JPH04370096A - Stabilized composition of lipase originated from microorganism and stabilizing method - Google Patents

Abstract

PURPOSE:To obtain a lipase composition containing a lipase originated from a microorganism and stable to a bile acid salt. CONSTITUTION:The objective composition containing a lipase originated from a microorganism, L-histidine and/or protein having L-histidine as a N-ended amino group. The objective stabilizing method is carried out by adding L- histidine and/or a protein having L-histidine as N-ended amino group to lipase originated from a microorganism. The objective composition is stabilized to a pile acid salt and can be applied to patient of indigestion syndrome or chronic pancreatitis or patient from which digestion function is lost by extraction of spleen.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a lipase composition and a method for stabilizing lipase of microbial origin against bile salts. More specifically, the gastrointestinal tract (duodenum)
In the presence of internally secreted bile salts, in a reaction in which the activity of microbial lipase is inhibited, the lipase is stabilized by the coexistence of L-histidine and/or a protein having it as an N-terminal group. The present invention relates to lipase compositions and stabilization methods.

[0002] The composition according to the present invention utilizes lipase of microbial origin, similar to pancreatic lipolytic enzyme (pancreatin) of animal origin, in patients with dyspepsia syndrome, chronic pancreatitis, or those who have lost their digestive function due to pancreatic removal. Can be applied to patients.

0003

[Prior Art] Lipase as a digestive enzyme is used in the digestive tract (
Fat is digested in the presence of bile components in the duodenum. Therefore, pancreatin, which contains pancreatic lipolytic enzyme of animal origin, has been widely used because its lipase activity is not inhibited even in the presence of bile components (bile salts).

[0004] However, pancreatin is mainly isolated and prepared from pig pancreas, and there are problems in terms of productivity and price. Therefore, there has been a need for a technology that uses lipase of microbial origin as a substitute for pancreatin.

[0005]

[Problems to be Solved by the Invention] Lipases of microbial origin whose reaction is promoted by bile components have been reported [Pharmacy, Vol. 19, p. 1956 (1966)].
, [Agric. Biol. Chem. , 46
Vol. 7, p. 1743 (1982)]. However, the properties of the obtained lipase are greatly influenced by the activity measurement method used for its investigation. For example, a measurement method using the most widely used polyvinyl alcohol (hereinafter referred to as PVA) emulsified substrate [Yakuzai Gazette, Vol. 1120, p. 153 (198
0)] However, it is known that the lipase activity value varies greatly when the type or composition ratio of PVA is changed. but,
It has been found that when bile salts are added to this reaction system, the measured lipase activity value is not affected even if the type or composition ratio of PVA changes [Research on lipase activity measurement method, p. 6 (1986) , published by the Fermentation Industry Association].

[0006] Therefore, it is inconvenient to examine the influence of bile components on lipase of microbial origin using a measurement system using a conventional emulsion-based measurement method, and it is difficult to firmly prove that the reaction acceleration as reported in the past is observed. Met.

On the other hand, it has been found that the products of digestion in the small intestine are a mixture of an oily white cloudy layer in the upper layer and a transparent micellar layer in the lower layer [J. Clin. Invest. , 39
vol. 809 (1960)], [same magazine, vol. 43, 247
(1964)], [Fed. Proc. , Volume 21, 4
3 pages (1962)].

[0008] That is, β-monoglyceride produced through hydrolysis by pancreatic lipase forms micelles with the help of bile salts, fatty acids are dissolved into the micelles, and an enzymatic reaction proceeds in the oil phase. In this way, the lipase reaction involves water (enzyme side)
It is carried out in a two-phase system of oil and oil (substrate side), and there is no artificial emulsifier such as PVA.

[0009] Therefore, it is desirable that the measurement method for determining appropriate lipase characteristics in the gastrointestinal tract be a non-emulsifying system. Therefore, the present inventor developed a non-emulsifying measurement method [Science and Industry, 43
Vol., p. 577 (1969)]. Regarding the accuracy of this measurement method, see [Pharmacology, Vol. 48, No. 4,
277 (1988)], and its measurement accuracy is at least as good as that of the emulsion-based measurement method described above.

