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Beta-agaropectionase gene aga B, its preparation method and application

A technology of agarase and pet24-agab, which is applied in the direction of biochemical equipment and methods, enzymes, and the use of vectors to introduce foreign genetic materials, can solve the problems of severe reaction conditions, poor specificity, and unsatisfactory production and scientific research, and achieve The effect of high expression level, low cost and easy purification

Inactive Publication Date: 2003-12-10
青岛中国海洋大学控股有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the preparation of oligosaccharides is still a worldwide technical problem. The traditional method of acid-degrading agar has severe reaction conditions, poor specificity, and the hydrolyzate is easily destroyed, which is not conducive to the analysis and recovery of the product. The conditions are easy to control, which is conducive to the large-scale preparation of specific oligosaccharides, so enzymatic hydrolysis instead of acid hydrolysis has become a trend
Most of the β-agarase degradation products found so far are new agarose, new agarotetraose and new agarosexose, so now there are only standard products of di-, tetra-, and hexaose in the market, which are far from meeting the needs of production and scientific research. need

Method used

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  • Beta-agaropectionase gene aga B, its preparation method and application

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Embodiment 1

[0017] The β-agarase gene agaB of Pseudomonas CY24 was cloned by the shotgun method, and the specific operation was to screen the agarase gene agaB from the Pseudomonas genome library constructed in our laboratory using agar as the only carbon source. Cloning and sequencing of agar activity. Using the biological software Primer 5.0 to analyze the reading frame and the appropriate restriction site, the reading frame was digested and subcloned respectively, and the active target clone was screened on the agar plate. Design upstream primers (5'-CATATGTTAAAGCGCCACCAAGC-3') and downstream primers (5'-CTCGAGTTGGCAAGTATAACCT-3') according to the sequencing results, and use genomic DNA as a template to carry out PCR reactions. The PCR reaction composition is as follows (10 μl reaction system): 0.8 μl dNTP, 1 μl 10×PCR buffer, 0.05 μl (5 U / μl) Taq enzyme, 1 μl DNA template, 0.1 μl each of upstream and downstream primers. The reaction conditions are: pre-denaturation at 94°C for 2 minu...

Embodiment 2

[0018] Connection of β-agarase agaB gene and carrier pBS-T, 20 μl reaction system: pBS-T 0.5 μl, PCR product 10 μl obtained in Example 1, T 4 DNA ligase 1.0 μl, 10×T 4 DNA ligase buffer 2.0 μl, ddH 2 O 6.5 μl of the connection liquid obtained by connecting overnight at 16°C can obtain the E. coli cloning vector pBS-agaB, which is used to transform E. coli DH5α. Example 3: Construction of Escherichia coli recombinant strain DH5α-pBS-agaB

Embodiment 3

[0019] Add 100 μl of thawed competent E.coli DH5α and 10 μl of the connection solution obtained in Example 2 to a 1.5 ml Eppendorf tube, ice-bath for 30 min, 42 °C for 90 seconds, ice-bath for 3 min, add 900 μl of LB medium, and shake at 37 °C for 1 hour. The bacterial liquid was mixed with 4 μl IPTG and 40 μl X-gal, spread on the LB plate containing 50 μg / ml ampicillin, and cultured overnight at 37°C. Pick a single white colony for PCR detection, and the one showing a 1.4kb specific band in agarose gel electrophoresis is a positive transformed colony, that is, Escherichia coli DH5α-pBS-agaB containing pBS-agaB. The plasmid was extracted from DH5α-pBS-agaB by alkaline lysis method, and the nucleotide sequence of the exogenous insertion sequence agaB was determined with the general sequencing primer of pBluescript II KS(+). Example 4: Construction of Escherichia coli expression vector pET24-agaB

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Abstract

The present invention relates to a beta-gelase gene agaB. It is a gene of beta-gelase of coded pseudoallomonad CY24. Said beta-gelase coded by beta-gelase gene agaB can specifically degrade agar to produce new agar oligose whose degree of polymerization is 8-14, said new agar oligose content content in the degraded product is greater than 20%. The recombinant beta-glase expressed by colibacillus recombinant strain BL21-pET24-agaB is 25% of total protein, its expressing level is high, and its purification is easy, so that it can be used for large-scale production of beta-gelase.

Description

technical field [0001] The present invention relates to a β-agarase gene agaB, in particular to a β-agarase gene agaB derived from Pseudoalteromonas sp. CY24 and its cloning, expression, preparation and application of the expression product . Background technique [0002] Agarase is mainly derived from marine microorganisms, and various agarases have been isolated and purified from marine Pseudomonas and Vibrio. According to the different modes of action of agarase, it can be divided into two categories: α-agarase and β-agarase. α-agarase cleaves the α-1,3 glucosidic bond of agarose to generate β-D-galactose as the non-reducing end and 3,6-endether-α-L-galactose as the reducing end Agar oligosaccharide series; β-agarase cleaves the β-1,4 glucosidic bond of agarose to generate β-D-galactose as the reducing end and 3,6-inside ether-α-L-semi Lactose is a new series of agar-oligosaccharides with non-reducing ends. Agarase has a wide range of applications, and it was first us...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N9/00C12N15/52C12N15/70C12P19/00
Inventor 于文功马翠萍路新枝韩峰褚艳石超
Owner 青岛中国海洋大学控股有限公司
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