CN114317498A - Alpha-transglucosidase mutant and application thereof - Google Patents
Alpha-transglucosidase mutant and application thereof Download PDFInfo
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Abstract
The invention relates to an alpha-transglucosidase mutant, belonging to the technical field of genetic engineering. The amino acid sequence of the alpha-glucosidase mutant is shown as SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO. 5. On the basis of alpha-transglucosidase from deep-sea bacillus licheniformis, two mutant strains S204L and A307E with improved single-point thermal stability and a combined mutant S204L/A307E are obtained by screening, and the activity of the enzyme is not influenced while the thermal stability of the mutant is obviously improved. Compared with natural alpha-glucosyltransferase, the mutants of the invention are more suitable for industrial production, and have huge application prospect and industrial value.
Description
Technical Field
The invention relates to an alpha-transglucosidase mutant and application thereof, belonging to the technical field of genetic engineering.
Background
Isomaltooligosaccharides (IMOs) are functional oligosaccharides, and comprise 2-10 glucosyl groups connected with alpha-1 and 6-glycosidic bonds, and comprise main functional components such as isomaltose, panose and isomaltotriose. The IMOs can promote the proliferation of probiotics such as bifidobacterium, lactobacillus and the like in human intestinal tracts and regulate intestinal flora; has low calorie value, promotes gastrointestinal motility, improves constipation and lipid metabolism, prevents dental caries, and the like, and thus, IMOs are widely used as prebiotics, food additives and feed ingredients.
Currently, IMOs are generally produced by enzymatic conversion, and are catalytically produced from starch by using α -amylase, β -amylase, pullulanase and glucosidase, and a typical production process generally comprises 3 steps: one is liquefaction, in which starch is liquefied by a thermostable alpha-amylase to produce oligosaccharides and dextrins; secondly, saccharification, wherein oligosaccharide and dextrin are saccharified by beta-amylase, pullulanase and other saccharifying enzymes; thirdly, transglycosylation, α -glucosidase (ec3.2.1.20) catalyzes the release of glucose from the non-reducing end of the substrate and transfers the glucosyl residues to the 6-OH group of the non-reducing glucose units, yielding IMOs. At present, the research and industrial application of commercial alpha-amylase, beta-amylase, pullulanase and the like are mature, while the alpha-glucosidase applied in industry is mostly derived from Aspergillus niger (Aspergillus niger), and the application of the alpha-glucosidase in the large-scale production of the IMOs is limited due to the problems of poor thermal stability, low reaction rate, high cost of enzyme purification and recycling and the like.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide an alpha-glucosidase mutant with improved thermal stability.
Technical scheme
An alpha-glucosidase mutant is obtained by performing site-directed mutagenesis on the 204 th amino acid and/or the 307 th amino acid of alpha-glucosyltransferase with the amino acid sequence shown as SEQ ID NO.1, wherein serine at the 204 th position is mutated into leucine, alanine at the 307 th position is mutated into glutamic acid, and the amino acid sequence of the alpha-glucosidase mutant is shown as SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO. 5; the nucleotide sequence for coding the alpha-transglucosidase is shown as SEQ ID NO. 2.
The application of the alpha-glucosidase mutant in producing isomaltooligosaccharide.
A method for preparing the alpha-glucosidase mutant comprises the following steps:
(1) using the nucleotide sequence shown in SEQ ID NO.2 as a template, designing a site-directed mutagenesis primer according to a mutated site, carrying out PCR amplification to obtain a gene containing the mutated site, and then constructing a vector containing a coding mutant gene.
(2) The gene vector containing the encoding mutant is transformed into a host cell.
(3) Screening and verifying the recombinant cells constructed in the last step to obtain positive clones, then producing enzyme through culture and fermentation, centrifugally collecting cells, crushing the cells by using an ultrasonic cell crusher, and centrifuging to obtain the alpha-glucosyltransferase mutant.
The nucleotide sequence of the coding gene is shown as SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO. 8.
A recombinant expression vector carrying the gene.
The recombinant expression vector uses pET-28a (+) vector as an original expression vector.
A gene engineering bacterium obtained by transforming the recombinant expression vector.
The genetic engineering bacteria take escherichia coli as a host. The E.coli strain comprises BL21(DE 3).
The invention has the beneficial effects that:
1) on the basis of natural alpha-transglucosidase, the invention modifies the molecular structure of the alpha-transglucosidase by rational design and combining site-directed mutagenesis biotechnology, analyzes the influence of the mutated residues on the thermal stability of the enzyme, and finally obtains mutant strains with improved stability (S204L, A307E and S204L/A307E).
2) The half-life of the natural alpha-transglucosidase is 18.3h, and the half-life of the alpha-transglucosidase mutant S204L (the serine at the 204 th site is mutated into leucine) provided by the invention reaches 37.4h at 65 ℃, which is 2.06 times of the half-life of the natural alpha-transglucosidase; the half-life of the alpha-transglucosidase mutant A307E (alanine at position 307 is mutated into glutamic acid) at 65 ℃ reaches 28.6h, which is 1.56 times of the half-life of natural alpha-transglucosidase; S204L/A307E (serine at position 204 is mutated into leucine, alanine at position 307 is mutated into glutamic acid) has a half-life of 56.9h at 65 ℃, which is 3.1 times of that of natural alpha-glucosyltransferase.
3) The alpha-transglucosidase mutant provided by the invention has obviously improved thermal stability and simultaneously has no influence on the activity of the enzyme. Wherein, after the heat treatment at 65 ℃ for 40h under the condition that the enzyme catalytic activity is basically unchanged, the mutants S204L (the serine at the 204 th site is mutated into the leucine) and A307E (the alanine at the 307 th site is mutated into the glutamic acid) and the combined mutant S207L/A307E respectively retain 49.1 percent, 43.2 percent and 91.2 percent of relative enzyme activity, and the control group only retains 15.7 percent of relative enzyme activity.
4) Compared with the wild type, the alpha-transglucosidase mutant obtained by the invention is more suitable for catalyzing maltose to generate isomaltooligosaccharide, and is more beneficial to the flexibility of the production process.
Drawings
FIG. 1 is a SDS-PAGE electrophoresis of pure enzyme solutions of wild type alpha-transglucosidase and alpha-transglucosidase mutants;
FIG. 2 shows the residual activity test results of wild-type α -transglucosidase and its mutants after incubation at 65 ℃ and pH7.0 for 40 h;
FIG. 3 shows half-lives of wild-type α -transglucosidase and α -transglucosidase mutants S204L, A307E, S204L/A307E at 65 ℃;
FIG. 4 shows the enzyme activities of wild-type α -transglucosidase and α -transglucosidase mutants S204L, A307E, S204L/A307E at different pH;
FIG. 5 shows the enzyme activities of wild-type alpha-transglucosidase and alpha-transglucosidase mutants S204L, 307E, S204L/A307E at different temperatures.
Detailed Description
The technical solution of the present invention is further explained with reference to the accompanying drawings and specific embodiments. In the examples described below, the pET-28a (+) vector referred to was purchased from Invitrogen; the related culture medium and the formula are as follows:
LB liquid medium: 10g/L peptone, 5g/L yeast powder and 10g/L NaCl.
LB solid medium: 2% agar was added based on LB liquid medium.
The detection methods referred to in the following examples are as follows:
the method for measuring the enzyme activity of the alpha-glucosyltransferase comprises the following steps: mu.L of 0.05mg/ml pure enzyme was added to 900. mu.L of a reaction system containing 200g/L maltose in 50mM sodium dihydrogenphosphate-disodium hydrogenphosphate buffer solution of pH 7.0; reacting for 10min under the condition of 45 ℃ water bath, then terminating the enzymatic reaction in 100 ℃ boiling water bath for 10min, centrifuging, taking the supernatant, diluting by 20 times, and detecting the content of oligoisomaltose 0 in the reaction solution by using HPLC.
Definition of enzyme activity: the enzyme activity unit U is defined as the amount of enzyme required for catalyzing maltose to generate l mu mol of isomaltooligosaccharide per minute at 45 ℃ and pH 7.0.
Specific enzyme activity: is defined as the unit protein enzyme activity U/mg.
EXAMPLE 1 construction of recombinant plasmid containing alpha-transglucosidase mutant
Constructing a recombinant plasmid containing the alpha-transglucosidase mutant:
(1) construction of recombinant plasmid containing wild-type alpha-transglucosidase GSJ
Chemically synthesizing wild alpha-glucosyltransferase GSJ with nucleotide sequence shown as SEQ ID NO.2, carrying out enzyme digestion on the wild alpha-glucosyltransferase GSJ and pET-28a (+) vector by Nde I enzyme and Mlu I enzyme, and then connecting to prepare the recombinant vector pET-28a (+) -GSJ.
