Alpha-galactosidase with enhanced activity

文档序号：802961 发布日期：2021-03-26 浏览：25次中文

阅读说明：本技术 具提升活性的α-半乳糖苷酶 (Alpha-galactosidase with enhanced activity ) 是由郑雅珊吴姿慧林正言赖惠琳黄婷沅郑成彬于 2019-09-25 设计创作，主要内容包括：本申请涉及一种具提升活性的α-半乳糖甘酶,其氨基酸序列是将SEQ ID NO：2第450个位置的精氨酸改为苯丙氨酸、缬氨酸、丝氨酸、丙氨酸、或甘氨酸取代修饰的序列。(The application relates to alpha-galactose glazyme with activity improvement, and the amino acid sequence of the alpha-galactose glazyme is shown in SEQ ID NO: 2 arginine at position 450 is changed to a phenylalanine, valine, serine, alanine, or glycine substitution modified sequence.)

1. An alpha-galactosyl glycase, the amino acid sequence of which is shown in SEQ ID NO: 2 arginine at position 450 is changed to a phenylalanine, valine, serine, alanine, or glycine substitution modified sequence.

2. The α -galactosyl glycinase of claim 1, wherein the polypeptide encoding the amino acid sequence of SEQ ID NO: 2 is the AnBgl36 gene isolated from Aspergillus niger (Aspergillus niger).

3. The α -galactosyl glycinase as claimed in claim 1, wherein the amino acid sequence thereof is as shown in SEQ ID NO: shown at 9.

4. The α -galactosyl glycinase as claimed in claim 1, wherein the amino acid sequence thereof is as shown in SEQ ID NO: shown at 11.

5. The α -galactosyl glycinase as claimed in claim 1, wherein the amino acid sequence thereof is as shown in SEQ ID NO: shown at 13.

6. The α -galactosyl glycinase as claimed in claim 1, wherein the amino acid sequence thereof is as shown in SEQ ID NO: shown at 15.

7. The α -galactosyl glycinase as claimed in claim 1, wherein the amino acid sequence thereof is as shown in SEQ ID NO: shown at 17.

Technical Field

The present application relates to an alpha-galactosidase, and more particularly to an alpha-galactosidase with enhanced activity.

Background

Alpha-galactosidase (alpha-galactosidase; EC 3.2.1.22) is an exohydrolase that is widely found in nature in microorganisms, plants, and animals. It can catalyze the hydrolysis of 1, 6-glycosidic bond on different alpha-galactoside oligosaccharides such as raffinose (raffinose) and stachyose (stachyyose), and further release galactose at a non-reducing end. In addition to hydrolyzing galactoside oligosaccharides that are widely present in legumes, such glycosidases also break down galactomannans (galactomannans), galactolipids (galactolipids), and glycoproteins (glycoprotens). Galactosidases can be divided into two broad classes according to substrate specificity: the first type is that which acts only on small substrates, such as synthetic substrates or oligosaccharides. The second type is the release of galactose from highly polymeric galactomannan polysaccharides. On the other hand, the amino acid sequences can be classified into the 4, 27, 36, 57, 97 and 110 families of glycoside hydrolases (glycoside hydrolases) according to their similarity. For the complete breakdown of galactomannans, galactosidases can have a synergistic effect with other glycosidases, like mannanases and mannosidases. And therefore, the application range of the galactosidase is quite wide, such as in the fields of feed, food, paper making and even medicine.

The feed raw material mainly comprising the soybean meal is added with galactosidase which can decompose galactoside substances incapable of being metabolized by animals, so that the utilization rate of the feed is increased, and the adverse effects of flatulence or diarrhea caused by anti-nutrient substances are relieved. In the food industry, galactosidase can be added to soybeans or other legume products. It can decompose galactoside oligosaccharide such as stachyose which can cause flatulence. In the paper industry, galactosidase can decompose the branched chains of galactoside in cork, and further help other subsequent enzyme species decompose main chain polysaccharide and fiber matrix. In the aspect of medical application, the galactosidase can be used for treating Fabry's disease and converting B-O blood type. In these many different industrial applications, galactosidases are also required to meet different industrial requirements. In addition to the properties and stability of the enzyme protein itself, its specific activity is also an important point for improving industrial enzymes. The higher the specific activity of the enzyme protein, the better the substrate decomposition efficiency, and further the cost of the industrial process is reduced, and the profit is improved. Therefore, the present application intends to improve the specific activity of the galactosidase by genetic engineering, thereby increasing the economic value of the galactosidase in industrial applications.

Disclosure of Invention

The application aims to modify the existing alpha-galactosidase, and effectively improve the activity of the galactosidase by using structural analysis and point mutation technology, so that the industrial application value of the galactosidase is increased.

To achieve the above object, a broader embodiment of the present application provides an α -galactosyl glycinase having an amino acid sequence represented by SEQ ID NO: 2 arginine at position 450 is changed to a phenylalanine, valine, serine, alanine, or glycine substitution modified sequence.

