CN111718921A

CN111718921A - Maltotriamylase mutant for producing maltotriose with high specificity

Info

Publication number: CN111718921A
Application number: CN202010507573.9A
Authority: CN
Inventors: 吴敬; 王蕾; 胡凡; 陈晟; 宿玲恰
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2020-06-05
Filing date: 2020-06-05
Publication date: 2020-09-29
Anticipated expiration: 2040-06-05
Also published as: JP2022539641A; WO2021243821A1; CN111718921B; JP7238158B2

Abstract

The invention discloses a maltotriose amylase mutant for producing maltotriose with high specificity, belonging to the field of genetic engineering and enzyme engineering. The invention modifies TfAmyA derived from Thermobifidafuusca NTU22, truncates the TfAmyA, and performs site-specific mutagenesis on leucine at position 47, glycine at position 101, glycine at position 103 and glycine at position 153 on the basis. The obtained mutant enzyme has the advantages of increased specificity of the main product maltotriose and increased yield, and is more suitable for the production of maltotriose.

Description

Maltotriamylase mutant for producing maltotriose with high specificity

Technical Field

The invention relates to a maltotriose amylase mutant for producing maltotriose with high specificity, belonging to the field of genetic engineering and enzyme engineering.

Background

Maltotriose (Maltotriose, α -D-Glc- (1 → 4))α -D-Glc- (1 → 4) -D-Glc, G3 for short, is a functional oligosaccharide, which is formed by connecting three glucose units through α - (1 → 4) glycosidic bond, and has molecular formula of C₁₈H₃₂O₁₆A relative molecular mass of 504.438 and a density of 1.8. + -. 0.1 g/cm^-3The melting point is 132-135 ℃, and the powder is white solid powder at normal temperature. G3 has a variety of excellent physicochemical and physiological properties, is a digestible, nutritious sweet product, has non-cariogenic, low sweetness, low viscosity, low calorie, anti-crystallization, and low osmotic pressure characteristics, and can be used to change the freezing temperature of frozen foods and control the browning amount caused by maillard reactions in thermally processed foods. G3 provides high moisture retention, prevents excessive drying and low water activity, facilitates control of microbial contamination, has strong stability, and is a strong inhibitor of starch retrogradation. G3 is widely used in food, beverage, chemical industry, medicine and other industries. The G3 product on the market is mostly from japan. "Fuji oligo G3" from Nippon food chemical Co., Ltd "," novel malto-oligosaccharide "from Nippon corn starch Co., Ltd", "Linear oligosaccharide origo" from Mitsubishi chemical Co., Ltd "," puretose "from Jurong chemical industry Co., Ltd" and the main component is G3.

Maltotriose amylase (EC 3.2.1.116, amylo forming alpha-amylase, AmyA for short) is encoded by gene tfa, and is an alpha-amylase having maltotriose-forming ability. AmyA takes starch or dextrin as a substrate, and hydrolyzes alpha- (1 → 4) glycosidic bond in sugar chain by endo-or exo-action to generate maltotriose and a small amount of byproducts. The discovery of the enzyme provides a new idea for preparing G3.

The preparation process of G3 is divided into chemical method and enzymatic method. The chemical method takes pullulan as a starting material and prepares G3 through two steps of acetyl hydrolysis and deacetylation. In the two-step reaction, high-risk reagents such as concentrated sulfuric acid, petroleum ether, methanol, sodium methoxide and the like are needed, and although the steps are short and the operation is simple, the substrate cost is high and the environmental hazard is large. The enzyme method is used as a green, energy-saving, efficient and safe alternative method, can obviously reduce the production cost, improve the product quality and meet the consumption requirements of the public, and is a prime power for promoting the sustainable development of the maltotriose industry. Enzymatic preparations are divided into two categories due to the different enzymes used. The enzymatic preparation is divided into two types because of the difference of the enzymes used. The first enzyme method is a method in which pullulan is hydrolyzed by pullulanase to prepare pharmaceutical-grade G3 (purity 99% or more) continuously and automatically, but this method has a problem of high substrate cost. Compared with pullulan, starch is an abundant natural renewable resource, is a main storage carbohydrate in seeds, roots or tubers of many important grain crops (such as rice, corn, wheat, rice, potatoes and cassava), and is more economical and practical. Maltodextrin is a liquefied product of starch gelatinization followed by the addition of alpha-amylase. Due to the action of the alpha-amylase, the maltodextrin has low polymerization degree and high solubility, and is beneficial to the action of other enzymes. The second method is to produce G3 using AmyA as the main enzyme and cheap raw materials such as starch or maltodextrin as the substrate. In general, pullulanase is also added to the reaction system to perform debranching. However, AmyA has the problem of more by-products of glucose and maltose, and the difficulty and cost of separation and purification are increased. Therefore, AmyA with high G3 specificity is obtained by means of molecular modification and the like, the yield of G3 can be improved, the production process is simplified, and the production cost is reduced.

