KR20160000205A - A NOVEL SALT-RESISTANT α-GALACTOSIDASE GENE FROM HERMETIA ILLUCENS AND USES THEREOF - Google Patents

Abstract

The present invention relates to an α-galactosidase gene isolated from a rarely culturable microorganism present in the intestine of Hermetia illucens larvae, and uses of the same. The present invention isolates a novel enzyme gene, which degrades α-1,6-glycosidic bonds at the terminal of unreduced α-galactoside, from the rarely culturable microorganism in the intestine of Hermetia illucens larvae, and develops a novel strain expressing the same massively, thereby being effectively applied in various industrial processes using a phytomacromolecule hemicellulose. In addition, an effect on hydrolysis of carbohydrate of the novel enzyme gene in accordance with the present invention can be used in the sugar industry, bean products (soymilk, syrup) processes, the paper/pulp industry, the feed industry, the drug development industry, etc.

Description

The present invention relates to a novel α-galactosidase gene Agas2 and a recombinant enzyme produced therefrom, which have stable activity under a high salt level, and a novel salt-resistant α-galactosidase gene from Hermetia illucens and uses thereof.

The present invention relates to an α-galactosidase gene isolated from an egg-culturing microorganism existing in a larva of a larva and the use thereof.

Extraction of first generation biofuels, mainly from edible crop biomass, has many problems, including net energy loss, greenhouse gas emissions, and rising food prices.

On the other hand, second generation bioethanol production from renewable lignocellulosic biomass has many advantages in terms of energy and environment.

In the related hemicellulosic biomass process, microorganisms or fungi that can be separated conventionally due to the complex structure characteristic of hemicellulose have been directly used, or some known alpha-galactosidase gene has been heterologously expressed in E. coli.

However, the hemicellulosic biomass process has a high salt concentration, a water-soluble organic solvent, a protease, a nonionic surfactant and a wide acidity, which are inadequate for enzyme activity, and have many purification steps and economic losses.

Korean Patent Publication No. 10-2013-0117550

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

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

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 method for producing alpha -galactosidase having salt tolerance.

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

The polypeptide may be a salt tolerant alpha-galactosidase.

Alpha-galactosidase is an enzyme widely found in bacteria, yeast, mold, plant (seed) and animal, hydrolyzing allyl and alkyl-D-galactoside to produce galactose and converting the produced galactose residue into another Transfer to sugar, alcohol and phenol. It exists as two enzymes of α and β type, and α-galactosidase is also called melibiase · raffinase and acts on melisis, lipinose, and other oligosaccharides in addition to α-galactoside.

The present invention provides a gene encoding said polypeptide.

The gene may be derived from Hermetia illucens , 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 . It is distributed in Korea (naturalized), Japan (naturalized), USA, Canada, Central and South America, and Hawaii. 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.

In addition, the gene includes both the gene consisting of the nucleotide sequence of SEQ ID NO: 2 and the corresponding cDNA and DNA.

"% Of sequence homology" for polynucleotides according to the cDNA or DNA is confirmed by comparing the comparison region with two optimally arranged sequences, and a portion of the polynucleotide sequence in the comparison region is the (I.e., a gap) relative to a reference sequence (not including additions or deletions).

The present invention provides a vector containing the gene.

The term " vector " means a DNA construct that retains a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in the appropriate host. The vector may be a plasmid, phage particle, or simply a potential genome insert. Once transformed into the appropriate host, the vector can replicate and function independently of the host genome, or, in some cases, integrate into the genome itself.

The term " expression vector " means a recombinant DNA molecule comprising a desired coding sequence and a suitable nucleic acid sequence necessary for expressing a coding sequence operably linked in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotic cells are known.

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 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 vector according to the present invention may be pET21a, but is not limited thereto.

The present invention provides a transformant transformed with said vector.

The transformant is preferably Escherichia coli, more preferably a transformant deposited under Accession No. KACC95128P, but is not limited thereto.

