KR20160049859A - A NOVEL β-MANNOSIDASE AND PRODUCING METHOD THEREOF - Google Patents
A NOVEL β-MANNOSIDASE AND PRODUCING METHOD THEREOF Download PDFInfo
- Publication number
- KR20160049859A KR20160049859A KR1020140147592A KR20140147592A KR20160049859A KR 20160049859 A KR20160049859 A KR 20160049859A KR 1020140147592 A KR1020140147592 A KR 1020140147592A KR 20140147592 A KR20140147592 A KR 20140147592A KR 20160049859 A KR20160049859 A KR 20160049859A
- Authority
- KR
- South Korea
- Prior art keywords
- mannosidase
- beta
- nucleic acid
- activity
- asp
- Prior art date
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2477—Hemicellulases not provided in a preceding group
- C12N9/2488—Mannanases
- C12N9/2491—Beta-mannosidase (3.2.1.25), i.e. mannanase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/02—Monosaccharides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01025—Beta-mannosidase (3.2.1.25), i.e. mannanase
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
The present invention relates to a novel beta-mannosidase, a nucleic acid molecule encoding the same, a recombinant vector comprising the nucleic acid molecule, a recombinant microorganism in which the nucleic acid molecule or the recombinant vector is inserted into a host cell, Mannosidase, a method of decomposing a saccharide containing D-mannose as a main component using the beta-mannosidase, a method of producing D-mannose by the method, Mannosidase is added to the biomass to convert the biomass into a sugar.
Mannan polysaccharide is a major constituent of hemicellulose and is a fairly branched beta-1,4-linked D-mannose polymer with substituents containing acetyl and galactosyl groups. Endo-β-1,4-mannanase, β-mannosidase, and α-galactosidase in order to completely resolve the mannan. . Of these enzymes, endomannase and beta-mannosidase play an important role in degrading the mannan skeleton. Endomannanase attacks only mannosidic linkages inside the metamorphosed skeleton and breaks down into mannooligosaccharides. Subsequently, beta-mannosidase is shortened to mannobiose, including mannobiose Hydrolysis of mannooligosaccharides to D-mannose.
In recent years, mannanase has been reported to be produced in numerous microorganisms, while beta-mannosidase has not been reported in microorganisms.
The object of the present invention is to provide novel β-mannosidase, a nucleic acid molecule encoding the β-mannosidase, a recombinant vector comprising the nucleic acid molecule, a nucleic acid molecule or the recombinant vector isolated from a Penny Bacillus strain, A method for producing the beta-mannosidase, a method for decomposing a saccharide containing D-mannose as a main component using the beta-mannosidase, a method for producing the beta-mannosidase, Mannose according to the method of the present invention, or a method of converting the biomass into a sugar by adding the beta-mannosidase to the biomass.
In order to solve the above-mentioned problems, the present invention provides, in one aspect, a beta-mannosidase comprising the amino acid sequence of SEQ ID NO: 1.
The inventors of the present invention have obtained a novel beta-mannosidase enzyme from Penny Bacillus subtilis (Accession No. KCTC 3953, Deposit No. 20070502), and the novel beta-mannosidase enzyme is a para-nitrophenyl- It has an activity to catalyze the hydrolysis reaction of two or more substrates including the catalytic activity of hydrolysis of para- nitrophenyl-β-mannopyranoside (pNPM). It has a high pH at neutral pH and middle temperature, Not only shows reactivity but also exhibits thermal stability at room temperature and exhibits a high reaction rate at the same time, thus confirming that the hydrolyzate can be efficiently obtained, and the present invention has been completed.
In the present specification, the term " beta-mannosidase "refers to mannose, including mannobiose, mannotriose, mannotetraose, mannopentaose or mannohexaose, Quot; means an enzyme that catalyzes the hydrolysis reaction.
The beta-mannosidase according to the present invention may include beta-mannosidase that can be naturally induced, as well as synthetic beta-mannosidase as described in a specific embodiment of the present invention, It should be homologous to the sequence. More specifically, beta-mannosidase, which can be naturally induced, is preferably obtained from the strain P. woosongensis (accession number KCTC 3953) and is isolated from the strain by conventional methods known in the art For example, by centrifuging the culture broth, recovering the supernatant to obtain an extracellular fraction, or recovering the supernatant, washing the remaining pellet, resuspending and centrifuging to obtain pellet binding fractions But is not limited thereto. The term "strain" includes live cells, dead cells, dry bacteria, or culture medium. The term "culture medium" refers to a culture medium itself obtained by culturing the strain in a suitable culture medium, a filtrate (filtrate or centrifuged supernatant) from which the culture is removed by filtration or centrifugation, A cell lysate obtained by ultrasonication of the culture solution or by treating lysozyme with the culture solution, and the like.
In the present invention, "beta-mannosidase" includes functional equivalents thereof. The "functional equivalents" have at least 85%, more preferably at least 90%, even more preferably at least 95% sequence homology with the amino acid sequence of SEQ ID NO: 1, and the beta-mannose Quot; refers to a protein that exhibits substantially the same activity as an agent.
In the present specification, the term "homology" refers to the similarity of the amino acid sequence of beta-mannosidase or the nucleotide sequence of the nucleic acid molecule encoding the same to the amino acid sequence or base sequence of the present invention , More preferably at least 85%, even more preferably at least 90%, most preferably at least 95%. This comparison of homology can be performed using a visual program or a comparison program that is easy to purchase. Commercially available computer programs can calculate homology between two or more sequences as a percentage, and homology (%) can be calculated for adjacent sequences.
"Substantially homogeneous activity" means that it has the same functional properties as beta-mannosidase, especially having the functional properties of the enzyme. The functional equivalents also include protein derivatives in which the basic structure of beta-mannosidase and some chemical structures of the protein are modified while maintaining their activity. These include, for example, fusion proteins made by fusion with other proteins, such as GFP, while retaining structural modification and physiological activity to alter the stability, shelf-life, volatility, or solubility of the protein of the present invention. In addition, polypeptides having substantially equivalent activity as beta-mannosidase as a fragment of a protein form another preferred group of functional equivalents of beta-mannosidase. The term "fragment" refers to an amino acid sequence corresponding to a part of a protein, which has a common origin element, structure and action mechanism falling within the scope of the present invention, and is a substance existing in beta-mannosidase, And those that have been cut chemically.
