JP5080797B2 - Method for producing useful substance using immobilized enzyme - Google Patents
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- JP5080797B2 JP5080797B2 JP2006337889A JP2006337889A JP5080797B2 JP 5080797 B2 JP5080797 B2 JP 5080797B2 JP 2006337889 A JP2006337889 A JP 2006337889A JP 2006337889 A JP2006337889 A JP 2006337889A JP 5080797 B2 JP5080797 B2 JP 5080797B2
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Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Description
本発明は、固定化酵素を充填した固定床型反応塔を用いた反応による有用物質の製造方法に関する。 The present invention relates to a method for producing a useful substance by a reaction using a fixed bed type reaction column packed with an immobilized enzyme.
液体を固定床型反応塔に通液して行う反応として、L−アスパラギン酸生成、エステル交換油脂生成、乳糖加水分解、油脂類の加水分解等に利用されている固定化酵素を用いた反応が知られている。これらの反応は、いずれも発熱量が比較的小さい為、通常、最も単純なドラム型反応器が使用されている。 As a reaction performed by passing the liquid through a fixed bed type reaction tower, a reaction using an immobilized enzyme used for L-aspartic acid production, transesterified oil production, lactose hydrolysis, fats hydrolysis, etc. Are known. Since these reactions have a relatively small calorific value, the simplest drum reactor is usually used.
固定化酵素を用いた反応のうち、油脂類の加水分解のように2種類以上の液を反応塔に流通させる場合には、反応効率向上の観点から、反応液を均一に混合した状態で通液することが好ましい。この場合、加水分解に用いる油相基質と水相基質は、本来混合しても一相にならないものであるため、エマルションとするのが一般的である。一方で、エマルション粒子は担体の細孔内に吸着した酵素に到達し難いため、通液速度を、反応液が乳化しない範囲とした技術もある(特許文献1参照)。 Among the reactions using immobilized enzymes, when two or more kinds of liquids are circulated through the reaction tower as in the hydrolysis of fats and oils, the reaction liquids are passed in a uniformly mixed state from the viewpoint of improving reaction efficiency. It is preferable to liquefy. In this case, since the oil phase substrate and the water phase substrate used for the hydrolysis do not become a single phase even if they are originally mixed, it is generally used as an emulsion. On the other hand, since emulsion particles are unlikely to reach the enzyme adsorbed in the pores of the carrier, there is a technique in which the liquid passing speed is within a range where the reaction liquid is not emulsified (see Patent Document 1).
また、固定床に油相基質と水相基質を流通させる方法としては、向流で流通させる方法(特許文献1、2参照)、及び並流で流通させる方法(特許文献3参照)があるが、前者は特殊な仕組みと運転方法が必要となるため、一般的には並流で流通させる方法が採られている。
前記固定化酵素を充填した固定床型反応塔に、2液相を形成する液体混合物を流通させて反応を行う方法においては、反応塔の径が小さい場合は固定化酵素の活性が有効に発現するものの、特に当該液体混合物を乳化させずに流通させる場合、反応塔の径が大きくなるに従って、酵素の発現活性が低下し、結果として反応性が低下するという問題があることを見出した。この場合、反応性を高めるために、単に固定化酵素と反応液の接触時間を長くしようとすると、生産性(流量)が低下するという問題もある。 In the method of carrying out the reaction by circulating a liquid mixture that forms a two-liquid phase in the fixed bed type reaction tower packed with the immobilized enzyme, the activity of the immobilized enzyme is effectively expressed when the diameter of the reaction tower is small. However, in particular, when the liquid mixture is circulated without being emulsified, it has been found that there is a problem that as the diameter of the reaction tower increases, the expression activity of the enzyme decreases and, as a result, the reactivity decreases. In this case, in order to increase the reactivity, simply increasing the contact time between the immobilized enzyme and the reaction solution has a problem that the productivity (flow rate) decreases.
従って、本発明は、固定化酵素を充填した固定床型反応塔に、2液相を形成する液体混合物を流通させて反応を行う有用物質の製造方法において、流量を低下させずに反応性を高め、生産性を向上させることにより、より効率的に有用物質を製造する方法を提供するものである。 Accordingly, the present invention provides a method for producing a useful substance in which a reaction is carried out by circulating a liquid mixture that forms a two-liquid phase through a fixed bed type reaction column packed with an immobilized enzyme. It is intended to provide a method for producing a useful substance more efficiently by enhancing and improving productivity.
そこで本発明者は、固定化酵素を充填した固定床型反応塔における酵素の発現活性について種々検討したところ、管径が大きな反応塔を用いる場合において、反応塔の管径と固定化酵素の粒子径との比率を規定することにより、酵素活性を有効に発現させることができ、高い反応性を維持したまま、生産性を向上させることができることを見出した。通常、固定化酵素を用いる場合には、酵素の発現活性を向上させようとすれば、当業者は固定化担体の比表面積を大きくするために担体の粒子径を小さくするものである。本発明者は、固定床型反応塔の管径が小さい場合には本手法は有効であるが、管径を大きくした場合には、全く意外にも担体粒子径については逆の手段を採らなければ効果が得られないことを見出し、本発明を完成した。 Therefore, the present inventor has conducted various studies on the enzyme expression activity in a fixed bed type reaction column packed with an immobilized enzyme. When a reaction column having a large tube diameter is used, the tube diameter of the reaction column and the immobilized enzyme particles It has been found that by defining the ratio with the diameter, the enzyme activity can be effectively expressed, and the productivity can be improved while maintaining high reactivity. Usually, when an immobilized enzyme is used, a person skilled in the art reduces the particle diameter of the carrier in order to increase the specific surface area of the immobilized carrier if the expression activity of the enzyme is to be improved. The present inventor is effective when the tube diameter of the fixed bed type reaction column is small. However, when the tube diameter is increased, the present inventor should surprisingly take the opposite means for the carrier particle size. Thus, the present invention has been completed.