(2) Obtaining of recombinant vectors containing mutants:
and (2) carrying out site-directed mutagenesis by using the recombinant vector pET-28a (+) -GSJ prepared in the step (1) as a template by using a whole plasmid PCR technology to obtain recombinant plasmids pET-28a (+) -S204L, pET-28a (+) -I209M, pET-28a (+) -S360L, pET-28a (+) -N441M, pET-28a (+) -G108P, pET-28a (+) -S132F, pET-28a (+) -A307E and pET-28a (+) -S204L/A307E containing mutant genes.
The designed primer sequences are as follows:
S204L-F：AACTGGGAGAATCTCGAAGTG
S204L-R：CTTGGCGCACTTCGAGAT
I209M-F：GCCGAGCTGTACGACATGTTACGCTTCTGGCTG
I209M-R：CAGCCAGAAGCGTAACATGTCGTACAGCTCGGC
S360L-F：CTGCAAGAAAATGCCTTAACCTTAACTTTAGCCCC
S360L-R：GCTAAAGTTAAGGTTAAGGCATTTTCTTGCAGACTCGG
N441M-F：GGCATTTACAAATTAATGCACCACCACCACCA
N441M-R：TGGTGGTGGTGGTGCATTAATTTGTAAATGCC
G108P-F：TTTTGGCGCGACCCGAAACAAGGTCAAGCTCCGAAT
G108P-R：TTGACCTTGTTTCGGGTCGCGCCAAAAGTAGTAGTC
S132F-F：TTCCGCCAAGTTATCTTCCAAACCGATCGTGC
S132F-R：GCACGATCGGTTTGGAAGATAACTTGGCGGAA；
A307E-F：ATCTTTGGTGTTCCGCTGAGCGCCATGCCCGAT；
A307E-R：ATCGGGCATGGCGCTCAGCGGAACACCAAAGAT。
wherein, the PCR amplification program is set as follows: firstly, pre-deforming for 5min at 95 ℃; then 30 cycles are carried out; denaturation at 95 ℃ for 30S, annealing at 72 ℃ for 30S, extension at 58 ℃ for 3.5min, and heat preservation at 4 ℃. The PCR product was detected by 0.8% agarose gel electrophoresis.
The final amplified fragment was exposed to Dpn I enzyme in a 37 ℃ water bath for 1h for template removal, then the PCR mixture was chemically transformed into e.coli JM109 competent cells, the transformation solution was plated on LB solid medium containing ampicillin (50 μ g/mL), plasmids were extracted and sequenced, and the sequencing work was done by jinwei, su.
Example 2: construction of recombinant escherichia coli engineering bacteria for producing alpha-transglucosidase mutant and expression, separation and purification of alpha-transglucosidase
The method comprises the following specific steps:
(1) the recombinant plasmids pET-28a (+) -GSJ, pET-28a (+) -S204L, pET-28a (+) -I209M, pET-28a (+) -S360L, pET-28a (+) -N441M, pET-28a (+) -G108P, pET-28a (+) -S132F, pET-28a (+) -A307E and pET-28a (+) -S204L/A307E obtained in example 1 were transformed into E.coli BL21 competent cells, and genetically engineered bacteria were prepared: e.coli BL21/pET-28a (+) -GSJ, E.coli BL21/pET-28a (+) -S204L, E.coli BL21/pET-28a (+) -I209M, E.coli BL21/pET-28a (+) -S360L, E.coli BL21/pET-28a (+) -N441M, E.coli BL21/pET-28a (+) -G108P, E.coli BL21/pET-28a (+) -S132F, E.coli BL21/pET-28a (+) -A307E, E.coli BL21/pET-28a (+) -S204L/A307E.
(2) Respectively inoculating the genetically engineered bacteria prepared in the step (1) into 10mL of LB liquid culture medium containing 50 mug/mL kanamycin sulfate, and culturing at 37 ℃ and 200rpm overnight to prepare seed liquid;
the prepared seed solution was inoculated into 100mL of LB liquid medium containing 50. mu.g/mL kanamycin sulfate in an inoculum size of 2% (v/v), and further cultured at 30 ℃ for 20 hours to obtain a fermentation broth. The prepared fermentation broth was centrifuged at 8000 Xg at 4 ℃ for 5min to obtain cell bodies, and the cells were washed 3 times and resuspended in 10mL of disodium hydrogenphosphate-sodium dihydrogenphosphate buffer (pH 7.0).
Treating the resuspended cells with ultrasonicator under ice bath condition for 30min, centrifuging for 30min (8000 Xg, 4 deg.C), and removing supernatant to obtain crude enzyme solution;
the supernatant fraction was filtered through a 0.22- μm filter and then further loaded onto a 1mL Ni affinity column, which was pre-equilibrated with 50mM wash buffer (20mM Tris and 500mM NaCl, pH 7.4), and then elution buffer (20mM Tris, 500mM NaCl and 500mM imidazole, pH 7.4) eluted unbound protein and α -transglucosidase with a linear gradient; respectively preparing pure enzyme liquid containing wild GSJ, pure enzyme liquid containing S204L, pure enzyme liquid containing I209M, pure enzyme liquid containing S360L, pure enzyme liquid containing N441M, pure enzyme liquid containing G108P, pure enzyme liquid containing S132F, pure enzyme liquid containing A307E and pure enzyme liquid containing S204L/A307E;
the pure enzyme solutions were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the electrophoretogram is shown in FIG. 1, wherein: m, protein maker; 1, wild type GSJ pure enzyme solution; 2-9 are respectively: contains S204L, I209M, A307E, S360L, S132F, G108P, N441M, S207L/A307E pure enzyme liquid. The results show that: a band was evident at 63kDa, demonstrating that alpha-transglucosidase was expressed.
Performing a thermal stability experiment on the pure enzyme prepared in the step (2), and performing primary screening, wherein the method comprises the following steps: respectively incubating the pure enzyme prepared in the step (2) in a water bath kettle at 65 ℃ for 40 hours, taking 1mL, determining the residual enzyme activity of the residual enzyme according to an alpha-transglucosidase enzyme activity determination method, taking the enzyme activity of the pure enzyme solution which is not subjected to high-temperature treatment as a blank control, and obtaining the percentage of the residual enzyme activity, wherein the residual activity test results of the wild alpha-transglucosidase and the mutant thereof are shown in a figure 2.
As can be seen from FIG. 2, the mutant S204L retained 49.1% of relative enzyme activity, while the control group retained only 9.7% of relative enzyme activity; the relative enzyme activity of other mutants is below 18 percent; therefore, the thermostability of the mutant S204L of the present invention was significantly higher than that of the other mutants.
Measuring specific enzyme activity of the pure enzyme solution prepared in the step (2)
The pure enzyme solution containing the wild-type GSJ, the pure enzyme solution containing S204L, the pure enzyme solution containing A307E and the pure enzyme solution containing S204L/A307E prepared in the step (2) are respectively detected, and the results are shown in Table 1:
TABLE 1 specific enzyme activities of different alpha-transglucosidase enzymes
Example 3: enzymatic properties of alpha-transglucosidase mutants
1. Thermal stability
The pure enzyme solution containing the wild-type GSJ, the pure enzyme solution containing S204L, the pure enzyme solution containing A307E and the pure enzyme solution containing S204L/A307E, which are prepared in the step (2) of the example 2, are respectively put in a constant-temperature water bath at 65 ℃, samples are taken at intervals, the residual enzyme activity is measured according to an alpha-glucosyltransferase activity measuring method, the thermal stability is compared, and the half-life results of the wild-type GSJ and the mutant thereof are shown in figure 3.
2. Optimum pH
The enzyme activity was measured by placing the pure enzyme solution containing wild-type GSJ, the pure enzyme solution containing S204L, the pure enzyme solution containing A307E, and the pure enzyme solution containing S204L/A307E, which were prepared in step (2) of example 2, in a 50mM buffer solution containing citric acid/sodium phosphate (pH 5.0-9.0) with the initial enzyme activity without incubation as 100%, and the results are shown in FIG. 4.
As can be seen from FIG. 4, the optimum pH of the mutant was 7.0, similar to that of the wild type.
3. Optimum temperature
The enzyme activity was measured by placing the pure enzyme solution containing wild-type GSJ, the pure enzyme solution containing S204L, the pure enzyme solution containing A307E, and the pure enzyme solution containing S204L/A307E, which were prepared in step (2) of example 2, in a 50mM buffer solution containing citric acid/sodium phosphate (pH7.0), at a reaction temperature of 30 to 60 ℃ with the initial enzyme activity without incubation being 100%, and the results are shown in FIG. 5.
As can be seen from FIG. 5, the optimum temperature of the mutant was 45 ℃ and was similar to that of the wild type.
