Starch branching enzyme derived from myxobacteria, gene of starch branching enzyme, engineering bacterium containing gene and application of starch branching enzyme

A starch branching enzyme and gene technology, applied in genetic engineering, application, bacteria, etc., can solve the problems of low enzyme activity and catalytic efficiency, and low enzyme production of natural strains

Active Publication Date: 2022-04-12
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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 reported starch branching enzymes have low enzyme production in natural strains, low enzyme activity and catalytic activity. Low efficiency and other problems make the development of starch branching enzyme resources with high activity and excellent performance of great significance

Method used

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  • Starch branching enzyme derived from myxobacteria, gene of starch branching enzyme, engineering bacterium containing gene and application of starch branching enzyme
  • Starch branching enzyme derived from myxobacteria, gene of starch branching enzyme, engineering bacterium containing gene and application of starch branching enzyme
  • Starch branching enzyme derived from myxobacteria, gene of starch branching enzyme, engineering bacterium containing gene and application of starch branching enzyme

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Expression purification and activity determination of embodiment 1 starch branching enzyme

[0031] 1.1 pCR amplification of starch branching enzyme gene

[0032] According to the completion map of the Corallococcus sp.strain EGB genome and combined with NCBI genome information for gene function prediction, starch branching enzyme primers were designed using the full length of the SEQ ID NO. Open) genome as a template, carry out the pCR amplification of the full-length starch branching enzyme gene, 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%, and it encodes 725 amino acids. The amino acid sequence is SEQ ID NO.2 without signal peptide. The primers used for pCR amplification are F and R, and the amplification results are shown in electrophoresis figure 1 , the construction process of the expression plasmid refers to figure ...

Embodiment 2

[0043] Example 2. Study on Enzymatic Characteristics of Starch Branching Enzyme CcGBE

[0044] 3.1 Effect of temperature on enzyme activity

[0045] The determination scheme of the optimum reaction temperature 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 recombinase, set the highest enzyme activity as 100% ( Figure 4 A). Determination of thermal stability: Incubate the recombinant enzyme at 20°C, 30°C, 35°C, 40°C, 45°C, 50°C and 60°C, 20mM Tris-HCl buffer (pH9.0) for 1-9h, and place on ice Rapid cooling on the surface, the residual enzyme activity was determined respectively, and the enzyme activity without incubation was taken as 100% ( Figure 4 B). It has been determined that the optimum reaction temperature of the starch branching enzyme CcGBE is 40°C, and it remains relatively stable between 20°C and 45°C.

[0046] 3.2 Effect of pH on enzyme activity

[0047]...

Embodiment 3

[0053] Embodiment 3 Utilizes starch branching enzyme CcGBE to prepare highly branched modified starch

[0054] Prepare 0.5% cassava starch for 30 minutes in a boiling water bath to fully gelatinize. After cooling to room temperature, add starch branching enzyme CcGBE in an amount of 25000 U / kg starch, react at 40°C for different times, and terminate the reaction in a boiling water bath. Add isoamylase (100 U / g starch) to the prepared modified starch, react at 38° C. for 24 hours for debranching reaction, and terminate the reaction in a boiling water bath. Polymerization analysis of branched chains was performed using high performance anion exchange chromatography (Thermo ICS 5000+, Thermo Fisher Scientific, USA). The results showed that the content of branched chains with a degree of polymerization DP>25 in the modified starch after CcGBE treatment for 15 minutes increased significantly, and the content of branched chains with a degree of polymerization DP<7 in the modified st...

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Abstract

The invention discloses a myxobacteria-derived starch branching enzyme and a gene thereof, an engineering bacterium containing the gene and application of the engineering bacterium. The nucleotide sequence of the starch branching enzyme gene provided by the invention is SEQ ID NO. 1, and the amino acid sequence of the coded protein is SEQ ID NO. 2. The recombinant starch branching enzyme obtained by using an engineering strain constructed by the gene takes cassava starch as an activity detection substrate, and the specific activity of the recombinant starch branching enzyme is 19439.9 +/-62.4 U/mg through iodine solution detection under the optimal reaction condition. 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 modified starch with anti-aging characteristic, preparation of cold water soluble starch and the like. The prepared modified starch can be widely applied to the industries of food, brewing, fermentation, textile industry, medicine and the like, and 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 a starch branching enzyme and its gene derived from myxobacteria Corallococcus sp. Application of starch. Background technique [0002] As one of the most abundant carbohydrates in nature, starch 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, wherein amylose is composed of α-1,4-glycosidic bonds, and amylopectin is composed of α-1,4-glycosidic bonds. chains and branched chains linked by α-1,6-glycosidic linkages. According to the digestion rate and degree of digestion of starch in animals after eating, starch can be divided into fast digestible starch ( r apidly d igestible s tarch, RDS), slowly digestible starch ( s lowly d igestible s tarch, SDS) and resistant starch ( r resistant s tarch, RS). The formation of slow-digesting starch and resistant starc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/10C12N15/70C12N1/21C12P19/18C12P19/04C12R1/19
CPCY02P60/87
Inventor 崔中利叶现丰刘威黄彦李周坤
Owner NANJING AGRICULTURAL UNIVERSITY
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