Cloning of an alkaline lipase gene from <i>Penicillium cyclopium</i> and its expression in <i>Escherichia coli</i>

Wu, Minchen; Zhou, Qian; Pin, Jiang; Min, Taishan; Sun, Chao; Wei-da, Huang

doi:10.1007/s11745-003-1051-7

Cited by 18 publications

(11 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…2). However, the number of introns and amino acid residues varies among fungal lipases (Toida et al 2000;Eddine et al 2001;Wu et al 2003), and A. nidulans encodes several putative lipases (Galagan et al 2005). The predicted LipA protein from A. nidulans encodes a lipase with the amino acid sequence consensus GVSIS and WIFGGG as the catalytic signature.…”

Section: Resultsmentioning

confidence: 99%

A Single Amino Acid Substitution in One of the Lipases of Aspergillus nidulans Confers Resistance to the Antimycotic Drug Undecanoic Acid

et al. 2008

View full text Add to dashboard Cite

A plausible approach to evaluate the inhibitory action of antifungals is through the investigation of the fungal resistance to these drugs. We describe here the molecular cloning and initial characterization of the A. nidulans lipA gene, where mutation (lipA1) conferred resistance to undecanoic acid, the most fungitoxic fatty acid in the C(7:0)-C(18:0) series. The lipA gene codes for a putative lipase with the sequence consensus GVSIS and WIFGGG as the catalytic signature. Comparison of the wild-type and LIP1 mutant strain nucleotide sequences showed a G --> A change in lipA1 allele, which results in a Glu(214) --> Lys substitution in LipA protein. This ionic charge change in a conserved LipA region, next to its catalytic site, may have altered the catalytic properties of this enzyme resulting in resistance to undecanoic acid.

show abstract

Section: Resultsmentioning

confidence: 99%

A Single Amino Acid Substitution in One of the Lipases of Aspergillus nidulans Confers Resistance to the Antimycotic Drug Undecanoic Acid

et al. 2008

View full text Add to dashboard Cite

show abstract

“…cyclopium PG37 was cultured in a liquid medium containing 4.0% (w/v) soybean meal, 3.0% (v/v) corn steep liquor, 0.75% (w/v) soybean phospholipids, 1.0% (w/v) K 2 HPO 4 , 0.1% (w/v) MgSO 4 and 0.05% (w/v) trisodium citrate, pH 7.5 (Wu et al 2003). E. coli JM109 and E. coli DH5a were grown in the Luria-Bertani medium, containing 1.0% (w/v) tryptone, 0.5% (w/v) yeast extract and 1.0% (w/v) NaCl, pH 7.2 (Sambrook and Russell 2001).…”

Section: Methodsmentioning

confidence: 99%

“…One transformant expressing the highest rLipI activity was preserved and used for subsequent studies. rLipI activity assays A qualitative assay for rLipI activity was carried out according to the method described previously (Wu et al 2003) with appropriate modification. A brief protocol was as follows: 3.75 g agar, 7.5 ml tributyrin, 19.0 ml 3% (w/ v) polyvinyl alcohol (PVA), 2.5 ml 1.0% (w/v) Victoria blue and 221 ml 50 mM glycine-NaOH buffer (pH 10.0) were added to a 500 ml flask and mixed under heating until homogeneity.…”

Section: Transformation and Rlipi Expressionmentioning

confidence: 99%

“…The temperature and pH optima of the purified LipI were 25°C and 10.0, respectively, belonging to a low-temperature alkaline enzyme (Wu et al 1999). Subsequently, the cDNA encoding P. cyclopium PG37 LipI was cloned and expressed functionally in Escherichia coli (Wu et al 2003;Deng et al 2008), but failed in P. pastoris. In this work, we report the high-level heterologous expression of the LipI gene (lipI) from P. cyclopium PG37 in P. pastoris GS115.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

High-level heterologous expression of an alkaline lipase gene from Penicillium cyclopium PG37 in Pichia pastoris

