CN116426503A - Acid alpha-galactosidase KAG, and coding gene and application thereof - Google Patents
Acid alpha-galactosidase KAG, and coding gene and application thereof Download PDFInfo
- Publication number
- CN116426503A CN116426503A CN202310500336.3A CN202310500336A CN116426503A CN 116426503 A CN116426503 A CN 116426503A CN 202310500336 A CN202310500336 A CN 202310500336A CN 116426503 A CN116426503 A CN 116426503A
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- Prior art keywords
- galactosidase
- alpha
- acid alpha
- kag
- acid
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Abstract
The invention relates to the field of genetic engineering, in particular to an acid alpha-galactosidase KAG, and a coding gene and application thereof. The amino acid sequence is shown as SEQ ID NO. 1. The acid alpha-galactosidase can reach higher production level after being fermented by high-density liquid, and can be widely applied to the fields of feed, medicine, food and the like.
Description
Technical Field
The invention relates to the field of genetic engineering, in particular to an acid alpha-galactosidase KAG, and a coding gene and application thereof.
Background
Alpha-galactosidase (EC3.2.1.22) is capable of specifically hydrolysing non-reducing terminal alpha-1, 6-galactoside bonds of the class of galactosides. The enzymatic hydrolysis substrate mainly comprises raffinose oligosaccharides such as stachyose, raffinose and the like, and has good application prospect in the fields of feed, food, medicine and the like.
The feed raw materials such as soybean meal and miscellaneous meal usually contain alpha-galactoside substances, and because monogastric animals cannot produce enzymes for decomposing the alpha-galactoside, the existence of the alpha-galactoside substances increases the water holding capacity and permeability of the content of small intestine and reduces the digestion and absorption rate of nutrient components, and on the other hand, the alpha-galactoside substances are metabolized by anaerobic bacteria in the rear section of the digestive tract of the animals to produce carbon dioxide, hydrogen and a small amount of methane gas, thereby causing the symptoms such as dyspepsia, abdominal distension, diarrhea and the like of the animals. By exogenously adding alpha-galactosidase, the adverse effects of these alpha-galactosides can be effectively removed. The alpha-galactosidase is used as a novel enzyme preparation for feed, can effectively promote the decomposition of alpha-galactoside substances in cake and meal feed raw materials, improves the feed utilization rate, eliminates the anti-nutritional effect caused by the substances, and has good application prospect in the feed industry.
The alpha-galactosidase is used as an enzyme preparation for food to be applied to soymilk fermentation, and substances such as raffinose, stachyose and the like which cause gastric distention in the soymilk can be hydrolyzed to obtain a soymilk product with low alpha-galactosyl oligosaccharide content, which is beneficial to digestion and absorption of human bodies.
In clinic, galactosidase is useful for the treatment of briy's disease and blood group switching.
At present, the alpha-galactosidase is mainly prepared by microbial fermentation, but the fermentation level of the alpha-galactosidase is low at present, and the stability of the enzyme is also poor, so that the fermentation level of the enzyme and the stability of the enzyme are required to be improved.
Disclosure of Invention
The object of the present invention is to provide an acid alpha-galactosidase KAG.
Another object of the present invention is to provide a gene KGal encoding the above acid alpha-galactosidase.
It is another object of the present invention to provide a recombinant vector comprising the above-mentioned acid alpha-galactosidase.
It is another object of the present invention to provide a recombinant strain comprising the acid α -galactosidase gene KGal as described above.
It is another object of the present invention to provide a process for preparing the above acid alpha-galactosidase KAG.
It is a further object of the present invention to provide the use of an acid alpha-galactosidase KAG as described above.
The amino acid sequence of the acid alpha-galactosidase KAG is shown as SEQ ID NO. 1:
the acid alpha-galactosidase enzyme protein consists of 431 amino acids, has a molecular weight of 47.48kD and an isoelectric point of 4.68.
The nucleotide sequence of the gene KGal for encoding the acid alpha-galactosidase is shown as SEQ ID NO. 2:
the invention also provides recombinant vectors, preferably KGal-pPICZ alpha A, comprising the acid alpha-galactosidase gene KGal described above. As a most preferred embodiment of the present invention, the optimized acid alpha-galactosidase gene KGal of the present invention is inserted between EcoRI and NotI restriction sites on plasmid pPICZ alpha A, and the gene sequence is located downstream of and regulated by the AOX1 promoter to obtain recombinant yeast expression plasmid KGal-pPICZ alpha A.
The invention also provides a recombinant strain containing the acid alpha-galactosidase gene KGal, and the preferred expression host is a wild Pichia pastoris strain X33.
