JPH0533034B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a method for decomposing alginic acid alkaline earth metal salts by the action of alginate lyase, and in particular, to decomposing alginic acid salts in the presence of an alkaline earth metal chelating substance and/or a surfactant in the above method. This invention relates to an improved method for decomposing alkaline earth metal salts. [Prior Art] Alginate lyase is an enzyme that acts on alginic acid and its salts to decompose them and reduce their molecular weight. Alginic acid and its salts are used in many applications. For example, its applications include warp thread agents, food processing, paints, and dental impression materials. In these applications, it may be necessary to remove alginic acid and/or its salt after use. For example, calcium alginate gel, which is used as a dental impression material, must be removed from the tray after use. In such cases, alginic acid and/or its salts have conventionally been chemically solubilized using a solution containing a chelating substance such as ethylenediaminetetraacetic acid (EDTA) as a main component. However, EDTA is not preferred because it has the effect of corroding metals and may damage the base material. There is also a method of gel removal of alginic acid and/or its salt using an acid such as citric acid or phosphoric acid or a salt thereof, but these methods are not efficient because their dissolving action is weak. As an example of using alginate lyase as an alternative to the chemical method described above, a method for measuring the titer of alginate lyase that decomposes alginic acid is known (for example, Methods in Engineering Vol. 8, pp. 641-644). p. 1966). Unlike the chemical methods mentioned above, alginate lyase acts only on alginic acid and/or its salts to decompose it and reduce its molecular weight to make it easier to remove. This is very advantageous because it does not damage the material. Microorganisms that produce such alginate lyase include the genus Pseudomonas (Journal of Biochemistry, Vol. 66, pp. 503-512).
(1969), Aeromonas (Science and Industry, Vol. 43, pp. 213-218, 1969), Klebsiella (Carbohydride Research, Vol. 59, No. 163)
Microorganisms such as the genus Bacillus (Applied Endo-Environmental Microbiology, Vol. 47, pp. 704-709) are known. [Problems to be Solved by the Invention] Although alginate lyase is more advantageous in decomposing alginic acid and/or its salts than the above-mentioned chemical methods, its decomposition ability, especially its decomposition rate, is still insufficient. However, the current situation is that it is desired to improve the decomposition rate, especially when decomposing alginic acid alkaline earth metal salts. Accordingly, it is an object of the present invention to provide an improved method for increasing the rate of decomposition of alginic acid alkaline earth metal salts by alginate lyase. [Means for Solving the Problems] The present invention provides the following steps when decomposing alginic acid alkaline earth metal salts by the action of alginate lyase.
A method for decomposing an alkaline earth metal salt of alginic acid in the presence of an alkaline earth metal chelating substance and/or a surfactant. The origin of the alginate lyase used in the method of the present invention is not particularly limited, and can be obtained by culturing the aforementioned microorganisms such as Pseudomonas, Aeromonas, Klebsiella, and Bacillus by a known method. The resulting alginate lyase can be used. In addition, alginate lyase produced by culturing alginate lyase-producing bacteria belonging to the genus Xanthomonas, which was newly discovered by the present inventor, can also be used. It is preferable because there is. This method for producing alginate lyase from microorganisms of the genus Xanthomonas is described in a patent application (Japanese Patent Application Laid-open No. 39581/1983) filed on the same date as the present application.
Therefore, the description of the patent application specification is hereby incorporated by reference. The microorganisms belonging to the genus Xanthomonas are advantageous because they can produce alginate lyase in significant amounts in culture. This will be further explained below. Such strains may be any strains belonging to the genus Xanthomonas that have the ability to produce anginate lyase, or may be mutants thereof. Specific examples of strains that belong to the genus Xanthomonas and have the ability to produce alginate lyase include, for example, Xanthomonas sp.
(Xanthomonas spN-26). This bacterial strain was isolated from the soil of a field in Fukuchiyama City, Kyoto Prefecture. The mycological properties of this strain are as follows. a. Morphological properties Microscopic observation (cultured at 30°C in broth liquid medium) (1) Cell shape and size: Normal cell size is 4-5 μm x 0.6-0.7 μm. (2) Presence or absence of cell pleomorphism: None, isolated. (3) Motility: Yes, with polar flagella. (4) Presence or absence of spores: None. (5) Gram staining: Negative. b Growth status in each medium (1) Broth agar plate culture: Form convex circular colonies with a diameter of 2 to 5 mm by culturing at 30°C for 48 hours. The surface is smooth and slightly yellow. (2) Juicy agar slant culture: When cultured at 30°C, the growth is normal, with thread-like appearance and smooth and glossy edges. (3) Meat juice liquid culture: Grows in 24 hours when cultured at 30℃, and growth is normal. (4) Meat juice gelatin puncture culture: Grows in 24 hours when cultured at 30°C, and growth is normal. Liquefaction is funnel-shaped. (5) Litmus milk culture: It does not coagulate when cultured at 30℃, and the color remains bluish-purple with no change. c Physiological properties (1) Nitrate reduction: Negative (2) Denitrification reaction: Negative (3) MR test: Positive (4) VP test: Negative (5) Indole production: Negative (6) Hydrogen sulfide production: Positive (7) Starch hydrolysis: Positive (8) Auxotrophy: Methionine (9) Utilization of citric acid: Positive (10) Utilization of inorganic nitrogen source: Positive (11) Urease: Negative (12) Oxidase: Negative ( 13) Catalase: Positive (14) Growth pH: 5-10 (15) Growth temperature: 15-40℃ (16) Attitude towards oxygen: Aerobic (17) O-F test: Oxidative (18) Growth from sugars Gas production: positive (19) Acid production from sugars: positive; maltose, lebulose, lactose, glucose, cellobiose, fructose, sucrose, salicin negative; sorbitol, xylose, arabinose, anthole, galactose, rhamnose,
Raffinose, Inositol The above properties are summarized in "Bergie's Manual of Determinative Bacteriology" No. 8.
(1974), it was found that it is similar to Xanthomonas chiambestris, but since it differs in the production of acid from sugars, it was judged to be a new species of the genus Xanthomonas and was named Xanthomonas S.B. N-26. I named it. This strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology under microbial accession number 8800. This strain contains alginic acid and alginic acid salts of 0.01 to 20% in nutrients commonly used for culturing microorganisms, such as meat extract, peptone, yeast extract, malt extract, corn steep liquor, and casamino acids.
%, preferably 0.1% to 1% at 20°C.
It grows well under aerobic conditions at ~40°C, preferably 27°C to 36°C, and produces alginate lyase after 5 to 40 hours of culture. Since the alginate lyase produced by the bacterium of the present invention exists within the bacterium, alginate lyase can be extracted from the bacterium by cell disruption using beads or ultrasound, organic solvent extraction, etc., but preferably the following methods are used: Do it in a method. A surfactant such as Triton
Add 0.01% to 5%, preferably 0.05% to 2%. At this time, adding 0.001 to 10 mg/ml, preferably 0.01 to 1 mg/ml of lysozyme improves the extraction effect. After the treatment, a crude enzyme solution can be obtained by allowing the mixture to stand for 1 minute to 5 hours, and then removing bacterial residue by flocculation with a flocculant, centrifugation, etc., although this varies depending on the amount added and the bacterial cell concentration. The alginate lyase used in the present invention is not limited to the crude enzyme solution described above, but also processed products of the crude enzyme solution, such as enzyme solutions purified by known methods such as organic solvent precipitation, salting out, chromatography, etc. They may be dried, fixed, or even cell suspensions. Next, the enzymatic and physicochemical properties of alginate lyase obtained from Xanthomonas S.B. N-26 are as follows. (1) Effect: When reacting with alginic acid as a substrate, an increase in specific absorption at 235 nm derived from double bonds, which are reaction products of alginate lyase, was confirmed. (2) Substrate specificity:

【table】 (3) Optimal PH and stable PH range: The optimum pH is around PH6.1 (see Figure 1) Stable at pH5.5-6.5 (see Figure 2) (4) Optimal temperature and thermal stability: The optimum temperature is around 50℃ (see Figure 3) Thermal stability is stable up to around 40℃ (see Figure 4) (5) Inhibition, activation

【table】 (6) Stabilization (45℃, 30 minutes treatment)

[Effect]

According to the present invention, the use of an alkaline earth metal chelating substance and/or a surfactant together with alginate lyase, which is not theoretically known,
Compared to the conventional case where alginate lyase is used alone, the decomposition reaction rate of the alginic acid alkaline earth metal salt is increased, and other coexisting materials are not damaged due to corrosion or the like. [Example] The present invention will be described below with reference to Examples. Example 1 Dental impression block (2.5×2.5×2.5cm19.5
g) was left to react in an alginate lyase aqueous solution containing an alkaline earth metal chelating substance and/or a surfactant adjusted to pH 6.3 at 30° C. for 20 hours. The weight of the undissolved block was measured using a balance, and the effect was determined by the proportion of the impression agent block dissolved (dissolution rate (%)) according to the following formula.
However, pH adjustment was performed using an aqueous NaH ₂ PO ₄ solution. The results are shown in Table 2. Dissolution rate (%) = 19.5g - Weight of undissolved block / 19.5 x 100 (%)

〔Effect of the invention〕

When decomposing alginic acid alkaline earth metal salts, by using alginate lyase together with an alkaline earth metal chelating substance and/or a surfactant, equipment corrosion is not caused compared to conventional decomposition agents. When decomposing alginic acid alkaline earth metal salts, it has become possible to significantly increase the decomposition power and improve efficiency.

[Brief explanation of drawings]

Figure 1 is a graph showing the activity of alginate lyase obtained from Xanthomonas sp. N-26 at each pH, and Figure 2 is a graph showing the activity of alginate lyase obtained from Xanthomonas sp.
3 is a graph showing the PH stability after treatment at each temperature for 1 hour, FIG. 3 is a graph showing the activity at each temperature, and FIG. 4 is a graph showing the thermal stability after 10 minutes treatment at each temperature.

Claims (1)

[Claims] 1. An alginic acid alkaline earth metal salt is decomposed by the action of alginate lyase in the presence of an alkaline earth metal chelating substance and/or a surfactant. How to take it apart.