KR100664582B1 - Chitinase-producing trichoderma viride ????-?41 strain, chitinases purified therefrom and a method for producing ?-acetylglucosamine using the chitinases - Google Patents

Abstract

The present invention is a Trichoderma viride AGCC-M41 strain that produces exo-chitinase and endo-chitinase at the same time, exo-kitty separated and purified from the strain the better and endo- relates to a method of producing acetylglucosamine (N -acetylgucosamine) - kitty better claim, and N using a first come the kitty. The chitinase produced from the Trichoderma viride AGCC-M41 strain of the present invention has a high yield of enzyme and effectively converts chitin into N -acetylglucosamine, so that N − by enzymatic hydrolysis of chitin. It can be usefully used for the production of acetylglucosamine.

Description

CHITINASE-PRODUCING TRICHODERMA VIRIDE AVCC-M41 STRAIN, CHITINASES PURIFIED THEREFROM AND A METHOD FOR PRODUCING N-ACETYLGLUCOSAMINE USING THE CHITINASES}             

1 shows the productivity of exo-chitinases according to the incubation time of Trichoderma viride AGCC-M41 strain,

-■-: exo-chitinase activity (U / ml),-▲-: dry cell weight (g / l),

-◆-: pH

FIG. 2 is a result of purifying chitinase fractions showing endo-chitinase and exo-chitinase activity from phenylo-Toyopearl column chromatography from trichoderma viride AGCC-M41 strain-derived chitinase enzyme.

FIG. 3 shows Q-Sepharose column chromatography of endo-chitinase and exo-chitinase-containing chitinase active fractions purified from Trichoderma viride AGCC-M41 strain by phenyl-Toyopearl column chromatography in FIG. 2 . Purification with exo-chitinase and endo-chitinase is the result of separation,

Figure 4 is the result of measuring the molecular weight of exo-chitinase and endo-chitinase derived from Trichoderma viride AGCC-M41 strain by SDS-PAGE,

M: protein standard molecular weight size marker,

Lane 1: endo-chitinase fraction, lane 2: exo-chitinase fraction

5 is a result of TLC analysis of the hydrolysis products of chitin by exo-chitinase and endo-chitinase derived from Trichoderma viride AGCC-M41 strain (wherein N1 to N4 are monosaccharides of N-acetylglucosamine to Tetrasaccharides)

Figure 6 shows the relative activity (▼) and stability (○) of the exo- chitinase derived from Trichoderma viride AGCC-M41 strain at various pH conditions,

7 is a graph showing the relative activity (▼) and stability (○) of the exo-chitinase derived from Trichoderma viride AGCC-M41 strain at various temperature conditions,

8 shows the relative activity (▼) and stability (○) of endo-chitinase derived from Trichoderma viride AGCC-M41 strain at various pH conditions,

Figure 9 is a graph showing the relative activity (▼) and stability (○) of endo-chitinase derived from Trichoderma viride AGCC-M41 strain at various temperature conditions.

The present invention is Trichoderma viride AGCC-M41 strain that produces exo-chitinase and endo-chitinase at the same time, exo-kitty separated and purified from the strain the better and endo- relates to a method of producing acetylglucosamine (N -acetylgucosamine) - kitty better claim, and N using a first come the kitty.

Chitin fiber, and then the rich raw material (biomass) is N - acetylglucosamine (N -acetylgucosamine, NAG) to the basic unit as a molecular weight polysaccharide linked by β-1,4 bonds is about 1 × 10 ⁶ Da or so. Chitin is a major component of the shells of sea crustaceans such as crabs, krill, lobsters, and insects such as spiders, ants, and grasshoppers.

Such chitin is an important natural resource recently, its application is gradually increasing. In particular, D-glucosamine (DGA) produced by hydrolysis of chitin and NAG, an acetylated form of DGA, not only have excellent effects on degenerative arthritis, but also promote anti-inflammatory, cardioprotective, hepatoprotective and mobilization It is known to be effective. NAG, a monomer of chitin, acts as a major component to make glycoproteins such as connective tissues, tendons and cartilage tissues in vivo. Glycoprotein containing NAG is a binding material that connects cells to cells and maintains complete tissue or organs. Is essential. In addition, NAG is a major component of the mucous membrane of the digestive tract and helps treat ulcers in the digestive tract, and it has been found that NAG is used in mucosal cells much faster than DGA in the body. In view of these facts, NAG is an improved biological material than DGA and its utility value is very high.

As described above, NAG having a large useful value as a medicinal and functional food material is produced by acid hydrolysis which hydrolyzes chitin at 6 to 12 N HCl at 60 to 80 ° C. In this acid hydrolysis, chitin is decomposed into monosaccharides and deacetylation occurs to simultaneously generate DGA and NAG. The relative ratio of DGA and NAG may vary depending on acid hydrolysis conditions. However, when the acid concentration and temperature are lowered to reduce deacetylation, there is a problem in that the efficiency of NAG obtained from chitin is lowered. In addition, the conventional NAG manufacturing process by acid hydrolysis method has a high concentration of acid, there is a problem such as corrosion of the device, risk of operation, generation of impurities harmful to the human body and the need for a complex purification process for removal thereof.

As an alternative to solve this problem of acid hydrolysis, a method of producing NAG using an enzyme has been proposed. Enzymatic hydrolysis converts chitin into monomeric NAG by synergistic hydrolysis of endo-chitinases and exo-chitinases, chitinases. This enzymatic hydrolysis method has the advantage that the purification process is very simple and the NAG can be produced with high yield because no deacetylation of NAG, a chitin decomposition product, and no impurities are generated. However, in order to produce NAG from chitin by enzymatic hydrolysis, exo-chitinase which completely hydrolyzes chitin oligosaccharides thus formed with NAG, together with endo-chitinase which decomposes crystalline chitin into chitin oligosaccharides. need. However, the technology for producing these enzymes efficiently and economically producing NAG using them has not yet been applied industrially.