[0010] An example of studying the influence of bile salts on microbial lipase activity using a non-emulsifying measurement method can be found in [Science and Industry, Vol. 39, p. 415 (1965)].
Lipases of microbial origin have not been extensively investigated, although a decrease in activity due to cholic acid has been reported.

[0011] First of all, the present inventor used a non-emulsification measurement method to understand the influence of bile salts on lipases derived from various microorganisms. The results revealed that all microbial lipases are inhibited by bile salts, although there are differences in degree as described below.

[0012] Secondly, we have intensively investigated methods for preventing or recovering the reaction of microbial lipases from being inhibited by bile salts at the site of digestion in the intestinal tract.

[0013]

[Means for Solving the Problems] In order to prevent the activity of microbial lipase from decreasing due to bile salts, the inventors have utilized a non-emulsification measurement method to develop biologically relevant components and foods that can be used in biological reactions. After conducting extensive research on related ingredients, we discovered that by adding certain additives, we could reduce the effects of bile salts on lipases of microbial origin, and even reduce the effects of bile salts on lipases of microbial origin whose activity was reduced by bile salts. completed the present invention by discovering that the activity could be restored by coexisting the additive.

Lipases originating from microorganisms include filamentous fungi,
A wide range of lipases, such as those of bacterial or yeast origin, are of interest to the present invention. More specifically, Rhizopus oryzae (R
hizopus oryzae), Rhizopus delemar, Mucor japanicus
, Pseudomonas cepacia
cepacia), Candida rugosa (Ca.
Examples include lipases originating from N. ndida rugosa) and the like.

[0015] Amino acids and/or their related proteins are used as additives. L-histidine can be used as the amino acid. Any protein having L-histidine as its N-terminal amino group can be used as the related protein. Specific examples of biologically relevant proteins include serum albumin and lactalbumin.

[0016] The amount added varies depending on the type of microbial lipase used, but it may be sufficient as long as the effect of the present invention is exhibited. Generally, the degree of recovery and stabilization of activity increases with increasing concentration of additive.

[0017] As for the method of addition, it can be added during the enzyme purification process or the formulation process, and any method can be used as long as the lipase is in a form that can coexist when it comes into contact with bile salts.

The present invention also provides a lipase composition of microbial origin prepared by the method described above.

A non-emulsifying method was used to measure lipase activity to judge the effectiveness of the present invention. Namely, 1 ml of olive oil (substrate), Tris-HCl buffer (pH 7.0)
After preheating 5ml at 37℃ for 5 minutes, add 1ml of diluted enzyme solution.
and react at 600 rpm for 30 minutes. Next, 20 ml of acetone/ethanol mixture was added and titrated with 0.1N potassium hydroxide solution using 2 drops of phenolphthalein reagent as an indicator.

Mixed bile salts [K. W. Hea
``Bile acids, their organization and pathology'' by Kazuaki Kamisaka et al. 1
8 (1977)]. Namely, sodium glycocholate: sodium glycodeoxycholate:
Mixed bile salts of sodium taurocholate: sodium taurodeoxycholate = 3.1:4.5:1:1.4 were added to 2mM-10mM [Digestion and Absorption, Modern Medicine, Vol. 20, No. 11, No. 12, Separate volume, p. 116 (1966)] was used as the final reaction concentration.

[0021] Amino acids and proteins were used in the range of 0.5% to 10% of olive oil as a substrate (in this reaction system, the maximum addition was 5% of the olive oil for amino acids and 10% for protein). If it exceeds this, it will be difficult to determine the end point during titration, and the additive will precipitate without dissolving, making measurement difficult.)
.

Experimental Example 1 Effect of bile salts on lipase activity of microbial origin According to the above measurement method, 0-10 m of mixed bile salts was added to the reaction system.
M and added, Rhizopus ose, Rhizopus
Deremer, Mucor javanicus (these are filamentous fungi),
Pseudomonas cepacia (bacteria) and Candida
The effect on the activity of lipase derived from Rugosa (yeast) (both manufactured by Amano Pharmaceutical Co., Ltd.) was investigated (shown in Figure 1).

As a result, the activity of all lipases was reduced by bile salts. The concentration of mixed bile salts on lipase activity is 2mM-1, except for lipases of Mucor origin.
Since it was almost the same between 0mM and 4mM as the mixed bile salt concentration for subsequent experiments, it was determined that there was no problem and it was adopted.

