CN118086257A - Alpha-galactosidase mutants - Google Patents
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- CN118086257A CN118086257A CN202310595815.8A CN202310595815A CN118086257A CN 118086257 A CN118086257 A CN 118086257A CN 202310595815 A CN202310595815 A CN 202310595815A CN 118086257 A CN118086257 A CN 118086257A
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Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
The invention relates to the technical field of genetic engineering and protein modification, in particular to an 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.
Description
Technical Field
The invention belongs to the technical field of genetic engineering and protein modification, and particularly relates to an 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 an alpha-galactosidase mutant for solving the problems in the prior art; the mutant with obviously improved heat resistance is finally obtained through a large number of mutation screening on the basis of alpha-galactosidase AG2, and a foundation is laid for the wide use of the mutant in the feed field.
The present invention provides an alpha-galactosidase mutant comprising an amino acid sequence having at least 90% identity to SEQ ID NO. 2 and comprising a substitution of an amino acid at least one position selected from the group consisting of SEQ ID NO. 1 ：41、108、146、174、176、201、202、206、211、230、233、239、268、328、347、358、395、397、407、459、462、478、487、502、539、552、577、581、587、601、613、654、659、681、710.
In some embodiments of the invention, the amino acid sequence of the mutant has at least 91%,92%,93%,94%,95%,96%,97%,98%, or at least 99% identity as compared to SEQ ID NO. 2.
In some more specific embodiments, the amino acid sequence of the mutant has at least 99.1%,99.2%,99.3%,99.4%,99.5%,99.6%,99.7%,99.8%, or at least 99.9% identity as compared to SEQ ID NO. 2.
In some embodiments of the invention, the mutant comprises a substitution of at least one amino acid in the group consisting of ：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.
In some embodiments of the invention, the mutant comprises a combination of substitutions of at least one amino acid of the group consisting of:
S41H/I108V；
S41H/S146M；
S41H/A174W；
S41H/A176M；
S41H/N201Y；
S41H/V202P；
S41H/K206Y；
S41H/V211W；
S41H/A230L；
S41H/A268I；
S41H/T347K；
S41H/H358E；
S41H/T395K；
S41H/N407A；
S41H/N487L；
S41H/Q502C；
S41H/Q539Y；
S41H/A552P；
S41H/V587T；
S41H/G613F；
S41H/S654L；
S41H/Q659I；
S41H/Q659M；
S41H/Q681D；
S41H/S710R；
V211W/I108V；
V211W/S146M；
V211W/A174W；
V211W/A176M；
V211W/N201Y；
V211W/V202P；
V211W/K206Y；
V211W/A230L；
V211W/A268I；
V211W/T347K；
V211W/H358E；
V211W/T395K；
V211W/N407A；
V211W/N487L；
V211W/Q502C；
V211W/Q539Y；
V211W/A552P；
V211W/A581R；
V211W/V587T；
V211W/G613F；
V211W/S654L；
V211W/Q659I；
V211W/Q659M；
V211W/Q681D；
V211W/S710R；
A459C/I108V；
A459C/S146M；
A459C/A174W；
A459C/A176M；
A459C/N201Y；
A459C/V202P；
A459C/K206Y；
A459C/A230L；
A459C/A268I；
A459C/T347K；
A459C/H358E；
A459C/T395K；
A459C/N407A；
A459C/N487L；
A459C/Q502C；
A459C/Q539Y；
A459C/A552P；
A459C/A581R；
A459C/V587T；
A459C/G613F；
A459C/S654L；
A459C/Q659I；
A459C/Q659M；
A459C/Q681D；
A459C/S710R；
N487L/I108V；
N487L/S146M；
N487L/A174W；
N487L/A176M；
N487L/N201Y；
N487L/V202P；
N487L/K206Y；
N487L/A230L；
N487L/A268I；
N487L/T347K；
N487L/H358E；
N487L/T395K；
N487L/N407A；
N487L/Q502C；
N487L/Q539Y；
N487L/A552P；
N487L/A581R；
N487L/V587T；
N487L/G613F；
N487L/S654L；
N487L/Q659I；
N487L/Q659M；
N487L/Q681D；
N487L/S710R；
A581R/I108V；
A581R/S146M；
A581R/A174W；
A581R/A176M；
A581R/N201Y；
A581R/V202P；
A581R/K206Y；
