KR101791564B1 - A nobel β-glucosidase gene Bgls6 with persistent activity in the aqueous organic solvents and the recombinant β-glucosidase from transformed strain using thereof - Google Patents

Abstract

The present invention relates to a strong? -Glucosidase gene and its use in an organic solvent isolated from an egg-culturing microorganism existing in a larva of an adult.
The present invention relates to a method for isolating a novel enzyme gene which degrades the? -1,4-glycosidic bond at the terminal of a non-reducing? -Glycoside from an egg-culturing microorganism in a larva of a Hermetia illucens larvae, It is possible to efficiently apply the present invention to a variety of industrial processes using vegetable polymer polysaccharides. In addition, the decomposition effect of the novel enzyme gene according to the present invention can be used in the food fermentation industry, the paper / textile industry, the alcohol production process, the manufacture of medicines and the glycosylation of plant polysaccharides in addition to the industrial process.

Description

A novel β-glucosidase gene Bgls6 capable of maintaining a sustained activity in a water-soluble organic solvent and the use of a recombinant enzyme produced therefrom {A nobel β-glucosidase gene Bgls6 with persistent activity in the aqueous organic solvents and the recombinant β-glucosidase from transformed strain using thereof}

The present invention provides a recombinant vector comprising a β-glucosidase gene isolated from egg-bearing microorganisms present in a black soldier fly (BSF) larvae , a recombinant vector containing the gene, a transformant transformed with the recombinant vector, Recombinant? -Glucosidase proteins expressed from transformants and their use.

Cellulose is the most abundant organic material on Earth and a renewable resource that does not have to worry about depletion like coal or oil. These resources are also a major source of environmental pollution for agricultural wastes overflow. Therefore, the development of an efficient saccharification process for these polysaccharides is expected to greatly contribute to food, energy and environmental problems. Therefore, studies have been made to saccharify cellulose for a long time, and a lot of studies have been made especially focusing on the development of glycosylation enzymes. As a result, many uses of glycosylated enzymes have been developed variously, commercialization has been made in textile, detergent, and feed business, and researches on new application fields are actively carried out.

Cellulase is a complex enzyme and is classified into three kinds of endoglucanase (EG), exoglucanase (CBH) and? -Glucosidase according to the hydrolysis characteristics depending on the substrate, It is known that these enzymes are involved in the degradation of cellulose.

In the biomass recycling process involving conventional polymeric polysaccharides, due to the complex structure of polymeric polysaccharides such as starch, the microorganisms or fungi themselves capable of chemical treatment or conventional separation have been used directly. However, this method has a disadvantage in that a separate step of culturing a large amount of microorganisms is required. Currently, some known β-glucosidase genes have been used to overcome this problem. However, the characteristics of known enzymes have been limited due to the lack of various characteristics required for the industry. Therefore, in order to carry out the process more efficiently, there has been a demand for an enzyme which maintains a stable resolution in a high concentration of a water-soluble organic solvent and a wide acidity change and a nonionic surfactant, various protein reducing agents and denaturants.

Korean Registered Patent KR 1183114

The object of the present invention is to provide a protein consisting of the amino acid sequence of SEQ ID NO: 1.

Another object of the present invention is to provide a gene encoding the protein.

Another object of the present invention is to provide a vector carrying the gene.

Another object of the present invention is to provide a transformant transformed with said vector.

Another object of the present invention is to provide a process for producing recombinant? -Glucosidase having water-soluble organic solvent stability.

The present invention provides a protein consisting of the amino acid sequence of SEQ ID NO: 1.

The protein may be? -Glucosidase having glucose polysaccharide-degrading activity.

The range of the? -Glucosidase protein according to the present invention includes the protein having the amino acid sequence represented by SEQ ID NO: 1 and the functional equivalent of the protein.

As used herein, the term "functional equivalent"

Refers to a protein having at least 50%, at least 70%, and at least 90% sequence homology with the amino acid sequence represented by SEQ ID NO: 1, and having substantially the same physiological activity as the protein represented by SEQ ID NO: 1. "Substantially homogenous physiological activity" means a glucose degrading activity that is highly hydrolyzable in an organism.

The term "glucosidase" is a generic term for enzymes that catalyze the reaction of hydrolysis of glucosides to generate sugars and aglycones. The enzyme that hydrolyzes glycosides is collectively referred to as glycosidase. Especially when the sugar moiety is glucose Called glucosidase.

Glucosidase is classified into? -Glucosidase and? -Glucosidase depending on whether the anomeric form of the glucosidic bond is? Or?. Therefore, "? -Glucosidase" refers to an enzyme that degrades? -Glucosidic linkage, and includes cellulase.

The protein is derived from the intestinal microorganism metagenomic gene bank which can not be artificially cultured in larvae, such as a dysentery which effectively decomposes food waste, and is a polymer polysaccharide such as starch or a β-1,4- Or a? -Glucosidase protein that degrades glycosidic bonds.

The protein may be a? -Glucosidase protein exhibiting stable activity in a water-soluble organic solvent.

The β-glucosidase according to the present invention may be a protein characterized in that the concentration of the water-soluble organic solvent is stable at 10% to 20% (vol% or wt%).

The term " water soluble organic solvent " includes all organic solvents conventionally used in the art. For example, it can be DMSO (dimethyl sulfoxide), DMF (dimethylformamide), methanol, acetonitrile, ethanol, acetone, 2-propanol and the like.

