LU503704B1

LU503704B1 - Type iii pullulan hydrolase mutant for preparing corn resistant starch, preparation method and application thereof

Info

Publication number: LU503704B1
Application number: LU503704A
Authority: LU
Inventors: Jing Zeng
Original assignee: Inst Of Microbiology Jiangxi Academy Of Sciences Jiangxi Inst Of Watershed Ecology
Priority date: 2023-03-21
Filing date: 2023-03-21
Publication date: 2023-09-21

Abstract

Disclosed is a type III pullulan hydrolase mutant for preparing corn-resistant starch, and relates to the technical field of biological enzyme engineering and genetic engineering. An amino acid sequence of the type III pullulan hydrolase mutant is shown in SEQ ID NO.1. The invention carries out saturation mutation on isoleucine at the 500th position and leucine at the 538th position adjacent to the catalytic active center in the type III pullulan hydrolase TK-PUL, and constructs the type III pullulan hydrolase mutant with the amino acid sequence as shown in SEQ ID NO.1. The specific activity of the mutant type III pullulan hydrolase provided by the invention to common corn starch increases from 48.08 U/mg before the mutation to 133.66 U/mg, and the yield of corn resistant starch prepared by the mutant type III pullulan hydrolase system increases from 30.87 percent before the mutation to 59.15 percent.

Description

DESCRIPTION 509704

TYPE II PULLULAN HYDROLASE MUTANT FOR PREPARING

CORN RESISTANT STARCH, PREPARATION METHOD AND

APPLICATION THEREOF

TECHNICAL FIELD

The invention relates to the technical field of biological enzyme engineering and genetic engineering, and in particular to a type III pullulan hydrolase mutant for preparing corn resistant starch, a preparation method and an application thereof.

BACKGROUND

Resistant starch (RS), as a new dietary fiber, may not be digested and absorbed by human stomach and small intestine, but may be fermented and utilized by intestinal flora in colon, thus exerting its physiological functions of lowering blood sugar, lowering blood fat, preventing cardiovascular diseases and colon cancer.

According to different sources, structures, digestion characteristics and applications,

RS is divided into five categories: RS1 resistant starch (physically embedded starch),

RS2 resistant starch (resistant starch granules), RS3 resistant starch (aged retrogradation starch), RS4 resistant starch (chemically modified starch) and RSS resistant starch (starch-lipid complex). RS3 resistant starch is the main component of resistant starch in diet, and has important physiological functions for human body. As a new functional food raw material, the preparation method of RS3 resistant starch has become a hot topic in carbohydrate science in recent years.

In recent years, researchers have adopted different preparation methods for different starch raw materials to obtain high-content RS3 resistant starch products.

Different treatment methods and condition parameters have different effects on the yield of RS3 resistant starch. In the preparation of RS3 resistant starch, an important factor affecting the yield of resistant starch is the amylose content in starch. Because amylose molecules in starch are more prone to aging than amylopectin molecules, it is easy to form orderly arrangement between molecules to form resistant starch; the greater the ratio of the two, the higher the content of resistant starch. From this point 509704 of view, high amylose corn starch has obvious raw material advantages in preparing

RS3 resistant starch. Because there is no large-scale planting production of high amylose corn starch in China, the price of the amylose corn starch is high. The RS3 resistant starch needs to be obtained after repeated high-temperature treatment in the process of preparing RS3 resistant starch from high amylose corn starch. This raw material and processing conditions increase the cost of the product, so it is urgent to develop a method for preparing high-content RS3 resistant starch from ordinary corn starch (low amylose corn starch).

RS3 resistant starch is usually prepared by heat treatment and debranching, or a combination of the two. During the gelatinization process of starch by heating, starch particles swell and break when absorbing water, releasing amylose, and then cool and coagulate; long-chain polymers form resistant starch through double helix superposition. In the preparation of RS3 resistant starch, there are two existing debranching methods, namely acid debranching and enzymatic debranching. Acid debranching is to treat starch with acid, but its debranching effect and safety are not as good as those of enzymatic debranching, and the high corrosiveness of acid to equipment is a technical problem that must be considered in actual production.

Enzymatic debranching preparation of resistant starch greatly reduces the amount of chemical reagents used in the preparation process, improves the quality of resistant starch and reduces environmental pollution, but it has the disadvantages of long enzymolysis time and low yield of resistant starch preparation. At present, it has been reported that the highest yield of corn resistant starch is 58.87% by combining heat treatment with enzymatic debranching. Zhang et al. used 10% common corn starch milk as raw material, treated corn starch milk at 80°C for 20 min, then added 4.0 U/g of a-amylase at 90°C and pH value of 5.5, hydrolyzed for 15 min, and then continuously treated with pullulanase (an enzyme dosage is 12.8 U/g, a reaction time is 32 h, a reaction temperature is 46.2°C, and pH value is 5.0) to prepare the RS3 resistant starch product with resistant starch content of 58.87%. That is, when corn starch is gelatinized, thermophilic a-amylase is added, and a-amylase acts on a-1,4-glycosidic bond in starch to change the chain length and structure of starch; 7503704 then pullulanase is added, and it acts on the a-1,6-glycosidic bond to make the starch hydrolysate contain more free amylose molecules; the reaction system coagulates at low temperature, different amylose molecules form a double helix through hydrogen bonds, and finally RS3 resistant starch is obtained. Thermophilic acidic pullulan hydrolase TK-PUL hydrolyzes a-1,4-glycosidic bond and a-1,6-glycosidic bond in starch at the same time. The application of TK-PUL in the preparation of RS3 resistant starch gives full play to the functions of thermophilic o-amylase and pullulanase. By selecting and controlling the reaction conditions, the preparation process of RS3 resistant starch is simplified, the enzymolysis time is shortened and the preparation efficiency is improved. Therefore, it is of great significance to develop the type III pullulan hydrolase TK-PUL mutant with high enzymolysis efficiency and high yield of resistant starch for improving the preparation process of resistant starch.

SUMMARY

The invention aims to provide a type III pullulan hydrolase mutant for preparing corn resistant starch, a preparation method and an application thereof, so as to solve the problems existing in the prior art. The type III pullulanase mutant prepared by the invention has high enzyme activity, and the preparation yield is effectively improved by using the mutant to prepare corn resistant starch.

In order to achieve the above objectives, the present invention provides the following schemes.

The invention provides a type III pullulan hydrolase mutant for preparing corn resistant starch, and an amino acid sequence of the type III pullulan hydrolase mutant is shown in SEQ ID NO.1.

The invention also provides a coding gene for coding the type III pullulan hydrolase mutant.

Further, a nucleotide sequence of the coding gene is shown in SEQ ID NO 2.

The invention also provides a recombinant vector, including the coding gene.

