Alkaline protease mutant as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of gene engineering and enzyme engineering, and discloses an alkaline protease mutant as well as a preparation method and application thereof. The alkaline protease mutant has alkaline protease activity, and compared with parent alkaline protease with an amino acid sequence as shown in SEQ ID NO: 1, the alkaline protease mutant comprises mutation Q200Y. Compared with parent alkaline protease, the alkaline protease mutant provided by the invention has the advantages that the enzyme activity, heat resistance and / or laundry detergent tolerance are obviously improved, and the requirements of the daily chemical washing industry can be better met.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and enzyme engineering technology, specifically relating to alkaline protease mutants, their preparation methods, and applications. Background Technology

[0002] Subtilisin (EC number 3.4.21.62), first obtained from Bacillus subtilis, belongs to the S8 peptidase family. It has a catalytic triplet in its amino acid sequence of Asp, His, and Ser. It is also known as a serine protease or alkaline protease.

[0003] Alkaline protease is currently the best-selling detergent additive on the market. The main organic components of stains (such as blood stains, milk stains, sweat stains, food residue, grass stains, and sebum) are proteins, greases, and starches. It can cleave the carboxyl ends of hydrophobic, aromatic, and some uncharged neutral amino acids. This broad substrate spectrum is highly advantageous for treating stains with complex compositions. It can hydrolyze large, water-insoluble proteins firmly attached to fibers (such as blood, egg, and milk stains) into smaller peptides or amino acids. Furthermore, many stains (such as grease and pigments) are "encapsulated" or "fixed" to the fabric by proteins. Protease disrupts these protein "skeletons," making other stains easier to remove by surfactants and mechanical force, thus significantly improving overall cleaning power. However, maintaining high enzyme concentration and stability, especially stability in detergents, remains a crucial technical challenge in the development of alkaline protease.

[0004] Therefore, modifying alkaline proteases using genetic engineering to develop alkaline proteases that can further improve enzyme activity, heat resistance, and / or detergent resistance is of great significance to the detergent industry. Summary of the Invention

[0005] This invention aims to solve at least one of the technical problems existing in the prior art. To this end, this invention proposes an alkaline protease mutant, its preparation method, and its applications. The alkaline protease provided by this invention exhibits high enzymatic activity, as well as excellent heat resistance and detergent resistance.

[0006] This invention provides an alkaline protease mutant.

[0007] Specifically, the alkaline protease mutant has alkaline protease activity and, compared with the parental alkaline protease with the amino acid sequence shown in SEQ ID NO:1, the alkaline protease mutant includes the mutant Q200Y.

[0008] SEQ ID NO:1: AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGISTHPDLNIRGGASFVPGEPSTQDGNGHGTHVAGTIAALNNSIGVLGVAPSAELYAVKVLGASGSGSSVSSIAQGLEWAGNNGMHVANLSLGSPSPSATLEQ AVNSATSRGVLVVAASGNSGAGSISYPARYANAMAVGATDQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGCAALVKQKNPSWSNVQIRNHLKNTATSLGSTDLYGSGLVNCEAATR.

[0009] In some embodiments of the invention, the alkaline protease mutant has improved properties, including increased heat resistance and / or increased detergent tolerance.

[0010] In some embodiments of the invention, the increased thermal stability includes increased stability at 50°C, and / or enhanced stability after heat storage at 37°C in detergent-containing conditions. More specifically, the alkaline protease mutant retains ≥50% of its enzyme activity after heat treatment at 50°C for 24 hours, and / or a commercial detergent containing the alkaline protease mutant retains ≥30% of its enzyme activity after heat storage at 37°C for 4 weeks.

[0011] In some embodiments of the present invention, the parental alkaline protease is derived from Bacillus tarda (Bacillus repens). Bacillus slow ).

[0012] In some embodiments of the invention, the mutant of the alkaline protease has at least 93% sequence identity with the polypeptide of SEQ ID NO:1, such as at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% but less than 100% sequence identity.

[0013] In some embodiments of the present invention, the alkaline protease variants include the following mutant combinations: Q200Y, V199I.

[0014] In some embodiments of the present invention, the alkaline protease variant further includes at least one of the following mutations: N179E, N198D, A209R, T218S, S9I, I41P, P51S, N74D, N74M, N74S, S76D, L80V, E87D, V102L, L109I, G116M, H118D, P127S, S128Y, T141W, R143K, L146M, T174V, A188P, A209L, L211M, Q230E, N232H, N237P, N242Q, S250P, S253D, L256E, S259A, N263D, C264A.