Experimental Example 2 Effects of various amino acids on lipase originating from Rhizopus oryzae in the presence of bile salts First, the effects of various amino acids (manufactured by Ajinomoto Takara Corporation) on lipase originating from Rhizopus oryzae were investigated. did. Mixed bile salts were added at 4mM, amino acids were added at 1% based on olive oil, and the measurements were performed using the method described above. The results are shown in Table 1.

[0025]

[Table 1]

That is, 4mM of mixed bile salt alone reduces lipase activity by about 30%, and the activity can be restored by adding various amino acids, such as L-
Histidine showed a degree of recovery (relative value when the activity when 4 mM of mixed bile salts was added was set as 100) of 118%. However, other amino acids either had no effect or, on the contrary, showed results that the activity was further reduced.

Based on Experimental Example 2, the present inventors focused on L-histidine and biological components and food components having L-histidine as a functional group as a means of preventing the activity of lipase from decreasing due to bile salts. An example of a biologically relevant protein having L-histidine at its N-terminal group is serum albumin [References
Shiro Akahori et al., Protein Chemistry III, p. 364 (1961
α-lactalbumin, which is a biological component derived from serum albumin and has very similar physical and chemical properties [References: Shiro Akahori et al., Protein Chemistry III
, 9 pages (1961, Kyoritsu Shuppan)] was considered to be equivalent to this.

Experimental Example 3 Effects of L-histidine, serum albumin (bovine origin), and α-lactalbumin (bovine origin) on microbial lipase in the presence of mixed bile salts L
- Histidine (manufactured by Wako Pure Chemical Industries, Ltd.), serum albumin,
α-lactalbumin (both manufactured by Sigma) was added in the presence of 4mM mixed bile salts, Rhizopus oryzae,
The effect on the activity of each lipase originating from Rhizopus delemer, Mucor javanicus, Pseudomonas cepacia, and Candida rugosa was investigated. The results are shown in FIGS. 2, 3 and 4, respectively.

That is, addition of 4mM of mixed bile salts reduces the activity of lipase of any origin, and addition of L-histidine and α-lactalbumin to 5% and serum albumin to 10% of olive oil to this decreases the activity of lipase of any origin. As the concentration increased, lipase activity recovered.

Serum albumin and α-lactalbumin have L-methionine, L-methionine, and L-histidine as terminal groups.
- has aspartic acid [Reference: Shiro Akahori et al., Protein Chemistry III, p. 364 (1961, Kyoritsu Shuppan)],
In addition, in addition to L-histidine, L-arginine and L-lysine are amino groups that cause positive charge in the molecule [References Koichiro Aoki et al., Serum Albumin, p. 12 (1989, No. 3).
Edition, Kodansha Scientific)]. Therefore, the present inventors also investigated these amino acids in addition to the above experimental examples.

Experimental Example 4 Effects of L-arginine, L-lysine, L-methionine, and L-aspartic acid on microbially derived lipase in the presence of bile acids According to the above measurement method, in the presence of 4mM of mixed bile salts, L-arginine, L-lysine, L-methionine, and L-aspartic acid Addition of 0-5% (based on olive oil) of L-lysine, L-methionine or L-aspartic acid to each of Rhizopus oryzae, Rhizopus deremer, Mucor javanicus, Pseudomonas cepacia and Candida rugosa origin. Figures 5 and 6 show the effects on lipase.
, shown in FIGS. 7 and 8.

Regarding L-arginine and L-lysine, which are positively charged factors in serum albumin and α-lactalbumin molecules, the activity of all lipases decreased significantly as the added concentration increased.

[0033]Also, L-aspartic acid, which is the N-terminal group of serum albumin and lactalbumin, showed decreased activity in two lipases originating from the genus Rhizopus, but exerted activity on lipases originating from the other three origins. There wasn't.

[0034] From the above, it exists in serum albumin and lactalbumin molecules, becomes a positively charged factor, and N
It was found that only L-histidine present at the -terminus prevents the decrease in microbial lipase activity caused by bile salts or restores the activity.

