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CN-122012484-A - Cellobiose 2-epimerase mutant with high catalytic efficiency and application thereof

CN122012484ACN 122012484 ACN122012484 ACN 122012484ACN-122012484-A

Abstract

The invention relates to a cellobiose 2-epimerase mutant with high catalytic efficiency and application thereof. The present invention obtains mutants by altering specific amino acid positions of wild-type enzymes, wherein double mutants psyCE-FR exhibit optimal performance, and catalytic efficiency (Kcat/Km) is improved by about 40% compared with that of the wild-type. The mutant has excellent catalytic efficiency and substrate affinity at low temperature (4-10 ℃), and is suitable for processing low-temperature dairy products and preparing functional sugar. In addition, the mutant maintains the characteristic that psychrophilic enzyme is easy to inactivate by heat, can inactivate instantly at 50 ℃, is compatible with pasteurization flow in dairy industry, reduces downstream treatment cost, and improves product purity and safety. The invention also provides a gene for coding the mutant, a recombinant expression vector, recombinant engineering bacteria and a method for producing bad lactose by using mutant enzyme, and the method has wide application prospect and commercial value.

Inventors

  • LIU YONGQIN
  • XU HU

Assignees

  • 兰州大学

Dates

Publication Date
20260512
Application Date
20251225

Claims (8)

  1. 1. A cellobiose 2-epimerase mutant with high catalytic efficiency, which is characterized in that the mutant is obtained by mutating Asn at 309 th position of an amino acid sequence of a wild-type psychrophilic cellobiose 2-epimerase with an amino acid sequence of sequence 1 into Phe or His or mutating Lys at 189 th position into Arg.
  2. 2. A cellobiose 2-epimerase mutant with high catalytic efficiency, which is characterized in that the mutant is obtained by mutating Asn at 309 th position of an amino acid sequence of a wild-type psychrophilic cellobiose 2-epimerase with an amino acid sequence of sequence 1 into Phe or His and mutating Lys at 189 th position into Arg.
  3. 3. The cellobiose 2-epimerase mutant according to claim 1 or 2, characterized in that the amino acid sequence of the mutant is a double mutation of mutant N309F, of which the 309 th Asn is mutated to Phe and the amino acid sequence is sequence 2, of mutant N309H, of which the 309 th Asn is mutated to His and the amino acid sequence is sequence 3, of mutant K189R, of which the 189 th Lys is mutated to Arg and the amino acid sequence is sequence 4, of which the mutant psyCE-FR, of which the 309 th Asn is mutated to Phe and of which the 189 th Lys is mutated to Arg, of which the amino acid sequence is sequence 5, of which the mutant psyCE-HR, of which the 309 th Asn is mutated to His and of which the 189 th Lys is mutated to Arg, and of which the amino acid sequence is sequence 6.
  4. 4. A cellobiose 2-epimerase mutant according to claim 3, characterized in that said mutant enzyme is used in a low temperature environment for the preparation of epalactose.
  5. 5. The cellobiose 2-epimerase mutant according to claim 3, wherein the low temperature environment is 4-10 ℃.
  6. 6. A cellobiose 2-epimerase mutant according to claim 3, characterized in that the low temperature environment is 8 ℃.
  7. 7. A method for producing epinasin by using the cellobiose 2-epimerase mutant according to claim 3, wherein lactose or lactose-containing materials are used as substrates, and the cellobiose 2-epimerase mutant is used as a catalyst to catalyze epimerization of lactose at C-2 position under low temperature condition to produce epinasin.
  8. 8. A recombinant expression vector and recombinant engineering bacteria comprising the cellobiose 2-epimerase mutant gene of claim 3.

