JPS59173080A - Stabilization of lipase - Google Patents

Abstract

PURPOSE:To improve the stability of lipase exposed to a high temperature in aqueous solution, and to prevent the lowering of the enzymatic activity at about the optimum temperature, by adding a polyoxyalkylene ether of a monohydric or polyhydric alcohol. CONSTITUTION:An aqueous solution containing lipase to be used for the hydrolysis of oil or fat is added with 0.1-50wt%, preferably 1-20wt% of a polyoxyalkylene ether of a monohydric or polyhydric alcohol.

Description

[Detailed description of the invention] This invention relates to a method for stabilizing lipase.

For information on how to hydrolyze fats and oils using lipase,
Although many studies have been conducted in the past, most research has focused on hydrolyzing liquid oils at room temperature (25-37°C), and there have been very few examples of hydrolyzing fats with high melting points. .

The main reason for this is that lipase is a proteinaceous catalyst produced by living cells, and when it exceeds the optimum temperature of approximately 37°C, it undergoes thermal denaturation and becomes inactive, reducing its ability to hydrolyze fats and oils. This is because even if attempts were made to hydrolyze high melting point fats at such high temperatures, a high hydrolysis rate could not be obtained. Also,
In order to prevent the above lipase from deactivating, when high melting point fats are hydrolyzed at low temperatures, part of the fat and solid fatty acids produced by hydrolysis precipitate as crystals, reducing the viscosity of the reaction system. As the amount increases, stirring becomes difficult, and contact between the lipase aqueous solution and the fat becomes insufficient, and the hydrolysis rate does not improve even after several days of reaction.

In order to overcome the above-mentioned problem and improve the hydrolysis rate of high melting point fats to 97-98 or higher, the applicant has already mixed melted fat and cooled lipase aqueous solution to increase the melting point of fat. Discloses a method for achieving the above objectives by obtaining an emulsified dispersion that is stable at the following temperatures, and hydrolyzing it by allowing it to stand still, thereby preventing lipase from deactivating and effectively retaining lipase activity. (Unexamined Japanese Patent Publication No. 57-577)
No. 99]. However, in this method, the reaction temperature is 30
The reaction took 48 hours due to the relatively low temperature of 'C.

On the other hand, regarding the method of hydrolyzing oil that is liquid at room temperature, which is different from the above-mentioned high melting point fat, using an aqueous lipase solution,
As already mentioned, many studies have been conducted. In this case, a method generally employed is to separate the oil phase by centrifugation or the like after the completion of the hydrolysis reaction at room temperature, and collect and reuse the lipase aqueous solution. However, it is difficult to hydrolyze at room temperature, for example, 37° which is the optimal temperature for lipase.
The reaction time to achieve a decomposition rate of 97 to 98% with C is a long time of 76 to 96 hours due to the low dispersion effect, so there is a problem that the enzyme activity of the lipase aqueous solution decreases to about 90% during this time. there were. Therefore, when recovering and reusing the lipase aqueous solution, it was necessary to replenish lipase equivalent to about 10 times the enzyme activity after one hydrolysis reaction was completed.

The inventors sought to avoid the two drawbacks mentioned above, namely that the hydrolysis reaction by lipases cannot be carried out at relatively high temperatures, such as those targeted at high melting point fats;
As a result of extensive research to prevent lipase aqueous solutions from gradually losing their enzymatic activity near the optimum temperature for lipase (usually 37°C), we have found that a substance containing a large number of ether bonds is added to lipase aqueous solutions. The present invention was completed based on the discovery that the stability of the aqueous lipase solution can be improved by coexisting with the lipase, thereby avoiding the above-mentioned drawbacks.

That is, the present invention relates to a method of stabilizing lipase by adding a polyoxyalkylene ether of a monohydric or polyhydric alcohol to an aqueous solution containing lipase that is used for hydrolyzing fats and oils.

In this invention, -hydric alcohol refers to a carbon number of 1 to 18
Polyhydric alcohols are alcohols that have two or more hydroxyl groups in one molecule, and the three types include ethylene glycol, propylene glycol, and neopentyl glycol, and the three types include glycerol, Examples include trimethylolpropane, and those having four or more include pentaerythritol, sorbitol, and glucose.

The oxyalkylene group of the polyoxyalkylene ether (hereinafter referred to as polyether) of monohydric or polyhydric alcohol is an oxyethylene group or an oxyfuropylene group, and these may be used alone or together, and when they coexist, block addition The number of oxyalkylene groups per molecule is 2 to 40, preferably 5.
~20. Outside this range, the stabilizing effect of lipase decreases.

