KR20160051932A - A NOVEL α-GLUCOSIDASE GENE FROM HERMETIA ILLUCENS WITH STABLE ACTIVITY IN HIGH-CONCENTRATION ORGANIC SOLVENT, AND USES THEREOF - Google Patents

Abstract

The present invention relates to an α-glucosidase gene isolated from uncultured microorganisms present in the intestines of Ptecticus tenebrifer larvae, and to a use thereof. The present invention isolates, from uncultured microorganisms present in the intestines of Ptecticus tenebrifer larvae, a novel enzymatic gene that breaks an alpha-1,4-glycosidic bond at the non-reducing end of α-glucoside; and develops a novel strain that expresses the same in a large amount. Therefore, the present invention can be used in various industrial processes involving plant-derived polysaccharides. Also, the breakdown effect of the novel enzymatic gene of the present invention can be used in food fermentation industries, paper/fabric industries, alcohol manufacture processes, medicine production, and saccharification of plant polysaccharides.

Description

A novel α-glucosidase gene Agls6 having stable activity in a high concentration organic solvent and the use of a recombinant enzyme produced therefrom {A novel α-glucosidase gene from Hermetia illucens with stable activity in high-concentration organic solvent,

The present invention relates to an α-glucosidase gene isolated from an egg-culturing microorganism existing in a larval gland and the use thereof.

In the biomass recycling process involving the existing polymeric polysaccharides, the microorganisms or fungi themselves, which can be chemically treated or separated normally, were directly used because of the complex structure of the polymeric polysaccharides such as starch.

However, in order to perform the process more efficiently, it has been required to maintain a stable resolution in a high concentration of a water-soluble organic solvent and a wide acidity change and in a non-ionic surfactant, various protein reducing agents and denaturants.

In order to solve such a problem, the present inventors have constructed a metagenome bank derived from intestinal microorganisms which can not be artificially cultured in larvae, such as dysentery which effectively decomposes food waste, and from this, a polymeric polysaccharide such as starch or a non- A novel enzyme gene which degrades the α-1,4-glycosidic bond at the seed end is isolated and purified by recombinant α-glucosidase enzyme which can be efficiently applied to various industrial processes using vegetable high molecular polysaccharide Thereby completing the invention.

Korean Patent Publication No. 10-2003-0088963

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-glucosidase having solvent stability.

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

The polypeptide may be alpha-glucosidase which exhibits stable activity in a high concentration organic solvent.

The alpha glucosidase according to the present invention may exhibit stable activity at 20-50 ° C, or pH 5.0-8.0.

The term "high concentration organic solvent " means a solution in which the organic solvent is contained at a relatively high concentration of 10-20% (wt% or volume%), and the organic solvent is any organic solvent commonly used in the art . For example, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), methanol, acetonitrile, ethanol, acetone, 2-propanol and the like.

Alpha glucosidase is a generic term for enzymes that hydrolyze glycosidic bonds between oligosaccharides (small sugars) and glycosides (glycosides) to liberate monosaccharides, and glucosidase, mannosidase , Galactosidase, fructosidase, and xylosidase. There are also? -Glucosidase and? -Glucosidase depending on the anomeric form of the hydrolyzed glycosidic bond.

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 (FIG. 2) and cDNA and DNA accordingly.

"% 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 with Accession No. KACC95129P, 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 obtain a solvent-stable alpha-glucosidase.

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 Dongae and others. From this, a novel clone capable of degrading the glucose 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 1,923 bp gene and thus the 640 amino acid sites were a novel gene that breaks down the polymer polysaccharide.

In addition, the present inventors have invented a novel strain capable of efficiently expressing the above gene in E. coli and effectively degrading the alpha-1,4-glycosidic bond at the terminal of the non-reducing alpha-glycoside. The α-glucosidase enzyme purified from the strain showed high enzyme activity stability against EDTA, nonionic surfactant (1%), and showed at least 60% activity stability in the protein reducing agent and the denaturant.

