CN114317565A

CN114317565A - Starch branching enzyme from myxobacteria, gene thereof, engineering bacterium containing gene and application thereof

Info

Publication number: CN114317565A
Application number: CN202111177948.0A
Authority: CN
Inventors: 崔中利; 叶现丰; 刘威; 黄彦; 李周坤
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2021-10-09
Filing date: 2021-10-09
Publication date: 2022-04-12
Anticipated expiration: 2041-10-09
Also published as: CN114317565B

Abstract

The invention discloses a starch branching enzyme from myxobacteria, a gene thereof, engineering bacteria containing the gene and application thereof. The invention provides a starch branching enzyme gene, the nucleotide sequence of which is as follows: SEQ ID NO.1, the amino acid sequence of the encoded protein is: SEQ ID NO. 2. The recombinant starch branching enzyme obtained by utilizing the engineering strain constructed by the gene takes cassava starch as a substrate for activity detection, and the specific activity of the recombinant starch branching enzyme is 19439.9 +/-62.4U/mg under the optimal reaction condition through iodine solution detection. The starch branching enzyme produced by using the gene can be used for starch modification, including preparation of slowly digestible starch or resistant starch, preparation of high-branching-degree modified starch with anti-aging characteristic, preparation of cold water soluble starch and the like. The prepared modified starch can be widely applied to industries such as food, brewing, fermentation, textile industry and medicine, and can obtain considerable economic benefit while solving practical problems.

Description

Starch branching enzyme from myxobacteria, gene thereof, engineering bacterium containing gene and application thereof

Technical Field

The invention belongs to the field of applied industrial microorganisms, and discloses a starch branching enzyme derived from Corallococcus sp.

Background

Starch, one of the most abundant carbohydrates in nature, is the most important source of nutrition for humans and animals. Starches can be classified into amylose and amylopectin, according to the type of glycosidic bond, wherein amylose is composed of α -1, 4-glycosidic bonds and amylopectin is composed of an amylose formed by α -1, 4-glycosidic bonds and an α -1, 6-glycosidic-bonded branch. Starch can be classified into fast-digestible starch (according to its digestion rate and digestion degree in animal body after eating itrapidly digestible starch, RDS), slowly digestible starch(s) ((R)slowly digestible starch, SDS) and resistant starch (resistant starch, RS). The formation of slowly digestible and resistant starch is critically linked to the ratio of the types of glycosidic bonds in the starch and the length of the starch chain. Wherein the more the proportion of short chains with DP < 13 (DP) in amylopectin, the more easily it forms slowly digestible starch, and when the length of short chains with DP < 20 in amylose, the more easily it forms resistant starch. Long-term consumption of food containing large amount of RDS can significantly increase incidence of diabetes, obesity and cardiovascular and cerebrovascular diseases, while SDS and RS can significantly decrease due to their slow digestion propertiesLow risk of the above diseases. Starch retrogradation is another important problem faced by starch in the application process, and the essence of the starch retrogradation is a process in which starch molecules gradually go from a high energy state to a low energy state under the action of molecular potential energy. The retrogradation of starch is also closely related to the degree of branching and chain length of the starch. For example, a chain length of between 14 and 24 may be beneficial for retrogradation of starch. Therefore, the preparation of modified starch by changing the degree of branching and the chain length of starch can improve the application range and application potential of starch in the food industry.

