JPH03168084A - Immobilized lipase and hydrolysis of fat and oil with same lipase - Google Patents
Immobilized lipase and hydrolysis of fat and oil with same lipaseInfo
- Publication number
- JPH03168084A JPH03168084A JP1308167A JP30816789A JPH03168084A JP H03168084 A JPH03168084 A JP H03168084A JP 1308167 A JP1308167 A JP 1308167A JP 30816789 A JP30816789 A JP 30816789A JP H03168084 A JPH03168084 A JP H03168084A
- Authority
- JP
- Japan
- Prior art keywords
- oils
- lipase
- fats
- immobilized lipase
- group
- 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
- 102000004882 Lipase Human genes 0.000 title claims abstract description 48
- 108090001060 Lipase Proteins 0.000 title claims abstract description 48
- 239000004367 Lipase Substances 0.000 title claims abstract description 48
- 235000019421 lipase Nutrition 0.000 title claims abstract description 48
- 230000007062 hydrolysis Effects 0.000 title description 6
- 238000006460 hydrolysis reaction Methods 0.000 title description 6
- 239000003925 fat Substances 0.000 claims abstract description 34
- 239000003921 oil Substances 0.000 claims abstract description 31
- 229920001661 Chitosan Polymers 0.000 claims abstract description 17
- 230000003301 hydrolyzing effect Effects 0.000 claims abstract description 13
- 238000000034 method Methods 0.000 claims abstract description 10
- 125000001165 hydrophobic group Chemical group 0.000 claims abstract description 8
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 claims abstract description 6
- 125000003277 amino group Chemical group 0.000 claims abstract description 5
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims abstract description 5
- 125000000217 alkyl group Chemical group 0.000 claims abstract description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 3
- 244000144992 flock Species 0.000 abstract 5
- 235000019198 oils Nutrition 0.000 description 15
- 230000000694 effects Effects 0.000 description 10
- 102000004190 Enzymes Human genes 0.000 description 9
- 108090000790 Enzymes Proteins 0.000 description 9
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- 239000000872 buffer Substances 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 102000004195 Isomerases Human genes 0.000 description 2
- 108090000769 Isomerases Proteins 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 239000004006 olive oil Substances 0.000 description 2
- 235000008390 olive oil Nutrition 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 230000002152 alkylating effect Effects 0.000 description 1
- 210000001557 animal structure Anatomy 0.000 description 1
- KCXMKQUNVWSEMD-UHFFFAOYSA-N benzyl chloride Chemical compound ClCC1=CC=CC=C1 KCXMKQUNVWSEMD-UHFFFAOYSA-N 0.000 description 1
- 229940073608 benzyl chloride Drugs 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Fats And Perfumes (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は、固定化リパー ゼ、および固定化リパーゼを
用いた油脂の加水分解方法に関する。DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to an immobilized lipase and a method for hydrolyzing fats and oils using the immobilized lipase.
従来のリパーゼによる油脂の加水分解は、リパーゼ水溶
液と油脂とを混合撹拌するか、または両者を混合乳化し
たスラリー状態で反応させる方法により行われている。Conventional hydrolysis of fats and oils by lipase is carried out by mixing and stirring an aqueous lipase solution and fats and oils, or by mixing and emulsifying the two to react in a slurry state.
また酵素の再利用によるコスト低減を目的として酵素を
固定化することが試みられ、イソメラーゼ等においては
既に実用化されて，いる。Furthermore, attempts have been made to immobilize enzymes for the purpose of reducing costs by reusing enzymes, and this has already been put into practical use for isomerase and the like.
しかしながら、前記イソメラーゼの場合の反応は水溶系
であって、これに対し、リパーゼの場合には基質となる
油脂が水不溶性であるため、固定化リパーゼを基質中に
充分に分散することができず、このため充分な加水分解
率が得られずにリパーゼによる油脂の工業的製造の難点
となっている。However, the reaction in the case of isomerase is a water-soluble system, whereas in the case of lipase, the substrate fat or oil is water-insoluble, so the immobilized lipase cannot be sufficiently dispersed in the substrate. Therefore, a sufficient hydrolysis rate cannot be obtained, which is a problem in the industrial production of fats and oils using lipase.
本発明者は、かかる欠点を改良すべく鋭意研究を重ねた
結果、塩基性多糖類であるキトサンのアミノ基、水酸基
の一部にアルキル基、ベンジル基などの疎水基を導入し
てキトサンの疎水性をある程度高め、マクロポーラスな
担体とともにこれを用いてフロックを形或させ、さらに
リパーゼを吸着させることにより、油脂の加水分解を良
好に達威できる固定化リパーゼの作或に成功した。As a result of extensive research in order to improve these drawbacks, the present inventor has introduced hydrophobic groups such as alkyl groups and benzyl groups into some of the amino groups and hydroxyl groups of chitosan, which is a basic polysaccharide, to make chitosan hydrophobic. We succeeded in creating an immobilized lipase that can successfully hydrolyze fats and oils by increasing its properties to some extent, forming flocs using it together with a macroporous carrier, and adsorbing lipase.
