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Transeliminase encoding gene and enzyme and preparation and application

A technology of pectate lyase and gene, applied in the direction of lyase, carbon-oxygen lyase, application, etc., can solve the problems of weak degradation activity of pectin polysaccharides and unsuitable for large-scale application, and achieve good activity and efficient degradation effect

Inactive Publication Date: 2020-06-09
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The reported pectate lyases have weak degradation activities on pectin polysaccharides and are not suitable for large-scale application.

Method used

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  • Transeliminase encoding gene and enzyme and preparation and application
  • Transeliminase encoding gene and enzyme and preparation and application
  • Transeliminase encoding gene and enzyme and preparation and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Pectate lyase Ap Pel full-length gene cloning

[0044] The total RNA of Aspergillus parasitica was extracted according to the operating procedure of the Column Fungal Total RNA Extraction and Purification Kit (Shanghai Sangong), and the first-strand cDNA was synthesized according to the operating procedure of the RevertAid Frist Strand cDNA Synthesis Kit (Thermo Scientific). After performing multiple sequence alignment analysis on the pectate lyase gene sequence in The National Center for Biotechnology Information (NCBI) database, degenerate primers were designed:

[0045] Ap Pel-F: 5'-CAGTCTCGAGAAAAAGACAGAAAGTCTCTGGTGCC-3';

[0046] Ap Pel-R: 5'-ATCTTCTAGAGATCTTGCCCGCACCAGCATTC-3',

[0047] Using the first-strand cDNA of Aspergillus parasitica as a template, the gene sequence encoding pectate lyase protein (excluding the signal peptide gene) was amplified. The PCR reaction conditions were: 98°C for 3min, 1 cycle; 98°C for 10s, 55°C for 15s, 72°C for 2min, 3...

Embodiment 2

[0048] Example 2 Analysis of pectate lyase gene sequence

[0049] The sequencing results were analyzed using Basic Local Alignment Search Tool (BLAST) in the GenBank database, DNAMAN software was used for multiple sequence alignment, and Vector NTI was used to analyze sequence information.

[0050] The coding region of the obtained pectate lyase gene (named Ap Pel) is 1086 bp long, and its nucleotide sequence is shown in SEQ ID NO 1. Ap Pel encodes 361 amino acids and a stop codon, its amino acid sequence is shown in SEQ ID NO 2, the theoretical molecular weight of the protein is 38.10kDa, and the predicted isoelectric point is 8.90. The amino acid encoded by ApPel has a pectate lyase central domain, which indicates that ApPel is a member of the polysaccharide lyase family.

Embodiment 3

[0051] Example 3 Heterologous expression and purification of pectate lyase Ap Pel in Pichia pastoris

[0052] Pectate lyase Ap Pel was induced and expressed according to the method of Pichia pastoris expression manual. The expression and purification of pectate lyase Ap Pel were detected by polyacrylamide gel electrophoresis, and the results were as follows: figure 2 As shown, the purified pectate lyase Ap Pel presents a single band on the electrophoresis gel, and its position (about 38 kDa) coincides with the predicted molecular weight (38.10 kDa).

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Abstract

The invention discloses a preparation and application of a transeliminase encoding gene derived from Aspergillus parasiticus, and an enzyme of the transeliminase encoding gene, that is, a gene of transeliminase is cloned to a pichia pastoris expression vector by using a technical method of gene engineering, then a pichia pastoris recombinant strain for heterologous expression of the enzyme can beobtained, the strain is capable of heterologously expressing the prepared transeliminase and efficiently degrading pectin polysaccharides and polygalacturonic acid (PGA). The transeliminase disclosedby the invention can be widely applied to fields such as agriculture, industry, foods, feed additives, medicines and pectin oligosaccharide preparation.

Description

technical field [0001] The present invention relates to a gene sequence of pectate lyase, its preparation method and application. The invention provides the recombinant plasmid and recombinant genetic engineering strain of the pectate lyase and its application in polysaccharide degradation. The pectate lyase provided by the invention can be widely used in the fields of agriculture, food, feed addition, medicine, oligosaccharide preparation and the like. Background technique [0002] Pectin is the polysaccharide with the most complex structure and function in the plant cell wall, and it exists widely in nature; pectin is distributed in the soft tissue cell wall, intercellular layer, Cell and secondary wall connection area, etc.; pectin polysaccharide is a high molecular compound with an average molecular weight between 20,000 and 400,000. The structure of pectin polysaccharides is very complex, mainly including homogalacturonan (HG), accounting for about 65% of pectin; rham...

Claims

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Application Information

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IPC IPC(8): C12N15/60C12N9/88C12P19/00C12P19/02
CPCC12N9/88C12P19/00C12P19/02C12Y402/02002
Inventor 尹恒杨国军
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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