The invention also provides a recombinant microbial strain, including the recombinant vector. 509704

The invention also provides an application of the coding gene, the recombinant vector or the recombinant microbial strain in preparing the type III pullulan hydrolase mutant.

The invention also provides an application of the type III pullulan hydrolase mutant in preparing corn resistant starch.

The invention discloses the following technical effects.

In order to obtain a type III pullulan hydrolase mutant for efficiently preparing corn resistant starch, the invention carries out saturation mutation on the 500% isoleucine (I) and the 538" leucine (L) those are adjacent to the catalytic active center in the type III pullulan hydrolase TK-PUL, and constructs a corresponding saturated mutant library. By comparing the specific activity of recombinant enzyme with common corn starch as substrate and the yield of corn resistant starch with common corn starch as raw material, the mutant TP-ISOOE/L538D of type III pullulan hydrolase with significantly improved yield of corn resistant starch 1s screened out.

The specific activity of the type III pullulan hydrolase mutant TP-ISOOE/L538D provided by the invention to common corn starch is increased from 48.08 U/mg before the mutation to 133.66 U/mg, and this is increased by 1.78 times. The technological conditions for preparing corn resistant starch by using type III pullulan hydrolase TK-PUL and mutant TP-ISOOE/L538D are as follows: a concentration of common corn starch milk is 15%, a dosage of type III pullulan hydrolase is 15 U/g of corn starch, a reaction time is 12 h, a reaction temperature is 80°C and a pH value is 5.0. Under these conditions, the yield of corn resistant starch of type III pullulan hydrolase mutant TP-ISOOE/L538D is increased from 30.87% of the control (before the mutation) to 59.15%, an increase of 0.92 times.

BRIEF DESCRIPTION OF THE FIGURES

In order to explain the embodiment of the invention or the technical scheme in the prior art more clearly, the drawings used in the embodiment will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the invention. For ordinary technicians in the field, other drawings 509704 may be obtained according to these drawings without making creative efforts.

FIG. 1 is an SDS-PAGE detection chart of the type III pullulan hydrolase

TK-PUL and mutants TP-I500E/L538D, TP-I500Q/L538K and TP-IS00E/L538H.

DESCRIPTION OF THE INVENTION

Various exemplary embodiments of the present invention will now be described in detail, which should not be regarded as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics and embodiments of the present invention.

It should be understood that the terms described in the present invention are only for describing specific embodiments, and are not intended to limit the present invention. In addition, as for the numerical range in the present invention, it should be understood that every intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Intermediate values within any stated value or stated range and every smaller range between any other stated value or intermediate values within the stated range are also included in the present invention.

The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

Unless otherwise stated, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the present invention relates. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe methods and/or materials related to the documents. In case of conflict with any incorporated documents, the contents of this specification shall prevail.

Without departing from the scope or spirit of the invention, it is obvious to those skilled in the art that many modifications and changes may be made to the specific embodiments of the specification of the invention. Other embodiments derived from the description of the present invention will be apparent to the skilled person. The 509704 specification and examples of this application are only exemplary.

As used herein, the terms “including”, “comprising”, “having”, “containing”, etc. are all open terms, and they mean including but not limited to.

The experimental materials used in the following embodiments are as follows: 1. Strains and vectors

Escherichia coli JM109 (purchased from Huayueyang Biotechnology (Beijing)

Co., Ltd.), Bacillus subtilis WB600 (purchased from BeNa Culture Collection), and

Bacillus subtilis expression vector pSTOP1622 (purchased from MoBi Tec Company). 2. Enzymes and other biochemical reagents

Gene site-directed mutation kits are purchased from Shanghai Beyotime

Biotechnology Co., Ltd., KOD-Plus-neo DNA polymerase is purchased from Toyobo

Company, DNA restriction endonuclease and T4 DNA ligase are purchased from

Fermentase Company, DNA gel recovery kits and plasmid extraction kits E.Z N A. are purchased from Omega Bio-tek Company, Chelating SepharoseTM Fast Flow is purchased from GE Healthcare Company of the United States, ordinary corn starch, corn amylose and corn amylopectin are purchased from J&K Scientific and

Technology Co., Ltd, o-amylase and glucoamylase are purchased from Sigma

Company of the United States, and other chemical reagents are domestic or imported analytical pure. 3. Culture medium

LB culture medium (g/L): tryptone 10, yeast extract 5, NaCl 10, and pH 7.0. A screening culture medium is LB culture medium containing 50 pg/mL ampicillin.

Molecular cloning technology and protein detection technology used in the present invention are both conventional technologies in this field. Techniques not described in detail in the following examples are all carried out according to the relevant parts in the following experimental manual: Green M R, Sambrook J.

Molecular Cloning: a laboratory manual [M]. New York: Cold Spring Harbor

Laboratory Press, 2012.

Embodiment 1 Construction of saturated mutant library

(1) Synthesis of gene fk-pul 509704

According to the Gene ID (No. 3235344) of #k-pul in NCBI database, the gene sequence of tk-pul is obtained by searching, as shown in SEQ ID NO 4, and it is submitted to Shanghai Boyi Biotechnology Co., Ltd. for complete gene synthesis of tk-pul. (2) Construction of recombinant expression vector pSTOP1622- #k-pul h

According to the base sequence of gene fk-pul, PCR primers F1 and RI are designed (Table 1), and the synthetic gene #k-pu/ is used as a template and F1 and R1 are used as primers for PCR amplification. The PCR amplification conditions are: 95°C for 10 min; 98°C for 30 sec, 60°C for 30 sec, 74°C for 1 min, and 30 cycles; 74°C for 5 min. The amplified products are digested by Spe I and BamH I, and connected to the vector pSTOP1622 to construct the recombinant vector pSTOP1622- tk-pul h.

ne ie es

F1 AGTACTAGTATGAAAAAAGGTGGTCTGCTGCTCATTCTC SEQ ID NO.5

R1 TGGGATCCACCCCGCTCAAGGATGATTATC SEQ ID NO.6 1500F CTTTGACGGCnnkAGGGTGGATGTG SEQ ID NO.7

I500R AAGCCGAACTCTATCCAGTG SEQ ID NO.8

L538F GATATGGACGnnkTCCCCGGAGTG SEQ ID NO.9

L538R ATCTCGCCGACGACGAGGTAC SEQ ID NO.10

Note: the underlined part is the restriction enzyme cleavage site, and the lowercase letter “nnk” is the introduced degenerate codon.

Table 1 Primers used to construct recombinant plasmid (3) Construction of saturated mutant library

Similarity analysis of amino acid sequence of TK-PUL is conducted by using online software BLASTP of NCBI database, and starch hydrolase with high similarity to its amino acid sequence is selected. The amino acid sequences of TK-PUL and these starch hydrolases are compared by Clustal-Omega software. According to the results of multi-sequence alignment, amino acid residues 1500 and L538 adjacent to the catalytic activity center of TK-PUL are selected as the sites of saturation mutation.