Transformants capable of continuously cloning and expressing the vector of the present invention in a stable manner can be any transformants known in the art such as E. coli JM109, E. coli BL21, E. coli RR1, E . coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus strains such as Bacillus Chuo ringen sheath, and Salmonella typhimurium, Serratia, and various Pseudomonas species and CL Marseille And the like.

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 CaCl2 method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9: 2110-2114 (1973) ), One method (Hanahan, D., J. Mol. Biol., 166: 557-580 (1983)) and electroporation (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 (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

(1) isolating the gene consisting of the nucleotide sequence of SEQ ID NO: 2 from the gene derived from Donghae and the like;

(2) cloning the gene into a vector;

(3) transforming the vector into a transformant;

(4) purifying a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 from the transformant, to produce a salt tolerant alpha -galactosidase.

In a specific embodiment of the present invention, the present inventors have constructed a metagenome bank derived from intestinal microorganisms that can not be artificially cultured in larvae of Donga and others. From this, a novel clone capable of degrading the cellulose substrate using a unique high- Respectively. By analyzing the gene sequence derived from the egg culturing microorganism contained in the clone, it was found that the 1,932 bp gene and thus the 643 amino acid sites are a novel gene that breaks hemicellulose.

In addition, the inventors of the present invention invented a novel strain capable of mass-expressing the gene in E. coli and effectively degrading the alpha-1, 6-galactosidic bond at the terminal of the non-reducing alpha-galactoside. The α-galactosidase enzyme purified from the strain showed more than 30% enzyme activity and more than 90% enzyme activity stability at a high salt concentration (4M), and the water-soluble organic solvent (10%, v / v) High enzyme activity was maintained even in the active agent (1%, v / v), proteolytic enzyme (1:10, w / w) and wide pH range (pH 4.0 to 11.0).

The alpha-galactosidase enzyme provided by the present invention has a hydrolytic function for various kinds of carbohydrates that form a side chain at the alpha-1, 6-galactosidic bond end. Therefore, the sugar industry such as candy sugar- · It can be used for soybean and food processing such as molasses, paper and pulp industry, animal feed, drug development and so on.

In addition, the alpha-galactosidase enzyme provided by the present invention exerts an excellent function in the decomposition of garbage of high saltiness and can be industrially used in this regard.

On the other hand, in the existing hemicellulose biomass process, microorganisms or fungi which can be separated by conventional methods due to the complex structure characteristic of hemicellulose were directly used or some known alpha-galactosidase gene was expressed in Escherichia coli. However, , Which is inadequate for enzyme activity, has many purification steps and is complicated, resulting in economic loss. The alpha-galactosidase enzyme provided by the present invention exhibits a stable decomposing ability in the presence of a high salt concentration, a water-soluble organic solvent, a proteolytic enzyme, a nonionic surfactant and a wide acidity in the expression of Escherichia coli, The number of purification steps can be minimized and thus the economic loss can be reduced.

The invention (such Hermetia dongae The present inventors isolated a novel enzyme gene capable of degrading the alpha-1, 6-galactosidic bond at the terminal of non-reducing alpha-galactoside from an egg-culturing microorganism in a larva of a larva and developed a new strain expressing it in large quantities, Can be efficiently applied in various industrial processes using hemicellulose. In addition, the carbohydrate hydrolyzing effect of the novel enzyme gene according to the present invention can be utilized in the sugar industry, soybean and food (soymilk, molasses) process, paper and pulp industry, animal feed industry, drug development industry and the like.

