Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Endo-1, 3-fucoidan enzyme and application thereof

A fucoidanase and enzyme activity technology, applied in the field of endo-1,3-fucoidanase, can solve the problems of low activity, high preparation cost, and difficult purification, and achieve high expression activity and high efficiency The effect of preparation

Active Publication Date: 2020-09-22
OCEAN UNIV OF CHINA
View PDF0 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved in the present invention is that wild endo-1,3-fucoidanase has low yield, low activity, and difficult purification, and the enzyme-producing strain needs to be induced by a fucoidan substrate to produce enzyme The cost of enzyme preparation is high, and there is no enzyme available for large-scale production of low molecular weight fucoidan and oligosaccharides

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Endo-1, 3-fucoidan enzyme and application thereof
  • Endo-1, 3-fucoidan enzyme and application thereof
  • Endo-1, 3-fucoidan enzyme and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Heterologous expression of endo-1,3-fucoidanase in Escherichia coli

[0023] Culture of W. fucanilytica CZ1127 in 2216E medium TThe whole genome DNA was extracted until the end of the logarithm, and the upstream and downstream primers were designed according to the target gene, and PCR was carried out using the whole genome as a template. The PCR conditions were 94°C for 5 minutes, then 94°C for 30s, 50°C for 30s, 72°C for 60s, and 25 cycles. The PCR product was identified by nucleic acid electrophoresis, recovered and purified by tapping the gel to obtain the endo-1,3-fucoidanase gene fragment (such as figure 1 ). The pET-28a(+) plasmid and the endo-1,3-fucoidanase gene fragment obtained above were digested with BamHI and XhoI at the same time, digested at 37°C for 2 hours, verified by nucleic acid electrophoresis, and recovered by tapping gel Genes and target plasmids, the recovered products were ligated at 16°C for 1 hour using DNA ligase, the ligated pr...

Embodiment 2

[0024] Example 2: Heterologous expression of endo-1,3-fucoidanase in Bacillus subtilis

[0025] Design upstream and downstream primers, and retrieve the target sequence by PCR. The PCR process is the same as 1). The pMA5 vector and the target sequence were double-digested with HindIII and EcoRI at the same time, and the digested target gene was connected to the same digested vector, and the constructed expression vector was transformed into the expression host of Bacillus subtilis BS WB600 . Positive recombinants were screened on kanamycin resistance plates. The correct recombinant bacteria were inoculated in LB medium containing kanamycin resistance, and cultured at 37°C for 12h. Then transfer to LB medium containing kanamycin resistance with 1% inoculum amount, and culture at 37°C for 16h. The bacteria were collected by centrifugation, the resuspended bacteria were washed with buffer, and ultrasonically disrupted to obtain an intracellular enzyme solution, that is, a crud...

Embodiment 3

[0026] Example 3: Heterologous expression of endo-1,3-fucoidanase in Pichia pastoris

[0027] Design upstream and downstream primers, and retrieve the target sequence by PCR. The PCR process is the same as 1). The pPIC9K vector and the target sequence were digested with EcoRI and NotI at the same time, and the digested target gene was connected to the pPIC9K vector. The constructed recombinant plasmid was digested overnight with SalI enzyme and recovered, and the linearized and non-linearized plasmids were detected by electrophoresis. Linearized intelligence was electrotransformed into GS115 competent cells, and a single colony was extracted for PCR verification. The single clones with correct sequencing and antibiotic selection were cultured in YPD liquid medium at 30°C and 220rpm for 12h. Then inoculated in BMGY medium with pH 6.0, cultured at 30°C and 220rpm until OD600 was 5.0, centrifuged, then added BMMY medium with pH 6.0 for cultivation, added 0.5% methanol for 12h t...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
molecular weightaaaaaaaaaa
Login to View More

Abstract

The invention relates to the technical field of biotechnology, in particular to an endo-1, 3-fucoidan enzyme and application thereof. The amino acid sequence of the endo-1, 3-fucoidan enzyme is SEQ IDNO.1, and the endo-1, 3-fucoidan enzyme is an enzyme which is derived from 1 through substitution, deletion or addition of one or more amino acids and has enzymatic activity of the SEQ ID NO.1. The invention provides an enzyme with the novel sequence, and breaks through the key bottleneck of efficient acquisition and practical application of the endo-1, 3-fucoidan enzyme and large-scale preparation of fucoidan low-molecular-weight polysaccharide and oligosaccharide.

Description

technical field [0001] The invention relates to the technical field of biotechnology, in particular to an endo-1,3-fucoidanase and its application. Background technique [0002] Fucoidan, also known as fucoidan sulfate, fucoidan, is an important class of marine food polysaccharides. Fucoidan has rich physiological regulation functions and good biomaterial properties, and its huge application potential is widely recognized. It is mainly composed of fucose and sulfate ester groups. According to the current classification, fucoidan can be divided into type I and type II. Type I fucoidan is composed of repeating unit structures connected by fucose through α1,3 glycosidic bonds. Sugars are composed of repeating unit structures in which fucose is alternately linked by α1,3 and α1,4 glycosidic bonds. Such structures are mainly distributed in brown algae of the order Fucus. [0003] The molecular weight of fucoidan is huge, the absorption rate and bioavailability are not high; it...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N15/56C12N15/70C12N15/75C12N15/81C12N1/21C12N1/19C12P19/04C12P19/00C12P19/12C12P19/14C12R1/19C12R1/125C12R1/84
CPCC12N9/24C12N15/70C12N15/75C12N15/815C12P19/04C12P19/00C12P19/12C12P19/14
Inventor 常耀光申晶晶薛长湖张玉莹梅轩伟陈广宁唐庆娟王玉明
Owner OCEAN UNIV OF CHINA
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com