Neoagarobiose hydrolase and application thereof

A new technology of agarobiose and hydrolase, applied in the direction of hydrolase, application, and introduction of foreign genetic material using carriers, etc., can solve the problems of no chemical synthesis reports, etc., and achieve good application potential and prospects, good application value, and reaction Environmentally friendly effect

Active Publication Date: 2013-12-25
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, even in the form of reagents, L-AHG is not commercially available, and there is no report of chemical synthesis

Method used

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  • Neoagarobiose hydrolase and application thereof
  • Neoagarobiose hydrolase and application thereof
  • Neoagarobiose hydrolase and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0018] Cloning of embodiment 1 new agarobiohydrolase gene

[0019] A new agarobiohydrolase gene from Agarophora chrysanthemum WH0801 (=NRRLB-59247(T)=CGMCC1.10131(T)) was cloned by PCR with degenerate primers and nested PCR. Specific operation: Cultivate Agarophore WH0801 in 2216E seawater medium until the end of the logarithm, and extract the DNA genome. Use specific degenerate primers for amplification. The sequence of the degenerate primers is as follows: 5'-GGYDCSTAYGATGATCGYAGCGTKTTYACC-3'; 5'-GGTRTTTTKTTCCGGRCCATCGGTG-3', using the genome as a template, the conditions are: 94°C for 5 minutes, then 94°C for 30 minutes Seconds, 30 seconds at 55°C, 1 minute at 72°C for 30 cycles, PCR was performed, and the product was a fragment of the new agarobiohydrolase gene, and then primers were designed based on the end sequence of the fragment, and three rounds of nesting were carried out A PCR product fragment with a size of 500Kb-2000Kb was recovered by formula PCR (using the chr...

Embodiment 2

[0020] Embodiment 2 Contains the construction of new agarobiohydrolase gene expression plasmid pETNA

[0021] According to the full-length sequence of the new agarobiohydrolase gene, the upstream primers and downstream primers of the gene were designed (see Example 1), and the genome of Agarophagus pale yellowus WH0801 was used as a template to amplify the new agarobiohydrolase The full-length sequence of the enzyme gene. Conditions were 94°C for 2 minutes, followed by 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 1.5 minutes, and 30 cycles of extension at 72°C for 10 minutes. Agarose gel electrophoresis showed an obvious single band at the position of 1.0kb. The single band was recovered by cutting the gel, and double-digested with Xho1 and BamH1, and the expression vector pET21a was also double-digested with Xho1 and BamH1, and then the PCR product and Restriction vectors are purified using kits. The DNA ligation reaction is then carried out at a certain molar ratio. ...

Embodiment 3

[0022] Example 3 Construction of engineering bacteria pETNA / BL21 highly expressing new agarobiohydrolase gene NABHwh

[0023] The expression vector plasmid pETna was transformed into BL21 (DE3) according to standard calcium chloride heat shock transformation, and positive transformants with ampicillin resistance were screened. The plasmid was extracted by standard alkaline lysis method, and the plasmid was double digested with Xho1 and BamH1, and the gel electrophoresis bands showed two clear bands of 1.0Kb and 5.3Kb, corresponding to the full length of the new agarobiohydrolase gene and the length of the plasmid . It proved that the expression vector plasmid pETNA containing the new agarobiohydrolase gene had been transferred into BL21 (DE3).

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Abstract

The invention provides a neoagarobiose hydrolase. The amino acid sequence of the neoagarobiose hydrolase is SEQ IDNO:1. The neoagarobiose hydrolase can degrade neoagarobiose to generate 3,6-anhydro-L-galactose (L-AHG) and D-galactose, and therefore the neoagarobiose hydrolase can be used for preparing pure L-3,6-inner ether galactose. Serving as a biocatalyst used for producing the L-AHG, the neoagarobiose hydrolase has a user-friendly reaction environment, high catalytic activity, a certain advantage over a chemical method and good application potential and prospects. The L-AHG is a kind of levoglucosan, has good skin whitening performance and anti-inflammatory activity and has very good potential application value in the aspects of medicine and cosmetics. According to recombined escherichia coli strains, recombined agarase expressed by pETNA/BL21 accounts for 28% of total protein. The expression level is high, the recombined agarase is easily purified and expression quantity is not reduced after continuous passage is carried out. The neoagarobiose hydrolase is adopted for preparing recombined neoagarobiose hydrolase and the L-AHG in a mass mode.

Description

technical field [0001] The invention belongs to the technical field of functional gene screening, and specifically relates to a new agarobiohydrolase and its application. technical background [0002] New agarobiose hydrolase is a hydrolase that degrades new agarobiose, hydrolyzes and breaks the α-1,3 bond of new agarobiose, and generates L-3,6-endether galactose (L-AHG) and D - Galactose. Neoagarose is the hydrolysis product of certain agarases that hydrolyze agar. L-AHG occurs naturally as a monomer as an alternating unit of L-AHG and galactose in agarose, which is a major constituent of red macroalgae (Rhodophyta). So far, even in the form of reagents, L-AHG is not commercially available, nor is there any report on its chemical synthesis. As a kind of inner ether sugar, L-AHG has good whitening and anti-inflammatory activities, and has good potential application value in medicine and cosmetics. As a biocatalyst that can produce L-AHG, the new agarobiohydrolase has cer...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N15/56C12N15/70C12N1/21C12P19/14C12P19/02
Inventor 毛相朝刘楠杨孟魏东芝
Owner OCEAN UNIV OF CHINA
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