The β-glucosidase according to the present invention may be a protein characterized in that it exhibits stable activity continuously for 24 hours in a water-soluble organic solvent having a concentration of 10% (weight% or volume).

The β-glucosidase according to the present invention may be a protein characterized in that it exhibits stable activity even after being left at pH 5.0 to pH 10.0.

The term " stable activity " refers to whether or not residual activity is maintained after exposure to a certain condition and range of specific conditions that inhibit activity, to produce a constant result.

The β-glucosidase according to the present invention may be a protein characterized by exhibiting stable activity in a protein denaturant or a nonionic surfactant.

The term " protein denaturant " refers to all natural or chemical substances that alter the structure of a protein to lose its activity, including all protein denaturants commonly used in the art. For example, it may be β-mercaptoethanol, Dithiothreitol (DTT), Urea, Guanidium hydrochloride, Guanidium thiocyanate, and the like.

The term " nonionic surfactant " refers to a substance that alleviates the boundary of an interface that does not have an electric charge. The term " nonionic surfactant " generally includes all nonionic surfactants conventionally used in the art. For example, Tween 20, Tween 40, Tween 60, Tween 80, Triton X-100, and the like.

The present invention provides a gene encoding the protein.

The gene may be derived from Dongae and the like, but is not limited thereto. More specifically, it may be derived from an egg-culturing microorganism existing in the intestines of a human being.

Hermetia illucens is an insect belonging to Diptera and is distributed in Korea (naturalized), Japan (naturalized), USA, Canada, Central and South America, Hawaii and so on. It has a slender, long black body with a length of 12-20mm and a large, translucent pattern of white or tan on the second leg. Food waste and various organic matter are fed, and research on eco-friendly food waste disposal and recycling using Donghae has been conducted.

The gene includes both the gene consisting of the nucleotide sequence of SEQ ID NO: 2 and the corresponding cDNA and RNA.

Also, variants of the nucleic acid sequences are included within the scope of the present invention. Specifically, the gene may include a nucleotide sequence having 70% or more, 80% or more, 90% or more, or 95% or more of sequence homology with the nucleotide sequence of SEQ ID NO: 2, respectively. "% Of sequence homology to polynucleotides" is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the < / RTI >

The present invention provides a vector containing the gene.

The recombinant vector may be an E. coli expression vector or a yeast expression vector, but is not limited thereto. Examples of the E. coli expression vector include a pET21b vector. Examples of the yeast expression vector include, but are not limited to, a pKBN-IFN vector. In the present invention, the gene was cloned into a protein expression vector pET21a to prepare a recombinant vector pBgls6.

The term "recombinant" refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, heterologous peptide or heterologous nucleic acid. Recombinant cells can express genes or gene segments that are not found in the native form of the cells. Recombinant cells can also express genes found in cells in their natural state, but the gene has been modified and reintroduced into cells by artificial means.

The term "vector" refers to a DNA fragment (s) that transfers into a cell,

. The vector replicates the DNA and can be independently regenerated in the host cell. for

A "carrier" is often used interchangeably with a "vector ". The term "expression vector"

Sequence and a coding sequence operably linked to a particular host organism

Quot; means a recombinant DNA molecule comprising a suitable nucleotide sequence.

The expression vector may comprise one or more selectable markers. The marker is typically a nucleic acid sequence having a property that can be selected by a chemical method, and includes all genes capable of distinguishing a transformed cell from a non-transformed cell. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, antibiotics such as kanamycin, G418, Bleomycin, hygromycin, chloramphenicol, Resistant gene, but are not limited thereto, and can be appropriately selected by those skilled in the art.

The term "promoter " refers to a region of DNA upstream from a structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription. In a vector according to an embodiment of the present invention, the promoter may be, but is not limited to, CaMV 35S, actin, ubiquitin, pEMU, MAS or histone promoter.

The gene of the present invention is typically a vector for cloning or protein expression,

It can be shaken. In addition, the vector of the present invention may be produced by using prokaryotic cells or eukaryotic cells as a host

Can be constructed. For example, vectors that may be used in the present invention are often used in the art

(For example,? Gt4.λB,? -Charon and M13, etc.) or viruses (for example, plasmids such as pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pGEX series, pET series and pUC19) For example, SV40 or the like). On the other hand, when the gene of the present invention is converted into a codon optimized for a plant and a plant cell is used as a host, a vector commonly used in the art (for example, pBI vectors, pCAMBIA vectors, pSB vectors) Can be produced.

The vector is preferably used for large-scale expression of proteins, but is not limited thereto, and may be appropriately selected by those skilled in the art.

The present invention provides a transformant transformed with said recombinant vector.

The transformant is preferably Escherichia coli, more preferably a transformant deposited under accession number KACC95130P, but is not limited thereto.

Any host cell known in the art may be used as a host cell capable of cloning and expressing the vector of the present invention in a stable manner continuously, for example, E. coli JM109, E. coli BL21, E. coli RR1, E. coli . such as E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus strains, and Salmonella typhimurium, Serratia marcesensus and various Pseudomonas spp. Enterobacteriaceae and strains. The host cell is preferably Escherichia coli.

In addition, when the vector of the present invention is transformed into eukaryotic cells, yeast ( Saccharomyce cerevisiae), and the like insect cells, human cells (e.g., CHO cells (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines) and plant cell may be used.

The method of delivering the vector of the present invention into a transformant can be carried out by the CaCl ₂ method (Cohen, SN et al., Proc . Natl . Acac . Sci . USA , 9: 2110-2114 )), a method (Hanahan, D., J. Mol Biol , 166:.. 557-580 (1983)) and electroporation method (Dower, WJ et al, Nucleic Acids Res, 16:... 6127- 6145 (1988)).