A581R/A230L；
A581R/A268I；
A581R/T347K；
A581R/H358E；
A581R/T395K；
A581R/N407A；
A581R/A462Q；
A581R/Q502C；
A581R/Q539Y；
A581R/A552P；
A581R/V587T；
A581R/G613F；
A581R/S654L；
A581R/Q659I；
A581R/Q659M；
A581R/Q681D；
A581R/S710R；
S41H/I108V/S146M；
S41H/I108V/A174W；
S41H/S146M/A174W；
S41H/S146M/D233S；
S41H/S146M/S239H
I108V/S146M/N407A/V587T；
I108V/S146M/D233S/V587T；
I108V/S146M/V328I/H358E；
I108V/S146M/D233S/V397M；
I108V/S146M/A459C/Q502C；
A268I/H358E/N407A；
A268I/T347K/H358E/N407A；
A268I/T347K/H358E/T395K；
A268I/T347K/H358E/V587T；
A268I/T347K/H358E/N407A/A459C；
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；
S146M/H358E/N407A/A459C/Q502C/V587T；
S146M/A176M/H358E/N407A/A459C/Q502C/V587T；
S146M/N201Y/V202P/H358E/N407A/A459C/Q502C/V587T；
S146M/K206Y/H358E/N407A/A459C/Q502C/V587T；
S146M/V211W/H358E/N407A/A459C/Q502C/V587T；
S146M/A230L/H358E/N407A/A459C/Q502C/V587T；
S146M/T347K/H358E/N407A/A459C/Q502C/V587T；
A268I/H358E/N407A/A459C/Q502C/V587T；
A268I/T347K/H358E/N407A/A459C/Q502C/V587T；
A268I/T347K/H358E/T395K/N407A/A459C/Q502C/V587T；
A268I/T347K/H358E/N407A/A459C/A462D/Q502C/V587T；
A268I/T347K/H358E/N407A/A459C/T478K/Q502C/V587T；
A268I/T347K/H358E/N407A/A459C/N487L/Q502C/V587T；
A268I/T347K/H358E/N407A/A459C/Q502C/Q539Y/V587T；
A268I/T347K/H358E/N407A/A459C/Q502C/A552P/V587T；
H358E/N407A/A459C/Q502C/V587T；
H358E/T395K/N407A/A459C/Q502C/V587T；
H358E/N407A/A459C/A462D/Q502C/V587T；
H358E/N407A/A459C/T478K/Q502C/V587T；
H358E/N407A/A459C/N487L/Q502C/V587T；
H358E/N407A/A459C/Q502C/Q539Y/V587T；
H358E/N407A/A459C/Q502C/A552P/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/V587TK/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/V58
7TK/Q659M；
I108V/S146M/V211W/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/V
587T/Q681D；
I108V/S146M/V211W/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/V
587T/S710R；
I108V/S146M/V211W/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/V
587T/S654L/S710R；
I108V/S146M/V211W/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/V
587T/Q659I/S710R；
I108V/S146M/V211W/A268I/T347K/H358E/T395/N407A/A459C/Q502C/A552P/V58
7TK/Q659M/S710R；
I108V/S146M/V211W/A268I/T347K/H358E/T395K/N407A/A459C/Q502C/A552P/V
587T/Q681D/S710R；
S41H/I108V/S146M/N201Y/V202P/K206Y/V211W/H358E/N407A/A459C/Q502C/A5
52P/A581R/V587T/Q681D；
S41H/I108V/S146M/N201Y/V202P/K206Y/V211W/H358E/N407A/A459C/Q502C/A5
52P/V587T/Q659I/S710R。
the invention also relates to DNA molecules encoding the above alpha-galactosidase mutants.
The invention also relates to a recombinant expression vector comprising the DNA molecule.
The invention also relates to a host cell comprising the recombinant expression vector.
The host cell is Pichia pastoris.
The host cell is Aspergillus niger (Aspergillus niger).
The host cell is Trichoderma reesei (Trichoderma reesei).
The invention provides an alpha-galactosidase mutant 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 based on wild-type alpha-galactosidase AG 2. Compared with alpha-galactosidase AG2, the heat resistance of the mutant provided by the invention is obviously improved. The mutant containing the single mutation site provided by the invention has the enzyme activity residual rate as high as 40.42-72.06% after being treated for 3min at 67 ℃, and is improved by 10.47-96.94% compared with a wild type; the mutant containing at least two mutation sites provided by the invention has the advantage that the enzyme activity residual rate reaches 24.92-64.21% after being treated for 3min at 68 ℃, and is improved by 113.4-449.7% compared with a wild type.
