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Isoamylase, gene of isoamylase, engineering bacterium with gene and application of gene

A technology of isoamylase and starch processing, which is applied in the direction of genetic engineering, application, plant gene improvement, etc., and can solve problems such as unrealized industrial production

Active Publication Date: 2016-08-24
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, although a lot of progress has been made in the research of isoamylases from different sources and the properties of isoamylases in China, industrial production has not yet been realized.

Method used

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  • Isoamylase, gene of isoamylase, engineering bacterium with gene and application of gene
  • Isoamylase, gene of isoamylase, engineering bacterium with gene and application of gene
  • Isoamylase, gene of isoamylase, engineering bacterium with gene and application of gene

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Experimental program
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Embodiment 1

[0035] The cloning of embodiment 1 isoamylase gene and the construction of expression vector

[0036] 1.1 PCR amplification of isoamylase gene

[0037] Referring to the Myxobacteria EGB genome sequence and combined with NCBI genome information for ORF prediction, design primers for the isoamylase gene based on the full-length sequence, and use the genomic DNA of EGB bacteria (CCTCC NO: M2012528) as a template to perform PCR for the full length of the isoamylase gene Amplify to obtain the full-length sequence of the isoamylase gene, the primers used are F and R, the results are shown in figure 1 . Refer to the specific process figure 2 .

[0038] F: ctcgagAAAAGAGAGGCTATGACCCCCTCCCCGTCGTCAC(XhoI) (SEQ ID NO. 5)

[0039] R: tctagaCTTGGCCACCAACACCAGC (XbaI) (SEQ ID NO. 6)

[0040] 1.2 Preparation of E.coli DH10B electroporation competent

[0041] Streak the strain E.coli DH10B from the ‐80°C refrigerator on a fresh LB plate, culture overnight, pick a colony with a diameter ...

Embodiment 2

[0068] Example 2. High expression of isoamylase gene in P.pastoris GS115 (pEFaA-Iam)

[0069] 2.1 Expression of isoamylase IaM

[0070] Culture the expression strain P.pastoris GS115 (pEFαA‐IaM) on a streak plate, pick a single colony into a 50mL liquid YPD flask, culture at 28°C, 200rpm for 24h; then centrifuge at 4000rpm for 5min at room temperature, and discard the supernatant , resuspend the bacteria with 25mL BMMY medium, and start to induce the expression of yeast cells; continue to culture at 28°C, 200rpm, add methanol every 24h to a final concentration of 0.5% (v / v), and co-cultivate for 96h; After the culture was over, the enzyme activity of the target protein was detected, and it was found that the expression level of the isoamylase reached 0.5 μg / mL.

[0071] 2.2 Protein electrophoresis SDS-PAGE identification and renaturation zymogram

[0072] Protein samples were subjected to 12% polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, the lanes con...

Embodiment 3

[0073] Example 3. Research on Enzymatic Properties of Isoamylase

[0074] 3.1 Effect of temperature on enzyme activity

[0075] Determination of the optimum reaction temperature: at different temperatures (20°C, 30°C, 40°C, 45°C, 50°C, 55°C, 60°C, 70°C), the activity of the purified enzyme of the recombinant enzyme was measured under the conditions of pH 7.0, Set the highest enzyme activity to 100% ( Figure 5 a). Determination of thermal stability: Incubate the recombinant enzyme purified enzyme solution at 20°C, 30°C, 40°C, 45°C, 50°C, 60°C, 70°C, and pH 7.0 for 1 hour, take samples every 10 minutes, and quickly place on ice After cooling, measure the residual enzyme activity respectively, and take the unincubated enzyme activity as 100% ( Figure 5 b). It has been determined that the optimum reaction temperature of the amylase is 45°C, and it remains stable between 20°C and 45°C.

[0076] 3.2 The effect of pH on enzyme activity

[0077] Determination of the optimum re...

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Abstract

The invention belongs to the field of application industrial microorganisms, and discloses isoamylase, a gene of the isoamylase, an engineering bacterium with the gene and an application of the gene. The isoamylase gene serving as a key enzyme in the amylolytic enzyme system is provided, the full-length of the gene is 2,436 bp, the content of G and C is 66%, 811 amino acids are encoded, the nucleotide sequence of the gene is SEQ ID NO.1, and the amino acid sequence of protein of an encoded incision enzyme is SEQ ID NO.2. Engineering strains constructed with the gene can efficiently express the isoamylase, potato starch serves as an activity testing substrate of the isoamylase, the activity of the isoamylase is detected through an iodine solution, and the specific activity of the isoamylase is as high as 70,600 U / mg. An enzymic preparation produced with the gene can be used for industries such as grain processing, the food industry, making, fermenting, the textile industry and the medicine industry. Considerable economic benefits can be obtained while practical problems are solved.

Description

technical field [0001] The invention belongs to the field of applied industrial microorganisms, and discloses an isoamylase gene, engineering bacteria containing the gene and applications thereof. Background technique [0002] As one of the most abundant polymers on earth, starch is composed of glucose monomer units connected by glucosidic bonds. According to the difference of glycosidic bonds, it can be divided into two important components: amylose (linear α‐1, glucans linked by 4 bonds) and amylopectin (linear glucans linked by α‐1,4 bonds with branched chains of α‐1,6 bonds), although α‐1,6 bonds only accounted for the total glycosides About 6% of the bonds make the starch sugar chains form a branched structure. Due to the complexity of starch structure, it is difficult for a single enzyme to completely hydrolyze it. Therefore, the synergistic action of multiple enzymes is usually required in the starch processing process, including α‐amylase and starch debranching enzy...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N9/44C12N15/81C12N1/19C12P19/16C12P19/14C12P19/04A23L29/30A23K20/189A23K10/14C12R1/84
CPCA23V2002/00C12N9/246C12N15/815C12N2800/102C12P19/04C12P19/14C12P19/16C12Y302/01068A23V2250/5118A23V2300/28
Inventor 崔中利李周坤冀凯黄彦
Owner NANJING AGRICULTURAL UNIVERSITY
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