CN117165548A - Method for preparing branched starch by using mutant glycogen branching enzyme - Google Patents
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Abstract
The invention discloses a method for preparing branched starch by using mutant glycogen branching enzyme, belonging to the field of biological modified starch. The invention discloses a mutant glycogen branching enzyme H415W with an optimal reaction temperature of 70 ℃. The mutant glycogen branching enzyme H415W directly acts on starch, so that the conventional modification step of cooling and enzyme adding after high-temperature pregelatinization treatment is omitted, the effect of directly adding the mutant glycogen branching enzyme H415W to act on granular starch at 70 ℃ is realized, the synchronous proceeding of branching reaction and starch swelling is realized, the water consumption and the energy consumption required by drying required by production are reduced, and adverse effects on the texture and the property of starch products caused by high-temperature gelatinization are avoided. And the method can treat the starch milk with higher concentration, improves the production strength and efficiency, and provides a new environment-friendly idea and means with low carbon, energy conservation and consumption reduction for preparing branched starch by biological modification.
Description
Technical Field
The invention relates to a method for preparing branched starch by using mutant glycogen branching enzyme, belonging to the field of biological modified starch.
Background
Starch is one of the main sources of energy supplied to the human body, consisting of amylose and amylopectin. In the food processing and application process, most of natural starches can bring about a series of structural and property changes through high temperature, cooking or shearing, and the changes have great influence on the digestibility, aging speed and solubility of the starches, which is against the current development trend of the food industry. The amylose content and the side chain distribution of amylopectin are critical in the change of starch structure and properties, and are closely related to digestibility, retrogradation, solubility and the like. The modified branched starch has more branching degree than natural starch, and can obviously improve the solubility, viscosity, retrogradation, rheology, digestion and other characteristics of the original starch.
At present, the research on branched starch mainly modifies the molecular structure of starch by means of genes and enzyme methods. The enzymatic modification has the characteristics of high reaction efficiency, mild reaction conditions and the like, is suitable for large-scale industrial production, and has wide application. Glycogen branching enzyme (Glycogen branching enzyme, GBE, EC 2.4.1.18) is an important enzyme for preparing branched starch by biological enzyme modification, and can catalyze the rupture of alpha-1, 4 glycosidic bonds in starch molecules to generate short chains with non-reducing ends, and the obtained short chains are connected to straight chains or branched chains through the alpha-1, 6 glycosidic bonds, so that the starch structure is effectively modified, the branching degree is improved, the amylose content is reduced, and branched starch with low digestion performance and slow lifting speed is formed.
At present, most branched starch researches are focused on the high-temperature (above 90 ℃) gelatinization treatment of starch, then cooling and enzyme adding for modification, or the direct modification of granular starch at low-medium temperature (about 60 ℃ or below), and the problems of long action time, lower substrate concentration, poor action effect and the like exist.
Disclosure of Invention
In order to solve the technical problems existing at present, the invention provides a mutant glycogen branching enzyme. And (3) at 70 ℃, starch which is not subjected to pregelatinization is used as a substrate, and mutant glycogen branching enzyme is used for starch branching modification. The concentration of starch is improved, the production strength is enhanced, and the method is beneficial to industrialized production of branched starch.
The technical scheme of the invention is as follows:
the invention provides a glycogen branching enzyme mutant with improved thermostability, which is based on a starting sequence and has mutation of valine at 140 th or histidine at 415 th.
In one embodiment of the present invention, the mutant is a mutation of valine at position 140 of a glycogen branching enzyme having an amino acid sequence shown in SEQ ID NO.2 to isoleucine; the name is: V140I.
In one embodiment of the invention, the mutant is characterized in that histidine at 415 th position of glycogen branching enzyme with an amino acid sequence shown as SEQ ID NO.2 is mutated into tryptophan; the name is: H415W.
In one embodiment of the invention, the starting sequence has the amino acid sequence shown in SEQ ID NO.2 or an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% homology to the sequence shown in SEQ ID NO. 2.
The invention also provides genes encoding the mutants.
The invention also provides a recombinant plasmid carrying the gene or expressing the mutant.
The invention also provides recombinant microbial cells expressing the mutants.
The invention also provides an enzyme preparation containing the glycogen branching enzyme mutant.