1 is the amino acid sequence of a novel alpha-galactosidase: the underlined red portion is a signal peptide sequence derived from a gram negative / positive microorganism, and the novel alpha-galactosidase amino acid sequence according to the present invention is the Part.
Figure 2 shows the result of analysis of the domain conservation domain of the novel alpha-galactosidase.
Figure 3 is a phylogenetic diagram of the novel alpha-galactosidase.
Fig. 4 shows the results of the large-scale expression of the novel α-galactosidase E. coli heterogeneous: 1 is the E. coli crude protein before induction, 2 is the E. coli crude protein expressing the large amount of new α-galactosidase, 3 is water-soluble protein, New alpha-galactosidase.
FIG. 5 is a graph showing the biochemical characteristics of novel alpha-galactosidase: A is the optimum temperature of the new alpha-galactosidase, B is the heat resistance of the novel alpha-galactosidase, C is the new alpha-galactosidase PH, D is the pH stability of the novel α-galactosidase.
Figure 6 is a graphical analysis of the activity stability of novel alpha-galactosidase for chemical additives: A is the activity stability of the novel alpha-galactosidase to the organic solvent, B is the fresh alpha-galactosidase activity for the surfactant, Activity stability of cidase, and C is activity stability of novel alpha-galactosidase to metal ion.
FIG. 7 is a graph showing the enzyme activity and activity stability of novel α-galactosidase under high salt concentration: A is stability of enzyme residual activity of novel α-galactosidase pretreated with sodium chloride and potassium chloride, B is sodium chloride and potassium chloride, Inhibition of New Alpha - Galactosidase Enzyme Activity in Potassium Phosphate Buffer Included.
8 is a graph showing the stability of novel alpha-galactosidase to proteolytic enzymes.

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.

< Example  1> 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 isolated larval metagenomes were prepared by adding loading buffer (60% glycerol, 0.025% promophenol blue, 0.025% xylenesianol) for electrophoresis in order to remove intestinal impurities mixed with DNA, and mixing them with 0.8% low melting point agarose DNA samples were loaded into a gel (0.5 × TBE: 45 mM Tris base, 45 mM boric acid, 1 mM EDTA) and then analyzed using a 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 T4 polymerase, 2 T4 polynucleotide kinase, and 6 Klenow fragments (Takara Co., Japan) to 25 μl (5 μg) of the purified DNA, The volume of the solution was adjusted to 50 μl and incubated 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 end-repaired DNA was added to the EpiFOS5 phosphide vector (Epicenter, USA) and subjected to a ligation reaction at 16 ° C for 16 hours. The samples reacted for 16 h at 16 ° C for in vitro packaging were heat treated at 70 ° C for 10 min to inactivate the ligase. To the 7.5 μl of the ligation reaction solution, 25 μl of the concentrate of the MAX lambda packaging extract kit (Epicenter, USA) was carefully mixed so as not to cause air bubbles and reacted 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. Centrifugation was carefully performed, and the mixture was stored at 4 ° C. The supernatant was infected with Escherichia coli Epi100 to complete a metagenome gene bank composed of 92,000 individual transformed strains.

< Example  2> Cellulase digestion active gene screening

In order to test the metagenomic clones showing degradative activity in the cellulose 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 antibiotic chloramphenicol (20 μg / ml) were inoculated with each individual metagenome using a replica pin and cultured at 28 ° C for 5 days. The culture medium was then stained with Congo Red dyeing reagent at room temperature for 5 minutes and then washed with sterile water to determine the ability of cellulose degrading enzyme production to form yellow rings around the cells. Respectively. In order to confirm contamination of the meta-genomic clone or 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 [alpha] 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. Using this, a clone that repeatedly stained the cellulosic medium and exhibited cellulolytic ability was finally selected as a positive strain.

< Example  3> New Alpha Galactosidase  Gene analysis

A total nucleotide sequence of 2,007 bp was obtained from the clone showing the cellulolytic activity 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 a gram negative / positive microorganism was found to exist in 25 amino acids at the N-terminal (red color). It is assumed that the 643 amino acid sequence corresponding to the 1,932-bp base sequence from which this site was deleted has an alpha-galactosidase enzyme function, and the corresponding 643 amino acids are named as Agas2 (alpha galactosidase S2, alpha -galactosidase S2) Respectively. The size of the protein derived from Agas2 was 73.3 kDa and the pI was estimated to be 5.33.

< Example  4> New Alpha Galactosidase  Analysis of conserved domains of genes

As a result of conserved domain analysis using the amino acid sequence of Agas2 (alpha-galactosidase S2, alpha -galactosidase S2) as shown in FIG. 2, glycosyl hydrolase (GH ) Belonging to the family GH97 (http://www.cazy.org/fam/acc_GH.html). A comparison of homology with the amino acid sequences known to GenBank in the Agas2 base sequence was performed using the BalstX algorithm. The highest homology is Dysgonomonas Hypothetical protein derived from gadei showed 70% identity and 82% similarity.

