CN-121991931-A - Cellulase mutant and application thereof

Abstract

The invention relates to the technical field of genetic engineering and protein transformation, in particular to a high specific activity cellulase mutant and application thereof. Compared with wild type cellulase, the specific activity of the mutants respectively containing P14A, P19T, S75A, S109N, H P, M123I, M123L, I130L, I Q, Y167E, Y167F, Y167W, A176S, A176F, D177S, L192I single mutation sites under neutral conditions is generally improved by 10.4% -76.1%. Wherein, the specific activity of the single-point mutant containing I130L is highest and reaches 183.64U/mg. The production cost of the cellulase mutant is obviously reduced, thereby being beneficial to promoting the wide application of the cellulase mutant.

Inventors

• Ma Juanzhen
• LIU ANBANG
• ZHANG QING
• WANG HUAMING

Assignees

• 青岛蔚蓝生物集团有限公司

Dates

Publication Date

20260508

Application Date

20241108

Claims (9)

1. 1. A cellulase mutant, characterized in that the mutant comprises an amino acid sequence having at least 95% identity to SEQ ID No. 1 and comprises a substitution of an amino acid at least one position selected from the group consisting of 14, 19, 75, 109, 118, 123, 130, 167, 176, 177, 192 compared to SEQ ID No. 1.
2. 2. The mutant of claim 1, which has an amino acid sequence at least 96%,97%,98%, or at least 99% identity to SEQ ID No. 1.
3. 3. The mutant of claim 2, wherein the amino acid sequence of the mutant has at least 99.1%,99.2%,99.3%,99.4%,99.5%,99.6%,99.7%,99.8%, or at least 99.9% identity to SEQ ID No. 1.
4. 4. The mutant of claim 1, wherein the mutant comprises a substitution of at least one amino acid of the group consisting of P14A, P19T, S75A, S109N, H P, M I/L, I L/Q, Y167E/F/W, A176S/F, D177S, L I.
5. 5. The mutant of claim 4, wherein the mutant comprises a substitution selected from the group consisting of P14A, P19T, S75A, S109N, H118P, M123I, M123L, I130Q, Y167E, Y167F, Y167W, A176S, A176F, D177S, L192I; P14A/P19T; P14A/M123I; P19T/I130L; P19T/Y167E; P19T/A176S; S75A/S109N; S75A/M123I; S75A/I130L; S75A/A176S; S75A/L192I; S109N/M123I; S109N/I130L; S109N/Y167W; S109N/A176S; S109N/L192I; M123I/I130L; M123I/Y167W; M123I/A176S; M123I/L192I; I130L/Y167W; I130L/A176S; I130L/L192I; Y167W/A176S; Y167W/L192I; Y167E/A176F; Y167F/L192I; D177S/L192I; A176S/L192I; P14A/M123I/A176F; P14A/M123L/A176S; P19T/I130L/L192I; S75A/I130L/A176F; S109N/I130L/D177S; M123I/A176S/L192I; Y167E/A176F/L192I; Y167W/A176S/D177S; S75A/I130Q/A176F; S109N/I130Q/D177S; P19T/I130L/Y167W/A176S; I130L/A176S/D177S/L192I; I130L/A176F/D177S/L192I; I130Q/A176S/D177S/L192I; I130Q/A176F/D177S/L192I; P14A/P19T/I130L/Y167E/A176F; P19T/S75A/M123I/A176S/L192I; P14A/S75A/M123I/A176F/L192I; S75A/I130Q/A176F/D177S/L192I。
6. 6. a DNA molecule encoding the cellulase mutant of claim 5.
7. 7. A recombinant expression plasmid comprising the DNA molecule of claim 6.
8. 8. A host cell comprising the recombinant expression plasmid of claim 7, which is a non-plant cell or an animal cell.
9. 9. The host cell of claim 8, wherein the host cell is trichoderma reesei (Trichoderma reesei).

