CN114317565A - Myxobacter-derived starch branching enzyme and gene thereof, engineering bacteria containing the gene and application thereof - Google Patents

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

A kind of myxobacter-derived starch branching enzyme and gene thereof, engineering bacteria containing the gene and application thereof

technical field

The invention belongs to the field of applied industrial microorganisms, and discloses a starch branching enzyme derived from the myxobacteria Corallococcus sp. strain EGB (Corallococcus sp. strain EGB) and its gene, an engineered bacteria containing the gene and its application in starch modification and preparation of antibiotics The application of sex starch.

Background technique

Starch, as one of the most abundant carbohydrates in nature, is the main source of nutrition for humans and animals. According to the type of glycosidic bonds, starch can be divided into amylose and amylopectin, where amylose is composed of α-1,4-glycosidic bonds, while amylopectin is composed of α-1,4-glycosidic bonds. Chain and branched chains linked by α-1,6-glycosidic bonds. Starch can be divided into rapidly digestible starch (rapidly digestible tarch , RDS ), slowly digestible starch (slowly digestible tarch , SDS ) and resistant starch ( r esistant tarch, RS). The formation of slow-digestible starch and resistant starch is crucially related to the ratio of glycosidic bond types in starch and the length of starch chain. Among them, the more the proportion of short chains with DP<13 (Degree of polymerization, DP) in amylopectin, the easier it is to form slow-digesting starch, and when the chain length in amylose starch is DP<20, it is easier to form resistant starch. Long-term consumption of foods containing a large amount of RDS can significantly increase the incidence of diabetes, obesity, and cardiovascular and cerebrovascular diseases, while SDS and RS can significantly reduce the risk of these diseases due to their slow-digesting properties. Starch retrogradation is another important problem faced by starch in the application process. The starch retrogradation process is also closely related to the branching degree and chain length of starch. For example, the chain length between 14-24 is beneficial to the retrogradation of starch. Therefore, the preparation of modified starch by changing the degree of starch branching and chain length can improve the application range and application potential of starch in the food industry.

Biological enzymatic method is an important method in starch modification research and preparation because of its safety, high efficiency and specificity. Starch branching enzyme (α-1,4-glucan branching enzyme; EC 2.4.1.18) belongs to a class of glycosyltransferases of glycoside hydrolase 13 and 57 families, which can catalyze the cleavage of intermolecular α-1,4-glycosidic bonds and The formation of α-1,6-glycosidic bonds in turn forms new branches on the main chain of starch molecules. At present, starch branching enzymes derived from plants, animals and microorganisms have been reported in the preparation of modified starch. For example, glycogen branching enzymes derived from Thermomonospora curvata can catalyze the conversion of long amylose substrates into highly soluble hyperbranched pastes. Essence product (patent number: CN 108300750 A); starch branching enzyme derived from Thermobifidafusca is expressed in Escherichia coli to prepare resistant dextrin (patent number: CN 107384989 A) with resistant components up to 60% and the use of cyclodextrin glucosyl transfer Enzyme and starch branching enzyme treatment to prepare slow-digesting starch (patent number: CN 108251475 A) and so on. Although starch branching enzymes derived from microorganisms have become the main industrial enzymes due to their high substrate specificity and large branching degree of catalytic products, however, the currently reported starch branching enzymes exist in natural strains with low enzyme production, enzyme activity and catalytic activity. Problems such as low efficiency make the development of starch branching enzyme resources with high activity and excellent performance of great significance.

SUMMARY OF THE INVENTION

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

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

Another object of the present invention is to provide applications of the protein and its encoding gene.

The nucleotide sequence of the starch branching enzyme gene 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 it encodes 725 amino acids.

The amino acid sequence of the starch branching enzyme protein encoded by the starch branching enzyme gene of the present invention is: SEQ ID NO.2.

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

The recombinant plasmid containing the starch branching enzyme gene of the present invention.

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

Recombinant microorganisms containing the recombinant plasmids of the present invention.

The recombinant microorganism preferably uses Escherichia coli BL21 (DE3) as the host bacteria.

The application of the starch branching enzyme gene, recombinant plasmid and recombinant microorganism of the present invention in genetic engineering in the fields of starch processing, food or feed.

