KR101236558B1 - A novel isolated Phoma sp. from rotten tangerine peel and cellulolytic enzymes and β-glucosidase produced by thereof - Google Patents

Abstract

The present invention is a novel forma species strain, a fibrinolytic enzyme derived from fibrin-based biomass including red algae produced in the strain, a composition for fibrin degradation isolated from fibrin-based biomass including red algae comprising the same as an active ingredient, using the same It relates to a method for breaking down cellulose separated from cellulose-based biomass including red algae, and a method for producing bioethanol by the above-described method. It also relates to beta glucosidase isolated and purified from the strain. Since the novel strain according to the present invention can develop an enzymatic digestion system capable of efficiently hydrolyzing the remaining fiber after acid saccharification for high efficiency ethanol fermentation from fibrous biomass including red algae gelium amansi, biofuel It can be very useful in industry.

Description

Novel strains of forma isolated from the surface of tangerines and fibrinolytic enzymes and betaglucosidases produced therefrom [A novel isolated Phoma sp. from rotten tangerine peel and cellulolytic enzymes and β-glucosidase produced by conjugate}

The present invention is a novel forma species strain, a fibrinolytic enzyme derived from fibrin-based biomass including red algae produced in the strain, a composition for fibrin degradation isolated from fibrin-based biomass including red algae comprising the same as an active ingredient, using the same It relates to a method for breaking down cellulose separated from cellulose-based biomass including red algae, and a method for producing bioethanol by the above-described method. It also relates to beta glucosidase isolated and purified from the strain. The fibrinolytic enzyme produced by the Phoma sp. (Poma sp.) Developed by the present invention is a red algae, Moroccan loot, Galidium amansii , Morocco) are very useful for developing enzymatic digestion system and mass production technology of enzymes that can efficiently hydrolyze remaining cellulose after acid saccharification for high efficiency ethanol fermentation.

A recent solution to the problem of global warming due to exhaustion and an increase in CO ₂ emissions from fossil fuels and the importance of transport bio-ethanol production technology for the emerging worldwide.

As a carbon source (glucose, etc.) that is a raw material of bioethanol, starch-based systems (corn, potato, sweet potato, etc.) can produce ethanol relatively easily, but since the supply and demand of raw materials is unstable in the long term because it is related to food resources, raw material cost increases and secures raw materials. There are a lot of problems. In the case of sugar-based sugar (sugar cane, sugar beet, etc.), it is possible to secure glucose, which is a carbon source that can be fermented at a relatively low cost, so that hydrogen ethanol can be manufactured at low cost. In case of Korea, which is applicable only to the plant growth conditions (temperature, etc.), it is not suitable for domestic situation because mass production is difficult. On the other hand, wood-based biomass (wood, rice straw, waste paper, etc.) must be accompanied by a pretreatment process required for the lignin removal process as a raw material that can ensure the stability of raw material supply and demand, due to an increase in process cost and a low yield of glycation. Low economic feasibility In order to solve these problems, it is necessary to explore new resources to secure fermentable carbon sources.

Red algae, a marine biomass, has been rarely studied, but it can be produced inexpensively, easily processed, and has excellent growth potential (comparable to rainforests in the coastal waters). It is expected to be very economical than conventional sugar system (4 times a year can be harvested offshore of our country and up to 6 times a year using Southeast Asian subtropical climate). In addition, there is no lignin component that must be removed as in the case of the wood-based system, so the manufacturing process is simple and it is possible to increase the yield of ethanol because it is made of high quality carbohydrates.

The major carbohydrate constituents of red algae are galactan and fibrin, which are decomposed into galactose and unhydrogalactose (3,6-anhydrogalactose (AHG)) by saccharification, and fibrin is broken down into glucose.

Fibrin of red algae is a glucose polymer that forms β-1,4- bonds, including endo-β-1,4-glucanase (EG) and cellobiohydrolase. Hydrolyzed by enzymatic systems such as CBH) and β-glucosidase (BGL), and these enzymes can efficiently hydrolyze natural fibrin if they function complementarily.

EG randomly hydrolyzes β-1,4-linkages of the fibrin inner chain, and CBH gradually hydrolyzes the reducing and non-reducing ends of the fibrillar polymer produced by hydrolysis by EG, thereby producing two molecules of glucose. Produce combined cellobiose. Since cellobiose acts as a potent inhibitor for EG and CBH, BGL hydrolyzes these cellobiose to eliminate the strong inhibition of EG and CBH, respectively (Wong, KKY, LUL Tan, JN Saddler. 1988. Multiplicity of β- 1,4-xylanase in microorganisms: Functions and applications.Microbiological Reviews. 52: 305-317; Medve, J., J. Karlsson, D. Lee, and F. Tjerneld. 1998. Hydrolysis of microcrystalline cellulose by cellobiohydrolase I and endoglucanase II from Trichoderma reesei : Adsorption, sugar production pattern, and synergism of the enzymes.Biotechnology and Bioengineering. 59: 621-634). In general, EG can hydrolyze amorphous regions of fibrin molecules and CBH can bind to the crystal regions to produce soluble reducing sugars from the reducing and non-reducing ends of the fibrin chain (Enari, TM and ML Niku-Paavula). 1987. Enzymatic hydrolysis of cellulose: Is the current theory of mechanism of hydrolysis valid? .Critical Reviews in Biotechnology. 5: 67-87).

