KR20010037633A - Method and composition for increasing stability and activity of enzymes - Google Patents

Abstract

PURPOSE: Provided is a method for increasing the stability and activity of enzymes including polygalacturonase by adding gums such as xanthan gum, guar gum, locust bean gum, gum arabic, and carrageenan. CONSTITUTION: Stability and activity of enzymes in particular, hydrophilic enzyme, is increased by adding gums selected form the group consisting of xanthan gum, guar gum, locust bean gum and gum arabic. The enzyme is selected from the group consisting of polygalacturonase, thrombin, urokinase and superoxide dismutase, and is preferably polygalacturonase that is isolated and purified from Kluyveromyces marxianus IFO 0288. A method for increasing the stability and activity of enzymes comprises the following steps of: i) mixing 4.29*10¬(-4) nM of purified polygalacturonase with 20 mM of sodium acetate buffer solution being pH 5.0 or buffer solution including 0.2%(w/v) of xanthan gum, guar gum, locust bean gum, gum arabic, or k-carrageenan at 30 deg.C; and ii) measuring the activity of polygalacturonase during the cultivation to calculate k value in connection with the inactivation of enzyme. The value shows that xanthan gum, guar gum, locust bean gum, and gum arabic considerably increase the activity of the enzyme but k-carrageenan inhibits the activity.

Description

Method and composition for increasing stability and activity of enzymes

The present invention relates to novel methods and compositions that can enhance the stability and activity of enzymes, and more particularly, to hydrophilic enzymes, including, for example, polygalacturonase isolated and purified from microorganisms. The present invention relates to a method and composition capable of simultaneously non-specifically enhancing the stability and activity by adding a rubbery substance which is a colloidal substance.

Polygalacturonase is known as an enzyme that hydrolyzes the α-1,4-linkage of polygalacturonic acid, a kind of pectin. Such polygalacturonases are also present in plants and are known to increase their activity as immature fruits ripen, and are also present in various microorganisms [J. Ind. Microbiol. Biotechnol., 20 (1), 34 (1998); FEMS Microbiol. Lett., 158 (1), 133 (1998); Can. J. Microbiol., 43 (5), 417 (1997); Biosci. Biotechnol. Biochem., 60 (4), 603 (1996); Yeast, 10 (10), 1311 (1994); Agric. Biol. Chem., 48 (8), 1951 (1984); Agric. Biol. Chem., 44 (3), 473 (1980)]. Such polygalacturonases are used for the purpose of the prevention of clouding by the pectin of the juice, ie clarification [J. Food Sci., 53 (4), 1236 (1988)], which is used for the production of plant single cells that can selectively break down the intercellular materials of plants and maintain them without destroying the natural components of each unit cell. [J. Food Sci., 60 (3), 468 (1995); J. Food Biochem., 9, 325 (1985)] is an enzyme whose application value is increasing.

On the other hand, thrombin is a type of endopeptidase that specifically hydrolyzes -Arg-Gly- bonds in the α and β chains in the fibrinogen molecule to generate four fibrinopeptides and fibrin monomers. Lipase (lipase, triacylglycerol acylhydrolase) is an enzyme that catalyzes the reaction of hydrolysis of triacylglycerides into fatty acids and glycerin. This enzymatic reaction proceeds in an emulsified state. Present in filamentous fungi. Eurokinase is an enzyme present in the urine of mammals and other vertebrates such as humans. It is formed by human renal parenchymal cells and acts as a plasminogen activator, and is a thrombolytic or fibrinolytic substance. is used, and superoxide dismutase (superoxide dismutase) are ^{_{O 2 - · (superoxide) +}} O 2 - · + 2H + → O 2 + H 2 as a kind of a redox enzyme that catalyzes the chemical reaction of O ₂ superoxide Is an enzyme that protects cells when they reach dangerous concentrations.

Conventional enzymes, including polygalacturonase as described above, have a problem in that their activity gradually decreases over time when they are separated from nature due to the nature of proteins, that is, their stability is deteriorated. Have been tried. As a method for enhancing the stability of the enzyme activity, (i) albumin [Biotechnol. Appl. Biochem. 22 (2), 203 (1995)] or a substance capable of acting as a stabilizer, such as dextrin, or ii) as a more selective method, using the information of the enzyme protein at the molecular level to remove the molecule itself. Method of securing stability by modifying [Biotechnol. Bioeng., 40 (6), 650 (1992).

However, the stabilizers such as albumin and dextrin are known to play a role as stabilizers of enzymes, but it has not been revealed at all whether they play a role as activity enhancers for enhancing the activity of enzymes themselves. In addition, there are reports that the activity of some enzymes is enhanced when stability is enhanced by inducing variation of the strain or molecular modification of the protein, which is a more selective method [Enzyme Microb. Technol., 16 (5), 420 (1994)], in which case the stability of the mutated strains or modified proteins should be ensured, and the safety of these enzymes for human-related purposes should also be verified. As an alternative method, there is a problem that the establishment of specific information at the molecular level of a specific enzyme must be preceded.

On the other hand, rubbery materials obtained from nature are used in foods as binders, calorie adjusters, emulsifiers, film forming agents, stabilizers, etc., which utilizes the property that almost all water-soluble rubbery materials form hydrophilic colloids. Rubbery materials include, for example, polysaccharides such as xanthan gum, guar gum, locust bean gum, gum arabic, carrageenan. Xanthan rubber and locust bean rubber are stabilizers or emulsifiers in foodstuffs or cosmetics, guar rubber is a protective colloid, stabilizer, thickener, film forming agent, binder, disintegrant, flocculant, filter or coagulant, and gum arabic is a lubricant In addition, it is used for medical purposes for gastrointestinal catarrh and the like, and is used for binders such as tablets, emulsifiers or ashes and adhesives. In general, the stability of the enzyme in the presence of colloids seems to be enhanced, but there is no known method for enhancing the activity of the enzyme using a rubbery material.

As a result of continuous research to develop new methods for enhancing the stability and activity of enzymes, the present inventors have found that a variety of enzymes, including polygalacturonase, isolated and purified from microorganisms, can be used for pelleting rubber, guar gum and locust bean. The surprising fact that by adding rubbery materials such as rubber and gum arabic can simultaneously non-specifically enhance its stability and activity, has led to the completion of the present invention.

Accordingly, it is an object of the present invention to provide methods and compositions that can simultaneously nonspecifically enhance the stability and activity of an enzyme.

1 is a graph showing the thermal stability with or without the treatment of the rubbery material of the purified polygalacturonase;

Figure 2 is a graph showing the change in activity with or without the treatment of the rubbery substance of the purified polygalacturonase; And

Figure 3 is a graph showing the change in activation energy with or without the treatment of the rubbery substance of the purified polygalacturonase.

In order to achieve the object as described above, the present invention provides a method for enhancing the stability and activity of the enzyme, characterized in that the addition of a rubbery substance to the enzyme. In the process according to the invention, the rubbery material is preferably selected from the group consisting of pelleted rubber, guar rubber, locust bean rubber and gum arabic, the enzyme is preferably a hydrophilic enzyme, more preferably polygal Lacturonases, thrombin, urokinase or superoxide dismutase, most preferably polygalacturonases. Among them, polygalacturonase is more preferably isolated and purified from Kluyveromyces maxilianus IFO (Institute of Fermentation in Osaka) 0288 (the same strain is the same strain as KCCM 50555 of the Korean spawn association).

The present invention also provides an enzyme composition characterized by containing a rubbery material as a stability and activity enhancer. In the composition according to the invention, the rubbery material is preferably selected from the group consisting of pelletized rubber, guar rubber, locust bean rubber and gum arabic, the enzyme is preferably a hydrophilic enzyme, more preferably polygal Lacturonases, thrombin, urokinase or superoxide dismutase, most preferably polygalacturonases. Among them, polygalacturonase is more preferably isolated and purified from Cluyveromyces maximus IFO 0288.

Hereinafter, the present invention will be described in detail.

In the method of enhancing the stability and activity of the polygalacturonase which is a representative example of the present invention, the polygalacturonase is isolated and purified from microorganisms in the following manner. In other words, the Kluyveromyces maxianus IFO 0288 was stirred at a speed of 200 rpm at 30 ° C. using a liquid medium consisting of 20 mM sodium acetate buffer solution at pH 5.0 containing 3% glucose, 0.6% peptone and 0.2% yeast extract. Shake culture for 20 hours in the incubator. After incubation, the culture medium was centrifuged to remove the cells, the supernatant was taken and concentrated, and the concentrated culture solution was then subjected to fast protein liquid chromatography using a mono S HR 5/5 [Pharmacia] ion exchange column. separation by liquid chromatography (FPLC). As chromatographic conditions, a 20 mM sodium acetate buffer solution of pH 5.0 was used, and the salt concentration during protein elution was a linear concentration gradient of 0 to 0.2 M NaCl. Among the proteins eluted from the column, fractions showing polygalacturonase activity were collected and purified by SDS PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) to confirm the purity of the purified polygalacturonase. Hereinafter, the polygalacturonase sample used in Examples 1 to 3 of the present invention is a pure enzyme isolated by the above method.

Other enzymes, such as polygalacturonases from Aspergillus niger, thrombin, urokinase, and superoxide dismutases from Bacillus thermophilus, are commercially available reagents (Sigma, USA). The lipase uses partially purified coenzyme secreted by Bacillus thermoleovorans.