The α-glucosidase enzyme provided by the present invention decomposes the high-molecular polysaccharide forming an α-1,4-glycosidic bond, and thus can be efficiently applied to various industrial processes using vegetable high-molecular 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.

In the conventional biomass recycling process, due to the complex structure of polymeric polysaccharides such as starch, in order to perform the process more efficiently in the chemical treatment process or the usual separation, a high concentration of the water-soluble organic solvent, It has been desired to maintain stable resolution in non-ionic surfactants, various protein reducing agents and denaturants. The alpha-glucosidase enzyme provided by the present invention exhibits a stable decomposing ability in the presence of organic solvents, a nonionic surfactant, a protein reducing agent / denaturant and a wide acidity when Escherichia coli is expressed, and the number of purification steps in the biomass recycling process And thus the economic loss can be reduced.

The invention (such Hermetia dongae illucens ) A novel enzyme gene that cleaves alpha-1, 4-glycosidic bonds at the non-reducing alpha-glycoside ends from egg culture microorganisms in the intestinal tract was developed and a new strain expressing this enzyme was developed in large quantities to develop a vegetable polymer polysaccharide And can be efficiently applied to various industrial processes used. In addition, the decomposition effect of the novel polysaccharide of the novel enzyme gene according to the present invention can be utilized for the food fermentation industry, the paper / textile industry, the alcohol production process, the manufacture of medicines, and the glycosylation of plant polysaccharides.

Figure 1 is the amino acid sequence of the novel alpha glucosidase (Agls6): the underlined red portion is the signal peptide sequence from the gram negative / positive microorganism, the novel alpha glucosidase amino acid sequence according to the invention The rest.
Figure 2 is the DNA sequence of the novel alpha glucosidase
Figure 3 shows the result of analysis of the domain conservation domain of the novel alpha glucosidase.
Figure 4 is a phylogenetic diagram of the novel alpha glucosidase.
FIG. 5 shows the results of the large-scale expression of the novel α-glucosidase from Escherichia coli: 1 is the E. coli crude protein before the induction, 2 is the E. coli crude protein expressing the large amount of new α-glucosidase, 3 is the water-soluble protein, Alpha glucosidase.
FIG. 6 is a graph showing the analysis of the biochemical properties of novel alpha glucosidase: A is the optimum activity temperature of the novel alpha glucosidase, B is the thermal stability of the novel alpha glucosidase, C is the optimum of the novel alpha glucosidase Active pH, D is the pH stability of the novel alpha glucosidase.
Figure 7 is a graphical analysis of the activity stability of novel alpha glucosidase for chemical additives: A is the activity stability of the novel alpha glucosidase for the metal ion, B is the activity of the novel alpha glucosidase for the surfactant, Activity stability, C is the activity stability of novel alpha glucosidase for various protein reducing agents and denaturants.
8 is a graph showing the activity stability of novel alpha glucosidase according to changes in organic solvent concentration: A is the activity stability of a novel alpha glucosidase in an organic solvent at a concentration of 10%, B is an activity stability of a novel alpha glucosidase in an organic solvent Activity stability of novel alpha glucosidase in.

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> Glycosidase-degrading active gene screening

In order to test the metagenomic clones showing degrading activity in glucose medium, 1% carboxymethyl glucose (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 completely dyed at room temperature for 5 minutes using a Congo Red dyeing reagent and washed with sterilized water to determine the ability to produce glucosease with or without ring formation around the cells. 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. A clone that repeatedly streaks on the glucose medium and exhibits glucose degrading ability was finally selected as a positive strain.

< Example  3> New Alpha Glucose Sidase  Gene analysis

A total nucleotide sequence of 2,022 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 a Gram-negative / positive microorganism was found in 33 amino acids of N-terminal (red color). The 640 amino acid sequence corresponding to the 1,923-bp nucleotide sequence from which the region was deleted was presumed to have an alpha-glucosidase enzyme function, and the 640 amino acids corresponding thereto were named Agls6 (alpha-glucosidase 6, alpha-glucosidase 6). The size of the protein derived from Agls6 was 73.0 kDa and the pI was estimated to be 4.82.

