CN-122012470-A - Thermophilic debranching enzyme mutant with improved debranching efficiency and activity and application thereof

Abstract

The invention discloses a thermophilic debranching enzyme mutant with improved debranching efficiency and activity and application thereof, belonging to the field of enzyme preparations. The residue 358 of the binding force field of 0 '-Glc-C2/3-OH and-1' -Glc-C2-OH which participates in the non-reducing end side of the branch point main chain in the thermophilic debranching enzyme T2P is a key site affecting the debranching efficiency of the enzyme, the mutation of the site can realize the effects of maintaining the heat-adapting property and heat resistance of the enzyme and improving the debranching efficiency and activity of the enzyme, the maximum debranching specific enzyme activity (V max ) of K358D/E/N/H is improved to 2 times before mutation, and the debranching efficiency V max /K m of K358D/E is respectively improved to 3.3 times and 2.5 times before mutation. Thereby providing a method guide for improving the debranching efficiency of T2P and debranching enzymes with similar structures, and being beneficial to improving the industrial application value of thermophilic debranching enzymes.

Inventors

• WANG JIANFENG

Assignees

• 东华理工大学

Dates

Publication Date

20260512

Application Date

20260204

Claims (10)

1. 1. A thermophilic debranching enzyme mutant with improved debranching efficiency and activity is characterized in that the thermophilic debranching enzyme mutant is characterized in that the 358 th lysine of the thermophilic debranching enzyme with an amino acid sequence shown as SEQ ID NO.1 is mutated into aspartic acid, glutamic acid, asparagine or histidine.
2. 2. A gene encoding the thermophilic debranching enzyme mutant according to claim 1.
3. 3. A recombinant vector expressing the thermophilic debranching enzyme mutant of claim 1.
4. 4. The recombinant vector according to claim 3, wherein the expression vector of the recombinant vector includes, but is not limited to, plasmid, cosmid, phage and retrovirus.
5. 5. A recombinant cell expressing the thermophilic debranching enzyme mutant of claim 1.
6. 6. The recombinant cell of claim 5, wherein the host cell of the recombinant cell is a bacterial, fungal or animal plant cell.
7. 7. Use of a thermophilic debranching enzyme mutant according to claim 1 or a recombinant cell according to claim 5 or 6 in starch debranching.
8. 8. The use according to claim 7, wherein the debranching reaction is performed under high temperature conditions by adding the thermophilic debranching enzyme mutant or the recombinant cell to a reaction system containing starch, wherein the high temperature conditions are at a temperature of not less than 70 ℃.
9. 9. The use according to claim 8, wherein the pH of the reaction system is 5.0-8.0.
10. 10. The method according to claim 8, wherein the starch-containing reaction system is obtained by gelatinizing and liquefying starch, and the starch content in the reaction system is 1% -33% by mass/volume.

Description

Thermophilic debranching enzyme mutant with improved debranching efficiency and activity and application thereof Technical Field The invention relates to a thermophilic debranching enzyme mutant with improved debranching efficiency and activity and application thereof, belonging to the field of enzyme preparations. Background Starch is an important food, pharmaceutical and chemical raw material, the molecular structure is tree-shaped alpha-1.4 glucan, and shorter alpha-1.4 glucan branches are connected with longer alpha-1.4 glucan main chains through alpha-1, 6 glycosidic bonds. Starch debranching, namely hydrolyzing alpha-1, 6 glycosidic bonds of starch to make the starch become chain molecules easy to crystallize, is widely used for manufacturing health-care food (slowly digestible starch for controlling sugar and weight), medicine sustained release agent, degradable biological base film and other products, and simultaneously, the starch debranching is also an important link in starch sugar production, and can obviously improve the yield and quality of target sugar. The existing debranching starch is mainly prepared by an acid hydrolysis method, and has the defects of poor hydrolysis selectivity, high process control difficulty, incomplete branch point removal, short alpha glucan chain and the like, and the enzymatic debranching method has the advantages of strong glycosidic bond selectivity, mild acting condition and the like, and is widely applied to starch sugar production. Starch debranching enzyme can specifically hydrolyze alpha-1, 6 glycosidic bonds of starch, at present commercial starch debranching enzyme is mainly isoamylase and pullulanase, both are suitable for weak acid and medium temperature environment, wherein Pseudomonas amyloderamosa isoamylase is suitable for pH 3.5 and 40 ℃, xia Chengpu pullulanase FDY-2224 is suitable for pH 3.5-6.0 and 40-65 ℃, and meanwhile, commercial pullulanase has enzyme activity defined by pullulanase hydrolysis activity, and starch debranching activity is unknown. Raw starch is insoluble at low temperature, high temperature gelatinization is needed for starch processing, and industrial gelatinization temperature is often higher than 90 ℃. If the existing starch debranching enzyme is utilized for debranching, the temperature of the starch paste needs to be reduced to 60 ℃, the pH value needs to be reduced to about 4.5, and the starch paste needs to be properly diluted to reduce the viscosity so as to promote mass transfer. Obviously, the heat-resistant efficient debranching enzyme combines the gelatinization debranching of starch into a whole, so that the operation can be simplified, the working hours can be shortened, and the cost and the efficiency can be reduced. Disclosure of Invention In order to solve the defects of the prior art, the heat-resistant efficient starch debranching enzyme is developed, enzyme function prediction is firstly carried out through protein sequence homology comparison and homology modeling, thermusthermophilusGH protein is screened from NCBI database to be a potential high-temperature debranching enzyme, and the protein ThermusthermophilusGH (T2P) is confirmed to be thermophilic starch debranching enzyme through heterologous expression of the protein gene and functional identification of recombinant protein, and the amino acid sequence of the protein ThermusthermophilusGH (T2P) is shown as SEQ ID NO. 1. The optimal temperature interval of T2P debranching is 76.0-100 ℃, the optimal reaction pH is 6.0, the enzyme is preserved at 97 ℃ for 30min, the residual debranching enzyme activity is 94.3% +/-0.9%, the industrial pasting liquefying temperature requirement is completely met, and the one-step pasting liquefying debranching can be realized. However, the debranching efficiency and activity of T2P are low, which is difficult to meet the requirement of industrial application, and the debranching efficiency and activity of T2P are required to be improved through protein rational design so as to be convenient for cost reduction and synergy in industrial application. Therefore, the invention further carries out protein engineering on T2P, and discovers that the end group of the K358 side chain participates in the bonding force field of 0 '-Glc-C2/3-OH and-1' -Glc-C2-OH at the non-reducing end side of the main chain of the branch point through SWISS-MODEL on-line homologous modeling and molecular docking, and the strength and the direction of the force field can be obviously influenced to change the debranching efficiency V max/Km of the enzyme, so that the K358M/R/H/E/Q/D/N mutant is designed to investigate the influence of the polarity and the steric hindrance of the side chain group of the 358 residue on the debranching performance of the enzyme. The invention is realized by the following technical scheme: The first object of the present invention is to provide a thermophilic debranching enzyme mutant with improved debranching efficienc