In the present specification, the term "activity" means quantitatively converting a given substrate, and the term "multifunctional enzyme " means an enzyme in which one enzyme catalyzes two or more reactions. Lt; RTI ID = 0.0 > quantitatively < / RTI > According to a specific embodiment, the beta-mannosidase according to the present invention is administered to para- nitrophenyl-beta-mannopyranoside (pNP-beta-D-mannopyranoside (pNPM) (L-arabinopyranoside), pNP-beta-D-galactopyranoside, pNP-alpha-D-galactopyranoside The activity to catalyze the hydrolysis reaction of at least one substrate selected from the group consisting of galactopyranoside, pNP-α-D-glucopyranoside and pNP-β-D-glucuronide Bar, and multifunctional enzymes.
Preferably, the beta-mannosidase according to the invention may comprise one or more of the following characteristics:
(a) isoelectric point PI 4.5 to 5.5, more preferably 4.5 to 5;
(b) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) measurement Molecular weight 95-98 kDa, more preferably 96-97 kDa;
(c) exhibits a maximal activity for pNPM at 42-52 ° C, more preferably for pNPM at 45-50 ° C;
(d) exhibits a maximal activity for pNPM at pH 5 - 6.5, more preferably for pNPM at pH 5.5;
(e) exhibits thermal stability at < RTI ID = 0.0 > 30 C < / RTI >
(f) K m for pNPM is 2.14 mM and V max is 2.21 U / mg;
(g) increased K + , Mg 2+ or Fe 2+ enzyme activity;
(h) reduced enzyme activity upon addition of Ni 2+ , Mn 2+ , Fe 3+ , Cu 2+ , EDTA, SDS, Hg 2+ , PMSF or 8-hydroxyquinoline; or
(i) the activity of the enzyme is reduced upon addition of at least one saccharide selected from the group consisting of glucose, galactose, ribose, mannose and xylose.
As can be seen from the above characteristics (c) - (e), the beta-mannosidase according to the present invention exhibits high reactivity at neutral pH and middle temperature and exhibits thermal stability at room temperature, high.
In addition, according to the characteristics (a) to (i), it is possible to utilize the purpose of the reaction, the reaction rate / the reaction rate, and the like.
The beta-mannosidase of the present invention may comprise an exogenous amino acid at its N- or C-terminus and the "exogenous amino acid" is an amino acid that is not present in the native (naturally occurring) beta-mannosidase, Quot; refers to a stretch of at least about 1, at least about 3, at least about 5 at least about 6, or at least about 7 contiguous amino acids. A preferred extension of the foreign amino acid may include, but is not limited to, a "tag " that facilitates purification of the recombinantly produced beta-mannosidase. Examples of such tags include, but are not limited to, His 6 tags, FLAG tags, myc tags, and the like.
The polypeptides and polynucleotides of the present invention, including beta-mannosidase, can be provided in isolated form, and are preferably provided homogeneously. As used herein, the term "isolated " means that a substance is removed from its native environment (e.g., the natural environment if naturally occurring). For example, naturally occurring polynucleotides or polypeptides present in living microorganisms are not isolated, but the same polynucleotides or polypeptides isolated from some or all coexisting materials in a natural system are isolated. Such polynucleotides can be part of a vector and / or part of a composition, and can be isolated such that the vector or composition is not part of its natural environment. The isolated polypeptide is preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, most preferably at least 99% pure. The purity can be determined according to known methods in the art, for example, by SDS-PAGE and subsequent protein staining, and the protein bands can be quantified by a density meter, but are not limited thereto.
In another aspect, the invention relates to nucleic acid molecules encoding beta-mannosidase or isolated or purified nucleic acid molecules thereof.
In the present specification, the term "nucleic acid molecule" means any single or double helical nucleic acid molecule of cDNA, genomic DNA, synthetic DNA or RNA, PNAS or LNA origin, or a mixture thereof. The "nucleic acid molecule" includes a) a nucleic acid base sequence encoding the beta-mannosidase and its functional equivalents according to the present invention, or b) sequences hybridizing to these sequences under a high stringent condition . Conditions for high tightening are as described in the well known literature (Molecular Cloning, Cold Spring Harbor, New York, Cold Spring Harbor Laboratory Press, 1989). Also, a nucleotide sequence having at least 70% homology, more preferably at least 80% homology, more preferably at least 90% homology, and most preferably at least 95% homology with the nucleotide sequence of a) -b) . Also, fragments or complement of any of the nucleic acid molecules of a) -b) above may be included.
The nucleic acid molecule encoding the beta-mannosidase of the present invention may have a nucleotide sequence encoding a beta-mannosidase represented by the amino acid sequence of SEQ ID NO: 1 and a nucleotide sequence having homology thereto, 2 or a nucleotide sequence having homology thereto.
Nucleic acid molecules encoding beta-mannosidase of the present invention can be obtained by the method for constructing nucleic acid molecules known in the art from the sequence information disclosed in the present invention. For example, a nucleic acid molecule as a starting material can be isolated from a P. vasospasm strain and / or can be prepared synthetically based on the nucleotide sequence disclosed in the present invention.
Nucleic acid molecules encoding the beta-mannosidase of the present invention can be isolated and purified from isolated polynucleotides, including, but not limited to, other chromosomal DNA and RNA, and polynucleotides that are substantially free of other base sequences such as extrachromosomal DNA and RNA Lt; / RTI > Methods of nucleic acid molecule purification known to those of ordinary skill in the art can be used to obtain isolated nucleic acid molecules. Alternatively, the nucleic acid molecule of the present invention relates to a promoter, a functional polynucleotide to which a ribosome-binding site and a terminator are operatively linked if necessary in the case of a bacterial cell.
In another aspect, the invention provides a recombinant vector comprising a nucleic acid molecule according to the invention.
