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Methods and compositions for stabilizing trans-splicing intein modified proteases

A trans-splicing and intein technology, applied in detergent compositions, fusion with post-translational modification motifs, chemical instruments and methods, etc., can solve the problems of increasing chemical load and cost

Active Publication Date: 2017-08-18
AGRIVIDA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Current stabilization techniques are often based on the use of high concentrations of chemical protease inhibitors, which increases chemical load and cost

Method used

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  • Methods and compositions for stabilizing trans-splicing intein modified proteases
  • Methods and compositions for stabilizing trans-splicing intein modified proteases
  • Methods and compositions for stabilizing trans-splicing intein modified proteases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach

[0059]One embodiment includes a composition comprising the precursors (NI and IC) of any of the intein-modified proteases herein. The first precursor NI may contain, consist essentially of, or consist of at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97 of the reference sequence of SEQ ID NO: 1 , 98, 99 or 100% identity amino acid sequence composition. The first precursor may be capable of trans-splicing with the second precursor to form the target protease. The second precursor IC may contain, consist essentially of, or consist of at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97 of the reference sequence of SEQ ID NO: 2 , 98, 99 or 100% identity amino acid sequence composition. The second precursor may be capable of trans-splicing with the first precursor to form the target protease. The concentration of the first precursor or the second precursor may be in the range of 0.01% (v:v) to 10% (v:v). Concentration can be 0.02%(v:v), 0.03%(v:v), 0.04%(v:v),...

Embodiment 1

[0169] Example 1. Trans-splicing Intein Technology for Modulating Protease Activity

[0170] Trans-splicing intein technology can be used to modulate protease activity. In this approach, the protease is split between the catalytic residues into two inactive fragments, each of which is expressed as a fusion to a trans-spliced ​​intein. Mixing in an aqueous environment triggers trans-intein-mediated conjugation of the inactive fragment, splicing and seamless ligation of the inactive portion to form a fully functional active protease.

[0171] The construction, expression and detergent dilution-inducing properties of the trans-spliced ​​intein-regulated liquid laundry protease Savinase have been described in PCT patent application PCT / US2013 / 063304, which is incorporated herein by reference as if fully set forth. Described herein are methods to improve formulation stability and detergent dilution-inducible protease activity to levels relevant for commercial liquid laundry applic...

Embodiment 2

[0172] Example 2. Molecules optimized for solubility

[0173] Detergent refers to liquid laundry detergent. Detergents-1 and -4 are standard liquid detergent formulations without enzymes, optical brighteners and colorants. Detergent-1 can contain Deionized Water, Neodol 25_7, LAS Acid, Prifac 5908, Dequest 2010, SLES 3EO, Sodium Sulfite, Sokalan HP20, MPG, MEA, TEA, Glycerin, Tinopal 5BMGX and other common liquid laundry detergent ingredients . Detergent-1 had a pH of ~8.3±0.2 and a conductivity of ~3890 μSimens. Detergent-4 had a pH of ~7.3±0.2 and a conductivity of ~22400 μSimens.

[0174] Detergents-2 and -3 were commercial brand detergents with formulated protease free fluorescers and colorants. Detergent-2 had a pH of ~8.8±0.2 and a conductivity of ~3900 μSimens. Detergent-3 had a pH of ~8.3±0.2 and a conductivity of ~7090 μSimens. Proteases were inactivated by heat treatment at 80°C for 90 min. After cooling to room temperature, verify the absence of proteases as ...

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Abstract

Methods for producing a trans-splicing intein-modified protease with enhanced solubility and regulating its activity are described. Intein-modified proteases having enhanced solubility and polynucleotides encoding the same are provided. Methods of storing trans-splicing proteases are also described.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Provisional Application No. 62 / 069,653, filed October 28, 2014 and incorporated herein by reference as if fully set forth. [0003] The Sequence Listing entitled "Sequence Listing," filed electronically with this application, was created on October 28, 2015, has a size of 59,091 bytes, and is incorporated herein by reference as if fully set forth. technical field [0004] The present invention relates to methods and compositions capable of tightly regulating the activity of trans-spliced ​​intein-modified proteases in liquid detergents and home care products for stable storage and rapid and efficient restoration of protease activity in formulations with reduced cleaning efficacy Loss. Background technique [0005] Proteases are common components in various liquid detergents, laundry detergents, dishwashing liquids, paints, coatings and industrial cleaners. Proteases are notoriou...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/50C12N9/54C12N15/62C11D3/386
CPCC11D3/386C07K2319/00C07K2319/92C12N9/54C07K14/00C11D3/38663
Inventor M·R·莱布B·沈G·拉扎尔
Owner AGRIVIDA
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