CN117431227A - Starch branching enzyme mutant and application thereof - Google Patents
Starch branching enzyme mutant and application thereof Download PDFInfo
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- CN117431227A CN117431227A CN202311158233.XA CN202311158233A CN117431227A CN 117431227 A CN117431227 A CN 117431227A CN 202311158233 A CN202311158233 A CN 202311158233A CN 117431227 A CN117431227 A CN 117431227A
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1051—Hexosyltransferases (2.4.1)
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
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- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/01—Hexosyltransferases (2.4.1)
- C12Y204/01018—1,4-Alpha-glucan branching enzyme (2.4.1.18), i.e. glucan branching enzyme
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Abstract
The invention discloses a starch branching enzyme mutant, which is obtained by mutating amino acid residues of at least one of 306 th, 416 th, 429 th, 557 th and 588 th sites of a starch branching enzyme TrGBE shown in SEQ ID NO.1, nucleic acid for encoding the starch branching enzyme mutant, a recombinant expression vector containing the nucleic acid and genetically engineered bacteria containing the recombinant expression vector. And the application of the starch branching enzyme mutant, the gene for encoding the starch branching enzyme mutant, the recombinant expression vector containing the gene and the genetic engineering bacteria containing the recombinant expression vector in preparing resistant dextrin products or preparing foods or medicines. The starch branching enzyme mutant has excellent activity, so that the efficiency of preparing resistant dextrin is greatly improved, and the starch branching enzyme mutant has wide industrial application prospect.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a starch branching enzyme mutant and application thereof.
Background
The resistant dextrin is a low-molecular soluble dietary fiber formed by taking starch as a raw material and carrying out partial degradation and glycosylation transfer, and is also called indigestible dextrin because the resistant dextrin contains alpha-1, 6, alpha-1, 3, alpha-1, 2 and other glycosidic bonds which are difficult or impossible to hydrolyze by human digestive enzymes. The resistant dextrin not only has excellent processing characteristics of heat resistance, acid resistance, freezing resistance and the like, but also can exert good health efficacy, and is easy to produce satiety after a proper amount of dextrin is taken. It can slow down saccharide absorption in upper digestive tract and inhibit postprandial blood glucose rise, and promote intestinal peristalsis in lower digestive tract. In addition, resistant dextrins are widely used in low calorie foods because they also produce short chain fatty acids after fermentation, which help to improve intestinal flora. The resistant dextrin is listed as common food at present, and is applied to various foods such as beverages, dairy products, wines, meat products, flour products and the like, so that the resistant dextrin not only can partially replace fat, but also can improve the texture, the yield and the taste.
The study of resistant dextrins was originally initiated in japan, the pine and cereal chemical industry co.ltd, which first prepared resistant dextrins by the acid heating method and named Fibersol, AOAC defined it as "resistant" dextrins in 2002, distinguished from normal maltodextrins. Research on resistant dextrin is started in the nineties of the 20 th century in China, and in 1995, lin Qinbao and the like, pyrodextrin is prepared from starch as a raw material, and is treated with alpha-amylase and then treated with glucoamylase to prepare the resistant dextrin, and factors influencing the composition of the product are explained. In 2006, the Guangdong food institute applied for a patent on a preparation process of resistant maltodextrin, which comprises the steps of hydrolyzing the maltodextrin with alpha-amylase, treating with pullulanase, decoloring, ion-exchanging and spray-drying to obtain the resistant maltodextrin, wherein the yield is not high and is basically about 40% -45%.
Starch branching enzymes (4-alpha-glucanbranching enzyme, GBE for short, EC 2.4.1.18) are a class of glycosyltransferases belonging to the alpha-amylase family, which are capable of catalyzing the hydrolysis of alpha-1, 4 glycosidic linkages of starch molecules to produce free short chains with non-reducing ends, which are linked to acceptor chains in the form of alpha-1, 6 glycosidic linkages by transglycosidation, thereby forming new alpha-1, 6 branch points. The characteristic of starch branching enzyme makes it of great value in starch modification and preparation of starch with high branching degree, including high branching dextrin, resistant starch and dextrin, starch gum preparation, slow digestion starch preparation, starch modification and other applications.
Based on the application value of starch branching enzyme in preparing starch products with high branching degree and starch modification, starch branching enzyme of plant, animal and microorganism sources are reported in starch modification, such as the application of glycogen branching enzyme of Thermomonospora curvata source in preparing high branching dextrin and synthesizing multi-branching starch (application number: 201410579597.X; 201810116999.8); use of Thermuobifidafusca WSH03-11 derived branching enzyme Tfu0582 in the preparation of resistant dextrins (application number: 201710594597.0); use of a Rhodothermus obamensis derived starch branching enzyme to increase the clarity of starch liquefaction products (application No. 201810219838.8); geobacillus thermoglucosidans STB02 in starch ageing and resistant starch preparation (application number: 201810531948.8).
Although starch branching enzymes derived from microorganisms are a major industrial enzyme because of their high substrate specificity and high branching degree of catalytic products. However, the starch branching enzyme with independent intellectual property rights in China is less, and the reported starch branching enzyme has the problems of low enzyme yield, low enzyme activity, low catalytic efficiency and the like of a natural strain, so that the development of starch branching enzyme resources with high activity and excellent performance has important value.