Firstly, the recombinant plasmid pSTOP1622- #k-pulh is used as a template, 1500F and ISOOR in Table 1 are used as primers, and the 1500 site is saturated and mutated by gene site-directed mutation kits. The PCR amplification conditions are: 94°C for 5 min; 94°C for 30 sec, 55°C for 20 sec, 68°C for 4 min, and 35 cycles; 68°C for min. The amplified products are transformed into competent cells of £. coli IM109 by electric shock after treated with Dpn I enzyme, coated with LB plates containing 100 pg/mL ampicillin, and cultured overnight at 37°C. A single transformant is picked on LB solid plate, the recombinant plasmid contained in it is extracted, and it is sent to Sangon Biotech Engineering (Shanghai) Co., Ltd. for DNA sequencing. The measured base sequence is compared with the base sequence of gene #k-pul, and the 509704 comparison results show that the recombinant plasmid with successful mutation of the corresponding base sequence is the recombinant vector pSTOP1622-tk-pul hI500.

Then, the recombinant plasmid pSTOP1622-tk-pul hI500 is used as a template,

L538F and L538R in Table 1 are used as primers, and the L538 site is saturated and mutated by gene site-directed mutation kits. The PCR amplification conditions are: 94°C for 5 min; 94°C for 30 sec, 55°C for 20 sec, 68°C for 4 min, and 35 cycles; 68°C for min. The amplified products are transformed into competent cells of £. coli IM109 by electric shock after treated with Dpn I enzyme, coated with LB plates containing 100 pg/mL ampicillin, and cultured overnight at 37°C. A single transformant is picked on LB solid plate, the recombinant plasmid contained in it is extracted, and it is sent to Sangon Biotech Engineering (Shanghai) Co., Ltd. for DNA sequencing. The measured base sequence is compared with the base sequence of gene #k-pul, and the comparison results show that the recombinant plasmid with successful mutation of the corresponding base sequence is the recombinant vector pSTOP1622-tk-pul hI500L538.

The obtained recombinant plasmid pSTOP1622-tk-pulhI500L538 is transformed into B. subtilis WB600, and the transformant is coated on an LB solid plate containing 100 pg/mL kanamycin, and cultured overnight at 37°C to obtain a saturated mutant library.

Embodiment 2 Primary screening of saturated mutant library (1) High throughput culture of saturated mutant library

Taking sterilized and dried 96-well plates, and adding 500 ul LB culture medium to each well; picking up the single colony on the LB solid with a sterile toothpick, transferring it to a culture well, and repeating in turn until the selection is over; covering and culturing overnight at 37°C in a shaker; transferring the activated seed solution to a new 96-well plate containing 500 uL of LB culture medium with 1% inoculation amount in sequence by a pipette, culturing at 37°C for 5 h, then adding the xylose solution to each well until its final concentration is 0.5%(m/v), and then culturing at 37°C for 20 h. 509704 (2) High throughput screening of saturated mutant library

Centrifuging the cultured 96-well plate at 4000xg for 10 min at 4°C, and using the supernatant after centrifugation for enzyme activity detection; taking the sterilized and dried 96-well plate, adding 100 uL of fermentation broth supernatant to each well, then adding 100 uL of 50 mmol/L MES containing 1% (m/v) corn starch and buffer solution with pH of 4.5, reacting at 80°C for 20 min, adding 300 pL of 3,5-dinitrosalicylic acid to it, reacting in boiling water bath for 10 min and then transferring to ice water bath for rapid cooling; taking clean enzyme label plate, adding 150 uL of sterile water and 50 uL of reaction solution and mixing well, and measuring the absorbance value at 540 nm with an enzyme marker. The light absorption value of the reaction solution reflects the enzyme activity of the mutants, and three mutants with higher light absorption value of the reaction solution are selected from the saturated mutant library.

The recombinant Bacillus subtilis corresponding to the mutant with improved absorbance of the reaction solution is inoculated into 20 mL LB liquid culture medium containing 100 pg/mL ampicillin, and is rapidly shaken at 37°C overnight.

After the culture is finished, the thalli are collected and the recombinant plasmid is extracted. The recombinant plasmid is sequenced and compared with the gene sequence of fk-pul, and the amino acid mutation of the mutant is deduced by triplet codon. The amino acid mutation in the mutant and the absorbance of the corresponding reaction solution are shown in Table 2. TK-PUL has an absorption value of 0.460 at 540 nm; the mutant TP-IS00F/L538D has an absorption value of 0.865 at 540 nm; the mutant TP-I500Q/LS38K has an absorption value of 0.648 at 540 nm; and the mutant TP-ISOOE/L538H has an absorption value of 0.510 at 540 nm.

Recombinant enzyme A540nm

TK-PUL 0.460+0.021

TP-1500F/L538D 0.865+0.051

TP-1500Q/L538K 0.648+0.045

TP-1500FE/L538H 0.510+0.046

Note: TP-I500E/L538D: in TK-PUL, the 500" isoleucine (I) is mutated into glutamic acid (E), and the 538" leucine (L) is mutated into aspartic acid (D);

TP-I500Q/L538K: in TK-PUL, the 500% isoleucine (I) is mutated into glutamine (Q), and the 538% leucine (L) is mutated into lysine (K); TP-I500E/L538H: in TK-PUL, the 500% isoleucine (I) is mutated into glutamic acid (E), and the 538% leucine (L) is mutated into histidine (H).

Table 2 Absorption values of mutants and corresponding reaction solutions

Embodiment 3 Re-screening of mutants (1) Induced expression and purification of recombinant enzyme

Inoculating B. subtilis WB600 strains containing recombinant plasmid into 20 mL LB liquid culture medium containing 100 pg/mL ampicillin, and culturing overnight with rapid shaking at 37°C; transferring the overnight culture to 50 mL LB liquid culture medium containing 100 pg/mL ampicillin with 1% inoculation amount, and rapidly shaking at 37°C until the ODé6oonn of the bacterial liquid reaches about 0.8; adding xylose with a final concentration of 0.5%(m/v), and then culturing at 37°C for h, and then centrifuging at 12000 r/min for 10 min to collect fermentation supernatant.

