ含有組成物中の油性成分量を低下させた後、上述した湿潤気体をリパーゼ含有組成物中に通気させてもよい。油性成分量の低減のための通気に使用する気体は、空気、窒素、炭酸ガス、アルゴン等を使用することができるが、特に乾燥窒素ガスが好ましい。通気は、例えば、10分以下、好ましくは7分以下、より好ましくは5分以下とすることが適当である。
さらに、湿潤気体をリパーゼ含有組成物に通気させることにより、リパーゼ含有組成物に含まれる油性成分量を低下させるとともに、リパーゼ含有組成物に対して吸湿を行ってもよい。この通気に使用する湿潤気体は、上述した吸湿工程で使用する湿潤気体と同じ気体を使用することができる。湿潤気体の通気は、例えば、10分以下、好ましくは7分以下、より好ましくは1分以上5分以下とすることが適当である。なお、リパーゼ含有組成物中に含まれる油性成分の質量は、リパーゼ含有組成物中の油性成分を除く成分の質量に対して1.5倍以下になるまで低下させることが好ましい。より好ましくは1倍(即ち、リパーゼ含有組成物中の油性成分の質量とリパーゼ含有組成物中の油性成分を除く成分の質量とが等しい)以下であり、最も好ましくは0.1~0.7倍である。 <Method for producing lipase whose activity has been restored>
Another aspect of the present invention is to provide a method for producing a lipase whose activity has been restored.
<Preparation of lipase with reduced activity>
First, prepare a lipase with reduced activity. The lipase with reduced activity can be obtained by repeatedly performing esterification reaction and transesterification reaction. For example, in the case of transesterification, the mass of the lipase-containing composition is 0.05 to 10% by mass, preferably 0.1 to 5% by mass, more preferably 1 to 2% by mass, based on the starting substrate. The lipase-containing composition is added to the mixture and transesterification is performed for 10 to 50 hours while stirring at 20 to 80.degree. By repeating this reaction 10 to 50 times, preferably 20 to 50 times, more preferably 30 to 50 times, a lipase with reduced activity can be obtained. In addition, the good activity of the lipase is continuously maintained by alternately carrying out the esterification reaction or transesterification reaction of 1 to 10 times, preferably about 1 to 5 times, and the method for recovering lipase activity of the present invention. May be
Moisture absorption process
The lipase whose activity has been reduced recovers its activity by contacting the above-mentioned lipase-containing composition in a wet gas, and the activity can be recovered to produce a lipase whose activity has been restored. Specifically, production of lipase is carried out, for example, by placing the lipase with reduced activity in a container such as a tray and exposing it to a wet gas atmosphere as described above. At this time, the thickness of the lipase whose activity has been reduced in the container is, for example, 5 cm or less in thickness, preferably 3 cm or less, more preferably 0.1 to 1 cm.
In addition, it is preferable to aerate the moist gas into the lipase-containing composition because the activity can be recovered efficiently. For example, it is preferable to hold the lipase-containing composition in a column or a filter and to force a wet gas into the lipase-containing composition. The moist gas used for this forced aeration can use the same gas as the moist gas used in the above-mentioned moisture absorption process. The forced aeration of the moist gas is suitably, for example, 10 minutes or less, preferably 7 minutes or less, more preferably 1 minute or more and 5 minutes or less.
<Oil component adjustment process>
Since the hygroscopic effect of the lipase is affected by the amount of the oil component in the lipase-containing composition, it is preferable to reduce the amount of the oil component contained in the lipase-containing composition. In that case, the mass of the oily component contained in the lipase-containing composition is not more than 1.5 times the mass of the component excluding the oily component in the lipase-containing composition before the moisture absorption step, ie, before contacting with the wet gas. It is preferable to More preferably, it is 1 times or less (ie, the mass of the oil component in the lipase-containing composition and the mass of the components excluding the oil component in the lipase-containing composition are equal) or less, most preferably 0.1 to 0.7. It is a double. In order to reduce the amount of the oily component in the lipase-containing composition, the gas is passed through the lipase-containing composition in a column or a filter to reduce the amount of the oily component in the lipase-containing composition, and then the aforementioned wetting is performed. A gas may be bubbled into the lipase containing composition. Air, nitrogen, carbon dioxide gas, argon or the like can be used as the gas used for aeration for reducing the amount of the oily component, and dry nitrogen gas is particularly preferable. The aeration is suitably, for example, 10 minutes or less, preferably 7 minutes or less, more preferably 5 minutes or less.
Furthermore, by allowing the lipase-containing composition to aerate the moist gas, the amount of the oil component contained in the lipase-containing composition may be reduced, and moisture absorption may be performed on the lipase-containing composition. The moist gas used for this aeration can use the same gas as the moist gas used by the moisture absorption process mentioned above. The aeration of the wet gas is suitably, for example, 10 minutes or less, preferably 7 minutes or less, more preferably 1 minute to 5 minutes. The mass of the oil component contained in the lipase-containing composition is preferably reduced to 1.5 times or less the mass of the component excluding the oil component in the lipase-containing composition. More preferably, it is 1 times or less (ie, the mass of the oil component in the lipase-containing composition and the mass of the components excluding the oil component in the lipase-containing composition are equal) or less, most preferably 0.1 to 0.7. It is a double.
<リパーゼを利用した反応>
本発明の他の一つの態様は、活性が回復されたリパーゼを用いてエステル化反応又はエステル交換反応することにより得られるエステル合成物(エステル化物又はエステル交換物)の製造方法にある。
本発明の活性が回復されたリパーゼを使用して行うエステル化反応は、例えば、脂肪酸の部分エステルと脂肪酸とのエステル化反応、又は一価又は多価アルコールと脂肪酸とのエステル化反応が挙げられる。好ましくは、グリセリンと脂肪酸とのエステル化反応等が挙げられる。
また、本発明の活性が回復されたリパーゼを使用して行うエステル交換反応は、脂肪酸エステル、脂肪酸、及びアルコールから選ばれる1種以上と、油脂とのエステル交換反応である。好ましいエステル交換反応としては、例えば、常法による油脂と油脂のエステル交換反応、脂肪酸エステルと油脂とのエステル交換反応、アルコリシスやアシドリシスのエステル交換反応が挙げられる。中でも、エステル交換反応の原料基質としては、脂肪酸エステル及び油脂が好ましい。
脂肪酸エステルとしては、例えば、炭素数6~30、好ましくは8~22の、直鎖又は分枝鎖の、飽和又は不飽和の脂肪酸エステルが好適である。ここで脂肪酸には、例えば、オクタン酸、デカン酸、ラウリン酸、ミリスチン酸、パルミチン酸、ステアリン酸、オレイン酸、リノール酸、リノレイン酸、及びベヘン酸などが挙げられる。特に好ましい脂肪酸エステルとしては、パルミチン酸エチル、パルミチン酸メチル、ステアリン酸エチル、ステアリン酸メチル、ベヘン酸エチル、ベヘン酸メチルが挙げられる。
油脂としては、例えば、構成脂肪酸の炭素数が8~24のトリアシルグリセリドが好ましく、特に植物油、例えば、菜種油、大豆油、ヒマワリ油、紅花油、コーン油からなる群から選ばれる植物油が好ましい。
脂肪酸エステルと油脂とのエステル交換反応の場合、原料基質中の脂肪酸エステルと油脂の質量比は、例えば、9:1~1:9、好ましくは、8:2~2:8、より好ましくは、7:3~5:5が適当である。また、原料基質に水が含まれていてもよい。原料基質に含まれる水の量は、原料基質全体の質量に対し、例えば、10~1000ppm、好ましくは、100~500ppm、より好ましくは200~400ppmであることが適当である。 <Reaction using lipase>
Another aspect of the present invention is a method for producing an ester compound (esterified product or transesterified product) obtained by carrying out an esterification reaction or a transesterification reaction using a lipase whose activity is restored.
The esterification reaction carried out using the lipase having recovered activity of the present invention includes, for example, an esterification reaction of a partial ester of fatty acid with a fatty acid, or an esterification reaction of a monohydric or polyhydric alcohol with a fatty acid . Preferably, the esterification reaction of glycerol and a fatty acid etc. are mentioned.
Moreover, the transesterification reaction performed using the lipase by which the activity was recovered | restored of this invention is a transesterification reaction between 1 or more types chosen from fatty acid ester, a fatty acid, and alcohol, and fats and oils. Examples of preferable transesterification reactions include transesterification between fats and oils by a conventional method, transesterification between fatty acid esters and fats and oils, transesterification with alcoholysis and acidolysis. Among them, fatty acid esters and fats and oils are preferable as raw material substrates for transesterification.
As the fatty acid ester, for example, a linear or branched, saturated or unsaturated fatty acid ester having 6 to 30, preferably 8 to 22 carbon atoms is suitable. Here, examples of the fatty acid include octanoic acid, decanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, and behenic acid. Particularly preferred fatty acid esters include ethyl palmitate, methyl palmitate, ethyl stearate, methyl stearate, ethyl behenate and methyl behenate.
As fats and oils, for example, triacylglycerides having 8 to 24 carbon atoms of the constituent fatty acids are preferable, and vegetable oils such as vegetable oils selected from the group consisting of rapeseed oil, soybean oil, sunflower oil, safflower oil and corn oil are particularly preferable.
In the case of transesterification reaction of fatty acid ester and fat or oil, the mass ratio of fatty acid ester to fat or oil in the raw material substrate is, for example, 9: 1 to 1: 9, preferably 8: 2 to 2: 8, more preferably 7: 3 to 5: 5 is suitable. In addition, the raw material substrate may contain water. The amount of water contained in the starting substrate is, for example, 10 to 1000 ppm, preferably 100 to 500 ppm, more preferably 200 to 400 ppm, based on the total mass of the starting substrate.