In conclusion, compared with the alpha-galactosidase AG2, the heat resistance of the single-point mutant and the combined mutant provided by the invention is obviously improved, so that the wide application of the alpha-galactosidase in feeds is facilitated.
Drawings
Fig. 1: recombinant plasmid pPIC9K-AG2 map.
Detailed Description
The process according to the invention is further illustrated by the following examples. The experimental procedures, which are not specified in the following examples, may be generally performed under conventional conditions, such as those described in the molecular cloning experimental guidelines written by j. The present invention may be better understood and appreciated by those skilled in the art by reference to the examples. The protection and scope of the claims is not limited to the specific examples provided, but rather includes the protection that can be extended by a person skilled in the art without the inventive effort on the basis of the present description.
Experimental materials and reagents:
Strains and vectors: coli DH 5. Alpha., pichia pastoris GS115, vector pPIC9k were purchased from Invitrogen.
Enzyme and kit: the PCR enzyme and the ligase were purchased from Takara, the restriction enzyme from Fermentas, the plasmid extraction kit and the gel purification recovery kit from Omega, and the GeneMorph II random mutagenesis kit from Beijing Bomeis Biotechnology Co.
The formula of the culture medium comprises:
Coli medium (LB medium): 0.5% yeast extract, 1% peptone, 1% NaCl, pH7.0;
LB+Amp Medium: 100 mug/mL ampicillin was added to LB medium;
Lb+kana medium: LB medium plus 50. Mu.g/mL kanamycin;
Yeast Medium (YPD Medium): 1% yeast extract, 2% peptone, 2% glucose;
Yeast screening medium (MD medium): 2% glucose, 1.34% YNB, 4× -5% biotin and 2% agar powder;
BMGY medium: 2% peptone, 1% yeast extract, 100mM potassium phosphate buffer (pH 6.0), 1.34% YNB, 4X 10 -5% biotin, 1% glycerol;
BMMY medium: 2% peptone, 1% yeast extract, 100mM potassium phosphate buffer (pH 6.0), 1.34% YNB, 4X 10 -5% biotin, 0.5% methanol;
The present invention will be described in detail with reference to examples.
EXAMPLE 1 Synthesis of alpha-galactosidase Gene and obtaining of recombinant plasmid
The alpha-galactosidase gene was designated AG2, the nucleotide sequence of which was SEQ ID NO:1, which encodes an amino acid sequence of SEQ ID NO:2. the gene was synthesized by Shanghai JieRui bioengineering Co.
Designing a PCR primer containing an EcoRI endonuclease site according to the 5 'end of the gene, and designing a PCR primer containing a NotI endonuclease site according to the 3' end, wherein the primer sequences are as follows:
5' -terminal primer AG2-F: GGCGAATTCCCCCTGCTATTGGTGCTTCTAA (restriction enzyme EcoRI recognition site underlined);
3' -terminal primer AG2-R: ATAGCGGCCGCTTATTGTCTTTCAAGAAAAACAAC (underlined are restriction endonuclease NotI recognition sites).
The synthesized alpha-galactosidase AG2 gene SEQ ID NO:1 as a template, and carrying out PCR amplification by using the primer, wherein a PCR amplification system is as follows: 1. Mu.L of template, 1. Mu.L of upstream primer AG 2-F1. Mu.L of downstream primer AG 2-R1. Mu.L, 5X PSBuffer, 10. Mu. L, dNTPs (2.5 mM) 4. Mu. L, primer-StarDNA polymerase 1. Mu. L, ddH 2 O32. Mu.L, and 50. Mu.L of total reaction system. The PCR cycling program was: pre-denaturation at 95 ℃ for 5min,30cycles:94 ℃ for 30sec, 55 ℃ for 30sec, 72 ℃ for 2min and 72 ℃ for 10min; and (3) recovering PCR products by using glue, carrying out enzyme digestion treatment on EcoRI and NotI, then connecting the PCR products with pPIC-9K vectors subjected to the same enzyme digestion at 16 ℃ overnight, converting the PCR products into escherichia coli DH5a, coating the escherichia coli DH5a on LB+Amp plates, carrying out inversion culture at 37 ℃, verifying positive clones by using colony PCR after the transformants appear, and finally obtaining the correct recombinant plasmid pPIC9K-AG2 after sequencing verification (figure 1).
