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Streptomyces-sourced broad-spectrum polysaccharide degrading enzyme rAly16-1 as well as coding gene and application thereof

A technology of polysaccharide degrading enzyme and coding gene, which is applied in the field of broad-spectrum polysaccharide degrading enzyme rAly16-1 and its coding gene and application, can solve the problems of precise application and directional molecular enzymatic transformation, lack of mechanism and cognition, etc. Achieve the effect of stable physical and chemical properties and high activity

Active Publication Date: 2021-07-20
JINAN ACTIVE BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the whole, there is no in-depth study on the catalytic properties and corresponding mechanisms of M-specific broad-spectrum alginate endonucleases, so there is a lack of relevant regular mechanisms and cognition, and the precise use and orientation of the enzymes Molecular enzymatic modification brings difficulties
[0006] In the prior art, it is found that an enzyme contains a multifunctional module, thereby catalyzing the characteristics of different polysaccharide substrates to generate oligosaccharides, but this discovery is limited to neutral polysaccharides (such as agarose, starch, and carrageenan, etc.), and acidic polysaccharides None reported in

Method used

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  • Streptomyces-sourced broad-spectrum polysaccharide degrading enzyme rAly16-1 as well as coding gene and application thereof
  • Streptomyces-sourced broad-spectrum polysaccharide degrading enzyme rAly16-1 as well as coding gene and application thereof
  • Streptomyces-sourced broad-spectrum polysaccharide degrading enzyme rAly16-1 as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0114] Extraction of Genomic DNA from Streptomyces sp. CB16 Strain

[0115] Streptomyces sp. CB16 strain was inoculated into TSB liquid medium, and cultured with shaking at 28°C and 200 rpm until the absorbance value at 600 nm (OD 600 ) was 1.21; take 20 mL of the cultured bacteria, centrifuge at 28°C and 12,000×g (g, the earth’s gravitational constant) for 20 minutes, and collect the bacterial sediment.

[0116] The above TBS liquid medium is composed of: tryptone 17g / L, plant peptone 3g / L, sodium chloride 5g / L, potassium dihydrogen phosphate 2.5g / L, glucose 2.5g / L, pH 7.2.

[0117] Add 12.0 mL of lysozyme buffer solution to the above cell precipitation to obtain about 14.0 mL of bacterial liquid, add 560 μL of lysozyme with a concentration of 20 mg / mL respectively, and the final concentration is about 800 μg / mL; place in an ice-water bath for 1.0 h , and then transferred to a 37°C water bath, warmed for 2h until the reaction system was viscous; added 0.82mL of sodium hexade...

Embodiment 2

[0119] Genome Scanning and Sequence Analysis of Streptomyces sp. CB16 Strain

[0120] The scanning and sequencing of the genomic DNA prepared in Example 1 was carried out by pyrosequencing technology, which was completed by Shanghai Meiji Biotechnology Company. The DNA sequencing results were analyzed using the online software of the NCBI (National Center for Biotechnology Informationh, ttp: / / www.ncbi.nlm.nih.gov / ) website. The analysis software used on the NCBI website is Open Reading Frame Finder (ORF Finder, http: / / www.ncbi.nlm.nih.gov / gorf / gorf.html) and Basic Local Alignment Search Tool (BLAST, http: / / blast.ncbi.nlm.nih.gov / Blast.cgi).

[0121] The results analyzed by the above biological software showed that Streptomyces sp. (Streptomyces sp.) CB16 strain genomic DNA carried a candidate polysaccharide degrading enzyme gene orf03870, the nucleotide sequence of which was shown in SEQ ID NO. There are 809 amino acids in the enzyme rAly16-1, and the 1-38 amino acids at th...

Embodiment 3

[0123] Recombinant expression of gene orf03870 in Escherichia coli BL21(DE3) strain

[0124] Using the genomic DNA prepared in Example 1 as a template, PCR amplification was performed. The primer sequences are as follows:

[0125] Forward primer orf03870-F: 5'-g catatg TCCGGCACGGCACGGACCG-3' (Nde I) SEQ ID NO.4;

[0126] Reverse primer orf03870-R: 5'-g tctaga GCTCCTCTTCAGGGTGAGGCC-3' (Xba I) SEQ ID NO. 5;

[0127] The underlined mark in the forward primer orf03870-F is the restriction endonuclease Nde I site, and the underlined mark in the reverse primer orf03870-R is the restriction endonuclease Xba I site. The high-fidelity DNA polymerase PrimeSTAR HSDNA Polymer used was purchased from China Dalian Biotech Co., Ltd., and the PCR reaction reagents used were operated according to the product instructions provided by the company.

[0128] PCR reaction conditions: pre-denaturation at 94°C for 5 minutes; denaturation at 98°C for 30 seconds; annealing at 65°C for 30 seconds...

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Abstract

The invention relates to a broad-spectrum polysaccharide degrading enzyme rAly16-1 derived from a streptomyces CB16 strain. The amino acid sequence of the broad-spectrum polysaccharide degrading enzyme rAly16-1 is shown as SEQ ID NO.3. The nucleotide sequence of a coding gene orf03870 of the broad-spectrum polysaccharide degrading enzyme rAly16-1 is as shown in SEQ ID NO. 1. The nucleotide sequence of the coding gene YH03870 of the broad-spectrum polysaccharide degrading enzyme rAly16-1 after codon optimization is shown as SEQ ID NO.2, and the amino acid sequence of the coding gene YH03870 is consistent with the original amino acid sequence. The broad-spectrum polysaccharide degrading enzyme rAly16-1 is the algin lyase which is found for the first time and belongs to the PL-8 family, the enzyme can degrade various acidic polysaccharides including chondroitin sulfate A (CSA), chondroitin sulfate C (CSC), dermatan sulfate (HS / DS), xanthan gum and algin, and the algin is taken as an optimal substrate.

Description

technical field [0001] The invention relates to a broad-spectrum polysaccharide degrading enzyme rAly16-1 derived from the genus Streptomyces and its coding gene and application, belonging to the field of biotechnology. [0002] technical background [0003] Biomass polysaccharides are widely distributed in nature and exist in almost all organisms [1] , such as peptidoglycan and cellulose that make up the cell walls of animals and plants [2] ; glycogen and starch as storage nutrients for plants and animals; chitin, the structural polysaccharide in the exoskeleton of crustaceans [3] . Studies have shown that some polysaccharides and their series of oligosaccharides have anticoagulant, antioxidative [4,5] ,Antitumor [6,7] ,Antiviral [8] , Anti-radiation and other special biological activities, participate in physiological processes such as molecular recognition, cell signal transduction and virus infection [9 , 10] . Polysaccharide degrading enzymes can catalyze the cl...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21C12P19/00C12P19/02C12R1/19
CPCC12N9/88C12N15/70C12P19/00C12P19/02C12Y402/02C12P19/12C12P19/26C12N2800/101C12N2800/22
Inventor 韩文君曾良欢古静燕李俊鸽李新卫洁
Owner JINAN ACTIVE BIOTECHNOLOGY CO LTD
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