The term "plasmid "," vector "or" expression vector ", used interchangeably herein, refers to a construct for in vivo or in vitro expression, . These constructs can be used to insert beta-mannosidase encoding nucleic acid molecules into host cells. In these constructs, the beta-mannosidase-encoding nucleic acid molecule is operably linked to a suitable regulatory sequence so that it can be expressed in the host cell and, when inserted into the host cell, Replicate and function, or in some cases may be integrated into the host genome itself. Typically, the plasmid vector includes a cloning start point that allows replication to be efficiently made to include several hundred plasmid vectors per host cell, an antibiotic resistance gene that allows the host cell inserted with the plasmid vector to be selected, And a restriction enzyme cleavage site for allowing restriction enzyme cleavage. Although there is no suitable restriction enzyme cleavage site, using a synthetic oligonucleotide adapter or linker according to conventional methods can facilitate ligation of the vector and the foreign nucleic acid molecule.
Operatively linked "means that the appropriate nucleic acid molecule is linked in such a way as to enable gene expression when it is coupled to a regulatory sequence.
Refers to a cell in which a cell replicates a heterologous nucleic acid molecule, expresses the nucleic acid molecule, or expresses a protein encoded by a peptide, heterologous peptide or heterologous nucleic acid molecule. Recombinant cells can express genes or gene segments that are not found in the wild-type form of the cells, either in sense or antisense form. In addition, recombinant cells can express genes found in wild-type cells, but these genes have been modified and reintroduced intracellularly by artificial means. The "vector" delivers the nucleic acid molecule into the cell. Said "vector" may further comprise any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site and a sequence regulating the termination of transcription and translation.
Vectors that can be used for expression in host cells are known in the art, and in particular suitable vectors that can be used to express in E. coli are also known in the art. According to a specific embodiment of the present invention, pET23a (+) was used as a vector, and a recombinant vector cleavage map was specifically disclosed in FIG. 1, but it is not limited thereto.
In another aspect, the present invention provides a recombinant microorganism in which the above-described nucleic acid molecule or recombinant vector according to the present invention is inserted into a host cell.
In the present specification, the term "host cell" includes any cell comprising the nucleic acid molecule or vector described above, and may be used in the recombinant production of beta-mannosidase. The host cell usually has high introduction efficiency of the nucleic acid molecule and high expression efficiency of the introduced nucleic acid molecule. The host cells include prokaryotic or progressive cells. As the recombinant microorganisms including such host cells, for example, bacteria, enzymes, fungi, etc. may be used. In the examples of the present invention, E. coli was used, Any kind of microorganism may be used as long as the beta-mannosidase variant according to the present invention can be sufficiently expressed.
In the present invention, conventionally known manipulation methods for inserting nucleic acid molecules into host cells may be used. For example, microprojectile bombardment, particle gun bombardment, silicon carbide whiskers, sonication, electroporation, PEG-mediated fusion (PEG- mediated fusion, microinjection, liposome-mediated method, In planta transformation, Vacuum infiltration method, floral meristem dipping method, ), Agrobacteria spraying method can be used.
In another aspect, the present invention provides a composition comprising the beta- mannosidase according to the present invention described above.
The composition may be a liquid or solid phase. The composition may include beta-mannosidase alone, may contain other proteins or enzymes, and may contain additional additives that will complement the stability and / or activity of beta-mannosidase, other proteins, or enzymes in the composition . ≪ / RTI > Examples include, but are not limited to, glycerol, sorbitol, propylene glycol, salt, sugar, pH-buffer, preservative and carbohydrate. Typically, the liquid composition is a water or oil based slurry, suspension or solvent. Solid phase compositions can be prepared by any method known in the art such as spray-drying, lyophilisation, down-draft evaporation, thin-layer evaporation, centrifugal evaporation, Or a combination thereof. The solid phase composition may be granulated to a size suitable for food or feed.
The composition can be used in various industrial fields based on the characteristics of the beta-mannosidase according to the present invention, for example, but not limited to, the following fields:
- Mannan decomposition,
- Alcohol fermentation or production,
- food or feed processing,
- coffee extraction or processing of coffee waste,
- food or feed supplements,
- Pulp bleaching assistant for paper,
Bleaching agents or desizing agents in the textile industry,
- Stimulation of wells or wells by hydraulic fracturing,
- Cleaner,
- processing or harvesting of renewable resources intended for biological fuel production,
- Baking ingredients,
- Biofilm removal,
- delivery system,
- Biomass resource processing,
- Fabrics, oil drilling, washing, washing, detergents and cellulose fiber processing,
- Mannooligosaccharide degradation, or
- D-mannose production and so on.
Depending on the use of the composition, various components may be added to the composition according to the present invention. For example, an enzyme may be added to the composition. The enzyme may include endomannase, alpha-galactosidase, cellobiohydrolase, , Glucoamylase, alpha amylase, protease, cellulase, and mixtures thereof.
In another aspect, the present invention provides a method for industrially utilizing beta-mannosidase according to the present invention as described above. More specifically, the present invention provides a method for industrially utilizing beta-mannosidase according to the present invention, A method for decomposing a saccharide is provided. The saccharide containing mannose as a main component may preferably be mannooligosaccharide. Further, the present invention provides a method for preparing mannose by adding a beta-mannosidase according to the present invention and decomposing a saccharide containing mannose as a main component. Also provided is a method for converting a biomass into a sugar by adding a beta-mannosidase according to the present invention to the biomass. The term "biomass" in the context of the present invention means a subject including hemicellulose, especially mannan, and includes, for example, plant seeds, kernels, tubers, corn (including spruce, (Indian Grass, Switchgrass, etc.), water streams, sugar cane gas, and the like, and may include but are not limited to plant wastes, food processing or by-products of industrial processing. The above-described methods can be used in an industrial field in which the composition according to the present invention is used, but the present invention is not limited thereto.
In another aspect, the present invention provides a method for producing a recombinant microorganism, comprising: culturing the recombinant microorganism according to the present invention to express beta-mannosidase; And recovering the expressed beta-mannosidase. The present invention also provides a method for producing beta-mannosidase.
More preferably, the following method can be used:
The beta-mannosidase is prepared by culturing E. coli BL21 (DE3) Codon Plus-RIL as a fusion protein tagged with 6 histidines at the C-terminus; And recovering the expressed beta-mannosidase from the cell lysis supernatant.