EXAMPLE 2 alpha-galactosidase mutant screening
To further increase the thermostability of α -galactosidase AG2, the synthesized α -galactosidase AG2 gene was subjected to protein structural analysis, which has three domains: the 285 amino acid residues at the N end form a structural domain 1, the 349 amino acid residues at the middle form a structural domain 2, the 74 amino acid residues at the C end form a structural domain 3, the conserved sequence and the active center are both positioned in the structural domain 2, a large number of mutation sites of the gene are further screened on the premise of not damaging the secondary structure and the active center of the protein, and the effect is verified by experiments:
PCR primers AG2-F1, AG2-R1 were designed:
AG2-F1: GGCCCATGGCCCCTGCTATTGGTGCTTCTAA (underlined is the restriction endonuclease NcoI recognition site);
AG2-R1: GGCCTCGAGTTATTGTCTTTCAAGAAAAACAAC (restriction enzyme XhoI recognition site underlined).
The method comprises the steps of taking an alpha-galactosidase gene AG2 as a template, carrying out PCR amplification by using a GeneMorph II random mutation PCR kit (Stratagene) by using the primers, recovering PCR products by using glue, carrying out enzyme digestion treatment on NcoI and XhoI, connecting with a pET-28a carrier subjected to the same enzyme digestion, converting into escherichia coli BL21 (DE 3), coating the escherichia coli with an LB+kana plate, carrying out inversion culture at 37 ℃, after the transformant appears, picking up the transformant into 96-well plates one by using toothpicks, adding 150ul of LB+kana culture medium containing 0.1mM IPTG into each well, carrying out culture at 37 ℃ at 220rpm for about 6 hours, centrifuging, discarding supernatant, carrying out heavy suspension on thalli by using a buffer solution, and carrying out repeated freeze thawing wall breaking to obtain escherichia coli cell lysate containing alpha-galactosidase.
40Ul of lysate was removed separately to two new 96 well plates, one of which was treated at 65℃for 10min, 40ul of substrate (3 mg/ml p-nitrophenol-. Alpha. -D-galactopyranoside solution) was added to both 96 well plates, reacted at 37℃for 10min, 160ul of stop solution (0.5 mol/L Na 2CO3 reagent) was added, absorbance was measured at 400nm, and the activities maintained after high temperature treatment of the different mutants were different.
Experimental results show that some mutations have no effect on the heat resistance of the alpha-galactosidase AG2 protein, and some mutations even make the heat resistance or the enzyme activity worse; in addition, some mutations, although improving the temperature tolerance of the alpha-galactosidase AG2 protein, have significantly changed enzymatic properties after mutation, and all are not satisfactory. Finally, the mutation site which can not only remarkably improve the heat resistance of the alpha-galactosidase AG2 but also not affect the enzyme activity and the original enzymatic properties of the alpha-galactosidase AG2 is obtained ：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.
The invention provides mutants respectively comprising 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 single mutation sites on the basis of alpha-galactosidase AG 2.
The invention also provides mutants comprising a combination of two or more mutation sites selected from the above heat resistant mutation sites, which have a further improved heat resistance than the corresponding single point mutants.
EXAMPLE 3 construction of Pichia pastoris engineering strains
3.1 Construction of expression vectors
The gene sequences of the alpha-galactosidase AG2 and mutants thereof are respectively optimized according to the password preference of pichia pastoris, the alpha-galactosidase AG2 and mutants thereof are synthesized by Shanghai Jierui bioengineering Co., ltd, and two enzyme cutting sites of EcoRI and NotI are respectively added at the 5 'end and the 3' end of the synthesized sequence.
The mutants are respectively used as templates, primers AG2-F, AG2-R are used for amplification, PCR products are recovered by gel, ecoRI and NotI are subjected to enzyme digestion treatment and then are connected with pPIC-9k carriers subjected to the same enzyme digestion at 16 ℃ overnight, escherichia coli DH5a is transformed, the obtained product is coated on LB+Amp plates, the product is subjected to inversion culture at 37 ℃, positive cloning is verified by colony PCR after the appearance of the transformant, and the correct recombinant plasmid is finally obtained after sequencing verification.