Disclosure of Invention

In order to solve the problems existing at present, the present invention provides maltogenic glucoamylase mutants. The maltogenic glucoamylase TfAmyA from Thermobifidafuuca NTU22 used in the invention generates products of G1-G3 at the initial stage of enzyme conversion reaction, belongs to an endonuclease, and reduces the yield of substrates when the TfAmyA is used for producing G3, and simultaneously increases the difficulty of separation and purification. Therefore, the capability and specificity of the enzyme for producing G3 are further improved, and the reduction of the production cost of G3 and the large-scale production are facilitated.

The invention provides a maltotriose-producing maltotriose amylase mutant which is truncated at the carboxyl terminal relative to the parent TfAmyA or has mutation on the amino acid at any one of the 47 th, 101 th, 103 th and 153 th positions of the truncated mutant amino acid.

In one embodiment of the invention, the amino acid sequence of the parent TfAmyA is shown in SEQ ID No. 1.

In one embodiment of the present invention, the mutant is any one of the following (a) to (e):

(a) the 451-572 th amino acid of the parent is cut off, and the amino acid sequence is shown as SEQ ID NO. 4;

(b) mutating leucine at position 47 of amino acid shown as SEQ ID NO.4 into lysine or arginine, and respectively naming the mutants as L47K and L47R;

(c) glycine at position 101 of amino acid shown as SEQ ID NO.4 is mutated into serine, threonine, aspartic acid, asparagine, glutamic acid or glutamine, and the mutants are named as G101S, G101T, G101D, G101N, G101E and G101Q respectively;

(d) glycine at the 103 th site of the amino acid with the sequence shown as SEQ ID NO.4 is mutated into histidine, and the mutant is named as G103H;

(e) glycine at position 153 of amino acid shown in SEQ ID NO.4 is mutated into serine, aspartic acid, asparagine, glutamic acid, glutamine or histidine, and the mutants are named as G153S, G153D, G153N, G153E, G153Q and G153H respectively.

The present invention provides a gene encoding the mutant.

The invention provides a recombinant expression vector carrying the gene, wherein the expression vector is any one of pET series, Duet series, pGEX series, pHY300PLK, pPIC3K or pPIC9K series.

The invention provides microbial cells expressing the mutants, or carrying the genes.

In one embodiment of the invention, the microbial cell is a prokaryotic cell or a eukaryotic cell.

The invention provides a method for improving the production specificity of maltotriose, which takes dextrin as a substrate and the mutant as a catalyst to produce the maltotriose.

In one embodiment of the invention, the dextrin is prepared from rice, corn, wheat, rice, potato, sweet potato, kudzu root and/or cassava.

The invention also protects the application of the mutant, the gene, the recombinant expression vector, the microbial cell of claim 6 or 7, or the method for improving the production specificity of maltotriose in preparing maltotriose.

The invention has the beneficial effects that: the invention obtains various mutants by carrying out truncation mutation and site-directed mutation on TfAmyA, and the activity of maltotrioylase is enhanced; the proportion of the maltotriose in the product is improved, which is beneficial to simplifying the subsequent purification process of the maltotriose and realizing large-scale production, and the production cost of the maltotriose can be obviously reduced, thereby having wide industrial application prospect.

Drawings

FIG. 1 is a SDS-PAGE image before and after truncation; a is wild type, b is truncated mutant.

Detailed Description

The technical solution of the present invention is further described below with reference to the specific embodiments, but the scope of the present invention is not limited thereto.

The media formulations described in the examples are as follows:

LB medium (g/L): peptone 10, yeast extract 5, NaCl 10.

TB medium (g/L): peptone 10, yeast powder 24, glycerol 5, K₂HPO₄·3H₂O 16.43，KH₂PO₄2.31。

The pullulanase described in the examples is any debranching enzyme having a starch debranching effect.