本発明の他の一つの態様は、活性が回復されたリパーゼを用いてエステル化反応又はエステル交換反応することにより得られるエステル合成物(エステル化物又はエステル交換物)の製造方法にある。
本発明の活性が回復されたリパーゼを使用して行うエステル化反応は、例えば、脂肪酸の部分エステルと脂肪酸とのエステル化反応、又は一価又は多価アルコールと脂肪酸とのエステル化反応が挙げられる。好ましくは、グリセリンと脂肪酸とのエステル化反応等が挙げられる。
また、本発明の活性が回復されたリパーゼを使用して行うエステル交換反応は、脂肪酸エステル、脂肪酸、及びアルコールから選ばれる1種以上と、油脂とのエステル交換反応である。好ましいエステル交換反応としては、例えば、常法による油脂と油脂のエステル交換反応、脂肪酸エステルと油脂とのエステル交換反応、アルコリシスやアシドリシスのエステル交換反応が挙げられる。中でも、エステル交換反応の原料基質としては、脂肪酸エステル及び油脂が好ましい。
脂肪酸エステルとしては、例えば、炭素数6~30、好ましくは8~22の、直鎖又は分枝鎖の、飽和又は不飽和の脂肪酸エステルが好適である。ここで脂肪酸には、例えば、オクタン酸、デカン酸、ラウリン酸、ミリスチン酸、パルミチン酸、ステアリン酸、オレイン酸、リノール酸、リノレイン酸、及びベヘン酸などが挙げられる。特に好ましい脂肪酸エステルとしては、パルミチン酸エチル、パルミチン酸メチル、ステアリン酸エチル、ステアリン酸メチル、ベヘン酸エチル、ベヘン酸メチルが挙げられる。
油脂としては、例えば、構成脂肪酸の炭素数が8~24のトリアシルグリセリドが好ましく、特に植物油、例えば、菜種油、大豆油、ヒマワリ油、紅花油、コーン油からなる群から選ばれる植物油が好ましい。
脂肪酸エステルと油脂とのエステル交換反応の場合、原料基質中の脂肪酸エステルと油脂の質量比は、例えば、9:1~1:9、好ましくは、8:2~2:8、より好ましくは、7:3~5:5が適当である。また、原料基質に水が含まれていてもよい。原料基質に含まれる水の量は、原料基質全体の質量に対し、例えば、10~1000ppm、好ましくは、100~500ppm、より好ましくは200~400ppmであることが適当である。 <Reaction using lipase>
Another aspect of the present invention is a method for producing an ester compound (esterified product or transesterified product) obtained by carrying out an esterification reaction or a transesterification reaction using a lipase whose activity is restored.
The esterification reaction carried out using the lipase having recovered activity of the present invention includes, for example, an esterification reaction of a partial ester of fatty acid with a fatty acid, or an esterification reaction of a monohydric or polyhydric alcohol with a fatty acid . Preferably, the esterification reaction of glycerol and a fatty acid etc. are mentioned.
Moreover, the transesterification reaction performed using the lipase by which the activity was recovered | restored of this invention is a transesterification reaction between 1 or more types chosen from fatty acid ester, a fatty acid, and alcohol, and fats and oils. Examples of preferable transesterification reactions include transesterification between fats and oils by a conventional method, transesterification between fatty acid esters and fats and oils, transesterification with alcoholysis and acidolysis. Among them, fatty acid esters and fats and oils are preferable as raw material substrates for transesterification.
As the fatty acid ester, for example, a linear or branched, saturated or unsaturated fatty acid ester having 6 to 30, preferably 8 to 22 carbon atoms is suitable. Here, examples of the fatty acid include octanoic acid, decanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, and behenic acid. Particularly preferred fatty acid esters include ethyl palmitate, methyl palmitate, ethyl stearate, methyl stearate, ethyl behenate and methyl behenate.
As fats and oils, for example, triacylglycerides having 8 to 24 carbon atoms of the constituent fatty acids are preferable, and vegetable oils such as vegetable oils selected from the group consisting of rapeseed oil, soybean oil, sunflower oil, safflower oil and corn oil are particularly preferable.
In the case of transesterification reaction of fatty acid ester and fat or oil, the mass ratio of fatty acid ester to fat or oil in the raw material substrate is, for example, 9: 1 to 1: 9, preferably 8: 2 to 2: 8, more preferably 7: 3 to 5: 5 is suitable. In addition, the raw material substrate may contain water. The amount of water contained in the starting substrate is, for example, 10 to 1000 ppm, preferably 100 to 500 ppm, more preferably 200 to 400 ppm, based on the total mass of the starting substrate.
また、油脂と脂肪酸によるアシドリシスを使ったエステル交換反応としては、リパーゼの持つ1,3-特異性リパーゼを大いに利用した構造油脂の製造をすることができる。グリセリン骨格の2位に特定の脂肪酸を残して1,3位の脂肪酸を目的の脂肪酸に置き換えるものである。得られたものはチョコレート等に使用する油脂へ利用でき、また特定の栄養効果を持つ油脂へ利用できる。
Moreover, as transesterification using acidolysis with fats and oils and fatty acids, it is possible to produce structural fats and oils using the 1,3-specific lipase possessed by lipases to a large extent. It leaves the specific fatty acid at the 2-position of the glycerin skeleton, replacing the fatty acid at the 1-3 position with the target fatty acid. The obtained product can be used for fats and oils used for chocolate etc., and can be used for fats and oils having specific nutritional effects.
本発明の活性が回復されたリパーゼを用いたエステル化反応やエステル交換反応の条件については、特に限定するものではなく、常法により行うことができる。
一般的には、加水分解の原因となる水分の混入を避けながら、常圧又は減圧下にて行なわれる。反応温度としては、使用する各原料基質の凝固点にもよるが、20~80℃程度で行うことが好ましく、凝固点により限定されなければ、40~60℃で行うことがより好ましい。
また、活性が回復されたリパーゼを含むリパーゼ含有組成物の原料基質への添加量としては、リパーゼ含有組成物と原料基質の合計質量に対し、0.05~10質量%が好ましく、0.1~5質量%がより好ましい。最適な量は、反応温度、設定する反応時間、活性が回復されたリパーゼの活性等により決定される。反応終了後、リパーゼは濾過及び/又は遠心分離等により除かれ、製造が不可能な活性に低下するまで繰り返し使用される。活性が低下したリパーゼは、本発明の方法により再活性化することができる。
したがって、通常、高価であるリパーゼは、本発明の方法により、繰り返し活性を回復することができる。
また、得られたエステル化物又はエステル交換物は、特に限定されないが、食品分野に使用されるエステル交換油脂又はエステル化油脂としても有用である。
次に本発明を製造例及び実施例により詳細に説明する。 The conditions for the esterification reaction and the transesterification reaction using the lipase of which the activity is recovered according to the present invention are not particularly limited, and the reaction can be performed by a conventional method.
Generally, it is carried out under normal pressure or reduced pressure while avoiding the inclusion of water which causes hydrolysis. As the reaction temperature, although depending on the freezing point of each raw material substrate to be used, it is preferable to carry out at about 20 to 80 ° C., and it is more preferable to carry out at 40 to 60 ° C. if not limited by the freezing point.
Further, the addition amount of the lipase-containing composition containing lipase whose activity has been restored to the starting substrate is preferably 0.05 to 10% by mass with respect to the total mass of the lipase-containing composition and the starting substrate. -5% by mass is more preferable. The optimum amount is determined by the reaction temperature, the reaction time set, the activity of the lipase whose activity has been recovered, and the like. After completion of the reaction, the lipase is removed by filtration and / or centrifugation and the like, and is repeatedly used until it is reduced to an activity which can not be produced. The reduced activity lipase can be reactivated by the method of the present invention.
Thus, lipases, which are usually expensive, can be repeatedly restored by the method of the present invention.
In addition, the obtained esterified product or transesterified product is not particularly limited, but is also useful as a transesterified oil or esterified oil or fat used in the food field.
The present invention will now be described in more detail by way of production examples and examples.
一般的には、加水分解の原因となる水分の混入を避けながら、常圧又は減圧下にて行なわれる。反応温度としては、使用する各原料基質の凝固点にもよるが、20~80℃程度で行うことが好ましく、凝固点により限定されなければ、40~60℃で行うことがより好ましい。
また、活性が回復されたリパーゼを含むリパーゼ含有組成物の原料基質への添加量としては、リパーゼ含有組成物と原料基質の合計質量に対し、0.05~10質量%が好ましく、0.1~5質量%がより好ましい。最適な量は、反応温度、設定する反応時間、活性が回復されたリパーゼの活性等により決定される。反応終了後、リパーゼは濾過及び/又は遠心分離等により除かれ、製造が不可能な活性に低下するまで繰り返し使用される。活性が低下したリパーゼは、本発明の方法により再活性化することができる。
したがって、通常、高価であるリパーゼは、本発明の方法により、繰り返し活性を回復することができる。
また、得られたエステル化物又はエステル交換物は、特に限定されないが、食品分野に使用されるエステル交換油脂又はエステル化油脂としても有用である。
次に本発明を製造例及び実施例により詳細に説明する。 The conditions for the esterification reaction and the transesterification reaction using the lipase of which the activity is recovered according to the present invention are not particularly limited, and the reaction can be performed by a conventional method.
Generally, it is carried out under normal pressure or reduced pressure while avoiding the inclusion of water which causes hydrolysis. As the reaction temperature, although depending on the freezing point of each raw material substrate to be used, it is preferable to carry out at about 20 to 80 ° C., and it is more preferable to carry out at 40 to 60 ° C. if not limited by the freezing point.
Further, the addition amount of the lipase-containing composition containing lipase whose activity has been restored to the starting substrate is preferably 0.05 to 10% by mass with respect to the total mass of the lipase-containing composition and the starting substrate. -5% by mass is more preferable. The optimum amount is determined by the reaction temperature, the reaction time set, the activity of the lipase whose activity has been recovered, and the like. After completion of the reaction, the lipase is removed by filtration and / or centrifugation and the like, and is repeatedly used until it is reduced to an activity which can not be produced. The reduced activity lipase can be reactivated by the method of the present invention.
Thus, lipases, which are usually expensive, can be repeatedly restored by the method of the present invention.
In addition, the obtained esterified product or transesterified product is not particularly limited, but is also useful as a transesterified oil or esterified oil or fat used in the food field.
The present invention will now be described in more detail by way of production examples and examples.
