Beta-agarase with homogeneous degradation products and application thereof

A technology of degradation products and agarase, which is applied in the biological field, can solve problems such as complex components, and achieve the effects of uniform product, good industrial application prospects, and stable properties

Inactive Publication Date: 2019-01-11
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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  • Beta-agarase with homogeneous degradation products and application thereof
  • Beta-agarase with homogeneous degradation products and application thereof
  • Beta-agarase with homogeneous degradation products and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Artificial design and sequence analysis of β-agarase AgaB1

[0027] β-agarase gene of the present invention agaB1 Artificially designed, fully synthetic sequence (synthesized by Huada Gene Company), including 1,620 base sequences, encoding 540 amino acid sequences, the N-terminal is the catalytic region (R1-V392), and the C-terminal contains a 148-amino acid sequence Carbohydrate binding domain (G393-Y540), this domain region can enhance the substrate recognition ability of β-agarase enzyme and change the distribution of degradation products. Conserved domain (CDD, https: / / www.ncbi.nlm.nih.gov / cdd) was analyzed using the conserved domain in National Center for Biotechnology Information (NCBI, https: / / www.ncbi.nlm.nih.gov / ) and Multiple Sequence Alignment Basic Local Alignment SearchTool (Blast, https: / / blast.ncbi.nlm.nih.gov / ) found that the C-terminus of the sequence contained a β-agarase polysaccharide hydrolase family 50 (GH family 50), the N-terminus c...

Embodiment 2

[0028] Example 2 Gene cloning and recombinant expression of β-agarase AgaB1

[0029] Fully synthesized in Example 1 agaB1 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 3: PAgaB1EF:

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

[0032] Reverse primer: SEQ ID 4: PAgaB1ER:

[0033] 5'- CCG CTCGAG GTAAATGTTCCATTCCCAAAA-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 for 3 mi...

Embodiment 3

[0037] Example 3 Fermentation process and purification preparation method of β-agarase AgaB1

[0038] The Escherichia coli BL21(DE3) / pET22b-AgaB1 constructed in Example 2 and stored at -80°C was streaked on the LB solid plate, and after culturing at 37°C for 16 hours, single clones were picked; 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 the inducer isopropyl-β-D-thiogalactopyranoside (IPTG) at...

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Abstract

The invention relates to an endo-cleaved Beta-agarase with homogeneous degradation products and an application thereof. Beta-Agarase is a new Beta-Agarase AgaB1, amino acid sequence shown in SEQ ID NO: 1. Beta. Provided by the present invention-Agarase is a new Beta-amylase designed artificially. Beta-agarase AgaB1 has a catalytic region (R1-V392) at the N terminal and a carbohydrate binding domain (G393-Y540) at the C terminal. Its amino acid sequence is identical to that of Beta-The sequence similarity of agarase is only 71%. Beta-Agarase AgaB1 is endo-cleaved and has homogeneous characteristics. The proportion of neo-agarose in the degradation product is up to 90.5%. Beta-Agarase AgaB1 has a stable property and a large yield, and has a certain potential for industrial application.

Description

technical field [0001] The invention relates to a beta-agarase AgaB1 with product uniformity and 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 acts on the α-1...

Claims

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Application Information

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IPC IPC(8): C12N9/38C12N15/56C12P19/14C12P19/00
CPCC12N9/2468C12P19/00C12P19/14C12Y302/01081
Inventor 吴中宝
Owner 吴中宝
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