Tan

et al. 2011

World J Microbiol Biotechnol

Self Cite

View full text Add to dashboard Cite

A 777-bp cDNA fragment encoding a mature alkaline lipase (LipI) from Penicillium cyclopium PG37 was amplified by RT-PCR, and inserted into the expression plasmid pPIC9 K. The recombinant plasmid, designated as pPIC9 K-lipI, was linearized with SalI and transformed into Pichia pastoris GS115 (his4, Mut ? ) by electroporation. MD plate and YPD plates containing G418 were used for screening of the multi-copy P. pastoris transformants (His ? , Mut ? ). One transformant resistant to 4.0 mg/ml of G418, numbered as P. pastoris GSL4-7, expressing the highest recombinant LipI (rLipI) activity was chosen for optimizing expression conditions. The integration of the gene LipI into the P. pastoris GS115 genome was confirmed by PCR analysis using 5 0 -and 3 0 -AOX1 primers. SDS-PAGE and lipase activity assays demonstrated that the rLipI, a glycosylated protein with an apparent molecular weight of about 31.5 kDa, was extracellularly expressed in P. pastoris. When the P. pastoris GSL4-7 was cultured under the optimized conditions, the expressed rLipI activity was up to 407 U/ml, much higher than that (10.5 U/ml) expressed with standard protocol. The rLipI showed the highest activity at pH 10.5 and 25°C, and was stable at a broad pH range of 7.0-10.5 and at a temperature of 30°C or below.

show abstract

“…The promoter region of E001 xynI was amplified according to the ligation-mediated PCR amplification approach as reported previously (Wu et al 2003). …”

Section: Reverse Transcription Of Total Rnamentioning

confidence: 99%

Cloning and sequence analysis of an acidophilic xylanase (XynI) gene from Aspergillus usamii E001

Wang

Zhang

et al. 2010

World J Microbiol Biotechnol

Self Cite

View full text Add to dashboard Cite

The full-length cDNA gene that encodes an acidophilic endo-1,4-b-xylanase XynI of Aspergillus usamii E001 was amplified by rapid amplification of cDNA 3 0 and 5 0 ends (RACE) using the total RNA as template and then cloned onto the pUCm-T vector, followed by sequencing. The cloned cDNA is 881 bp in length including 5 0 and 3 0 non-encoding regions, as well as a 678 bp of open reading frame (ORF) which encodes an E001 XynI of 188 amino acid residues together with a signal peptide of 37 amino acid residues. The homologies of E001 XynI with xylanases of Aspergillus niger, Aspergillus kawachii, Emericella nidulans and Penicillium funiculosum are 97.8, 92.0, 74.6 and 60.5%, respectively. From a BLAST search result, we concluded that E001 XynI belongs to the glycoside hydrolase family 11. Its three-dimensional structure was predicted using http://swiss-model.expasy.org/on-line programs based on that of the P. funiculosum xylanase (1TE1B) from the family 11. In addition, the complete DNA gene xynI encoding E001 XynI was cloned from the genomic DNA of A. usamii E001 by conventional PCR and ligation-mediated PCR amplification. The cloned xynI is 1,206 bp in length, composed of a promoter region, a 68 bp of intron and two exons when compared with the cDNA of E001 XynI.

show abstract

Cloning of an alkaline lipase gene from Penicillium cyclopium and its expression in Escherichia coli

Cited by 18 publications

References 28 publications

A Single Amino Acid Substitution in One of the Lipases of Aspergillus nidulans Confers Resistance to the Antimycotic Drug Undecanoic Acid

A Single Amino Acid Substitution in One of the Lipases of Aspergillus nidulans Confers Resistance to the Antimycotic Drug Undecanoic Acid

High-level heterologous expression of an alkaline lipase gene from Penicillium cyclopium PG37 in Pichia pastoris

Cloning and sequence analysis of an acidophilic xylanase (XynI) gene from Aspergillus usamii E001

Contact Info

Product

Resources

About