Cloning, sequence analysis and expression of bacterial lipase-coding DNA fragments from environment in Escherichia coli

Fan, Zhaoxin; Yue, Changwu; Tang, Yang; Zhang, Yizheng

doi:10.1007/s11033-008-9344-y

Cited by 10 publications

(5 citation statements)

References 14 publications

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“…The enzymes catalyze the hydrolysis and synthesis of fatty acid esters [3,4]. Despite sharing a common catalytic mechanism and a similar structure, the amino acid sequences of different lipases/esterases are greatly variable [5], which endow them potential specific catalytic activities on different ester substrates or functions under various tolerable conditions. Although extensively present in different organisms, lipases/esterases from microorganisms are more commercially significant for their versatile application potential in laundry, food, oil chemistry, fine chemistry, pharmaceutical and paper industries, as well as in biodiesel production, waste treatment and other biotechnological applications [6–9].…”

Section: Introductionmentioning

confidence: 99%

A Novel Cold-Adapted Lipase from Sorangium cellulosum Strain So0157-2: Gene Cloning, Expression, and Enzymatic Characterization

Cheng

Qian

et al. 2011

IJMS

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Genome sequencing of cellulolytic myxobacterium Sorangium cellulosum reveals many open-reading frames (ORFs) encoding various degradation enzymes with low sequence similarity to those reported, but none of them has been characterized. In this paper, a predicted lipase gene (lipA) was cloned from S. cellulosum strain So0157-2 and characterized. lipA is 981-bp in size, encoding a polypeptide of 326 amino acids that contains the pentapeptide (GHSMG) and catalytic triad residues (Ser114, Asp250 and His284). Searching in the GenBank database shows that the LipA protein has only the 30% maximal identity to a human monoglyceride lipase. The novel lipA gene was expressed in Escherichia coli BL21 and the recombinant protein (r-LipA) was purified using Ni-NTA affinity chromatography. The enzyme hydrolyzed the p-nitrophenyl (pNP) esters of short or medium chain fatty acids (≤C10), and the maximal activity was on pNP acetate. The r- LipA is a cold-adapted lipase, with high enzymatic activity in a wide range of temperature and pH values. At 4 °C and 30 °C, the Km values of r-LipA on pNP acetate are 0.037 ± 0.001 and 0.174 ± 0.006 mM, respectively. Higher pH and temperature conditions promoted hydrolytic activity toward the pNP esters with longer chain fatty acids. Remarkably, this lipase retained much of its activity in the presence of commercial detergents and organic solvents. The results suggest that the r-LipA protein has some new characteristics potentially promising for industrial applications and S. cellulosum is an intriguing resource for lipase screening.

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Section: Introductionmentioning

confidence: 99%

A Novel Cold-Adapted Lipase from Sorangium cellulosum Strain So0157-2: Gene Cloning, Expression, and Enzymatic Characterization

Cheng

Qian

et al. 2011

IJMS

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“…To improve the recombinant protein yield, the Rosetta singles strain was used for the expression of recombinant ACLyz (Figure 1). The Rosetta (DE3) singles strain, which provides tRNAs for six rare codons, was designed to enhance the expression of eukaryotic proteins containing codons rarely used in E. coli (Fan et al 2009).…”

Section: Discussionmentioning

confidence: 99%

Overexpression and purification of recombinant lysozyme fromAgrius convolvuliexpressed as inclusion body inEscherichia coli

Park

Yoe

2012

Animal Cells and Systems

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Amongst the various antimicrobial peptides, lysozyme plays a central role in initiating and maintaining the antibacterial defense response of insect. Here we propose the biosynthesis and refolding of recombinant lysozyme in Escherichia coli expressed in inclusion body form. The Agrius lysozyme gene was amplified using gene specific primers and then ligated into the pGEX-4T-1 vector, which contained the glutathione S-transferase (GST) gene as a fusion partner. A recombinant lysozyme was expressed in E. coli Rosetta cells using a pGEX-4T-1 expression vector, and the fusion protein was induced by ioporpyl-b-D-thiogalactopyranoside (IPTG). The recombinant protein produced as an inclusion body was resolubilized in solubilization buffer, and the resultant solution was dialyzed in refolding buffer. After thrombin cleavage, the recombinant lysozyme was purified by ion exchange chromatography and reverse phase chromatography. The recombinant lysozyme was subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) analysis and immunoreactivity against the anti-Agrius lysozyme was observed by western blot analysis of this protein. The recombinant lysozyme displayed antibacterial activity against Bacillus megaterium and Micrococcus luteus, which was confirmed by the inhibition zone assay.

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“…Results from sequence analyses using E. coli codon usage analyzer (Table 1) obtained before protein overexpression revealed that mature SLLyz gene contains 17 rare codons (14% of a total of 121 codons), 11 of which include AGG, AGU, AUA, CCC, CGA, CGG, CUA, GGA, GGG, UUA, and UUG, a feature that would lead to inefficient expression. Rosetta(DE3) Singles strain providing tRNAs for six rare codons (AGA, AGG, AUA, CCC, CUA, and GGA) was designed to enhance the expression of eukaryotic proteins containing codons rarely used in E. coli (Fan et al 2009). In order to avoid translational problems and improve yield and purity of rSLLyz, we have used Rosetta(DE3) Singles strain harbouring five of the rare tRNAs (underlined) from the SLLyz for expression of the rSLLyz.…”

Section: Discussionmentioning

confidence: 99%

Recombinant expression and refolding of the c-type lysozyme from Spodoptera litura in E. coli

Kim

Yoe

Lee

et al. 2011

Electro Journal of Biotech

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The chicken-type lysozyme of the insect Spodoptera litura (SLLyz) is a polypeptide of 121 amino acids containing four disulfide bridges and 17 rare codons and participates in innate defense as an anti-bacterial enzyme. The recombinant S. litura lysozyme (rSLLyz) expressed as a C-terminal fusion protein with glutathione S-transferase (GST) in Rosetta(DE3) Singles. The protein was produced as an inclusion body which was solubilized in 8 M urea, renatured by on-column refolding, and purified by reversed-phase chromatography to 95% purity. The purified rSLLyz demonstrated antibacterial activity against B. megaterium confirmed by inhibition zone assay. The overexpression and refolding strategy described in this study will provide a reliable technique for maximizing production and purification of proteins expressed as inclusion bodies in E. coli.

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Cloning, sequence analysis and expression of bacterial lipase-coding DNA fragments from environment in Escherichia coli

Cited by 10 publications

References 14 publications

A Novel Cold-Adapted Lipase from Sorangium cellulosum Strain So0157-2: Gene Cloning, Expression, and Enzymatic Characterization

A Novel Cold-Adapted Lipase from Sorangium cellulosum Strain So0157-2: Gene Cloning, Expression, and Enzymatic Characterization

Overexpression and purification of recombinant lysozyme fromAgrius convolvuliexpressed as inclusion body inEscherichia coli

Recombinant expression and refolding of the c-type lysozyme from Spodoptera litura in E. coli

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