CN112391365B

CN112391365B - Starch branching enzyme mutant with improved catalytic activity and application thereof

Info

Publication number: CN112391365B
Application number: CN202011393059.3A
Authority: CN
Inventors: 李兆丰; 江海旻; 顾正彪; 李才明; 班宵逢; 程力; 洪雁
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2020-11-30
Filing date: 2020-11-30
Publication date: 2022-04-15
Anticipated expiration: 2040-11-30
Also published as: CN112391365A

Abstract

The invention discloses a starch branching enzyme mutant with improved catalytic activity and application thereof, belonging to the technical field of enzyme engineering. Compared with wild type starch branching enzyme, the starch branching enzyme mutants with improved catalytic activity are obtained by adopting the technical scheme of the invention, the specific enzyme activities of the starch branching enzyme mutants G160R and G160T are 12482U/mg and 10497U/mg respectively, and are improved by 88% and 58% respectively compared with the wild type starch branching enzyme; the starch branching enzyme is added into a reaction system taking DE11 maltodextrin as a substrate, compared with the wild starch branching enzyme, the stability of the product is obviously improved, and after the starch branching enzyme is placed at 4 ℃ for 30 days, the clarity of the products obtained after the starch branching enzyme mutants G160T and G160R provided by the invention are respectively 10.7 times and 12 times of that of the products obtained after the wild starch branching enzyme is added.

Description

Starch branching enzyme mutant with improved catalytic activity and application thereof

Technical Field

The invention relates to a starch branching enzyme mutant with improved catalytic activity and application thereof, belonging to the technical field of enzyme engineering.

Background

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. Through the transglycosylation reaction, the starch branching enzyme can increase the branching degree of starch and derivatives thereof and improve the use performance. Starch branching enzymes are therefore important amylases in the field of modification of starch by biological enzymatic methods.

Maltodextrin has the advantages of strong thickening property, good solubility and the like, but is easy to aggregate and age in the storage process, so that some products containing maltodextrin have the defects of poor transparency stability and the like, and the phenomenon of turbidity occurs after one month of storage, thereby limiting the application range of maltodextrin to a certain extent. Thus, researchers will typically modify maltodextrins with starch branching enzymes. In the previous studies, the inventors of the present invention carried out enzymatic modification of maltodextrin with starch branching enzyme derived from Rhodothermus obamensis STB05 (Wangchi. secretion expression of starch branching enzyme in Escherichia coli and modification of maltodextrin [ D ]: Master thesis, Wuxi: Jiangnan university, 2019.), and as a result, it was found that the transparency stability of maltodextrin could be significantly improved after 12 hours of modification of maltodextrin with an enzyme addition amount of 100U/g. However, the method has the problems of large enzyme adding amount, long reaction time and the like, which limit the effective modification of maltodextrin by the starch branching enzyme.

Therefore, how to improve the modification efficiency of maltodextrin to meet the requirement of industrial production becomes a problem to be solved urgently.

Disclosure of Invention

In order to solve the above problems, the inventors have found that, when the catalytic activity of a starch branching enzyme is increased, the amount of the enzyme to be added or the reaction time can be reduced, thereby improving the efficiency of maltodextrin modification.

The invention firstly provides a starch branching enzyme mutant, which is obtained by mutating the 160 th amino acid of the starch branching enzyme with the amino acid sequence shown as SEQ ID NO. 1.

In one embodiment of the invention, the mutant is obtained by mutating the 160 th amino acid of the starch branching enzyme with the amino acid sequence shown as SEQ ID NO.1 from glycine to arginine;

or the mutant is obtained by mutating the 160 th amino acid of the starch branching enzyme with the amino acid sequence shown as SEQ ID NO.1 from glycine to threonine.

In one embodiment of the invention, the starch branching enzyme is derived from Rhodothermus obamensis STB 05.

In one embodiment of the invention, the nucleotide sequence of the starch branching enzyme is shown as SEQ ID No. 2.

The invention also provides a gene for coding the mutant.

The invention also provides a recombinant plasmid carrying the gene.

In one embodiment of the present invention, the recombinant plasmid is a starting plasmid selected from the group consisting of a pET-series vector, a pGEX-series vector, and a pKD-series vector.

