KR20140048904A - Cellulase fusion protein and method of degrading cellulosic material using the same - Google Patents

Abstract

The present invention provides cellulase with improve properties, and a method of degrading a cellulosic material using the same. The cellulase is fusion cellulase in which: a C terminal of one or more CBMs selected from the group consisting of a family 3 cellulose binding domain (CBM) derived from Clostridium thermocellum 35319, 4 CBM, and 30 CBM; and an N terminal of a glycosyl hydrolase family 9 (GH9) are fused. [Reference numerals] (AA) Genome DNA of C, thermocellum; (BB) Cel9A endoglucanase synthesis DNA

Description

Cellulase fusion protein and method of degrading cellulosic material using the same

It relates to a cellulase and a method for decomposing a cellulosic substance using the same.

Crop-derived cellulose substrates can be used in the production of biofuels such as ethanol. Cellulose is the most abundant carbon source in nature, but it is most difficult to degrade because of its insoluble and quasi-crystalline structure, and the presence of plant cell walls in the matrix with other polymers that limit access to the cellulose surface. Efficient decomposition of cellulose is a major limiting step for biofuel production. Thus, cellulase, which acts on insoluble cellulose, is a key enzyme in this process.

GH9 contains many cellulases isolated from various microorganisms. GH9 exhibits a wide range of substrate specificities and can be classified into subgroups A, B, C and D based on the protein molecular structure. Subgroup A contains enzymes consisting solely of catalytic modules without a cellulose binding module (CBM). Subgroup B has Family 3 CBM just downstream of the catalytic module. Subgroup C contains an N-terminal immunoglobulin (Ig)-like domain followed by one catalytic module. Subgroup D contains the Ig-like domain as well as one or more additional CBMs. Glycoside hydrolases using insoluble cellulose substrates are modular proteins comprising a catalytic module and at least one non-catalytic cellulose binding module (CBM). CBM plays an important role in the enzymatic hydrolysis of plant structure and storage polysaccharides. Currently, 43 families of CBMs are known, and these CBMs show substantial variations in ligand specificity. CBM is composed of 20-200 amino acids and is present in one, two or three domains adjacent to the C-terminus or N-terminus of the cellulase catalytic module.

Family 3 CBM, Family 4 CBM, and Family 30 CBM are commonly found with the GH9 catalytic module and, when removed from the catalytic module, result in a significant decrease in enzyme activity.

As described above, there is still a need for a cellulase having an improved effect compared to the cellulase known in the art.

One aspect is to provide a fusion cellulase with improved properties.

Another aspect is to provide a method of decomposing a cellulosic material using the cellulose.

One aspect of the present invention is Clostridium thermocellum thermocellum ) 35319-derived family 3 cellulose binding module (CBM), 4 CBM, and at least one CBM consisting of 30 CBM C-terminus and N of glycosyl hydrolase family 9: GH9 It provides a fused cellulase in which the ends are fused.

Family 3 CBM, family 4 CBM, and family 30 CBM may be those having the amino acid sequence of SEQ ID NO: 1, 2, and 3, respectively. GH9 is Alicyclobacillus acidocaldrius acidocaldrious ) It may be a GH9 endoglucanase (Cel9A) derived from ATCC27009. Cel9A may have the amino acid sequence of SEQ ID NO: 4. Cel9A is a 537 amino acid residue monomer consisting of two domains: an Ig-like domain at residues 1-85 followed by a catalytic domain at residues 86-537.

The fusion cellulase may have an amino acid sequence of any one of SEQ ID NOs: 5 to 7.

GH9 contains many cellulases isolated from various microorganisms. GH9 exhibits a wide range of substrate specificities and can be classified into subgroups A, B, C and D based on the protein molecular structure. Subgroup A contains enzymes consisting solely of catalytic modules without a cellulose binding module (CBM). Subgroup B has Family 3 CBM just downstream of the catalytic module. Subgroup C contains an N-terminal immunoglobulin (Ig)-like domain followed by one catalytic module. Subgroup D contains an Ig-like domain as well as one or more additional CBMs.

The fusion cellulase may include a sequence or moiety that can be used for separation of a fusion cellulase, such as a His6 tag sequence, at one or more ends. The sequence or moiety that can be used for the separation of the fusion cellulase does not substantially affect the activity of the fusion cellulase, but may be one that specifically binds to a specific substance. In addition, it has been shown that the (Ig)-like domain serves as a junction sequence between the catalytic modules of CBM and GH9 cellulase in fusion cellulase.

Family 3 CBM, Family 4 CBM, and Family 30 CBM are commonly found with the GH9 catalytic module and, when removed from the catalytic module, result in a significant decrease in enzyme activity.

Another aspect of the present invention provides a nucleic acid encoding the fusion cellulase or a vector comprising the nucleic acid. The vector includes anything that can be replicated in a host cell, including a plasmid or viral genome. In addition, the nucleic acid may be operably linked to a regulatory sequence. The regulation may be selected from ribosome binding sequences, Kozak sequences, promoters, operators, polyA signals, and combinations thereof.

In addition, another aspect of the present invention provides a host cell comprising the nucleic acid or vector. The host cell may be any one capable of expressing the nucleic acid. For example, the host cell may be a prokaryotic cell or a eukaryotic cell. The prokaryotic cells may be bacteria such as E. coli. The eukaryotic cells include, but are not limited to, fungal cells such as yeast cells.

Another aspect of the present invention provides a method for producing the cellulase, comprising the step of culturing the host cell. The method may further include separating the cellulase from the obtained culture. In addition, it may include the use of the obtained culture as it is or by disrupting the cells.

Another aspect of the present invention provides a method of decomposing a cellulosic material comprising the step of contacting the cellulase and a cellulosic material described above.

The contact may be made in a solid or liquid phase. The contact is at a temperature of 10° C. to 90° C., for example, a temperature of 40° C. to 90° C., a temperature of 50° C. to 90° C., a temperature of 40° C. to 80° C., a temperature of 50° C. to 80° C. or 55° C. to 80° C. It can be done in. In addition, the contacting may be performed under normal pressure or pressurized conditions.

In the contacting step, the cellulase is as described above. The cellulase may be homogeneously separated, concentrated, or contained in a culture or cell lysate.

In the contacting step, the cellulose material represents a material containing cellulose in whole or in part. The cellulosic material may be soluble or water insoluble in an aqueous medium, for example an aqueous solution. The cellulosic material may be crushed or powdered. The cellulose material may be a fibrous material, a plant used as animal feed, a wood-derived pulp, or a secondary fiber.

In the contacting step, the contacting may be performed in the presence of another glycolytic enzyme. The enzyme may be an enzyme or a combination of enzymes capable of decomposing cellulose or its degradation products into monosaccharides, for example, glucose. The enzyme may be selected from cellobiohydrolase, endoglucanase, cellodextrinase, β-glucosidase, and combinations thereof. By contacting in the presence of other enzymes, cellulose can be decomposed into fermentable sugars, and by culturing the obtained fermentable sugar as a carbon source in the presence of microorganisms, fuel materials such as ethanol can be produced.

Accordingly, the method may include converting the obtained fermentable sugar into a fuel material by culturing the obtained fermentable sugar in the presence of microorganisms. Fermentable sugars may be disaccharides or monosaccharides. The fermentable sugar may be glucose. The microorganism may be a microorganism capable of alcoholic fermentation such as methanol or ethanol, or a microorganism capable of producing a substance such as butanediol. The microorganism may be yeast or bacteria such as E. coli. The fuel material may be selected from methanol, ethanol, butanediol, and combinations thereof.