In one embodiment of the invention, a stabilizer is included in the enzyme preparation.
In one embodiment of the invention, the enzyme preparation is a liquid preparation or a lyophilized powder.
The invention also provides a method for preparing branched starch by catalyzing starch with the mutant or the enzyme preparation.
In one embodiment of the invention, the method comprises the steps of dispersing starch in a buffer solution to obtain starch milk, adding the mutant or the enzyme preparation into the starch milk for constant temperature reaction to obtain a product, boiling and drying the product, grinding and sieving to obtain the branched starch.
In one embodiment of the invention, the concentration of the starch milk is 20% (w/w).
In one embodiment of the present invention, the buffer has a pH of 5.0 to 7.0.
In one embodiment of the invention, the buffer comprises sodium acetate buffer.
In one embodiment of the invention, the reaction temperature of the constant temperature reaction is 60-80 ℃ and the reaction lasts for 8-20 hours.
In one embodiment of the invention, the glycogen branching enzyme mutant is added in an amount of 50U/g dry basis starch.
In one embodiment of the invention, the freeze-drying is performed after boiling for 30 min.
In one embodiment of the invention, the starch comprises a starch having an amylose content of greater than 1%.
In one embodiment of the invention, the starch comprises one or more of common corn starch, tapioca starch and pea starch.
The invention also provides application of the mutant, the gene, the recombinant plasmid, the recombinant microbial cell, the enzyme preparation or the method for preparing branched starch in preparing foods, feeds and medicines.
The invention has the beneficial effects that
1. The branched starch is prepared by utilizing the modified starch by the biological enzyme method, the raw materials are easy to obtain, the process is simple, the operation is convenient, and the product yield is high. The glycogen branching enzyme is utilized to modify starch, other chemical groups are not introduced, other types of glycosidic bonds are not generated, and only the recombination assembly of alpha-1, 4 glycosidic bonds and alpha-1, 6 glycosidic bonds in starch molecules is generated, so that the product safety is high.
2. The optimal reaction temperature of the mutant glycogen branching enzyme H415W provided by the invention is raised to 70 ℃. Because the gelatinization temperature of the common starch is reached at 70 ℃, the step of pre-gelatinizing the starch by using high temperature (above 90 ℃) is avoided, and the activity of H415W can be kept 100%, so that the synchronous proceeding of branching reaction and starch swelling is realized, and the modification step is shortened. The modified starch prepared by using the mutant glycogen branching enzyme H415W has an amylose content reduced by about 4.42% as compared with the modified starch prepared by the wild type and about 3.89% as compared with the modified starch prepared by the mutant V140I.
3. Compared with natural corn starch, the branched starch prepared by the invention has the highest amylose content reduced by 8.27%; the branch length of DP <13 increases significantly; the quick digestion rate in the in vitro digestion rate is reduced by 20.35 percent; and the retrogradation enthalpy value (delta H) is reduced by 4.34J/g at the highest in inhibiting retrogradation.
4. The mutant glycogen branching enzyme H415W acts on starch, so that the conventional high-temperature pre-gelatinization treatment is simplified, the modification step of cooling and enzyme adding is realized, the effect that the H415W is directly added at 70 ℃ to act on granular starch is realized, the synchronous proceeding effect of branching reaction and starch swelling is realized, the energy consumption required by production and drying is reduced, and adverse effects on the texture and property of starch products caused by high-temperature (above 90 ℃) gelatinization are avoided. And the method can treat the starch milk with higher concentration, improves the production strength and efficiency, and provides a new environment-friendly idea and means with low carbon, energy conservation and consumption reduction for preparing branched starch by biological modification.
Drawings
Fig. 1: the amylose measurement wavelength was analyzed with respect to the reference wavelength.
Fig. 2: amylose standard curve.
Fig. 3: amylose content of wild-type and mutant glycogen branching enzyme products.
Detailed Description
Technical terms:
glycogen branching enzyme: the term "glycogen branching enzyme" refers to an enzyme in class EC 2.4.1.18 as defined by the enzyme nomenclature. For the purposes of the present invention, "activity of glycogen branching enzyme" is determined according to the procedure described in the examples. In one aspect, the glycogen branching enzyme of the present invention is a glycogen branching enzyme having an amino acid sequence as shown in SEQ ID NO. 1; or a glycogen branching enzyme having at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to the amino acid sequence shown in SEQ ID No. 1; or a glycogen branching enzyme gene having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% sequence with the coding sequence shown in SEQ ID NO. 2.
