Methods of using archaeal serine protease

A serine protease and amino acid technology, applied in the field of thermophilic protease, can solve problems such as stability or harmful activity

Pending Publication Date: 2019-12-20
DUPONT NUTRITION BIOSCIENCES APS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0013] In such industrial processes, uses or products, proteases may be exposed to environmental conditions such as extremes in temperature and / or pH as described above, which may be detrimental to the stability or activity of the enzyme

Method used

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  • Methods of using archaeal serine protease
  • Methods of using archaeal serine protease
  • Methods of using archaeal serine protease

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[0427] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton et al., DICTIONARY OFMICROBIOLOGY AND MOLECULAR BIOLOGY, 2nd ed., John Wiley and Sons, New York (1994), and Hale and Marham, THE HARPER COLLINSDICTIONARY OF BIOLOGY [ HarperCollins Biological Dictionary], Harper Perennial, New York (1991) provides the skilled artisan with a general dictionary of many of the terms used in this disclosure.

[0428] The disclosure is further defined in the examples below. It should be understood that these Examples, while illustrating certain embodiments, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this disclosure, and without departing from the spirit and scope of this disclosure, can make various changes and modifications to a...

example 1

[0430] Analysis of the TnaPro1 sequence

[0431] The nucleotide sequence of the full-length TnaPro1 gene identified in the NCBI database is provided in SEQ ID NO: 1 (Thermophilus nautilus, NCBI Reference Sequence: NZ_CP007264.1, 1327825-1329105, complement). The corresponding full-length protein sequence encoded by the TnaPro1 gene is shown in SEQ ID NO: 2 (GenBank accession number: AHL23118.1).

[0432] The propeptide and mature regions of the TnaPro1 protein were predicted based on the protein sequence alignment with the homologous enzyme Thermococcus kodakaraensis KOD1 TK-subtilisin protein (Pulido et al. (2006) Applied and Environmental Microbiology, 72( 6): 4154-4162). Using SignalP v4.0 (Nordahl Petersen et al. (2011) Nature Methods [Nature Methods] 8:785-786) software, the TnaPro1 signal peptide is predicted to have the sequence shown in SEQ ID NO: 10, and the zymogen is predicted to have the sequence shown in SEQ ID NO: 10 The sequence shown in ID NO:8. The N-termin...

example 2

[0435] Cloning of TnaPro1

[0436] The DNA sequence encoding the proenzyme of TnaPro1 was synthesized and inserted into the Bacillus subtilis expression vector p2JM103BBI (Vogtentanz, Protein Expr Purif [protein expression and purification], 55:40-52, 2007) to obtain the name pGX706 (aprE- TnaPro1) plasmid ( figure 1 ).

[0437] Ligation of the gene encoding the TnaPro1 protein into the digested vector resulted in the addition of three codons (encoding Ala-Gly-Lys ). This gene has an alternative start codon (GTG). Such as figure 1 As shown, pGX706(aprE-TnaPro1) contains an AprE promoter, an AprE signal sequence for directing Bacillus subtilis to secrete the target protein, and a synthetic nucleotide sequence encoding the predicted TnaPro1 zymogen. SEQ ID NO: 4 shows the gene sequence of the aprE-TnaPro1 construct, including three additional codons introduced during ligation of the fusion sequence. SEQ ID NO:5 shows the amino acid sequence of the AprE-TnaPro1 fusion pro...

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Abstract

Methods of using archaeal serine protease sequences are disclosed.

Description

[0001] Cross References to Related Applications [0002] This application claims priority from PCT / CN 2017 / 076770 filed March 15, 2017, which is hereby incorporated by reference in its entirety. [0003] Incorporated by reference into the sequence listing [0004] The Sequence Listing provided as a file named NB41217WOPCT_SequenceListing_ST25 with a size of 29 KB to be created and submitted concurrently on March 2, 2018 is hereby incorporated by reference in its entirety. technical field [0005] The field relates to thermostable archaeal serine proteases, and in particular to the identification and characterization of said protein, referred to herein as TnaPro1, which is a thermophilic protease suitable for use in feed or grain applications. Background technique [0006] Proteases (also known as peptidases or proteinases) are enzymes capable of cleaving peptide bonds. Proteases have evolved many times, and different kinds of proteases can perform the same reaction through...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/64A23K10/18
CPCC12N9/6408C12N9/6424A23K10/18A23K20/147A23J3/346C12N9/52C12N15/75C12Y304/21
Inventor 顾晓岗Z·邹S·于S·希普夫斯科夫M·范布鲁塞尔-兹维内
Owner DUPONT NUTRITION BIOSCIENCES APS
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