CN-122012466-A - Heat-resistant alpha-galactosidase mutant

Abstract

The invention relates to the technical field of genetic engineering and protein modification, in particular to a heat-resistant alpha-galactosidase mutant. The invention provides alpha-galactosidase mutants, based on alpha-galactosidase AG2, each comprising at least one mutation site selected from S41H, I108V, S146M, A174W, A176M, N201Y, V202P, K206Y, V211W, A230L D233S, S239H, A268I, V328I, T347K, T347L, T347Q, H358E, T395K, T395R, V397M, N407A, A459C, A462D, A462Q, T478K, N487L, Q502C, Q539M, Q539Y, A552P, A577S, A581P, A581R, V587T, S601C, G613F, S654L, Q659I, Q659K, Q659M, Q681D, Q681S, S710R . Compared with the wild type, the heat resistance of the mutant is obviously improved, and the mutant is favorable for wide application in feed.

Inventors

• CAO TISHUANG
• LU NA
• XU XIAODONG
• WU XIUXIU

Assignees

• 青岛蔚蓝生物集团有限公司

Dates

Publication Date

20260512

Application Date

20230525

Priority Date

20221125

Claims (6)

1. 1. An alpha-galactosidase mutant, characterized in that the mutant is an alpha-galactosidase having the amino acid sequence of SEQ ID No. 2 comprising a substitution or combination of substitutions of any one of the amino acids of the group: I108V; S41H/I108V; V211W/I108V; A459C/I108V; N487L/I108V; A581R/I108V; S41H/I108V/S146M; S41H/I108V/A174W; I108V/S146M/N407A/V587T; I108V/S146M/D233S/V587T; I108V/S146M/V328I/H358E; I108V/S146M/D233S/V397M; I108V/S146M/A459C/Q502C; S41H/I108V/S146M/A174W; S41H/I108V/S146M/A174W/D233S; S41H/I108V/S146M/A174W/D233S/S239H; S41H/I108V/S146M/D233S; S41H/I108V/S146M/V328I; S41H/I108V/S146M/H358E; S41H/I108V/S146M/N407A; S41H/I108V/S146M/V587T; S41H/I108V/S146M/N407A/V587T; S41H/I108V/S146M/D233S/V587T; S41H/I108V/S146M/V328I/H358E; S41H/I108V/S146M/D233S/V397M; S41H/I108V/S146M/A459C/Q502C; S41H/I108V/S146M/V328I/A459C/Q502C; S41H/I108V/S146M/H358E/A459C/Q502C; S41H/I108V/S146M/V328I/H358E/A459C/Q502C; S41H/I108V/S146M/D233S/V397M/V587T; S41H/I108V/S146M/N407A/A459C/Q502C; S41H/I108V/S146M/D233S/V397M/A459C/Q502C; S41H/I108V/S146M/H358E/N407A/V587T; S41H/I108V/S146M/N407A/A459C/Q502C/V587T; S41H/I108V/S146M/H358E/N407A/A459C/Q502C/V587T ; I108V/S146M/V328I/A459C/Q502C; I108V/S146M/H358E/A459C/Q502C; I108V/S146M/V328I/H358E/A459C/Q502C; I108V/S146M/D233S/V397M/V587T; S41H/T95K/I108V/S146M/T347K/H358E/N407A/A459C/Q502C/V587T; S41H/I108V/S146M/T347K/H358E/N407A/A459C/A462D/Q502C/V587T; S41H/I108V/S146M/T347K/H358E/N407A/A459C/T478K/Q502C/V587T; S41H/I108V/S146M/T347K/H358EN407A/A459C/N487L/Q502C/V587T; S41H/I108V/S146M/T347K/H358E/N407A/A459C/Q502C/Q539Y/V587T; S41H/I108V/S146M/T347K/H358E/N407A/A459C/Q502C/A552P/V587T; S41H/I108V/S146M/A176M/H358E/N407A/A459C/Q502C/V587T; S41H/I108V/S146M/N201Y/V202P/H358E/N407A/A459C/Q502C/V587T; S41H/I108V/S146M/K206Y/H358E/N407A/A459C/Q502C/V587T; S41H/I108V/S146M/V211W/H358E/N407A/A459C/Q502C/V587T; S41H/I108V/S146M/A230L/H358E/N407A/A459C/Q502C/V587T; S41H/I108V/S146M/A268I/H358E/N407A/A459C/Q502C/V587T; S41H/I108V/S146M/T347K/H358E/N407A/A459C/Q502C/V587T; S41H/I108V/S146M/A268I/T347K/H358E/N407A/A459C/Q502C/V587T; S41H/I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/V587T; S41H/I108V/S146M/A268I/T347K/H358E/N407A/A459C/A462D/Q502C/V587T; S41H/I108V/S146M/A268I/T347K/H358E/N407A/A459C/T478K/Q502C/V587T; S41H/I108V/S146M/A268I/T347K/H358E/N407A/A459C/N487L/Q502C/V587T; S41H/I108V/S146M/A268I/T347K/H358E/N407A/A459C/Q502C/Q539Y/V587T; S41H/I108V/S146M/A268I/T347K/H358E/N407A/A459C/Q502C/A552P/V587T; S41H/I108V/S146M/N201