The detection method of the content of G3 in the examples comprises the following steps:

the enzyme-converted sample was boiled in a water bath for 10min at 12000 r.min^-1Centrifuging for 10min, co-precipitating with acetonitrile with final concentration of 50% (volume ratio), standing for 2h, and standing at 12000 r.min^-1Centrifuge for 10min, collect the supernatant, and filter through 0.22. mu.M filter using syringe. The mobile phase was prepared using acetonitrile/water (74:26) ratio, and the samples were examined using Agilent 1200HPLC chromatograph and HYPERSILAPS2 amino column, with the column temperature set at 40 ℃ and the flow rate set at 0.8 mL/min^-1. According to absorption peak areaAnd calculating the peak area of the maltotriose standard sample to obtain the generation amount of the maltotriose.

Example 1: preparation of truncated enzyme

1) Construction of truncation and wild-type vectors

Firstly, according to an amino acid sequence and a nucleotide sequence (the nucleotide sequence is shown as SEQ ID NO. 2) of maltotrioylase from Thermobifidafuuca NTU22, a truncated gene fragment Tftfa-delta ML (the nucleotide sequence is shown as SEQ ID NO. 3) is connected with a vector pET20b (+) (a signal peptide for promoting gene expression is arranged on an expression vector) by utilizing a one-step cloning enzyme Exnase II (Nanjing Nozac Toxae Biotech Co., Ltd.), and a recombinant plasmid pET20b (+) -Tftfa-delta ML is constructed.

Secondly, the gene fragment with the nucleotide sequence shown as SEQ ID NO.2 is connected with a vector pET20b (+) by the same method, and the recombinant plasmid pET20b (+) -Ttfa is constructed.

2) Expression and purification of truncation and wild enzymes

① the recombinant plasmid pET20b (+) -Tftfa-Delta ML is transformed into host escherichia coli BL21(DE3) competent cells, cultured at 37 ℃ for 12-14h until monoclonals grow out, the monoclonals are selected for sequencing verification, the positive transformants verified to be correct are inoculated into LB liquid culture medium (containing 100 mug/mL ampicillin), cultured at 37 ℃ and 200rpm for 8-10 h, the seed fermentation liquid is inoculated into TB liquid culture medium (containing 100 mug/mL ampicillin and 7.5g/L glycine) according to the inoculation amount of 5 percent, and the escherichia coli is cultured by shaking at 37 ℃ until OD is achieved₆₀₀And (3) adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.134mM to induce extracellular expression, continuously culturing and fermenting for 48 hours at 25 ℃ by using a shaking table, centrifuging the fermentation liquor at 4 ℃ and 10000g for 15min, removing thalli, collecting supernatant, purifying, and obtaining the purified truncated enzyme.

Secondly, the recombinant plasmid pET20b (+) -Ttfa is transformed into host escherichia coli BL21(DE3) competent cells by the same method of the step (r), and the purified wild enzyme is obtained after culture and induction.

Example 2: preparation of mutant enzymes

1) Site-directed mutagenesis

The mutant was constructed by PCR using plasmid pET20b (+) -Tftfa- Δ ML as a template and a synthetic mutation primer.

TABLE 1 primers

PCR system 2 × Phanta Max Master Mix 25 uL, template 1 uL, upstream primer 2 uL, downstream primer 2u L, ddH2O 20 uL, PCR parameters 94 ℃ pre-denaturation 4min, PCR cycle 98 ℃ denaturation 10s, 55 ℃ annealing 30s, 72 ℃ extension (length of time is calculated according to the length of amplified fragment, 1000bp min)^-1) Circulating for 25-30 times; finally, the temperature is 72 ℃ for 10min, and the temperature is kept at 4 ℃. The PCR product was digested with Dpn I (Fermentas corporation), E.coli JM109 competent cells were transformed, the transformed competent cells were cultured overnight in LB solid medium (containing 100. mu.g/mL ampicillin), then cultured in LB liquid medium (containing 100. mu.g/mL ampicillin), plasmids were extracted, and sequencing was performed.

