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Proteases

A protease activity, amino acid technology, applied in the direction of enzymes, hydrolases, enzymes, etc., can solve the problem of no sequence information, strains can not be obtained and so on

Inactive Publication Date: 2014-01-15
NOVOZYMES AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] DD20043218 discloses a proteolytic preparation derived from Nocardiopsis d'Arsonville strain ZIMET43647, however no sequence information
It appears that the strain is no longer available

Method used

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  • Proteases
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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0421] Example 1: Cloning and expression of the protease from Nocardiopsis darsonville subsp. darsonville subsp. DSM43235

[0422] Reagents and media

[0423]

[0424] 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.

[0425] NH 4 Ac 7.5M CH 3 COONH 4

[0426] TER 1 μg / ml RNase A in TE-buffer

[0427] CIA Chloroform / Isoamyl Alcohol 24:1

[0428] Experimental procedure

[0429] SEQ ID NO: 1 is the DNA sequence encoding the proform of the protease from Nocardiopsis darsonville subsp. darsonville subsp. DSM 43235. Nucleotides 499-1062 correspond to the mature peptide coding portion.

[0430] SEQ ID NO:2 is the deduced amino acid sequence of SEQ ID NO:1. Amino acids -166 to 1 are the propeptide, while amino acids 1 to 188 are the mature peptide.

[0431] Clone SEQ ID NO:1

[0432] The wild type was grown at 30°C f...

Embodiment 2

[0452] Example 2: Purification and Characterization of Protease from Nocardiopsis darsonville subspecies DSM43235

[0453] protease assay

[0454] 1) pNA determination:

[0455] pNA substrate: Suc-AAPF-pNA (Bachem L-1400)

[0456] Temperature: Room temperature (25°C)

[0457] 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.

[0458] 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.

[0459] 2) Protazyme AK determination:

[0460] Substrate: Protazyme AK sheet (cross-linked and stained casein; from Megazyme)

[0461] Temperature : Controlled (measurement tempera...

Embodiment 3

[0481] Example 3: Performance of Nocardiopsis darsonville subsp. darsonville subsp. DSM43235 protease in a monogastric in vitro digestion model

[0482] Purified preparations of the mature fraction of the protease 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...

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Abstract

The present invention relates to thermostable proteases having an amino acid sequence which homologous to the amino acid sequence of proteases derived from Nocardiopsis, and the production thereof by wild-type and recombinant host cells including transgenic plants and non-human transgenic animals. The proteases are effective in animal feed, in particular fish feed, and detergents. The proteases are capable of degrading the soybean Bowman-Birk inhibitor, and other antinutritional factors such as soybean agglutinin and the Kunitz trypsin inhibitor, as well as the isolated soy storage proteins glycinin and beta-conglycinin. Characteristic structural features of relevance for the thermostability of these proteases of peptidase family S2A or S1E are disclosed.

Description

[0001] The application of the present invention is based on the application date of June 21, 2004, the application number is "200480017141.9" (the international application number is "PCT / DK2004 / 000432"), and the divisional application of the invention patent application named "protease". technical field [0002] The present invention relates to isolated polypeptides having protease activity and homology to Nocardiopsis proteases, 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 . [0003] The proteases of the invention are thermostable and disclose characteristic structural features associated with the thermostability of proteases of peptidase family S2A or S1E. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/52C12N15/57C12N15/75C12N1/21A23K1/165A23K1/18C11D3/386C12N1/20C12R1/01A23K3/00C07H21/04C12N9/08C12N9/54C12N9/58C12N15/74
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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