According to the fused cellulase of the present invention, it is thermophilic, has broad substrate specificity, and can have high decomposition activity even for insoluble cellulose substances.

According to the method for decomposing a cellulose substance of the present invention, the cellulose substance can be decomposed efficiently.

1 schematically shows the manufacturing process of the chimeric enzyme. CBM3 from C. thermocellum was fused to the Ig-like domain of Cel9A endoglucanase from A. acidocaldrious. The fusion of CBM4 derived from C. thermocellum with CBM30 and Cel9A endoglucanase followed the same strategy.
Figure 2 is a view showing the molecular structure of the chimeric enzyme of the present invention and other family 9 cerulases.
3 is a diagram showing the results of SDS-PAGE analysis on the expression and purification of chimeric enzymes. (A) CBM3-Cel9A, (B) CBM4-Cel9A, and (C) CBM30-Cel9A. Lanes are as follows: (W) total cell protein, (S) soluble protein, (P) cell pellet protein, (HP) His-tag purified protein, and (M) molecular weight marker.
4 is a diagram showing the optimum pH and temperature of the chimeric enzyme. Symbols indicate: Cel9A (square), CBM3-Cel9A (circle), CBM4-Cel9A (triangle), and CBM30-Cel9A (diamond). The standard deviation error is within 10%.
5 is a diagram showing the TLC analysis results of hydrolysis products for PASC, CMC, filter paper, Avicel, cellotetraose, and p-NPC. A: Extensive (5 hours) hydrolysis products of CMC and PASC, B: Time course of hydrolysis products of PASC from 0 to 150 minutes, C: Extensive (16 hours) hydrolysis products of filter paper and Avicel, D: Extensive (5 hour) hydrolysis products of cellotetraose, E: p- NPC and extensive hydrolysis products of M: As standard markers, G1 to G4 represent glucose, cellobiose, cellotriose, and cellotetraose, respectively. Show.
PASC, CMC, cellotetraose and p- NPC were treated with 0.1 nmol CBM3-Cel9A at 60°C. The same reaction was carried out on Avicel and filter paper with 0.1 nmol CBM3-Cel9A. The reaction was carried out in the absence (-) and in the presence of enzymes.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Materials and methods

Materials and methods used in the examples are as follows.

(1) Chemical substances

Avicel PH101, carboxymethyl cellulose (CMC), barley β-glucan, p- nitrophenyl-β-D-glucoside ( p- NPG) and p- nitrophenyl-β-D- cellobioside (p- NPC) Was purchased from Sigma Chemicals (St. Louis, MO, USA). TLC plates were purchased from Merck Laboratory (Germany).

(2) bacterial strains, plasmids and growth conditions

A codon optimized synthetic gene encoding Cel9A endoglucanase in plasmid pUCel9A was obtained from GenScript (NJ, USA). Seed. Thermocellum ( C. thermocellum ) Genomic DNA of ATCC 35319 was used as the family 3, 4 and 30 CBM gene source. Saccharophagus degradans ) The genomic DNA of strain 2-40 ATCC43961 was used as the processive endoglucanase Cel5H. The expression vector pET-28a(+) was purchased from Novagen (Madison, WI, USA). E. coli DH5a and E. coli BL21 (DE3) was used as a host for cloning and expression, respectively. Luria-Bertani (LB) medium (50 μg/ml) supplemented with kanamycin was used for the culture of E. coli cells.

(3) chimera The production of genes, Cloning And sequencing

It was prepared using chimeric gene 3-step overlapping PCR (Zhang et al., 2010, Appl Environ Microbiol , 76, 6870-6). 1 shows the manufacturing process of the chimeric gene. The pUCel9A plasmid was used as a template for amplification of full-length Cel9A endoglucanase.

Cellulosome-integrating protein CipA from family CBM3, cellobiohydrolase A (CbhA) from family CBM4 and advanced endoglucanase CelJ from family CBM30 from C. thermocellum 's genomic DNA by PCR. Used to amplify. The full-length gene of the progressive endoglucanase Cel5H is S. degradans It was amplified from genomic DNA of strains 2-40. The CbhA (GH9-Fn3,2-CBDIII) gene was cloned from genomic DNA of C. thermocellum using PCR. The sequence of the primers used in this example is shown in Table 1. Restriction sites were introduced at the 5'and 3'ends of each primer. The primer was Bioneer Co., Ltd. (South Korea).

primer Sequence (5'->3') ^a Restriction enzyme P1 TTGTCATATG AATTTGAAGGTTGAATTCTACAACAGCAATC (SEQ ID NO: 8) NdeI P2 GATTTAGGCACACGGCTCGG GGGTTCTTTACCCCATACAAG (SEQ ID NO: 9) P3 GGGTAAAGAACCC CCGAGCCGTGTGCCTAAATC (SEQ ID NO: 10) P1' ATGTTCATATG TTAGAAGATAATTCTTCGAC (SEQ ID NO: 11) NdeI P2' GATTTAGGCACACGGCTCGG AGGCTGCGGAAGTATATATT (SEQ ID NO: 12) P3' AATATATACTTCCGCAGCCT CCGAGCCGTGTGCCTAAATC (SEQ ID NO: 13) P1" ATTGTCATATG GCTCCTGAAGGCTACAGGAAGC (SEQ ID NO: 14) NdeI P2" GATTTAGGCACACGGCTCGG GATTGCAGGAGCGGACTTTTC (SEQ ID NO: 15) P3" CCGCTCCTGCAATC CCGAGCCGTGTGCCTAAATC (SEQ ID NO: 16) P4 CCGATCTCGAG TTAACGGCCACGAGCCTCCAG (SEQ ID NO: 17) XhoI Cel5H-F CCCGGATCC AATTCTTAGCGGTGGCCAGCAA (SEQ ID NO: 18) BamHI Cel5H-R CCCGCTCGAG CCAGCTACCAAATTGCAGGGTGT (SEQ ID NO: 19) XhoI CbhA-F CTAGCTAGC TATACTTCCGCAGCCTGAT (SEQ ID NO: 20) NheI CbhA-R TATATAAGCTT AACCGCCCGGCGGCGTTCCCCA (SEQ ID NO: 21) HindIII

a: Restriction enzyme sites are shown in bold. The portion of the overlap PCR region (14-20 bp) is underlined. P1-P3, P1'-P3' and P1"-P3" primers were used for the generation of CBM3-Cel9A, CBM4-Cel9A, and CBM30-Cel9A chimeric genes. P4 primers were commonly used for the generation of all chimeric genes.

PCR was performed using 30 consecutive cycles as follows: denaturation at 98° C. for 0.5 min, annealing at 56° C. for 0.5 min, and extension at 72° C. for a time depending on the length of the PCR product.

Phusion high-fidelity DNA polymerase (Finnzymes, USA) was used during the procedure. The PCR product was separated by 1% agarose gel electrophoresis, and extracted from the gel using a gel extraction kit. The extracted DNA fragment was digested with a restriction enzyme and ligated to the vector pET-28a(+). E. coli, chemically competent in the connecting mixture It was used to transform DH5a. Plasmids isolated from these transformants were verified by restriction analysis, and gene sequences were confirmed by DNA sequencing. Plasmids with matching sequences for Cel5H, CbhA, Cel9A, CBM3-Cel9A, CBM4-Cel9A and CBM30-Cel9A were designated as pECel5H, pECbhA, pECel9A, pECBM3-Cel9A, pECBM4-Cel9A and pECBM30-Cel9A, respectively.

