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Monooxygenase DszC mutant and preparation method and application thereof

A monooxygenase and mutant technology, applied in the field of genetic engineering, can solve problems such as binding sites and binding mechanisms that have not yet been resolved

Inactive Publication Date: 2019-10-22
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the binding site and binding mechanism of HBP on DszC have not yet been resolved

Method used

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  • Monooxygenase DszC mutant and preparation method and application thereof
  • Monooxygenase DszC mutant and preparation method and application thereof
  • Monooxygenase DszC mutant and preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1, use pSB4A5-BADC plasmid to construct the mutant library of dszC gene

[0034] (1) Design random mutation primers EP-S / EP-A.

[0035] EP-S:5'-ATCTGTTGTTTGTCGGTGAACGCTCTCTAC-3' (SEQ NO:5)

[0036] EP-A: 5'-TTTACAAAAAACCCCTCAAGACCCGT-3' (SEQ NO: 6)

[0037] Using the nucleotide sequence of wild-type DszC shown in Seq1 as a template, DszC was randomly mutated by using error-prone PCR primers EP-S / EP-A, using low-fidelity DNA amplicon rTaq enzyme, and adding MnSO4. The mutant gene of dszC and the expression vector pSB4A5-BAD containing the other three genes dszA, dszB and dszD of the 4S pathway were digested with Nhe I and Hind III and ligated with T4 ligase to transform the cloning host E.coli BL21 (DE 3 ). The obtained mutants were picked one by one into a sterile 96-well plate, and cultured overnight in LB medium containing ampicillin with shaking at 35-38°C to obtain the mother plate. Copy the bacteria in the master plate to a deep-well 96-well plate to...

Embodiment 2

[0038] Embodiment 2, the re-screening of the mutant shake flask fermentation obtained by orifice screening

[0039] Pick A from Motherboard 610 / A 600The strains with higher yield than wild-type HBP were sequenced, and the strains with amino acid changes were selected for re-screening in shake flasks. The cells were collected and washed after induced fermentation, and a certain amount of cells and a certain amount of DBT were controlled in a 5mL reaction system as whole-cell catalytic substrates. After shaking and reacting at 27-35°C for 24 hours, take a certain amount of reaction solution, extract it with an equal volume of ethyl acetate, and use HPLC to detect the content of each component in the reaction system. Through high-throughput screening and simulated molecular docking, the mutant strain E.coli BL21(DE3) / A101V with the highest HBP production was obtained, which was 28.81% higher than the strain containing wild-type DszC. The result is as figure 1 shown.

Embodiment 3

[0040] Example 3, iterative saturation mutation of DszC

[0041] The wild-type DszC was subjected to saturation mutation of amino acid A101 by site-directed mutagenesis primers, and the obtained mutants were expressed and purified, and their enzymatic activities were measured under the inhibitory effect of a certain concentration of HBP. Among them, A101K has the highest enzyme activity, which is 29.51% higher than that of WT.

[0042] Using A101K as a template, DszC was subjected to iterative saturation mutation with site-directed mutagenesis primers. After the obtained mutants were expressed and purified, their enzyme activities were measured under the inhibitory effect of a certain concentration of HBP. Such as figure 2 As shown, A101K / W327C (marked as AKWC) has the highest enzyme activity, which is 3.3 times higher than that of WT. The enzymatic activity of purified AKWC was measured under HBP inhibition, and its IC 50 The increase from μM level to mM level indicates ...

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Abstract

The invention discloses a monooxygenase DszC mutant, and the monooxygenase DszC mutant desensitizes the feedback inhibition of a final product HBP of a 4S pathway. The monooxygenase DszC mutant mutates the alanine at position 101 of DszC derived from Rhodococcus erythropolis IGTS8 to lysine, The 327th tryptophan is mutated to cysteine, and an amino acid sequence is shown in SEQ NO: 4. The invention also provides a method for preparing the monooxygenase DszC mutant and an application of the monooxygenase DszC mutant in preparing a college desulfurizer. The technical scheme of the present invention desensitizes the feedback inhibition of the final product HBP of the 4S pathway by obtaining the DszC enzyme mutant AKWC with the highest enzyme activity under the inhibition of HBP, and significantly improves the desulfurization efficiency.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a monooxygenase DszC mutant and its preparation method and application. Background technique [0002] In the microbial 4S desulfurization pathway, the DszC enzyme is the first enzyme involved in the reaction to oxidize DBT to DBTO in two steps 2 . DszA, DszB, and DszC enzymes were all inhibited by the metabolic end product HBP, and DszC was most severely inhibited. Therefore, it is very important to improve the desulfurization efficiency of the 4S desulfurization pathway by releasing the inhibition of HBP on DszC enzymes to improve the catalytic activity of DszC enzymes and obtaining preferred DszC enzymes. [0003] The 4S pathway of microbial desulfurization can specifically cleave the C-S bonds of refractory aromatic S-heterocyclic compounds remaining after hydrodesulfurization (HDS) without losing their combustion value. The four Dsz enzymes of this pathway conv...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N15/70C12N15/10C12P7/22
CPCC12N9/0083C12N15/1031C12N15/70C12P7/22C12Q2531/113
Inventor 梁书利林影李璐廖一波
Owner SOUTH CHINA UNIV OF TECH
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