CN110684751B - Starch branching enzyme mutant with improved catalytic capability - Google Patents
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Abstract
The invention discloses a starch branching enzyme mutant with improved catalytic capability, and belongs to the technical field of enzyme engineering. The invention obtains the starch branching enzyme mutant with improved catalytic capability by mutating uncharged amino acid residues near a substrate binding site in the starch branching enzyme into exogenous amino acid residues with negative charges, and compared with wild type starch branching enzyme, the starch branching enzyme mutant has the relative contents of alpha-1, 6 glycosidic bonds of modified products of R158T and Q489E which are respectively 8.2 percent and 10.7 percent, and are improved by 10.81 percent and 44.59 percent compared with the wild type modified product of the starch branching enzyme.
Description
Technical Field
The invention relates to a starch branching enzyme mutant with improved catalytic capability, belonging to the technical field of enzyme engineering.
Background
Starch is widely existed in nature and has wide application potential in the industries of food, medical treatment and the like, however, the problems of easy retrogradation, poor stability, low solubility and the like exist in natural starch, and the application of the starch in the industries is limited. The fundamental reason for these problems is that native starch contains many linear long-chain macromolecules or branched molecules with low branching degree, which are easily associated with each other by the action of hydrogen bonds, and thus retrogradation occurs. The degree of easy mutual association between starch molecules is related to the branching degree of the starch molecules, and the starch molecules with high branching degree have larger steric hindrance compared with the starch molecules with low branching degree, so that the starch molecules are not easy to mutually associate. Therefore, if the branching degree of starch molecules can be increased, the defects in the use of starch can be effectively improved.
Starch branching enzymes (1, 4-alpha-glucan branching enzymes; EC 2.4.1.18) are a class of glycosyltransferases belonging to the alpha-amylase family that catalyze the hydrolysis of alpha-1, 4-glycosidic linkages in starch molecules to produce free short chains with non-reducing ends, which are then linked to acceptor chains in the form of alpha-1, 6-glycosidic linkages by transglycosidation, thereby forming new alpha-1, 6-branch points.
By the transglycosylation reaction, the starch branching enzyme can increase the branching degree of starch, improve the anti-digestibility and slow digestibility of the starch, delay the retrogradation process of the starch, enhance the stability of the starch and improve the usability of the starch, and is used for producing starch derivatives with good application value. Therefore, starch branching enzymes are important amylases in the field of modification of starch by a biological enzyme method due to their characteristic transglycosylation.
At present, there are many reports on modifying starch with starch branching enzyme to increase the degree of starch branching, however, the degree of starch branching increase is limited, and therefore modification of the enzyme is required to further enhance its catalytic ability. Methods commonly used to modify starch branching enzymes include physical, chemical and biological methods. Compared with physical and chemical methods, the biological method mainly changes the spatial conformation of enzyme protein molecules through technical means such as protein engineering, genetic engineering and the like, improves the service performance of the enzyme protein, and has higher stability and better safety. However, the existing starch branching enzyme can only lead the branching degree of the product to reach about 7 percent. Therefore, there is a need for starch branching enzyme mutants with improved catalytic ability, which act on starch to produce products with higher branching degree.
Disclosure of Invention
In order to solve the above problems, the present invention provides a starch branching enzyme mutant with improved catalytic ability, which contains an amino acid sequence shown in SEQ ID NO.2 or SEQ ID NO. 3.
The starch branching enzyme mutant takes starch branching enzyme with an amino acid sequence shown as SEQ ID NO.1 as parent enzyme, and the 158 th arginine is mutated into threonine, or the 489 th glutamine is mutated into glutamic acid.
In one embodiment of the invention, the parent enzyme is derived from hyperthermophilic (Rhodothermus obamensis) STB05, which is referenced from the literature as STB 05: the expression, purification and enzymological property research of extreme thermophilic starch branching enzyme [ J ] 2019.
The present invention provides a gene encoding the starch branching enzyme mutant with improved catalytic ability.
In one embodiment of the invention, the nucleotide sequence of the gene is shown as SEQ ID NO.9 or SEQ ID NO. 10.
The invention provides a plasmid or a vector containing the gene.
The present invention provides a cell carrying the above gene.