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

Conserved domain analysis using the amino acid sequence of Agls6 as shown in FIG. 3 confirmed that it belonged to GH97 among the glycosyl hydrolase (GH) families classified in the CAZy database http://www.cazy.org/fam/acc_GH.html). A homology comparison with the amino acid sequence known to GenBank in the Agls6 base sequence was performed by the BalstX algorithm. The highest homologues are Bacteroides sp. The α-glucosidase derived from HPS0048 showed 77% identity.

< Example  5> New Alpha Glucosidase  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. 4, and as a result, Bacteroides sp. It has been confirmed that the enzyme is an alpha - glucosidase enzyme which is evolutionarily similar to 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> New Alpha Glucosidase  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 Agls6 protein in E. coli in a large amount. (induction). To isolate and purify the expressed protein, only 50 ml of a culture solution of Escherichia coli transformed with the Agls6 gene was centrifuged to obtain cells, which were 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 crude enzyme solution, Agls6 protein was mixed with TALON metal affinity resin (BD Bioscience Clontech) using a 6XHis-Tag randomly attached to the C-terminal of the amino acid and bound to the column. After binding to the coenzyme resin, it was washed with 10 volumes of wash buffer solution (50 mM K ₂ HPO ₄ (pH 7.0), 300 mM sodium chloride, 10 mM imidazole) and Agls6 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 large amount of Agls6 protein of 74.5 kDa was expressed (FIG. 5).

< Example  7> New Alpha Glucosidase  Biochemical characterization

0.5 μg of the purified Agls6 protein was incubated with 1 mM p -nitrophenyl-α-D (5 mM) for 5 minutes in sodium phosphate buffer solution (50 mM Na ₂ HPO ₄ (pH 6.5), 200 μl) ( P- Nitrophenyl-α-D-glucophyranoside), and the amount of p -nitrophenyl produced was measured by a spectrophotometer at 405 nm. As a result, the Agls6 protein showed enzyme activity at 20-50 ° C and showed the highest activity at 40 ° C (Fig. 6A).

In order to confirm the thermostability of the Agls6 protein, a standard assay reaction (sodium phosphate buffer solution (50 mM Na ₂ HPO ₄ (pH 6.5), pH 7.0, 0.5 μg of the purified Agls6 protein was reacted with 1 mM p -nitrophenyl-α-D-glucopyranoside at 40 ° C. for 10 minutes). As a result, the Agls6 protein maintained a stable enzyme activity of 55% or more for 2 hours even at an appropriate temperature of 40 DEG C (Fig. 6B).

To determine the optimal active pH of the Agls6 protein, sodium acetate (pH 4.0-6.0), sodium phosphate (pH 6.0-7.5), HEPES (4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid , 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (pH 7.5-8.5), CHES (N-cyclohexyl-2-aminoethanesulfonic acid) ) Buffer solution was used to measure the decomposing activity of p -nitrophenyl-alpha-D-glucopyranoside at 40 DEG C for 10 minutes. As a result, the Agls6 protein exhibited enzyme activity in the range of pH 5.0-8.0, and showed optimal activity at pH 6.5 (FIG. 6C).

To confirm the pH stability of the Agls6 protein, the Agls6 protein was allowed to stand at 4 [deg.] C for 15 hours under different pHs, and the amount of the degradation products was measured according to the standard assay method. As a result, the Agls6 protein exhibited an enzyme activity of 75% or more at various pH ranges of pH 4.0-10.0 (FIG. 6D).

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

To analyze the enzymatic activity of the Agls6 protein on the chemical additives, purified Agls6 protein (0.25 μg / μl, final 10 μg) without inhibitor was preincubated for 30 minutes at 30 ° C. and then 2 μl (final 0.5 μg) Was reacted under the standard assay reaction method and the amount of p - nitrophenol produced was measured by a spectrophotometer at 405 nm. This value was set as the standard reaction 100%.