For the (large) culture of recombinant microorganisms, various culture methods can be applied, for example, large scale production of gene products expressed or over expressed from recombinant microorganisms can be achieved by batch or continuous culture methods. Batch and fed-batch cultivation methods are conventional and known in the art. Techniques for controlling nutrients and growth factors for continuous culture processes as well as techniques for maximizing product formation rates are known in the microbiological industry. In addition, as a culture medium, a medium composed of carbon source, nitrogen source, vitamins and micellar may be used. Preferably, carbon source may include mannan, but it is not limited thereto and can be constituted as known in the art.
The novel beta-mannosidase enzyme according to the present invention is capable of separating from microorganisms and has an activity of catalyzing the hydrolysis reaction of two or more substrates including the hydrolysis activity of pNPM and has a neutral pH And exhibits high reactivity at a middle temperature, exhibits thermal stability at room temperature, and exhibits a high reaction rate at the same time, so that a hydrolyzate can be efficiently obtained. Therefore, the beta-mannosidase according to the present invention can decompose saccharides containing D-mannose as a main component and can produce D-mannose by this method, The biomass is converted into a sugar by the addition of a biomass, which can be effectively utilized in various industrial fields.
Figure 1 shows the recombinant vector pYEM28N of the beta-mannosidase gene.
FIGS. 2A-2B show SDS-PAGE results of purified His-tagged beta-mannosidase from recombinant E. coli BL21 (DE3) or recombinant E. coli BL21 (DE3) Codon Plus-RIL. T: cell lysate, S: cell lysate supernatant (water soluble protein), P: cell lysate precipitate (insoluble protein)
Figure 3 shows the optimal temperature and optimal pH of the purified His-tagged beta-mannosidase for hydrolysis of pNPM.
Figure 4 shows the thermal stability of the purified His-tagged beta-mannosidase. x axis: incubation time (min), y axis: residual activity (%)
Figure 5 shows the effect of metal ions or other agents on the degrading activity of beta-mannosidase on pNPM. The y-axis of the graph represents relative activity (%).
A; No addition, B: KCl, C: NiCl 2 D: MgCl 2 , E: MnCl 2 , F: CaCl 2 , G: CoCl, H: FeCl 2 , I: FeCl 3 , J: CuCl 2 , K: EDTA, L: SDS, M: HgCl 2 , , O: 8-hydroxy quinoline
Figure 6 shows the kinetic parameters for pNPM of the purified His-tagged beta-mannosidase.
Figure 7 shows the hydrolysis products of mannooligosaccharides.
M3: mannotriose, M4: mannotetraose, M5: mannopentaose, M6: mannohexaose, E: purified His-tagged beta-mannosidase
Figure 8 shows the substrate specificity of beta-mannosidase.
Figures 9a and 9b show the effect of addition of sugar on beta-mannosidase degrading activity against pNPM.
Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.
[Experimental Method]
Bacterial strains, plasmids and culture medium
Penny Bacillus mail N-Sys (accession number KCTC 3953, deposit 20,070,502) is a beta-coding gene was used as a source of let manno claim, E. coli DH5α was used as a host for recombinant plasmid injection, E. coli BL21 (DE3) Codon Plus-RIL was used as a host for E. coli gene expression. These E. coli were cultured at 37 ° C in LB broth (10 g tryptone, 5 g yeast extract, 10 g NaCl per liter, pH 7.0). Ampicillin (100 g / ml) and chloramphenicol (30 g / ml) were used for E. coli transformant selection and culture. The penny bacillus strains were incubated at 37 ° C in a cryptic sobros (TSB; 17 g tryptone, 3 g soytone, 2.5 g dextrose, 5 g NaCl, 2.5 g K 2 HPO 4 per liter, pH 7.2). Plasmid pUC19 was used for cloning and sequencing experiments, and pET23a + was used as an expression vector for the beta-mannosidase gene.
Cloning and sequencing of the beta-mannosidase gene
In order to clone the gene (SEQ ID NO: 2) coding for beta-mannosidase (SEQ ID NO: 1), the following experiment was carried out.
Genomic DNA isolated from Penny Bacillus Pneumocystis KCTC 3953 cells was isolated. The separated genomic DNA was partially digested with Sau 3AI and a DNA fragment of 1.5 to 10 kb in length was separated from the agarose gel. The Sau 3AI-treated chromosome DNA fragment was introduced into the phosphorylated BamHI site of pUC19 and the ligation mixture was transformed into E. coli DH5a. After the transformed host colonies were formed, a solution containing 0.2 mM Meybepperyl beta-mannopyranoside, 20 mM sodium phosphate buffer (pH 6.5) and 0.8% agar was layered on a plate medium and incubated at 37 ° C for 5 hours. Was selected for E. coli transgenic plants in which the β-mannosidase gene was cloned by selecting ultraviolet light to identify fluorescent colonies. After transfection of the transformed strain in which the beta-mannosidase gene was cloned in an LB liquid medium overnight, the plasmid was separated and the nucleotide sequence of the cloned DNA fragment was analyzed using a DNA sequencer (ABI Prism 377, Perkin Elmer Co., Foster, Calif. , USA).
Preparation of Recombinant Plasmid for High Expression of Beta-Mannosidase Gene
To construct a recombinant plasmid that will highly express beta-mannosidase in E. coli , the gene was subcloned into pET23a (+). The oligonucleotide primer was designed according to the nucleotide sequence of the gene as follows:
Forward primer (5'-GGAGACATATGAAAAGATTTGATTTAAACGACGC-3 ': SEQ ID NO: 3) and
Reverse primer (5'-AATACTCGAGTCTGACCGAAGCAAATTTTAGCGC-3 ': SEQ ID NO: 4)
Appropriate restriction enzyme recognition sites were added to forward primer ( Nde I site) and reverse primer ( Xho I site), respectively. In addition, the reverse primer was designed so that pET23a (+) contained no stop codon at the C-terminus to contain a gene product fused to the 6-His tag. PCR amplification was performed by adding 50 μl of the reaction mixture containing 95 μl of the clone gene (10 ng), 40 pmol of each primer, 1.5 mM MgCl 2 , 0.3 mM dNTPs, PCR buffer and 2.5 U Pfu DNA Lt; 0 > C for 40 seconds, and 72 < 0 > C for 3 minutes. The PCR products of the Nde I and Xho I-treated DNA fragments were introduced into the same region of pET23a (+) and the resulting plasmid was designated pYEM28N (Fig. 1).
