title: Cloning, characterization and expression of the extracellular lipase gene from Aureobasidium pullulans HN2-3 isolated from sea saltern.
abstract: The extracellular lipase structural gene was isolated from cDNA of Aureobasidium pullulans HN2-3 by using SMART(TM) RACE cDNA amplification kit. The gene had an open reading frame of 1245 bp long encoding a lipase. The coding region of the gene was interrupted by only one intron (55 bp). It encodes 414 amino acid residues of a protein with a putative signal peptide of 26 amino acids. The protein sequence deduced from the extracellular lipase structural gene contained the lipase consensus sequence (G-X-S-X-G) and three conserved putative N-glycosylation sites. According to the phylogenetic tree of the lipases, the lipase from A. pullulans was closely related to that from Aspergillus fumigatus (XP_750543) and Neosartorya fischeri (XP_001257768) and the identities were 50% and 52%, respectively. The mature peptide encoding cDNA was subcloned into pET-24a (+) expression vector. The recombinant plasmid was expressed in Escherichia coli BL21(DE3). The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular mass of about 47 kDa was found. Enzyme activity assay verified the recombinant protein as a lipase. A maximum activity of 0.96 U/mg was obtained from cellular extract of E. coli BL21(DE3) harboring pET-24a(+)LIP1. Optimal pH and temperature of the crude recombinant lipase were 8.0 and 35 degrees C, respectively and the crude recombinant lipase had the highest hydrolytic activity towards peanut oil.
authors.affiliation: UNESCO Chinese Center of Marine Biotechnology, Ocean University of China, Yushan Road, No. 5, Qingdao, China.
authors.forename: Zhiqiang
authors.initials: Z
authors.lastname: Liu
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Li
authors.forename: Zhenming
authors.initials: Z
authors.lastname: Chi
authors.forename: Lin
authors.initials: L
authors.lastname: Wang
authors.forename: Jing
authors.initials: J
authors.lastname: Li
authors.forename: Xianghong
authors.initials: X
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D001203:Ascomycota / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006868:Hydrolysis; D008049:Lipase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D012623:Seawater / Q000382:microbiology*; D016415:Sequence Alignment; D013696:Temperature
chemical_list: D005656:Fungal Proteins; D008049:Lipase