JPS59213390A - Immobilized enzyme and its preparation - Google Patents

Abstract

PURPOSE:To immobilize lipase which catalyses important reactions in the oil and fat industry, e.g. hydrolysis, esterification, ester-exchange, etc., in high efficiency, by using a carrier composed of a porous solid and a chitosan derivative. CONSTITUTION:Lipase is immobilized to a carrier composed of a porous solid and a chitosan derivative and prepared by dispersing the porous solid in a gelled chitosan derivative and drying the dispersion. The porous solid is selected from florisil, diatomaceous earth, Celite, silica gel, China clay, corn cob, and sawdust or their mixture, and the chitosan derivative is selected from chitosan, N- acylchitosan, N-(mixed acyl)chitosan, N,O-acylchitosan, N-allylidenechitosan, N-alkylidenechitosan, a chitosan salt, their partial reaction product, or their mixture.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an immobilized enzyme, more specifically, an immobilized enzyme effective for major reactions in the oil and fat industry such as hydrolysis reactions, esterification reactions, and transesterification reactions, and a method for producing the same. The present invention also relates to the above reaction method using the immobilized enzyme.

What is important when immobilizing an enzyme is that it not only maintains stable activity of the enzyme, but also that the reaction product is easy to separate and purify, and that there is no change in form when reused and that recovery is easy. There are certain things, etc.

The main reactions in the oil and fat industry include 5-hydrolysis reactions, esterification reactions, and transesterification reactions, but practical immobilization of the enzymes that catalyze these reactions has not yet been developed. Therefore, commercial reactions using immobilized enzymes have not yet been carried out.

In the case of the hydrolysis reaction of fats and oils by enzymes, the reaction usually takes place in a heterogeneous substrate consisting of a substrate, that is, fats and oils, water, and an enzyme or an immobilized enzyme. In a reaction system like this,
However, an emulsion is formed at the interface, and the amount of decomposed products becomes F211t or F211t, which lowers the recovery rate of the 4W product.

Furthermore, in the case of esterification reactions and transesterification reactions using enzymes, non-aqueous reaction systems are often used, but in the case of non-aqueous systems, there are problems different from those of ordinary aqueous enzyme reaction systems.

That is, for non-aqueous systems, the apparent reduction in enzyme activity is a
・In sushi, it is not the case that the enzyme is inactivated, but the problem is that it is caused by a temporary stoppage of the enzyme activity due to a decrease in water in the reaction site, which greatly affects the results. In order to solve this problem, it is very important to maintain or control the amount of water necessary to maintain the stable appearance of enzyme activity inside the enzyme or immobilized enzyme that is the reaction site. Become.

In order to solve these problems, the inventors have conducted various studies on immobilized enzymes useful for hydrolysis reactions, esterification reactions, transesterification reactions, etc., which are the main reactions mainly in the oil and fat industry. and maintenance of stable enzyme activity and separation of reaction products 1'? We have now invented a useful immobilized enzyme from the viewpoints of ease of use, no change in form upon use, and ease of recovery.

Accordingly, the present invention provides an immobilized enzyme characterized in that the enzyme is immobilized on a carrier made of a porous solid and a cylindrical derivative, and a method for producing the same.

Porous solids constituting the carrier in the present invention include florisil, diatomaceous earth, celite, silica gel, white clay,
It is preferable to use one or more selected from the group consisting of corn corn and sawdust.

The chitosan derivatives constituting the carrier in the present invention include chitosan, N-acyl chitosan, N-mixed acyl chitosan, N,O-acyl chitosan, N-allylidene],
A compound consisting of one or more selected from the group consisting of chitozan, N-alkylidene chitozan, chitozan salts, and partial reactants thereof is preferably used. These partial reactants refer to compounds formed by the reaction of a part of the functional groups of chitozan, that is, the amino groups or hydroxyl groups.

The chitosan derivative is also a deacetylated product in a homogeneous chitin reaction system, with a deacetylation rate of 4.
0 to 60% may also be used.