3.2 Yeast competent preparation
Performing YPD plate activation on Pichia pastoris GS115 strain, culturing at 30 ℃ for 48 hours, inoculating activated GS115 monoclonal in 5mL YPD liquid culture medium, culturing at 30 ℃ for about 18 hours at 220rpm, transferring bacterial liquid into a triangular flask filled with 50mL YPD liquid culture medium, culturing at 30 ℃ for about 5 hours at 220rpm, detecting bacterial density by an ultraviolet spectrophotometer, respectively collecting 5mL bacterial bodies into a sterilized EP tube after OD600 value is in a range of 1.1-1.3, centrifuging at 6000rpm for 3 minutes at 4 ℃, slightly discarding supernatant, sucking residual supernatant with sterilized filter paper, re-suspending bacterial bodies with pre-cooled 2mL sterilized water, centrifuging at 6000rpm for 3 minutes at 4 ℃, slightly discarding supernatant, re-suspending bacterial bodies with pre-cooled 2mL sorbitol (1 mol/L); centrifuge at 6000rpm for 3min at 4℃and gently discard the supernatant, gently resuspend the cells with 100-150. Mu.l sorbitol (1 mol/L) pre-chilled.
3.3 Transformation and screening
The recombinant expression plasmids obtained in example 2 were linearized with Sac I, the linearized fragments were purified and recovered, and transformed into Pichia pastoris GS115 by electroporation, and the recombinant Pichia pastoris strains were obtained by screening on MD plates. The single transformant was picked up and transferred to BMGY medium, after shaking culture at 30℃and 250rpm for 1d, transferred to BMMY medium, shaking culture at 30℃and 250rpm, and 0.5% methanol was added every day. After 3d of induction expression, the cells were removed by centrifugation to obtain a supernatant containing α -galactosidase, and the activity of α -galactosidase was measured.
The result shows that the activity of the alpha-galactosidase in the fermentation supernatant of the recombinant pichia pastoris strain for recombinant expression of the alpha-galactosidase AG2 and the mutant thereof constructed by the invention is 1140-2020U/ml.
Method for measuring enzyme activity of alpha-galactosidase
(1) Definition of enzyme Activity Unit
The amount of enzyme required to release 1. Mu. Mol of p-nitrophenol per minute from a 1.5mg/ml p-nitrophenol-alpha-D-galactopyranoside solution at 37℃and pH 5.0 is one enzyme activity unit U.
(2) Measurement method
Taking 0.5ml of p-nitrophenol-alpha-D-galactopyranoside solution with the concentration of 3mg/ml, adding the solution into a colorimetric tube, balancing for 5min at 37 ℃, adding 0.5ml of alpha-galactosidase enzyme solution which is properly diluted by disodium hydrogen phosphate-citric acid buffer solution with the pH of 5.0 and balanced at 37 ℃, uniformly mixing, and accurately preserving the temperature at 37 ℃ for reaction for 10min. After the reaction was completed, 4ml of Na 2CO3 reagent at a concentration of 0.5mol/L was added and mixed well to terminate the reaction. Then cooling to room temperature, taking a standard blank as a blank control, measuring an absorbance value A E at 400nm, and calculating a corresponding ug number R= (A E -b)/K of the p-nitrophenol by using a regression equation according to a p-nitrophenol standard curve.
Enzyme activity calculation formula: x D = (r×n)/(10×0.5× 139.11).
Wherein: x D is the activity of alpha-galactosidase in the enzyme solution, U/ml; r is calculated p-nitrophenol ug number; 10 is the reaction time of 10min;0.5 ml of added enzyme liquid is 0.5ml; n is the dilution factor of the enzyme solution; 139.11 is the molar mass of p-nitrophenol, 139.11g/mol.
EXAMPLE 4 thermostable assay of alpha-galactosidase mutants
The fermentation supernatant of the recombinant Pichia pastoris strain expressing the alpha-galactosidase mutant constructed in example 3 was diluted to 200U/mL of alpha-galactosidase enzyme activity with a pre-heated disodium hydrogen phosphate-citric acid buffer solution of pH5.0 for 10min, and the mixture was uniformly mixed, treated at 67℃for 3min, sampled and cooled to room temperature at the end, and then the alpha-galactosidase activity was measured, and the enzyme activity residual rate was calculated as 100% of the enzyme activity of the untreated sample. The specific results are shown in Table 1.
