JPH0439994B2 - - Google Patents

Description

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

A lot of research has been done on methods of hydrolyzing fats and oils using lipase, but most of the research has focused on hydrolyzing liquid oils at around room temperature (25-37℃); There are very few examples of fat hydrolysis.

The main reason for this is that lipase is a proteinaceous catalyst produced by living cells, and when the temperature exceeds the optimum temperature of 37°C, it is thermally denatured and deactivated, reducing its ability to hydrolyze fats and oils. This was because even if attempts were made to hydrolyze high melting point fats at such high temperatures, a high hydrolysis rate could not be obtained. In addition, when high melting point fats are hydrolyzed at low temperatures to prevent the lipase from deactivating, some of the fats and solid fatty acids produced by hydrolysis crystallize and precipitate, causing the reaction system to deteriorate. The viscosity increases, making stirring difficult, and contact between the aqueous lipase 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 problem and improve the hydrolysis rate of high melting point fats to 97-98% or more, the applicant has already mixed melted fat with a cooled lipase aqueous solution to improve the hydrolysis rate of fats below the melting point of the fats. Disclosed is a method for achieving the above objective by obtaining a temperature-stable emulsified dispersion and hydrolyzing it by allowing it to stand still to prevent lipase deactivation, that is, to effectively maintain lipase activity (particularly (No. 57-57799). However, in this method, the reaction required 48 hours because the reaction temperature was relatively low at 30°C.

On the other hand, as mentioned above, many studies have been conducted on methods of hydrolyzing oils that are liquid at room temperature, which is different from the high melting point fats mentioned above, using an aqueous lipase solution. 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 recover and reuse the lipase aqueous solution. However, when hydrolyzing at room temperature, for example, at 37℃, which is the optimal temperature for lipase,
The reaction time to achieve a decomposition rate of 97-98% is a long time of 76-96 hours due to the low dispersion effect, so during this time the enzyme activity of the lipase aqueous solution increases by approximately 90
There was a problem where the value decreased to %. Therefore, when recovering and reusing the lipase aqueous solution, it was necessary to replenish lipase equivalent to about 10% of 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 that can target high-melting point fats, and that the optimal temperature for lipases (usually As a result of extensive research in order to prevent the lipase aqueous solution from gradually decreasing its enzymatic activity at temperatures around 37°C, we found that by coexisting a substance containing a large number of ether bonds in the lipase aqueous solution,
The present invention was completed based on the discovery that the stability of the lipase aqueous solution is improved, thereby avoiding the above-mentioned drawbacks.

That is, the present invention provides an aqueous solution containing lipase that is used for hydrolyzing fats and oils.
This method involves adding polyoxyalkylene ether of 13 monohydric alcohols or polyhydric alcohols to stabilize lipase.

In this invention, a monohydric alcohol is an alcohol having 1 to 13 carbon atoms, and a polyhydric alcohol is an alcohol having two or more hydroxyl groups in one molecule,
Those having two such molecules include ethylene glycol, propylene glycol, and neopentyl glycol, those having three atoms such as glycerol and trimethylolpropane, and those having four or more molecules such as 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 oxypropylene group, and these may be used alone or together, and when they coexist, block addition is performed. It may be a substance or a random addition. The number of oxyalkylene groups per molecule is 2 to 40, preferably 5 to 20. Outside this range, the stabilizing effect of lipase decreases.

Polyether is used after being dissolved or decomposed in water, but the amount added is 0.1 to 0.1 in the lipase aqueous solution.
The amount amounts to 50% by weight, preferably 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 0.1% by weight, the stabilizing effect is insufficient.

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

By the method for stabilizing lipase according to the present invention,
It becomes possible to use lipase whose optimal 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 decrease of about 10% of the original oxygen activity after 2 hours, and a decrease in enzyme activity of about 20% even after 4 hours. It only decreases by %.

Therefore, as an advantage of the present invention, for example, when beef tallow, which is a triglyceride with a melting point of 42°C, is left to decompose at 30°C using the method previously proposed by this applicant, the reaction time is 48 hours. However, at 50°C, the decomposition rate reaches 97-98% in 4 hours, which is less than 1/10 of the reaction time, so we can expect a significant improvement in production efficiency by shortening the reaction time. .