< Example  5> New Alpha Galactosidase  Systematic analysis of genes

The amino acid sequence identified in Example 4 was systematically analyzed using Mega5 program (www.megasoftware.net) as shown in FIG. 3, and as a result, Dysgonomonas but it was confirmed that it is a novel α - galactosidase enzyme which has not yet been known. In the BlastP search, the galactosidase gene and the GenBank number showing the significance were confirmed. The number on the branch is the bootstrap analysis (1,000 resampling).

< Example  6> New Alpha Galactosidase  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 in order to mass-express Agas2 (alpha-galactosidase S2, alpha -galactosidase S2) protein in E. coli Followed by induction with IPTG 0.5 mM at 20 &lt; 0 &gt; C for 20 hours. To isolate and purify the expressed protein, only 50 ml of a culture solution of Escherichia coli transformed with Agas2 was centrifuged to obtain cells. To this, 10 ml of lysis buffer (50 mM K ₂ HPO ₄ (pH 7.0), 300 mM sodium chloride, ), And 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. The Agas2 protein was bound to the column by mixing with a TALON metal affinity resin (BD Bioscience Clontech) using a 6XHis-Tag randomly attached to the C-terminus of the amino acid in the crude enzyme solution. 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 Agas2 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 it was confirmed that a 75 kDa Agas2 protein was mass-expressed (FIG. 4).

< Example  7> New Alpha Galactosidase  Biochemical characterization

To determine the temperature at which the optimal activity of the Agas2 (alpha-galactosidase S2, alpha -galactosidase S2) protein was determined, 0.1 μg of the purified Agas2 protein was dissolved in potassium phosphate buffer solution (50 mM K ₂ HPO ₄ (pH 7.0) ) under five minutes 1mM p-nitrophenyl was measured at 405nm with a spectrophotometer. Yield-nitrophenyl-alpha -D- galacto pyrano seed (p -Nitrophenyl-aD-galactophyranoside) reacting with p. As a result, the Agas2 protein showed enzyme activity at 25 to 50 DEG C and showed the highest activity at 40 DEG C (FIG. 5, (A)).

In addition, in order to determine the naeyeolseongreul Agas2 protein, respectively, to each other under the purified left standing for a total of 4 hours at different temperatures, the standard assay reaction (potassium phosphate buffer solution at regular intervals _{(50mM K 2 HPO 4 (pH7.0} ), 200μl) 0.1 g of Agas2 protein was reacted with 1 mM p -nitrophenyl-alpha-D-galactopyranoside at 40 DEG C for 5 minutes). As a result, the Agas2 protein maintained a stable enzyme activity of 85% or more for 4 hours at an appropriate temperature of 40 ° C (FIG. 5, (B)).

To determine the optimum active pH of the Agas2 protein, sodium acetate (pH 4.0-6.0), potassium phosphate (pH 6.0-7.5), HEPES (4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid , 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (pH 7.5 to 8.5), CHES (N-cyclohexyl-2-aminoethanesulfonic acid) ) And glycine-NaOH (pH 10.0 to 11.0) buffer solution, the activity of decomposing p -nitrophenyl-alpha-D-galactopyranoside was measured at 40 DEG C for 5 minutes. As a result, the Agas2 protein exhibited an enzyme activity in the range of pH 6.0 to 9.0, and showed optimal activity at pH 7.0 (FIG. 5, (C)).

To confirm the pH stability of the Agas2 protein, the Agas2 protein was allowed to stand at 40 DEG C for 15 hours under different pH, and the amount of the degradation product was measured according to the standard assay method. As a result, the Agas2 protein showed more than 70% enzyme activity at various pH ranges of pH 4.0 to 11.0 (Fig. 5, (D)).