[リパーゼ含有組成物の作製]
粉末リパーゼとして、天野エンザイム株式会社の商品:リパーゼDF “Amano” 15-K (リパーゼDともいう)の酵素溶液(150000U/ml)を準備した。
この酵素溶液中の水の量は、リパーゼDの質量に対し、15倍の量であった。また、穀物粉末として脱臭全脂大豆粉末(商品名:アルファプラスHS-600、日清オイリオグループ株式会社製)10質量%水溶液を準備した。上記脱臭全脂大豆粉末の質量は、上記リパーゼDの質量に対して500質量%であった。上記脱臭全脂大豆粉末を、上記リパーゼDの酵素溶液に、該溶液を攪拌しながら加えた。得られた混合溶液に0.5N NaOH溶液を1ml加えて混合溶液全体をpH7.8に調整後、ノズル並流型のスプレードライ(噴霧乾燥、上記混合溶液の温度30℃、送風の入口温度100℃、出口温度50℃、東京理科器械株式会社、SD-1000型)を行ってリパーゼ粉末製剤を得た。このリパーゼ粉末製剤にろ過助剤としてセルロースパウダー(平均粒子径30μm)を加えリパーゼ含有組成物を得た。平均粒子径は、株式会社堀場製作所の粒度分布測定装置(LA-500)を用いて測定した。得られたリパーゼ含有組成物に含まれる粉末リパーゼとろ過助剤との質量比は、1/1であった。 [Preparation of lipase-containing composition]
As a powdery lipase, an enzyme solution (150,000 U / ml) of a product of Amano Enzyme Co., Ltd .: Lipase DF “Amano” 15-K (also referred to as Lipase D) was prepared.
The amount of water in this enzyme solution was 15 times the mass of lipase D. Further, a 10% by mass aqueous solution of deodorized whole fat soybean powder (trade name: Alpha Plus HS-600, manufactured by Nisshin Oillio Group, Inc.) was prepared as grain powder. The mass of the above-mentioned deodorized whole fat soybean powder was 500 mass% with respect to the mass of the above-mentioned lipase D. The above deodorized whole fat soybean powder was added to the above enzyme solution of Lipase D while the solution was being stirred. 1 ml of 0.5 N NaOH solution is added to the obtained mixed solution to adjust the whole mixed solution to pH 7.8, and then nozzle cocurrent spray drying (spray drying, temperature of the above mixed solution of 30 ° C., inlet temperature of air 100) C., outlet temperature 50.degree. C., Tokyo Scientific Instruments Co., Ltd., Model SD-1000) was carried out to obtain a lipase powder preparation. Cellulose powder (average particle diameter 30 μm) was added to the lipase powder preparation as a filter aid to obtain a lipase-containing composition. The average particle size was measured using a particle size distribution analyzer (LA-500) manufactured by Horiba, Ltd. The mass ratio of the powdered lipase to the filter aid contained in the obtained lipase-containing composition was 1/1.
粉末リパーゼとして、天野エンザイム株式会社の商品:リパーゼDF “Amano” 15-K (リパーゼDともいう)の酵素溶液(150000U/ml)を準備した。
この酵素溶液中の水の量は、リパーゼDの質量に対し、15倍の量であった。また、穀物粉末として脱臭全脂大豆粉末(商品名:アルファプラスHS-600、日清オイリオグループ株式会社製)10質量%水溶液を準備した。上記脱臭全脂大豆粉末の質量は、上記リパーゼDの質量に対して500質量%であった。上記脱臭全脂大豆粉末を、上記リパーゼDの酵素溶液に、該溶液を攪拌しながら加えた。得られた混合溶液に0.5N NaOH溶液を1ml加えて混合溶液全体をpH7.8に調整後、ノズル並流型のスプレードライ(噴霧乾燥、上記混合溶液の温度30℃、送風の入口温度100℃、出口温度50℃、東京理科器械株式会社、SD-1000型)を行ってリパーゼ粉末製剤を得た。このリパーゼ粉末製剤にろ過助剤としてセルロースパウダー(平均粒子径30μm)を加えリパーゼ含有組成物を得た。平均粒子径は、株式会社堀場製作所の粒度分布測定装置(LA-500)を用いて測定した。得られたリパーゼ含有組成物に含まれる粉末リパーゼとろ過助剤との質量比は、1/1であった。 [Preparation of lipase-containing composition]
As a powdery lipase, an enzyme solution (150,000 U / ml) of a product of Amano Enzyme Co., Ltd .: Lipase DF “Amano” 15-K (also referred to as Lipase D) was prepared.
The amount of water in this enzyme solution was 15 times the mass of lipase D. Further, a 10% by mass aqueous solution of deodorized whole fat soybean powder (trade name: Alpha Plus HS-600, manufactured by Nisshin Oillio Group, Inc.) was prepared as grain powder. The mass of the above-mentioned deodorized whole fat soybean powder was 500 mass% with respect to the mass of the above-mentioned lipase D. The above deodorized whole fat soybean powder was added to the above enzyme solution of Lipase D while the solution was being stirred. 1 ml of 0.5 N NaOH solution is added to the obtained mixed solution to adjust the whole mixed solution to pH 7.8, and then nozzle cocurrent spray drying (spray drying, temperature of the above mixed solution of 30 ° C., inlet temperature of air 100) C., outlet temperature 50.degree. C., Tokyo Scientific Instruments Co., Ltd., Model SD-1000) was carried out to obtain a lipase powder preparation. Cellulose powder (average particle diameter 30 μm) was added to the lipase powder preparation as a filter aid to obtain a lipase-containing composition. The average particle size was measured using a particle size distribution analyzer (LA-500) manufactured by Horiba, Ltd. The mass ratio of the powdered lipase to the filter aid contained in the obtained lipase-containing composition was 1/1.
[活性が失われたリパーゼ含有組成物(リパーゼろ過ケーキ)の作製]
上述のようにして得られたリパーゼ含有組成物の活性を失わせるため、得られたエステル含有組成物を使用したエステル交換反応を繰り返し行った。
エステル交換反応は、まず、上述のようにして得られたリパーゼ含有組成物にエステル交換反応に使用する原料基質を混合した。原料基質には、ステアリン酸エチル(商品名:エチルステアレート、株式会社井上香料製造所製)及びハイオレイックヒマワリ油(日清オイリオグループ株式会社製)を6:4の質量比で混合し、原料基質全体中の水分が質量で300ppmとなるように調整したものを用いた。
このようにして得られた原料基質に対し、リパーゼ含有組成物が1質量%となるように、リパーゼ含有組成物を原料基質に加え、50℃で撹拌しながら16時間反応を行った。
反応終了後、リパーゼ含有組成物をろ過によって回収した。回収したリパーゼ含有組成物は再度反応に利用した。上記反応操作を30回繰返し行った後、ろ過・回収したリパーゼ含有組成物(リパーゼろ過ケーキA)を以下の実施例及び比較例に用いた。 [Preparation of lipase-containing composition (lipase filter cake) whose activity has been lost]
In order to lose the activity of the lipase-containing composition obtained as described above, transesterification was repeated using the obtained ester-containing composition.
In the transesterification reaction, first, the lipase-containing composition obtained as described above was mixed with the starting substrate used for the transesterification reaction. As a raw material substrate, ethyl stearate (trade name: ethyl stearate, manufactured by Inoue Perfume Manufacturing Co., Ltd.) and high oleic sunflower oil (manufactured by Nisshin Oillio Group, Inc.) are mixed at a mass ratio of 6: 4, What adjusted so that the water | moisture content in the whole raw material board | substrate might be set to 300 ppm by mass was used.
With respect to the raw material substrate thus obtained, the lipase-containing composition was added to the raw material substrate such that the composition containing lipase was 1% by mass, and the reaction was carried out for 16 hours while stirring at 50 ° C.
After completion of the reaction, the lipase-containing composition was recovered by filtration. The recovered lipase-containing composition was used again for the reaction. The above reaction procedure was repeated 30 times, and then the filtered and recovered lipase-containing composition (lipase filter cake A) was used in the following examples and comparative examples.
上述のようにして得られたリパーゼ含有組成物の活性を失わせるため、得られたエステル含有組成物を使用したエステル交換反応を繰り返し行った。
エステル交換反応は、まず、上述のようにして得られたリパーゼ含有組成物にエステル交換反応に使用する原料基質を混合した。原料基質には、ステアリン酸エチル(商品名:エチルステアレート、株式会社井上香料製造所製)及びハイオレイックヒマワリ油(日清オイリオグループ株式会社製)を6:4の質量比で混合し、原料基質全体中の水分が質量で300ppmとなるように調整したものを用いた。
このようにして得られた原料基質に対し、リパーゼ含有組成物が1質量%となるように、リパーゼ含有組成物を原料基質に加え、50℃で撹拌しながら16時間反応を行った。
反応終了後、リパーゼ含有組成物をろ過によって回収した。回収したリパーゼ含有組成物は再度反応に利用した。上記反応操作を30回繰返し行った後、ろ過・回収したリパーゼ含有組成物(リパーゼろ過ケーキA)を以下の実施例及び比較例に用いた。 [Preparation of lipase-containing composition (lipase filter cake) whose activity has been lost]
In order to lose the activity of the lipase-containing composition obtained as described above, transesterification was repeated using the obtained ester-containing composition.
In the transesterification reaction, first, the lipase-containing composition obtained as described above was mixed with the starting substrate used for the transesterification reaction. As a raw material substrate, ethyl stearate (trade name: ethyl stearate, manufactured by Inoue Perfume Manufacturing Co., Ltd.) and high oleic sunflower oil (manufactured by Nisshin Oillio Group, Inc.) are mixed at a mass ratio of 6: 4, What adjusted so that the water | moisture content in the whole raw material board | substrate might be set to 300 ppm by mass was used.
With respect to the raw material substrate thus obtained, the lipase-containing composition was added to the raw material substrate such that the composition containing lipase was 1% by mass, and the reaction was carried out for 16 hours while stirring at 50 ° C.