In one embodiment of the invention, the cell comprises a genetically engineered bacterium.
In one embodiment of the present invention, the genetically engineered bacterium is a host escherichia coli or bacillus subtilis.
In one embodiment of the invention, the genetically engineered bacterium takes pET-20b (+) as an expression vector and Escherichia coli BL21(DE3) as an expression host.
The invention provides a method for improving catalytic ability of starch branching enzyme, which is characterized in that arginine at position 158 of starch branching enzyme from extreme thermophilic bacteria (Rhodothermus obamensis) STB05 is mutated into threonine, or glutamine at position 489 is mutated into glutamic acid, the amino acid sequence of the starch branching enzyme from extreme thermophilic bacteria (Rhodothermus obamensis) STB05 is shown as SEQ ID NO.1, and the nucleotide sequence of a gene for coding the starch branching enzyme is shown as SEQ ID NO. 8.
The invention provides application of the starch branching enzyme mutant in the aspect of hydrolyzing starch.
The invention provides application of the genetic engineering bacteria in the aspect of hydrolyzing starch.
The invention provides application of the starch branching enzyme mutant in the field of food.
The invention provides application of the genetic engineering bacteria in the field of food.
The invention has the beneficial effects that:
the starch branching enzyme mutant with improved catalytic capability is obtained by mutating uncharged amino acid residues near a substrate binding site in the starch branching enzyme into exogenous amino acid residues with negative charges. Compared with wild starch branching enzyme, the starch branching enzyme mutant obtained by the invention takes 5% (w/w) corn starch solution as a substrate, the branching degrees of products respectively reach 8.2% (R158T) and 10.7% (Q489E), and are respectively improved by 10.81% and 44.59% compared with the wild starch branching enzyme.
Drawings
FIG. 1 shows the wild-type and the modified products of each starch branching enzyme before and after modification1H NMR spectrum.
FIG. 2 shows the relative alpha-1, 6-glycosidic bond contents of the wild-type and mutant starch branching enzyme products before and after modification in the examples of the present invention.
Detailed Description
The examples of the present invention are provided only for further illustration of the present invention and should not be construed as limitations or limitations of the present invention.
The detection method comprises the following steps:
method for measuring relative content of alpha-1, 6 glycosidic bond
Accurately weighing 20mg of sample, dissolving in 1mL of heavy water, gelatinizing in boiling water bath, and passing through1H NMR (hydrogen nuclear magnetic resonance) was measured. And obtaining the relative content of the alpha-1, 6-glycosidic bond according to the peak area ratio of the absorption peaks corresponding to the alpha-1, 4-glycosidic bond and the alpha-1, 6-glycosidic bond in the calculated spectrogram.
Method for measuring activity of starch branching enzyme
0.25% (w/v) potato amylopectin solution prepared from 50mM phosphate buffer solution (pH 7.0) is used as substrate for enzyme reaction, 900 μ L substrate is taken and kept at 65 ℃ for 10min, 100 μ L starch branching enzyme is added and mixed uniformly, and the mixture is placed in a water bath condition at 65 ℃ for reaction for 15 min. The reaction was terminated by inactivating the enzyme in a bath of boiling water for 30 min. After cooling to room temperature, 300. mu.L of the reaction mixture was added to 5mL of a developing solution (0.05% (w/v) KI, 0.005% (w/v) I2pH 6.0) was left standing at room temperature to sufficiently develop color. Absorbance at 530nm was measured after development for 15 min.
One unit of enzyme activity (U/mL) is defined as: at 530nm, the absorbance decreased by 1% per minute as one unit of enzyme activity.
(III) culture Medium
LB culture medium: 5g/L of yeast powder, 10g/L of tryptone, 10g/L of NaCl and 7.0 of pH.