Accordingly, the present inventors have made extensive efforts to efficiently produce NAG by enzymatic hydrolysis of chitin, and as a result, the enzyme activity is higher than other chitinases previously reported from a novel mutant strain whose enzyme activity is improved by artificial mutation. The present invention was completed by confirming that it is possible to produce chitinase, which is extremely high and excellent in NAG production capacity, and developing a method of producing high purity NAG using the chitinase produced from the strain.

Accordingly, it is an object of the present invention to provide a Trichoderma viride strain that produces exo-chitinases and endo-chitinases simultaneously.

Another object of the present invention is to provide an exo-chitinase and an endo-chitinase isolated and purified from the Trichoderma viride strain.

Still another object of the present invention is to provide a method for producing NAG using the exo-chitinases and endo chitinases.

In order to achieve the above object, the present invention provides a novel Trichoderma Biride AGCC-M41 strain that produces exo-chitinases and endo-chitinases at the same time.

Enzymes involved in the production of NAG by decomposing chitin are endo-chitinase which decomposes chitin into chitin oligosaccharides and exo-chitinase which completely decomposes chitin oligosaccharide into NAG. Both of these enzymes are important for the production of NAG, but since the determination of NAG production rate is the latter titer of exo-chitinase, in the present invention, the criteria for selection and improvement of the chitinase producing strain for NAG production are used. As an indicator, the titer of exo-chitinase is used.

A total of 500 microorganisms, including microorganisms obtained from domestic and overseas strain storage and microorganisms collected from nature, are selected for strains that have high exo-chitinase enzymatic activity and produce large amounts of NAG when decomposing chitin. This confirms that Trichoderma viride AGCC-27 strain exhibits high exo-chitinase activity and NAG productivity (see Table 1 ).

However, the Trichoderma viride AGCC-27 strain is relatively high in exo-chitinase activity compared to other strains, but still lacks in activity for industrial purposes. In the present invention, N -methyl- N' -nitro- N '' -nitroso- to increase the exo-chitinase productivity of the Trichoderma viride AGCC-27 strain selected as a chitinase producing strain for NAG production. Random mutations with guanidine ( N- methyl- N'- nitro- N ''-nitroso-guanidine, NTG) are performed. Specifically, Trichoderma viride AGCC-27 is treated with NTG and plated on chitin agar medium, and mutant strains having increased exo-chitinase productivity are first selected by observing the size of chitin cleavage rings of colonies. These strains are then cultured in chitin liquid medium to compare exo-chitinase productivity to select mutant strains that exhibit the highest exo-chitinase productivity among the primary mutants derived from the Trichoderma viride AGCC-27 strain. . In the present invention, a strain showing an exo-chitinase productivity of about 8.3 U / ml is selected in this manner and named AGCC-M8 (see Table 2 ).

Secondary NTG mutations were carried out in the same manner as described above for the primary strains selected for enzyme activity improvement. Tricoderma Biride Secondary mutations using AGCC-M8 resulted in the selection of a strain of exo-chitinase productivity of 18.5 U / ml, with a 2.2-fold improvement in the kinase productivity of the parent strain Trichoderma biride AGCC-M8, which was AGCC-M41. (See Table 3 ).

Trichoderma viride whose chitinase activity is the parent strain through the second NTG mutation process as described above. Trichoderma viride, about 53-fold more than AGCC-27 (0.35 U / mL) The AGCC-M41 strain is finally selected as chitinase high productivity strain. This enzymatic activity is very high activity that has not been reported so far, the strain was deposited on December 1, 2004 to the Korea Biotechnology Research Institute affiliated Gene Bank (Accession No .: KCTC 10734BP).

In order to obtain the maximum chitinase productivity by culturing the Trichoderma viride AGCC-M41 strain selected as the high productivity of chitinase as described above, 0.1 to 5.0% of chitin, corn-steeped powder ) 0.1-5.0%, ammonium sulfate 0.05-2.0%, potassium phosphate 0.1-3.0%, calcium chloride 0.01-1.0%, magnesium sulfate 0.01-1.0%, triton X-100 0.05-3.0% and glucose 0.01-2.0% It is preferable to incubate in the medium for 20 to 30 ° C., initial pH of 2.5 to 5.0, aeration condition of 0.1 to 2.0 vvm, agitation speed of 100 to 500 rpm, spawn inoculation amount of 0.5 to 10.0%, and culture time of 72 to 168 hours. When culturing Trichoderma viride AGCC-M41 under the above conditions, the cell mass was increased as the incubation time elapsed, and exo-chitinase production was 25 U / ml at 132 hours of cultivation (see FIG. 1 ). .

The present invention also provides a method for producing exo-chitinases and endo-chitinases from the Trichoderma viride AGCC-M41 strain.

To this end, in the present invention, first, a chitinase enzyme capable of producing NAG from Trichoderma viride AGCC-M41 strain is prepared.

First, Tricoderma viride AGCC-M41 strain is inoculated in potato dextrose agar medium (2% glucose, 20% potato extract, 1.5% agar, pH 7.0) and incubated for 1 to 4 days at 20 to 30 ° C. The cultured mycelium block (width × length = 0.5 × 0.5 cm) is inoculated into 100 to 500 ml of chitin liquid medium having the optimum medium composition established above, and then for 1 to 4 days at 20 to 30 ° C. and 100 to 300 rpm. Shake culture to obtain a seed culture solution.