2) Expression and purification of mutant enzymes

Transforming plasmid with correct sequencing mutation into competent cells of expression host Escherichia coli BL21(DE3), selecting a monoclonal transformed into expression host Escherichia coli BL21(DE3), culturing in LB liquid culture medium (containing 100 mug/mL ampicillin) at 37 ℃ and 200rpm for 8-10 h, inoculating the seed fermentation liquid to TB liquid culture medium (containing 100 mug/mL ampicillin and 7.5 g.L) according to 5% inoculation amount^-1Glycine); escherichia coli was shake-cultured at 37 ℃ to OD₆₀₀And (3) adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.134mM to induce extracellular expression, continuously culturing and fermenting for 48 hours at 25 ℃ by using a shaking table, centrifuging the fermentation liquor at 4 ℃ and 10000g for 15min to remove thalli, collecting supernatant and purifying to respectively obtain mutant enzyme samples.

Example 3: enzyme activity assay for maltogenic glucoamylase

The enzyme activity is measured by a DNS method, and the system is as follows.

Preparing a substrate: suspending soluble starch with the mass concentration of 1% in a corresponding Buffer, and heating, stirring and pasting.

Reaction system: blank group: 1mL substrate (2g/100mL soluble starch) +1mL Buffer.

Control group: 1mL of substrate +0.9mL of Buffer +0.1mL of enzyme solution diluted by an appropriate amount.

The substrate and Buffer components were added in advance to a stoppered tube and incubated in a constant temperature water bath for 10 min. Diluting the enzyme to a proper ratio, adding a control group for reaction, and accurately timing for 10 min. After 10min, 3mL DNS is added, all test tubes with plugs are placed in a boiling water pot at the same time, the time is accurately timed for 7min, and the test tubes are taken out quickly and placed in an ice bath basin for cooling. After cooling to low temperature, 10mLH was added₂And O, mixing uniformly. Measuring absorbance Abs of the blank with 540nm spectrophotometer₅₄₀Substituting the standard curve to calculate the enzyme activity.

Standard curve: abs was measured with different concentrations of glucose according to the above system₅₄₀And the linear regression equation is a standard curve according to the relation between the concentration of the reducing sugar and the concentration of the reducing sugar.

Definition of enzyme activity: the amount of enzyme required to release 1. mu. mol of reducing sugar per minute was defined as one enzyme activity unit (U).

After the shaking table is continuously cultured and fermented for 48 hours at 25 ℃, the supernatant is collected, and the shake flask enzyme activity results of the wild enzyme and the mutant enzyme are shown in table 1. The activity of the truncated TfAmyA-delta ML enzyme is not changed greatly, and except that the mutant enzyme activity of L47 and G103 is improved, most of mutant enzyme activity of G101 site and G153 site is reduced or slightly reduced.

TABLE 2 comparison of Shake flask enzyme activities of mutant and wild type

Example 4: analysis of Maltotriose formation by HPLC method

Preparing 5% maltodextrin solution (pH 5.5) with DE value of 5-7, adding certain amount of wild enzyme and mutant enzyme of maltogenic glucoamylase respectively, wherein the enzyme addition amount is 60 U.g^-1Substrate, pullulanase in an amount of 32 U.g^-1Adding the substrate into the reaction system, placing inThe reaction is carried out for 11 hours in a water bath shaking table with the rotating speed of 150rpm and the temperature of 55 ℃. Sampling, boiling in a boiling water bath for 10min to inactivate enzyme, centrifuging and taking supernatant to obtain product solution. mu.L of the product solution was mixed with acetonitrile 1:1, left at room temperature for 2 hours to precipitate high molecular weight dextrins or limit dextrins, and then centrifuged at 12000rpm for 20 minutes, and the supernatant was passed through a 0.22. mu.M filter using a syringe to obtain a sample capable of HPLC analysis. The mobile phase was prepared using acetonitrile/water (74:26) ratio, and the samples were examined using Agilent 1200HPLC chromatograph and HYPERSIL APS2 amino column, with the column temperature set at 40 ℃ and the flow rate set at 0.8 mL/min^-1. The amount of G3 produced was calculated from the area of the absorption peak and the area of the peak of the G3 standard sample.

The results of the enzymatic conversion analysis of the wild-type enzyme and the mutant enzyme are shown in Table 2. The truncation TfAmyA-. DELTA.ML increased the yield of G3 with a small increase in the ratio of G3. In addition to the mutations G103V and G103P of G103, the other mutations L47, G101, G103 and G153 gave a marked increase in the G3 ratio of the enzymatic conversion products. The single mutant improves the specificity of G3, reduces other sugar in the product and increases the main product G3. The ratio refers to the mass concentration of G3 in the product to the mass concentration of the total product. Yield refers to the ratio of the mass concentration of G3 of the product to the mass concentration of the substrate.