Enzymes used for DNA manipulation were purchased from New England Biolabs (UK). DNA sequencing was performed by SolGent (South Korea). Genomic DNA extraction was performed according to the previously described method (Sambrook and Russell, 2001). Plasmid DNA was extracted using the Qiagen Spin Column plasmid Mini-Preps kit (USA). Restricted plasmids and PCR products were recovered from agarose gels using a Qiagen gel extraction kit (USA).

(4) chimera Protein expression and purification

E. coli with pECel5H, pECbhA, pECel9A, pECBM3-Cel9A, pECBM4-Cel9A and pECBM30-Cel9A BL21(DE3) cells were grown at 37° C. in LB medium containing kanamycin (50 μg/ml). When the culture reached A600 0.7, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.2 mM. Next, the culture was incubated overnight at 22° C. under stirring conditions (180 rpm). After induction overnight, the culture was centrifuged (10,000xg, 20 minutes, 4°C), and the cell pellet was recovered. Proteins were extracted using the CelLyticB system (Sigma) according to the manufacturer's instructions. Chimeric proteins were purified on a nickel Sepharose column pre-filled under native conditions (HisTrap, GE Healthcare/Amersham, Piscataway, NJ). The chimeric protein includes a (His) ₆ sequence at the N-terminus.

(5) enzyme analysis

Most analyzes for chimeric enzymes were performed at pH 5.0 and 70° C. with 0.1-1.0 nmol purified enzyme in reaction buffer containing 50 mM sodium acetate (pH 5.0), 200 mM NaCl, and 10 mM CaCl _2. One unit of enzyme activity was defined as the amount of enzyme that releases 1 μmol of reducing sugar per minute. The effect of pH and temperature on catalytic activity was tested in the pH range 4.0-8.0 and 10-90°C, respectively. Sodium acetate (4.0 to 5.6) and piperazine- N , N′ -bead (2-ethanesulfonic acid) (6.0 to 8.0) buffers were used to maintain the pH at a final concentration of 50 mM.

Carboxymethyl cellulose (CMC) and barley β-glucan assays were carried out for 10 minutes reaction time with 1% (w/v) substrate in 0.5 ml total volume. Phosphoric acid-swollen cellulose (PASC) was prepared according to the method described in the previous report (Wood, 1971,Biochem J, 121, 353-62; Zhang et al., 2006,Biomacromolecules, 7, 644-8). PASC (0.1%, w/v) degradation assay was performed in 0.5 ml total volume for 10 minutes. Dinitrosalicylic acid (DNS) analysis was performed to detect product formation during these reactions (Ghose, 1987,Pure Appl Chem, 59, 257-268).

Disintegration of Avicel (1%, w/v) or Whatman No. 1 filter paper (3 mg) was performed at 60° C. for 2 hours in 0.25 ml of a total volume. The reaction was stopped by incubation at 95° C. for 15 minutes, and the remaining substrate was separated by centrifugation at 10,000×g. Next, the product of Avicel or filter paper digestion was digested with 50 U of β-glucosidase (Sigma, USA) in a total volume of 0.35 ml at 37° C. for 1 hour. β-glucosidase was inactivated by incubation at 95° C. for 15 minutes. The glucose concentration in the final reaction mixture was measured using a glucose oxidase kit (GAGO-20 from Sigma, USA) according to the manufacturer's instructions. The release of cellobiose was calculated as 50% of the glucose accumulation rate (Watson et al., 2009, J Bacteriol , 191, 5697-705). Hydrolysis of p -nitrophenyl-β-D-glycoside (10 mM) and p -nitrophenyl-β-D-cellobioside (10 mM) was released at 410 nm after NaOH was added to a final concentration of 0.1 M. The concentration of p -nitrophenol was monitored and analyzed (Eckert et al., 2002, Appl Microbiol Biotechnol , 60, 428-36). The viscosity of the CMC solution was measured using a Brookfield DV-III viscometer (USA). Measurements were performed using a second number spindle at 25° C. with 100 rpm motor speed. Cellulase was added to a 0.5% (wt/v) CMC solution in an amount of 0.1 nmol, incubated at 60° C. for 30 minutes, and used for viscosity measurement.

(6) protein quantification and SDS - PAGE analysis

Protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermoscientific, USA) using purified bovine serum albumin (BSA) as a standard. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed on a vertical polyacrylamide slab gel.

Electrophoresis was performed on 5% stacking gel and 12% polyacrylamide gel in denaturing conditions. The buffer solutions for the stacking gel and polyacrylamide gel were 1 M Tris-HCl (pH 6.8) and 1.5 M Tris-HCl (pH 8.8), respectively.

(7) Cellulase Binding analysis

Avicel binding properties of chimeric enzymes were studied as previously described with minor modifications (Li et al., 2007, Appl. Environ Microbiol , 73, 3165-72). Binding assays were performed in 2.0 ml tubes. 1 mg of Avicel was mixed with 0.5% BSA in sodium acetate buffer (20 mM, pH 5). The mixture was incubated at 25° C. for 30 minutes to prevent non-specific binding. To this solution an equal amount (2 nmol) of natural or chimeric enzyme was added.

Next, it was placed in an intelli-mixer RM-2 (Rose Scientific Ltd., Canada) for 1 hour at a rotational speed of 20 rmp. The reaction mixture was centrifuged at 10,000×g for 5 minutes, and the amount of unbound enzyme was estimated from the residual cellulase activity in the supernatant. The amount of Avicel binding enzyme was calculated from the difference between the initial and unbound enzyme activity. The control reaction was performed under the same conditions, except that Avicel was not present. In addition, Avicel binding protein was qualitatively analyzed using SDS-PAGE.

(8) Cellulase Progress ( processivity ) Analysis and synergistic interaction

Cellulase progression was evaluated by measuring the ratio of soluble reducing sugar to insoluble reducing sugar according to the process of Watson et al. (Watson et al., 2009, J Bacteriol , 191, 5697-705). The ratio was obtained as μmol of soluble reducing sugar (cellobiose standard) divided by μmol of insoluble sugar (glucose standard). Filter paper was used as a substrate for the cellulase progression test. After hydrolysis and centrifugation of filter paper, the soluble reducing sugar in the supernatant fraction was measured using the glucose oxidase method as described above. Insoluble reducing sugar in the precipitate fraction was analyzed by modified 2,2-bicinchoninate (Doner and Irwin, 1992, Anal. Biochem , 202, 50-3). The precipitated filter paper was washed with 6 M guanidine hydrochloride to remove unbound cellulase, and then washed again 4 times with assay buffer and water. The reducing sugar of the treated filter paper was measured using Pierce microBCA reagent (USA) using glucose as a standard. 1 nmol of CBM3-Cel9A chimeric enzyme and S. degradans It was investigated using Cel5H endoglucanase. The synergistic interaction between CBM3-Cel9A and CbhA on filter paper was investigated. The reaction mixture consisted of 7 mg filter paper disks, and an equal molar mixture (1:1) of CBM3-Cel9A and CbhA in reaction buffer containing 50 mM sodium acetate (pH 5.0), 200 mM NaCl, and 10 mM CaCl _2. The reaction was carried out at 60° C. for 10 hours, and the reducing sugar product was measured by the DNS method. As a control, the same reaction was carried out using Cel9A instead of CBM3-Cel9A. The degree of synergistic effect (DSE) was defined as the ratio of the observed activity of the combined enzyme to the sum of the observed individual activities. All enzyme activity assays were performed in triplicate, and data are presented as mean±standard deviation.