Further, the carrier in the immobilized enzyme of the present invention can further contain a super absorbent resin, and the super absorbent 46" resin includes:
Water-absorbing polyurethane resin, Koxyethyl methacrylate I for Borich l, polyacrylic acid type 4i + fat, forging powder - acrylic acid rough 1 to commercial product (allylic acid to starch graph 1)
- polymerized, neutralized and crosslinked with a small amount of crosslinking agent),
Starch--acrylonitrile graph 1-polymerized product (graph 1-polymerized acrylonitrile to starch using ceric salt radiation, hydrolysis, purification, and drying),
starch carboxymethylated with monochloroacetic acid and cross-linked with formalin; cellulose-acrylonitrile graft polymer; cellulose carboxymethylated with monochloroacetic acid and cross-linked with formalin;
Alternatively, a copolymer of vinyl alcohol and acrylic acid or a copolymer of vinyl and methyl methacrylate is hydrolyzed to form a self-crosslinker, polyvinyl alcohol intermolecularly crosslinked with dialdehyde or radiation, crosslinked polyethylene oxy-F etc. These super absorbent resins may be used alone or in combination of two or more. Among these super absorbent resins, those obtained by hydrolyzing and self-crosslinking a θ powder-acrylic acid graft polymer, a vinyl alcohol and acrylic acid copolymer, or a vinyl acetate and methyl methacrylate copolymer are preferably used. It is possible. The former is Sanyo Chemical Co., Ltd.'s product specification, San Ueno h1M 300, and the latter is Sumitomo Chemical Co., Ltd., product specification Sumikagel 5-50.
It is also available as a commercial product.

In the present invention, lipase can be particularly effectively immobilized as an enzyme to be immobilized on the carrier described above, but other enzymes include bosvolipase, crucose schisomerase, inhertase, conisterase, lactate racemase, Kimo1-Lipsin, papain, promelain, pecranase, amino[1f/! Kisikuse etc. are also mentioned.

Examples of lipases that can be used include rhizopus, asbeno (barrus thread, candida, mucor, pancreatic perse, etc.), and many of these are commercially available. In the case of specifically transesterifying the polymer, Sopus de Lemer, Rhinopus jabonicus, and Mucor-Tobonicus having characteristics matching L1 may be used.

The usage ratio (weight) of the porous solid and the chitosan derivative is 0.05 of the chitosan derivative per 1 part of the porous solid.
Preferably from 1 part to 1 part, more preferably from 0.1 to 0 parts.
．． There are 5 parts. When using super absorbent resin, 0.05 part to 1815 parts of super absorbent resin is used for 1 part of porous solid.
The amount is preferably 0.1 part to 0.5 part, more preferably 0.1 part to 0.5 part.

The immobilized enzyme of the present invention can be obtained by forming a gel of a chitosan derivative, dispersing a porous solid in a gel, drying this dispersion to obtain a carrier, and holding the enzyme in the carrier. It is produced by the method of grinding an immobilized enzyme of the present invention, which is characterized by:

In order to retain the enzyme on the carrier, the above dispersion must be dried,
The enzyme can be immobilized more effectively each time a carrier is obtained by pulverization and mixed with an aqueous enzyme solution or an enzyme buffer solution. Furthermore, after thoroughly mixing the dried and pulverized product with the enzyme powder, each time water or a buffer solution is sufficiently added and mixed, more effective immobilization can be achieved. Methods for drying after dispersing a porous solid in a gel made of a chitosan derivative include adding and stirring the dispersion in acetone, forming a thin film and air drying it, spray-trying, and freeze drying. For example, methods such as

Alternatively, the immobilized enzyme of the present invention can also be produced by adding and mixing a super absorbent resin to a dried dispersion consisting of a chitosan derivative and a porous solid, and then adsorbing the enzyme in the same manner as in Section 2. I can do it.

In the IIl'I structure 4J of the immobilized enzyme of the present invention, the surface of the porous solid is coated with a gel made of a xane derivative, and the
It is assumed that the l'14' element is immobilized on the chitozan gel by adsorption, inclusion, ionic bonding, or the like. In addition, in systems containing super absorbent resin,
The porous solid surface is coated with a gel made of a xane derivative and a super absorbent resin, and the enzyme is immobilized on the gel by adsorption, entrapment, ionic bonding, etc. It seems that there are. Because of this structure, the immobilized enzyme of the present invention has a large surface area, making it a commercially available immobilized enzyme. Furthermore, by selecting the particle size of the porous solid, it has the characteristic that it can be made into an immobilized enzyme that can be easily separated and recovered. In addition, the surface portion and the inside of the pores of the immobilized enzyme of the present invention are composed of a chitosan derivative gel or a chitosan derivative and a super absorbent resin gel,
It has the characteristic of being able to freely control the water content on the surface and inside of the immobilized enzyme, which is the reaction site.
-ru. In other words, the immobilized enzyme of the present invention has the characteristic that it can retain not only water necessary and sufficient for the appearance of enzyme activity, but also water necessary and sufficient for the reaction.

