An acidic protease mutant, its encoding gene, preparation method and application

By genetically modifying the acidic protease of Trichoderma reesei, the resulting acidic protease mutant AQAP52 maintains high enzyme activity over a wide range of pH and temperature, solving the problem of poor thermal stability of existing acidic proteases and making it suitable for industrial applications such as brewing, feed processing, and leather softening.

CN120944859BActive Publication Date: 2026-01-09HUBEI UNIV +1
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Patent Information

Application Number
CN202511476612.2
Authority / Receiving Office
CN · China
Patent Type
Patents(China)
Current Assignee / Owner
Filing Date
2025-10-16
Publication Date
2026-01-09
Estimated Expiration
2045-10-16

AI Technical Summary

Technical Problem

Existing acidic proteases have low enzyme activity and/or poor thermal stability, making them unsuitable for high-temperature environments and limiting their widespread application in industrial fields.

Method used

A mutant acidic protease, AQAP52, was prepared by genetically modifying the Trichoderma reesei acidic protease. The amino acid sequence was point-mutated at specific sites, and combined with a specific expression vector and recombinant strain, the enzyme activity and thermostability were improved.

Benefits of technology

The acidic protease mutant AQAP52 maintains high enzyme activity over a wide range of pH and temperature, is not easily inactivated in high-temperature environments, and is suitable for applications such as brewing, feed processing, and leather softening, thereby improving the flexibility of production processes and the value of industrial applications.

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Abstract

The application belongs to the technical field of genetic engineering, and particularly relates to an acid protease mutant, a coding gene thereof, a preparation method and application. The amino acid sequence of the acid protease mutant is shown as SEQ ID NO. 2. The acid protease mutant provided by the application has the advantages of high enzyme activity, strong pH and temperature adaptability, good high-temperature resistance and the like, can react in a wide pH value (pH 2.0-6.0) and temperature range (30-70 DEG C), which is beneficial to improve the flexibility of a production process, especially can adapt to higher temperature for industrial production and can meet a high-temperature catalytic environment for a long time, is not easy to be inactivated in a high-temperature environment, can maintain more than 70% of enzyme activity at 50 DEG C for 1 h, can maintain more than 55% of enzyme activity at 55 DEG C for 1 h, and has a good market prospect and industrial application value in the fields of brewing, feed processing, leather softening and the like.
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Claims

1. An acidic protease mutant, characterized in that, The amino acid sequence is shown in SEQ ID NO.

2.

2. A nucleic acid molecule, characterized in that, Includes the gene sequence encoding the acidic protease mutant as described in claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that, The gene sequence of the acidic protease mutant is shown in SEQ ID NO.

3.

4. The nucleic acid molecule according to claim 2, characterized in that, The nucleic acid molecule also includes gene sequences encoding signal peptides, promoters, terminators, resistance markers, and / or replication initiation sites.

5. The nucleic acid molecule according to claim 4, characterized in that, The nucleotide sequences of the signal peptide gene, promoter gene, terminator gene, replication initiation site gene, and resistance marker gene are shown in SEQ ID NO.4-8, respectively.

6. An expression carrier, characterized in that, The expression vector comprises the nucleic acid molecule as described in any one of claims 3-5.

7. A recombinant bacterial strain, characterized in that, The recombinant strain comprises the expression vector as described in claim 6.

8. The recombinant strain according to claim 7, characterized in that, The recombinant strain originated from Trichoderma reesei QM 9414.

9. The method for preparing the acidic protease mutant according to claim 1, characterized in that, Includes the following steps: Cultivate the recombinant strain as described in any one of claims 7-8, centrifuge the fermentation culture broth, filter the supernatant, and collect the filtrate to obtain the crude enzyme solution containing the acidic protease mutant.

10. The use of the acidic protease mutant as described in claim 1 in brewing, feed processing, or leather softening.

Citation Information

Patent Citations

  • Acid protease mutant and application thereof

    CN119265166A

  • Acid protease mutant as well as coding gene and application thereof

    CN119654409A