In one embodiment, the nucleic acid sequence encoding the SEQ ID NO: 2 is the AnBgl36 gene isolated from Aspergillus niger (Aspergillus niger).

In one embodiment, the amino acid sequence of the α -galactosyltransferase is as set forth in SEQ ID NO: shown at 9.

In one embodiment, the amino acid sequence of the α -galactosyltransferase is as set forth in SEQ ID NO: shown at 11.

In one embodiment, the amino acid sequence of the α -galactosyltransferase is as set forth in SEQ ID NO: shown at 13.

In one embodiment, the amino acid sequence of the α -galactosyltransferase is as set forth in SEQ ID NO: shown at 15.

In one embodiment, the amino acid sequence of the α -galactosyltransferase is as set forth in SEQ ID NO: shown at 17.

Drawings

FIG. 1 shows the nucleotide sequence as well as the amino acid sequence of wild-type α -galactosyltransferase Angal 36.

FIG. 2 shows the primer sequences used in the point mutation technique.

FIG. 3 shows the nucleotide sequence and amino acid sequence of R450F mutant alpha-galactoglycase Angal 36.

FIG. 4 shows the nucleotide sequence and amino acid sequence of R450V mutant alpha-galactoglycase Angal 36.

FIG. 5 shows the nucleotide sequence and amino acid sequence of R450S mutant alpha-galactoglycase Angal 36.

FIG. 6 shows the nucleotide sequence and amino acid sequence of R450A mutant alpha-galactoglycase Angal 36.

FIG. 7 shows the nucleotide sequence and amino acid sequence of R450G mutant alpha-galactoglycase Angal 36.

FIG. 8 shows the analysis of the alpha-galactosidase activity of the wild-type protein and the muteins R450F, R450V, R450S, R450A and R450G.

Detailed Description

Some exemplary embodiments that embody features and advantages of the present application will be described in detail in the description that follows. It should be understood that the application is capable of various modifications in various obvious respects, all without departing from the scope of the application, and that the description and drawings are to be regarded as illustrative in nature, and not as restrictive.

The alpha-galactosidase gene Angal36 of the present application was isolated from the fungus Aspergillus niger. The alpha-galactosidase gene is constructed into a vector and is transformed into an expression protein in Pichia pastoris (Pichia pastoris) which is commonly used in industry. In order to increase the catalytic efficiency and further improve the application value, the method uses a known protein structure with high similarity as a template, simulates a structural model of the alpha-galactosyl glycase by using software, analyzes the structural model, selects potential amino acids from the structural model, and carries out gene modification by using a site-directed mutagenesis technology (site-directed mutagenesis). Through structural analysis, the arginine (arginine) at the 450 th position of the amino acid sequence is picked out from an active region, and is respectively mutated into phenylalanine (phenylalanine), valine (valine), serine (serine), alanine (alanine) and glycine (glycine), and the mutant proteins are found to increase the activity of alpha-galactosamine.

The method for modifying alpha-galactosyl glycinase and the modified alpha-galactosyl glycinase obtained by the method are described in detail below.

FIG. 1 shows the nucleotide sequence as well as the amino acid sequence of wild-type α -galactosyltransferase Angal 36. As shown in FIG. 1, the wild-type α -galactosyltransferase gene Angal36 contains 2178 bases (containing a stop codon, the nucleotide sequence is shown by SEQ ID NO: 1) and 725 amino acids (the amino acid sequence is shown by SEQ ID NO: 2). The Angal36 gene was constructed in pPICZ α A vector using EcoRI and XbaI and delivered to Pichia pastoris by electroporation. Successfully transfected strains were selected using zeocin antibiotics, after which single colonies were selected and inoculated into YPD medium to alternate days. In order to amplify the bacterial load, the cells were inoculated into BMGY medium for every other day. After the cell suspension was separated by centrifugation, the cells were transferred to BMMY medium containing 0.5% methanol to induce protein expression. And finally, collecting the induced sample for subsequent experiments.

Five mutant genes of the alpha-galactosyltransferase gene Angal36 were obtained by point mutation technology, using wild-type gene Angal36 as template for polymerase chain reaction, and the mutation primers used therein are shown in FIG. 2, wherein R450F means that the amino acid at the 450 th position of alpha-galactosyltransferase gene Angal36 is mutated from arginine (arginine) to phenylalanine (phenylalanine), and the sequence of the R450F mutation primer is represented by SEQ ID NO: marking 3; R450V means that the amino acid at position 450 of α -galactosyltransferase Angal36 is mutated from arginine (arginine) to valine (valine), and the R450V mutation primer sequence is represented by SEQ ID NO: 4, marking; R450S means that the amino acid at position 450 of α -galactosyltransferase Angal36 is mutated from arginine (arginine) to serine (serine), and the R450S mutation primer sequence is represented by SEQ ID NO: 5, marking; R450A means that the amino acid at position 450 of α -galactosyltransferase Angal36 is mutated from arginine (argine) to alanine (alanine), and the R450A mutant primer sequence is represented by SEQ ID NO: marking 6; R450G means that the amino acid at position 450 of alpha-galactosyltransferase Angal36 is mutated from arginine (arginine) to glycine (glycine), and the R450G mutant primer sequence is represented by SEQ ID NO: and 7, marking. Therefore, the five mutant genes of alpha-galactosyl glycinase Angal36 obtained by the point mutation technology are R450F, R450V, R450S, R450A and R450G respectively.

