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Alginate lyase mutant with high catalytic activity and application thereof

A technology of alginate lyase and mutants, which is applied in the field of alginate lyase mutants with high catalytic activity, can solve the problems of long cycle, time-consuming, high blindness, etc., to reduce reaction time, improve catalytic efficiency, accelerate The effect of industrial development and application

Active Publication Date: 2021-06-18
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method has many disadvantages, such as time-consuming, high blindness, long cycle, etc.

Method used

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  • Alginate lyase mutant with high catalytic activity and application thereof
  • Alginate lyase mutant with high catalytic activity and application thereof
  • Alginate lyase mutant with high catalytic activity and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1. Analysis of the crystal structure of alginate lyase AlgAT5.

[0054] 1.1 Protein expression and purification

[0055] Defluviitalea phaphyphila sp.Alg1[Ji S Q,Wang B,Lu M,etal.Defluviitalea phaphyphila sp.nov.,a novel thermophilic bacterium that degrades brown algae[J].Applied and environmental microbiology,2016,82(3 ):868-877.] Source alginate lyase AlgAT5 using pET-30a (+) vector for protein expression. Pick a single clone of BL21(DE3) expression strain containing AlgAT5 gene from the solid medium plate cultivated overnight in LB with kanamycin resistance in a 37°C constant temperature incubator, and add kanamycin to 5 mL of LB liquid Cultured overnight at 200 rpm in medium at 37°C as seeds. The next day, transfer the activated cells to 500mL LB liquid medium containing kanamycin, culture at 200rpm at 37°C until the OD600 is 0.5-0.8, add IPTG with a final concentration of 1mM, and place at 22-25°C The culture was shaken at 200rpm in a constant temperatur...

Embodiment 2

[0073] Example 2. Based on the sequence and structure analysis of protein, the catalytic amino acid mutation of the active center is studied and its influence on AlgAT5 enzymatic activity

[0074] Based on the protein sequence and structure analysis of AlgAT5, it was found that the enzyme is the same as the typical three-dimensional structure of the PL7 family alginate lyase. AlgAT5 is also a β-jelly roll fold, which contains two antiparallel β-fold sheets (sheet A and sheet B ). The structure includes 1 α-helix, 16 β-sheets, and 2 η-helices. Sheet A contains 9 β-strands (β1, residues 13-16; β4, residues66-73; β7, residues 106-113; β8, residues 119-125; β9, residues 128-132; β10, residues 136-142; β14, residues 182-185; and β15, residues 187-189), sheet B contains 7 β-strands (β3, residues 48-53; β6, residues 88-95; β11, residues 151-158; β12, residues 161 -166; β13, residues 169-175 and β16, residues 202-215). The remaining β-strands (β2, residues 39-41; andβ5, residues 77...

Embodiment 3

[0079] 1) Based on protein sequence and structure analysis, the acquisition of alginate lyase mutants:

[0080] The template used for point mutation was plasmid template pET30a-AlgAT5. The PCR reaction system for point mutations is as follows:

[0081] Forward primer (10pM) 1μL Reverse primer (10pM) 1μL template DNA 1μL 2×KAPA PCR SuperMix 50μL h 2 o

43μL

[0082] PCR amplification conditions are as follows: pre-denaturation, denaturation, annealing, extension, cycle 33 times, extension.

[0083] 1 94℃ 10min 2 94℃ 30sec 3 67℃ 30sec 4 72℃ 6min 5 72℃ 10min

[0084] To obtain different mutants, refer to the following table for the primers used:

[0085]

[0086] According to the above process, different mutants are obtained as follows:

[0087] In the mutant R94A, the arginine R at the 94th position of the amino acid sequence of alginate lyase AlgAT5 is mutated into alanine A, and t...

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Abstract

The invention relates to the technical field of protein engineering, in particular to an alginate lyase mutant with high catalytic activity and application thereof. The alginate lyase mutant is obtained by mutation of any one or more sites of cysteine at the 82<nd> site, cysteine at the 95 site, aspartic acid at the 146 site, cysteine at the 209 site and amino acids forming hydrogen bonds with the cysteine at the 82<nd> site, the 95 site or the 209 site of alginate lyase AlgAT5. The alginate lyase mutant or the expression microorganism of the alginate lyase mutant can catalyze algin to be subjected to beta elimination reaction to prepare alginate oligosaccharide, so that the catalysis efficiency of algin lyase is remarkably improved, the reaction time is shortened, and industrial development and application of the algin lyase are accelerated.

Description

technical field [0001] The invention relates to the technical field of protein engineering, in particular to a mutant of alginate lyase with high catalytic activity and its application. Background technique [0002] Alginate is the main structural component of the algae of the Phaeophyta in the marine kelp, similar to the lignocellulose of terrestrial plants, it mainly plays the role of structural support and stabilizing cell morphology. As the most important polysaccharide component in brown algae cells, its content will change dynamically according to different seasons, different growth cycles, and different growth parts. The degradation product of alginate is alginate oligosaccharide, because it has many excellent biological activities, it is widely used in many fields such as pharmaceuticals, feed additives, food additives, and plant protection. At present, the traditional production of alginate oligosaccharides mainly adopts chemical degradation, which mainly uses stro...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/70C12N1/21C12P19/00C12R1/19
CPCC12N9/88C12N15/70C12Y402/02C12P19/00Y02E50/10
Inventor 李福利苏航马小清吕明
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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