Starch branching enzyme mutant with improved catalytic capability
阅读说明：本技术 一种催化能力提高的淀粉分支酶突变体 (Starch branching enzyme mutant with improved catalytic capability ) 是由 李兆丰 顾正彪 江海旻 李才明 班宵逢 程力 洪雁 于 2019-10-23 设计创作，主要内容包括：本发明公开了一种催化能力提高的淀粉分支酶突变体,属于酶工程技术领域。本发明通过将淀粉分支酶中的底物结合位点附近不带电的氨基酸残基突变为带负电荷的外源性氨基酸残基,得到催化能力提高的淀粉分支酶突变体,与野生型淀粉分支酶相比,本发明所得的淀粉分支酶突变体R158T、Q489E改性产物α-1,6糖苷键相对含量分别为8.2％和10.7％,较淀粉分支酶野生型改性产物提高了10.81％和44.59％。(The invention discloses a starch branching enzyme mutant with improved catalytic capability, and belongs to the technical field of enzyme engineering. The invention obtains the starch branching enzyme mutant with improved catalytic capability by mutating uncharged amino acid residues near a substrate binding site in the starch branching enzyme into exogenous amino acid residues with negative charges, and compared with wild type starch branching enzyme, the starch branching enzyme mutant has the relative contents of alpha-1, 6 glycosidic bonds of modified products of R158T and Q489E which are respectively 8.2 percent and 10.7 percent, and are improved by 10.81 percent and 44.59 percent compared with the wild type modified product of the starch branching enzyme.)
1. A starch branching enzyme mutant is characterized by comprising an amino acid sequence shown in SEQ ID NO.2 or SEQ ID NO. 3.
2. A gene encoding the starch branching enzyme mutant of claim 1.
3. A plasmid or vector comprising the gene of claim 2.
4. A cell expressing the starch branching enzyme mutant of claim 1.
5. The cell of claim 4, wherein the cell is a host selected from the group consisting of E.coli and Bacillus subtilis.
6. The cell of claim 4 or 5, wherein the cell has pET-20b (+) as an expression vector and Escherichia coli BL21(DE3) as an expression host.
7. A method for improving branching capability of starch branching enzyme is characterized in that arginine at position 158 of the starch branching enzyme with an amino acid sequence shown as SEQ ID NO.1 is mutated into threonine, or glutamine at position 489 is mutated into glutamic acid.
8. Use of a starch branching enzyme mutant as defined in claim 1 for the hydrolysis of starch.
9. Use of a cell according to any one of claims 4 to 6 for the hydrolysis of starch.
10. Use of the starch branching enzyme mutant of claim 1 in the field of food products.
Technical Field
The invention relates to a starch branching enzyme mutant with improved catalytic capability, belonging to the technical field of enzyme engineering.
Background
Starch is widely existed in nature and has wide application potential in the industries of food, medical treatment and the like, however, the problems of easy retrogradation, poor stability, low solubility and the like exist in natural starch, and the application of the starch in the industries is limited. The fundamental reason for these problems is that native starch contains many linear long-chain macromolecules or branched molecules with low branching degree, which are easily associated with each other by the action of hydrogen bonds, and thus retrogradation occurs. The degree of easy mutual association between starch molecules is related to the branching degree of the starch molecules, and the starch molecules with high branching degree have larger steric hindrance compared with the starch molecules with low branching degree, so that the starch molecules are not easy to mutually associate. Therefore, if the branching degree of starch molecules can be increased, the defects in the use of starch can be effectively improved.
Starch branching enzymes (1, 4-alpha-glucan branching enzymes; EC 2.4.1.18) are a class of glycosyltransferases belonging to the alpha-amylase family that catalyze the hydrolysis of alpha-1, 4-glycosidic linkages in starch molecules to produce free short chains with non-reducing ends, which are then linked to acceptor chains in the form of alpha-1, 6-glycosidic linkages by transglycosidation, thereby forming new alpha-1, 6-branch points.
By the transglycosylation reaction, the starch branching enzyme can increase the branching degree of starch, improve the anti-digestibility and slow digestibility of the starch, delay the retrogradation process of the starch, enhance the stability of the starch and improve the usability of the starch, and is used for producing starch derivatives with good application value. Therefore, starch branching enzymes are important amylases in the field of modification of starch by a biological enzyme method due to their characteristic transglycosylation.
At present, there are many reports on modifying starch with starch branching enzyme to increase the degree of starch branching, however, the degree of starch branching increase is limited, and therefore modification of the enzyme is required to further enhance its catalytic ability. Methods commonly used to modify starch branching enzymes include physical, chemical and biological methods. Compared with physical and chemical methods, the biological method mainly changes the spatial conformation of enzyme protein molecules through technical means such as protein engineering, genetic engineering and the like, improves the service performance of the enzyme protein, and has higher stability and better safety. However, the existing starch branching enzyme can only lead the branching degree of the product to reach about 7 percent. Therefore, there is a need for starch branching enzyme mutants with improved catalytic ability, which act on starch to produce products with higher branching degree.
