Biochemical properties and structure analysis of a DAG-Like lipase from Malassezia globosa.
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.
College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, China. xuhuanscut@163.com.
Huan
H
Xu
College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, China. xuhuanscut@163.com.
Dongming
D
Lan
School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China. yangbo@scut.edu.cn.
Bo
B
Yang
College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, China. yonghw@scut.edu.cn.
Yonghua
Y
Wang
D000595:Amino Acid Sequence; D001665:Binding Sites; D002210:Caprylates / Q000737:chemistry / Q000378:metabolism; D004075:Diglycerides / Q000378:metabolism; D000431:Ethanol / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008071:Lipoprotein Lipase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008290:Malassezia / Q000201:enzymology*; D008670:Metals / Q000737:chemistry; D000432:Methanol / Q000737:chemistry; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D010843:Pichia / Q000378:metabolism; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature
D002210:Caprylates; D004075:Diglycerides; D008670:Metals; D011994:Recombinant Proteins; C034106:4-nitrophenyloctanoate; D000431:Ethanol; D008071:Lipoprotein Lipase; D000432:Methanol