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Application of arabinogalactosidase coding gene 00578 in preparation of recombinant peach gum polysaccharide hydrolase

A technology of arabinogalactose and arabinogalactan, which is applied in the field of peach gum polysaccharide lyase, can solve the problems of unsuitable commercial preparation of peach gum hydrolytic enzyme preparations and low purity, and achieve good peach gum polysaccharide degradation activity and large The effect of applying the foreground

Pending Publication Date: 2022-03-04
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although the Chinese patent CN104651340A discloses the peach gum polysaccharide lyase derived from Microbacteria, it is obtained by separating and purifying the culture of fermented culture after domestication of Microbacterium A5, and what it obtains is that the peach gum polysaccharide lyase is metabolized by microorganisms. The compound product is not high in purity and is not suitable for commercial preparation of peach gum hydrolase preparations

Method used

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  • Application of arabinogalactosidase coding gene 00578 in preparation of recombinant peach gum polysaccharide hydrolase
  • Application of arabinogalactosidase coding gene 00578 in preparation of recombinant peach gum polysaccharide hydrolase
  • Application of arabinogalactosidase coding gene 00578 in preparation of recombinant peach gum polysaccharide hydrolase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Cloning of the gene orf-00578 encoding enzymes related to the degradation of peach gum polysaccharides and construction of recombinant plasmids

[0047] 1. Method

[0048] 1. Design of PCR primers

[0049] According to the ORF 00578 gene sequence obtained from Microbacterium genome sequencing, PCR primers were designed using the software Primer premier5.0, and the primer sequences are shown in Table 1 below. The primers were synthesized by Shanghai Sangon Bioengineering Company.

[0050] Table 1

[0051]

[0052] 2. PCR reaction

[0053] (1) Using Microbacterium genomic DNA as a PCR template, add each component as shown in Table 2, and mix;

[0054] Table 2

[0055]

[0056]

[0057] (2) Set the following program for PCR reaction

[0058] PCR reaction conditions: pre-denaturation at 98°C for 10 min; denaturation at 98°C for 45 sec, annealing at 55°C for 40 sec, extension at 72°C for 1 min, a total of 35 cycles; extension at 72°C for 8 min.

[0...

Embodiment 2

[0091] Example 2 Expression Analysis of Peach Gum Polysaccharide Degradation Related Enzyme Encoding Gene orf-00578 Recombinant Plasmid

[0092] 1. The growth curve of recombinant Escherichia coli BL21 was measured, and the logarithmic growth phase of Escherichia coli BL21 (DE3) was measured between 2 and 2.5 hours, and the corresponding A600nm was between 0.5 and 0.8, so this time period was selected It is the starting point for subsequent IPTG induction.

[0093] 2. Induced expression of recombinant target protein

[0094] (1) Escherichia coli BL21 expression bacteria and plasmid empty bacteria were inoculated into liquid LB medium containing corresponding antibiotics (Kan) at a 1% inoculum size, and activated overnight in a constant temperature shaker at 37°C and 220 rpm.

[0095] (2) The next day, transfer the transfer bacteria solution to fresh LB medium at 1%, and cultivate until the OD of Escherichia coli is detected by the spectrophotometer 600 When the value is 0.5-...

Embodiment 3

[0129] Example 3 Verification of polysaccharide hydrolysis function of 00578 target protein

[0130] 1. Preparation of standard curve for the determination of reducing sugar by DNS colorimetric method

[0131] (1) Take 6 EP tubes and number them sequentially;

[0132] (2) Add 200 μL of standard glucose solutions with different concentrations, the concentrations are 0, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL;

[0133] (3) Add 100 μL 3,5-dinitrosalicylic acid reagent and shake well;

[0134](4) Heating in a boiling water bath for 5 minutes, taking it out immediately after the water bath to cool, and measuring the change of the absorbance value of each system at 540nm;

[0135] (5) Draw a standard curve according to the absorbance value and the concentration of the reducing sugar solution.

[0136] 2. DNS chromogenic method to determine the enzyme activity of arabinogalactosidase

[0137] Add 40 μL 6% peach gum solution and 40 μL crude enzyme solution (that is, ...

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Abstract

The invention discloses an application of an arabinogalactosidase coding gene 00578 in preparation of recombinant peach gum polysaccharide hydrolase, which is characterized in that the arabinogalactosidase coding gene shown as SEQ ID NO: 1 is connected to an expression vector pET22b (+) with a 6 * His tag, and is converted to escherichia coli to obtain recombinant expression bacteria; the method comprises the following steps: preparing a recombinant peach gum polysaccharide hydrolase, performing IPTG (isopropyl-beta-d-thiogalactoside) induced expression on the recombinant expression bacteria to obtain a recombinant protein, and performing nickel affinity chromatography purification to obtain the recombinant peach gum polysaccharide hydrolase which has good peach gum splitting decomposition activity and arabinogalactan and galactan hydrolysis activity. The peach gum polysaccharide hydrolase can be used for preparing peach gum polysaccharide hydrolase and polysaccharide hydrolase containing arabinogalactan and galactan, and has a great application prospect.

Description

technical field [0001] The invention relates to the technical field of peach gum polysaccharide lyase, in particular to the application of arabinogalactosidase coding gene 00578 in the preparation of recombinant peach gum polysaccharide, arabinogalactan and galactan hydrolase. Background technique [0002] Peach gum belongs to Rosaceae plant gum, which is a brown gelatinous substance secreted by the bark of peach trees after mechanical damage. Because of its similar properties to Arabic gum, peach gum is also known as "domestic Arabic gum". Compared with gum arabic, peach gum with the same concentration has higher transparency and viscosity than gum arabic. It can replace gum arabic in adhesives, setting agents, protective glue, thickeners, film-forming materials, emulsifiers and other industries. It also has a unique homologous property of medicine and food. It is recorded in ancient Chinese medical books that peach gum is effective for bloody stranguria, thirst, stone str...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N9/24C12N15/70C12P19/14C12P19/02
CPCC12N9/2402C12N15/70C12P19/14C12P19/02C12Y302/01089C12Y302/01023
Inventor 冉艳红邓进丰蔡雨薇王婷杨晓苹李弘剑
Owner JINAN UNIVERSITY
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