The application of the starch branching enzyme of the present invention in the preparation of hyperbranched starch and/or starch modification; preferably in the preparation of slow-digesting starch or resistant starch, the preparation of highly branched modified starch, the optimization of starch gel properties or Application in the optimization of starch antiaging properties.

Compared with the untreated control, the starch branching enzyme treated tapioca starch at 40℃ for 10 minutes, the relative content of amylose in the enzyme-treated tapioca starch decreased to 23.4% of the control, and the degree of polymerization DP>25 branched chains in the modified starch decreased. The content increased significantly; when the reaction time was extended to 40 minutes, the relative content of amylose in the enzyme-treated tapioca starch decreased to 4.2% of the control, and the content of branch chains with polymerization degree DP<7 in the modified starch was significantly rise. After the enzyme was completely reacted, the content of resistant starch in tapioca starch increased from 3.31% in the control group to 12.80%, and the transparency and cold water solubility of tapioca starch increased from 31.8% and 23.1% in the control group to 96.1% and 96.6%, respectively.

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

Beneficial effects of the present invention:

(1) The present invention uses Corallococcus sp. strain EGB whose deposit number is CCTCC NO: M2012528 as a material, and with reference to the genome sequence information and combined with PCR amplification, to successfully obtain the gene sequence of starch branching enzyme. The full length of the gene (from the start codon to the stop codon) is 2178 bp, the G+C content is 69.65%, and it encodes 725 amino acids without signal peptide.

(2) The product of the gene expression of the starch branching enzyme, the enzyme activity of the starch branching enzyme is measured by the iodine solution method, and the starch branching enzyme can efficiently act on tapioca starch, amylopectin, soluble starch, wheat starch, potato starch, For corn starch, pea starch and amylose, the optimal catalytic substrate is tapioca starch. Under the optimal reaction conditions, the specific enzyme activity of the enzyme is as high as 19439.9±62.4U/mg.

(3) The recombinant starch branching enzyme of SEQ ID NO.1 obtained by using pET-29a as a carrier showed high catalytic efficiency and special catalytic properties in starch modification. In the catalytic reaction with tapioca starch as the substrate, the blue value of the reaction product within 3 min was significantly higher than that of the control group; within 15 min of the reaction, the content of branched chains with a polymerization degree of DP>25 in the modified starch prepared by the enzyme increased significantly, The content of resistant starch increased from 3.17% in the control group to 3.95%; after the reaction was complete, the branched chains with a polymerization degree of DP<7 in the modified starch prepared by the enzyme increased significantly, while the content of branched chains with DP>25 increased significantly decreased, and the content of resistant starch increased from 3.17% in the control group to 12.80%. The modified starch prepared by the enzyme showed strong anti-aging properties, which was directly related to the marked increase in the content of resistant starch and slow-digestible starch in the modified starch and the change of starch gel properties.

Description of drawings

Fig. 1 The pCR amplification electrophoresis of the gene encoding the starch branching enzyme

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

Figure 2 Schematic diagram of starch branching enzyme gene cloning and expression

Figure 3 SDS-PAGE electrophoresis of recombinant starch branching enzymes

M: Standard Protein Marker; 1: Supernatant of E. coli cell disruption containing pET-29a(+) empty vector; 2: Supernatant of pET-29a(+)-CcGBE engineering strain disruption (crude enzyme solution); 3: After purification CcGBE protein

Figure 4 Enzymatic properties of starch branching enzymes

A: Optimum temperature of starch branching enzyme CcGBE; B: Temperature stability; C: Optimum pH of starch branching enzyme; D: pH stability

Figure 5. Analysis of transparency and cold water solubility of modified starch prepared by starch branching enzyme CcGBE

Detailed ways

Example 1 Expression, purification and activity determination of starch branching enzymes

1.1 pCR amplification of starch branching enzyme gene

According to the completion map of Corallococcus sp.strain EGB genome and combined with NCBI genome information to predict gene function, the full length of SEQ ID NO. The genome of disclosed) was used as a template, and pCR amplification of the full-length starch branching enzyme gene was performed 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 2178 bp, the sequence GC content is 69.65%, and it encodes 725 amino acids, the amino acid sequence of which is SEQ ID NO. 2, without signal peptide. The primers used for pCR amplification are F and R, the amplification results are shown in Figure 1, and the construction process of the expression plasmid is shown in Figure 2.