The biological enzyme method is an important method in the research and preparation of starch modification due to the safety, high efficiency and specificity of the biological enzyme method. Starch branching enzymes (alpha-1, 4-glucan branching enzymes; EC 2.4.1.18) belong to a class of glycosyltransferases of glycoside hydrolases 13 and 57 family, and are capable of catalyzing the cleavage of alpha-1, 4-glycosidic bonds and the formation of alpha-1, 6-glycosidic bonds between molecules to form new branches on the backbone of starch molecules. Currently, starch branching enzymes derived from plants, animals and microorganisms have been reported in the preparation of modified starches, for example, glycogen branching enzyme derived from Thermomonospora curvata can catalyze the conversion of long amylose substrates into highly branched dextrin products with good solubility (patent No. CN 108300750A); thermobifidafuuca-derived starch branching enzyme is expressed in Escherichia coli to prepare resistant dextrin with 60% of resistant components (patent number: CN 107384989A), and is treated by cyclodextrin glucosyltransferase and starch branching enzyme to prepare slowly digestible starch (patent number: CN 108251475A). Although starch branching enzyme derived from microorganisms is a main industrial enzyme due to the characteristics of high substrate specificity, high branching degree of catalytic products and the like, the currently reported starch branching enzyme has the problems of low enzyme yield of natural strains, low enzyme activity, low catalytic efficiency and the like, so that the development of starch branching enzyme resources with high activity and excellent performance has important significance.

Disclosure of Invention

The object of the present invention is to provide a novel starch branching enzyme gene and a protein encoded by the same.

Another object of the present invention is to provide a genetically engineered bacterium containing the starch branching enzyme gene.

The invention also aims to provide application of the protein and the coding gene thereof.

A starch branching enzyme gene, the nucleotide sequence of which is: SEQ ID NO.1, the full length of the gene (from the start codon to the stop codon) is 2178bp, the G + C content is 69.65%, and 725 amino acids are coded.

The amino acid sequence of the starch branching enzyme protein coded by the starch branching enzyme gene is as follows: SEQ ID NO. 2.

The optimum reaction pH of the starch branching enzyme is 9.0, the optimum reaction temperature is 40 ℃, and the activity is kept relatively stable between 20-45 ℃ (9h) and pH 6.0-9.0(24 h).

The recombinant plasmid containing the starch branching enzyme gene is disclosed.

The recombinant plasmid is preferably obtained by cloning the starch branching enzyme gene into pET-29a plasmid.

A recombinant microorganism comprising the recombinant plasmid of the present invention.

The recombinant microorganism preferably takes Escherichia coli BL21(DE3) as a host bacterium.

The starch branching enzyme gene, the recombinant plasmid and the recombinant microorganism are applied to the genetic engineering in the fields of starch processing, food or feed.

The starch branching enzyme is applied to the preparation and/or starch modification of high-branch starch; preferably in the preparation of slowly digestible starch or resistant starch, the preparation of high-branching-degree modified starch, the optimization of starch gel characteristics or the optimization of starch anti-aging characteristics.

Compared with an untreated control, the starch branching enzyme treats the cassava starch for 10 minutes at 40 ℃, the relative content of amylose in the enzyme treated cassava starch is reduced to 23.4 percent of that of the control, and the content of branch chains with the degree of polymerization DP of more than 25 in the modified starch is obviously increased; when the relative amylose content of the enzyme-treated tapioca starch was reduced to 4.2% of that of the control when the reaction time was extended to 40 minutes, the content of branched chains having a degree of polymerization DP <7 in the modified starch was significantly increased. After the enzyme completely reacts, the content of the resistant starch in the cassava starch is increased from 3.31 percent of the control group to 12.80 percent, and the transparency and the cold water solubility of the cassava starch are respectively increased from 31.8 percent and 23.1 percent of the control group to 96.1 percent and 96.6 percent.

The starch branching enzyme is used as an enzyme preparation for production and application in the fields of starch processing, food or feed.

The invention has the beneficial effects that:

(1) the invention uses Corallococcus sp.strain EGB with the preservation number of CCTCC NO of M2012528 as a material, refers to genome sequence information and combines PCR amplification to successfully obtain an amylobranching enzyme gene sequence. The full length of the gene (from the start codon to the stop codon) is 2178bp, the content of G + C is 69.65%, 725 amino acids are coded, and the gene does not contain a signal peptide.

(2) The product of the starch branching enzyme gene expression is used for measuring the enzyme activity of the starch branching enzyme by an iodine solution method, the starch branching enzyme can efficiently act on cassava starch, amylopectin, soluble starch, wheat starch, potato starch, corn starch, pea starch and amylose, the most suitable catalytic substrate is the cassava starch, and the specific enzyme activity of the starch branching enzyme is up to 19439.9 +/-62.4U/mg under the most suitable reaction condition.