The activity of polygalacturonase is described by Honda et al. [Anal. Biochem., 119, 194 (1982)], and the index of the stability of polygalacturonase is obtained by calculating the k value (min ⁻¹ ), which is the first-order rate constant for the inactivation rate of the enzyme. do.

The activity of thrombin is quantified by measuring the change in absorbance at 405 nm using the synthetic substrate S-2238 (D-Phenylalanine-Pipecolyl-Arginine-Paranitroanilide). [J. Biochem., 119 (5), 835 (1996).

The activity of the lipase is quantified by measuring the change in absorbance at 405 nm of the enzyme reaction product produced using paranitrophenyl butylate as a substrate [Biosci. Biotechnol. Biochem., 62 (1), 66 (1998).

The activity of urokinase is quantified by measuring the change in absorbance at 405 nm for the colorimetric reaction of paranitroanilide resulting from reaction with the synthetic substrate V-7127 (D-valine-leucine-lysine-paranitroanilide).

The activity of superoxide dismutase is quantified by measuring the level at which reduction of Nitro Blue Tetrazolium is inhibited by enzymes in the xanthine-santhoxidase system.

In the present invention, the term "rubber material" refers to pelleted rubber obtained by fermenting Xanthomonas campestris, guar gum derived from seeds of legumes, and carob tree (Mediterranean coastal evergreen trees). Locust bean rubber derived, gum arabic derived from acacia, etc., but carrageenan derived from red algae are excluded. Carrageenan is a negatively charged molecule, so the protein is positively charged at a pH lower than the pI (isoelectric point) of a protein, so it has a structural feature that easily binds to other proteins and loses its properties. In addition to carrageenan, the other rubbery materials have no molecular charge unlike carrageenan, and thus, do not bind to enzymes and lose their activity, and thus can function as enzyme activity enhancers.

On the other hand, in the present invention, the function of the rubbery substances as an enzyme activity enhancer is not due to the molecular structural aspects of the materials but due to their physical properties, and has been found to have a very close relationship with the viscosity among the physical properties. Therefore, rubbery materials increase the viscosity of the solution even at very low concentrations, and thus exhibit enzymatic activity enhancement functions even at extremely low concentrations, for example, concentrations below 0.0005% (w / v). On the other hand, it is 0.6% or less for pellets with relatively high viscosity, 1% or less for guar and locust bean rubber (because of gel-like properties above 1%), and 40% or less for Arabian rubber. (A characteristic of Arabian gum is that a high concentration of solution can be prepared.) It is more preferable to use.

EXAMPLE

Hereinafter, the present invention will be described in more detail with reference to specific examples, which are intended to aid the understanding of the present invention and are not intended to limit the scope of the present invention in any way.

Example 1

Purified polygalacturonase was dissolved in 20 mM sodium acetate buffer solution at pH 5.0 or 0.2% (w / v) pelleted rubber, 0.2% (w / v) guar rubber and 0.2% (w / v) locust bean rubber. Incubated at 30 ° C by mixing well in the same buffer containing 0.2% (w / v) Arabian rubber or 0.2% (w / v) κ-carrageenan. At this time, the concentration of polygalacturonase was 4.29 × 10 ⁻⁴ nM. A constant amount of enzyme was taken at regular time intervals during the cultivation to measure the activity of the remaining polygalacturonasase to determine the primary reaction rate constant k value for enzyme inactivation.

The results are shown in FIG. 1, and the following Table 1 shows the first order rate constant k values for inactivation of polygalacturonasase in the presence of each rubbery material.

Primary Reaction Rate Constants for Inactivation of Polygalacturonases in the Treatment of Gummy Materials k (min ^-1 ) Control 0.0082 Pellets Rubber Treatment Group 0.0003 Guar rubber treatment group <0.0001 Locust hollow rubber treatment group 0.0001 Arabian rubber treatment group 0.0001

As shown in Table 1, the stability of the enzyme is significantly increased when pelleted rubber, guar rubber, locust bean rubber or gum arabic is added, but when κ-carrageenan is added, the activity of the enzyme is immediately lost, and κ-carrageenan is an enzyme stabilizer. It was found to act as an inhibitor of activity of the enzyme rather than as an enzyme.

Example 2

The rubbery material was treated in the same conditions and methods as in Example 1, and after 30 minutes, the relative activity of the purified polygalacturonase was compared. The results are shown in FIG. 2 and Table 2 below.

Relative Activity of Polygalacurolonase in the Treatment of Gummy Materials Relative activity (%) Control 100 Pellets Rubber Treatment Group 189 Guar rubber treatment group 197 Locust hollow rubber treatment group 190 Arabian rubber treatment group 181

As shown in Table 2, the relative activity of polygalacturonase was 89% when pelleted rubber was added, 97% when guar rubber was added, 90% when locust bean rubber was added, and 81 when Arabian rubber was added. Increasing by%, it was confirmed that rubbery substances except κ-carrageenan acted as enzyme activity promoters.