The fermentation broth of the same composition is put into the fermentor and sterilized, and the seed culture solution cultured above is inoculated in an amount of 0.5 to 10.0%. For example, in the case of a 500 L fermenter (culture volume 300 L), the culture temperature is 20 to 30 ° C., an initial pH of 2.5 to 5.0, aeration condition of 0.1 to 2.0 vvm, agitation speed of 100 to 500 rpm, and inoculation amount of 1.5 to 30 L. Incubate for 3-7 days. At this time, it is preferable to properly adjust the stirring speed and aeration conditions according to the volume of the fermenter. Normally cultured exo-chitinase titers are 20-30 U / mL.

The mycelium is removed from the culture broth obtained by the above method by centrifugation or membrane filtration, and the enzyme is concentrated by ultrafiltration. The pore size of the membrane during membrane filtration is preferably 0.45 to 2.0 μm, and the molecular weight cut-off value during ultrafiltration is 5,000 to 30,000 Da. The enzyme concentrate is obtained by removing the media components in the culture solution through diafiltration from the obtained enzyme concentrate. In this case, as the dialysis buffer, sodium phosphate, citric acid, sodium phosphate or ultrapure distilled water may be used. The enzyme concentrate may be diluted to make a liquid enzyme or lyophilized to prepare a powdered enzyme. When purified normally, 10-15 U / mg of powdered enzyme can be obtained in an amount of 1.5-2.0 g per liter of culture. The chitinase enzymes purified from the culture medium of Tricoderma viride AGCC-M41 strain include endo-chitinases and exo-chitinases, and the content of endo-chitinases and exo-chitinases in the enzyme is 3.0 to 15.0% and 85.0 to 99.0%, respectively.

The method for separating and purifying exo-chitinase and endo-chitinase from the chitinase enzyme prepared as described above is as follows.

The chitinase enzyme obtained by the above-mentioned method was subjected to phenyl-Toyopearl column chromatography to separate fractions showing exo-chitinase activity, and the fractions were collected and subjected to Q-Sepharose column chromatography. Gual endo-chitinase active fractions are isolated (see FIGS. 2 and 3 ). The molecular weights of the isolated exo-chitinases and endo-chitinases were measured by SDS-PAGE. The exo-chitinases derived from the Tricoderma viride AGCC-M41 strain had a molecular weight of 65,000 Da and endo-kinase. Is found to have a molecular weight of 42,000 Da (see FIG . 4 ).

The enzymatic properties of the exo-chitinase and endo chitinase isolated and purified as described above can be investigated using swelled chitin as a substrate for enzymatic reaction. First, thin layer chromatography (TLC) of the hydrolysis products of chitin by exo-chitinase derived from Trichoderma viride AGCC-M41 strain revealed that only NAG was detected at both the beginning and the end of the reaction. N -acetyl-β-D-glucosaminidase ( N -acetyl-), wherein the exo-chitinase derived from the Trichoderma viride AGCC-M41 strain according to the present invention produces only NAG by cleaving chitin into exo-type. β-D-glucosaminidase) (see A of FIG. 5 ). As a result of analyzing the hydrolysis products of chitin by endo-chitinases in the same manner as described above, polysaccharides more than disaccharides were detected as well as monosaccharides, and endo-kitty from chitinase-producing strain Trichoderma viride AGCC-M41 strain of the present invention. It was confirmed that the naase was an endo-chitinase of endo type that randomly decomposed chitin to produce chitin oligosaccharides (see B of FIG. 5 ).

In addition, the optimum pH of the exo-chitinase derived from Trichoderma viride AGCC-M41 is 5.0, relatively stable in the range of pH 4.0 to 6.0 (see Fig. 6 ), the optimum temperature is 50 ℃, stable in the range 40 to 50 ℃ Enzyme activity is shown as (see FIG . 7 ). In addition, the optimal reaction pH of Trichoderma viride AGCC-M41-derived endo-chitinase is 5.0, relatively stable in the range of pH 4.0-7.0 (see FIG. 8 ), the optimum temperature is 45 ℃, in the range of 40-50 ℃ The enzyme activity was stably shown (see FIG. 9 ).

The present invention also provides a method for producing NAG using the exo-chitinases and endo chitinases.

According to the optimum fermentation conditions established as described above, a method for preparing NAG using a chitinase enzyme containing tricoderma viride AGCC-M41 derived exo-chitinase and endo-chitinase of the present invention is as follows. Includes:

1) swelling chitin to prepare an enzyme substrate;

2) hydrolyzing the swelling chitin obtained in step 1) with a chitinase enzyme;

3) vacuum concentrating the hydrolysis solution obtained in step 2);

4) decoloring and filtering the concentrate obtained in step 3);

5) crystallizing the NAG from the filtrate obtained in step 4); And

6) drying the NAG crystals obtained in step 5).

First, step 1) is a process of swelling chitin, adding 2 to 4 times the volume of 50 to 85%, preferably 85% phosphoric acid, and mixing well, followed by 10 to 100 rpm at room temperature for 4 to 12 hours. The chitin is swollen with stirring. After adding 5-20 times the volume of purified water to the swollen chitin and stirring at 10-100 rpm, the swollen chitin was washed several times with purified water to adjust the pH to 3.0-7.0 and then used as a substrate for enzymatic reaction.