すなわち本発明は、管径が35mmφ以上である固定化酵素を充填した固定床型反応塔に2液相を形成する液体混合物を供給し、同一方向に並流させて反応を行うことにより有用物質を製造する方法であって、固定床型反応塔として、固定床型反応塔の管径(mm)の固定化酵素の平均粒子径(mm)に対する比率(管径/平均粒子径)が135(mm/mm)以下となるように固定化酵素を充填した固定床型反応塔を用いる有用物質の製造方法を提供するものである。 That is, the present invention provides a useful substance by supplying a liquid mixture that forms a two-liquid phase to a fixed bed type reaction tower packed with an immobilized enzyme having a tube diameter of 35 mmφ or more, and carrying out the reaction in the same direction. The ratio of the tube diameter (mm) of the fixed bed type reaction tower to the average particle diameter (mm) of the immobilized enzyme (tube diameter / average particle diameter) is 135 (fixed bed type reaction tower). The present invention provides a method for producing a useful substance using a fixed bed type reaction column packed with an immobilized enzyme so as to be equal to or less than (mm / mm).
本発明によれば、固定化酵素を充填した固定床型反応塔へ2液相を形成する液体混合物を供給して行う反応において、管径が大きい反応塔を用いても、酵素活性を有効に発現させることができ、有用物質を効率的に製造することができる。 According to the present invention, in a reaction performed by supplying a liquid mixture that forms a two-liquid phase to a fixed bed type reaction tower packed with an immobilized enzyme, the enzyme activity is effectively improved even when a reaction tower having a large tube diameter is used. It can be expressed, and useful substances can be produced efficiently.
本発明においては、固定化酵素を充填した固定床型反応塔に2液相を形成する液体混合物(反応液)を供給することが必要である。固定床型反応塔(以下「酵素塔」ともいう)とは、固定化酵素をカラム等に充填し、固定化酵素間の空隙及び固定化担体の細孔に反応液を流通させ得るようにしたものをいう。2液相とは、2種類の液体が混合後にも1相にならない状態をいい、分相しているものから、均一であっても乳化状態となっているものも含む。 In the present invention, it is necessary to supply a liquid mixture (reaction liquid) that forms a two-liquid phase to a fixed bed type reaction tower packed with an immobilized enzyme. A fixed bed type reaction tower (hereinafter also referred to as “enzyme tower”) is a column in which immobilized enzyme is packed so that the reaction solution can be circulated through the gap between the immobilized enzymes and the pores of the immobilized carrier. Say things. The two-liquid phase means a state in which two types of liquids do not become one phase even after mixing, and includes those in which they are in an emulsified state even if they are uniform from those that are phase-separated.
本発明の態様としては、固定化酵素として油脂分解酵素を固定化担体に吸着させたものを用い、これを充填した酵素塔に、2液相として油相基質と水相基質を流通させることによる油脂類の加水分解反応により、有用物質として脂肪酸類を製造する方法であることが好ましい。なお、本発明において「固定化酵素」とは、固定化担体に酵素を吸着させた後の固定化担体を含めた全体を指す。 As an aspect of the present invention, an enzyme in which an oleolytic enzyme is adsorbed on an immobilization carrier is used as an immobilized enzyme, and an oil phase substrate and an aqueous phase substrate are circulated as two liquid phases in an enzyme tower packed with the enzyme. A method of producing fatty acids as useful substances by a hydrolysis reaction of fats and oils is preferable. In the present invention, the term “immobilized enzyme” refers to the whole including the immobilized carrier after the enzyme is adsorbed on the immobilized carrier.
本発明においては、2液相を同一方向に並流させることが必要である。この場合、2液相を予め混合して乳化状態として供給しても良く、分相したまま供給しても良い。また、2液相を一定時間毎に交互に供給しても良い。酵素塔への各基質の供給は、塔頂から塔底へ下方流で行っても、塔底から塔頂へ上方流で行っても良い。 In the present invention, it is necessary to cause two liquid phases to flow in the same direction. In this case, the two liquid phases may be mixed in advance and supplied as an emulsified state, or may be supplied while being separated. Further, the two liquid phases may be alternately supplied at regular intervals. Each substrate may be supplied to the enzyme tower in a downward flow from the tower top to the tower bottom or in an upward flow from the tower bottom to the tower top.
本発明で用いる固定化酵素は、固定化担体に酵素を吸着等により担持させたものである。固定化担体としては、セライト、ケイソウ土、カオリナイト、シリカゲル、モレキュラーシーブス、多孔質ガラス、活性炭、炭酸カルシウム、セラミックス等の無機担体、セラミックスパウダー、ポリビニルアルコール、ポリプロピレン、キトサン、イオン交換樹脂、疎水吸着樹脂、キレート樹脂、合成吸着樹脂等の有機高分子等が挙げられるが、特に保水力が高い点からイオン交換樹脂が好ましい。また、イオン交換樹脂の中でも、大きな表面積を有することにより酵素の吸着量を高くできるという点から、多孔質であることが好ましい。 The immobilized enzyme used in the present invention is one in which an enzyme is supported on an immobilized carrier by adsorption or the like. Immobilization carriers include celite, diatomaceous earth, kaolinite, silica gel, molecular sieves, porous glass, activated carbon, calcium carbonate, ceramics and other inorganic carriers, ceramic powder, polyvinyl alcohol, polypropylene, chitosan, ion exchange resin, hydrophobic adsorption Examples thereof include organic polymers such as resins, chelate resins, and synthetic adsorption resins, and ion exchange resins are particularly preferable from the viewpoint of high water retention. Of the ion exchange resins, a porous material is preferable because it has a large surface area and can increase the amount of adsorbed enzyme.
