Proteases and methods for producing them

Inactive Publication Date: 2006-10-19
NOVOZYMES AS
View PDF4 Cites 21 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0007] It is a well-known problem in the art of expressing polypeptides having proteolytic activity, that many of such polypeptides are inherently unstable, they may be subject to autoproteolysis, or they may be targeted for degradation by other proteases already during their

Problems solved by technology

It is a well-known problem in the art of expressing polypeptides having proteolytic activity, that many of such polypeptides are inherently unstable, they ma

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Proteases and methods for producing them
  • Proteases and methods for producing them

Examples

Experimental program
Comparison scheme
Effect test

example 1

Construction of Synthetic 10R Tail-Variant Genes With Savinase Signal

[0280] A synthetic 10R gene (10RS) encoding a S2A protease denoted 10R from Nocardiopsis sp. NRRL 18262 (WO 01 / 58276) was constructed which has the nucleotide sequence shown in SEQ ID NO: 1. This synthetic gene was fused by PCR in frame to the DNA coding for the signal peptide from SAVINASE™ (Novozymes) resulting in the coding sequence Sav-10RS which is shown in SEQ ID NO: 2. Several tail-variants of this construct were made. Compared to the Sav-10RS protease encoded by SEQ ID NO:2 the tail variant construct Sav-10RS HV0 was constructed to have 8 amino acids extra in the C-terminus: QSHVQSAP (SEQ ID NO: 3) which were encoded by the following DNA sequence extension inserted in front of the TAA stopcodon of SEQ ID NO: 2:

[0281] (SEQ ID NO: 4): caatcgcatgttcaatccgctcca

[0282] Tail variant Sav-10RS HV1 was constructed to have 4 amino acids extra in the C-terminus: QSAP (SEQ ID NO: 5), with the following DNA sequence e...

example 2

Fermentation Yields of 10R Tail-Variants With Savinase Signal

[0289] Fermentations for the production of the tail-variant enzymes of the invention were performed on a rotary shaking table in 500 ml baffled Erlenmeyer flasks each containing 100 ml TY supplemented with 6 mg / l chloramphenicol.

[0290] Six Erlenmeyer flasks for each of the five B. subtilis strains from example 1 were fermented in parallel. Two of the six Erlenmeyer flasks were incubated at 37° C. (250 rpm), two at 30° C. (250 rpm), and the last two at 26° C. (250 rpm). A sample was taken from each shake flask at day 1, 2 and 3 and analyzed for proteolytic activity. The results are shown in tables 1-3. As it can be seen from tables 1-3, the effect of the tails is a surprisingly high improvement on the expression level of the protease, as measured by activity in the culture broth. The effect is most pronounced at 26° C. and 30° C., but is also evident at 37° C. as an effect observed especially at the early stage of the fer...

example 3

Chromosomal Integration of Tail-Variant Genes

[0293] The following construct was used for the chromosomal integration of the tail-variant encoding genes. The coding sequence of the well-known subtilisin BPN′ protease was operationally linked to a triple promoter, a marker gene was fused to this (a spectinomycin resistance gene surrounded by resolvase res-sites), and pectate lyase encoding genes from Bacillus subtilis were fused to the construct as flanking segments comprising the 5′ polynucleotide region upstream [yfmD-ytmC-yfmB-yfmA-Pel-start], and the 3′ polynucleotide region downstream [Pel-end-yflS-citS(start)] of the tail-variant encoding polynucleotide, respectively. The integrational cassette was made by the joining of several different PCR fragments. After the final PCR reaction the PCR product was used for transformation of naturally competent B. subtilis cells. One done denoted PL3598-37 was selected and confirmed by sequencing to contain the correct construct.

[0294] The ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
Fractionaaaaaaaaaa
Fractionaaaaaaaaaa
Fractionaaaaaaaaaa
Login to view more

Abstract

A secreted mature polypeptide derived from an S2A or S1E protease which after maturation has protease activity, which polypeptide when expressed and before maturation comprises a heterologous pro-region.

Description

FIELD OF INVENTION [0001] A number of microbially derived related proteases are notably difficult to produce in industrially relevant yields, they may be prone to various types of degradation and / or instabilities. The present invention provides methods for producing such proteases by expressing them as proteases comprising a heterologous pro-sequence. The invention further provides the resulting proteases comprising such heterologous prosequences. [0002] The present invention relates to isolated polypeptides having protease activity related to a Nocardiopsis sp. protease, and isolated nucleic acid sequences encoding such proteases. The invention furthermore relates to nucleic acid constructs, vectors, and host cells comprising these nucleic acid sequences as well as methods for producing and using the proteases, in particular within animal feed. BACKGROUND [0003] Polypeptides having protease activity, or proteases, are sometimes also designated peptidases, proteinases, peptide hydro...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): A01K67/027C07H21/04C12P21/06C12N9/64A01H1/00C12N1/21A23K1/16A23K1/165A23K1/175C11D3/386C12N9/58
CPCA23K1/1603A23K1/1653C12N9/58C11D3/38609C11D3/38618A23K1/1758A23K20/174A23K20/189A23K20/30
Inventor LASSEN, SOREN
Owner NOVOZYMES AS
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap