KR101239757B1 - Novel paenibacillus lautus gd-a2 producing breaking down alginate lyase, biocatalyst for alginic acid and method for manufacturing alginic acid oligosaccaride by using the same - Google Patents

Abstract

PURPOSE: A method for preparing alginic acid oligosaccharides using a novel mircroorganism Paenibacillus lautus GD-A2 and a biocatalyst is provided to cheaply produce a low molecular compound of alginic acid. CONSTITUTION: A catalyst for decomposing alginate contains one or more ingredients selected from a culture of Paenibacillus lautus GD-A2(deposit number KFCC 11538P) and a concentrate of the culture as an active ingredient. The culture is prepared by removing a bacterium. The bacterium is cultured by adding a yeast extract and/or peptone as a nitrogen source.

Description

NOVEL PAENIBACILLUS LAUTUS GD-A2 PRODUCING BREAKING DOWN ALGINATE LYASE, BIOCATALYST FOR ALGINIC ACIDACT MEUFOD ALGINIC ACID OLIGOSACCARIDE BY USING THE SAME}

The present invention relates to a novel microorganism producing alginic acid degrading enzyme and a method for producing alginic acid oligosaccharides, and more particularly, to a novel mass production of alginic acid low molecular weight by enzymatic hydrolysis of alginic acid, a polysaccharide contained in seaweed. It relates to a microorganism.

In general, alginic acid, also called seaweed, is a polysaccharide that constitutes the cell wall of seaweed. It is a polymer of two kinds of uronic acid, and has a polymerization degree of 80 and a molecular weight of about 1500. It is a thin alkaline salt of brown algae washed with dilute sulfuric acid. It is a precipitate formed by extracting from warm water and making the extract acidic.

Such alginic acid exhibits acid properties because of the carboxyl group of uronic acid in its molecule, and is usually treated with sodium salts. In addition, calcium salts of alginic acid are insoluble in water, so they react with calcium ions in the blood to form insoluble salts, and because they block the blood vessels, they are not toxic by oral administration, but are known as harmful substances when injected into the blood.

Alginic acid was thought to consist only of mannuronic acid until 1957, but recently it was found that guluronic acid was also a component of alginic acid, and as shown in FIG. Hundreds of lonic acids are linked by β-1 and 4 bonds, and only some of the molecules of alginic acid are bound to only mannuronic acid or glulonic acid, and the ratio of the amount of manneuronic and gluronic acid depends on the type of seaweed. different.

In addition, alginic acid is insoluble, but sodium salt is well soluble in water and its viscosity is very high, so its use is various, and such sodium salt is treated with dilute acid or alkali, dried by crushing brown algae in the wind, and precipitated, air-floating. Remove impurities such as protein and fiber by the method and centrifugation method, dehydrate and dry it with alcohol, make it into powder and use it as a fabric paste, an aqueous paint, an emulsifier, and use it as a surgical thread by fiberizing it. Is used to increase the viscosity of ice cream, jam and mayonnaise.

In addition, since there is no enzyme that degrades alginic acid in mammals, alginic acid cannot be used on its own. Therefore, alginic acid must be low molecular weight to lower the degree of polymerization so that it can be used in vivo. It is known to have an anti-obesity effect by inhibiting and promoting fat metabolism and immune function enhancement by promoting secretion of cytokines such as IL-6 and TNF-α.

Alginic acid is also known to have excellent pharmaceutical efficacy, such as reducing cholesterol concentration, inhibiting adipocyte differentiation and reducing the content of leptin, a protein of the intracellular adipocyte gene.

In addition, there is a growing interest in renewable energy due to environmental pollution from fossil fuel combustion and the depletion of underground resources such as crude oil. Recently, research on bioenergy production using natural plants has been attracting attention.

In particular, research on the production of bioenergy using seaweed has attracted attention as an alternative to overcome the problems of bioenergy production using food resources such as corn.Alginate, which forms the cell wall of algae, is used for bioenergy production using seaweed. There is a need for the development of alginate degrading enzyme having high resolution to easily decompose.

In the meantime, several researchers have reported that the function of the macromolecular alginic acid, which is the polymerization of mannuronic acid and gluronic acid, to the low molecular weight of polymannuronate and polyguluronate is significantly higher. As a method, there is a method of firstly softening by high temperature, high pressure, high pressure or radiation, and secondly, using acid and alkali treatment, but most of carbohydrates derived from seaweed are relatively stable to acid or alkali, and thus it is not easy to decompose alginic acid. Due to excessive acid treatment, acid-resistant devices are required and neutralized to be discharged. Therefore, a large amount of chemicals are consumed, causing excessive cost and environmental pollution. Thus, enzymes using enzymes rather than physical and chemical decomposition of alginic acid There is increasing interest in biodegradation methods. .

Alginate lyase, which decomposes alginic acid, is known to perform alginic acid degradation by β-elimination, although its mechanism of degradation is not clear. These alginate degrading enzymes are classified according to which positions are decomposed by acting on pM-block or pG-block, and one type is an alginate degrading enzyme that decomposes β-1,4 sugar bonds between mannuronate and mannuronate. And the other is an alginate degrading enzyme that breaks down α-1,4 sugar bonds between gluronate and gluronate.

Microorganisms currently known to produce such alginate degrading enzymes include Pseudomonas aeruginosa (Franklin et al., J. Bacteriol., 186: 4759-4773; 2004 ), Streptomyces sp. (Cao et al., J. Agric. Food Chem , 55: 5113-5117; 2007), Bacillus sp. ATB-1015 (Nakagawa et al., J. Appl. Microbiol., 84: 328-335), Alteromonas sp. strain no. 272 (Iwamoto et al., Biosci Biotechnol Biochem., 65: 133-142; 2001) is known.

As such, a method of using microorganisms and enzymes is known as a decomposition method using alginic acid degrading enzymes recently, but it is possible to economically mass-produce alginic acid degrading enzymes to make alginic acid low molecular weight and to use it more efficiently in the manufacture of foods and pharmaceuticals. There is a continuing need for microorganisms and alginic acid degrading enzymes.

The present invention has been made to solve the above-mentioned problems, not only is it possible to recognize a significantly superior alginate resolution compared to other microorganisms having previously reported alginate resolution, and to economically mass-produce alginic acid oligosaccharides, and to lower molecular weight of alginic acid. It can be used more efficiently in the manufacture of health foods and medicines, so that it is possible to manufacture high value-added alginic acid oligosaccharides having various functions related to human health and disease treatment, as well as decomposing cell walls of algae It is an object of the present invention to provide a novel microorganism Penivacillus Lautus GD-A2 (Accession No. KFCC 11538P) that can increase the production efficiency of energy to produce alginic acid degrading enzyme that can be applied to various industrial fields.

In order to solve the above problems, the present invention provides a novel microorganism Penibacillus Lautus GD-A2 (Accession No. KFCC 11538P) producing alginic acid degrading enzyme.

In addition, the present invention is an alginate degradation comprising at least one selected from the group consisting of a culture medium or a concentrate of the culture of a novel microorganism Penivacillus Lautus GD-A2 producing alginic acid degrading enzymes in a culture medium. Provide a biocatalyst for

At this time, the culture is characterized in that the cells are removed.

In addition, the culture of the culture is characterized in that at least one of yeast extract and peptone is added to the culture medium as a nitrogen source.

In addition, the yeast extract is first added to the culture medium for the growth of the strain, the peptone is characterized in that it is added to the culture medium to be used as a biocatalyst after the growth of the strain.

In addition, the culture of the culture is added to the culture medium sodium alginate in the range of 0.1% (w / v) to 1.5% (w / v), NaCl 2.0% (w / v) or less (excluding 0) It is also characterized by being added to.

Further, the culture of the culture is characterized in that the reaction temperature is in the range of 20 ℃ to 60 ℃, the incubation time is more than 36 hours, the reaction pH is carried out in the range of 4 to 8.

In addition, the present invention provides a culture step of culturing the novel microbial Penicillus lautus GD-A2 producing alginic acid degrading enzyme in a culture medium; Obtaining a culture after the culture; And reacting the culture with algae or alginic acid to prepare alginic acid oligosaccharides.

In addition, the present invention is a culturing step of culturing a novel microorganism Penivacillus Lautus GD-A2 in the culture medium to produce alginic acid degrading enzyme; Obtaining step of obtaining a culture after the culture; An extraction and purification step of removing the cells from the culture to extract alginate degrading enzyme and then purifying; It provides a method for producing an alginic acid oligosaccharide comprising; and reacting the alginic acid degrading enzyme with algae or alginic acid to produce alginic acid oligosaccharides.

The novel microorganism Phenibacillus Lautus GD-A2 (Accession No. KFCC 11538P), which produces alginic acid degrading enzymes according to the present invention, is not only recognized for its outstanding alginate resolution compared to other microorganisms having previously reported alginate degradation, but also alginic acid oligosaccharides. It can be economically mass-produced and low molecular weight of alginic acid can be used more efficiently in the manufacture of health foods and medicines, so that it is possible to manufacture high value-added alginic acid oligosaccharides having various functions related to human health and disease treatment. Of course, it is possible to increase the production efficiency of bioenergy using the algae by decomposing the cell wall of the algae, there is an effect that can be applied to various industrial fields.

1 is a structural formula of alginic acid.
Figure 2 is a culture colony photograph of the strain Penivacillus Lautus GD-A2 finally selected as a strain having excellent alginic acid resolution.
Figure 3 is a result of 16S rRNA gene transmission sequence 1,368 bp analysis of the penivacillus Lactus VD-A2 (Accession Number KFCC 11538P) of the present invention.
4A and 4B show the homology of genes of Bacillus subtilis DSM10 (T) AJ276351 and GDYA-1.
Figure 5 is a schematic diagram showing the phylogenetic position of the novel microorganism Penivacillus Lautus vd-A2 (Accession No. KFCC 11538P) producing the alginic acid degrading enzyme of the present invention.
Fig. 6 shows the results of analysis of the cell fatty acid composition of Penivacillus lautus vd-A2 (Accession No. KFCC 11538P).
Figure 7 is a graph showing the proliferative capacity of the Penivacillus Lautus GD-A2 strain according to the nitrogen source of the culture medium.
Figure 8 is a graph showing the relative activity of alginate degrading enzymes of the Penivacillus Lautus GD-A2 strain according to the nitrogen source of the culture medium.
Figure 9 is a graph showing the proliferative capacity of the Penivacillus Lautus GD-A2 strain according to the mixed culture of the nitrogen source of the culture medium.
10 is a graph showing the relative activity of alginic acid degrading enzymes of the Penivacillus Lautus GD-A2 strain according to the culture time of the culture medium.
Figure 11 is a graph showing the proliferative capacity of the Penivacillus Lautus GD-A2 strain according to the alginic acid content of the culture medium.
Figure 12 is a graph showing the relative activity of alginic acid degrading enzymes of the Penivacillus Lautus GD-A2 strain according to the concentration of alginic acid in the culture medium.
Figure 13 is a graph showing the proliferative capacity of the Penivacillus Lautus GD-A2 strain according to the NaCl content of the culture medium.
Figure 14 is a graph showing the relative activity of alginic acid degrading enzymes of the Penivacillus Lautus GD-A2 strain according to the NaCl content of the culture medium.
15 is a graph showing the relative activity of alginic acid degrading enzymes of the Penivacillus lautus GD-A2 strain according to the reaction temperature of the culture medium.
Figure 16 is a graph showing the relative activity of the alginate degrading enzyme of the Penivacillus Lautus GD-A2 strain according to the reaction pH of the culture medium.
Figure 17 is a TLC photograph confirming the production of alginic acid oligosaccharides after treatment of the supernatant obtained after centrifugation of the penivacillus Lautus GD-A2 culture solution in aqueous sodium alginate solution for food.

The present inventors have isolated and identified new microorganisms producing alginic acid degrading enzymes, and research and efforts have been made to reduce alginic acid using these new microorganisms. As a result, the novel microorganisms of albinic acid degrading enzymes are produced. -A2 (Accession No. KFCC 11538P) was isolated, and the alginate degrading enzyme produced by the new microorganism can economically mass-produce alginic acid low molecular weight by hydrolyzing alginic acid, a polysaccharide contained in algae, and lower molecular weight of alginic acid. It was confirmed that there is an effect that can be used more efficiently in the production of food, pharmaceuticals and the like, the present invention was completed.

Hereinafter, the present invention will be described in detail with reference to the accompanying drawings. These examples are only illustrated to explain the present invention in more detail, but the scope of the present invention is not limited to the scope of the description of these embodiments, the present invention can be implemented in various forms.

The strain search for the alginate degradation microorganisms according to the present invention was carried out in two stages, firstly isolates strains having alginic acid degrading from samples collected from the tidal flats of Gwangyang-gun, Jeollanam-do, and secondly selected alginate of each strain The resolution was examined and analyzed to select the strain having the highest resolution.

In addition, through the search process, strains selected as having the highest alginate resolution were identified as Paenibacillus lautus GD-A2 as a result of 16S rDNA sequencing analysis. (Korean Federation of Culture Collections) Affiliated to the Korean Culture Center of Microorganisms under the deposit number of KFCC 11538P.

And, in order to confirm the strain growth ability and the enzyme activity of the growth degree according to the culture conditions of the identified Penivacillus Lautus GD-A2, Penivacillus Lautus GD-A2 was cultured, and nitrogen source ( The addition or content of yeast extract, peptone, soy protein isolate, urea and (NH ₄ ) ₂ SO ₄ ), sodium alginate and NaCl were adjusted.

Here, the yeast extract is composed of water-soluble components such as amino acids, peptides, carbohydrates and salts of yeast cells as natural additives, and a method of hydrolyzing polypeptides of edible yeast by adding edible enzymes to edible yeast. It is prepared by the addition, it may be added to the salt.

The peptone is an inducible protein, which is an acid, alkali, or a degradation product decomposed by a protease or peptidase, and includes a degradation product of pepsin of a protein, which is a lower molecule than a protease in water, and is water-soluble and does not coagulate with heat. Has the nature.

That is, the optimum culture medium was confirmed by confirming the strain growth capacity and enzyme activity according to the nitrogen source in relation to the culture medium of the strain, and the optimum culture time was confirmed by checking the strain growth capacity and enzyme activity according to the culture time, alginic acid concentration and NaCl concentration The optimum alginic acid content and the optimal NaCl content were confirmed by confirming the strain propagation ability and enzyme activity according to. In addition, by checking the enzyme activity according to the reaction temperature and the reaction pH to determine the reaction temperature and the reaction pH for the optimum reaction.

In addition, the present invention includes a biocatalyst for degrading alginic acid, which comprises a novel microorganism Phenibacillus lautus GD-A2 producing alginic acid degrading enzyme or a culture thereof or a concentrate of the culture as an active ingredient. do.

In this case, the culture refers to a culture medium obtained by culturing Penivacillus Lactus GD-A2, which may be a culture medium containing a strain or a cell-free culture medium from which the cells are removed, and mainly a penny in a liquid culture medium. It may be a culture medium obtained after culturing Bacillus Lautus GD-A2, but the properties thereof are not limited to the liquid phase.

Here, the culture medium may be a conventional medium for culturing the tissues of microorganisms or animals, preferably yeast extract or peptone may be included, more preferably yeast extract and peptone may be included together.