固定化担体として用いる樹脂の粒子径は0.1〜10mmが好ましく、更に0.2〜6mm、特に0.25〜4mm、殊更0.3〜2mmが好ましい。細孔径は10〜150nmが好ましく、更に10〜100nmが好ましい。材質としては、フェノールホルムアルデヒド系、ポリスチレン系、アクリルアミド系、ジビニルベンゼン系等が挙げられ、特にフェノールホルムアルデヒド系樹脂(例えば、Rohm and Hass社製Duolite A−568)が酵素吸着性向上の点から好ましい。なお、酵素を固定化した後の固定化酵素の粒子径は、「mm単位」においては酵素を固定化する前の担体粒子径と同じである。 The particle diameter of the resin used as the immobilization carrier is preferably 0.1 to 10 mm, more preferably 0.2 to 6 mm, particularly 0.25 to 4 mm, and particularly preferably 0.3 to 2 mm. The pore diameter is preferably 10 to 150 nm, more preferably 10 to 100 nm. Examples of the material include phenol formaldehyde, polystyrene, acrylamide, divinylbenzene, and the like, and phenol formaldehyde resin (for example, Dulite A-568 manufactured by Rohm and Hass) is particularly preferable from the viewpoint of improving the enzyme adsorptivity. The particle diameter of the immobilized enzyme after immobilizing the enzyme is the same as the carrier particle diameter before immobilizing the enzyme in “mm units”.
本発明の固定化酵素に使用する酵素は特に限定はされないが、生産性の向上効果が大きい点から、油脂類分解用酵素としてのリパーゼが好ましい。リパーゼは、動物由来、植物由来のものはもとより、微生物由来の市販リパーゼを使用することもできる。微生物由来リパーゼとしては、リゾプス(Rizopus)属、アスペルギルス(Aspergillus)属、ムコール(Mucor)属、シュードモナス(Pseudomonas)属、ジオトリケム(Geotrichum)属、ペニシリウム(Penicillium)属、キャンディダ(Candida)属等の起源のものが挙げられる。 The enzyme used for the immobilized enzyme of the present invention is not particularly limited, but lipase is preferred as an enzyme for decomposing oils and fats from the viewpoint of a large productivity improvement effect. As the lipase, not only those derived from animals and plants but also commercially available lipases derived from microorganisms can be used. Examples of microorganism-derived lipases include the genus Risopus, the genus Aspergillus, the genus Mucor, the genus Pseudomonas, the genus Geotrichum and the genus Penicillium and the genus Penicillium The thing of origin is mentioned.
酵素の固定化を行う温度は、酵素の特性によって決定することができるが、酵素の失活が起きない0〜60℃、特に5〜40℃が好ましい。また固定化時に使用する酵素溶液のpHは、酵素の変性が起きない範囲であればよく、温度同様酵素の特性によって決定することができるが、pH3〜9が好ましい。このpHを維持するためには緩衝液を使用するが、緩衝液としては、酢酸緩衝液、リン酸緩衝液、トリス塩酸緩衝液等が挙げられる。上記酵素溶液中の酵素濃度は、固定化効率の点から酵素の飽和溶解度以下で、かつ十分な濃度であることが好ましい。また酵素溶液は、必要に応じて不溶部を遠心分離で除去した上澄や、限外濾過等によって精製したものを使用することもできる。また用いる酵素質量はその酵素活性によっても異なるが、担体質量に対して5〜1000質量%、特に10〜500質量%が好ましい。 The temperature at which the enzyme is immobilized can be determined depending on the properties of the enzyme, but is preferably 0 to 60 ° C., particularly 5 to 40 ° C. at which the enzyme is not deactivated. Moreover, the pH of the enzyme solution used at the time of immobilization may be in a range where no denaturation of the enzyme occurs, and can be determined by the characteristics of the enzyme as well as the temperature. In order to maintain this pH, a buffer solution is used. Examples of the buffer solution include an acetate buffer solution, a phosphate buffer solution, and a Tris-HCl buffer solution. The enzyme concentration in the enzyme solution is preferably not more than the saturation solubility of the enzyme and sufficient from the viewpoint of immobilization efficiency. Moreover, the enzyme solution can also use what was refine | purified by the supernatant obtained by removing the insoluble part by centrifugation, ultrafiltration, etc. as needed. Moreover, although the enzyme mass to be used varies depending on the enzyme activity, it is preferably 5 to 1000 mass%, particularly preferably 10 to 500 mass%, based on the mass of the carrier.
