JP2008043274A - Immobilized lipase and method for producing the same - Google Patents
Immobilized lipase and method for producing the same Download PDFInfo
- Publication number
- JP2008043274A JP2008043274A JP2006223031A JP2006223031A JP2008043274A JP 2008043274 A JP2008043274 A JP 2008043274A JP 2006223031 A JP2006223031 A JP 2006223031A JP 2006223031 A JP2006223031 A JP 2006223031A JP 2008043274 A JP2008043274 A JP 2008043274A
- Authority
- JP
- Japan
- Prior art keywords
- lipase
- transesterification
- silica
- immobilized lipase
- immobilized
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 239000004367 Lipase Substances 0.000 title claims abstract description 161
- 102000004882 Lipase Human genes 0.000 title claims abstract description 161
- 108090001060 Lipase Proteins 0.000 title claims abstract description 161
- 235000019421 lipase Nutrition 0.000 title claims abstract description 161
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 33
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 151
- 239000002245 particle Substances 0.000 claims abstract description 99
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 52
- 239000005909 Kieselgur Substances 0.000 claims abstract description 45
- 239000003921 oil Substances 0.000 claims description 64
- 239000003925 fat Substances 0.000 claims description 47
- 239000000843 powder Substances 0.000 claims description 38
- 238000000034 method Methods 0.000 claims description 35
- 238000003756 stirring Methods 0.000 claims description 11
- 230000000694 effects Effects 0.000 abstract description 57
- 235000019626 lipase activity Nutrition 0.000 abstract description 16
- 238000005809 transesterification reaction Methods 0.000 description 74
- 235000019198 oils Nutrition 0.000 description 61
- 235000019197 fats Nutrition 0.000 description 44
- 238000006243 chemical reaction Methods 0.000 description 34
- 238000011084 recovery Methods 0.000 description 16
- 230000000052 comparative effect Effects 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 238000009826 distribution Methods 0.000 description 7
- 238000001914 filtration Methods 0.000 description 6
- 230000000977 initiatory effect Effects 0.000 description 6
- PVNIQBQSYATKKL-UHFFFAOYSA-N tripalmitin Chemical compound CCCCCCCCCCCCCCCC(=O)OCC(OC(=O)CCCCCCCCCCCCCCC)COC(=O)CCCCCCCCCCCCCCC PVNIQBQSYATKKL-UHFFFAOYSA-N 0.000 description 6
- 238000001035 drying Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 239000002994 raw material Substances 0.000 description 5
- 229960001947 tripalmitin Drugs 0.000 description 5
- 239000007788 liquid Substances 0.000 description 4
- 239000012299 nitrogen atmosphere Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- PHYFQTYBJUILEZ-IUPFWZBJSA-N triolein Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC(OC(=O)CCCCCCC\C=C/CCCCCCCC)COC(=O)CCCCCCC\C=C/CCCCCCCC PHYFQTYBJUILEZ-IUPFWZBJSA-N 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108010093096 Immobilized Enzymes Proteins 0.000 description 2
- 235000010724 Wisteria floribunda Nutrition 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 235000014593 oils and fats Nutrition 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 238000010298 pulverizing process Methods 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 235000012239 silicon dioxide Nutrition 0.000 description 2
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- 241000588986 Alcaligenes Species 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 235000004936 Bromus mango Nutrition 0.000 description 1
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 240000007228 Mangifera indica Species 0.000 description 1
- 235000014826 Mangifera indica Nutrition 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000235395 Mucor Species 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 235000019482 Palm oil Nutrition 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 235000019484 Rapeseed oil Nutrition 0.000 description 1
- 241000235527 Rhizopus Species 0.000 description 1
- 235000019485 Safflower oil Nutrition 0.000 description 1
- 235000009184 Spondias indica Nutrition 0.000 description 1
- 235000019486 Sunflower oil Nutrition 0.000 description 1
- 235000018936 Vitellaria paradoxa Nutrition 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 239000010775 animal oil Substances 0.000 description 1
- 239000003957 anion exchange resin Substances 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 239000012752 auxiliary agent Substances 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 239000000828 canola oil Substances 0.000 description 1
- 235000019519 canola oil Nutrition 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 239000003729 cation exchange resin Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 239000013522 chelant Substances 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 235000019868 cocoa butter Nutrition 0.000 description 1
- 229940110456 cocoa butter Drugs 0.000 description 1
- 239000003240 coconut oil Substances 0.000 description 1
- 235000019864 coconut oil Nutrition 0.000 description 1
- 235000005687 corn oil Nutrition 0.000 description 1
- 239000002285 corn oil Substances 0.000 description 1
- 235000012343 cottonseed oil Nutrition 0.000 description 1
- 239000002385 cottonseed oil Substances 0.000 description 1
- GVJHHUAWPYXKBD-UHFFFAOYSA-N d-alpha-tocopherol Natural products OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-UHFFFAOYSA-N 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000010419 fine particle Substances 0.000 description 1
- 235000021323 fish oil Nutrition 0.000 description 1
- 238000005984 hydrogenation reaction Methods 0.000 description 1
- 239000012051 hydrophobic carrier Substances 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 235000021243 milk fat Nutrition 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 239000004006 olive oil Substances 0.000 description 1
- 235000008390 olive oil Nutrition 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 239000003346 palm kernel oil Substances 0.000 description 1
- 235000019865 palm kernel oil Nutrition 0.000 description 1
- 239000002540 palm oil Substances 0.000 description 1
- 239000005011 phenolic resin Substances 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000011085 pressure filtration Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000002407 reforming Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 235000005713 safflower oil Nutrition 0.000 description 1
- 239000003813 safflower oil Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 239000003549 soybean oil Substances 0.000 description 1
- 235000012424 soybean oil Nutrition 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 239000002600 sunflower oil Substances 0.000 description 1
- 239000003760 tallow Substances 0.000 description 1
- 229960001295 tocopherol Drugs 0.000 description 1
- 229930003799 tocopherol Natural products 0.000 description 1
- 235000010384 tocopherol Nutrition 0.000 description 1
- 239000011732 tocopherol Substances 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- GVJHHUAWPYXKBD-IEOSBIPESA-N α-tocopherol Chemical compound OC1=C(C)C(C)=C2O[C@@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-IEOSBIPESA-N 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
本発明は、固定化リパーゼ及びその製造方法に関する。 The present invention relates to an immobilized lipase and a method for producing the same.
従来からリパーゼを触媒として使用した油脂のエステル交換反応によって油脂の改質を行なう方法が広く行なわれている。この油脂のエステル交換反応には回分反応と連続反応があり、また、リパーゼを固定化して使用する方法と粉末のまま使用する方法がある。
従来はリパーゼを何らかの担体、例えば陰イオン交換樹脂（特許文献１）、フェノール樹脂（特許文献２）、疎水性担体（特許文献３）、陽イオン交換樹脂（特許文献４）、キレート樹脂（特許文献５）等に固定化して使用する方法が行なわれてきたが、これらに記載の方法は、リパーゼを担体に固定することでリパーゼ活性が低下する欠点に加え、高価な担体を使用するためコストが高くなる欠点、さらには、リパーゼをいったん水溶液とした後に担体に含浸させ、後に乾燥する必要があるため、製造工程が煩雑であるという問題があった。
Conventionally, a method for reforming fats and oils by transesterification of fats and oils using lipase as a catalyst has been widely performed. This transesterification reaction of fats and oils includes a batch reaction and a continuous reaction, and there are a method in which lipase is immobilized and a method in which it is used as a powder.
Conventionally, lipase is used as a carrier such as anion exchange resin (Patent Document 1), phenol resin (Patent Document 2), hydrophobic carrier (Patent Document 3), cation exchange resin (Patent Document 4), chelate resin (Patent Document). 5) etc. have been used, but the methods described in these methods are costly because they use expensive carriers in addition to the disadvantage that the lipase activity is reduced by fixing the lipase to the carrier. In addition, there is a problem that the manufacturing process is complicated because it is necessary to make the lipase into an aqueous solution, impregnate the carrier, and then dry it.