[0015] In some embodiments of the present invention, the alkaline protease mutant, compared with the parental alkaline protease with the amino acid sequence shown in SEQ ID NO:1, includes at least one of the mutations (1) to (208): (1) Q200Y, (2) S9I / Q200Y, (3) I41P / Q200Y, (4) N74S / Q200Y, (5) E87D / Q200Y, (6) G116M / Q200Y, (7) N179E / Q200Y, (8) V199I / Q200Y, (9) Q200Y / A209R, (10) Q200Y / A209L, (11) P51S / V199I / Q200Y, (12) L80V / V199I / Q200Y, ( 13) E87D / V199I / Q200Y, (14) V102L / V199I / Q200Y, (15) L109I / V199I / Q 200Y, (16) H118D / V199I / Q200Y, (17) N198D / V199I / Q200Y, (18) N179E / V199I / Q200Y, (19) V199I / Q200Y / A209R, (20) V199I / Q200Y / T218S, (21 ) V199I / Q200Y / N232H, (22) V199I / Q200Y / N242Q, (23) V199I / Q200Y / S2 53D, (24) V199I / Q200Y / L256E, (25) N74D / V199I / Q200Y / A209R, (26) N1 79E / V199I / Q200Y / A209R, (27) N198D / V199I / Q200Y / A209R, (28) V199I / Q200Y / A209R / T218S, (29) N74D / N179E / V199I / Q200Y / A209R, (30) N74 D / N198D / V199I / Q200Y / A209R, (31) N74D / V199I / Q200Y / A209R / T218S, (32) N74D / V199I / Q200Y / A209R / L256E, (33) N179E / N198D / V199I / Q200 Y / A209R, (34) N179E / V199I / Q200Y / A209R / T218S, (35) N179E / V199I / Q 200Y / A209R / L256E, (36) N198D / V199I / Q200Y / A209R / T218S, (37) N198 D / V199I / Q200Y / A209R / L256E, (38) V199I / Q200Y / A209R / T218S / L256E,(39) N74D / N179E / N198D / V199I / Q200Y / A209R, (40) N74D / N179E / V199I / Q200Y / A209R / T218S, (41) N74D / N179E / V199I / Q200Y / A209R / L256E, (42) N1 79E / N198D / V199I / Q200Y / A209R / T218S、(43)N179E / N198D / V199I / Q200Y / A209R / L256E、(44)N198D / V199I / Q200Y / A209R / T218S / L256E、(45)N74D / N 179E / N198D / V199I / Q200Y / A209R / T218S、(46) N74D / N179E / N198D / V199I / Q200Y / A209R / L256E、(47) N179E / N198D / V199I / Q200Y / A209R / T218S / L25 6E、(48)N74D / S76D / N179E / N198D / V199I / Q200Y / A209R / T218S、(49)N74D / E87D / N179E / N198D / V199I / Q200Y / A209R / T218S、(50)N74D / H118D / N179E / N198D / V199I / Q200Y / A209R / T218S、(51)N74D / R143K / N179E / N198D / V199 I / Q200Y / A209R / T218S、(52)N74D / T174V / N179E / N198D / V199I / Q200Y / A20 9R / T218S、(53)N74D / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E、(5 4)N74D / N179E / N198D / V199I / Q200Y / A209R / T218S / S250P、(55)N74D / N179 E / N198D / V199I / Q200Y / A209R / T218S / L256E、(56) N74D / N179E / N198D / V199I / Q200Y / A209R / T218S / S259A、(57) N74D / N179E / N198D / V199I / Q200Y / A2 09R / T218S / N263D、(58)N74D / N179E / N198D / V199I / Q200Y / A209R / T218S / C 264A、(59)N74D / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P、(60)N74D / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / L256E、(61)N74D / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S259A、(62)N74D / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / N263D、(63)N74D / N179E / N198D / V199I / Q200Y / A209R / T218S / S250P / S259A、(64)N74D / N179E / N198D / V199I / Q200Y / A209R / T218S / S250P / N263D、(65)N74D / N179E / N198D / V199I / Q200Y / A209R / T218S / S250P / C264A、(66)N74D / N179E / N198D / V199I / Q200Y / A209R / T218S / S259A / N263D、(67)N74D / N179E / N198D / V199I / Q200Y / A209R / T218S / S259A / C264A、(68)N74D / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(69)N74D / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / N263D、(70)N74D / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / C264A、(71)N74D / N179E / N198D / V199I / Q200Y / A209R / T218S / S250P / S259A / N263D、(72)N74D / N179E / N198D / V199I / Q200Y / A209R / T218S / S250P / S259A / C264A、(73)N74M / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(74)N74D / P127S / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(75)N74D / S128Y / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(76)N74D / T141W / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(77)N74D / L146M / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(78)N74D / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(79)N74D / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(80)N74D / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A / N263D、(81)N74D / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A / C264A、(82)N74M / P127S / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(83)N74M / S128Y / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(84)N74M / T141W / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(85)N74M / L146M / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(86)N74M / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(87)N74M / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(88)N74D / P127S / T141W / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(89)N74D / P127S / L146M / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(90)N74D / P127S / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(91)N74D / P127S / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(92)N74D / P127S / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(93)N74D / S128Y / T141W / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(94)N74D / S128Y / L146M / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(95)N74D / S128Y / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(96)N74D / S128Y / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(97)N74D / S128Y / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(98)N74D / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(99)N74D / T141W / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(100)N74D / T141W / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(101)N74D / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(102)N74D / L146M / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(103)N74D / L146M / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(104)N74D / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(105)N74D / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(106)N74D / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(107)N74M / P127S / T141W / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(108)N74M / P127S / L146M / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(109)N74M / P127S / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(110)N74M / P127S / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(111)N74M / P127S / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(112)N74M / S128Y / T141W / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(113)N74M / S128Y / L146M / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(114)N74M / S128Y / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(115)N74M / S128Y / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(116)N74M / S128Y / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(117)N74M / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(118)N74M / T141W / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(119)N74M / T141W / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(120)N74M / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(121)N74M / L146M / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(122)N74M / L146M / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(123)N74M / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(124)N74M / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(125)N74M / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(126)N74D / P127S / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(127)N74D / P127S / T141W / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(128)N74D / P127S / T141W / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(129)N74D / P127S / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(130)N74D / P127S / L146M / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(131)N74D / P127S / L146M / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(132)N74D / P127S / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(133)N74D / P127S / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(134)N74D / P127S / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(135)N74D / S128Y / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(136)N74D / S128Y / T141W / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(137)N74D / S128Y / T141W / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(138)N74D / S128Y / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(139)N74D / S128Y / L146M / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(140)N74D / S128Y / L146M / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(141)N74D / S128Y / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(142)N74D / S128Y / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(143)N74D / S128Y / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(144)N74D / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(145)N74D / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(146)N74D / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(147)N74D / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(148)N74D / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(149)N74M / P127S / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(150)N74M / P127S / T141W / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(151)N74M / P127S / T141W / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(152)N74M / P127S / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(153)N74M / P127S / L146M / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(154)N74M / P127S / L146M / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(155)N74M / P127S / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(156)N74M / P127S / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(157)N74M / P127S / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(158)N74M / S128Y / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(159)N74M / S128Y / T141W / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(160)N74M / S128Y / T141W / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(161)N74M / S128Y / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(162)N74M / S128Y / L146M / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(163)N74M / S128Y / L146M / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(164)N74M / S128Y / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(165)N74M / S128Y / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(166)N74M / S128Y / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(167)N74M / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(168)N74M / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(169)N74M / T141W / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(170)N74M / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(171)N74M / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(172)N74M / L146M / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(173)N74M / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(174)N74D / P127S / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(175)N74D / P127S / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(176)N74D / P127S / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(177)N74D / P127S / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(178)N74D / P127S / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(179)N74D / S128Y / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(180)N74D / S128Y / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(181)N74D / S128Y / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(182)N74D / S128Y / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(183)N74D / S128Y / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(184)N74D / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(185)N74D / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(186)N74M / P127S / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(187)N74M / P127S / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(188)N74M / P127S / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(189)N74M / P127S / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(190)N74M / P127S / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(191)N74M / S128Y / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(192)N74M / S128Y / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(193)N74M / S128Y / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(194)N74M / S128Y / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(195)N74M / S128Y / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(196)N74M / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(197)N74M / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(198)N74D / P127S / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(199)N74D / P127S / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(200)N74D / S128Y / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(201)N74D / S128Y / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(202)N74M / P127S / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(203)N74M / P127S / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(204)N74M / S128Y / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(205)N74M / S128Y / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(206)N74D / P127S / S128Y / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(207)N74D / P127S / T141W / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(208) N74D / P127S / S128Y / T141W / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A. ,

[0016] The present invention also provides a nucleic acid molecule.

[0017] Specifically, a nucleic acid molecule that encodes the aforementioned alkaline protease mutant.

[0018] The present invention also provides a recombinant expression vector.

[0019] Specifically, a recombinant expression vector contains the aforementioned nucleic acid molecules.

[0020] In some embodiments of the present invention, the vector of the recombinant expression vector is a plasmid; preferably, the plasmid includes the pBE-S plasmid.

[0021] The present invention also provides a recombinant bacterium.

[0022] Specifically, a recombinant bacterium contains the aforementioned nucleic acid molecules or recombinant expression vectors.

[0023] In some embodiments of the present invention, the recombinant bacteria are selected from Escherichia coli cells or Bacillus cells.

[0024] The present invention also provides an enzyme-containing composition.

[0025] Specifically, an enzyme-containing composition comprising the above-mentioned alkaline protease mutant and containing one or more additional enzymes; said additional enzymes include aminopeptidase, α-amylase, β-amylase, isoamylase, carboxylase, carboxypeptidase, catalase, cellulase, chitinase, carrageenanase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, α-galactosidase, β-galactosidase, glucosylamylase, α-glucosidase, β-glucosidase, haloperoxidase, invertase, laccase, mannanase, α-galactosidase, β-galactosidase, glucosylamylase, α-glucosidase, β-glucosidase, haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase, pectinase, peptidylglutaminase, peroxidase, glucosylamylase, polyphenol oxidase, cellulase, proteolytic enzyme, ribonuclease, transglutaminase, or xylase.

[0026] The present invention also provides applications of the above-mentioned alkaline protease mutant.

[0027] Specifically, the application of the aforementioned alkaline protease mutant in detergents. These detergents include, but are not limited to, laundry liquids, such as those effective at removing protein stains.

[0028] The present invention also provides a detergent comprising the above-mentioned alkaline protease mutant.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows: Compared with the parental alkaline protease, the alkaline protease mutants provided by this invention have significantly improved enzyme activity, heat resistance and / or detergent tolerance. Most alkaline protease mutants also have excellent heat resistance and detergent tolerance, and have better cleaning effect on protein-stained cloths, thus better meeting the needs of the daily chemical washing industry. Detailed Implementation

[0030] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.

[0031] Unless otherwise specified, the biological materials, reagents, or devices used in the following examples are all available from conventional commercial sources or can be obtained by existing known methods. Molecular biology experimental methods not specifically described in the following examples were performed according to the specific methods listed in J. Sambrook's *Molecular Cloning: A Laboratory Manual* (3rd Edition), or according to the kit and product instructions.

[0032] definition Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings: The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that, in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0033] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0034] The terms “combinations thereof,” “any combination thereof,” and “any combination thereof” as used in this application include all suitable combinations of any two or more of the listed items.

[0035] In this application, terms such as "preferred," "better," "more suitable," and "ideal" are merely used to describe implementation methods or embodiments that achieve better results, and should be understood not to limit the scope of protection of this application.

[0036] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0037] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0038] A first aspect of the present invention is to provide an alkaline protease variant, relative to the parental alkaline protease sequence shown in SEQ ID NO.1, the alkaline protease variant comprising the mutation Q200Y.

