Keratinase mutant as well as coding gene, expression vector and application thereof

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a keratinase mutant as well as a coding gene, an expression vector and application thereof. The amino acid sequence of the keratinase mutant is as shown in SEQ ID NO. 2. The keratinase mutant provided by the invention can effectively degrade a keratin substrate, has good catalytic degradation activity on keratin, and has greatly improved enzyme activity compared with wild type protein. Moreover, the keratinase mutant can basically decompose the whole feather within 24 hours by taking the keratin waste such as the feather as a direct substrate under the matching of a reducing agent DTT, so that the degradation efficiency of the keratin-containing waste such as the feather is remarkably improved, the production cost of the keratinase and the degradation cost of the keratin waste are remarkably reduced, and the production cost of the keratinase is greatly reduced. The method has a wide market prospect and industrial application value in the aspects of promoting degradation of feather wastes, conversion, recovery and utilization of keratin resources and the like.

Claims

1. A keratinase mutant, characterized in that: The amino acid sequence is shown in SEQ ID NO.

2.

2. A nucleic acid molecule, characterized in that It comprises a gene sequence encoding the keratinase mutant as claimed in claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that The gene sequence of the keratinase 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 a gene sequence encoding a signal peptide, a promoter and / or a terminator, the keratinase mutant is located downstream of the signal peptide, the promoter is located upstream of the signal peptide, and the terminator is located downstream of the keratinase mutant.

5. The nucleic acid molecule according to claim 4, characterized in that The signal peptide is a vsi signal peptide, and the gene sequence of the vsi signal peptide is shown in SEQ ID NO.

4. The promoter is a kasOp* promoter, and the gene sequence of the kasOp* promoter is shown in SEQ ID NO.

5. The terminator is a T1 terminator, and the gene sequence of the T1 terminator is shown in SEQ ID NO.

6.

6. An expression vector or a recombinant strain, characterized in that: The recombinant strain comprises the expression vector, and the expression vector comprises the nucleic acid molecule according to any one of claims 3 to 5.

7. The expression vector or recombinant strain according to claim 6, characterized in that: The expression vector is the Streptomyces integration vector pSET152, and the recombinant strain is Streptomyces lividans TK24.

8. Use of the keratinase mutant according to claim 1 in treating keratin-containing materials.

9. Use of the keratinase mutant according to claim 8 in treating keratin-containing substances, characterized in that: The keratin-containing material is selected from feathers, hair or wool.

10. Use of the keratinase mutant according to claim 9 in treating keratin-containing substances, characterized in that: The method for treating keratin-containing materials with the keratinase mutant comprises: The keratinase mutant or its crude enzyme solution is mixed with keratin-containing material, a reducing agent is added, and a catalytic reaction is carried out at 50-60° C. wherein the reducing agent is dithiothreitol.

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