A low-M r lipase activation factor cooperating with lipase modulator protein LimL in Pseudomonas sp. strain 109

Tanaka, Junko; Ihara, Fumio; Nihira, Takuya; Yamada, Yasuhiro

doi:10.1099/00221287-145-10-2875

Cited by 4 publications

(2 citation statements)

References 18 publications

(7 reference statements)

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“…and then identified as Glutathione, which could facilitate the dissociation of LipA/LipB complex and liberate free active lipase [44,45]. In this work, E. coli Origami 2 (DE3) was selected as the expression host, which would enhance disulfide bond formation.…”

Section: Discussionmentioning

confidence: 99%

High-level soluble expression and enzymatic characterization of Burkholderia sp. lipase with steryl ester hydrolysis activity in E. coli

Shu

Jia

et al. 2019

Preprint

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Background Burkholderia cepacia lipase is an important industrial biocatalyst for biodiesel production and chiral pharmaceutical synthesis. Heterologous soluble expression of lipase lipA gene from B. cepacia in Escherichia coli highly depends on co-expression of its cognate foldase gene, lipB. However, the interaction between recombinant lipase LipA and chaperonin LipB is rather complicated and confusing. In this research, various systems of lipA/lipB co-expression combinations are investigated to obtain high-level soluble expression of lipA, respectively. Results The best co-expression combination system for lipA and lipB is E. coli Origami 2 (DE3)/pETDuet-lipB(MCS1)/lipA(MCS2). The soluble expression level of lipA is 100.4 U/OD600 towards 4-nitrophenyl laurate hydrolysis. The recombinant LipA can be rapidly isolated from cell-free supernatant of recombinant E. coli lysate using HisTrap HP affinity chromatography column, and the lipA/LipB complex is obtained. Enzymatic characterization analysis shows that the purified LipA is a mesothermal and alkaline enzyme. LipA displays preference for medium-chain-length acyl groups (C10-C12) and sn-1,3 regioselectivity. Besides triacylglycerol hydrolase activity (EC. 3.1.1.3), LipA also displays steryl ester hydrolase activity (EC. 3.1.1.13). The specific activity of LipA towards 4-nitrophenyl decanoate and cholesterol linoleate are 638.9 U/mg and 1111.5 mU/mg, respectively. Conclusions Host strain E. coli Origami 2 (DE3), lipB locus at MCS1 on the dual expression cassette plasmid pETDuet, and low-temperature induction contribute to the soluble expression of lipA. Recombinant LipA displays both triacylglycerol hydrolase activity and steryl ester hydrolase activity.

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Section: Discussionmentioning

confidence: 99%

High-level soluble expression and enzymatic characterization of Burkholderia sp. lipase with steryl ester hydrolysis activity in E. coli

Shu

Jia

et al. 2019

Preprint

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“…The activation mechanisms of the lipases from Burkholderia cepacia DSM3959 [9][10][11][12], Pseudomonas sp. 109 [13][14][15][16], and P. aeruginosa TE3285 [17][18][19][20][21] have been relatively well investigated and discussed. The lipase and foldase form a 1:1 complex [10][11][12].…”

Section: Introductionmentioning

confidence: 99%

Refolding of a recombinant organic solvent-stable lipase, which is overexpressed and forms an inclusion body, and activation with lipase-specific foldase

Ogino

Inoue

Akagi

et al. 2008

Biochemical Engineering Journal

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Cloning and expression of gene, and activation of an organic solvent-stable lipase from Pseudomonas aeruginosa LST-03

et al. 2007

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Organic solvent-tolerant Pseudomonas aeruginosa LST-03 secretes an organic solvent-stable lipase, LST-03 lipase. The gene of the LST-03 lipase (Lip9) and the gene of the lipase-specific foldase (Lif9) were cloned and expressed in Escherichia coli. In the cloned 2.6 kbps DNA fragment, two open reading frames, Lip9 consisting of 933 nucleotides which encoded 311 amino acids and Lif9 consisting of 1,020 nucleotides which encoded 340 amino acids, were found. The overexpression of the lipase gene (lip9) was achieved when T7 promoter was used and the signal peptide of the lipase was deleted. The expressed amount of the lipase was greatly increased and overexpressed lipase formed inclusion body in E. coli cell. The collected inclusion body of the lipase from the cell was easily solubilized by urea and activated by using lipase-specific foldase of which 52 or 58 amino acids of N-terminal were deleted. Especially, the N-terminal methionine of the lipase of which the signal peptide was deleted was released in E. coli and the amino acid sequence was in agreement with that of the originally-produced lipase by P. aeruginosa LST-03. Furthermore, the overexpressed and solubilized lipase of which the signal peptide was deleted was more effectively activated by lipase-specific foldase.

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A low-M r lipase activation factor cooperating with lipase modulator protein LimL in Pseudomonas sp. strain 109

Cited by 4 publications

References 18 publications

High-level soluble expression and enzymatic characterization of Burkholderia sp. lipase with steryl ester hydrolysis activity in E. coli

High-level soluble expression and enzymatic characterization of Burkholderia sp. lipase with steryl ester hydrolysis activity in E. coli

Refolding of a recombinant organic solvent-stable lipase, which is overexpressed and forms an inclusion body, and activation with lipase-specific foldase

Cloning and expression of gene, and activation of an organic solvent-stable lipase from Pseudomonas aeruginosa LST-03

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