Disclosure of Invention
The first object of the present invention is to provide a starch branching enzyme (TrGBE) mutant, a nucleic acid encoding the starch branching enzyme mutant, a recombinant expression vector containing the nucleic acid, and a genetically engineered bacterium containing the recombinant expression vector.
The invention also aims to provide the starch branching enzyme mutant, a gene for encoding the starch branching enzyme mutant, a recombinant expression vector containing the gene and application of genetically engineered bacteria containing the recombinant expression vector in preparing resistant dextrin products or preparing foods or medicines.
It is a final object of the present invention to provide a process for the preparation of resistant dextrins.
The first object of the present invention can be achieved by the following technical means: a starch branching enzyme mutant obtained by mutating an amino acid residue at least one of positions 306, 416, 429, 557 and 588 of a starch branching enzyme TrGBE as shown in SEQ ID No.1, wherein leucine L at position 306 is mutated to alanine a, tryptophan W at position 416 is mutated to glutamic acid E, tyrosine Y at position 429 is mutated to aspartic acid D, isoleucine I at position 557 is mutated to D aspartic acid or proline P at position 588 is mutated to asparagine N.
The starch branching enzyme mutants can be used for preparing resistant dextrins.
Alternatively, the starch branching enzyme mutant is obtained by mutating the amino acid residue at position 306, 416, 429, 557 or 588 of the starch branching enzyme TrGBE shown in SEQ ID NO. 1.
More preferably, the starch branching enzyme mutant is obtained by mutating the 416 th position of the starch branching enzyme TrGBE shown in SEQ ID NO. 1.
Optionally, the starch branching enzyme mutant is obtained by simultaneously mutating amino acid residues at any two positions of 306 th, 416 th, 429 th, 557 th and 588 th of the starch branching enzyme TrGBE shown in SEQ ID NO. 1.
More preferably, the starch branching enzyme mutant is obtained by simultaneously mutating the 416 th site and the 306 th site, 429 th site, 557 th site or 588 th site of the starch branching enzyme TrGBE shown in SEQ ID NO. 1.
Optimally, the starch branching enzyme mutant is obtained by simultaneously mutating the 416 th position and the 429 th position of the starch branching enzyme TrGBE shown in SEQ ID NO. 1.
According to an embodiment of the invention, the amino acid sequence of the mutant comprises a mutation of the amino acid residues at any two positions in L306, W416, Y429, I557, P588.
In a preferred embodiment, the amino acid sequence of the mutant comprises a mutation at both of the above sites, and at least one of the mutation sites is any one of W416, Y429, I557, P588.
In a more preferred embodiment, the amino acid sequence of the mutant comprises mutations at two positions: L306/W416, W416/Y429D, W/I557, W416/P588.
Alternatively, the starch branching enzyme mutant is obtained by simultaneously mutating amino acid residues at any three of 306 th, 416 th, 429 th, 557 th and 588 th sites of the starch branching enzyme TrGBE shown in SEQ ID NO. 1.
More preferably, the starch branching enzyme mutant is obtained by simultaneously mutating the 416 th, 429 th and 306 th, 557 th or 588 th positions of the starch branching enzyme TrGBE shown in SEQ ID NO. 1.
Optimally, the starch branching enzyme mutant is obtained by simultaneously mutating the 416 th, 429 th and 557 th positions of the starch branching enzyme TrGBE shown in SEQ ID NO. 1.
According to an embodiment of the invention, the amino acid sequence of the mutant comprises a mutation of the amino acid residues at any three of positions L306, W416, Y429, I557, P588.
In a preferred embodiment, the amino acid sequence of the mutant comprises a mutation at position W416/Y429, and a mutation at any of positions L306, I557, P588.
According to a more preferred embodiment of the invention, the amino acid sequence of the mutant comprises a mutation at any of the combination of mutation sites W416E/Y429D/L306A, W E/Y429D/I557D, W E/Y429D/P588N, preferably the W416E/Y429D/I557D site.
As a best embodiment of the invention, the starch branching enzyme mutant is obtained by mutating the 416 th site of the starch branching enzyme TrGBE shown in SEQ ID NO.1, and the amino acid sequence of the starch branching enzyme mutant is mutated into E at the W416 site corresponding to SEQ ID NO. 1; or the starch branching enzyme mutant is obtained by simultaneously mutating the 416 th site and the 429 th site of the starch branching enzyme TrGBE shown in SEQ ID NO.1, wherein the amino acid sequence of the starch branching enzyme mutant is mutated into E at the W416 site corresponding to the SEQ ID NO.1, and simultaneously mutated into D at the Y429 site of the SEQ ID NO. 1; or the starch branching enzyme mutant is obtained by simultaneously mutating the 416 th, 429 th and 557 th positions of the starch branching enzyme TrGBE shown in SEQ ID NO.1, wherein the amino acid sequence is mutated into E at the W416 position corresponding to SEQ ID NO.1, the Y429 position of SEQ ID NO.1 is mutated into D, and the I557 position of SEQ ID NO.1 is mutated into D.
According to an embodiment of the invention, the mutant has a homology of more than 70%, for example more than 80%, further for example more than 90%, more than 95%, more than 98% with the amino acid sequence shown in SEQ ID NO. 1.