In addition, when the host cell is a eukaryotic cell, microinjection (Capecchi, MR, Cell , 22: 479 (1980)), calcium phosphate precipitation (Graham, FL et al., Virology , 52: 456 electroporation (Neumann, E. et al, EMBO J., 1:. 841 (1982)), liposome-mediated transfection method (Wong, TK et al, Gene , 10:87 (1980).), DEAE- (Yang et al., Proc . Natl . Acad . Sci . , 87: 9568-9572 (1990)) and dextran treatment (Gopal, Mol . Cell Biol . , 5: 1188-1190 ) Or the like can be injected into the transformant. Such methods are well known in the art.

The present invention

(a) culturing the transformant;

(b) expressing a recombinant? -glucosidase protein from the cultivated transformant; And

(c) recovering the expressed recombinant? -glucosidase protein. The present invention also provides a method for producing a recombinant? -glucosidase protein exhibiting stable activity in a water-soluble organic solvent.

The culture of (a) can be performed by culturing the transformant using a known technique

It can be cultured in a medium suitable for production. For example, cells can be incubated with appropriate medium and protein

Laboratory or industrial foot performed under conditions permitting the quality to be expressed or separated

Can be cultured in a small or large scale fermentation, shake flask culture

All. Cultivation is carried out using a well-known technique, using a suitable medium containing carbon, nitrogen source and inorganic salt

. Suitable media are commercially available or commercially available from, for example, American Type

Depending on the ingredients and composition ratio listed in publications such as the Culture Collection catalog,

I can handle it.

The recombinant? -Glucosidase can recover the protein expressed by the target gene using a protein separation method known in the art. For example, the protein may be isolated from the nutrient medium by conventional methods including, but not limited to, centrifugation, sonication, filtration, extraction, spray drying, evaporation or precipitation.

Furthermore, the proteins may be purified by a variety of methods known in the art, including chromatography (e.g., ion exchange, affinity, hydrophobicity and size exclusion), electrophoresis, fractional salting (e.g. ammonium sulfate precipitation), SDS- Can be purified.

 In one embodiment of the present invention, a metagenome bank derived from an intestinal microorganism, which can not be artificially cultured in a larva, is constructed, and a novel clone capable of degrading the cellulose substrate using the high-speed selection system of the present invention is selected therefrom. We found that the ORF region of 2,232 bp is a novel gene that breaks down starch by analyzing the gene sequence derived from egg culture microorganism including the clone of the sugar chain. The 2,154 bp gene, 717 amino acids, which removed 26 N-terminal amino acids involved in protein targeting at the ORF site, was named 'Bgls6'.

In addition, the inventors of the present invention invented a novel strain that efficiently expresses the above gene in E. coli to effectively decompose the? -1,4-glycosidic bond at the non-reducing? -Glycoside terminal. The recombinant? -Glucosidase enzyme protein expressed and purified from the above-mentioned strain was referred to as "Bgls6." The? -Glycosidase enzyme purified from the strain was dissolved in EDTA, a nonionic surfactant (1%) Showed high enzyme activity stability and showed at least 70% activity stability in protein reducing agent and denaturant.

Since the recombinant? -Glycosidase Bgls6 of the present invention has a property of decomposing? -1,4-glycosidic bonds at the non-reducing? -Glycoside terminal, it can be efficiently applied to various industrial processes using vegetable high molecular polysaccharides have. Especially, it is expressed in Escherichia coli and can maintain its activity even at a high organic solvent concentration. It exhibits a decomposition ability of 85% or more even at a wide range of acid and base concentrations. Therefore, unnecessary purification in the process of using alkali decomposition and a large amount of organic solvent in cellulosic biomass recycling Step and economic losses can be reduced. In addition to this industrial process, β-glycosidase Bgls6 is also used in the production process of ethanol fuel, in the perfume industry (separation of aromatic compounds) and in the fruit juice extraction process, and in the process of ginseng preparation (conversion of ginsenosides) Can be used.

1 is the amino acid sequence of the novel? -Glucosidase Bgls6 protein: the underlined red portion is a signal peptide sequence derived from a gram negative / positive microorganism, and the Bgls6 amino acid sequence according to the present invention is the remaining portion.
2 shows the result of analysis of the domain conservation domain of the novel? -Glucosidase Bgls6.
3 is a phylogenetic diagram of the novel? -Glucosidase Bgls6.
Fig. 4 shows the results of the large-scale expression of the novel 棺 -glucosidase Bgls6: E. coli crude protein before induction; 2, E. coli crude protein expressing large quantities of Bgls6; 3 water-soluble protein;
Fig. 5 is a graph showing the biochemical characteristics of the novel? -Glucosidase Bgls6: A is the optimum activity temperature of Bgls6, B is the thermal stability of Bgls6, C is the optimum activity pH of Bgls6, D is the pH stability of Bgls6 Show.
Figure 6 is a graph of the activity stability of Bgls6 on chemical additives: A is the activity stability of Bgls6 on metal ions, B is the activity stability of Bgls6 on surfactants, C is on various protein reducing and denaturing agents Bgls6. ≪ / RTI >
FIG. 7 is a graph showing the activity stability of Bgls6 according to changes in organic solvent concentration: A is a comparison of the activity stability of Bgls6 in organic solvents at 10% and 20% concentration, B is time at various organic solvents at 10% Showing the activity stability of Bgls6 with the passage of time.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