Enzyme activity residual ratio (%) =enzyme activity after heat treatment/enzyme activity before heat treatment×100%.
TABLE 1 alpha-galactosidase Single Point mutant thermotolerance assay
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As shown in Table 1, compared with the wild type alpha-galactosidase AG2, the heat resistance of the alpha-galactosidase mutants respectively containing 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 single mutation sites is obviously improved. After the treatment is carried out at 67 ℃ for 3min, the enzyme activity residual rate of the galactosidase single-point mutant reaches 40.42-72.06%, and is improved by 10.47-96.94% compared with a wild type, so that unexpected technical effects are achieved.
In addition, based on the wild type alpha-galactosidase AG2, the S41H/I108V, S H/S146M, A459C/Q502C, S H/A174W two-point mutant provided by the invention; S41H/I108V/S146M, S H/I108V/A174W, S H/S146M/A174W three-point mutant ;S41H/I108V/S146M/A174W、S41H/I108V/S146M/D233S、S41H/I108V/S146M/N407A、S41H/I108V/S146M/V328I、S41H/I108V/S146M/A174W four-point mutant; S41H/I108V/S146M/A174W/D233S, S H/I108V/S146M/D233S/V397M, S H/I108V/S146M/A459C/Q502C five-point mutant; S41H/I108V/S146M/A174W/D233S/S239H six-point mutant; seven-point mutants of S41H/I108V/S146M/D233S/V397M/A459C/Q502C, after being treated for 3min at 68 ℃, the residual rate of the enzyme activity reaches 24.92-64.21%, and the heat resistance is obviously improved by 113.4-449.7% compared with a wild type.
In conclusion, compared with the alpha-galactosidase AG2, the heat resistance of the single-point mutant and the combined mutant provided by the invention is obviously improved, so that the wide application of the alpha-galactosidase in feeds is facilitated.
Claims (10)
1. An alpha-galactosidase mutant, characterized in that said mutant comprises an amino acid sequence having at least 90% identity to SEQ ID No. 2 and comprises a substitution of an amino acid at least one position selected from the group consisting of SEQ ID No. 2 ：41、108、146、174、176、201、202、206、211、230、233、239、268、328、347、358、395、397、407、459、462、478、487、502、539、552、577、581、587、601、613、654、659、681、710.
2. The α -galactosidase mutant of claim 1, wherein the amino acid sequence of said mutant is at least 91%,92%,93%,94%,95%,96%,97%,98%, or at least 99% identical to SEQ ID No. 2.
3. The α -galactosidase mutant of claim 2, wherein the amino acid sequence of said mutant has at least 99.1%,99.2%,99.3%,99.4%,99.5%,99.6%,99.7%,99.8%, or at least 99.9% identity as compared to SEQ ID No. 2.
4. The α -galactosidase mutant of claim 1, wherein the mutant comprises a substitution of at least one amino acid from the group consisting of ：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.
5. The α -galactosidase mutant of claim 1, wherein the mutant comprises a combination of substitutions of at least one amino acid from the group consisting of:
S41H/I108V；
S41H/S146M；
S41H/A174W；
S41H/A176M；
S41H/N201Y；
S41H/V202P；
S41H/K206Y；
S41H/V211W；
S41H/A230L；
S41H/A268I；
S41H/T347K；
S41H/H358E；
S41H/T395K；
S41H/N407A；
S41H/N487L；
S41H/Q502C；
S41H/Q539Y；
S41H/A552P；
S41H/V587T；
S41H/G613F；
S41H/S654L；
S41H/Q659I；
S41H/Q659M；
S41H/Q681D；
S41H/S710R；
V211W/I108V；
V211W/S146M；
V211W/A174W；
V211W/A176M；
V211W/N201Y；
V211W/V202P；
V211W/K206Y；