The method for efficiently expressing the acid alpha-galactosidase comprises the following steps:
1) Transforming pichia pastoris competent cells X33 by using the recombinant vector to obtain a recombinant strain KGal-pPICZ alpha A-X33;
2) Fermenting the recombinant strain obtained by screening, and inducing the expression of the acid alpha-galactosidase; and
3) And after the fermentation is finished, recovering and purifying the expressed acid alpha-galactosidase.
Wherein, the fermentation process of the recombinant strain can be divided into 3 stages:
the first stage is a thallus culturing stage, inoculating seeds according to the proportion of 10%, and culturing for 18-24 hours, wherein the rising of glycerol, pH and DO is marked;
the second stage is starvation stage, after glycerol is supplemented, no carbon source is added, and when dissolved oxygen rises to more than 80%, the stage is ended for about 30-60min;
the third stage is induction expression stage, and the induction culture medium is fed in, and dissolved oxygen is maintained at 20% or more, and the whole culture time is 180-200 hr.
After fermentation, removing thalli from the fermentation liquid, concentrating by an ultrafiltration membrane to obtain crude enzyme liquid, and then carrying out subsequent treatment.
The method of the invention can be used for efficiently expressing the acid alpha-galactosidase, can reach 1552U/mL in fermentation liquor, is greatly higher than the fermentation level of natural strains adopted in China, has good enzyme stability, greatly reduces the production cost of the alpha-galactosidase, and is beneficial to popularization and application of the alpha-galactosidase. The effective pH of the alpha-galactosidase is 1.5-7.0, the optimal reaction pH is 4.0, and the stability is good. Compared with some alpha-galactosidase products in the market, which are easy to inactivate and have poor stability, the alpha-galactosidase produced by the mutant strain has great advantages, can be suitable for the fields of feed, food, medicine and the like, and has good market application prospect.
Drawings
FIG. 1 is a graph of the fermentation process of recombinant acid alpha-galactosidase in a 50L tank;
FIG. 2 is an SDS-PAGE electropherogram of recombinant acid alpha-galactosidase;
FIG. 3 is a graph of the optimal reaction temperature for recombinant acid alpha-galactosidase;
FIG. 4 is an optimal reaction pH profile for recombinant acid alpha-galactosidase;
FIG. 5 shows the results of storage stability measurements of recombinant acid alpha-galactosidase at ambient temperature;
FIG. 6 shows the effect of various ions on the enzyme activity of recombinant acid alpha-galactosidase.
Detailed Description
Molecular biology experimental methods not specifically described in the following examples were carried out with reference to the specific methods listed in the guidelines for molecular cloning experiments (third edition), or according to the kit and product instructions; the reagents and biological materials, unless otherwise specified, are commercially available.
Experimental materials and reagents:
1. strain and vector
Coli strain Topl0, pichia X33, vector ppiczαa were all purchased from Invitrogen.
2. Enzyme and kit
RNA extraction kit and reverse transcriptase SuperScript TM III was purchased from Invitrogen. Plasmid DNA extraction kit, gel recovery kit, PCR purification kit, ligase, restriction enzyme were purchased from Shanghai Biotechnology.
3. Culture medium
Basal Salt Medium (BSM): ammonium dihydrogen phosphate 5%, potassium dihydrogen phosphate 0.5%, magnesium sulfate heptahydrate 1.5%, potassium sulfate 1.95%, calcium sulfate 0.1%, potassium hydroxide 0.1%, and defoamer 0.03%.
Sterilizing at 121deg.C under 0.1MPa for 30min, cooling, and adding 4.4 ml inorganic salt solution (PTM 1) per liter.
Microelement (PTM 1) preparation: copper sulfate 0.6%, potassium iodide 0.018%, manganese sulfate monohydrate 0.3%, sodium molybdate dihydrate 0.02%, boric acid 0.002%, cobalt chloride hexahydrate 0.05%, zinc chloride 2%, ferric sulfate heptahydrate 6.5%, concentrated sulfuric acid 0.5%, biotin 0.02%.
EXAMPLE 1 mutagenesis and fermentation culture of the naturally occurring species of alpha-galactosidase
Selecting AN aspergillus niger strain AN0803 (strain preservation number: ACCC 30132) for producing alpha-galactosidase, streaking on a PDA plate, culturing at 32 ℃ for 2-3 days until a large number of black spores grow, washing out spores with sterile water, performing multiple single and compound mutagenesis treatment by adopting ultraviolet rays, nitrosoguanidine, high-energy ion beams, diethyl sulfate (DES) and the like, and screening to obtain a mutant strain AN0803-M for producing the alpha-galactosidase.