The invention also provides a recombinant cell carrying the gene or the recombinant plasmid.

In one embodiment of the present invention, the recombinant cell is a bacterial or fungal expression host.

In one embodiment of the invention, the expression host is Escherichia coli or Bacillus subtilis.

In one embodiment of the invention, the expression host is Escherichia coli BL21(DE3) or Bacillus subtilis WB 600.

The invention also provides a method for improving the stability of maltodextrin, which is to add the mutant or the recombinant cell into a reaction system containing maltodextrin for reaction.

In one embodiment of the present invention, the method comprises adding at least 1. mu.g/g of the above mutant to a reaction system containing maltodextrin, and reacting at 60 to 70 ℃ for 3 to 5 hours.

In one embodiment of the present invention, the starch branching enzyme mutant is added in an amount of 8. mu.g/g.

In one embodiment of the invention, the maltodextrin is 30% (w/w) DE11 maltodextrin on a dry basis.

The invention also provides a method for hydrolyzing starch, which is to add the mutant or the recombinant cell into a reaction system containing starch for reaction.

In one embodiment of the present invention, the method comprises adding 5 to 25. mu.g/g of the mutant or 0.1 to 0.5G (DCW)/mL of the recombinant cell to a reaction system containing starch, and reacting at 60 to 70 ℃ for 12 to 24 hours.

The invention also provides the application of the mutant, the gene, the recombinant plasmid or the recombinant cell in modifying maltodextrin.

Advantageous effects

(1) Compared with wild starch branching enzyme, the starch branching enzyme mutants G160R and G160T obtained by the invention have specific enzyme activities of 12482U/mg and 10497U/mg respectively, and are respectively increased by 88% and 58% compared with wild starch branching enzyme.

(2) The starch branching enzyme provided by the invention can improve the stability of maltodextrin, compared with the wild starch branching enzyme, the starch branching enzyme provided by the invention is added into a reaction system taking DE11 maltodextrin as a substrate, the product stability is obviously improved, and after the starch branching enzyme is placed at 4 ℃ for 30 days, the clarity of the products obtained after the starch branching enzyme mutants G160T and G160R provided by the invention are respectively 10.7 times and 12 times of that of the products obtained after the wild starch branching enzyme is added.

Drawings

FIG. 1: and (3) electrophoresis picture of starch branching enzyme crude enzyme protein.

FIG. 2: thermostability of starch branching enzyme.

FIG. 3: real-time profiles of starch branching enzyme wild-type and mutant modified maltodextrins stored at 4 ℃ for 30 days.

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 media involved in the following examples are as follows:

LB liquid medium: 5g/L of yeast powder, 10g/L of tryptone, 10g/L of NaCl and 7.0 of pH.

LB solid medium: 5g/L of yeast powder, 10g/L of tryptone, 10g/L of NaCl, 15g/L of agar and 7.0 of pH.

TB liquid medium: 24g/L yeast powder, 12g/L tryptone, 5g/L glycerol and KH₂PO₄ 2.3136g/L，K₂HPO₄ 16.4318g/L，pH 7.0。

The detection methods referred to in the following examples are as follows:

the method for measuring the activity of the starch branching enzyme comprises the following steps:

0.25% (w/v) potato amylopectin solution prepared from 50mM phosphate buffer solution (pH7.0) is used as substrate for enzyme reaction, 900 μ L of the substrate is incubated at 65 deg.C for 10min, 100 μ L of 2mg/mL starch branching enzyme is added, mixed uniformly and placed in a water bath at 65 deg.C for reaction for 15 min. The reaction is stopped by inactivating the enzyme in boiling water bath. After cooling to room temperature, 300. mu.L of the reaction mixture was added to 5mL of 0.05% (w/v) KI and 0.005% (w/v) I developing solution₂pH 6.0, left at room temperature for sufficient color development. Absorbance at 530nm was measured after development for 15 min.

One unit of enzyme activity (U/mL) is defined as: at 530nm, the amount of enzyme added required to reduce absorbance by 1% per minute was one unit of enzyme activity.

