KR100205247B1 - A trichoderma sp which decompose cellulose - Google Patents

Abstract

The present invention relates to a trichoderma genus strain (Accession No .: KFCC-10906) having cellulose degradability, wherein the strain producing cellulose degrading enzyme according to the present invention exhibits superior degradability compared to known strains and thus is an additive in feed. It can be used as a solution, and can be widely used in the field of breaking down cellulose to produce useful products.

Description

Microorganisms of the genus Cricoderma with cellulose resolution

It is an object of the present invention to provide a novel Trichoderma genus strain that produces high cellulose degrading enzymes.

Another object of the present invention is to provide a high cellulose degrading enzyme obtained from the trichoderma mutant microorganism.

Still another object of the present invention is to provide a mixed microbial agent mixed with the new strain of the present invention and known strains.

Another object of the present invention is to provide a feed for animals incorporating the new strain of the present invention. The present invention relates to a microorganism of the genus Crycoderma, and more specifically, to be able to decompose cellulose with high efficiency The present invention relates to a microorganism of the genus Crycoderma that produces a degrading enzyme and a degrading enzyme thereof.

Cellulose (cellulose) is a polysaccharide formed by connecting D-glucopyranose to β-1, 4 bonds and constitutes the main component of the plant cell membrane. Cellulose obtained in nature is a large molecule in which about 3000 or more D-glucose units are bound and have high stability and generally have a strong resistance to enzymatic degradation.

Cellulose is the most abundant organic substance produced in nature, and the total amount of biomass available in Korea's natural resources is estimated to be about 150 million tons, of which forest and agricultural by-products account for about 96% of the total. Therefore, efforts have been widely attempted to effectively break down cellulose and convert it into a resource available for food, feed and other industrial resources. Indeed, some practical applications have been made in improving animal feed using cellulose degrading enzymes or their secreting microorganisms, alcohol or sugar fermentation, and the like.

However, known cellulose degrading microorganisms have not yet reached a satisfactory level in terms of cellulose degradation efficiency, and thus, further improvement is required.

As one of the attempts of the present invention, new strains of microorganisms have been isolated and repeated to obtain the microorganisms having high cellulose degradation efficiency.

1a to f are photographs of the medium showing the halo size of the strain by the Congo red test according to Example 1.

Figure 2 is a graph showing the halo size by the Congo red test of Example 1 and Comparative Example 1.

Figures 3a to 3h is a photograph of the medium showing the halo size of the strain by the Congo red test according to 1 in comparison.

Figure 4 is a graph showing the halo size by the Congo red test of Comparative Example 1.

5a to c is a graph showing the activity of CMCase, Avicelase and β- glucosidase of the strain over time in Example 2.

Figure 5d is a graph showing the cellulase activity of the strain in Example 2.

6a to c is a graph showing the activity of CMCase, Avicelase and β-glucosidase of the strain over time by culturing the strain by supplying filter paper as a carbon source in medium-2 of Example 3.

Figure 6d is a graph showing the cellulase activity of the strain in medium-2 of Example 3,

7a to b is a graph showing the CMCase and Avicelase activity of the strain over time after culturing the strain with a carbon source filter medium in the medium-1 of Example 3.

7c is a graph showing cellulase activity of strains in Medium-1 of Example 3. FIG.

8 is a graph of enzyme induction conditions according to ph of strain c-4.

Figure 9a is a graph of the enzyme induction according to the change in the culture temperature of the c-4 strain.

Figure 9b is a graph of the thermal stability of the c-4 strain enzyme.

Figure 9c is a graph of the ph stability of the c-4 strain enzyme.

10 is a graph showing the resistance of the c-4 strain enzyme to the protease.

Figure 11a is a photograph of the c-4 strain.

Figure 11b is a photograph taken of the T. harzianum strain.

Figure 12a is a picture of the stained after electrophoresis of the protein of the c-4 strain.

Figure 12b is a picture stained after electrophoresis of the protein of T. harzianum strain.

Figure 12c shows the activity staining for CMCase after PAGE.

Figure 12d shows the activity staining for β-glucosidase after PAGE.