In order to confirm the stability of enzyme activity against metal ions, 0.25 μg / μl of Agls6 protein was added to 1 mM of each metal ion, and the cells were preincubated at 30 ° C. for 30 minutes. Then, 2 μl (final 0.5 μg) was added thereto and 1 mM p- - The amount of p -nitrophenol produced according to the standard assay method using alpha-D-glucopyranoside as substrate was compared. As a result, the activity of the enzyme was not influenced by the Agls6 protein for EDTA, but Cu / Zn / Hg-2 was found to be inhibited for the ion (Fig. 7A).

To confirm the activity stability of the enzyme on the surfactant, the Agls6 protein was pre-incubated for 30 minutes at 30 ° C under 1% ([v / w] or [v / v] The amount of p - nitrophenol produced was compared. As a result, the Agls6 protein showed very high activity stability to the nonionic surfactant (Fig. 7B).

To confirm the stability of the enzyme activity against various protein reducing agents / modifiers, the Agls6 protein was added to each chemical additive and preincubated at 30 DEG C for 30 minutes. Then, p -nitro The amounts of phenol were compared with each other. As a result, the Agls6 protein showed at least 60% activity stability in the protein reducing agent and the denaturant (Fig. 7C).

< Example  9> Stability Analysis of Enzymatic Activity of New Alpha Glucosidase in High Concentration Organic Solvents

To confirm the stability of the enzyme activity of the Agls6 protein according to the organic solvent concentration, various kinds of organic solvents were added in an amount of 10% ([v / w] or [v / v]) or 20% ([v / w] / v]) (0.25 μg / μl of the purified Agls6 protein was added to 50 mM Na ₂ HPO ₄ (pH 6.5), 200 μl) and then pre-cultured at 30 ° C. for 30 minutes. Subsequently, 2 μl (final 0.5 μg) sample was dispensed, and after the standard assay reaction with 1 mM p -nitrophenyl-α-D-glucopyranoside substrate, the relative amount of p -nitrophenol produced was measured with a spectrophotometer 405 nm and compared. As a result, the Agls6 protein exhibited a high enzyme activity stability of 85% or more in most of the water-soluble organic solvent of 10% concentration and a high enzyme activity stability of 70% or more even in the 20% concentration of the water-soluble organic solvent (FIG.

< Example  10> New Alpha Glucosidase  Substrate specificity analysis

Standard Assay Reaction Method (0.5 μg of purified Agls6 protein in 50 mM sodium phosphate buffer (pH 6.5, 200 μl), reaction at 40 ° C. for 10 minutes) was performed to determine the substrate specificity of the Agls6 protein (ND, not detected).

In the case of the p - nitrophenyl group - substituted sugar derivative, 0.5 μg of the purified Agls6 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.

For oligosaccharides, 0.5 μg of purified Agls6 was reacted with 1 mM oligosaccharide substrate under standard assay reaction conditions, and the amount of glucose produced was measured at 540 nm using a glucose assay kit (Sigma, USA).

For the polymeric polysaccharide (starch), 10 μg of purified Agls6 protein was reacted with 0.5% starch substrate under standard assay reaction conditions, and the amount of glucose produced was measured by DNS (3,5-dinitrosalicylic acid, 3,5 -dinitrosalicylic acid method (Miller, 1959).

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

As a result, Agls6 showed a specific activity for short chain maltooligosaccharides such as maltose was higher than that of aryl glucosides (ρ-Nitrophenyl-α-D-glucophyranoside) , Indicating that Agls6 is a type II alpha glucosidase.