The method for measuring the concentration of the starch branching enzyme protein comprises the following steps:

protein concentration was determined using the Bradford protein concentration determination kit (detergent compatible type) for shanghai Biyun, which employs the Bradford method, according to the instructions. Bovine serum albumin was used to draw a standard curve.

The method for measuring the specific enzyme activity of the starch branching enzyme comprises the following steps:

a standard curve is drawn by taking bovine serum albumin as a standard substance, and the protein concentration of the wild type and the mutant of the starch branching enzyme is determined by a Bradford method. The specific activity of the enzyme refers to the number of enzyme activity units of unit weight (mg) of protein under a specific condition, and the specific activity can be used for comparing the catalytic capacity of the protein per unit mass in an enzyme preparation by utilizing the specific activity, so that the specific activity is calculated by the enzyme activity and the protein concentration.

The method for detecting the clarity of the maltodextrin solution comprises the following steps:

respectively weighing 15g (calculated by dry basis) of original maltodextrin (control) and modified samples thereof, dissolving in 50mL of deionized water, storing the prepared maltodextrin aqueous solution in a 40mL sample bottle at constant temperature (4 ℃), measuring the transparency of the maltodextrin aqueous solution at intervals, and photographing to record the real-time state of the maltodextrin aqueous solution. The transparency is represented by the light transmittance (%) measured at 620nm with a spectrophotometer.

Example 1: construction of genetically engineered bacterium containing starch branching enzyme mutant

1. Construction of recombinant plasmid containing starch branching enzyme mutant

The method comprises the steps of taking Rhodothermus obamensis genome DNA as a template, amplifying by a PCR method to obtain gbe genes (nucleotide sequences are shown as SEQ ID NO. 2) with Nde I and Xho I restriction enzyme sites at two ends, inserting the gbe genes into a pMD 18-T simple plasmid to obtain a recombinant plasmid pMD 18-T simple-gbe, carrying out double enzyme digestion on the recombinant plasmid, recovering a target gene fragment containing a sticky end, and inserting the target gene fragment into a pET-20b plasmid treated by the same endonuclease to obtain a recombinant vector pET-20 b-gbe. Complementary Primer chains (see table 1) required for experiments were designed using expression vector pET-20b-gbe as a template, and primers were synthesized by jinzhi biotechnology limited, and site-directed mutagenesis was performed according to the method shown in the kit manual of STAR Primer GXL of TaKaRa corporation.

PCR reaction system according to conditions set in STAR Primer kit instructions: 5 × PrimeSTAR Buffer (Mg)²⁺Plus) 10. mu.L, template DNA 1. mu.L, forward and reverse primers (10. mu.M) 1. mu.L, PrimeSTAR HS DNA Polymerase (2.5U/. mu.L) 0.5. mu.L, dNTPs (2.5 mM each) 4. mu.L, and finally ultrapure water 32.5. mu.L.

The PCR amplification conditions were: pre-denaturation at 98 deg.C for 3 min; pre-denaturation at 98 ℃ for 3 min; followed by denaturation at 98 ℃ for 10 s; annealing at 60 ℃ for 15 s; extension at 68 ℃ for 5.5min, and 30 cycles; finally, the temperature is kept for 10min at 68 ℃.

TABLE 1 introduction of starch branching enzyme mutation sites

Remarks, underlined bases correspond to the corresponding mutated amino acids.

2. Construction of genetically engineered bacterium containing starch branching enzyme

Respectively treating the PCR products obtained in the step 1 by DpnI for 2 hours at 37 ℃, then respectively transforming the treated PCR products into E.coli JM 109 competent cells to obtain transformation products, respectively coating the obtained transformation products into LB solid culture medium containing 100 mu G/mL of ampicillin, culturing for 12 hours in a 37 ℃ incubator, selecting a single colony, inoculating the single colony into LB liquid culture medium containing 100 mu G/mL of ampicillin, culturing overnight at 37 ℃ at 200r/min, extracting plasmids according to a method shown in a plasmid extraction kit specification, identifying and sequencing, wherein the sequencing is correct, namely the successfully-constructed recombinant plasmids containing the mutants, namely pET-20b-G160R, pET-20b-G160T, pET-20b-G160A and pET-20 b-G160W; respectively transferring the successfully constructed recombinant plasmid containing the wild type starch branching enzyme and the recombinant plasmid containing the mutant into an expression host E.coli BL21(DE3) by a chemical conversion method; finally, genetically engineered bacteria E.coli BL21(DE3)/pET-20b-gbe, E.coli BL21(DE3)/pET-20b-G160R, E.coli BL21(DE3)/pET-20b-G160T, E.coli BL21(DE3)/pET-20b-G160A and E.coli BL21(DE3)/pET-20b-G160W are obtained.