In addition, alginic acid may be added to the culture medium, and the content of the alginic acid is 0.1% (w / v) to 1.25% (w / v), preferably 0.3% (w / v), based on the content of the total medium volume. v) to 1.15% (w / v), more preferably 0.5% (w / v) to 1.0% (w / v).

In addition, the NaCl content of the culture medium is 0.75% (w / v) to 2.75% (w / v), preferably 0.75% (w / v) to 1.75% (w / v), more preferably 0.75% (w / v) to 1.0% (w / v).

In addition, the extract obtained by culturing Penivacillus Lactus GD-A2 may be included as an active ingredient.

And, such an active ingredient may be included in 0.001 to 99.99% by weight, preferably 0.1 to 99% by weight, it is possible to appropriately adjust the content of the active ingredient according to the method of use and purpose of use.

On the other hand, the alginic acid oligosaccharide production method according to the present invention performs a culture step (S10) of culturing a novel microorganism Penicillus lautus GD-A2 to produce alginic acid degrading enzyme in a culture medium.

Then, after performing the step S10 performs the step of obtaining (S20) to obtain a culture after the culture, and extracting and purifying the alginic acid degrading enzyme by removing the cells from the culture to perform the extraction and purification step (S30) do.

At this time, the step S30 is to destroy the cells from the culture to remove, and extract the alginate degrading enzyme, a cell-free extract, it is preferable to perform at low temperature in order to prevent deactivation and denaturation of the alginate degrading enzyme.

The methods include mechanical grinding, ultrasonic grinding, self-extinguishing, Lysozyme treatment and freeze thawing.

In addition, since the cell-free extract from which the cells are removed from the culture contains the nucleic acid of the cells, the nucleic acid is removed through a purification process.

The methods include salting and dialysis, precipitation with organic solvents, adsorption, ion exchange chromatography, gel filtration, crystallization, and the like.

Furthermore, after the steps S20 and S30 are performed, the alginic acid oligosaccharide is reacted with algae or alginic acid to prepare alginic acid oligosaccharides, thereby preparing alginic acid oligosaccharides which are alginic acid low molecular weight compounds.

[ Example  1] Alginate Decomposition

First, in order to separate microorganisms capable of decomposing alginic acid, a tidal flat was taken from the coast of Yeonggwang-gun, Jeollanam-do, and the sample was sterilized diluent (NaCl 2.5g, KH ₂ PO ₄ 0.1g, FeSO ₄ · 7H ₂ O 0.05). g, KCl 0.05g, NH ₄ Cl 0.1g, distilled water 1L, pH 6.5) was diluted 1,000 times by dilution to prepare a dilution solution, 0.1mL of the diluent was plated on a multi-layer plate medium to confirm the alginate resolution. It was carried out by the method.

At this time, the multi-layered flat medium is composed of two layers of a lower medium and an upper medium, the lower medium is each component of NaCl 2.5% (w / v), KH ₂ PO ₄ 0.1% (w / v) to the total medium composition volume ), FeSO ₄ .7H ₂ O 0.05% (w / v), KCl 0.05% (w / v), NH ₄ Cl 0.1% (w / v) and agar 2.0% (w / v), pH 7, The upper medium was 1.0% (w / v) of sodium alginate and 2.0% (w / v) of agar, pH 6.5, relative to the total medium composition volume.

Here, 0.1 mL of the sample diluted 1,000-fold by serial dilution was plated on the multi-layered plate medium and incubated at 25 ° C. for 3 days. Among the bacterial colonies, strains having a colony size of 1 mm or more were collected using platinum teeth. The strain was purely separated by the method of separating the plate repeatedly on a new multilayer flat medium.

[ Example  2] Determination of alginic acid resolution of isolated strain

The strains with the highest alginate resolution were isolated from the pure isolates. Alginate resolution was determined by plate assay and DNS method.

That is, each strain isolated from pure PYS medium (peptone 0.5% (w / v), yeast extract 0.3% (w / v), sodium alginate 0.5% (w / v), NaCl 1.0% (w / v), pH 6.5), shaking culture for 30 hours at 30 ℃ 180rpm conditions after the primary confirmation of alginic acid resolution was performed by plate assay.

Plate assay was performed by dropping 30 μL of strain cultured in PS agar medium (peptone 0.5% (w / v), sodium alginate 1.0% (w / v), agar 1.5% (w / v)) at 25 ° C for 2 days. After stationary culture, 10% CPC (cetylpyridinium chloride monohydrate) was administered to the medium in which the strains were grown so that the medium was submerged. After 10 minutes, the alginate degrading enzyme activity was confirmed. As a result, 15 strains isolated from the cells were 1 It was selected as the tea strain.

[ Example  3] Determination of alginic acid resolution of isolated strains

The selected primary strains identified by the activity of alginate degrading enzyme were PYS medium (peptone 0.5% (w / v), yeast extract 0.3% (w / v), sodium alginate 0.5% (w / v), NaCl 1.0%). (w / v), pH 6.5) was analyzed by measuring the reducing sugar production ability by time period while shaking culture at 180 ℃ shaking conditions at 30 ℃.

The DNS method was used to measure the reducing sugar, a product of the enzymatic reaction, and the experimental method was performed as follows.