(3) The recombinant starch branching enzyme of SEQ ID NO.1 obtained by taking pET-29a as a carrier shows high catalytic efficiency and special catalytic characteristics in the aspect of starch modification. In the catalytic reaction taking the cassava starch as a substrate, the blue value of a reaction product within 3min is obviously higher than that of a control group; within 15min of reaction, the content of the branch chain with the degree of polymerization DP greater than 25 in the modified starch prepared by the enzyme is obviously increased, and the content of resistant starch is increased to 3.95 percent from 3.17 percent of a control group; after the reaction is completed, the branched chain with the degree of polymerization DP <7 in the modified starch prepared by the enzyme is obviously increased, the content of the branched chain with the degree of polymerization DP >25 is obviously reduced, and the content of the resistant starch is increased to 12.80 percent from 3.17 percent of the control group. The modified starch prepared by the enzyme shows strong anti-aging property, and the property is directly related to the remarkable improvement of the content of resistant starch and slowly digestible starch in the modified starch and the change of the starch gel property.

Drawings

FIG. 1 pCR amplification electrophoretogram of the starch branching enzyme coding gene

1: a nucleic acid Marker; 2: PCR amplification of starch branching enzyme CcGBE gene

FIG. 2 schematic representation of cloning and expression of starch branching enzyme gene

FIG. 3 SDS-PAGE electrophoresis of recombinant starch branching enzyme

M is a standard protein Marker; 1: coli cell disruption supernatant containing pET-29a (+) empty vector; 2: breaking supernatant (crude enzyme solution) of pET-29a (+) -CcGBE engineering strain; 3: purified CcGBE protein

FIG. 4 enzymatic Properties of starch branching enzyme

A: the optimum temperature of starch branching enzyme CcGBE; b: temperature stability; c: the optimum pH value of the starch branching enzyme; d: stability of pH

FIG. 5 clarity and Cold Water solubility analysis of modified starches prepared by starch branching enzyme CcGBE

Detailed Description

Example 1 expression purification and Activity determination of starch branching enzyme

1.1 pCR amplification of starch branching enzyme genes

According to a Corallococcus sp.strain EGB genome completion diagram and gene function prediction by combining NCBI genome information, a starch branching enzyme primer is designed by taking the full length of a SEQ ID NO.1 gene as a template, and pCR amplification of the full length of a starch branching enzyme gene is carried out by taking the genome of a strain EGB (CCTCC NO: M2012528, disclosed in Chinese patent CN 103103152A) as a template to obtain the full length sequence of the starch branching enzyme gene. The full length of the gene (from the start codon to the stop codon) is 2178bp, the GC content of the sequence is 69.65%, 725 amino acids are coded, the amino acid sequence is SEQ ID NO.2, and the gene does not contain a signal peptide. The primers used for pCR amplification are F and R, the amplification result is shown in an electrophoresis chart 1, and the construction process of the expression plasmid is shown in a figure 2.

F：GGAGATATACATATGGTGGACGCGGAGCTGCAG(SEQ ID NO.3)

R：GTGGTGGTGGTGGTGCCCCGGCGTGAACCACACC(SEQ ID NO.4)