4. Kinetic parameters of alpha-transglucosidase
The kinetic parameters of the pure enzyme solution containing wild-type GSJ, the pure enzyme solution containing S204L, the pure enzyme solution containing A307E and the pure enzyme solution containing S204L/A307E prepared in step (2) of example 2 were measured under standard measurement conditions using maltose as a substrate. Wherein the maltose substrate concentration is 14.6, 29.2, 58.4, 102, 146, 234, 292 and 584mM respectively; adding 10 mu g of pure enzyme solution, reacting for 10min at 45 ℃, and after the reaction is finished, performing regression analysis on experimental data by using GraphPad Prism 8.0 to determine Vmax and Km values; the results are shown in table 2:
TABLE 2 kinetic parameters of different alpha-transglucosidase enzymes
The results show that the mutant showed similar activity compared to the wild type. The Km, Kcat/Km and other kinetic parameters of the mutants are slightly changed, which indicates that the mutation or the thermal stability has little influence on the catalytic property of the enzyme.
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
SEQ ID NO.1
Amino acid sequence of alpha-transglucosidase
KKTWWKEGVAYQIYPRSFMDANGDGIGDLRGIIEKLDYLVELGVDIVWICPIYRSPNADNGYDISDYYAIMDEFGTMDDFDELLAQAHRRGLKVILDLVINHTSDEHPWFIESRSSRDNPKRDWYIWRDGKDGREPNNWESIFGGSAWQYDERTGQYYLHIFDVKQPDLNWENSEVRQALYEMVNWWLDKGIDGFRIDAISHISKKPGLPDLPNPKGLKYVPSFAGHMNQPGIMEYLRELKEQTFARYDIMTVGEANGVTVDEAEQWVGEENGVFNMIFQFEHLGLWERRADGSIDVRRLKRTLTKWQKGLENRGWNALFLENHDLPRSVSTWGNDRDYWAESAKALGALYFFMQGTPFIYQGQEIGMTNVRFDDIRDYRDVSALRLYELERAKGRTHEEAMTIIWKTGRDNSRTPMQWSGASNAGFTTGTPWIKVNENYRTINVEAERRDPNSVWSFYRQMIQLRKANELFVYGTYDLLLENHPSIYAYTRTLGRDRALVVVNLSDRPSLYRYDGFRLQSSDLALSNYPVRPHKNATRFKLKPYEARVYIWKE
SEQ ID NO.2
Nucleotide sequence coding for alpha-transglucosidase
aaaaaaacgtggtggaaagaaggcgtggcgtatcagatctacccgcgtagctttatggacgccaatggcgacggcatcggcgatctgcgtggcatcatcgagaagctcgactatctggttgaactgggcgtggacatcgtttggatctgtccgatctatcgcagtccgaatgccgataacggctacgatatcagcgactactacgccatcatggacgagttcggcaccatggatgacttcgatgagctgctggcgcaagcccatcgtcgtggtctcaaggtgatcctcgatctggtgatcaatcacaccagcgatgaacacccgtggtttatcgaaagtcgcagcagtcgcgacaacccgaagcgcgattggtacatttggcgtgacggcaaagacggccgcgaaccgaacaactgggaaagcattttcggtggcagcgcgtggcagtacgatgaacgcacgggccagtactatctgcacattttcgacgtgaagcagccagatctgaactgggagaatagcgaagtgcgccaagcgctgtacgaaatggtgaactggtggctcgacaagggtatcgatggcttccgcatcgacgccatcagccatatcagcaagaagccgggtctgccggatctgccaaatccgaaaggtctgaaatacgtgccgagctttgccggccacatgaatcagccgggcatcatggaatatctgcgcgagctgaaagagcagaccttcgcccgctatgatatcatgaccgtgggcgaagcgaacggtgtgaccgttgacgaagccgaacagtgggtgggcgaggaaaacggtgtgttcaatatgatcttccaatttgagcatctgggtctctgggaacgtcgcgccgatggcagcatcgatgttcgtcgtctgaaacgcacgctgaccaaggcgcagaaaggcctcgaaaaccgcggttggaatgcgctgtttctggaaaaccacgatctcccgcgcagcgtgagtacgtggggcaatgaccgtgattactgggccgaaagcgcgaaagcgctcggtgcgctctatttcttcatgcaaggcaccccgtttatctaccaaggccaagagatcggcatgaccaatgttcgctttgacgacatccgcgactaccgcgatgttagcgccctccgcctctacgaactggaacgtgccaagggccgcacgcatgaagaggccatgaccatcatctggaaaacgggtcgcgacaacagtcgcacgccgatgcagtggagcggtgccagtaatgccggcttcacgaccggcaccccatggattaaggttaatgaaaactaccgcaccatcaatgtggaagcggaacgtcgcgacccgaacagcgtgtggagcttttatcgccagatgatccagctccgtaaagcgaacgagctgtttgtttacggcacgtacgatctgctgctggagaaccatccgagcatttacgcctatacccgtacgctgggtcgtgatcgtgcgctggtggttgttaatctcagcgaccgcccaagtctgtaccgctatgacggcttccgtctgcagagcagtgatctggcgctgagtaattacccagtgcgcccgcacaagaatgccacgcgcttcaaactcaagccgtacgaggcccgcgtgtacatctggaaggagtaa
SEQ ID NO.3
Amino acid sequence of alpha-transglucosidase mutant S204L
KKTWWKEGVAYQIYPRSFMDANGDGIGDLRGIIEKLDYLVELGVDIVWICPIYRSPNADNGYDISDYYAIMDEFGTMDDFDELLAQAHRRGLKVILDLVINHTSDEHPWFIESRSSRDNPKRDWYIWRDGKDGREPNNWESIFGGSAWQYDERTGQYYLHIFDVKQPDLNWENSEVRQALYEMVNWWLDKGIDGFRIDAISHILKKPGLPDLPNPKGLKYVPSFAGHMNQPGIMEYLRELKEQTFARYDIMTVGEANGVTVDEAEQWVGEENGVFNMIFQFEHLGLWERRADGSIDVRRLKRTLTKWQKGLENRGWNALFLENHDLPRSVSTWGNDRDYWAESAKALGALYFFMQGTPFIYQGQEIGMTNVRFDDIRDYRDVSALRLYELERAKGRTHEEAMTIIWKTGRDNSRTPMQWSGASNAGFTTGTPWIKVNENYRTINVEAERRDPNSVWSFYRQMIQLRKANELFVYGTYDLLLENHPSIYAYTRTLGRDRALVVVNLSDRPSLYRYDGFRLQSSDLALSNYPVRPHKNATRFKLKPYEARVYIWKE
SEQ ID NO.4
Amino acid sequence of alpha-transglucosidase mutant A307E
KKTWWKEGVAYQIYPRSFMDANGDGIGDLRGIIEKLDYLVELGVDIVWICPIYRSPNADNGYDISDYYAIMDEFGTMDDFDELLAQAHRRGLKVILDLVINHTSDEHPWFIESRSSRDNPKRDWYIWRDGKDGREPNNWESIFGGSAWQYDERTGQYYLHIFDVKQPDLNWENSEVRQALYEMVNWWLDKGIDGFRIDAISHISKKPGLPDLPNPKGLKYVPSFAGHMNQPGIMEYLRELKEQTFARYDIMTVGEANGVTVDEAEQWVGEENGVFNMIFQFEHLGLWERRADGSIDVRRLKRTLTKEQKGLENRGWNALFLENHDLPRSVSTWGNDRDYWAESAKALGALYFFMQGTPFIYQGQEIGMTNVRFDDIRDYRDVSALRLYELERAKGRTHEEAMTIIWKTGRDNSRTPMQWSGASNAGFTTGTPWIKVNENYRTINVEAERRDPNSVWSFYRQMIQLRKANELFVYGTYDLLLENHPSIYAYTRTLGRDRALVVVNLSDRPSLYRYDGFRLQSSDLALSNYPVRPHKNATRFKLKPYEARVYIWKE
SEQ ID NO.5
Amino acid sequence of alpha-transglucosidase mutant S204L/A307E
KKTWWKEGVAYQIYPRSFMDANGDGIGDLRGIIEKLDYLVELGVDIVWICPIYRSPNADNGYDISDYYAIMDEFGTMDDFDELLAQAHRRGLKVILDLVINHTSDEHPWFIESRSSRDNPKRDWYIWRDGKDGREPNNWESIFGGSAWQYDERTGQYYLHIFDVKQPDLNWENSEVRQALYEMVNWWLDKGIDGFRIDAISHILKKPGLPDLPNPKGLKYVPSFAGHMNQPGIMEYLRELKEQTFARYDIMTVGEANGVTVDEAEQWVGEENGVFNMIFQFEHLGLWERRADGSIDVRRLKRTLTKEQKGLENRGWNALFLENHDLPRSVSTWGNDRDYWAESAKALGALYFFMQGTPFIYQGQEIGMTNVRFDDIRDYRDVSALRLYELERAKGRTHEEAMTIIWKTGRDNSRTPMQWSGASNAGFTTGTPWIKVNENYRTINVEAERRDPNSVWSFYRQMIQLRKANELFVYGTYDLLLENHPSIYAYTRTLGRDRALVVVNLSDRPSLYRYDGFRLQSSDLALSNYPVRPHKNATRFKLKPYEARVYIWKE
SEQ ID NO.6
Nucleotide sequence for coding alpha-transglucosidase mutant S204L