Y/V202P/K206Y/V211W/H358E/N407A/A459C/Q502C/V587T; S41H/I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/N487L/Q502C/A552P/V587T; I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/A462D/Q502C/V587T; I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/V587T; I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/V587T/S710R; I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/N487L/Q502C/A552P/V587T; I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/V587T/G613F/S710R; I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/V587T/S654L/S710R; I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/V587T/Q659I/S710R; I108V/S146M/A268I/T347K/H358E/T395/N407A/A459C/Q502C/V587TK/Q659M/S710R; I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/V587T/Q681D/S710R; S41H/I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/N487L/Q502C/V587T; S41H/I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/A462D/Q502C/V587T; S41H/I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/V587T; S41H/I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/V587T/G613F; S41H/I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/V587T/S654L; S41H/I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/V587T/Q659I; S41H/I108V/S146M/A268I/T347K/H358E/T395/N407A/A459C/Q502C/V587TK/Q659M; S41H/I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/V587T/Q681D; S41H/I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/V587T/S710R; S41H/I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/V587T/Q659I; S41H/I108V/S146M/A268I/T347K/H358E/T395/N407A/A459C/Q502C/A552P/V587T/Q659M; S41H/I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/V587T/Q681D; S41H/I108V/S146M/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/V587T/S710R; I108V/S146M/V211W/A268I/T347K/H358E/T395K/N407A/A459C/N487L/Q502C/A552P/V587T; I108V/S146M/V211W/A268I/T347K/H358E/T395K/N407A/A459C/A462D/Q502C/A552P/V587T; I108V/S146M/V211W/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/A552P/V587T; I108V/S146M/V211W/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/V587T/G613F; I108V/S146M/V211W/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/V587T/S654L; I108V/S146M/V211W/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/V587T/Q659I; I108V/S146M/V211W/A268I/T347K/H358E/T395/N407A/A459C/Q502C/A552P/V587T/Q659M; I108V/S146M/V211W/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/V587T/Q681D; I108V/S146M/V211W/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/V587T/S710R; I108V/S146M/V211W/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/V587T/S654L/S710R; I108V/S146M/V211W/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/V587T/Q659I/S710R; I108V/S146M/V211W/A268I/T347K/H358E/T395/N407A/A459C/Q502C/A552P/V587T/Q659M/S710R; I108V/S146M/V211W/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/V587T/Q681D/S710R; S41H/I108V/S146M/N201Y/V202P/K206Y/V211W/H358E/N407A/A459C/Q502C/A552P/A581R/V587T/Q681D; S41H/I108V/S146M/N201Y/V202P/K206Y/V211W/H358E/N407A/A459C/Q502C/A552P/V587T/Q659I/S710R。
2. 2. A DNA molecule encoding the α -galactosidase mutant of claim 1.
3. 3. A recombinant expression plasmid comprising the DNA molecule of claim 2.
4. 4. A host cell comprising the recombinant expression plasmid of claim 3, wherein the host cell is a non-plant cell.
5. 5. The host cell of claim 4, wherein the host cell is any one of Pichia pastoris, aspergillus niger (Aspergillus niger) or Trichoderma reesei (Trichoderma reesei).
6. 6. Use of the α -galactosidase mutant of claim 1 in feed production.