Preparation of E.coli BL21(DE3) electrotransferase competence

Strain e.coli BL21(DE3) was streaked onto fresh LB plates from a-80 ℃ freezer, cultured overnight, and picked straightColony with diameter of about 2mm is inoculated without adding Mg²⁺The SOB test tube is cultured at 37 ℃ until the OD600 reaches 1.0, then the SOB test tube is inoculated into a 0.5L shake flask filled with 100ml of SOB culture medium in an inoculation amount of 1/100, and the SOB test tube is cultured at 18 ℃ and 220rpm until the OD600 reaches 0.7-0.8; placing the shake flask in an ice bath, cooling for 10min, centrifuging at 4000rpm at 4 ℃ for 5min, and collecting thallus precipitate; resuspending and washing thallus with sterilized ultrapure water of the same volume, centrifuging at 4 ℃ and 4000rpm for 5min, and collecting thallus precipitate; washing is repeated once; 100ml of 10 percent glycerol is used for resuspending the thalli, and the thalli is centrifuged for 5min at the temperature of 4 ℃ and the rpm is 4000 to collect thalli sediment; washing is repeated once; the supernatant was carefully discarded and the flask inverted and drained on sterile absorbent paper for about 1 min. Each 1000ml culture was carefully resuspended in 2ml 10% glycerol, 100. mu.l of each tube was dispensed into a centrifuge tube and quickly stored in a-80 ℃ freezer for further use.

1.3 construction and transformation of recombinant plasmids

The pCR amplification product of the gene SEQ ID NO.1 was recovered, and the target fragment and pET-29a expression vector were recombined in vitro using the Clonexpress II One Step Cloning Kit of Novozan, in the following recombination system:

gently sucking and beating the mixture by using a pipettor (do not shake the mixture for mixing), centrifuging the mixture for a short time to collect reaction liquid to the bottom of the tube, and reacting the reaction liquid for 30min at 37 ℃; cooled to 4 ℃ or immediately placed on ice to cool. The recombinant plasmid was added to E.coli BL21(DE3), left on ice for 30min, transferred to a 42 ℃ water bath for 90s in an annealing process, further transferred to ice water for cooling for 2min, and then added with 800. mu.l of a room temperature LB medium for recovery for 45min, and then applied to an LB plate containing 50mg/L kanamycin, and the single colony was selected, verified to be free of gene sequence by sequencing, and then stored in a-80 ℃ low temperature refrigerator with 15% glycerol at the final concentration.

1.4 measurement of the Activity of recombinantly expressed starch branching enzyme

E.coli BL21(DE3) expression strain containing recombinant expression plasmid was inoculated into LB medium and cultured at 37 ℃ to 0D600nm between 0.5 and 0.6, IPTG was added to a concentration of 0.2mM, and culture was continued at 18 ℃ for 24 hours. After the collected bacteria are resuspended by Tris-HCl (pH7.0), the bacteria cells are broken by ultrasonic treatment and centrifuged for 15min at 20000 Xg, and the obtained supernatant is the starch branching enzyme crude enzyme liquid. The recombinant starch branching enzyme was detected after purification by Ni-nta affinity chromatography and concentration by ultrafiltration, and the SDS-PAGE electrophoretogram is shown in fig. 3. The living body measuring system comprises the following steps: mu.l cassava starch solution (0.2%, w/w, 20mM Tris-HCl buffer (pH 9.0)) + 30. mu.l starch branching enzyme, reacted at 40 ℃ for 10min, quenched in a boiling water bath, 500. mu.l iodine solution added, left to stand at room temperature for 20min and then absorbance measured at 660 nm. One activity unit is defined as the amount of enzyme required to decrease by 1% per minute at 660 nm.

Example 2 study of enzymatic Properties of starch branching enzyme CcGBE

3.1 Effect of temperature on enzyme Activity

The protocol for determining the optimum reaction temperature is as follows: the activity of the recombinase was measured at various temperatures (20 ℃, 30 ℃, 35 ℃,40 ℃, 45 ℃, 50 ℃, 60 ℃) under the condition of 20mM Tris-HCl buffer (pH9.0), and the highest enzyme activity was set to 100% (FIG. 4A). Determination of thermal stability: the recombinant enzyme was incubated at 20 ℃, 30 ℃, 35 ℃,40 ℃, 45 ℃, 50 ℃ and 60 ℃ for 1-9h under 20mM Tris-HCl buffer (pH9.0), rapidly cooled on ice, and the residual enzyme activities were each determined with the uninsulated enzyme activity as 100% (FIG. 4B). The optimum reaction temperature of the starch branching enzyme CcGBE is determined to be 40 ℃ and is kept relatively stable between 20 ℃ and 45 ℃.