Polyether is used by dissolving or dispersing it in water,
The amount added is 01 to 50 in the lipase aqueous solution.
% by weight, preferably from 1 to 20% by weight. Even if the amount added is greater than 50% by weight, the stabilizing effect will not improve much, which is economically disadvantageous. Further, if it is less than 0.01% by weight, the stabilizing effect is insufficient.

Lipases used in this invention include lipases sourced from microorganisms such as Candida, Chromobacterium, Aspergillus, Penicillium, Mucor, Geotrichum, Rhizopus, Arthrobacter, and Hycomyces; There are lipases obtained from animal vases such as, and lipases obtained from plant seeds such as castor seeds.

By the method for stabilizing lipase according to this invention.

It becomes possible to use lipase whose optimum temperature is 37°C at 50'C. That is, at 50°C, an aqueous solution of lipase to which no polyether is added loses about 80% of its initial enzymatic activity in one hour. However, in an aqueous solution of lipase to which polyether has been added, the decrease in enzyme activity is small, with only a 10% decrease in the original enzyme activity after 2 hours, and a decrease in enzyme activity of about 20% even after 4 hours.
It only decreases by q6.

Therefore, as an advantage of this invention, for example, melting point 42°
When beef tallow, which is a triglyceride of C, was statically decomposed at 306C using the method previously proposed by this applicant, a decomposition rate of 97 to 98C was reached in a reaction time of 48 hours.
At 50°C, the reaction time is 1.

Since the decomposition rate reaches 97 to 98% in the following 4 hours, it can be expected that the production efficiency will be greatly improved by shortening the reaction time.

Another advantage of stabilizing lipase according to the present invention is that when kept at 37°C, which is the optimum temperature for lipase, conventional lipase aqueous solutions lose about 10% of their enzymatic activity in 4 to 5 days.
% is deactivated, whereas the aqueous solution of lipase according to the present invention containing polyether loses its enzyme activity by about 10% after 10 days at 37°C, which is the same as that of the conventional one. , the recycling efficiency of lipase is approximately doubled, which is very economically advantageous.

Next, the present invention will be specifically explained using examples. The enzymatic activity of the lipase aqueous solution is measured as follows.

Add 1 x of lipase aqueous solution to a mixture of PT (5 mff1 of 7,Q phosphate buffer and 1 m of olive oil),
The reaction mixture was stirred at 5QQr-pm at °C and allowed to react for 1 hour. After completion of one reaction, the amount of liberated fatty acids was determined by titration with an alkali. The activity of lipase was defined as 1 unit of enzyme activity that releases 1 μmol of fatty acid per minute.

Example 1 A 5-weight multi-aqueous solution of polyether in which 4 moles of propioxide and 6 moles of ethylene oxide were randomly added to ethanol was prepared and heated to 37°C.

In addition to this, 1,172 units/m obtained from Geotrichum sp.
A lipase aqueous solution of 7°C with an initial enzyme activity of 150 units was prepared by adding Pros. As a result, the enzyme activity was 150 units/m after 1 day, 150 units/m after 3 days, 144 units/m after 7 days, and 136 units/m after 11 days. That is, only 93% of the activity was lost after 11 days.

Comparative Example 1 A test was conducted under the same conditions as in Example 1 except that polyether was not used. The results were 150 units/m after 1 day, 139 units/m after 3 days, and 131 units/m after 5 days. After 10 days, the activity became 120 units/m, and at the 51st stage, 127% of the activity had already been lost.

Example 2 The same polyether as in Example 1 was used, using a polyether in which 4 moles of propylene oxide and 6 moles of ethylene oxide of a synthetic primary alcohol having 12 to 13 carbon atoms (linear chain ratio: 80%) were block-added in this order. The test was conducted under the following conditions. As a result, after one day, 150 units/mJ2.
145 units/h1 after 7 days, 13 after 11 days
It showed an enzyme activity of 3 units/m. That is, only 11.3% of the activity was lost after 11 days.

Example 3 A 10% by weight aqueous solution of polyether in which 2 moles of ethylene oxide and 37 rutoflopylene oxide were randomly added to ethylene glycol was prepared and heated to 50°C. In addition to this, 30,000 units/unit obtained from Candida sp.
g of lipase powder was added to prepare a lipase aqueous solution containing 150 units/m of initial activity.
The mixture was maintained at 50°C while stirring at 50°C, and samples were taken at appropriate times to measure enzyme activity. As a result, 148 units/m after 1 hour, 136 units/m after 2 hours,
After 4 hours, it was 121 units/mi. That is, only 193% of the activity was lost after 4 hours.

Comparative Example 2 A test was conducted under the same conditions as in Example 3 except that polyether was not used, and as a result, only 28 units/m2 of enzyme activity was exhibited after 1 hour. In other words, 81.00 in 1 hour.
3% activity was lost.