By using the immobilized enzyme of the present invention, hydrolysis reactions, esterification reactions of fats and oils, ester group exchange reactions of fats and oils, etc. can be carried out as effectively as 9J.

In the case of hydrolysis reactions of fats and oils, the system is usually a heterogeneous substrate reaction system consisting of a substrate and water, or an emulsion is formed at the interface in such a reaction system, and the separation of the decomposition products is difficult. However, if the weight of the scale is 1 ton and the workpiece is A1f, the product recovery rate will decrease and the process cost will be higher.

However, in the case of using the immobilized enzyme of the present invention/111 for hydrolysis of fats, sufficient water necessary for the hydrolysis reaction can be retained inside the immobilized enzyme, and the amount of water that is necessary and sufficient for the hydrolysis reaction can be retained within the immobilized enzyme. The reaction can be carried out in a system that does not substantially contain water, and there is almost no emulsion formation, making it easy to separate the decomposition products and increasing the product recovery rate. In the case of reactions and transesterification reactions, they are often non-aqueous, or if they are non-aqueous,
As for the enzyme activity, the low θ of J does not necessarily correspond to the essential inactivation of the enzyme, as it is due to temporary cessation of the enzyme's activity due to a decrease in water in the reaction site. However, the immobilized enzyme of the present invention is characterized in that it is possible to easily replenish the inside of the immobilized enzyme with water necessary and sufficient for the appearance of enzyme activity, so that stable enzyme activity can be maintained.

In the case of hydrolysis reaction, the amount of the immobilized enzyme of the present invention to be used is:
It is preferable to use 3 to 40%, more preferably 6 to 20%, based on the substrate. In addition, the enzyme amount is preferably 5 to 20001/g relative to the substrate, and more preferably,
It is 50-500 U/g. In the case of esterification reaction or transesterification reaction, the amount of the immobilized enzyme of the present invention is preferably 0.5 to 10%, preferably 1.0 to 5.0%, based on the substrate. It is. In addition, the amount of enzyme is
It is preferably 20-t0000 U/g, more preferably 100-100 OU/g, relative to the substrate. However, the activity unit (U) of the enzyme is 5ml of olive oil emulsion and 0.
Add the enzyme to 4 ml of 1 M phosphate solution for 1 hour and heat at 37°C.
One activity unit (U) was defined as each fatty acid produced corresponding to 0.06 ml of 0.05 N sodium hydroxide aqueous solution when 30 ml of reaction was carried out. The same applies to the enzyme activity units in the Examples shown below.

Substrates for the reaction method using the immobilized enzyme of the present invention include:
Oils and fats, fatty acids, fatty acid esters, polyols, alcohols, etc. can be used as appropriate.

The above-mentioned oils and fats include general vegetable oils, animal oils, processed oils and fats, or mixtures thereof, such as soybean oil, cottonseed oil, rapeseed oil, olive oil, corn oil, palm oil, flower oil, beef tallow, These include lard and fish oil. In addition, glycerides with specific compositions that are raw materials for cocoa butter substitutes, namely, 1,3-cystearo-2-oleoglyceride, l-palmi-2-oleo-3-stearoglyceride, 1,3-balmite -2 ~ When using oleoglyceride 1 as the object of the transesterification reaction, oils and fats containing a large amount of oleic acid at the 2-position of the glyceride, such as olive oil, camellia oil, sasanqua oil, panimu butter, colander fat,
Examples include irinope butter, kokum butter, shea butter, maua butter, fursola butter, porneotallow butter, and fractionated fats and oils thereof.

Further, as the fatty acid, a linear saturated or unsaturated fatty acid having 2 to 22 carbon atoms can be used. For example, palmitic acid, stearic acid, oleic acid, etc. can be used.

Examples of alcohol esters of fatty acids include esters of the above-mentioned fatty acids and linear saturated -(i11i) alcohols having 1 to G carbon atoms, such as ethyl valmitate,
Mention may be made of ethyl valmitate, methyl stearate, and nityl stearate.

As described above, the immobilized enzyme of the present invention has characteristics such as maintaining stable enzyme activity, easy separation and purification of reaction products, no change in form upon reuse, and easy recovery. It is extremely useful for various reactions using as a substrate.