First, the culture supernatant was centrifuged at 14,000 × g and 4 ° C. for 5 minutes to obtain a separated supernatant. The obtained supernatant was used as a coenzyme solution, and 1.0 mL of a substrate solution containing 1.0% sodium alginate. After mixing 500 μL of the coenzyme solution, the reaction mixture was reacted for 30 minutes at a temperature of 180 rpm at 30 ° C. in a thermostat, and then the reaction solution was centrifuged at 14,000 × g and 4 ° C. for 5 minutes, and 500 μL of the supernatant was separated. 2 mL of DNS solution was added thereto, followed by heating for 10 minutes, and the absorbance was measured at 540 nm.

Reducing sugars were quantified using standard calibration curves prepared with mannuronic acid, and the reducing sugars were considered to be proportional to the alginate degrading enzyme activity.

As a result, GD-A2 strain was finally selected as the strain having the most reducing sugar producing ability, that is, the alginate degrading ability, which is shown in FIG. 2.

[ Example  4] Alginate Degradation New Microorganism GD Of A-A2

(1) Sequence analysis of selected antagonists

Strain identification for selected antagonistic microorganisms was carried out using 16S rDNA sequencing. 16S chromosomal DNA sequencing of the isolated strain was performed in the following manner.

First, the isolated strains were inoculated in Nutrient Broth Aga r (Difco) medium, incubated at 28 ° C. for 2 days, and then extracted with chromosomal DNA using a modified method of Benzyl chloride method, which was used for 16S rRNA sequencing. A primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3 ') and 1492R (5'-GGTTACCTTGTTACGACTT-3') primers were used to amplify by PCR (Polymerase Chain Reaction) under the following conditions.

PCR  Condition

Then, the purified 16S rDNA PCR Product Purification Kit (Qiagen) was purified, and the purified 1368 bp 16S chromosomal DNA was analyzed using a Genetic analyzer 310A (Applied Biosystems), the base sequence of 16S rDNA The analysis results of (1368 bp) are shown in FIG. 3.

In addition, a homology search was performed using the BLASTIN program as shown in FIGS. 4A and 4B as compared to chromosomal RNA sequencing of GENEBANK and Ribosomal Database Project II (RDPII).

(2) Analysis of fatty acid composition of selected antagonists

In the case of higher organisms, since the cell fatty acid composition is simple and has the same components, it is difficult to use as a classification index, but in the case of microorganisms, the cell fatty acid composition is used as a useful information in terms of chemical taxonomy.

First, the isolated strains were inoculated in TSA (Trypticase Soy Broth Agar, Difco) medium, and then cultured at 28 ° C. for 3 days, and 50 mg (wet weight) of the cells were subjected to the methyl fatty acid methyl esterification and extraction by Ikemoto & Miyagawa method. .

And, it was analyzed by 6890N Gas Chromatograph (Agilent Technologies) using a Microbial Identification System (MIDI; Microbial ID, Inc., Newark, Del., USA), and the analysis results are shown in FIG.

(3) Strain Identification Results of Selected Antagonists

A molecular phylogenetic based on 16S rDNA base sequence of the selected microbe antagonistic results, as shown in Figure 5, Penny Bacillus (Paenibacillus) as a strain belonging to the phylogenetic group comprising the genus species, Paenibacillus lautus JCM 9073 ^T (AB073188) was found to have a 99.56% flexibility.

Therefore, the selected strains were identified as Paenibacillus lautus , and the alginate-degrading novel microorganism of the present invention was identified as Penivacillus lautus GD-A2 ( Paenibacillus lautus). GD-A2) and was deposited on August 21, 2012 with the deposit number of KFCC 11538P to the Korean Culture Center of Microorganisms affiliated with the Korean Federation of Culture Collections.

[ Example  5] Penny Bacillus Lautus GD Of Al-A2 Production Alginate Degrading Enzyme

(1) Experimental conditions

In order to confirm the growth degree according to the culture conditions of the identified Penivacillus Lautus GD-A2, that is, the strain growth ability and the enzyme activity according to the culture conditions of the alginate degrading enzyme produced by Penibacillus Lautus GD-A2, GD-A2 was incubated. The composition of the culture medium for cultivation was basically 0.1% (w / v) of KH ₂ PO _4, 0.05% (w / v) of FeSO ₄ · 7H ₂ O, 0.05% (w / v) of KCl and 2.0% (w of agar). / v) and the addition of nitrogen sources (yeast extract, peptone, isolated soy protein, urea and (NH ₄ ) ₂ SO ₄ ), sodium alginate and NaCl, depending on what is to be confirmed through culture or The content was adjusted.

Specifically, the optimum medium composition was confirmed by confirming the strain growth ability and enzyme activity according to the nitrogen source in relation to the culture medium of the strain, and the optimum culture time was confirmed by checking the strain growth ability and enzyme activity according to the culture time, alginic acid concentration and NaCl The optimum alginic acid content and the optimal NaCl content were confirmed by confirming the strain growth ability and enzyme activity according to the concentration. In addition, by checking the enzyme activity according to the reaction temperature and the reaction pH to determine the optimum reaction temperature and reaction pH. The following experiments were performed three times each, and the average value of each result was used.

