Uml2 is a novel CalB-type lipase of Ustilago maydis with phospholipase A activity

CalB of Pseudozyma aphidis (formerly named Candida antarctica ) is one of the most widely applied enzymes in industrial biocatalysis. Here, we describe a protein with 66 % sequence identity to CalB, designated Ustilago maydis lipase 2 (Uml2), which was identified as the product of gene um01422 of th...

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Bibliographic Details
Published in:Applied microbiology and biotechnology 2014-06, Vol.98 (11), p.4963-4973
Main Authors: Buerth, Christoph, Kovacic, Filip, Stock, Janpeter, Terfrüchte, Marius, Wilhelm, Susanne, Jaeger, Karl-Erich, Feldbrügge, Michael, Schipper, Kerstin, Ernst, Joachim F., Tielker, Denis
Format: Article
Language:English
Subjects:
Amino Acid Motifs
Analysis
Biomedical and Life Sciences
Biotechnologically Relevant Enzymes and Proteins
Biotechnology
Candida antarctica
Catalytic Domain
Cloning, Molecular
DNA Mutational Analysis
Enzymes
Fatty acids
Fungi
Gene Deletion
Gene Expression
Genetic aspects
Genetic recombination
Genomes
Life Sciences
Lipase
Microbial Genetics and Genomics
Microbiology
Pathogens
Phospholipases - genetics
Phospholipases - metabolism
Physiological aspects
Pichia - genetics
Pichia - metabolism
Pichia pastoris
Proteins
Pseudozyma
Recombinant Proteins - genetics
Recombinant Proteins - isolation & purification
Recombinant Proteins - metabolism
Sequence Analysis, DNA
Smut fungi
Studies
Substrate Specificity
Ustilago - enzymology
Ustilago - genetics
Ustilago maydis
Yeast
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Summary:CalB of Pseudozyma aphidis (formerly named Candida antarctica ) is one of the most widely applied enzymes in industrial biocatalysis. Here, we describe a protein with 66 % sequence identity to CalB, designated Ustilago maydis lipase 2 (Uml2), which was identified as the product of gene um01422 of the corn smut fungus U. maydis . Sequence analysis of Uml2 revealed the presence of a typical lipase catalytic triad, Ser-His-Asp with Ser125 located in a Thr-Xaa-Ser-Xaa-Gly pentapeptide. Deletion of the uml2 gene in U. maydis diminished the ability of cells to hydrolyse fatty acids from tributyrin or Tween 20/80 substrates, thus demonstrating that Uml2 functions as a lipase that may contribute to nutrition of this fungal pathogen. Uml2 was heterologously produced in Pichia pastoris and recombinant N -glycosylated Uml2 protein was purified from the culture medium. Purified Uml2 released short- and long-chain fatty acids from p -nitrophenyl esters and Tween 20/80 substrates. Furthermore, phosphatidylcholine substrates containing long-chain saturated or unsaturated fatty acids were effectively hydrolysed. Both esterase and phospholipase A activity of Uml2 depended on the Ser125 catalytic residue. These results indicate that Uml2, in contrast to CalB, exhibits not only esterase and lipase activity but also phospholipase A activity. Thus, by genome mining, we identified a novel CalB-like lipase with different substrate specificities.
ISSN:0175-7598
1432-0614
DOI:10.1007/s00253-013-5493-6