On the other hand, in order to efficiently produce ethanol from fibrous biomass including red algae, efficient glycosylation of fibrin should be made. However, the fibrin separated from red algae has a high crystallinity and a hard structure, so that it is not easily decomposed by acid.

Thus, the present inventors conducted a thorough study to overcome the conventional problems as described above, as a result of the new separation of the fungus decomposing the tangerine peel, the new fungal strain can produce fibrinase and betaglucosidase By confirming that the present invention was completed, the present invention was completed.

One object of the present invention is the deposition No. KCTC 11825BP poma strains producing fiber Subtotal cellulolytic enzyme of the biomass derived from red algae, including (Phoma sp.).

It is another object of the present invention to provide a fiber-derived composition for fibrin derivation including red algae containing the supernatant of the culture medium in which the strain is cultured as an active ingredient.

Another object of the present invention is to culture the strain; Filtering the strain culture to obtain a supernatant; And it provides a method for decomposing the fiber by adding the supernatant to the fiber separated from the fiber-based biomass including red algae, and a method for producing bioethanol by decomposing the fiber.

Another object of the present invention is derived from fibrin-based biomass including a novel beta glucosidase isolated from the forma species strain, nucleic acid encoding the beta glucosidase and red algae containing the beta glucosidase as an active ingredient It is to provide a composition for decomposition of cellulose.

Still another object of the present invention is to add the betaglucosidase to fibrin isolated from fibrous biomass containing red algae to decompose the fibrin-derived cellobiose and to decompose cellobiose to produce bioethanol. To provide.

Another object of the present invention is to provide a method for producing fibrinolytic enzyme derived from the forma species strain.

In some embodiments for achieving the above object, the present invention provides a strain producing the fiber sub-total cellulolytic enzyme of the biomass derived from red algae, including the deposit number KCTC 11825BP of poma strains (Phoma sp.).

The present inventors have separated the one from the fungal strain rotten tangerine surface, the strain of ITS (internal transcribed spccer) was analyzed for phylogenetic relationships using the sequence analysis, poma Formosan column bar sulfonic cine itaconic (Phoma pomorum var. circinata CBS 286.76) and 99.4%, Poma Pomorum Ba Cyane ( Phoma pomorum var. cyanea CBS 388.80), Phoma Pomorum Bar Pomorum pomorum var. pomorum CBS 539.66) and 99.6% homology was confirmed (Fig. 2). As a result, the isolated fungus is Phoma sp.) and was deposited with the KCTC 11825BP at the Korea Institute of Bioscience and Biotechnology Center (Korea Biotechnology Research Institute, 111, Sci-ro, Yuseong-gu, Daejeon, Korea) named Phoma sp. on December 9, 2010.

As used herein, the term "fibrous biomass" is composed of cellulose, hemicellulose, and lignin, and cellulose refers to a biopolymer that is present in abundance in nature, which accounts for 35% or more of most fibrous biomass components. . It can be obtained from the by-products of various agricultural, forestry and aquatic products, and such organic waste is spotlighted as an energy source including bioethanol production. Such fibrin-based biomass includes a material capable of producing energy such as bioethanol, without limitation, but for the purposes of the present invention, wood-based, red algae, etc., which can separate fibrin.

In the present invention, the term "red algae" is one of the largest groups living in the deepest of the algae, about 600 species of about 600 genera are known around the world, and include Gelidium amansii , snails , kotoni , and the like. Most preferably, the red alga is Gelidium amansii ), but is not limited thereto.

The major carbohydrate constituents of the "red algae" are galactan and fibrin, the galactan component is broken down into galactose and unhydrogalactose (3,6-anhydrogalactose (AHG)) by glycosylation, and fibrin is broken down into glucose. More specifically, the chemical components of red algae are shown in Table 1 below.

Fiber, a carbohydrate component of red algae, is a resource that can be converted into useful sugars, ethanol and industrially useful chemicals. The fibrin is a glucose polymer that forms β-1,4-bonds. Fibrin isolated from red algae has high crystallinity and hard structure, so it is not easily decomposed by acid. Therefore, an efficient enzymatic saccharification process is required to produce bioethanol using the fibers isolated from such red algae. Fibrin of red algae is a glucose polymer that forms β-1,4-bonds, including endo-β-1,4-glucosidase and exo-β-1,4-glu. It is hydrolyzed by enzyme systems such as kinase (exo-β-1,4-glucosidase) and β-glucosidase, and if these enzymes function complementarily, they naturally hydrolyze natural cellulose. can do. Therefore, there is a need for a group of enzymes having endoglucanase, exoglucanase, and betaglucosidase activity, and the present inventors have newly developed a forma species, a strain capable of producing such a group of enzymes.

The supernatant produced from the forma species includes a group of enzymes having fibrinolytic activity.

As used herein, the term "fibrinolytic activity" refers to an activity that catalyzes the production of glucose by hydrolyzing fibrin and endo-beta-1,4-glucanase (endo-β-1, 4-glucanase, EC 3.2.1.4, endoglucanase), cellobiohydrolase (CBH) and β-glucosidase (βGL) activity. More preferably, it means endoglucanase activity, exoglucanase activity, and betaglucosidase activity.