The target protein in the fermentation supernatant is purified by Ni** affinity chromatography column, and eluted with 250 mmol/L imidazole elution buffer to obtain the purified recombinant enzyme. The purity of recombinant enzyme is detected by SDS-PAGE, and the concentration of recombinant enzyme is determined by Bradford method. The SDS-PAGE detection chart of type III pullulan hydrolase

TK-PUL and mutants TP-ISOOE/L538D, TP-IS00Q/L538K and TP-ISOOE/LS38H is shown in FIG. 1. (2) Determination of specific activity of recombinant enzyme

Determination of specific activity of recombinant enzyme: the specific activity of recombinant enzyme is determined with corn starch, corn amylose or corn 7503704 amylopectin as substrates; adding 10 uL of enzyme solution and 490 uL of 50 mmol/L

MES containing 1% (m/v) substrate, mixing with pH 5.0 buffer solution, reacting at 80°C for 20 min, quickly putting into ice water bath to terminate the reaction, and then using 3,5-dinitrosalicylic acid method to determine the amount of reducing sugar in the reaction system. The reduced sugar generated is converted into maltose mass by the standard working curve of maltose. Definition of enzyme activity unit (U): under certain reaction conditions, the amount of enzyme that catalyzes the production of 1 umol maltose per minute is one activity unit (U).

The results of specific activity determination of recombinant enzyme are shown in Table 3. With corn starch as substrate, the specific activity of TK-PUL is 48.08

U/mg; the specific activity of the mutant TP-ISOOE/L538D is 133.66 U/mg, increased by 1.78 times; the specific activity of the mutant TP-ISOOE/L538D is 68.66 U/mg, increased by 0.43 times; and the specific activity of the mutant TP-ISOOE/L538D is 55.20 U/mg, increased by 0.15 times. Among the four recombinant enzymes, the specific activity of mutant TP-ISOOE/L538D is the highest, and the specific activity of mutant TP-ISOOE/L538D using corn amylose or corn amylopectin as substrate is also the highest.

Corn starch Corn amylose Corn amylopectin

TK-PUL 48.08+1.67 32.14+0.98 62.75+1.39

TP-1500F/L538D 133.66+4.15 73.833,21 188.25+4 31

TP-1500Q/L538K 68.16+3.76 39.76+2.19 91.62+2.34

TP-1500FE/L538H 55.20+1.78 34 881.23 80.6+2.96

Table 3 Specific activities of TK-PUL and mutants

Embodiment 4 Application of mutant in preparation of corn resistant starch

Type III pullulan hydrolase mutants TP-ISOOE/L538D, TP-I500Q/LS38K,

TP-IS00E/L538H are used to prepare corn resistant starch respectively, and type III pullulan hydrolase TK-PUL is used as control.

Preparation of resistant starch: putting a certain amount of common corn starch 509704 (M) in a triangular flask, adding distilled water, and preparing the corn starch milk with a concentration of 15%(m/v); adding 1 mol/L. HCI solution to 15% corn starch milk to adjust the pH value to 5.0, and adding type III pullulan hydrolase for treatment (the amount of enzyme added is 15 U/g corn starch, the reaction time is 12 h, the reaction temperature is 80°C, and the pH value is 5.0); heating the treated samples in boiling water bath for 5 min, then cooling to room temperature, and then storing at 4°C for 24 h to regenerate; adding distilled water to the fully regenerated sample, adjusting the pH value to 5.0-6.0 with disodium hydrogen phosphate-citric acid buffer (pH 6.0), adding excess a-amylase and taking a water bath at 60°C for 24 h; heating the hydrolyzed sample in boiling water bath for 5 min, and then treating with 4 times of 95% ethanol at room temperature for 12 h; centrifuging the treated sample at 4000 r/min for 20 min, discarding the supernatant, adding 10 ml of 95% ethanol to wash the precipitate twice, drying the precipitate at 50°C, weighing it (MO), crushing it and passing through a 100-mesh sieve to obtain the corn resistant starch.

Determination of resistant starch content: accurately weighing 1.0 g (M1) of crushed resistant starch sample into a centrifuge tube, adding 6 mL of water and 6 mL of 4 mol/L KOH solution, and stirring at room temperature for 30 min; adding 11.5 mL of 2 mol/L HCI solution and 6 ml of 0.4 mol/L sodium acetate buffer (pH 4.75) to adjust the pH to 4.5; adding 1 ml of 1% (m/v) glucoamylase (1500 U/mL) and hydrolyzing at 60°C for 45 min; heating in boiling water bath for 5 min, centrifuging at 4000 r/min for 20 min, collecting supernatant, and washing the precipitate twice with 10 mL of distilled water, combining the supernatants and fixing the volume to 100 mL. The reducing sugar content in the supernatant was determined by Fehling titration, and the data multiplied by 0.9 is the resistant starch content (M2). The content (X) of resistant starch is calculated according to formula (1), and it is substituted into formula (2) to calculate the yield (Y) of resistant starch.

X (%) = M1/M2x100% (1)

Y (%) = (MOxX)/Mx100% 2)

The technological conditions of preparing corn resistant starch by using type III pullulan hydrolase TK-PUL and mutant are as follows: the concentration of common 7503704 corn starch milk is 15% (m/v), the dosage of type III pullulan hydrolase is 15 U/g corn starch, the reaction time is 12 h, the reaction temperature is 80°C and the pH value is 5.0. Under the above preparation conditions, the preparation rate of corn resistant starch of type III pullulan hydrolase TK-PUL and mutants is shown in Table 4, wherein the preparation rate of corn resistant starch of mutant TP-ISOOE/L538D is the highest, and the preparation rate of corn resistant starch of mutant TP-ISOOE/L538D is increased from 30.87% of the control (before the mutation) to 59.15%, 0.92 times higher.

TK-PUL 30.87+1.28

TP-1500F/L538D 59.15+0.76

TP-1500Q/L538K 33.42+0.81

TP-I500E/L538H 31.98+0.95

Table 4 Preparation rates of corn resistant starch of TK-PUL and mutants

The amino acid sequence of TP-I5S00E/L538D (SEQ ID NO 1):

MKKGGLLLILLILVSIASGCISESNENQTATASTVPPTSVTPSQSSTPTTSTS

TYGPSERTELKLPSVNYTPIYVGIEKGCPSGRVPVKFTYNPGNKTVKSVSLRGS

FNNWGEWPMELKNGTWETTVCLRPGRYEYK YFINGQOWVKDMSDDGTGRPY

DPDADAYAPDGYGGKNAVRVVEGREAFYVEFDPRDPAYLSIADKRTVVRFEA

KRDTVESAVLVTDHGNYTMKLQVWWDFGETWRAEMPVEPADYYILVTSSDG

GKFAVLNTSESPFFHFDGVEGFPQLEWVSNGITYQIFPDRFNNGNKSNDALAL

DHDELILNQOVNPGQPILSNW SSDPITPLHCCHQYFGGDIKGITEKLDYLQSLGVT

ITYINPIFLSGSAHGYDTYDY YRLDPKFGTEDELREFLDEAHRRGMRVIFDFVP

NHCGIGNPAFLDVWEKGNESPYWDWFFVKKWPFKLGDGSAY VGWWGFGSL

PKLNTANQEVREYLIGAALHWIEFGFDGERVDVPNEVLDPGTFFPELRKAVKE

KKPDAYLVGEIWTDSPEWVKGDRFDSLMNYALGRDILLNYAKGLLSGESAM

KMMGRY YASYGENVVAMGFNLVDSHDTSRVLTDLGGGKLGDTPSNESIQRL

KLLSTLLYALPGTPVTFQGDERGLLGDKGHYDEQRYPIQWDTVNEDVLNHYR

ALAELRKRVPALRSSAMRFYTAKGGVMAFFRGHHDEVLVVANSWKKPALLE

LPEGEWKVIWPEDFSPELLRGTVEVPAIGIIILERG. 509704

The nucleotide sequence of TP-IS00E/L538D (SEQ ID NO.2):

