# US 9,434,934 B2 · Heat stable keratinase and use thereof > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 9,434,934 B2 - Document ID: US9434934B2 - Status: Granted - Kind: B2 - Application number: 14/903,194 - Filed: 2014-07-17 - Published: 2016-05-26 - Granted: 2016-09-06 - Earliest claimed: 2013-07-29 - Assignees: Academia Sinica - Applicants: Academia Sinica - Inventors: Meng-Chiao Ho; Shih-Hsiung Wu; Wan-Ling Wu - Web page: https://patentgrep.com/patent/US9434934B2 ## CPC - C12N 9/52: derived from bacteria {or Archaea} - C07K 14/39: from yeasts - C12N 15/52: Genes encoding for enzymes or proenzymes - C12P 21/06: produced by the hydrolysis of a peptide bond, e.g. hydrolysate products (preparing foodstuffs by protein hydrolysis A23J3/00) - C07K 2319/00: Fusion polypeptide - C07K 2319/02: containing a signal sequence - C07K 2319/21: containing a His-tag - C07K 2319/22: containing a Strep-tag - C07K 2319/50: containing protease site - C12Y 304/21: Serine endopeptidases (3.4.21) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)} - C12Y: ENZYMES ## Abstract A fusion gene encoding M. taiwanensis WR-220 keratinase is disclosed. The fusion comprises: (a) a first DNA sequence encoding a protein secretion signal peptide, located at the N-terminus of the fusion gene; (b) a second DNA sequence encoding an inhibitory domain of M. taiwanensis WR-220 keratinase, linked in translation frame with the first DNA sequence; and (c) a third DNA sequence encoding a catalytic domain of M. taiwanensis WR-220 keratinase, linked in translation frame with the second DNA sequence, wherein the fusion gene is a non-naturally occurring chimeric DNA. Also disclosed are a method for preparation of the catalytic domain of M. taiwanensis WR-220 keratinase, and use of the M. taiwanensis WR-220 keratinase. ## Claims ### Claim 1 A fusion gene comprising: (a) a first DNA sequence encoding a protein secretion signal peptide, located at the N-terminus of the fusion gene; (b) a second DNA sequence encoding an inhibitory domain of Meiothermus taiwanensis WR-220 keratinase, linked in translation frame with the first DNA sequence; and (c) a third DNA sequence encoding a catalytic domain of M. taiwanensis WR-220 keratinase, linked in translation frame with the second DNA sequence, wherein the fusion gene is a non-naturally occurring chimeric DNA. ### Claim 2 (depends on 1) The fusion gene of claim 1, wherein the first DNA sequence encodes a yeast alpha-factor signal peptide. ### Claim 3 (depends on 1) A protein expression vector comprising: (a) the fusion gene of claim 1; and (b) a promoter, linked in translation frame with the fusion gene. ### Claim 4 (depends on 3) The protein expression vector of claim 3, wherein the promoter is selected from the group consisting of alcohol oxidase (AOX) promoter, glyceraldehyde phosphate dehydrogenase promoter, translational elongation factor 1-α promoter, Na+-coupled phosphate symproter promoter, and formaldehyde dehydrogenase promoter. ### Claim 5 (depends on 3) A host cell comprising the expression vector of claim 3. ### Claim 6 (depends on 5) A cell culture comprising: (a) an artificial medium; (b) the host cell of claim 5, the host cell secreting the catalytic domain of M. taiwanensis WR-220 keratinase into the artificial medium. ### Claim 7 An isolated protease comprising a catalytic domain of M. taiwanensis WR-220 keratinase, the protease lacking an inhibitory domain of M. taiwanensis WR-220 keratinase, and being in a dosage form of tablet, caplet, pellet, capsule, granule, pill, powder or sachet, or in a dosage form of solution without containing a cell culture supplement. ### Claim 8 (depends on 7) The protease of claim 7, which comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 14. ### Claim 9 (depends on 7) A method for degrading a proteinaceous material, comprising: exposing the proteinaceous material to an effective amount of the isolated protease of claim 7. ### Claim 10 (depends on 9) The method of claim 9, wherein the protease is pretreated at a temperature above 40° C. but below 95° C. and remains its activity. ### Claim 11 (depends on 9) The method of claim 9, wherein the proteinaceous material is selected from the group consisting of animal feed, food, milk, casein, elastin, skin, hair, wool, silk, nails, scales, fiber, leather, and feathers. ### Claim 12 (depends on 9) The method of claim 9, wherein the protease is pretreated with a solution having a pH value ranging from 3 to 10 and remains its activity. ### Claim 13 (depends on 5) A method for preparation of a catalytic domain of M. taiwanensis WR-220 keratinase, comprising: (ai) growing the host cell of claim 5 in a culture medium under conditions that permits expression of M. taiwanensis WR-220 keratinase and secretion of the catalytic domain thereof into the medium; or (aii) growing a host cell transformed with an expression plasmid comprising a DNA insert encoding an inhibitory domain and a catalytic domain of M. taiwanensis WR-220 keratinase under conditions that permits expression of M. taiwanensis WR-220 keratinase and secretion of the catalytic domain thereof into the medium; and (b) removing the host cell to obtain a supernatant containing the catalytic domain of the M. taiwanensis WR-220 keratinase; and (c) isolating the catalytic domain from the supernatant or drying the supernatant to obtain the catalytic domain in solid form. ### Claim 14 (depends on 13) The method of claim 13, wherein the inhibitory domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 13; and the catalytic domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 14. ### Claim 15 (depends on 1) The fusion gene of claim 1, wherein the inhibitory domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 13; and the catalytic domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 14. ### Claim 16 (depends on 6) An isolated protease comprising the catalytic domain of M. taiwanensis WR-220 keratinase secreted by the cell culture of claim 6. ### Claim 17 (depends on 16) The isolated protease of claim 16, which is in a dosage form of tablet, caplet, pellet, capsule, granule, pill, powder, or sachet, or in a dosage form of solution without containing a cell culture supplement. ### Claim 18 (depends on 8) A method for degrading a proteinaceous material, comprising: exposing the proteinaceous material to an effective amount of the protease of claim 8. ### Claim 19 (depends on 2) A protein expression vector comprising: (a) the fusion gene of claim 2; and (b) a promoter, linked in translation frame with the fusion gene. ### Claim 20 (depends on 15) A protein expression vector comprising: (a) the fusion gene of claim 15; and (b) a promoter, linked in translation frame with the fusion gene.