E. coli High expression and purification of His-tagged enzyme
Recombinant E. coli BL21 (DE3) Codon Plus-RIL cells containing the plasmid pYEM28N were cultured at 37 ° C in LB culture medium containing antibiotics. When the culture reached 0.6 optical density at 600 nm, IPTG was added to give a final concentration of 0.5 mM. After 5 hours of induction at 25 ° C, the cells were removed and suspended in cell lysing buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 10 mM imidazole, pH 8.0), sonicated and pulverized and centrifuged to obtain a cell lysate supernatant . The cell lysate supernatant was added to Ni-NTA equilibrated with the same buffer. After removing the unbound or weakly bound proteins using wash buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 20 mM imidazole, pH 8.0), the His-tagged enzyme was extracted using extraction buffer containing 250 mM imidazole. The active fractions were combined and dialyzed in 10 mM sodium phosphate buffer (pH 6.5).
Enzyme analysis and epidemiological analysis
Beta-mannosidase activity was determined by measuring the release of para- nitrophenol (pNP) in pNPM. A 0.5 ml reaction mixture containing 1 mM substrate in 50 mM sodium phosphate buffer (pH 6.5) was incubated for 10 minutes at 50 < 0 > C. The reaction was terminated by the addition of 1 ml of 1 M sodium carbonate. The absorbance was measured at 405 nm. Steady-state epidemiological measurements were performed at the optimum temperature and pH of pNPM, varying the substrate concentration (0.125 - 4 mM). The values of K m and V max were calculated from the initial pNP free ratios. One unit enzyme activity was defined as the amount of enzyme producing 1 μmol of pNP or reducing sugar per minute.
Effect of pH, temperature and various agents on enzyme activity
The effect of pH on the reaction rate can be determined by using a fixed temperature (45 ° C) and various pHs buffer (50 mM sodium citrate (pH 4.0 - 6.0), sodium phosphate (pH 6.0 - 8.0) buffer) Lt; RTI ID = 0.0 > pNPM < / RTI > The activity of the purified enzyme was assayed in 50 mM sodium citrate buffer (pH 5.5) at various temperatures ranging from 30-65 占 폚. To investigate the effect of the compound, beta-mannosidase was measured by adding some reagents (5 mM) in the standard reaction mixture. To test the effect of the sugar on the enzyme activity, the monosaccharide was added to the standard reaction mixture to a final concentration of 25 mM to 400 mM, followed by a hydrolysis reaction to pNPM.
[Experiment result]
Cloning and nucleotide sequence of penny Bacillus mutant gene bS-mannosidase gene
E. coli transformants containing the penny Bacillus pneumocystis genomic DNA were transferred to LB agar plates for overnight culture. To screen for E. coli exhibiting beta-mannosidase activity, 0.2 mM 4-methylumbelliferyl beta-D-mannopyranoside (MUM) was applied onto the plate with soft agar. After incubating at 40 ° C for 2 h, MUM was hydrolyzed through a strong fluorescence observation at 360 nm to select a colony that could favor 4-methylumbelliferone. The entire 2,517 bp nucleotide sequence of the Pennibacillus spp. Genome DNA was determined in the recombinant plasmid. A sequence comparison of the amino acid sequence (SEQ ID NO: 1) of the penis Bacillus subsp. Mannisis beta-mannosidase with other protein sequences in the NCBI database using the BLAST search program confirmed that it was a novel protein.
E-coli Production and purification of His-tagged beta-mannosidase
To simplify purification of the enzyme, recombinant E. coli produced many fusion proteins. His-tagged beta-mannosidase was prepared with a 6-histidine tail at the C-terminus. Fusion protein E. coli BL21 (DE3) expression was rather high by rare (rare) to have a modified E. coli tRNA genes BL21 (DE3) IPTG at Codon Plus-RIL. The enzyme produced soluble protein not only in the recombinant E. coli cells cultured at 25 DEG C but also in the cells cultured at 37 DEG C, but a considerable amount was produced as insoluble protein in the cells cultured at 37 DEG C (FIG. 2A).
His-tagged beta-mannosidase was purified to 90% homology from the cell lysate supernatant in one step using Ni-NTA resin. The purified enzyme showed, via SDS-PAGE, a single protein band corresponding to a molecular weight of about 96.5 kDa. The molecular weight of the purified enzyme was consistent with the molecular weight of the His-tagged beta-mannosidase predicted by the beta-mannosidase gene nucleotide sequence (Figure 2b).
Reaction characteristics of purified His-tagged beta-mannosidase
PNPM was used as a substrate to investigate the effect of temperature and pH on enzyme activity. The purified His-tagged beta-mannosidase showed maximal activity against pNPM at 45 ° C and 5.5 pH and activity over 80% at 42 ° C to 52 ° C (FIG. 3).
To confirm the thermal stability and pH stability of the purified His-tagged beta-mannosidase, pNPM hydrolysis was carried out after pre-incubation at various temperatures (30-60 ° C) or in various pHs (4.0-8.0) Was determined. As a result, heat stability was shown at 30 캜 for 2 hours of incubation (Fig. 4).
His-tagged beta-mannosidase showed significant activity in pNPM, and NP-α-L-arabinopyranoside, pNP-β-D-galactopyranoside, pNP-α -D-galactopyranoside, pNP-? -D-glucopyranoside and pNP-? -D-glucuronide (Fig. 7 - 8).
The kinetic parameters of His-tagged beta-mannosidase for pNPM, K m and V max, were confirmed by steady-state kinetic analysis. For pNPM, K m was 2.14 mM and V max was 2.21 U / mg (Fig. 6).
