Endo-type beta-agarase with main degradation product being neoagarobiose as well as application thereof

A new technology of agarobiose and agarase, applied in the application, hydrolase, glycosylase and other directions, can solve the problems of complex components, and achieve the effects of uniform product, stable enzyme properties and large yield

Inactive Publication Date: 2018-12-18
吴中宝
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, currently reported β-agarase hydrolysis products have complex components, usually containing n

Method used

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  • Endo-type beta-agarase with main degradation product being neoagarobiose as well as application thereof
  • Endo-type beta-agarase with main degradation product being neoagarobiose as well as application thereof
  • Endo-type beta-agarase with main degradation product being neoagarobiose as well as application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Artificial design and sequence analysis of β-agarase AgaB10

[0027] β-agarase gene of the present invention agaB10 Artificially designed, fully synthetic sequence (synthesized by Huada Genomics Co., Ltd.), containing 1,050 base sequences, encoding 350 amino acid sequences. Using the conserved domain analysis in NCBI, https: / / www.ncbi.nlm.nih.gov / Conserved domain (CDD, https: / / www.ncbi.nlm.nih.gov / cdd) and multiple sequence alignment Basic Local Alignment Search Tool (Blast, https: / / blast.ncbi.nlm.nih.gov / ) found that the C-terminus of this sequence contains a conserved region of β-agarase polysaccharide hydrolase family 50 (GHfamily 50), The N-terminus contains a carbohydrate-binding domain. Blast analysis found that the agarase sequence with the highest amino acid sequence similarity to AgaB10 was only 67%.

Embodiment 2

[0028] Example 2 Gene cloning and recombinant expression of β-agarase AgaB10

[0029] Fully synthesized in Example 1 agaB10 restriction endonuclease Nco I and xho I (purchased from Dalian Bao Biological Co., Ltd.) is the enzyme cutting site and the protection base of the enzyme cutting site is designed, and the recombinant primers are designed as follows (the underline is the restriction endonuclease site, and the italic is the restriction endonuclease protection base) :

[0030] Forward primer: SEQ ID NO.3: PAgaB10EF:

[0031] 5'- CATG CCATGG GTATGAACTTAAAAAAAACATTC-3’ ( Nco I)

[0032] Reverse primer: SEQ ID NO.4: PAgaB10ER:

[0033] 5'- CCG CTCGAG GAAATCGTAACCTGTAAT-3’ ( xho I)

[0034] The β-agarase gene was amplified by PCR using the above-mentioned recombinant primers, and the high-fidelity DNA polymerase PrimerstarHS used in PCR was purchased from Dalian Bao Biology Company. The specific PCR amplification conditions are: pre-denaturation at 94°C ...

Embodiment 3

[0038] Example 3 Fermentation process and purification preparation method of β-agarase AgaB10

[0039] The Escherichia coli BL21(DE3) / pET22b-AgaB10 constructed and stored at -80°C in Example 2 was streaked on the LB solid plate, and after culturing at 37°C for 16 hours, single clones were picked; the single clones were transferred to cells containing 50 μg / mL ampicillin in LB liquid medium (500 mL Erlenmeyer flask loaded with 50 mL liquid medium), cultured in a shaker at 37°C at 180 rpm to OD 600 =0.6. The 5 L fermenter was loaded with 60% (3 L) Terrific Broth (TB) medium, and sterilized in advance; 50 μg / mL ampicillin was added to the fermenter, and the cultured bacteria in the Erlenmeyer flask The solution was inoculated into a 5 L fermenter according to the inoculum amount of 2%. Adjust the initial ventilation rate to 50 L / h, the initial rotation speed to 350 rpm, the temperature at 37°C, and the dissolved oxygen at 15-40%; when the bacteria grow to OD 600 When =5.0, add ...

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Abstract

The invention relates to endo-type beta-agarase with a main degradation product being neoagarobiose as well as an application thereof. The beta-agarase is a novel beta-agarase AgaB10, and an amino acid sequence isas shown byas SEQ ID NO. 1. The beta-agarase is an artificially-designed novel beta-agarase and comprises 350 amino acids. The similarity of the beta-agarase provided inby the invention to the existing beta-agarase sequence is only 67 percent. The degradation way of the beta-agarase AgaB10 is an endo-incision way and has the characteristic that the product is homogenous, and the ratioof the neoagarobiose in the degradation product reaches 86.2 percent. In addition, the beta-agarase AgaB10 ofprovided by the invention is stable in performance, high in yield, and great in industrialized application potential.

Description

technical field [0001] The invention relates to an endo-type β-agarase whose main product is new agarose, and an application thereof, belonging to the field of biotechnology. Background technique [0002] Agar is a component of the cell walls of red algae such as Geliflower, and is one of the most abundant seaweed polysaccharides in the ocean. The structure of agar is complex, consisting of 1,3-linked β-D-galactopyranose and 1,4-linked 3,6-endether-α-L-galactopyranose residues alternately linked to form a backbone , and contains substituents such as sulfate group and methyl group. Agarase refers to a type of glycoside hydrolase that can degrade agar and produce agar oligosaccharides. It mainly comes from marine bacteria, and a small part comes from bacteria and marine molluscs in the terrestrial environment. According to the different glycosidic bonds and products when agarase degrades agarose, agarase can be divided into two categories: α-agarase and β-agarase. α-agarase...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12P19/14C12P19/12
CPCC12N9/2434C12P19/12C12P19/14C12Y302/01081
Inventor 吴中宝
Owner 吴中宝
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