一方、リパーゼを固定化せずに粉末のまま使用する方法も行なわれてきた。例えば、超音波処理などの方法で反応時の粉末リパーゼの粒径を９０％以上が１〜１００μｍとなるように調整して回分反応をおこなう方法や、濾過器内に粉末状リパーゼを単独または濾過助剤とともに層を形成するように封入し、その濾過器に油脂を通油させる方法（特許文献７）が提案されている。
しかしながら、これらの方法は、固定化して用いるよりリパーゼ活性は高まるものの、粉末リパーゼは極めて微細であるため、再利用のために回収する際に粉末リパーゼが流出して失われるため回収率が低く、エステル交換活性が短期間で低下してしまう問題があった。さらに、特許文献７の方法は、通油時の圧力損失が高く偏流が生じやすい問題もあった。
On the other hand, a method of using lipase as powder without immobilization has also been performed. For example, a method of performing batch reaction by adjusting the particle size of the powder lipase during the reaction so that 90% or more becomes 1 to 100 μm by a method such as sonication, or filtering powder lipase alone or in a filter There has been proposed a method (Patent Document 7) in which oil and fat are encapsulated so as to form a layer together with an auxiliary agent and oil is passed through the filter.
However, although these methods have higher lipase activity than that used by immobilization, the powder lipase is extremely fine, so that the recovery rate is low because the powder lipase flows out and is lost when recovered for reuse. There was a problem that the transesterification activity decreased in a short period of time. Furthermore, the method of Patent Document 7 has a problem that the pressure loss at the time of oil passing is high and the drift tends to occur.
従って、本発明の目的は、固定化によるリパーゼ活性の損失が少ないため、従来の固定化リパーゼに比べてエステル交換活性が高く、且つ長期にわたってエステル交換活性を高く保持することができる固定化リパーゼ、及びこのような固定化リパーゼを簡単な操作で製造することができる、固定化リパーゼの製造方法を提供することにある。 Accordingly, an object of the present invention is to provide an immobilized lipase that has a high transesterification activity compared to conventional immobilized lipases and that can maintain a high transesterification activity over a long period of time, since there is little loss of lipase activity due to immobilization Another object of the present invention is to provide a method for producing an immobilized lipase, which can produce such an immobilized lipase by a simple operation.
本発明者らは、固定化リパーゼの製造方法を検討する過程で、偶然にも、シリカも珪藻土も吸着力の弱い担体であるが、併用した場合には吸着力が改善されることを知見した。さらに検討をすすめたところ、リパーゼを水溶液として担体に吸着させる方法の場合は、シリカ単独、又は珪藻土単独であっても固定化が可能であるが、油脂中でリパーゼ粉末を担体に固定化する方法の場合は、おどろくべきことに、シリカ単独又は珪藻土単独では、ほとんど固定化されず、シリカ及び珪藻土を担体として併用してはじめて固定化が可能となることを知見した。そして、この固定化リパーゼの粒径を特定範囲とした場合、従来の固定化リパーゼに比べてエステル交換活性が高く、且つ長期にわたってエステル交換活性を高く保持することができることを知見した。 In the course of studying the method for producing an immobilized lipase, the present inventors, by chance, found that silica and diatomaceous earth are weakly adsorbing carriers, but when used together, the adsorbing power is improved. . As a result of further studies, in the case of a method for adsorbing lipase on a carrier as an aqueous solution, it is possible to immobilize silica alone or diatomaceous earth alone, but a method of immobilizing lipase powder on a carrier in oil or fat. In this case, surprisingly, it has been found that silica alone or diatomaceous earth is hardly immobilized, and that immobilization is possible only when silica and diatomaceous earth are used together as a carrier. And when the particle size of this fixed lipase was made into the specific range, it discovered that transesterification activity was high compared with the conventional fixed lipase, and transesterification activity could be kept high over a long period of time.
本発明は、上記知見に基づきなされたものであり、シリカ及び珪藻土を担体とし、粒径１〜１００μｍの粒子が９０％以上(体積基準）であり、且つ粒径０．４５μm以下の粒子が１％以下（個数基準）であることを特徴とする固定化リパーゼを提供することによって、上記目的を達成したものである。 The present invention has been made on the basis of the above-mentioned knowledge, and has silica and diatomaceous earth as a carrier, particles having a particle diameter of 1 to 100 μm are 90% or more (volume basis), and particles having a particle diameter of 0.45 μm or less are 1 The above-mentioned object is achieved by providing an immobilized lipase characterized by being not more than% (number basis).
また、本発明は、上記固定化リパーゼの製造方法であって、シリカ、珪藻土、及びリパーゼ粉末を、油脂中に分散し、撹拌することにより、シリカ及び珪藻土にリパーゼ粉末を固定化することを特徴とする固定化リパーゼの製造方法を提供することで上記目的を達成したものである。 Further, the present invention is a method for producing the above-described immobilized lipase, wherein silica, diatomaceous earth, and lipase powder are dispersed in oil and fat, and the lipase powder is immobilized on silica and diatomaceous earth by stirring. The above-mentioned object is achieved by providing a method for producing an immobilized lipase.
さらに、本発明は、上記固定化リパーゼを使用する油脂のエステル交換方法を提供することで上記目的を達成したものである。 Furthermore, this invention achieves the said objective by providing the transesterification method of the fats and oils which use the said fixed lipase.
本発明の固定化リパーゼは、固定化によるリパーゼ活性の損失が少ないため、従来の固定化リパーゼに比べてエステル交換活性が高く、且つ、反応・回収・再使用時の損失が少ないため、長期にわたってエステル交換活性を高く保持することができる。また、本発明の固定化リパーゼの製造方法によれば、固定化リパーゼの乾燥の工程を必要とせず、ただちにエステル交換反応に用いることができ、また、油脂への分散性が良好である。 Since the immobilized lipase of the present invention has a low loss of lipase activity due to immobilization, the transesterification activity is higher than that of the conventional immobilized lipase, and the loss during reaction, recovery, and reuse is small. High transesterification activity can be maintained. Moreover, according to the manufacturing method of the immobilized lipase of this invention, the process of drying an immobilized lipase is not required, but it can use for transesterification immediately and the dispersibility to fats and oils is favorable.
以下、本発明の固定化リパーゼについて詳細に説明する。
本発明で用いられるリパーゼとしては、特に限定されず、例えば、リゾプス属、ムコール属、アスペルギルス属、シュードモナス属、アルカリゲネス属、キャンディダ属等の微生物由来のもの、及び、動植物由来のものを挙げることができる。本発明においては、エステル交換能が最も高いことからアルカリゲネス属由来のリパーゼを使用することが好ましい。
Hereinafter, the immobilized lipase of the present invention will be described in detail.
The lipase used in the present invention is not particularly limited, and examples thereof include those derived from microorganisms such as Rhizopus genus, Mucor genus, Aspergillus genus, Pseudomonas genus, Alcaligenes genus, Candida genus, and those derived from animals and plants. Can do. In the present invention, it is preferable to use a lipase derived from the genus Alkaligenes because of its highest transesterification ability.
本発明の固定化リパーゼにおける、リパーゼの含有量は、求める活性のレベルや由来の違いにより一概には言えないが、好ましくは、固形分を基準として、２〜５０質量％が好ましく、５〜３０質量%がさらに好ましい。２質量％未満では得られる固定化リパーゼのエステル交換活性が低くなるので好ましくなく、５０質量％を超えるとリパーゼが脱離しやすくなってしまうので好ましくない。 The content of the lipase in the immobilized lipase of the present invention cannot be generally stated depending on the level of activity to be obtained and the difference in origin, but preferably 2 to 50% by mass, preferably 5 to 30%, based on the solid content. More preferred is mass%. If it is less than 2% by mass, the transesterification activity of the obtained immobilized lipase becomes low, which is not preferable. If it exceeds 50% by mass, the lipase tends to be detached, which is not preferable.
本発明の固定化リパーゼは、シリカ及び珪藻土を担体とし、リパーゼを該担体に担持させたものである。
担体が、シリカのみであるか又は珪藻土のみであると、本発明の効果は得られない。
まず本発明で使用するシリカについて述べる。
The immobilized lipase of the present invention comprises silica and diatomaceous earth as a carrier and the lipase supported on the carrier.
If the support is only silica or only diatomaceous earth, the effect of the present invention cannot be obtained.
First, the silica used in the present invention will be described.
シリカとは、合成二酸化珪素のことであり、連続的に網の目のような微細な孔を形成しており、微粉末状、及び粒子状のようなものが使用できる。
シリカは硬度が高く、固定化時の撹拌程度では微細化しないため、求める固定化リパーゼの粒度と同じ粒度を有するシリカを使用することが好ましい。すなわち本発明では、粒径１〜１００μｍの粒子が９０％以上(体積基準）であり、且つ粒径０．４５μm以下の粒子が１％以下（個数基準）であるシリカを用いることが好ましく、より好ましくは、粒径１〜１００μｍの粒子が９５％以上(体積基準）であり、且つ粒径０．４５μm以下の粒子が１％以下（個数基準）であるシリカを用いる。
Silica is synthetic silicon dioxide, which continuously forms fine pores such as meshes, and fine powder and particles can be used.