(2) Culture medium  According to the nitrogen source Penny Bacillus Lautus GD Strains of -A2 Proliferative capacity  And resolution of alginic acid resolution

Phenibacillus Lactus GD-A2 was added to the basal medium composition consisting of 0.1% (w / v) KH ₂ PO _4, 0.05% (w / v) FeSO ₄ .7H ₂ O and 0.05% (w / v) KCl. NaCl 1.5% (w / v) and sodium alginate 1.0% (w / v) and a nitrogen source (pH 6.5) was inoculated and incubated respectively.

Nitrogen sources include yeast extract 0.3% (w / v), peptone 0.5% (w / v), isolated soy protein 0.5% (w / v), urea 0.5% (w / v) and (NH ₄ ) ₂ SO ₄ 0.5% (w / v) was added.

In addition, the cultivation of Penivacillus Lautus GD-A2 was performed by shaking culture for 2 days at 30 ° C. and 180 rpm using a thermostat.

The strain propagation capacity was determined by measuring the OD value at 600 nm, and is shown in FIG. 7, and the alginic acid resolution was measured by the DNS method of Example 3, after measuring the reducing sugar production capacity, and then the optimum activity was 100%. Relative activity (%) is shown in FIG. 8.

As a result, the yeast extract was remarkably excellent in the growth ability of the strain, and thus the optimum nitrogen source for the cultivation of Penivacillus lautus GD-A2 was confirmed to be the yeast extract.

Also, peptone was the most excellent in the alginate degradation associated with enzyme activity, and yeast extract was somewhat lower than peptone, but the enzyme activity was similar to that of peptone, and that of other inorganic nitrogen sources was relatively low. It became.

If so, in the process of preparing a reaction solution, that is, a biocatalyst using Penibacillus Lautus GD-A2, it is preferable to add a yeast extract as a nitrogen source to the culture medium for strain propagation of Penivacillus Lautus GD-A2. It was evaluated that it is preferable to add peptone as a nitrogen source to the culture medium to be used as the final biocatalyst after strain propagation.

(3) according to the incubation time Penny Bacillus Lautus GD Strains of -A2 Proliferative capacity  And resolution of alginic acid resolution

Penivacillus Lactose GD-A2 was 0.1% (w / v) KH ₂ PO _4, 0.05% (w / v) FeSO ₄ .7H ₂ O, 0.05% (w / v) KCl, 1.5% (w / v) NaCl. ) And 0.5% (w / v) of peptone and 0.5% (w / v) of yeast extract and 0.3% (w / v) of yeast extract, respectively, in the medium composition composed of 1.0% (w / v) and sodium alginate. The culture was inoculated in the added medium (pH 6.5).

Cultivation of Penivacillus Lactus GD-A2 was performed by shaking culture for 2 days at a temperature of 180 rpm at 30 ° C using a thermostat.

The strain propagation capacity was measured by measuring the OD value at 600 nm and is shown in FIG. 9, and the alginic acid resolution was measured by the DNS method of Example 3, after measuring the reducing sugar production capacity, and then the optimum activity was 100%. Relative activity (%) is shown in FIG. 10.

Regarding the growth ability and alginic acid resolution of the strain, the group added with both peptone and yeast extract (PSY) was superior to the group added with only peptone and sodium alginate (PSY). It was evaluated that adding together was preferable. These results were interpreted to be due to the growth of the microorganism smoothly by the addition of yeast extract in the growth stage of the microorganism.

In addition, in relation to the incubation time, the growth of the strain was significantly increased up to 2 days after inoculation, but was found to decrease slightly on the third day, the optimum incubation time was confirmed to be 2 days. In addition, regarding the alginic acid resolution, the highest activity was shown on the second day, and even after that, the activity change was maintained without much occurrence. In addition, in the group to which only peptone was added, the number of cells was decreased on the second day of culture compared to the first day of culture, but the activity was significantly increased.

From the above results, the maximum reported alginate degradation activity was 144 hours in the case of other previously reported strains, whereas the penibacilli lautus GD-A2 improved cell growth and alginate resolution even when cultured for a short time. Since its activity is maintained, it is expected to be applicable to various industrial fields.

In addition, from the above results, in terms of cell growth rate and alginic acid resolution, it was expected to be most preferable to incubate up to 2 days in the group containing both peptone and yeast extract (PSY).

(4) according to the alginic acid content Penny Bacillus Lautus GD Strains of -A2 Proliferative capacity  And resolution of alginic acid resolution

Penivacillus Lactose GD-A2 was 0.1% (w / v) KH ₂ PO _4, 0.05% (w / v) FeSO ₄ .7H ₂ O, 0.05% (w / v) KCl, 1.5% (w / v) NaCl. ) Was inoculated in a medium (pH 6.5) added with a controlled content of sodium alginate in a medium composition consisting of peptone 0.5% (w / v) and yeast extract 0.3% (w / v).

Cultivation of Penivacillus Lactus GD-A2 was performed by shaking culture for 2 days at a temperature of 180 rpm at 30 ° C using a thermostat.

Determination of strain propagation ability was performed in a method of measuring the OD value at 600 nm, and is shown in FIG. 11. Relative activity (%) is shown in FIG. 12.