According to one embodiment of the present invention, after culturing the strain and separating the supernatant and sediment, the activity was measured using the supernatant as a coenzyme solution, endoglucanase group of cellulose degrading enzymes of fibrinase (Example 4), it was confirmed to produce exoglucanase (Example 5) and betaglucosidase (Example 5). In particular, it showed high betaglucosidase activity, and its activity increased with the incubation period (Table 2). Therefore, the enzyme group derived from the Forma species strain of Accession No. KCTC 11825BP as described above, the enzyme glycosylation of the fibrinous biomass including the algae gelidium amansi ( Gelidium amansii , Morocco) occurs, two molecules of glucose combined cellobiose It is possible to produce bioethanol as a result of the decomposition.

As another aspect, the present invention relates to a fibrinolytic composition for fibrin degradation derived from fibrin-based biomass including red algae containing the supernatant of the culture medium cultured the strain as an active ingredient.

KCTC 11825BP strain, a strain newly identified in the present invention can produce endoglucanase, exoglucanase, or betaglucosidase, so the supernatant of the culture cultured the strain is a fibrin or fibrin-based biomass derived from red algae. It can be used as a composition capable of decomposing the mass. The composition may include without limitation a substance capable of increasing the activity of the enzyme.

As another aspect, the present invention provides a method for producing a culture, comprising the steps of: (a) culturing the strain; (b) filtering the strain culture to obtain a supernatant; And (c) adding the supernatant to the fiber separated from the fibrous biomass including red algae to decompose the fiber.

As described above, since the supernatant of the culture solution of the strain of the present invention is secreted endoglucanase, exoglucanase, or betaglucosidase, the supernatant of the culture solution was obtained to obtain a crystalline form and an amorphous form separated from red algae It is useful to decompose the fiber if it is added to the fiber of the.

As another aspect, the present invention provides a method for producing a culture, comprising the steps of: (a) culturing the strain; (b) filtering the strain culture to obtain a supernatant; And (c) adding the supernatant to cellulose separated from cellulose-based biomass including red algae to decompose the cellulose.

In yet one aspect, the invention poma strains (Phoma to purified betaglucosidase obtained by culturing sp.).

As used herein, the term "poma species (Phoma sp.)" Is ascorbyl MY to be within the cotta (Ascomycota), by the inventors of endo-glucanase newly cellulolytic enzymes in the poma species, exo-glucanase or beta Glucosidase was isolated and particularly high activity beta glucosidase was purified. The strain belonging to the species poma poma species include, but without limitation, and preferably the deposit number KCTC 11825BP of poma strains (Phoma sp.).

(A) comprising the amino acid sequences of GIQDAGVIA (SEQ ID NO: 2), AGTGSIM (SEQ ID NO: 3) and IMAAYYLVGR (SEQ ID NO: 4), by culturing a Forma species strain having Accession No. KCTC 11825BP; (b) optimum pH 3.5 to 6.5; (c) an optimum temperature of 40 ° C. to 70 ° C .; (d) stable for at least one month at 30 ° C. to 60 ° C .; And (e) Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) measurement Purified betaglucosidase having characteristics of 105 to 115 kDa can be obtained.

The inventors of the present invention not only isolated a strain of Forma species, which is a novel strain Accession No. KCTC 11825BP, but also isolated and purified beta glucosidase having excellent enzymatic activity, thereby revealing the characteristics of the purified enzyme. In addition, the partial amino acid sequence was determined to secure the gene of the present enzyme.

Betaglucosidase of the present invention may comprise SEQ ID NOs: 2, 3 and 4 as partial amino acid sequences. In addition, the optimum pH may have a pH of 3.5 to 6.5, preferably pH 4.0 to 5.0, most preferably pH 4.5. According to one embodiment of the present invention, the highest activity was shown at pH 4.5, and the enzyme activity was measured to be 97% and 81% at pH 4.0 and 5.0, respectively (FIG. 5).

The betaglucosidase of the present invention may be 40 ° C to 70 ° C as the optimum temperature for the activity, preferably 60 ° C to 70 ° C, and most preferably 65 ° C. According to one embodiment of the present invention, it was confirmed that the most optimal activity at 65 ℃ (Fig. 6).

Beta glucosidase of the present invention may be stable for more than one month at 30 ℃ to 60 ℃ by temperature stability, preferably the half life at 30 ℃ to 50 ℃ 3629, 2776 hours, half life 53 hours at 60 ℃ This may be better than the previously reported temperature stability of fungal derived betaglucosidase and may be more efficient in producing bioethanol. According to one embodiment of the present invention, it was confirmed that it is stable for at least one month at 30 ℃ to 60 ℃ (Fig. 7).

The betaglucosidase of the present invention may be sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) measurement molecular weight 105 to 115 kDa, preferably 110 kDa. In addition, the betaglucosidase of the present invention may be a tetramer having a molecular weight of 440 kDa. According to one embodiment of the present invention, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis confirmed that the protein having a size of about 110 kDa (FIG. 3a), as measured by gel filtration chromatography, It was confirmed that it was a tetramer having a molecular weight of 440 kDa (FIG. 3B).

In addition, the beta glucosidase of the present invention exhibits the highest activity against p -nitrophenyl glucopyranoside and cellobiose, and shows little activity against other p NP-glycosides and carboxymethylcellulose, xylan, and avicel. This property supports that the enzymes of the present invention have high substrate specificity only for p -nitrophenylglucopyranoside and cellobiose. In addition, the beta glucosidase of the present invention has a 10-fold higher affinity for p -nitrophenylglucopyranoside than cellobiose, and thus the purified beta glucosidase has an aryl-betaglucose having cellobiase activity. It may be a cedase. Further, if the addition of Mg ^{2 +,} because the enzyme activity of the present invention can be increased, it can be added to the Mg ^{2 +} in order to increase the beta-glucosidase activity of the present invention.