TB culture medium: 24g/L yeast powder, 12g/L tryptone, 5g/L glycerol and KH2PO4 2.3136g/L，K2HPO416.4318g/L，pH 7.0。
Example 1: preparation of starch branching enzyme mutants
A starch Branching Enzyme derived from Rhodothermus obamensis STB05 was combined with a Branching Enzyme derived from Escherichia coli binding maltoheptaose (cited in the literature: Feng L, Fawaz R, Hovde S, et al. Crystal Structures of Escherichia coli Branching Enzyme in Complex with Linear Oligosaccharides [ J ]. Biochemistry,2015,54(40):6207-18.) crystal structure overlap, using the latter as a template, by comparing the distances between the amino acids of the substrate binding site and the substrate, 158 sites located farther away from the substrate and 489 sites located closer to the substrate were selected, respectively, R158T (the amino acid sequence is shown in SEQ ID NO.2, and the nucleotide sequence of the gene encoding it is shown in SEQ ID NO. 9) and Q489E mutant (the amino acid sequence is shown in SEQ ID NO.3, and the nucleotide sequence of the gene encoding it is shown in SEQ ID NO. 10) were designed.
The expression vector pET-20b (+)/gbe is obtained by using Rhodothermus obamensis STB05 genome DNA as a template, amplifying by a PCR method to obtain gbe gene (nucleotide sequence is shown in SEQ ID NO. 8) with Nde I and Xho I restriction enzyme sites at two ends, inserting the gbe gene into a pMD 18-T simple plasmid to obtain a cloning vector pMD 18-T simple/gbe, carrying out double digestion on the vector, 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.
Complementary Primer strands (see Table 1) were designed using expression vector pET-20b (+)/gbe as a template, and primers were synthesized by Kinzhi Biotechnology Ltd, and site-directed mutagenesis was performed according to the method shown in the TAKaRa STAR Primer GXL kit (Takara Shuzo Co., Ltd.). 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 5 min; then 35 cycles are carried out under the conditions of one cycle of 98 ℃ for 10s, 55 ℃ for 10s and 72 ℃ for 7 min; finally, the temperature is kept for 15min at 72 ℃.
TABLE 1 primers for starch branching enzyme mutation sites
1The underlined bases correspond to the corresponding mutated amino acids.
Example 2: construction of genetically engineered bacteria containing gene expressing starch branching enzyme mutant of the present invention
The PCR product was treated with DpnI at 37 ℃ for 2 hours, followed by transformation of the treated PCR product into E.coli JM 109, spreading of the transformed E.coli JM 109 on LB agar medium containing 100. mu.g/mL ampicillin, overnight culture in 37 ℃ incubator for 12 hours, selection of single colonies therefrom, inoculation into LB liquid medium containing 100. mu.g/mL ampicillin, overnight culture at 37 ℃ at 200r/min and plasmid-identifying sequencing by extraction according to the method indicated in the plasmid extraction kit instructions. The constructed target plasmid is transformed into an expression host E.coli BL21(DE3) competence by a chemical transformation method. The genetically engineered bacterium E.coli BL21(DE3) (pET-20b (+)/gbe) was finally obtained.
Example 3: expression of starch branching enzyme mutants of the invention
Activating and culturing host bacteria: e.coli BL21(DE3) (pET-20b (+)/gbe) was streaked on LB solid medium, incubated overnight at 37 ℃ in an incubator, and positive single colonies were picked and inoculated into a 250mL Erlenmeyer flask containing 50mL of LB liquid medium. The centrifuge tube is placed in a rotary shaker at 200r/min and incubated at 37 ℃ for 8-10 h.
Fermentation culture: 1mL of activated inoculum broth was inoculated into a 250mL Erlenmeyer flask containing 50mL of TB liquid medium and incubated at 37 ℃ for 96h in a rotary shaker at a rate of 200 r/min.
Ampicillin was added to each medium at a final concentration of 100. mu.g/mL prior to use.
Example 4: enzyme activity detection of starch branching enzyme mutant
A0.25% (w/v) solution of potato amylopectin was prepared in 50mM phosphate buffer (pH 7.0), and a given amount of each of wild-type and mutants of starch branching enzyme R158T and Q489E was added to carry out the reaction at 65 ℃ and the activity of the starch branching enzyme mutant was measured as described above. The results of enzyme activity detection are shown in table 2, the enzyme activities of the mutants R158T and Q489E have no significant difference compared with the wild type, which indicates that the site-specific mutations do not cause the reduction of the total activity of the starch branching enzyme.
TABLE 2 enzymatic Activities of wild type and mutant GBE
1Each value is the average of 3 replicates.
