Novel subtilases

A technology of subtilase and subtilisin, which is applied in the directions of hydrolase, detergent compounding agent, chemical instruments and methods, etc., can solve the problems of inaccurate results and the like

Inactive Publication Date: 2006-05-03
NOVOZYMES AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, according to the present invention SSII subtilases belong to the new group of TY145-like subtilases, so SSII modeling based on the 3D structure of BPN'-like subtilases is likely to give inaccurate results

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0395] Construction of Savinase variant library

[0396] Libraries based on subtilase positions V28, I35, T71, I72, A73, M175 and T224 (BPN' numbering) have been synthesized. The library specifically contains the changes suggested by TY145 and covers the mutations introduced in the oligopeptides V28I, A, L; I35V, A, L; T71S; I72A, G, v; A73L, G; M175V, A; T224S, A, where Some mutations for doping. The nucleotide base doping required for doping from individual amino acid residues used in the following examples can be calculated as described on page 18 herein.

[0397] In the accompanying sequence listing, the following doped nucleotides are given the nucleotide designations recommended by WIPO standard ST25.

[0398] The list of constructed oligopeptide primers is as follows. Primers are named by where the modification occurs, so 28-35-CN could have changes at positions 28 and 35, 71-72-73-NC could have changes at positions 71, 72 and 73, etc.

[0399] 28-35-CN, SEQ ID NO: ...

Embodiment 2

[0435] Transfer of a region from TY145 to BPN' subtilase

[0436] It has been chosen to transfer the hyperactive regions in TY145 mentioned below from TY145 to Savinase. The TY145 region (SEQ ID NO: 1) was inserted after deletion of the Savinase region (BPN' numbering) instead. Alternatively, regions can be selected for transfer between psychrophilic TA41 / TA39 and BPN'-type protease-like Savinases, or from TA39 / TA41 to TY145-type non-psychrophilic subtilases.

[0437] Fragment I

[0438] TY145 SAKDSLIASAVD, position 144-155 (SEQ ID NO: 22)

[0439] Savinase PSPSATLEQAVN, position 129-140 (SEQ ID NO: 23)

[0440] Fragment II

[0441] TY145 AGNSGSGSNTIGFPGGLV, position 168-185 (SEQ ID NO: 24)

[0442] Savinase SGNSGAGSISYPARYA, position 153-172 (SEQ ID NO: 25)

[0443] Fragment IV

[0444] TY145 ASVESTWYTGGYNTIS, position 233-248 (SEQ ID NO: 26)

[0445] Savinase VNVQSTYPGSTYASLN, position 203-218 (SEQ ID NO: 27)

[0446] Savinase modified to receive TY145 fragment II (...

Embodiment 3

[0448] Transfer of regions from S39 and S41 to BPN' subtilases

[0449] The high activity regions in the TA39 subtilase S39 and TA41 subtilase S41 mentioned below have been selected for transfer to Savinase, the high activity regions of which were identified by the homology building procedure described above. Delete the Savinase region (BPN' number) and insert the S39 region or S41 region instead. Hereinafter the areas S39 and S41 are numbered according to FIG. 1 . Alternatively selectable regions can be transferred between the psychrophilic TA41 / TA39 and TA145 non-psychrophilic subtilases. Savinase variant V194S was used as acceptor for S39 fragment II.

[0450] Fragment I

[0451] S39 MSLGSSG, position 137-143 (SEQ ID NO: 28)

[0452] Savinase2 LSLGSPS, position 124-130 (SEQ ID NO: 29)

[0453] Fragment II

[0454] S39 MSLGSSGESSLI, position 137-148 (SEQ ID NO: 30)

[0455] Savinase variant V104S LSLGSPSPSATL, position 124-135 (SEQ ID NO: 31)

[0456] Fragment III

...

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Abstract

The present invention relates to methods of producing parental TY145 subtilase variants and parental BPN' subtilase variants, and to TY145 and BPN' variants having altered properties compared to the parental TY145 / BPN' subtilase variants.

Description

field of invention [0001] The present invention relates to variants of TY145 subtilase and BPN' subtilase and methods of constructing these variants with altered properties, such as stability (e.g. thermostability or storage stability), Ca 2+ dependent, pH dependent activity. Background of the invention [0002] Enzymes have been used in the detergent industry for over 30 years as part of washing formulations. Proteases are the most relevant enzymes in these preparations from a commercial point of view, but other enzymes including lipases, amylases, cellulases, hemicellulases or enzyme mixtures are also frequently used. [0003] To improve the cost and / or performance of proteases, proteases with altered properties are being sought such as: higher low temperature activity, higher thermostability, higher specific activity at a given pH, altered Ca 2+ dependence, greater stability in the presence of other detergent ingredients (eg bleach, surfactants, etc.). [0004] The sea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/54C11D3/386
Inventor A·斯文森H·德拉伯格N·廷德贝克
Owner NOVOZYMES AS
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