After completion of the reaction, the lipase-containing composition was recovered by filtration. The recovered lipase-containing composition was used again for the reaction. The above reaction procedure was repeated 30 times, and then the filtered and recovered lipase-containing composition (lipase filter cake A) was used in the following examples and comparative examples.
[エステル交換反応の活性:POS+SOS値]
リパーゼ活性の回復もしくは活性の程度をトリグリセリド中のPOSとSOSの合計質量を比較することで評価した。具体的には、比較対照の未処理(吸湿工程前)のリパーゼ含有組成物を使用したエステル交換反応において、1時間反応した後に得られたトリグリセリドの質量に対する該トリグリセリド中のPOS及びSOSの合計質量%を100とした場合の、処理後(吸湿工程後)のリパーゼ含有組成物サンプルを使用したエステル交換反応において、1時間反応した後に得られたトリグリセリド中のPOS及びSOSの合計質量%の相対量:(POS+SOS値)を求めた。
(POS+SOS値)=(リパーゼ含有組成物サンプルのエステル交換反応1時間後のトリグリセリドの質量に対する該トリグリセリド中のPOS及びSOSの合計質量%)/(比較対照リパーゼ含有組成物のエステル交換反応1時間後のトリグリセリドの質量に対する該トリグリセリド中のPOS及びSOSの合計質量%)×100
(POS+SOS値)が100より高いと、リパーゼ活性が回復したと評価でき、100より低いと、リパーゼ活性が低下したと評価できる。
なお、実施例及び比較例において、トリグリセリド中のPOS及びSOSの合計質量%の相対量(POS+SOS値)はガスクロマトグラフィーを用いて測定した。具体的には、反応溶液7.5μlを取り、これをヘキサン1.5mlで希釈し、リパーゼ粉末製剤等を含む固形物をろ過して得たろ液をガスクロマトグラフィー(GC)で測定した。ガスクロマトグラフィーとしては、アジレント・テクノロジー株式会社製、製品番号6890Nと、カラム 65TG(株式会社島津ジーエルシー製)を用いた。ガスクロマトグラフィーの条件は、カラム温度:350℃、昇温:1℃/分、最終温度:365℃とした。 [Activity of transesterification: POS + SOS value]
The degree of recovery or activity of lipase activity was evaluated by comparing the total mass of POS and SOS in triglyceride. Specifically, the total mass of POS and SOS in the triglyceride relative to the mass of triglyceride obtained after reacting for 1 hour in the transesterification using the untreated (before the moisture absorption step) lipase-containing composition as a comparison control % Relative to the total mass% of POS and SOS in the triglyceride obtained after reaction for 1 hour in the transesterification using the lipase-containing composition sample after treatment (after the moisture absorption step), where 100% is taken as 100 : (POS + SOS value) was determined.
(POS + SOS value) = (total mass% of POS and SOS in the triglyceride relative to mass of triglyceride after 1 hour of transesterification reaction of lipase-containing composition sample) / (transesterification reaction of comparative lipase-containing composition 1) The total mass% of POS and SOS in the triglyceride relative to the mass of triglyceride after time) x 100
When (POS + SOS value) is higher than 100, it can be evaluated that the lipase activity has recovered, and when it is lower than 100, it can be evaluated that the lipase activity has decreased.
In Examples and Comparative Examples, the relative mass (POS + SOS value) of the total mass% of POS and SOS in triglyceride was measured using gas chromatography. Specifically, 7.5 μl of the reaction solution was taken, diluted with 1.5 ml of hexane, and the filtrate obtained by filtering a solid containing a lipase powder preparation and the like was measured by gas chromatography (GC). As gas chromatography, Agilent Technologies Co., Ltd. product number 6890N and column 65 TG (manufactured by Shimadzu LC Co., Ltd.) were used. The conditions for gas chromatography were column temperature: 350 ° C., temperature rise: 1 ° C./min, and final temperature: 365 ° C.
リパーゼ活性の回復もしくは活性の程度をトリグリセリド中のPOSとSOSの合計質量を比較することで評価した。具体的には、比較対照の未処理(吸湿工程前)のリパーゼ含有組成物を使用したエステル交換反応において、1時間反応した後に得られたトリグリセリドの質量に対する該トリグリセリド中のPOS及びSOSの合計質量%を100とした場合の、処理後(吸湿工程後)のリパーゼ含有組成物サンプルを使用したエステル交換反応において、1時間反応した後に得られたトリグリセリド中のPOS及びSOSの合計質量%の相対量:(POS+SOS値)を求めた。
(POS+SOS値)=(リパーゼ含有組成物サンプルのエステル交換反応1時間後のトリグリセリドの質量に対する該トリグリセリド中のPOS及びSOSの合計質量%)/(比較対照リパーゼ含有組成物のエステル交換反応1時間後のトリグリセリドの質量に対する該トリグリセリド中のPOS及びSOSの合計質量%)×100
(POS+SOS値)が100より高いと、リパーゼ活性が回復したと評価でき、100より低いと、リパーゼ活性が低下したと評価できる。
なお、実施例及び比較例において、トリグリセリド中のPOS及びSOSの合計質量%の相対量(POS+SOS値)はガスクロマトグラフィーを用いて測定した。具体的には、反応溶液7.5μlを取り、これをヘキサン1.5mlで希釈し、リパーゼ粉末製剤等を含む固形物をろ過して得たろ液をガスクロマトグラフィー(GC)で測定した。ガスクロマトグラフィーとしては、アジレント・テクノロジー株式会社製、製品番号6890Nと、カラム 65TG(株式会社島津ジーエルシー製)を用いた。ガスクロマトグラフィーの条件は、カラム温度:350℃、昇温:1℃/分、最終温度:365℃とした。 [Activity of transesterification: POS + SOS value]
The degree of recovery or activity of lipase activity was evaluated by comparing the total mass of POS and SOS in triglyceride. Specifically, the total mass of POS and SOS in the triglyceride relative to the mass of triglyceride obtained after reacting for 1 hour in the transesterification using the untreated (before the moisture absorption step) lipase-containing composition as a comparison control % Relative to the total mass% of POS and SOS in the triglyceride obtained after reaction for 1 hour in the transesterification using the lipase-containing composition sample after treatment (after the moisture absorption step), where 100% is taken as 100 : (POS + SOS value) was determined.
(POS + SOS value) = (total mass% of POS and SOS in the triglyceride relative to mass of triglyceride after 1 hour of transesterification reaction of lipase-containing composition sample) / (transesterification reaction of comparative lipase-containing composition 1) The total mass% of POS and SOS in the triglyceride relative to the mass of triglyceride after time) x 100
When (POS + SOS value) is higher than 100, it can be evaluated that the lipase activity has recovered, and when it is lower than 100, it can be evaluated that the lipase activity has decreased.
In Examples and Comparative Examples, the relative mass (POS + SOS value) of the total mass% of POS and SOS in triglyceride was measured using gas chromatography. Specifically, 7.5 μl of the reaction solution was taken, diluted with 1.5 ml of hexane, and the filtrate obtained by filtering a solid containing a lipase powder preparation and the like was measured by gas chromatography (GC). As gas chromatography, Agilent Technologies Co., Ltd. product number 6890N and column 65 TG (manufactured by Shimadzu LC Co., Ltd.) were used. The conditions for gas chromatography were column temperature: 350 ° C., temperature rise: 1 ° C./min, and final temperature: 365 ° C.
[水分量、油性成分量比]
リパーゼ含有組成物を、基準油脂分析試験法(Standard Methods for Analysis of Fats, oils and Related Materials)にかけ、同試験法の1.4.1-1996水分Moisture(加熱乾燥法Air oven)(以下a(%)とする)および1.5-1996油分(Oil content)(以下b(%)とする)を測定し、以下の算式から、リパーゼ含有組成物中に含まれる水分量と油性成分量比を算出した。
リパーゼ含有組成物中に含まれる油性成分を除く成分の質量に対する水分量(質量%)=(a/(100-b))×100
リパーゼ含有組成物中に含まれる油性成分を除く成分の質量に対する油性成分量比(質量比)=b/(100-b) [Water content, oil content ratio]
The lipase-containing composition was subjected to the Standard Methods for Analysis of Fats, oils and Related Materials, and the 1.4.1-1996 Moisture Moisture (heat-drying method Air oven) of the same test method (hereinafter referred to as a (a %) And 1.5-1996 oil content (hereinafter referred to as b (%)), and from the following formula, the ratio of water content to the amount of oil component contained in the lipase-containing composition Calculated.
Water content (mass%) = (a / (100-b)) × 100 with respect to the mass of the components excluding the oily component contained in the lipase-containing composition
The ratio of the amount of the oil component to the mass of the component excluding the oil component contained in the lipase-containing composition (mass ratio) = b / (100-b)
リパーゼ含有組成物を、基準油脂分析試験法(Standard Methods for Analysis of Fats, oils and Related Materials)にかけ、同試験法の1.4.1-1996水分Moisture(加熱乾燥法Air oven)(以下a(%)とする)および1.5-1996油分(Oil content)(以下b(%)とする)を測定し、以下の算式から、リパーゼ含有組成物中に含まれる水分量と油性成分量比を算出した。
リパーゼ含有組成物中に含まれる油性成分を除く成分の質量に対する水分量(質量%)=(a/(100-b))×100
リパーゼ含有組成物中に含まれる油性成分を除く成分の質量に対する油性成分量比(質量比)=b/(100-b) [Water content, oil content ratio]
The lipase-containing composition was subjected to the Standard Methods for Analysis of Fats, oils and Related Materials, and the 1.4.1-1996 Moisture Moisture (heat-drying method Air oven) of the same test method (hereinafter referred to as a (a %) And 1.5-1996 oil content (hereinafter referred to as b (%)), and from the following formula, the ratio of water content to the amount of oil component contained in the lipase-containing composition Calculated.
