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Novel lysine decarboxylase mutant and application thereof

A technology of lysine decarboxylase and amino acid is applied in the application field of producing 1,5-pentanediamine, which can solve the problems of limited application value and the like

Active Publication Date: 2017-09-15
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The US2015132808 patent of Mitsui Chemicals Corporation of Japan protects multiple Escherichia coli CadA mutants with increased activity. However, the activities of these CadA mutants are all lower than 20%, and even most of the CadA mutants have an activity improvement of less than 10%. , so the application value of these CadA mutants in actual production is very limited

Method used

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  • Novel lysine decarboxylase mutant and application thereof
  • Novel lysine decarboxylase mutant and application thereof
  • Novel lysine decarboxylase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Construction of embodiment 1.CadA wild-type bacterial strain

[0084] E.coli MG1655 (obtained from ATCC 700926, can refer to Blattner FR et al., The completegenome sequence of Escherichia coli K-12.Science 277:1453-62 (1997)) in LB medium (tryptone 10g / L, yeast powder 5g / L, sodium chloride 10g / L, pH 7.0), 37°C, 200rpm, after culturing for 12-16h, the cells were collected, and the genomic DNA was extracted using the Biomiga genome mini-prep kit. Using the Escherichia coli genome as a template, with cadA-F (as shown in SEQ ID NO: 3) and cadA-R (as shown in SEQ ID NO: 4) as primers, amplify the cadA gene (sequence as shown in SEQ ID NO: 2 shown), cloned into the vector pET-21a(+) (purchased from NOVAGEN) by 5'NdeI and 3'XhoI to obtain the pET21-cadA expression plasmid, and then transformed it into E.coli BL21(DE3) to obtain E.coli BL21(DE3). coli BL21(DE3) / pET21-cadA wild type engineering strain.

Embodiment 2

[0085] The acquisition of embodiment 2.CadA mutant strain

[0086] Utilize the Stratagene Series XL-II site-directed mutagenesis kit, respectively designed 6 pairs of primers (see Table 1), using the constructed pET21-cadA wild-type plasmid as a template, respectively used 6 pairs of primers for PCR amplification, respectively, the 9th amino acid of CadA Histidine is mutated to arginine, lysine at 44 is mutated to arginine, threonine at 88 is mutated to serine, glutamic acid at 111 is mutated to glycine, methionine at 176 is mutated to valine , the tyrosine at position 230 was mutated to histidine, and the resulting plasmids expressing the mutants were named pET21-H9R, pET21-K44R, pET21-T88S, pET21-E111G, pET21-M176V and pET21-Y230H, respectively. The PCR reaction conditions were: 95° C. for 5 min, 25 cycles (95° C. for 30 s, 50° C. for 30 s, 68° C. for 9 min), and 68° C. for 10 min. PCR amplification system (50 μL): template 1 μL, upstream and downstream primers 2 μL, dNTP ...

Embodiment 3

[0089] The production of pentamethylenediamine of embodiment 3.CadA wild type and mutant engineering strain

[0090] The thalline culture of engineering strain and the expression of lysine decarboxylase: the bacterial lawn of the appropriate plate activation of each engineering bacterium is inoculated in 100ml liquid LB culture medium (peptone 1%, yeast powder 0.5%, sodium chloride 1% , pH 7.0) in the 500ml seed bottle, add 0.1g / L ampicillin, shake culture at 37 ℃, 220rpm for 12 hours; , ammonium chloride 8g / L, potassium dihydrogen phosphate 5g / L, magnesium sulfate heptahydrate 0.5g / L, ferrous sulfate heptahydrate 0.2g / L) in the 5L fermentation tank, add 0.1g / L ampicillin, ferment The temperature is 37°C, the stirring speed is 300rpm-900rpm, the air flow rate is 1-4vvm, the ammonia solution is used to control the pH to 7.0 during the fermentation process, the added glucose is controlled at about 10g / L, and the OD 600 When growing to 30, add 0.1mM IPTG to induce the expression...

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Abstract

The invention discloses a novel lysine decarboxylase, the amino acid sequence of which is from the mutation of a sequence shown by SEQ ID NO:1, and one or more amino acid residue sites chosen from the following group are mutated: site 9, site 44, site 88, site 111, site 176 and site 230. The activity of 1,5-pentanediamine produced from lysine catalyzed by lysine decarboxylase disclosed by the invention is remarkably increased, consequently, the dosage of a catalyst can be reduced, and ultimately the production cost is reduced. The invention further provides an expression vector containing a coded sequence of the lysine decarboxylase, a host cell capable of expressing the lysine decarboxylase, application of the expression vector and the host cell in the production of the 1,5-diaminopentane and production methods.

Description

technical field [0001] The present invention relates to the field of biotechnology. Specifically, the present invention relates to a highly active lysine decarboxylase, an expression vector comprising the gene encoding the lysine decarboxylase and a genetically engineered bacterium capable of expressing the lysine decarboxylase, and the lysine decarboxylase Application of enzyme, expression vector and genetically engineered bacteria in the production of 1,5-pentanediamine. Background technique [0002] 1,5-Pentanediamine, also known as cadaverine, 1,5-diaminopentane, pentamethylenediamine and cadaveric toxin, is a nitrogenous base with biological activity widely present in organisms, and it is a lysine Produced by decarboxylation under the action of lysine decarboxylase (E.C.4.1.1.18), the reaction is L-lysine+H + →CO 2 +cadaverine. [0003] 1,5-Pentanediamine has various functions and uses. For example, in agriculture, 1,5-pentanediamine can be used to regulate the agi...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12P13/00C12N15/63
CPCC12N9/88C12P13/001C12Y401/01018
Inventor 孙际宾赵晶刘娇孙村民郑平马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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