すなわち、本発明の固定化リパーゼは、キトサンのアミ
ン基、水酸基の一部にアルキル基、ベンジル基などの疎
水基を導入し、これにフロックを形成させた後リパーゼ
を吸着させたことを特徴とし、また本発明は併せて、前
記固定化リパーゼを用いて油脂を加水分解することを特
徴とする固定リパーゼによる油脂の加水分解方法を提供
する。That is, the immobilized lipase of the present invention is characterized by introducing hydrophobic groups such as alkyl groups and benzyl groups into some of the amine groups and hydroxyl groups of chitosan, forming flocs, and then adsorbing the lipase. The present invention also provides a method for hydrolyzing fats and oils using immobilized lipase, which comprises hydrolyzing fats and oils using the immobilized lipase.
第３請求項は、固定化リパーゼを油脂中に分散させて油
脂を加水分解すること、第４請求項は、固定化リパーゼ
を充填したカラム中に油脂を通液して油脂７を加水分解
することを特徴とする。The third claim is to hydrolyze the fat by dispersing the immobilized lipase in the fat, and the fourth claim is to hydrolyze the fat by passing the fat through a column packed with the immobilized lipase. It is characterized by
リパーゼの固定化については種々の方法が提案されてい
る。すなわち、寒天由来のアガロースをアルキル化、フ
ェニル化またはトリチル化した多種類ゲルにリパーゼを
固定化した例（特開昭５８−１４６２８４号）もあるが
、本発明の固定化リパーゼは、ゲルとは異り、親水性お
よび疎水性の両基を有するフロックを用いて構成されて
いるため、油脂、水の両方とも簡単に固定化リパーゼ中
に浸潤することが可能である。また油脂の加水分解に際
して油脂を乳化する必要がなく、この固定化リパーゼを
油脂と緩衝液を含む反応液中に加えて撹拌するか、また
は固定化リパーゼを充填したカラム中に油脂と緩衝液と
の混合液を通液するなどの方法により容易に油脂の加水
分解を実施することができる。Various methods have been proposed for immobilizing lipase. That is, although there are examples in which lipase is immobilized on various types of gels made by alkylating, phenylating, or tritylating agarose derived from agar (Japanese Patent Application Laid-Open No. 146284/1984), the immobilized lipase of the present invention is different from gel. On the contrary, since it is constructed using flocs having both hydrophilic and hydrophobic groups, both oil and water can easily infiltrate into the immobilized lipase. In addition, there is no need to emulsify fats and oils when hydrolyzing fats and oils, and this immobilized lipase is added to a reaction solution containing fats and oils and a buffer solution and stirred, or the fats and oils and buffer solution are mixed in a column packed with immobilized lipase. Hydrolysis of fats and oils can be easily carried out by a method such as passing a mixed solution of.
本発明の特徴とするところは上述したようにフロックの
形成とキトサンへの疎水基の導入である。すなわち塩基
性多糖類であるキトサンは酢酸、蟻酸などの稀有機酸に
溶解するが、アルカリ性にすることにより糸状の塊であ
るフロックを形戊する。このフロックはリパーゼを良く
吸着し、リパーゼを固定化することができるが、この固
定化リパーゼでは充分な酵素活性を発揮させることは困
難である。そこで上述したようにキトサンに疎水基を導
入することによって酵素活性を高めることができた。As mentioned above, the features of the present invention are the formation of flocs and the introduction of hydrophobic groups into chitosan. That is, chitosan, which is a basic polysaccharide, dissolves in rare organic acids such as acetic acid and formic acid, but when made alkaline, it forms flocs, which are thread-like lumps. Although this floc adsorbs lipase well and can immobilize lipase, it is difficult to exhibit sufficient enzymatic activity with this immobilized lipase. Therefore, as mentioned above, we were able to increase the enzyme activity by introducing hydrophobic groups into chitosan.
本発明で用いるリパーゼはその起源を問わず、微生物、
動物臓器または植物種子を起源とする何れのリパーゼを
も固定化し得るものである。Regardless of its origin, the lipase used in the present invention may be a microorganism,
Any lipase originating from animal organs or plant seeds can be immobilized.
キトサンへのベンジル基の導入を常法通リピリジン中で
塩化ベンジルを用いて行なった。Benzyl groups were introduced into chitosan using benzyl chloride in lipyridine in a conventional manner.