Water content (mass%) = (a / (100-b)) × 100 with respect to the mass of the components excluding the oily component contained in the lipase-containing composition
The ratio of the amount of the oil component to the mass of the component excluding the oil component contained in the lipase-containing composition (mass ratio) = b / (100-b)
[実施例1]
リパーゼろ過ケーキA10g(リパーゼ含有組成物中に含まれる油性成分を除く成分の質量に対する水分量8質量%)をガラスシャーレ上に広げ、温度60℃、相対湿度70%の湿潤気体(湿潤空気)雰囲気下に1日間保持した。保持後のリパーゼろ過ケーキの水の吸収量は、リパーゼろ過ケーキAの質量に対して10質量%(リパーゼ含有組成物中に含まれる油性成分を除く成分の質量に対する水分量18質量%)であり、リパーゼ含有組成物中の油性成分量の変化はなかった。その後、得られたリパーゼろ過ケーキを用いて前述のエステル交換反応を1回行った。また、リパーゼ活性の回復もしくは活性の程度(POS+SOS値)を調べるため、吸湿保持前のリパーゼろ過ケーキAを使用して上記同様にエステル交換反応を実施した(試行1-1~試行1-5)。吸湿保持前のリパーゼろ過ケーキAを使用したエステル交換反応において、1時間反応した後に得られたトリグリセリド中のPOS及びSOSの合計質量%を100とした場合における、吸湿保持後のリパーゼろ過ケーキを使用したエステル交換反応において、1時間反応した後に得られたトリグリセリド中のPOS及びSOSの合計質量%の相対量(POS+SOS値)を表1に示す。
表1
Example 1
10 g of lipase filter cake A (water content 8% by mass with respect to the mass of components excluding the oil component contained in the lipase-containing composition) is spread on a glass petri dish, and a temperature of 60 ° C. and a humidified gas (wet air) atmosphere of 70% relative humidity It kept under for 1 day. The water absorption capacity of the lipase filter cake after retention is 10% by mass (the water content is 18% by mass with respect to the mass of components excluding the oil component contained in the lipase-containing composition) with respect to the mass of lipase filter cake A There was no change in the amount of oil component in the lipase-containing composition. Then, the above-mentioned transesterification was performed once using the obtained lipase filter cake. In addition, in order to examine the recovery of lipase activity or the degree of activity (POS + SOS value), transesterification was performed in the same manner as described above using lipase filter cake A before moisture absorption retention (trial 1-1 to trial 1-5) . In transesterification using lipase filter cake A before moisture absorption retention, use lipase filter cake after moisture absorption retention when the total mass% of POS and SOS in triglyceride obtained after reaction for 1 hour is 100 Table 1 shows the relative mass (POS + SOS value) of the total mass% of POS and SOS in triglyceride obtained after reacting for 1 hour in the transesterification reaction.
Table 1
リパーゼろ過ケーキA10g(リパーゼ含有組成物中に含まれる油性成分を除く成分の質量に対する水分量8質量%)をガラスシャーレ上に広げ、温度60℃、相対湿度70%の湿潤気体(湿潤空気)雰囲気下に1日間保持した。保持後のリパーゼろ過ケーキの水の吸収量は、リパーゼろ過ケーキAの質量に対して10質量%(リパーゼ含有組成物中に含まれる油性成分を除く成分の質量に対する水分量18質量%)であり、リパーゼ含有組成物中の油性成分量の変化はなかった。その後、得られたリパーゼろ過ケーキを用いて前述のエステル交換反応を1回行った。また、リパーゼ活性の回復もしくは活性の程度(POS+SOS値)を調べるため、吸湿保持前のリパーゼろ過ケーキAを使用して上記同様にエステル交換反応を実施した(試行1-1~試行1-5)。吸湿保持前のリパーゼろ過ケーキAを使用したエステル交換反応において、1時間反応した後に得られたトリグリセリド中のPOS及びSOSの合計質量%を100とした場合における、吸湿保持後のリパーゼろ過ケーキを使用したエステル交換反応において、1時間反応した後に得られたトリグリセリド中のPOS及びSOSの合計質量%の相対量(POS+SOS値)を表1に示す。
表1
Example 1
10 g of lipase filter cake A (water content 8% by mass with respect to the mass of components excluding the oil component contained in the lipase-containing composition) is spread on a glass petri dish, and a temperature of 60 ° C. and a humidified gas (wet air) atmosphere of 70% relative humidity It kept under for 1 day. The water absorption capacity of the lipase filter cake after retention is 10% by mass (the water content is 18% by mass with respect to the mass of components excluding the oil component contained in the lipase-containing composition) with respect to the mass of lipase filter cake A There was no change in the amount of oil component in the lipase-containing composition. Then, the above-mentioned transesterification was performed once using the obtained lipase filter cake. In addition, in order to examine the recovery of lipase activity or the degree of activity (POS + SOS value), transesterification was performed in the same manner as described above using lipase filter cake A before moisture absorption retention (trial 1-1 to trial 1-5) . In transesterification using lipase filter cake A before moisture absorption retention, use lipase filter cake after moisture absorption retention when the total mass% of POS and SOS in triglyceride obtained after reaction for 1 hour is 100 Table 1 shows the relative mass (POS + SOS value) of the total mass% of POS and SOS in triglyceride obtained after reacting for 1 hour in the transesterification reaction.
Table 1
[比較例1]
リパーゼろ過ケーキA10gをガラスシャーレ上に広げ、霧吹きを用いてまんべんなく水を噴霧した。水の噴霧量はリパーゼろ過ケーキ(リパーゼ含有組成物)の質量に対して10質量%(リパーゼ含有組成物中に含まれる油性成分を除く成分の質量に対する水分量18質量%)であった。噴霧後、リパーゼろ過ケーキをよく混ぜて水を馴染ませた後、1時間静置した。その後、得られたリパーゼろ過ケーキを用いて前述のエステル交換反応を1回行った。また、リパーゼ活性の回復もしくは活性の程度(POS+SOS値)を調べるため、吸湿保持前のリパーゼろ過ケーキを使用して上記同様にエステル交換反応を実施した(試行1-6~試行1-10)。吸湿保持前のリパーゼろ過ケーキAを使用したエステル交換反応において、1時間反応した後に得られたトリグリセリド中のPOS及びSOSの合計質量%を100とした場合における、吸湿保持後のリパーゼろ過ケーキを使用したエステル交換反応において、1時間反応した後に得られたトリグリセリド中のPOS及びSOSの合計質量%の相対量(POS+SOS値)を表2に示す。
表2
表1の全ての試行1-1~1-5においてリパーゼの活性(POS+SOS値)の上昇が見られた。比較例の試行1-7と1-10では活性化されたが、その他の試行1-6、1-8及び1-9では水を噴霧することで逆に活性が低下した。試行1-1~1-5において反応率は、比較例の試行1-7と1-10の反応率よりも大きかった。実施例の試行1-1~1-5の結果は、ばらつきが少なく、自然吸湿では安定的に再活性化することがわかった。 Comparative Example 1
10 g of lipase filter cake A was spread on a glass petri dish and sprayed uniformly with water using a mister. The spray amount of water was 10% by mass (the amount of water relative to the mass of components excluding the oil component contained in the lipase-containing composition: 18% by mass) based on the mass of the lipase filter cake (lipase-containing composition). After spraying, the lipase filter cake was thoroughly mixed to adjust the water, and then allowed to stand for 1 hour. Then, the above-mentioned transesterification was performed once using the obtained lipase filter cake. In addition, in order to examine the recovery of lipase activity or the degree of activity (POS + SOS value), transesterification was carried out in the same manner as described above using lipase filter cake before moisture absorption retention (trial 1-6 to trial 1-10). In transesterification using lipase filter cake A before moisture absorption retention, use lipase filter cake after moisture absorption retention when the total mass% of POS and SOS in triglyceride obtained after reaction for 1 hour is 100 Table 2 shows the relative amounts (POS + SOS value) of the total mass% of POS and SOS in triglyceride obtained after reacting for 1 hour in the transesterification reaction.
Table 2
An increase in lipase activity (POS + SOS value) was observed in all trials 1-1 to 1-5 in Table 1. In the trials 1-7 and 1-10 of the comparative example, activation was achieved, but in the other trials 1-6, 1-8 and 1-9, the activity was reduced by spraying water. In the trials 1-1 to 1-5, the reaction rate was larger than that of the trials 1-7 and 1-10 of the comparative example. The results of trials 1-1 to 1-5 in the example were found to be stable and to be reactivated stably with natural moisture absorption.
リパーゼろ過ケーキA10gをガラスシャーレ上に広げ、霧吹きを用いてまんべんなく水を噴霧した。水の噴霧量はリパーゼろ過ケーキ(リパーゼ含有組成物)の質量に対して10質量%(リパーゼ含有組成物中に含まれる油性成分を除く成分の質量に対する水分量18質量%)であった。噴霧後、リパーゼろ過ケーキをよく混ぜて水を馴染ませた後、1時間静置した。その後、得られたリパーゼろ過ケーキを用いて前述のエステル交換反応を1回行った。また、リパーゼ活性の回復もしくは活性の程度(POS+SOS値)を調べるため、吸湿保持前のリパーゼろ過ケーキを使用して上記同様にエステル交換反応を実施した(試行1-6~試行1-10)。吸湿保持前のリパーゼろ過ケーキAを使用したエステル交換反応において、1時間反応した後に得られたトリグリセリド中のPOS及びSOSの合計質量%を100とした場合における、吸湿保持後のリパーゼろ過ケーキを使用したエステル交換反応において、1時間反応した後に得られたトリグリセリド中のPOS及びSOSの合計質量%の相対量(POS+SOS値)を表2に示す。
表2
表1の全ての試行1-1~1-5においてリパーゼの活性(POS+SOS値)の上昇が見られた。比較例の試行1-7と1-10では活性化されたが、その他の試行1-6、1-8及び1-9では水を噴霧することで逆に活性が低下した。試行1-1~1-5において反応率は、比較例の試行1-7と1-10の反応率よりも大きかった。実施例の試行1-1~1-5の結果は、ばらつきが少なく、自然吸湿では安定的に再活性化することがわかった。 Comparative Example 1
10 g of lipase filter cake A was spread on a glass petri dish and sprayed uniformly with water using a mister. The spray amount of water was 10% by mass (the amount of water relative to the mass of components excluding the oil component contained in the lipase-containing composition: 18% by mass) based on the mass of the lipase filter cake (lipase-containing composition). After spraying, the lipase filter cake was thoroughly mixed to adjust the water, and then allowed to stand for 1 hour. Then, the above-mentioned transesterification was performed once using the obtained lipase filter cake. In addition, in order to examine the recovery of lipase activity or the degree of activity (POS + SOS value), transesterification was carried out in the same manner as described above using lipase filter cake before moisture absorption retention (trial 1-6 to trial 1-10). In transesterification using lipase filter cake A before moisture absorption retention, use lipase filter cake after moisture absorption retention when the total mass% of POS and SOS in triglyceride obtained after reaction for 1 hour is 100 Table 2 shows the relative amounts (POS + SOS value) of the total mass% of POS and SOS in triglyceride obtained after reacting for 1 hour in the transesterification reaction.