TABLE 3 comparison of G3 yields and ratios between mutant and wild type

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

SEQUENCE LISTING

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Val Val Phe Val Val Asn His Asp Thr Gln Arg Tyr Glu Pro Ile Leu

275 280 285

Thr His Thr Asp Gly Ala Arg Tyr Asp Leu Ala Gln Lys Phe Met Leu

290 295 300

Ala His Pro Tyr Gly Thr Pro Lys Val Met Ser Ser Tyr Thr Trp Ser

305 310 315 320

Gly Asp Asp Lys Ala Gly Pro Pro Met His Ser Asp Gly Thr Thr Arg

325 330 335

Pro Thr Asp Cys Ser Ala Asp Arg Trp Leu Cys Glu His Arg Ala Val

340 345 350

Ala Gly Met Val Gly Phe His Asn Ala Val Ala Gly Gln Gly Ile Gly

355 360 365

Ser Ala Val Thr AspGly Asn Gly Arg Leu Ala Phe Ala Arg Gly Ser

370 375 380

Ala Gly Tyr Ala Ala Phe Asn Ala Thr Asn Thr Ala Trp Thr Arg Thr

385 390 395 400

Phe Thr Thr Ser Leu Pro Asp Gly Val Tyr Cys Asp Val Ala Asn Gly

405 410 415

Thr Phe Val Asp Gly Val Cys Asp Gly Pro Ser Tyr Gln Val Ser Gly

420 425 430

Gly Lys Phe Thr Ala Thr Val Pro Ala Asn Gly Ala Val Ala Leu His

435 440 445

Val Glu

450

<210>5

<211>36

<212>DNA

<213> Artificial sequence

<400>5

aacatgttgt gaaaccggcc gaagattatc cgtggt 36

<210>6

<211>33

<212>DNA

<213> Artificial sequence

<400>6

tcttcggccg gtttcacaac atgttcctgc ggc 33

<210>7

<211>34

<212>DNA

<213> Artificial sequence

<400>7

aacatgttgt gcgtccggcc gaagattatc cgtg 34

<210>8

<211>33

<212>DNA

<213> Artificial sequence

<400>8

tcttcggccg gacgcacaac atgttcctgc ggc 33

<210>9

<211>29

<212>DNA

<213> Artificial sequence

<400>9

tcacatgacc agcaccggct cagcaggcg 29

<210>10

<211>33

<212>DNA

<213> Artificial sequence

<400>10

tgctgagccg gtgctggtca tgtgattaat aac 33

<210>11

<211>30

<212>DNA

<213> Artificial sequence

<400>11

tcacatgacc accaccggct cagcaggcgc 30

<210>12

<211>33

<212>DNA

<213> Artificial sequence

<400>12

tgctgagccg gtggtggtca tgtgattaat aac 33

<210>13

<211>29

<212>DNA

<213> Artificial sequence

<400>13

tcacatgacc gataccggct cagcaggcg 29

<210>14

<211>36

<212>DNA

<213> Artificial sequence

<400>14

tgctgagccg gtatcggtca tgtgattaat aacggc 36

<210>15

<211>32

<212>DNA

<213> Artificial sequence

<400>15

tcacatgacc aataccggct cagcaggcgc ag 32

<210>16

<211>37

<212>DNA

<213> Artificial sequence

<400>16

ctgctgagcc ggtattggtc atgtgattaa taacggc 37

<210>17

<211>26

<212>DNA

<213> Artificial sequence

<400>17

tcacatgacc gaaaccggct cagcag 26

<210>18

<211>33

<212>DNA

<213> Artificial sequence

<400>18

tgctgagccg gtttcggtca tgtgattaat aac 33

<210>19

<211>32

<212>DNA

<213> Artificial sequence

<400>19

tcacatgacc cagaccggct cagcaggcgc ag 32

<210>20

<211>36

<212>DNA

<213> Artificial sequence

<400>20

tgctgagccg gtctgggtca tgtgattaat aacggc 36

<210>21

<211>27

<212>DNA

<213> Artificial sequence

<400>21

accggaaccc cttcagcagg cgcaggt 27

<210>22

<211>34

<212>DNA

<213> Artificial sequence

<400>22

gcgcctgctg aaggggttcc ggtcatgtga ttaa 34

<210>23

<211>25

<212>DNA

<213> Artificial sequence

<400>23

accggaaccg tttcagcagg cgcag 25

<210>24

<211>24

<212>DNA

<213> Artificial sequence

<400>24

gcgcctgctg aaacggttcc ggtc 24

<210>25

<211>26

<212>DNA

<213> Artificial sequence

<400>25

gaccggaacc cactcagcag gcgcag 26

<210>26

<211>30

<212>DNA

<213> Artificial sequence

<400>26

cgcctgctga gtgggttccg gtcatgtgat 30

<210>27

<211>41

<212>DNA

<213> Artificial sequence

<400>27