(9) thin layer chromatography

Sample 1 microliter was spotted on a silica 60 TLC plate (Merck, USA) and air dried. The chromatogram was developed using nitromethane, 1-propanol, and water (2:5:1.5) (v/v/v) (Watson et al., 2009, J Bacteriol , 191, 5697-705). The TLC plate was immersed in a mixture of 0.3% (w/v) α-naphthol and 5% (v/v) sulfuric acid in methanol and heated to 110° C. for 10 minutes to visualize the partitioned product.

Example One: chimera Preparation and purification of enzymes

C. thermocellum Derived families 3, 4 and 30 CBM were separately fused to the N-terminus of Cel9A endoglucanase through overlap PCR technique as described in FIG. 1. The molecular architecture of the chimeric enzyme and the comparison with the previously reported GH9 family cellulase are shown in FIG. 2. Next, the fused gene was inserted into the expression vector pET-28a(+) encoding the _{N-terminal His 6 tag.} E. coli It was used for protein expression in BL21 (DE3). The expressed protein was extracted using CelLyticB reagent. Soluble expression of the protein was confirmed by SDS-PAGE (Fig. 3). These proteins were purified using a nickel Sepharose column pre-filled in natural conditions. Binding was achieved in the presence of 20 mM imidazole and washed with 1× sodium phosphate buffer (pH 7.5-8.0) containing 40 mM imidazole. Finally, it was eluted using 250 mM imidazole. Fractions containing the desired protein were pooled and dialyzed overnight at 4° C. against 50 mM sodium phosphate buffer (pH 7.0). Finally, the enzyme was stored in 50 mM piperazine-N , N′ -bis(2-ethanesulfonic acid) buffer (pH 7.0) containing 10% glycerol. Each of the purified preparations showed a single band on SDS-PAGE and the molecular weight was in good agreement with that calculated from the nucleotide sequence (FIG. 3 ).

Example 2: chimera Enzymatic properties of enzymes

The optimum temperature and pH of the chimeric enzyme were determined in a temperature range of 10 to 90°C and a pH range of 4.0 to 8.0 (FIG. 4). The temperature change experimental conditions of FIG. 4A used 1% (w/v) CMC as a substrate at pH 5.0, and the reaction solution composition included 50 mM sodium acetate (pH 5.0), 200 mM NaCl, and 10 mM CaCl ₂ . As for the pH change experimental conditions of FIG. 4B, a substrate 1% (w/v) CMC was used at 70° C. and a corresponding buffer was used according to the pH range. For comparison purposes, Cel9A endoglucanase was also used in a similar assay. As a result, all chimeric enzymes showed an optimum pH of 5.0, while natural Cel9A endoglucanase showed an optimum pH of 5.5. Not surprisingly, all natural and chimeric enzymes showed the same optimum temperature of 70°C. When stored overnight at 4° C., the chimeric enzyme was stable in the pH range 5.0-8.0. On the other hand, there was a slight loss of enzyme activity after 1 hour at 60°C. The half-life of native Cel9A endoglucanase was 30 minutes at 75° C., whereas the half-life of chimeric enzymes was 20 minutes at the same temperature.

The chimeric enzyme had broad substrate specificity and efficiently hydrolyzed both soluble as well as insoluble cellulose substrates (Table 2).

temperament Inert (U/μmol) Cel9A
(59kDa) CBM3-Cel9A
(76kDa) CBM4-Cel9A
(80kDa) CBM30-Cel9A
(80kDa) CMC 4,325±70 6,600±90 7,320±80 7,720±90 Barley β-glucan 11,800±200 12,800±190 15,000±170 15,000±180 PASC 649±50 1,000±60 1,260±70 1,280±70 Avicel 0.50±0.09 6.0±0.9 4.0±0.7 5.0±0.8 Filter paper 0.70±0.08 7.0±1.0 5.4±1.0 6.0±0.9 pNPG ND ^a ND ^a ND ^a ND ^a pNPC 118±30 120±30 130±20 135±20

^a : no activity is detected

The catalytic activity of CBM4-Cel9A and CBM30-Cel9A chimeric enzymes on CMC increased 1.7 and 1.8 times compared to that of native Cel9A endoglucanase. Compared to the natural enzyme, the catalytic activity of the chimeric enzyme for PASC was increased by 2 times. The catalytic activity of chimeric enzymes against insoluble polysaccharides (Avicel and filter paper) was significantly increased by 7.7-12.0 times compared to that of natural enzymes. Among all chimeric enzymes, CBM3-Cel9A chimeric enzyme showed the highest catalytic activity against insoluble polysaccharides. However, there was no significant increase in the enzymatic activity of the chimeric enzyme against p-NPC. There was no detectable activity against p- NPG in all enzymes.

A viscometric assay of CMC degradation was performed to investigate the endoglucanase activity of the chimeric protein. The viscosity of the CMC solution (0.5%, w/v) decreased after incubation with CBM3-Cel9A chimeric enzyme. The viscosity of CMC decreased from 80 cP to 10 cP within 30 minutes of incubation. A significant decrease in CMC viscosity indicates the endoglucanase activity of the chimeric protein. The Avicel binding affinity of chimeric proteins was determined to find a correlation with their increased catalytic activity for insoluble polysaccharides. Compared to the native Cel9A endoglucanase, the chimeric enzyme showed the highest affinity for Avicel. The binding rates of Cel9A, CBM3-Cel9A, CBM4-Cel9A and CBM30-Cel9A were 5%, 50%, 35% and 37%, respectively.

Example 3: Analysis of hydrolysis products and progression

Hydrolysis products of CMC, PASC, filter paper, Avicel, cellotetraose, and p- NPC by CBM3-Cel9A were qualitatively analyzed by TLC (FIG. 5). Cellobiose and cellotetraose were detected after hydrolysis of CMC, PASC, filter paper, and Avicel. In the time-dependent reaction of PASC hydrolysis (Fig. 5B), both cellobiose and cellotetraose were observed at all times. It is believed that chimeric enzymes simultaneously produce cellobiose and cellotetraose as cleavage products. In the reaction with time, the intensity of the cellobiose spot on TLC increased with the increase from 30 minutes to 150 minutes of the reaction time, while there was no significant change in the intensity of the cellotetraose spot. This indicates that cellotetraose is further decomposed into cellobiose by the chimeric enzyme. When cellotetraose and p- NPC were used as substrates (Figs. 5D and E), the chimeric enzyme hydrolyzed the substrate efficiently and simultaneously released cellobiose. These results suggest that, unlike the conventional properties of endoglucanase, which produces cellodextrin as the main hydrolysis product, chimeric enzymes tend to hydrolyze cellulose to cellobiose. The production of cellobiose can be an indication of processivity. To evaluate the progression of the CBM3-Cel9A chimeric enzyme, the concentrations of soluble and insoluble sugars were measured after hydrolysis of filter paper, and S. degradans It was compared to the known advanced endoglucanase Cel5H from. CBM3-Cel9A chimeric enzyme produced 81% soluble reducing sugar and 19% insoluble reducing sugar 2 hours after hydrolysis of the filter paper. The Cel5H reference enzyme produced 80% soluble reducing sugar and 20% insoluble reducing sugar. The progression of chimeric enzyme and Cel5H was 4.26 and 4.0, respectively.