Disclosure of Invention
In order to solve the above problems, the present invention provides a starch branching enzyme mutant with improved catalytic ability, which contains an amino acid sequence shown in SEQ ID NO.2 or SEQ ID NO. 3.
The starch branching enzyme mutant takes starch branching enzyme with an amino acid sequence shown as SEQ ID NO.1 as parent enzyme, and the 158 th arginine is mutated into threonine, or the 489 th glutamine is mutated into glutamic acid.
In one embodiment of the invention, the parent enzyme is derived from hyperthermophilic (Rhodothermus obamensis) STB05, which is referenced from the literature as STB 05: the expression, purification and enzymological property research of extreme thermophilic starch branching enzyme [ J ] 2019.
The present invention provides a gene encoding the starch branching enzyme mutant with improved catalytic ability.
In one embodiment of the invention, the nucleotide sequence of the gene is shown as SEQ ID NO.9 or SEQ ID NO. 10.
The invention provides a plasmid or a vector containing the gene.
The present invention provides a cell carrying the above gene.
In one embodiment of the invention, the cell comprises a genetically engineered bacterium.
In one embodiment of the present invention, the genetically engineered bacterium is a host escherichia coli or bacillus subtilis.
In one embodiment of the invention, the genetically engineered bacterium takes pET-20b (+) as an expression vector and Escherichia coli BL21(DE3) as an expression host.
The invention provides a method for improving catalytic ability of starch branching enzyme, which is characterized in that arginine at position 158 of starch branching enzyme from extreme thermophilic bacteria (Rhodothermus obamensis) STB05 is mutated into threonine, or glutamine at position 489 is mutated into glutamic acid, the amino acid sequence of the starch branching enzyme from extreme thermophilic bacteria (Rhodothermus obamensis) STB05 is shown as SEQ ID NO.1, and the nucleotide sequence of a gene for coding the starch branching enzyme is shown as SEQ ID NO. 8.
The invention provides application of the starch branching enzyme mutant in the aspect of hydrolyzing starch.
The invention provides application of the genetic engineering bacteria in the aspect of hydrolyzing starch.
The invention provides application of the starch branching enzyme mutant in the field of food.
The invention provides application of the genetic engineering bacteria in the field of food.
The invention has the beneficial effects that:
the starch branching enzyme mutant with improved catalytic capability is obtained by mutating uncharged amino acid residues near a substrate binding site in the starch branching enzyme into exogenous amino acid residues with negative charges. Compared with wild starch branching enzyme, the starch branching enzyme mutant obtained by the invention takes 5% (w/w) corn starch solution as a substrate, the branching degrees of products respectively reach 8.2% (R158T) and 10.7% (Q489E), and are respectively improved by 10.81% and 44.59% compared with the wild starch branching enzyme.
Drawings
FIG. 1 shows wild-type and mutant forms of starch branching enzymesOf products before and after modification1H NMR spectrum.
FIG. 2 shows the relative alpha-1, 6-glycosidic bond contents of the wild-type and mutant starch branching enzyme products before and after modification in the examples of the present invention.
Detailed Description
The examples of the present invention are provided only for further illustration of the present invention and should not be construed as limitations or limitations of the present invention.
The detection method comprises the following steps:
method for measuring relative content of alpha-1, 6 glycosidic bond
Accurately weighing 20mg of sample, dissolving in 1mL of heavy water, gelatinizing in boiling water bath, and passing through1H NMR (hydrogen nuclear magnetic resonance) was measured. And obtaining the relative content of the alpha-1, 6-glycosidic bond according to the peak area ratio of the absorption peaks corresponding to the alpha-1, 4-glycosidic bond and the alpha-1, 6-glycosidic bond in the calculated spectrogram.
Method for measuring activity of starch branching enzyme
0.25% (w/v) potato amylopectin solution prepared from 50mM phosphate buffer solution (pH 7.0) is used as substrate for enzyme reaction, 900 μ L substrate is taken and kept at 65 ℃ for 10min, 100 μ L starch branching enzyme is added and mixed uniformly, and the mixture is placed in a water bath condition at 65 ℃ for reaction for 15 min. The reaction was terminated by inactivating the enzyme in a bath of boiling water for 30 min. After cooling to room temperature, 300. mu.L of the reaction mixture was added to 5mL of a developing solution (0.05% (w/v) KI, 0.005% (w/v) I2pH 6.0) was left standing at room temperature to sufficiently develop color. Absorbance at 530nm was measured after development for 15 min.
One unit of enzyme activity (U/mL) is defined as: at 530nm, the absorbance decreased by 1% per minute as one unit of enzyme activity.
(III) culture Medium
LB culture medium: 5g/L of yeast powder, 10g/L of tryptone, 10g/L of NaCl and 7.0 of pH.
TB culture medium: 24g/L yeast powder, 12g/L tryptone, 5g/L glycerol and KH2PO42.3136g/L，K2HPO416.4318g/L，pH 7.0。