Example 5: product branching degree analysis of starch branching enzyme mutant of the present invention
Accurately weighing Na (dissolved in pH 7.0) of corn starch2HPO4-NaH2PO4And (3) preparing a buffer solution (50mmol/L) into starch milk with the concentration of 5% w/v (calculated on a dry basis), uniformly mixing, carrying out boiling water bath for 30min, and then placing in a water bath shaking table with the temperature of 65 ℃ (the rotating speed is 160r/min) for heat preservation for 15 min. 100U/g Ro-GBE wild type, mutant R158T and Q489E were added to the mixture, and the mixture was reacted for 12 hours, followed by boiling water bath for 30 min. Placing the sample in an ultralow temperature refrigerator at minus 80 ℃ for 12h, freeze-drying for 72h, and grinding and sieving with a 100-mesh sieve for later use. 20mg of raw corn starch (control) and its modified sample were each weighed out and dissolved in 1mL of heavy water and measured by 1H NMR (hydrogen nuclear magnetic resonance).
The relative content of α -1, 6-glycosidic bond was obtained by calculating the peak area of the absorption peak corresponding to α -1, 4-glycosidic bond and α -1, 6-glycosidic bond in the spectra, and the results are shown in table 3, fig. 1, and fig. 2. The relative content of alpha-1, 6 glycosidic bonds of the modified products of the mutants R158T and Q489E is 8.2 percent and 10.7 percent respectively, and compared with the wild type, the branching degree of the modified products of the mutants is improved by 10.81 percent and 44.59 percent respectively, which shows that although the activity of the starch branching enzyme is not changed by site-specific mutation, the transglycosylation catalysis capability of the starch branching enzyme is improved. This is probably because the total activity of the starch branching enzyme comprises hydrolysis activity and branching activity, and the mutation at the sites R158T and Q489E improves the proportion of the branching activity in the total activity under the condition of keeping the total activity, so that the starch branching enzyme is more prone to transglycosylation reaction in the starch modification process, thereby further improving the branching degree of the product.
TABLE 3 degree of product branching before and after modification of starch branching enzyme
1Each value is the average of 3 replicates. The relative content of alpha-1, 6 glucosidic bonds of the starch itself.
Comparative example 1
The starch branching enzyme with the amino acid sequence shown as SEQ ID NO.1 is used as parent enzyme, the 489 th glutamine is mutated into glycine, and the rest steps are consistent with the embodiment. The results show that the relative alpha-1, 6 glycosidic bond content of the sample after Q489G modification is 7.27%, which is reduced by 1.89% compared with the wild type.
Comparative example 2
The starch branching enzyme with the amino acid sequence shown as SEQ ID NO.1 is used as parent enzyme, the glutamine at the 489 th position is mutated into arginine, and the rest steps are consistent with the embodiment. The result shows that the relative content of alpha-1, 6 glycosidic bonds of the sample after Q489R modification is 7.41%, and compared with the wild type, the sample has no significant difference.