<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION GYENGSANG UNIVERSITY <120> Cellulase fusion protein and method of degrading cellulosic material using the same <130> PN0104715 <160> 21 <170> KopatentIn 2.0 <210> 1 <211> 155 <212> PRT <213> Clostridium thermocellum <400> 1 Asn Leu Lys Val Glu Phe Tyr Asn Ser Asn Pro Ser Asp Thr Thr Asn 1 5 10 15 Ser Ile Asn Pro Gln Phe Lys Val Thr Asn Thr Gly Ser Ser Ala Ile 20 25 30 Asp Leu Ser Lys Leu Thr Leu Arg Tyr Tyr Tyr Thr Val Asp Gly Gln 35 40 45 Lys Asp Gln Thr Phe Trp Cys Asp His Ala Ala Ile Ile Gly Ser Asn 50 55 60 Gly Ser Tyr Asn Gly Ile Thr Ser Asn Val Lys Gly Thr Phe Val Lys 65 70 75 80 Met Ser Ser Ser Thr Asn Asn Ala Asp Thr Tyr Leu Glu Ile Ser Phe 85 90 95 Thr Gly Gly Thr Leu Glu Pro Gly Ala His Val Gln Ile Gln Gly Arg 100 105 110 Phe Ala Lys Asn Asp Trp Ser Asn Tyr Thr Gln Ser Asn Asp Tyr Ser 115 120 125 Phe Lys Ser Arg Ser Gln Phe Val Glu Trp Asp Gln Val Thr Ala Tyr 130 135 140 Leu Asn Gly Val Leu Val Trp Gly Lys Glu Pro 145 150 155 <210> 2 <211> 175 <212> PRT <213> Clostridium thermocellum <400> 2 Met Leu Glu Asp Asn Ser Ser Thr Leu Pro Pro Tyr Lys Asn Asp Leu 1 5 10 15 Leu Tyr Glu Arg Thr Phe Asp Glu Gly Leu Cys Tyr Pro Trp His Thr 20 25 30 Cys Glu Asp Ser Gly Gly Lys Cys Ser Phe Asp Val Val Asp Val Pro 35 40 45 Gly Gln Pro Gly Asn Lys Ala Phe Ala Val Thr Val Leu Asp Lys Gly 50 55 60 Gln Asn Arg Trp Ser Val Gln Met Arg His Arg Gly Leu Thr Leu Glu 65 70 75 80 Gln Gly His Thr Tyr Arg Val Arg Leu Lys Ile Trp Ala Asp Ala Ser 85 90 95 Cys Lys Val Tyr Ile Lys Ile Gly Gln Met Gly Glu Pro Tyr Ala Glu 100 105 110 Tyr Trp Asn Asn Lys Trp Ser Pro Tyr Thr Leu Thr Ala Gly Lys Val 115 120 125 Leu Glu Ile Asp Glu Thr Phe Val Met Asp Lys Pro Thr Asp Asp Thr 130 135 140 Cys Glu Phe Thr Phe His Leu Gly Gly Glu Leu Ala Ala Thr Pro Pro 145 150 155 160 Tyr Thr Val Tyr Leu Asp Asp Val Ser Leu Tyr Asp Pro Glu Tyr 165 170 175 <210> 3 <211> 185 <212> PRT <213> Clostridium thermocellum <400> 3 Ala Pro Glu Gly Tyr Arg Lys Leu Leu Asp Val Gln Ile Phe Lys Asp 1 5 10 15 Ser Pro Val Val Gly Trp Ser Gly Ser Gly Met Gly Glu Leu Glu Thr 20 25 30 Ile Gly Asp Thr Leu Pro Val Asp Thr Thr Val Thr Tyr Asn Gly Leu 35 40 45 Pro Thr Leu Arg Leu Asn Val Gln Thr Thr Val Gln Ser Gly Trp Trp 50 55 60 Ile Ser Leu Leu Thr Leu Arg Gly Trp Asn Thr His Asp Leu Ser Gln 65 70 75 80 Tyr Val Glu Asn Gly Tyr Leu Glu Phe Asp Ile Lys Gly Lys Glu Gly 85 90 95 Gly Glu Asp Phe Val Ile Gly Phe Arg Asp Lys Val Tyr Glu Arg Val 100 105 110 Tyr Gly Leu Glu Ile Asp Val Thr Thr Val Ile Ser Asn Tyr Val Thr 115 120 125 Val Thr Thr Asp Trp Gln His Val Lys Ile Pro Leu Arg Asp Leu Met 130 135 140 Lys Ile Asn Asn Gly Phe Asp Pro Ser Ser Val Thr Cys Leu Val Phe 145 150 155 160 Ser Lys Arg Tyr Ala Asp Pro Phe Thr Val Trp Phe Ser Asp Ile Lys 165 170 175 Ile Thr Ser Glu Asp Asn Glu Lys Ser 180 185 <210> 4 <211> 537 <212> PRT <213> Clostridium thermocellum <400> 4 Met Pro Ser Arg Val Pro Lys Ser Ile Phe Tyr Asn Gln Val Gly Tyr 1 5 10 15 Leu Ile Ser Gly Asp Lys Arg Phe Trp Ile Gln Ala His Glu Pro Gln 20 25 30 Pro Phe Ala Leu Arg Thr Pro Glu Gly Gln Ala Val Phe Ala Gly Met 35 40 45 Thr Lys Pro Val Gly Gly Asn Trp Tyr Val Gly Asp Phe Thr Ala Leu 50 55 60 Arg Val Pro Gly Thr Tyr Thr Leu Thr Val Gly Thr Leu Glu Ala Arg 65 70 75 80 Val Val Ile His Arg Arg Ala Tyr Arg Asp Val Leu Glu Ala Met Leu 85 90 95 Arg Phe Phe Asp Tyr Gln Leu Cys Gly Val Val Leu Pro Glu Asp Glu 100 105 110 Ala Gly Pro Trp Ala His Gly Ala Cys His Thr Ser Asp Ala Lys Val 115 120 125 Phe Gly Thr Glu Arg Ala Leu Ala Cys Pro Gly Gly Trp His Asp Ala 130 135 140 Gly Asp Tyr Gly Lys Tyr Thr Val Pro Ala Ala Lys Ala Val Ala Asp 145 150 155 160 Leu Leu Leu Ala His Glu Tyr Phe Pro Ala Ala Leu Ala His Val Arg 165 170 175 Pro Met Arg Ser Val His Arg Ala Pro His Leu Pro Pro Ala Leu Glu 180 185 190 Val Ala Arg Glu Glu Ile Ala Trp Leu Leu Thr Met Gln Asp Pro Ala 195 200 205 Thr Gly Gly Val Tyr His Lys Val Thr Thr Pro Ser Phe Pro Pro Leu 210 215 220 Asp Thr Arg Pro Glu Asp Asp Asp Ala Pro Leu Val Leu Ser Pro Ile 225 230 235 240 Ser Tyr Ala Ala Thr Ala Thr Phe Cys Ala Ala Met Ala His Ala Ala 245 250 255 Leu Val Tyr Arg Pro Phe Asp Pro Ala Leu Ser Ser Cys Cys Ala Asp 260 265 270 Ala Ala Arg Arg Ala Tyr Ala Trp Leu Gly Ala His Glu Met Gln Pro 275 280 285 Phe His Asn Pro Asp Gly Ile Leu Thr Gly Glu Tyr Gly Asp Ala Glu 290 295 300 Leu Arg Asp Glu Leu Leu Trp Ala Ser Cys Ala Leu Leu Arg Met Thr 305 310 315 320 Gly Asp Ser Ala Trp Ala Arg Val Cys Glu Pro Leu Leu Asp Leu Asp 325 330 335 Leu Pro Trp Glu Leu Gly Trp Ala Asp Val Ala Leu Tyr Gly Val Met 340 345 350 Asp Tyr Leu Arg Thr Pro Arg Ala Ala Val Ser Asp Asp Val Arg Asn 355 360 365 Lys Val Lys Ser Arg Leu Leu Arg Glu Leu Asp Ala Leu Ala Ala Met 370 375 380 Ala Glu Ser His Pro Phe Gly Ile Pro Met Arg Asp Asp Asp Phe Ile 385 390 395 400 Trp Gly Ser Asn Met Val Leu Leu Asn Arg Ala Met Ala Phe Leu Leu 405 410 415 Ala Glu Gly Val Gly Val Leu His Pro Ala Ala His Thr Val Ala Gln 420 425 430 Arg Ala Ala Asp Tyr Leu Phe Gly Ala Asn Pro Leu Gly Gln Cys Tyr 435 440 445 Val Thr Gly Phe Gly Gln Arg Pro Val Arg His Pro His His Arg Pro 450 455 460 Ser Val Ala Asp Asp Val Asp His Pro Val Pro Gly Met Val Val Gly 465 470 475 480 Gly Pro Asn Arg His Leu Gln Asp Glu Ile Ala Arg Ala Gln Leu Ala 485 490 495 Gly Arg Pro Ala Met Glu Ala Tyr Ile Asp His Gln Asp Ser Tyr Ser 500 505 510 Thr Asn Glu Val Ala Val Tyr Trp Asn Ser Pro Ala Val Phe Val Ile 515 520 525 Ala Ala Leu Leu Glu Ala Arg Gly Arg 530 535 <210> 5 <211> 692 <212> PRT <213> Artificial Sequence <220> <223> CBM3-Cel9A cellulase fusion protein <400> 5 Asn Leu Lys Val Glu Phe Tyr Asn Ser Asn Pro Ser Asp Thr Thr Asn 1 5 10 15 Ser Ile Asn Pro Gln Phe Lys Val Thr Asn Thr Gly Ser Ser Ala Ile 20 25 30 Asp Leu Ser Lys Leu Thr Leu Arg Tyr Tyr Tyr Thr Val Asp Gly Gln 35 40 45 Lys Asp Gln Thr Phe Trp Cys Asp His Ala Ala Ile Ile Gly Ser Asn 50 55 60 Gly Ser Tyr Asn Gly Ile Thr Ser Asn Val