Hereinafter, the present invention will be further explained in detail with reference to Examples.

Example 1 8 g of Ki1-Zan (manufactured by Kyowa Yushi Kogyo Co., Ltd., trade name Fronac N) was added and mixed in 60 g of a 10% acetic acid aqueous solution to form chitosan acetate gel, and then 440 g of water was added to this.
After adding 32 g of Serai and 100 g of Serai to make a homogeneous mixture, this was added dropwise to 2000 g of acetone and mixed, and the insoluble matter was collected by centrifugation.
Added to 00g of acetone, mixed, filtered, 1 sieve,
After air-drying, deacetone was removed under vacuum to obtain 1 υ of a carrier consisting of KiI-zanacetate-Celai-1-.

After thoroughly mixing 10 g of the carrier with 2 ty of pulverized products of Sanweso 1 to IM-300 (super absorbent resins manufactured by Sanyo Chemical Industries, Ltd.), 10 g of water was added and mixed, thoroughly air-dried, and dried in a colander under vacuum. A carrier for immobilized enzyme was obtained by drying at -1. This carrier and 6,000 U of Lipase OF (Lipaase, manufactured by Meito Sangyo Co., Ltd.) were thoroughly mixed, and 25 g of 0.1M phosphate buffer was further added and mixed to obtain an immobilized enzyme.

- 30 g of Oliveura (saponification value 196.2) and 30 g of n-hexane were prepared iM as described above.
Add to substrate mixture, reaction temperature 37゛C1,)00rp
The hydrolysis 1'1X reaction was carried out at a continuous speed of 1 mm. A small amount of the reaction mixture was separated transversely, the solvent n-hex 97 was removed, and the mixture was neutralized! 1lli was measured and the progress of the reaction was evaluated six times. - After the reaction has progressed to a certain extent, the immobilized enzyme is collected by decantation from the reaction mixture (-7
, similar new group Sr! (Repeat anti-1+i in the mixture
> was done. There is almost no change in the form of the immobilized enzyme when reused, and it can be easily separated by decantation.
I was able to recover it. These results are shown in Table 1.

Table 1: Immobilized Lipase OI? (Meito Sangyo) Hydrolysis reaction example 2 Chitosan (Fronac N) 8g and 0.5% acetic acid aqueous solution 1
Add 132 g of 1,000 g of 1000 g of choline on a foot stool, and without stirring, gradually add 25% glutaral alcohol 1-f solution to loosen the gelled product, and thoroughly dissolve with ion-exchanged water. It was air-dried. The air-dried product was further dried under vacuum to obtain a carrier for immobilized enzyme. Rhizpus derma (R)
lipase (9) derived from Hizopus delemar)
8000U/g)O, 1.71g was dissolved in 1.2g of water, and the above carrier 2. The immobilized enzyme was prepared by gradually adding it to Og and mixing and leaving it for day and night.

20g of the immobilized enzyme, 20g of oleic acid and 1-
It was added to a substrate mixture consisting of 20 g of off-cool and 40 g of n-hexane, and an esterification reaction was carried out at a reaction temperature of 37° C. and 300 rpm. A small amount of the reaction mixture was separated over time, the solvent n-hexane was removed, and the neutralization value was decreased (1 constant,
The degree of progress of the reaction was examined. After the reaction has progressed to a certain extent,
The immobilized enzyme was recovered from the reaction mixture by decantation, and the reaction was repeated using the same new substrate mixture. The results shown in Table 2 were obtained (-!I). There was almost no change in the feeling, and 1η1 could be easily recovered by tectonics.

Table 2 Esterification reaction example 3 using immobilized Rh1zopus delemdr lipase Chitin (manufactured by Tokyo Kasei Kogyo Co., Ltd.) was
(Makromol Chem 177 3589
(1976) and Makromol Chem 17
8. 3197 (1977) in a homogeneous system to obtain a randomly deacetylated product with a deacetylation rate of 48%. 2 g of the deacetylated product was added to 200 g of a 5% malic acid solution with N, 8 g of ground corn cob was added and mixed, poured onto a glass plate, air-dried, and the air-dried product was separated from the glass plate and ground. The carrier for the immobilized enzyme was completely dehydrated under vacuum. Rhizopus derma (R)
lipase (98
000U/g) 0.171g to 1.2g of water/g I
jZ L was gradually added to and mixed with the above-mentioned carrier at 2° Otz, and left to stand day and night to prepare immobilized lipase (immobilized enzyme).