[0039] Alkaline protease: The term "alkaline protease" refers to an enzyme that, under alkaline conditions, can cleave peptide bonds within a protein molecule, breaking it down into smaller polypeptides and amino acids. Alkaline protease activity can be determined using methods known in the art (e.g., the Folin method in GB / T 23527-2009, "Protein Preparations").

[0040] In this invention, "parental alkaline protease," "parental protease," and "parental polypeptide" refer to an alkaline protease whose amino acid sequence is shown in SEQ ID NO.1. This parental polypeptide is mutated to produce the alkaline protease mutant of this invention.

[0041] SEQ ID NO:1: AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGISTHPDLNIRGGASFVPGEPSTQDGNGHGTHVAGTIAALNNSIGVLGVAPSAELYAVKVLGASGSGSSVSSIAQGLEWAGNNGMHVANLSLGSPSPSATLE QAVNSATSRGVLVVAASGNSGAGSISYPARYANAMAVGATDQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGCAALVKQKNPSWSNVQIRNHLKNTATSLGSTDLYGSGLVNCEAATR In this invention, "alkaline protease mutant," "alkaline protease variant," "protease mutant," and "protease variant" refer to a protease that differs from the parental protease due to at least one amino acid mutation. Optionally, the protease variant has at least one amino acid mutation compared to the parental alkaline protease; for example, it has 1 to 17 amino acid mutations (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17) compared to the parental protease.

[0042] Amino acid modifications include substitution, insertion, and deletion. Substitution means replacing an amino acid occupying a position with a different amino acid; deletion means removing an amino acid occupying a position; insertion means adding an amino acid adjacent to or immediately following an amino acid occupying a position.

[0043] The protease variant sequence of the present invention has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and at least 99.9% sequence similarity to the parent protease sequence, optionally at least about 93%, and further optionally at least about 94%, 95%, 96%, 97%, 98%, and 99%. The protease variant of the present invention has alkaline protease activity.

[0044] In this context, for the purposes of this application, homology between two amino acid sequences is described by the parameter “identity,” and the degree of identity between the two amino acid sequences is determined using the Needleman-Wunsch algorithm as described above. In addition to amino acid alignment, the results from the program also calculate the “percentage similarity” between the two sequences. Regarding this application, sequence similarity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, *J. Mol. Biol.*, 48: 443-453), as implemented in the Needle program of the EMBOSS software package (“EMBOSS: The European Molecular Biology Open Software Suite”, Rice et al., 2000, *Trends Genet.*, 16: 276-277), preferably version 6.1.0 or later. The parameters used are: 10 for open vacancy penalty, 0.5 for extended vacancy penalty, and an EBLOSUM62 (EMBOSS version of BLOSUM62) replacement matrix. The Needle output marked "Longest Consistency" (obtained using the -nobrief option) is used as the consistency percentage and calculated as follows: (Equivalent residues × 100) / (Alignment length - total number of vacancies in alignment).

[0045] This comparison, used to determine the percentage of consistency, is performed using the entire length of the sequence in this application.

[0046] According to the present invention, "improved property" refers to a property associated with the alkaline protease variant described herein, which is improved compared to the parental alkaline protease. For the purposes of the invention, in some embodiments, the improved property is increased enzyme activity. In some embodiments of the invention, at pH 10.5, the relative fermentation activity of the alkaline protease variant is greater than 1.0 compared to the fermentation activity of the parental alkaline protease.

[0047] In some embodiments of the present invention, to meet the requirements of the daily chemical detergent industry, the improved characteristic is increased heat resistance. In some embodiments of the present invention, compared with the parental alkaline protease, the alkaline protease mutant of the present invention exhibits a higher enzyme activity retention rate after heat treatment at 50°C for 24 hours.

[0048] In some embodiments of the present invention, the improved property is increased detergent tolerance. In some embodiments of the present invention, after being stored in a constant temperature oven at 37°C for 4 weeks, the alkaline protease mutant of the present invention exhibits a higher enzyme activity retention rate compared to the parental alkaline protease.

[0049] In some embodiments of the invention, the improved properties include increased enzyme activity and / or increased heat resistance and / or increased detergent tolerance. It is understood that, compared to the parental alkaline protease, the alkaline protease mutant of the present invention has at least one advantage in terms of increased enzyme activity, heat resistance, or detergent tolerance.

[0050] A second aspect of the present invention is to provide an alkaline protease mutant comprising the mutant Q200Y relative to the parent protease SEQ ID NO:1, and the mutant having at least 93%, such as at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% but less than 100% sequence identity with the polypeptide of SEQ ID NO:1, and the variant of the present application having increased enzyme activity and / or increased heat resistance and / or increased detergent tolerance.

[0051] In some embodiments of the present invention, the alkaline protease variants include the following mutant combinations: Q200Y, V199I.

[0052] In some embodiments of the present invention, the alkaline protease variant further includes at least one of the following mutations: N179E, N198D, A209R, T218S, S9I, I41P, P51S, N74D, N74M, N74S, S76D, L80V, E87D, V102L, L109I, G116M, H118D, P127S, S128Y, T141W, R143K, L146M, T174V, A188P, A209L, L211M, Q230E, N232H, N237P, N242Q, S250P, S253D, L256E, S259A, N263D, C264A.

[0053] In some embodiments of the invention, the alkaline protease variant has any one of the groups of mutants described in Table 1.

[0054] Table 1

[0055] Variations of the invention may further include one or more additional modifications. These amino acid changes may be minor in nature, i.e., conserved amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions typically of 1 to 30 amino acids; small or carboxyl-terminal extensions, such as N-terminal methionine residues; small linker peptides of up to 20 to 25 residues; or small extensions that facilitate purification by altering net charge or another function, such as poly-histidine tracts, antigenic epitopes, or binding domains.

[0056] Examples of conserved substitutions are listed in the following group: basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions that do not typically alter specific reactivity are known in the art. Common substitutions are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0057] A second aspect of this application provides a nucleic acid that encodes the alkaline protease variant.

[0058] A third aspect of this application provides a carrier comprising the aforementioned nucleic acid.

[0059] The terms “vector,” “expression vector,” and “recombinant expression vector” refer to linear or circular DNA molecules that contain polynucleotides encoding variants and are operatively linked to a control sequence that provides the means for their expression.

[0060] The recombinant expression vectors involved in this application comprise nucleotides encoding variants of this application, a promoter, and transcription and translation termination signals. Various nucleotides and control sequences can be linked together to produce a recombinant expression vector, which may contain one or more suitable restriction sites to allow the insertion or substitution of polynucleotides encoding variants at such sites. Alternatively, polynucleotides can be expressed by inserting the polynucleotide or a nucleic acid construct containing the polynucleotide into a suitable expression vector. In creating the expression vector, the coding sequence is located within the vector such that the coding sequence is operatively linked with suitable control sequences for expression.

[0061] Recombinant expression vectors can be any vector (e.g., plasmids or viruses) that can readily undergo recombinant DNA processes and enable the expression of polynucleotides. The choice of vector typically depends on its compatibility with the host cell into which it will be introduced. Vectors can be linear plasmids or closed circular plasmids.

[0062] The vector can be a self-replicating vector (i.e., a vector existing as an extrachromosomal entity) whose replication is independent of chromosome replication, such as a plasmid, extrachromosomal element, small chromosome, or artificial chromosome. The vector may contain any means to ensure self-replication. Alternatively, the vector may be such that, upon introduction into a host cell, it integrates into the genome and replicates along with the chromosome (in which the vector is already integrated). Furthermore, a single vector or plasmid, or two or more vectors or plasmids, may be used, collectively containing a transposon or the entire DNA to be introduced into the host cell genome. Vectors contemplated for use with the methods of this application include integrating and non-integrating vectors.

[0063] In some embodiments, the vector contains one or more optional markers that allow for easy selection of cells for transformation, transfection, transduction, etc. Optional markers are genes whose products provide resistance to biocides or viruses, heavy metal resistance, prototrophic and auxotrophic traits, etc. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Optional markers for filamentous fungal host cells include, but are not limited to, amdS (acetamipase), argB (ornithine carbamoyltransferase), bar (phosphinicotinic acid acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), PYRG (guanosine-5′-phosphate decarboxylase), sC (adenosyl sulfate transferase), and their equivalents. Preferred genes for Aspergillus cells are the amdS and pyrG genes from *Aspergillus nidus* or *Aspergillus oryzae*, and the bar gene from *Streptomyces* hygroscopic fungi.