<Examples>

Example  One. Dong Ae  Larva intestinal microorganism Meta genome  Construction of gene bank

The larvae were fixed with a pin on the dorsal tail, and the whole field was removed with tweezers and separated from the body by a sterilized anatomical knife. The isolated intestinal tissues were collected in a container, and then the entire metachronous intestinal microorganism was isolated using a bacterial DNA isolation kit (MoBio Laboratories, USA). The separated larval metagenomes were prepared by adding loading buffer (60% glycerol, 0.025% bromophenol blue, 0.025% xylenesianol) for electrophoresis in order to remove intestinal impurities mixed with DNA, and mixing 0.8% DNA samples were loaded into a liposome (0.5 × TBE: 45 mM Tris base, 45 mM boric acid, 1 mM EDTA), and the CHEF III system (BioRad, USA) of Pulsed Field Gel Electrophoresis (PFGE) And electrophoresed at 6 V / cm for 14 hours. Agarose block containing DNA of 35 kb or more was cut, agarose degrading enzyme was added, and reaction was carried out for 1 hour, and agarose degrading enzyme was inactivated at 70 ° C for 10 minutes. 2.5 times of ethanol was added to the reaction solution, After separation, the precipitated DNA pellet was dissolved in TE buffer to an appropriate concentration and used to make a metagenome gene bank.

The Fosmid library was prepared by adding 5 units of T4 polymerase, 2 units of T4 polynucleotide phosphorylase and 6 units of Klenow fragment (Takara Co., Japan ) Was added, and the amount of the reaction solution was adjusted to 50 μl, followed by reaction at 37 ° C for 30 minutes to make the DNA ends smoothed. After the reaction was completed, the same amount of phenol / chloroform / isoamyl alcohol (25: 24: 1, v / v / v) was added and the mixture was centrifuged for 10 minutes. The supernatant was collected and washed with an equal volume of chloroform / isoamyl alcohol , v / v) were added and mixed. Then, the mixture was centrifuged at 12,000 rpm for 10 minutes, and the supernatant was taken. 2.5 times 100% ethanol was added and incubated at room temperature for 1 hour. The supernatant was discarded and DNA was extracted with 70% DNA was prepared by washing. The larvae metagenomic DNA was added to the EpiFOS5 phosphide vector (Epicenter, USA) for end-repaired homozygous and the ligation reaction was carried out at 16 ° C for 16 hours. Samples ligation-treated at 16 ° C for 16 hours for in vitro packaging were heat-treated at 70 ° C for 10 minutes to inactivate the ligase. Twenty-five microliters of the MAX lambda packaging extract kit (Epicenter, USA) was carefully mixed with 7.5 μl of the ligation reaction solution so as to avoid air bubbles, followed by reaction at 30 ° C for 90 minutes. Again, 25 μl of the phage concentrate was added, carefully mixed with a pipette to prevent air bubbles, and then incubated at 30 ° C for 1 hour and 30 minutes for in vitro packaging. 1 ml of SM buffer solution was added and mixed well. Subsequently, 25 μl of chloroform was added to inactivate the unpackaged materials. The supernatant was infected with Escherichia coli Epi100, and a metagenomic gene bank composed of 92,000 individually transformed strains was completed.

Example  2. Glycosidase-degrading active gene screening

In order to test the metagenomic clones showing degrading activity in glucose medium, 1% carboxymethylcellulose (CMC, Sigma Co.) was added to a basal medium containing 0.1% sodium chloride, 0.1% bacto-tryptone and 0.05% bacto-yeast extract. USA) and each antibiotic chloramphenicol (20 μg / ml) were inoculated with replica pins in a plate medium and incubated at 28 ° C. for 5 days. The culture medium was completely dyed at room temperature for 5 minutes using Congo Red dyeing reagent, and then washed with sterilized water to determine the ability to produce glucose polysaccharide degrading enzymes with or without a ring around the cells . In order to confirm the contamination of the meta-genomic clone or the false-positive result of Escherichia coli host, the fosmid isolated from the metagenome selected through the primary screening was transformed into the E. coli host DH5 [beta] strain and repeatedly subjected to the same screening , And the separated phosmid was cut into an average 4-kb fragment using HydroShear (DigiLab, USA), and the whole fragment was further purified by smoothing the DNA end as indicated in Example 1 The vector was cloned into the pUC118 / EcoRV / CIP-treated vector and electroporation was carried out on E. coli DH5α to prepare a shot-gun subclone library. A clone showing repeated degradation of glucose polysaccharide was repeatedly selected as a positive strain in the above cellulose culture medium.

Example  3. Bgls6  Gene analysis

A total nucleotide sequence of 2,232 bp was obtained from the clone showing the activity of degrading the glucose selected in Example 2. As shown in FIG. 1, the peptide was decoded into an amino acid sequence, and a signal peptide sequence derived from the Gram-negative / positive microorganism was found to exist in the N-terminal 26 amino acids (red color). It is assumed that 717 amino acid sequences corresponding to the 2,154 bp nucleotide sequence from which this region is deleted have a β-glucosidase enzyme function, and the corresponding 717 amino acids are named Bgls6 (β-glucosidase 6, β-glucosidase 6) Respectively. The size of the protein derived from Bgls6 was estimated to be 80.5 kDa and the pI to be 5.04.