The invention also provides nucleic acids encoding the starch branching enzyme mutants. The sequence is shown in EQIDNO. 2.
Recombinant expression vectors containing said nucleic acids.
Alternatively, the expression vector is pET-28a (+).
The invention also provides a genetic engineering bacterium containing the recombinant expression vector.
Alternatively, the genetically engineered bacterium is a recombinant strain obtained by connecting the nucleic acid with a vector to obtain a recombinant expression vector and then introducing the recombinant expression vector into a host bacterium, wherein the host bacterium is escherichia coli, bacillus subtilis or yeast.
Alternatively, the E.coli is E.coli (E.coli).
More preferably, the E.coli is selected from E.coli BL21 (DE 3) or E.coli DH 5. Alpha.
The mutant is prepared by carrying out single-point mutation or combined mutation on starch branching enzyme TrGBE with branching enzyme activity of Thermococcus radiotolerans bacteria which are derived from thermophilic cocci and have the amino acid sequence shown in SEQ ID NO. 1. The invention also discloses the starch branching enzyme mutant coding nucleic acid, a recombinant expression vector, a genetic engineering bacterium and application thereof in preparing resistant dextrin. The starch branching enzyme mutant has excellent activity, so that the efficiency of preparing resistant dextrin is greatly improved, and the starch branching enzyme mutant has wide industrial application prospect.
The second object of the present invention can be achieved by the following technical means: the starch branching enzyme mutant, the gene, the recombinant expression vector and the genetic engineering bacteria are applied to the preparation of resistant dextrin products.
The invention further provides the starch branching enzyme mutant, the gene, the recombinant expression vector and the application of the genetically engineered bacterium in preparing food or medicine.
The last object of the invention can be achieved by the following technical scheme: a preparation method of resistant starch is characterized in that starch is taken as a substrate, the substrate is subjected to acidolysis at high temperature, and then the starch branching enzyme mutant is added for enzyme conversion reaction to prepare the resistant dextrin.
Compared with the prior art, the invention has the following advantages:
(1) In the process of screening a large amount of starch branching enzyme, the invention firstly digs the starch branching enzyme TrGBE from Thermococcus radiotolerans bacteria of thermophilic cocci, on the basis, the cloning, the expression and the enzymatic property are researched, a process of preparing resistant dextrin by taking starch as a substrate, carrying out acidolysis on the substrate at high temperature, adding the starch branching enzyme, and carrying out directional transformation on the enzyme activity by utilizing site-directed mutation, thereby improving the catalytic efficiency of the enzyme in producing the resistant dextrin;
(2) Compared with starch branching enzyme in the prior art, the starch branching enzyme mutant W416E/Y429D/I557D has higher enzyme activity and catalytic efficiency: the E.coli is utilized to express the TrGBE mutant W416E/Y429D/I557D gene, and the optimal pH7.0 and the optimal temperature of starch branching enzyme produced by the recombinant genetically engineered bacterium strain are 75 ℃.
(3) The starch branching enzyme is utilized, corn starch is used as a substrate, after high-temperature acidolysis, the substrate concentration of 400g/L-500g/L is prepared, then enzyme conversion is carried out at 65 ℃, the reaction time is 6-8h, and the content of resistant dextrin reaches 70%;
(4) The starch branching enzyme mutant has excellent activity, so that the efficiency of preparing the resistant dextrin is greatly improved, and the starch branching enzyme mutant has wide industrial application prospect.
Drawings
The invention will be further described with reference to the accompanying drawings, in conjunction with examples.
FIG. 1 is an expression of starch branching enzyme in E.coli BL21 (DE 3) in example 5, lanes illustrate: lane M is a protein molecular weight standard Marker; lane 1 is the supernatant of mutant W416E/Y429D/I557D;
FIG. 2 is the effect of pH on the enzymatic activity of mutant W416E/Y429D/I557D in example 7;
FIG. 3 is the effect of temperature on the enzymatic activity of mutant W416E/Y429D/I557D in example 7.
Detailed Description
The following describes the invention in more detail. The description of these embodiments is provided to assist understanding of the present invention, but is not intended to limit the present invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The experimental methods used in the following implementation methods are conventional experimental methods unless otherwise specified.
The terms used in the following methods and examples, unless otherwise indicated, generally have meanings that are commonly understood by those of ordinary skill in the art.
The present invention relates to nucleic acids encoding starch branching enzyme mutants (TrGBE mutants) according to the invention, or comprising a sequence encoding a TrGBE mutant according to the invention. The invention also relates to expression cassettes for the nucleic acids of the invention. It further relates to vectors comprising the nucleic acids or expression cassettes of the invention. Preferably, the vector is an expression vector. Preferably, the vector is a plasmid vector. Furthermore, the invention relates to a host cell comprising a nucleic acid according to the invention, an expression cassette of a nucleic acid of the invention or a vector comprising a nucleic acid or an expression cassette of the invention. The nucleic acid encoding the TrGBE mutants of the invention may be present in the host cell as an episomal sequence or may be incorporated into its chromosome. The nucleic acid encoding the TrGBE mutant according to the invention may be present in the host cell in one copy or in several copies.
