JP3489789B2 - Novel laminarinase and method for producing the same - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel laminarinase and a method for producing the same. More particularly, the present invention relates to a novel laminarinase derived from Antarctic krill (Eupasia superba) and a method for producing the same.

[0002]

BACKGROUND OF THE INVENTION Conventionally, the development of glycolytic enzymes has been carried out for the purpose of utilizing biomass resources, for example, as a utilization of unused agricultural and forestry wastes, using cellulase, delignification of wood, cellulosic materials (wood etc.). It is expected that the seaweed polysaccharide can be used for food production of various oligosaccharides produced by such degrading enzymes. Laminarinase is also one of the cellulase enzymes.
The existence of the laminarinase enzyme in krill has already been reported [Suzuki et al., Nisui, 53 (2), 311].
-317 (1987)], the enzyme molecule of Antarctic krill has not been identified as a single component, and there are still many unclear points regarding the structure and biochemical properties, and its clarification is awaited.

[0003]

SUMMARY OF THE INVENTION It is an object of the present invention to provide an enzyme laminarinase isolated as a single component from Antarctic krill (Euphasia superba) and a method for producing the same.

[0004]

[Means for Solving the Problems] In order to solve the above-mentioned problems, the present inventor diligently conducted research on extraction, purification and properties of laminarinase from Antarctic krill, and succeeded in isolating and purifying laminarinase. The inventors have further found the properties and completed the present invention. The Antarctic krill laminarinase of the present invention produces glucose and various oligosaccharides when it acts on a polysaccharide containing β-1,3-glucan.

Effective use of a large amount of β-1,3-glucan existing as a renewable resource in the natural world, particularly efficient use of sugars such as glucose, laminaribiose and laminaritriose by enzymatic hydrolysis of the substance. It can be used for recovery and utilization. Further, various oligosaccharides can be produced by using in combination with a polysaccharide degrading enzyme other than laminarinase.

In addition to utilizing the obtained useful oligosaccharide as a food additive, other nutrients are mixed with one of the forms of food which can be efficiently ingested and which is well suited to the current diversified lifestyles. Can be provided as capsules, granules, tablets, etc. containing oligosaccharides. Further, by adopting such a form, deterioration of oligosaccharides can be prevented, and by blending with other nutrient components, the nutrient components necessary for the body can be taken in a well-balanced manner. Capsules combine other nutrients to harden the contents of oils, emulsions and granules,
Alternatively, it is filled in a soft capsule.

In the present invention, the laminarin degrading activity was measured by quantifying reducing sugars by the Somogyi Nelson method. Regarding the enzyme titer, 1 Unit was defined as the amount of enzyme that produced a reducing sugar corresponding to 1 μmol of glucose per minute under constant conditions.

The novel laminarinase of the present invention has the following physicochemical properties. (A) Action It often acts on laminarin (β-1,3-glucan) and decomposes it to produce reducing sugar. It is an endo-type degrading enzyme. (B) Substrate specificity Laminarin is used as a substrate. The substrate is an oligosaccharide having a β-1,3 bond of glucose and a laminaritetraose or higher.

(C) Working pH and optimum pH The working pH is in a wide range of 4 to 9, and the optimum pH is 5.0. (D) pH stability When kept at 4 ° C for 1 hour at various pHs, it is stable in the range of pH 4-9.

(E) Working temperature and optimum temperature The working temperature is 5 to 60 ° C., and the optimum temperature is 40 ° C. (F) Temperature stability It is stable at 40 ° C. for 10 minutes and has a residual activity of 90% or more even at 50 ° C. for 10 minutes.

(G) Effect of chelating agent Even if EDTA and EGTA which are chelating agents are made to coexist during the activity measurement, the activity is hardly inhibited. (H) Effect of metal ion It is inhibited by Hg ²⁺ , Cu ²⁺ and Fe ³⁺ . (I) Molecular weight of about 27,000 by SDS-PAGE method

Separation and purification methods for krill laminarinase include various anion exchange chromatography such as DEAE- and MonoQ chromatography, gel filtration,
Various chromatography methods such as various affinity chromatography methods, various chromatography methods such as ammonium sulfate fractionation method, salting out methods such as ammonium sulfate fractionation method, and organic solvent fractionation methods such as ethanol fractionation method. Can be obtained as a single enzyme.

[0013]

The details of the present invention will be described with reference to examples. The invention is in no way limited by these examples.