[0035] When using lipase of microbial origin suitable for lipolysis in the intestinal tract (duodenum), the present inventor has investigated the effects of bile salts, which had not been clearly clarified due to the incompatibility of conventional activity assay methods. This was revealed using an emulsion-based measurement method. As a result, it was found that the activity of microbial lipase was decreased in the presence of bile salts. The decrease in activity is L
- The present inventors have discovered for the first time that the activity can be prevented or restored by a protein having histidine or L-histidine as the N-terminal group. Hereinafter, the present invention will be made clearer by way of examples.

[0036]

[Example] Example 1 Using olive oil as a substrate, L-histidine was added at 5% to olive oil in the presence of 4mM mixed bile salts (final reaction concentration).
%, and to this, lipase originating from Rhizopus delemer (
When 1 ml of 0.33 mg/ml) was added and reacted at 37°C for 30 minutes at 600 rpm, the residual activity of lipase derived from Rhizopus deremer in the control (no L-histidine added) was 73%. The activity of lipase added with L-histidine showed a residual activity rate of 93% (recovery rate of 27%).
%)showed that.

Example 2 Using olive oil as a substrate, in the presence of 4mM mixed bile salts (final reaction concentration), L-histidine was added at 5% to olive oil.
1 ml of lipase originating from Mucor javanicus (0.33 mg/ml) was added to this, and the reaction was carried out at 37°C for 30 minutes at 600 rpm. The residual activity rate of the original lipase was 82%, whereas the activity of the lipase to which L-histidine was added showed a residual activity rate of 95% (recovery rate of 16%).

Example 3 A reaction was carried out under the same reaction conditions as in Example 1 with the addition of 1 ml of lipase (0.067 mg/ml) originating from Pseudomonas cepacia. The remaining activity rate of lipase is 85%, whereas the remaining activity rate of lipase derived from Pseudomonas cepacia added with L-histidine is 101% (
The recovery rate was 19%).

Example 4 Using olive oil as a substrate, in the presence of 4mM mixed bile salts (final reaction concentration), bovine serum albumin was added to olive oil at 1%
0%, add 1 ml of lipase originating from Rhizopus oryzae (0.033 mg/ml), and incubate at 37°C for 30
When reacted at 600 rpm for 1 minute, the residual activity of the lipase derived from Rhizopus oryzae as a control (no bovine serum albumin added) was 73%, whereas the activity of the lipase to which L-histidine was added was 95%. % (recovery rate 30%).

Example 5 A reaction was carried out under the same reaction conditions as in Example 4 by adding 1 ml of lipase originating from Candida rugosa (0.04 mg/ml), resulting in a control (no bovine serum albumin added).
Candida rugosa lipase residual activity rate 41%
In contrast, lipase derived from Candida rugosa supplemented with bovine serum albumin had a residual activity rate of 60% (recovery rate of 46%).
%)showed that.

Example 6 Using olive oil as a substrate, in the presence of 4mM mixed bile salts (final reaction concentration), 5% α-lactalbumin derived from cattle was added to the olive oil, and to this Add 1 ml of lipase (0.033 mg/ml) and
When the reaction was carried out at 7°C for 30 minutes at 600 rpm, the residual activity of the lipase originating from Rhizopus oryzae as a control (without the addition of α-lactalbumin) was 78%, whereas that of the lipase to which α-lactalbumin was added was 78%. The activity showed a residual activity rate of 88% (recovery rate of 13%).

Example 7 When 1 ml of lipase (0.033 mg/ml) originating from Rhizopus deremer was added and reacted under the same reaction conditions as in Example 6, a control (no α-lactalbumin added) Rhizopus deremer Residual activity rate of original lipase 6
7%, the lipase to which α-lactalbumin was added showed a residual activity rate of 79% (recovery rate of 18%).

[0043]

Effects of the Invention According to the present invention, a method for preventing the decrease in lipase activity due to bile salts or recovering the activity when using microbially derived lipase for digestion in the intestinal tract has been discovered, and this method can be used to prevent indigestion syndrome, chronic pancreatitis, etc. This could greatly contribute to the possibility of using pancreatic lipolytic enzyme (pancreatin) substitutes in the treatment of patients with cancer.

[Brief explanation of drawings]

[Figure 1] Lipase activity when no mixed bile salts are added
This figure shows the influence of bile salts on the activity of various lipases when the activity is set to 00%. The results are shown for lipase originating from C. javanicus, -△- for lipase originating from Pseudomonas cepacia, and -□- for lipase originating from Candida rugosa.