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.
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Gly Val Pro Ile Gly Gly Pro Trp His Glu Val Leu Asn Ser Asp Ala
565 570 575
Val Ala Tyr Gly Gly Ser Gly Met Gly Asn Phe Gly Arg Val Glu Ala
580 585 590
Val Pro Glu Ser Trp His Gly Arg Pro Phe His Leu Glu Leu Thr Leu
595 600 605
Pro Pro Leu Ala Ala Leu Ile Leu Glu Pro Glu His Gly
610 615 620
<210> 4
<211> 45
<212> DNA
<213> Artificial sequence
<400> 4
tggcgtcaca aaactcccgg cgagtccttc tcttaccggg agatt 45
<210> 5
<211> 42
<212> DNA
<213> Artificial sequence
<400> 5
actcgccggg agttttgtga cgccaggagc ccagatgtac ct 42
<210> 6
<211> 37
<212> DNA
<213> Artificial sequence
<400> 6
ctgctggacg aaccctacca tcgaggtatt cagctgt 37
<210> 7
<211> 35
<212> DNA
<213> Artificial sequence
<400> 7
atggtagggt tcgtccagca ggtgccattc gagct 35
<210> 8
<211> 1890
<212> DNA
<213> Rhodothermus obamensis
<400> 8
catatgagct ggctcacgga agaagacatc cggcgctggg aaagcggtac gttctacgac 60
agttaccgaa agctgggcgc ccatcccgac gacgaaggca cctggttctg cgtctgggcg 120
ccgcatgccg atggcgtctc ggtgctcgga gcgttcaacg actggaatcc ggaggccaac 180
ccgctggagc gctacggcgg cggcctgtgg gccggttacg taccgggagc gcgcccgggc 240
cacacctaca agtatcgcat ccggcacggc ttctatcagg ccgacaagac ggatccctac 300
gccttcgcca tggagccgcc taccggcagt cccatcgaag ggctggcctc catcatcacg 360
cggctcgact acacctggca cgacgacgaa tggatgcggc gccggaaggg tccggccagc 420
ctttacgagc cggtttccat ctacgaggta catctgggct cctggcgtca caaacggccc 480
ggcgagtcct tctcttaccg ggagattgcc gagccgctgg ccgactacgt gcaggagatg 540
ggcttcacgc acgtggagct gctgcccgtc atggaacatc cctactacgg ctcctggggc 600
tatcaggtgg tgggctacta cgccccaacg tttcgctacg gatcacccca ggacctgatg 660
tacctgatcg actacctgca ccagcgcggc atcggcgtca tcctcgactg ggtcccgagc 720
cactttgcgg ccgatcccca gggactggtt ttcttcgacg ggaccacact cttcgaatac 780
gacgatccca agatgcgcta tcaccctgac tggggtacgt atgtgttcga ttacaacaag 840
ccgggcgtac gcaactttct gatttccaac gcacttttct ggctcgaaaa gtaccacgtc 900
gacgggctgc gcgtcgatgc ggtggcttct atgctctacc gggactactc acgcaaggag 960
tggacaccca acatcttcgg cggccgtgaa aacctggagg ccattgattt catcaagaaa 1020
ttcaacgaaa cggtctacct gcacttcccc gaggccatga cgatcgccga ggagtcgacg 1080
gcctggcccg gcgtgtcggc ccccacctac aacaacggtc tgggcttcct ctacaagtgg 1140
aacatgggct ggatgcacga cacgctggac tacatccagc gcgatcccat ctaccgcaag 1200
tatcaccacg acgagctgac cttctcgctc tggtacgcct tttcggagca ctacgtcctg 1260
ccgctctcgc acgacgaggt ggtgcacggc aagggctcgc tctggggtaa aatgcccggc 1320
gacgactggc agaaggcagc caacttgcgc ctgctctttg gccacatgtg gggccatccg 1380
ggcaaaaaac tgctcttcat gggcggcgag ttcggccagc accacgagtg gaaccacgac 1440
acgcagctcg aatggcacct gctggaccag ccctaccatc gaggtattca gctgtgggtg 1500
tgcgatctga accacctcta ccgtacgaat ccggccctct ggcacgacgg accggaaggg 1560
ttcgagtgga tcgacttcag cgaccgcgac cagagcgtga tctgttacct gcgcaagaat 1620
gccggccgca tgctgctgtt cgtgctgaac tttacgcccg tgccacgcga gcactaccgc 1680
gtgggcgtgc cgatcggtgg cccctggcac gaggtgctca acagcgacgc ggtggcctac 1740
ggcgggagcg ggatgggcaa cttcggccgc gtcgaggcgg tgcccgagtc ctggcacggc 1800
cgccccttcc acttagagct gacgcttccc ccgctggccg ccctcatcct ggagccggag 1860