< Example  8> Chemicals  New Alpha for Additives Galactosidase  Active Stability Analysis

To analyze the enzyme activities of Agas2 (alpha-galactosidase S2, alpha -galactosidase S2) proteins on chemical additives, purified Agas2 protein (0.05 μg / μl, final 10 μg) After 30 minutes of preincubation, 2 μl (final 0.1 μg) was dispensed and reacted under standard assays to determine the amount of p - nitrophenol produced by the spectrophotometer at 405 nm. This value was set as the standard reaction 100%.

In order to confirm the stability of the enzyme activity against the organic solvent, 0.05 μg / μl of Agas2 protein was added to different organic solvents (10%) and the cells were pre-cultured at 30 ° C. for 30 minutes. The amount of p - nitrophenol was compared with each other. As a result, the Agas2 protein showed a very high activity stability in a water-soluble organic solvent (Fig. 6 (A)).

To confirm the activity stability of the enzyme against the surfactant, the Agas2 protein was pre-incubated at 30 占 폚 for 30 minutes under 1% ([v / w] or [v / v] The amount of p - nitrophenol produced was compared. As a result, Agas2 protein showed very high activity stability in nonionic surfactants. It was found that the ionic surfactant CTAB (Cetyltrimethyl ammonium bromide) (cationic) and SDS (sodium dodecyl sulfate ) (Anionic) showed activity of 50% and 20%, respectively (Fig. 6, (B)).

To confirm the stability of the enzyme activity against metal ions, Agas2 protein was added to 1 mM of each metal ion and incubated at 30 ℃ for 30 min. Then, the amount of p - nitrophenol produced by the standard assay method was compared. As a result, the Agas2 protein exhibited activity of 35% or more with respect to magnesium chloride, manganese chloride, copper chloride, zinc chloride, ferrous sulfate, cadmium chloride, cobalt chloride, nickel chloride and calcium chloride except for mercury chloride and EDTA (FIG. (Fig.

< Example  9> High saltiness  Under the new alpha Galactosidase  Enzyme activity stability analysis

In order to confirm the stability of the enzyme activity of Agas2 (alpha-galactosidase S2, alpha -galactosidase S2) protein according to the salt concentration, potassium phosphate buffer solution (50 mM K ₂ HPO ₄ (pH 7.0)) and incubated at 30 ° C for 30 minutes. Subsequently, 2 μl (final 0.1 μg) sample was dispensed and reacted with 1 mM p -nitrophenyl-α-D-galactopyranoside at 40 ° C. for 5 minutes, and the relative amount of p- The residual activity of the enzyme was compared by measuring with a photometer at 405 nm. As a result, the Agas2 protein exhibited a high enzyme activity stability of 90% or more even at a salt concentration of 4M under sodium chloride or potassium chloride (Fig. 7 (A)).

To confirm the degree of inhibition of the Agas2 enzyme activity under high salt loading, sodium chloride and potassium chloride were added to the potassium phosphate buffer solution (50 mM K ₂ HPO ₄ (pH 7.0), 200 μl) at different concentrations (0 to 4 M) Respectively. To this was added 0.1 μg of purified Agas2 and 1 mM p -nitrophenyl-alpha-D-galactopyranoside to compare the amount of p -nitrophenol produced according to the standard assay method. As a result, specifically, the Agas2 protein showed an enzyme activity of 30% or more even under 4M of high salt (Fig. 7, (B)).

These properties are not well documented in conventional alpha-galactosidases.

< Example  10> New alpha for proteolytic enzymes Galactosidase  Stability analysis

In order to confirm the stability of Agas2 (alpha-galactosidase S2, alpha -galactosidase S2) protein to proteolytic enzymes, the presence of excess protease (Agas2: protease = 1:10, [w / ]) Buffer (0.05 μg / μl of Agas2 protein) and incubated at 30 ° C for 30 minutes. Here, 2 μl (final 0.1 μg) was dispensed to compare the amount of p -nitrophenol produced by a standard assay reaction method using 1 mM p -nitrophenyl-α-D-galactopyranoside as a substrate. As a result, it was confirmed that the Agas2 protein was not degraded by various proteolytic enzymes and showed very high stability of 70% or more (FIG. 8).