Example  4. Bgls6  Analysis of conserved domains of genes

As a result of conserved domain analysis using the amino acid sequence of Bgls6 as shown in Fig. 2, the glycosyl hydrolase classified in the CAZy database (http://www.cazy.org/fam/acc_GH.html) (GH) among the glycosyl hydrolase (GH) families. A homology comparison of the Bgls6 base sequence with the amino acid sequence known in GenBank was performed through the BalstX algorithm. The highest homologues are Bacteroides sp. It was 81% identical to? -Glucosidase derived from HPS0048.

Example  5. Bgls6  Systematic analysis of genes

The amino acid sequence identified in Example 4 was analyzed by the Mega5 program (www.megasoftware.net) as shown in FIG. 3, and as a result, Bacteroides sp. Glucosidase enzyme which is evolutionarily similar to that of HPS0048 but has a novelty that has not yet been known. In the BlastP search, the glucosidase gene and the GenBank number showing significance were confirmed. The number on the branch is the bootstrap analysis (1,000 resampling).

Example  6. Bgls6  E. coli heterogeneous expression of genes

An open reading frame (ORF) of the gene was cloned into pET21a and introduced into Escherichia coli BL21 (DE3) strain, followed by induction with IPTG 0.5 mM at 20 DEG C for 20 hours in order to express Bgls6 protein in E. coli in a large amount. (induction). In order to isolate and purify the expressed protein, only 50 ml of a culture solution of Escherichia coli transformed with Bgls6 gene was obtained by centrifugation and re-expressed in 10 ml of lysis buffer (50 mM K ₂ HPO ₄ (pH 7.0), 300 mM sodium chloride) After thawing, the cells were disrupted using an ultrasonic disintegrator. The disrupted cells were centrifuged at 15,000 rpm for 15 minutes, and then the supernatant was obtained and used as the crude enzyme solution necessary for the cellulase separation. In the obtained crude enzyme solution, Bgls6 protein was bound to a column by mixing with a TALON metal affinity resin (BD Bioscience Clontech) using 6XHis-Tag optionally attached at the C-terminal of the amino acid. After binding to the coenzyme resin, the cells were washed with 10 volumes of wash buffer (50 mM K ₂ HPO ₄ (pH 7.0), 300 mM sodium chloride, 10 mM imidazole) and then the Bgls 6 protein was eluted with 5 volumes of elution buffer (50 mM K ₂ HPO ₄ (pH 7.0), 300 mM sodium chloride, 150 mM imidazole). Protein concentration and imidazole dialysis were concentrated by one-time filtration through a YM10 membrane (Amicon Ultra, Millipore Co.) capable of concentrating above a molecular weight of 10 kDa. The final purified protein was subjected to SDS-PAGE electrophoresis, and a large amount of 82 kDa Bgls6 protein was expressed (FIG. 4).

Example  7. Bgls6 Biochemical characterization of

0.3 μg of purified Bgls6 protein was incubated with 1 mM p -nitrophenyl-β-D-glucopyranoside for 10 minutes in potassium phosphate buffer (50 mM K ₂ HPO ₄ , pH 7.0, 200 μl) -D- glucopyranoside (p -Nitrophenyl-β-D- glucopyranoside) reacting with p - nitro phenol amount was measured with a spectrophotometer at 405nm. As a result, Bgls6 showed the highest activity at 35 DEG C with enzyme activity at 15 to 40 DEG C (FIG. 5A).

Further, in order to confirm the thermostability of Bgls6, the above standard assay reaction (potassium phosphate buffer (50 mM K ₂ HPO the Bgls6 0.3μg protein purified under _{4, pH7.0, 200μl)) 1mM} p - nitrophenyl -β-D- glucopyranoside (p -Nitrophenyl-β-D- glucopyranoside) and 35 ℃, 10min. The amount of decomposition products was determined according to the method. The Bgls6 protein retained more than 90% of stable enzyme activity for 2 hours at the optimal temperature of 35 DEG C (FIG. 5B).

Bgls6 In order to confirm the optimum pH of the activity, sodium acetate (pH 4.0-6.5), potassium phosphate (pH 6.5-7.5), HEPES (pH 7.5-8.5), CHES 0.5 to 10.0) using a buffer p - decomposing activity nitrophenyl -β-D- glucopyranoside (p -Nitrophenyl-β-D- glucopyranoside) was measured at 35 ℃ for 10 minutes. As a result, the Bgls6 protein exhibited an enzyme activity in the range of pH 6.0 to 9.0, and showed optimal activity at pH 7.0 (FIG. 5C).

In order to confirm the pH stability of Bgls6, the Bgls6 protein was allowed to stand at 4 DEG C for 15 hours under different pH, and the amount of the degradation product was measured according to the above standard assay reaction method. The Bgls6 protein showed over 85% enzyme activity over various pH ranges of pH 5.0-10.0 (FIG. 5D).

Example  8. Chemicals  For additives Bgls6 Activity stability analysis

In order to analyze the enzymatic activity of the Bgls6 protein on the chemical additives, first purified Bgls6 protein (0.15 mu g / mu l, final 10 mu g) without inhibitor was pre-incubated at 30 DEG C for 30 minutes, Then, 2 μl (final 0.3 μg) was dispensed and reacted under the above standard assay to measure the amount of p -nitrophenol produced by a spectrophotometer at 405 nm. This value was set as the standard reaction 100%.