ATGAAAAAAGGTGGTCTGCTGCTCATTCTCCTGATTCTGGTCTCAATCG

CCAGCGGATGTATCTCGGAGAGCAACGAAAATCAAACTGCAACGGCTTCG

ACCGTTCCACCGACTTCAGTGACACCCTCACAGTCTTCCACTCCCACAACC

TCGACCTCGACGTACGGCCCTTCCGAAAGAACGGAGCTTAAACTTCCTTCG

GTTAACTACACTCCCATCTACGTCGGCATAGAGAAAGGCTGTCCCTCCGGA

AGAGTCCCGGTGAAGTTCACGTACAACCCCGGAAACAAGACCGTAAAGTC

TGTCAGCCTCCGCGGGAGCTTCAACAACTGGGGAGAGTGGCCGATGGAGC

TGAAGAACGGCACGTGGGAGACGACCGTCTGTCTCCGCCCTGGAAGGTAT

GAGTATAAGTACTTCATCAACGGCCAGTGGGTCAAGGACATGTCCGACGAC

GGGACGGGAAGGCCCTACGACCCCGATGCAGACGCCTATGCCCCCGATGG

CTACGGGGGAAAGAACGCCGTGAGGGTAGTTGAGGGCCGCGAAGCGTTCT

ACGTGGAGTTCGATCCAAGAGACCCAGCCTACCTCAGCATCGCGGACAAA

AGAACCGTGGTCAGGTTCGAGGCTAAGAGAGACACCGTCGAGTCTGCGGT

TCTCGTTACGGATCACGGGAACTACACGATGAAGCTTCAGGTCTGGTGGGA

CTTCGGCGAAACCTGGCGCGCCGAGATGCCAGTTGAACCCGCTGATTATTA

CATTCTCGTAACCTCCTCCGACGGCGGGAAGTTTGCCGTCCTAAACACAAG

CGAAAGCCCGTTCTTCCACTTTGATGGCGTTGAGGGGTTCCCCCAGCTGGA

GTGGGTGAGCAACGGGATAACCTACCAGATATTCCCCGACAGGTTCAACAA

CGGCAATAAAAGCAACGATGCCCTAGCTTTGGATCACGACGAGCTAATTTT

GAACCAGGTTAATCCAGGGCAGCCAATCCTCTCCAACTGGAGCGACCCGAT

AACGCCCCTCCACTGCTGCCACCAGTACTTCGGCGGCGACATAAAGGGAAT

AACGGAGAAGCTCGACTACCTTCAGAGCCTAGGTGTTACTATAATCTACATC

AACCCGATTTTCCTCTCGGGAAGCGCCCACGGCTACGACACCTACGACTAC

TACCGGCTCGACCCCAAGTTCGGGACCGAGGATGAGCTGAGAGAGTTCCT

CGATGAGGCCCACAGGAGGGGAATGAGGGTAATCTTCGATTTCGTGCCCAA

CCACTGCGGCATAGGGAATCCAGCCTTCCTCGACGTCTGGGAGAAGGGCA

ACGAAAGCCCATACTGGGACTGGTTCTTCGTCAAGAAGTGGCCCTTCAAG

CTCGGCGATGGGAGCGCCTACGTCGGCTGGTGGGGCTTTGGGAGCCTTCC

GAAGCTCAACACTGCCAACCAGGAGGTCAGGGAGTACCTGATAGGAGCGG 1008706

CCCTCCACTGGATAGAGTTCGGCTTTGACGGCGAAAGGGTGGATGTGCCG

AACGAAGTCCTCGACCCGGGGACGTTCTTCCCGGAGCTGAGAAAGGCAGT

TAAGGAGAAAAAGCCCGACGCGTACCTCGTCGGCGAGATATGGACGGACT

CCCCGGAGTGGGTGAAGGGAGACCGCTTCGACTCCCTCATGAACTACGCC

CTCGGGAGGGACATCCTCCTGAACTACGCTAAGGGCCTGCTCAGCGGAGA

AAGTGCAATGAAAATGATGGGACGTTACTACGCTTCCTACGGCGAGAACGT

AGTTGCGATGGGCTTCAACCTCGTTGATTCGCACGACACTTCGAGGGTTCT

CACTGACCTCGGTGGTGGCAAACTGGGAGACACACCGTCAAACGAGTCAA

TTCAGAGGCTCAAGCTCCTCTCAACGCTCCTCTATGCCCTGCCCGGAACTC

CCGTCACCTTCCAGGGGGACGAGAGGGGACTGCTCGGAGACAAGGGACA

CTACGATGAGCAACGCTATCCGATACAGTGGGATACTGTGAACGAGGACGT

CCTGAACCACTACAGGGCACTGGCGGAGCTCAGAAAAAGAGTTCCCGCAT

TGAGGAGCAGCGCAATGAGGTTCTACACTGCCAAAGGCGGCGTTATGGCC

TTCTTCAGGGGACATCATGACGAGGTTCTCGTCGTTGCCAACAGCTGGAAG

AAGCCAGCCCTACTGGAGCTTCCCGAGGGAGAGTGGAAAGTAATCTGGCC

TGAGGATTTCAGCCCGGAACTGCTTCGCGGCACAGTTGAAGTGCCAGCCAT

AGGGATAATCATCCTTGAGCGGGGTTGA.