Step 2) is a hydrolysis process, in which 1-10% (w / v) of swelling chitin, preferably 2-5% of swelling chitin, prepared in step 1) is dissolved in water to prepare a substrate solution. At this time, it is preferable to adjust pH to 3.0-6.0, Preferably it is 4.0-5.0. The substrate solution was transferred to a reactor equipped with an agitator and a thermostat, and chitinase derived from Trichoderma viride AGCC-M41 strain of 1 to 100 U, preferably 5 to 30 U, per chitin was added and the temperature was 30 to 30. Hydrolysis is carried out at 60 ° C., preferably at 40-50 ° C., for 24 to 72 hours. At this time, the chitinases used are chitinase enzymes containing exo- and endo-chitinases purified from the culture solution of Trichoderma viride AGCC-M41 strain. After the hydrolysis reaction, the enzyme and protein are recovered from the reaction solution by using an ultrafiltration membrane (molecular weight cut-off value: 8-30 kDa), and the hydrolysis solution is purified.

On the other hand, chitin undergoes about 10% deacetylation in the manufacturing process so that D-glucosamine residues exist in the chitin molecule. Therefore, in order to more effectively decompose D-glucosamine present in the chitin molecule in the acid hydrolysis step, it is preferable to add exo-chitosanase in an amount of 5 to 30 U per gram in addition to the chitinase. Additional addition of exo-chitosanase increases NAG productivity by 10-20% compared to the addition of chitinase alone (see Table 10 ).

Step 3) is a concentration step, wherein the hydrolyzate purified as described above is concentrated in vacuo until the total solid content reaches 40 to 60%. In order to prevent coloring by heating, vacuum concentration is preferably carried out at a low temperature or a thin film or centrifugal concentrator is used.

Step 4) is a decolorization and filtration step, and decolorization is carried out at a temperature of 30 to 60 ° C. by adding 1 to 10%, preferably 2 to 5%, of activated carbon to the concentrate obtained in step 3). At this time, when 0.1-1.0%, preferably 0.3-0.5% of diatomaceous earth is added, the decolorization effect will become more favorable. After decolorization is complete, activated carbon and diatomaceous earth are removed using a suitable filter.

Step 5) is a crystallization process. After the decolorization is completed, the reaction solution is left at a temperature of 10 ° C. or lower for at least 12 hours to generate NAG crystals, and the crystals are recovered by using a centrifuge or a filter. After recovering the crystals, the mother liquor may be concentrated to 50% of a solid content, and then left at a low temperature of 10 ° C. or lower for at least 12 hours to obtain secondary crystals. Small amounts of NAG crystals may be added to promote the formation of crystals or ethanol in a volume of 1 to 2 times the concentration.

Step 6) is a drying process, the NAG crystals obtained in step 5) are put into a vacuum dryer maintained at a temperature of 40 ℃ or less and dried for more than 12 hours.

Through the above steps, NAG is produced using trichoderma viride AGCC-M41 strain-derived chitinase according to the present invention to obtain NAG crystal powder having a purity of 95% or more in a 50 to 90% yield.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Selection of Chitinase Producing Strains

To select chitinase producing strains, we investigated the activity of chitinase from microorganisms collected from domestic and overseas strain archives. At this time, the endo-chitinase activity is determined by the amount of reducing sugars produced from the swelling chitin in the dinitro-salicylic acid (DNS) method (Miller. GL, Anal. Chem. 31: 426-428, 1959). It was measured as follows. First, 0.3 ml of endo-chitinase enzyme solution diluted appropriately to fall in the range of 550 nm wavelength (0.1 to 0.9) was added to 0.3 ml of 2% (w / v) swelling chitin (50 M sodium phosphate buffer, pH 5.0). After reacting at 50 ° C. for 30 minutes. After the reaction solution was centrifuged at 12,000 rpm and 4 ° C for 10 minutes to separate the supernatant, 0.3 ml of the supernatant was mixed well with 1 ml of DNS and heated at 100 ° C for 5 minutes. After cooling the reaction solution in cold water, the absorbance was measured at 550 nm to determine the absolute amount of NAG produced using a standard curve quantified by NAG, from which the enzyme titer was calculated. In this case, the endo-chitinase enzyme unit (1 U) was defined as the amount of enzyme that produces 1 μmole NAG in 1 minute.

In addition, exo-order to determine the activity better Kitty, ρ--nitro-phenyl-N-acetylglucosamine (ρ-nitro-phenyl- N -acetylglucosamine ) solution (1 mM, 50 mM Na- phosphate, pH 5.0) 0.2 After adding 0.2 ml of exo-chitinase enzyme solution diluted appropriately to fall within the range of 420 nm wavelength (0.1 to 0.5) to ㎖ and reacting at 50 ° C for 30 minutes, 1 ml of 0.1 M glycine-NaOH buffer solution (pH 10.5) was added. Was added to stop the enzyme reaction. The absorbance of the reaction solution was measured at 420 nm to determine the amount of p-nitrophenol liberated from the standard curve. Exo-chitinase enzyme unit (1 U) was defined as the amount of enzyme that produced 1 μmole of p-nitrophenol in 1 minute.

The enzyme titers measured as above are shown in Table 1 below.

As shown in Table 1 , seven strains were selected from 500 species of microorganisms having high chitinase activity and generating large amounts of NAG when decomposing chitin. Among these, Trichoderma viride AGCC-27 strain showed the highest chitinase activity and yield of NAG, and this strain was selected as a chitinase producing strain.

Example 2: Improvement of Trichoderma viride AGCC-27 Strain

Randomization using NTG ( N- methyl- N'- nitro- N '' -nitroso-guanidine) to enhance the kinase enzyme activity of Trichoderma viride AGCC-27 strain selected as chitinase producing strain for NAG production Mutations were performed. Trichoderma viride AGCC-27 culture solution was treated with NTG at a concentration of 0.5 mg / ml and plated on chitin agar medium (1.0% swelling chitin, 0.5% yeast extract, 0.2% potassium monophosphate, 0.2% ammonium sulfate, 1.5% agar). Then incubated for 5 days at 25 ℃. About 50 species of mutant strains with increased chitinase enzyme activity were selected by observing the size of the chitin cleavage ring formed from the colonies. These strains were then cultured in chitin liquid medium to compare chitinase enzyme activity.

