Glucan branching enzyme and coding gene and application thereof

A technology encoding gene and glucan, applied in application, genetic engineering, glycosyltransferase and other directions, can solve the problems of difficulty in being degraded, accelerated aging rate of starch products, and reduced effective utilization of starch raw materials, and achieves good low temperature. Adaptability, excellent enzymatic properties, and strong substrate specificity

Active Publication Date: 2014-05-14
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Industrially produced starch raw materials contain about 18-33% amylose (Buleon A, Colonna P, Planchot V, et al. Starch granules: structure and biosynthesis. International J Biol Macromol1998, 23: 85-112), until The special linear structure of amylopectin makes it difficult to be degraded, resulting in a reduction in the effective utilization of starch raw materials in industrial production
In addition, amylose is easy to retrograde, which accelerates the aging rate of starch products, resulting in a decline in product quality

Method used

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  • Glucan branching enzyme and coding gene and application thereof
  • Glucan branching enzyme and coding gene and application thereof
  • Glucan branching enzyme and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1, the acquisition of glucan branching enzyme gene

[0043] 1. PCR amplification of the conserved region of the glucan branching enzyme gene genome

[0044] Genomic DNA of Rhizomucor miehei (Rhizomucor miehei) CAU432 was used as a template, with degenerate primers GBEDF and GBEDR, and Ex Taq DNA polymerase was used for PCR amplification to obtain PCR amplification products.

[0045] PCR reaction conditions: 94°C for 5min; 94°C for 30s, 60°C to 55°C (0.5°C decrease for each cycle) for 30s, 72°C for 1min, a total of 10 cycles; 94°C for 30s, 55°C for 30s, 72°C for 1min, 20 cycles Cycle; 72°C, 10min.

[0046] The PCR amplification product was detected by 1% agarose gel electrophoresis, and there was a specific band at about 460bp, which was recovered and connected to the pMD18-T vector, transformed into E.coli DH5α by heat shock method, and identified by colony PCR Subsequent sequencing. The sequencing results were searched and compared by NCBI BLAST, and the ...

Embodiment 2

[0064] Embodiment 2, the preparation of recombinant dextran branching enzyme

[0065] 1. Construction of recombinant bacteria

[0066] (1) Design and synthesize the following primers

[0067] RmGBEF: 5'-ATTCCG GAATTC ATGCTTACCGACGACTTTG-3'

[0068] (The underlined sequence is the recognition site for EcoRI digestion)

[0069] RmGBER: 5'-ATTCCG CTCGAG CTAATCTGCTTTTTCCCAATACA-3'

[0070] (The underlined sequence is the XhoI restriction recognition site)

[0071] (2) Extract the mRNA of Rhizomucor miehei CAU432 and reverse transcribe it into cDNA, use the cDNA as a template, and use RmGBEF and RmGBER as primers to carry out PCR amplification to obtain PCR amplification products.

[0072] PCR reaction conditions: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 2 min and 30 s, a total of 30 cycles; total extension at 72°C for 10 min.

[0073](3) EcoRI and XhoI double-digest the PCR amplification product t...

Embodiment 3

[0088] Example 3, Properties of Recombinant Glucan Branching Enzyme (RmGBE)

[0089] 1. Determination of the optimal pH of RmGBE: Glucan branching enzyme (RmGBE) enzyme solution and amylose (purchased from Sigma) were dissolved in five buffer systems with different pH values ​​as follows: citric acid / sodium citrate Buffer (pH3.0-6.0), MES (2-(N-morpholino)ethanesulfonic acid) buffer (pH5.5-6.5), disodium hydrogen phosphate / sodium dihydrogen phosphate buffer (pH6.0 -8.0), Tris-HCl buffer (pH7.0-9.0), glycine / sodium hydroxide buffer (pH8.6-10.6), and then measure the enzyme activity of dextran branching enzyme at 25°C. The highest activity point was taken as 100%, and the relative enzyme activity at each pH was calculated. The result is as figure 2 As shown in A.

[0090] 2. Determination of the pH stability of RmGBE: Dilute the glucan branching enzyme (RmGBE) enzyme solution with the above five buffer solutions with different pH values, and treat the diluted enzyme solution...

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Abstract

The invention discloses a glucan branching enzyme and a coding gene and an application thereof. A protein disclosed by the invention is shown as a formula (1) or (2): (1) a protein shown as SEQ ID No.2; (2) a protein which is obtained by substituting and / or deleting and / or adding the amino acid sequence shown as SEQ ID No.2 by one or more amino acid residues and is same in function. The glucan branching enzyme RmGBE disclosed by the invention has excellent enzymatic characteristics, and has a greater application potential in industries such as foods, feed and papermaking.

Description

technical field [0001] The invention relates to a glucan branching enzyme, its coding gene and application. Background technique [0002] Glucan branching enzyme (1,4-α-glucan branching enzyme, branching enzyme, α-1,4-glucan-6-glycosyltransferase; EC2.4.1.18), also known as Q enzyme, belongs to the GH13 family, is the A special class of enzymes in the family of enzymes, which can act on both α-1,4 and α-1,6-glycosidic bonds, and is the only one known in the GH13 family that can form α-1 between glucose polymers , 6-glycosidic bond enzyme. Glucan branching enzyme can cleave the α-1,4-glycosidic bond in the α-glucan molecule, and at the same time link the excised oligosaccharide to the glucan in the form of α-1,6-glycosidic bond to form a branch , is an essential enzyme in the biochemical synthesis of glycogen and starch, and has an important impact on the synthesis of starch and glycogen as well as its final structural and functional properties (Thiemann V, Saake B, Vollste...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21A21D8/04C12R1/645
CPCA21D8/042A21D13/00C12N9/107C12Y204/01018
Inventor 江正强吴淑朋杨绍青鲍庆丹闫巧娟刘昱
Owner CHINA AGRI UNIV
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