Lys Gly Thr Phe Val Lys 65 70 75 80 Met Ser Ser Ser Thr Asn Asn Ala Asp Thr Tyr Leu Glu Ile Ser Phe 85 90 95 Thr Gly Gly Thr Leu Glu Pro Gly Ala His Val Gln Ile Gln Gly Arg 100 105 110 Phe Ala Lys Asn Asp Trp Ser Asn Tyr Thr Gln Ser Asn Asp Tyr Ser 115 120 125 Phe Lys Ser Arg Ser Gln Phe Val Glu Trp Asp Gln Val Thr Ala Tyr 130 135 140 Leu Asn Gly Val Leu Val Trp Gly Lys Glu Pro Met Pro Ser Arg Val 145 150 155 160 Pro Lys Ser Ile Phe Tyr Asn Gln Val Gly Tyr Leu Ile Ser Gly Asp 165 170 175 Lys Arg Phe Trp Ile Gln Ala His Glu Pro Gln Pro Phe Ala Leu Arg 180 185 190 Thr Pro Glu Gly Gln Ala Val Phe Ala Gly Met Thr Lys Pro Val Gly 195 200 205 Gly Asn Trp Tyr Val Gly Asp Phe Thr Ala Leu Arg Val Pro Gly Thr 210 215 220 Tyr Thr Leu Thr Val Gly Thr Leu Glu Ala Arg Val Val Ile His Arg 225 230 235 240 Arg Ala Tyr Arg Asp Val Leu Glu Ala Met Leu Arg Phe Phe Asp Tyr 245 250 255 Gln Leu Cys Gly Val Val Leu Pro Glu Asp Glu Ala Gly Pro Trp Ala 260 265 270 His Gly Ala Cys His Thr Ser Asp Ala Lys Val Phe Gly Thr Glu Arg 275 280 285 Ala Leu Ala Cys Pro Gly Gly Trp His Asp Ala Gly Asp Tyr Gly Lys 290 295 300 Tyr Thr Val Pro Ala Ala Lys Ala Val Ala Asp Leu Leu Leu Ala His 305 310 315 320 Glu Tyr Phe Pro Ala Ala Leu Ala His Val Arg Pro Met Arg Ser Val 325 330 335 His Arg Ala Pro His Leu Pro Pro Ala Leu Glu Val Ala Arg Glu Glu 340 345 350 Ile Ala Trp Leu Leu Thr Met Gln Asp Pro Ala Thr Gly Gly Val Tyr 355 360 365 His Lys Val Thr Thr Pro Ser Phe Pro Pro Leu Asp Thr Arg Pro Glu 370 375 380 Asp Asp Asp Ala Pro Leu Val Leu Ser Pro Ile Ser Tyr Ala Ala Thr 385 390 395 400 Ala Thr Phe Cys Ala Ala Met Ala His Ala Ala Leu Val Tyr Arg Pro 405 410 415 Phe Asp Pro Ala Leu Ser Ser Cys Cys Ala Asp Ala Ala Arg Arg Ala 420 425 430 Tyr Ala Trp Leu Gly Ala His Glu Met Gln Pro Phe His Asn Pro Asp 435 440 445 Gly Ile Leu Thr Gly Glu Tyr Gly Asp Ala Glu Leu Arg Asp Glu Leu 450 455 460 Leu Trp Ala Ser Cys Ala Leu Leu Arg Met Thr Gly Asp Ser Ala Trp 465 470 475 480 Ala Arg Val Cys Glu Pro Leu Leu Asp Leu Asp Leu Pro Trp Glu Leu 485 490 495 Gly Trp Ala Asp Val Ala Leu Tyr Gly Val Met Asp Tyr Leu Arg Thr 500 505 510 Pro Arg Ala Ala Val Ser Asp Asp Val Arg Asn Lys Val Lys Ser Arg 515 520 525 Leu Leu Arg Glu Leu Asp Ala Leu Ala Ala Met Ala Glu Ser His Pro 530 535 540 Phe Gly Ile Pro Met Arg Asp Asp Asp Phe Ile Trp Gly Ser Asn Met 545 550 555 560 Val Leu Leu Asn Arg Ala Met Ala Phe Leu Leu Ala Glu Gly Val Gly 565 570 575 Val Leu His Pro Ala Ala His Thr Val Ala Gln Arg Ala Ala Asp Tyr 580 585 590 Leu Phe Gly Ala Asn Pro Leu Gly Gln Cys Tyr Val Thr Gly Phe Gly 595 600 605 Gln Arg Pro Val Arg His Pro His His Arg Pro Ser Val Ala Asp Asp 610 615 620 Val Asp His Pro Val Pro Gly Met Val Val Gly Gly Pro Asn Arg His 625 630 635 640 Leu Gln Asp Glu Ile Ala Arg Ala Gln Leu Ala Gly Arg Pro Ala Met 645 650 655 Glu Ala Tyr Ile Asp His Gln Asp Ser Tyr Ser Thr Asn Glu Val Ala 660 665 670 Val Tyr Trp Asn Ser Pro Ala Val Phe Val Ile Ala Ala Leu Leu Glu 675 680 685 Ala Arg Gly Arg 690 <210> 6 <211> 712 <212> PRT <213> Artificial Sequence <220> <223> CBM4-Cel9A cellulase fusion protein <400> 6 Met Leu Glu Asp Asn Ser Ser Thr Leu Pro Pro Tyr Lys Asn Asp Leu 1 5 10 15 Leu Tyr Glu Arg Thr Phe Asp Glu Gly Leu Cys Tyr Pro Trp His Thr 20 25 30 Cys Glu Asp Ser Gly Gly Lys Cys Ser Phe Asp Val Val Asp Val Pro 35 40 45 Gly Gln Pro Gly Asn Lys Ala Phe Ala Val Thr Val Leu Asp Lys Gly 50 55 60 Gln Asn Arg Trp Ser Val Gln Met Arg His Arg Gly Leu Thr Leu Glu 65 70 75 80 Gln Gly His Thr Tyr Arg Val Arg Leu Lys Ile Trp Ala Asp Ala Ser 85 90 95 Cys Lys Val Tyr Ile Lys Ile Gly Gln Met Gly Glu Pro Tyr Ala Glu 100 105 110 Tyr Trp Asn Asn Lys Trp Ser Pro Tyr Thr Leu Thr Ala Gly Lys Val 115 120 125 Leu Glu Ile Asp Glu Thr Phe Val Met Asp Lys Pro Thr Asp Asp Thr 130 135 140 Cys Glu Phe Thr Phe His Leu Gly Gly Glu Leu Ala Ala Thr Pro Pro 145 150 155 160 Tyr Thr Val Tyr Leu Asp Asp Val Ser Leu Tyr Asp Pro Glu Tyr Met 165 170 175 Pro Ser Arg Val Pro Lys Ser Ile Phe Tyr Asn Gln Val Gly Tyr Leu 180 185 190 Ile Ser Gly Asp Lys Arg Phe Trp Ile Gln Ala His Glu Pro Gln Pro 195 200 205 Phe Ala Leu Arg Thr Pro Glu Gly Gln Ala Val Phe Ala Gly Met Thr 210 215 220 Lys Pro Val Gly Gly Asn Trp Tyr Val Gly Asp Phe Thr Ala Leu Arg 225 230 235 240 Val Pro Gly Thr Tyr Thr Leu Thr Val Gly Thr Leu Glu Ala Arg Val 245 250 255 Val Ile His Arg Arg Ala Tyr Arg Asp Val Leu Glu Ala Met Leu Arg 260 265 270 Phe Phe Asp Tyr Gln Leu Cys Gly Val Val Leu Pro Glu Asp Glu Ala 275 280 285 Gly Pro Trp Ala His Gly Ala Cys His Thr Ser Asp Ala Lys Val Phe 290 295 300 Gly Thr Glu Arg Ala Leu Ala Cys Pro Gly Gly Trp His Asp Ala Gly 305 310 315 320 Asp Tyr Gly Lys Tyr Thr Val Pro Ala Ala Lys Ala Val Ala Asp Leu 325 330 335 Leu Leu Ala His Glu Tyr Phe Pro Ala Ala Leu Ala His Val Arg Pro 340 345 350 Met Arg Ser Val His Arg Ala Pro His Leu Pro Pro Ala Leu Glu Val 355 360 365 Ala Arg Glu Glu Ile Ala Trp Leu Leu Thr Met Gln Asp Pro Ala Thr 370 375 380 Gly Gly Val Tyr His Lys Val Thr Thr Pro Ser Phe Pro Pro Leu Asp 385 390 395 400 Thr Arg Pro Glu Asp Asp Asp Ala Pro Leu Val Leu Ser Pro Ile Ser 405 410 415 Tyr Ala Ala Thr Ala Thr Phe Cys Ala Ala Met Ala His Ala Ala Leu 420 425 430 Val Tyr Arg Pro Phe Asp Pro Ala Leu Ser Ser Cys Cys Ala Asp Ala 435 440 445 Ala Arg Arg Ala Tyr Ala Trp Leu Gly Ala His Glu Met Gln Pro Phe 450 455 460 His Asn Pro Asp Gly Ile Leu Thr Gly Glu Tyr Gly Asp Ala Glu Leu 465 470 475 480 Arg Asp Glu Leu Leu Trp Ala Ser Cys Ala Leu Leu Arg Met Thr Gly 485 490 495 Asp Ser Ala Trp Ala Arg Val Cys Glu Pro Leu Leu Asp Leu Asp Leu 500 505 510 Pro Trp Glu Leu Gly Trp Ala Asp Val Ala Leu Tyr Gly Val Met Asp 515 520 525 Tyr Leu Arg Thr Pro Arg Ala Ala Val Ser Asp Asp Val Arg Asn Lys 530 535 540 Val Lys Ser Arg Leu Leu Arg Glu Leu Asp Ala Leu Ala Ala Met Ala 545 550 555 560 Glu Ser His Pro Phe Gly Ile Pro Met Arg Asp Asp Asp Phe Ile Trp 565 570 575 Gly Ser Asn Met Val Leu Leu Asn Arg Ala Met Ala Phe Leu Leu Ala 580 585 590 Glu