Since silica has high hardness and does not become fine by the degree of stirring during immobilization, it is preferable to use silica having the same particle size as the desired immobilized lipase. That is, in the present invention, it is preferable to use silica in which particles having a particle size of 1 to 100 μm are 90% or more (volume basis) and particles having a particle size of 0.45 μm or less are 1% or less (number basis). Preferably, silica having a particle size of 1 to 100 μm of 95% or more (volume basis) and a particle size of 0.45 μm or less of 1% or less (number basis) is used.
なお、本発明では、使用するリパーゼの至適ｐＨから１．０ｐＨ単位以上は偏位していないシリカを使用することが好ましい。リパーゼの至適ｐＨと１．０ｐＨ単位以上偏位したシリカを使用した場合は高活性な固定化リパーゼを得ることができない。 In the present invention, it is preferable to use silica in which 1.0 pH unit or more is not displaced from the optimum pH of the lipase to be used. When silica having a deviated optimum pH of lipase and 1.0 pH unit or more is used, highly active immobilized lipase cannot be obtained.
次に、本発明で使用する珪藻土について述べる。
珪藻土とは、二酸化珪素質の殻をもつ珪藻の化石を破砕、乾燥、焼成などの処理をしたものであり、シリカと異なり、多様・独特な構造と高次の微細な孔を有するものである。その粒径は最大で百数十μm、最小ではサブミクロン粒子も存在し、微粉末シリカよりは幅広い粒度分布を有するという特徴を有する。本発明では、粒径１〜１００μｍの粒子が９０％以上(体積基準）であり、且つ、粒径０．４５μm以下の粒子が１％以下（個数基準）である珪藻土を用いることが好ましく、より好ましくは、粒径１〜１００μｍの粒子が９５％以上(体積基準）、且つ粒径０．４５μm以下の粒子が１％以下（個数基準）である珪藻土を用いる。
Next, the diatomaceous earth used in the present invention will be described.
Diatomaceous earth is a diatom fossil with a silicon dioxide shell that has been crushed, dried, fired, etc., and unlike silica, it has a diverse and unique structure and high-order fine pores. . Its particle size is a few hundreds of μm at the maximum, submicron particles are also present at the minimum, and it has a feature that it has a wider particle size distribution than fine powder silica. In the present invention, it is preferable to use diatomaceous earth in which particles having a particle diameter of 1 to 100 μm are 90% or more (volume basis) and particles having a particle diameter of 0.45 μm or less are 1% or less (number basis), Preferably, diatomaceous earth in which particles having a particle size of 1 to 100 μm are 95% or more (volume basis) and particles having a particle size of 0.45 μm or less are 1% or less (number basis) is used.
この範囲外の珪藻土を使用すると、エステル交換活性が高く、長期にわたってエステル交換活性を高く保持することができる固定化リパーゼが得られにくい。例えば、１μｍ未満の粒子が１０％より多いと、固定化リパーゼの回収の際に超微細なメッシュの濾過装置を必要とし、その場合、すぐに目詰まりしてしまい、実質的に固定化リパーゼの回収、再利用が困難である。また、１００μｍを超える粒子が１０％より多いと、高いエステル交換活性を得ることができない。 When diatomaceous earth outside this range is used, it is difficult to obtain an immobilized lipase that has high transesterification activity and can maintain high transesterification activity over a long period of time. For example, if the number of particles less than 1 μm is more than 10%, an ultrafine mesh filtration device is required for the recovery of the immobilized lipase. Collection and reuse are difficult. Moreover, when there are more than 10% of particles exceeding 100 μm, high transesterification activity cannot be obtained.
本発明の固定化リパーゼは、上記シリカ及び珪藻土を合計した含有量が５０〜９８質量％であることが好ましく、より好ましくは７０〜９５質量％である。５０質量％未満であると、リパーゼの脱離が起こりやすく、９８質量％を超えると、相対的に酵素含量が減ってしまい、得られる固定化リパーゼの活性が低下してしまうため好ましくない。 In the immobilized lipase of the present invention, the total content of the silica and diatomaceous earth is preferably 50 to 98% by mass, more preferably 70 to 95% by mass. If it is less than 50% by mass, lipase is likely to be detached, and if it exceeds 98% by mass, the enzyme content is relatively reduced and the activity of the resulting immobilized lipase is undesirably reduced.
また、上記シリカ及び珪藻土の比率は、２０：８０〜８０：２０（質量比）であることが好ましく、より好ましくは３０：７０〜７０：３０（質量比）である。シリカの割合が２０％未満、又は、８０％を超えると、極端に固定化率が低下するので好ましくない。 Moreover, it is preferable that the ratio of the said silica and diatomaceous earth is 20: 80-80: 20 (mass ratio), More preferably, it is 30: 70-70: 30 (mass ratio). If the ratio of silica is less than 20% or exceeds 80%, the immobilization rate is extremely lowered, which is not preferable.
また、本発明の固定化リパーゼは、本発明の効果に影響のない限り、上記シリカ及び珪藻土以外の担体や、有機酸や脂肪酸などのリパーゼの活性化物質、さらには、トコフェロールなどの酸化防止剤などのその他の物質を含むものであってもよい。その他の物質の含有量は、好ましくは、２０質量％未満とすることが好ましく、より好ましくは１０質量％未満とする。 Further, the immobilized lipase of the present invention is a carrier other than the silica and diatomaceous earth, a lipase activator such as an organic acid or a fatty acid, and an antioxidant such as tocopherol, as long as the effects of the present invention are not affected. It may contain other substances such as. The content of other substances is preferably less than 20% by mass, and more preferably less than 10% by mass.
本発明の固定化リパーゼは、上記シリカ及び珪藻土を担体として、上記リパーゼを担持したものであって、粒径１〜１００μｍの粒子が９０％以上(体積基準）であり、且つ粒径０．４５μm以下の粒子が１％以下（個数基準）、好ましくは、粒径１〜１００μｍの粒子が９５％以上(体積基準）であり、且つ、粒径０．４５μm以下の粒子が１％以下（個数基準）である。この範囲外の粒径であると、エステル交換活性が高く、反応・回収・再使用時の損失がなく、長期にわたってエステル交換活性を高く保持することができる固定化リパーゼが得られない。 The immobilized lipase of the present invention carries the above lipase using the above silica and diatomaceous earth as a carrier, the particles having a particle size of 1 to 100 μm are 90% or more (volume basis), and the particle size is 0.45 μm. The following particles are 1% or less (number basis), preferably, particles having a particle size of 1 to 100 μm are 95% or more (volume basis), and particles having a particle size of 0.45 μm or less are 1% or less (number basis). ). If the particle size is outside this range, the transesterification activity is high, there is no loss during reaction, recovery and reuse, and an immobilized lipase that can maintain high transesterification activity over a long period of time cannot be obtained.
例えば、粒径１μｍ未満の粒子が１０％（体積基準）より多いか、又は粒径０．４５μm以下の粒子が１％（個数基準）を超えるような微細なものであると、固定化リパーゼの回収の際に超微細なメッシュの濾過装置を必要とし、その場合、すぐに目詰まりしてしまい、実質的に固定化リパーゼの回収、再利用が困難である。また、１００μｍを超える粒子が１０％より多いと、固定化時のリパーゼ活性の損失が多く、高いエステル交換活性を得ることができない。 For example, if the particles having a particle size of less than 1 μm are more than 10% (volume basis) or the particles having a particle size of 0.45 μm or less are more than 1% (number basis), the immobilized lipase An ultra-fine mesh filtration device is required for collection, and in this case, clogging occurs immediately, making it substantially difficult to collect and reuse the immobilized lipase. Moreover, when there are more than 10% of particles exceeding 100 μm, the loss of lipase activity at the time of immobilization is large, and high transesterification activity cannot be obtained.