To confirm the stability of the enzyme activity against metal ions, 0.15 μg / μl of Bgls6 was added to each metal ion (1 mM) and preincubated at 30 ° C. for 30 minutes. After the standard reaction in the substrate nitrophenyl -β-D- glucopyranoside (p -Nitrophenyl-β-D- glucopyranoside), the resulting p - - Here 2μl (final 0.3μg) of the frequency divider by p 1mM nitro The amounts of phenol were compared with each other. As a result, β-glucosidase Bgls6 was presumed to be a non-metalloenzyme because the activity of the enzyme was not affected by EDTA and was greatly inhibited by Cd ²⁺ / Hg ²⁺ (FIG. 6A ).

Bgls6 protein was pre-cultured at 30 캜 for 30 minutes under 1% ([v / w] or [v / v]) concentration to confirm the activity stability of the enzyme against the surfactant. The relative amounts of p - nitrophenol produced were compared. As a result, the Bgls6 protein showed very high enzyme activity stability in the nonionic surfactant (1%) and less than 15% residual activity in the ionic surfactants SDS (anionic) and CTAB (cationic) 6B).

Bgls6 protein was added to each chemical additive and incubated for 30 min at 30 ° C in order to confirm the enzyme activity stability against various protein reducing agents / modifiers. The relative p - The amounts of nitrophenol were compared with each other. As a result, the Bgls6 protein showed an activity stability of at least 70% or more in the protein reducing agent and the denaturant (Fig. 6C).

Example  9. Concentration of organic solvent Bgls6 Enzyme activity stability analysis

(V / w) or 20% (v / w) or [v / v], respectively, / v]) concentration was incubated with potassium (potassium phosphate) buffer solution _{(50mM K 2 HPO 4 (pH7} ), 200μl) and then the protein Bgls6 0.15μg / μl purified in addition, from 30 ℃ 30 bungan former comprising of . Subsequently, 2 μl (final 0.3 μg) sample was dispensed, and after the standard assay reaction with 1 mM p -nitrophenyl-β-D-glucopyranoside substrate, the relative amount of p -nitrophenol produced was measured with a spectrophotometer 405 nm and compared. As a result, the Bgls6 protein showed 95% or higher enzyme activity stability in most of the water-soluble organic solvent having a concentration of 10%, and a high enzyme activity stability of 85% or more even in the water-soluble organic solvent having the concentration of 20% (FIG. 7A).

The purified Bgls6 protein (0.15 μg / μl) was added to a potassium phosphate buffer (50 mM, pH 7.0, 200 μl) containing various organic solvents at a concentration of 10% and pre- incubation) at regular time intervals, 2μl (final 0.3μg) of the sample by the dispensing 1mM p - nitrophenyl -β-D- glucopyranoside (p -Nitrophenyl-β-D- glucopyranoside) after a reaction with the standard substrate , And the amount of p - nitrophenol produced were compared with each other. As a result, in the case of the water-soluble organic solvent at the concentration of 10%, the activity of the enzyme was maintained at 75% or more for 24 hours (FIG. 7B).

Example  10. Bgls6 Of substrate specificity

In Table 1, the substrate specificity of the Bgls6 protein was determined according to the standard assay reaction method (0.3 μg of purified Bgls6 protein was added to 50 mM potassium buffer (pH 7, 200 μl), followed by reaction at 40 ° C. for 10 minutes) , Not detected).

In the case of the p - nitrophenyl group - substituted sugar derivative, 0.3 μg of purified Bgls6 protein was reacted with 1 mM different p - nitrophenyl - based substrate under standard assay reaction conditions, and the amount of p - And measured with a spectrophotometer at 405 nm.

In the case of oligosaccharides, 0.3 μg of purified Bgls6 and several oligosaccharide substrates of 1 mM were reacted under standard assay reaction conditions, and the amount of glucose produced was measured using a glucose assay kit (Sigma, USA) at 540 nm Respectively. In the case of sucrose, lactose, meliobiose and maltose, which showed a low specific activity, 5 μg of Bgls 6 enzyme was used. One unit of enzyme activity (Unit, U) was defined as the amount of enzyme required to produce 1 μmol of product (o / p - nitrophenol, glucose) for 1 minute.

As a result, Bgls6 exhibited particularly high activity in the natural glucosides cellobiose, cellotriose, cellotetraose and cellopentaose, and showed high activity in the case of sucrose, lactose, , And maltose (Maltose). This shows that it specifically degrades the glucose polysaccharide having a? -1,4-glycosidic bond. Bgls6 has substrate specificity that acts only on glucosidic bonds and shows excellent activity in natural-glycoside or natural aryl-glycoside compounds, which is highly expected of industrial application (Table 1).