Gly Val Gly Val Leu His Pro Ala Ala His Thr Val Ala Gln Arg 595 600 605 Ala Ala Asp Tyr Leu Phe Gly Ala Asn Pro Leu Gly Gln Cys Tyr Val 610 615 620 Thr Gly Phe Gly Gln Arg Pro Val Arg His Pro His His Arg Pro Ser 625 630 635 640 Val Ala Asp Asp Val Asp His Pro Val Pro Gly Met Val Val Gly Gly 645 650 655 Pro Asn Arg His Leu Gln Asp Glu Ile Ala Arg Ala Gln Leu Ala Gly 660 665 670 Arg Pro Ala Met Glu Ala Tyr Ile Asp His Gln Asp Ser Tyr Ser Thr 675 680 685 Asn Glu Val Ala Val Tyr Trp Asn Ser Pro Ala Val Phe Val Ile Ala 690 695 700 Ala Leu Leu Glu Ala Arg Gly Arg 705 710 <210> 7 <211> 722 <212> PRT <213> Artificial Sequence <220> <223> CBM30-Cel9A cellulase fusion protein <400> 7 Ala Pro Glu Gly Tyr Arg Lys Leu Leu Asp Val Gln Ile Phe Lys Asp 1 5 10 15 Ser Pro Val Val Gly Trp Ser Gly Ser Gly Met Gly Glu Leu Glu Thr 20 25 30 Ile Gly Asp Thr Leu Pro Val Asp Thr Thr Val Thr Tyr Asn Gly Leu 35 40 45 Pro Thr Leu Arg Leu Asn Val Gln Thr Thr Val Gln Ser Gly Trp Trp 50 55 60 Ile Ser Leu Leu Thr Leu Arg Gly Trp Asn Thr His Asp Leu Ser Gln 65 70 75 80 Tyr Val Glu Asn Gly Tyr Leu Glu Phe Asp Ile Lys Gly Lys Glu Gly 85 90 95 Gly Glu Asp Phe Val Ile Gly Phe Arg Asp Lys Val Tyr Glu Arg Val 100 105 110 Tyr Gly Leu Glu Ile Asp Val Thr Thr Val Ile Ser Asn Tyr Val Thr 115 120 125 Val Thr Thr Asp Trp Gln His Val Lys Ile Pro Leu Arg Asp Leu Met 130 135 140 Lys Ile Asn Asn Gly Phe Asp Pro Ser Ser Val Thr Cys Leu Val Phe 145 150 155 160 Ser Lys Arg Tyr Ala Asp Pro Phe Thr Val Trp Phe Ser Asp Ile Lys 165 170 175 Ile Thr Ser Glu Asp Asn Glu Lys Ser Met Pro Ser Arg Val Pro Lys 180 185 190 Ser Ile Phe Tyr Asn Gln Val Gly Tyr Leu Ile Ser Gly Asp Lys Arg 195 200 205 Phe Trp Ile Gln Ala His Glu Pro Gln Pro Phe Ala Leu Arg Thr Pro 210 215 220 Glu Gly Gln Ala Val Phe Ala Gly Met Thr Lys Pro Val Gly Gly Asn 225 230 235 240 Trp Tyr Val Gly Asp Phe Thr Ala Leu Arg Val Pro Gly Thr Tyr Thr 245 250 255 Leu Thr Val Gly Thr Leu Glu Ala Arg Val Val Ile His Arg Arg Ala 260 265 270 Tyr Arg Asp Val Leu Glu Ala Met Leu Arg Phe Phe Asp Tyr Gln Leu 275 280 285 Cys Gly Val Val Leu Pro Glu Asp Glu Ala Gly Pro Trp Ala His Gly 290 295 300 Ala Cys His Thr Ser Asp Ala Lys Val Phe Gly Thr Glu Arg Ala Leu 305 310 315 320 Ala Cys Pro Gly Gly Trp His Asp Ala Gly Asp Tyr Gly Lys Tyr Thr 325 330 335 Val Pro Ala Ala Lys Ala Val Ala Asp Leu Leu Leu Ala His Glu Tyr 340 345 350 Phe Pro Ala Ala Leu Ala His Val Arg Pro Met Arg Ser Val His Arg 355 360 365 Ala Pro His Leu Pro Pro Ala Leu Glu Val Ala Arg Glu Glu Ile Ala 370 375 380 Trp Leu Leu Thr Met Gln Asp Pro Ala Thr Gly Gly Val Tyr His Lys 385 390 395 400 Val Thr Thr Pro Ser Phe Pro Pro Leu Asp Thr Arg Pro Glu Asp Asp 405 410 415 Asp Ala Pro Leu Val Leu Ser Pro Ile Ser Tyr Ala Ala Thr Ala Thr 420 425 430 Phe Cys Ala Ala Met Ala His Ala Ala Leu Val Tyr Arg Pro Phe Asp 435 440 445 Pro Ala Leu Ser Ser Cys Cys Ala Asp Ala Ala Arg Arg Ala Tyr Ala 450 455 460 Trp Leu Gly Ala His Glu Met Gln Pro Phe His Asn Pro Asp Gly Ile 465 470 475 480 Leu Thr Gly Glu Tyr Gly Asp Ala Glu Leu Arg Asp Glu Leu Leu Trp 485 490 495 Ala Ser Cys Ala Leu Leu Arg Met Thr Gly Asp Ser Ala Trp Ala Arg 500 505 510 Val Cys Glu Pro Leu Leu Asp Leu Asp Leu Pro Trp Glu Leu Gly Trp 515 520 525 Ala Asp Val Ala Leu Tyr Gly Val Met Asp Tyr Leu Arg Thr Pro Arg 530 535 540 Ala Ala Val Ser Asp Asp Val Arg Asn Lys Val Lys Ser Arg Leu Leu 545 550 555 560 Arg Glu Leu Asp Ala Leu Ala Ala Met Ala Glu Ser His Pro Phe Gly 565 570 575 Ile Pro Met Arg Asp Asp Asp Phe Ile Trp Gly Ser Asn Met Val Leu 580 585 590 Leu Asn Arg Ala Met Ala Phe Leu Leu Ala Glu Gly Val Gly Val Leu 595 600 605 His Pro Ala Ala His Thr Val Ala Gln Arg Ala Ala Asp Tyr Leu Phe 610 615 620 Gly Ala Asn Pro Leu Gly Gln Cys Tyr Val Thr Gly Phe Gly Gln Arg 625 630 635 640 Pro Val Arg His Pro His His Arg Pro Ser Val Ala Asp Asp Val Asp 645 650 655 His Pro Val Pro Gly Met Val Val Gly Gly Pro Asn Arg His Leu Gln 660 665 670 Asp Glu Ile Ala Arg Ala Gln Leu Ala Gly Arg Pro Ala Met Glu Ala 675 680 685 Tyr Ile Asp His Gln Asp Ser Tyr Ser Thr Asn Glu Val Ala Val Tyr 690 695 700 Trp Asn Ser Pro Ala Val Phe Val Ile Ala Ala Leu Leu Glu Ala Arg 705 710 715 720 Gly Arg <210> 8 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> P1 primer <400> 8 ttgtcatatg aatttgaagg ttgaattcta caacagcaat c 41 <210> 9 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> P2 primer <400> 9 gatttaggca cacggctcgg gggttcttta ccccatacaa g 41 <210> 10 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> P3 primer <400> 10 gggtaaagaa cccccgagcc gtgtgcctaa atc 33 <210> 11 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> P1' primer <400> 11 atgttcatat gttagaagat aattcttcga c 31 <210> 12 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> P2' primer <400> 12 gatttaggca cacggctcgg aggctgcgga agtatatatt 40 <210> 13 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> P3' primer <400> 13 aatatatact tccgcagcct ccgagccgtg tgcctaaatc 40 <210> 14 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> P1'' primer <400> 14 attgtcatat ggctcctgaa ggctacagga agc 33 <210> 15 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> P2'' primer <400> 15 gatttaggca cacggctcgg gattgcagga gcggactttt c 41 <210> 16 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> P3'' primer <400> 16 ccgctcctgc aatcccgagc cgtgtgccta aatc 34 <210> 17 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> P4 primer <400> 17 ccgatctcga gttaacggcc acgagcctcc ag 32 <210> 18 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Cel5H-F primer <400> 18 cccggatcca attcttagcg gtggccagca a 31 <210> 19 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Cel5H-R primer <400> 19 cccgctcgag ccagctacca aattgcaggg tgt 33 <210> 20 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> CbhA-F primer <400> 20 ctagctagct atacttccgc agcctgat 28 <210> 21 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> CbhA-R primer <400> 21 tatataagct taaccgcccg gcggcgttcc cca 33