Example 1 Hereinafter, a method for purifying Antarctic krill laminarinase will be described. (1) Antarctic krill 10 stored frozen at -30 ° C
0 g to 50 mM sodium acetate (pH 5.0), 1 mM
A 5 times volume of a solution containing PMSF and 5 mM 2-mercaptoethanol was added, and the mixture was homogenized under ice cooling for 1 minute. Further, the mixture was stirred at 4 ° C. for 1 hour to extract the enzyme. After 1 hour, centrifugation was performed at 10,000 × g for 20 minutes to obtain 400 ml of a crude enzyme solution as a supernatant. (2) Solid ammonium sulfate was added to this supernatant so as to be 40% saturated. After centrifugation at 10,000 × g for 20 minutes to remove the precipitate, solid ammonium sulfate was further added to 420 ml of the obtained supernatant so as to be 60% saturation,
Centrifugation was performed at 00 × g for 20 minutes.

(3) The obtained precipitate was suspended in 20 ml of the buffer solution shown in (1) above to obtain Sephadex G-2.
Desalination was performed by treatment with 5 columns (20 mm × 250 mm). (4) The fraction obtained in (3) above was previously added to 50 mM.
DEAE-Toyopearl 650M column (2) equilibrated with sodium acetate (pH 5.0) buffer
0 mm × 300 mm (manufactured by Tosoh Corporation), 0-0.
Protein extraction was performed with a linear gradient of 6M NaCl. As a result, one active fraction peak was obtained at around 0.4-0.5M.

(5) The active fraction was concentrated and desalted by ultrafiltration (fraction molecular weight 10,000, manufactured by Filtron). (6) 50m of the enzyme solution obtained in (5) above
Laminarin-Sepharose 6B affinity column (10 mm × 30 mm Epoxy-acti) equilibrated with M sodium acetate (pH 5.0) solution
loaded Sepharose 6B (made by Pharmacia, with laminarin immobilized), and the non-adsorbed fraction was added to 5
The adsorbed fraction was eluted with 0 mM sodium acetate (pH 5.0) solution and 0.1 or 0.5 M Nacl, 50 mM sodium acetate (pH 5.0) solution, respectively. As a result, a peak of one active fraction was obtained in the 0.5 M NaCl elution fraction.

(7) Next, the enzyme solution obtained in (6) above was loaded on a Superose 12 gel filtration column (Pharmacia) to obtain 50 mM sodium acetate (pH 5.
0) Elute with solution. As a result, a peak of one active fraction was confirmed. (8) The fraction obtained in (7) above was applied to SDS-polyacrylamide gel electrophoresis, and after electrophoresis, stained with Coomassie Brilliant Blue. As shown in FIG. confirmed. The molecular weight is about 27,000.
It was shown to be 0. When laminarin is used as a substrate, the activity at pH 5.0 and 30 ° C is 25 per mg.
It was 0 Unit.

Example 2 The physicochemical properties of the Antarctic krill laminarinase isolated and recovered in Example 1 were measured. (1) Laminarinolytic activity The laminarinolytic activity was measured according to the following method. 0.2% 1% laminarin (Nacalai Tesque)
ml, 50 mM sodium acetate solution (pH 5.0)
Add 2 ml and 0.1 ml of enzyme solution to 1 at 37 ° C.
Reacted for hours. The reaction was stopped by heating at 100 ° C. for 10 minutes. After stopping the reaction, Somogyi Nelson
The reducing sugar was quantified by the method.

That is, after the reaction, 0.5 ml of the copper reagent was added to 0.5 ml, heated at 100 ° C. for 10 minutes, cooled, then 0.5 ml of the Nelson reagent was added, and 12.
It was diluted to 5 ml with distilled water. After 15 minutes, wavelength 660nm
Colorimetrically determined by. Regarding the enzyme titer, 1 Unit was defined as the amount of enzyme that produced a reducing sugar equivalent to 1 μmol of glucose per minute under the above conditions.

Laminalin, Laminaribiose, Laminaritriose, Laminaritetraose, Laminaripentaose, Laminarihexaose, Laminariheptaose,
CM-cellulose, liqueran, Burley β-glucan,
The decomposition activity against Baker's yeast glucan and gentiobiose was examined. Table 1 shows the reaction for each substrate. It acts only on laminarin and has no degradative activity against carboxymethylcellulose, cellobiose liqueran, and Burley β-glucan.

