Alginate lyase and preparation method and application thereof

A technology of alginate lyase and brown algae lyase, applied in the directions of lyase, carbon-oxygen lyase, biochemical equipment and methods, etc., can solve the problems of narrow enzyme substrate spectrum, poor enzyme stability, low enzyme activity, etc. The properties are stable, the product is uniform, and the effect of improving the enzyme activity

Active Publication Date: 2017-08-01
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the product development of alginate lyase still faces problems such as unsafe, low enzyme activity, poor enzyme stability, and narrow substrate spectrum of the enzyme.

Method used

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  • Alginate lyase and preparation method and application thereof
  • Alginate lyase and preparation method and application thereof
  • Alginate lyase and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1 Purification of brown algae lyase in the original bacteria

[0070] The purification of brown algae lyase in the original bacteria comprises the following steps:

[0071] (1) Preparation of crude enzyme solution:

[0072] (a) Take Bacillus Alg07 stored at -80°C, streak on the optimized solid medium, and culture in a 30°C incubator;

[0073] (b) Pick a single colony, inoculate it into a 30 mL test tube containing 5 mL of fermentation medium, and incubate with shaking at 30°C for 16 hours;

[0074] (c) Inoculate into a 250mL Erlenmeyer flask containing 40mL of fermentation medium with an inoculum size of 0.5% (v / v), and cultivate for 24h at 30°C and 180r / min;

[0075] (d) Collect the bacteria cultured in step (c), centrifuge at 4°C and 12000r / min for 30min, take the supernatant, discard the bacteria precipitate, and obtain the crude enzyme solution.

[0076] (2) Concentrate the crude enzyme solution obtained in step (1): take 1000mL of fermentation supernatan...

Embodiment 2

[0083] Example 2 N-terminal sequencing of brown algae lyase

[0084] (1) Establish standard amino acid map

[0085] ① Using the mixed amino acid standard (PTH-AA), run under normal conditions to generate a standard chromatogram

[0086] ② Correct the retention time of the mixed amino acid standard and generate a standard method file

[0087] (2) On-board inspection

[0088] The PVDF membrane is sandwiched between two PTFE filter membranes, placed in the reactor of the protein sequencer PPSQ-31A, and the number of cycles is set.

[0089] (3) Transfer the brown algae lyase SEQ ID NO.2 to PVDF membrane.

Embodiment 3

[0090] Example 3 Gene mining of alginate lyase

[0091] The results analyzed by the RAST software showed that the genome DNA of the Bacillus weihaiensis Alg07 strain carried the coding gene No.2004 of alginate lyase. Analyzed by the biological software DNAMAN, it was shown that the theoretical molecular weight of the protein (AlgA) encoded by gene No.2004 was about 143kD. The signal peptide online prediction software SignalP4.1Server was used to analyze, and the result showed that the amino acid sequence of protein 2004 contained secreted signal peptide.

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Abstract

The invention relates to the field of genetic engineering technology, especially to alginate lyase and a preparation method and application thereof. Maturation protein of the alginate lyase contains an amino acid sequence as shown in the SEQ ID NO. 1, and the precursor protein contains an amino acid sequence as shown in the SEQ ID NO.4. The alginate lyase has stable properties, and specific enzyme activity reaches up to 4600 U/mL and above. Alginate can be subjected to enzymolysis to obtain micromolecular alginate-derived oligosaccharides with special biological activity. The product has the potential for industrial application.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to an alginate lyase, its preparation method and application. Background technique [0002] Alginate is composed of β-D-mannuronic acid (β-D-1,4-mannuronic acid, referred to as M) and its 5-position epimer α-L-guluronic acid (α-L- 1,4-guluronic acid, referred to as G) is a linear molecule formed by non-homopolymerization of C-1,4 glycosidic bonds. , the intramolecular poly M segments, poly G segments and M / G mixed segments are arranged alternately. Usually, alginate is processed from edible brown algae such as kelp and sargassum. Microorganisms such as Pseudomonas aeruginosa and brown nitrogen-fixing bacteria can secrete alginate with acetyl modification. [0003] Studies have found that the degradation product of alginate, alginate oligosaccharides, has many physiological activities such as anti-tumor, anti-coagulation, enhancing plant stress resistance, and promotin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/63C12N1/21C12N1/19C12P19/00A23L29/00A61K8/66A61Q19/00C12R1/19C12R1/125C12R1/85
CPCA61K8/66A61K2800/10A61Q19/00C12N9/88C12P19/00C12Y402/02003C12Y402/02011
Inventor 朱玥明陈朋曾艳门燕孙媛霞
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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