Expression: the term "expression" includes any step involving the production of glycogen branching enzyme mutants, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
Expression vector: the term "expression vector" means a linear or circular DNA molecule comprising a polynucleotide encoding a glycogen branching enzyme mutant of the invention and operably linked to control sequences that provide for expression thereof.
Host cell: the term "host cell" means any cell type that is readily transformed, transfected, transduced, or the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" encompasses any parent cell progeny that are not identical to the parent cell due to mutations that occur during replication.
The host cell may be any cell useful in the recombinant production of glycogen branching enzyme mutants, such as prokaryotic or eukaryotic cells.
The prokaryotic host cell may be any gram-positive or gram-negative bacterium. Gram positive bacteria include, but are not limited to: bacillus, clostridium, enterococcus, geobacillus (Geobacillus), lactobacillus, lactococcus, bacillus, staphylococcus, streptococcus and streptomyces. Gram-negative bacteria include, but are not limited to, campylobacter, escherichia, flavobacterium, fusobacterium, helicobacter, mirobacter, neisseria, pseudomonas, salmonella, and ureaplasma.
The host cell may also be a eukaryotic organism, such as a mammalian, insect, plant or fungal cell.
Amylose: is a linear long chain molecule formed by connecting hundreds to thousands of glucose monomers through alpha-1, 4 glycosidic bonds (99%), and contains a small amount of alpha-1, 6 branched chains (1.0%), wherein the alpha-1, 6 glycosidic bonds of branch points account for 0.3 to 0.5 percent of the total glycosidic bonds, and the polymerization degree is 324 to 4920.
Branched starch: the branched starch and the modified starch can be used interchangeably, and are a multi-branched soluble multi-carbohydrate formed by D-glucose, wherein the main chain is formed by connecting D-glucose groups through alpha-1, 4 glycosidic bonds, one branch exists in every 24-30D-glucose groups, and the branch point is connected with the main chain through alpha-1, 6 glycosidic bonds.
The degree of polymerization (degree ofpolymerization, DP) refers to the average number of anhydrous glucose units in the molecule.
Rapidly digestible starch refers to the fraction of starch that increases rapidly in blood glucose levels after ingestion.
Slowly digestible starch refers to the starch component that is fermented in the large intestine.
The main reagents involved in the following examples: the E.coli BL21 (DE 3) and BCA concentration assay kit were purchased from Biyun biotechnology Co., ltd, isoamylase (EC 3.2.1.68,200U/mL) was purchased from Megazyme Co., U.S.A., pepsin (P7000, EC 3.4.23.1,250U/mg) and amyloglucosidase (A7095, EC 3.2.1.3,260U/mL) were purchased from SigmaAldrich Co., U.S.A., porcine pancreatin (P110505, EC 232-468-9) and common cornstarch were purchased from Shanghai Albumin Biotechnology Co., ltd, and other common reagents were all of domestic analytical purity.
The following examples relate to the following media: the culture medium is prepared by ultrapure water, and the culture medium is sterilized by an autoclave at 121 ℃ for 20min after the preparation.
LB liquid medium: yeast extract 0.5g/100mL, tryptone 1.0g/100mL, naCl 1.0g/100mL.
LB solid medium: on the basis of LB liquid medium, agar powder is 1.5g/100mL.
LB liquid resistant Medium: based on LB liquid medium, 100mL of liquid medium was added with kanamycin at a final concentration of 30. Mu.g.mL -1 。
LB solid medium: on the basis of LB solid medium, 100mL of solid medium is added with kanamycin, and the final concentration is 30 mug.mL -1 。
The following examples relate to methods for measuring enzyme activity:
200 μl of 1mg/mL amylose solution is taken to be placed in a 5mL centrifuge tube, the centrifuge tube is placed in a water bath shaking table for heat preservation for 5min, 14 μg enzyme is added, shaking and mixing are carried out uniformly, the mixture is reacted for 10min under the conditions of the corresponding optimal temperature and pH6.0 of mutant enzyme and wild type enzyme respectively at 150rpm of the water bath shaking table, 500 μl of 1mmol/L hydrochloric acid is added for quenching reaction, 1mL iodine solution is added for color development for 5min under dark condition, absorbance change is measured at 660nm, and enzyme activity is calculated.