Effects of metal ions and other agents on enzyme activity
The effect of various agents including metal salts, PMSF and 8-hydroxyquinoline on the activity of His-tagged beta-mannosidase was investigated. The enzymatic activity of β-mannosidase was measured by measuring the degree of hydrolysis of pNPM in the presence of various preparations. It was confirmed that the enzyme activity was increased by metal ions such as K + , Mg 2+ or Fe 2+ . On the contrary, it was confirmed that Ni 2+ , Mn 2+ , Fe 3+ , Cu 2+ , EDTA, SDS, Hg 2+ , PMSF or 8-hydroxyquinoline suppress the hydrolytic activity of the enzyme. In addition, Ca 2+ and Co + showed little effect on the hydrolytic activity (Fig. 5).
Most of the glycosidases studied so far have been reported to be inhibited by sugar addition, the resultant product of an enzyme reaction catalyzed by them, and have been reported to be inhibited by other monosaccharides. In order to investigate the effect of the product resulting from the hydrolysis reaction on the enzyme activity, the pNPM hydrolyzing activity of His-tagged beta-mannosidase in the presence of various concentrations of glucose, galactose, ribose, mannose and xylose was tested. Hydrolytic activity against pNPM was weakly inhibited by glucose, galactose and mannose, and hydrolytic activity against pNPM was inhibited by xylose and ribose, which are pentoses, and in particular, the concentration of ribose was increased to 50 mM or more , The enzyme activity was abruptly decreased (Figs. 9A to 9B).
≪ 110 > CTC Bio. <120> A novel beta-mannosidase and producing method thereof <130> P14-056 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 838 <212> PRT <213> Paenibacillus woosongensis <400> 1 Met Lys Arg Phe Asp Leu Asn Asp Ala Trp Leu Leu His Glu Ala Pro 1 5 10 15 Leu His Trp Gly Arg Asp Ser Leu Ala Ala Val Lys Ala Leu Lys Glu 20 25 30 Gly Trp Tyr Pro Cys Ala Leu Pro Thr Asp Val Arg Ile Pro Leu Ile 35 40 45 Glu His Gly Ile Ile Gln Glu Pro Leu Glu Ser Asp His Ala Leu Ala 50 55 60 Ser Glu Trp Ile Glu Gln Arg Ser Trp Trp Tyr Thr Lys Glu Phe Asp 65 70 75 80 Gly Ala Gly Ile Asp Phe Asp Ser Asp Ile Ile Glu Leu Val Ile Glu 85 90 95 Thr Ile Asp Thr Asn Ser Asp Ile Phe Val Asn Asp Gln Tyr Val Gly 100 105 110 Ser His Arg Asn Val His Tyr Pro Phe Val Arg Asn Ile Lys Asp Ile 115 120 125 Leu Thr Ala Gly Lys Asn Thr Leu Thr Val Arg Thr Thr Gly Leu 130 135 140 Glu Asp Val Thr Asp Ser Asp Leu Ser Glu Leu Asn Trp Ala Thr Cys 145 150 155 160 Arg Glu Tyr Asp Asn Gly Gly Lys Asp Arg Gly Asp Tyr Arg Arg Ser 165 170 175 Tyr Val Arg Arg Pro Gln Tyr Thr Val Gly Trp Asp Trp Gly Pro Arg 180 185 190 Val Val Thr Cys Gly Leu Gly Gly Asn Ala Tyr Leu Arg Cys Glu Lys 195 200 205 Glu Ile Ala Val Glu Val Lys Leu Val Thr Val Ser Ala Ala Gln 210 215 220 Thr Ala Arg Leu Lys Ala Thr Val Asn Ile Glu Asn Leu Asp Ile Ile 225 230 235 240 Gly Thr Ile Asn Cys Asp Leu Gln Ile Asp Ile Ser Tyr Asp Gly Gln 245 250 255 Ser Cys Val Thr Lys Lys Leu Glu Asn Leu Leu Leu Thr Ser Gly Thr 260 265 270 Asn Tyr Phe Asp Val Asp Leu Glu Val Glu Asn Ala Arg Leu Trp Trp 275 280 285 Pro Ala Gly His Gly Asp His Pro Leu Tyr Asp Val Arg Val Ser Ala 290 295 300 Thr Ser Ala Asn Ala Lys Thr Glu Tyr Pro Ala Phe Gln Phe Gly Ile 305 310 315 320 Arg Thr Val Glu Leu Asp Thr Ser Ile Leu Arg Gly Glu Glu Arg Asn 325 330 335 Phe Arg Leu Ile Val Asn Gly Val Pro Ile Phe Ser Lys Gly Gly Asn 340 345 350 Trp Val Pro Ala Asp Phe Ile His Ala Arg Val Thr Asp Glu Lys Tyr 355 360 365 Glu Thr Leu Ile Arg Glu Ala Val Glu Ala Asn Phe Asn Met Leu Arg 370 375 380 Val Trp Gly Gly Gly Leu Phe Glu Arg Asp Ile Phe Tyr Asp Leu Cys 385 390 395 400 Asp Arg Asn Gly Leu Leu Val Trp Gln Asp Phe Met Met Ala Cys Ser 405 410 415 Thr Tyr Pro Asp His Lys Arg Glu Phe Arg Asp Glu Met Arg Ala Glu 420 425 430 Met Asp Tyr Gln Thr Lys Arg Leu Arg Asn Arg Ala Ser Ile Ala Leu 435 440 445 Phe Cys Gly Thr Asn Glu Val His Trp Ile Phe Asn Lys Tyr Asp Asn 450 455 460 Pro Arg Trp Gln Ile Glu Phe Lys His Glu Lys Gln Tyr Gly Met Tyr 465 470 475 480 Ile Ala Asn Ile Leu Ala Lys Glu Ile Met Tyr Asn Asn Cys Ser His 485 490 495 Ile Pro Tyr Trp Asn Ser Ser Pro Tyr Gly Gly Ala Leu Pro Asn Asp 500 505 510 Asp Thr Val Gly Asp Val His Arg Trp His Asn Ala Phe Met Ser Leu 515 520 525 Asn Met Asp Glu Arg Ile Glu Pro Met Asp Phe Asp Thr Val Asn Ser 530 535 540 Lys Phe Val Ser Glu Tyr Gly Phe Val Gly Pro Cys Ser Leu Glu Ser 545 550 555 560 Thr Lys Lys Tyr Leu Gly Gly Glu Glu Ile Asp Phe Asp Ser Glu Val 565 570 575 Trp Gln Met His Cys Asn Val Phe Glu Lys Gly Thr Val Val Thr Gly 580 