The nucleic acid may be DNA (cDNA or gDNA), RNA, or a mixture of both. It may be in single-stranded form or in duplex form or a mixture of both. It may comprise modified nucleotides, for example comprising modified linkages, modified purine or pyrimidine bases, or modified sugars. It can be prepared by any method known to those skilled in the art, including chemical synthesis, recombination, mutagenesis, and the like.
The expression cassette comprises all the elements required for expression of the TrGBE mutants according to the invention, in particular elements required for transcription and translation in a host cell, in particular in a host cell under consideration.
The host cell may be prokaryotic or eukaryotic, preferably prokaryotic or lower eukaryotic, more preferably prokaryotic. In particular, the expression cassette comprises a promoter and a terminator, optionally an enhancer. Promoters may be prokaryotic or eukaryotic, depending on the host cell chosen. Examples of preferred eukaryotic promoters include the RNA polymerase promoters of Lacl, lacZ, pLacT, ptac, pARA, pBAD, phage T3 or T7. In general, to select a suitable promoter, one skilled in the art can advantageously review the work of Sambrook et al (1989) or the techniques described by Fuller et al (1996; immunology in current protocols in molecular biology (Immunology in Current Protocols in Molecular Biology)).
The present invention relates to a vector comprising a nucleic acid or expression cassette encoding a TrGBE mutant according to the invention. Preferably, the vector is an expression vector, that is, it comprises the elements necessary for expression of the variant in a host cell. The vector is a self-replicable vector. The host cell may be a prokaryotic cell, such as E.coli (E.coli), or a eukaryotic cell. Eukaryotic cells may be lower eukaryotic cells, such as yeasts (e.g. Saccharomyces cerevisiae) or fungi (e.g. from the genus Aspergillus or Actinomyces) or higher eukaryotic cells, such as insect, mammalian or plant cells.
The vector may be a plasmid, phage, phagemid, cosmid, virus, YAC, BAC, pTi plasmid from agrobacterium, etc. Preferably, the vector may comprise one or more elements selected from the group consisting of an origin of replication, a multiple cloning site and a selection gene. In a preferred embodiment, the vector is a plasmid. The vector is a self-replicable vector. Examples of prokaryotic vectors include, but are not limited to, the following: pER322, pQE70, pMA5, pUC18, pQE60, pUB110, pQE-9 (Qiagen), pbs, pTZ4, pC194, pD10, pHV14, yep7, phagescript, psiX174, pbluescriptSK, pbsks, pNH8A, pNH16A, pNH18A, pNH A (Stratagene); ptrC99a, pKK223-3, pKK233-3, pDR540, pBR322 and pRIT5 (Pharmacia), pET (Novagen). Examples of eukaryotic vectors include, but are not limited to, the following: pWLNEO, pSV2CAT, pPICZ, pcDNA3.1 (+) Hyg (Invi trogen), pOG44, pXT1, pSG (Stratagene); pSVK3, pBPV, pCI-neo (Stratagene Co.), pMSG, pSVL (Pharmacia Co.) and pQE-30 (QLAexpress Co.). Preferably, the expression vector is a plasmid vector.
The amino acid is represented by single letter or three letter codes, and has the following meaning A: ala (alanine); arg (arginine); asn (asparagine); asp (aspartic acid); cys (cysteine); gln (glutamine); glu (glutamic acid); gly (glycine); h, his (histidine); ile (isoleucine); leu (leucine); lys (lysine); met (methionine); phe (phenylalanine); pro (proline); ser (serine); t: thr (threonine); trp (tryptophan); y: tyr (tyrosine); val (valine).
"homology" in the present invention has the meaning conventional in the art, referring to "identity" between two nucleic acid or amino acid sequences, the percentage of which means the statistically significant percentage of identical nucleotide or amino acid residues between the two sequences to be compared obtained after optimal alignment (best alignment), the differences between the two sequences being randomly distributed over their entire length.
In the present invention, the variants are described in terms of their mutations at specific residues, the position of which is determined by comparison with the wild-type enzyme sequence SEQ ID NO.1 or by reference to the enzyme sequence SEQ ID NO. 1. In the context of the present invention, it also relates to any variant carrying these same mutations at functionally equivalent residues.
In the present invention, the terms "primer" and "primer strand" are used interchangeably to refer to an initial nucleic acid fragment, typically an RNA oligonucleotide, DNA oligonucleotide or chimeric sequence that is complementary to all or part of a primer binding site of a target nucleic acid molecule. The primer strand may comprise natural, synthetic or modified nucleotides. The lower limit of primer length is the minimum length required to form a stable duplex under the conditions of the nucleic acid amplification reaction.
As used herein, the terms "wild type", "wild-type enzyme" and "wild-type enzyme" are intended to mean the same meaning and refer to the starch branching enzyme SEQ ID NO.1 which has not been genetically engineered.