cacgggctcg agcaccacca ccaccaccac 1890
<210> 9
<211> 1890
<212> DNA
<213> Artificial sequence
<400> 9
catatgagct ggctcacgga agaagacatc cggcgctggg aaagcggtac gttctacgac 60
agttaccgaa agctgggcgc ccatcccgac gacgaaggca cctggttctg cgtctgggcg 120
ccgcatgccg atggcgtctc ggtgctcgga gcgttcaacg actggaatcc ggaggccaac 180
ccgctggagc gctacggcgg cggcctgtgg gccggttacg taccgggagc gcgcccgggc 240
cacacctaca agtatcgcat ccggcacggc ttctatcagg ccgacaagac ggatccctac 300
gccttcgcca tggagccgcc taccggcagt cccatcgaag ggctggcctc catcatcacg 360
cggctcgact acacctggca cgacgacgaa tggatgcggc gccggaaggg tccggccagc 420
ctttacgagc cggtttccat ctacgaggta catctgggct cctggcgtca caaaactccc 480
ggcgagtcct tctcttaccg ggagattgcc gagccgctgg ccgactacgt gcaggagatg 540
ggcttcacgc acgtggagct gctgcccgtc atggaacatc cctactacgg ctcctggggc 600
tatcaggtgg tgggctacta cgccccaacg tttcgctacg gatcacccca ggacctgatg 660
tacctgatcg actacctgca ccagcgcggc atcggcgtca tcctcgactg ggtcccgagc 720
cactttgcgg ccgatcccca gggactggtt ttcttcgacg ggaccacact cttcgaatac 780
gacgatccca agatgcgcta tcaccctgac tggggtacgt atgtgttcga ttacaacaag 840
ccgggcgtac gcaactttct gatttccaac gcacttttct ggctcgaaaa gtaccacgtc 900
gacgggctgc gcgtcgatgc ggtggcttct atgctctacc gggactactc acgcaaggag 960
tggacaccca acatcttcgg cggccgtgaa aacctggagg ccattgattt catcaagaaa 1020
ttcaacgaaa cggtctacct gcacttcccc gaggccatga cgatcgccga ggagtcgacg 1080
gcctggcccg gcgtgtcggc ccccacctac aacaacggtc tgggcttcct ctacaagtgg 1140
aacatgggct ggatgcacga cacgctggac tacatccagc gcgatcccat ctaccgcaag 1200
tatcaccacg acgagctgac cttctcgctc tggtacgcct tttcggagca ctacgtcctg 1260
ccgctctcgc acgacgaggt ggtgcacggc aagggctcgc tctggggtaa aatgcccggc 1320
gacgactggc agaaggcagc caacttgcgc ctgctctttg gccacatgtg gggccatccg 1380
ggcaaaaaac tgctcttcat gggcggcgag ttcggccagc accacgagtg gaaccacgac 1440
acgcagctcg aatggcacct gctggaccag ccctaccatc gaggtattca gctgtgggtg 1500
tgcgatctga accacctcta ccgtacgaat ccggccctct ggcacgacgg accggaaggg 1560
ttcgagtgga tcgacttcag cgaccgcgac cagagcgtga tctgttacct gcgcaagaat 1620
gccggccgca tgctgctgtt cgtgctgaac tttacgcccg tgccacgcga gcactaccgc 1680
gtgggcgtgc cgatcggtgg cccctggcac gaggtgctca acagcgacgc ggtggcctac 1740
ggcgggagcg ggatgggcaa cttcggccgc gtcgaggcgg tgcccgagtc ctggcacggc 1800
cgccccttcc acttagagct gacgcttccc ccgctggccg ccctcatcct ggagccggag 1860
cacgggctcg agcaccacca ccaccaccac 1890
<210> 10
<211> 1890
<212> DNA
<213> Artificial sequence
<400> 10
catatgagct ggctcacgga agaagacatc cggcgctggg aaagcggtac gttctacgac 60
agttaccgaa agctgggcgc ccatcccgac gacgaaggca cctggttctg cgtctgggcg 120
ccgcatgccg atggcgtctc ggtgctcgga gcgttcaacg actggaatcc ggaggccaac 180
ccgctggagc gctacggcgg cggcctgtgg gccggttacg taccgggagc gcgcccgggc 240
cacacctaca agtatcgcat ccggcacggc ttctatcagg ccgacaagac ggatccctac 300
gccttcgcca tggagccgcc taccggcagt cccatcgaag ggctggcctc catcatcacg 360
cggctcgact acacctggca cgacgacgaa tggatgcggc gccggaaggg tccggccagc 420
ctttacgagc cggtttccat ctacgaggta catctgggct cctggcgtca caaacggccc 480
ggcgagtcct tctcttaccg ggagattgcc gagccgctgg ccgactacgt gcaggagatg 540
ggcttcacgc acgtggagct gctgcccgtc atggaacatc cctactacgg ctcctggggc 600
tatcaggtgg tgggctacta cgccccaacg tttcgctacg gatcacccca ggacctgatg 660
tacctgatcg actacctgca ccagcgcggc atcggcgtca tcctcgactg ggtcccgagc 720
