An extracellular lipase from the dimorphic yeast Arxula adeninivorans: molecular cloning of the ALIP1 gene and characterization of the purified recombinant enzyme.
The lipase-encoding Arxula adeninivorans ALIP1 gene was isolated using fragments of lipase isolates obtained by trypsin digestion for the definition of oligonucleotide primers in a PCR screening approach. The gene harbours an ORF of 1347 bp encoding a 420 amino acid protein of some 50 kDa preceded by an N-terminal 28 prepro-secretion sequence. The deduced amino acid sequence was found to be similar to the lipases from Candida albicans and C. parapsilosis (34-38% identity) and more distantly related to other lipases. The sequence contains the consensus pentapeptide motif (-Gly-X-Ser-X-Gly-) that forms a part of the interfacial lipid recognition site in lipases. The expression of the gene is regulated by carbon source. In media supplemented with Tween 20, induction of the ALIP1 gene and accumulation of the encoded lipase in the medium is observed, thus demonstrating gene regulation by lipophilic compounds. The enzyme characteristics are analysed from isolates of native strains as well as from those of recombinant strains expressing the ALIP1 gene under control of the strong A. adeninivorans-derived TEF1 promoter. For both proteins a molecular mass of 100 kDa was determined, indicating a dimeric structure, a pH optimum at pH 7.5 and a temperature optimum at 30 degrees C. The enzyme hydrolyses all ester bonds in all triglyceride substrates tested. Middle-sized chain fatty acids are more efficiently hydrolysed than short- and long-chain fatty acids, with the highest activity on C8/C10 fatty acid esters pNP-caprylate, pNP-caprate and tricaprylin.
Böer
Erik
E
Institut für Pflanzengenetik und Kulturpflanzenforschung, Corrensstrasse 3, D-06466 Gatersleben, Germany.
Mock
Hans Peter
HP
Bode
Rüdiger
R
Gellissen
Gerd
G
Kunze
Gotthard
G
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D005227:Fatty Acids / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D008049:Lipase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004718:Saccharomycetales / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D017422:Sequence Analysis, DNA
D005227:Fatty Acids; D005656:Fungal Proteins; D011994:Recombinant Proteins; D008049:Lipase