Enzyme activity is defined as the amount of enzyme that causes a decrease in absorbance of 0.01 optical density units per minute at 660nm absorbance as one enzyme activity unit (U).
Iodine solution preparation: 0.5mL stock iodine solution (containing 0.26. 0.26g I) 2 And 2.6g of an aqueous solution of KI) and 1mL of 1mol/L HCl were brought to a volume of 130mL using ultrapure water.
The purification solutions of the enzymes involved in the following examples were as follows:
(1) Loading buffer solution: 2.42g of Tris-HCl,29.22gNaCl,1.36g imidazole was weighed, dissolved in ultrapure water and fixed to a volume of 1L (pH=7.4).
(2) Elution buffer: 2.42g of Tris-HCl,29.22gNaCl,34.04g imidazole was weighed, dissolved in ultrapure water and fixed to a volume of 1L (pH=7.4).
(3) And (3) heavy suspension: 2.42g of Tris-HCl,29.22g of NaCl, ultrapure water were weighed out and dissolved and the volume was set to 1L (pH=7.4).
The modification solutions and assay required solutions referred to in the examples below:
(1) Sodium acetate buffer: weigh 4.101g CH 3 COOH (anhydrous), ultrapure water was dissolved and fixed to a volume of 1L (ph=6.0).
(2) Amylose mother liquor: 10mg of amylose was weighed, 1mL of 1mol/LNaOH solution was added thereto, and the volume was set to 10mL with ultrapure water.
(3) Amylopectin mother liquor: 10mg of pullulan was weighed, 1mL of 1mol/LNaOH solution was added thereto, and the volume was set to 10mL with ultrapure water.
(4) Iodine solution: 200mg KI and 20mg I were weighed 2 Ultrapure water was set to a volume of 10mL.
The detection method involved in the following examples is as follows:
(1) Amylose content determination
The amylose content of the raw starch and the modified starch is determined by a dual-wavelength iodine colorimetry. A1 mg/mL stock solution was prepared using pure amylose and amylopectin standards. 1.5mL of amylose mother liquor and 6mL of amylopectin mother liquor are taken in a beaker respectively, the pH is regulated to 3.0 by 0.1mol/LHCl, 0.5mL of iodine solution is added, and the volume is fixed to 50mL. Selecting the determination wavelength lambda of amylose by wavelength scanning in the range of 400-800nm 1 And lambda (lambda) 2 . The amylose mother liquors of 0.25, 0.50, 1.00, 1.25 and 1.50mL are respectively absorbed, the pH value is regulated to 3.0 by 0.1mol/LHCl, and 0.5mL of iodine solution is added to fix the volume to 50mL. Amylose solution was prepared at 5, 10, 15, 20, 25, 30. Mu.g/mL, and 1mL NaOH (0.1 mol/L) was used as a blank. At lambda 1 And lambda (lambda) 2 Respectively measuring absorbance A 1 And A 2 By means of absorbance difference (A 1 -A 2 ) And (5) drawing an amylose standard curve. A sample solution was prepared at 1mg/mL, and 50mL of the sample solution was subjected to the above-described procedure to determine the amylose content.
(2) Branch distribution measurement step
The chain length distribution was by anion chromatography (HPAEC-PAD). 10mg (dry basis) of raw corn starch and modified corn starch are accurately weighed, 2mL of sodium acetate buffer (50 mmol/L, pH=4.5) is added, after 30min of vortex gelatinization in a boiling water bath, 1.5mL of gelatinized solution is taken to be placed in a water bath with 40 ℃ for oscillation and heat preservation for 10min, isoamylase (5U) is added, and reaction is carried out for 24h at 40 ℃. Stopping the reaction in boiling water bath for 30min, centrifuging at 10000 Xg for 10min, passing supernatant through 0.22 μm water-based membrane, and detecting by ion chromatography.