cactttgcgg ccgatcccca gggactggtt ttcttcgacg ggaccacact cttcgaatac 780
gacgatccca agatgcgcta tcaccctgac tggggtacgt atgtgttcga ttacaacaag 840
ccgggcgtac gcaactttct gatttccaac gcacttttct ggctcgaaaa gtaccacgtc 900
gacgggctgc gcgtcgatgc ggtggcttct atgctctacc gggactactc acgcaaggag 960
tggacaccca acatcttcgg cggccgtgaa aacctggagg ccattgattt catcaagaaa 1020
ttcaacgaaa cggtctacct gcacttcccc gaggccatga cgatcgccga ggagtcgacg 1080
gcctggcccg gcgtgtcggc ccccacctac aacaacggtc tgggcttcct ctacaagtgg 1140
aacatgggct ggatgcacga cacgctggac tacatccagc gcgatcccat ctaccgcaag 1200
tatcaccacg acgagctgac cttctcgctc tggtacgcct tttcggagca ctacgtcctg 1260
ccgctctcgc acgacgaggt ggtgcacggc aagggctcgc tctggggtaa aatgcccggc 1320
gacgactggc agaaggcagc caacttgcgc ctgctctttg gccacatgtg gggccatccg 1380
ggcaaaaaac tgctcttcat gggcggcgag ttcggccagc accacgagtg gaaccacgac 1440
acgcagctcg aatggcacct gctggacgaa ccctaccatc gaggtattca gctgtgggtg 1500
tgcgatctga accacctcta ccgtacgaat ccggccctct ggcacgacgg accggaaggg 1560
ttcgagtgga tcgacttcag cgaccgcgac cagagcgtga tctgttacct gcgcaagaat 1620
gccggccgca tgctgctgtt cgtgctgaac tttacgcccg tgccacgcga gcactaccgc 1680
gtgggcgtgc cgatcggtgg cccctggcac gaggtgctca acagcgacgc ggtggcctac 1740
ggcgggagcg ggatgggcaa cttcggccgc gtcgaggcgg tgcccgagtc ctggcacggc 1800
cgccccttcc acttagagct gacgcttccc ccgctggccg ccctcatcct ggagccggag 1860
cacgggctcg agcaccacca ccaccaccac 1890
Claims (10)
1. A starch branching enzyme mutant is characterized in that the amino acid sequence of the starch branching enzyme mutant is shown as SEQ ID NO.2 or SEQ ID NO. 3.
2. A gene encoding the starch branching enzyme mutant of claim 1.
3. A vector comprising the gene of claim 2.
4. A cell expressing the starch branching enzyme mutant of claim 1.
5. The cell of claim 4, wherein the cell is a host selected from the group consisting of E.coli and Bacillus subtilis.
6. The cell of claim 4 or 5, wherein the cell uses pET-20b (+) as an expression vector, and wherein the expression vector is pET-20b (+) or a mixture thereofEscherichia coliBL21(DE3) is an expression host.
7. A method for improving branching capability of starch branching enzyme is characterized in that arginine at position 158 of the starch branching enzyme with an amino acid sequence shown as SEQ ID NO.1 is mutated into threonine, or glutamine at position 489 is mutated into glutamic acid.
8. Use of a starch branching enzyme mutant as defined in claim 1 for the hydrolysis of starch.
9. Use of a cell according to any one of claims 4 to 6 for the hydrolysis of starch.
10. Use of the starch branching enzyme mutant of claim 1 in the field of food products.
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| CN112391365B (en) * | 2020-11-30 | 2022-04-15 | 江南大学 | Starch branching enzyme mutant with improved catalytic activity and application thereof |
| CN113481177B (en) * | 2021-08-03 | 2023-08-25 | 江南大学 | Starch branching enzyme mutant with enhanced extracellular secretion capacity |
| CN114574459B (en) * | 2022-03-09 | 2024-04-16 | 中国科学院天津工业生物技术研究所 | Glycogen branching enzyme mutant with improved catalytic activity and application thereof |
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