According to an embodiment of the present invention, the beta glucosidase isolated in the present invention is K _m for p -nitrophenyl glucopyranoside and cellobiose The values were 0.31 mM and 3.24 mM, respectively. Inhibition of glucose and glucono-δ-lactone confirmed that the two substances were competitive inhibitors of betaglucosidase. Each inhibition constant was 1.69 mM and 0.09 mM (Table 4). Also stylized various metal ions are a result of analyzing the effects on beta-glucosidase, Mg ^{2 +,} on the other hand to increase the activity of the enzyme 120%, Cu ^{2 +} decreases the enzyme activity ^{58%, Mn 2 +, Fe} 2 ^It was confirmed that the ⁺ , Co ^{2 +} , Ca ^{2 +} ions did not significantly affect the enzyme activity (FIG. 8). In addition, as a result of analyzing the relative ratio of hydrolysis to various substrates, the highest activity was shown for p -nitrophenylglucopyranoside and cellobiose, and other p NP-glycosides, carboxymethyl cellulose, xylan, It confirmed that it showed little activity with Avicel, and it was confirmed that it has high substrate specificity only for p -nitrophenyl glucopyranoside and cellobiose (Table 5).

As described above, the betaglucosidase of the present invention has a characteristic of degrading cellobiose derived from fibrin-based biomass including red algae, and the red algae is as described above.

As another aspect, the present invention relates to a nucleic acid molecule encoding the betaglucosidase.

As used herein, the term "nucleic acid molecule" is meant to encompass DNA and RNA molecules inclusive, and the nucleotides that are the basic structural units in nucleic acid molecules are not only natural nucleotides but also analogs in which sugar or base sites are modified. Include.

As another aspect, the present invention relates to a fibrinolytic composition for fibrin degradation derived from fibrin-based biomass including red algae containing the beta glucosidase as an active ingredient.

As another aspect, the present invention relates to a method for producing bioethanol by decomposing the fiber-derived cellobiose by adding the betaglucosidase to the fiber separated from the fibrous biomass separated from the red algae.

In another aspect, the present invention provides a method for producing a cell, comprising: (a) culturing a forma species strain; And (b) filtrating the culture to obtain a supernatant, and a method for producing fibrinolytic enzyme isolated from fibrin-based biomass including red algae from forma species strains.

The preparation method preferably comprises the steps of (c) concentrating the supernatant; (d) adsorbing said concentrate to a column; And (e) eluting the adsorbate. The step of concentrating the supernatant of step (c) may be any known method for concentrating the supernatant, without limitation. Preferably, the supernatant is equipped with an extrafiltration apparatus equipped with a polyethersulfone membrane of 10 kDa. Can be used to concentrate.

The forma species strain is as described above, preferably may be a forma species strain with accession number KCTC 11825BP.

The column may use 16/10 DEAE FF, Sephacryl 300S HR, and MonoQ HR5 / 5 comum, but is not limited thereto. Various columns known in the art may be used, and the column may be adsorbed at least once. have.

According to the present invention, the biofuel industry can be efficiently hydrolyzed after acid saccharification for high-efficiency ethanol fermentation from various cellulose-based biomass, including red algae, Gelidium amansii and Morocco. This can be very useful in.

1 is a diagram showing the nucleotide sequence of the novel fungi ITS region isolated from the rotting tangerine surface.
2 is a diagram showing a systematic relationship based on the ITS region nucleotide sequence.
Figure 3 is a diagram showing the result of measuring the molecular weight of the purified protein. (a) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis result (SDS-PAGE) and (M: standard molecular weight marker, No. 1: enzyme concentrate before purification, No. 2: concentrate after application of MonoQ ion exchange column HR5 / 50 ), (b) Native molecular weight measurement using Sephacryl S-300 HR 16/60.
4 is a diagram comparing and analyzing amino acid sequences of purified enzymes and registered fungal derived proteins.
5 is a diagram showing the optimum pH of the purified enzyme.
6 is a diagram showing the optimum temperature of the purified enzyme.
7 is a diagram showing the stability of purified enzymes at different temperatures.
8 is a diagram showing the effect of the effect of metal ions on the purified enzyme activity.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

Example  1: Identification of Mold Isolated from Rotten Tangerine Surfaces

The mold on the rotten tangerine surface was plated on a potato dextrose agar plate and incubated at 28 ° C. for 7 days, followed by continuous subculture to isolate one pure mold. Deoxyribonucleic acid was extracted from the isolated mold (AccuPrep Genomic DNA extraction kit) and the ITS sequence was identified by primers ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG-3 ', SEQ ID NO: 5) and ITS4 (5'-TCCTCCGCTATATGATATGC-3', SEQ ID NO: 6) was used to amplify the polymerase chain reaction (polymerase chain reaction, primer pair ITS4 &ITS5; White et al., 1990). The conditions of the polymerase chain reaction are as follows. Initial denaturation; 95 ° C., 5 minutes, denaturation; 95 ° C., 30 minutes, annealing; 48 ° C., 30 seconds, polymerization; 72 ° C., 80 seconds, final extension; 72 ° C., 7 minutes (40 cycles of denaturation-bond-polymerization). The product obtained by the polymerase chain reaction was subjected to electrophoresis using a 1% agarose gel containing 1% ethyl bromide and purification of deoxyribonucleic acid fragments (AccuPrep PCR Purification kit). ). Sequence analysis was submitted to Macrogen (Macrogen, Seoul). The result is shown in [Fig. 1, SEQ ID NO: 1].