Table 2
An increase in lipase activity (POS + SOS value) was observed in all trials 1-1 to 1-5 in Table 1. In the trials 1-7 and 1-10 of the comparative example, activation was achieved, but in the other trials 1-6, 1-8 and 1-9, the activity was reduced by spraying water. In the trials 1-1 to 1-5, the reaction rate was larger than that of the trials 1-7 and 1-10 of the comparative example. The results of trials 1-1 to 1-5 in the example were found to be stable and to be reactivated stably with natural moisture absorption.
[実施例2]
リパーゼろ過ケーキA10gをガラスシャーレ上に広げ、30℃、40℃、50℃及び60℃の各温度;50%、70%及び90%の相対湿度の湿潤気体(湿潤空気)雰囲気下に1時間又は1日間保持した。リパーゼ含有組成物中に含まれる油性成分を除く成分の質量に対する水分量(質量%)を表3に示す。その後、リパーゼろ過ケーキを回収して上述したエステル交換反応を1回行った。このときの吸湿保持前のリパーゼろ過ケーキAを使用したエステル交換反応において、1時間反応した後に得られたトリグリセリド中のPOS及びSOSの合計質量%を100とした場合における、吸湿保持後のリパーゼろ過ケーキを使用したエステル交換反応において、1時間反応した後に得られたトリグリセリド中のPOS及びSOSの合計質量%の相対量(POS+SOS値)を表4に示す。
表3
*値は、リパーゼ含有組成物中に含まれる油性成分を除く成分の質量に対する水分量(質量%)
表4
*値は、リパーゼ含有組成物のPOS+SOS値
この結果、50℃で相対湿度70%に1日放置した場合が最も効果的であった。60℃で相対湿度70%に1日放置した場合、リパーゼ含有組成物の容器内の厚さに関係なく良好な活性が得られたが、その他では厚さによる影響が観察された。1時間の放置時間で良好な効果を出すためには、60℃で相対湿度90%とすることが好ましいことがわかった。 Example 2
10 g of lipase filter cake A is spread on a glass petri dish, and each temperature of 30 ° C., 40 ° C., 50 ° C. and 60 ° C .; 50 hours, 70% and 90% relative humidity in wet gas (wet air) atmosphere or 1 hour It was kept for 1 day. The water content (% by mass) based on the mass of the components excluding the oil-based component contained in the lipase-containing composition is shown in Table 3. Thereafter, the lipase filter cake was recovered and subjected to the above-described transesterification reaction once. In transesterification using lipase filter cake A before moisture absorption retention at this time, lipase filtration after moisture absorption retention when the total mass% of POS and SOS in triglyceride obtained after reacting for 1 hour is 100 The relative amount (POS + SOS value) of the total mass% of POS and SOS in triglyceride obtained after reacting for 1 hour in the ester exchange reaction using a cake is shown in Table 4.
Table 3
* Value is the amount of water (mass%) with respect to the mass of components excluding the oily component contained in the lipase-containing composition
Table 4
* Value: POS + SOS value of lipase containing composition
As a result, it was most effective when left at 50 ° C. and 70% relative humidity for 1 day. When left at 60 ° C. and 70% relative humidity for 1 day, good activity was obtained regardless of the thickness of the lipase-containing composition in the container, but otherwise the effect of thickness was observed. It was found that it is preferable to set the relative humidity to 90% at 60 ° C. in order to obtain a good effect after leaving for 1 hour.
リパーゼろ過ケーキA10gをガラスシャーレ上に広げ、30℃、40℃、50℃及び60℃の各温度;50%、70%及び90%の相対湿度の湿潤気体(湿潤空気)雰囲気下に1時間又は1日間保持した。リパーゼ含有組成物中に含まれる油性成分を除く成分の質量に対する水分量(質量%)を表3に示す。その後、リパーゼろ過ケーキを回収して上述したエステル交換反応を1回行った。このときの吸湿保持前のリパーゼろ過ケーキAを使用したエステル交換反応において、1時間反応した後に得られたトリグリセリド中のPOS及びSOSの合計質量%を100とした場合における、吸湿保持後のリパーゼろ過ケーキを使用したエステル交換反応において、1時間反応した後に得られたトリグリセリド中のPOS及びSOSの合計質量%の相対量(POS+SOS値)を表4に示す。
表3
*値は、リパーゼ含有組成物中に含まれる油性成分を除く成分の質量に対する水分量(質量%)
表4
*値は、リパーゼ含有組成物のPOS+SOS値
この結果、50℃で相対湿度70%に1日放置した場合が最も効果的であった。60℃で相対湿度70%に1日放置した場合、リパーゼ含有組成物の容器内の厚さに関係なく良好な活性が得られたが、その他では厚さによる影響が観察された。1時間の放置時間で良好な効果を出すためには、60℃で相対湿度90%とすることが好ましいことがわかった。 Example 2
10 g of lipase filter cake A is spread on a glass petri dish, and each temperature of 30 ° C., 40 ° C., 50 ° C. and 60 ° C .; 50 hours, 70% and 90% relative humidity in wet gas (wet air) atmosphere or 1 hour It was kept for 1 day. The water content (% by mass) based on the mass of the components excluding the oil-based component contained in the lipase-containing composition is shown in Table 3. Thereafter, the lipase filter cake was recovered and subjected to the above-described transesterification reaction once. In transesterification using lipase filter cake A before moisture absorption retention at this time, lipase filtration after moisture absorption retention when the total mass% of POS and SOS in triglyceride obtained after reacting for 1 hour is 100 The relative amount (POS + SOS value) of the total mass% of POS and SOS in triglyceride obtained after reacting for 1 hour in the ester exchange reaction using a cake is shown in Table 4.
Table 3
* Value is the amount of water (mass%) with respect to the mass of components excluding the oily component contained in the lipase-containing composition
Table 4
* Value: POS + SOS value of lipase containing composition
As a result, it was most effective when left at 50 ° C. and 70% relative humidity for 1 day. When left at 60 ° C. and 70% relative humidity for 1 day, good activity was obtained regardless of the thickness of the lipase-containing composition in the container, but otherwise the effect of thickness was observed. It was found that it is preferable to set the relative humidity to 90% at 60 ° C. in order to obtain a good effect after leaving for 1 hour.
[実施例3]
リパーゼろ過ケーキA4gを前述の反応基質(ステアリン酸エチル:ハイオレイックヒマワリ油=6:4(質量比))400gに添加し、50℃で18時間反応した。反応終了後、加圧濾過器(アドバンテック KST-142-JA)を用いて乾燥窒素ガスによって加圧ろ過し(加圧条件0.1MPa)、反応済ろ過ケーキを得た(比較対照のろ過ケーキ)。さらに、乾燥窒素ガスを通気し(最大5分)、反応済ろ過ケーキ中の油性成分を除去し、試行3-1~3-6の通気済ろ過ケーキを得た。この通気済ろ過ケーキの油性成分量を、通気済ろ過ケーキ(リパーゼ含有組成物)中に含まれる油性成分の質量が該ろ過ケーキ中の油性成分を除く成分の質量に対して何倍であるかを基準として測定した。また、この通気済ろ過ケーキの水分量を、吸湿済ろ過ケーキ(リパーゼ含有組成物)中に含まれる油性成分を除く成分の質量に対する水分の質量(質量%)を基準として測定した。上記の方法で油性成分量の調整を行った通気済ろ過ケーキをそれぞれガラスシャーレ上に広げ、50℃、相対湿度70%(湿潤空気)の雰囲気下で1日保管した。吸湿保持前の通気済ろ過ケーキを使用したエステル交換反応において、1時間反応した後に得られたトリグリセリド中のPOS及びSOSの合計質量%を100とした場合における、吸湿保持後のリパーゼろ過ケーキを使用したエステル交換反応において、1時間反応した後に得られたトリグリセリド中のPOS及びSOSの合計質量%の相対量(POS+SOS値)を表5に示す。
表5
*油性成分量は、比較対照及び試行3-1~3-6のろ過ケーキ(リパーゼ含有組成物)中に含まれる油性成分の質量が該ろ過ケーキ中の油性成分を除く成分の質量に対して何倍かを示す値
リパーゼ粉末製剤含有ろ過ケーキ中の油性成分量が少ないほうが、吸湿による再活性化度合いが高かった。 [Example 3]
4 g of lipase filter cake A was added to 400 g of the above reaction substrate (ethyl stearate: hyoleic sunflower oil = 6: 4 (mass ratio)) and reacted at 50 ° C. for 18 hours. After completion of the reaction, pressure filtration was performed with dry nitrogen gas using a pressure filter (ADVANTEC KST-142-JA) (pressure condition 0.1 MPa) to obtain a reacted filter cake (filter cake for comparison) . Furthermore, dry nitrogen gas was passed through (maximum 5 minutes) to remove the oily component in the reacted filter cake, to obtain trials 3-1 to 3-6 of aerated filter cake. The amount of the oil component of the aerated filter cake is such that the mass of the oil component contained in the aerated filter cake (lipase-containing composition) is multiple times the mass of the component excluding the oil component in the filter cake It measured on the basis of. Moreover, the water content of this aerated filter cake was measured based on the mass (mass%) of the water with respect to the mass of the component except the oil-based component contained in a hygroscopic filter cake (lipase containing composition). The aerated filter cakes adjusted in the amount of the oil component by the above method were each spread on a glass petri dish, and stored at 50 ° C. under an atmosphere of 70% relative humidity (wet air) for 1 day. In transesterification using aerated filter cake before moisture absorption, use lipase filter cake after moisture absorption when the total mass% of POS and SOS in triglyceride obtained after reaction for 1 hour is 100 Table 5 shows the relative amounts (POS + SOS value) of the total mass% of POS and SOS in triglycerides obtained after the reaction for 1 hour in the transesterification reaction.