In the present invention, the terms "mutant" and "variant" are used interchangeably and "modification" or "mutation" are used interchangeably and these expressions refer to an amino acid relative to a wild type protein, such as the wild type sequence SEQ ID NO:1 starch branching enzyme derived from Thermococcus Thermococcus radiotolerans, or a modification at one or more positions, i.e. substitution, insertion and/or deletion, which is/are included on the basis of such an enzyme, and still retain its activity. Mutants may be obtained by various techniques known in the art. Exemplary techniques for modifying a DNA sequence encoding a wild-type protein include, but are not limited to, site-directed mutagenesis, random mutagenesis, and construction of synthetic oligonucleotides, whereby the modified DNA sequence is expressed in a host bacterium to produce mutants in which substitutions, insertions, and/or deletions of the amino acid sequence have occurred. In the present invention, the expression "the mutant includes a mutation at position … …" or "the amino acid sequence of the mutant includes a mutation at position …" has the same meaning, and each means that substitution, insertion and/or deletion occurs at a specific position of the amino acid sequence of the mutant protein. The term "substitution" with respect to an amino acid position or residue refers to the replacement of an amino acid at a particular position with another amino acid. Substitutions may be conservative or non-conservative.
In particular embodiments, the homology or sequence identity may be more than 90%, preferably more than 95%, more preferably 98% homology. The mutation site and its substitution are expressed herein by the position number of the mutation site and the amino acid type of the site, for example L306A indicates that leucine at the position corresponding to position 306 of SEQ ID NO.1 is substituted with alanine in comparison with SEQ ID NO. 1. In the present invention, the combination of mutation sites is indicated by "/", for example, "L306/W416" indicates that both leucine at position 306 and tryptophan at position 416 are mutated, and the mutation sites include two mutation sites, and are double mutants. By analogy, "W416/Y429/I557" indicates that the corresponding three sites are mutated simultaneously, as a triple mutant.
When used as biocatalysts for the preparation of resistant dextrins, the starch branching enzyme of the present invention may take the form of an enzyme or a bacterial form. The enzyme forms include free enzymes, immobilized enzymes, including purified enzymes, crude enzymes, fermentation broths, vector immobilized enzymes, and the like, and the bacterial forms include viable bacterial and dead bacterial.
Example 1: transformation of recombinant plasmid pET-28a-TrGBE
The wild-type TrGBE gene was obtained by synthesizing (codon optimized) a gene derived from a polypeptide of Thermococcus radiotolerans (NCBI: WP_ 088866673.1) of the genus Thermococcus of the microorganism from the general biosystems (Anhui Co., ltd.). The obtained wild-type TrGBE enzyme gene was inserted into the expression plasmid pET-28a (+) using restriction enzymes Nde I and BamH I, thereby producing a recombinant expression vector 28a-TrGBE. The recombinant expression vector is transformed into E.coli BL21 (DE 3) by a conventional transformation method to obtain the genetically engineered bacterium E.coli BL21 (DE 3) -28a-TrGBE containing the wild TrGBE gene, and the genetically engineered bacterium E.coli BL21 (DE 3) -28a-TrGBE is preserved in an ultralow temperature refrigerator at the temperature of minus 80 ℃.
Example 2: establishment of mutant high-throughput screening methods
The invention establishes a strategy for enzyme screening of TrGBE mutant library, optimizes conditions such as reaction mode, measurement mode, reaction temperature and the like, and finally realizes high-throughput screening of starch branching enzyme mutants, and the specific method of the process is as follows:
(1) Selecting transformation from mutant library (a mutant library obtained by single-point saturation mutation of 306 th, 416 th, 429 th, 557 th and 588 th of specific sites is selected according to the analysis method of example 3), inoculating the transformation into 96-well deep well plates, culturing the transformation in LB culture medium containing 30 mug/mL kanamycin at 37 ℃ and 200rpm for 3 hours, adding 0.1mM IPTG, and continuously inducing and culturing at 30 ℃ for 8 hours;
(2) Removing the supernatant by low-temperature high-speed centrifugation, adding a proper amount of lysozyme after the wet thalli are resuspended by 0.1M sodium phosphate with pH of 7.0, incubating for 20min at room temperature by a shaking table, and collecting a supernatant crude enzyme solution by centrifugation at 12000rpm at 4 ℃;
(3) Preparing a reaction substrate solution: weighing 5g of starch, adding 0.1M sodium phosphate buffer with pH of 7.0, stirring uniformly, and fixing the volume to 1L;
(4) Adding 900 mu L of a crude enzyme solution, reacting for 10min at 60 ℃, terminating the reaction by boiling water, adding 5mL of iodine solution and hydrochloric acid solution with the final concentration of about 3.8 mu mol, standing for 20min at room temperature, adding 200 mu L of the reaction solution into a 96-well plate, detecting the light absorption value of 660nm or 530nm in an enzyme labeling instrument, defining an enzyme activity unit as the enzyme quantity required for 1% reduction per minute at 660nm, calculating the mutant enzyme activity, and screening the high-enzyme activity mutant.
Example 3: increasing TrGBE enzyme activity by site-directed saturation mutagenesis
Homology alignment of the starch branching enzyme of the thermophilic genus Thermococcus radiotolerans with the amino acid sequences of starch branching enzymes reported in the Genbank database was analyzed: meanwhile, the TrGBE is subjected to structure prediction, homologous modeling is carried out on wild type proteins of the enzyme by using software such as Swiss-Model, phyre2, discovery Studio and the like, the catalytic site and the substrate binding site of the wild type enzyme are predicted, and the action of amino acid residues near the sites is analyzed, so that the amino acid sequence of the TrGBE mutant is designed. Through the analysis, the 306 th, 416 th, 429 th, 557 th and 588 th sites corresponding to SEQ ID NO.1 are selected as mutation sites for saturation mutation, and a single site saturation mutation library is established.