V211W/A230L；
V211W/A268I；
V211W/T347K；
V211W/H358E；
V211W/T395K；
V211W/N407A；
V211W/N487L；
V211W/Q502C；
V211W/Q539Y；
V211W/A552P；
V211W/A581R；
V211W/V587T；
V211W/G613F；
V211W/S654L；
V211W/Q659I；
V211W/Q659M；
V211W/Q681D；
V211W/S710R；
A459C/I108V；
A459C/S146M；
A459C/A174W；
A459C/A176M；
A459C/N201Y；
A459C/V202P；
A459C/K206Y；
A459C/A230L；
A459C/A268I；
A459C/T347K；
A459C/H358E；
A459C/T395K；
A459C/N407A；
A459C/N487L；
A459C/Q502C；
A459C/Q539Y；
A459C/A552P；
A459C/A581R；
A459C/V587T；
A459C/G613F；
A459C/S654L；
A459C/Q659I；
A459C/Q659M；
A459C/Q681D；
A459C/S710R；
N487L/I108V；
N487L/S146M；
N487L/A174W；
N487L/A176M；
N487L/N201Y；
N487L/V202P；
N487L/K206Y；
N487L/A230L；
N487L/A268I；
N487L/T347K；
N487L/H358E；
N487L/T395K；
N487L/N407A；
N487L/Q502C；
N487L/Q539Y；
N487L/A552P；
N487L/A581R；
N487L/V587T；
N487L/G613F；
N487L/S654L；
N487L/Q659I；
N487L/Q659M；
N487L/Q681D；
N487L/S710R；
A581R/I108V；
A581R/S146M；
A581R/A174W；
A581R/A176M；
A581R/N201Y；
A581R/V202P；
A581R/K206Y；
A581R/A230L；
A581R/A268I；
A581R/T347K；
A581R/H358E；
A581R/T395K；
A581R/N407A；
A581R/A462Q;
A581R/Q502C；
A581R/Q539Y；
A581R/A552P；
A581R/V587T；
A581R/G613F；
A581R/S654L；
A581R/Q659I；
A581R/Q659M；
A581R/Q681D；
A581R/S710R；
S41H/I108V/S146M；
S41H/I108V/A174W；
S41H/S146M/A174W；
S41H/S146M/D233S；
S41H/S146M/S239H
I108V/S146M/N407A/V587T；
I108V/S146M/D233S/V587T；
I108V/S146M/V328I/H358E；
I108V/S146M/D233S/V397M；
I108V/S146M/A459C/Q502C；
A268I/H358E/N407A；
A268I/T347K/H358E/N407A；
A268I/T347K/H358E/T395K；
A268I/T347K/H358E/ V587T；
A268I/T347K/H358E/N407A/A459C；
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；
S146M/H358E/N407A/A459C/Q502C/V587T ；
S146M/A176M/H358E/N407A/A459C/Q502C/V587T；
S146M/N201Y/V202P/H358E/N407A/A459C/Q502C/V587T；
S146M/K206Y/H358E/N407A/A459C/Q502C/V587T；
S146M/V211W/H358E/N407A/A459C/Q502C/V587T；
S146M/A230L/H358E/N407A/A459C/Q502C/V587T；
S146M/T347K/H358E/N407A/A459C/Q502C/V587T；
A268I/H358E/N407A/A459C/Q502C/V587T；
A268I/T347K/H358E/N407A/A459C/Q502C/V587T；
A268I/T347K/H358E/T395K/N407A/A459C/Q502C/V587T；
A268I/T347K/H358E/N407A/A459C/A462D/Q502C/V587T ；
A268I/T347K/H358E/N407A/A459C/T478K/Q502C/V587T；
A268I/T347K/H358E/N407A/A459C/N487L/Q502C/V587T；
A268I/T347K/H358E/N407A/A459C/Q502C/Q539Y/V587T；
A268I/T347K/H358E/N407A/A459C/Q502C/A552P/V587T；
H358E/N407A/A459C/Q502C/V587T；
H358E/T395K/N407A/A459C/Q502C/V587T；
H358E/N407A/A459C/A462D/Q502C/V587T ；
H358E/N407A/A459C/T478K/Q502C/V587T；
H358E/N407A/A459C/N487L/Q502C/V587T；
H358E/N407A/A459C/Q502C/Q539Y/V587T；
H358E/N407A/A459C/Q502C/A552P/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/V587TK/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/V587TK/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/V587TK/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。
6. a DNA molecule encoding the α -galactosidase mutant according to any one of claims 1 to 5.
7. A recombinant expression plasmid comprising the DNA molecule of claim 6.
8. A host cell comprising the recombinant expression plasmid of claim 7; the host cell is a non-plant cell.
9. The host cell of claim 8, wherein the host cell is any one of Pichia pastoris, aspergillus niger (Aspergillus niger) or trichoderma reesei (Trichoderma reesei).
10. Use of the α -galactosidase mutant according to any one of claims 1 to 5 in feed production.
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