Proteases

A technology of protease activity and nucleic acid sequence, applied in the field of isolated polypeptides, can solve the problems of no sequence information and unobtainable strains

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

[0006] DD 20043218 discloses a proteolytic preparation derived from Nocardiopsis dassonvillei strain ZIMET 43647, however no sequence information
It appears that the strain is no longer available

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0390] Example 1: Cloning and expression of a protease from Nocardiopsis dassonvillei subspecies dassonvillei DSM 43235

[0391] Reagents and media

[0392] LB agar is described in Ausubel, F.M. et al. (eds.) "Current protocols in Molecular Biology". John Wiley and Sons, 1995

[0393] LB-PG Agar LB agar supplemented with 0.5% glucose and 0.05M potassium phosphate, pH7.0

[0394] PS-1 10% sucrose, 4% soybean flour, 1% Na 3 PO 4 -12H 2 O, 0.5% CaCO 3 , and 0.01% polyoxyacrylic acid (pluronic acid)

[0395] TE 10mM Tris-HCl, pH7.4 1mM EDTA, pH8.0

[0396] TEL 50mg / ml Lysozym in TE-buffer

[0397] Thiocyanate 5M Guanidium thiocyanate

[0398] 100mM EDTA

[0399] 0.6% w / v N-laurylsarcosine, sodium salt

[0400] 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 H 2 0 to 100ml and filter through a 0.2μ sterile filter.

[0401] NH 4 Ac 7.5M CH 3 COONH 4

[0402] TER 1 μg / ml RNa...

Embodiment 2

[0432] Example 2: Purification and characterization of protease from Nocardiopsis dassonvillei subspecies dassonvillei DSM 43235

[0433] protease assay

[0434] 1) pNA determination:

[0435] pNA substrate: Suc-AAPF-pNA (Bachem L-1400)

[0436] Temperature: Room temperature (25°C)

[0437] Assay buffer: 100mM succinic acid, 100mM HEPES, 100mM CHES, 100mMCABS, 1mM CaCl 2, 150mm KCl, 0.01% Triton X-100, adjusted to pH 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 with HCl or NaOH.

[0438] 20 μl protease (diluted in 0.01% Triton X-100) was mixed with 100 μl assay buffer. The assay was started by adding 100 μl of pNA substrate (50 mg dissolved in 1.0 ml DMSO and further diluted 45× with 0.01% TritonX-100). Monitor OD 405 The increase in is used as a measure of protease activity.

[0439] 2) Determination of Protazyme AK:

[0440] Substrate: Protazyme AK tablet (cross-linked and stained casein; from Megazyme)

[0441] Temperature: Controlled (me...

Embodiment 3

[0457] Example 3: Performance of Nocardiopsis dassonvillei subspecies dassonvillei DSM 43235 protease in an in vitro digestion model of monogastric

[0458] Purified preparations of the protease mature fraction having SEQ ID NO: 2 (prepared as described in Examples 1 and 2) were tested in an in vitro model simulating digestion in monogastric animals. Specifically, the ability of the proteases to improve solubilization and digestion of corn / -SBM (corn / -soybean meal) proteins was tested. In the table below, this protease is referred to as "protease of the invention". The in vitro system consisted of 15 shake flasks in which the maize / -SBM substrate was initially incubated with HCl / pepsin - simulating gastric digestion - and subsequently incubated with trypsin - simulating Intestinal digestion. Ten of the shake flasks were formulated with protease at the beginning of the stomach phase, while the remaining shake flasks served as blanks. At the end of the incubation period of th...

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PUM

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Abstract

Disclosed is a thermostable protease congenetic with the protease from Nocardiopsis and the preparation in the wild type reconstructed host-cells which contain the transgenic plant and the no-human transgenic animal cells. The protease is effective in animal feed, in particular to fish feed and washing agent. The protease can degrade soybean Bowman-Birk stayer, other anti-nutritional factors such as soybean agglutinin and Kunitz trypsinase stayer, and separated soybean storage protein, glycinin and beta-conglycinin. The structural characteristics related to the thermostability of the proteases of the peptidase family S2A or S1E.

Description

technical field [0001] The present invention relates to isolated polypeptides having protease activity and homology to Nocardiopsis protease, and isolated nucleic acid sequences encoding said polypeptides. The present invention further relates to nucleic acid constructs, vectors and host cells containing these nucleic acid sequences, including transgenic plants and non-human animals, as well as methods for the production and application of said proteases, in particular in animal feed, such as fish feed . [0002] The proteases of the invention are thermostable and disclose characteristic structural features associated with the thermostability of proteases of peptidase family S2A or S1E. [0003] The protease of the present invention is further effective in degrading soybean Bowman-Birk inhibitors, as well as other anti-nutritional factors, such as soybean agglutinin and Kunitz trypsin inhibitors, and isolated soybean storage Protein (soy storage protein), such as glycinin (g...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/58C12N9/52C12N15/57A23J3/34A23K3/00C07H21/04C11D3/386C12N9/08C12N9/54C12N15/74
CPCA01K2217/05C12N9/52C12N9/58C12N2310/111A23K20/189
Inventor 索伦·F·拉森卡斯滕·肖霍姆彼得·R·奥斯特加德卡斯藤·安德森莫滕·费希尔
Owner NOVOZYMES AS
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