aaaaaaacgtggtggaaagaaggcgtggcgtatcagatctacccgcgtagctttatggacgccaatggcgacggcatcggcgatctgcgtggcatcatcgagaagctcgactatctggttgaactgggcgtggacatcgtttggatctgtccgatctatcgcagtccgaatgccgataacggctacgatatcagcgactactacgccatcatggacgagttcggcaccatggatgacttcgatgagctgctggcgcaagcccatcgtcgtggtctcaaggtgatcctcgatctggtgatcaatcacaccagcgatgaacacccgtggtttatcgaaagtcgcagcagtcgcgacaacccgaagcgcgattggtacatttggcgtgacggcaaagacggccgcgaaccgaacaactgggaaagcattttcggtggcagcgcgtggcagtacgatgaacgcacgggccagtactatctgcacattttcgacgtgaagcagccagatctgaactgggagaatagcgaagtgcgccaagcgctgtacgaaatggtgaactggtggctcgacaagggtatcgatggcttccgcatcgacgccatcagccatatcctgaagaagccgggtctgccggatctgccaaatccgaaaggtctgaaatacgtgccgagctttgccggccacatgaatcagccgggcatcatggaatatctgcgcgagctgaaagagcagaccttcgcccgctatgatatcatgaccgtgggcgaagcgaacggtgtgaccgttgacgaagccgaacagtgggtgggcgaggaaaacggtgtgttcaatatgatcttccaatttgagcatctgggtctctgggaacgtcgcgccgatggcagcatcgatgttcgtcgtctgaaacgcacgctgaccaaggcgcagaaaggcctcgaaaaccgcggttggaatgcgctgtttctggaaaaccacgatctcccgcgcagcgtgagtacgtggggcaatgaccgtgattactgggccgaaagcgcgaaagcgctcggtgcgctctatttcttcatgcaaggcaccccgtttatctaccaaggccaagagatcggcatgaccaatgttcgctttgacgacatccgcgactaccgcgatgttagcgccctccgcctctacgaactggaacgtgccaagggccgcacgcatgaagaggccatgaccatcatctggaaaacgggtcgcgacaacagtcgcacgccgatgcagtggagcggtgccagtaatgccggcttcacgaccggcaccccatggattaaggttaatgaaaactaccgcaccatcaatgtggaagcggaacgtcgcgacccgaacagcgtgtggagcttttatcgccagatgatccagctccgtaaagcgaacgagctgtttgtttacggcacgtacgatctgctgctggagaaccatccgagcatttacgcctatacccgtacgctgggtcgtgatcgtgcgctggtggttgttaatctcagcgaccgcccaagtctgtaccgctatgacggcttccgtctgcagagcagtgatctggcgctgagtaattacccagtgcgcccgcacaagaatgccacgcgcttcaaactcaagccgtacgaggcccgcgtgtacatctggaaggagtaa
SEQ ID NO.7
Nucleotide sequence for coding alpha-transglucosidase mutant A307E
aaaaaaacgtggtggaaagaaggcgtggcgtatcagatctacccgcgtagctttatggacgccaatggcgacggcatcggcgatctgcgtggcatcatcgagaagctcgactatctggttgaactgggcgtggacatcgtttggatctgtccgatctatcgcagtccgaatgccgataacggctacgatatcagcgactactacgccatcatggacgagttcggcaccatggatgacttcgatgagctgctggcgcaagcccatcgtcgtggtctcaaggtgatcctcgatctggtgatcaatcacaccagcgatgaacacccgtggtttatcgaaagtcgcagcagtcgcgacaacccgaagcgcgattggtacatttggcgtgacggcaaagacggccgcgaaccgaacaactgggaaagcattttcggtggcagcgcgtggcagtacgatgaacgcacgggccagtactatctgcacattttcgacgtgaagcagccagatctgaactgggagaatagcgaagtgcgccaagcgctgtacgaaatggtgaactggtggctcgacaagggtatcgatggcttccgcatcgacgccatcagccatatcagcaagaagccgggtctgccggatctgccaaatccgaaaggtctgaaatacgtgccgagctttgccggccacatgaatcagccgggcatcatggaatatctgcgcgagctgaaagagcagaccttcgcccgctatgatatcatgaccgtgggcgaagcgaacggtgtgaccgttgacgaagccgaacagtgggtgggcgaggaaaacggtgtgttcaatatgatcttccaatttgagcatctgggtctctgggaacgtcgcgccgatggcagcatcgatgttcgtcgtctgaaacgcacgctgaccaaggaacagaaaggcctcgaaaaccgcggttggaatgcgctgtttctggaaaaccacgatctcccgcgcagcgtgagtacgtggggcaatgaccgtgattactgggccgaaagcgcgaaagcgctcggtgcgctctatttcttcatgcaaggcaccccgtttatctaccaaggccaagagatcggcatgaccaatgttcgctttgacgacatccgcgactaccgcgatgttagcgccctccgcctctacgaactggaacgtgccaagggccgcacgcatgaagaggccatgaccatcatctggaaaacgggtcgcgacaacagtcgcacgccgatgcagtggagcggtgccagtaatgccggcttcacgaccggcaccccatggattaaggttaatgaaaactaccgcaccatcaatgtggaagcggaacgtcgcgacccgaacagcgtgtggagcttttatcgccagatgatccagctccgtaaagcgaacgagctgtttgtttacggcacgtacgatctgctgctggagaaccatccgagcatttacgcctatacccgtacgctgggtcgtgatcgtgcgctggtggttgttaatctcagcgaccgcccaagtctgtaccgctatgacggcttccgtctgcagagcagtgatctggcgctgagtaattacccagtgcgcccgcacaagaatgccacgcgcttcaaactcaagccgtacgaggcccgcgtgtacatctggaaggagtaa
SEQ ID NO.8
Nucleotide sequence for coding alpha-transglucosidase mutant S204L/A307E
aaaaaaacgtggtggaaagaaggcgtggcgtatcagatctacccgcgtagctttatggacgccaatggcgacggcatcggcgatctgcgtggcatcatcgagaagctcgactatctggttgaactgggcgtggacatcgtttggatctgtccgatctatcgcagtccgaatgccgataacggctacgatatcagcgactactacgccatcatggacgagttcggcaccatggatgacttcgatgagctgctggcgcaagcccatcgtcgtggtctcaaggtgatcctcgatctggtgatcaatcacaccagcgatgaacacccgtggtttatcgaaagtcgcagcagtcgcgacaacccgaagcgcgattggtacatttggcgtgacggcaaagacggccgcgaaccgaacaactgggaaagcattttcggtggcagcgcgtggcagtacgatgaacgcacgggccagtactatctgcacattttcgacgtgaagcagccagatctgaactgggagaatagcgaagtgcgccaagcgctgtacgaaatggtgaactggtggctcgacaagggtatcgatggcttccgcatcgacgccatcagccatatcctgaagaagccgggtctgccggatctgccaaatccgaaaggtctgaaatacgtgccgagctttgccggccacatgaatcagccgggcatcatggaatatctgcgcgagctgaaagagcagaccttcgcccgctatgatatcatgaccgtgggcgaagcgaacggtgtgaccgttgacgaagccgaacagtgggtgggcgaggaaaacggtgtgttcaatatgatcttccaatttgagcatctgggtctctgggaacgtcgcgccgatggcagcatcgatgttcgtcgtctgaaacgcacgctgaccaaggaacagaaaggcctcgaaaaccgcggttggaatgcgctgtttctggaaaaccacgatctcccgcgcagcgtgagtacgtggggcaatgaccgtgattactgggccgaaagcgcgaaagcgctcggtgcgctctatttcttcatgcaaggcaccccgtttatctaccaaggccaagagatcggcatgaccaatgttcgctttgacgacatccgcgactaccgcgatgttagcgccctccgcctctacgaactggaacgtgccaagggccgcacgcatgaagaggccatgaccatcatctggaaaacgggtcgcgacaacagtcgcacgccgatgcagtggagcggtgccagtaatgccggcttcacgaccggcaccccatggattaaggttaatgaaaactaccgcaccatcaatgtggaagcggaacgtcgcgacccgaacagcgtgtggagcttttatcgccagatgatccagctccgtaaagcgaacgagctgtttgtttacggcacgtacgatctgctgctggagaaccatccgagcatttacgcctatacccgtacgctgggtcgtgatcgtgcgctggtggttgttaatctcagcgaccgcccaagtctgtaccgctatgacggcttccgtctgcagagcagtgatctggcgctgagtaattacccagtgcgcccgcacaagaatgccacgcgcttcaaactcaagccgtacgaggcccgcgtgtacatctggaaggagtaa
Sequence listing
<110> Shandong Henren Industrial and trade Co Ltd
<120> alpha-transglucosidase mutant and application thereof
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 554
<212> PRT
<213> Isomaltooligosaccharides (IMOs)
<400> 1
Lys Lys Thr Trp Trp Lys Glu Gly Val Ala Tyr Gln Ile Tyr Pro Arg
1 5 10 15
