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L-proline 4-hydroxylase and application thereof

A technology of proline and hydroxylase, applied in the direction of application, oxidoreductase, enzyme, etc., can solve the problems of limited industrial application, low catalytic activity, low yield, etc.

Pending Publication Date: 2020-09-15
HEBEI BOLUNTE PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the proline-4-hydroxylase produced by the cyst fungus RH1 is used the most, but the catalytic activity of this enzyme is low and the yield is not high, which seriously limits its application in industry. Further improvement and optimization

Method used

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  • L-proline 4-hydroxylase and application thereof
  • L-proline 4-hydroxylase and application thereof
  • L-proline 4-hydroxylase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Homologous Modeling and Evaluation

[0041] Using the SWISS-MODEL homology modeling server (https: / / www.swissmodel.expasy.org / ) to perform homology modeling on proline hydroxylase (BP4H) from Bacillus cereus HBU-AI, specifically The steps of the method are as follows: input the target sequence, search for a suitable template, and then select a suitable template for homology modeling. The experimental results of P4H structure research are few, and the ectoine hydroxylase (PDB: 4q5o) with high homology to the target sequence is selected as a template, and the automatic mode (Automated Mode) is used for modeling. The Ramachandran plot of the Procheck program and the verify3D model structure were used to score, and the compatibility of the three-dimensional structure with its primary sequence was evaluated.

[0042] The results show that: inputting the BP4H protein sequence to the SWISS-MODEL homology modeling server for sequence comparison, it is found that the ...

Embodiment 2

[0043] Example 2 Sequence Conservation Analysis and Molecular Docking

[0044] Sequence homology comparison search found that proline hydroxylase gene and ectoine hydroxylase gene ( ect D) have high homology and belong to Fe 2+ Binds Fe to the α-ketoglutarate-dependent dioxygenase superfamily 2+ The conserved 2-His-1-carboxylate facet triplet (see image 3 ) and the conserved site RXS motifs that bind α-ketoglutarate (see Figure 4 ). The enzymatic functions of the non-ferrorubin- and α-ketoglutarate-dependent dioxygenase families are largely dependent on highly reactive iron ions. Proteins Superimposing reveals that in the active center of proline hydroxylase BP4H, the functional groups (2 imidazole groups and 1 carboxyl group) of amino acid residues His109, Asp111 and His215 interact with Fe 2+ Combine to form the so-called 2-His-1-carboxylate triad. In all non-ferrirubin- and α-ketoglutarate-dependent enzymes of the dioxygenase family, the co-substrate α-ketoglutarat...

Embodiment 3

[0046] Example 3 BP4H enzyme site-directed half-saturation mutation

[0047] To introduce a single-base site-directed mutation into a plasmid, it is only necessary to design a pair of primers to amplify the plasmid by reverse PCR. The sequences of the designed primers are shown in Table 1 below. The degenerate primers encoding all polar amino acids designed at a key amino acid site were used to amplify the plasmid containing the BP4H gene by PCR, and the amplified product was detected by agarose gel electrophoresis. Finally, we successfully obtained the BP4H gene containing the mutation plasmid with clear bands.

[0048] Table 1 Primer sequences

[0049] .

[0050] Note: n stands for four nucleotides; v stands for A / C / G; k stands for G / T, n, v, k encode almost all polar amino acids, suitable for us to verify the substitution of these five amino acid positions sex.

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Abstract

The invention relates to an L-proline 4-hydroxylase with high catalytic activity. The amino acid sequence of the L-proline 4-hydroxylase has one or more of the following mutations in a sequence shownin SEQ ID NO.1: the 92th amino acid is mutated into serine, threonine, cysteine, lysine, aspartic acid or glutamic acid from glutamine; the 94th amino acid is mutated into serine, cysteine, histidineor aspartic acid from lysine; the 115th amino acid is mutated into glutamine, lysine or glutamic acid from tryptophan; the 217th amino acid is mutated into threonine, cysteine, glutamine, arginine, histidine or aspartic acid from serine; and the 226th amino acid is mutated into threonine, tyrosine, aspartic acid or glutamic acid from arginine. The invention also relates to application of the L-proline 4-hydroxylase.

Description

technical field [0001] The invention belongs to the field of biocatalysis, and in particular relates to an L-proline 4-hydroxylase and its application in synthesizing trans-4-hydroxyl-L-proline. Background technique [0002] L-hydroxyproline (L-hydroxyproline, Hyp) is an imino acid produced by the hydroxylation modification of L-proline by L-proline hydroxylase (L-prolinehydroxylase). Due to the different addition positions of hydroxyl groups in the hydroxylation of L-proline, four isomers are formed—trans-4-hydroxyproline, trans-3-hydroxyproline, cis-3 -Hydroxyproline and cis-4-hydroxyproline. Among them, trans-4-hydroxyproline (trans-4-hydroxy-L-proline, trans-Hyp) is ubiquitous in animals and plants in nature, and it exists in the cell wall protein of plants in the form of glycoprotein, and in the form of polypeptide Exist in the form of animal collagen (such as gelatin, bone glue). It plays a key role in some physiological and pathological processes of organisms, and ...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12P13/24
CPCC12N9/0071C12P13/24C12Y114/11002
Inventor 李玮张红蕾王春田
Owner HEBEI BOLUNTE PHARMA
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