20g of oleic acid and 1 liter of oleic acid.
The mixture was added to a substrate mixture consisting of 20 g of -occour and 40 g of n=hexane, and an esterification reaction was carried out at a reaction temperature of 37° C. and 130 Orpm. A small amount of the reaction mixture was separated over time, the solvent was removed, the acid value was measured, and the progress of the reaction was examined. After the reaction had progressed to a certain extent, the immobilized enzyme was recovered from the reaction mixture by decantation, and the reaction was repeated using the same new substrate mixture to obtain the results shown in Table 3. There was almost no change in the form of the immobilized enzyme upon reuse, and it could be easily separated and recovered by decantation.

Table 3 Esterification reaction using immobilized 11hizopus delemar lipase Example 4 8 g of chi-1-zan (Fronac N) was added and mixed in 60 g of a 0.5% acetic acid aqueous solution to form a chitosan acetate gel.
Furthermore, 440 g of water and 32 g of celite were added to make a homogeneous mixture, and this was mixed with 2000 g of celite.
- The insoluble matter was collected by centrifugation, and the insoluble matter was further added to 1000 g of acetin, mixed, filtered, air-dried, and then deacetonized under vacuum. After drying, the carrier for the immobilized enzyme consisting of chitozan acetate-Celite turned blue. Rhizopus thelema (Rhizopus)
0.171 g of lipase derived from dela wholesaler) in 1 portion of water
．． The lipase aqueous solution was gradually added to and mixed with 1.7 g of the carrier made of chitozan acetate-Celite described in Section 2 to prepare immobilized lipase.

The entire amount of the immobilized lipase was added to a substrate solution consisting of 31.6 g of balm soft part oil, 28.4 g of stearic acid, and 100 g of n-hexano, and transesterification reaction was carried out at 40'C for 8 hours. After 8 hours of reaction, the lid-fixed fermentation enzyme was collected from the reaction system, and the reaction was repeated under the same reaction conditions. To determine the composition of triclyceride 1 by Craffey, the sum of the amount of change in carbon number 50 (ΔCso) and the change L1 (8C51) in triclyceride with 54 carbons (Δ
The relationship between Cso (ΔC54) and the number of repeated reactions is represented by a solid line (---) in FIG. 1.

Rhizopus delema
r) derived from River-11 (8000LJ/g) 0.1
71 g was dissolved in 1.8 g of water, and the solution was gradually added to and mixed with 1.7 g of Celite. Cellai 1-adsorbed Verse was subjected to the same transesterification reaction system as in Example 4, and the same analysis was conducted. The iM results are shown in FIG. 1 by a dotted line (-).

The results of Comparative Example 1 show that the reaction rate is much slower than that of Example 4. The Celite-adsorbed lipase is finely dispersed in the system and is easily separated by decantation.
- However, it was difficult to collect and store the items.

Example 5 8 g of Gitozan (Fronac N) was added to 10% acetic acid aqueous solution 6
Add and mix into 0g of acetone to form a chitosan acetate gel, then add 440g of water and 32g of Q clay to make a homogeneous mixture, then drop this into 2000g of acetone and mix. The insoluble matter was collected by centrifugation, and the insoluble matter was added to 1.000 g of acetone, mixed, separated into 6 portions, air-dried, and deacetone-dried under vacuum. A carrier for immobilized enzyme consisting of salt - Serai I was obtained. Rizupustelema (I draw 1zo
pus delemrlr) derived from lipase (913
000 [J/g) 1.258LX was dissolved in 1.2 g of water, and the Lipase aqueous solution was added to 1.7 g of the above-mentioned chitosan acetate-Celite carrier and mixed. The immobilized lipase was prepared and air-dried.

The above immobilized Reverse company was mixed with palm central oil 34°:31
3 and O-stearic acid 2.57g and RL-Hegyoman 1
A group consisting of 00g)! The mixture was dispersed and added to the liquid, and the ester group reaction was carried out at a reaction temperature of 37° and a stirring speed of 300 rpm. After the reaction has proceeded for a constant f2 times, the immobilized enzyme is recovered by reaction 71M compound, J, region) = conversion, and n - = ' = Kizanyo < ? 3
14 L'ft Ly tag, same c no see ζW', rfJ
+1 volume of the solution, and the process of converting anti-L'i> to fi', + was repeated under the same conditions. The reaction mixture of 1,000 ml was heated over time, and the charcoal was heated using a heated caster 1,1 phthalate.The composition was measured to show the reaction. The immobilized enzyme recovered in the reaction was air-dried 6: J, excluding rehegisan, and then water 1.2
g was added, mixed, sawed, and air-dried day and night, and the process for 110J4 was repeated. There is almost no change in the shape of the immobilized enzyme after reuse, and it can be easily separated by canting.
I was able to recover it.