< Example  11> New Alpha Galactosidase  Substrate specificity analysis

As shown in Table 1, the substrate specificity of Agas2 (alpha-galactosidase S2, alpha -galactosidase S2) protein was confirmed according to the standard assay reaction method ( ^b ND, not detected).

In the case of the p -nitrophenyl group-substituted sugar derivative, 0.1 μg of purified Agas2 protein and 1 mM p -nitrophenyl-alpha-D-galactopyranoside were reacted under the standard assay reaction conditions, The amount of p - nitrophenol was measured with a spectrophotometer at 405 nm.

In the case of oligosaccharides, 0.1 μg of purified Agas2 and 1 mM of p -nitrophenyl-alpha-D-galactopyranoside 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.

In the case of polymeric polysaccharides, 10 μg of purified Agas2 protein was reacted with 0.5% p - nitrophenyl - α - D - galactopyranoside under standard assay reaction conditions and the amount of galactose produced was measured by DNS (3, 5-dinitrosalicylic acid) method (Miller, 1959).

One unit was defined as the amount of enzyme required to produce 1 μmol of product ( p - nitrophenol, glucose, galactose) for 1 minute.

Substrate Specific activity (U / mg) ^a p- Nitrophenyl-a-D-galactopyranoside 128.37 + - 2.93 alpha-D-Melibiose 53.02 + - 3.58 Locust bean gum 0.32 ± 0.02 Guar gum 0.24 ± 0.01 p- Nitrophenyl-ss-D-galactopyranoside ND ^b p- Nitrophenyl-a-D-glucopyranoside ND p- Nitrophenyl-ss-D-glucopyranoside ND p- Nitrophenyl-a-D-mannopyranoside ND p- Nitrophenyl-ss-D-mannopyranoside ND Lactose ND Maltose ND Cellobiose ND

