A method for producing phospholipase D by recombinant Escherichia coli

A technology of recombinant Escherichia coli and phospholipase, which is applied in the field of genetic engineering and microbial fermentation, can solve the problems of short synthesis time, cytotoxicity, cell lysis, etc.

Active Publication Date: 2020-12-04
XIAMEN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In Escherichia coli and yeast expression systems, recombinant PLD exhibited severe cytotoxicity, causing plasmid instability, cell lysis, short synthesis time and enzyme leakage, making it difficult to achieve high-density fermentation expression (Iwasaki et al.1995; Mishima et al. al.1997; Zambonelli et al.2003)

Method used

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  • A method for producing phospholipase D by recombinant Escherichia coli
  • A method for producing phospholipase D by recombinant Escherichia coli
  • A method for producing phospholipase D by recombinant Escherichia coli

Examples

Experimental program
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Embodiment 1

[0038] Embodiment 1. Construction of recombinant bacteria TOP10 / pBADKP-PLD

[0039] Using the plasmid pUC57-par (containing the par region sequence of pSC101 shown in SEQ ID No.1) as a template, by primer 1 and primer 2 (Table 1), the par sequence was amplified by PCR; to contain the arabinose promoter P BAD The plasmid pBADK was used as a template, and the plasmid pBADK backbone was amplified by PCR with primers 3 and 4 (Table 1); the par sequence and pBADK backbone obtained by the above two amplifications were used as primers and templates, respectively, for POE-PCR, and the product was directly Transformed into E.coli DH5α, colony PCR verification and sequencing, to obtain the vector pBADKP; the plasmid pUC57-PLD containing the codon-optimized Streptomycesantibioticus PLD gene sequence shown in SEQ ID No.2 and the vector pBADKP were passed through Nco I and Xba I double digestion, T4 ligase ligation, transformed into E.coli TOP10 (recA - ), the colony PCR was verified and ...

Embodiment 2

[0050] Example 2. Effect of promoter on phospholipase D expression.

[0051] Four promoters P T7 ,P lac / ara-1 a ,P lac / ara-1 b and P BAD It was used to investigate the expression of phospholipase D, and its activation strength decreased sequentially. where P lac / ara-1 a Partially activated by IPTG induction, P lac / ara-1 b Fully activated by IPTG and arabinose. Recombinant bacteria TOP10 / pBADKP-PLD was induced at 25°C for 16 hours, the results were as follows figure 1 shown. Under saturation induction, in the expression of PLD, P lac / ara-1 b and P BAD better than P T7 and P lac / ara-1 a , where P BAD Produces maximum PLD enzyme activity and specific yield. During the induction process, the PLD enzyme activities under all promoters almost reached the maximum after 1 hour of induction; after 16 hours of induction, the proportions of extracellular enzyme activities were 100%, 25%, 34% and 26%, respectively; process, the increase of extracellular enzyme activity ...

Embodiment 3

[0052] Example 3. Effect of Induction Temperature on Cell Growth and Phospholipase D Expression

[0053] In this experiment, TOP10 / pBADKP-PLD was cultured in TB medium at 37°C to OD 600 5 to 6, and then at 12°C, 18°C, 22°C, 25°C, 30°C and 37°C, respectively, to induce expression for 16 hours, and investigate the effect of induction temperature on the expression of PLD and biomass of TOP10 / pBADKP-PLD, the results are as follows figure 2 shown. Induction temperature significantly affected the expression of PLD and cell growth. The expression level was higher at 16-22°C, and the highest expression level appeared at 18°C. The specific productivity at 18°C ​​was 3.8 times that at 30°C, and 92% of PLD appeared in the cell. Meanwhile, when the temperature was greater than 18°C, the expression of PLD decreased. The cell growth was significantly better at a moderate low temperature than at a high temperature, which reflected that a certain low temperature weakened the toxicity of P...

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Abstract

The invention discloses a method for producing phospholipase D through recombinant escherichia coli. According to the method, a phospholipase D gene is in preciseness-type starting sub control, a pararea of pSC101 is inserted in an expression plasmid, an escherichia coli mutant strain recA is used for keeping the genetic stability of the plasmid, cells containing the plasmid at the growth periodare cultured to high density, and saturation induction is conducted at the induction period; meanwhile, the temperature is decreased, alkali metal salt stress is applied, the cytotoxicity of PLD to ahost is reduced, cell lysis is inhibited, the synthesis time of PLD is prolonged, and accordingly PLD expression is improved.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and microbial fermentation, and in particular relates to a method for producing phospholipase D by recombinant Escherichia coli. Background technique [0002] Phospholipase D (EC 3.1.4.4, PLD) uses phospholipids as substrates, acts on phosphodiester bonds, and undergoes hydrolysis and transphosphatidyl reactions depending on the receptors (water and alcohol). Among them, through the transphosphatidyl reaction, various alcohol groups can be introduced into the substrate phospholipids to generate a variety of phospholipids with biological activity and medicinal value, which are widely used in the food and pharmaceutical industries. For example, when the substrate is phosphatidylcholine (PC) and the acceptor is serine, glycerol and ethanolamine, PLD converts PC into phosphatidylserine (PS), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) respectively ( and Iwasaki 2013). Am...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/16C12N15/70C12N1/21C12R1/19
CPCC12N9/16C12N15/70C12Y301/04004
Inventor 卢英华熊维德曾宪海姚传义沈亮陈翠雪
Owner XIAMEN UNIV
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