F: GGAGATATACATATGGTGGACGCGGAGCTGCAG (SEQ ID NO. 3)

R: GTGGTGGTGGTGGTGCCCCGGCGTGAACCACACC (SEQ ID NO. 4)

1.2 Preparation of E.coli BL21(DE3) electrocompetent

Take the strain E.coli BL21(DE3) from the -80℃ refrigerator and streak it on a fresh LB plate, culture it overnight, pick a colony with a diameter of about 2mm and transfer it to SOB test tube without Mg ²⁺ , and cultivate it at 37℃ to OD600 After reaching 1.0, the inoculum amount of 1/100 was inserted into a 0.5L shake flask containing 100ml SOB medium, and cultured at 18°C and 220rpm until the OD600 reached 0.7-0.8; the shake flask was placed in an ice bath and cooled for 10min Afterwards, centrifuge at 4°C and 4000rpm for 5min to collect the cell pellet; after resuspending and washing the cells with an equal volume of sterile ultrapure water, centrifuge at 4°C for 5min at 4000rpm to collect the cell pellet; repeat the washing once; resuspend the cells in 100ml 10% glycerol , 4 °C 4000rpm centrifugation for 5min to collect the bacterial pellet; repeat washing once; carefully discard the supernatant, invert the centrifuge bottle and drain on sterile absorbent paper for about 1min. Each 1000ml of culture was carefully resuspended with 2ml of 10% glycerol, and 100μl of each tube was dispensed into centrifuge tubes and then quickly placed in a -80°C refrigerator for future use.

1.3 Recombinant plasmid construction and transformation

The pCR amplification product of the SEQ ID NO.1 gene was recovered, and the ClonExpress II OneStep Cloning Kit of Novozymes was used to carry out the in vitro recombination of the target fragment and the pET-29a expression vector. The recombination system was as follows:

Use a pipette to gently mix (do not shake and mix), centrifuge briefly to collect the reaction solution to the bottom of the tube, and react at 37°C for 30 minutes; drop to 4°C or immediately place on ice to cool. The recombinant plasmid was added to E.coli BL21 (DE3), placed on ice for 30 min, transferred to a 42°C water bath for heat shock for 90 s, then transferred to ice water for 2 min, and 800 μl of room temperature LB medium was added to recover for 45 min. Spread it onto LB plate containing 50 mg/L kanamycin, pick a single colony, verify the gene sequence is correct by sequencing, and store it in a -80°C low-temperature refrigerator with a final concentration of 15% glycerol.

1.4 Determination of enzyme activity of recombinantly expressed starch branching enzymes

Inoculate the E.coli BL21 (DE3) expression strain containing the recombinant expression plasmid into LB medium at 37°C and culture until the OD600nm is between 0.5-0.6, add IPTG to the concentration of 0.2mM, and continue to culture at 18°C for 24h. After the cells were collected and resuspended with Tris-HCl (pH 7.0), the cells were disrupted by ultrasonication, and centrifuged at 20 000 × g for 15 min. The supernatant obtained was the crude starch branching enzyme solution. After purification by Ni-NTA affinity chromatography and concentration by ultrafiltration, the recombinant starch branching enzyme was detected, and the SDS-PAGE electropherogram was shown in Figure 3. The activity measurement system is: 70 μl tapioca starch solution (0.2%, w/w, 20mM Tris-HCl buffer (pH9.0)) + 30 μl starch branching enzyme, react at 40°C for 10 min, stop the reaction in a boiling water bath, add 500 μl iodine solution, room temperature After standing for 20 min, the absorbance was measured at 660 nm. One unit of activity is defined as the amount of enzyme required to drop 1% per minute at 660nm.