3.2 Effect of pH on enzyme Activity

Determination of optimum reaction pH: the activity of the recombinant starch branching enzyme CcGBE was measured at 40 ℃ at different buffer pH values [20mM Citrate buffer (pH 3.0-6.0), PBS buffer (pH 6.0-8.0), Tris-HCl buffer (pH 7.0-9.0) and glycine-NaOH buffer (pH 9.0-10.0) ], setting the highest activity to 100% (FIG. 4C). Determination of pH stability: the recombinant starch branching enzyme was maintained at 4 ℃ for 24 hours in different buffer systems of pH 3.0-10.0, and then the residual activity was measured, taking the enzyme activity of pH9.0 as 100% (FIG. 4D). The starch branching enzyme CcGBE is determined to have the optimum reaction pH of 9.0 and to be relatively stable at the pH of 6.0-9.0.

3.3 substrate specificity of starch branching enzyme

Is prepared from cassava starch, amylopectin and soluble starchThe substrate specificity of the expressed starch branching enzyme was analyzed by using native starch, wheat starch, potato starch, corn starch, pea starch and amylose as substrates (see table 1), and as a result, it was found that the starch branching enzyme CcGBE had activity on both the substrates tested, with the highest activity being observed when tapioca starch and amylopectin were used as substrates. The activity of starch branching enzyme is detected by iodine solution with cassava starch as substrate, and the enzyme activity unit is defined as OD per minute₆₆₀The enzyme amount required for 1% reduction is one activity unit, and the specific activity of the starch branching enzyme CcGBE is 19439.9 +/-62.4U/mg when cassava starch is taken as a substrate.

TABLE 1 substrate specificity analysis of recombinant starch branching enzyme CcGBE

Example 3 preparation of highly branched modified starch Using starch branching enzyme CcGBE

Preparing 0.5% cassava starch, gelatinizing in boiling water bath for 30min, cooling to room temperature, adding starch branching enzyme CcGBE in the amount of 25000U/kg starch, reacting at 40 deg.C for different times, and stopping reaction in boiling water bath. Isoamylase (100U/g starch) is added into the prepared modified starch, the modified starch reacts for 24 hours at the temperature of 38 ℃ to carry out debranching reaction, and the reaction is stopped by boiling water bath. The degree of polymerization of the branched chains was analyzed by high performance anion exchange chromatography (Thermo ICS 5000+, Thermo Fisher Scientific, USA). The results show that the content of amylopectin with a degree of polymerization DP >25 in the modified starch treated by CcGBE for 15min is remarkably increased, and the content of the amylopectin with a degree of polymerization DP <7 in the modified starch treated by CcGBE for 40min is remarkably increased (Table 2). The results show that the starch branching enzyme CcGBE can produce two different types of high-branching degree modified starch with high polymerization degree and low polymerization degree according to the acting time of the starch branching enzyme CcGBE on starch.

TABLE 2 analysis of branched chain polymerization degree after treatment of tapioca starch with starch branching enzyme CcGBE

Example 4 analysis of the anti-aging Properties of modified starch prepared Using starch branching enzyme CcGBE

Cassava starch treated for 0min, 5min, 15min, 60min and fully reacted (360min) using the starch branching enzyme prepared in example 3 was stored at 4 ℃ for 14 days and the enthalpy change values of the modified starch treated for different times by CcGBE were analyzed using differential scanning calorimetry (DSC823E, Mettler Toledo, Switzerland) to determine the effect of starch branching enzyme CcGBE treatment on starch aging resistance. As a result, as shown in Table 3, the enthalpy value of the modified starch was reduced to 1.146J/g after 5 minutes of the branching enzyme treatment, as compared with that of the original starch (Δ Hg) of 1.66J/g, and the enthalpy value of the modified starch was continuously decreased as the CcGBE treatment time was prolonged, and the enthalpy value of the modified starch after the complete reaction was 0.40J/g, which was significantly lower than that of the original starch sample. The result shows that the high-branching-degree modified starch generated after the starch branching enzyme CcGBE acts on the cassava starch shows stronger anti-aging characteristic, and the characteristic shows great potential in the anti-aging industrial application of the modified starch.