[0021]

[Table 1]

(2) Working pH and optimum pH As shown in FIG. 2, the working pH is in a wide range of 4 to 9,
The optimum pH is 5.0. (3) pH stability As shown in FIG. 3, when various pH values were kept at 4 ° C. for 1 hour, they were stable in the pH range of 4 to 9.

(4) Working temperature and optimum temperature As a result of measuring the working temperature in a 50 mM sodium acetate (pH 5.0) buffer solution, as shown in FIG.
Over a wide range, the optimum temperature was 40 ° C. (5) Temperature stability 1 in 50 mM sodium acetate (pH 5.0) buffer
After treatment for 0 minutes at each temperature, the residual activity was measured,
As shown in FIG. 5, it was stable at 40 ° C. and had a residual activity of 90% or more even at 50 ° C.

(6) Effect of chelating agent As a result of examining the effect of chelating agents, EDTA and EGTA, which were coexisting at a final concentration of 1 mM when measuring the activity, the activity was hardly inhibited. (7) Effects of metal ions Various metal ions (Mn ²⁺ , Ca ²⁺ , Co ²⁺ , Hg
²⁺ , Cu ²⁺ , Fe ²⁺ ; 1 mM) were made to coexist during the activity measurement, and the effect was examined. As a result, Hg ²⁺ , C
It was inhibited by u ²⁺ and Fe ³⁺ .

(8) Amino acid composition The amino acid composition of the enzyme was measured. The results are shown in Table 2.

[0026]

[Table 2]

(9) Trypsin 1/100 (w /
w) Add and hold at 15 ° C for 0 to 60 minutes to give 0, 5, 10,
The activity was measured at 30 and 60 minutes (40 ° C.). as a result,
90% or more of the activity was retained at any time point.

[0028]

Various laminarinases can be provided in the form of a single molecule as a glycolytic enzyme. β-1,3
It can be used in the field of producing glucose and various oligosaccharides by acting on a polysaccharide containing glucan, and in the field of producing various oligosaccharides in combination with a polysaccharide degrading enzyme other than laminarinase.

[Brief description of drawings]

FIG. 1 shows the results of SDS-polyacrylamide gel electrophoresis of laminarinase.

FIG. 2 shows the relationship between the enzyme reaction pH and the relative residual activity of the same enzyme.

FIG. 3 shows the relationship between the treatment pH of the enzyme and the relative residual activity.

FIG. 4 shows the relationship between reaction temperature and relative residual activity of the same enzyme.

FIG. 5 shows the relationship between the treatment temperature and the relative residual activity of the same enzyme.

─────────────────────────────────────────────────── ─── Continuation of front page (58) Fields surveyed (Int.Cl. ⁷ , DB name) C12N 9/14-9/46 BIOSIS (DIALOG) WPI (DIALOG) JSTPlus (JOIS)

Claims (4)

(57) [Claims] 1. A laminarinase derived from Antarctic krill (Euphasia superba), which has a physicochemical property as follows: New laminarinase (a) action Laminalin (β-1,3-glucan) acts well and decomposes it. It produces a reducing sugar. It is an endo-type degrading enzyme. (B) Substrate specificity Laminarin is used as a substrate. An oligosaccharide having a β-1,3 bond of glucose and a laminaritetraose or higher is used as a substrate. (C) Working pH and optimum pH The working pH is in a wide range of 4 to 9, and the optimum pH is 5.0. (D) pH stability When kept at 4 ° C for 1 hour at various pHs, it is stable in the range of pH 4-9. (E) Working temperature and optimum temperature The working temperature is 5 to 60 ° C, and the optimum temperature is 40 ° C. (F) Temperature stability It is stable at 40 ° C. for 10 minutes and has a residual activity of 90% or more even at 50 ° C. for 10 minutes. (G) Effect of chelating agent Even if EDTA and EGTA, which are chelating agents, coexist during the activity measurement, the activity is hardly inhibited. (H) Effect of metal ion It is inhibited by Hg ²⁺ , Cu ²⁺ and Fe ³⁺ . (I) Molecular weight of about 27,000 by SDS-PAGE method 2. The method for producing laminarinase according to claim 1 , wherein the crude enzyme solution extracted from Antarctic krill (Euphasia superba) or a frozen product thereof is supplied to an affinity column on which at least laminarin is immobilized and purified by gel filtration. A process for producing a novel laminarinase, which comprises isolating and recovering the laminarinase. 3. Glucose and novel laminarinase of claim 1, wherein the enzyme for various oligosaccharide production. According to claim 4 is a health food production for degrading enzyme according to claim 1,
The novel laminarinase described.