Example 2: expression of starch branching enzyme mutants

The method comprises the following specific steps:

(1) activating and culturing genetically engineered bacteria:

the genetically engineered bacteria E.coli BL21(DE3)/pET-20b-gbe, E.coli BL21(DE3)/pET-20b-G160R, E.coli BL21(DE3)/pET-20b-G160T, E.coli BL21(DE3)/pET-20b-G160A, and E.coli BL21(DE3)/pET-20b-G160W obtained in example 1 were streaked on LB solid medium, and cultured in a 37 ℃ incubator for 12 hours, and then positive single colonies were each selected and inoculated into a sterilized 250mL triangular flask containing 50mL of LB liquid medium (ampicillin was added to 100. mu.mg/mL before use), and the flask was placed in a 200r/min rotary shaker at 37 ℃ for 8 to 10 hours to obtain a seed solution.

(2) Fermentation culture:

the seed solutions obtained in step (1) were inoculated in an amount of 2% (v/v) into 250mL flasks containing 50mL of TB liquid medium (ampicillin was added to a final concentration of 100. mu.mg/mL before use) and incubated at 37 ℃ for 96 hours in a rotary shaker at a rate of 200r/min to obtain a fermentation broth.

(3) Expression of starch branching enzyme mutants:

centrifuging the fermentation liquor obtained in the step (2) for 20min at 10000 Xg respectively, and taking supernate to obtain crude enzyme liquid containing wild type starch branching enzyme WT, crude enzyme liquid containing mutant G160R, crude enzyme liquid containing mutant G160T, crude enzyme liquid containing mutant G160A and crude enzyme liquid containing mutant G160W respectively; the crude enzyme solution was analyzed by SDS-PAGE (FIG. 1), and both the wild-type and mutant crude enzyme solutions showed a molecular band at 73kDa, indicating successful expression.

Example 3: separation and purification of starch branching enzyme mutant

The crude enzyme solution containing the wild-type starch branching enzyme WT, the crude enzyme solution containing the mutant G160R, the crude enzyme solution containing the mutant G160T, the crude enzyme solution containing the mutant G160T, the crude enzyme solution containing the mutant G160A, which were obtained in example 2, were filtered through a 0.45 μm aqueous membrane, and then HisTrap was used^TMAnd (4) carrying out nickel column affinity chromatography purification by using an HP column (specification of 5 mL). The method comprises the steps of balancing a nickel column (about 5 column volumes) by using a buffer solution A at a flow rate of 2mL/min, configuring a sample into a system with the same solution A, keeping the flow rate unchanged, loading 60mL of the sample, eluting by using a 60% (v/v) buffer solution B, and collecting eluent to obtain pure enzymes, namely the wild type starch branching enzyme WT pure enzyme, mutant G160R pure enzyme, mutant G160T pure enzyme, mutant G160A pure enzyme and mutant G160W pure enzyme.

And (3) buffer solution A: 20mM imidazole, 10mM Tris-HCl, 500mM NaCl, pH7.0

And (3) buffer solution B: 500mM imidazole, 10mM Tris-HCl, 500mM NaCl, pH7.0

Crude enzyme solution: 20mM imidazole, 500mM NaCl, pH7.0

Example 4: analysis of specific enzyme Activity of starch branching enzyme mutant

Adding potato amylopectin into 50mM phosphate buffer solution (pH7.0), preparing 0.25% (w/v) potato amylopectin solution as reaction substrate, and preheating the reaction substrate at 65 deg.C for 15 min;

0.9mL of the preheated substrate was placed in a reaction vessel, 0.1mL of 10. mu.g/mL of the pure wild-type starch branching enzyme WT obtained in example 3, the pure mutant G160R, the pure mutant G160T, the pure mutant G160A and the pure mutant G160W were added to the reaction vessel, respectively, to obtain reaction systems, and the respective reaction systems were reacted at 65 ℃ to determine the activity of the starch branching enzyme mutants.