TABLE 3 analysis of gel characteristics of samples of tapioca starch treated with starch branching enzyme CcGBE at different times

Note: to: an initial temperature; tp: a peak temperature; tc: the temperature is terminated.

Example 5 analysis of the use of starch branching enzyme CcGBE for the preparation of resistant starch

Cassava starch treated by starch branching enzyme CcGBE in example 3 for 0min, 5min, 15min, 60min and complete reaction (360min) is placed in a refrigerator at 4 ℃ for storage for 24h, then 3000U of pig trypsin is added, the mixture is placed in a water bath kettle at 37 ℃ for reaction for 12h, and the content of reducing sugar released by hydrolysis is determined by a DNS method. Simultaneous reaction with DNS using glucose of different concentrationsAbsorbance value (OD) measured by spectrophotometer₅₄₀) And establishing a standard curve with the concentration of the glucose standard solution, calculating the content of reducing sugar released by the cassava starch after the porcine pancreatin treatment, and further evaluating the potential application value of the starch branching enzyme CcGBE in the preparation of the slowly digestible starch and the resistant starch.

The determination formula is as follows: RDS% ((D-E)/F × 100%), SDS% ((G-D)/F × 100%, and RS% ((F-G)/G × 100%; wherein D represents the amount of glucose released at 1 hour, E represents the amount of glucose released at the beginning of the reaction (0 hour), F represents the mass of each sample, and G represents the maximum amount of reducing sugars that can be produced by hydrolysis of the modified starch prepared by CcGBE treatment with porcine trypsin.

Results are shown in table 4 that the contents of both Slowly Digestible Starch (SDS) and Resistant Starch (RS) in the modified starch after the branching enzyme CcGBE treatment increased gradually with increasing enzyme treatment time. The content of slowly digestible starch and resistant starch in the original tapioca starch were 15.39% and 3.31%, respectively, while the content of slowly digestible starch and resistant starch increased to 17.40% and 12.80%, respectively, after complete reaction of CcGBE and tapioca starch. Compared with the original cassava starch, the content of resistant starch in the modified starch is increased by 286.7%, which shows great application potential of CcGBE in preparing the resistant starch.

TABLE 4 analysis of the variation in the content of nutritive starch in samples treated with starch branching enzyme CcGBE at different times

Example 6 analysis of the application of starch branching enzyme CcGBE in altering starch gel characteristics and water solubility

After cooling the tapioca starch treated by starch branching enzyme CcGBE in example 3 for 0min, 5min, 15min, 60min and completely reacted (360min) to room temperature, the transparency of the modified tapioca starch was measured at a wavelength of 620nm by an ultraviolet spectrophotometer with deionized water as a blank control (light transmittance of 100%). Meanwhile, freeze-drying the prepared starch samples, accurately weighing 0.15g of each group of freeze-dried starch samples, preparing 15ml of 1% (w/v) cassava starch by using deionized water, uniformly stirring at a low speed for 30s, then stirring at a high speed for 5min, transferring the starch paste into a centrifuge tube after stabilizing for 1h, and centrifuging at a low speed for 10min at the rotating speed of 4000 rpm/min. And pouring the supernatant into a weighed culture dish, putting the culture dish into a high-temperature oven, and drying to constant weight. The cold water solubility of the modified starch is calculated by the formula: solubility (CWS)%, oven-dried sample mass (g)/total sample mass (g) × 100% in the supernatant.

The transparency and cold water solubility of cassava starch modified by the branching enzyme CcGBE are shown in fig. 5, the transparency and cold water solubility of the cassava starch are both remarkably increased along with the prolonging of the treatment time of the CcGBE, and after the CcGBE is treated for 60min, the transparency of the cassava starch reaches 94.6%, and the cold water solubility of the cassava starch is 93.8%. Therefore, the cassava starch modified by the starch branching enzyme CcGBE has high transparency and good cold water solubility, and the fluidity and the stability of the starch stored at room temperature are improved, which also shows that the starch branching enzyme has great potential application value in preparing the modified starch with high transparency and cold water solubility.

