Proteases

A protease, protease activity technology, applied in the field of protease

Inactive Publication Date: 2007-08-08
NOVOZYMES AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The strain is no longer available because the deposit was withdrawn

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0372] Example 1: Cloning and expression of three proteases (L1a, L1b and L1c)

[0373] Reagents and media

[0374] LB agar is described in Ausubel, F.M. et al. (eds.) "Current protocols in Molecular

[0375]Biology "John Wiley and Sons, 1995;

[0376] LB-PG Agar LB agar supplemented with 0.5% glucose and 0.05 M potassium phosphate, pH7.0

[0377] PS-1 10% sucrose, 4% soybean flour, 1% Na3PO4-12H2O, 0.5% CaCO3,

[0378] and 0.01% pluronic acid

[0379] TE 10mM Tris-HCl, pH7.4

[0380] 1 mM EDTA, pH 8.0

[0381] TEL 50mg / ml Lysozyme (Lysozym) in TE-buffer

[0382] Thiocyanate 5M Guanidine Thiocyanate

[0383] 100mM EDTA

[0384] 0.6% w / v N-Lauryl Sarcosine, Sodium Salt,

[0385] 60g thiocyanate, 20ml 0.5M EDTA, pH8.0, 20ml H 2 o

[0386] Dissolve at 65°C. Cool to room temperature (RT) and add 0.6 g N-lauryl

[0387] creatine. Add H 2 0 to 100ml and filter through a 0.2μ sterile filter

[0388...

Embodiment 2

[0426] Example 2: Cloning and expression of protease L2a

[0427] The precursor form (pro-form) of the protease coding gene (88-1152 nucleotides of SEQ ID NO: 7) was isolated from Nocardioides species DSM 16424 by the steps described in Example 1, The difference is that the following primers are used:

[0428] 1718 (SEQ ID NO: 20): 5'-gttcatcgatcgcatcggctgcgcccggccccgtcccccag-3'

[0429] 1720 (SEQ ID NO: 21): 5'-ggagcggattgaacatgcgatcagctggtgcggatgcgaac-3'.

[0430] The corresponding protease (SEQ ID NO: 8) was named L2a.

[0431] Also as generally described in Example 1, a B. subtilis host strain was constructed, designated Sav-L2a, and chloramphenicol-resistant, protease-positive colonies were selected and analyzed by DNA sequencing of the insert.

Embodiment 3

[0432] Example 3: Cloning of two additional proteases

[0433]By the method described in Example 1, two additional protease-encoding genes (respectively nucleotides of SEQ ID NO: 9 88-1149 nucleotides and 88-1152 nucleotides of SEQ ID NO: 11), the difference is that primers 1728 and 1763 are used respectively; and 1747 and 1749:

[0434] 1728 (SEQ ID NO: 22): 5'-gttcatcgatcgcatcggctgcccccgccccccagtc-3';

[0435] 1763 (SEQ ID NO: 23):

[0436] 5'-ggagcggattgaacatgcgattaggtgcgcagacgcaggcccca-3';

[0437] 1747 (SEQ ID NO: 24): 5'-gttcatcgatcgcatcggctggaaccgtacccaccccccagg-3';

[0438] 1749 (SEQ ID NO: 25): 5'-ggagcggattgaacatgcgattagctggtgcgcagtcgcac-3'

[0439] The corresponding proteases (SEQ ID NO: 10 and 12, respectively) were named L2b and L2c, respectively.

[0440] Also as generally described in Example 1, B. subtilis host strains were constructed, designated Sav-L2b and Sav-L2c, respectively, and chloramphenicol-resistant, protease-positive colonies were selected and...

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PUM

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Abstract

Proteases derived from Nocardiopsis dassonvillei subsp. dassonvillei DSM 43235, Nocardiopsis prasina DSM 15649, Nocardiopsis prasina (previously alba) DSM 14010 Nocardiopsis sp. DSM 16424, Nocardiopsis alkaliphila DSM 44657 and Nocardiopsis lucentensis DSM 44048, as well as homologous proteases; their recombinant production in various hosts, including transgenic plants and non-human animals, and their use in animal feed and detergents. The proteases are acid-stable, alkali-stable, and/or thermostable.

Description

technical field [0001] The present invention relates to isolated polypeptides having protease activity and isolated nucleic acid sequences encoding the polypeptides. The present invention also relates to nucleic acid constructs, vectors and host cells containing the nucleic acid sequence, including plant and animal cells, and the present invention also relates to methods for producing and using said polypeptide, especially the use of said polypeptide in animal feed and detergents use. Background technique [0002] Proteases from Nocardiopsis sp. NRRL 18262 and Nocardiopsis dassonvillei NRRL 18133 are disclosed in WO 88 / 03947. The DNA and amino acid sequence of the protease from Nocardiopsis NRRL 18262 is shown in DK application no.1996 00013. WO 01 / 58276 discloses the use of acid-stable proteases related to proteases from Nocardiopsis alba NRRL 18262 and from Nocardiopsis alba DSM 14010 in animal feed. [0003] JP 2-255081-A discloses a protease from Nocardiopsis strain O...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/52C12N15/57C12N5/10C12N15/82A23K1/165A23L1/305C11D3/386A23J3/34A23K1/18
CPCA23J3/346A23K1/1826A23K1/184C11D3/38618A23K1/1893C12N9/52C11D3/38609A23K1/1653A23K20/189A23K50/30A23K50/60A23K50/75
Inventor 索伦·F·拉森卡斯滕·肖霍姆彼得·R·奥斯特加德莫滕·费希尔
Owner NOVOZYMES AS
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