To analyze the phylogenetic relationship, the sequence was retrieved from blast (http://www.ncbi.nlm.nih.gov/BLAST) and phylogenetic tree by the neighbor-joining analysis method. ) Was established. As a result, the separation from the mold surface tangerine poma Formosan Rum Bar Cine stand Ita (Phoma pomorum var. Circinata CBS 286.76) and 99.4%, Phoma pomorum var. cyanea CBS 388.80), Phoma Pomorum Bar Pomorum pomorum var. pomorum CBS 539.66) and 99.6% homology was confirmed (FIG. 2). As a result, the isolated fungus is Phoma sp.). This is not the same as the existing strain, for the first time isolated from tangerine surface poma species (Phoma sp.) and the Korea Institute of Bioscience and Biotechnology, Korea Biotechnology Center, Korea Research Institute of Bioscience and Biotechnology, Phoma on December 9 It was named sp. and deposited under accession number KCTC 11825BP.

Example  2: Media composition and culture for fibrinase production

The fungus isolated in Example 1, incubated on a potato dextrose agar plate for 7 days at 28 ° C., was inoculated into a flask containing 200 ml potato dextrose broth and subjected to 150 rpm in a rotary shake incubator. Incubation was carried out at 7 ° C. for 7 days to form a preculture. 200 ml of the preculture was inoculated into a 4 L bioreactor containing 2 L of the main culture aseptically and incubated at 28 ° C. and pH 4.5 for 14 days. The medium composition of this culture medium was 1% (w / v) peptone, 0.005% (w / v) NaCl, 0.1% (w / v) KH ₂ PO ₄ , 0.05% (w / v) MgSO ₄ 7H ₂ O, 1% (w / v) dextrose and 10% (w / v) cellulose powder were used as a carbon source for culturing the fungus.

Example  3: Measurement of Enzyme Protein Concentration Related to Fibrin Degradation

In order to investigate the concentration of enzymes related to cellulose degradation, the culture solution of Example 2 was taken at intervals of 3, 6, and 10 days, and the supernatant and the precipitate were separated using a centrifuge, and the supernatant was used as a coenzyme solution.

Protein concentration in the enzyme solution is the standard protein using bovine serum albumin (Bradford, MM 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding) Analytical Biochemistry.72: 248-254).

Example  4 : Endoglucanase  Enzyme activity measurement

As a substrate used for endoglucanase enzyme activity measurement, 1.0% carboxymethyl cellulose (Carboxylmethylcellulose, Sigma / Aldrich) dissolved in 100 mM sodium acetate buffer (pH 4.5) was used. Enzymatic reaction was performed by using a 100 mM sodium acetate buffer solution (pH 4.5) containing 45 μl of substrate solution and 5 μl of purified enzyme for 30 minutes at 60 ° C., followed by heating at 100 ° C. for 10 minutes. Stopped. The reaction mixture was used in Sobiegyi, M. 1952. Notes on sugar determination.Journal of biological chemistry. 195: 19-23), the reducing sugar produced was measured. One unit (U) of endoglucanase enzyme activity is defined as the amount of enzyme that promotes the release of 1 μmol equivalent of glucose per minute.

Example  5: Exoglucanase  Enzyme activity measurement

In order to measure the exo-glucanase activity 10 mM p - nitrophenyl galactosyl side (p -nitrophenyl-D-lactoside, pNPL) of 100 ㎕ a substrate solution of 100 mM sodium phosphate buffer solution acetamide (pH 4.5) containing 900 ㎕ The crude enzyme solution was added and reacted at 60 ° C for 30 minutes. Thereafter, 100 μl of 2M Na ₂ CO ₃ was added to measure exoglucanase enzyme activity at 405 nm. The enzyme activity unit was expressed as 1 unit (U) the amount of enzyme required to release 1 μmol of p -nitrophenol per 1 ml of the reaction solution for 1 minute under certain conditions.

Example  6: Betaglucosidase  Enzyme activity measurement

Nitrophenyl glue nose Llano side (p -nitrophenyl-D-glucopyranoside, pNPG) 100 mM sodium phosphate buffer solution acetamide (pH 4.5) 100 in a substrate solution containing 900 ㎕ - 10 mM p to measure the beta-glucosidase activity in Put the co-enzyme solution of ㎕ was reacted for 30 minutes at 60 ℃. Thereafter, 100 μl of 2M Na ₂ CO ₃ was added to measure the enzyme activity of betaglucosidase at 405 nm. Enzyme activity unit was expressed as 1 unit (U) the amount of enzyme required to release 1 μmol of p-nitrophenol per 1 ml of the reaction solution for 1 minute under certain conditions.

As a result, the changes in enzyme activity according to the culture period measured in Examples 4 to 6 are shown in Table 2 below.

Incubation period
(Work)

Endoglucanase
(Unit / mg protein)

Exoglucanase
(Unit / mg protein)

Betaglucosidase
(Unit / mg protein)
3 58.2 1.3 25.5 6 24.3 1.7 108.9 10 10.3 2.0 163.3

Example  7: Newly  Isolated Poma  Bell ( Phoma sp Generated from.) Betaglucosidase  refine

The present inventors are betaglucosids having high activity against oligosaccharide substrates such as cellobiose or cellotetrose from the culture medium of the fungus isolated in Example 1 grown in a medium containing cellulose. The aze was purified.