Another advantage of stabilizing lipase according to this invention is that when kept at 37°C, which is the optimal temperature for lipase, approximately 10% of the enzyme activity of conventional lipase aqueous solutions is deactivated in 4 to 5 days. In contrast, the aqueous solution of lipase according to the present invention containing polyether loses about 10% of the same enzyme activity as the conventional solution after 10 days at 37°C, so the recycling efficiency of lipase is reduced to about 10%. This will double the amount and become 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 ml of lipase aqueous solution to a mixture of 5 ml of phosphate buffer at pH 7.0 and 1 ml of olive oil, stir at 500 r.p.m. at 37°C and react for 1 hour. After the reaction, the amount of released fatty acids was titrated with alkali. I asked for it. Lipase activity was defined as the amount of enzyme that released 1 μmol of fatty acid per minute as 1 unit.

Example 1 A 5% by weight aqueous solution of polyether in which 4 moles of propylene oxide and 6 moles of ethylene oxide were randomly added to ethanol was prepared and heated to 37°C. To this, 1172 units/ml of broth obtained from Diotrichum was added, and the initial enzyme activity was 150 units/ml.
ml of lipase aqueous solution was prepared, kept at 37°C while stirring at 500 rpm, and sampled at appropriate times to measure enzyme activity. As a result, after one day
The enzyme activity was 150 units/ml, 150 units/ml after 3 days, 144 units/ml after 7 days, and 136 units/ml after 11 days. i.e. 9.3 after 11 days
Only % of activity was lost.

Comparative Example 1 As a result of testing under the same conditions as Example 1 except that polyether was not used, the test result was 150 units/day after one day.
ml, 139 units/ml after 3 days, 131 after 5 days
units/ml, 120 units/ml after 10 days, 5
Already on the first day, 12.7% of the activity was lost, and on the 10th day, 20% of the activity was lost.

Example 2 Propylene oxide 4 of synthetic primary alcohol (linear chain ratio 80%) having 12 to 13 carbon atoms as polyether
A test was conducted under the same conditions as in Example 1 using a polyether in which 6 moles of ethylene oxide and 6 moles of ethylene oxide were added in this order. As a result, 150 units/ml after 1 day, 145 units/ml after 7 days, 11
After one day, the enzyme activity was 133 units/ml. 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 3 moles of ethylene oxide and 2 moles of propylene oxide were randomly added to ethylene glycol was prepared and heated at 50°C.
It was heated to This was obtained from the genus Candeida.
Add 30,000 units/g of lipase powder to determine initial activity.
Prepare a lipase aqueous solution containing 150 units/ml,
The mixture was maintained at 50° C. while stirring at 500 r.p.m., and the enzyme activity was measured by sampling at appropriate times. As a result, the concentration was 148 units/ml after 1 hour, 136 units/ml after 2 hours, and 121 units/ml after 4 hours. That is, only 19.3% 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/ml of enzyme activity was exhibited after 1 hour. That is,
81.3% of the activity was lost in 1 hour.

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 glycerol. As a result, the enzyme activity was 148 units/ml after 1 hour, 138 units/ml after 2 hours, and 120 units/ml after 4 hours. That is, only 20% of the activity was lost after 4 hours.

Example 5 A 20% by weight aqueous dispersion of polyether in which 5 moles of ethylene oxide and 10 moles of propylene oxide were block-added to pentaerythritol in this order was prepared and heated to 37°C. Add 15,000 units/g of lipase powder obtained from Mucor to this,
An aqueous enzyme solution containing an initial activity of 150 units/ml was prepared and maintained at 37°C while stirring at 500 rpm. Enzyme activity was measured by sampling at appropriate times.
As a result, 150 units/ml after 1 day, 148 units/ml after 3 days, 145 units/ml after 7 days, 10
After one day, the enzyme activity was 135 units/ml. That is, only 10% of the activity was lost after 10 days.

Comparative Example 3 As a result of testing under the same conditions as Example 5 except that polyether was not used, the test result was 150 units/day after one day.
ml, 137 units/ml after 3 days, 135 after 5 days
units/ml, 122 units/ml after 10 days, 5
Already 10% of the activity was lost on day 1, and 18.7% on day 10.

Example 6 A 1% by weight aqueous dispersion of a 5 mole adduct of glucose with propylene oxide was prepared and tested under the same conditions as in Example 5 except for the above. The results were 150 units/ml after 1 day, 146 units/ml after 3 days, 140 units/ml after 7 days, and 137 units/ml after 10 days.
showed the enzyme activity of i.e. after 10 days
Only 8.7% of activity was lost.

