CN-122012474-A - Alkaline protease mutant and preparation method and application thereof

CN122012474ACN 122012474 ACN122012474 ACN 122012474ACN-122012474-A

Abstract

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

Inventors

JIN XING
SUN FEIYE
LI YANGYUAN
WANG JUN
WU YUPENG
ZHANG ZHILIANG
TAN YAOYAO

Assignees

广东溢多利生物科技股份有限公司

Dates

Publication Date: 20260512
Application Date: 20260211

Claims (10)

1. An alkaline protease mutant, characterized in that the alkaline protease mutant has alkaline protease activity, and the alkaline protease mutant comprises mutation Q200Y compared with a parent alkaline protease having an amino acid sequence as shown in SEQ ID NO. 1.
2. Alkaline protease mutants according to claim 1, characterized in that they have improved properties, including increased thermostability and/or increased laundry liquor tolerance.
3. Alkaline protease variant according to claim 1 or 2, characterized in that the alkaline protease variant comprises the following combination of mutations Q200Y, V199I.
4. The alkaline protease variant of claim 3, further comprising 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 of claim 1, wherein the alkaline protease mutant comprises at least one of mutations (1) - (208) as compared to a parent alkaline protease having the amino acid sequence shown in SEQ ID No. 1 ：（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/Q200Y、（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/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）N74D/N179E/V199I/Q200Y/A209R、（30）N74D/N198D/V199I/Q200Y/A209R、（31）N74D/V199I/Q200Y/A209R/T218S、（32）N74D/V199I/Q200Y/A209R/L256E、（33）N179E/N198D/V199I/Q200Y/A209R、（34）N179E/V199I/Q200Y/A209R/T218S、（35）N179E/V199I/Q200Y/A209R/L256E、（36）N198D/V199I/Q200Y/A209R/T218S、（37）N198D/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）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/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/V199I/Q200Y/A209R/T218S、（52）N74D/T174V/N179E/N198D/V199I/Q200Y/A209R/T218S、（53）N74D/N179E/N198D/V199I/Q200Y/A209R/T218S/Q230E、（54）N74D/N179E/N198D/V199I/Q200Y/A209R/T218S/S250P、（55）N74D/N179E/N198D/V199I/Q200Y/A209R/T218S/L256E、（56）N74D/N179E/N198D/V199I/Q200Y/A209R/T218S/S259A、（57）N74D/N179E/N198D/V199I/Q200Y/A209R/T218S/N263D、（58）N74D/N179E/N198D/V199I/Q200Y/A209R/T218S/C264A、（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 encoding the alkaline protease mutant of any one of claims 1 to 5.
7. A recombinant expression vector comprising the nucleic acid molecule of claim 6.
8. A recombinant bacterium comprising the nucleic acid molecule of claim 6 or the recombinant expression vector of claim 7.
9. An enzyme-containing composition comprising the alkaline protease mutant of any one of claims 1-5 and one or more additional enzymes, wherein the additional enzymes comprise aminopeptidase, alpha-amylase, beta-amylase, isoamylase, carboxylase, carboxypeptidase, catalase, cellulase, chitinase, carrageenase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, haloperoxidase, invertase, laccase, mannanase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, peroxidase, beta-glucosidase, haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase, pectolytic enzyme, peptidase, peroxidase, glucoamylase, polyphenol oxidase, cellulase, proteolytic enzyme, ribonuclease, transglutaminase, or transglutaminase.
10. The use of the alkaline protease mutant according to any one of claims 1 to 5 in detergents.

Description

Alkaline protease mutant and preparation method and application thereof Technical Field The invention belongs to the technical fields of genetic engineering and enzyme engineering, and particularly relates to an alkaline protease mutant, a preparation method and application thereof. Background Subtilisins (EC number 3.4.21.62), originally obtained from bacillus subtilis, belong to the family of S8 peptidases, having in their amino acid sequence catalytic triplets in the order Asp, his and Ser, also called serine proteases or alkaline proteases. Alkaline proteases are the most popular detergent additives in the market today, and the main organic components of stains (e.g. blood stains, milk stains, sweat stains, food residues, grass stains, sebum of the human body, etc.) are proteins, oils and starches. It cleaves the carboxy terminus of hydrophobic, aromatic and some uncharged neutral amino acids, and this broad substrate spectrum is very advantageous for the treatment of complex stains. It can hydrolyze large proteins (such as blood, egg, milk, etc.) which are insoluble in water and firmly attached to fibers into small polypeptides or amino acids. In addition, many stains (e.g., grease, pigments) are "coated" or "immobilized" on fabrics by proteins. Proteases break these protein "skeletons" making other stains easier to remove by surfactants and mechanical forces, thereby significantly improving overall detergency. However, how to maintain high concentration and high stability of enzymes, especially in detergents, is a technical problem that alkaline proteases are not well developed. Therefore, the alkaline protease is modified by utilizing genetic engineering, and the alkaline protease which can further improve the enzyme activity, heat resistance and/or detergent resistance is researched, so that the alkaline protease has great significance in the washing industry. Disclosure of Invention The present invention aims to solve at least one of the technical problems in the prior art described above. To this end, the invention proposes alkaline protease mutants, a process for their preparation and their use. The alkaline protease provided by the invention has higher enzyme activity and simultaneously has excellent heat resistance and detergent resistance. The invention provides an alkaline protease mutant. Specifically, the alkaline protease mutant has alkaline protease activity, and compared with the parent alkaline protease with the amino acid sequence shown as SEQ ID NO. 1, the alkaline protease mutant comprises mutation Q200Y. SEQ ID NO:1: AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGISTHPDLNIRGGASFVPGEPSTQDGNGHGTHVAGTIAALNNSIGVLGVAPSAELYAVKVLGASGSGSVSSIAQGLEWAGNNGMHVANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGAGSISYPARYANAMAVGATDQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGCAALVKQKNPSWSNVQIRNHLKNTATSLGSTDLYGSGLVNCEAATR. In some embodiments of the invention, the alkaline protease mutants have improved properties including increased thermostability and/or increased laundry detergent tolerance. In some embodiments of the invention, the increased thermal stability includes increased stability at 50 ℃ and/or increased stability after heat storage at 37 ℃ in a laundry detergent containing condition. More specifically, the alkaline protease mutant has an enzyme activity retention rate of not less than 50% after heat treatment at 50 ℃ for 24 hours, and/or a commercial laundry detergent containing the alkaline protease mutant has an enzyme activity retention rate of not less than 30% after heat storage at 37 ℃ for 4 weeks. In some embodiments of the invention, the parent alkaline protease is derived from Bacillus lentus (Bacillus lentus). In some embodiments of the invention, the mutant of the alkaline protease has at least 93% sequence identity, 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, to the polypeptide of SEQ ID NO. 1. In some embodiments of the invention, the alkaline protease variant comprises a combination of mutations of Q200Y, V199I. In some embodiments of the invention, the alkaline protease variant further comprises 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. In some embodiments of the invention, the alkaline protease mutants comprise at least one of mutations (1) - (208) as compared to the parent alkaline protease having the amino acid sequence shown in SEQ ID NO. 1 ：（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、（1