The culture solution incubated for 10 days was separated from the supernatant and the precipitate using a centrifuge, and the supernatant was concentrated in an ultrafiltration apparatus equipped with a 10 kDa polyethersulfone membrane, and 20 mM Tris buffer solution (Tris-HCl) was used. buffer, pH7.0). This solution was adsorbed on a DEAE Sepharose ^™ Fast Flow column equilibrated with the same buffer (GEhealthcare, Sweden) and purified primarily betaglucosidase by applying a step gradient (1 mM-500 mM) with sodium chloride. . Thereafter, fractions having betaglucosidase activity were collected, concentrated, and dissolved in 50 mM sodium acetate buffer (pH 5.0) containing 0.15 M sodium chloride. The solution was eluted with protein using Fast Protein Liquid Chromatography (FPLC) equipped with a HiPrep 16/60 Sephacry S-300 HR column (GEhealthcare, Sweden). Among them, the fractions showing betaglucosidase activity were once again collected, concentrated and dissolved in 20 mM sodium acetate buffer (pH 4.5). The solution was adsorbed onto MonoQ ion exchange column 5/50 (GEhealthcare, Sweden) equilibrated with the same buffer, and a fraction containing betaglucosidase activity was recovered by applying a linear gradient with 0.5 M sodium chloride. Concentrated.

The yield at each purification step is shown in Table 3 below.

Example  8: refined Betaglucosidase  Determination of the molecular weight of enzyme

Sodium according to the Laemmli method (Laemmli, UK, 1970, Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227: 680-685) to determine the subunit molecular weight of the enzyme purified in Example 7. Dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed. Molecular weight measurements of native enzymes were performed with Sephacry S-300 HR using thyroglobulin (669 kDa), apoferritin (443 kDa), β-amylase (200 kDa) and albumin (66 kDa) as standard proteins. Measured by column.

3 shows a protein having a molecular weight of about 110 kDa as a result of conducting sodium dodecyl sulfate-polyacrylamide gel electrophoresis to measure molecular weight of a purified protein using a column chromatography system. (M: standard molecular weight marker, No. 1: enzyme concentrate before purification, No. 2: concentrate after MonoQ ion exchange column HR5 / 50 application). (b) was a result of measuring the native molecular weight using a gel filtration chromatography device Sephacryl S-300 HR 16/60 as a result of the purified enzyme was confirmed that the tetramer (tetramer) having a molecular weight of 440 kDa.

Example  9: Identification of Purified Enzyme by Amino Acid Sequence Analysis

In order to identify the enzyme purified in Example 7, the protein band supposed to be beta glucosidase shown on the SDS-PAGE gel was cut and decolorized, and then the probion was analyzed to analyze the N-terminal and partial amino acid sequences of the protein. (Probiond, Seoul). The purified enzyme sequenced peptide fragments cut by trypsine treatment using the MS data analysis program SEQUEST (ThermoFinnigan, USA), and homologous to other proteins based on the protein database of fungi registered with NCBI. It was confirmed.

As a result, the amino acid sequence between the purified enzyme and the fungal-derived protein registered in the NCBI was analyzed and compared to Phaeosphaeria. nodorum , Aspergillus It was confirmed that the homology with the beta-1,4-glucosidase of glycosyl hydrolase family 3 (Glycosyl hydrolase family 3) produced from aculeatus (Fig. 4).

Example  10: Optimal Purification Enzyme pH , Temperature and temperature stability

<10-1> Optimal Purification Enzyme pH

Experiments on the optimum pH for the enzyme activity purified in Example 7 used different types of 100 mM buffers in the range of pH 3 to pH 10; pH 3-6 sodium acetate buffer, pH 6-8 phosphate buffer, pH 8-10 Tris buffer.

As a result, the purified enzyme showed the highest activity at pH 4.5. The enzyme activity at pH 4.0 and 5.0 was measured to be 97% and 81% of the maximum activity, and showed no activity below pH 3.0 and above 7.0 (FIG. 5). In general, the optimal pH range of fungal-derived betaglucosidase in nature ranges from 4.0 to 6.5 (Leite RSR, Gomes E, Da-Silva R, 2007. Characterization and comparison of thermostability of purified β-glucosidases from a mesophilic Aureobasidium pullulans and a thermophilic Thermoascus aurantiacus .Process Biochemistry.42: 1101; Saha BC, Bothast RJ, 1996. Production, purification, and characterization of a highly glucose-tolerant novel β-glucosidase from Candida peltata.Applied and Environmental Microbiology. 62: 3165-3170; Yoon JJ, Kim KY, Cha CJ, 2008. Purification and characterization of thermostable β-glucosidase from the brown-rot basidiomycete Fomitopsis palustris grown on microcrystalline cellulose.Journal of Microbiology. 46: 51-55). This suggests that the enzyme isolated in the present invention is betaglucosidase.

<10-2> Optimum Temperature of Purified Enzyme

To determine the optimum temperature, the purified enzyme was reacted at different temperature ranges (30 ° C. to 90 ° C.) in 100 mM sodium acetic acid buffer (pH 4.5) containing 10 mM p -nitrophenylglucopyranoside as substrate. It measured and the result is shown in [FIG. 6].

