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CN-115698280-B - Novel serine protease variants

CN115698280BCN 115698280 BCN115698280 BCN 115698280BCN-115698280-B

Abstract

The present application relates to novel serine protease variants.

Inventors

  • LI ZHOUJI
  • Cao Ala
  • Chi Changjun

Assignees

  • CJ第一制糖株式会社

Dates

Publication Date
20260505
Application Date
20220311
Priority Date
20210312

Claims (7)

  1. 1. A serine protease variant consisting of: (a) An amino acid sequence shown in SEQ ID NO. 52, wherein the amino acid at position 12 corresponding to the amino acid sequence shown in SEQ ID NO. 52 is substituted with tyrosine (Y), (B) An amino acid sequence shown in SEQ ID NO. 53, wherein the amino acid corresponding to position 12 of the amino acid sequence shown in SEQ ID NO. 53 is substituted with tyrosine (Y), or (C) The amino acid sequence shown in SEQ ID NO. 54, wherein the amino acid at position 12 corresponding to the amino acid sequence shown in SEQ ID NO. 54 is substituted with tyrosine (Y).
  2. 2. A serine protease variant consisting of: (a) An amino acid sequence shown in SEQ ID NO. 70, wherein the amino acid at position 203 corresponding to the amino acid sequence shown in SEQ ID NO. 70 is substituted with tyrosine (Y), (B) An amino acid sequence shown in SEQ ID NO. 71, wherein the amino acid corresponding to position 201 of the amino acid sequence shown in SEQ ID NO. 71 is substituted with tyrosine (Y), or (C) The amino acid sequence shown in SEQ ID NO. 72, wherein the amino acid at position 201 corresponding to the amino acid sequence shown in SEQ ID NO. 72 is substituted with tyrosine (Y).
  3. 3. A composition comprising the serine protease variant of any one of claims 1 to 2.
  4. 4. A polynucleotide encoding the serine protease variant of any one of claims 1 to 2.
  5. 5. A vector comprising the polynucleotide of claim 4.
  6. 6. A host cell comprising at least one of the serine protease variants of any one of claims 1 to 2, a polynucleotide encoding the serine protease variants, and a vector comprising the polynucleotide.
  7. 7. A composition comprising at least one of the serine protease variant of any one of claims 1 to 2 and a microorganism expressing the serine protease variant.

Description

Novel serine protease variants Technical Field The present application relates to novel serine protease variants. Background Proteases are involved in various functions such as digestion, absorption and defense in organisms, and are classified into serine proteases, cysteine proteases, aspartic proteases and metalloproteases according to the structure of the active site. Among these enzymes, serine proteases (or serine endopeptidases) are characterized by having in common in their active site an active serine residue that cleaves a peptide bond in a protein, with serine as a nucleophilic amino acid at the active site of the protease (Hedstrom, 2002.Chem Rev 102:4501-4524). Serine proteases have been used in a variety of applications. In addition to therapeutic applications for treating human diseases (e.g., dissolving blood clots), serine protease is used not only as a component of laundry detergents and contact lens cleaners, but also for modification of milk proteins, silk degumming, leather soaking, dehairing, synthesis of oligopeptides, recovery of silver from lung X-ray film, production and improvement of feeds and foods, etc. (korean patent laid-open No. 10-2005-0068750). Disclosure of Invention Technical problem There is a need in the art to develop serine proteases with improved thermostability, increased activity, etc., to achieve greater industrial cost effectiveness and efficiency. Technical proposal It is an object of the present application to provide serine protease variants. It is another object of the present application to provide polynucleotides encoding serine protease variants and vectors comprising said polynucleotides. It is another object of the present application to provide a microorganism comprising at least one of a serine protease variant, a polynucleotide encoding a serine protease variant, and a vector comprising the polynucleotide. It is another object of the present application to provide a feed composition comprising at least one of a serine protease variant and a microorganism expressing a serine protease variant. Advantageous effects The serine protease variant of the present application has excellent activity as compared with the existing serine protease, and thus can be industrially used. Brief description of the drawings FIG. 1 shows the residue positions of the introduced mutations in the tertiary structure of serine protease variants derived from Thermobifida fusca. Detailed Description The following describes the content of the present application in detail. Meanwhile, each of the descriptions and embodiments disclosed in the present application may be applied to different descriptions and embodiments herein. In other words, all combinations of the various components disclosed in the present application are included within the scope of the present application. Furthermore, the scope of the application should not be limited by the description provided below. Furthermore, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific embodiments of the application. Such equivalents are intended to be encompassed within the scope of the present application. One aspect of the application provides serine protease variants. The term "serine protease" as used herein refers to an enzyme belonging to the protease subgroup and having proteolytic activity. In particular, serine proteases may be enzymes that degrade proteins by hydrolyzing peptide bonds and have active serine residues substantially at their active sites, and more particularly, enzymes having a spatial arrangement of amino acid residues of histidine, aspartic acid and serine (which may be referred to as a catalytic triplet), but are not limited thereto. Serine proteases according to the present application may be derived from microorganisms of the genus Thermobifida, nocardia (Nocardiopsis), actinorugispora or Spinactinospora, but are not limited thereto. Specifically, in the present application, the wild-type serine protease may be nocardia pseudolaris (Nocardiopsis composta) or Nocardiopsis potens composted with serine protease ：Thermobifida fusca,Thermobifida celulosilytica,Thermobifida halotolerans,Actinorugispora endohytica,Spinactinospora alkalitolerans, derived from the following, but is not limited thereto. In one embodiment, the serine protease of the present application may be a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence shown in SEQ ID NO. 31, but not limited to the amino acid sequence shown in SEQ ID NO. 31. In one embodiment, the amino acid sequence of SEQ ID NO. 31 may be an amino acid sequence derived from SEQ ID NO. 40 or SEQ ID NO. 2, but is not limited thereto. In one embodiment, serine proteases of the application can comprise, consist essentially of, or consist of any of the amino acid sequences of SEQ ID NOs 49 to 54, but are not limited to. In one embodiment, th