Example 2. Study on the enzymatic properties of starch branching enzyme CcGBE

3.1 The effect of temperature on enzyme activity

The optimal reaction temperature determination scheme is as follows: at different temperatures (20°C, 30°C, 35°C, 40°C, 45°C, 50°C, 60°C), under the condition of 20mM Tris-HCl buffer (pH9.0) The activity of the recombinase, the highest enzyme activity was set to 100% (Fig. 4A). Determination of thermal stability: Incubate the recombinant enzyme at 20 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃ and 60 ℃, 20 mM Tris-HCl buffer (pH 9.0) for 1-9 h, in ice After rapid cooling, the residual enzyme activity was determined for each, and the enzyme activity without incubation was taken as 100% (Fig. 4B). The optimum reaction temperature of the starch branching enzyme CcGBE was determined to be 40°C, and it remained relatively stable between 20°C and 45°C.

3.2 The effect of pH on enzyme activity

Determination of the optimum reaction pH: in 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)], the activity of the recombinant starch branching enzyme CcGBE was determined at 40°C, and the highest activity was set as 100% (Fig. 4C). Determination of pH stability: The recombinant starch branching enzymes were kept in different buffer systems of pH 3.0-10.0 at 4°C for 24 hours, and then their residual activities were measured respectively, and the enzyme activity at pH 9.0 was 100% (Fig. 4D). The optimum reaction pH of the starch branching enzyme CcGBE was determined to be 9.0, and it remained relatively stable at pH 6.0-9.0.

3.3 Substrate specificity of starch branching enzymes

Using tapioca starch, amylopectin, soluble starch, wheat starch, potato starch, corn starch, pea starch and amylose as substrates, the substrate specificity analysis of the expressed starch branching enzymes was carried out (as shown in Table 1), and it was found that The starch branching enzyme CcGBE has activity on all the tested substrates, among which tapioca starch and amylopectin have the highest activity. Tapioca starch was used as the substrate for testing, and the activity of starch branching enzymes was detected by iodine solution. The unit of enzyme activity was defined as the amount of enzyme required to decrease OD ₆₆₀ value by 1% per minute as an activity unit. The specific activity of the starch branching enzyme CcGBE was 19439.9±62.4U/mg.

Table 1 Substrate specificity analysis of recombinant starch branching enzyme CcGBE

Example 3 Utilize starch branching enzyme CcGBE to prepare highly branched modified starch

Prepare 0.5% tapioca starch in boiling water bath for 30min to fully gelatinize, add starch branching enzyme CcGBE at 25000U/kg starch after cooling to room temperature, react at 40°C for different times, and stop the reaction in boiling water bath. Add isoamylase (100U/g starch) to the prepared modified starch, react at 38°C for 24h to carry out debranching reaction, and terminate the reaction in a boiling water bath. Analysis of the degree of polymerization of the branched chains was performed using high performance anion exchange chromatography (Thermo ICS 5000+, Thermo Fisher Scientific, USA). The results showed that the content of branch chains with polymerization degree DP>25 in modified starch treated with CcGBE for 15 min increased significantly, and the content of branch chains with polymerization degree DP<7 in modified starch after CcGBE treatment for 40 min increased significantly (Table 2). The results indicated that starch branching enzyme CcGBE could produce two types of modified starch with high degree of polymerization and low degree of polymerization according to the time it acts on starch.

Table 2 Analysis of branched chain polymerization degree of tapioca starch treated with starch branching enzyme CcGBE

Example 4. Analysis of anti-aging properties of modified starch prepared by starch branching enzyme CcGBE

The tapioca starch of 0min, 5min, 15min, 60min and complete reaction (360min) was treated with the starch branching enzyme prepared in Example 3, and stored at 4°C for 14 days. Differential scanning calorimetry (DSC823E, MettlerToledo, Switzerland) was used. ) to analyze the enthalpy change of modified starch treated with CcGBE at different times to judge the effect of starch branching enzyme CcGBE treatment on starch anti-aging. The results are shown in Table 3. Compared with the original starch’s enthalpy (ΔHg) of 1.66 J/g, the enthalpy of modified starch was reduced to 1.146 J/g after 5 minutes of branching enzyme treatment, and with the extension of CcGBE treatment time , the enthalpy of modified starch continued to decrease, and the enthalpy of modified starch after complete reaction was 0.40 J/g, which was significantly lower than that of the original starch sample. The results showed that the modified starch with high branching degree produced by starch branching enzyme CcGBE acting on tapioca starch showed strong anti-aging properties, which showed great potential in the anti-aging industrial application of modified starch.