Substrate Major glycosidic bond Specific activity (U / mg) ^a Maltose Glucose-a-1,4-Glucose 101.17 + - 7.69 Maltotriose (Glucose-a-1,4) _2- Glucose 90.54 ± 6.33 Maltotetraose (Glucose-a-1,4) _3- Glucose 76.88 + - 3.72 Maltopentaose (Glucose-a-1,4) _4- Glucose 66.41 + - 2.58 Maltohexaose (Glucose-a-1,4) _5- Glucose 63.25 + - 2.84 Maltoheptaose (Glucose-a-1,4) _6- Glucose 61.74 + 2.05 Lactose Glucose-β-1,4-Galactose ND ^b Cellobiose Glucose-beta-1,4-Glucose ND Sucrose Glucose-β-1,2-Fructose ND p- Nitrophenyl-a-D-glucopyranoside Nitrophenol-a- (4) -D-Glucose 8.95 ± 1.19 p- Nitrophenyl-ss-D-glucopyranoside Nitrophenol-β- (4) -D-Glucose ND p -Nitrophenyl-a-D-galactopyranoside Nitrophenol-a- (4) -D-Galactose ND p- Nitrophenyl-ss-D-galactopyranoside Nitrophenol-β- (4) -D-Galactose ND Starch (Glucose-a-1,4) _n- Glucose &Lt; 0.1