[0064] In some embodiments, the vector includes one or more elements that allow the vector to be integrated into the host cell genome or to replicate autonomously within the cell independently of the genome.

[0065] Regarding integration into the host cell genome, the vector may rely on a polynucleotide sequence encoding a variant or any other element of the vector (for integration into the genome via homologous or non-homologous recombination). Alternatively, the vector may contain additional polynucleotides to guide integration into the host cell genome at one or more precise locations on one or more chromosomes via homologous recombination. To increase the likelihood of integration at precise locations, the integrative element should contain sufficient nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, that have a high sequence identity with the corresponding target sequence to enhance the likelihood of homologous recombination. The integrative element can be any sequence homologous to the target sequence in the host cell genome. Furthermore, the integrative element can be a non-coding or coding polynucleotide. On the other hand, the vector can integrate into the host cell genome via non-homologous recombination.

[0066] The present application allows for the insertion of one or more copies of the nucleic acid into a host cell to increase the yield of variants, including the use of multiple genes encoding variant alkaline proteases in a vector, multiple vectors transformed into cells, or multiple integrations of a vector into the genome. An increase in the number of polynucleotide copies can be obtained by integrating at least one additional copy of the sequence into a host cell or by including an amplifiable optional marker gene with a polynucleotide; wherein, in the presence of a suitable selector, cells containing an amplified copy of the optional marker gene and thus an additional copy of the polynucleotide can be selected.

[0067] The process for connecting the elements described above to construct the recombinant expression vector of this application is well known to those skilled in the art.

[0068] Regarding expression in yeast, one example utilizes the Saccharomyces cerevisiae INSCV1 strain (Thermo Fisher Scientific, catalog number V8251-20) and the pYES2 / CT vector (Thermo Fisher Scientific, catalog number V8251-20). Both are commercially available and are discussed in the examples below.

[0069] A fourth aspect of this application provides a cell that expresses the alkaline protease variant, or includes the nucleic acid, or includes the vector.

[0070] The term “expression” includes any step involved in the production of the variant alkaline protease described herein, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0071] The terms "cell" and "host cell" refer to any cell type that is readily transformable, transfected, transduced, etc., using nucleic acid constructs or expression vectors containing the polynucleotides of this application and allowing enzyme expression. The term "host cell" encompasses any progeny of the parent cell; the progeny is not equivalent to the parent cell due to mutations that occur during replication.

[0072] As those skilled in the art will understand, there are a wide variety of host organisms available for the recombinant production of the alkaline protease variants of this application, including but not limited to bacterial and fungal cells (including yeast).

[0073] The host cell of this application contains a polynucleotide encoding a variant alkaline protease of this application, the polynucleotide being operatively linked to one or more control sequences that direct the production of the variant. A construct or vector containing the polynucleotide is introduced into the host cell such that the construct or vector remains a chromosomal integrase or a self-replicating extrachromosomal vector as previously described. The choice of host cell will depend largely on the gene encoding the variant and the host production organism's ability to produce high titers of expressed and / or secreted proteins. In some embodiments, the host cell exhibits transient expression of the variant alkaline protease. In some embodiments, the host cell is a stably transfected host or a host cell that stably (i.e., permanently) expresses the variant alkaline protease. In some embodiments, the host cell is a production host cell. The host cell is run to transform and / or transfect an expression vector containing the coding region of the variant alkaline protease of this application, as is known in the art.

[0074] The host cell can be any cell used for the recombinant production of variants, such as prokaryotic or eukaryotic cells. Such host cells include, but are not limited to, bacterial, fungal, and yeast cells. The host cell can also be a eukaryotic cell, such as a mammalian, insect, plant, or fungal cell.

[0075] The host cell may be a fungal cell. In some embodiments, the fungal host cell may be a yeast cell. As used in this application, "yeast" includes Ascospore-producing yeasts (Endosporales), Basidiospore-producing yeasts, and yeasts belonging to the Deuteromycetes (Blastomycetes). The yeast host cell may be a cell of the genera *Candida*, *Hansenula*, *Kluyveromyces*, *Pichia*, *Saccharomyces*, *Saccharomyces*, or *Yersinia*, such as *Kluyveromyces lactis*, *Karlsbergia*, *Saccharomyces cerevisiae*, *Saccharomyces cerevisiae*, *Saccharomyces sacchariformis*, *Douglas yeast*, *Kluyveromyces cerevisiae*, *Norbae*, *Ovoyces*, or *Yersinia lipolytica* cells.

[0076] Generally, variants can be prepared by constructing genes encoding protein sequences using well-known techniques, including site-directed mutagenesis of parental genes and synthetic gene construction. As is known in the art, alkaline proteases are typically manufactured using a signal sequence and a progenitor sequence, the signal sequence directing the secretion of the protein from the cell, and the progenitor sequence keeping the protein inactive until removal. As is known in the art, secretory proteases (such as the variant alkaline protease of this application) also contain a signal sequence (also referred to as a "pre" sequence), in addition to the progenitor sequence removed during or after secretion. As those skilled in the art will understand, the variant protease of this application can be manufactured using an endogenous signal sequence and a progenitor sequence or an exogenous signal sequence and a progenitor sequence.

[0077] A fifth aspect of this application provides a method for constructing the cell described above, the method comprising the step of introducing the nucleic acid or the vector into the cell to be modified.

[0078] This application also relates to nucleic acid constructs comprising a polynucleotide encoding a variant of this application, the polynucleotide being operatively linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

[0079] The control sequence may include a promoter or a polynucleotide that is recognized by the host cell for expression of that polynucleotide. The promoter contains a transcriptional control sequence that mediates variant expression. The promoter may be any polynucleotide that indicates transcriptional activity in the host cell, including mutant, truncated, and hybrid promoters; and may be derived from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to that of the host cell.

[0080] Examples of suitable promoters for directing the transcription of the nucleic acid constructs of this application in filamentous fungal host cells are promoters derived from Aspergillus genes (including Aspergillus nidus, Aspergillus niger, and Aspergillus oryzae) as known in the art, and from Rhizobium genes (such as Rhizopus oryzae), Mucor genes (including Trichoderma reesei), and Fusarium genes (including Fusarium venetum). Yeast control sequences (including promoters) are also well known in Saccharomyces cerevisiae.

[0081] Suitable promoter sequences (and other control sequences) for these genera include promoters of amylases (especially α-amylase), glucosylamylases, proteases, phosphatases, endoglucanases, cellulases, etc., as are known in the art. Furthermore, for codon optimization, it is desirable to use promoters (and other control sequences) homologous to the host producing strain that are operatively linked to nucleic acids encoding variant alkaline proteases.

[0082] Control sequences can also be transcription terminators, which are recognized by the host cell to terminate transcription. The terminator sequence is operationally linked to the 3' end of the polynucleotide encoding the variant. Any terminator that functions in the host cell can be used.

[0083] In some embodiments, the terminator (and other control sequences, such as promoters) of the filamentous fungal host cell acquires genes for autogenic Aspergillus nidulans anthraquinone synthase, Aspergillus niger glucosidase, Aspergillus niger α-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium trypsin-like protease.

[0084] In some embodiments, the terminator of the yeast host cell is derived from the genes of Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase.

[0085] Control sequences can also be mRNA stabilization regions downstream of the promoter and upstream of the gene coding sequence, which increase gene expression.

[0086] The control sequence can also be a leader sequence (the untranslated region of mRNA), which is important for translation in the host cell. The leader sequence is operationally linked to the 5' end of a polynucleotide encoding the variant. Any leader sequence that functions in the host cell can be used.

[0087] In some embodiments, the leader sequence of the filamentous fungal host cell is obtained from the genes of Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

[0088] In some embodiments, a suitable leader sequence from a yeast host cell is obtained from the genes of *Saccharomyces cerevisiae* enolase (ENO-1), *Saccharomyces cerevisiae* 3-phosphoglycerate kinase, *Saccharomyces cerevisiae* α-factor, and *Saccharomyces cerevisiae* alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0089] The control sequence can also be a polyadenylation sequence, a sequence operatively linked to the 3′ end of the variant coding sequence; and during transcription, this sequence is recognized by the host cell as a signal to add polyadenylation residues to the transcribed mRNA. Any polyadenylation sequence that functions in the host cell can be used.