The nucleotide and amino acid sequences of the five mutants constructed in the present application are shown in FIGS. 3 to 7. Figure 3 shows the nucleotide sequence and amino acid sequence of R450F mutant α -galactosyltransferase Angal36, wherein the nucleotide sequence is represented by SEQ ID NO: 8, the amino acid sequence is represented by SEQ ID NO: 9, and the amino acid at position 450 thereof is mutated from arginine (arginin) to phenylalanine (phenylalanine). Figure 4 shows the nucleotide sequence and amino acid sequence of R450V mutant alpha-galactoglycase Angal36, wherein the nucleotide sequence is represented by SEQ ID NO: 10, the amino acid sequence is represented by SEQ ID NO: 11, and the amino acid at position 450 thereof is mutated from arginine (arginine) to valine (valine). Figure 5 shows the nucleotide sequence and amino acid sequence of R450S mutant α -galactosyltransferase Angal36, wherein the nucleotide sequence is represented by SEQ ID NO: 12, the amino acid sequence is represented by SEQ ID NO: 13, and the amino acid at position 450 thereof is mutated from arginine (arginin) to serine (serine). Figure 6 shows the nucleotide sequence and amino acid sequence of R450A mutant α -galactosyltransferase Angal36, wherein the nucleotide sequence is represented by SEQ ID NO: 14, the amino acid sequence is represented by SEQ ID NO: 15, and the amino acid at position 450 thereof is mutated from arginine (arginine) to alanine (alanine). Figure 7 shows the nucleotide sequence and amino acid sequence of R450F mutant α -galactosyltransferase Angal36, wherein the nucleotide sequence is represented by SEQ ID NO: 16, the amino acid sequence is represented by SEQ ID NO: 17, and the amino acid at position 450 thereof is mutated from arginine (arginin) to glycine (glycine).

After the mutation reaction is completed, DpnI is added to remove the original template, and then the plasmid DNA is sent into escherichia coli competent cells and screened by antibiotics. The success of the sequence mutation was confirmed by DNA sequencing. Finally, as described in the protein expression step, the successfully mutated genes are individually delivered into pichia pastoris for expression, and protein amount measurement and alpha-galactosamine activity detection are performed.

The method for detecting the activity of the alpha-galactosyltransferase comprises the steps of carrying out catalytic hydrolysis on nitrophenyl galactoside (p-nitrophenyl-beta-D-galactosyltranside) serving as a substrate by utilizing the alpha-galactosyltranside, releasing nitrophenol (nitrophenol) with a color development effect, and further calculating the activity of the alpha-galactosyltranside. The experimental procedure was as follows: after mixing the substrate at 7mM with the diluted enzyme protein solution, it was allowed to stand in a water bath at 37 ℃ for 15 minutes, followed by addition of a 0.2M boric acid solution to terminate the reaction. Finally, the absorbance was measured at OD405 and then converted to α -galactosidase activity.

In order to be close to industrial application, the wild gene and the mutant gene are respectively sent into a pichia pastoris system commonly used in industry to express enzyme protein, and the activity detection of alpha-galactosidase is carried out under the same protein concentration. FIG. 8 shows the analysis of the alpha-galactosidase activity of the Wild type (Wild type) Angal36 enzyme protein and the muteins R450F, R450V, R450S, R450A and R450G, wherein the different enzyme activities were compared on the basis of the enzyme activity of the Wild type protein as 100%. From the results shown in fig. 8, it can be seen that the activity of the five mutant proteins is significantly higher than that of the wild protein, wherein the activity of R450G is greatly increased up to about 207%, and then R450F is also increased to about 191% of the activity. The activities of the other three muteins R450V, R450S and R450A were also significantly increased to about 150%. In addition, the expression amount of the mutant protein in a pichia pastoris system is not much different from that of the wild protein. Thus, the total activity of the mutant protein was also significantly higher than the original protein, in particular R450G.

In summary, in order to enhance the activity of α -galactosyl glycase AnBgl36, the present application further analyzed the protein structure, and selected arginine (Arg450) at the 450 th position of the sequence in the active region to be engineered and mutated individually to phenylalanine (R450F), valine (R450V), serine (R450S), alanine (R450A), and glycine (R450G). The results show that the activity of the five mutant proteins is obviously higher than that of the wild protein. Therefore, the mutant protein of the alpha-galactosyl glargine AnBgl36 provided by the application can effectively improve the activity of the alpha-galactosyl glargine, so that the production cost can be further reduced, and the industrial application value of the mutant protein can be increased.

While the present invention has been described in detail with respect to the above embodiments, it will be apparent to those skilled in the art that various modifications can be made without departing from the scope of the invention as defined in the appended claims.

Sequence listing

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