The amino acid sequence of type III pullulan hydrolase TK-PUL (SEQ ID

NO.3):

MKKGGLLLILLILVSIASGCISESNENQTATASTVPPTSVTPSQSSTPTTSTS

TYGPSERTELKLPSVNYTPIY VGIEKGCPSGRVPVKFTYNPGNKTVKSVSLRGS

FNNWGEWPMELKNGTWETTVCLRPGRYEYKYFINGQWVKDMSDDGTGRPY

DPDADAYAPDGYGGKNAVRVVEGREAFYVEFDPRDPAYLSIADKRTVVRFEA

KRDTVESAVLVTDHGNYTMKLQVWWDFGETWRAEMPVEPADYYILVTSSDG

GKFAVLNTSESPFFHFDGVEGFPQLEWVSNGITYQIFPDRFNNGNKSNDALAL

DHDELILNQOVNPGQPILSNW SSDPITPLHCCHQYFGGDIKGITEKLDYLQSLGVT

ITYINPIFLSGSAHGYDTYDY YRLDPKFGTEDELREFLDEAHRRGMRVIFDFVP

NHCGIGNPAFLDVWEKGNESPYWDWFFVKKWPFKLGDGSAY VGWWGFGSL

PKLNTANQEVREYLIGAALHWIEFGFDGIRVDVPNEVLDPGTFFPELRKAVKE

KKPDAYLVGEIWTLSPEWVKGDRFDSLMNYALGRDILLNYAKGLLSGESAMK 509704

MMGRYYASYGENVVAMGFNLVDSHDTSRVLTDLGGGKLGDTPSNESIQRLKL

LSTLLYALPGTPVTFQGDERGLLGDKGHYDEQRYPIQWDTVNEDVLNHYRAL

AELRKRVPALRSSAMRFYTAKGGVMAFFRGHHDEVLVVANSWKKPALLELPE

GEWK VIWPEDFSPELLRGTVEVPAIGIILERG.

The nucleotide sequence of type III pullulan hydrolase TK-PUL (SEQ ID NO 4):

ATGAAAAAAGGTGGTCTGCTGCTCATTCTCCTGATTCTGGTCTCAATCG

CCAGCGGATGTATCTCGGAGAGCAACGAAAATCAAACTGCAACGGCTTCG

ACCGTTCCACCGACTTCAGTGACACCCTCACAGTCTTCCACTCCCACAACC

TCGACCTCGACGTACGGCCCTTCCGAAAGAACGGAGCTTAAACTTCCTTCG

GTTAACTACACTCCCATCTACGTCGGCATAGAGAAAGGCTGTCCCTCCGGA

AGAGTCCCGGTGAAGTTCACGTACAACCCCGGAAACAAGACCGTAAAGTC

TGTCAGCCTCCGCGGGAGCTTCAACAACTGGGGAGAGTGGCCGATGGAGC

TGAAGAACGGCACGTGGGAGACGACCGTCTGTCTCCGCCCTGGAAGGTAT

GAGTATAAGTACTTCATCAACGGCCAGTGGGTCAAGGACATGTCCGACGAC

GGGACGGGAAGGCCCTACGACCCCGATGCAGACGCCTATGCCCCCGATGG

CTACGGGGGAAAGAACGCCGTGAGGGTAGTTGAGGGCCGCGAAGCGTTCT

ACGTGGAGTTCGATCCAAGAGACCCAGCCTACCTCAGCATCGCGGACAAA

AGAACCGTGGTCAGGTTCGAGGCTAAGAGAGACACCGTCGAGTCTGCGGT

TCTCGTTACGGATCACGGGAACTACACGATGAAGCTTCAGGTCTGGTGGGA

CTTCGGCGAAACCTGGCGCGCCGAGATGCCAGTTGAACCCGCTGATTATTA

CATTCTCGTAACCTCCTCCGACGGCGGGAAGTTTGCCGTCCTAAACACAAG

CGAAAGCCCGTTCTTCCACTTTGATGGCGTTGAGGGGTTCCCCCAGCTGGA

GTGGGTGAGCAACGGGATAACCTACCAGATATTCCCCGACAGGTTCAACAA

CGGCAATAAAAGCAACGATGCCCTAGCTTTGGATCACGACGAGCTAATTTT

GAACCAGGTTAATCCAGGGCAGCCAATCCTCTCCAACTGGAGCGACCCGAT

AACGCCCCTCCACTGCTGCCACCAGTACTTCGGCGGCGACATAAAGGGAAT

AACGGAGAAGCTCGACTACCTTCAGAGCCTAGGTGTTACTATAATCTACATC

AACCCGATTTTCCTCTCGGGAAGCGCCCACGGCTACGACACCTACGACTAC

TACCGGCTCGACCCCAAGTTCGGGACCGAGGATGAGCTGAGAGAGTTCCT

CGATGAGGCCCACAGGAGGGGAATGAGGGTAATCTTCGATTTCGTGCCCAA 7503704

CCACTGCGGCATAGGGAATCCAGCCTTCCTCGACGTCTGGGAGAAGGGCA

ACGAAAGCCCATACTGGGACTGGTTCTTCGTCAAGAAGTGGCCCTTCAAG

CTCGGCGATGGGAGCGCCTACGTCGGCTGGTGGGGCTTTGGGAGCCTTCC

GAAGCTCAACACTGCCAACCAGGAGGTCAGGGAGTACCTGATAGGAGCGG

CCCTCCACTGGATAGAGTTCGGCTTTGACGGCATTAGGGTGGATGTGCCGA

ACGAAGTCCTCGACCCGGGGACGTTCTTCCCGGAGCTGAGAAAGGCAGTT

AAGGAGAAAAAGCCCGACGCGTACCTCGTCGGCGAGATATGGACGCTCTC

CCCGGAGTGGGTGAAGGGAGACCGCTTCGACTCCCTCATGAACTACGCCC

TCGGGAGGGACATCCTCCTGAACTACGCTAAGGGCCTGCTCAGCGGAGAA

AGTGCAATGAAAATGATGGGACGTTACTACGCTTCCTACGGCGAGAACGTA

GTTGCGATGGGCTTCAACCTCGTTGATTCGCACGACACTTCGAGGGTTCTC

ACTGACCTCGGTGGTGGCAAACTGGGAGACACACCGTCAAACGAGTCAAT

TCAGAGGCTCAAGCTCCTCTCAACGCTCCTCTATGCCCTGCCCGGAACTCC

CGTCACCTTCCAGGGGGACGAGAGGGGACTGCTCGGAGACAAGGGACAC

TACGATGAGCAACGCTATCCGATACAGTGGGATACTGTGAACGAGGACGTC

CTGAACCACTACAGGGCACTGGCGGAGCTCAGAAAAAGAGTTCCCGCATT

GAGGAGCAGCGCAATGAGGTTCTACACTGCCAAAGGCGGCGTTATGGCCT

TCTTCAGGGGACATCATGACGAGGTTCTCGTCGTTGCCAACAGCTGGAAG

AAGCCAGCCCTACTGGAGCTTCCCGAGGGAGAGTGGAAAGTAATCTGGCC

TGAGGATTTCAGCCCGGAACTGCTTCGCGGCACAGTTGAAGTGCCAGCCAT

AGGGATAATCATCCTTGAGCGGGGTTGA.

The above-mentioned embodiments only describe the preferred mode of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, all kinds of modifications and improvements made by ordinary technicians in the field to the technical scheme of the present invention should fall within the protection scope determined by the claims of the present invention.