As a result, the optimum activity was found to be 65 ° C. (FIG. 6), and the optimum temperature of 40 ° C. to 65 ° C. is a general feature of betaglucosidase derived from various fungi (Daroit DJ, Simonetti A, Hertz PF, Brandelli A, Purification and characterization of an extracellular β-glucosidase from Monascus purpureus.Journal of microbiology and Biotechnology.18: 933-941; Decker CH, Visser J, Schreier P, 2000.β-glucosidases from five black Aspergillus species: study of their physico-chemical and biocatalytic properties.Journal of Agricultural and Food Chemistry.48: 4929-4936; Murray P, Aro N, Collins C, Grassick A, Penttila M, Saloheimo M, Tuohy M, 2004.Expression in Trichoderma reesei and characterization of a thermostable family 3 β-glucosidase from the moderately thermophilic fungus Talaromyces emersonii .Protein Expression and Purification.38: 248-257; Yoon JJ, Kim KY, Cha CJ, 2008. Purification and characterization of thermostable β-glucosidase from the brown-rot basidiomycete Fomitopsis palustris grown on microcrystalline cellulose. Journal of Microbiology. 46: 51-55).

<10-3> Temperature Stability of Purified Enzyme

In order to test the temperature stability, the enzyme purified in Example 7 was stored at 30 ° C., 40 ° C., 50 ° C. and 60 ° C. for various periods of time, and then its activity was measured.

As a result, it was confirmed that the purified enzyme at each temperature was stable for at least one month (FIG. 7). The half-lives at each temperature are 3629, 2776 and 53 hours, which is higher than the previously reported temperature stability of fungal derived betaglucosidase (Montero MA, Romeu A, 1992. Kinetic study on the β-glucosidase. catalysed reaction of Trichoderma viride cellulase.Applied Microbiology and Biotechnology. 38: 350-353; Parry NJ, Beever DE, Owen E, Vandenberghe I, Van BJ, Bhat MK, 2001.Biochemical characterization and mechanism of action of a thermostable β-glucosidase purified from Thermoascus aurantiacus . Biochemical Journal. 353: 117-127; Riou C, Salmon JM, Vallier MJ, GZ, Barre P, 1998. Purification, characterization, and substrate specificity of a novel highly glucose-tolerant β-glucosidase from Aspergillus oryzae . Applied and Environmental Microbiology. 64: 3607-3614). Thichoderma The virided beta glucosidase also shows stability at 60 ° C., but has a half-life of 2.3 hours when compared to the beta glucosidase produced by the strain of the present invention.

As such, the beta glucosidase isolated from the present invention may have higher temperature stability than the conventional beta glucosidase, and thus may be more efficiently used to prepare bioethanol.

Example  11: Kinetics of Purification Enzyme and Inhibition of Enzyme Reaction

To test the kinetics ( K _m and K _cat ) of the purified enzyme in Example 7, various concentrations of p -nitrophenylglucopyranoside (0.05 mM to 1 mM) and cellobiose (0.05 mM to 1 mM) were added. The reaction was carried out at 60 ° C. for 30 minutes on 100 mM sodium acetic acid buffer (pH 4.5). Inhibition constant ( K _i ) for glucose and glucono-δ-lactone is 0-10 mM glucose and 0-1 mM with p -nitrophenylglucopyranoside as substrate. The experiment was performed by adding glucono-δ-lactone.

The reaction rate constants and inhibition constants of the resulting purified enzymes are shown in Table 4 below.

As shown in Table 4 above, K _m for p -nitrophenylglucopyranoside and cellobiose Values were 0.31 mM and 3.24 mM, respectively. This demonstrates that the betaglucosidase of the present invention has a 10-fold higher affinity for p -nitrophenylglucopyranoside than cellobiose. The results suggest that the purified betaglucosidase can be classified as aryl-betaglucosidase (aryl-BGL) with cellobiase activity.

Inhibition experiments on glucose and glucono-δ-lactone confirmed that the two substances are competitive inhibitors of betaglucosidase. Each inhibition constant is 1.69 mM and 0.09 mM (Table 4), which means that glucose acts as a stronger inhibitor of betaglucosidase than glucono-δ-lactone (Gueguen Y, Chemardin P, Arnaud A, Galzy P, 1995. Purification and characterization of an intracellular β-glucosidase from Botrytis cinerea . Enzyme and microbial technology. 17: 900-906; Lymar ES, Li B, Renganathan V, 1995. Purification and Characterization of a Cellulose-Binding β-Glucosidase from Cellulose-Degrading Cultures of Phanerochaete chrysosporium . Applied and Environmental Microbiology. 61: 2976-2980).

Example  12: Analysis of the effect on metal ions

In order to confirm the effect of various kinds of metal ions on the enzyme activity purified in Example 7, 5 mM and 10 mM of MgCl ₂ , MnCl ₂ , ZnCl ₂ , FeCl ₂ , CuCl ₂ , CoCl ₂ , and CaCl ₂ were purified. After reacting with the enzyme in advance, its activity was measured at the optimum temperature and pH.

The result was Mg ^{2 +,} on the other hand to increase the activity of the enzyme 120%, Cu ^{2 +} decreases the enzyme activity 58%. In addition, Mn ^{2 +} , Fe ^{2 +} , Co ^{2 +} , Ca ^{2 +} ions did not significantly affect the enzyme activity (FIG. 8).