(3) In vitro digestibility determination step
Preparing 0.75mol/L HCl, taking 150mL, adding 10mg pepsin and 50mg guar gum to form pepsin solution, and storing in ice bath. 1g of porcine pancreatin was thoroughly mixed with 20mL of ultrapure water, magnetically stirred for 15min, and centrifuged at 6000rpm for 10min. Taking 15mL of supernatant, adding 0.2mL of amyloglucosidase, fully mixing to prepare a mixed enzyme solution, and storing in an ice bath. 200mg (dry basis) of the sample was taken in 15mL of 0.2mol/L sodium acetate buffer (pH=5.2), and after 30min of swirling gelatinization in a boiling water bath, it was preheated in a 37℃water bath shaker (160 rpm) for 15min, and 10 glass beads were added. 2mL of pepsin solution was added, the reaction was allowed to proceed for 30min with shaking, followed by 1mL of the mixed enzyme solution. And taking 0.2mL of reaction liquid respectively at the reaction time of 20min and 120 min. The enzyme was deactivated by adding 8 times 66.6% ethanol, and the resulting mixture was centrifuged at 4000rpm for 10 minutes, and 0.1mL of the supernatant was collected and the glucose content was measured by the glucokinase method.
The determination of the glucose content is carried out by the glucokinase method described in the literature of Luxiyu, haocun Kong, zhengbiao Gu, caiming Li, xiaofeng Bans, li Cheng, yan Hong, zhaoeng Li, two 1, 4-. Alpha. -glucan branching enzymes successively rearrange glycosidic bonds: A novel synergistic approach for reducing starch digestibility, carbohydrate Polymers, volume 262,2021,117968,ISSN 0144-8617.
The specific formula is as follows:
RDS(％)＝(G20-FG)×0.9/TS
SDS(％)＝(G120-G20)×0.9/TS
RS(％)＝100-RDS(％)-SDS(％)
wherein:
free glucose content (mg) in starch before FG-enzymatic hydrolysis treatment;
glucose content (mg) produced after 20min of G20-amylase hydrolysis;
glucose content (mg) produced after 120min of G120-amylase hydrolysis;
total starch content (mg) in TS-samples.
(4) Retrogradation property determination step
The retrogradation property determination was characterized using Differential Scanning Calorimeter (DSC). Weighing a certain amount of samples, and mixing the samples with deionized water according to a ratio of 1:2 (mass ratio), carrying out aluminum crucible tabletting, placing a sample tray at 4 ℃ for standing for 12 hours to balance water, and taking an empty crucible as a control group. Setting test conditions: the temperature range is 20-100 ℃, and the temperature rising rate is 10 ℃/min. The sample was gelatinized and stored at 4℃for 7 days, and the initial temperature (T) of the sample after 7 days of storage at 4℃was measured o ) Peak temperature (T) p ) Termination temperature (T) c ) And retrogradation enthalpy (Δh). Retrogradation of starch refers to naturally cooling completely gelatinized starch at a lower temperature to break down starch fraction during gelatinizationThe sub-hydrogen bonds are recombined and the molecules become ordered. The retrograded starch is heated again with a concomitant change in energy, which appears as an endothermic peak on the DSC profile. At this time, the peak shows the retrogradation enthalpy value ΔH, and the initial, peak and end temperatures during heating are correspondingly denoted as T o 、T p 、T c 。
Example 1: construction of mutants
(1) Construction of recombinant vector containing wild glycogen branching enzyme Ph GBE Gene
According to the accession number PDB:5WU7 (the amino acid sequence is shown as SEQ ID NO. 1) on NCBI of the wild glycogen branching enzyme Ph GBE, the gene is optimized by escherichia coli coding genes, and the gene is sent to the Anshengda biotechnology Co-Ltd for gene synthesis and plasmid recombination. The vector is plasmid pET29b (+), and restriction enzyme sites are NdeI and XhoI respectively, so as to obtain a wild recombinant plasmid pET29b-GBE.
(2) Construction of mutant recombinant vector
Site mutation primers are designed, and site-directed mutagenesis is carried out by taking a wild recombinant plasmid pET29b-GBE as a template to obtain recombinant plasmids pET29b-V140I and pET29b-H415W containing mutants V140I and H415W respectively.