Ser Phe Met Asp Ala Asn Gly Asp Gly Ile Gly Asp Leu Arg Gly Ile
20 25 30
Ile Glu Lys Leu Asp Tyr Leu Val Glu Leu Gly Val Asp Ile Val Trp
35 40 45
Ile Cys Pro Ile Tyr Arg Ser Pro Asn Ala Asp Asn Gly Tyr Asp Ile
50 55 60
Ser Asp Tyr Tyr Ala Ile Met Asp Glu Phe Gly Thr Met Asp Asp Phe
65 70 75 80
Asp Glu Leu Leu Ala Gln Ala His Arg Arg Gly Leu Lys Val Ile Leu
85 90 95
Asp Leu Val Ile Asn His Thr Ser Asp Glu His Pro Trp Phe Ile Glu
100 105 110
Ser Arg Ser Ser Arg Asp Asn Pro Lys Arg Asp Trp Tyr Ile Trp Arg
115 120 125
Asp Gly Lys Asp Gly Arg Glu Pro Asn Asn Trp Glu Ser Ile Phe Gly
130 135 140
Gly Ser Ala Trp Gln Tyr Asp Glu Arg Thr Gly Gln Tyr Tyr Leu His
145 150 155 160
Ile Phe Asp Val Lys Gln Pro Asp Leu Asn Trp Glu Asn Ser Glu Val
165 170 175
Arg Gln Ala Leu Tyr Glu Met Val Asn Trp Trp Leu Asp Lys Gly Ile
180 185 190
Asp Gly Phe Arg Ile Asp Ala Ile Ser His Ile Ser Lys Lys Pro Gly
195 200 205
Leu Pro Asp Leu Pro Asn Pro Lys Gly Leu Lys Tyr Val Pro Ser Phe
210 215 220
Ala Gly His Met Asn Gln Pro Gly Ile Met Glu Tyr Leu Arg Glu Leu
225 230 235 240
Lys Glu Gln Thr Phe Ala Arg Tyr Asp Ile Met Thr Val Gly Glu Ala
245 250 255
Asn Gly Val Thr Val Asp Glu Ala Glu Gln Trp Val Gly Glu Glu Asn
260 265 270
Gly Val Phe Asn Met Ile Phe Gln Phe Glu His Leu Gly Leu Trp Glu
275 280 285
Arg Arg Ala Asp Gly Ser Ile Asp Val Arg Arg Leu Lys Arg Thr Leu
290 295 300
Thr Lys Trp Gln Lys Gly Leu Glu Asn Arg Gly Trp Asn Ala Leu Phe
305 310 315 320
Leu Glu Asn His Asp Leu Pro Arg Ser Val Ser Thr Trp Gly Asn Asp
325 330 335
Arg Asp Tyr Trp Ala Glu Ser Ala Lys Ala Leu Gly Ala Leu Tyr Phe
340 345 350
Phe Met Gln Gly Thr Pro Phe Ile Tyr Gln Gly Gln Glu Ile Gly Met
355 360 365
Thr Asn Val Arg Phe Asp Asp Ile Arg Asp Tyr Arg Asp Val Ser Ala
370 375 380
Leu Arg Leu Tyr Glu Leu Glu Arg Ala Lys Gly Arg Thr His Glu Glu
385 390 395 400
Ala Met Thr Ile Ile Trp Lys Thr Gly Arg Asp Asn Ser Arg Thr Pro
405 410 415
Met Gln Trp Ser Gly Ala Ser Asn Ala Gly Phe Thr Thr Gly Thr Pro
420 425 430
Trp Ile Lys Val Asn Glu Asn Tyr Arg Thr Ile Asn Val Glu Ala Glu
435 440 445
Arg Arg Asp Pro Asn Ser Val Trp Ser Phe Tyr Arg Gln Met Ile Gln
450 455 460
Leu Arg Lys Ala Asn Glu Leu Phe Val Tyr Gly Thr Tyr Asp Leu Leu
465 470 475 480
Leu Glu Asn His Pro Ser Ile Tyr Ala Tyr Thr Arg Thr Leu Gly Arg
485 490 495
Asp Arg Ala Leu Val Val Val Asn Leu Ser Asp Arg Pro Ser Leu Tyr
500 505 510
Arg Tyr Asp Gly Phe Arg Leu Gln Ser Ser Asp Leu Ala Leu Ser Asn
515 520 525
Tyr Pro Val Arg Pro His Lys Asn Ala Thr Arg Phe Lys Leu Lys Pro
530 535 540
Tyr Glu Ala Arg Val Tyr Ile Trp Lys Glu
545 550
<210> 2
<211> 1665
<212> DNA
<213> Isomaltooligosaccharides (IMOs)
<400> 2
aaaaaaacgt ggtggaaaga aggcgtggcg tatcagatct acccgcgtag ctttatggac 60
gccaatggcg acggcatcgg cgatctgcgt ggcatcatcg agaagctcga ctatctggtt 120
gaactgggcg tggacatcgt ttggatctgt ccgatctatc gcagtccgaa tgccgataac 180
ggctacgata tcagcgacta ctacgccatc atggacgagt tcggcaccat ggatgacttc 240
gatgagctgc tggcgcaagc ccatcgtcgt ggtctcaagg tgatcctcga tctggtgatc 300
aatcacacca gcgatgaaca cccgtggttt atcgaaagtc gcagcagtcg cgacaacccg 360
aagcgcgatt ggtacatttg gcgtgacggc aaagacggcc gcgaaccgaa caactgggaa 420
agcattttcg gtggcagcgc gtggcagtac gatgaacgca cgggccagta ctatctgcac 480
attttcgacg tgaagcagcc agatctgaac tgggagaata gcgaagtgcg ccaagcgctg 540
tacgaaatgg tgaactggtg gctcgacaag ggtatcgatg gcttccgcat cgacgccatc 600
agccatatca gcaagaagcc gggtctgccg gatctgccaa atccgaaagg tctgaaatac 660
gtgccgagct ttgccggcca catgaatcag ccgggcatca tggaatatct gcgcgagctg 720
aaagagcaga ccttcgcccg ctatgatatc atgaccgtgg gcgaagcgaa cggtgtgacc 780
gttgacgaag ccgaacagtg ggtgggcgag gaaaacggtg tgttcaatat gatcttccaa 840
tttgagcatc tgggtctctg ggaacgtcgc gccgatggca gcatcgatgt tcgtcgtctg 900
aaacgcacgc tgaccaaggc gcagaaaggc ctcgaaaacc gcggttggaa tgcgctgttt 960
ctggaaaacc acgatctccc gcgcagcgtg agtacgtggg gcaatgaccg tgattactgg 1020
gccgaaagcg cgaaagcgct cggtgcgctc tatttcttca tgcaaggcac cccgtttatc 1080
taccaaggcc aagagatcgg catgaccaat gttcgctttg acgacatccg cgactaccgc 1140
gatgttagcg ccctccgcct ctacgaactg gaacgtgcca agggccgcac gcatgaagag 1200
gccatgacca tcatctggaa aacgggtcgc gacaacagtc gcacgccgat gcagtggagc 1260
ggtgccagta atgccggctt cacgaccggc accccatgga ttaaggttaa tgaaaactac 1320
cgcaccatca atgtggaagc ggaacgtcgc gacccgaaca gcgtgtggag cttttatcgc 1380
cagatgatcc agctccgtaa agcgaacgag ctgtttgttt acggcacgta cgatctgctg 1440
ctggagaacc atccgagcat ttacgcctat acccgtacgc tgggtcgtga tcgtgcgctg 1500
gtggttgtta atctcagcga ccgcccaagt ctgtaccgct atgacggctt ccgtctgcag 1560
agcagtgatc tggcgctgag taattaccca gtgcgcccgc acaagaatgc cacgcgcttc 1620
aaactcaagc cgtacgaggc ccgcgtgtac atctggaagg agtaa 1665
<210> 3
<211> 554
<212> PRT
<213> Isomaltooligosaccharides (IMOs)
<400> 3
Lys Lys Thr Trp Trp Lys Glu Gly Val Ala Tyr Gln Ile Tyr Pro Arg
1 5 10 15
Ser Phe Met Asp Ala Asn Gly Asp Gly Ile Gly Asp Leu Arg Gly Ile
20 25 30
Ile Glu Lys Leu Asp Tyr Leu Val Glu Leu Gly Val Asp Ile Val Trp
35 40 45
Ile Cys Pro Ile Tyr Arg Ser Pro Asn Ala Asp Asn Gly Tyr Asp Ile
50 55 60
Ser Asp Tyr Tyr Ala Ile Met Asp Glu Phe Gly Thr Met Asp Asp Phe
65 70 75 80
Asp Glu Leu Leu Ala Gln Ala His Arg Arg Gly Leu Lys Val Ile Leu
85 90 95
Asp Leu Val Ile Asn His Thr Ser Asp Glu His Pro Trp Phe Ile Glu
100 105 110
Ser Arg Ser Ser Arg Asp Asn Pro Lys Arg Asp Trp Tyr Ile Trp Arg
115 120 125
Asp Gly Lys Asp Gly Arg Glu Pro Asn Asn Trp Glu Ser Ile Phe Gly
130 135 140
Gly Ser Ala Trp Gln Tyr Asp Glu Arg Thr Gly Gln Tyr Tyr Leu His
145 150 155 160
Ile Phe Asp Val Lys Gln Pro Asp Leu Asn Trp Glu Asn Ser Glu Val