Sequence listing

<110> Nanjing university of agriculture

<120> a starch branching enzyme from myxobacteria and gene thereof, engineering bacteria containing the gene and application thereof

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Val Asp Ala Glu Leu Gln Arg Val Val Glu Leu Arg His Pro Glu Pro

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His Ser Val Leu Gly Val His Pro Asp Gly Asp Ala Val Val Val Arg

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Ala Tyr Arg Pro Asp Ala Val Ala Ile His Val Leu Pro Glu Phe Gly

35 40 45

Gly Lys Val Pro Met Gln His Arg Thr Gly Gly Val Phe Glu Ala Arg

50 55 60

Ile Asn Gly Arg Thr Glu Pro Phe Ser Tyr Leu Leu Glu Val Glu Tyr

65 70 75 80

Pro Gly Lys Lys Val Phe Thr Leu Arg Asp Pro Tyr Ser Phe Leu Pro

85 90 95

Thr Ile Gly Glu Met Asp Leu Tyr Phe Ala Gly Glu Gly Arg His Glu

100 105 110

Arg Leu Trp Glu Arg Met Gly Ala His Leu Ile His His Asn Gly Val

115 120 125

Lys Gly Thr Ser Phe Ala Val Trp Ala Pro Thr Ala Arg Gly Val Ser

130 135 140

Val Val Gly Asp Phe Asn Gly Trp Asp Gly Arg Leu His Ala Met Arg

145 150 155 160

Arg Met Gly Ser Ser Gly Ile Trp Glu Leu Phe Ile Pro Glu Val Gly

165 170 175

Glu Gly Thr Arg Tyr Lys Phe Glu Ile Arg Pro Gly His Gly Gly Gly

180 185 190

Pro Leu Leu Lys Ser Asp Pro Phe Ala Phe Arg Thr Glu Thr Pro Pro

195 200 205

Ala Thr Ala Ser Val Val His Asp Leu Arg Arg Tyr Asn Trp Gly Asp

210 215 220

Asp Ala Trp Leu Glu Gly Arg Asp Arg Arg Gly Glu Ala Ala Gln Gln

225 230 235 240

Pro Trp Ser Val Tyr Glu Val His Leu Gly Ser Trp Arg Arg Val Val

245 250 255

Glu Asp Gly Asp Arg Pro Met Thr Tyr Arg Glu Leu Ala Pro Glu Leu

260 265 270

Ser Arg Tyr Val Lys Glu Leu Gly Phe Thr His Val Glu Phe Leu Pro

275 280 285

Val Ala Glu His Pro Tyr Gly Gly Ser Trp Gly Tyr Gln Val Gly Gly

290 295 300

Tyr Tyr Ala Pro Thr Ser Arg Phe Gly His Pro Asp Asp Phe Arg Tyr

305 310 315 320

Leu Val Asp Tyr Leu His Gln Glu Gly Ile Gly Val Ile Val Asp Trp

325 330 335

Val Pro Gly His Phe Pro Arg Asp Ser His Ala Leu Gly Gln Phe Asp

340 345 350

Gly Thr Ala Leu Tyr Glu His Ala Asp Pro Arg Gln Gly Ser Gln Pro

355 360 365

Asp Trp Gly Thr Leu Val Phe Asn Phe Gly Arg Asn Glu Val Arg Asn

370 375 380

Phe Leu Ile Ala Asn Ala Leu Phe Trp Leu Glu Glu Tyr His Ile Asp

385 390 395 400

Gly Leu Arg Val Asp Ala Val Ala Ser Met Leu Tyr Leu Asp Tyr Ser

405 410 415

Arg Lys Gln Gly Glu Trp Ile Pro Asn Arg Trp Gly Gly Arg Glu Asn

420 425 430

Glu Glu Ala Ile Gln Phe Leu Arg Glu Leu Asn Glu Thr Ile Arg Arg

435 440 445

Lys His Pro Gly Val Val Val Ile Ala Glu Glu Ser Thr Ala Trp Pro

450 455 460

Lys Val Ser Gln Pro Val Ser Glu Gly Gly Leu Gly Phe Thr Phe Lys

465 470 475 480

Trp Asn Met Gly Trp Met His Asp Thr Leu Ser Tyr Phe Ser Lys Asp

485 490 495

Ala Val Tyr Arg Gln Tyr His His Asn Gln Leu Thr Phe Gly Leu Leu

500 505 510