Agriculture Gene Resource Center KACC95128P 20140409

<110> Republic of Korea <120> a-galactosidase gene from Hermetia illucens and uses thereof <130> P14R12D0268 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 643 <212> PRT <213> a-galactosidase S2 protein from Hermetia illucens <400> 1 Gln Lys Gln Phe Leu Val Gln Ser Pro Asp Asn Glu Leu Lys Val Asn   1 5 10 15 Ile Ser Val Gly Asn Thr Ile Glu Tyr Ser Val Ser His Asn Gly Asp              20 25 30 Leu Met Ile Ala Asn Ser Pro Ile Ser Ser Ser Leu Thr Asn Gly Lys          35 40 45 Ser Phe Gly Ile Asn Pro Arg Leu Ala Gly Leu Ser Ser Lys Thr Leu      50 55 60 Asn Asp Thr Ile Thr Ala Ser Val Tyr Lys Arg Lys Glu Ile Ala Asp  65 70 75 80 Thr Tyr Asn Glu Leu Ile Leu Arg Phe Lys Glu Gly Phe Ser Leu Val                  85 90 95 Phe Arg Ala Tyr Asn Asp Gly Ile Ala Tyr Arg Phe Val Ser Asn Leu             100 105 110 Lys Lys Pro Phe Glu Val Lys Ser Glu Glu Val Asn Phe Asn Phe Pro         115 120 125 Thr Asp Gln Lys Ala Tyr Ile Pro Tyr Val Gln Ile Ile Lys Glu Pro     130 135 140 Ile Glu Lys Gln Phe Tyr Asn Ser Phe Glu Asn Thr Tyr Gln His Ile 145 150 155 160 Asn Ile Ser Gln Trp Asp Lys Lys Arg Leu Ala Phe Leu Pro Leu Val                 165 170 175 Val Glu Gly Val Asn Gly Lys Lys Val Cys Ile Thr Glu Ser Asp Leu             180 185 190 Thr Asn Tyr Pro Gly Met Tyr Leu Tyr Asn Gly Asn Gly Ser Thr Thr         195 200 205 Leu Ser Gly Val Phe Ala Pro Tyr Pro Lys Asp Ile Lys Gln Gly Gly     210 215 220 His Asn Met Leu Gln Gly Glu Val Gln Ser Ala Glu Pro Tyr Ile Ala 225 230 235 240 Arg Tyr Asn Gly Ala Thr Asn Phe Pro Trp Arg Ile Ile Ile Val Ser                 245 250 255 Glu Asn Asp Lys Glu Leu Ala Asp Asn Asp Met Val Tyr Lys Leu Ala             260 265 270 Thr Pro Asn Lys Leu Ala Asp Ala Ser Trp Ile Lys Pro Gly Lys Val         275 280 285 Ala Trp Asp Trp Trp Asn Asp Trp Asn Leu Tyr Asp Val Asp Phe Arg     290 295 300 Ser Gly Ile Asn Asn Glu Thr Tyr Lys Tyr Tyr Ile Asp Phe Ala Ser 305 310 315 320 Gln His Gly Ile Glu Tyr Val Ile Leu Asp Glu Gly Trp Ala Val Asn                 325 330 335 Leu Gln Ala Asp Leu Phe Gln Val Val Pro Glu Ile Asp Leu Lys Glu             340 345 350 Leu Val Asp Tyr Ala Asn Lys Lys Asn Val Gly Leu Ile Leu Trp Ala         355 360 365 Gly Tyr Tyr Ala Phe Asn Lys Asp Ile Glu Gly Ile Cys Lys His Tyr     370 375 380 Ser Glu Met Gly Ile Lys Gly Phe Lys Val Asp Phe Met Asp Arg Asp 385 390 395 400 Asp Gln Pro Met Val Asp Phe His Tyr Arg Ala Ala Glu Ile Ala Ala                 405 410 415 Lys Tyr His Met Met Leu Asp Tyr His Gly Thr Tyr Lys Pro Thr Gly             420 425 430 Leu Gln Arg Thr Phe Pro Asn Val Ile Asn Phe Glu Gly Val His Gly         435 440 445 Leu Glu Gln Met Lys Trp Ser Pro Glu Ser Val Asp Gln Val Thr Tyr     450 455 460 Asp Val Thr Ile Pro Phe Ile Arg Met Val Ala Gly Pro Leu Asp Tyr 465 470 475 480 Thr Gln Gly Ala Met Arg Asn Ala Ser Lys Gly Asn Tyr Arg Pro Val                 485 490 495 Asn Ser Glu Pro Met Ser Gln Gly Thr Arg Cys Arg Gln Leu Ala Glu             500 505 510 Tyr Ile Ile Phe Glu Ser Pro Leu Asn Met Leu Cys Asp Asn Pro Ser         515 520 525 Asn Tyr Met Arg Glu Lys Glu Cys Thr Glu Phe Ile Ala Asn Val Pro     530 535 540 Thr Val Trp Asp Asn Thr Ile Ala Leu Asn Gly Lys Ile Gly Glu Tyr 545 550 555 560 Ile Thr Ile Ala Arg Glu Lys Asp Asp Val Trp Tyr Val Gly Ser Leu                 565 570 575 Thr Asn Trp Asp Ala Arg Thr Leu Asp Val Asp Leu Ser Phe Leu Gly             580 585 590 Glu Gly Ser Phe Lys Ala Glu Val Phe Lys Asp Gly Ala Asn Ala Asp         595 600 605 Arg Ser Gly His Asp Tyr Lys Lys Glu Val Ile Asp Ile Pro Ser Asp     610 615 620 Arg Lys Leu Ser Ile