165 170 175
Arg Gln Ala Leu Tyr Glu Met Val Asn Trp Trp Leu Asp Lys Gly Ile
180 185 190
Asp Gly Phe Arg Ile Asp Ala Ile Ser His Ile Leu Lys Lys Pro Gly
195 200 205
Leu Pro Asp Leu Pro Asn Pro Lys Gly Leu Lys Tyr Val Pro Ser Phe
210 215 220
Ala Gly His Met Asn Gln Pro Gly Ile Met Glu Tyr Leu Arg Glu Leu
225 230 235 240
Lys Glu Gln Thr Phe Ala Arg Tyr Asp Ile Met Thr Val Gly Glu Ala
245 250 255
Asn Gly Val Thr Val Asp Glu Ala Glu Gln Trp Val Gly Glu Glu Asn
260 265 270
Gly Val Phe Asn Met Ile Phe Gln Phe Glu His Leu Gly Leu Trp Glu
275 280 285
Arg Arg Ala Asp Gly Ser Ile Asp Val Arg Arg Leu Lys Arg Thr Leu
290 295 300
Thr Lys Trp Gln Lys Gly Leu Glu Asn Arg Gly Trp Asn Ala Leu Phe
305 310 315 320
Leu Glu Asn His Asp Leu Pro Arg Ser Val Ser Thr Trp Gly Asn Asp
325 330 335
Arg Asp Tyr Trp Ala Glu Ser Ala Lys Ala Leu Gly Ala Leu Tyr Phe
340 345 350
Phe Met Gln Gly Thr Pro Phe Ile Tyr Gln Gly Gln Glu Ile Gly Met
355 360 365
Thr Asn Val Arg Phe Asp Asp Ile Arg Asp Tyr Arg Asp Val Ser Ala
370 375 380
Leu Arg Leu Tyr Glu Leu Glu Arg Ala Lys Gly Arg Thr His Glu Glu
385 390 395 400
Ala Met Thr Ile Ile Trp Lys Thr Gly Arg Asp Asn Ser Arg Thr Pro
405 410 415
Met Gln Trp Ser Gly Ala Ser Asn Ala Gly Phe Thr Thr Gly Thr Pro
420 425 430
Trp Ile Lys Val Asn Glu Asn Tyr Arg Thr Ile Asn Val Glu Ala Glu
435 440 445
Arg Arg Asp Pro Asn Ser Val Trp Ser Phe Tyr Arg Gln Met Ile Gln
450 455 460
Leu Arg Lys Ala Asn Glu Leu Phe Val Tyr Gly Thr Tyr Asp Leu Leu
465 470 475 480
Leu Glu Asn His Pro Ser Ile Tyr Ala Tyr Thr Arg Thr Leu Gly Arg
485 490 495
Asp Arg Ala Leu Val Val Val Asn Leu Ser Asp Arg Pro Ser Leu Tyr
500 505 510
Arg Tyr Asp Gly Phe Arg Leu Gln Ser Ser Asp Leu Ala Leu Ser Asn
515 520 525
Tyr Pro Val Arg Pro His Lys Asn Ala Thr Arg Phe Lys Leu Lys Pro
530 535 540
Tyr Glu Ala Arg Val Tyr Ile Trp Lys Glu
545 550
<210> 4
<211> 554
<212> PRT
<213> Isomaltooligosaccharides (IMOs)
<400> 4
Lys Lys Thr Trp Trp Lys Glu Gly Val Ala Tyr Gln Ile Tyr Pro Arg
1 5 10 15
Ser Phe Met Asp Ala Asn Gly Asp Gly Ile Gly Asp Leu Arg Gly Ile
20 25 30
Ile Glu Lys Leu Asp Tyr Leu Val Glu Leu Gly Val Asp Ile Val Trp
35 40 45
Ile Cys Pro Ile Tyr Arg Ser Pro Asn Ala Asp Asn Gly Tyr Asp Ile
50 55 60
Ser Asp Tyr Tyr Ala Ile Met Asp Glu Phe Gly Thr Met Asp Asp Phe
65 70 75 80
Asp Glu Leu Leu Ala Gln Ala His Arg Arg Gly Leu Lys Val Ile Leu
85 90 95
Asp Leu Val Ile Asn His Thr Ser Asp Glu His Pro Trp Phe Ile Glu
100 105 110
Ser Arg Ser Ser Arg Asp Asn Pro Lys Arg Asp Trp Tyr Ile Trp Arg
115 120 125
Asp Gly Lys Asp Gly Arg Glu Pro Asn Asn Trp Glu Ser Ile Phe Gly
130 135 140
Gly Ser Ala Trp Gln Tyr Asp Glu Arg Thr Gly Gln Tyr Tyr Leu His
145 150 155 160
Ile Phe Asp Val Lys Gln Pro Asp Leu Asn Trp Glu Asn Ser Glu Val
165 170 175
Arg Gln Ala Leu Tyr Glu Met Val Asn Trp Trp Leu Asp Lys Gly Ile
180 185 190
Asp Gly Phe Arg Ile Asp Ala Ile Ser His Ile Ser Lys Lys Pro Gly
195 200 205
Leu Pro Asp Leu Pro Asn Pro Lys Gly Leu Lys Tyr Val Pro Ser Phe
210 215 220
Ala Gly His Met Asn Gln Pro Gly Ile Met Glu Tyr Leu Arg Glu Leu
225 230 235 240
Lys Glu Gln Thr Phe Ala Arg Tyr Asp Ile Met Thr Val Gly Glu Ala
245 250 255
Asn Gly Val Thr Val Asp Glu Ala Glu Gln Trp Val Gly Glu Glu Asn
260 265 270
Gly Val Phe Asn Met Ile Phe Gln Phe Glu His Leu Gly Leu Trp Glu
275 280 285
Arg Arg Ala Asp Gly Ser Ile Asp Val Arg Arg Leu Lys Arg Thr Leu
290 295 300
Thr Lys Glu Gln Lys Gly Leu Glu Asn Arg Gly Trp Asn Ala Leu Phe
305 310 315 320
Leu Glu Asn His Asp Leu Pro Arg Ser Val Ser Thr Trp Gly Asn Asp
325 330 335
Arg Asp Tyr Trp Ala Glu Ser Ala Lys Ala Leu Gly Ala Leu Tyr Phe
340 345 350
Phe Met Gln Gly Thr Pro Phe Ile Tyr Gln Gly Gln Glu Ile Gly Met
355 360 365
Thr Asn Val Arg Phe Asp Asp Ile Arg Asp Tyr Arg Asp Val Ser Ala
370 375 380
Leu Arg Leu Tyr Glu Leu Glu Arg Ala Lys Gly Arg Thr His Glu Glu
385 390 395 400
Ala Met Thr Ile Ile Trp Lys Thr Gly Arg Asp Asn Ser Arg Thr Pro
405 410 415
Met Gln Trp Ser Gly Ala Ser Asn Ala Gly Phe Thr Thr Gly Thr Pro
420 425 430
Trp Ile Lys Val Asn Glu Asn Tyr Arg Thr Ile Asn Val Glu Ala Glu
435 440 445
Arg Arg Asp Pro Asn Ser Val Trp Ser Phe Tyr Arg Gln Met Ile Gln
450 455 460
Leu Arg Lys Ala Asn Glu Leu Phe Val Tyr Gly Thr Tyr Asp Leu Leu
465 470 475 480
Leu Glu Asn His Pro Ser Ile Tyr Ala Tyr Thr Arg Thr Leu Gly Arg
485 490 495
Asp Arg Ala Leu Val Val Val Asn Leu Ser Asp Arg Pro Ser Leu Tyr
500 505 510
Arg Tyr Asp Gly Phe Arg Leu Gln Ser Ser Asp Leu Ala Leu Ser Asn
515 520 525
Tyr Pro Val Arg Pro His Lys Asn Ala Thr Arg Phe Lys Leu Lys Pro
530 535 540
Tyr Glu Ala Arg Val Tyr Ile Trp Lys Glu
545 550
<210> 5
<211> 554
<212> PRT
<213> Isomaltooligosaccharides (IMOs)
<400> 5
Lys Lys Thr Trp Trp Lys Glu Gly Val Ala Tyr Gln Ile Tyr Pro Arg
1 5 10 15
Ser Phe Met Asp Ala Asn Gly Asp Gly Ile Gly Asp Leu Arg Gly Ile
20 25 30
Ile Glu Lys Leu Asp Tyr Leu Val Glu Leu Gly Val Asp Ile Val Trp
35 40 45
Ile Cys Pro Ile Tyr Arg Ser Pro Asn Ala Asp Asn Gly Tyr Asp Ile
50 55 60
Ser Asp Tyr Tyr Ala Ile Met Asp Glu Phe Gly Thr Met Asp Asp Phe
65 70 75 80
Asp Glu Leu Leu Ala Gln Ala His Arg Arg Gly Leu Lys Val Ile Leu
85 90 95
Asp Leu Val Ile Asn His Thr Ser Asp Glu His Pro Trp Phe Ile Glu
100 105 110
Ser Arg Ser Ser Arg Asp Asn Pro Lys Arg Asp Trp Tyr Ile Trp Arg
115 120 125
Asp Gly Lys Asp Gly Arg Glu Pro Asn Asn Trp Glu Ser Ile Phe Gly
130 135 140
Gly Ser Ala Trp Gln Tyr Asp Glu Arg Thr Gly Gln Tyr Tyr Leu His
145 150 155 160
Ile Phe Asp Val Lys Gln Pro Asp Leu Asn Trp Glu Asn Ser Glu Val
165 170 175