The mutant strain is adopted for shake flask fermentation culture, and the culture medium comprises the following components:
0.5% of peptone, 2.5% of soybean meal powder, 2% of glucose, 0.25% of potassium dihydrogen phosphate and 0.10% of magnesium sulfate, and the fermentation culture is sterilized for 30min at 121 ℃ and 0.1MPa, and after cooling, AN0803-M strain is inoculated, and the culture is carried out for 4-5 days at 30 ℃ and 200 rpm.
After fermentation, filtering or centrifuging to remove thalli to obtain filtrate or centrifuging supernatant, and detecting the enzyme activity of the alpha-galactosidase. The result shows that the effective action pH of the alpha-galactosidase is 1.5-7.0, the optimal reaction pH is 4.0, and the stability is good.
EXAMPLE 2 cloning of alpha-galactosidase Gene from Aspergillus niger mutant (AN 0803-M)
Extracting total RNA of Aspergillus niger mutant strain (AN 0803-M) by RNA extraction kit according to reverse transcriptase SuperScript TM III Reverse Transcriptase procedure illustrates the synthesis of first strand cDNA. The cDNA is used as a template, and a primer is designed for PCR amplification.
The primers used for amplification were as follows:
GAL-F(EcoRI)：
5’GACAGAATTCATGCGGTGGCTTCTCGCACT 3’
GAL-R(NotI)：
5’GACAGCGGCCGCCTACACCACCAACGCGACAT3’
after the PCR is finished, the PCR product purification kit is adopted for purification, ecoRI and NotI double digestion are carried out on the purified product, then the purified product is connected with a vector pPICZ alpha A subjected to the same double digestion, the connected product is transformed into competent cells of escherichia coli Topl0, and positive clones are obtained through screening of antibiotics Zeocin.
The plasmid of the positive clone is extracted and sequenced, and the sequencing result shows that the full length of the gene encoding the alpha-galactosidase is 1296bp, and the gene encodes 431 amino acids. Meanwhile, the homology comparison of the gene sequence shows that the enzyme has 92% homology with an alpha-galactosidase gene derived from Aspergillus niger (CBS 101883). Recombinant expression vectors were constructed and designated KGal-pPICZαA.
EXAMPLE 3 construction of Pichia pastoris engineering bacteria containing acid alpha-galactosidase Gene KGal
Linearizing the recombinant expression vector KGal-pPICZ alpha A by adopting endonuclease SacI, performing electric shock transformation on pichia pastoris competent cells X33 by using the linearized recombinant vector, performing electric transformation, coating the electric shock transformation on a YPD plate containing Zeocin (addition concentration: 150-200 mu g/mL), and screening to obtain a pichia pastoris recombinant strain KGal-pPICZ alpha A-X33 with high yield of acid alpha-galactosidase.
EXAMPLE 4 high Density liquid fermentation culture of acid alpha-galactosidase recombinant engineering bacteria
The acid alpha-galactosidase recombinant engineering strain KGal-pPICZ alpha A-X33 obtained by screening in the example 3 is adopted for high-density liquid fermentation culture.
Preparing 20L basic salt culture medium (BSM culture medium), sterilizing at 121deg.C and 0.1MPa in 50L liquid fermenter for 30min, and cooling to room temperature. Ammonia water is used for regulating the pH value of fermentation liquor to be 4.60-4.80, 2L of shake flask liquid seeds are inoculated, the fermentation temperature is set to be 30 ℃, the rotating speed and the air flow of a fermentation tank are regulated, and the dissolved oxygen in the whole fermentation process is controlled to be more than 20%.
The whole fermentation process is divided into 3 stages:
the first stage is a thallus culturing stage, namely, 4L of sterilized glycerol with concentration of 50% is fed in, and the culturing is carried out for 20-24 hours, and the culturing is finished after the complete glycerol is fed in;
the second stage is a starvation stage, glycerin is fed completely, dissolved oxygen rises to more than 80%, and the beginning of the rising of pH indicates that the stage is finished for about 30 min;
the third stage is the induction expression stage, and the induction culture medium is fed in, and the dissolved oxygen is maintained at more than 20%.
The whole fermentation culture time is 180-200 hours.
After the induction starts, the enzyme activity detection is carried out by sampling at fixed time, the expression condition of the acid alpha-galactosidase in the whole fermentation process is shown as a figure 1, and the enzyme activity of fermentation liquor fermented for 190h is 1552U/mL.
Example 5 determination of enzymatic Properties of acid alpha-galactosidase
Definition of enzyme activity unit: 1mL of the enzyme solution or 1g of the solid enzyme powder was reacted at 37℃and pH5.5 for 5 minutes, and the amount of the enzyme required for releasing 1. Mu.M of p-nitrophenol per minute was defined as one enzyme activity unit (U).