Example  13: Relative Ratio Analysis of Hydrolysis of Various Substrates

p NP-glycoside family, cellobiose, carboxymethylcellulose, xylan (avilan) was used to measure the hydrolytic activity of the enzyme purified in Example 7 on various substrates. activity of p NP series is 100 mM sodium acetate buffer (pH 4.5) at 60 ℃, was reacted measurement p NP that is released during the 30 minutes, the cellobiose hydrolysis activity of 100 to cellobiose in the 5 ㎕ enzyme and 10 mM The amount of glucose produced from cellobiose by reaction in mM sodium acetate buffer (pH 4.5) was measured at 505 nm using a glucose kit (Wakp Pure Chem., Japan). One unit of cellobiose hydrolysis activity was expressed as the amount of 1 μmol of glucose released per minute under reaction conditions. Carboxymethyl cellulose, xylan and Avicel showed activity by measuring reducing sugars according to dinitrosalicylic acid (3,5-dinitrosalicylic acid, DNS) method.

The relative activity of the various substrates hydrolyzed by the resulting purification enzymes is shown in Table 5 below.

As shown in Table 5, purified beta glucosidase showed the highest activity against p -nitrophenylglucopyranoside and cellobiose, and other p NP-glycosides, carboxymethylcellulose, xylan, and avicel. It showed little activity for. These results show that the enzyme of the present invention has high substrate specificity only for p -nitrophenylglucopyranoside and cellobiose, which is a general feature of betaglucosidase in nature (Woodward J, Lima M, Lee NE, 1988.The role of cellulase concentration in determining the degree of synergism in the hydrolysis of microcrystalline cellulose.Biochemical Journal.255: 895-899).

These results suggest that the purified enzyme of the present invention acts as a betaglucosidase, and has a longer half-life than the existing betaglucosidase, thereby stably producing bioethanol.

Korea Biotechnology Research Institute KCTC11825BP 20101209

<110> KOREA INSTITUTE OF INDUSTRIAL TECHNOLOGY <120> A novel isolated Phoma sp. from rotten tangerine peel and          cellulolytic enzymes and beta-glucosidase produced by <130> PA100794 / KR <150> KR10-2009-0127178 <151> 2009-12-18 <160> 6 <170> Kopatentin 1.71 <210> 1 <211> 485 <212> DNA <213> ITS of KCTC11825BP <400> 1 atcattacct agagttgtag gctttgcctg ctatctctta cccatgtctt ttgagtacct 60 tcgtttcctc ggcgggtccg cccgccgatt ggacaattta aaccatttgc agttgcaatc 120 agcgtctgaa aaaacttaat agttacaact ttcaacaacg gatctcttgg ttctggcatc 180 gatgaagaac gcagcgaaat gcgataagta gtgtgaattg cagaattcag tgaatcatcg 240 aatctttgaa cgcacattgc gccccttggt attccatggg gcatgcctgt tcgagcgtca 300 tttgtacctt caagctctgc ttggtgttgg gtgtttgtct cgcctctgcg cgtagactcg 360 cctcgaaaca attggcagcc ggcgtattga tttcggagcg cagtacatct cgcgctctgc 420 actcataacg acgacgtcca aaagtacatt tttacactct tgacctcgga tcaggtaggg 480 atacc 485 <210> 2 <211> 9 <212> PRT <213> part of Beta-glucosidase derived from KCTC11825BP <400> 2 Gly Ile Gln Asp Ala Gly Val Ile Ala   1 5 <210> 3 <211> 7 <212> PRT <213> part of Beta-glucosidase derived from KCTC11825BP <400> 3 Ala Gly Thr Gly Ser Ile Met   1 5 <210> 4 <211> 10 <212> PRT <213> part of Beta-glucosidase derived from KCTC11825BP <400> 4 Ile Met Ala Ala Tyr Tyr Leu Val Gly Arg   1 5 10 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> ITS5 primer for ITS <400> 5 ggaagtaaaa gtcgtaacaa gg 22 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ITS4 primer for ITS <400> 6 tcctccgcta tatgatatgc 20

Claims (16)

Phoma sp. Sp., Which is an accession number KCTC 11825BP that produces fibrinase derived from fibrinous biomass including red algae, the fibrinase being an enzyme having endoglucanase or betaglucosidase activity Phosphorus strains.
delete The strain of claim 1, wherein the strain is isolated from a tangerine peel.
Accession No. KCTC 11825BP of poma strains (Phoma A composition for fibrin degradation derived from fibrin-based biomass including red algae, comprising the supernatant of the culture solution in which sp.) is cultured.
(a) culturing the strain of claim 1;
(b) filtering the strain culture to obtain a supernatant; And
(c) adding the supernatant to the fiber separated from the fibrous biomass including red algae to decompose the fiber.
(a) culturing the strain of claim 1;
(b) filtering the strain culture to obtain a supernatant; And
(c) a method for producing bioethanol by adding the supernatant to the fibers separated from the fibrous biomass including red algae to decompose the fibers.
delete delete delete delete delete delete delete (a) culturing the forma species strain having accession number KCTC 11825BP; And
(b) filtering the culture to obtain a supernatant, a method for producing an endoglucanase or betaglucosidase from a forma species strain.
delete (a) culturing the forma species strain having accession number KCTC 11825BP;
(b) filtering the culture to obtain a supernatant;
(c) separating betaglucosidase from the supernatant; And
(d) adding the isolated betaglucosidase to the fibers separated from the fibrous biomass including red algae to decompose the fibrin-derived cellobiose.

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