[0090] In some embodiments, the polyadenylated sequences of filamentous fungal host cells yield the genes for autogenic Aspergillus nidulans anthraquinone synthase, Aspergillus niger glucosidase, Aspergillus niger α-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium trypsin-like protease.

[0091] The control sequence can also be a signal peptide coding region, which encodes a signal peptide linked to the N-terminus of the variant and directs the expression of the variant alkaline protease into the cellular secretory pathway. In many cases, the signal sequence is an endogenous G1P signaling sequence.

[0092] The effective signal peptide coding sequences for filamentous fungal host cells are obtained from the genes of neutral amylase, glucosylamylase, TAKA amylase, cellulase, endoglucanase V, lipase, and aspartic protease of Rhizopus oryzae.

[0093] The useful signal peptides of yeast host cells are obtained from the genes of Saccharomyces cerevisiae α-factor and Saccharomyces cerevisiae invertase.

[0094] When both the signal peptide and the propeptide sequence are present, the propeptide sequence is located near the N-terminus of the variant, and the signal peptide sequence is located near the N-terminus of the propeptide sequence.

[0095] It is also desirable to add regulatory sequences that regulate the expression of variants relative to host cell growth. Examples of regulatory systems are those that cause gene expression to turn on or off in response to chemical or physical stimuli, including the presence of regulatory compounds. Regulatory systems in prokaryotes include the lac, tac, and trp operon systems. In yeast, the ADH2 or GAL1 system can be used. In filamentous fungi, Gpd (glyceraldehyde-3-phosphate dehydrogenase) from Ascomycetes can be used, such as the Aspergillus, Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA α-amylase promoter, and Aspergillus oryzae glucoamylase promoter. Other examples of regulatory sequences are those that allow gene amplification. In eukaryotic systems, these regulatory sequences include dihydrofolate reductase genes amplified in the presence of methotrexate, and metallothionein genes amplified with heavy metals. In these cases, the polynucleotide encoding the variant will be operatively linked to the regulatory sequence.

[0096] A sixth aspect of this application provides a method for producing the alkaline protease variant, the method comprising the steps of culturing the cells and isolating the alkaline protease variant from the resulting culture.

[0097] This application relates to a method for producing a variant alkaline protease, the method comprising: (a) culturing the host cell of the application under conditions suitable for the expression of the variant alkaline protease polypeptide; and (b) optionally recovering the variant alkaline protease polypeptide.

[0098] Using methods known in the art, host cells are cultured in a nutrient medium suitable for producing the variant alkaline protease peptide. For example, cells can be cultured using shake-flask culture or via small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid-state fermentation) in a laboratory or industrial fermenter, performed in a suitable medium and under conditions that allow for variant expression and / or isolation. Culture is carried out using a process known in the art in a suitable nutrient medium containing carbon and nitrogen sources, as well as inorganic salts. The suitable medium is available from a commercial supplier or can be prepared according to a published composition (e.g., the U.S. Type Culture Collection catalogue). If the variant alkaline protease peptide is secreted into the nutrient medium, it can be recovered directly from that medium. If the variant is not secreted, it can be recovered from cell lysates.

[0099] Variant alkaline protease peptides can be detected using variant-specific methods known in the art. These methods include, but are not limited to, the use of specific antibodies, enzyme product formation, or enzyme substrate disappearance. For example, enzyme assays as outlined herein can be used to determine the activity of variant alkaline protease peptides.

[0100] Variant alkaline protease peptides can be recovered using methods known in the art. For example, variant alkaline protease peptides can be recovered from nutrient media through conventional processes, including but not limited to collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.

[0101] Variants can be purified by a variety of processes known in the art, including but not limited to chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic chromatography, chromatographic focusing and size exclusion chromatography), electrophoresis (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction to obtain substantially pure variants.

[0102] Alternatively, the variant was not recycled, but the host cell of this application expressing the variant was used as the source of the variant.

[0103] A seventh aspect of this application provides the use of the alkaline proteasome in the preparation of enzyme-containing products.

[0104] The definition of enzyme-containing products is described below.

[0105] An eighth aspect of this application provides an enzyme-containing product, the enzyme-containing product comprising the alkaline protease variant described above.

[0106] The enzyme-containing product may be a composition comprising a variant alkaline protease of the present application. In some embodiments, the composition includes a carrier and / or excipients. In some embodiments, the composition is enriched with such a variant alkaline protease of the present application. The term "enrichment" indicates that the alkaline protease activity of the composition has been increased, for example, having an enrichment factor of at least 1. In some embodiments, the composition is formulated to provide desired properties of a detergent additive, such as high detergency against stains, high detergency against different ionic strengths (e.g., both soft and hard water), tolerance to oxidants and chelating agents in the formulated detergent, low color, low odor, and long shelf life at room temperature.

[0107] In some embodiments, the composition comprises a variant alkaline protease of the present application as the main enzyme component, such as a single-component composition.

[0108] In some embodiments, depending on the end use, the composition may comprise one or more additional enzymes, including but not limited to aminopeptidase, α-amylase, β-amylase, isoamylase, carboxylase, carboxypeptidase, catalase, cellulase, chitinase, carrageenanase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, α-galactosidase, β-galactosidase, glucosidase, α-glucosidase, β-glucosidase, haloperoxidase, invertase, laccase, mannanase, α-galactosidase, β-galactosidase, glucosidase, α-glucosidase, β-glucosidase, haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase, pectinase, peptidylglutaminase, peroxidase, glucosylamylase, polyphenol oxidase, cellulase, proteolytic enzyme, ribonuclease, transglutaminase, and / or xylase.

[0109] In some embodiments, the composition comprises amylase and a variant alkaline protease according to the present application. In some embodiments, the composition comprises lipase and a variant alkaline protease according to the present application. In another embodiment, the composition comprises amylase, lipase, and a variant alkaline protease according to the present application.

[0110] In some embodiments, the composition comprises a variant alkaline protease according to the present application, and further comprises acidic, neutral, and / or alkaline proteases. In another embodiment, the composition comprises a variant alkaline protease according to the present application and one or more enzymes, including amylase, protease, peptidase, lipase, cellulose, etc.

[0111] In one aspect of this application, the enzyme-containing product is an enzyme preparation, specifically a formulation of a variant alkaline protease. As those skilled in the art will understand, the formulation of the variant alkaline protease in this application depends on its end use and associated conditions. Suitable formulations of the variant alkaline protease in this application include liquid formulations, dried formulations (including spray-dried formulations), powder formulations, granule formulations, microparticle formulations, and pellet formulations. The variant alkaline protease can be stabilized according to methods known in the art.

[0112] In some embodiments, the composition of this application (i.e., the polypeptide composition) may be in any form suitable for use, such as, for example, crude fermentation broth (with or without cell removal) as an enzyme source, cell lysate (with or without cell debris), semi-purified or purified enzyme composition, or host cell.

[0113] In some embodiments, the composition may be a dry powder or granules, non-dust-removing granules, a liquid, a stabilized liquid, or a stabilized protective enzyme. For example, a liquid enzyme composition may be stabilized according to an established process by adding a stabilizer (such as sugar, sugar alcohol, or other polyols, and / or lactic acid or other organic acids).

[0114] In some embodiments, the amount of composition and other conditions of use of the composition may be determined based on methods known in the art.

[0115] In another aspect of the embodiments of this application, the enzyme-containing product is a detergent composition, such as a detergent composition comprising a protease variant of the present application. In one embodiment, the composition is a liquid or powder laundry detergent suitable for washing, for example, at high temperatures and / or high pH, ​​such as at 50°C or above and / or at pH 10 or above. The detergent may also be formulated as a unit-dose detergent, and / or formulated as a concentrated detergent optionally having minimal or no water. The detergent may also be a dishwashing detergent, which is preferably phosphate-free.

[0116] In some embodiments, the variants according to this application have improved stability in detergents compared to the parent enzyme or compared to the protease of SEQ ID NO. 1, wherein detergent stability is measured in the examples.