gtgaattagt ggaattagca gacctgaaaa cctctagtcc g 41

<210>28

<211>39

<212>DNA

<213> Artificial sequence

<400>28

ggtctgctaa ttccactaat tcacaatgct gaacttccc 39

<210>29

<211>41

<212>DNA

<213> Artificial sequence

<400>29

gtgaattagt gcagttagca gacctgaaaa cctctagtcc g 41

<210>30

<211>41

<212>DNA

<213> Artificial sequence

<400>30

caggtctgct aactgcacta attcacaatg ctgaacttcc c 41

<210>31

<211>40

<212>DNA

<213> Artificial sequence

<400>31

tgaattagtg gacttagcag acctgaaaac ctctagtccg 40

<210>32

<211>35

<212>DNA

<213> Artificial sequence

<400>32

ggtctgctaa gtccactaat tcacaatgct gaact 35

<210>33

<211>40

<212>DNA

<213> Artificial sequence

<400>33

tgaattagtg aatttagcag acctgaaaac ctctagtccg 40

<210>34

<211>35

<212>DNA

<213> Artificial sequence

<400>34

ggtctgctaa attcactaat tcacaatgct gaact 35

<210>35

<211>40

<212>DNA

<213> Artificial sequence

<400>35

tgaattagtg agtttagcag acctgaaaac ctctagtccg 40

<210>36

<211>34

<212>DNA

<213> Artificial sequence

<400>36

ggtctgctaa actcactaat tcacaatgct gaac 34

<210>37

<211>41

<212>DNA

<213> Artificial sequence

<400>37

gtgaattagt gcatttagca gacctgaaaa cctctagtcc g 41

<210>38

<211>40

<212>DNA

<213> Artificial sequence

<400>38

aggtctgcta aatgcactaa ttcacaatgc tgaacttccc 40

Claims

1. The mutant of maltogenic glucoamylase is characterized in that the mutant takes the maltogenic glucoamylase with the amino acid sequence shown as SEQ ID NO.1 as a parent, and the end of the mutant is truncated, or the amino acid at any position of 47 th position, 101 th position, 103 th position and 153 th position of the parent is mutated.

2. The mutant according to claim 1, wherein the mutant is any one of the following (a) to (e):

(a) truncating the 451-572 th amino acid of the parent;

(b) mutating leucine with the sequence shown as SEQ ID NO.4 at 47 th position into lysine or arginine;

(c) glycine at position 101 of amino acid with a sequence shown as SEQ ID NO.4 is mutated into serine, threonine, aspartic acid, asparagine, glutamic acid or glutamine;

(d) mutating glycine at 103 th position of amino acid shown as SEQ ID NO.4 into histidine;

(e) glycine at position 153 of amino acid shown in SEQ ID NO.4 is mutated into serine, aspartic acid, asparagine, glutamic acid, glutamine or histidine.

3. A gene encoding the mutant of claim 1 or 2.

4. A recombinant expression vector carrying the gene of claim 3.

5. The recombinant expression vector according to claim 4, wherein the expression vector is any one of pET series, Duet series, pGEX series, pHY300PLK, pPIC3K or pPIC9K series.

6. A microbial cell expressing the mutant of claim 1 or 2 or carrying the gene of claim 3.

7. The microbial cell of claim 6, wherein the microbial cell is a prokaryotic cell or a eukaryotic cell.

8. A method for improving the production specificity of maltotriose, which is characterized in that maltotriose is produced by using dextrin as a substrate and the mutant as described in any one of claims 1 to 2 as a catalyst.

9. The method of claim 8, wherein the dextrin is derived from rice, corn, wheat, rice, potato, sweet potato, kudzu and/or tapioca.

10. Use of the mutant according to claim 1 or 2, or the gene according to claim 3, or the recombinant expression vector according to claim 4 or 5, or the microbial cell according to claim 6 or 7, or the method according to claim 8 or 9 for preparing maltotriose.