TECHNICAL FIELD

The present disclosure generally relates to a truncated and modified Serratiopeptidase (also called Serralysin, Serrapeptase, and Serratiapeptidase), polynucleotides encoding the same, methods for production thereof, and compositions comprising the truncated and modified Serratiopeptidase; more particularly, the present disclosure relates to the truncated and modified Serratiopeptidase having about 344 to about 380 amino acid residues.

BACKGROUND

Serratiopeptidase (also known as Serralysin, Serrapeptase, Serratiapeptidase, etc.) is a member of Serralysin family metalloproteases, secreted from a member of Enterobacteriace familySerratia marcesence. This enzyme has been widely used for clinical applications including orthopedics, surgery, dentistry and otorhinolaryngology owing to its analgesic, anti-edema, and anti-inflammatory properties. Fibrinolytic and caseinolytic activities of Serratiopeptidase has made it a promising candidate for treating atherosclerosis and cystic fibrosis. Meanwhile, anti-biofilm activity of this metalloprotease prevents bacterial adhesion to abiotic surfaces, in turn, decreasing their invasive power. It's proven that co-administration of Serratiopeptidase with antibiotics such as ampicillin, ciclacillin, cephalexin, minocycline, cefotiam, etc. may improve antibiotic activity by enhancing their dispersion through an infection site.

Despite of the wide range of applications and actions reported for Serratiopeptidase, this enzyme has raised a number of negative issues when employed in different pharmaceutical and industrial applications. Such issues include sensitivity to environmental tensions (such as high temperature and suboptimal pH), low membrane permeability and mucosal penetration, and low bioavailability.

Thereby, production of a stable form of Serratiopeptidase with high functionality, high thermal and pH stability, improved mucosal and membrane permeability, and enhanced anti-biofilm activity is of critical importance to industrial and pharmaceutical applications.

SUMMARY

This summary is intended to provide an overview of the subject matter of the present disclosure, and is not intended to identify essential elements or key elements of the subject matter, nor is it intended to be used to determine the scope of the claimed implementations. Its sole purpose is to present some concepts of one or more aspects in a simplified form as a prelude to the more detailed description that is presented later. The proper scope of the present disclosure may be ascertained from the claims set forth below in view of the detailed description below and the drawings.

In one general aspect, the present disclosure describes a truncated and modified Serratiopeptidase. The truncated and modified Serratiopeptidase may have an amino acid sequence with about 344 to about 380 amino acid residues. In exemplary embodiments, the amino acid sequence may include amino acids 1 to 344, and amino acids 1 to 380 of SEQ ID NO: 1. The truncated and modified Serratiopeptidase may further include a first Cysteine (C) residue at a N-terminus of the truncated and modified Serratiopeptidase that may be substituted for at least one of Alanine 8 and Leucine 12 of SEQ ID NO: 1, and a second Cysteine residue at a C-terminus of the truncated and modified Serratiopeptidase that may be substituted for at least one of Valine 339 and Arginine 302 of SEQ ID NO: 1.

In exemplary embodiments, the first and the second Cysteine residues may be adapted to form at least one disulfide bond between the N-terminus and the C-terminus of the truncated and modified Serratiopeptidase. In an exemplary embodiment, said at least one disulfide bond may include C8-C339, and C12-C302 disulfide bonds.

In one or more exemplary embodiments, the truncated and modified Serratiopeptidase may have an amino acid sequence as set forth in SEQ ID NO: 2 that includes Cysteine 8 and Cysteine 339. The Cysteine 8 and 339 may be adapted to form the C8-C339 disulfide bond between the N-terminus and the C-terminus of the truncated and modified Serratiopeptidase. In exemplary embodiments, the truncated and modified Serratiopeptidase set forth in SEQ ID NO: 2 may have a molecular weight between 37 and 37.5 kDa (kilo Daltons). The truncated and modified Serratiopeptidase as set forth in SEQ ID NO: 2 may have at least 80% residual activity at 90° C. after 10 minutes.

In one or more exemplary embodiments, the truncated and modified Serratiopeptidase may have an amino acid sequence as set forth in SEQ ID NO: 3 that includes Cysteine 8 and Cysteine 339. The Cysteines 8 and 339 may be adapted to form the C8-C339 disulfide bond between the N-terminus and the C-terminus of the truncated and modified Serratiopeptidase. In exemplary embodiments, the truncated and modified Serratiopeptidase set forth in SEQ ID NO: 3 may have a molecular weight between 40.5 and 41 kDa. The truncated and modified Serratiopeptidase with the amino acid sequence set forth in SEQ ID NO: 3 may have at least 70% residual activity at 90° C. after 10 minutes.

In one or more exemplary embodiments, the truncated and modified Serratiopeptidase may have an amino acid sequence as set forth in SEQ ID NO: 4 that includes Cysteine 12 and Cysteine 302. The Cysteine 12 and 302 may be adapted to form the C12-C302 disulfide bond between the N-terminus and the C-terminus of the truncated and modified Serratiopeptidase. In exemplary embodiments, the truncated and modified Serratiopeptidase set forth in SEQ ID NO: 4 may have a molecular weight between 40.2 and 40.8 kDa. The truncated and modified Serratiopeptidase with the amino acid sequence set forth in SEQ ID NO: 4 may have at least 70% residual activity at 90° C. after 10 minutes.

In another aspect, the present disclosure relates to a polynucleotide encoding the truncated and modified Serratiopeptidase. In exemplary embodiments, the polynucleotide may include an open reading frame that encodes one of the amino acid sequences set forth in SEQ ID NOs: 2, 3, and 4. In an exemplary embodiment, the polynucleotide may have a nucleic acid sequence as set forth in SEQ ID NO: 6. The polynucleotide may further include at least one of TGC or TGT at positions 22 to 24, and positions 1015 to 1017 of SEQ ID NO: 6.

In another exemplary embodiment, the polynucleotide may have a nucleic acid sequence as set forth in SEQ ID NO: 7. The polynucleotide may further include at least one of TGC or TGT at positions 22 to 24, and positions 1015 to 1017 of SEQ ID NO: 7.

In another exemplary embodiment, the polynucleotide may have a nucleic acid sequence as set forth in SEQ ID NO: 8. The polynucleotide may further include at least one of TGC or TGT at positions 34 to 36, and positions 904 to 906 of SEQ ID NO: 8. In exemplary embodiments, the at least one of TGC or TGT may encode for Cysteine.