次に本発明の固定化リパーゼの製造方法について述べる。
リパーゼを水溶液とし、これを、シリカ及び珪藻土の混合物に吸着させた後、乾燥、続いて粉末化する方法など公知の方法を採ることもできるが、本発明の固定化リパーゼは、シリカ、珪藻土及びリパーゼ粉末を、油脂中に分散し、撹拌することにより、シリカ及び珪藻土にリパーゼ粉末を固定化する方法を採ることが、固定化の際にダマを生じることなく、上記粒径の固定化リパーゼを容易に得ることができることに加え、固定化酵素の乾燥の工程を必要とせず、ただちに油脂のエステル交換反応に用いることができる点、さらには、得られた固定化リパーゼが極めて親油性の高い状態であるため、エステル交換時に原料油脂への分散性が極めて高く、簡単に分散させることが可能な点で好ましい。
Next, a method for producing the immobilized lipase of the present invention will be described.
A known method such as a method of making lipase into an aqueous solution and adsorbing the lipase to a mixture of silica and diatomaceous earth, followed by drying and then pulverizing can be adopted. However, the immobilized lipase of the present invention comprises silica, diatomaceous earth and The method of fixing the lipase powder to silica and diatomaceous earth by dispersing the lipase powder in oil and fat and stirring it is possible to fix the immobilized lipase having the above particle size without causing lumps during the fixation. In addition to being easily obtainable, it does not require a step of drying the immobilized enzyme and can be used immediately for transesterification of fats and oils. The obtained immobilized lipase is extremely lipophilic. Therefore, the dispersibility in the raw oil and fat is extremely high at the time of transesterification, which is preferable in that it can be easily dispersed.
そして、本発明の固定化リパーゼの製造方法においては、担体として、シリカ単独又は珪藻土単独ではほとんど固定化されず、担体として、シリカ及び珪藻土を併用してはじめて固定化が可能となるものである。 In the method for producing an immobilized lipase of the present invention, silica is hardly immobilized alone or diatomaceous earth alone as a carrier, and immobilization is possible only when silica and diatomaceous earth are used together as a carrier.
本発明の固定化リパーゼの製造方法では、リパーゼは粉末の形態で使用する。このリパーゼの粉末化方法は特に限定されず、例えば、リパーゼを含む微生物培養液をアセトン沈殿、精製、乾燥等の工程により粉末化したものを用いることができ、また、市販の粉末リパーゼを使用することができる。 In the method for producing an immobilized lipase of the present invention, the lipase is used in the form of a powder. The lipase pulverization method is not particularly limited. For example, a lipase-containing microorganism culture solution powdered by acetone precipitation, purification, drying, or the like can be used, or a commercially available powder lipase is used. be able to.
なお、上記リパーゼ粉末の粒径は、リパーゼ剤の種類にもよるが、粒径１〜１００μｍの粒子の占める割合が５０〜１００％（体積基準）であり、且つ粒径０．１〜０．５μｍの粒子の占める割合が８０％〜１００％（個数基準）であるような微細なものを使用することが好ましい。 In addition, although the particle size of the said lipase powder is based also on the kind of lipase agent, the ratio for which a particle | grain with a particle size of 1-100 micrometers occupies is 50-100% (volume basis), and particle size is 0.1-0. It is preferable to use a fine one in which the proportion of 5 μm particles is 80% to 100% (number basis).
本発明の固定化リパーゼの製造方法で使用する油脂は特に限定されず、例えばパーム油、大豆油、菜種油、コーン油、ヒマワリ油、サフラワー油、オリーブ油、キャノーラ油、綿実油、米油、カカオ脂、サル脂、シア脂、マンゴー核油、ヤシ油、パーム核油、牛脂、豚脂、魚油、乳脂等の各種植物油脂、動物油脂、並びにこれらを水素添加、及び／又は、エステル交換、及び／又は、分別処理を施した加工油脂から選ばれた１種又は２種以上を挙げることができる。 The fats and oils used in the method for producing the immobilized lipase of the present invention are not particularly limited. For example, palm oil, soybean oil, rapeseed oil, corn oil, sunflower oil, safflower oil, olive oil, canola oil, cottonseed oil, rice oil, cocoa butter , Monkey oil, shea fat, mango kernel oil, coconut oil, palm kernel oil, beef tallow, lard, fish oil, milk fat and other vegetable oils, animal oils, and hydrogenation and / or transesterification, and / or Or the 1 type (s) or 2 or more types chosen from the processing fats and oils which performed the classification process can be mentioned.
本発明の固定化リパーゼの製造方法では、エステル交換油脂を使用することが、固定化時の油脂の物性変化がほとんどないことから好ましい。
なお、本発明の油脂のエステル交換油脂を使用するに際しては、上記原料油脂の水分を常法により、１００〜１０００ｐｐｍに調整することが好ましく、より好ましくは２００〜５００ｐｐｍに調整する。１００ｐｐｍ未満であると、固定化率が低下してしまう問題があり、１０００ｐｐｍを超えると大きなダマが生成しやすくなり、その場合、エステル交換活性が大きく低下してしまう問題がある。
In the method for producing an immobilized lipase of the present invention, it is preferable to use transesterified oils and fats because there is almost no change in the physical properties of the oils and fats during immobilization.
In addition, when using the transesterified oil of fats and oils of this invention, it is preferable to adjust the water | moisture content of the said raw material fats and oils to 100-1000 ppm by a conventional method, More preferably, it adjusts to 200-500 ppm. If it is less than 100 ppm, there is a problem that the immobilization rate is reduced, and if it exceeds 1000 ppm, large lumps are likely to be generated. In this case, there is a problem that transesterification activity is greatly reduced.
なお、本発明の固定化リパーゼの製造方法で使用するシリカの粒度や、珪藻土の粒度、シリカと珪藻土の比率は上述のとおりである。 In addition, the particle size of the silica used by the manufacturing method of the fixed lipase of this invention, the particle size of diatomaceous earth, and the ratio of a silica and diatomaceous earth are as above-mentioned.
本発明の固定化リパーゼの製造方法では、リパーゼ粉末１重量部に対し、シリカ及び珪藻土を合計して１〜１９重量部の割合で添加することが好ましく、より好ましくは２．３〜１９重量部である。１重量部未満であると、リパーゼの脱離が起こりやすくなるという問題があり、１９重量部を超えると相対的に酵素含量が減ってしまい、得られる固定化リパーゼの活性が低下してしまうため好ましくない。 In the method for producing an immobilized lipase of the present invention, it is preferable to add silica and diatomaceous earth in a ratio of 1 to 19 parts by weight, more preferably 2.3 to 19 parts by weight, relative to 1 part by weight of the lipase powder. It is. If the amount is less than 1 part by weight, there is a problem that lipase is likely to be detached. If the amount exceeds 19 parts by weight, the enzyme content is relatively reduced, and the activity of the resulting immobilized lipase is decreased. It is not preferable.
また、本発明の固定化リパーゼの製造方法では、油脂１００重量部に対し、リパーゼ粉末、シリカ及び珪藻土を合計して０．５〜３０重量部の割合で添加することが好ましく、より好ましくは１〜２５重量部である。０．５重量部未満であると、固定化に長時間を要するため好ましくなく、３０重量部を超えると、流動性が低下し、固定化率が低下しやすくなるため好ましくない。 Moreover, in the manufacturing method of the fixed lipase of this invention, it is preferable to add lipase powder, a silica, and a diatomaceous earth in the ratio of 0.5-30 weight part with respect to 100 weight part of fats and oils, More preferably, 1 ~ 25 parts by weight. If it is less than 0.5 part by weight, it is not preferred because it takes a long time for immobilization, and if it exceeds 30 parts by weight, the fluidity is lowered and the immobilization rate is liable to be lowered.
以下、具体的な好ましい製造方法を述べる。
まず水分含有量を好ましくは１００〜１０００ｐｐｍに調整した油脂を加温し、溶解する。もちろん液状油を使用した場合は特に加温する必要はない。次いで、リパーゼ粉末、シリカ及び珪藻土を添加し、分散させ、パドル式撹拌機等を用いて好ましくは５０〜１０００ｒｐｍ、より好ましくは１００〜５００ｒｐｍで、好ましくは３０〜２００分撹拌する。
Hereinafter, specific preferred production methods will be described.
First, fats and oils whose water content is preferably adjusted to 100 to 1000 ppm are heated and dissolved. Of course, when liquid oil is used, it is not necessary to heat. Next, lipase powder, silica and diatomaceous earth are added and dispersed, and the mixture is stirred preferably at 50 to 1000 rpm, more preferably at 100 to 500 rpm, and preferably for 30 to 200 minutes using a paddle type stirrer or the like.