Example 4 A test was conducted under the same conditions as in Example 3 using a polyether prepared by adding 20 moles of ethylene oxide to chrycerol.

As a result, 148 units/m after 1 hour, 138 units/m after 2 hours, 120 units/m after 44 hours.
It showed the enzyme activity of . In other words, after 4 hours, 20
Only % of activity was lost.

Example 5 A 20% by weight aqueous dispersion of a polyether in which 5 moles of ethylene oxide and 10 moles of propylene oxide were block-added to pentaerythritol in this order was prepared.
It was heated to C. In addition to this, 15. O
Add OO units/g of lipase powder to prepare an enzyme aqueous solution containing an initial activity of 150 units/m2, and incubate at 500 r-p.
The temperature was maintained at 370C while stirring at -m. Enzyme activity was measured by sampling at appropriate times. As a result, 150 units/rf after 1 day 148 units/m after 3 days
145 units/m after 7 days, 1 after 10 days
It showed an enzyme activity of 35 units/m. That is, only 10% of the activity was lost after 10 days.

Comparative Example 3 Tested under the same conditions as Example 5 except that polyether was not used. Results were 150 units/m after 1 day, 137 units/m after 3 days, and 135 units/m after 5 days.
After 10 days, the activity was 122 units/m2, and 10% of the activity had already been lost at the 5th mouth weight, which was 187 units at the 10th day's mouth weight.

Example 6 A 1-weight polyaqueous dispersion of a 5-mol adduct of glucose with propylene oxide was prepared, and otherwise tested under the same conditions as in Example 5. As a result, 150 units/mi after 1 day,
The enzyme activity was 146 units/m after 3 days, 140 units/m after 7 days, and 137 units/m after 10 days. That is, only 8.7% of the activity was lost after 10 days.

Example 7 A 10% by weight aqueous solution of polyether in which 2 moles of propylene oxide and 3 moles of ethylene oxide were randomly added to ethylene glycol was prepared, and to this was added 30,000 units/g of lipase powder obtained from the genus Candida. was added to prepare an aqueous lipase solution containing an initial activity of 500 units/m2. Add 100 g of beef tallow (acid value 02, saponification value 192.3, melting point 42°C) to this,
The temperature was maintained at 50°C for 4 hours while stirring at 00rPm. After the reaction was completed, the oil layer was separated and dehydrated. This one has an acid value of 199.0. The saponification value was 2039, and the decomposition rate [(acid value/saponification value) x 100] was 97.6.

Comparative Example 4 As a result of testing under the same conditions as Example 7 except that polyether was not used, the oil layer part had an acid value of 123.2 degrees and a saponification value of 1.
At 998, the decomposition rate was 617.

Example 8 5 double ft of polyether in which 4 moles of propylene oxide and 6 moles of ethylene oxide were randomly added to ethanol
ft% aqueous solution was prepared, and 1,172 units/m kalpa broth obtained from Diol-IJ Cam was added to this to prepare an aqueous lipase solution with an initial activity of 500 units/m2. Add this to olive oil (acid value 05, saponification value 189.1) 100
g and stirred at 37° while stirring at 5QQr-pm.
It was kept at C for 4 days. After the reaction was completed, the oil layer was centrifuged and dehydrated. This product has an acid value of i94.4. The saponification value is 2003, the decomposition rate is 97.1%, and the enzyme activity in the aqueous phase is 490.
The unit was /m. 0.01 ml of the above-mentioned broth was added to 100 ml of this aqueous phase to give an enzyme activity of 500 units/mi, and this was used to repeatedly hydrolyze olive oil.

After the reaction was completed, the oil layer was centrifuged and dehydrated. This product had an acid value of 193.0, a saponification value of 2000, and a decomposition rate of 965%.

Comparative Example 5 A test was conducted under the same conditions as in Example 8 except that polyether was not used, and the acid value of the oil layer portion was 190.5.

The saponification value was 1995 and the decomposition rate was 955. The enzyme activity in the aqueous phase was 450 units/m. This water phase 100
0.05 m of the above-mentioned pros was added to mff1 to give an enzyme activity of 500 units Mr1 mi, and this was used to repeatedly hydrolyze olive oil. After the reaction was completed, the oil layer was centrifuged and dehydrated. This item has an acid value of 190.0 and a saponification value of 198.
．． 5, the decomposition rate was 95.7%.

Patent applicant: NOF Corporation

Claims (1)

[Claims] (1) A method for stabilizing lipase, which comprises adding a polyoxyalkylene needle of monohydric or polyhydric alcohol to an aqueous solution containing lipase.