As indicated above, in relation to the growth ability and alginic acid resolution of the cells, it was confirmed that the growth of the cells was smooth when the concentration of sodium alginate was 0.1% (w / v) or more. In addition, with respect to the alginic acid resolution, it was gradually increased until the alginic acid concentration was 1.0% (w / v), and it was confirmed that the alginic acid concentration was 0.5% (w / v), and the alginic acid concentration was 1.5% (w). / v) was found to decrease rather than activity.

In addition, even when no alginic acid was added, it showed a certain degree of activity, and it was confirmed that a certain amount of alginic acid degrading enzyme was expressed even when culturing Penivacillus lautus GD-A2 in a medium without adding alginic acid. In addition, even when comparing the enzyme activity per unit cell by dividing the enzyme activity to cell growth, it was confirmed that the enzyme activity was the best when the alginic acid concentration was 0.5% (w / v).

(5) NaCl  According to the content Penny Bacillus Lautus GD Strains of -A2 Proliferative capacity  And resolution of alginic acid resolution

Penivacillus Lactose GD-A2 was 0.1% (w / v) KH ₂ PO ₄ , FeSO ₄ .7H ₂ O 0.05% (w / v), KCl 0.05% (w / v), sodium alginate 1.0% (w / v), NaCl content was adjusted from 0% (w / v) to 3% (w / v) in the medium composition consisting of peptone 0.5% (w / v) and yeast extract 0.3% (w / v) The culture was inoculated in the added medium (pH 6.5).

Cultivation of Penivacillus Lactus GD-A2 was performed by shaking culture for 2 days at a temperature of 180 rpm at 30 ° C using a thermostat.

Determination of strain propagation ability was performed in a method of measuring the OD value at 600 nm, and is shown in FIG. 13, and the alginate resolution was measured for each reducing sugar generation ability (g / L) by the DNS method of Example 3, and the activity of the optimum conditions was determined. At 100%, relative activity (%) is shown in FIG. 14.

In addition, in order to confirm the alginate resolution per cell, the measured amount of reducing sugar produced was divided by the OD600 value.

Regarding the proliferation ability of the cells, it was confirmed that the NaCl content is optimal at 1.0% (w / v), and also excellent at 0.5 (w / v) and 1.5% (w / v).

In addition, with respect to the alginic acid resolution, it was confirmed that the reduced sugar equivalent and the enzyme activity per unit cell (Reduced sugar per Biomass) produced when the NaCl content is 1.0% (w / v). Therefore, it was confirmed that the optimal NaCl content for the preparation of the biocatalyst through the cultivation of the Penivacillus Lautus GD-A2 was 1.0% (w / v).

(6) Penny Bacillus Lautus GD Degradation of Alginate with Temperatures of Alginate Degrading Enzyme Produced by A-A2

Penivacillus Lactose GD-A2 was 0.1% (w / v) KH ₂ PO _4, 0.05% (w / v) FeSO ₄ .7H ₂ O, 0.05% (w / v) KCl, 0.5% (w / v) sodium alginate. v), incubated in a medium (pH 7) consisting of NaCl 1.0% (w / v), peptone 0.5% (w / v) and yeast extract 0.3% (w / v) content.

Cultivation of Penivacillus Lactus GD-A2 was performed by shaking culture for 2 days at 25 ° C. and 150 rpm using a thermostat.

Each reducing sugar production capacity (g / L) was measured by the DNS method of Example 3 while adjusting the reaction temperature to 20 ℃ to 60 ℃, the relative activity (Relative activity (%)) to the optimum activity of 100% 15 is shown.

The alginic acid resolution of the biocatalyst solution was found to show the highest value at 40 ° C. Therefore, it was confirmed that the optimum reaction temperature of the biocatalyst prepared using the Penivacillus Lautus GD-A2 was 40 ° C.

(7) Penny Bacillus Lautus GD Of alginate degrading enzymes pH Resolution of alginic acid according to

Penivacillus Lactose GD-A2 was 0.1% (w / v) KH ₂ PO _4, 0.05% (w / v) FeSO ₄ .7H ₂ O, 0.05% (w / v) KCl, 0.5% (w / v) sodium alginate. v), incubated in a medium (pH 7) consisting of NaCl 1.0% (w / v), peptone 0.5% (w / v) and yeast extract 0.3% (w / v) content.

Cultivation of Penivacillus Lactus GD-A2 was performed by shaking culture for 2 days at 25 ° C. and 150 rpm using a thermostat.

In addition, each reducing sugar generating ability (g / L) was measured by the DNS method of Example 3 while adjusting the reaction pH at 3 to 10, and the relative activity (Relative activity (%)) was set to 100% of the optimum conditions. 16 is shown.

Specifically, the pH is adjusted in the reaction solution 200mM sodium acetate buffer (pH 3 to pH 6), 40mM sodium phosphate buffer (pH 6 to pH 8) and 100 mM glycine- NaOH buffer (pH 8 to pH 11) was used.

Alginate resolution according to the pH showed the highest value at pH 6.5, it was confirmed that the optimum pH of the alginic acid degrading enzyme produced by Penibacillus Lautus GD-A2 is 6.5.