酵素を固定化する場合、担体と酵素を直接吸着してもよいが、高活性を発現するような吸着状態にするため、酵素吸着前に予め担体を脂溶性脂肪酸又はその誘導体で処理することが好ましい。脂溶性脂肪酸又はその誘導体と担体の接触法としては、水又は有機溶剤中にこれらを直接加えてもよいが、分散性を良くするため、有機溶剤に脂溶性脂肪酸又はその誘導体を一旦分散、溶解させた後、水に分散させた担体に加えてもよい。この有機溶剤としては、クロロホルム、ヘキサン、エタノール等が挙げられる。脂溶性脂肪酸又はその誘導体の使用質量は、担体質量に対して1〜500質量%、特に10〜200質量%が好ましい。接触温度は0〜100℃、特に20〜60℃が好ましく、接触時間は5分〜5時間程度が好ましい。この処理を終えた担体は、ろ過して回収するが、乾燥してもよい。乾燥温度は室温〜100℃が好ましく、減圧乾燥を行ってもよい。 When the enzyme is immobilized, the carrier and the enzyme may be directly adsorbed. However, in order to obtain an adsorption state that expresses high activity, the carrier may be treated with a fat-soluble fatty acid or a derivative thereof in advance before the enzyme adsorption. preferable. As a method for contacting the fat-soluble fatty acid or derivative thereof with the carrier, these may be added directly to water or an organic solvent, but in order to improve dispersibility, the fat-soluble fatty acid or derivative thereof is once dispersed and dissolved in the organic solvent. And then added to a carrier dispersed in water. Examples of the organic solvent include chloroform, hexane, ethanol, and the like. The used mass of the fat-soluble fatty acid or derivative thereof is preferably 1 to 500 mass%, particularly preferably 10 to 200 mass%, based on the mass of the carrier. The contact temperature is preferably 0 to 100 ° C, particularly preferably 20 to 60 ° C, and the contact time is preferably about 5 minutes to 5 hours. The carrier after this treatment is collected by filtration, but may be dried. The drying temperature is preferably room temperature to 100 ° C., and drying under reduced pressure may be performed.
予め担体を処理する脂溶性脂肪酸又はその誘導体のうち、脂溶性脂肪酸としては、炭素数4〜24、好ましくは炭素数8〜18の飽和又は不飽和の、直鎖又は分岐鎖の、水酸基を有していてもよい脂肪酸が挙げられる。具体的には、カプリン酸、ラウリン酸、ミリスチン酸、オレイン酸、リノール酸、α−リノレン酸、リシノール酸、イソステアリン酸等が挙げられる。また前記脂溶性脂肪酸の誘導体としては、これらの脂溶性脂肪酸と一価若しくは多価アルコール又は糖類とのエステル、リン脂質、及びこれらのエステルにエチレンオキサイドを付加したもの等が挙げられる。具体的には、上記脂肪酸のメチルエステル、エチルエステル、モノグリセライド、ジグリセライド、それらのエチレンオキサイド付加体、ポリグリセリンエステル、ソルビタンエステル、ショ糖エステル等が挙げられる。これら脂溶性脂肪酸及びその誘導体はいずれも常温で液状であることが酵素を担体に固定化する工程上好ましい。これら脂溶性脂肪酸又はその誘導体としては、上記2種以上を併用してもよく、菜種脂肪酸、大豆脂肪酸等の天然由来の脂肪酸を用いることもできる。 Among the fat-soluble fatty acids or derivatives thereof for treating the carrier in advance, the fat-soluble fatty acid has a saturated or unsaturated, linear or branched, hydroxy group having 4 to 24 carbon atoms, preferably 8 to 18 carbon atoms. Fatty acids that may be used are listed. Specific examples include capric acid, lauric acid, myristic acid, oleic acid, linoleic acid, α-linolenic acid, ricinoleic acid, isostearic acid and the like. Examples of the fat-soluble fatty acid derivatives include esters of these fat-soluble fatty acids with mono- or polyhydric alcohols or saccharides, phospholipids, and those obtained by adding ethylene oxide to these esters. Specific examples include methyl esters, ethyl esters, monoglycerides, diglycerides, ethylene oxide adducts thereof, polyglycerin esters, sorbitan esters, and sucrose esters of the above fatty acids. These fat-soluble fatty acids and derivatives thereof are preferably liquid at room temperature in terms of immobilizing the enzyme on the carrier. As these fat-soluble fatty acids or derivatives thereof, two or more of the above may be used in combination, and naturally derived fatty acids such as rapeseed fatty acids and soybean fatty acids may be used.
固定化酵素の加水分解活性は20U/g以上、更に100〜10000U/g、特に500〜5000U/gの範囲であることが好ましい。ここで酵素の1Uは、40℃において、油脂類:水=100:25(質量比)の混合液を攪拌混合しながら30分間加水分解をさせたとき、1分間に1μmolの遊離脂肪酸を生成する酵素の分解能を示す。油脂類の単位質量当りに付与した固定化酵素の加水分解活性(U/g−oil)と、ある加水分解率に到達するまでの所要時間は、略反比例の関係にある。 The hydrolysis activity of the immobilized enzyme is preferably 20 U / g or more, more preferably 100 to 10,000 U / g, and particularly preferably 500 to 5000 U / g. Here, 1 U of enzyme produces 1 μmol of free fatty acid per minute when hydrolyzed at 40 ° C. for 30 minutes while stirring and mixing a mixture of fats and oils: water = 100: 25 (mass ratio). The resolution of the enzyme is shown. The hydrolysis activity (U / g-oil) of the immobilized enzyme imparted per unit mass of fats and oils and the time required to reach a certain hydrolysis rate are in a substantially inverse relationship.
固定化酵素を充填した充填層(酵素塔)を用いて加水分解を行う場合、送液条件(通液速度、温度等)により分解速度は異なるが、酵素充填層出口における油脂の加水分解率、加水分解所要時間(充填層内の滞留時間)、充填層内に存在する油脂類の質量(g−oil)及び固定化酵素の充填質量(g)から固定化酵素の見かけ活性(発現活性)(U/g)が求められる。なお、充填層内に存在する油脂類の質量を求めるためには、固定化酵素充填部の容積に充填部の空隙率、反応液中の油脂類の容量比及び油脂類の比重を乗ずることにより求める。 When hydrolysis is carried out using a packed bed (enzyme tower) filled with an immobilized enzyme, the decomposition rate varies depending on the liquid feeding conditions (liquid passing speed, temperature, etc.), but the hydrolysis rate of fats and oils at the outlet of the enzyme packed bed, The apparent activity (expression activity) of the immobilized enzyme from the time required for hydrolysis (residence time in the packed bed), the mass of fats and oils (g-oil) present in the packed bed and the packed mass (g) of the immobilized enzyme ( U / g). In order to determine the mass of the fats and oils present in the packed bed, the volume of the immobilized enzyme packed part is multiplied by the porosity of the packed part, the volume ratio of the fats and oils in the reaction solution, and the specific gravity of the fats and oils. Ask.
本発明における2液相を形成する液体混合物のうちの好ましい一種は油相基質である。油相基質とは主に植物油、動物油又はこれらを組み合わせた油脂類をいうが、油脂類とはトリアシルグリセロールの他、ジアシルグリセロール、モノアシルグリセロール、又は脂肪酸類を含んでいても良く、加水分解の結果得られる脂肪酸を含んでいても良い。油相基質の具体例としては、菜種油、大豆油、ヒマワリ油、パーム油及びアマニ油等の植物油、牛脂、豚脂及び魚油等の動物油等、又はこれらの組み合わせの油脂類が挙げられる。これら油脂類は、脱臭油の他、予め脱臭されていない未脱臭油脂を用いることができるが、これら油脂類の一部又は全部に未脱臭油脂を使用することが、トランス不飽和脂肪酸、共役不飽和脂肪酸を低減し、原料油脂由来の植物ステロール、植物ステロール脂肪酸エステル、トコフェロールを残存させることができる点から好ましい。油相基質中には、前記油脂類の他に脂肪酸等の油溶性成分が混合されていても良い。脂肪酸類とは、加水分解の結果得られる脂肪酸の他、上記グリセリドの1種以上を含むものも指す。 A preferred type of the liquid mixture forming the two liquid phases in the present invention is an oil phase substrate. The oil phase substrate mainly refers to vegetable oils, animal oils, or oils and combinations thereof. The oils and fats may contain diacylglycerol, monoacylglycerol, or fatty acids in addition to triacylglycerol. The fatty acid obtained as a result may be included. Specific examples of the oil phase substrate include vegetable oils such as rapeseed oil, soybean oil, sunflower oil, palm oil and linseed oil, animal oils such as beef tallow, pork tallow and fish oil, or a combination of these. These fats and oils can be deodorized oil and non-deodorized fats and oils that have not been deodorized beforehand. However, the use of non-deodorized fats and oils for a part or all of these fats and oils may be trans Saturated fatty acids are preferred, and the plant sterols, plant sterol fatty acid esters, and tocopherols derived from raw oils and fats are preferred. In the oil phase substrate, oil-soluble components such as fatty acids may be mixed in addition to the oils and fats. Fatty acids refer to fatty acids obtained as a result of hydrolysis, as well as those containing one or more of the above glycerides.
本発明における2液相を形成する液体混合物のうちの他の好ましい一種は水相基質である。水相基質は水であるが、加水分解の結果得られるグリセリン等、その他の水溶性成分が混合されていても良い。 Another preferred one of the liquid mixtures forming the two liquid phases in the present invention is an aqueous phase substrate. The aqueous phase substrate is water, but other water-soluble components such as glycerin obtained as a result of hydrolysis may be mixed.
本発明において使用する酵素塔の形状は使用するポンプの押し込み圧に耐えられるものであれば良い。また、酵素塔の周囲にジャケットを設け、酵素塔内に流通する反応液を酵素反応に適した温度に調整できるものであることが好ましい。 The shape of the enzyme tower used in the present invention is not particularly limited as long as it can withstand the pushing pressure of the pump used. Moreover, it is preferable that a jacket be provided around the enzyme tower so that the reaction liquid flowing in the enzyme tower can be adjusted to a temperature suitable for the enzyme reaction.
本発明においては、管径が35mmφ以上である酵素塔を用いて反応を行う。酵素塔の管径が35mmφ未満の場合は、酵素活性の低下が起こりにくく、反応性も良好であるが、酵素塔の径が35mmφより大きくなるに従って、酵素の発現活性が低下し、その結果反応性が低下するという問題が起こる。酵素塔の管径は、35〜1000mmφ、更に35〜800mmφ、特に40〜600mmφ、殊更50〜300mmφとすることが、固定化酵素の充填し易さ等の作業性、反応性、生産性の点から好ましい。 In the present invention, the reaction is carried out using an enzyme tower having a tube diameter of 35 mmφ or more. When the tube diameter of the enzyme tower is less than 35 mmφ, the enzyme activity is less likely to decrease and the reactivity is good. However, as the diameter of the enzyme tower becomes larger than 35 mmφ, the enzyme expression activity decreases, resulting in a reaction. The problem of decreased sex occurs. The tube diameter of the enzyme tower should be 35 to 1000 mmφ, more preferably 35 to 800 mmφ, particularly 40 to 600 mmφ, and particularly 50 to 300 mmφ, in terms of workability, reactivity, productivity, etc. To preferred.