Agriculture Gene Resource Center KACC95129 20140925

<110> Republic of Korea <120> A novel alpha-glycosidase gene from Hermetia illucens with stable          activity in high-concentration organic solvents, and uses thereof <130> P14R12D1053 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 640 <212> PRT &Lt; 213 > amino acids of Agls6 protein <400> 1 Ser Pro Asp Glu Asn Leu Glu Ile Lys Ile Glu Val Gly Gln Asn Ile   1 5 10 15 Thr Tyr Thr Leu Thr His Gln Gly Asp Leu Leu Leu Ser Pro Ser Met              20 25 30 Ile Ala Met Glu Ile Glu Asp Gly Val Asp Phe Gly Ile Asn Ser Arg          35 40 45 Val Val Lys Glu Lys Arg Asn Gln Val Asn Gln Thr Ile Leu Ser Pro      50 55 60 Phe Tyr Lys Lys Glu Gln Val Glu Asp Val Tyr Asn Glu Leu Ile Leu  65 70 75 80 Ser Phe Lys Asp Gly Phe Glu Leu Val Leu Arg Ala Tyr Asn Glu Gly                  85 90 95 Ala Ala Tyr Arg Phe Val Ser Ser Arg Lys Gln Asp Phe Ile Val Arg             100 105 110 Asn Glu Ile Ala Glu Phe Asn Val Gly Ala Thr Asn Gln Ala Tyr Ile         115 120 125 Pro Tyr Val Lys Thr Asp Lys Val Ser Lys Ile Asp Gln Leu Phe Asn     130 135 140 Ser Phe Glu Asn Thr Tyr Thr Tyr Ser Ala Val Asp Gln Trp Glu Gly 145 150 155 160 Asp Arg Leu Ala Phe Val Pro Leu Leu Val Glu Thr Leu Ser Gly Lys                 165 170 175 Lys Ile Val Ile Thr Glu Ala Asp Leu Glu Asp Tyr Pro Gly Met Tyr             180 185 190 Leu Glu Asn Ala Asp Lys Asp Ser Ile Leu Asn Gly Phe Phe Ala Pro         195 200 205 Tyr Pro Lys Lys Ile Glu Gln Gly Gly His Asn Met Leu Gln Glu Ile     210 215 220 Val Thr Glu Arg Glu Glu Tyr Ile Ala Gln Cys Lys Gly Arg Thr Ser 225 230 235 240 Phe Pro Trp Arg Ile Ile Thr Val Ala Ser Lys Asp Ala Asp Leu Leu                 245 250 255 Asp Asn Leu Val Tyr Gln Leu Ala Ser Pro Ser Arg Ile Glu Asp             260 265 270 Val Ser Trp Val Lys Pro Gly Lys Val Ala Trp Glu Trp Trp Asn Asp         275 280 285 Trp Asn Leu Tyr Gly Val Asp Phe Glu Thr Gly Val Asn Asn Ala Thr     290 295 300 Tyr Lys Tyr Tyr Ile Asp Phe Ala Ser Glu His Gly Ile Glu Tyr Val 305 310 315 320 Ile Leu Asp Glu Gly Trp Ser Val Gly Gln Lys Ala Asp Leu Phe Gln                 325 330 335 Val Ile Pro Gly Ile Asn Leu Pro Glu Leu Val Glu Tyr Ala Glu Asp             340 345 350 Arg Asn Val Gly Ile Ile Leu Trp Ala Gly Tyr His Ala Phe Asn Lys         355 360 365 Asp Ile Glu Gly Ile Cys Lys His Tyr Ala Glu Met Gly Val Lys Gly     370 375 380 Phe Lys Val Asp Phe Met Asp Arg Asp Asp Gln Pro Met Val His Phe 385 390 395 400 His Tyr Glu Ala Ala Lys Ile Ala Ala Lys Tyr Lys Leu Leu Leu Asp                 405 410 415 Tyr His Gly Thr Tyr Lys Pro Thr Gly Leu Gln Arg Thr Tyr Pro Asn             420 425 430 Val Ile Asn Phe Glu Gly Val His Gly Leu Glu Thr Met Lys Trp Ser         435 440 445 Glu Asn Thr Val Asp Gln Val Thr Tyr Asp Val Thr Ile Pro Phe Ile     450 455 460 Arg Met Leu Ala Gly Pro Met Asp Tyr Thr Gln Gly Ala Met Lys Asn 465 470 475 480 Ala Thr Lys Lys Asn Tyr His Pro Val Phe Ser Glu Ala Met Ser Gln                 485 490 495 Gly Thr Arg Cys Arg Gln Leu Ala Gln Tyr Ile Ile Phe Glu Ala Pro             500 505 510 Leu Thr Met Leu Cys Asp Ser Pro Ser Asn Tyr Glu Arg Glu Glu Glu         515 520 525 Ser Thr Glu Phe Ile Ala Ser Val Pro Thr Val Trp Asp Glu Thr Ile     530 535 540 Ala Leu Gly Gly Lys Val Ser Glu Tyr Val Thr Ile Ala Arg Arg Lys 545 550 555 560 Asp Asp Val Trp Tyr Val Gly Ser Met Thr Asn Trp Asp Lys Arg Pro                 565 570 575 Val Glu Leu Asp Leu Ser Phe Leu Gly Ala Gly Lys Tyr Gln Ala Glu             580 585 590 Ile Phe Lys Asp Gly Ile Asn Ala Gly Lys Val Ala Lys Asp Tyr Lys         595 600 605 Lys Glu Val Ile Asp Ile Pro Ala Asp Arg Lys Leu Glu Ile Ser Leu     610 615 620 Ala Ala Gly Gly Cys Ala Ile Lys Ile Tyr Lys Leu Asn Asp Glu 625 630 635 640 <210> 2 <211> 1923 <212> DNA <213> DNA of Agls6 gene <400> 2 tccccggatg aaaatttgga aataaagata gaagtcggac aaaatataac ctataccttg 60 acccatcagg gcgatctcct tctctcccct tcaatgatcg cgatggaaat agaagatgga 120 gtagatttcg gaataaattc cagggtagtc aaagagaaac ggaaccaagt caatcaaacg 180 attttatctc ctttttataa aaaagaacag gttgaagatg tctacaacga actgatcctc 240 agtttcaagg atggttttga actggtgctc agggcgtata atgagggagc cgcatatcgc 300 ttcgtctctt cccgtaaaca ggattttatt gtcaggaatg aaattgcaga attcaatgtg 360 ggggcaacaa atcaagctta tatcccttat gtaaagacgg ataaggtctc caaaatagat 420 caactgttta attctttcga aaatacatac acttactctg ctgtggatca atgggaggga 480 gatcgtttgg cttttgtgcc tttattggtg gaaaccctta gcggtaagaa aatcgtaatc 540 acagaggctg atcttgaaga ttatcccgga atgtacctgg aaaatgcaga taaagattct 600 atcctgaacg gtttttttgc cccttatcct aaaaaaatag agcagggtgg acataacatg 660 ttgcaggaaa ttgtaacaga aagagaagaa tatatcgctc aatgtaaagg aagaacatca 720 tttccatgga ggattatcac ggtagcatca aaagatgctg atttactcga taatgatttg 780 gtttatcagt tggcgtctcc aagccggata gaagatgtat cctgggtcaa acccggaaaa 840 gttgcatggg aatggtggaa tgattggaac ttgtatggcg ttgattttga aacaggggta 900 aacaatgcaa cctataagta ttacattgat tttgcttctg aacatggaat cgaatatgtt 960 attctggatg aaggatggtc tgttggccaa aaagccgatt tattccaagt gattcccggg 1020 atcaatcttc ccgaattggt ggaatatgca gaagatcgca atgtaggtat cattctttgg 1080 gcaggatatc atgcgttcaa taaggatatt gagggaattt gcaagcatta tgccgaaatg 1140 ggtgtgaaag gatttaaggt tgattttatg gacagggatg accagcccat ggttcatttc 1200 cattatgaag cggcaaagat tgctgcaaaa tataagctgt tattggatta tcacgggaca 1260 tacaaaccga caggattgca gagaacctat cctaacgtga taaactttga aggcgtacat 1320 ggtttggaaa ccatgaagtg gtccgaaaac acagtggacc aggttacgta tgatgtaacc 1380 attcctttta tcagaatgct ggcaggtccg atggattaca cacaaggcgc gatgaagaat 1440 gcgacaaaga aaaactacca tccggttttc tcggaagcga tgagccaggg gacacgctgc 1500 cgtcaattag cccagtatat tatttttgaa gcgccgttga caatgttatg cgacagtcct 1560 tccaactatg agagagaaga ggaaagtacg gagttcatag cttctgtacc gaccgtatgg 1620 gatgaaacca tagcattggg gggaaaggtg agtgaatatg taacaatagc ccgccggaaa 1680 gatgacgtat ggtatgtagg ttccatgact aactgggata aacgacctgt tgaattggat 1740 ttgtcatttt taggtgccgg caaatatcag gctgaaatct tcaaggatgg aatcaatgcc 1800 gggaaagtag caaaagatta taagaaggaa gttattgata ttcccgcaga tagaaaactg 1860 gagatatcct tagctgccgg gggcggatgt gccataaaaa tatacaagtt gaatgatgaa 1920 taa 1923