Description

Heat-resistant alpha-galactosidase mutant Technical Field The invention belongs to the technical field of genetic engineering and protein modification, and particularly relates to a heat-resistant alpha-galactosidase mutant. Background Alpha-galactosidase, also known as melibiase (alpha-galactosidase, EC 3.2.1.22), catalyzes the removal of alpha-1, 6-linked galactose residues from different galactose substrates. Alpha-galactosidase is widely found in nature, and is most widely distributed among microorganisms such as bacteria, fungi, yeasts, and the like. The bacterial sources of the alpha-galactosidase include Bacillus thermophilus, lactobacillus acidophilus, lactobacillus fermentum, bifidobacterium adolescentis, bifidobacterium breve, etc. In addition, alpha-galactosidase is also isolated from the extremely thermophilic bacterium Dunaliella and the marine bacterium Pseudomonas. It has been reported that the filamentous actinomycetes also contain an alpha-galactosidase, which is isolated from, for example, streptomyces rubrum and Saccharopolyspora rubra. Brouns and the like can also be separated and purified to obtain alpha-galactosidase from the hyperthermophilic archaea sulphur mine sulfolobus P2 found in high acidity land volcanic areas, and the alpha-galactosidase has extremely high heat stability. In addition, among fungi, aspergillus oryzae, aspergillus fumigatus, gibberella caner, penicillium simplicissimum, mucor thermophilus, and Rhizopus oligosporus can be used as sources of alpha-galactosidase. Wang et al isolated an alpha-galactosidase from Fishcet's disease, and the results of the study showed that it had highly specific hydrolytic activity towards bean products. Soybeans and other beans are used as rich protein sources in feeds, and contain high concentration of soluble oligosaccharides such as raffinose, stachyose and the like, and the soluble oligosaccharides cannot be thoroughly digested in the gastrointestinal tract of animals, and after the undigested complete saccharides enter the intestinal tract of the animals, harmful flora growth is promoted, flatulence and gastrointestinal disturbance are caused, the health of the animals is seriously damaged, and the feeding efficiency of the animal feeds is reduced. Therefore, the alpha-galactosidase preparation is added into the animal feed to thoroughly remove the redundant sugar, inhibit the fermentation in the intestinal tracts, relieve the flatulence symptom of the animal gastrointestinal tracts, avoid the damage of harmful pathogenic bacteria to the animal intestinal tracts, improve the immunity of the animal, increase the absorption of nutrient substances by the animal intestinal tracts and improve the utilization rate of the feed. For example, baucells and the like are added into the feed of growing pigs and fattening pigs, and research results show that compared with a control group, the weight of the growing pigs and the fattening pigs of a test group is obviously increased, and various indexes are obviously improved. Ghazi and the like are added into the soybean feed of the broiler chickens, and the result shows that the digestion efficiency of the broiler chickens on the soybean feed is improved, and the nutritional value of the soybean feed is increased. Dai Qiuzhong and the like are added into the feed to study the influence of the feed on the production performance of yellow-feather broilers, and the result shows that the feed can degrade the alpha-galactoside in the feed, change the composition of intestinal substrates, promote the growth of beneficial bacteria, inhibit the growth of harmful bacteria, improve the growth condition of broilers, obviously reduce the feed-to-weight ratio of the later and whole stages of broilers and improve the production performance of experimental chickens after the enzyme preparation is added. Miao Zhijun and the like are added into the feed to research the influence of the alpha-galactosidase preparation on the weight gain and the production performance of the muscovy ducks, and the result shows that the alpha-galactosidase preparation can improve the weight gain and the production performance of the muscovy ducks. At present, a short high-temperature stage exists in the production process of pellet feed, alpha-galactosidase is directly added into animal feed for pelleting, the residual enzyme activity is extremely low, and after pelleting, the alpha-galactosidase is sprayed onto the feed or mixed into the feed, so that the equipment investment is increased, and the stability of an enzyme preparation and the uniformity of distribution in the feed cannot be well ensured. Therefore, it is important to improve the temperature resistance of α -galactosidase. Disclosure of Invention The invention provides a heat-resistant alpha-galactosidase mutant for solving the problems in the prior art, which is obtained by a large number of mutation screening on the basis of alpha-galactosidase AG2, and lays a fou