As a result, as shown in Table 2 , among the first mutants derived from the Trichoderma viride AGCC-27 strain 8 mutants showed the highest chitinase productivity of about 8.3 U / ㎖ on the 7th day of culture It was named AGCC-M8.

Primary strain Tricoderma viride Secondary NTG mutations were performed using AGCC-M8. Secondary mutations and screening methods were performed in the same manner as the first mutation method, and about 50 species of chitin-degrading rings were selected in chitin agar medium, and these were incubated in chitin liquid medium to compare the enzyme activity of chitinase.

As a result, as shown in Table 3 , the strain was selected as the chitinase productivity of 18.5 U / ml about 2.2 times improved than the parent strain Trichoderma viride AGCC-M8, which was named AGCC-M41. Trichoderma viride through the second NTG mutation process Trichoderma viridede, approximately 53-fold higher in chitinase activity than AGCC-27 The AGCC-M41 strain was finally selected. The Trichoderma Biride The AGCC-M41 strain was deposited on December 1, 2004 to the Gene Bank of Korea Research Institute of Bioscience and Biotechnology (Accession No .: KCTC 10734BP).

Example 3: Optimization of fermentation conditions and preparation of chitinase enzyme

<3-1> Medium Composition Optimization

A medium composition optimization study for mass fermentation of Trichoderma viride AGCC-M41 strain finally selected as chitinase producing strain was performed. Culture experiments using medium by changing carbon source, nitrogen source, phosphoric acid source, trace element, etc. in the medium by type and concentration, high activity of chitinase, easy to industrialize in consideration of cost and supply of raw materials Fermentation medium conditions were determined. The composition of the finally confirmed medium is shown in Table 4 below.

<3-2> Fermentation Conditions Optimization

In order to increase the productivity of chitinase of Trichoderma viride AGCC-M41 strain, fermentation condition optimization study was performed based on the medium composition of Table 4 . As a result, the optimum fermentation conditions for the preparation of chitinase were 25 ℃, initial pH 3.2, aeration condition 0.5 vvm, agitation speed 120 rpm, spawn inoculation 15 L at 500 L fermenter scale (culture volume 300 L). The time was determined to be about 144 hours. As a result of culturing Trichoderma viride AGCC-M41 strain under the above conditions, as shown in FIG. 1 , the cell mass increased as the incubation time elapsed, and the exo-chitinase productivity reached 25 U / ml at 132 hours of culture. Maximum productivity was shown.

<3-3> Preparation of chitinase enzyme

260 ℓ culture medium of Trichoderma viride AGCC-M41 strain was removed by continuous centrifugation at 15,000 rpm to remove mycelium, and by primary membrane filtration (membrane pore 1.0 μm) and ultrafiltration (molecular weight cut-off value: 30 kDa). The culture supernatant was concentrated. Medium components were removed from the concentrate by diafiltration of about 25 times the volume of the concentrate using ultrapure purified water. The filtrate obtained therefrom was subjected to secondary membrane filtration (membrane pore 0.45 μm) for sterilization and finally freeze-dried to prepare a chitinase enzyme.

As a result, as shown in Table 5 above, 410 g of enzyme could be prepared from 260 L of culture, and the purification yield of enzyme from the culture was 92%. As described above, the enzyme kinases purified from the culture medium of the Trichoderma viride AGCC-M41 strain were endo-chitinase and exo-chitinase in a mixed form, and the exo-chitinase activity was 13.5 U / mg powder. -Chitinase activity was 195.08 U / mg powder.

Example 4: Properties of Chitinase

Exo-chitinases and endo-chitinases were purified from chitinase enzymes obtained by culturing the Trichoderma viride AGCC-M41 strain in Example 3, and their enzymatic properties were examined as follows. .

<4-1> Purification of exo-chitinases and endo-chitinases

The chitinase enzyme prepared in Example <3-3> was purified by column chromatography on phenyl-Toyopearl (Tocho Co., Japan). First, the enzyme powder was dissolved in 15 ml of 10 mM sodium phosphate buffer (pH 6.8) at a concentration of 1%. After 15 ml of phenyl-Toyopearl resin was charged to the column, the solution was eluted by back-cultivation using 10 mM sodium phosphate buffer containing 1.5 M ammonium sulfate as buffer A and 10 mM sodium phosphate buffer as buffer B. I was. The dissolution rate was fractionated by 4 mL at 60 mL per hour. FIG. 2 shows the results of purification of exo-chitinases and endocytase by phenyl-Toyopearl column chromatography. Most of the chitinase activity was observed in fractions 138 to 152, in which the endo-chitinase activity and the exo-chitinase activity were detected simultaneously.

