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CN-121991929-A - Alpha-galactosidase mutant and preparation method and application thereof

CN121991929ACN 121991929 ACN121991929 ACN 121991929ACN-121991929-A

Abstract

The invention relates to the technical field of genetic engineering and enzyme engineering, and discloses an alpha-galactosidase mutant, a preparation method and application thereof, wherein the amino acid sequence of the mutant is based on a wild alpha-galactosidase amino acid sequence shown in SEQ ID NO.1, and the mutant is obtained through mutation of amino acid residues at one or more positions, namely 74 th, 145 th, 432 th, 560 th and 615 th, wherein the amino acid position number corresponds to the sequence shown in SEQ ID NO. 1. The enzyme is extracted from bacillus megatherium, adopts single-point mutation and saturation mutation technology, performs enzyme modification through free energy virtual screening coupling internal topology cavity engineering modification strategy, obtains double-point mutants K74R and L432V with remarkably improved enzyme activity, can rapidly degrade anti-nutritional oligosaccharides and polysaccharides in plant raw materials, can be used as flavoring enzymes to efficiently hydrolyze glycosidic bonds, releases aroma-submerged substances, and is applied to tobacco processing, plant extraction and essence and spice industries.

Inventors

  • Sun Lanqian
  • TAO WENSHENG
  • FAN ZHAOCHENG
  • LIU RUCAN
  • WANG PENG
  • WANG YI

Assignees

  • 重庆中烟工业有限责任公司

Dates

Publication Date
20260508
Application Date
20260320

Claims (10)

  1. 1. An alpha-galactosidase mutant is characterized in that the amino acid sequence of the mutant is obtained by mutating amino acid residues at one or more positions 74, 145, 432, 560 and 615 based on the amino acid sequence of wild-type alpha-galactosidase shown in SEQ ID NO.1, wherein the amino acid position numbers correspond to the sequence shown in SEQ ID NO. 1.
  2. 2. The α -galactosidase mutant of claim 1, wherein the mutation comprises any one or a combination of K74R, A145,145, 145T, L432,432, 432V, Q560,560, 560I, D615E, wherein the K74R site mutation is a mutation of lysine at position 74 of SEQ ID NO.1 to arginine, the A145T site mutation is a mutation of alanine at position 145 of SEQ ID NO.1 to threonine, the L432V site mutation is a mutation of leucine at position 432 of SEQ ID NO.1 to valine, the Q560I site mutation is a mutation of glutamine at position 560 of SEQ ID NO.1 to isoleucine, and the D615E site mutation is a mutation of aspartic acid at position 615 of SEQ ID NO.1 to glutamic acid.
  3. 3. The alpha-galactosidase mutant according to claim 2, wherein the mutant is a double-point mutant comprising K74R and L432V, and the amino acid sequence of the mutant is shown as SEQ ID NO. 2.
  4. 4. A nucleic acid molecule encoding an alpha-galactosidase mutant according to any one of claims 1 to 3.
  5. 5. A recombinant expression vector comprising the nucleic acid molecule of claim 4, wherein the recombinant expression vector is pET-28a (+).
  6. 6. A recombinant host cell comprising the nucleic acid molecule of claim 4 or the recombinant expression vector of claim 5.
  7. 7. The recombinant host cell of claim 6 wherein said recombinant host cell is E.coli BL21 (DE 3).
  8. 8. A method for producing the alpha-galactosidase mutant according to any one of claims 1 to 3, comprising the steps of (1) culturing the recombinant host cell according to claim 6 in a suitable medium, (2) inducing expression of the alpha-galactosidase mutant, and (3) isolating and purifying the alpha-galactosidase mutant from the culture.
  9. 9. Use of an α -galactosidase mutant according to any one of claims 1 to 3, a nucleic acid molecule according to claim 4, a recombinant expression vector according to claim 5 or a recombinant host cell according to claim 6 or 7 for the preparation of an enzyme preparation for the hydrolysis of a substrate comprising an α -galactosidase linkage.
  10. 10. The use according to claim 9, characterized in that the use is: (1) The application in tobacco processing or tobacco extract preparation is particularly used for degrading raffinose family oligosaccharides and/or galactomannans in tobacco raw materials, and can improve the sensory quality of the tobacco extract; (2) The method is applied to natural plant spice processing, and is particularly used for hydrolyzing glycosidic bonds bonded on aroma substances to promote the conversion of combined aroma substances into free aroma substances.