Description

Cellobiose 2-epimerase mutant with high catalytic efficiency and application thereof Technical Field The invention belongs to the technical field of enzyme engineering and biocatalysis, and particularly relates to a mutant with high catalytic activity and high substrate affinity based on modification of psychrophilic cellobiose 2-epimerase and application of the mutant in preparing epinastine by an enzyme method under a low-temperature condition. Background Lactose is a carbohydrate that is widely available and of low value in nature. As the C-2 epimer of lactose, the epinastine (epilactose) not only has excellent prebiotic characteristics (such as bifidobacterium proliferation and calcium absorption promotion), but also has physiological functions similar to dietary fibers and good physicochemical stability, and has wide application prospect in the fields of dairy products and health-care foods. Cellobiose epimerase Cellobiose-EPIMERASE (CE) is a currently known biocatalyst that specifically catalyzes the epimerization of lactose at the C-2 position to produce epinaste. Compared with a chemical method, the enzymatic conversion has the advantages of mild reaction conditions, no byproducts, environmental friendliness and the like. In the dairy industry, low temperature processing (typically 4-10 ℃) is the preferred process in order to maximize the preservation of heat sensitive nutrients and inhibit spoilage microorganism growth. However, most CE enzymes reported so far are derived from thermophilic or mesophilic microorganisms, and their optimum temperature is usually above 45 ℃, and their catalytic activity drops sharply under low temperature conditions, and cannot meet the actual demands of cold chain processing. The prior art has obtained a psychrophilic cellobiose 2-epimerase from the Qinghai-Tibet plateau glacier environment by metagenomic mining techniques (psyCE). Although the enzyme shows good low temperature adaptability and has the highest catalytic conversion number (Kcat) among the cold active CEs reported so far. But are limited by the unique structure-function tradeoff of psychrophilic enzymes (Activity-Stability-AFFINITY TRADE-off), psyCE has a low affinity for the substrate lactose (higher Km). This defect leads to the limitation of the overall catalytic efficiency (Kcat/Km) in low substrate concentration or industrial practical application systems, and the difficulty in realizing high-efficiency low-temperature continuous conversion limits the industrialized popularization of the catalyst. Disclosure of Invention Aiming at the defects of low substrate affinity and limited comprehensive catalytic efficiency of the psychrophilic cellobiose 2-epimerase in the prior art, the invention obtains a series of psychrophilic cellobiose 2-epimerase mutants psyCE-FR with improved performance and application thereof through semi-rational design and high-throughput screening. A first object of the present invention is to provide cellobiose 2-epimerase mutants having high catalytic activity and high substrate affinity. The mutant is obtained by carrying out at least one mutation on the amino acid sequence of wild-type psychrophilic cellobiose 2-epimerase psyCE (the amino acid sequence is the sequence 1 and the gene sequence is the sequence 7): Asparagine (Asn) at position 309 is mutated to phenylalanine (Phe) or histidine (His); lysine (Lys) at position 189 is mutated to arginine (Arg). Preferably, the amino acid sequence of the mutant is selected from any one of the following sequences: mutant N309F, comprising only the mutation of Asn at position 309 to Phe, the amino acid sequence being sequence 2; mutant N309H only comprises 309 th Asn mutation to His, and the amino acid sequence is sequence 3; mutant K189R only comprises the mutation of Lys at position 189 to Arg, and the amino acid sequence is sequence 4; mutant psyCE-FR comprising a double mutation of Asn 309 to Phe and Lys 189 to Arg, the amino acid sequence being sequence 5; mutant psyCE-HR comprising a double mutation of Asn 309 to His and Lys 189 to Arg, the amino acid sequence being sequence 6. A second object of the present invention is to provide a gene encoding the mutant, the amino acid sequence of which is shown in the sequences 2 to 6. The third object of the present invention is to provide a recombinant expression vector and a recombinant engineering bacterium each comprising the above-mentioned gene. A fourth object of the present invention is to provide the use of said mutant enzyme in the preparation of epinastine, in particular in a low temperature dairy processing environment. Preferably, the low temperature is 4-10 ℃. Preferably, the indicated low temperature is 8 ℃. The fifth object of the invention is to provide a method for producing epinastine by using lactose or lactose-containing materials as substrates, and using the cellobiose 2-epimerase mutant as a catalyst to catalyze epimerization of lactose at the C-2 position under th