Description

Cellulase mutant and application thereof Technical Field The invention relates to the technical field of genetic engineering and protein modification, in particular to a cellulase mutant and application thereof. Background Cellulose is macromolecular polysaccharide formed by connecting glucose through beta-1, 4 glycosidic bond, the chemical formula is (C 6H10O5)n, which is the main component of plant cell wall, is the polysaccharide with the widest distribution and the highest content in nature, in nature, the resource reserves of cellulose are very large, but only a very small part of natural cellulose is recycled due to the lack of low-cost and effective utilization technologies and means. The cellulase research provides an effective way for the resource utilization of cellulose. Cellulase is a complex enzyme system mainly comprising 3 components of beta-1, 4-endoglucanase, exoglucanase and beta-glucosidase. Wherein endoglucanases act randomly on amorphous regions within the cellulose polysaccharide chains to produce oligosaccharides of different lengths and new chain ends, exoglucanases act on these cellulose polysaccharide chain ends to produce glucose or cellobiose, and beta-glucosidase hydrolyzes cellobiose to produce glucose. The degradation of cellulose involves the synergistic effect of the 3 cellulases, and converts macromolecular cellulose in nature into biological products such as micromolecular sugar. Because the cellulase has the advantages of environmental protection, capability of playing a role together with other proteins, high treatment efficiency and the like, the cellulase has taken an important role in production and application. At present, cellulose or raw materials rich in cellulose are degraded or modified by using cellulase, and the method is widely applied to industries such as textile, papermaking, feed, food, energy and the like. However, as protein, cellulase has the problems of low stability, poor extreme temperature resistance and pH value resistance, high production cost and the like, and limits the application range of the cellulase. Disclosure of Invention The invention aims to provide a neutral cellulase mutant. According to the invention, through protein engineering modification of cellulase, mutant protein with remarkably improved specific activity is obtained, so that wide application of the mutant protein is facilitated. In order to achieve the above object, the present invention provides the following technical solutions: the present invention relates to a cellulase mutant comprising an amino acid sequence having at least 90% identity to SEQ ID NO. 1 and comprising an amino acid substitution at least one position selected from the group consisting of 14, 19, 75, 109, 118, 123, 130, 167, 176, 177, 192 compared to SEQ ID NO. 1. In some embodiments of the invention, the amino acid sequence of the mutant has at least 91%,92%,93%,94%,95%,96%,97%,98%, or at least 99% identity as compared to SEQ ID NO. 1. In some more specific embodiments, the amino acid sequence of the mutant has at least 99.1%,99.2%,99.3%,99.4%,99.5%,99.6%,99.7%,99.8%, or at least 99.9% identity compared to SEQ ID No. 1. In some embodiments of the invention, the mutant comprises a substitution of at least one amino acid of the group consisting of P14A, P19T, S75A, S109N, H P, M I/L, I L/Q, Y167E/F/W, A176S/F, D177S, L I. In some embodiments of the invention, the mutant comprises a substitution selected from the group consisting of P14A, P19T, S75A, S109N, H118P, M123I, M123L, I130Q, Y167E, Y167F, Y167W, A176S, A176F, D177S, L192I; P14A/P19T; P14A/M123I; P19T/I130L; P19T/Y167E; P19T/A176S; S75A/S109N; S75A/M123I; S75A/I130L; S75A/A176S; S75A/L192I; S109N/M123I; S109N/I130L; S109N/Y167W; S109N/A176S; S109N/L192I; M123I/I130L; M123I/Y167W; M123I/A176S; M123I/L192I; I130L/Y167W; I130L/A176S; I130L/L192I; Y167W/A176S; Y167W/L192I; Y167E/A176F; Y167F/L192I; D177S/L192I; A176S/L192I; P14A/M123I/A176F; P14A/M123L/A176S; P19T/I130L/L192I; S75A/I130L/A176F; S109N/I130L/D177S; M123I/A176S/L192I; Y167E/A176F/L192I; Y167W/A176S/D177S; S75A/I130Q/A176F; S109N/I130Q/D177S; P19T/I130L/Y167W/A176S; I130L/A176S/D177S/L192I; I130L/A176F/D177S/L192I; I130Q/A176S/D177S/L192I; I130Q/A176F/D177S/L192I; P14A/P19T/I130L/Y167E/A176F; P19T/S75A/M123I/A176S/L192I; P14A/S75A/M123I/A176F/L192I; S75A/I130Q/A176F/D177S/L192I。 The present invention relates to DNA molecules encoding the cellulase mutants described above. The present invention relates to recombinant expression vectors comprising the above-described DNA molecules. The present invention relates to a host cell comprising the recombinant expression vector described above. In an embodiment of the invention, the host cell is Trichoderma reesei (Trichoderma reesei). The recombinant expression vector constructed above is transferred into Trichoderma reesei host cells for recombinant expression, and the specific activity of the obtained cellulase mutant is obviously improved. Comp