本発明においては、上記管径(mm)の固定化酵素の平均粒子径(mm)に対する比率(管径/平均粒子径)が、135(mm/mm)以下であることが必要である。管径の大きい酵素塔を用いる場合も、酵素塔の管径と固定化酵素の平均粒子径との比率を規定することで、スケールアップが可能になると共に、酵素の活性低下を防止できるので、有用物質を効率的に製造できる。
管径/平均粒子径は、5〜135(mm/mm)、更に15〜130 (mm/mm)、特に30〜125 (mm/mm)とすることが、反応性向上の点から好ましい。なお、本発明における酵素を担持させた固定化酵素の平均粒子径は、レーザー散乱回折法粒度分布測定装置LS 13 320(ベックマン・コールター(株)製)により測定した値をいう。
In the present invention, the ratio of the tube diameter (mm) to the average particle size (mm) of the immobilized enzyme (tube diameter / average particle diameter) needs to be 135 (mm / mm) or less. Even when using an enzyme tower with a large tube diameter, by regulating the ratio between the tube diameter of the enzyme tower and the average particle diameter of the immobilized enzyme, it becomes possible to scale up and prevent a decrease in enzyme activity. Useful substances can be produced efficiently.
The tube diameter / average particle diameter is preferably 5 to 135 (mm / mm), more preferably 15 to 130 (mm / mm), and particularly preferably 30 to 125 (mm / mm) from the viewpoint of improving the reactivity. In addition, the average particle diameter of the immobilized enzyme carrying the enzyme in the present invention is a value measured by a laser scattering diffraction particle size distribution analyzer LS 13 320 (manufactured by Beckman Coulter, Inc.).
酵素塔内の温度は、固定化酵素の活性をより有効に引き出すために、0〜60℃、更に20〜40℃とすることが好ましい。酵素塔の長さは、所望の分解率を得るのに必要な長さとすれば良いが、反応性、塔内圧力損失等の点から0.01〜10m、好ましくは0.1〜5mの範囲とすることが好ましい。 The temperature in the enzyme tower is preferably 0 to 60 ° C., more preferably 20 to 40 ° C., in order to extract the activity of the immobilized enzyme more effectively. The length of the enzyme column may be a length necessary for obtaining a desired decomposition rate, but it is in the range of 0.01 to 10 m, preferably 0.1 to 5 m from the viewpoint of reactivity, pressure loss in the column, etc. It is preferable that
酵素塔へ反応液を供給する方法としては、それぞれ別々に酵素塔へ直結する配管にて行ってもよく、又は共有の配管にて供給を行っても良いが、水相と油相の乳化回避の点及び操作性の点から、別々に酵素塔へ直結する配管にて行うことが好ましい。
酵素塔内における反応液の通液線速度は、好ましくは1〜400mm/分、更に5〜200mm/分であるのが好ましい。この通液線速度(mm/分)は、1分間当りの送液量(mm3/分)(又は送液速度(10-3mL/分)ともいう)を充填層断面積(mm2)で除した商で表わされる値をいう。通液線速度を上げることによる充填塔内圧力の増大に伴ない、通液が困難となり、耐圧性の高い酵素充填塔が必要となる他に、固定化酵素が塔内圧力増加により破砕される場合が生じることもあるため、通液線速度は400mm/分以下とすることが好ましい。また、生産性の点から通液線速度は1mm/分以上とすることが好ましい。固定化酵素の発現活性は、通液線速度により変化するため、最適な通液線速度を選定して反応条件を決定することで、所望の生産能力、製造コストに見合った反応を行うことができる。
As a method of supplying the reaction liquid to the enzyme tower, it may be carried out separately by piping directly connected to the enzyme tower, or may be supplied by a common pipe, but avoiding emulsification of the water phase and the oil phase. From the viewpoints of operability and operability, it is preferably carried out separately by piping directly connected to the enzyme tower.
The flow rate of the reaction liquid in the enzyme tower is preferably 1 to 400 mm / min, more preferably 5 to 200 mm / min. The liquid flow rate (mm / min) is the amount of liquid fed per minute (mm 3 / min) (or liquid feed speed (10 −3 mL / min)) as the packed layer cross-sectional area (mm 2 ). The value expressed by the quotient divided by. As the pressure in the packed column increases by increasing the liquid flow rate, liquid passing becomes difficult and an enzyme packed column with high pressure resistance is required. In addition, the immobilized enzyme is crushed by the increased pressure in the column. Since a case may arise, it is preferable that the liquid passing velocity is 400 mm / min or less. Further, from the viewpoint of productivity, it is preferable that the liquid flow rate is 1 mm / min or more. Since the expression activity of the immobilized enzyme changes depending on the flow rate, the reaction can be performed according to the desired production capacity and manufacturing cost by selecting the optimal flow rate and determining the reaction conditions. it can.
酵素塔内の反応液の滞留時間は、加水分解反応の平衡状態を回避し、固定化酵素の活性をより有効に引き出し、生産性を向上させる点から30秒〜60分、更に1分〜40分とすることが好ましい。滞留時間(分)とは、充填層の厚み(mm)に空隙率を乗じ、これを通液線速度(mm/分)で除した値で表わされる。 The residence time of the reaction liquid in the enzyme tower is 30 seconds to 60 minutes, and further 1 minute to 40 minutes from the viewpoint of avoiding the equilibrium state of the hydrolysis reaction, more effectively drawing out the activity of the immobilized enzyme, and improving productivity. Minutes are preferred. The residence time (minute) is represented by a value obtained by multiplying the thickness (mm) of the packed bed by the porosity and dividing this by the liquid linear velocity (mm / minute).