FIG. 2 shows the influence of L-histidine on the activity of various microbial lipases in the presence of mixed bile salts.
○− is a lipase originating from Rhizopus oryzae, −●− is a lipase originating from Rhizopus deremer, −◆− is a lipase originating from Rhizopus deremer.
lipase originating from Pseudomonas javanicus, -△- is lipase originating from Pseudomonas cepacia, and -□- is lipase originating from Pseudomonas cepacia.
The results for lipase originating from Rugosa are shown. Note that the downward arrow in the figure indicates the addition of mixed bile salts.

FIG. 3 shows the influence of bovine serum albumin on various microbial lipase activities in the presence of mixed bile salts.
-○- is a lipase originating from Rhizopus oryzae, -●- is a lipase originating from Rhizopus deremer, -◆- is a lipase originating from Mucor javanicus, -△- is a lipase originating from Pseudomonas cepacia, and -□- is a lipase originating from Candida. - Shows the results of lipase originating from Rugosa. Note that the downward arrow in the figure indicates the addition of mixed bile salts.

Figure 4 shows the influence of α-lactalbumin on the activity of various microbial lipases in the presence of mixed bile salts, where -○- indicates lipase originating from Rhizopus oryzae, -●
- indicates the results for lipase originating from Rhizopus delemer, -◆- indicates lipase originating from Mucor javanicus, -Δ- indicates lipase originating from Pseudomonas cepacia, and -□- indicates lipase originating from Candida rugosa. Note that the downward arrow in the figure indicates the addition of mixed bile salts.

FIG. 5 shows the influence of L-arginine on various microbial lipase activities in the presence of mixed bile salts.
○− is a lipase originating from Rhizopus oryzae, −●− is a lipase originating from Rhizopus deremer, −◆− is a lipase originating from Rhizopus deremer.
lipase originating from Pseudomonas javanicus, -△- is lipase originating from Pseudomonas cepacia, and -□- is lipase originating from Pseudomonas cepacia.
The results for lipase originating from Rugosa are shown. Note that the downward arrow in the figure indicates the addition of mixed bile salts.

FIG. 6 shows the influence of L-lysine on various microbial lipase activities in the presence of mixed bile salts, −○−
is a lipase originating from Rhizopus oryzae, -●- is a lipase originating from Rhizopus delemer, -◆- is a lipase originating from Mucor javanicus, -△- is a lipase originating from Pseudomonas cepacia, and -□- is originating from Candida rugosa. The results for lipase are shown. Note that the downward arrow in the figure indicates the addition of mixed bile salts.

FIG. 7 shows the influence of L-methionine on various microbial lipase activities in the presence of mixed bile salts;
○− is a lipase originating from Rhizopus oryzae, −●− is a lipase originating from Rhizopus deremer, −◆− is a lipase originating from Rhizopus deremer.
lipase originating from Pseudomonas javanicus, -△- is lipase originating from Pseudomonas cepacia, and -□- is lipase originating from Pseudomonas cepacia.
The results for lipase originating from Rugosa are shown. Note that the downward arrow in the figure indicates the addition of mixed bile salts.

FIG. 8 shows the influence of L-aspartic acid on various microbial lipase activities in the presence of mixed bile salts, where -○- is lipase originating from Rhizopus oryzae, -●-
shows the results for lipase originating from Rhizopus delemer, -◆- for lipase originating from Mucor javanicus, -△- for lipase originating from Pseudomonas cepacia, and -□- for lipase originating from Candida rugosa. Note that the downward arrow in the figure indicates the addition of mixed bile salts.

Claims (4)

[Claims] 1. A lipase composition stabilized against bile salts, comprising a lipase of microbial origin and a protein having L-histidine and/or L-histidine as an N-terminal amino group. 2. A method for stabilizing lipase against bile salts by coexisting a lipase derived from a microorganism with L-histidine and/or a protein having L-histidine as an N-terminal amino group. 3. A lipase composition stabilized against bile salts, comprising a lipase of microbial origin and at least one of L-histidine, serum albumin, or lactalbumin. 4. A method of stabilizing the lipase against bile salts by coexisting a lipase of microbial origin with at least one of L-histidine, serum albumin, or lactalbumin.