[0117] In addition to enzymes, detergent compositions may also contain additional components. The selection of additional components is within the skill of a person skilled in the art; and includes conventional ingredients, thereby including exemplary non-limiting components as explained below. Regarding fabric care, the selection of components may include considerations of the following: the type of fabric to be cleaned, the type and / or extent of soiling, the temperature at which cleaning is performed, and the formulation of the detergent product. As a person skilled in the art will understand, components may include additional functionalities.

[0118] In one embodiment, according to a variant of this application, it is combined with one or more enzymes, such as at least two enzymes, more preferably at least three, four, or five enzymes. Preferably, the enzymes have different substrate specificities, such as proteolytic activity, starch-degrading activity, lipolytic activity, hemicellulose-degrading activity, or pectin-degrading activity. Additional enzymes, such as carbohydrate-active enzymes, are similar to carbohydrate enzymes, pectinases, mannanases, amylases, cellulases, arabinases, galactanases, xylanases, proteases, lipases, keratinases, oxidases such as laccases, and / or peroxidases.

[0119] Generally speaking, the properties of one or more selected enzymes should be compatible with the selected detergent (i.e., optimal pH, compatibility with other enzymes and non-enzyme components, etc.), and one or more enzymes should be present in an effective amount.

[0120] A ninth aspect of this application provides a method for using an alkaline protease variant, the method comprising contacting the alkaline protease variant or the enzyme-containing product with a substrate-containing target to induce an enzyme-catalyzed reaction.

[0121] (A) Industrial applications of variant alkaline proteases The variant alkaline protease of this application has important properties that allow for a variety of industrial applications, such as leather processing, food processing, pharmaceutical use, chemical industry, and detergent industry.

[0122] 1. Detergent industry Compared to the parent alkaline protease, variant alkaline proteases offer numerous benefits for use in the detergent industry, including increased activity, thermal activity, thermal stability, pH stability, superior stain removal performance, high performance under varying ionic strengths (e.g., both soft and hard water), tolerance to oxidants and chelating agents in formulated detergents, and longer shelf life at room temperature.

[0123] In some embodiments, the variant alkaline protease of this application is formulated and added to a detergent, or may be formulated as a component of a detergent. In some embodiments, the detergent is a laundry detergent. In some embodiments, the detergent is a dishwasher detergent. In some embodiments, the detergent is a contact lens detergent.

[0124] In one embodiment, an alkaline protease is added to the detergent (as known in the art) such that the detergent is formed using the alkaline protease therein. In other embodiments, the alkaline protease may be sprayed or quantitatively added to the detergent in liquid form.

[0125] In some embodiments, this application includes a method for cleaning one or more surfaces of clothing, tableware, and / or contact lenses, the method comprising contacting the one or more surfaces with one or more variants of an alkaline protease according to this application.

[0126] In some embodiments, this application includes a method for cleaning one or more surfaces of clothing, tableware, and / or contact lenses, the method comprising contacting the one or more surfaces with one or more detergents according to this application.

[0127] Depending on the application, the variant enzymes of this application can be combined with other enzymes, including but not limited to carboxypeptidase, α-amylase, lactase, sucrase, maltase, lipase, cellulase, mannanase, pectinase, etc.

[0128] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0129] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0130] Example 1: Construction and expression of alkaline enzymes and their variants 1.1 Materials and Reagents 1.1.1 Strains and Vectors: The expression strains containing the alkaline protease apr gene and its variants, Escherichia coli TOP10, Bacillus WB600, vector pBE-S, antibiotics, kanamycin, and ampicillin were all purchased from Shanghai Sangon Biotech Co., Ltd., and the wild-type alkaline protease gene of Bacillus tarda was purchased from General Biotechnology (Anhui) Co., Ltd.

[0131] 1.1.2 Enzymes and Kits: The Q5® Ultra Fidelity 2×Master Mix PCR polymerase and restriction endonucleases were purchased from NEB, and the plasmid extraction kit and purification kit were purchased from Shanghai Sangon Biotech Co., Ltd.

[0132] 1.1.3 Culture medium: Escherichia coli was cultured on LB medium (1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0). LB+Amp medium consisted of LB medium with ampicillin added to a final concentration of 100 μg / mL. Bacillus was cultured on TB medium (11.8 g / L tryptone, 23.6 g / L yeast extract, 9.4 g / L K₂HPO₄, 2.2 g / L KH₂PO₄), and TB+Kan medium consisted of TB medium with kanamycin added to a final concentration of 20 μg / mL. 1.1.4 Chemical reagents: Casein was purchased from Sinopharm Chemical Reagent Co., Ltd., and other reagents were purchased from Guangzhou Chemical Reagent Factory.

[0133] 1.2 Alkaline protease activity assay method The activity of alkaline protease was determined using the Folin-Ciocalteu method as specified in the national standard GB / T 23527-2009, "Alkaline Protease Preparations". Under specific temperature and pH conditions, the protease hydrolyzes casein substrates to produce amino acids containing phenolic groups (such as tyrosine and tryptophan). Under alkaline conditions, the Folin-Ciocalteu reagent is reduced to produce molybdenum blue and tungsten blue. The absorbance of the solution was measured at 680 nm using a spectrophotometer. Enzyme activity is proportional to absorbance, and the enzyme activity of the product can be calculated from this.

[0134] Protease activity is expressed in protease activity units, defined as the amount of 1 µg of tyrosine produced by hydrolyzing casein in 1 minute under certain temperature and pH conditions from 1 g of solid enzyme powder (or 1 mL of liquid enzyme), which is expressed as U / g (U / mL).

[0135] For gene mutations, the parental sequence and its species are disclosed, and subsequent site numbering is based on that parental sequence. The amino acid sequence of the mutated alkaline protease (apr) gene of Bacillus lentus is shown in SEQ ID NO.1.

[0136] 1.3 Expression of alkaline proteases and their variants 1.3.1 Synthesis of alkaline protease apr gene and vector construction Bacillus retardans ( Bacillus lentus The wild-type alkaline protease gene (WT, synthesized by General Biotechnology (Anhui) Co., Ltd.) was further mutated to obtain the parental alkaline protease gene, the amino acid sequence of which is shown in SEQ ID NO.1. Compared with the wild-type alkaline protease of Bacillus tarda, the parental alkaline protease has mutations at sites A224C, N255D, and A264C.

[0137] NdeI and SalI restriction sites were introduced at the 5' and 3' ends of the alkaline protease apr gene, respectively, and ligated into the pUC57-amp vector. pUC57-amp was seeded into LB+Amp medium and cultured overnight. The plasmid was extracted, digested with NdeI and SalI, and the target gene fragment was recovered by gel excision and ligated into the expression vector pBE-S to obtain the expression vector pBE-apr.

[0138] 1.3.2 Introduction of Mutations Using pBE-apr as a template, the first mutation site Q200Y was introduced into the parental alkaline protease apr gene using PCR. After digestion with DpnI (DpnI endonuclease), the target fragment was recovered by gel extraction. The digested product was then transformed into *E. coli* TOP10 competent cells using a chemical transformation heat shock method. The recombinant transformants were verified by colony PCR, and plasmids of the verified transformants were extracted and sequenced to identify the corresponding mutants, thus obtaining the expression vector pBE-apr-M1. This vector was then chemically transformed into WB600 chemically transformed competent cells to obtain the *Bacillus* recombinant transformant APR-M1.

[0139] Using pBE-apr-M1 as a template, multiple sites (second, third, or fourth) of the parental alkaline protease apr gene were introduced via PCR. After digestion with DpnI (DpnI endonuclease), the target fragment was recovered via gel extraction. The digested product was then transformed into *E. coli* TOP10 competent cells using a chemical transformation heat shock method. Recombinant transformants were verified by colony PCR. Plasmids from verified transformants were extracted and sequenced to identify the corresponding mutants and obtain the appropriate expression vectors. Finally, these vectors were chemically transformed into WB600 chemically transformed competent cells.

[0140] 1.3.3 Mutant strains used in shake-flask fermentation Using toothpicks, each recombinant transformant obtained in step 2 was picked up and transferred to a 250 mL shake flask. 40 mL of TB medium was added to each shake flask. The flasks were incubated at 37°C, 220 rpm, and 85% humidity for about 48 hours. The supernatant was collected by centrifugation, and the parental alkaline protease and mutants were purified by affinity chromatography to obtain enzyme solutions of each alkaline protease mutant.

