Biochemical Properties and Structure Analysis of a DAG-Like Lipase from Malassezia globosa

Abstract

Diacylglycerol (DAG)-like lipases are found to play an important role in the life sciences and industrial fields. A putative DAG-like lipase (MgMDL2) from Malassezia globosa was cloned and expressed in recombinant Pichia pastoris. The recombinant MgMDL2 was expressed as a glycosylated protein and purified into homogeneity by anion exchange chromatography. The activity of recombinant MgMDL2 was optimal at 15 °C and pH 6.0, and it keeps over 50% of relative activity at 5 °C, suggesting that MgMDL2 was a cold active lipase. MgMDL2 retained over 80% of initial activity after incubation at 30 and 40 °C for 2.5 h, but it was not stable at 50 °C. Incubation of methanol and ethanol at a concentration of 30% for 2 h did not affect the recombinant enzyme activity, while metal ions, including Ca2+, Mn2+ and Ni2+, sharply inhibited the MgMDL2 activity at 5 mM by 42%, 35% and 36%, respectively. MgMDL2 exhibited a preference for medium chain-length esters with highest activity toward p-nitrophenyl caprylate, while it was active on mono- and diacylglycerol but not on triacylglycerol, indicating that it was a typical DAG-like lipase. By homology modeling, Phe278 was predicted to be involved in the preference of MgMDL2 for monoacyl- and diacyl-glyceride substrates, but not triglycerides.

Secreted lipases from Malassezia globosa: recombinant expression and determination of their substrate specificities

“…However, no family class 3 lipase showed a significant activity with triolein. This observation is not surprising, as MgLip1 and MgMDL2, belonging to the family class 3 lipases, have been reported to be active with MAGs and DAGs only Xu et al, 2015). As TAG is a key component of human sebum, these results suggest…”

“…During expression in P. pastoris, a majority (70 to 90 %) of the Asn residues in potential N-glycosylation sites will be modified by glycosylation (Skropeta et al, 2007). N-Glycosylation was previously shown for heterologously expressed MgLip1 and MgMDL2 from P. pastoris cells (Xu et al, 2012b(Xu et al, , 2015, and N-glycosylation has also been shown to be a post-translation modification in M. globosa as MgLip2 purified from M. globosa also exhibited a larger molecular mass than expected (55 vs 49.5 kDa). MALDI-TOF MS analysis of the purified protein confirmed the identity as MgLip2 (Juntachai et al, 2011).…”

“…2 exclusively for the M. globosa lipases. These two families are LIP (PF03583), containing MgLip2 (Juntachai et al, 2011), and family class 3 (PF01764), containing MgLip1 (DeAngelis et al, 2007) and MgMDL2 (Xu et al, 2015) (Fig. 2).…”

“…However, monoolein appeared in assays using MgMDL2 (Fig. 4) but, as already shown, MgMDL2 is able to hydrolyse both 1,2-and 1,3-DAGs (Xu et al, 2015).…”

“…Three lipases from M. globosa have recently been characterized. Biochemical characterization of MgLip1 and MgMDL2 (Xu et al, 2015) revealed that these lipases utilized MAGs and DAGs as substrates, but not TAGs. In addition, MgLip2 was identified, purified and published as a further extracellular lipase from M. globosa (Juntachai et al, 2011).…”