Transformants in the mutant library were then screened using the high throughput screening strategy of example 2, from which 5 mutants with increased enzyme activity were obtained and sequenced. After sequence analysis, the corresponding mutant expression strain was cultured, induced by adding 0.1mM1PTG, and then the mutant protein was purified by using Ni column, and the mutants with improved enzyme activity were re-screened.
Enzyme activity detection: taking 100 mu L of pure enzyme solution, adding 900 mu L of reaction substrate solution (prepared by buffering sodium phosphate with pH 7.0), reacting for 10min at 60 ℃, terminating the reaction by boiling water, adding 5mL of iodine solution and hydrochloric acid solution with the final concentration of about 3.8 mu mol, standing for 20min at room temperature, adding 200 mu L of reaction solution into a 96-well plate, detecting the light absorption value of 660nm in an enzyme label instrument, and defining the enzyme activity: the amount of enzyme required was reduced by 1% per minute at 660nm at 60℃and pH7.0. The relative enzyme activities of the mutants were compared with the enzyme activity of the wild type TrGBE as 100%, and the results are shown in Table 1.
Method for measuring protein concentration: protein concentration was determined according to the instructions using the Bradford protein concentration determination kit (detergent compatible) from shanghai bi-cloud, which uses the Bradford method and uses bovine blood albumin to draw a standard curve.
The specific enzyme activity measuring method comprises the following steps: a standard curve was drawn using bovine serum albumin as a standard, and the protein concentrations of the starch branching enzyme wild type and mutant were determined by the Bradford method. The specific activity of the enzyme refers to the number of units of enzyme activity of a unit weight (mg) of protein under specific conditions, and the specific activity can be used for comparing the catalytic ability of the unit mass of protein in the enzyme preparation, so that the specific enzyme activity is calculated through the enzyme activity and the protein concentration, and 5 mutants with the enzyme activity obviously improved compared with that of a wild type are obtained through screening as shown in the following table 1.
TABLE 1 relative enzyme Activity and specific Activity of starch branching enzyme wild type and mutant
Example 4: site directed mutagenesis and multiple point combination mutagenesis of wild-type TrGBE
The combined mutant is obtained by adopting a site-directed mutagenesis technology, and a mutant primer is designed by adopting CE Design V1.04 provided by Norwegian company. The mutant primers are shown in Table 2.
TABLE 2 mutant primers
| Mutation | Nucleotide sequence of mutant primer | Corresponding serial number |
| L306A-F | ATGCGGGCGAGAAAGAATTCTATGATCCGGAAA | SEQ ID NO:3 |
| L306A-R | ATTCTTTCTCGCCCGCATCCACGTTCTTACCAGTAACACG | SEQ ID NO:4 |
| W416E-F | AGCACCGAGTGGAATGAAGAGACGGAATGGACC | SEQ ID NO:5 |
| W416E-R | TTCATTCCACTCGGTGCTGTGATCCGCGTTTGC | SEQ ID NO:6 |
| Y429D-F | TTGATCGTGCGGAGGAACGCATGGTGGCGCTG | SEQ ID NO:7 |
| Y429D-R | TTCCTCCGCACGATCAACGTGACCCCAGGTCCATT | SEQ ID NO:8 |
| I557D-F | AGATGCTGTGAAGTCTCCACGTAAAGTTAAGCG | SEQ ID NO:9 |
| I557D-R | GGAGACTTCACAGCATCTTCTTTAACCACCTCGGTCGC | SEQ ID NO:10 |
| P588N-F | TTAAGCGCAATTCTCCGGAAGAGTCCCGTAAGC | SEQ ID NO:11 |
| P588N-R | CCGGAGAATTGCGCTTAACTTTACGTGGAGACT | SEQ ID NO:12 |
Site-directed mutagenesis the whole plasmid was amplified using Vazyme 2xphanta master mix with the selected recombinant plasmid of 5 mutants as a template, and the reaction system was set up as shown in Table 3.
TABLE 3 site-directed mutagenesis System
| Name of the name | Volume (mu L) |
| Vazyme 2xphanta master mix | 12.5μL |
| pET-28a-TrGBE mutant recombinant plasmid | 1μL |
| primer-F | 1μL |
| primer-R | 1μL |
| ddH2O | To 25. Mu.L |
The PCR procedure was: pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing at 66 ℃ for 30s, extension at 72 ℃ for 5min, reaction for 30 cycles, extension at 72 ℃ for 10min, and finally heat preservation at 4 ℃. After the PCR reaction is finished, 1 mu L of DpnI digestive enzyme is added, the template is digested for 2 hours at 37 ℃, and the purified PCR product is recovered and converted into E.coli BL21 (DE 3) competent cells.