Pro Met Ala Ser Gly Gly Gly Tyr Val Met Lys 625 630 635 640 Ile Tyr Lys             <210> 2 <211> 1932 <212> DNA <213> a-galactosidase S2 gene from Hermetia illucens <400> 2 caaaagcagt tcttggtaca gtctcctgat aacgagctta aagtaaacat atctgttggt 60 aatacgatag aatattctgt atcccacaat ggagatctaa tgatagcaaa ctcacctata 120 tcaatgtctt tgaccaacgg gaaatctttc ggaataaacc ccagattagc aggcttatct 180 tctaagacat tgaatgatac aataactgct tcggtatata aacgaaaaga aattgcagat 240 acttataatg agcttattct tagattcaaa gaaggattca gccttgtatt cagggcatac 300 aacgatggta tagcctatcg cttcgtttca aacttgaaaa aaccttttga agtaaaaagt 360 gaagaggtaa atttcaattt cccgaccgat cagaaagcgt atattcctta tgtacagatc 420 ataaaagaac ctattgagaa gcaattttac aattcgttcg aaaatacgta ccagcatata 480 aatatatctc agtgggacaa aaaacgtctg gcttttctgc ccttggtagt agagggtgta 540 aacggcaaga aagtatgtat tacggaatct gacctgacaa attatccggg gatgtatcta 600 tataatggta atgggtctac tactttaagc ggggtatttg caccttatcc taaggatata 660 aaacaaggtg gtcacaatat gctacagggt gaagtacagt cggcagaacc ttatattgca 720 cgatacaacg gagcaactaa tttcccttgg cgcatcataa tcgtttcgga gaatgataag 780 gaactggctg ataatgacat ggtatataag ctggctaccc ccaataagct ggcagatgct 840 tcgtggatca aacccggaaa agtggcttgg gattggtgga atgactggaa tctgtacgat 900 gttgatttcc gttcgggtat caataatgaa acgtataaat attatattga ttttgcttcg 960 caacatggta tcgaatatgt aatattggac gagggttggg ctgtaaatct gcaagccgac 1020 ttatttcagg tagttcccga aatagatcta aaagagctgg ttgattatgc caataaaaag 1080 aacgtaggac ttatcctatg ggcaggatac tatgctttta ataaagacat cgaaggtatc 1140 tgcaaacatt attcggaaat gggtatcaaa ggctttaagg ttgactttat ggacagggac 1200 gatcagccaa tggtagactt ccactatcgg gcagccgaga ttgccgcaaa ataccacatg 1260 atgtcgatt atcacggcac atataaaccg acgggattgc aacgtacatt ccccaatgtt 1320 atcaattttg aaggtgttca cggattggag caaatgaaat ggtcgcccga aagcgtagat 1380 caggttacat acgatgttac tatacctttt atcagaatgg tagccggacc tcttgattat 1440 actcaggggg ctatgcgtaa tgcttctaaa ggcaattatc gccctgtcaa cagtgagccg 1500 atgagtcagg gtacacgctg ccgtcagtta gcggaatata ttatattcga atcgcctctg 1560 aatatgctat gcgataaccc gtccaattat atgcgtgaga aagaatgtac cgagttcata 1620 gccaatgtgc caacagtatg ggacaatact atagctttga atggaaaaat aggtgaatac 1680 atcactattg cacgagagaa agacgatgtg tggtatgtag gttcgcttac caactgggat 1740 gcaagaacac ttgatgttga tttgtcattt ctgggagaag gcagctttaa ggctgaggtc 1800 tttaaagacg gtgcgaatgc cgaccgatcg ggacatgatt ataaaaaaga agtaattgac 1860 ataccatctg accgaaaact aagtattcca atggcttcgg gcggtggtta tgtgatgaaa 1920 atttacaagt ag 1932

Claims (10)

1. A polypeptide consisting of the amino acid sequence of SEQ ID NO: 1. The polypeptide according to claim 1, wherein the polypeptide is a salt tolerant alpha-galactosidase. A gene encoding the polypeptide of claim 1. 4. The gene according to claim 3, wherein the gene is derived from Donghae or the like. 4. The gene according to claim 3, wherein the gene comprises the nucleotide sequence of SEQ ID NO: 2. A vector comprising the gene according to claim 3. A transformant transformed with the vector of claim 6. 8. The transformant according to claim 7, wherein the transformant is Escherichia coli. 9. The transformant according to claim 8, wherein said transformant is deposited under Accession No. KACC95128P. (1) isolating the gene consisting of the nucleotide sequence of SEQ ID NO: 2 from the gene derived from Donghae and the like;
(2) cloning the gene into a vector;
(3) transforming the vector into a transformant; And
(4) purifying a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 from the transformant;
A method for producing a salt tolerant alpha -galactosidase.

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