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Compositions and methods comprising variant microbial proteases

A technology of protease and protease variant, applied in the field of variant protease, can solve problems such as time-consuming and time-consuming

Inactive Publication Date: 2011-05-04
DANISCO US INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Thus, most methods are labor-intensive and time-consuming

Method used

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  • Compositions and methods comprising variant microbial proteases
  • Compositions and methods comprising variant microbial proteases
  • Compositions and methods comprising variant microbial proteases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0211] Assay

[0212] In the following examples, for ease of reading, various assays are used as given below. Any deviations from the protocols provided below are indicated in the examples.

[0213] A. TCA assay for protein content determination in 96-well microtiter plates

[0214]For BPN' (e.g., reference protease) and BPN' variants, the assay was started using filtered culture supernatant from microtiter plates grown for 3-4 days at 33°C with shaking at 230 rpm and humidified ventilation . A new 96-well flat bottom microtiter plate (MTP) was used for the assay. First, place 100 µL / well of 0.25N HCl in each well. Then, 50 μL of filtered culture solution was added. Light scatter / absorbance at 405 nm (using 5 second mixing mode in the plate reader) was then measured to provide a "blank" reading. For testing, 100 [mu]L / well of 15% (w / v) trichloroacetic acid (TCA) was plated and incubated at room temperature for 5 to 30 minutes. Light scatter / absorption at 405 nm (using...

Embodiment 2

[0301] Production of BPN' protease in Bacillus subtilis

[0302] In this example, experiments performed to produce BPN' protease in Bacillus subtilis are described. Transformation of plasmid pHPLT-BPN' into Bacillus subtilis is performed as known in the art (see eg, WO 02 / 14490, incorporated herein by reference). The DNA sequence provided below (aprE-BPN' hybrid leader sequence, BPN' pre- and BPN' mature DNA sequence from Bacillus amyloliquefaciens) encodes the BPN' precursor protein:

[0303]

[0304] In the above sequences, bold indicates the DNA encoding the mature protease, standard type indicates the leader sequence (aprE-BPN' hybrid leader sequence), and underlined indicates the pre-sequence (BPN'). The amino acid sequence of the BPN' precursor protein (aprE-BPN' hybrid leader, BPN' pro and BPN' mature DNA sequences) is provided below. In this sequence, the mature BPN' protease is indicated in bold and underlined.

[0305] VRSKKLWISLLFALALIFTMAFGSTSSAQAAGKSNGEKKYIV...

Embodiment 3

[0346] Generation of BPN' site evaluation libraries and multiple mutation libraries

[0347] In this example, the construction of BPN' variants is described.

[0348] BPN' site evaluation library (SEL) construction

[0349] The pHPLT-BPN' vector containing the BPN' expression cassette was used as template DNA. This vector contains a unique BglII restriction enzyme site, which is used for SEL construction. Invitrogen Primers were synthesized (desalted, 50 nmol scale) and listed in Table 7-1 for library generation.

[0350] To construct the BPN'SEL, three PCR amplifications were performed: two mutagenesis PCRs to introduce the mutated codon of interest in the mature BPN' DNA sequence, and a third PCR fused with two mutagenesis PCRs to build in the mature BPN' sequence pHPLT-BPN' expression vector with desired mutated codons. The mutagenesis method is based on the codon-specific mutation method. In this method, generation of all possible mutations at once in a specific DNA...

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Abstract

The present invention provides methods for protein engineering. Specifically, the invention provides methods utilizing site evaluation libraries to design libraries that optimize two or more properties of a protein. The present invention also provides variant subtilisins suitable for various uses.

Description

field of invention [0001] The present invention provides methods and variant proteases for protein engineering. In particular, the invention provides methods for evaluating libraries using loci. The invention also provides subtilisin variants for various uses. Background of the invention [0002] Serine proteases are a subclass of glycosyl hydrolases comprising distinct classes of enzymes with a wide range of specificities and biological functions. Subtilisins have been extensively studied, mainly for their use in cleaning and feed applications. Other work has looked at adverse environmental conditions (eg, exposure to oxidizing agents, chelating agents, extremes in temperature and / or pH) that can negatively affect the function of these enzymes in various applications. Nonetheless, there remains a need in the art for enzyme systems that are resistant to these adverse conditions and retain or have enhanced activities currently known in the art. [0003] Various methods of...

Claims

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Application Information

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IPC IPC(8): C12N9/54A23K1/165A23K20/195
CPCA23K20/189C11D3/38681C12N9/54C12Y304/21062C12N9/52C11D2111/12C11D3/386
Inventor L·G·卡斯康-佩雷拉D·A·伊斯泰尔F·齐德戈伯J·T·小凯利斯
Owner DANISCO US INC
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