本発明においては、反応性、生産性等の兼ね合いから、酵素塔を通過した反応液をそのまま反応終了物としても良く、また、反応液を一旦油水分離し、油相を分取した後に新しい水を加えて上記と同様の方法で再度同一の酵素塔へ供給し、所望の反応率が得られるまで繰り返し通過させても良い。また、反応液を一旦油水分離し、油相を分取した後に新しい水を加えて上記と同様の方法で再度、別の酵素塔へ供給して連続反応を行っても良い。また、複数の酵素塔を用いて反応液の油水分離を行いながら、油相を次の酵素塔へ、水相を前の酵素塔へ供給する事により、より分解率の高い油相を新鮮な水相と反応させる擬似向流法で行っても良い。反応液の油水分離法としては、自然沈降型、遠心分離型等の油水分離器が一般に使用されるが、特に限定されない。 In the present invention, the reaction solution that has passed through the enzyme tower may be used as it is as a reaction-finished product in consideration of reactivity, productivity, and the like. May be added again to the same enzyme tower in the same manner as described above, and may be repeatedly passed until a desired reaction rate is obtained. Alternatively, the reaction solution may be once separated into oil and water, and after separating the oil phase, fresh water may be added and supplied again to another enzyme tower by the same method as described above to perform a continuous reaction. In addition, by separating the reaction liquid into water using multiple enzyme towers, the oil phase is supplied to the next enzyme tower and the aqueous phase is supplied to the previous enzyme tower, so that the oil phase with a higher decomposition rate is fresh. You may carry out by the pseudo countercurrent method made to react with an aqueous phase. As an oil-water separation method of the reaction liquid, an oil-water separator such as a natural sedimentation type or a centrifugal separation type is generally used, but is not particularly limited.
〔固定化酵素の調製〕
DuoliteA−568(ダイヤモンドシャムロック社製、粒径分布100〜1000μm)1質量部をN/10のNaOH溶液10質量部中で1時間攪拌した。ろ過した後10質量部のイオン交換水で洗浄し500mMの酢酸緩衝液(pH7)10質量部でpHの平衡化を行なった。その後50mMの酢酸緩衝液(pH7)10質量部で2時間ずつ2回pHの平衡化を行なった。この後ろ過を行ない担体を回収した後、エタノール5質量部でエタノール置換を30分行なった。ろ過した後、リシノール酸を1質量部含むエタノール5質量部を加え30分間、リシノール酸を担体に吸着させた。ろ過によって担体を回収した後、50mMの酢酸緩衝液(pH7)5質量部で30分ずつ4回洗浄し、エタノールを除去し、ろ過して担体を回収した。その後市販のリパーゼ(リパーゼAY、アマノ天野製薬(株))1質量部を50mMの酢酸緩衝液(pH7)9質量部に溶解した酵素液と5時間接触させ、固定化を行なった。ろ過し、固定化酵素を回収して50mMの酢酸緩衝液(pH7)10質量部で洗浄を行なうことにより、固定化していない酵素やタンパクを除去した。その後実際に分解を行なう大豆油を4質量部加え12時間攪拌した。以上の操作はいずれも20℃で行なった。その後ろ過して油脂と分離し、固定化酵素とした(以下、固定化酵素Aと表記する)。更に、DuoliteA−568を粉砕して分級した固定化担体、及びDuoliteA−568を分級して425μm以下の微粒子を除去した固定化担体を用いて、上記と同様の方法で固定化酵素を調製した(それぞれ固定化酵素B、固定化酵素Cとする)。固定化酵素A〜Cの加水分解活性(発現すべき活性)、及び質量基準の平均粒子径を表1に示す。
[Preparation of immobilized enzyme]
1 part by weight of Duolite A-568 (manufactured by Diamond Shamrock, particle size distribution: 100 to 1000 μm) was stirred in 10 parts by weight of N / 10 NaOH solution for 1 hour. After filtration, it was washed with 10 parts by mass of ion exchange water, and the pH was equilibrated with 10 parts by mass of 500 mM acetate buffer (pH 7). Thereafter, the pH was equilibrated twice for 2 hours with 10 parts by mass of 50 mM acetate buffer (pH 7). Thereafter, filtration was performed and the carrier was recovered, and then ethanol substitution with 5 parts by mass of ethanol was performed for 30 minutes. After filtration, 5 parts by mass of ethanol containing 1 part by mass of ricinoleic acid was added and ricinoleic acid was adsorbed on the carrier for 30 minutes. After collecting the carrier by filtration, it was washed with 5 parts by mass of 50 mM acetate buffer (pH 7) four times for 30 minutes, ethanol was removed, and the carrier was collected by filtration. Thereafter, 1 part by mass of commercially available lipase (Lipase AY, Amano Amano Pharmaceutical Co., Ltd.) was contacted with an enzyme solution dissolved in 9 parts by mass of 50 mM acetate buffer (pH 7) for 5 hours for immobilization. By filtration, the immobilized enzyme was recovered and washed with 10 parts by mass of 50 mM acetate buffer (pH 7) to remove unimmobilized enzyme and protein. Thereafter, 4 parts by mass of soybean oil to be actually decomposed was added and stirred for 12 hours. All the above operations were performed at 20 ° C. Then, it filtered and isolate | separated from fats and oils, and it was set as the fixed enzyme (henceforth the fixed enzyme A). Furthermore, an immobilized enzyme was prepared in the same manner as described above using an immobilized carrier obtained by pulverizing and classifying Duolite A-568 and an immobilized carrier obtained by classifying Duolite A-568 and removing fine particles of 425 μm or less ( Respectively, immobilized enzyme B and immobilized enzyme C). Table 1 shows the hydrolysis activity (activity to be expressed) of the immobilized enzymes A to C and the average particle diameter based on mass.