Cellulase (cellulase) is composed of endoglucanase (exoglucanase), exoglucanase and (beta) -glucosidase, and the degree of secretion and enzymes for each strain The interaction between them is also different. Therefore, the resolution of cellulose is finally determined by the relative secretions and interactions of these enzymes.

The present inventors obtained various natural products in order to isolate cellulose degradation strains, and then inoculated these natural products into a medium having cellulose as the only carbon source as a sample, and investigated the resolution. Secondarily screening for the selected strains, the cellulolytic strains of the present invention were finally obtained.

The strain obtained in the present invention was found to be a strain of genus Tricodoma in view of morphological and biochemical properties, and is presumed to be a strain of Trichodoma harz ianum.

The Crycoderma genus strain obtained according to the present invention was found to exhibit high cellulose degradation activity compared to the known strains. This variation is governed by the present application. The Korean spawn association was deposited (JFCC-10906).

Hereinafter, the present invention will be described in more detail based on Examples.

Example 1

Strain First Screening

Humus, deciduous soil, bovine feces, larvae and mushrooms to collect cellulose as the sole carbon source-1 medium-cellulose secreting strain screen medium, Mandels, M. D. Sternberg, 1976. J. Ferment. 54, 267-286). The composition of this medium-1 is shown in Table 1 below.

1) CMC: carboxymethy1cellulose degree of sudstitution 0.7, MW 10 ⁵

2) Avicel: crystalline cellulos

3) [(NH 4) ₂ SO ₄ 14g, KH ₂ PO ₄ 20g, CaCI ₂ 3g, MgSO ₄ * 7H ₂ O 3g, FeSO ₄ * 7H ₂ O 50mg, MnSO ₄ * 6H ₂ O 22.8 mg, ZnSO ₄ * 14H of 7H ₂ O, 42.5mg of CoCI ₂ * 6H ₂ O and 1L of distilled water]

Each sample was incubated at 28 ° C. in an aerobic and anaerobic atmosphere. For aerobic cultivation, the sample was diluted in saline and plated or staked on a plate, and then incubated at 25 ° C. For anaerobic cultivation, nitrogen gas was flowed while flowing carbon dioxide and nitrogen gas into an anaerobic chamber. After diluting the sample with the charged saline, it was plated and cultured in a container filled with nitrogen gas.

After separating the bacteria growing on each plate. After 3 days of inoculation with fresh medium and 1 week after fungus, the fungal size was determined according to Congo-red: PJWood, JD Erflo and RM Teather, 1988, Methods in Enzymol., 160.59-74. halo size) was measured.

As a result of this test, growth was relatively active, and six symbols were given C-1, C-3, C-4, D-1, E-1 and E-2, and six bacteria The symbols are given as A-1, B-1, B-2, D-2, D-3, and D-1. The experimental results of these primary selection strains are shown in Table 2 below.

As shown in Table 2, it can be seen that the halo size of the C-4 strain was the largest and the most actively grown in the medium having cellulose as the only carbon source.

Among them, halo-size photographs of B-1, B-2, C-4, D-2, D-3, and D-4 are shown in FIGS. 1A to F. In addition, a graph showing the halo size of these strains in a bar graph is shown in FIG. 2.

Comparative Example 2

Congo red test of known microorganisms

Twenty four fungi and five bacteria known to have cellulose degradation were purchased from the depository and compared. These known strains were cultured in the same conditions and medium as in Example 1 for the same time period, and then subjected to the Congo red method to measure halo size. The results are shown in Table 3 below.

1) Symbols in parentheses are abbreviations used to denote microorganisms in this specification.

1) Symbols in Goraho are abbreviations used to refer to the microorganisms herein.

As can be seen in Table 3, the halo size of the known microorganisms was generally in the range of 0.8 to 7.0 cm. Among them, A. fungus, S. cellulophilum, A. terreus, T. harzianum. T. 1ongibrachiatum, V. albo-atrum, S. flavogriseus and cellulomonas sp. Photos showing halo sizes of strains are shown in FIGS. 3A-H. In addition, FIG. 4 shows a graph in which a halo size of a strain showing a relatively large halo size is plotted in a bar graph.