Table 5
* The amount of the oily component is based on the weight of the component excluding the oily component in the filter cake, the mass of the oily component contained in the filter cake (composition containing lipase) of comparative control and trial 3-1 to 3-6. A value indicating how many times
The lower the amount of the oil component in the lipase powder-containing filter cake, the higher the degree of reactivation due to moisture absorption.
リパーゼろ過ケーキA4gを前述の反応基質(ステアリン酸エチル:ハイオレイックヒマワリ油=6:4(質量比))400gに添加し、50℃で18時間反応した。反応終了後、加圧濾過器(アドバンテック KST-142-JA)を用いて乾燥窒素ガスによって加圧ろ過し(加圧条件0.1MPa)、反応済ろ過ケーキを得た(比較対照のろ過ケーキ)。さらに、乾燥窒素ガスを通気し(最大5分)、反応済ろ過ケーキ中の油性成分を除去し、試行3-1~3-6の通気済ろ過ケーキを得た。この通気済ろ過ケーキの油性成分量を、通気済ろ過ケーキ(リパーゼ含有組成物)中に含まれる油性成分の質量が該ろ過ケーキ中の油性成分を除く成分の質量に対して何倍であるかを基準として測定した。また、この通気済ろ過ケーキの水分量を、吸湿済ろ過ケーキ(リパーゼ含有組成物)中に含まれる油性成分を除く成分の質量に対する水分の質量(質量%)を基準として測定した。上記の方法で油性成分量の調整を行った通気済ろ過ケーキをそれぞれガラスシャーレ上に広げ、50℃、相対湿度70%(湿潤空気)の雰囲気下で1日保管した。吸湿保持前の通気済ろ過ケーキを使用したエステル交換反応において、1時間反応した後に得られたトリグリセリド中のPOS及びSOSの合計質量%を100とした場合における、吸湿保持後のリパーゼろ過ケーキを使用したエステル交換反応において、1時間反応した後に得られたトリグリセリド中のPOS及びSOSの合計質量%の相対量(POS+SOS値)を表5に示す。
表5
*油性成分量は、比較対照及び試行3-1~3-6のろ過ケーキ(リパーゼ含有組成物)中に含まれる油性成分の質量が該ろ過ケーキ中の油性成分を除く成分の質量に対して何倍かを示す値
リパーゼ粉末製剤含有ろ過ケーキ中の油性成分量が少ないほうが、吸湿による再活性化度合いが高かった。 [Example 3]
4 g of lipase filter cake A was added to 400 g of the above reaction substrate (ethyl stearate: hyoleic sunflower oil = 6: 4 (mass ratio)) and reacted at 50 ° C. for 18 hours. After completion of the reaction, pressure filtration was performed with dry nitrogen gas using a pressure filter (ADVANTEC KST-142-JA) (pressure condition 0.1 MPa) to obtain a reacted filter cake (filter cake for comparison) . Furthermore, dry nitrogen gas was passed through (maximum 5 minutes) to remove the oily component in the reacted filter cake, to obtain trials 3-1 to 3-6 of aerated filter cake. The amount of the oil component of the aerated filter cake is such that the mass of the oil component contained in the aerated filter cake (lipase-containing composition) is multiple times the mass of the component excluding the oil component in the filter cake It measured on the basis of. Moreover, the water content of this aerated filter cake was measured based on the mass (mass%) of the water with respect to the mass of the component except the oil-based component contained in a hygroscopic filter cake (lipase containing composition). The aerated filter cakes adjusted in the amount of the oil component by the above method were each spread on a glass petri dish, and stored at 50 ° C. under an atmosphere of 70% relative humidity (wet air) for 1 day. In transesterification using aerated filter cake before moisture absorption, use lipase filter cake after moisture absorption when the total mass% of POS and SOS in triglyceride obtained after reaction for 1 hour is 100 Table 5 shows the relative amounts (POS + SOS value) of the total mass% of POS and SOS in triglycerides obtained after the reaction for 1 hour in the transesterification reaction.
Table 5
* The amount of the oily component is based on the weight of the component excluding the oily component in the filter cake, the mass of the oily component contained in the filter cake (composition containing lipase) of comparative control and trial 3-1 to 3-6. A value indicating how many times
The lower the amount of the oil component in the lipase powder-containing filter cake, the higher the degree of reactivation due to moisture absorption.
[実施例4]
リパーゼろ過ケーキA6gを前述の反応基質(ステアリン酸エチル:ハイオレイックヒマワリ油=6:4(質量比))300gに添加し、50℃で16時間反応した。反応終了後、加圧濾過器(アドバンテック KST-90)を用いて乾燥窒素ガスによって加圧ろ過し(加圧条件0.1MPa)、反応済ろ過ケーキを得た(比較対照のろ過ケーキ)。得られた反応済ろ過ケーキをガラスシャーレ上に広げ、25℃、相対湿度50%(湿潤空気)の雰囲気下で1時間保管するか(試行4-1)、もしくはろ過器に真空ポンプを接続し、反応済ろ過ケーキに、25℃、相対湿度50%の湿潤空気を10分間(試行4-2)、又は30℃、相対湿度70%の湿潤空気を10分間(試行4-3)通気した。この吸湿済ろ過ケーキの油性成分量を、吸湿済ろ過ケーキ(リパーゼ含有組成物)中に含まれる油性成分の質量が該ろ過ケーキ中の油性成分を除く成分の質量に対して何倍であるかを基準として測定した。また、この吸湿済ろ過ケーキの水分量を、吸湿済ろ過ケーキ(リパーゼ含有組成物)中に含まれる油性成分を除く成分の質量に対する水分の質量(質量%)を基準として測定した。吸湿保持前の反応済ろ過ケーキを使用したエステル交換反応において、1時間反応した後に得られたトリグリセリド中のPOS及びSOSの合計質量%を100とした場合における、吸湿保持後のリパーゼろ過ケーキを使用したエステル交換反応において、1時間反応した後に得られたトリグリセリド中のPOS及びSOSの合計質量%の相対量(POS+SOS値)を表6に示す。
表6
*油性成分量は、比較対照及び試行4-1~4-3のろ過ケーキ(リパーゼ含有組成物)中に含まれる油性成分の質量が該ろ過ケーキ中の油性成分を除く成分の質量に対して何倍かを示す値
加湿した空気を通気することによる吸湿の効果は、放置よりも短時間で見られ、またその度合いは温度・湿度を上昇させることで更に上昇した。 Example 4
6 g of lipase filter cake A was added to 300 g of the above reaction substrate (ethyl stearate: hyoleic sunflower oil = 6: 4 (mass ratio)), and reacted at 50 ° C. for 16 hours. After completion of the reaction, pressure filtration was performed with dry nitrogen gas using a pressure filter (ADVANTEC KST-90) (pressure condition 0.1 MPa) to obtain a reacted filter cake (filter cake for comparison). The obtained reacted filter cake is spread on a glass petri dish, and stored at 25 ° C. under an atmosphere of 50% relative humidity (wet air) for 1 hour (trial 4-1), or a vacuum pump is connected to the filter. The reacted filter cake was aerated with moist air at 25 ° C. and 50% relative humidity for 10 minutes (trial 4-2), or wet air at 30 ° C. and relative humidity 70% for 10 minutes (try 4-3). The amount of the oil component of the absorbed filter cake is determined by multiplying the mass of the oil component contained in the absorbed filter cake (lipase-containing composition) with respect to the mass of the components excluding the oil component in the filter cake It measured on the basis of. In addition, the moisture content of this moisture absorbed filter cake was measured based on the mass (mass%) of moisture relative to the mass of the components excluding the oil component contained in the moisture absorbed filter cake (lipase-containing composition). In transesterification using a reacted filter cake before moisture absorption retention, use lipase filter cake after moisture absorption retention when the total mass% of POS and SOS in triglyceride obtained after reaction for 1 hour is 100 Table 6 shows the relative amounts (POS + SOS value) of the total mass% of POS and SOS in triglyceride obtained after reacting for 1 hour in the transesterification reaction.
Table 6
* The amount of the oily component is based on the weight of the component excluding the oily component in the filter cake, the mass of the oily component contained in the filter cake (composition containing lipase) of the comparative control and trials 4-1 to 4-3. A value indicating how many times
The effect of moisture absorption by ventilating humidified air was seen in a shorter time than leaving, and the degree was further increased by raising the temperature and humidity.