The transformation method comprises the following steps: mixing 10 μl of PCR product with 100 μl of Novozan commercial E.coli BL21 (DE 3) competence, ice-bathing for 20min, rapidly taking out after heat shock at 42deg.C for 90s, ice-bathing for 2min, adding 400 μl of LB liquid medium, recovering at 37deg.C and 200rpm for 60min, coating 100 μl of bacterial liquid on solid LB plate containing 30 μg/mL kana resistance, and culturing overnight at 37deg.C in incubator.
The next day three recombinant E.coli BL21 strains were selected from the plates, and the recombinant strains were inoculated from the plates into 50mL shaking tubes containing 5mL of liquid LB medium (LB (g/L): peptone 10, sodium chloride 10, yeast extract 5) containing the respective resistances, and the respective resistances were added, and incubated on a shaking table at 37℃for 12 hours at a rotation speed of 200rpm. After the completion of the culture, the plasmid was extracted and sent to general-purpose company for sequencing. Finally, comparing the sequencing result with a wild-type enzyme protein nucleic acid sequence to determine whether mutation is successful.
The above 5 sites were combined two by two according to the above method to construct 10 double mutant expression strains in total. The mutant proteins are purified and the activity is measured, the results show that the enzyme activity of 6 double mutants is obviously reduced compared with that of the single mutant in the first round, the enzyme activity of W416E/Y429D in the other 4 mutants is obviously improved, the relative enzyme activity is 2.7 times that of the wild type, and the specific results are shown in Table 4.
TABLE 4 relative enzyme Activity and specific Activity of 10 double mutant expression strains
| Catalytic enzymes | Relative enzyme Activity (%) | Specific activity (U/mg) |
| Wild-type TrGBE | 100 | 4263 |
| L306A/W416E | 247 | 10530 |
| L306A/Y429D | 136 | 5798 |
| L306A/I557D | 92 | 3922 |
| L306A/P588N | 126 | 5371 |
| W416E/Y429D | 272 | 11595 |
| W416E/I557D | 256 | 10913 |
| W416E/P588N | 210 | 8952 |
| Y429D/I557D | 129 | 5499 |
| Y429D/P588N | 88 | 3751 |
| I557D/P588N | 110 | 4689 |
The double mutants W416E/Y429D with the most obvious improvement of the enzyme activity are respectively combined with L306A, I557D, P588N to construct three mutants, the mutant proteins are purified and the activity is measured, and the enzyme activity is shown in Table 5. The three mutants are obviously improved compared with the wild type, and compared with the double mutants W416E/Y429D, the specific activity reaches 13726U/mg which is 3.2 times of that of the wild type.
Table 53 relative enzyme Activity and specific Activity of three mutant expression strains
| Catalytic enzymes | Relative enzyme Activity (%) | Specific activity (U/mg) |
| Wild-type TrGBE | 100 | 4263 |
| L306A/W416E/Y429D | 296 | 12618 |
| W416E/Y429D/I557D | 322 | 13726 |
| W416E/Y429D/P588N | 310 | 13215 |
Example 5: recombinant escherichia coli fermentation culture
Recombinant escherichia coli strains of three mutants with most obvious improvement of enzyme activity of 1 strain obtained by screening wild type TrGBE in the embodiment are selected: e.coli BL21 (DE 3) -28a-TrGBE and E.coli BL21 (DE 3) -28a-W416E/Y429D/I557D were inoculated into 5mL of LB medium containing kanamycin (30 ug/mL) resistance, cultured at 37℃for 8 hours, transferred to 200mL of TB fermentation medium in an inoculum size of 2%, and subjected to shake flask induction fermentation at 200rpm for 16 hours at 30℃before culturing at 37℃at 200rpm at a constant temperature until the cell OD600 reaches 0.6. After fermentation, crushing fermentation liquor, centrifuging, and obtaining supernatant which is wild starch branching enzyme TrGBE and mutant enzyme crude enzyme liquid. As a result of SDS-PAGE of the supernatant, a specific band having a molecular weight of about 77kDa was detected as shown in FIG. 1, which was consistent with the predicted protein size, indicating that the mutant was expressed efficiently and correctly.
Example 6: purification of enzymes
For purification of the above wild-type TrGBE and mutants thereof. An AKTAprime chromatography system from GE was used, and 5mL of High Affinity Ni-charge Resin FF nickel column (purchased from gold) was used for Affinity chromatography. The column was pre-equilibrated with pH 8.0, 300mM NaCl,10mM imidazole, 50mM sodium phosphate buffer (buffer A), elution buffer pH 8.0, 300mM NaCl,250mM imidazole, 50mM phosphate buffer (buffer B), gradient elution with 0% -100% buffer B, total elution time of 60min, then the collected active protein was further desalted by Sephacryl S-300 gel column to remove the contaminating proteins, monitored by UV detector, and the protein of interest was collected at high values. The purified enzyme-80 was stored for subsequent determination of protein concentration and activity.
Example 7: effect of pH and temperature on wild-type and mutant W416E/Y429D/I557D enzyme Activity
7.1 determination of optimum pH
Reaction substrate solutions were prepared using buffers 3.0, 4.0, 5.0, 6.0, 6.5, 7.0, 7.5, 8.0, 9.0, and 10.0 at different pH values, and the activity of the recombinant starch branching enzyme was measured at 60℃according to the enzyme activity detection method in example 3, and the highest activity was set to 100%, and the relative enzyme activities were compared. As can be seen from FIG. 2, the optimal reaction pH for the mutant W416E/Y429D/I557D enzyme was pH7.0.