Example 2

Second Screening of Strains

Isolated strains and commercially known strains were incubated at 28 ° C. for 8 days in a medium-2 broth. The composition of the medium used at this time is shown in Table 4.

During the incubation period, 1 ml of broth was taken daily, and enzyme activity was measured using CMC, Avicel, and PNPG (p-Nitropheny1-β-1) -G1 ucopyraboside) as substrates.

For the determination of enzyme activity, a reaction mixture of 0.5 mM CMC solution or 0.4 ml of Avicel suspension and 0.1 ml of enzyme solution in 50 mM acetic acid buffer (ph 5.0) was added for 15 minutes at 40 ° C (for CMC) or 4 The amount of reducing sugars produced was measured according to Rumorian-Nelson's method (M. Somogyi, Note on sugar determination, J. Biol. Chem., 195, 19-23). However, the amount of enzyme required to produce ¹ μmolemin ⁻¹ reducing sugar under standard reaction conditions was defined as 1 unit.

In addition, in order to measure the activity of β-glucosidase, 0.4 ml of 1 mM PNPG dissolved in acetic acid buffer (pH5.0) and 0.1 ml of a solution were mixed and reacted at 40 ° C for 30 minutes. Subsequently, 1 ml of 1M Na ₂ CO ₃ was added, diluted with 5 ml of distilled water, and the amount of free PNP was measured at 42 nm. However, the amount of enzyme required to generate ¹ μmol min ⁻¹ PNP under standard reaction conditions was generated in 1 unit.

Figure 5a is a graph showing the activity of CMCase of cultured microorganisms over time, Figure 5b is a graph showing the activity of Avicelase, Figure 5c is a graph showing the activity of β-glucosidase.

As shown in FIG. 5A, CMCase activity was high in S. cellulophi lum, A. tereus, M. verrucaria, S. cellulophilum, S. commune, T. reesei QM9414, T. harzianum and 1.1acteus among known strains. Among the isolates, C-1, C-4, D-4, D-1 and D-2 showed relatively high activity.

Avicelase activity was high in S. cellulophilum, T. harzianum and I. 1acteus among known strains, and high in D-1 and C-4 among isolates (FIG. 5B).

In β-glucosidase, high activity was measured in the known strains such as A. tereus, S. cellulophilum, T. ressei QM9419 and T. harzianum, and among the isolated strains, C-4 and D-2. It showed high activity in the strain of (Fig. 5c).

FIG. 5D is a graph showing comprehensively the activities of CMCase, Avicelase and β-glucosidase measured at the highest time of enzymatic activity of each strain. As can be seen from this graph, strains of C-4, S. cellulophilum, T. harzianum, etc., all showed high enzymatic activity, and A. terreus, in particular, showed that β-glucosidase activity was different from other enzyme activities. It was higher than that. D-1 showed higher CMCase activity than other enzyme activities. In particular, C-4 was found to be suitable for the present invention because all the enzyme activity is significantly higher than other strains.

Example 3

Tertiary Culture of Strains

In order to determine the significance level under different enzyme-induced conditions in the strains showing relatively high activity in the test of Examples 1 and 2, cultured by feeding filter paper crushed as a carbon source to medium-2. The enzyme activity was measured according to the method of Example 1. The filter paper as a substrate was intended to measure the resolution for cellulose under near-natural conditions. The results are shown in Figures 6A-6D.

Subsequently, filter paper ground as a carbon source was supplied to medium-1, and the strains were cultured, and the enzyme activity was measured according to the method of Example 1. The results are shown in 7a-7b. The activity of cellulase is shown in Figure 7c.

As described in the above examples, the culture of the strain and the measurement of enzyme activity, c-4 was found to be superior or at least equal to the three enzyme activity than the known fibrin degradation strain under all induction conditions. In particular, it showed high activity value in all the tested conditions, and showed a high activity value because it showed maximum activity in a relatively short time (3 to 6 days) as a fungus.

Example 4

Enzyme Induction Condition of c-4 Strain

Enzyme activity was measured while changing the culture conditions for the isolated strain c-4.