[0141] Example 2: Fermentation activity of wild-type alkaline protease and alkaline protease mutant Enzyme activity was tested at 40℃ and pH 10.5, respectively. The relative fermentation activity of wild-type and mutant was calculated by using the measured fermentation activity of parental alkaline protease as a control.

[0142] Relative fermentation activity refers to the ratio of the fermentation activity of a certain enzyme to that of its parental alkaline protease under specific conditions. The formula for calculating relative fermentation activity is as follows: Relative fermentation activity = (Fermentation activity of the tested mutant / Fermentation activity of the parental alkaline protease) × 100%. The results of relative fermentation activity are shown in Table 2.

[0143] Example 3: Thermostability of Alkaline Protease Mutants Wild-type alkaline protease, purified parental alkaline protease, and alkaline protease mutant enzyme solutions were heat-treated at 50°C for 24 hours, cooled on ice, and then their enzyme activity was measured at 40°C and pH 10.5. The enzyme activity retention rate was calculated using the following formula: Enzyme activity retention rate = (Enzyme activity after heat treatment / Enzyme activity before heat treatment) × 100%. The heat resistance test results are shown in Table 2. Table 2 shows that the wild-type alkaline protease was almost completely inactivated after heat treatment at 50°C for 24 hours; therefore, the parental alkaline protease (SEQ ID NO. 1) was used as a control. The alkaline protease mutant, after treatment at 50°C for 24 hours, showed a significantly higher enzyme activity retention rate than the parental alkaline protease, indicating a significant improvement in its heat resistance.

[0144] Example 4: Enzyme activity and stability test of wild-type alkaline protease, parental alkaline protease, and alkaline protease mutant in commercial laundry detergent. Wild-type alkaline protease, purified parental alkaline protease, and alkaline protease mutant enzyme solutions were added to commercial laundry detergent CM-1 in a certain proportion (CM-1 laundry detergent formula is shown in Table 3), and mixed evenly to ensure that the initial activity of alkaline protease in CM-1 was 560 u / mL. Samples of CM-1 containing wild-type alkaline protease, parental alkaline protease, and alkaline protease mutant were separately frozen at -18℃ as week 0 samples. The remaining laundry detergent was stored in a constant temperature oven at 37℃ for 4 weeks. Megazyme Protazyme AK Tablets (T-PRAK-1000T, 1000 tablets per package, repeatability error less than 5%) were used. The protease activity in each group of week 0 and week 4 samples was tested, and the enzyme activity retention rate was calculated. The formula for calculating the enzyme activity retention rate is as follows: Enzyme activity retention rate = (Enzyme activity after 4 weeks of storage / Enzyme activity of week 0 sample) × 100%. The results are shown in Table 2. As shown in Table 2, the wild-type alkaline protease used in commercial laundry detergent was almost completely inactivated after being stored at 37°C for 4 weeks; the alkaline protease mutant used in commercial laundry detergent had a significantly higher enzyme activity retention rate than the parental alkaline protease after being stored at 37°C for 4 weeks, and its heat storage stability was significantly improved.

[0145] Table 2

[0146] Table 3. CM-1 Laundry Detergent Formula

[0147] Example 5: Detergent stability test of wild-type alkaline protease, parental alkaline protease and some alkaline protease mutants in commercial laundry detergent. Wild-type alkaline protease, purified parental alkaline protease, and a portion of the alkaline protease mutant enzyme solution were added to a commercial laundry detergent (CM-1) formula at a specific ratio and mixed thoroughly to ensure that the initial activity of the alkaline protease in the laundry detergent formula was 560 u / mL. A sample of the laundry detergent containing wild-type alkaline protease, parental alkaline protease, and the alkaline protease mutant was stored at -18℃ as a 0-week sample. The remaining laundry detergent was then stored in a constant temperature oven at 37℃ for 4 weeks.

[0148] Referring to GB / T 13174-2021 standard, based on JB-02 protein-stained fabric, the detergency of laundry detergent was tested initially and after 4 weeks of heat storage, and the corresponding detergency ratio was obtained. This was used to investigate the detergency stability of wild-type alkaline protease, parental alkaline protease, and alkaline protease mutant in commercially available laundry detergent formulations. The results are shown in Table 4.

[0149] Table 4. Detergency stability of alkaline protease in commercially available laundry detergent formulations

[0150] As shown in Table 4, the initial detergency of the parental alkaline protease in this invention is basically the same as that of the WT wild-type alkaline protease, and its detergency stability is stronger after heat storage. The alkaline protease mutant provided in the embodiments of this invention has significantly higher detergency than both the wild-type alkaline protease and the parental alkaline protease, both in terms of initial detergency and detergency after 4 weeks of heat storage. The detergency after heat storage is 1.6-2.5 times that of the parental alkaline protease and 2.4-3.8 times that of the wild-type alkaline protease.

[0151] In summary, compared with the parental alkaline protease, the alkaline protease mutants provided in the embodiments of the present invention have at least one advantage in heat resistance or detergent stability. Moreover, most alkaline protease mutants have both excellent heat resistance and detergent tolerance, and higher catalytic activity for proteins, which can better meet the needs of the daily chemical washing industry.

[0152] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. An alkaline protease mutant, characterized in that, The alkaline protease mutant has alkaline protease activity and, compared with the parental alkaline protease with the amino acid sequence shown in SEQ ID NO:1, the alkaline protease mutant includes the mutant Q200Y.

2. The alkaline protease mutant according to claim 1, characterized in that, The alkaline protease mutant has improved properties, including increased heat resistance and / or increased detergent tolerance.

3. The alkaline protease mutant according to claim 1 or 2, characterized in that, The alkaline protease variants include the following mutant combinations: Q200Y, V199I.

4. The alkaline protease mutant according to claim 3, characterized in that, The alkaline protease variants also include at least one of the following mutations: N179E, N198D, A209R, T218S, S9I, I41P, P51S, N74D, N74M, N74S, S76D, L80V, E87D, V102L, L109I, G116M, H118D, P127S, S128Y, T141W, R143K, L146M, T174V, A188P, A209L, L211M, Q230E, N232H, N237P, N242Q, S250P, S253D, L256E, S259A, N263D, C264A.