7.2 determination of optimum reaction temperature
A reaction substrate solution was prepared using sodium phosphate buffer at pH7.0, and the activity of the recombinant starch branching enzyme was measured at different temperatures by controlling the reaction temperature to 40℃50℃60℃65℃70℃75℃80℃90℃according to the enzyme activity detection method in example 3, and the highest activity was set to 100% and the relative enzyme activities were compared. As can be seen from FIG. 3, the optimal reaction temperature of the mutant W416E/Y429D/I557D enzyme was 75℃which was 10℃higher than that of the wild type.
Example 8: preparation of resistant dextrins Using mutant W416E/Y429D/I557D enzyme
Corn starch is used as a substrate, 5% of HC1 solution with the concentration of 1mol/L is added, then high-temperature reaction is carried out at 160-200 ℃, and after cooling and sieving, the resistance component is 40% -45%. The high-temperature acidolysis is carried out to prepare the substrate with the concentration of 400g/L-500g/L, the enzyme quantity is 1000-1500U at the temperature of 65 ℃ and the pH value of 7.0, the enzyme conversion reaction is carried out for 6-8h, the content of the resistant dextrin reaches 70%, and the content of the resistant dextrin is improved by more than 20% after the high-temperature acidolysis.
Detecting the ratio change of alpha-1, 4 glycosidic bond and alpha-1, 6 glycosidic bond before and after the obtained crude resistant dextrin and starch are detected by a nuclear magnetic resonance method, and before starch branching enzyme is added, alpha-1, 4 glycosidic bond: α -1,6 glycosidic bond = 18.20:1, after addition of branching enzyme, alpha-1, 4 glycosidic bond: α -1.6 glycosidic bond = 6.74:1, a-1, 4 glycosidic linkages are decreasing and a-1, 6 glycosidic linkages are increasing.
The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, and yet fall within the scope of the invention.
Claims (10)
1. A starch branching enzyme mutant, characterized by: the starch branching enzyme mutant is obtained by mutating amino acid residues at least one of 306 th, 416 th, 429 th, 557 th and 588 th sites of starch branching enzyme TrGBE shown in SEQ ID NO.1, wherein leucine L at 306 th site is mutated into alanine A, tryptophan W at 416 th site is mutated into glutamic acid E, tyrosine Y at 429 th site is mutated into aspartic acid D, isoleucine I at 557 th site is mutated into D aspartic acid or proline P at 588 th site is mutated into asparagine N.
2. The starch branching enzyme mutant according to claim 1, characterized in that: the starch branching enzyme mutant is obtained by mutating amino acid residues at 306 th, 416 th, 429 th, 557 th or 588 th of a starch branching enzyme TrGBE shown in SEQ ID NO. 1; or the starch branching enzyme mutant is obtained by simultaneously mutating amino acid residues of any two positions of 306 th, 416 th, 429 th, 557 th and 588 th of the starch branching enzyme TrGBE shown in SEQ ID NO. 1; or the starch branching enzyme mutant is obtained by simultaneously mutating amino acid residues at any three of positions 306, 416, 429, 557 and 588 of the starch branching enzyme TrGBE shown in SEQ ID NO. 1.
3. The starch branching enzyme mutant according to claim 2, characterized in that: the starch branching enzyme mutant is obtained by mutating the 416 th position of the starch branching enzyme TrGBE shown in SEQ ID NO. 1; or the starch branching enzyme mutant is obtained by simultaneously mutating the 416 th site and the 306 th site, the 429 th site, the 557 th site or the 588 th site of the starch branching enzyme TrGBE shown in SEQ ID NO. 1; or the starch branching enzyme mutant is obtained by simultaneously mutating the 416 th, 429 th and 306 th, 557 th or 588 th of the starch branching enzyme TrGBE shown in SEQ ID NO. 1.
4. A nucleic acid encoding the starch branching enzyme mutant of any one of claims 1-3.
5. A recombinant expression vector comprising the nucleic acid of claim 4.
6. A genetically engineered bacterium comprising the recombinant expression vector of claim 5.
7. The genetically engineered bacterium of claim 6, wherein: the genetic engineering bacteria are recombinant strains obtained by introducing the recombinant expression vector obtained by connecting the nucleic acid with the vector of claim 4 into host bacteria, wherein the host bacteria are escherichia coli, bacillus subtilis or yeast.
8. Use of the starch branching enzyme mutant of any one of claims 1-3, the nucleic acid of claim 4, the recombinant expression vector of claim 5, the genetically engineered bacterium of claim 6 or 7 for the preparation of a resistant dextrin product.
9. Use of the starch branching enzyme mutant of any one of claims 1-3, the nucleic acid of claim 4, the recombinant expression vector of claim 5, the genetically engineered bacterium of claim 6 or 7 in the preparation of a food or pharmaceutical product.
10. A preparation method of resistant starch is characterized in that: the method comprises the steps of taking starch as a substrate, carrying out acidolysis on the substrate at high temperature, and then adding the starch branching enzyme mutant according to any one of claims 1-3 to carry out enzyme conversion reaction to prepare the resistant dextrin.
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