In order to check the error according to the type of buffer solution, again, 300 mM GTA buffer solution (3,3-dimehtylglutarid acid, Tris (hydrosymethyl) aminomethane, 2-amino-2-methyl-1,3-propanediol, pH 3 to pH 10 The same result was confirmed also in the result measured using). Under acidic or alkaline conditions based on the optimum pH of pH 6.5, the alginic acid resolution was lowered as the acidic or alkaline properties became stronger.

Therefore, it was confirmed that the optimum reaction pH of the biocatalyst prepared using Penivacillus Lautus GD-A2 was pH 6.5.

In addition, by using the biocatalyst prepared from the culture solution of Penivacillus lautus GD-A2 (supernatant removed from the culture medium), the degradation activity of the polysaccharides such as starch, carrageenan, pectin, and agar was measured. It was confirmed that the decomposing activity was not observed with respect to the alginic acid, and that the enzyme produced by the penivacillus lautus GD-A2 was alginate degrading enzyme.

[ Example  6] Penny Bacillus Lautus GD Alginate Oligosaccharides Using A-A2 Culture

Penivacillus Lactose GD-A2 was 0.1% (w / v) KH ₂ PO _4, 0.05% (w / v) FeSO ₄ .7H ₂ O, 0.05% (w / v) KCl, 0.5% (w / v) sodium alginate. v) pH 7 and 30 ° C. in a medium consisting of NaCl 1.0% (w / v), peptone 0.5% (w / v) and yeast extract 0.3% (w / v) (culture conditions are 30 ° C., The optimum conditions for enzyme activity were shaken for 2 days at 40 rpm) at 180 rpm, and the cultured shaker was centrifuged at 8,000 rpm for 30 minutes to obtain a supernatant.

Then, the supernatant was added to the aqueous sodium alginate solution diluted in 0.1%, and the alginate oligosaccharides produced at different times were confirmed by TLC while incubated at 40 ° C.

At this time, sucrose and maltodextrin were used as standard markers of TLC, and the developing solvent was a mixture of 1-propanol, nitromethane and water (5: 3: 2, v / v / v).

Figure 17 is a TLC photograph confirming the production of alginic acid oligosaccharides after treatment of the supernatant obtained after centrifugation of the penivacillus lautus GD-A2 culture in the aqueous sodium alginate solution for food supplements, the leftmost of the photograph is a size marker, the sequence therefrom As long as the decomposition reaction time is a sample, it shows excellent production of various types of alginic acid oligosaccharide according to the reaction time.

As a result, as can be seen from the above results, the present invention can be produced alginate degrading enzyme that can produce alginic acid oligosaccharides by digesting alginic acid alginate, alginic acid degrading enzyme from the penivacillus lautus GD-A2 Since the optimum conditions for producing the reaction conditions and the optimum conditions of the alginic acid degrading enzyme have been confirmed, the Penivacillus Lautus GD-A2 may be applied to various industrial fields, including industries related to pharmaceuticals and functional foods, and bioenergy related industries. It is expected.

Claims (9)

Novel microorganism Paenibacillus lautus GD-A2 (Accession No. KFCC 11538P) producing alginic acid degrading enzyme.
At least one selected from the group consisting of a culture medium or a concentrate of the new microorganism Paenibacillus lautus GD-A2 (Accession No. KFCC 11538P) that produces alginic acid degrading enzymes in a culture medium. Biocatalyst for decomposing alginic acid.
The method of claim 2,
The culture is a biocatalyst for degrading alginic acid, characterized in that the cells are removed.
The method of claim 2,
The culturing of the culture is a biocatalyst for degrading alginic acid, characterized in that at least one of yeast extract and peptone is added to the culture medium as a nitrogen source.
5. The method of claim 4,
The yeast extract is first added to the culture medium for the growth of the strain, the peptone is added to the culture medium to be used as a biocatalyst after the growth of the strain, characterized in that the biocatalyst for degradation of alginic acid.
The method of claim 2,
The culture of the culture is added to the culture medium sodium alginate in the range of 0.1% (w / v) to 1.5% (w / v), NaCl is added to 2.0% (w / v) or less (excluding 0) Biocatalyst for degrading alginic acid, characterized in that.
The method of claim 2,
The culture of the culture, the reaction temperature is in the range of 20 ℃ to 60 ℃, the incubation time is 36 hours or more, the reaction pH is a biocatalyst for degrading alginic acid, characterized in that carried out in the range of 4 to 8.
A culture step of culturing the new microorganism Paenibacillus lautus GD-A2 (Accession No. KFCC 11538P) producing alginic acid degrading enzyme in a culture medium;
Obtaining a culture after the culture; And
Reacting the culture with algae or alginic acid to prepare alginic acid oligosaccharides.
A culture step of culturing the new microorganism Paenibacillus lautus GD-A2 (Accession No. KFCC 11538P) producing alginic acid degrading enzyme in a culture medium;
Obtaining step of obtaining a culture after the culture;
An extraction and purification step of removing the cells from the culture to extract alginate degrading enzyme and then purifying; And
Reacting the alginic acid degrading enzyme with algae or alginic acid to prepare alginic acid oligosaccharide; Alginic acid oligosaccharide manufacturing method comprising a.

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