なお、本発明の固定化リパーゼの製造方法では、一度に上記リパーゼ粉末、シリカ及び珪藻土を添加、撹拌してもよいが、製造中のだまの発生を防止することが可能である点、及び、短時間で固定化酵素を得ることが可能な点で、まず、粉末リパーゼを添加、分散、撹拌した後、さらに、シリカ及び珪藻土を添加し、さらに撹拌する方法を採ることが好ましい。 In the method for producing an immobilized lipase of the present invention, the above lipase powder, silica and diatomaceous earth may be added and stirred at one time, but it is possible to prevent the occurrence of lumps during production, and From the viewpoint that an immobilized enzyme can be obtained in a short time, it is preferable to first employ a method in which powder lipase is added, dispersed and stirred, and then silica and diatomaceous earth are further added and further stirred.
この方法を採る場合、リパーゼ粉末を添加してからの撹拌は、好ましくはパドル式撹拌機、ホモミキサー等を用い、好ましくは５０〜１００００ｒｐｍ、より好ましくは１００〜５０００ｒｐｍで、好ましくは２０〜１８０分撹拌する。この撹拌により、リパーゼ粉末は微細化し、油脂中に均一に分散する。次にシリカ及び珪藻土を添加、分散した後、好ましくはパドル式撹拌機等を用い、好ましくは５０〜１０００ｒｐｍ、より好ましくは１００〜５００ｒｐｍで、好ましくは１０〜１８０分撹拌する。この撹拌により、先の撹拌により微細化し、均一に分散したリパーゼ粉末が、シリカ及び珪藻土に効率よく固定化され、遊離した微細なリパーゼ粉末が消失する。 In the case of adopting this method, stirring after adding the lipase powder is preferably performed using a paddle type stirrer, a homomixer or the like, preferably 50 to 10,000 rpm, more preferably 100 to 5000 rpm, preferably 20 to 180 minutes. Stir. By this stirring, the lipase powder is refined and uniformly dispersed in the fats and oils. Next, after adding and dispersing silica and diatomaceous earth, the mixture is preferably stirred using a paddle type stirrer or the like, preferably at 50 to 1000 rpm, more preferably at 100 to 500 rpm, and preferably for 10 to 180 minutes. By this stirring, the lipase powder that has been refined and uniformly dispersed by the previous stirring is efficiently fixed to silica and diatomaceous earth, and the released fine lipase powder disappears.
なお、固定化の完了を知るには、リパーゼ粉末を、油脂中で分散、撹拌した際の粒度分布（個数基準）をあらかじめ測定しておき、担体を添加、分散、撹拌後の粒度分布と比較し、粉末リパーゼに由来する微細粉末、特に粒径０．４５μｍ未満の微粒子が消失していることをもって簡単に確認することができる。なお、油脂中の粒度分布は、レーザー式粒度分布測定器などを用いて簡単に測定することができる。 In order to know the completion of immobilization, the particle size distribution (number basis) when the lipase powder is dispersed and stirred in fats and oils is measured in advance and compared with the particle size distribution after adding, dispersing and stirring the carrier. It can be easily confirmed by the disappearance of fine powder derived from powder lipase, particularly fine particles having a particle size of less than 0.45 μm. In addition, the particle size distribution in fats and oils can be easily measured using a laser type particle size distribution measuring instrument.
次に、密閉型加圧濾過、膜分離、遠心分離、有機溶媒による洗浄等の公知の方法で、油脂を濾別し、固定化リパーゼを得る。
なお、得られた固定化リパーゼは必要に応じ、乾燥してもよい。
Next, the fats and oils are separated by a known method such as hermetic pressure filtration, membrane separation, centrifugation, and washing with an organic solvent to obtain an immobilized lipase.
In addition, you may dry the obtained fixed lipase as needed.
上記のようにして得られた固定化リパーゼは、従来の固定化リパーゼとリパーゼ粉末の中間の粒度分布を示すため、従来の固定化リパーゼに比べ、固定化時の活性の損失が少ないため、エステル交換活性が高く、また、粉末リパーゼに比べ、反応・回収・再使用時の損失がなく、長期にわたってエステル交換活性を高く保持することができる。
さらに油脂中に分散して製造する本発明の固定化リパーゼの製造方法で得られた固定化リパーゼは、油脂中で製造されたものであるため、水分含量が極めて低く、そのため、水分が多いと反応が困難である、油脂のエステル交換反応に特に好ましく使用することができる。
The immobilized lipase obtained as described above exhibits an intermediate particle size distribution between the conventional immobilized lipase and the lipase powder, and therefore has less loss of activity during immobilization than the conventional immobilized lipase. The exchange activity is high, and compared with powder lipase, there is no loss during reaction, recovery and reuse, and transesterification activity can be kept high over a long period of time.
Furthermore, since the immobilized lipase obtained by the method for producing an immobilized lipase of the present invention produced by dispersing in fats and oils is produced in fats and oils, the moisture content is extremely low, and therefore there is a lot of moisture. It can be particularly preferably used for transesterification of fats and oils, which are difficult to react.
次に、本発明の油脂のエステル交換方法について述べる。
本発明の油脂のエステル交換方法は、本発明の固定化リパーゼを使用するものであり、高いエステル交換活性を有し、且つ反応・回収・再使用時の損失がなく、長期にわたってエステル交換活性を高く保持することができるという特徴を有する。特に、本発明の製造方法によって得られた固定化リパーゼを使用する場合は、固定化リパーゼの乾燥の工程を必要とせず、ただちにエステル交換反応に用いることができる点でより好ましいものである。
Next, the method for transesterification of fats and oils of the present invention will be described.
The method for transesterification of fats and oils of the present invention uses the immobilized lipase of the present invention, has high transesterification activity, has no loss during reaction, recovery and reuse, and has long-term transesterification activity. It has the feature that it can be kept high. In particular, when the immobilized lipase obtained by the production method of the present invention is used, it is more preferable in that it does not require a drying step of the immobilized lipase and can be immediately used for transesterification.
なお、本発明の油脂のエステル交換方法は、連続反応または回分反応のどちらでも問題なく使用できる。
連続反応の場合、カラム等に本発明の固定化リパーゼを充填し、原料油脂を通液するだけでよくプロセスが簡略であるという利点を有する。原料油脂の通液速度はエステル交換油の反応率を測定しながら、随時所望の反応率が得られるよう調整する。
回分反応の場合、本発明の固定化リパーゼ分散原料油脂を、タンク中で回分反応を行なうものである。温度条件は好ましくは２０〜８０℃、より好ましくは４０〜７０℃、さらに好ましくは５０〜６０℃であり、反応時間は好ましくは５分〜２４時間である。また、反応は好ましくは窒素雰囲気下で行なう。
In addition, the transesterification method of fats and oils of the present invention can be used without any problem in either a continuous reaction or a batch reaction.
In the case of continuous reaction, it is only necessary to fill the immobilized lipase of the present invention in a column or the like and pass the raw material fats and oils, so that the process is simple. The liquid feed rate of the raw material fat is adjusted so that a desired reaction rate can be obtained at any time while measuring the reaction rate of the transesterified oil.
In the case of batch reaction, the immobilized lipase-dispersed raw material fat of the present invention is subjected to batch reaction in a tank. The temperature condition is preferably 20 to 80 ° C, more preferably 40 to 70 ° C, still more preferably 50 to 60 ° C, and the reaction time is preferably 5 minutes to 24 hours. The reaction is preferably performed in a nitrogen atmosphere.
なお、本発明の油脂のエステル交換方法において、本発明の固定化リパーゼに加え、粉末のリパーゼや本発明の固定化リパーゼ以外の固定化リパーゼなどのその他のリパーゼを併用することを除外するものではないが、エステル交換活性が高く、長期にわたってそのエステル交換活性を保持するためには、その他のリパーゼの使用量は、好ましくは５０質量％以下とすることが好ましく、より好ましくは２０質量％以下、さらに好ましくは１０質量％以下、最も好ましくは使用しないことである。 In addition, in the transesterification method of fats and oils of the present invention, in addition to the immobilized lipase of the present invention, it is not excluded to use in combination with other lipases such as powdered lipase and immobilized lipase other than the immobilized lipase of the present invention. Although there is no transesterification activity, in order to maintain the transesterification activity over a long period of time, the amount of other lipase used is preferably 50% by mass or less, more preferably 20% by mass or less, More preferably, it is 10 mass% or less, Most preferably, it is not used.