The primer sequences used for the mutation were as follows:
the primers for introducing the V140I mutation points are as follows:
V140I-F:5’-GTTATGTGGAAATTATTACCAGCGCGGCCACCCATGG-3’；
V140I-R:5’-GGTAAATAACCATGGGTGGCCGCGCTGGTAATAATTTCC-3’；
the primers for introducing the H415W mutation point are as follows:
H415W-F:5’-GCTGGGGTATGTTTGGCACCCATTGGACCTGGTG-3’；
H415W-R:5’-CACTCAACCTCCGGATTCCACCAGGTCCAATGGG-3’；
the PCR reaction system is as follows:
mutation PCR reaction System (20. Mu.l): 0.25. Mu.l of original plasmid template (2 ng/. Mu.l), 0.5. Mu.l of upstream primer F (10. Mu.M), 0.5. Mu.l of downstream primer R (10. Mu.M), 10. Mu. l PrimeSTARMax DNAPolymerase, 8.75. Mu.l of ddH 2 O。
The PCR reaction conditions were: pre-denaturation at 95℃for 3min; then, the mixture was denatured at 98℃for 10s, annealed at 55℃for 5s, and extended at 72℃for 3min for 30s as one cycle for 30 cycles.
(3) Construction of engineering bacteria
The recombinant plasmid was verified by agarose gel nucleic acid electrophoresis, and the target fragment was recovered by gel transformation to competent E.coli BL21 (DE 3). The transformants were then plated on LB plates containing kanamycin (30. Mu.g/mL), cultured upside down at 37℃overnight, after colonies were grown, single colonies were picked up to liquid LB medium containing kanamycin (30. Mu.g/mL), cultured at 37℃at 200rpm for 10-16h, and the bacterial solutions were taken and sent to the Soviet Biotechnology Co., ltd. Respectively obtaining mutant engineering bacteria containing correct mutation points.
Example 2: expression and purification of enzymes
(1) Culturing the bacterial cells:
the recombinant plasmid-containing engineering bacteria constructed in example 1 were streaked on LB solid plates containing kanamycin (30. Mu.g/mL), cultured upside down at 37℃for 10 hours, single colonies were picked up and inoculated into LB liquid medium containing kanamycin (30. Mu.g/mL), shake cultured at 37℃and 200rpm for 10-16 hours to obtain activated strains, the activated strains were drawn into LB liquid medium containing kanamycin (30. Mu.g/mL) according to an inoculum size of 1% (v/v) for expansion culture, shake cultured at 37℃and 200rpm for about 2 hours for 20 minutes, then added with IPTG with a final concentration of 0.5mmol/L, induced at 30℃for 6 hours, and centrifuged for 1440 Xg and 10 minutes to collect the cells. Re-suspending the thallus with heavy suspension, ultrasonic crushing in ice bath for 30min at 40% power for 2s, stopping for 3s, centrifuging at 10000rpm for 10min after crushing to collect supernatant, and passing through 0.45 μm water system membrane to obtain coarse enzyme solution.
(2) Purification of enzyme:
using His Trap HP 1mL Ni 2+ Purifying protein by column affinity chromatography, balancing Ni with sample buffer solution 2+ And (3) carrying out affinity chromatography column, loading crude enzyme liquid at the flow rate of 1mL/min, and carrying out gradient elution by using an elution buffer solution after loading to obtain pure mutant enzyme.
Pure enzyme solutions containing wild glycogen branching enzyme and mutant enzymes V140I and H415W are prepared respectively. The enzyme activity of the wild-type glycogen branching enzyme is 226.60 +/-1.1U/mg, and the relative enzyme activities of V140I and H415W are 129.7 +/-3.0% and 115.9+/-2.2% respectively by taking the enzyme activity of the wild-type glycogen branching enzyme as 100%.
(3) Optimum reaction temperature: to further investigate the properties of the mutant enzymes, a further enzymatic property study was performed on mutant V140I, H415W. 1mg/ml amylose substrate was prepared with 50mmol/L sodium acetate buffer (pH=6.0), 14. Mu.g enzyme was added thereto, and the enzyme activities were measured after reacting at 30℃at 40℃at 50℃at 60℃at 70℃at 80℃at 90℃at 95℃for 10 minutes, respectively, and the optimal reaction temperature for wild-type enzyme and mutant V140I was 60℃and the optimal reaction temperature for H415W was 70 ℃.