Claims (10)

Glycosyl hydrolase family of the C-terminus and glycosyl hydrolase family of at least one CBM selected from the group consisting of Family 3 cellulose binding domain (CBM), 4 CBM, and 30 CBM from Clostridium termocelum 35319 9: fused cellulase fused to the N terminus of GH9). The cellulase of claim 1, wherein family 3 CBM, family 4 CBM, family 30 CBM, and GH9 have amino acid sequences of SEQ ID NOs: 1, 2, 3, and 4, respectively. The cellulase according to claim 1, having an amino acid sequence of any one of SEQ ID NOS: 5 to 7. The cellulase according to any one of claims 1 to 3, further comprising a His ₆ tag sequence at the end. A method of decomposing a cellulosic material, comprising contacting the cellulase of any one of claims 1 to 3 with the cellulosic material. The method according to claim 5, wherein the cellulose material is a fiber material, a plant used as animal feed, a wood-derived pulp or a secondary fiber. The method according to claim 5, wherein the contacting is carried out in the presence of another saccharide degrading enzyme. 8. The method of claim 7, wherein the enzyme is selected from cellobiose hydrolase, endoglucanase, cellodextrinase, beta -glucosidase, and combinations thereof. The method according to claim 5, wherein the contacting is performed at a temperature of 50 ° C to 90 ° C. The method according to claim 5, wherein the cellulase further comprises a His ₆ tag sequence at its end.

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