Arg Gln Ala Leu Tyr Glu Met Val Asn Trp Trp Leu Asp Lys Gly Ile
180 185 190
Asp Gly Phe Arg Ile Asp Ala Ile Ser His Ile Leu Lys Lys Pro Gly
195 200 205
Leu Pro Asp Leu Pro Asn Pro Lys Gly Leu Lys Tyr Val Pro Ser Phe
210 215 220
Ala Gly His Met Asn Gln Pro Gly Ile Met Glu Tyr Leu Arg Glu Leu
225 230 235 240
Lys Glu Gln Thr Phe Ala Arg Tyr Asp Ile Met Thr Val Gly Glu Ala
245 250 255
Asn Gly Val Thr Val Asp Glu Ala Glu Gln Trp Val Gly Glu Glu Asn
260 265 270
Gly Val Phe Asn Met Ile Phe Gln Phe Glu His Leu Gly Leu Trp Glu
275 280 285
Arg Arg Ala Asp Gly Ser Ile Asp Val Arg Arg Leu Lys Arg Thr Leu
290 295 300
Thr Lys Glu Gln Lys Gly Leu Glu Asn Arg Gly Trp Asn Ala Leu Phe
305 310 315 320
Leu Glu Asn His Asp Leu Pro Arg Ser Val Ser Thr Trp Gly Asn Asp
325 330 335
Arg Asp Tyr Trp Ala Glu Ser Ala Lys Ala Leu Gly Ala Leu Tyr Phe
340 345 350
Phe Met Gln Gly Thr Pro Phe Ile Tyr Gln Gly Gln Glu Ile Gly Met
355 360 365
Thr Asn Val Arg Phe Asp Asp Ile Arg Asp Tyr Arg Asp Val Ser Ala
370 375 380
Leu Arg Leu Tyr Glu Leu Glu Arg Ala Lys Gly Arg Thr His Glu Glu
385 390 395 400
Ala Met Thr Ile Ile Trp Lys Thr Gly Arg Asp Asn Ser Arg Thr Pro
405 410 415
Met Gln Trp Ser Gly Ala Ser Asn Ala Gly Phe Thr Thr Gly Thr Pro
420 425 430
Trp Ile Lys Val Asn Glu Asn Tyr Arg Thr Ile Asn Val Glu Ala Glu
435 440 445
Arg Arg Asp Pro Asn Ser Val Trp Ser Phe Tyr Arg Gln Met Ile Gln
450 455 460
Leu Arg Lys Ala Asn Glu Leu Phe Val Tyr Gly Thr Tyr Asp Leu Leu
465 470 475 480
Leu Glu Asn His Pro Ser Ile Tyr Ala Tyr Thr Arg Thr Leu Gly Arg
485 490 495
Asp Arg Ala Leu Val Val Val Asn Leu Ser Asp Arg Pro Ser Leu Tyr
500 505 510
Arg Tyr Asp Gly Phe Arg Leu Gln Ser Ser Asp Leu Ala Leu Ser Asn
515 520 525
Tyr Pro Val Arg Pro His Lys Asn Ala Thr Arg Phe Lys Leu Lys Pro
530 535 540
Tyr Glu Ala Arg Val Tyr Ile Trp Lys Glu
545 550
<210> 6
<211> 1665
<212> DNA
<213> Isomaltooligosaccharides (IMOs)
<400> 6
aaaaaaacgt ggtggaaaga aggcgtggcg tatcagatct acccgcgtag ctttatggac 60
gccaatggcg acggcatcgg cgatctgcgt ggcatcatcg agaagctcga ctatctggtt 120
gaactgggcg tggacatcgt ttggatctgt ccgatctatc gcagtccgaa tgccgataac 180
ggctacgata tcagcgacta ctacgccatc atggacgagt tcggcaccat ggatgacttc 240
gatgagctgc tggcgcaagc ccatcgtcgt ggtctcaagg tgatcctcga tctggtgatc 300
aatcacacca gcgatgaaca cccgtggttt atcgaaagtc gcagcagtcg cgacaacccg 360
aagcgcgatt ggtacatttg gcgtgacggc aaagacggcc gcgaaccgaa caactgggaa 420
agcattttcg gtggcagcgc gtggcagtac gatgaacgca cgggccagta ctatctgcac 480
attttcgacg tgaagcagcc agatctgaac tgggagaata gcgaagtgcg ccaagcgctg 540
tacgaaatgg tgaactggtg gctcgacaag ggtatcgatg gcttccgcat cgacgccatc 600
agccatatcc tgaagaagcc gggtctgccg gatctgccaa atccgaaagg tctgaaatac 660
gtgccgagct ttgccggcca catgaatcag ccgggcatca tggaatatct gcgcgagctg 720
aaagagcaga ccttcgcccg ctatgatatc atgaccgtgg gcgaagcgaa cggtgtgacc 780
gttgacgaag ccgaacagtg ggtgggcgag gaaaacggtg tgttcaatat gatcttccaa 840
tttgagcatc tgggtctctg ggaacgtcgc gccgatggca gcatcgatgt tcgtcgtctg 900
aaacgcacgc tgaccaaggc gcagaaaggc ctcgaaaacc gcggttggaa tgcgctgttt 960
ctggaaaacc acgatctccc gcgcagcgtg agtacgtggg gcaatgaccg tgattactgg 1020
gccgaaagcg cgaaagcgct cggtgcgctc tatttcttca tgcaaggcac cccgtttatc 1080
taccaaggcc aagagatcgg catgaccaat gttcgctttg acgacatccg cgactaccgc 1140
gatgttagcg ccctccgcct ctacgaactg gaacgtgcca agggccgcac gcatgaagag 1200
gccatgacca tcatctggaa aacgggtcgc gacaacagtc gcacgccgat gcagtggagc 1260
ggtgccagta atgccggctt cacgaccggc accccatgga ttaaggttaa tgaaaactac 1320
cgcaccatca atgtggaagc ggaacgtcgc gacccgaaca gcgtgtggag cttttatcgc 1380
cagatgatcc agctccgtaa agcgaacgag ctgtttgttt acggcacgta cgatctgctg 1440
ctggagaacc atccgagcat ttacgcctat acccgtacgc tgggtcgtga tcgtgcgctg 1500
gtggttgtta atctcagcga ccgcccaagt ctgtaccgct atgacggctt ccgtctgcag 1560
agcagtgatc tggcgctgag taattaccca gtgcgcccgc acaagaatgc cacgcgcttc 1620
aaactcaagc cgtacgaggc ccgcgtgtac atctggaagg agtaa 1665
<210> 7
<211> 1665
<212> DNA
<213> Isomaltooligosaccharides (IMOs)
<400> 7
aaaaaaacgt ggtggaaaga aggcgtggcg tatcagatct acccgcgtag ctttatggac 60
gccaatggcg acggcatcgg cgatctgcgt ggcatcatcg agaagctcga ctatctggtt 120
gaactgggcg tggacatcgt ttggatctgt ccgatctatc gcagtccgaa tgccgataac 180
ggctacgata tcagcgacta ctacgccatc atggacgagt tcggcaccat ggatgacttc 240
gatgagctgc tggcgcaagc ccatcgtcgt ggtctcaagg tgatcctcga tctggtgatc 300
aatcacacca gcgatgaaca cccgtggttt atcgaaagtc gcagcagtcg cgacaacccg 360
aagcgcgatt ggtacatttg gcgtgacggc aaagacggcc gcgaaccgaa caactgggaa 420
agcattttcg gtggcagcgc gtggcagtac gatgaacgca cgggccagta ctatctgcac 480
attttcgacg tgaagcagcc agatctgaac tgggagaata gcgaagtgcg ccaagcgctg 540
tacgaaatgg tgaactggtg gctcgacaag ggtatcgatg gcttccgcat cgacgccatc 600
agccatatca gcaagaagcc gggtctgccg gatctgccaa atccgaaagg tctgaaatac 660
gtgccgagct ttgccggcca catgaatcag ccgggcatca tggaatatct gcgcgagctg 720
aaagagcaga ccttcgcccg ctatgatatc atgaccgtgg gcgaagcgaa cggtgtgacc 780
gttgacgaag ccgaacagtg ggtgggcgag gaaaacggtg tgttcaatat gatcttccaa 840
tttgagcatc tgggtctctg ggaacgtcgc gccgatggca gcatcgatgt tcgtcgtctg 900
aaacgcacgc tgaccaagga acagaaaggc ctcgaaaacc gcggttggaa tgcgctgttt 960
ctggaaaacc acgatctccc gcgcagcgtg agtacgtggg gcaatgaccg tgattactgg 1020
gccgaaagcg cgaaagcgct cggtgcgctc tatttcttca tgcaaggcac cccgtttatc 1080
taccaaggcc aagagatcgg catgaccaat gttcgctttg acgacatccg cgactaccgc 1140
gatgttagcg ccctccgcct ctacgaactg gaacgtgcca agggccgcac gcatgaagag 1200
gccatgacca tcatctggaa aacgggtcgc gacaacagtc gcacgccgat gcagtggagc 1260
ggtgccagta atgccggctt cacgaccggc accccatgga ttaaggttaa tgaaaactac 1320
