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Branching enzyme and application of branching enzyme for preparing resistant dextrin

A technology of resistant dextrin and branched enzymes, applied in the field of bioengineering, can solve the problems of restriction potential and low enzyme production capacity of natural strains

Inactive Publication Date: 2017-11-24
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the low enzyme-producing ability of natural strains limits their potential for industrial applications

Method used

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  • Branching enzyme and application of branching enzyme for preparing resistant dextrin
  • Branching enzyme and application of branching enzyme for preparing resistant dextrin
  • Branching enzyme and application of branching enzyme for preparing resistant dextrin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1: the construction of genetically engineered bacteria

[0041] 1. Design a pair of primers P1 and P2 according to the gene sequence of Tfu_0582 (Genbank accession number NC_007333.1:663757-666006). The underlines are the enzyme cutting sites Nde Ⅰ and Hind Ⅲ,

[0042] P1: 5'-C CATATG ACCGCCCGGCCTGCAGT-3'

[0043] P2: 5'-C AAGCTT TCACGTCCCGTCGAACACCAGC-3'

[0044] Using the total DNA of Thermuobifidafusca WSH03-11 (published in 2008, in a paper titled Identification and characterization of bacterial cutinase) as a template, and using P1 and P2 as primers, PCR amplified the gene encoding the branching enzyme (Genbank accession number NC_007333.1: 663757 -666006), then cloned into the pMD18-T simple vector (commercialized tool vector), the ligation product was transformed into Escherichia coli JM109, and the transformation product was coated with an LB plate containing 100 mg / L ampicillin. After culturing overnight at 37°C, colonies were selected and inser...

Embodiment 2

[0047] Embodiment 2: fermentation produces branching enzyme

[0048] 1. Transfer the glycerol tube strain to LB medium for 37°C liquid culture overnight, and then insert it into TB fermentation liquid medium (glycerol 5g / L, peptone 12g / L, yeast extract 24g / L, K 2 HPO 4 12.54g / L, KH 2 PO 4 2.31g / L), cultured at 37℃ to OD 600 After reaching 0.6, induce with a final concentration of 0.12-0.2mm / L isopropylthio-β-D-galactoside (IPTG), then transfer to 25°C for 48 hours, centrifuge the bacteria, and use pH 6.5 0.05mol / L Cells were suspended in sodium phosphate buffer, ultrasonically disrupted, and centrifuged to measure the branching enzyme activity in the supernatant. The fermentation activity of the recombinant branching enzyme reached 2500 U / mL, and the SDS-PAGE electrophoresis of the recombinant branching enzyme protein was shown in figure 1 .

[0049] Using amylose as the substrate, the enzyme activity was measured at different temperatures, and the results showed that t...

Embodiment 3

[0050] Example 3: Application of starch branching enzyme in the preparation of resistant dextrin

[0051] Taking starch as a substrate, adding 5% 1mol / L HCl solution to the starch, and then performing high-temperature reaction at 160-200°C, the resistant components are 40%-45% after cooling and sieving. After high-temperature acid hydrolysis, the substrate concentration of 200g / L-300g / L was prepared for enzyme conversion at different temperatures. The temperature of enzyme conversion was 35-45°C, and enzyme conversion was carried out under different pH conditions. The pH of enzyme conversion was found. The efficiency is higher in 6-7, and the amount of enzyme added and the reaction time of enzyme conversion are detected, and the amount of enzyme added is 1000-1500U / g, and the reaction time of enzyme conversion is 8-12h, and the content of resistant dextrin reaches 60%. , increased by more than 10% after higher temperature acid hydrolysis.

[0052] The obtained resistant dextr...

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Abstract

The invention discloses branching enzyme and an application of branching enzyme for preparing resistant dextrin, which belongs to the technical field of bioengineering. According to recombinant escherichia coli provided in the invention, the enzyme activity of starch branching enzyme can reach 2500 U / mL, the optimum pH of the produced starch branching enzyme is 6.5, and the optimum temperature is 40 DEG C, and a half-life period at the temperature of 40 DEG C can reach 200 h. The starch branching enzyme takes starch as a substrate, the concentration of the substrate through high-temperature acidolysis is 200 g / L-300 g / L, when the temperature is 35-45 DEG C, the enzyme addition amount is 1000-1500 U / g, the enzymatic conversion is carried out at a reaction time of 8-12 h to prepare the resistant dextrin, a resistance component can reach 60%, and a base for industrial production of the resistant dextrin is established.

Description

technical field [0001] The invention relates to a branching enzyme and its application in the preparation of resistant dextrin, belonging to the technical field of bioengineering. Background technique [0002] Resistant dextrin is also called indigestible dextrin. It is made from cornstarch. Because it contains resistant ingredients, it can resist the action of human digestive enzymes. It will not be digested and absorbed in the digestive tract and can directly enter the large intestine. At the same time, as a low-molecular water-soluble dietary fiber, resistant dextrin has the physiological function of dietary fiber, and also has many characteristics of food raw materials, so it is easy to add to processed foods. Now resistant dextrin is mainly used in the production of cool drinks, fruit juice drinks, seasonings, jams, etc.; in recent years, research at home and abroad has found that it also has therapeutic and health effects in hyperglycemia and hyperlipidemia. The resea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/18C12P19/08C12N1/21C12N15/70C12N9/10C12R1/19
CPCC12N9/107C12N15/70C12P19/08C12P19/18C12Y204/01018
Inventor 吴敬陈晟刘军
Owner JIANGNAN UNIV
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