Table 4 Immobilization R) + esterification reaction with 1 zopus delemar lipase * 4 times 1. Water 1.2 + Z
After adding and mixing, and air drying, (1f was used, 7.

[Brief explanation of the drawing]

FIG. 1 shows the amount of change in tricryceride in the reaction mixture due to repeated reactions. It is a clan that shows the transition. Figure 1 diagonal'') Continuation of the anti-J Heart Conversion - June 29, 1981 1. Patent Application for Indication of the Case 1981-881 (: 71 2. Name of the Invention) Immobilized intoxicants and O, their manufacturing capacity (3-sleeve market, 1.4 tons + 1 person per head, N138), Asahi Denka, Inc. 41 4 Agents Aka 1 Le 9 J' IJ, Minato-ku, Tokyo (+ J No. 29-Bashifi, Rinogizaka 601, 107 Ju03 (d79) 2531 (7653)
Patent Attorney Hatori Rei 5.711i Inquiry 7 of the suspension order, contents of amendment (11, page 4, line 3, ``retain or J'' to ``retain, or amend J.'' (2) Page 5 1' Lines 9-20 "alrylic acid-1 has been corrected to 'acrylic acid'. (3) 1 acrylic on page 6, line 1, line 3, and line 6"
Corrected to 1acrylo. (4) Delete “wo” on page 6, line 3. (5) On page 6, line 4, "1 number decomposition" is replaced with "-water decomposition" and 7
Ichimasa. (7) Corrected “vinyl J” on page 6, line 10 as “hinyl acetate J.” (7) Corrected “glucose isomerase, inhertase,” on page 7, line 8 as “glucose isomerase, inhertase.” . (8) Page 7, line 10, 1 promelain, pectinase,
" was corrected to "fromelain, pectinase,". (9) Corrected "1 fat, 1 IJj acid combination" on page 7, line 15 to "1 fatty acid." (10) No. 1 in line 11 of O shell J7 - 1 is "workability"
and 7 cities i1-. (11) Correct "Niri desirably" in lines 3 and 4 of page 12 to [more desirably -1]. (12) On page 14, lines 1-2, 1. ``Isuke's stable appearance of enzyme activity'' was corrected to 1 - ``maintenance of stable appearance of enzyme activity''. (I3) 1' in the 8th row from the bottom of the 18th Q and the 7th row from the same G
chcm J to 1cberI1. J and correction. (14) Page 16-1 to 3 ii, page 18, line 2,
Page 19, lines 1-2fJ, page 20, lines 2, page 21, lines 1-2, line 19, page 22, line 20 to page 23, line 1 and 24
page 3 line l R1+1zoI+us dclemar-
1 each F+, r-1111i 4o, p cough, -d
e! ;,mar-,1 and correction. Tsugami

Claims (7)

[Claims] (1) An immobilized enzyme characterized in that the enzyme is immobilized on a carrier made of a porous solid and a chitosan derivative. (2) The immobilized hexagonal element according to claim (1), wherein the carrier contains a superabsorbent resin. (3) Porous solids are Florisil, Geiso Earth, Serai 1
-, silica gel, white clay, corncob, and sawdust (Claim 1, j or (2))
Immobilized enzymes as described in section. (4) Chitosan derivatives, mainly 1-sane, N-acyl chitosan, N-mixed acyl chitosan, N, O-acyl chitosan, N-allylene chitosan, N-alkylidene chitosan, Gitosan salt, and their moieties. The immobilized enzyme according to claim (1) or (2), characterized in that the immobilized enzyme is one or two selected from the group consisting of reactants. (5) The immobilized enzyme according to claim (1) or (2), wherein the enzyme is lipase. (6) An immobilized enzyme characterized in that a gel of a chitosan derivative is formed, a porous solid is dispersed in this gel, the dispersion is dried to obtain a carrier, and the enzyme is retained on the carrier. Production method. (7) The method for producing an immobilized enzyme according to claim (6), wherein the carrier is obtained by adding and mixing a superabsorbent resin to a dried dispersion.