上記物質１．５ｇを稀酢酸に溶解し、セライトとともに
アルρり溶液中に投入しフロックを形威せしめ、水洗し
担体を調整した。これにリパーゼ２ｇを吸着させ固定化
リパーゼを作成した。第ｌ図はキトサンおよびベンジル
化キトサンを用いて調整した固定化リパーゼをカラムに
充填しオリーブオイルを基質として酵素活性を比較した
ものである。ベンジル基を導入したものはキトサンに比
べ高い活性を示し、また流速２ｍｌ／ｍｉｎにおける酵
素活性はキトサンの場合の０．５ ｍｌ／ ｍｉｎにお
ける酵素活性と同程度を示し、加水分解効率としては約
４倍の活性が示された。図上、黒丸がペンジル化キトサ
ン、白丸がキトサンである。1.5 g of the above substance was dissolved in dilute acetic acid, poured into an alkali solution together with celite to form a floc, and washed with water to prepare a carrier. 2 g of lipase was adsorbed onto this to prepare immobilized lipase. Figure 1 shows a comparison of the enzyme activities of immobilized lipase prepared using chitosan and benzylated chitosan packed into a column and using olive oil as a substrate. The one with a benzyl group introduced shows higher activity than chitosan, and the enzyme activity at a flow rate of 2 ml/min is comparable to that of chitosan at 0.5 ml/min, and the hydrolysis efficiency is about 4. It showed twice the activity. In the figure, the black circles are pensylated chitosan and the white circles are chitosan.
〔実施例２〕
上記と同様に調整したカラムを用い、３０〜６０°Ｃま
での各温度で酵素活性を測定した結果を第２図に示した
。５０°Ｃにおいて最大活性を示したが、６０°Ｃまで
急激な酵素の失活は見られなかった。また、第３図はオ
リーブオイルと緩衝液との比を種々変化させ酵素活性を
比較したものである。流速２ ｍｌ／ ｍｉｎ ， ４
５℃で反応を行わせた。緩衝液の多いものほど高い分解
率を示したが、流速一定であるため緩衝液の量が少なく
なるにつれ油脂の量が増加するため加水分解量は、緩衝
液が少なくなるにつれて増加した。[Example 2] Using a column prepared in the same manner as above, the enzyme activity was measured at various temperatures from 30 to 60°C. The results are shown in FIG. The enzyme showed maximum activity at 50°C, but no rapid enzyme deactivation was observed up to 60°C. Furthermore, FIG. 3 shows a comparison of enzyme activity by varying the ratio of olive oil to buffer solution. Flow rate 2 ml/min, 4
The reaction was carried out at 5°C. The higher the amount of buffer, the higher the decomposition rate, but since the flow rate was constant, the amount of hydrolysis increased as the amount of buffer decreased, as the amount of fat and oil increased as the amount of buffer decreased.
第ｌ図ないし第３図はそれぞれ固定化リバーゼの活性特
性を示すグラフである。
ｍｌ／ｍｉｎFigures 1 to 3 are graphs showing the activity characteristics of immobilized reverse, respectively. ml/min
Claims (1)
ベンジル基などの疎水基を導入し、これにフロックを形
成させた後リパーゼを吸着させたことを特徴とする固定
化リパーゼ。 ２、請求項１の固定化リパーゼを用いて油脂を加水分解
することを特徴とする固定化リパーゼによる油脂の加水
分解方法。 ３、固定化リパーゼを油脂中に分散させて油脂を加水分
解することを特徴とする第２項記載の固定化リパーゼに
よる油脂の加水分解方法。 ４、固定化リパーゼを充填したカラム中に油脂を通液し
て油脂を加水分解することを特徴とする第２項記載の固
定化リパーゼによる油脂の加水分解方法。[Claims] 1. Alkyl group in part of the amino group and hydroxyl group of chitosan,
An immobilized lipase characterized by introducing a hydrophobic group such as a benzyl group, forming a floc, and then adsorbing the lipase. 2. A method for hydrolyzing fats and oils using immobilized lipase, which comprises hydrolyzing fats and oils using the immobilized lipase according to claim 1. 3. The method for hydrolyzing fats and oils using immobilized lipase according to item 2, which comprises dispersing the immobilized lipase in fats and oils to hydrolyze the fats and oils. 4. The method for hydrolyzing fats and oils using immobilized lipase according to item 2, which comprises hydrolyzing the fats and oils by passing the fats and oils through a column packed with immobilized lipase.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1308167A JPH03168084A (en) | 1989-11-28 | 1989-11-28 | Immobilized lipase and hydrolysis of fat and oil with same lipase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1308167A JPH03168084A (en) | 1989-11-28 | 1989-11-28 | Immobilized lipase and hydrolysis of fat and oil with same lipase |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH03168084A true JPH03168084A (en) | 1991-07-19 |
| JPH0523746B2 JPH0523746B2 (en) | 1993-04-05 |
Family
ID=17977707
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1308167A Granted JPH03168084A (en) | 1989-11-28 | 1989-11-28 | Immobilized lipase and hydrolysis of fat and oil with same lipase |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH03168084A (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS59213390A (en) * | 1983-05-19 | 1984-12-03 | Asahi Denka Kogyo Kk | Immobilized enzyme and its preparation |
-
1989
- 1989-11-28 JP JP1308167A patent/JPH03168084A/en active Granted
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS59213390A (en) * | 1983-05-19 | 1984-12-03 | Asahi Denka Kogyo Kk | Immobilized enzyme and its preparation |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0523746B2 (en) | 1993-04-05 |
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