Since endo-chitinase and exo-chitinase cannot be separated by the phenyl-toyopearl column chromatography method, in the present invention, fractions 138 to 152 having high chitinase activity in phenyl-toyopearl column chromatography ( Fractions having both endo-kinase and exo-chitinase enzymatic activity) were collected and dialyzed in 10 mM sodium phosphate buffer (pH 6.5) and filtered. The column was filled with 30 ml of Q-Separose (Q-Separose, Pharmacia, USA) resin, washed with the same buffer, and then loaded with the fractions 138-152. The protein adsorbed onto the Q-Sepharose column was eluted with a gradient of 0-1 M sodium chloride solution. At this time, the dissolution rate was fractionated by 4 ml at 1 ml per minute. Figure 3 shows the results of separation of endo-chitinase and exo-chitinase by Q-Sepharose column chromatography. Endo-chitinase activity was high in fractions 60 to 67, and exo-chitinase activity was high in fractions 81 to 85. As shown in FIG. 3 , exo-chitinase activity was simultaneously measured in the endo-chitinase fraction, which means that the endo-chitinase produced from the Trichoderma viride AGCC-M41 strain simultaneously had exo-chitinase activity. It suggests that there is. In addition, the endo-chitinase activity was also detected in the exo-chitinase fraction, which means that exo-chitinase decomposes swelling chitin, a substrate of endo-chitinase, by exo-cleavage. It is because it is generated. This presumption can be confirmed from the result of swelling chitin digestion of isolated endo-kinase and exo-chitinase ( FIG. 5 ). The exo-chitinase decomposed swelling chitin to form monosaccharides, and endo-kitinaa. Decomposing the swelling chitin to produce oligosaccharides more than disaccharides, including monosaccharides. From this, fractions 60 to 67 of the Q-Sepharose column were separated into endo-chitinase active fractions, and fractions 81 to 85 were separated into exo-chitinase active fractions and used in subsequent experiments.

<4-2> Molecular weight measurement

The exo-chitinase (fractions 81 to 85 of the Q-Sepharose column) and the endo-chitinase fraction (fractions 60 to 67 of the Q Sepharose column) purified in <4-1> were 12% polyacrylamide. The gel was loaded onto a polyacrylamaide gel and electrophoresed until the die ran out at 30 mA. The electrophoretic gel was stained with 0.05% Coomassie brilliant blue R-250 and then decolorized with a destaining solution. At this time, molecular weight was measured using protein size markers (protein size markers) having a range of 97,400 to 14,400 Da. As a result, it can be seen that the exo-chitinase derived from Trichoderma viride AGCC-M41 strain has a molecular weight of 65,000 Da and the endo-chitinase has a molecular weight of 42,000 Da ( FIG. 4 ).

Example 5: Properties of Chitinase Enzyme

<5-1> Preparation of swollen chitin

Enzymatic treatment with exo-chitinases requires pretreatment of hard chitin to produce swollen chitin. For this purpose, 40 ml of 85% phosphoric acid was added to 10 g of chitin and mixed well, and the chitin was swollen with stirring at 40 rpm for 12 hours at room temperature. 400 ml of distilled water was added to the swollen chitin and stirred at 40 rpm. The swollen chitin was washed several times with distilled water to adjust the pH to 4.0 and then used in subsequent experiments.

<5-2> Hydrolysis Product of Exo-Kittinase

100 U of exo-chitinase purified in Example <4-1> was added to 100 ml of the 2% swollen chitin solution, and the mixture was reacted at 40 ° C and pH 4.0. 0.5 and 15 hours after the start of the reaction, the reaction solution was collected and the reaction product was examined by TLC. TLC was developed fully using a mixed solvent of isopropanol: pyridine: acetic acid: water (10: 6: 6: 9) and then sprayed with a solution of 1% ninhydrin in saturated butanol and then 100 The color was developed until color appeared while being careful not to burn at ℃. The result was the same NAG only detected at both 0.5 and 15 hours, better kitty of the invention The producing strain Trichoderma irregularities having AGCC-M41 strain derived from exo-forth Kitty I cut produces only NAG chitin with exo-type N- Acetyl-β-glucosaminidase ( N- acetyl-β-glucosaminidase) to indicate that ( Fig. 5A ).

<5-3> Hydrolysis Product of Endo-Kitinase

To 100 mL of the 2% swollen chitin solution, 100 U of the endo-chitinase purified in Example <4-1> was added, and the mixture was reacted at 40 ° C and pH 4.0. 0.5 and 15 hours after the start of the reaction, the reaction solution was collected and the reaction product was examined by TLC. Then, as a result of inducing a color reaction in the same manner as in Example <5-2>, not only monosaccharides but polysaccharides or more were detected. This indicates that the endo-chitinase derived from the chitinase-producing strain Trichoderma viride AGCC-M41 strain of the present invention is an endo type endo-chitinase that randomly decomposes chitin to produce chitin oligosaccharides ( FIG. 5B ). .

<5-4> Effect of pH on Enzyme Activity

In order to investigate the effect of pH of exo-chitinase produced from Trichoderma viride AGCC-M41 strain, the enzyme activity was measured after variously adjusting the pH of swelling chitin as a substrate with 1 N hydrochloric acid or 1 N caustic soda. The optimum pH was determined, 100 mM sodium acetate buffer (pH 3-6), malate buffer (pH 6-7), Tris buffer (pH 7-9), glycine buffer (pH 9-11) The enzyme was added to a pH-adjusted solution and allowed to stand at 35 ° C. for 1 hour to measure pH stability by measuring residual enzyme activity. As a result, the optimum pH of the exo-chitinase derived from Trichoderma viride AGCC-M41 strain was 5.0, and appeared to be relatively stable in the pH range 4-6 ( Fig. 6 ). In order to investigate the effect of pH of endo-chitinase produced from Trichoderma viride AGCC-M41 strain, enzyme activity was measured using swelling chitin as a substrate in the same manner as above, and Trichoderma viride AGCC-M41 The optimum pH of the strain-derived endo-chitinase was 5.0 and was found to be relatively stable in the pH range of 4-7 ( FIG. 8 ).