Sequence Listing 508708 "sequence seq 1"

MKKGGLLLIL LILVSIASGC ISESNENQTA TASTVPPTSV TPSQSSTPTT

STSTYGPSER 60

TELKLPSVNY TPIYVGIEKG CPSGRVPVKF TYNPGNKTVK SVSLRGSFNN

WGEWPMELKN 120

GTWETTVCLR PGRYEYKYFI NGQWVKDMSD DGTGRPYDPD

ADAYAPDGYG GKNAVRVVEG 180

REAFYVEFDP RDPAYLSIAD KRTVVRFEAK RDTVESAVLV TDHGNYTMKL

QVWWDFGETW 240

RAEMPVEPAD YYILVTSSDG GKFAVLNTSE SPFFHFDGVE GFPQLEWVSN

GITYQIFPDR 300

FNNGNKSNDA LALDHDELIL NQVNPGQPIL SNWSDPITPL HCCHQYFGGD

IKGITEKLDY 360

LQSLGVTIY INPIFLSGSA HGYDTYDYYR LDPKFGTEDE LREFLDEAHR

RGMRVIFDFV 420

PNHCGIGNPA FLDVWEKGNE SPYWDWFFVK KWPFKLGDGS

AYVGWWGFGS LPKLNTANQE 480

VREYLIGAAL HWIEFGFDGE RVDVPNEVLD PGTFFPELRK AVKEKKPDAY

LVGEIWTDSP 540

EWVKGDRFDS LMNYALGRDI LLNYAKGLLS GESAMKMMGR

YYASYGENVV AMGFNLVDSH 600

DTSRVLTDLG GGKLGDTPSN ESIQRLKLLS TLLYALPGTP VTFQGDERGL

LGDKGHYDEQ 660

RYPIQWDTVN EDVLNHYRAL AELRKRVPAL RSSAMRFYTA KGGVMAFFRG

HHDEVLV VAN 720

SWKKPALLEL PEGEWKVIWP EDFSPELLRG TVEVPAIGII ILERG 765 "sequence seq 2"

atgaaaaaag gtggtctgct gctcattetc ctgattctgg tetcaatcge cageggatgt 60 509704 atctcggaga gcaacgaaaa tcaaactgca acggettega ccgttccacc gacttcagtg 120 acaccctcac agtcttccac teccacaacc tcgacctcga cgtacggecce ttecgaaaga 180 acggagctta aacttccttc ggttaactac actcccatct acgtcggcat agagaaagge 240 tgtcectceg gaagagtecc getgaagttc acgtacaacc ccggaaacaa gaccgtaaag 300 tctgtcagcc tecgcgggag cttcaacaac tggggagagt ggecgatgga gctgaagaac 360 ggcacgtggg agacgaccgt ctgtetcege cctegaaget atgagtataa gtacttcatc 420 aacggccagt gggtcaagga catgtccgac gacgggacgg gaaggeccta cgaccecgat 480 gcagacgcct atgcccccga tggetacggg ggaaagaacg cegtgagggt agttgaggge 540 cgcgaagegt tctacgtgga gttcgatcca agagacccag cctacctcag catcgeggac 600 aaaagaaccg tggtcaggtt cgaggctaag agagacaccg tcgagtetge ggttetegtt 660 acggatcacg ggaactacac gatgaagctt caggtctggt gggacttcgg cgaaacctgg 720 cgecgecgaga tgccagttga acccgctgat tattacatte tegtaaccte ctecgacggc 780 gggaagtttg ccgtectaaa cacaagegaa agcccettct tecactttga tggcettgag 840 gggttccccc agetggagtg ggtgageaac gggataacct accagatatt ccccgacagg 900 ttcaacaacg gcaataaaag caacgatgcc ctagctttgg atcacgacga gctaattttg 960 aaccaggtta atccagggca gccaatecte tccaactgga gcgaccegat aacgecccte 1020 cactectgcc accagtactt cggcggcgac ataaagggaa taacggagaa getcgactac 1080 cttcagagcc taggtgttac tataatctac atcaacccga ttttectetc gggaagegee 1140 cacggctacg acacctacga ctactaccgg ctcgacccca agttcgggac cgaggatgag 1200 ctgagagagt tcctcgatga ggcccacagg aggggaatga gggtaatctt cgatttcgtg 1260 cccaaccact gcggcatagg gaatccagec ttectcgacg tetgggagaa gggcaacgaa 1320 agcccatact gggactggtt cttcgtcaag aagtggccct tcaagetcgg cgatgggage 1380 gcctacgtcg getggtgggg ctitgggage cttccgaage tcaacactge caaccaggag 1440 gtcagggagt acctgatagg agcggeccte cactggatag agttcggctt tgacggegaa 1500 agggtggatg tgccgaacga agtectegac cecggggacgt tetteccgga getgagaaag 1560 gcagttaagg agaaaaagcc cgacgegtac ctegtcggeg agatatggac ggactccccg 1620 gagtggetga agggagaccg cttegactec ctcatgaact acgcectegg gagggacate 1680 ctectgaact acgctaaggg cctgetcage ggagaaagtg caatgaaaat gatgggacgt 1740 tactacgctt cctacggcga gaacgtagtt gcgatgggct tcaacctegt tgattegcac 1800 gacacttcga gggttctcac tgacctcggt ggtggcaaac tgggagacac accgtcaaac 1860 509704 gagtcaattc agaggctcaa geteetetea acgeteetet atgcectgec cggaactcce 1920 gtcaccttcc agggggacga gaggggactg ctcggagaca agggacacta cgatgagcaa 1980 cgctatccga tacagtggga tactgtgaac gaggacgtee tgaaccacta cagggcactg 2040 gcggagctca gaaaaagagt teccgcattg aggagcagcg caatgaggtt ctacactgec 2100 aaaggcggcg ttatggcett cttcagggga catcatgacg aggttctegt cgttgecaac 2160 agctggaaga agccagecct actggagcett cccgagggag agtggaaagt aatctggect 2220 gaggatttca gcccggaact gettcgegge acagttgaag tgecagecat agggataate 2280 atccttgage ggggttga 2298 "sequence seq 3"