そして、回分反応の場合、濾過終了後、定法により固定化リパーゼを濾別して再利用する。また、回分反応で得られた濾過液や、連続反応で得られた通液は定法により精製処理してエステル交換油脂を得る。
そして上記のエステル交換反応を繰り返すことで、長期にわたって高いエステル交換活性を保持したままの状態とすることができる。
In the case of a batch reaction, after completion of filtration, the immobilized lipase is filtered and reused by a conventional method. Moreover, the filtrate obtained by batch reaction and the liquid flow obtained by continuous reaction are refined by a conventional method to obtain transesterified fats and oils.
And by repeating said transesterification reaction, it can be set as the state which kept high transesterification activity over a long period of time.
以下、実施例を示して本発明を具体的に説明するが、本発明はこれらの実施例により何等制限されるものではない。なお、例中に示す％は、特に記載がない限り質量％を意味する。 EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited to these examples. In addition,% shown in an example means the mass% unless there is particular description.
〔実施例１〕
２００ｍｌの４つ口フラスコにパームオレインのエステル交換油脂（水分５００ｐｐｍに調整）５０ｇとアルカリゲネス属由来の粉末リパーゼ（リパーゼＱＬＭ：名糖産業（株）製、至適ｐＨ７〜９、）１ｇを添加、分散し、６０℃、２０分間、窒素雰囲気下でパドル式撹拌機を用いて撹拌した。ここで原料油脂中に存在する粒子の粒径を、（株）島津製作所製レーザー式粒度分布計により測定したところ、粒径１〜１００μmの粒子が８５％（体積基準）、粒径０．２０〜０．４５μmの粒子が８５％（個数基準）であった。シリカ（Silopute２０２：富士シリシア化学製、ｐＨ７．６、粒径１〜１００μmの粒子が９９％（体積基準）、粒径0.45μm以下の粒子は0%（個数基準））３ｇ、及び、珪藻土（Celite Standard super-cel：Celite社製、粒径１〜１００μmの粒子が９８％（体積基準）、粒径0.45μm以下は0%（個数基準））６ｇを添加、分散し、２時間撹拌した。ここで油脂中に存在する粒子の粒径を、再度測定し、固定化率を次式により求め、その結果を表１に記載した。
[Example 1]
To a 200 ml four-necked flask, 50 g of palm olein transesterified oil (adjusted to a water content of 500 ppm) and 1 g of powdered lipase derived from Alkaligenes (Lipase QLM: manufactured by Meito Sangyo Co., Ltd., optimum pH 7-9), The mixture was dispersed and stirred using a paddle type stirrer at 60 ° C. for 20 minutes under a nitrogen atmosphere. Here, when the particle size of the particles present in the raw oil and fat was measured with a laser particle size distribution meter manufactured by Shimadzu Corporation, 85% (volume basis) of particles having a particle size of 1 to 100 μm and a particle size of 0.20 were obtained. The particle size of ˜0.45 μm was 85% (number basis). 3 g of silica (Silopute 202: manufactured by Fuji Silysia Chemical, pH 7.6, 99% (volume basis) of particles having a particle size of 1 to 100 μm, 0% (number basis) of particles having a particle size of 0.45 μm or less), and diatomaceous earth (Celite) Standard super-cel: manufactured by Celite, 6 g of particles having a particle diameter of 1 to 100 μm (98% (volume basis) and 0% (number basis) for particle diameter of 0.45 μm or less)) were added and dispersed, and stirred for 2 hours. Here, the particle size of the particles present in the oil and fat was measured again, the immobilization rate was determined by the following equation, and the results are shown in Table 1.
固定化率（％）＝〔（固定化前の０．４５μm以下の粒子個数％−固定化後の０．４５μm以下の粒子個数％）／（固定化前の０．４５μm以下の粒子個数％−シリカ及び珪藻土由来の０．４５μm以下の粒子個数％）〕×１００ Immobilization rate (%) = [(number of particles of 0.45 μm or less before immobilization−number of particles of 0.45 μm or less after immobilization) / (number of particles of 0.45 μm or less before immobilization− (Number of particles of 0.45 μm or less derived from silica and diatomaceous earth))] × 100
次に、加圧密閉型濾過機（加圧ヌッチェ機・ＩＨＩ製：３６０メッシュ金網使用）により濾過を行い固定化リパーゼを得た。
２０００ｍｌの４つ口フラスコにパームオレイン１０００ｇ（水分３００ｐｐｍ）、上記で得られた固定化リパーゼを添加し、撹拌羽根を用いて６０℃、３００ｒｐｍ、窒素雰囲気下で回分反応を行った。
反応生成物については、ＧＬＣでトリパルミチン含量を測定し、下記に示す式で反応率を求めた。
Next, filtration was performed with a pressure sealed filter (pressure Nutsche machine, manufactured by IHI: 360 mesh wire mesh used) to obtain an immobilized lipase.
To a 2000 ml four-necked flask, 1000 g of palm olein (water content 300 ppm) and the immobilized lipase obtained above were added, and batch reaction was performed at 60 ° C., 300 rpm, under a nitrogen atmosphere using a stirring blade.
About the reaction product, the tripalmitin content was measured by GLC and the reaction rate was calculated | required by the formula shown below.
反応率＝（反応生成物のトリパルミチン含量−原料油脂のトリパルミチン含量）／（反応平衡組成物のトリパルミチン含量−原料油脂のトリパルミチン含量） Reaction rate = (tripalmitin content of reaction product−tripalmitin content of raw oil / fat) / (tripalmitin content of reaction equilibrium composition−tripalmitin content of raw oil / fat)
次いで、反応時間を横軸に、ｌｎ〔１／（１−反応率）〕を縦軸にプロットし、原点を通る一次近似式を設定して、１時間あたりのｌｎ〔１／（１−反応率）〕を読み取り、エステル交換活性能ａとした。 Next, the reaction time is plotted on the horizontal axis, and ln [1 / (1-reaction rate)] is plotted on the vertical axis, and a linear approximate expression passing through the origin is set, and ln [1 / (1-reaction] per hour is set. Rate)] was read and was defined as transesterification activity a.
このエステル交換活性能ａと、Ｘ＝油脂１０００ｇに対する固定化リパーゼの添加量（％）及び、Ｙ＝粉末リパーゼ使用量／（粉末リパーゼ＋シリカ＋珪藻土の合計使用量）から下記に示す式で算出した値を、エステル交換初期活性値（固定化に使用した単位粉末リパーゼ重量を基準とした）とし、表１に記載した。
エステル交換初期活性値＝〔ａ／Ｘ／Ｙ〕×１００
From this transesterification activity a, X = addition amount (%) of immobilized lipase to 1000 g of fat and oil, and Y = powder lipase use amount / (total use amount of powder lipase + silica + diatomaceous earth) The values obtained are shown in Table 1 as the initial transesterification activity values (based on the weight of the unit powder lipase used for immobilization).
Transesterification initial activity value = [a / X / Y] × 100
さらに、固定化時の活性損失率（％）については、下記の式で算出した。
固定化時の活性損失率（％）＝１−（エステル交換初期活性値／比較例４のエステル交換初期活性値）
Furthermore, the activity loss rate (%) at the time of immobilization was calculated by the following formula.
Activity loss rate during immobilization (%) = 1- (transesterification initial activity value / transesterification initial activity value of Comparative Example 4)
反応率が０．９以上になったところで、反応を終了し、加圧密閉型濾過機（加圧ヌッチェ機・ＩＨＩ製：３６０メッシュ金網使用）を使用して固定化リパーゼを回収し、その濾過液中の残存リパーゼ活性から活性回収率を算出し、表１に記載した。
次いで、回収した固定化リパーゼの全量を、１０００ｇの新たな原料油脂中に添加、分散し、２０００ｍｌの４つ口フラスコに再び投入し、上記同様の条件でエステル交換反応を再度行なった。
When the reaction rate reached 0.9 or higher, the reaction was terminated, and the immobilized lipase was recovered using a pressure sealed filter (pressurized Nutsche machine, manufactured by IHI: 360 mesh wire mesh used) and filtered. The activity recovery rate was calculated from the residual lipase activity in the liquid and listed in Table 1.
Next, the entire amount of the recovered immobilized lipase was added and dispersed in 1000 g of new raw material fats and oils, and again put into a 2000 ml four-necked flask, and the transesterification reaction was performed again under the same conditions as described above.