585 590 Ile Ala Lys Asn Tyr Leu Asp Arg Thr Asp Asn Leu Ser Ile Glu Asp 595 600 605 Tyr Leu Leu Tyr Gly Gly Met Val His Ser Leu Met Leu Glu Tyr Ser 610 615 620 Leu Glu Ala Ile Arg Phe Lys Glu Asp Cys Gly Gly Ala Leu Phe Trp 625 630 635 640 Met Tyr Asn Asp Ala Trp Gly Glu Val Gly Trp Thr Ile Ile Asp Tyr 645 650 655 Tyr Leu Ser Arg Lys Ile Pro Tyr Tyr Gly Val Lys Arg Ala Leu Ala 660 665 670 His Thr Lys Leu Ser Met Arg Val Val Asp Gly Asn Val Val Val Gln 675 680 685 Gly Met Asn Asp Thr Ala Glu Lys Val Ser Phe Gln Ala Glu Tyr Gly 690 695 700 Tyr Ile Ser Phe Asp Gly Thr Val Arg Gln Thr Arg Thr Leu Glu Ile 705 710 715 720 Ser Leu Glu Pro His Ser Arg Val Tyr Leu Leu Thr Glu Lys Leu Pro 725 730 735 Asp Gln Asp Tyr Thr Lys Gly Ser Met Met Leu Ile Pro Asp Ser Asp 740 745 750 Thr Val Asn Ser Ile Ser Leu Arg Thr Gly Asp Met Lys Thr Met Val 755 760 765 Phe Asp Pro Ser Pro Val Glu Ile Val Ser Asp Glu Gln Val Gly Ser 770 775 780 Asp Arg Lys Val Thr Leu Thr Ser Lys Gly Tyr Ala His Gly Val Tyr 785 790 795 800 Val Ala Gly Gly Tyr Asp Cys Ser Asp Leu Tyr Phe Asp Leu Leu Pro 805 810 815 Gly Glu Val Lys Thr Ile Thr Val Tyr Ser Ala Gly Ser Glu Ala Leu 820 825 830 Lys Phe Ala Ser Val Arg 835 <210> 2 <211> 2517 <212> DNA <213> Paenibacillus woosongensis <400> 2 atgaaaagat ttgatttaaa cgacgcatgg cttcttcacg aggcgccgct gcactggggc 60 agagacagcc tggcggctgt aaaggctttg aaggaaggct ggtatccctg cgcgcttcca 120 accgatgttc gtattcctct catcgagcat ggcattattc aggagccgct tgagtccgat 180 catgcgctcg ccagcgaatg gatcgaacag cgttcctggt ggtacaccaa ggaattcgat 240 ggcgcgggca tcgattttga cagtgacatt atcgagcttg tgatcgaaac gatcgacacg 300 aacagcgata ttttcgtcaa cgatcaatat gtcggcagcc atcgcaatgt gcattatcct 360 ttcgttcgca atatcaagga cattctgacc gccgggaaaa acacgcttac ggtgcgcgtc 420 actaccgggc tggaggacgt taccgatagc gacttgtcgg agctcaactg ggcgacctgc 480 cgtgaatacg ataacggcgg caaagaccgc ggcgactatc gccgttccta cgtaagacgt 540 ccgcaatata cggtgggctg ggactggggg ccgcgcgttg tcacctgcgg acttggcgga 600 aatgcatacc tgcgctgcga gaaagagatc gcggttcgcg aggtgaagct tgttaccgtt 660 agcgccgccc agacagcccg gcttaaagcg accgttaaca tcgagaacct ggacatcatc 720 gggacgatta actgcgatct gcagatcgac atttcctatg acgggcaatc ctgtgtgacg 780 aagaagctcg agaacctgct tctgacctcc ggcacgaact attttgacgt ggatctcgag 840 gtggagaatg cccggctgtg gtggcctgcg ggacatggcg atcatccgct gtacgatgtg 900 cgggtatctg caacgagtgc gaacgcgaag accgaatatc ctgcattcca atttggcatc 960 cggacggtcg agctggatac gtccatcctg cgcggcgaag agcgcaattt ccggctgatc 1020 gtcaacggcg ttcctatttt cagcaaaggg ggcaactggg ttccagccga tttcatccat 1080 gcccgcgtta ccgacgagaa gtatgaaacc ttgatccgcg aggcggtgga agcgaacttc 1140 aacatgctgc gcgtttgggg cggcggactg tttgagcggg atattttcta cgatctatgc 1200 gccggaacg gtctgctggt gtggcaggat ttcatgatgg cctgctccac ttatcctgac 1260 cacaaacggg aattccgcga cgagatgcgg gccgagatgg actatcagac caagcgcctg 1320 cgcaaccggg catccattgc cttgttctgc ggaaccaacg aagtccactg gatctttaac 1380 aaatatgaca atccgagatg gcagatcgag tttaagcatg agaagcagta tggcatgtac 1440 atcgcgaata ttctcgccaa agagatcatg tacaacaact gttcccacat tccttactgg 1500 aacagctcgc catacggcgg ggcgcttccg aacgacgata cggtcggcga tgtccaccgc 1560 tggcataacg ccttcatgag cctcaatatg gacgagcgca tcgagccgat ggacttcgat 1620 acagtgaact ccaaattcgt gagcgaatac ggcttcgttg gcccgtgctc tctggagagc 1680 acgaagaagt atttgggcgg ggaagagatc gacttcgaca gcgaagtgtg gcaaatgcac 1740 tgcaacgtgt ttgagaaagg aaccgtcgtc acgggcatcg ctaaaaacta tcttgaccgc 1800 accgacaatt tatccatcga ggactatctg ctgtacggcg gcatggtaca ctcgctcatg 1860 ctggagtatt cgctggaagc gatccgattc aaggaagatt gcggcggcgc gctgttctgg 1920 atgtacaacg atgcctgggg cgaggtcggc tggacgatca tcgattatta tctgagccgc 1980 aaaatcccgt attatggcgt aaaacgcgca ttggctcata cgaagctgtc gatgcgcgtg 2040 gtggatggca acgtcgttgt tcaagggatg aacgatacgg cagagaaggt gagcttccag 2100 gcggaatacg gctatatttc gttcgacgga acagttcggc agacgagaac gctggagatt 2160 tcccttgagc cgcatagccg ggtttacctg cttacggaga agctgccgga tcaggactac 2220 accaaaggca gcatgatgct gattccagat tcggataccg tgaactccat atccctgcgg 2280 acaggcgata tgaagacgat ggtattcgat ccatcgccgg ttgaaatcgt aagcgatgag 2340 caggtcggat cggatcgcaa agttacgctg accagcaaag ggtatgcgca cggtgtgtat 2400 gttgcgggtg gttatgactg ctccgacctc tactttgatt tgctgccggg tgaggtgaag 2460 acgattaccg tctattccgc aggcagtgaa gcgctaaaat ttgcttcggt cagataa 2517 <210> 3 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer <400> 3 ggagacatat gaaaagattt gatttaaacg acgc 34 <210> 4 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer <400> 4 aatactcgag tctgaccgaa gcaaatttta gcgc 34
Claims (15)
(a) isoelectric point PI 4.5 to 5.5;