cgcaccatca atgtggaagc ggaacgtcgc gacccgaaca gcgtgtggag cttttatcgc 1380
cagatgatcc agctccgtaa agcgaacgag ctgtttgttt acggcacgta cgatctgctg 1440
ctggagaacc atccgagcat ttacgcctat acccgtacgc tgggtcgtga tcgtgcgctg 1500
gtggttgtta atctcagcga ccgcccaagt ctgtaccgct atgacggctt ccgtctgcag 1560
agcagtgatc tggcgctgag taattaccca gtgcgcccgc acaagaatgc cacgcgcttc 1620
aaactcaagc cgtacgaggc ccgcgtgtac atctggaagg agtaa 1665
<210> 8
<211> 1665
<212> DNA
<213> Isomaltooligosaccharides (IMOs)
<400> 8
aaaaaaacgt ggtggaaaga aggcgtggcg tatcagatct acccgcgtag ctttatggac 60
gccaatggcg acggcatcgg cgatctgcgt ggcatcatcg agaagctcga ctatctggtt 120
gaactgggcg tggacatcgt ttggatctgt ccgatctatc gcagtccgaa tgccgataac 180
ggctacgata tcagcgacta ctacgccatc atggacgagt tcggcaccat ggatgacttc 240
gatgagctgc tggcgcaagc ccatcgtcgt ggtctcaagg tgatcctcga tctggtgatc 300
aatcacacca gcgatgaaca cccgtggttt atcgaaagtc gcagcagtcg cgacaacccg 360
aagcgcgatt ggtacatttg gcgtgacggc aaagacggcc gcgaaccgaa caactgggaa 420
agcattttcg gtggcagcgc gtggcagtac gatgaacgca cgggccagta ctatctgcac 480
attttcgacg tgaagcagcc agatctgaac tgggagaata gcgaagtgcg ccaagcgctg 540
tacgaaatgg tgaactggtg gctcgacaag ggtatcgatg gcttccgcat cgacgccatc 600
agccatatcc tgaagaagcc gggtctgccg gatctgccaa atccgaaagg tctgaaatac 660
gtgccgagct ttgccggcca catgaatcag ccgggcatca tggaatatct gcgcgagctg 720
aaagagcaga ccttcgcccg ctatgatatc atgaccgtgg gcgaagcgaa cggtgtgacc 780
gttgacgaag ccgaacagtg ggtgggcgag gaaaacggtg tgttcaatat gatcttccaa 840
tttgagcatc tgggtctctg ggaacgtcgc gccgatggca gcatcgatgt tcgtcgtctg 900
aaacgcacgc tgaccaagga acagaaaggc ctcgaaaacc gcggttggaa tgcgctgttt 960
ctggaaaacc acgatctccc gcgcagcgtg agtacgtggg gcaatgaccg tgattactgg 1020
gccgaaagcg cgaaagcgct cggtgcgctc tatttcttca tgcaaggcac cccgtttatc 1080
taccaaggcc aagagatcgg catgaccaat gttcgctttg acgacatccg cgactaccgc 1140
gatgttagcg ccctccgcct ctacgaactg gaacgtgcca agggccgcac gcatgaagag 1200
gccatgacca tcatctggaa aacgggtcgc gacaacagtc gcacgccgat gcagtggagc 1260
ggtgccagta atgccggctt cacgaccggc accccatgga ttaaggttaa tgaaaactac 1320
cgcaccatca atgtggaagc ggaacgtcgc gacccgaaca gcgtgtggag cttttatcgc 1380
cagatgatcc agctccgtaa agcgaacgag ctgtttgttt acggcacgta cgatctgctg 1440
ctggagaacc atccgagcat ttacgcctat acccgtacgc tgggtcgtga tcgtgcgctg 1500
gtggttgtta atctcagcga ccgcccaagt ctgtaccgct atgacggctt ccgtctgcag 1560
agcagtgatc tggcgctgag taattaccca gtgcgcccgc acaagaatgc cacgcgcttc 1620
aaactcaagc cgtacgaggc ccgcgtgtac atctggaagg agtaa 1665
Claims (8)
1. An alpha-glucosidase mutant is characterized in that the alpha-glucosidase mutant is obtained by performing site-directed mutagenesis on the 204 th amino acid and/or the 307 th amino acid of alpha-glucosyltransferase with the amino acid sequence shown as SEQ ID NO.1, wherein serine at the 204 th position is mutated into leucine, alanine at the 307 th position is mutated into glutamic acid, and the amino acid sequence of the alpha-glucosidase mutant is shown as SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO. 5; the nucleotide sequence for coding the alpha-transglucosidase is shown as SEQ ID NO. 2.
2. Use of the alpha-glucosidase mutant of claim 1 for the production of isomaltooligosaccharides.
3. The nucleotide sequence of the coding gene is shown as SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO. 8.
4. A recombinant expression vector carrying the gene of claim 3.
5. The recombinant expression vector of claim 4, wherein the recombinant expression vector uses the pET-28a (+) vector as an original expression vector.
6. A genetically engineered bacterium transformed with the recombinant expression vector of claim 3 or 4.
7. The genetically engineered bacterium of claim 6, wherein said genetically engineered bacterium is a host Escherichia coli.
8. The genetically engineered bacterium of claim 7, wherein said E.coli strain comprises BL21(DE 3).
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114015708A (en) * | 2021-11-16 | 2022-02-08 | 中南大学 | Deep sea bacterium-derived alpha-glucosidase QsGH13 and coding gene and application thereof |
| CN114752580A (en) * | 2022-05-25 | 2022-07-15 | 浙江工业大学 | Alpha-glycosidase mutant with improved transglycosylation activity, coding gene and application |
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| CN101392242A (en) * | 2007-09-18 | 2009-03-25 | 中国科学院微生物研究所 | Alpha-glucosidase, gene thereof, preparation method, vector and host cell |
| CN107267484A (en) * | 2017-07-18 | 2017-10-20 | 杭州师范大学 | A kind of α glycosidases, encoding gene, carrier, engineering bacteria and the application of grand gene source |
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| CN101392242A (en) * | 2007-09-18 | 2009-03-25 | 中国科学院微生物研究所 | Alpha-glucosidase, gene thereof, preparation method, vector and host cell |
| CN107267484A (en) * | 2017-07-18 | 2017-10-20 | 杭州师范大学 | A kind of α glycosidases, encoding gene, carrier, engineering bacteria and the application of grand gene source |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114015708A (en) * | 2021-11-16 | 2022-02-08 | 中南大学 | Deep sea bacterium-derived alpha-glucosidase QsGH13 and coding gene and application thereof |
| CN114015708B (en) * | 2021-11-16 | 2023-07-21 | 中南大学 | Deep sea bacteria-derived alpha-glucosidase QsGH13 and encoding gene and application thereof |
| CN114752580A (en) * | 2022-05-25 | 2022-07-15 | 浙江工业大学 | Alpha-glycosidase mutant with improved transglycosylation activity, coding gene and application |
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