Example 3: preparation of branched starch
(1) Preparation of branched starch
20% (w/w) corn starch milk was prepared with 50mmol/L sodium acetate solution (pH=6.0), and the wild-type or mutant glycogen branching enzyme (50U/g dry starch) prepared in example 2 was added, and reacted in a water bath shaker at the optimum reaction temperature (60℃or 70 ℃) for 10 hours at 200rpm, and the reaction was terminated with a boiling water bath for 30 minutes after the completion of the reaction. After cooling, three volumes of absolute ethanol were added for ethanol precipitation, at 8000rpm, and centrifuged for 20min. After the ethanol volatilizes, the ethanol is washed three times by deionized water. Freezing at-80deg.C for 12 hr, lyophilizing, pulverizing, grinding into powder, and sieving with 100 mesh sieve to obtain branched starch.
(2) Optimization of glycogen branching enzyme addition amount
In the step, the adding amount of glycogen branching enzyme is optimized, and in the specific embodiment, the adding amount of glycogen branching enzyme is only adjusted to be 20U/g dry starch, 30U/g dry starch, 40U/g dry starch, 50U/g dry starch and 60U/g dry starch in the same step (1), and the result shows that the adding amount of the glycogen branching enzyme is relatively good in modifying effect.
(3) Optimization of reaction time
In the step, the reaction time is optimized, and the specific implementation mode is the same as the step (1), and the reaction time is only adjusted to be 4h, 6h, 8h, 10h and 12h, so that the result shows that the modification time effect of 10h is relatively better.
(4) Optimization of corn starch milk concentration
In the step, the corn starch milk concentration is optimized, and the specific implementation mode is the same as the step (1), and only the corn starch milk concentration is adjusted to 10%, 15%, 20%, 25% and 30%, so that the result shows that the modification effect is relatively good at the corn starch milk concentration of 20%.
Example 4: determination of branched starch
The modified starch prepared in step (1) of example 3 was weighed and amylose content, branched chain distribution, in vitro digestibility and retrogradation properties were measured respectively using unmodified native starch as a control.
(1) Amylose content
In the determination of the amylose content, the results show (FIG. 1) that the amylose content in the modified starch prepared by enzymatic hydrolysis of mutant H415W is reduced by about 8.27% at 70℃compared with the native starch; the amylose content was reduced by about 4.42% compared to the wild type; the amylose content was reduced by about 3.89% compared to mutant V140I.
(2) Distribution of branch length
In the branched chain distribution, the results showed (shown in table 1) that the branched chain length distribution of mutant H415W modified starch was superior to that of wild type and V140I modified starches. The DP value of the mutant was significantly reduced at 13-24 compared to wild-type enzyme and mutant V140I, the chain length of DP >36 was also significantly reduced and the chain length of DP <13 was significantly increased. The chain transfer pattern of the mutant was similar to that of the wild-type enzyme studied previously, but the effect was more pronounced.
TABLE 1 branching Length distribution
(3) In vitro simulated digestion assay
In vitro simulated digestion assay, the results show (as shown in table 2) that the fast-digestion starch content ratio of the branched starch obtained by modifying mutant H415W is reduced to below 80%. Compared with the mutant V140I, the method has the advantages that the ratio is reduced by 10.42 percent, and compared with the wild type, the highest ratio is reduced by 11.71 percent; the highest reduction was 20.35% compared to the control group. The mutant H415W has remarkable promotion effect on slow digestion and digestion resistant starch.
TABLE 2 in vitro digestibility
(4) Retrogradation assay
In the retrogradation assay, the results show (as shown in Table 3) that the initial gelatinization temperature (T o ) And the retrogradation enthalpy value (delta H) is reduced, and the delta H is obviously reduced. And compared with the wild type and mutant V140I, the mutant H415W has significantly reduced delta H, which indicates that the mutant modified corn starch with the optimal reaction temperature of 70 ℃ has stronger regeneration inhibition capability. This result is associated with a reduced amylose content of the modified starch.
