Engineering bacterium for co-expressing L-threonine aldolase and PLP synthase and application

A technology of threonine aldolase and engineering bacteria is applied to engineering bacteria and application fields that co-express L-threonine aldolase and PLP synthase, and can solve the problem of large amount of coenzyme PLP and low utilization rate of free enzyme, etc. problem, to achieve the effect of low cost, high reuse efficiency and lower production cost

Active Publication Date: 2021-01-05
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] Aiming at the problems in the prior art that the L-threonine aldolase uses a large amount of coenzyme PLP and the low utilization rate of the free enzyme, the present invention provides a method for co-expressing L-threonine aldolase and PLP. Enzyme Engineering Bacteria and Its Application

Method used

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  • Engineering bacterium for co-expressing L-threonine aldolase and PLP synthase and application
  • Engineering bacterium for co-expressing L-threonine aldolase and PLP synthase and application
  • Engineering bacterium for co-expressing L-threonine aldolase and PLP synthase and application

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Experimental program
Comparison scheme
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Embodiment 1

[0034] Embodiment 1: construction of engineering bacteria

[0035] The NCBI accession number of L-threonine aldolase (hereinafter referred to as DdLTA) derived from Desulfitobacterium dichloroeliminans is WP_015261381.1. The NCBI accession number of L-threonine aldolase (hereinafter referred to as BnLTA) derived from Bacillus nealsonii is WP_016204489.1. The NCBI accession number of L-threonine aldolase (hereinafter referred to as CbLTA) derived from Clostridium beijerinckii is WP_023973138.1. The above three enzymes were optimized according to the gene sequence in Escherichia coli to obtain the gene sequences shown in SEQ ID No.1, SEQ ID No.2 or SEQ ID No.3 respectively.

[0036] The PLP synthase (hereinafter referred to as ST) derived from Bacillus subtilis has a heteromultimer structure, and the NCBI accession numbers of the two subunits are QJR44475.1 and QJR44476.1 respectively. The gene sequences of the two subunits are based on the After the gene sequence is optimized...

Embodiment 2

[0057] Embodiment 2: the preparation of the culture of thalline and crude enzyme liquid

[0058] 1. Bacteria culture

[0059] Composition of LB liquid medium: peptone 10g / L, yeast powder 5g / L, NaCl 10g / L, dissolved in deionized water and then constant volume, sterilized at 115°C for 30min, ready for use.

[0060] Streak the co-expression engineered bacteria on a plate, activate at 37°C for 12h, pick a single colony and inoculate into 5mL LB liquid medium containing the corresponding antibiotic (50μg / mL), and culture with shaking at 37°C for 8-10h. Transfer 1% of the inoculum into 50 mL of LB liquid medium containing the corresponding antibiotic (50 μg / ml), culture with shaking at 37 ° C for about 2 h until the OD600 reaches 0.6, add IPTG to induce, the final concentration is 0.5 mM, and induce at 18 ° C Cultivate for 16-18h. After the cultivation, the culture solution was centrifuged at 12,000 rpm for 2 minutes to collect the bacterial cells, the supernatant was discarded, a...

Embodiment 3

[0063] Example 3: Pretreatment of immobilized materials

[0064] First, use distilled water to clean the immobilized carrier: amino resin HA, epoxy resin HFA and Fe 3 o 4 Soak in distilled water for 3 hours, stirring from time to time, repeat 3 times. Then filter under reduced pressure to get the filter cake.

[0065] Amino resin HA and Fe 3 o 4 Need to use glutaraldehyde cross-linking activation, HA and Fe 3 o 4 Use 0.4% glutaraldehyde for 3 hours with continuous stirring, then filter under reduced pressure to get the filter cake, and wash it with distilled water for 3 times.

[0066] The cleaned epoxy resin HFA and activated HA, Fe 3 o 4 Use 0.5% polyethyleneimine (PEI), polyethylene glycol (PEG) and ethanolamine (EA) calcium bicarbonate solutions to soak for 3 hours to wrap, the reaction temperature is 20°C-40°C, and the stirring speed is 100rpm -200rpm. Then the filter cake was obtained by filtration under reduced pressure, and washed 3 times with distilled water...

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Abstract

The invention discloses an engineering bacterium for co-expressing L-threonine aldolase and PLP synthase and an application. The invention relates to an engineering bacterium for co-expressing L-threonine aldolase and PLP synthase. The engineering bacterium is obtained by transferring an L-threonine aldolase gene and a PLP synthase gene into an expression strain. The L-threonine aldolase gene andthe PLP synthase gene are introduced into the engineering bacterium in an exogenous manner, the two enzymes are co-expressed, the synthesis amount of endogenous coenzyme PLP of the strain can be increased through over-expression of the PLP synthase, and the co-expressed engineering bacterium is crushed to prepare crude enzyme liquid. According to the co-immobilized enzyme prepared by co-immobilizing the L-threonine aldolase and coenzyme PLP, the use amount of exogenous PLP in the production process of the L-syn-p-methyl sulfonyl phenylserine can be remarkably reduced, the use efficiency of theL-threonine aldolase and the PLP is improved, and the production cost is reduced. The method has the advantages of mild reaction conditions, environmental protection, simple production process, highreutilization efficiency and low cost, and has a wide application prospect in the production of L-syn-p-methyl sulfonyl phenylserine.

Description

technical field [0001] The invention relates to the technical field of enzyme engineering, in particular to an engineering bacterium co-expressing L-threonine aldolase and PLP synthase and its application. Background technique [0002] L-syn-p-thiamphenicol phenylserine is an important pharmaceutical intermediate, which is used in the synthesis of various antibiotics, such as thiamphenicol and florfenicol. [0003] Synthesize L-syn-p-thiamphenylphenylserine by chemical method, use a large amount of copper sulfate, and use the complexation of metal ions under the condition of high temperature and strong alkali to generate two products of syn formula: L-syn-p-thiamphenylphenylserine Copper salt and D-syn-p-thiamphenicol phenylserine copper salt, and then use chiral reagents for resolution to obtain optically pure L-syn-p-thiamphenicol phenylserine. The chemical method mainly has the disadvantages of theoretical yield, complex and harsh process conditions, and large environmen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N11/089C12N11/091C12N11/14C12N9/88C12N9/06C12P13/04C12R1/19
CPCC12N9/88C12Y401/02005C12Y403/03006C12N9/0022C12Y104/03005C12N11/089C12N11/091C12N11/14C12P13/04Y02P20/584
Inventor 吴坚平郑文隆陈开通徐刚杨立荣
Owner ZHEJIANG UNIV
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