<5-4> Effect of Temperature on Enzyme Activity

Enzyme activity was measured at temperatures of 30, 40, 50, 60 and 70 ° C. under pH 5.0 to investigate the effects of temperature of exo- and endo-chitinases derived from Trichoderma viride AGCC-M41 strain. In addition, the remaining titer of the enzyme was investigated at each temperature for 1 hour. As a result, the apparent optimum temperature of the exo-chitinase derived from Trichoderma viride AGCC-M41 strain was 50 ℃, the stability of the enzyme was found to be relatively low at a temperature above 40 ℃ ( Fig. 7 ). In addition, the apparent optimum temperature of the endo-chitinase derived from Trichoderma viride AGCC-M41 strain was 45 ℃, it was shown that the stability of the enzyme is relatively low at a temperature above 50 ℃ ( Fig. 9 ).

Example 6: Optimization of NAG Production Conditions Using Chitinase Enzyme

<6-1> Effect of Enzyme Amount

Kitty prepared in Example <3-3> corresponding to 5, 10, 15, 20, 25 and 30 U for 1 g of swelling chitin after adjusting the reaction pH of swelling chitin to 1 N HCl The hydrolase reaction was carried out at 42.5 ° C. for 48 hours after adding the enzyme enzyme. At this time, the chitinase enzyme agent includes an endo-chitinase and exo-chitinase in a ratio of about 1: 9. Thereafter, heat treatment was performed at 80 ° C. for 30 minutes to stop enzymatic activity, and then NAG conversion yield was determined by measuring NAG production. At this time, in order to measure the NAG content, the hydrolyzed solution was diluted so that the solid content was about 10 to 100 µg / ml, and 0.2 ml of 0.8 M potassium tetraborate was added to 1 ml of the diluted solution, followed by boiling water for 3 minutes. Heated at. After quenching the reaction solution, 6.0 g of a solution of 10 g of ρ-dimethylaminobenzaldehyde dissolved in 88 mL of acetic acid and 12 mL of 10N HCl was added thereto, and the mixture was heated at 37 ° C for 20 minutes. After cooling the reaction solution again, the absorbance was measured at 585 nm, and the NAG content in the test solution was measured by comparison with a standard curve prepared using a standard NAG. The conversion yield (%) in this example is expressed as a percentage (%) of the weight ratio of chitin used as a substrate and NAG produced therefrom.

As a result, as shown in Table 6 , NAG production increased as the amount of chitinase enzyme increased, and 1 g of chitin was 100% converted to NAG when 30 U of chitinase was added.

<6-2> Effect of Reaction pH

The reaction pH of the swelling chitin solution was adjusted to 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5 and 7.0 using 1 N HCl and 1 N NaOH, respectively, and for 1 g of swelling chitin. A chitinase enzyme equivalent to 30 U was added. This was hydrolyzed at 42.5 ° C. for 48 hours, followed by heat treatment at 80 ° C. for 30 minutes to stop the enzyme reaction, and the yield of NAG conversion was measured as in Example <6-1>.

As a result, as shown in Table 7 , the trichoderma viride AGCC-M41-derived strain chitinase of the present invention showed the highest conversion yield (about 100%) in the pH range of 3.5 to 5.0.

<6-3> Effect of reaction temperature

Adjust the reaction pH of the swelling chitin solution to 4.0 using 1 N HCl, and then add 30 U of chitinase enzyme to 1 g of swelling chitin and adjust the reaction temperature to 35, 37.5, 40, 42.5 and 45 ° C, respectively. After the hydrolysis reaction was performed for 48 hours. Thereafter, the reaction was stopped by heat treatment at 80 ° C. for 30 minutes, and then the NAG conversion yield was measured as in Example <6-1>.

As a result, as shown in Table 8 , the chitinase derived from the Trichoderma viride AGCC-M41 strain of the present invention showed a conversion yield of 90% or more between 40 and 45 ° C, in particular, almost 100% NAG swelling chitin at 42.5 ° C. Could be converted to

<6-4> Effect of Substrate Concentration

The chitin solution was prepared by varying the concentration of the swollen chitin at 1, 2, 3, 4, 5, 6, and 7%, and the reaction pH was adjusted to 4.0 using 1 N HCl. To the swelling chitin solution, 30 U of chitinase enzyme was added per 1 g of swelling chitin, and the enzyme reaction was performed at 42.5 ° C. for 48 hours. Thereafter, heat treatment was performed at 80 ° C. for 30 minutes to stop the enzyme reaction, and the yield of NAG conversion was measured as in Example <6-1>.

As a result, as shown in Table 9 , the chitinase derived from the Trichoderma viride AGCC-M41 strain of the present invention converted the swelling chitin at a concentration of 1 to 5% to almost 100% NAG, and at about 7% swelling chitin. NAG conversion yield of 91% was shown.

<6-5> Effects of Exo-chitosanase Addition on Enzyme Reaction

Chitin undergoes about 10% deacetylation during manufacture, resulting in the presence of D-glucosamine residues in the chitin molecule. In order to effectively decompose D-glucosamine present in the chitin molecule, the effect of adding exo-chitosanase in addition to chitinase was investigated. To the swelling chitin solution adjusted to pH 4.0, exo-chitosanase 3 U was added to 1 g of swelling chitin, and the enzyme was reacted for 48 hours at 42.5 ° C. after adding the chitinase enzyme in an amount of 5-30 U. Was performed. Thereafter, heat treatment was performed at 80 ° C. for 30 minutes to stop the enzymatic reaction, and the yield of NAG conversion was measured as in Example <6-1>.

As a result, as shown in Table 10 , NAG productivity increased by about 10 to 20% when additionally adding exo-chitosanase than when the reaction was performed by adding only chitinase enzyme.