(1) induction conditions for substrates

As shown in the above examples, c-4 exhibited higher or similar cellulose resolution than other strains in medium-1, medium-2, medium-1 (filter paper), and medium-2 (filter paper), so that the specific substrate specificity This was not observed.

(2) Enzyme induction condition according to pH

The pH of medium-1 was adjusted to 3.5, 4, 4.4, 5.5, 6.2, 6.5, 7.0, 7.5 and 7.8 using HCI and NaOH, respectively, and the enzyme induction was measured while culturing the c-4 strain for 8 days. . 8 is a graph showing the results at day 6 when the activity of the enzyme was maximized. As shown in this graph, strain c-4 showed the maximum activity at pH 6.2, and showed high enzyme activity at weak acidity, and the enzyme induction rate decreased drastically above pH7.0.

Example 5

Characterization of enzymes derived from C-4 strains

C-4 strains were incubated with a pH of 6.2 in medium-1 and the cultures were collected and used as coenzymes in the next test.

(1) optimum temperature

The crude enzyme was added to acetic acid buffer solution (pH5.0, 100 mM) and the enzyme activity was measured while changing the temperature to 30 to 60 ℃. The results are shown in Figure 9a. As shown in this graph, the optimum temperature was found to be 40 ° C.

(2) thermal stability

Coenzyme was added to acetic acid buffer (pH 5.0, 100 mM) and the enzyme activity was measured while changing the temperature. The results are shown in Figure 9b. Enzyme activity on CMC was maintained almost for 1 hour, but lost 60% of activity at 60 ° C. for 10 minutes. However, it is estimated that the enzyme having high thermal stability is present in view that the activity remaining after 1 hour at 60-80 ° C. is about 20%.

(3) pH stability

The crude enzyme was added to acetic acid buffer solution (pH5.0, 100mM) in the range of pH 3.0 to 8.0 and left at 5 ° C. for 24 hours, and adjusted to pH 5.0 by using acetic acid buffer solution (pH 5.0, 500mM). , Residual activity was examined according to the method of Example 1. The results are shown in Figure 9c. The coenzyme was very stable at pH 5.0 and overall showed stability in the range of pH 4-7.

(4) heavy metal and reducing agent effect

The coenzyme was finally added to a heavy metal solution (pH 5.0 acetic acid buffer solution, 100 mM) adjusted to 1 mM and allowed to stand at room temperature for 1 hour, after which residual activity was measured according to the method of Example 1.

As can be seen from the table, the coenzyme of the present invention was not largely affected by metal ions, but when Ca ions were present, the activity of about 15% showed a tendency to decrease for 1 hour. However, no decrease in activity was observed with other metal ions. In addition, CMCase activity increased in all cases treated with 1 mM of reducing agent.

(5) protease resistance test

The coenzyme was exchanged with phosphate buffer (pH7.8, 50 mM) and protease (trypsin or chymotrypsin) was added to 2% (dl / W). After standing at room temperature for 1 hour, the solution was collected at 5,10,30,60 minutes intervals and placed in acetic acid buffer (pH 5.0, 500mM) to measure residual CMCase activity. The results are shown in FIG.

The coenzyme was found to have lost 30% of activity by each of the two proteases 10 minutes after the reaction, but there was little decrease in activity for 1 hour thereafter. These results suggest that the coenzyme has more than one type of CMCase, among which there is an enzyme having high resistance to protease.

Example 6

Identification of c-4 strain

Through the above examples were identified for the c-4 strain found to be most suitable for the purpose of the present invention. The T. harzianum grown under the same conditions as the grown C-4 strain was observed with an optical microscope at 400 magnification. FIGS. 11A and 11B.

Figure 11a is a photograph of the C-4 strain, Figure 11b is Trichoderma harzianum, the morphological characteristics of the two strains were similar.

However, two strains incubated in PDA medium (potato (from infusio) 200g, 15 g of bacto dextrose 20 g bactoa, less than 1 l of distilled water) and MEA medium (20 g of malt extract, 5 g of peptone, 15 g of agar, 15 g of agar, 1 l or less) Differences were observed in the color of the spores and the color of the back of the badge.

