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Method of improving enzymatic thermostability via artificially designed glycosylation modification

A technology of thermal stability and glycosyl modification, applied in the field of bioengineering, can solve the problems of reduced heat resistance, low efficiency of enzymatic properties, limited improvement of enzymatic properties, etc., to simplify the preparation process and thermal inactivation of enzyme molecules. The effect of raising the threshold and reducing the cost

Active Publication Date: 2015-09-09
BEIJING INSTITUTE OF TECHNOLOGYGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, spontaneous glycosylation in organisms is highly random, and many glycosyl modifications have no significant effect on improving the thermal stability of enzymes. In some cases, glycosylation can even reduce the heat resistance of enzymes, causing sugar The efficiency of sylation to improve enzymatic properties is low, because random glycosyl modification sites may not be the key sites that affect the heat resistance of enzymes, and the improvement of enzymatic properties is limited

Method used

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  • Method of improving enzymatic thermostability via artificially designed glycosylation modification
  • Method of improving enzymatic thermostability via artificially designed glycosylation modification
  • Method of improving enzymatic thermostability via artificially designed glycosylation modification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1: Rational design of N-glycosylation modification sites

[0017] 1. Knock out potential glycosylation sites

[0018] Taking β-D-glucuronidase PGUS (Genbank registration serial number EU095019) as the verification object, the online software NetNGlyc was used to analyze its primary amino acid sequence, and the sequon Asn-X-Ser / Thr find out the potential glycosylation sites of PGUS, and use the site-directed mutagenesis method to mutate the amino acid residues Asn of the potential glycosylation sites N28, N231, N383 and N594 to Gln to obtain mutations of aglycosylation modification sites Enzyme PGUS-UN.

[0019] 2. Using the crystal analysis structure of PGUS as a template, use SWISS-MODEL to simulate PGUS-UN at the non-glycosylation modification site, use PyMOL to display the simulation results, and analyze the B-Factor value of the local loop of the protein, The size of the protein surface groove and the secondary structure information of key parts are analy...

Embodiment 2

[0021] Example 2: Pichia expresses enzyme molecules with glycosyl modifications

[0022] 1. Design the N-glycosylation recognition feature enhancement sequence EAS sequence at the glycosyl modification sites 35K and 206S finally determined in Example 1: Phe-X-Asn-Y-Ser / Thr (X is any one Amino acid, Y is any amino acid except Pro), using site-directed mutagenesis primers to carry out overlap extension PCR mutations corresponding to the gene sequences at 35K and 206S, and using EcoRI and NotI to perform double enzyme digestion on the PCR amplification results, after enzyme digestion The fragments were ligated to the pGAPZ a vector with the same restriction end sticky ends.

[0023] 2. Use BlnI to linearize the connected circular vector to obtain the linearized fragment and transform Pichia pastoris GS115 by electroporation, and coat the transformation product on a medium containing bleomycin resistance screening and 2% 5-bromo-4-chloro - On the 3-indolyl-β-D-glucuronide activit...

Embodiment 3

[0025] Example 3: Thermal Stability Verification of Enzyme Molecules with Glycosyl Modifications

[0026] The glycosyl-modifying enzymes PGUS-35K and PGUS-206S obtained in Example 2 were placed in a 65°C water bath and incubated for 30 minutes, 60 minutes, 90 minutes, 120 minutes, 150 minutes and 180 minutes respectively. Then pipette 10 μL enzyme solution into 40 μL acetic acid-sodium acetate buffer solution containing 1.25 mmol / L 4-nitrophenyl-β-D-glucopyranoside and pH 4.2, and react at 40°C for 5 After 2 minutes, add 200 μ L, the sodium carbonate of 0.4mol / L stops reaction, and sample solution detects the content of p-nitrophenol with microplate reader (405nm), to measure the relative enzymatic activity of β-D-glucuronidase, thereby Detect the thermal stability of glycosyl-modifying enzyme molecules, the results are as follows Figure 4 , the results showed that the artificially designed glycosyl hairpin structure PGUS-35K and glycosyl spacer structure PGUS-206S significa...

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Abstract

The invention provides a method of improving enzymatic thermostability via artificially designed glycosylation modification and belongs to the field of bioengineering. The method includes: subjecting a primary sequence of enzyme protein and its spatial structure to combinatory analysis, designing an N-glycosylation site on a subunit joint of the enzyme protein or on a joint of internal motifs of subunits of the enzyme protein, and utilizing site-directed mutagenesis to introduce an N-glycosylation modification feature recognition enhancer sequon so as to enable carbohydrate chains to form a specific glycosyl hairpin structure among subunits of the enzyme protein or form a specific glycosyl pad structure among the internal motifs of the subunits of the enzyme protein, thereby maximally improving the rigidity of the tertiary structure of the enzyme protein and stabilizing the spatial conformation of the enzyme protein to be free from stress of high-temperature environments. The method of enzyme protein modification has the advantages that thermostability of the enzyme protein is greatly improved, the improvement in the thermostability of the enzyme protein is under artificial control, enzyme activity is partly improved, and catalytic features of the enzyme protein are enhanced.

Description

technical field [0001] The invention relates to a method for artificially designing glycosyl modification to improve enzyme thermal stability, belonging to the field of bioengineering. Background technique [0002] Enzyme is a biomacromolecule with special catalytic function produced by living cells. Compared with chemical catalysts, it has two obvious advantages of high efficiency and specificity. It has played an important role in the fields of food, medicine and fine chemical industry. increasingly important role. However, natural enzymes have become an important bottleneck in the application of enzyme engineering due to poor thermal stability, easy inactivation, and reduced reaction efficiency under industrial catalytic conditions; in addition, increasing the reaction temperature within a certain range can accelerate molecular diffusion and increase the reaction rate. , so improving the heat resistance of the enzyme can not only prolong the service life of the enzyme, b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/96C12N9/24
CPCC12N9/2402C12N9/96C12Y302/01139
Inventor 李春王小艳冯旭东樊艳爽
Owner BEIJING INSTITUTE OF TECHNOLOGYGY
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