<実施例1>
ジャケット付きのステンレス製カラム(内径35mm、高さ1600mm)に、固定化酵素A350g(乾燥質量)を充填し(充填高さ1500mm)、ジャケットにて35℃に保温した。カラム上部より菜種油と蒸留水を質量比10:6で混合した液を1.1kg/Hrで送液し、加水分解反応を行った。その結果を表2に示す。なお、表中の分解率は、分析により求めた酸価をケン化価で除することにより算出した。なお、酸価は、American Oil Chemists.Society Official Method Ca 5a−40に記載の方法により、またケン化価はAmerican Oil Chemists.Society Official Method Cd 3a−94に記載の方法により測定した。
<Example 1>
A stainless steel column with a jacket (inner diameter 35 mm, height 1600 mm) was packed with 350 g of immobilized enzyme A (dry mass) (packing height 1500 mm) and kept at 35 ° C. with the jacket. A liquid in which rapeseed oil and distilled water were mixed at a mass ratio of 10: 6 was fed at 1.1 kg / Hr from the top of the column to conduct a hydrolysis reaction. The results are shown in Table 2. The decomposition rate in the table was calculated by dividing the acid value obtained by analysis by the saponification value. In addition, an acid value is American Oil Chemist. According to the method described in Society Official Method Ca 5a-40, the saponification value was determined by American Oil Chemists. It was measured by the method described in Society Official Method Cd 3a-94.
<実施例2>
ジャケット付きのステンレス製カラム(内径55mm、高さ1600mm)に、固定化酵素Aを乾燥ベースで865g充填し(充填高さ1500mm)、ジャケットにて35℃に保温した。カラム上部より菜種油と蒸留水を質量比10:6で混合した液を2.7kg/Hrで送液し、加水分解反応を行った。結果を表2に示す。
<Example 2>
A stainless steel column with a jacket (inner diameter 55 mm, height 1600 mm) was charged with 865 g of immobilized enzyme A on a dry basis (packing height 1500 mm), and kept at 35 ° C. with the jacket. A liquid in which rapeseed oil and distilled water were mixed at a mass ratio of 10: 6 was fed from the top of the column at 2.7 kg / Hr to conduct a hydrolysis reaction. The results are shown in Table 2.
<実施例3>
実施例1における固定化酵素Aを固定化酵素Bに替えた以外は実施例1と同一の方法で、加水分解反応を行った。結果を表2に示す。
<Example 3>
The hydrolysis reaction was performed in the same manner as in Example 1 except that the immobilized enzyme A in Example 1 was replaced with the immobilized enzyme B. The results are shown in Table 2.
<実施例4>
ジャケット付きのステンレス製カラム(内径70mm、高さ1600mm)に、固定化酵素Cを乾燥ベースで1400g充填し(充填高さ1500mm)、ジャケットにて35℃に保温した。カラム上部より菜種油と蒸留水を質量比10:6で混合した液を4.3kg/Hrで送液し、加水分解反応を行った。結果を表2に示す。
<Example 4>
A stainless steel column with a jacket (inner diameter 70 mm, height 1600 mm) was charged with 1400 g of immobilized enzyme C on a dry basis (packing height 1500 mm), and kept at 35 ° C. with the jacket. A liquid in which rapeseed oil and distilled water were mixed at a mass ratio of 10: 6 was fed from the top of the column at 4.3 kg / Hr to conduct a hydrolysis reaction. The results are shown in Table 2.
<比較例1>
実施例4における固定化酵素Cを固定化酵素Aに替えた以外は実施例4と同一の方法で、加水分解反応を行った。結果を表2に示す。
<Comparative Example 1>
A hydrolysis reaction was performed in the same manner as in Example 4 except that the immobilized enzyme C in Example 4 was replaced with the immobilized enzyme A. The results are shown in Table 2.
<比較例2>
実施例2における固定化酵素Aを固定化酵素Bに替えた以外は実施例2と同一の方法で、加水分解反応を行った。結果を表2に示す。
<Comparative example 2>
The hydrolysis reaction was performed in the same manner as in Example 2 except that the immobilized enzyme A in Example 2 was replaced with the immobilized enzyme B. The results are shown in Table 2.
<比較例3>
実施例4における固定化酵素Cを固定化酵素Bに替えた以外は実施例4と同一の方法で、加水分解反応を行った。結果を表2に示す。
<Comparative Example 3>
The hydrolysis reaction was performed in the same manner as in Example 4 except that the immobilized enzyme C in Example 4 was replaced with the immobilized enzyme B. The results are shown in Table 2.
表2に示した結果から、管径が35mmφ以上の固定床型反応塔を用いた場合も、反応塔内に、該管径の固定化酵素の平均粒子径に対する比率(管径/平均粒子径)が135以下となるように固定化酵素を充填することによって、分解率が向上し、固定化酵素の(見かけ)活性が有効に発現することが明らかとなった。 From the results shown in Table 2, even when a fixed bed type reaction tower having a tube diameter of 35 mmφ or more was used, the ratio of the tube diameter to the average particle diameter of the immobilized enzyme (tube diameter / average particle diameter) It has been clarified that the degradation rate is improved and the (apparent) activity of the immobilized enzyme is effectively expressed by filling the immobilized enzyme so as to be 135 or less.
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PCT/JP2007/001403 WO2008072382A1 (en) | 2006-12-15 | 2007-12-14 | Process for producing useful substance with immobilized enzyme |
US12/518,285 US8252560B2 (en) | 2006-12-15 | 2007-12-14 | Process for producing useful substance with immobilized enzyme |
CN200780046399.5A CN101558162B (en) | 2006-12-15 | 2007-12-14 | Process for producing useful substance with immobilized enzyme |
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