Agriculture Gene Resource Center KACC95130P 20150410

<110> Republic of Korea <120> A nobel beta-glucosidase gene Bgls6 with persistent activity in          the aqueous organic solvents and the recombinant beta-glucosidase          from transformed strain using it <130> P15R12D0318 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 717 <212> PRT &Lt; 213 > amino acids of Bgls6 protein <400> 1 Gln Thr Asp Lys Ala Asp Tyr Arg Phe Gln Val Asp Ser Ile Leu Asn   1 5 10 15 Met Met Thr Leu Glu Glu Lys Val Gly Gln Met Ile Gln Tyr Ser Asp              20 25 30 Asp Arg Leu Gln Thr Gly Pro Ser Ile Gln Asn Thr Asp Gln Phe Glu          35 40 45 Glu Ile Lys Lys Gly Arg Val Gly Ser Met Phe Asn Val Met Ser Val      50 55 60 Glu Arg Ile Arg Gly Tyr Gln Asp Ile Ala Met Gln Ser Arg Leu Lys  65 70 75 80 Ile Pro Leu Leu Phe Gly Leu Asp Val Val His Gly Met Arg Thr Thr                  85 90 95 Phe Pro Ile Pro Leu Ala Glu Ala Ala Ser Phe Asp Leu Ser Leu Met             100 105 110 Glu Glu Thr Ala Arg Val Ala Ala Asp Glu Thr Ser Ala Tyr Gly Ile         115 120 125 His Trp Thr Phe Ala Pro Met Val Asp Ile Ser Arg Asp Ala Arg Trp     130 135 140 Gly Arg Val Met Glu Gly Ala Gly Glu Asp Pro Trp Leu Gly Ser Arg 145 150 155 160 Ile Ala Ile Ala Arg Ile Lys Gly Phe Gln Gly Glu Asn Tyr Asp Asp                 165 170 175 Ala Lys Arg Val Ala Cys Ala Lys His Phe Ala Ala Tyr Gly Ala             180 185 190 Ala Val Ala Gly Lys Asp Tyr Ala Glu Ala Asp Ile Ser Glu Thr Thr         195 200 205 Leu His Gln Ile Tyr Leu Pro Pro Phe Lys Ala Ala Val Asp Ala Asn     210 215 220 Val Ala Thr Met Met Asn Gly Phe Asn Glu Ile Asn Gly Ile Pro Ala 225 230 235 240 Asn Ala His Lys Glu Leu Gln Arg Asp Ile Leu Lys Gly Glu Trp Gly                 245 250 255 Phe Glu Gly Phe Met Val Ser Asp Trp Gly Ser Ile Gly Glu Ile Ala             260 265 270 Thr His Gly Met Ala Lys Asp Asn Lys Glu Ala Thr Met Leu Ala Val         275 280 285 Leu Ala Gly Cys Asp Met Asp Met His Ser Met Ser Tyr Lys Arg Asn     290 295 300 Leu Val Asp Leu Val Asp Glu Gly Gln Val Asp Ile Gln Tyr Ile Asn 305 310 315 320 Asp Ala Ile Arg Arg Ile Leu Thr Lys Lys Phe Glu Leu Gly Leu Phe                 325 330 335 Glu Asp Pro Tyr Arg Tyr Cys Tyr Arg Glu Lys Asp Leu Ser Asn Pro             340 345 350 Ser Ile Ile Glu Glu His Arg His Thr Ala Arg Leu Met Gly Ala Lys         355 360 365 Ser Ile Val Leu Leu Lys Asn Glu Asp Val Leu Pro Ile Ser Pro Asp     370 375 380 Ile Lys Lys Ile Ala Leu Val Gly Pro Leu Asn Lys Ser Lys Met Asp 385 390 395 400 Met Leu Gly Asn Trp Asn Ala Ala Gly Glu Glu Lys Asp Val Ile Ser                 405 410 415 Val Tyr Glu Gly Leu Cys Thr Ala Met Pro Asp Ala Glu Ile Thr Tyr             420 425 430 Ile Glu Gly Tyr Asp Leu Glu Thr Asn Thr Leu Asn Pro Leu Pro Asn         435 440 445 Met Thr His Phe Asp Met Ile Ile Val Ser Val Gly Glu Arg Ala Leu     450 455 460 Asp Ser Gly Glu Ala Lys Ser Lys Val Asp Ile Asn Val His Val Asn 465 470 475 480 Gln Gln Ala Leu Ile Arg Ser Leu Lys Glu Glu Thr Asn Lys Pro Val                 485 490 495 Val Met Leu Leu Met Gly Gly Arg Pro Leu Ile Phe Ser Glu Ala Thr             500 505 510 Pro Phe Ala Asp Ala Ile Val Met Thr Trp Trp Leu Gly Thr Glu Ala         515 520 525 Gly Asn Ser Ile Asp Val Leu Thr Gly Gln Tyr Asn Pro Ser Gly     530 535 540 Lys Leu Pro Val Thr Phe Pro Arg His Val Gly Gln Cys Pro Ile Tyr 545 550 555 560 Tyr Asn His Lys Asn Thr Gly Arg Pro Trp Arg Pro Asn Glu Lys Tyr                 565 570 575 Val Ser Gly Tyr Val Asp Glu Thr Ile Leu Pro Ala Tyr Pro Phe Gly             580 585 590 Phe Gly Leu Ser Tyr Thr Gln Phe Glu Ile Gly Lys Pro Glu Leu Asp         595 600 605 Arg Asn Ser Tyr Ala Val Asp Asp Ser Val Asn Val Lys Val Arg Val     610 615 620 Lys Asn Ile Gly Glu Arg Thr Gly Ile Glu Thr Val Gln Trp Tyr Leu 625 630 635 640 Arg Asp Ile Val Ser Ser Ile Thr Arg Pro Val Ile Glu Leu Cys Gly                 645 650 655 Val Gln Gln Ile Glu Leu Lys Pro Gly Glu Glu Arg Trp Leu Glu Phe             660 665 670 Thr Leu Ser Ser Glu Ser Leu Gly Phe Tyr Asp Lys Ser Glu Phe Lys         675 680 685 Ile Glu Pro Gly Glu Phe Lys Ile Phe Ala Gly Asn Ser Ala Gln His     690 695 700 Leu Gln Glu Gln Ser Phe Tyr Leu Asn Ser Gly Gln Asn 705 710 715 <210> 2 <211> 2154 <212> DNA <213> DNA of Bgls6 gene <400> 2 caaacagata aggcagatta cagatttcag gttgattcaa tactgaatat gatgacattg 60 gaagagaagg tagggcagat gatccagtat tctgacgata ggttacaaac gggtccttcg 120 attcaaaata cagatcaatt tgaggaaata aaaaaaggaa gagtagggtc aatgtttaat 180 gttatgtctg ttgagcgtat aagaggttat caggacatag cgatgcaaag caggttaaaa 240 attcctctgt tatttggtct cgatgtagtt cacgggatga ggaccacttt ccccatccca 300 ttggcagaag cggcaagttt cgatttgtcg ctgatggaag aaacagcgag ggttgccgca 360 gacgaaacat ctgcctatgg cattcactgg acattcgcgc caatggtcga tatttcgagg 420 gatgcccgtt ggggaagagt gatggaagga gccggcgaag atccatggct ggggagccgg 480 atagctattg caaggataaa aggctttcag ggtgaaaact acgatgatgc aaaaagagta 540 atggcatgtg cgaaacattt tgctgcgtat ggagccgctg ttgccgggaa agattatgca 600 gaagcagata tctcggaaac gacgctacat cagatttatc ttcctccttt taaagctgcc 660 gttgatgcaa atgtggctac gatgatgaat ggttttaacg aaattaatgg aatcccggcc 720 aatgcacata aagaattaca aagagatata ctcaaaggag aatggggctt tgagggtttt 780 atggtcagcg attggggttc tatcggagaa atagccaccc atggaatggc caaagacaat 840 ggttgcgata tacaaaagga atttggttga tcttgtcgat gaaggacaag tggatatcca atacatcaat 960 gacgccataa gacgtattct tacaaaaaaa tttgaattgg gattatttga ggatccatac 1020 cgttattgct atcgggaaaa ggacctgagt aatccttcaa taatagagga acatcgccat 1080 acagcccgtt tgatgggagc aaaatctatc gtattgttga aaaatgagga tgtactacct 1140 ataagcccgg atattaaaaa gattgctttg gttggtcctt tgaataaatc aaaaatggat 1200 atgttaggaa attggaatgc tgccggagaa gaaaaagatg ttatttcggt ctatgagggg 1260 ctgtgtacgg caatgccgga tgcagaaatt acttacattg aagggtacga tttggagaca 1320 aatactctga atcctcttcc caatatgaca cattttgaca tgattattgt atcggttgga 1380 gaaagggctt tagactcggg cgaagccaaa tcgaaagttg atataaatgt tcatgttaac 1440 cagcaggcat tgatcaggag tctcaaggag gaaacgaata aaccggttgt aatgcttcta 1500 atgggaggaa ggcctctcat cttttcagaa gccacccctt tcgctgatgc aattgttatg 1560 acctggtggt taggtacaga agccggaaat tccatagcag atgttttgac ggggcaatat 1620 aatccttcag gaaaattacc ggtcacattt ccccgccatg tggggcaatg ccccatttat 1680 tataatcata aaaatacggg aagaccttgg aggccaaatg aaaaatatgt gtccggatat 1740 gttgatgaga caattttgcc ggcttatccc tttggttttg gattgagtta tactcaattt 1800 gaaataggga aacctgaatt ggaccggaat agttatgctg tggacgattc tgtaaatgtg 1860 aaggtgaggg tgaagaatat tggagaaagg acagggatag aaactgttca atggtattta 1920 cgggatattg tcagttctat tacgcgtcct gtaattgaat tgtgtggagt acagcaaata 1980 gaattaaaac cgggggaaga gcggtggctt gaatttacat tatcctctga atcattgggc 2040 ttttatgaca aaagtgaatt caaaattgag cccggtgagt ttaaaatctt tgcaggaaat 2100 agtgctcagc atttacaaga acaatctttt tatttgaatt cggggcaaaa ctga 2154