Example 7: NAG Production Using Chitinase

Chitinase enzyme 30 U / g chitin was added to 200 ml of 5% swollen chitin solution and hydrolyzed for 48 hours at 42.5 ° C. and pH 4.0 reaction conditions. After completion of the hydrolysis reaction, 2 g of activated carbon powder was added to decolorize at 50 ° C. for 20 minutes, and then activated carbon and impurities were removed by filtration. The filtrate was concentrated in vacuo to 60 briquettes at about 60 ° C., and then left at 4 ° C. for at least 12 hours to crystallize NAG according to the difference in solubility. 8.5 g of crystal powder were obtained.

Example 8: Recovery of chitinase enzyme using ultrafiltration

As in Example 7, 30 U / g chitin was added to 200 ml of 5% swollen chitin solution, and hydrolyzed for 48 hours at 42.5 ° C. and pH 4.0 reaction conditions. 30 kDa) was used to recover the chitinase enzyme. The titer of total chitinase used for NAG preparation was 300 U, from which the recovery of chitinase was shown in Table 11 .

As described above, the chitinase enzyme produced from the Tricoderma viride AGCC-M41 strain of the present invention has a high yield of enzyme and can effectively convert chitin to NAG, thereby enzymatic hydrolysis of chitin. It can be usefully used for the production of NAG by.

Claims (23)

Trichoderma viride AGCC-M41 (KCTC 10734BP), which produces exo-chitinases and endo-chitinases simultaneously. delete delete 1) culturing by inoculating Trichoderma viride AGCC-M41 (KCTC 10734BP) in chitin-containing medium; 2) removing the mycelium from the culture solution; 3) concentrating the enzyme by ultrafiltration of the filtrate from which the mycelium is removed; 4) removing the media component from the concentrate through diafiltration; And 5) purifying the filtrate using column chromatography, a method for producing an exo-chitinase. The method of claim 4, wherein In step 1), the chitin-containing medium was 0.1 to 5.0% of chitin, 0.1 to 5.0% of corn-steeped powder, 0.05 to 2.0% of ammonium sulfate, 0.1 to 3.0% of potassium phosphate, 0.01 to 1.0% of calcium chloride, and sulfuric acid. 0.01 to 1.0% of magnesium, 0.05 to 3.0% of Triton X-100 and 0.01 to 2.0% of glucose. The method of claim 4, wherein In step 1), Trichoderma viride AGCC-M41 (KCTC 10734BP) is incubated for 72 to 168 hours at 20 to 30 ℃, pH 2.5 to 5.0, aeration conditions 0.1 to 2.0 vvm and agitation speed 100 to 500 rpm Manufacturing method. The method of claim 4, wherein The column chromatography is performed by sequentially performing phenyl-Toyopearl column chromatography and Q-Sepharose column chromatography. delete delete 1) culturing by inoculating Trichoderma viride AGCC-M41 (KCTC 10734BP) in chitin-containing medium; 2) removing the mycelium from the culture solution; 3) concentrating the enzyme by ultrafiltration of the filtrate from which the mycelium is removed; 4) removing the media component from the concentrate through diafiltration; And 5) purifying the filtrate using column chromatography, a method for producing an endo-chitinase. The method of claim 10, In step 1), the chitin-containing medium was 0.1 to 5.0% of chitin, 0.1 to 5.0% of corn-steeped powder, 0.05 to 2.0% of ammonium sulfate, 0.1 to 3.0% of potassium phosphate, 0.01 to 1.0% of calcium chloride, and sulfuric acid. 0.01 to 1.0% of magnesium, 0.05 to 3.0% of Triton X-100 and 0.01 to 2.0% of glucose. The method of claim 10, In step 1), Trichoderma viride AGCC-M41 (KCTC 10734BP) is incubated for 72 to 168 hours at 20 to 30 ℃, pH 2.5 to 5.0, aeration conditions 0.1 to 2.0 vvm and agitation speed 100 to 500 rpm Manufacturing method. The method of claim 10, The column chromatography is performed by sequentially performing phenyl-Toyopearl column chromatography and Q-Sepharose column chromatography. 1) swelling chitin to prepare an enzyme substrate; 2) hydrolyzing the swelling chitin obtained in step 1) using chitianase obtained from Trichoderma viride AGCC-M41 (KCTC 10734BP); 3) vacuum concentrating the hydrolysis solution obtained in step 2); 4) decoloring and filtering the concentrate obtained in step 3); 5) crystallizing N -acetylglucosamine from the filtrate obtained in step 4); And 6) A method for producing N -acetylglucosamine using trichoderma viride AGCC-M41 (KCTC 10734BP) derived chitinase, comprising drying the N -acetylglucosamine crystal obtained in step 5). The method of claim 14, In step 1), the chitin is swelled by adding 2-4 times the volume of strong acid to chitin and stirring at room temperature for 4-12 hours. The method of claim 15, A swelling chitin is dissolved in water at a concentration of 1 to 10% (w / v) to prepare an enzyme substrate. The method of claim 14, The method according to claim 2, wherein the chitinase is used in an amount of 1 to 100 U per gram of swelling chitin. The method of claim 14, Process for producing a hydrolysis reaction is carried out for 30 to 60 ℃, pH 3.0 to 7.0 for 24 to 72 hours. The method of claim 14, Production method characterized in that the exo-chitosanase is further added in step 2). The method of claim 19, Exo-chitosanase is added in an amount of 5 to 30 U per gram of swelling chitin. The method of claim 14, After the hydrolysis reaction, the production method characterized by recovering the chitinase through ultrafiltration (molecular weight cut-off value: 8-30 kDa) to obtain a hydrolysis solution. The method of claim 14, 1 to 10% of activated carbon relative to the liquid amount of the concentrate in step 4), characterized in that the decolorization at a temperature of 30 ~ 60 ℃. The method of claim 22, 0.1-1.0% of diatomaceous earth is added further. The production method characterized by the above-mentioned.

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