Table 3 Analysis of gel properties of samples of tapioca starch treated with starch branching enzyme CcGBE at different times

Note: To: starting temperature; Tp: peak temperature; Tc: ending temperature.

Example 5. Application analysis of starch branching enzyme CcGBE in the preparation of resistant starch

The tapioca starch treated with starch branching enzyme CcGBE for 0min, 5min, 15min, 60min and complete reaction (360min) in Example 3 was placed in a 4°C refrigerator for 24h, then 3000U of porcine pancreatin was added, and placed in a 37°C water bath. After 12h of reaction, the content of reducing sugar released by hydrolysis was determined by DNS method. At the same time, different concentrations of glucose were reacted with DNS, the absorbance value (OD ₅₄₀ ) measured by spectrophotometer and the concentration of glucose standard solution were used to establish a standard curve, and the content of reducing sugar released from cassava starch after porcine pancreatin treatment was calculated, and then To evaluate the potential application value of starch branching enzyme CcGBE in the preparation of slow-digesting starch and resistant starch.

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

The results are shown in Table 4. The content of slow digestible starch (SDS) and resistant starch (RS) in modified starch after branching enzyme CcGBE treatment increased gradually with the prolongation of enzyme treatment time. The contents of slow-digestible starch and resistant starch in the original tapioca starch were 15.39% and 3.31%, respectively, while the contents of slow-digestible and resistant starch increased to 17.40% and 12.80%, respectively, after the complete reaction of CcGBE and tapioca starch. Compared with the original tapioca starch, the content of resistant starch in the modified starch increased by 286.7%, indicating the great application potential of CcGBE in the preparation of resistant starch.

Table 4 Analysis of the content change of nutritional starch in samples treated with starch branching enzyme CcGBE at different times

Example 6. Application analysis of starch branching enzyme CcGBE in changing starch gel properties and water solubility

After the tapioca starch treated with starch branching enzyme CcGBE for 0min, 5min, 15min, 60min and the complete reaction (360min) in Example 3 was cooled to room temperature, deionized water was used as a blank control (light transmittance was 100%), and ultraviolet spectroscopy was used. The transparency of the modified tapioca starch was measured by a photometer at a wavelength of 620 nm. At the same time, freeze-dry the starch samples prepared above, accurately weigh 0.15g of each group of starch freeze-dried samples, prepare 15ml of 1% (w/v) tapioca starch with deionized water, first stir and mix at low speed for 30s, and then high-speed After stirring for 5 min, the starch paste was transferred to a centrifuge tube after stabilization for 1 h, and centrifuged at low speed for 10 min at 4000 rpm/min. Pour the supernatant into a weighed petri dish, place the petri dish in a high temperature oven, and dry to constant weight. The calculation formula of the cold water solubility of the modified starch is: solubility (CWS)%=mass of the dried sample in the supernatant (g)/total mass of the sample (g)*100%.

The transparency and cold water solubility results of tapioca starch modified by branching enzyme CcGBE are shown in Figure 5. With the extension of CcGBE treatment time, the transparency and cold water solubility of tapioca starch increased significantly. After CcGBE treatment for 60 min, the transparency of tapioca starch reached 94.6 %, and the cold water solubility of tapioca starch is 93.8%. Therefore, tapioca starch modified with starch branching enzyme CcGBE has higher transparency, better cold water solubility, and improved fluidity and stability of starch stored at room temperature, which also indicates that starch branching enzyme has high transparency and cold water solubility in the preparation of The ability of modified starch has great potential application value.

sequence listing

<110> Nanjing Agricultural University

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

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2178

<212> DNA

<213> Corallococcus coralloides EGB (Corallococcus coralloides EGB)