Concentrating the fermentation supernatant by a 10kD ultrafiltration membrane, salting out, purifying the salting-out product by a Sephadex G-75 chromatographic column, collecting chromatographic liquid, concentrating, and measuring the following enzymatic properties:
1. SDS-PAGE electrophoresis
SDS-PAGE protein electrophoresis is carried out by adopting the chromatographic concentrate, after electrophoresis is finished, a clear band is displayed, the molecular weight is about 47kD, the molecular weight is consistent with the theoretical molecular weight of the enzyme, and the electrophoresis result is shown in figure 2.
2. Determination of optimum reaction temperature
According to the method for detecting alpha-galactosidase, 0.9mL of p-nitrophenol-alpha-D-galactopyranose (SIGMA) is respectively sucked into a plurality of test tubes, preheated for 3min at each set temperature, 0.1mL of enzyme solution is respectively added, uniformly mixed, reacted for 5min at each set temperature, 10% of sodium carbonate solution is added, uniformly mixed to finish the reaction, then enzyme activity is detected at 405nm by utilizing a spectrophotometer, and the highest enzyme activity is 100%, compared with the enzyme activity detected at other temperatures, so that the relative enzyme activity at the temperature is obtained.
The optimal reaction temperature curve of the acid alpha-galactosidase is shown in FIG. 3, and the optimal reaction temperature is about 40 ℃.
3. Determination of optimal reaction pH
A series of reaction pH values (1.5-7.0) were set, buffers of different pH values were prepared, and the enzyme solution was diluted with these buffers and 20mmol/L of p-nitrophenol-alpha-D-galactopyranose (p-NPG) was prepared as a reaction substrate. And respectively sucking 0.9mL of the p-NPG prepared under each pH condition into a plurality of test tubes, preheating for 3min at 37 ℃, shaking uniformly, adding 0.1mL of enzyme solution with each pH value, adding 10% sodium carbonate solution, mixing uniformly, ending the reaction, detecting the enzyme activity, and comparing other detection results with the highest enzyme activity of 100%, thus obtaining the relative enzyme activity under different pH detection conditions.
The optimal reaction pH curve of the acid alpha-galactosidase is shown in FIG. 4, and the result shows that the optimal reaction pH of the enzyme is about 4.0.
4. Determination of storage stability
The acidic alpha-galactosidase solid sample is stored at room temperature, sampled at fixed time according to month, and the enzyme activity of the sample with different preservation time is detected according to the detection method of the alpha-galactosidase, so as to determine the storage stability of the enzyme at room temperature.
The results of the stability measurement of the acid alpha-galactosidase are shown in FIG. 5. The detection result shows that: the residual enzyme activity of the acid alpha-galactosidase can still reach more than 85% after the acid alpha-galactosidase is preserved for one year at room temperature, which shows that the storage stability of the enzyme is good.
5. Effect of various ions on the Activity of acid alpha-galactosidase KAG
Preparation of 1mM K + 、Mg 2+ 、Ca 2+ 、Fe 2+ 、Fe 3+ 、Zn 2+ 、Mn 2+ 、Co 2+ 、Ag + 、Cu 2+ The test results are shown in FIG. 6, which shows that the acetic acid-sodium acetate buffer of (C) is mixed with an enzyme solution diluted in an appropriate ratio and left at room temperature for 1 hour, and the untreated enzyme solution is used as a control.
The results show that Ca 2+ 、Zn 2+ 、Mg 2+ Has certain activation effect on enzyme activity, fe 2+ 、K + 、Mn 2+ Has little influence on the enzyme activity, fe 3+ 、Co 2+ 、Ag + 、Cu 2+ The enzyme activity is inhibited to a certain extent.
The above embodiments are only used for explaining the technical solution of the present application, and do not limit the protection scope of the present application.
Claims (8)
1. The acid alpha-galactosidase KAG is characterized in that the amino acid sequence is shown as SEQ ID NO. 1.
2. An α -galactosidase gene encoding the acid α -galactosidase of claim 1.
3. The α -galactosidase gene of claim 2, wherein the nucleotide sequence of the α -galactosidase gene is shown in SEQ ID No. 2.
4. A recombinant vector comprising the α -galactosidase gene of claim 2.
5. A recombinant strain comprising the α -galactosidase gene of claim 2.
6. A process for preparing the acid α -galactosidase of claim 1, comprising the steps of:
1) The recombinant vector of claim 4 transformed into pichia pastoris X33, and the recombinant strain is obtained by screening;
2) Culturing the recombinant strain, and inducing acid alpha-galactosidase KAG to express; and
3) Recovering and purifying the expressed acid alpha-galactosidase KAG.
7. Use of an acid alpha-galactosidase KAG according to claim 1.
8. Use of the acid alpha-galactosidase KAG of claim 1 as a feed additive or as an enzyme preparation for food.
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