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Flavin monooxygenase mutant and preparation method thereof

A monooxygenase and mutant technology, applied in the field of bioengineering, can solve problems such as difficulty in obtaining satisfactory results, and achieve the effects of simplifying production links and reducing production costs

Inactive Publication Date: 2017-11-24
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is generally difficult to obtain satisfactory results for a gene mutated once, so a sequential error-prone PCR (Sequential Error-prone PCR) strategy has been developed.

Method used

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  • Flavin monooxygenase mutant and preparation method thereof
  • Flavin monooxygenase mutant and preparation method thereof
  • Flavin monooxygenase mutant and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Obtaining of wild-type flavin monooxygenase mature peptide gene

[0039] 1. Take fresh garlic bulbs and grind them in liquid nitrogen, extract total RNA according to the instructions of Trizol reagent and perform RT-PCR;

[0040] 2. Using the first strand of the synthesized cDNA as a template, design a pair of primers in the upstream and downstream of the ORF frame according to the flavin monooxygenase sequence registered in Genbank sequence number AB924383.1, and introduce restriction enzyme sites BamH I and Hind III respectively , design the amplification primers of the flavin monooxygenase gene of the present invention as follows:

[0041] Upstream primer P1 (SEQ ID NO.1):

[0042] 5'-CGC GGATCC ATGGTTTCTCTCATCTTGCTCGTC-3'

[0043] Downstream primer P2 (SEQ ID NO.2):

[0044] 5'-CCC AAGCTT TGAAAGAAATTTGCTGAAAGTTTCTCTTG-3'

[0045] Using P1 and P2 as upstream and downstream primers, and using the garlic bulb wild-type flavin monooxygenase genome as a...

Embodiment 2

[0050] Embodiment 2 Obtaining of flavin monooxygenase mutant gene

[0051] 1. Random mutation based on error-prone PCR technology to construct a novel flavin monooxygenase, and design primers as follows: upstream primer P1 (SEQ ID NO.1):

[0052] 5'-CGCGGATCCATGGTTTCTCTCATCTTGCTCGTC-3'

[0053] Downstream primer P2 (SEQ ID NO.2):

[0054] 5'-CCCAAGCTTTGAAAGAAATTTGCTGAAAGTTTCTCTTG-3'

[0055] In the error-prone PCR reaction system, P1 and P2 were used as upstream and downstream primers, and the wild-type flavin monooxygenase mature peptide gene was used as a template to perform error-prone PCR.

[0056] The reaction conditions for its amplification are:

[0057]

[0058]

[0059] The amplification conditions were: pre-denaturation at 95°C for 10 min; denaturation at 94°C for 30 s, annealing at 54°C for 45 s, 30 cycles of extension at 72°C for 1 min and 30 s; extension at 72°C for 10 min.

[0060] 2. Cloning the flavin monooxygenase mutant gene into the expression vect...

Embodiment 3

[0066] Example 3 Construction of recombinant bacteria for free expression of Pichia pastoris flavin monooxygenase mutant

[0067] 1. Construction of flavin monooxygenase mutant expression vector pPIC9K-fmom

[0068] The flavin monooxygenase mutant gene obtained by error-prone PCR was digested with EcoRI and NotI and then ligated with the same double-digested Pichia expression vector pPIC9K with ligase, and the ligated product was transformed into Escherichia coli JM109 (DH5α ) Competent cells, through Amp resistance screening, select positive transformants; extract positive transformant plasmids, extract plasmids after shaking tube culture at 37°C, and carry out preliminary verification of single and double enzyme digestion, and verify the correctness of enzyme digestion The recombinant plasmid was named pPIC9K-fmom; the positive clones verified by enzyme digestion were sent to Beijing Huada Gene Technology Co., Ltd. for sequencing to further ensure the correctness of the targ...

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Abstract

The invention belongs to the field of bioengineering and particularly relates to a flavin monooxygenase mutant and a preparation method thereof. The error-prone PCR technology is used to conduct random mutation on wild type flavin monooxygenase to obtain the flavin monooxygenase mutant. The specific enzyme activity of the flavin monooxygenase mutant is improved by 35% compared with that of the wild type flavin monooxygenase. Meanwhile, a yeast expression system is adopted, when the flavin monooxygenase is displayed on the surface of yeasts, yeast cells can serve as enzyme producers and can also serve as carriers in immobilized enzymes, operation of purification, fixation and the like of the enzymes are omitted, the production link is simplified, and the production cost is reduced.

Description

Technical field: [0001] The invention belongs to the field of bioengineering, and in particular relates to a flavin monooxygenase mutant and a preparation method thereof. technical background: [0002] Alliin (S-allyl-L-cysteine ​​sulfoxide, S-allyl cysteine ​​sulphoxide, SACS) is a tasteless and odorless hemihydrate. Alliin mainly exists in the cells of garlic bulbs, and is the most abundant sulfur-containing compound in garlic, accounting for more than 85% of all sulfur-containing compounds in garlic, and its content is as high as 0.5%-1.4%, up to 3.2% base calculation). [0003] In recent years, Western medical and biological researchers have done a lot of research on the pharmacological effects of alliin. The research found that alliin mainly has the following effects: broad-spectrum antibacterial effect. Alliin has different degrees of inhibitory and killing effects on Staphylococcus aureus, Staphylococcus saprophyticus, Staphylococcus albus, Shigella flexneri, Shige...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N15/81C12N1/19C12P13/04C12R1/84
CPCC12N9/0073C12N15/815C12N2800/102C12P13/04C12Y114/13008
Inventor 路福平刘逸寒贾雷博
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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