Tyr Ala Phe Ser Glu Asn Phe Met Leu Pro Leu Ser His Asp Glu Val

515 520 525

Val His Gly Lys Gly Ser Leu Tyr Gly Arg Met Pro Gly Asp Ala Trp

530 535 540

Gln Lys Arg Ala Asn Leu Arg Ala Leu Phe Ala Trp Met Trp Ala His

545 550 555 560

Pro Gly Lys Lys Leu Leu Phe Met Gly Gly Glu Phe Gly Gln Pro Ala

565 570 575

Glu Trp Asn His Asp Lys Ser Leu Asp Trp His Leu Leu His Asp Pro

580 585 590

Gly His Lys Gly Ile Gln Lys Leu Val Gly Asp Leu Asn Arg Val Tyr

595 600 605

Arg Asp Leu Pro Ala Leu Tyr Asp Cys Asp Ser Glu Pro Arg Gly Phe

610 615 620

Gln Trp Leu Gln Pro Asp Ala Ser Ala Ala Asn Val Leu Ala Phe Val

625 630 635 640

Arg Arg Ser Arg Thr Pro Gly Arg His Val Val Cys Val Ala Asn Leu

645 650 655

Ser Pro Val Pro Arg Glu Asp Tyr Arg Val Gly Phe Pro Leu His Gly

660 665 670

Arg Tyr Val Glu Leu Val Asn Thr Asp Ala Gly Glu Tyr Gly Gly Ser

675 680 685

Gly Leu Gly Asn Arg Gly Gln Val His Thr Glu Pro Thr Gly Trp Asp

690 695 700

Gly Gln Pro Ala Ser Ala Val Leu Thr Leu Pro Pro Leu Ser Val Val

705 710 715 720

Trp Phe Thr Pro Gly

725

<210> 3

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ggagatatac atatggtgga cgcggagctg cag 33

<210> 4

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gtggtggtgg tggtgccccg gcgtgaacca cacc 34

Claims

1. A starch branching enzyme gene, the nucleotide sequence of which is: SEQ ID NO. 1.

2. The starch branching enzyme encoded by the starch branching enzyme gene of claim 1 having the amino acid sequence: SEQ ID NO. 2.

3. A recombinant plasmid containing the starch branching enzyme gene according to claim 1.

4. The recombinant plasmid according to claim 3, wherein the recombinant plasmid is obtained by cloning the starch branching enzyme gene of claim 1 into the plasmid pET-29a (+).

5. A recombinant microorganism comprising the starch branching enzyme gene according to claim 1 or the recombinant plasmid according to claim 3 or 4.

6. The recombinant microorganism according to claim 5, wherein Escherichia coli or yeast is used as a host bacterium.

7. The starch branching enzyme gene according to claim 1, the recombinant plasmid according to claim 3 or 4, or the recombinant microorganism according to claim 5 or 6, for use in the field of starch processing, food or feed.

8. Use of a starch branching enzyme according to claim 2 for the preparation of highly branched starch and/or for the modification of starch.

9. Use according to claim 8, characterized in that the starch branching enzyme according to claim 2 is used in the preparation of slowly digestible or resistant starch, in the preparation of highly branched modified starch, in the preparation of cold water soluble starch and/or in the optimization of starch anti-aging properties.

10. Use of the starch branching enzyme according to claim 2 as an enzyme preparation for the production of starch, food or feed.