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atccacgtgc tgccggagtt cgggggcaag gtgcccatgc agcaccgcac cggcggcgtc 180

ttcgaggcgc gcatcaacgg ccgcacggag cccttcagct acctgctgga ggtggagtac 240

ccgggcaaga aggtcttcac gctgcgcgac ccgtacagct tcctgcccac catcggggag 300

atggacctgt acttcgccgg cgagggccgc cacgagcggc tctgggagcg catgggcgcg 360

cacctcatcc accacaacgg cgtgaagggc acgtccttcg cggtgtgggc gcccaccgcc 420

cggggcgtgt ccgtggtggg cgacttcaac ggctgggacg ggcgcctgca cgccatgcgg 480

cgcatgggct cctccggcat ctgggagctg ttcatccccg aggtcggcga gggcacccgc 540

tacaagttcg agatccgtcc cggccacggc ggtggcccgc tgctcaagtc ggatcccttc 600

gccttccgca cggagacccc gcccgccacc gcgtcggtgg tgcatgacct gcgccgctac 660

aactggggcg acgacgcgtg gctggagggg cgcgaccgcc gcggagaggc ggcccagcag 720

ccgtggagcg tctacgaggt gcacctgggc agctggcgcc gcgtggtgga ggacggcgac 780

cggcccatga cgtaccgcga gctggcgccg gagctgtccc ggtacgtgaa ggagctgggc 840

ttcacgcacg tggagttcct gcccgtggcg gagcacccct acggcggctc ctggggctac 900

caggtgggcg gctactacgc gcccacgtcg cgcttcggcc acccggacga cttccgctac 960

ctggtggact acctccacca ggagggcatt ggcgtcatcg tggactgggt gccgggccac 1020

ttcccgcgcg acagccatgc gctgggccag ttcgacggca cggcgctcta tgagcacgcg 1080

gatccacgcc agggttcgca gccggactgg ggcacgctcg tcttcaactt cggccgcaac 1140

gaggtgcgca acttcctcat cgccaacgcg ctgttctggc tggaggagta ccacatcgac 1200

gggctgcgcg tggacgccgt ggcctccatg ctctacctgg actacagccg caagcagggc 1260

gagtggatcc ccaaccgctg gggcggccgc gagaacgaag aggccatcca gttcctgcgt 1320

gagctcaacg agaccatccg ccgcaagcac ccgggcgtgg tggtcatcgc ggaggagtcc 1380

accgcgtggc ccaaggtgtc ccagcccgtc agcgagggcg gcctgggctt cacgttcaag 1440

tggaacatgg ggtggatgca cgacacgctg tcgtacttct ccaaggacgc ggtctaccgg 1500

cagtaccacc acaaccagct caccttcggc ctgctgtacg cgttcagcga gaacttcatg 1560

ttgcccttga gccacgacga ggtggtgcac ggcaagggca gcctctacgg gcgcatgccg 1620

ggagacgcgt ggcagaagcg cgccaacctg cgcgcgctgt tcgcgtggat gtgggcccac 1680

ccgggaaaga agctgctctt catggggggt gagttcggcc agcccgccga gtggaaccac 1740

gacaagagcc tggactggca cctgctccac gatccgggcc acaagggcat ccagaagctg 1800

gtgggtgacc tgaaccgcgt gtaccgcgac ctgcccgcgc tctacgactg cgacagcgag 1860

ccccggggct tccagtggct gcagccggac gcatccgcgg cgaacgtgct ggccttcgtg 1920

cgccgctcgc gcacgcccgg ccgccacgtg gtgtgcgtgg ccaacctgtc gccggtgccg 1980

cgcgaggatt atcgcgtggg cttcccgctc cacggccgtt acgtggagct cgtcaacacc 2040

gacgcggggg agtacggcgg cagcggcctg ggcaaccggg gacaggtgca cacggagccc 2100

acgggctggg acggacagcc cgcttccgcg gtgctcaccc tgcctccgct gtcggtggtg 2160

tggttcacgc cggggtag 2178

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<213> Corallococcus coralloides EGB (Corallococcus coralloides EGB)

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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

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

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

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

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

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

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

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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

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

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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

<400> 3

ggagatatac atatggtgga cgcggagctg cag 33

<210> 4

<211> 34

<212> DNA

<213> Artificial Sequence

<400> 4

gtggtggtgg tggtgccccg gcgtgaacca cacc 34

Claims (10)

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.

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