Example 3

In order to prove that the rubbery materials in Examples 1 and 2 promoted the activity of polygalacturonases, an Arennius graph for the enzymatic activity of each rubbery material was obtained and shown in FIG. 3. Here, T represents the absolute temperature value at the time of enzyme reaction. The activation energy of the enzymatic reaction of the polygalacturonic acid reacted with the polygalacturonic acid from the absolute value of the slope of the straight line of FIG. 3 is shown in Table 3 below.

Activation Energy of Polygalacturonase in the Treatment of Gummy Materials Activation energy (cal / mole) Apparent Viscosity (cP) * Control 13,300 2.9 Pellets Rubber Treatment Group - 94.0 Guar rubber treatment group 10,900 15.0 Locust hollow rubber treatment group 11,400 10.0 Arabian rubber treatment group 11,000 7.5

* Shear rate 6.1 sec ^-1 measured at Brookfield viscometer with UL adapter and No.00 spindle at temperature 28 ℃.

However, as shown in Table 3, the apparent viscosity of the reaction solution to which the pelletized rubber was added was relatively very high, and it was difficult to obtain the activation energy induced by the Areneus graph when the pelleted rubber was added. However, it was confirmed that the activation energy of the enzyme reaction was lower in the case of other rubbery substances than the control, which means that the rubbery substances play a role as an activity promoter of the enzyme.

Example 4

To ascertain the effect of rubbery substance on activity of enzymes other than polygalacturonases and polygalacturonases derived from Kluyveromyces maxilianus and other species and genera, i) Aspergillus Enzymes such as polygalacturonase derived from Niger, ii) thrombin, iv) lipase derived from Bacillus thermoeoborance, iv) urokinase, and iv) superoxide dismutase derived from Bacillus thermophilus. Their original activity and activity when treated with 0.2% guar rubber were analyzed by comparison, and the results are shown in Table 4 below.

Comparison of activities with and without guar gum (0.2%) treatment of different enzymes Relative activity (%) Control Guar rubber treatment group Polygalacturonase * 100 185 Thrombin 100 124 Lipase 100 100 Eurokinase 100 116 Superoxide dismutase 100 128

* Aspergillus Niger

As shown in Table 4, the rubbery material was found to exhibit activity enhancing effects on not only polygalacturonases derived from sources other than Kluyveromyces maximus, but also completely different enzymes.

These results demonstrate that the rubbery material does not selectively act on specific enzymes to specifically enhance activity, but can nonspecifically enhance the activity of conventional enzymes.

In particular, in the case of hydrophilic enzyme system, the activity enhancement effect was remarkable, in the case of non-hydrophilic enzyme system such as lipase was not as remarkable as the hydrophilic enzyme system, it could be confirmed that has the effect of maintaining the activity. In the case of the non-hydrophilic enzyme system, the activity enhancement effect is not as remarkable as the hydrophilic enzyme system is related to the inability of the rubbery material to form a hydrophilic colloidal state.

As in the present invention, the addition of rubbery materials such as pelleted rubber, guar rubber, locust bean rubber or gum arabic to various enzymes, including polygalacturonases, can simultaneously nonspecifically enhance their stability and activity.

Claims (12)

A method for enhancing the stability and activity of an enzyme, characterized by adding a rubbery substance to the enzyme. The method of claim 1 wherein the rubbery material is selected from the group consisting of xanthan gum, guar gum, locust bean gum and gum arabic. The method according to claim 1 or 2, wherein the enzyme is a hydrophilic enzyme. 4. The method of claim 3, wherein the enzyme is selected from the group consisting of polygalacturonase, thrombin, urokinase and superoxide dismutase. 5. The method of claim 4 wherein the enzyme is polygalacturonase. 6. The method of claim 5, wherein the polygalacturonase is isolated and purified from Kluyveromyces marxianus IFO 0288. An enzyme composition comprising a rubbery substance as a stability and activity enhancer. 8. The composition of claim 7, wherein the rubbery material is selected from the group consisting of pelleted rubber, guar rubber, locust bean rubber, and gum arabic. The composition according to claim 7 or 8, wherein the enzyme is a hydrophilic enzyme. 10. The composition of claim 9, wherein the enzyme is selected from the group consisting of polygalacturonase, thrombin, urokinase and superoxide dismutase. The composition of claim 10 wherein the enzyme is a polygalacturonase. 12. The composition according to claim 11, wherein the polygalacturonasase is isolated and purified from Kluyveromyces marxianus IFO 0288.