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 30,000 units/g of polyether obtained from Candeida sp.
of lipase powder was added to prepare an aqueous lipase solution containing an initial activity of 500 units/ml. Add this to 100g of beef tallow (acid value 0.2, saponification value 192.3, melting point 42℃)
was added and kept at 50°C for 4 hours while stirring at 500rpm. After the reaction was completed, the oil layer was separated and dehydrated.
This product had an acid value of 199.0, a saponification value of 203.9, and a decomposition rate [(acid value/saponification value) x 100] of 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,
The saponification value was 199.8 and the decomposition rate was 61.7%.

Example 8 A 5% by weight aqueous solution of polyether in which 4 moles of propylene oxide and 6 moles of ethylene oxide were randomly added to ethanol was prepared, and a broth obtained from Diotrichum spp. with a concentration of 1172 units/ml was added to give an initial activity of 500 units/ml. ml of lipase aqueous solution was prepared. Add this to olive oil (acid value 0.5, saponification value
189.1) 100g was added and kept at 37°C for 4 days while stirring at 500rpm. After the reaction was completed, the oil layer was centrifuged and dehydrated. This product had an acid value of 194.4, a saponification value of 200.3, a decomposition rate of 97.1%, and an enzyme activity in the aqueous phase of 490 units/ml. Add 0.01ml of the above broth to 100ml of this aqueous phase and adjust the enzyme activity to 500 units/ml.
This was used to repeatedly hydrolyze olive oil. After the reaction was completed, the oil layer was centrifuged and dehydrated.
This material has an acid value of 193.0, a saponification value of 200.0, and a decomposition rate of
It became 96.5%.

Comparative Example 5 As a result of testing under the same conditions as Example 8 except that polyether was not used, the oil layer part had an acid value of 190.5,
The saponification value was 199.5 and the decomposition rate was 95.5%. The enzyme activity in the aqueous phase was 450 units/ml. 100ml of this aqueous phase
0.05 ml of the above-mentioned broth was added to adjust the enzyme activity to 500 units/ml, and this was used to repeatedly hydrolyze olive oil. After the reaction was completed, the oil layer was centrifuged and dehydrated. This material had an acid value of 190.0, a saponification value of 198.5, and a decomposition rate of 95.7%.

Example 9 (Re-experiment of Example 3) A 10% by weight aqueous solution of polyether in which 3 moles of ethylene oxide and 2 moles of propylene oxide were randomly added to ethylene glycol was prepared.
Warmed to ℃. Add 30,000 units/g of lipase powder obtained from Candeida to this to prepare a lipase aqueous solution containing an initial activity of 150 units/ml, maintain at 50°C while stirring at 500 r.p.m. Samples were taken to measure enzyme activity.
As a result, 146 units/ml after 1 hour, 134 units/ml after 2 hours, and 120 units/ml after 4 hours.
It became ml. That is, only 20% of the activity was lost even after 4 hours.

Comparative Example 6 A 10% by weight aqueous solution of Triton X-100 (adduct of octylphenol with 9 to 10 moles of ethylene oxide) was prepared and heated to 50°C. Add 30,000 units/g of lipase powder obtained from Candeida to this to prepare a lipase aqueous solution containing 150 units/ml of initial activity, and while stirring at 500 r.p.m.
It was kept at 50°C and sampled at appropriate times to measure enzyme activity. As a result, the activity decreased over time to 130 units/ml after 1 hour, 110 units/ml after 2 hours, and 70 units/ml after 4 hours, and 53.3 units/ml after 4 hours. % of activity was lost.

Comparative example 7 ADEKA TOL 45-S-8 equivalent product (carbon number 14
Prepare a 10% aqueous solution of ethylene oxide (8 mole adduct of a secondary alcohol mixture of
Warmed to ℃. Add 30,000 units/g of lipase powder obtained from Candeida to this to prepare a lipase aqueous solution containing an initial activity of 150 units/ml, maintain at 50°C while stirring at 500 r.p.m. Samples were taken to measure enzyme activity.
As a result, 135 units/ml after 1 hour, 120 units/ml after 2 hours, and 90 units/ml after 4 hours.
The activity decreased with each ml and time, and 40% of the activity was lost after 4 hours.

Claims (1)

[Claims] 1. A method for stabilizing lipase, which comprises adding polyoxyalkylene ether of a monohydric alcohol or polyhydric alcohol having 1 to 13 carbon atoms to an aqueous solution containing lipase.