Claims (13)

1. A polypeptide consisting of the amino acid sequence of SEQ ID NO: 1. The polypeptide according to claim 1, wherein the polypeptide is alpha-glucosidase exhibiting stable activity in a high concentration organic solvent. 3. The method according to claim 2, wherein the concentration of the organic solvent is 10 to 20% by weight or 10 to 20% by volume, and the organic solvent is DMSO, , Methanol, acetonitrile, ethanol, acetone, and 2-propane. 3. The polypeptide according to claim 2, wherein the alpha-glucosidase exhibits stable activity at 20 to 50 &lt; 0 &gt; C. 3. The polypeptide according to claim 2, wherein the alpha-glucosidase exhibits stable activity at a pH of 5.0 to 8.0. A gene encoding the polypeptide of claim 1. The gene according to claim 6, wherein the gene is derived from Donghae. 7. The gene according to claim 6, wherein the gene comprises the nucleotide sequence of SEQ ID NO: 2. A vector comprising the gene according to claim 6. A transformant transformed with the vector of claim 9. 11. The transformant according to claim 10, wherein the transformant is Escherichia coli. 11. The transformant according to claim 10, wherein said transformant is deposited under accession number KACC95129P. (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;
(2) a method for producing alpha-glucosidase which exhibits stable activity in a high concentration organic solvent comprising

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