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

Abstract

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.

A Novel CalB-Type Lipase Discovered by Fungal Genomes Mining

“…For N-deglycosylation, the purified protein was dialyzed against sodium citrate buffer, pH 5.5, and then incubated with Endo H at 37°C for 24 h, following the manufacturer’s instructions. Protein concentration was determined by theBradford (Bio-Rad), bovine serum albumin was used as standard [ 6 ].…”

“…These closely related enzymes are distributed among animals, plants and microorganism. One of the most important lipases used in biotechnological applications is CalBfrom the basidiomycete Pseudozyma antarctica ,formerly known as Candida antarctica [ 4 ].This yeast belongs to the Ustilagomycetes class, which includes the plant pathogen Ustilago maydis , where few lipases have been recently characterized [ 5 , 6 ].CalB shows broad substrate specificity, stability, and high regio-and enantio-selectivity. Moreover, itis active under conditions that are unnatural to most other enzymeslike in non-aqueous organic solvents and at elevated temperatures[ 7 ].…”

Nitrogen Source Affects Glycolipid Production and Lipid Accumulation in the Phytopathogen Fungus <i>Ustilago maydis</i>

“…It has also been reported that the fatty acid profile of MEL contains C14:0, C16:0, C14:1 and C16:1 in U. nuda, but the enzymes involved in the synthesis of this molecule have not been identified [34]. If fatty acids are accumulated they can be the raw material to be processed and join to carbohydrate molecules involving enzymes as lipases [35]. So biosurfactant secreted into the culture medium containing lipids and carbo hydrate moieties involve a large amount of enzyme synthesis, but do not yet know how this happens.…”

Monitoring of the enzymatic activity of intracellular lipases of <em>Ustilago maydis</em> expressed during the growth under nitrogen limitation and its correlation in lipolytic reactions

“…This enzyme could participate in the mobilization of LDs. In silico analysis of the U. maydis genome reported 21 genes with codes to hypothetical lipases, but only two lipases, CalA and CalB, have been characterized, and neither of these two enzymes has been implicated in the mobilization of LDs in this yeast (Brundiek et al, 2012;Buerth et al, 2014). However, it is not unreasonable to think that TAGs stored in the LDs are degraded by a cascade of hydrolytic reactions that converts TAGs into diacylglycerol (DG), monoacylglycerol (MG) and finally glycerol and fatty acids (FA) that could be used for β-oxidation in mitochondria (Rambold et al, 2015), suggesting that the full degradation of TAGs might be carried out by a group of lipases.…”