Application of polysaccharide lyase coding gene 04147 in preparation of recombinant peach gum polysaccharide hydrolase

A coding gene, peach gum polysaccharide technology, applied in the direction of lyase, hydrolase, recombinant DNA technology, etc., can solve the problem of polysaccharide polymerization degree, composition, viscosity and quality of hydrolyzed products are difficult to control, the purity is not high, and it is not suitable for commercialization Preparation of peach gum hydrolyzing enzyme preparations and other issues to achieve good peach gum cracking activity and great application prospects

Active Publication Date: 2021-07-09
JINAN UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these treatments are non-directional hydrolysis of glycosidic bonds, and it is difficult to control the polysaccharide polymerization degree, composition, viscosity and quality of hydrolyzed products.
Bio-enzymatic degradation method, using glycosidic bond-specific enzymatic degradation of polysaccharides, can achieve directional hydrolysis, stably generate specific enzyme-catalyzed products, and has the advantages of mild reaction conditions and environmental friendliness, but at present, there is no commercial peach glue enzyme report
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 polysaccharide lyase coding gene 04147 in preparation of recombinant peach gum polysaccharide hydrolase
  • Application of polysaccharide lyase coding gene 04147 in preparation of recombinant peach gum polysaccharide hydrolase
  • Application of polysaccharide lyase coding gene 04147 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-04147 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-04147 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] (2) Set the following program for PCR reaction

[0057] 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.

[0058] (3) The...

Embodiment 2

[0091] Example 2 Expression Analysis of Peach Gum Polysaccharide Degradation Related Enzyme Encoding Gene orf-04147 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 (Amp) at a 1% inoculum size, and were 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...

Embodiment 4

[0139] Example 4 Study on Enzymatic Properties of Recombinant Peach Gum Polysaccharide Hydrolase 04147

[0140]Due to the complex structure and different degrees of polymerization of peach gum polysaccharides, even the same batch of products may have large differences in composition and structure. However, the study of enzymatic properties requires the use of substrates with clear components and stable quality for quantitative experiments. Therefore, this example uses the artificially synthesized glycoside structural analogue p-nitrophenyl-β-D-galactopyranoside (p-Nitrophenyl-β-D-Galactopyranoside, pNPGal) to replace peach gum polysaccharide to hydrolyze glycosides with recombinant enzymes. Key types for preliminary research.

[0141] 1. Specificity analysis of hydrolyzed glycosidic bond of gene recombinant peach gum polysaccharide

[0142] Add 90 μL of 1M pNPGal (p-nitrophenylgalactoside), 1M pNPRa (p-nitrophenylarabinoside) and 1M pNPGlu (p-nitrophenylglucoside) to the PCR ...

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Abstract

The invention discloses application of a polysaccharide lyase coding gene 04147 in preparation of recombinant peach gum polysaccharide hydrolase. According to the invention, the optimized polysaccharide lyase coding gene as shown in SEQ ID No. 3 is connected to an expression vector pET28a with a 6*His tag, and is transformed to escherichia coli to obtain a recombinant expression strain; then the recombinant expression strain is subjected to IPTG-induced expression of a recombinant protein; and then nickel affinity chromatography purification is performed to obtain the recombinant peach gum polysaccharide hydrolase and a single enzyme. The recombinant peach gum polysaccharide hydrolase has good peach gum cracking activity, can be used for preparing hydrolase of low-molecular-weight and small-molecular-weight peach gum, and has relatively great application prospects.

Description

technical field [0001] The invention relates to the technical field of peach gum polysaccharide lyase, in particular to the application of polysaccharide lyase coding gene 04147 in the preparation of recombinant peach gum polysaccharide hydrolase. Background technique [0002] Peach gum is peach, plum, apricot, cherry and other Rosaceae plants. After mechanical damage or microbial disease, a kind of gum is secreted by the bark. It is mostly light red or light yellow and transparent. It is a sticky substance. Acidic polysaccharides contain a small amount of protein and impurities at the same time, and have unique characteristics of homology of medicine and food. Ancient Chinese medical books record that peach gum is effective for bloody stranguria, thirst, stone stranguria and diarrhea. Modern medicine has proved that it can be used for cystitis, Treatment of type 2 diabetes. It has great development and application value. [0003] However, natural peach gum has a complex s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N9/88C12N15/70C12P19/14C12P19/00C12R1/19
CPCC12N9/2402C12N9/88C12N15/70C12P19/14C12P19/00
Inventor 冉艳红邓进丰杨晓苹王超张欣李弘剑
Owner JINAN UNIVERSITY
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