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Alginate lyase Alg2A and preparation method and application thereof

A technology of alginate lyase and alginic acid, applied in lyase, botany equipment and methods, biochemical equipment and methods, etc., can solve the unstable expression of free plasmid vectors, environmental pollution of resistance genes, increased difficulty, etc. problems, to achieve efficient and rapid hydrolysis activity, stable expression, and avoid pollution

Inactive Publication Date: 2019-05-14
ZHONGKE RUNXIN SUZHOU BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the further research and development process, it was found that the above-mentioned technical scheme for preparing alginate lyase Alg2A still has technical defects, for example, the resistance gene present in the bacterial residue produced by the industrial fermentation process of the recombinant strain pollutes the environment; the free plasmid The expression of the vector is unstable, and it is easy to lose after multiple passages; the alginate lyase Alg2A expressed in Escherichia coli is in the cell, and the target enzyme must be obtained through cell disruption, which increases the difficulty of large-scale preparation

Method used

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  • Alginate lyase Alg2A and preparation method and application thereof
  • Alginate lyase Alg2A and preparation method and application thereof
  • Alginate lyase Alg2A and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Codon optimization and total gene synthesis of alginate lyase gene

[0024] On the premise of not changing the amino acid sequence, the codons of the gene encoding alginate lyase derived from Flavobacterium sp.S20 (Flavobacteriumsp.S20) were optimized. See SEQ ID NO.2. The identity (identity) of the optimized gene sequence and the original sequence of alginate lyase Alg2A (as shown in sequence SEQ ID NO.3, GenBank accession number: JF412659) is 77%. At the same time, in order to enable efficient and stable secretion and expression of alginate lyase in Pichia pastoris, the optimized alginate lyase gene lacks 22 amino acids encoding the 5' terminal signal peptide sequence. The optimized gene sequence was entrusted to Beijing Qingke Xingye Biotechnology Co., Ltd. for the total synthesis, and the synthesized gene sequence was recorded as alg2A.

Embodiment 2

[0025] Example 2 Construction of expression vector of alginate lyase gene alg2A

[0026] First, use restriction endonucleases Xho I and Not I to double-enzyme digest the cloning vector containing the alginate lyase gene alg2A to obtain the target gene fragment, and use the same endonuclease to double-enzyme digest the expression vector pGBG1, recover large fragments. The two recovered products were connected to obtain a recombinant vector named alg2A-pGBG1. In order to confirm that the target alginate lyase gene has been constructed into the vector, the recombinant vector was double digested with Xho I and Not I, and the product was subjected to agarose gel electrophoresis. The results are as follows: figure 1 shown. according to figure 1 It can be seen that after double digestion, the target gene fragment appeared between 750bp and 1000bp, which was consistent with the fragment length of alg2A of 834bp.

Embodiment 3

[0027] Example 3 Screening of alginate lyase Pichia pastoris engineering bacteria and preparation of alginate lyase

[0028] After the obtained recombinant plasmid alg2A-pGBG1 was linearized by the restriction endonuclease BglII, it was separated by gel electrophoresis and the large nucleic acid fragment containing the target gene was cut out. Recombinant colonies were obtained by screening. Eight of the colonies were picked and streaked onto a BMMY agar plate containing 0.2% sodium alginate, cultured at 30°C for 48 hours, then poured into 10% cetylpyridinium chloride (Cetylpyridinium chloride, CPC) aqueous solution for color development, and found Among them, No. 2, 5, 6 and 7 clone strains showed alginate cracking activity, see figure 2 , is the primary screening chart for the enzymatic activity of the recombinant expression Pichia pastoris GS115 strain sodium alginate substrate plate. A single colony of strain No. 6 was picked, inoculated in 50ml of BMGY medium, cultured...

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Abstract

The invention discloses alginate lyase Alg2A and a preparation method and application thereof. According to the preference of pichia pastoris codon, an alginate lyase coding gene in Flavobacterium sp.S20 is optimized, and the optimized nucleic acid sequence is shown in SEQ ID NO. 2. Furthermore, the optimized alginate lyase coding gene is efficiently secreted and expressed by means of a pichia pastoris expression system, the alginate lyase is obtained, and the amino acid sequence of the alginate lyase is shown in SEQ ID NO.1. The obtained alginate lyase has high hydrolytic activity to a sodium alginate substrate, and crude enzyme liquid produced by shaking flask for fermentation has the capacity of completely degrading 1 g of sodium alginate by 0.1 ml (protein content is about 0.042 mg),and the alginate lyase Alg2A has the good industrial application prospect.

Description

technical field [0001] The invention belongs to the technical field of preparation of alginate lyase, in particular to a high-efficiency preparation method and application of alginate lyase. Background technique [0002] Alginic acid is a heteropolysaccharide formed by connecting guluronic acid and mannuronic acid through 1,4 glycosidic bonds, and is the main component of the cell wall of brown algae. Alginic acid and its salts have been widely used in food, chemical and pharmaceutical fields because of their high viscosity and easy gel formation. However, high molecular weight and limited water solubility limit the application of alginic acid and its salts. Relatively speaking, its degradation product, alginic acid oligosaccharide, has a broader application prospect because of its lower molecular weight and better biological activity. [0003] Alginate lyase can cleave the glycosidic bonds of alginic acid through β-elimination to produce alginic acid oligosaccharides cont...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/81C12P19/00C12R1/84
Inventor 杜昱光程功赵勇焦思明任立世王颖王倬孙明
Owner ZHONGKE RUNXIN SUZHOU BIOLOGICAL TECH CO LTD
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