Claims (14)

A protein consisting of the amino acid sequence of SEQ ID NO: 1 derived from intestinal microorganism,
Wherein the protein is exo-β-glucosidase having a stable activity in a water-soluble organic solvent and having a glucose-degrading activity. delete delete The method according to claim 1, wherein the concentration of the water soluble organic solvent is 10 to 20% by weight or 10 to 20% by volume, and the water soluble organic solvent is selected from the group consisting of dimethyl sulfoxide (DMF), dimethylformamide ), Methanol, acetonitrile, ethanol, acetone, and 2-propane. The protein according to claim 1, wherein the exo -? - glucosidase exhibits stable activity at pH 5.0 to pH 10.0. The protein according to claim 1, wherein the exo -? - glucosidase exhibits stable activity in a protein denaturant or a nonionic surfactant. A gene encoding the protein of claim 1. delete 8. The gene according to claim 7, wherein the gene comprises the nucleotide sequence of SEQ ID NO: 2. A recombinant vector comprising the gene of claim 7. A transformant transformed with the recombinant vector of claim 10. 12. The transformant according to claim 11, wherein the transformant is transformed into E. coli. 12. The transformant according to claim 11, wherein the transformant is KACC 95130P strain. (a) culturing the transformant according to any one of claims 11 to 13;
(b) expressing the recombinant exo-β-glucosidase protein from the cultured transformant; And
(c) recovering the expressed recombinant exo -? - glucosidase protein;
&Lt; / RTI &gt; wherein the recombinant exo-beta-glucosidase is a recombinant exo-beta-glucosidase.

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