リパーゼろ過ケーキA6gを前述の反応基質(ステアリン酸エチル:ハイオレイックヒマワリ油=6:4(質量比))300gに添加し、50℃で16時間反応した。反応終了後、加圧濾過器(アドバンテック KST-90)を用いて乾燥窒素ガスによって加圧ろ過し(加圧条件0.1MPa)、反応済ろ過ケーキを得た(比較対照のろ過ケーキ)。得られた反応済ろ過ケーキをガラスシャーレ上に広げ、25℃、相対湿度50%(湿潤空気)の雰囲気下で1時間保管するか(試行4-1)、もしくはろ過器に真空ポンプを接続し、反応済ろ過ケーキに、25℃、相対湿度50%の湿潤空気を10分間(試行4-2)、又は30℃、相対湿度70%の湿潤空気を10分間(試行4-3)通気した。この吸湿済ろ過ケーキの油性成分量を、吸湿済ろ過ケーキ(リパーゼ含有組成物)中に含まれる油性成分の質量が該ろ過ケーキ中の油性成分を除く成分の質量に対して何倍であるかを基準として測定した。また、この吸湿済ろ過ケーキの水分量を、吸湿済ろ過ケーキ(リパーゼ含有組成物)中に含まれる油性成分を除く成分の質量に対する水分の質量(質量%)を基準として測定した。吸湿保持前の反応済ろ過ケーキを使用したエステル交換反応において、1時間反応した後に得られたトリグリセリド中のPOS及びSOSの合計質量%を100とした場合における、吸湿保持後のリパーゼろ過ケーキを使用したエステル交換反応において、1時間反応した後に得られたトリグリセリド中のPOS及びSOSの合計質量%の相対量(POS+SOS値)を表6に示す。
表6
*油性成分量は、比較対照及び試行4-1~4-3のろ過ケーキ(リパーゼ含有組成物)中に含まれる油性成分の質量が該ろ過ケーキ中の油性成分を除く成分の質量に対して何倍かを示す値
加湿した空気を通気することによる吸湿の効果は、放置よりも短時間で見られ、またその度合いは温度・湿度を上昇させることで更に上昇した。 Example 4
6 g of lipase filter cake A was added to 300 g of the above reaction substrate (ethyl stearate: hyoleic sunflower oil = 6: 4 (mass ratio)), and reacted at 50 ° C. for 16 hours. After completion of the reaction, pressure filtration was performed with dry nitrogen gas using a pressure filter (ADVANTEC KST-90) (pressure condition 0.1 MPa) to obtain a reacted filter cake (filter cake for comparison). The obtained reacted filter cake is spread on a glass petri dish, and stored at 25 ° C. under an atmosphere of 50% relative humidity (wet air) for 1 hour (trial 4-1), or a vacuum pump is connected to the filter. The reacted filter cake was aerated with moist air at 25 ° C. and 50% relative humidity for 10 minutes (trial 4-2), or wet air at 30 ° C. and relative humidity 70% for 10 minutes (try 4-3). The amount of the oil component of the absorbed filter cake is determined by multiplying the mass of the oil component contained in the absorbed filter cake (lipase-containing composition) with respect to the mass of the components excluding the oil component in the filter cake It measured on the basis of. In addition, the moisture content of this moisture absorbed filter cake was measured based on the mass (mass%) of moisture relative to the mass of the components excluding the oil component contained in the moisture absorbed filter cake (lipase-containing composition). In transesterification using a reacted filter cake before moisture absorption retention, use lipase filter cake after moisture absorption retention when the total mass% of POS and SOS in triglyceride obtained after reaction for 1 hour is 100 Table 6 shows the relative amounts (POS + SOS value) of the total mass% of POS and SOS in triglyceride obtained after reacting for 1 hour in the transesterification reaction.
Table 6
* The amount of the oily component is based on the weight of the component excluding the oily component in the filter cake, the mass of the oily component contained in the filter cake (composition containing lipase) of the comparative control and trials 4-1 to 4-3. A value indicating how many times
The effect of moisture absorption by ventilating humidified air was seen in a shorter time than leaving, and the degree was further increased by raising the temperature and humidity.
Claims (12)
- リパーゼ含有組成物を湿潤気体に接触する吸湿工程を含む、リパーゼ活性の回復方法。 A method of recovering lipase activity, comprising a step of absorbing moisture in which the lipase-containing composition is contacted with a wet gas.
- 前記湿潤気体が、相対湿度50~90%である、請求項1に記載の方法。 The method of claim 1, wherein the humidified gas is at 50-90% relative humidity.
- リパーゼ含有組成物中に含まれる水分量が、前記リパーゼ含有組成物中の油性成分を除く成分中8.5~20質量%となるまで前記吸湿工程を行う、請求項1又は2に記載の方法。 The method according to claim 1 or 2, wherein the moisture absorption step is carried out until the amount of water contained in the lipase-containing composition is 8.5 to 20% by mass in the components excluding the oil component in the lipase-containing composition. .
- 前記吸湿工程が、相対湿度50~90%、温度30~60℃、保持時間0.5~36時間を満たす条件下で行われる、請求項1~3のいずれか1項に記載の方法。 The method according to any one of claims 1 to 3, wherein the moisture absorption step is performed under the conditions of a relative humidity of 50 to 90%, a temperature of 30 to 60 属 C, and a holding time of 0.5 to 36 hours.
- 前記吸湿工程が、前記湿潤気体を前記リパーゼ含有組成物中に通気させることを特徴とする請求項1~4のいずれか1項に記載の方法。 The method according to any one of claims 1 to 4, wherein the moisture absorption step comprises aerating the moist gas into the lipase-containing composition.
- 前記吸湿工程の前に、前記リパーゼ含有組成物中に含まれる油性成分の質量を、前記リパーゼ含有組成物中の油性成分を除く成分の質量に対して1.5倍以下に調節する油性成分調節工程を含む、請求項1~5のいずれか1項に記載の方法。 Oil component adjustment to adjust the mass of the oil component contained in the lipase-containing composition to not more than 1.5 times the mass of the component excluding the oil component in the lipase-containing composition before the moisture absorption step The method according to any one of claims 1 to 5, comprising a step.
- 前記吸湿工程が、前記湿潤気体を前記リパーゼ含有組成物に通気させることにより、前記リパーゼ含有組成物に含まれる油性成分量を低下させるとともに、前記リパーゼ含有組成物に対して吸湿を行なうことを特徴とする請求項1~5のいずれか1項に記載の方法。 The moisture absorption step reduces the amount of the oil component contained in the lipase-containing composition by aerating the moist gas into the lipase-containing composition, and performs moisture absorption on the lipase-containing composition. The method according to any one of claims 1 to 5, wherein
- 前記リパーゼ含有組成物が、粉末リパーゼ及びろ過助剤を含む、請求項1~7のいずれか1項に記載の方法。 The method according to any one of claims 1 to 7, wherein the lipase-containing composition comprises a powdered lipase and a filter aid.
- 前記リパーゼが、リゾプス オリザエ(Rhizopus oryzae)由来のリパーゼである、請求項1~8のいずれか1項に記載の方法。 The method according to any one of claims 1 to 8, wherein the lipase is a lipase derived from Rhizopus oryzae.
- 前記ろ過助剤が、セルロースである、請求項8に記載の方法。 9. The method of claim 8, wherein the filter aid is cellulose.
- 活性が低下したリパーゼを準備する工程、及び
請求項1~10のいずれか1項に記載の方法により前記活性が低下したリパーゼの活性を回復する工程、
を含む、活性が回復されたリパーゼの製造方法。 Preparing a lipase with reduced activity, and recovering the activity of the reduced lipase by the method according to any one of claims 1 to 10,
A process for producing a lipase with restored activity, comprising - 請求項11に記載の方法により製造された活性が回復されたリパーゼを用いてエステル化又はエステル交換することを特徴とする、エステル合成物の製造方法。 A process for producing an ester compound, which comprises esterification or transesterification using the lipase with recovered activity produced by the method according to claim 11.
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CN201280040264.9A CN103732741B (en) | 2011-08-19 | 2012-08-20 | The restoration methods of lipase activity |
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JP2011179720A JP5258941B2 (en) | 2011-08-19 | 2011-08-19 | Recovery method of lipase activity |
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JPS5840086A (en) * | 1981-08-31 | 1983-03-08 | Fuji Oil Co Ltd | Production of enzyme preparation |
JPS61149084A (en) * | 1984-12-21 | 1986-07-07 | Kao Corp | Method of activating enzyme |
JPH05137574A (en) * | 1991-11-21 | 1993-06-01 | Nisshin Oil Mills Ltd:The | Method for reactivating immobilized lipase |
JPH0956379A (en) * | 1995-08-18 | 1997-03-04 | Fuji Spinning Co Ltd | Regeneration of immobilized lipase |
JPH1175834A (en) * | 1997-09-16 | 1999-03-23 | Asahi Denka Kogyo Kk | Reactivation of immobilized lipase |
JP2006325465A (en) * | 2005-05-25 | 2006-12-07 | Nisshin Oillio Group Ltd | Composition containing lipase for functional group exchange reaction, method of producing the same and use of the same |
WO2007132775A1 (en) * | 2006-05-11 | 2007-11-22 | The Nisshin Oillio Group, Ltd. | Method for recovery of lipase activity |
WO2008114656A1 (en) * | 2007-03-16 | 2008-09-25 | The Nisshin Oillio Group, Ltd. | Powdery lipase preparation, method for production thereof, and use thereof |
-
2011
- 2011-08-19 JP JP2011179720A patent/JP5258941B2/en active Active
-
2012
- 2012-08-20 WO PCT/JP2012/070983 patent/WO2013027697A1/en active Application Filing
- 2012-08-20 CN CN201280040264.9A patent/CN103732741B/en not_active Expired - Fee Related
- 2012-08-20 MY MYPI2014000436A patent/MY162044A/en unknown
Patent Citations (8)
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JPS5840086A (en) * | 1981-08-31 | 1983-03-08 | Fuji Oil Co Ltd | Production of enzyme preparation |
JPS61149084A (en) * | 1984-12-21 | 1986-07-07 | Kao Corp | Method of activating enzyme |
JPH05137574A (en) * | 1991-11-21 | 1993-06-01 | Nisshin Oil Mills Ltd:The | Method for reactivating immobilized lipase |
JPH0956379A (en) * | 1995-08-18 | 1997-03-04 | Fuji Spinning Co Ltd | Regeneration of immobilized lipase |
JPH1175834A (en) * | 1997-09-16 | 1999-03-23 | Asahi Denka Kogyo Kk | Reactivation of immobilized lipase |
JP2006325465A (en) * | 2005-05-25 | 2006-12-07 | Nisshin Oillio Group Ltd | Composition containing lipase for functional group exchange reaction, method of producing the same and use of the same |
WO2007132775A1 (en) * | 2006-05-11 | 2007-11-22 | The Nisshin Oillio Group, Ltd. | Method for recovery of lipase activity |
WO2008114656A1 (en) * | 2007-03-16 | 2008-09-25 | The Nisshin Oillio Group, Ltd. | Powdery lipase preparation, method for production thereof, and use thereof |
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CN103732741B (en) | 2016-04-20 |
MY162044A (en) | 2017-05-31 |
JP2013039109A (en) | 2013-02-28 |
CN103732741A (en) | 2014-04-16 |
JP5258941B2 (en) | 2013-08-07 |
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