同一の操作を、エステル交換活性値が１／４となるまで繰り返し、その間に生産することができたエステル交換油の総生産量（ｋｇ）を、固定化時に投入した粉末リパーゼ量（ｇ）で除し、エステル交換活性値が１／４となるまでの期間における固定化時に使用した粉末リパーゼ１ｇあたりのエステル交換油の生産量（ｋｇ）を、エステル交換油脂の生産性の指標とし、表１に記載した。 The same operation was repeated until the transesterification activity value became 1/4, and the total production amount (kg) of the transesterification oil that could be produced during that time was the amount of powder lipase (g) charged at the time of immobilization. The amount of transesterified oil production (kg) per gram of powdered lipase used at the time of immobilization during the period until the transesterification activity value becomes 1/4 was used as an index of transesterified oil and fat productivity, and Table 1 It was described in.
〔実施例２〕
実施例１におけるシリカをSilopute３０３（富士シリシア化学製、ｐＨ７．５ 粒径粒子の９９％が１〜１００μmの粒子が９９％（体積基準）、粒径0.45μm以下の粒子は0%(個数基準)）２ｇに変更し、及び、珪藻土をCelpure S300 （アドバンスドミネラルズ社製、粒径１〜１００μmの粒子が９８％（体積基準）、粒径0.45μm以下は0%（個数基準））２ｇに変更した他は実施例１と同様にして固定化操作、及び、エステル交換反応を行なった。
なお、粒径は実施例１と同一の方法で測定し、固定化率、エステル交換初期活性値、リパーゼ活性回収率、エステル交換活性値が１／４となるまでの期間における固定化時に使用した粉末リパーゼ１ｇあたりのエステル交換油の生産量は実施例１と同一の方法で算出し、それぞれ結果を表１に記載した。
[Example 2]
Silopute 303 (produced by Fuji Silysia Chemical, 99% of pH 7.5 particle size is 99% (volume basis) of particles having a particle size of 1 to 100 μm, and 0% (number basis) of particles having a particle size of 0.45 μm or less is used. ) Change to 2g, and diatomaceous earth to Celpure S300 (manufactured by Advanced Minerals, particle size 1 to 100μm 98% (volume basis), particle size 0.45μm or less 0% (number basis)) 2g Otherwise, the immobilization operation and the transesterification reaction were carried out in the same manner as in Example 1.
The particle size was measured by the same method as in Example 1 and used for immobilization in a period until the immobilization rate, initial transesterification activity value, lipase activity recovery rate, transesterification activity value became 1/4. The amount of transesterified oil produced per gram of powdered lipase was calculated by the same method as in Example 1, and the results are shown in Table 1, respectively.
〔比較例１〕
シリカを無添加とした以外は実施例２と同様にして固定化操作、及び、エステル交換反応を行なった。
なお、粒径は実施例１と同一の方法で測定し、固定化率、エステル交換初期活性値、リパーゼ活性回収率、エステル交換活性値が１／４となるまでの期間における固定化時に使用した粉末リパーゼ１ｇあたりのエステル交換油の生産量は実施例１と同一の方法で算出し、それぞれ結果を表１に記載した。
[Comparative Example 1]
Immobilization operation and transesterification were carried out in the same manner as in Example 2 except that no silica was added.
The particle size was measured by the same method as in Example 1 and used for immobilization in a period until the immobilization rate, initial transesterification activity value, lipase activity recovery rate, transesterification activity value became 1/4. The amount of transesterified oil produced per gram of powdered lipase was calculated by the same method as in Example 1, and the results are shown in Table 1, respectively.
〔比較例２〕
珪藻土を無添加とした以外は実施例２と同様にして固定化操作、及び、エステル交換反応を行なった
なお、粒径は実施例１と同一の方法で測定し、固定化率、エステル交換初期活性値、リパーゼ活性回収率、エステル交換活性値が１／４となるまでの期間における固定化時に使用した粉末リパーゼ１ｇあたりのエステル交換油の生産量は実施例１と同一の方法で算出し、それぞれ結果を表１に記載した。
[Comparative Example 2]
The immobilization operation and transesterification were performed in the same manner as in Example 2 except that diatomaceous earth was not added. The particle size was measured by the same method as in Example 1, and the immobilization rate and the initial transesterification were measured. The production amount of transesterified oil per gram of powdered lipase used at the time of immobilization in the period until the activity value, the lipase activity recovery rate, and the transesterification activity value became 1/4 was calculated by the same method as in Example 1. The results are shown in Table 1, respectively.
〔比較例３〕
固定化操作時に水を対油５％添加した以外は実施例２と同様にして固定化操作を行った後、実施例２同様にしてエステル交換反応を行なった。
固定化操作終了時、実施例１と同様の方法で、粒度を測定したところ、粒径１００μｍ以上の粒子含量が３４％（体積基準）であり、得られた固定化リパーゼは粒度が大きいものであった。
なお、固定化率、エステル交換初期活性値、リパーゼ活性回収率、エステル交換活性値が１／４となるまでの期間における固定化時に使用した粉末リパーゼ１ｇあたりのエステル交換油の生産量は実施例１と同一の方法で算出し、それぞれ結果を表１に記載した。
[Comparative Example 3]
After performing the immobilization operation in the same manner as in Example 2 except that 5% of water was added to the oil during the immobilization operation, the transesterification reaction was performed in the same manner as in Example 2.
At the end of the immobilization operation, the particle size was measured in the same manner as in Example 1. As a result, the content of particles having a particle size of 100 μm or more was 34% (volume basis), and the obtained immobilized lipase had a large particle size. there were.
The production amount of transesterified oil per gram of powdered lipase used during immobilization during the period until the immobilization rate, the initial transesterification activity value, the lipase activity recovery rate, and the transesterification activity value becomes 1/4 is shown in the examples. 1 and the results are shown in Table 1.
〔比較例４〕
２０００ｍｌの４つ口フラスコにパームオレイン（水分５００ｐｐｍに調整）５０ｇとアルカリゲネス属由来の粉末リパーゼ（リパーゼＱＬＭ：名糖産業（株）製、至適ｐＨ７〜９、）１ｇを入れ、パドル式撹拌機を用いて６０℃、３００ｒｐｍ、窒素雰囲気下で回分反応を行なった。
なお、固定化率、エステル交換初期活性値、リパーゼ活性回収率、エステル交換活性値が１／４となるまでの期間における固定化時に使用した粉末リパーゼ１ｇあたりのエステル交換油の生産量は実施例１と同一の方法で算出し、それぞれ結果を表１に記載した。
ここで、エステル交換初期活性値の計算式におけるＸ＝油脂１０００ｇに対する粉末リパーゼの添加量、Ｙ＝１として算出した。
[Comparative Example 4]
A paddle type stirrer is charged with 50 g of palm olein (adjusted to a water content of 500 ppm) and 1 g of powdered lipase derived from the genus Alkagenes (Lipase QLM, manufactured by Meisei Sangyo Co., Ltd., optimum pH 7-9) in a 2000 ml four-necked flask. Was used for batch reaction at 60 ° C. and 300 rpm in a nitrogen atmosphere.
The production amount of transesterified oil per gram of powdered lipase used during immobilization during the period until the immobilization rate, the initial transesterification activity value, the lipase activity recovery rate, and the transesterification activity value becomes 1/4 is shown in the examples. 1 and the results are shown in Table 1.
Here, X was calculated in the formula for calculating the initial transesterification activity value as follows: X = addition amount of powder lipase with respect to 1000 g of fat and oil, Y = 1.
〔実施例３〕
実施例２にて得られた固定化リパーゼをステンレス製カラムに充填し、６０℃でパームオレイン（水分５００ｐｐｍに調整）を反応率が０．９以上となる速度ｖ（ｇ／ｈ）で通液し、エステル交換を行った。
エステル交換初期活性値は、次式により求めた。
Example 3
The immobilized lipase obtained in Example 2 was packed in a stainless steel column, and palm olein (adjusted to a water content of 500 ppm) was passed at 60 ° C. at a rate v (g / h) at which the reaction rate was 0.9 or more. Then, transesterification was performed.
The transesterification initial activity value was determined by the following formula.
