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Intein-modified protease, its preparation method and industrial use

A technology of intein and protease, which is applied in the field of preparation of protease and intein-modified protease, and can solve problems such as difficulty in producing enzymes

Active Publication Date: 2021-07-09
AGRIVIDA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Despite their many beneficial uses, proteases are very difficult to produce because they degrade not only other proteins but also themselves

Method used

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  • Intein-modified protease, its preparation method and industrial use
  • Intein-modified protease, its preparation method and industrial use
  • Intein-modified protease, its preparation method and industrial use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach

[0109] The following items include specific embodiments. However, it is not intended to limit and exclude embodiments described elsewhere herein or alternative embodiments.

[0110] 1. An intein-modified protease comprising a target protease and an intein fused to the target protease, the fusion position being such that the intein can control the activity of the target protease , wherein the intein enables splicing of an intein-modified protease.

[0111] 2. The intein-modified protease of embodiment 1, wherein the position of fusion is such that the intein substantially reduces or inhibits the activity of the target protease.

[0112] 3. The intein-modified protease of any one or more of embodiments 1-2, wherein the target protease is selected from the group consisting of: EC3.4.99 protease, EC3.4.21.62 protease, keratinase, Enzymes in the group consisting of serine proteases, alkaline proteases, metalloproteases, cysteine ​​proteases, aspartic proteases, ATP-dependent prot...

Embodiment 1

[0170] Example 1 Experiment overview

[0171] In order to express keratinase for use as animal feed, the following steps were carried out: Bacillus licheniformis PWD-1 keratinase (Q53521) can be expressed as a pro-enzyme in the endosperm or embryo of maize seeds. The expression cassette was optimized, different codon-optimized versions of the coding sequence were tested, and the promoter, 5'-UTR intron, and targeting signal were evaluated. Fertility, fruit set and seed viability were tested. Multiple T1 seeds from a single event were screened to identify high expressors. Molecular mass, cumulative level, and activity were examined and compared with microbially prepared enzymes for specific activity, MW, pH, and optimum temperature for seed-expressed keratinase. Seed-expressed keratinase can be assessed in feeding experiments as a surrogate for microbial keratinase. The inoculation of keratinase into elite inbred germplasm can be the start of a commercial feed product. As a...

Embodiment 2

[0172] Example 2 Expression of Q53521 keratinase precursor enzyme in maize seeds

[0173] A maize codon-optimized gene of Q53521 keratinase from Bacillus licheniformis was synthesized (gi998767) (SEQ ID NO: 12). The codon-optimized gene of Q53521 was cloned into pUC57 to construct pUC57:FPROTQ53. To aid in cloning between the EcoRI and XhoI sites of Uni ZAP XR (Agilent) of pBluescript and bacteriophage lambda, the XhoI site at bases 898-903 was removed by silencing mutation C900 to G (on the labeled sequence) with site-directed mutagenesis point (CTCGAG). The plant expression construct contained the complete XhoI site.

[0174] Nucleotide and protein sequences of the corn codon-optimized Q53521 keratinase of Bacillus licheniformis:

[0175]

[0176] The amino acid sequence and domain structure of Q53521 keratinase (http: / / www.uniprot.org) from Bacillus licheniformis PWD-1 shown below includes the amino terminus between amino acid residues 1-29 (underlined) Signal peptid...

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Abstract

The present invention provides methods for preparing intein-modified proteases. The present invention also describes expression cassettes and vectors for genetically engineering hosts. The invention also provides genetic engineering of hosts to express one or more intein-modified proteases using the expression cassettes and vectors of the invention. The invention also describes methods of making proteases and methods of modulating their activity.

Description

[0001] This application claims priority to US Provisional Application No. 61 / 744863, filed October 3, 2012, and US Provisional Application No. 61 / 783,424, filed March 14, 2013, which are incorporated by reference Incorporated herein as if fully set forth. [0002] The Sequence Listing entitled "Sequence Listing" that is filed electronically with this application was created on October 3, 2012, has a size of 3,246,349 bytes, and is also incorporated herein by reference as if fully set forth. technical field [0003] The present invention relates to intein-modified proteases, methods for producing intein-modified proteases, methods for producing proteases and uses of intein-modified proteases. Background technique [0004] Proteases are enzymes that hydrolyze proteins and polypeptides into smaller peptides and amino acids. Proteases have found widespread use in industry, especially in fabric care agents, detergents, dishwashing liquids, industrial cleaners, solutions for remo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/00C12P21/06C12N9/64
CPCC12N9/64C07K14/81C12N15/62C12N15/63C12N9/20C12N9/2437C12N9/248C12N9/54C12Y304/21062C12N9/52C12N9/58C12N9/503C12N9/62C12N15/635A23K20/189C07K2319/92C11D3/386
Inventor R·M·莱布B·沈G·拉扎尔H·德拉维加
Owner AGRIVIDA
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