Alginate lyase mutant capable of relieving dependence of divalent metal ions and application of alginate lyase mutant

A technology of alginate lyase and divalent metal ions, which is applied in the field of protein engineering and can solve problems such as limiting the wide use of alginate lyase

Active Publication Date: 2021-06-08
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

This shortcoming will severely limit

Method used

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  • Alginate lyase mutant capable of relieving dependence of divalent metal ions and application of alginate lyase mutant
  • Alginate lyase mutant capable of relieving dependence of divalent metal ions and application of alginate lyase mutant
  • Alginate lyase mutant capable of relieving dependence of divalent metal ions and application of alginate lyase mutant

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Experimental program
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Embodiment 1

[0032] Example 1. Analysis of metal ion-dependent alginate lyases of the PL7 family based on structure and catalytic properties

[0033] After analyzing the alginate lyases in the PL7 family, we found that most of the enzymes whose substrate specificity is PolyG have obvious divalent metal ion activation effects, and the addition of these divalent metal ions will significantly increase their enzyme activity. However, adding After metal ion chelating agents such as EDTA or EGTA remove these divalent metal ions in the reaction system, the enzyme activity is significantly reduced.

[0034] Take alyPG as an example, add Zn 2+ and Mn 2+ Afterwards, compared with the wild type, the enzyme activity increased to 131% and 175%, but after adding the metal ion chelating agent EDTA, the enzyme activity decreased to 17% of the wild type. For Alg_M3, Ca 2+ mn 2+ 、Co 2 + , Mg 2+ Can significantly increase enzyme activity; for Alg, Ca 2+ , Mn 2+ Can significantly increase the enzyme...

Embodiment 2

[0040] Embodiment 2, the induced expression of AlgAT5 in Escherichia coli

[0041] The expression plasmid containing pET-30a-AlgAT5 stored in a -80°C refrigerator [Qingdao Institute of Bioenergy and Biotechnology, Chinese Academy of Sciences, Li Fuli, Su Hang, Ji Shiqi, Lu Ming. The gene encoding alginate lyase and its application: China , 201810862414.3[P].2018-08-01.] BL21 protein expression strain, 5 μL was taken out and inoculated in 5 mL of LB plus kanamycin liquid medium at 200 rpm, and cultivated overnight 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 temperature shaker for 16-18h to induce protein expression. Alginate lyase AlgAT5 was purified with Ni-NTA Resin (purchased from TransGen Biotech). After the purification was completed, the purif...

Embodiment 3

[0052] Example 3. Construction and enzyme activity determination of D146E, D146H, D146K, D146R, D146A, D146P mutants.

[0053] Based on the analysis in Example 2, a site-directed mutation was performed on the amino acid at position 146, and further analysis of the amino acids near the substrate binding region in the PL7 family, combined with the results of multiple sequence alignments with different substrate specificities, we found that Asp146 is the key factor that determines PolyG and The key amino acid of PolyM substrate specificity, we speculate that Asp146 also plays an important role in the binding of metal ions.

[0054] According to the properties of amino acids, it can be known that aspartic acid D is an amino acid with a negative charge on the side chain, and glutamic acid E also has this property, so it is preferred to mutate it into one with the same negative charge but a slightly longer side chain length Glutamate E. And further choose to mutate it into positive...

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Abstract

The invention relates to the technical field of protein engineering, in particular to an alginate lyase mutant capable of relieving dependence of divalent metal ions and application of the alginate lyase mutant. The mutant is characterized in that in an amino acid sequence of alginate lyase which takes PolyG as a specific substrate during enzymolysis in PL7 family alginate lyase, amino acid at least located at the same three-dimensional structure position as aspartic acid D146 in AlgAT5 or at the D146 position based on multiple sequence alignment of structure is positively charged amino acid, glycine, alanine, valine, leucine or isoleucine formed by mutating aspartic acid D or glutamic acid E. The beneficial mutations can provide theoretical and technical support for industrial development and application of alginate lyase, and promote green and efficient preparation of alginate oligosaccharide.

Description

technical field [0001] The invention relates to the technical field of protein engineering, in particular to a mutant of alginate lyase capable of releasing dependence on divalent metal ions and its application. Background technique [0002] Phaeophyta are the most important part of marine macroalgae, not only of great significance in terms of marine carbon fixation, marine ecological environment and other ecological values, but also have important application value as biomass resources. There are many macroalgae belonging to the Phaeophyta, such as Laminaria hyperborean, Macrocystis pyrifera, Lamimria digitata, Ascophyllum nodosum, Laminaria japonica, Lessonia nigrescens, Antarctic seaweed (Durvillea antarctica). The main component of brown algae is alginate, which can reach up to 40% depending on the growth site and season. Its specific function is similar to that of cellulose, the structural substance in land plants. Alginate is the main biological agent in macroalgae. ...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/70C12N1/21C12P19/14C12R1/19
CPCC12N9/88C12N15/70C12P19/14C12Y402/02011
Inventor 苏航李福利马小清
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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