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Proteases

A technology of protease activity and hemoglobin, applied in the field of isolated nucleic acid sequence, production and use of this protease, can solve the problem of no sequence information

Inactive Publication Date: 2006-07-26
NOVOZYMES AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] DD20043218 discloses a proteolytic preparation from Nocardiopsis dassonvillei strain ZIMET 43647, however no sequence information

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0337] Example 1: Cloning and expression of protease derived from Nocardopsis albicans DSM 15647

[0338] Reagents and media

[0339] LB agar Described in Ausubel, F.M. et al. (eds.) "Current protocols in Molecular Biology". John Wiley and Sons, 1995

[0340] LB-PG Agar LB agar supplemented with 0.5% glucose and 0.05M potassium phosphate, pH 7.0

[0341] PS-1 10% sucrose, 4% soybean flour, 1% Na 3 PO 4 -12H 2 O, 0.5% CaCO 3 , and 0.01% pluronic acid

[0342] TE 10mM Tris-HCl, pH7.4

[0343] 1mM EDTA, pH8.0

[0344] Lysozym 50mg / ml in TEL TE-buffer

[0345] Thiocyanate 5M Guanidium thiocyanate

[0346] 100mM EDTA

[0347] 0.6% w / v N-Lauryl Sarcosine, Sodium Salt

[0348] 60g thiocyanate, 20ml 0.5M EDTA, pH8.0, 20ml H 2 O dissolves at 65°C. Cool to room temperature (RT) and add 0.6 g N-lauryl sarcosine. Add water to 100ml and filter through a 0.2μ sterilized filter.

[0349] NH 4 Ac 7.5M CH 3 COONH 4

[0350] 1μg / ...

Embodiment 2

[0380] Example 2: Purification and Characterization of Protease from Nocardiopsis alba DSM 15647

[0381] protease test

[0382] 1) pNA test:

[0383] pNA substrate: Suc-AAPF-pNA (Bachem L-1400).

[0384] Temperature: room temperature (25°C)

[0385] Assay Buffer: 100mM Succinic Acid, 100mM HEPES, 100mM CHES, 100mM CABS, 1mM CaCl 2, 150 mM KCl, 0.01% Triton X-100 with HCl or NaOH to adjust the pH-value to 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0.

[0386] 20 μl protease (diluted in 0.01% Triton X-100) was mixed with 100 μl assay buffer. 100 [mu]l of pNA substrate (50 mg dissolved in 1.0 ml DMSO, further diluted 45x with 0.01% Triton X-100) was added to start the assay. Monitor OD 405 An increase in , as a measure of protease activity.

[0387] 2) Protazyme AK test:

[0388] Substrate: Protazyme AK tablets (cross-linked and stained casein; from Megazyme)

[0389] Temperature: controlled temperature (test temperature)

[0390] Assay Buff...

Embodiment 3

[0403] Example 3: Specific activity of proteases derived from Nocardiopsis alba

[0404] Specific activity was determined using the purified protease preparation described in Example 2. SDS-PAGE (measured according to the method described in Example 2A of WO01 / 58275) analysis showed that the purity of the product was above 95%. Split the protease sample in two. One part was analyzed for its protein content by amino acid analysis, and the other part was analyzed for protease activity.

[0405] Amino Acid Analysis (AAA) / (mg / ml)

[0406] The peptide bonds of the protease samples were hydrolyzed with acid, and the released amino acids were then separated and quantified according to the manufacturer's instructions on a Biochrom 20 Plus amino acid analyzer commercially available from Bie & Berntsen A / S, Sandbaekvej 5-7, DK -2610 Roedovre, Denmark acquired. The protein samples were dried in a vacuum centrifuge, dissolved in 18.5% (v / v) HCl + 0.1% (v / v) phenol, and incubated at ...

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PUM

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Abstract

Disclosed are a high specific active protease congenetic with the protease from imitative Nocard's bacillus, and the production in the wild type and the reconstructed host-cells containing the transgenic plant and the no-human transgenic animal cells. The protease is effective in animal feed and washing agent. Peculiar structural characteristics related to the high specific activity of protease are also disclosed. The protease belongs to the peptidase family S2A or S1E.

Description

technical field [0001] The present invention relates to an isolated polypeptide having protease activity homologous to Nocardiopsis protease, and to an isolated nucleic acid sequence encoding the polypeptide. In addition, the present invention relates to nucleic acid constructs, vectors, and host cells including transgenic plants and non-human animals containing these nucleic acid sequences, as well as methods for producing and using the protease, especially in animal feed. [0002] The protease of the present invention has high specific activity. Disclosed are characteristic structural features associated with the high specific activity of proteases belonging to the peptidase family S2A or S1E. Background of the invention [0003] Proteases derived from Nocardiopsis sp. NRRL 18262 and Nocardiopsis dassonvillei NRRL 18133 are disclosed in WO88 / 03947. DK application no. 1996 00013 discloses the DNA and amino acid sequence of the protease derived from Nocardiopsis sp. NRRL 1...

Claims

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Application Information

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IPC IPC(8): C12N9/58C12N9/52C12N15/57A23J3/34
CPCA01K2217/05C12N9/52C12N9/58C12N2310/111
Inventor 索伦·F·拉森卡斯滕·肖霍姆彼得·R·奥斯特加德
Owner NOVOZYMES AS
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