Description

Alpha-galactosidase mutant and preparation method and application thereof Technical Field The invention relates to the technical fields of genetic engineering and enzyme engineering, in particular to an alpha-galactosidase mutant and a preparation method and application thereof. Background Alpha-galactosidase is an exoglycosidase capable of specifically catalyzing and hydrolyzing alpha-galactoside bonds, and is widely used in animals, plants and microorganisms. In the fields of tobacco industry, natural plant extraction and food bio-processing, the enzyme has extremely high application value due to the modification capability of the enzyme on specific carbohydrate. The processing quality and organoleptic properties of plant materials are largely related to the carbohydrate component contained therein, mainly including macromolecular polysaccharides, functional oligosaccharides and secondary metabolites in the form of glycosides. However, the naturally occurring state of these components often limits the efficiency of raw material utilization or negatively impacts product quality, requiring targeted improvement by enzymatic engineering means. In plant matrices, macromolecular polysaccharides such as galactomannans are the main components constituting the cell wall or the cell matrix. In the plant extraction or tobacco sheet preparation process, the existence of the high molecular weight polymers can obviously increase the viscosity of feed liquid, and form mass transfer resistance, so that the dissolution and diffusion of active ingredients are inhibited. More importantly, macromolecular polysaccharides often generate burnt smell due to incomplete pyrolysis during thermal processing or burning and sucking, and seriously interfere with the sensory experience of the product. By utilizing the enzyme digestion technology, the system viscosity can be effectively reduced, the processing efficiency can be improved, and the content of the precursor with poor flavor can be reduced from the source. Meanwhile, raffinose family oligosaccharides such as raffinose and stachyose are commonly contained in plants such as tobacco and beans (RFOs). The oligosaccharides are thoroughly degraded through enzymolysis to form hydrolysis products such as galactose, sucrose and the like, and the content of reducing sugar in the system can be obviously improved. The reducing sugar is used as a core substrate of Maillard reaction, and can promote the generation of flavoring substances such as pyrazine, furan and the like in the subsequent processing processes such as baking, alcoholization and the like, and change the caramelization reaction path, thereby endowing the product with stronger and natural baked sweet fragrance and realizing quality improvement. In addition, many of the precious aroma components in plants (e.g., monoterpenes, sesquiterpenes, aromatic alcohols, etc.) are not all present in free form, and a significant portion are bound to sugar groups to form non-volatile glycosides (bound aroma). These "fragrance-diving substances" cannot directly contribute to fragrance, resulting in waste of resources. The alpha-galactosidase has the potential of hydrolyzing glycosidic bonds, can be used as an aroma release agent, breaks through glycosyl package, and converts combined state substances into free state aroma components, so that the aroma total amount and the aroma richness of plant extracts or tobacco products are obviously improved. Although the application potential of the alpha-galactosidase is huge, the existing natural enzyme preparation applied industrially has the problems of low specific activity, poor thermal stability, insufficient substrate affinity in complex plant matrixes and the like, and is difficult to meet the industrial production requirements of high efficiency and low cost. Disclosure of Invention In view of the above, the invention aims to provide an alpha-galactosidase mutant, a preparation method and application thereof, the enzyme is extracted from bacillus megatherium, single-point mutation and saturation mutation technologies are adopted, and enzyme modification is performed through free energy virtual screening coupled with internal topology cavity engineering modification strategies, so that double-point mutants K74R and L432V with remarkably improved enzyme activity are obtained, the mutant has remarkably improved catalytic efficiency, can rapidly degrade anti-nutritional oligosaccharides and polysaccharides in plant raw materials, can be used as flavoring enzymes to efficiently hydrolyze glycosidic bonds, release latent fragrance substances, and are applied to tobacco processing, plant extraction and essence and spice industries. The invention solves the technical problems by the following technical means: In a first aspect, the invention provides an alpha-galactosidase mutant, wherein the amino acid sequence of the mutant is obtained by mutating one or more amino acid residues at 74 th, 145 th