エステル交換初期活性値＝ｌｎ〔１／（１−反応率）〕×〔ｖ／ｘ／ｙ〕×１００
ｘ：固定化リパーゼ使用量
ｙ：固定化リパーゼ調整に供した粉末リパーゼの割合＝粉末リパーゼ使用量／（粉末
リパーゼ使用量＋担体使用量）
また、リパーゼ活性回収率は、反応油中の残存リパーゼ活性から算出した。
なお、エステル交換活性値が１／４となるまでの期間における固定化時に使用した粉末リパーゼ１ｇあたりのエステル交換油の生産量は実施例１と同一の方法で算出し、それぞれ結果を表１に記載した。
Transesterification initial activity value = ln [1 / (1-reaction rate)] × [v / x / y] × 100
x: amount of immobilized lipase used y: proportion of powdered lipase used for preparation of immobilized lipase = amount of powdered lipase used / (powder
Lipase usage + carrier usage)
The lipase activity recovery rate was calculated from the residual lipase activity in the reaction oil.
The amount of transesterified oil produced per gram of powdered lipase used at the time of immobilization until the transesterification activity value became 1/4 was calculated by the same method as in Example 1, and the results are shown in Table 1. Described.
上記表１に示す結果から、シリカ及び珪藻土を含有し、粒径１〜１００μｍの粒子が９０％以上(体積基準）であり、且つ、粒径０．４５μm以下の粒子が１％以下（個数基準）である固定化リパーゼは、固定化によるリパーゼ活性の損失が少なく（固定化時の活性損失率参照）、また、該固定化リパーゼを使用して油脂のエステル交換反応を行なうと、エステル交換活性が高く（固定化リパーゼの初期活性値参照）、反応・回収・再使用時の損失がなく（固定化リパーゼの活性回収率参照）、長期にわたってエステル交換活性を高く保持することができるため、生産性が極めて高いことがわかる。（固定化リパーゼのエステル交換油生産量参照） From the results shown in Table 1 above, silica and diatomaceous earth are contained, particles having a particle size of 1 to 100 μm are 90% or more (volume basis), and particles having a particle size of 0.45 μm or less are 1% or less (number basis). ) Is less loss of lipase activity due to immobilization (refer to the activity loss rate at the time of immobilization), and when transesterification of fats and oils is performed using the immobilized lipase, transesterification activity Production (see the initial activity value of immobilized lipase), no loss during reaction / recovery / reuse (see activity recovery rate of immobilized lipase), and high transesterification activity can be maintained over a long period of time. It can be seen that the property is extremely high. (See transesterified oil production of immobilized lipase)
それに対し、水分を多く添加した油脂を使用したため粒子径の大きな固定化リパーゼとなってしまった比較例３の固定化リパーゼは、固定化によるリパーゼ活性の損失が大きく、また、該固定化リパーゼを使用して油脂のエステル交換反応を行なうと、エステル交換活性が極めて低く、そのため、生産性も極めて低くなってしまうことがわかる。 On the other hand, the immobilized lipase of Comparative Example 3 which has become an immobilized lipase having a large particle size due to the use of fats and oils to which a large amount of water is added has a large loss of lipase activity due to the immobilization. It can be seen that the transesterification of the fats and oils when used, has a very low transesterification activity and, therefore, a very low productivity.
また、固定化に際し珪藻土のみ使用した場合（比較例１）、又は、シリカのみを使用した場合（比較例２）、固定化リパーゼが得られず、そのため、濾過して得たリパーゼと担体の混合物を使用してエステル交換を行なうと、粉末リパーゼのみを用いてエステル交換を行なう場合（比較例４）と同様、初期のエステル交換活性は高いものの、反応・回収・再使用時の損失が大きいため、長期にわたってエステル交換活性を高く保持することができず、生産性は低くなってしまうことがわかる。
In addition, when only diatomaceous earth is used for immobilization (Comparative Example 1) or when only silica is used (Comparative Example 2), an immobilized lipase cannot be obtained. Therefore, a mixture of lipase and carrier obtained by filtration is used. When transesterification is carried out using the same as in the case of transesterification using only powdered lipase (Comparative Example 4), although the initial transesterification activity is high, the loss during reaction, recovery and reuse is large. It can be seen that the transesterification activity cannot be kept high over a long period of time, and the productivity is lowered.
Claims (6)
A method for transesterifying fats and oils using the immobilized lipase according to any one of claims 1 to 3.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2006223031A JP4942421B2 (en) | 2006-08-18 | 2006-08-18 | Immobilized lipase and method for producing the same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2006223031A JP4942421B2 (en) | 2006-08-18 | 2006-08-18 | Immobilized lipase and method for producing the same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2008043274A true JP2008043274A (en) | 2008-02-28 |
| JP4942421B2 JP4942421B2 (en) | 2012-05-30 |
Family
ID=39177635
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2006223031A Active JP4942421B2 (en) | 2006-08-18 | 2006-08-18 | Immobilized lipase and method for producing the same |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP4942421B2 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2010183873A (en) * | 2009-02-12 | 2010-08-26 | Kao Corp | Method for producing fat and oil highly containing monoacylglycerol |
| JP2012034622A (en) * | 2010-08-06 | 2012-02-23 | Kao Corp | Method for producing fat and oil with high diacylglycerol content |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002095471A (en) * | 2000-09-25 | 2002-04-02 | Toyota Central Res & Dev Lab Inc | Immobilized lipase, method for improving substrate specificity of lipase and method for performing optical resolution |
-
2006
- 2006-08-18 JP JP2006223031A patent/JP4942421B2/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002095471A (en) * | 2000-09-25 | 2002-04-02 | Toyota Central Res & Dev Lab Inc | Immobilized lipase, method for improving substrate specificity of lipase and method for performing optical resolution |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2010183873A (en) * | 2009-02-12 | 2010-08-26 | Kao Corp | Method for producing fat and oil highly containing monoacylglycerol |
| JP2012034622A (en) * | 2010-08-06 | 2012-02-23 | Kao Corp | Method for producing fat and oil with high diacylglycerol content |
Also Published As
| Publication number | Publication date |
|---|---|
| JP4942421B2 (en) | 2012-05-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Lage et al. | Preparation of a biocatalyst via physical adsorption of lipase from Thermomyces lanuginosus on hydrophobic support to catalyze biolubricant synthesis by esterification reaction in a solvent-free system | |
| EP2094824B1 (en) | A continuous process and apparatus for enzymatic treatment of lipids | |
| JP4942421B2 (en) | Immobilized lipase and method for producing the same | |
| Katiyar et al. | One-pot lipase entrapment within silica particles to prepare a stable and reusable biocatalyst for transesterification | |
| JP2007300855A (en) | Method for recovering activity of lipase | |
| JP5080771B2 (en) | Method for producing useful substance using immobilized enzyme | |
| Yeoh et al. | Influence of silica gel in production of diacylglycerol via enzymatic glycerolysis of palm olein | |
| JP4849967B2 (en) | Method for producing fatty acids | |
| WO2019063718A1 (en) | A process for the cell-free enzymatic production of 10-hydroxystearic acid (10-hsa) from bio-based oils for lubricant formulation | |
| JP5080797B2 (en) | Method for producing useful substance using immobilized enzyme | |
| EP2757158B1 (en) | Enzymatic process for the synthesis of estolides | |
| JP5284663B2 (en) | Method for transesterification of fats and oils | |
| JP4915732B2 (en) | Method for producing useful substance using immobilized enzyme | |
| JP4220957B2 (en) | Method for producing immobilized enzyme | |
| JP6990076B2 (en) | Method for producing fatty acids | |
| JP2006246808A (en) | Method for carrying out transesterification of oil and fat | |
| JP2012034622A (en) | Method for producing fat and oil with high diacylglycerol content | |
| Asih et al. | Pilot-scale production of lipase using palm oil mill effluent as a basal medium and its immobilization by selected materials | |
| CN106867989B (en) | Immobilized enzyme and preparation method thereof | |
| JP2657887B2 (en) | Preparation method of immobilized enzyme | |
| JP5242236B2 (en) | Method for producing fatty acids | |
| EP2821468B1 (en) | Method for producing fatty acid composition | |
| JP5080828B2 (en) | Method for producing useful substance using immobilized enzyme | |
| Yu et al. | Magnetic immobilisation of phospholipase C and its hydrolysis of phospholipids in crude soybean oil | |
| JP4842770B2 (en) | Method for producing immobilized enzyme reaction tower |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20090626 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20111220 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20120209 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20120228 |
|
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20120228 |
|
| R151 | Written notification of patent or utility model registration |
Ref document number: 4942421 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R151 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20150309 Year of fee payment: 3 |