The measured specific enzyme activity results are shown in table 2, compared with the wild type, the specific enzyme activities of the mutants G160R and G160T are respectively increased by 88% and 58%, and the specific enzyme activities of the mutants G160A and G160W are respectively reduced by 1% and 11% compared with the wild type.

TABLE 2 specific enzyme Activity of wild type and mutant GBE

Each value is the average of 3 replicates.

Example 5: thermostability assay of starch branching enzyme mutants

The wild type starch branching enzyme WT pure enzyme, the mutant G160R pure enzyme, the mutant G160T pure enzyme, the mutant G160A pure enzyme and the mutant G160W pure enzyme obtained in the example 3 are respectively subjected to heat preservation at 85 ℃ for 2 hours, enzyme liquid is taken out at intervals, the enzyme liquid is immediately placed in an ice-water mixture for cooling, the residual enzyme activity of the starch branching enzyme is determined, and the activity of the enzyme liquid which is not subjected to heat preservation is 100%.

As shown in fig. 2, mutants G160R and G160T have improved thermostability compared to the wild type, while mutants G160A and G160W have reduced thermostability compared to the wild type.

Example 6: starch branching enzyme mutant modified maltodextrin

Adding 8 μ G/G of the pure wild-type starch branching enzyme WT enzyme, the pure mutant G160R enzyme, the pure mutant G160T enzyme, the pure mutant G160A enzyme and the pure mutant G160W enzyme obtained in example 3 to an aqueous solution containing maltodextrin at a dry basis concentration of 30% (w/w) DE11 to obtain a reaction system; taking no starch branching enzyme as a blank control;

respectively placing the reaction systems at 65 ℃ for reacting for 4h, and inactivating enzymes in boiling water bath for 30min to obtain reaction products.

The reaction product was stored in a 40mL sample bottle at constant temperature (4 ℃) for 30 days, the clarity of the solution was measured every 2 days, and the real-time status and the clarity were recorded by photographing, and the specific results are shown in table 3 and fig. 3, in which Control in fig. 3 is a blank Control.

TABLE 3 clarity of different starch branching enzyme modified maltodextrins

As can be seen from Table 3 and FIG. 3, after standing at 4 ℃ for 30 days, the clarity of the products obtained after adding the starch branching enzyme mutants G160T and G160R provided by the present invention was 10.7 and 12 times that of the products obtained after adding the wild-type starch branching enzyme, respectively; the clarity of the products obtained by adding the starch branching enzyme mutants G160A and G160W is not much different from that of the products obtained by adding the wild type starch branching enzyme, and is even lower.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

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Claims

1. A starch branching enzyme mutant is characterized in that the mutant is obtained by mutating the 160 th amino acid of the starch branching enzyme with the amino acid sequence shown as SEQ ID NO.1 from glycine to arginine or threonine.

2. A gene encoding the mutant of claim 1.

3. A recombinant plasmid carrying the gene of claim 2.

4. The recombinant plasmid according to claim 3, wherein the recombinant plasmid is a starting plasmid selected from the group consisting of a pET-series vector, a pGEX-series vector, and a pKD-series vector.

5. A recombinant cell carrying the gene of claim 2, or the recombinant plasmid of claim 3 or 4.

6. The recombinant cell of claim 5, wherein the recombinant cell is a bacterial or fungal expression host.

7. A method for improving the stability of maltodextrin by adding the mutant according to claim 1 or the recombinant cell according to claim 5 or 6 to a reaction system containing maltodextrin for reaction.

8. A method for hydrolyzing starch, which comprises adding the mutant according to claim 1 or the recombinant cell according to claim 5 or 6 to a reaction system containing starch to carry out the reaction.

9. Use of the mutant of claim 1, or the gene of claim 2, or the recombinant plasmid of claim 3 or 4, or the recombinant cell of claim 5 or 6 for modifying maltodextrin.