Again, C-4 strains and Confucius strains were grown in the same medium-2 and then protein profiles were compared by electrophoresis. Figure 12a is a picture of the coomasic staining after the PAGE (polyacry lamide gel electrophoresis) for the protein of strain C-4, Figure 12b is a stain after the SDS-PAGE for the T. harzianum protein (coomasie staining) This is a picture. From these two figures, it can be seen that the electrophoretic images of the two strains are very similar but not completely identical.

For the analysis of enzymes, electrophoresis gels were activated for β-glucosidase and CMCase. Figure 12c is a photograph of the active staining for CMCase after PAGE, Figure 12d is a photograph of the active staining for β-glucosidase after PAGE. As a result of β-glucosidase activity staining, there is a slight difference in enzyme position between C-4 strain and T. harzianum strain. In addition, the results of CMCase staining showed that three types of endoglucanases were identified in C-4, two types were identified in T. harzianum, and their positions were slightly different.

In light of this, the C-4 strain isolated in the present invention is considered to be a genus of trichiderma, and more closely, a subspecies of Trichoerma harzianum.

In conclusion, the isolated C-4 strain was identified as a new strain capable of effectively degrading cellulose because cellulase activity is superior to known strains and stable against various environmental factors. The present inventors used this C-4 strain as a new microorganism, trichoderma spp. It was deposited with the Korean spawn association (DKC) under the name of DKC (Deposit date: June 24,199., No .: KFCC-10906).

Example 7

Synergy test of C-4 and other strains

Since the cellulose-degrading microorganisms are different from each other in the type and amount of the enzymes produced, synergistic effects can be obtained when different strains are combined. It was used as a coenzyme and adjusted to their activity similar to the experiment. The synergistic effect was obtained by dividing the value of each coenzyme with similar activity against CMC by the sum of the values of each coenzyme. The results are shown in Table 6 below.

As can be seen in the table, C-4 strains of the present invention, D-1, T. Reel, T. ree2, T. har, T. con, and S. cel were found to have synergistic effects.

Cellulose is not only present in nature in enormous quantities, but is produced by plants every year. Trichoderma spp. Of the present invention, which can dissolve this cellulose effectively and stably. DKC strains appear to have sufficient efficiency and commerciality and therefore can be used for a variety of applications.

In particular, the decomposition of cellulose to produce ethanol, or to make the animal feed more digestible form of trichoderma spp. DKC strains can be used. In addition, as a mixed microbial preparation in which a known microorganism that decomposes cellulose and the trichoderma spp.DKC strain of the present invention is mixed, the resolution may be further improved by synergy of cellulose degrading enzymes (eg, C-4 and T). reesi, etc.)

Recent studies have shown that cows fed starch-rich feeds such as corn have a lower pH, which inhibits the growth of microorganisms that degrade cellulose in these acidic environments (Burroughs et al. , The influnce of corn starch upon roughae digestion in cattle, j.Animal Sci., 8, 271-278,2949). Moreover, the intestinal pH of most cattle fed commercially available foods often drops below 6, and in this environment it is reported that the intestinal cellulose degradation is almost lost, causing the growth of cattle (Slyter, Influence of acidosis on rumen function, J). Animal Sci, 43, 920-929, 1976).

Thus, trichoderma spp. By feeding the DKC strains into the animal feed, cellulose degradation can be actively degraded to prevent and promote indigestion in animals. Therefore, the present invention provides a new type of feed that promotes the growth of an animal by mixing the strain of the present invention with an animal feed.

As described above, Trichoderma spp. Producing cellulose degrading enzyme according to the present invention. DKC strain can be used as an additive in the feed exhibiting superior resolution compared to known strains, and further can be widely used in the field of producing useful products by breaking down cellulose.

Gene Sequence of Cellulose Degrading Enzyme Produced in Trichoderma Genus Mutant (KFCC-10906) According to the Present Invention

Claims (3)

Trichoderma genus strain with cellulose resolution (Accession No .: KFFC-10906). Cellulose degrading enzyme isolated from Trichoderma sp. (Accession number: KFCC-10906) having the following gene sequence. Animal feed including Trichoderma spp. (Accession No .: KFCC-10906) having cellulose resolution and feed containing cellulose components.