MKKGGLLLIL LILVSIASGC ISESNENQTA TASTVPPTSV TPSQSSTPTT

STSTYGPSER 60

TELKLPSVNY TPIYVGIEKG CPSGRVPVKF TYNPGNKTVK SVSLRGSFNN

WGEWPMELKN 120

GTWETTVCLR PGRYEYKYFI NGQWVKDMSD DGTGRPYDPD

ADAYAPDGYG GKNAVRVVEG 180

REAFYVEFDP RDPAYLSIAD KRTVVRFEAK RDTVESAVLV TDHGNYTMKL

QVWWDFGETW 240

RAEMPVEPAD YYILVTSSDG GKFAVLNTSE SPFFHFDGVE GFPQLEWVSN

GITYQIFPDR 300

FNNGNKSNDA LALDHDELIL NQVNPGQPIL SNWSDPITPL HCCHQYFGGD

IKGITEKLDY 360

LQSLGVTIY INPIFLSGSA HGYDTYDY YR LDPKFGTEDE LREFLDEAHR

RGMRVIFDFV 420

PNHCGIGNPA FLDVWEKGNE SPYWDWFFVK KWPFKLGDGS

AYVGWWGFGS LPKLNTANQE 480

VREYLIGAAL HWIEFGFDGI RVDVPNEVLD PGTFFPELRK AVKEKKPDAY

LVGEIWTLSP 540

EWVKGDRFDS LMNYALGRDI LLNYAKGLLS GESAMKMMGR 509704

YYASYGENVV AMGFNLVDSH 600

DTSRVLTDLG GGKLGDTPSN ESIQRLKLLS TLLYALPGTP VTFOGDERGL

LGDKGHYDEQ 660

RYPIQWDTVN EDVLNHYRAL AELRKRVPAL RSSAMRFYTA KGGVMAFFRG

HHDEVLVVAN 720

SWKKPALLEL PEGEWKVIWP EDFSPELLRG TVEVPAIGII ILERG 765 "sequence_seq_4" atgaaaaaag gtggtctgct gctcattetc ctgattetgg tetcaatcge cageggatgt 60 atctcggaga gcaacgaaaa tcaaactgca acggcttcga ccgttccacc gacttcagtg 120 acaccctcac agtcttccac teccacaacc tcgacctega cgtacggecc tteccgaaaga 180 acggagctta aacttccttc ggttaactac actcccatct acgtcggcat agagaaagge 240 tgtcectceg gaagagtecc getgaagttc acgtacaacc ccggaaacaa gaccgtaaag 300 tctgtcagcc tecgcgggag cttcaacaac tggggagagt ggcegatgga gctgaagaac 360 ggcacgtggg agacgaccgt ctgtetccge cctggaaget atgagtataa gtacttcatc 420 aacggccagt gggtcaagga catgtccgac gacgggacgg gaaggeccta cgaccecgat 480 gcagacgcct atgcccccga tggetacggg ggaaagaacg cegtgagggt agttgaggge 540 cgcgaagegt tctacgtgga gttcgatcca agagacccag cctacctcag catcgeggac 600 aaaagaaccg tggtcaggtt cgaggctaag agagacaccg tcgagtetge ggttetegtt 660 acggatcacg ggaactacac gatgaagctt caggtctggt gggacttcgg cgaaacctgg 720 cgecgecgaga tgccagttga acccgctgat tattacatte tegtaaccte ctecgacggc 780 gggaagtttg ccgtectaaa cacaagegaa agcccgttet tccactttga tggegttgag 840 gggttccccc agetggagtg ggtgageaac gggataacct accagatatt ccccgacagg 900 ttcaacaacg gcaataaaag caacgatgcc ctagctttgg atcacgacga gctaattttg 960 aaccaggtta atccagggca gecaatccte tccaactgga gcgaccegat aacgecccte 1020 cactectgcc accagtactt cggeggegac ataaagggaa taacggagaa getcgactac 1080 cttcagagcc taggtgttac tataatctac atcaacccga ttttectete gggaagegee 1140 cacggctacg acacctacga ctactaccgg ctcgacccca agttcgggac cgaggatgag 1200 ctgagagagt tectcgatga ggcccacagg aggggaatga gggtaatctt cgatttegtg 1260 509704 cccaaccact gcggcatagg gaatccagec ttcctegacg tetgggagaa gggcaacgaa 1320 agcccatact gggactggtt cttcgtcaag aagtggccct tcaagetcgg cgatgggage 1380 gcctacgtcg getggtgggg ctitgggage cttccgaage tcaacactge caaccaggag 1440 gtcagggagt acctgatagg agcggeccte cactggatag agttcggcett tgacggcatt 1500 agggtggatg tgccgaacga agtectegac cecggggacgt tetteccgga getgagaaag 1560 gcagttaagg agaaaaagcc cgacgegtac ctegtcggeg agatatggac gctetecccg 1620 gagtggetga agggagaccg cttegactec ctcatgaact acgcectegg gagggacate 1680 ctectgaact acgctaaggg cctgetcage ggagaaagtg caatgaaaat gatgggacgt 1740 tactacgctt cctacggcga gaacgtagtt gcgatgggct tcaacctegt tgattegcac 1800 gacacttcga gggttctcac tgacctcggt ggtggcaaac tgggagacac accgtcaaac 1860 gagtcaattc agaggctcaa gctectctca acgctectet atgcectgec cggaactcee 1920 gtcaccttcc agggggacga gaggggactg ctcggagaca agggacacta cgatgagcaa 1980 cgctatccga tacagtggga tactgtgaac gaggacgtee tgaaccacta cagggcactg 2040 gcggagctca gaaaaagagt teccgcattg aggagcagcg caatgaggtt ctacactgec 2100 aaaggcggcg ttatggcett cttcagggga catcatgacg aggttctegt cgttgecaac 2160 agctggaaga agccagecct actggagcett cccgagggag agtggaaagt aatctggect 2220 gaggatttca gcccggaact gettcgegge acagttgaag tgecagecat agggataate 2280 atccttgage ggggttga 2298 "sequence seq 5S" agtactagta tgaaaaaagg tggtctgctg ctcattctc 39 "sequence seq 6" tgggatccac cecgctcaag gatgattate 30 "sequence seq_7"

ctttgacggc nnkagggtgg atetg 25 17508704 "sequence seq_8" aagccgaact ctatccagtg 20 "sequence seq 9" gatatggacg nnktccccgg agtg 24 "sequence seq 10" atctcgecga cgacgaggta c 21

Claims

CLAIMS LU503704

1. A type III pullulan hydrolase mutant for preparing corn resistant starch, characterized in that, an amino acid sequence of the type III pullulan hydrolase mutant is shown in SEQ ID NO.1.

2. A coding gene for coding the type III pullulan hydrolase mutant according to claim 1.

3. The coding gene according to claim 2, characterized in that, a nucleotide sequence of the coding gene is shown in SEQ ID NO.2.

4. A recombinant vector, characterized by comprising the coding gene according to claim 2 or claim 3.

5. A recombinant microbial strain, characterized by comprising the recombinant vector according to claim 4.

6. An application of the coding gene according to claim 2 or claim 3, the recombinant vector according to claim 4 or the recombinant microbial strain according to claim 5 in preparing the type III pullulan hydrolase mutant.

7. An application of the type III pullulan hydrolase mutant according to claim 1 in preparing corn resistant starch.