TABLE 3 retrogradation Properties
The optimal reaction temperature for mutant H415W was increased to 70℃whereas the wild-type and V140I mutants were 60 ℃. Starch swells to a greater extent at 70℃facilitating the entry of enzymes and more facilitating the action of mutant H415W on amylose or long-chain amylopectin of starch. Therefore, the mutant with the optimal reaction temperature of 70 ℃ improves the industrial production strength, reduces the energy consumption required by production, and simultaneously further and obviously improves the branching degree of the product, thereby having obvious advantages in the industrial enzymatic preparation of branched starch.
The starch which is not pre-gelatinized is directly modified by a high-temperature enzyme method, so that the enzyme method effect and the gelatinization of the starch are synchronously carried out, industrial resources are saved, the modification step is simplified, and the method has great value in industrial production of branched starch.
The glycogen branching enzyme mutant has improved heat stability, the optimal reaction temperature reaches 70 ℃, the direct high-temperature (70 ℃) enzymatic modification of starch which is not subjected to gelatinization pretreatment is realized, the branching reaction and the starch swelling are synchronously carried out, and the method has the application prospect of saving industrial resources and simplifying modification steps. Compared with the original starch, the branching degree of the starch is obviously increased, the amylose content of the original starch is reduced, the digestion and retrogradation characteristics are improved, and the starch can be well applied to low GI, dietary fiber and functional beverage and food, and can also be added into bread to delay aging. Has great application value for improving obesity, cardiovascular diseases, chronic diseases such as type II diabetes and the like and bread aging.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and 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.
Claims (15)
1. A glycogen branching enzyme mutant having improved thermostability, characterized by having a mutation of valine at position 140 or histidine at position 415 based on a starting sequence;
the starting sequence has an amino acid sequence shown as SEQ ID NO.2 or an amino acid sequence with 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% and 99.9% homology with the sequence shown as SEQ ID NO. 2.
2. The glycogen branching enzyme mutant according to claim 1, characterized in that the glycogen branching enzyme mutant is obtained by mutating valine at position 140 of a glycogen branching enzyme having an amino acid sequence shown in SEQ ID No.2 to isoleucine; the name is: V140I.
3. The glycogen branching enzyme mutant of claim 1, wherein the glycogen branching enzyme mutant is characterized in that histidine at position 415 of a glycogen branching enzyme having an amino acid sequence shown in SEQ ID No.2 is mutated to tryptophan; the name is: H415W.
4. A gene encoding the glycogen branching enzyme mutant according to any one of claims 1 to 3.
5. A recombinant plasmid carrying the gene of claim 4 or expressing the glycogen branching enzyme mutant of any one of claims 1 to 3.
6. A recombinant microbial cell expressing the glycogen branching enzyme mutant of any one of claims 1 to 3.
7. An enzyme preparation comprising the glycogen branching enzyme mutant according to any one of claims 1 to 3.
8. A method for preparing branched starch, characterized in that the method is to catalyze starch using a glycogen branching enzyme mutant according to any one of claims 1 to 3 or an enzyme preparation according to claim 7.
9. The method according to claim 8, wherein starch is dispersed in a buffer to obtain starch milk, a glycogen branching enzyme mutant according to any one of claims 1 to 3 or an enzyme preparation according to claim 7 is added to the starch milk to perform a constant temperature reaction to obtain a product, and the product is boiled and dried, ground and sieved to obtain branched starch.
10. The method according to claim 9, wherein the concentration of the starch milk is 10-30% (w/w).
11. The method according to claim 9 or 10, wherein the buffer has a pH of 5.0 to 7.0.
12. The method according to any one of claims 9 to 11, wherein the reaction temperature of the isothermal reaction is 60 to 80 ℃ for 8 to 20 hours.
13. The method according to any one of claims 8 to 12, wherein the glycogen branching enzyme mutants according to claims 1 to 3 are added in an amount of 20 to 60U/g dry basis starch.
14. The method of any one of claims 8 to 13, wherein the starch comprises a starch having an amylose content of greater than 1%.
15. Use of a glycogen branching enzyme mutant according to any one of claims 1 to 3, or a gene according to claim 4, or a recombinant plasmid according to claim 5, or a recombinant microbial cell according to claim 6, or an enzyme preparation according to claim 7, or a method for preparing a branched starch according to any one of claims 8 to 14, for the preparation of a food, feed, pharmaceutical product.
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