(b) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) measurement Molecular weight 94.5-96 kDa;
(c) exhibits a maximal activity for pNPM at 40 - 52 ° C;
(d) shows the maximal activity for pNPM at pH 5 - 6.5;
(e) exhibits thermal stability at < RTI ID = 0.0 > 30 C < / RTI >
(f) Km for pNPM is 2.14 mM and Vmax is 2.21 U / mg;
(g) increased K + , Mg 2+ or Fe 2+ enzyme activity;
(h) reduced enzyme activity upon addition of Ni 2+ , Mn 2+ , Fe 3+ , Cu 2+ , EDTA, SDS, Hg 2+ , PMSF or 8-hydroxyquinoline; or
(i) the activity of the enzyme is reduced upon addition of at least one saccharide selected from the group consisting of glucose, galactose, ribose, mannose and xylose.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020140147592A KR20160049859A (en) | 2014-10-28 | 2014-10-28 | A NOVEL β-MANNOSIDASE AND PRODUCING METHOD THEREOF |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020140147592A KR20160049859A (en) | 2014-10-28 | 2014-10-28 | A NOVEL β-MANNOSIDASE AND PRODUCING METHOD THEREOF |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020160179379A Division KR101834493B1 (en) | 2016-12-26 | 2016-12-26 | A novel β-Mannosidase and producing method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| KR20160049859A true KR20160049859A (en) | 2016-05-10 |
Family
ID=56020984
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020140147592A KR20160049859A (en) | 2014-10-28 | 2014-10-28 | A NOVEL β-MANNOSIDASE AND PRODUCING METHOD THEREOF |
Country Status (1)
| Country | Link |
|---|---|
| KR (1) | KR20160049859A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111122268A (en) * | 2019-12-31 | 2020-05-08 | 华南理工大学 | Method for preserving sulfate conjugate and glucuronide conjugate in liquid sample |
-
2014
- 2014-10-28 KR KR1020140147592A patent/KR20160049859A/en active Search and Examination
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111122268A (en) * | 2019-12-31 | 2020-05-08 | 华南理工大学 | Method for preserving sulfate conjugate and glucuronide conjugate in liquid sample |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Lee et al. | Purification and properties of a chitinase from Penicillium sp. LYG 0704 | |
| WO2017197546A1 (en) | Β-mannanase mrmman5a and encoding gene and application thereof | |
| EP3031926B1 (en) | Thermostable beta-glucosidase | |
| US20160201045A1 (en) | Endo-xylanase and Coding Gene and Use Thereof | |
| EP3031912B1 (en) | Thermostable ss-xylosidase | |
| Zahura et al. | cDNA cloning and bacterial expression of an endo-β-1, 4-mannanase, AkMan, from Aplysia kurodai | |
| KR101628856B1 (en) | Mutifunctional beta-Xylosidases | |
| He et al. | A novel xylanase from Streptomyces sp. FA1: purification, characterization, identification, and heterologous expression | |
| KR101718908B1 (en) | A novel Mannanase and its use | |
| CA2815905C (en) | Temperature-stable .beta.-pyranosidase | |
| CN107236719B (en) | Thermostable cellobiohydrolase | |
| KR20160049859A (en) | A NOVEL β-MANNOSIDASE AND PRODUCING METHOD THEREOF | |
| EP2393925B1 (en) | Protein and DNA sequence encoding a cold adapted xylanase | |
| KR101706451B1 (en) | Maltotrios-Producing Amylase derived from Microbulbifer sp. and the Process for Producing Maltotrios therewith | |
| KR100762410B1 (en) | Cold-active acidic beta-14-d-mannanase coding gene and use thereof | |
| WO2016175202A1 (en) | Heat-resistant xylanase | |
| KR101834493B1 (en) | A novel β-Mannosidase and producing method thereof | |
| KR101183114B1 (en) | -Glucosidase gene from Phanerochate chrysosporium, expression vector containing gene, transformant transformed by the vector, and method for preparation of transformant | |
| US11371032B2 (en) | Beta glucosidase with high glucose tolerance, high thermal stability and broad PH activity spectrum | |
| KR20140111848A (en) | A Novel Cellulase from Metagenomic Resources and Method for Preparing the Same | |
| EP3201333A1 (en) | Compositions comprising beta-mannanase and methods of use | |
| EP2982751B1 (en) | Hyperthermostable endoglucanase belonging to gh family 12 | |
| KR101615141B1 (en) | Beta agarase from Alteromonas sp. GNUM-1 | |
| Mesta et al. | Construction of a chimeric xylanase using multidomain enzymes from Neocallimastix frontalis | |
| KR102594729B1 (en) | Method for producing a thermo-tolerant κ-carrageenase via a food-grade host |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A201 | Request for examination | ||
| E902 | Notification of reason for refusal | ||
| AMND | Amendment | ||
| E601 | Decision to refuse application | ||
| AMND | Amendment |