5. The alkaline protease mutant according to claim 1, characterized in that, Compared with the parental alkaline protease with the amino acid sequence shown in SEQ ID NO:1, the alkaline protease mutant includes at least one of the mutations (1) to (208): (1) Q200Y, (2) S9I / Q200Y, (3) I41P / Q200Y, (4) N74S / Q200Y, (5) E87D / Q200Y, (6) G116M / Q200Y, (7) N179E / Q200Y, (8) V199I / Q200Y, (9) Q200Y / A209R, (10) Q200Y / A209L, (11) P51S / V199I / Q200Y, (12) L80V / V199I / Q200Y, (13) E87D / V1 99I / Q200Y, (14) V102L / V199I / Q200Y, (15) L109I / V199I / Q200Y, (16) H11 8D / V199I / Q200Y, (17) N198D / V199I / Q200Y, (18) N179E / V199I / Q200Y, (19 ) V199I / Q200Y / A209R, (20) V199I / Q200Y / T218S, (21) V199I / Q200Y / N232 H. (22) V199I / Q200Y / N242Q, (23) V199I / Q200Y / S253D, (24) V199I / Q200Y / L256E, (25) N74D / V199I / Q200Y / A209R, (26) N179E / V199I / Q200Y / A209R, (27) N198D / V199I / Q200Y / A209R, (28) V199I / Q200Y / A209R / T218S, (29) N7 4D / N179E / V199I / Q200Y / A209R, (30) N74D / N198D / V199I / Q200Y / A209R, ( 31) N74D / V199I / Q200Y / A209R / T218S, (32) N74D / V199I / Q200Y / A209R / L25 6E, (33) N179E / N198D / V199I / Q200Y / A209R, (34) N179E / V199I / Q200Y / A2 09R / T218S, (35) N179E / V199I / Q200Y / A209R / L256E, (36) N198D / V199I / Q2 00Y / A209R / T218S, (37) N198D / V199I / Q200Y / A209R / L256E, (38) V199I / Q 200Y / A209R / T218S / L256E, (39) N74D / N179E / N198D / V199I / Q200Y / A209R,(40) N74D / N179E / V199I / Q200Y / A209R / T218S, (41) N74D / N179E / V199I / Q200Y / A209R / L256E, (42) N179E / N198D / V199I / Q200Y / A209R / T218S (43) N179E / N198D / V199I / Q200Y / A209R / L256E, (44) N198D / V199I / Q200Y / A209R / T218S / L256E, (45) N74D / N179E / N198D / V199I / Q200Y / A209R / T218S, (46) N74D / N179E / N198D / V199I / Q200Y / A209R / L256E, (47) N179E / N198D / V199I / Q200Y / A209R / T218S / L256E, (48) N74D / S76D / N179E / N1 98D / V199I / Q200Y / A209R / T218S、(49) N74D / E87D / N179E / N198D / V199I / Q200Y / A209R / T218S、(50) N74D / H118D / N179E / N198D / V199I / Q200Y / A2 09R / T218S、(51)N74D / R143K / N179E / N198D / V199I / Q200Y / A209R / T218 S、(52)N74D / T174V / N179E / N198D / V199I / Q200Y / A209R / T218S、(53)N7 4D / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E、(54)N74D / N179E / N198D / V199I / Q200Y / A209R / T218S / S250P、(55)N74D / N179E / N198D / V1 99I / Q200Y / A209R / T218S / L256E、(56)N74D / N179E / N198D / V199I / Q200 Y / A209R / T218S / S259A、(57)N74D / N179E / N198D / V199I / Q200Y / A209R / T 218S / N263D、(58)N74D / N179E / N198D / V199I / Q200Y / A209R / T218S / C26 4A、(59)N74D / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P、(60)N74D / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / L256E、(61)N74D / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S259A、(62)N74D / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / N263D、(63)N74D / N179E / N198D / V199I / Q200Y / A209R / T218S / S250P / S259A、(64)N74D / N179E / N198D / V199I / Q200Y / A209R / T218S / S250P / N263D、(65)N74D / N179E / N198D / V199I / Q200Y / A209R / T218S / S250P / C264A、(66)N74D / N179E / N198D / V199I / Q200Y / A209R / T218S / S259A / N263D、(67)N74D / N179E / N198D / V199I / Q200Y / A209R / T218S / S259A / C264A、(68)N74D / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(69)N74D / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / N263D、(70)N74D / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / C264A、(71)N74D / N179E / N198D / V199I / Q200Y / A209R / T218S / S250P / S259A / N263D、(72)N74D / N179E / N198D / V199I / Q200Y / A209R / T218S / S250P / S259A / C264A、(73)N74M / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(74)N74D / P127S / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(75)N74D / S128Y / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(76)N74D / T141W / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(77)N74D / L146M / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(78)N74D / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(79)N74D / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(80)N74D / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A / N263D、(81)N74D / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A / C264A、(82)N74M / P127S / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(83)N74M / S128Y / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(84)N74M / T141W / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(85)N74M / L146M / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(86)N74M / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(87)N74M / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(88)N74D / P127S / T141W / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(89)N74D / P127S / L146M / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(90)N74D / P127S / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(91)N74D / P127S / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(92)N74D / P127S / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(93)N74D / S128Y / T141W / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(94)N74D / S128Y / L146M / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(95)N74D / S128Y / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(96)N74D / S128Y / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(97)N74D / S128Y / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(98)N74D / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(99)N74D / T141W / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(100)N74D / T141W / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(101)N74D / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(102)N74D / L146M / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(103)N74D / L146M / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(104)N74D / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(105)N74D / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(106)N74D / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(107)N74M / P127S / T141W / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(108)N74M / P127S / L146M / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(109)N74M / P127S / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(110)N74M / P127S / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(111)N74M / P127S / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(112)N74M / S128Y / T141W / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(113)N74M / S128Y / L146M / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(114)N74M / S128Y / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(115)N74M / S128Y / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(116)N74M / S128Y / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(117)N74M / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(118)N74M / T141W / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(119)N74M / T141W / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(120)N74M / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(121)N74M / L146M / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(122)N74M / L146M / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(123)N74M / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(124)N74M / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(125)N74M / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(126)N74D / P127S / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(127)N74D / P127S / T141W / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(128)N74D / P127S / T141W / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(129)N74D / P127S / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(130)N74D / P127S / L146M / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(131)N74D / P127S / L146M / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(132)N74D / P127S / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(133)N74D / P127S / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(134)N74D / P127S / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(135)N74D / S128Y / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(136)N74D / S128Y / T141W / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(137)N74D / S128Y / T141W / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(138)N74D / S128Y / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(139)N74D / S128Y / L146M / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(140)N74D / S128Y / L146M / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(141)N74D / S128Y / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(142)N74D / S128Y / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(143)N74D / S128Y / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(144)N74D / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(145)N74D / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(146)N74D / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(147)N74D / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(148)N74D / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(149)N74M / P127S / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(150)N74M / P127S / T141W / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(151)N74M / P127S / T141W / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(152)N74M / P127S / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(153)N74M / P127S / L146M / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(154)N74M / P127S / L146M / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(155)N74M / P127S / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(156)N74M / P127S / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(157)N74M / P127S / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(158)N74M / S128Y / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(159)N74M / S128Y / T141W / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(160)N74M / S128Y / T141W / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(161)N74M / S128Y / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / S250P / S259A、(162)N74M / S128Y / L146M / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(163)N74M / S128Y / L146M / N179E / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(164)N74M / S128Y / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(165)N74M / S128Y / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(166)N74M / S128Y / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(167)N74M / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(168)N74M / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(169)N74M / T141W / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(170)N74M / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(171)N74M / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(172)N74M / L146M / N179E / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(173)N74M / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(174)N74D / P127S / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(175)N74D / P127S / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(176)N74D / P127S / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(177)N74D / P127S / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(178)N74D / P127S / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(179)N74D / S128Y / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(180)N74D / S128Y / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(181)N74D / S128Y / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(182)N74D / S128Y / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(183)N74D / S128Y / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(184)N74D / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(185)N74D / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(186)N74M / P127S / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(187)N74M / P127S / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(188)N74M / P127S / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(189)N74M / P127S / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(190)N74M / P127S / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(191)N74M / S128Y / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(192)N74M / S128Y / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(193)N74M / S128Y / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / S250P / S259A、(194)N74M / S128Y / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / T218S / Q230E / N237P / S250P / S259A、(195)N74M / S128Y / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(196)N74M / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(197)N74M / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(198)N74D / P127S / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(199)N74D / P127S / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(200)N74D / S128Y / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(201)N74D / S128Y / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(202)N74M / P127S / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(203)N74M / P127S / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(204)N74M / S128Y / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(205)N74M / S128Y / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(206)N74D / P127S / S128Y / T141W / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(207)N74D / P127S / T141W / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A、(208)N74D / P127S / S128Y / T141W / L146M / N179E / A188P / N198D / V199I / Q200Y / A209R / L211M / T218S / Q230E / N237P / S250P / S259A。、 6. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the alkaline protease mutant according to any one of claims 1 to 5.

7. A recombinant expression vector, characterized in that, It includes the nucleic acid molecule as described in claim 6.

8. A recombinant bacterium, characterized in that, It comprises the nucleic acid molecule of claim 6 or the recombinant expression vector of claim 7.

9. An enzyme-containing composition, characterized in that, The enzyme-containing composition comprises the alkaline protease mutant of any one of claims 1 to 5, and contains one or more additional enzymes; said additional enzymes include aminopeptidase, α-amylase, β-amylase, isoamylase, carboxylase, carboxypeptidase, catalase, cellulase, chitinase, carrageenanase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, α-galactosidase, β-galactosidase, glucosylamylase, α-glucosidase, β-glucosidase, haloperoxidase, invertase, laccase, mannanase, α-galactosidase, β-galactosidase, glucosylamylase, α-glucosidase, β-glucosidase, haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase, pectinase, peptidylglutaminase, peroxidase, glucosylamylase, polyphenol oxidase, cellulase, proteolytic enzyme, ribonuclease, transglutaminase, or xylase.

10. The use of the alkaline protease mutant according to any one of claims 1 to 5 in detergents.

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