Secretory phospholipase A(2) (sPLA(2)) catalyzes the hydrolysis of sn-2 linkage in the glycerophospholipid, thereby releasing fatty acid and 1-acyl lysophospholipid. Among sPLA(2)s from various organisms and tissues, group XIV fungal/bacterial sPLA(2)s are relatively less characterized compared to their mammalian counterparts. Here we report cloning, recombinant expression, refolding, and enzymatic characterization of two sPLA(2)s, NCU06650 and NCU09423, from the filamentous fungus Neurospora crassa. The hexahistidine-tagged putative mature region of both proteins was expressed in Escherichia coli. Inclusion bodies were solubilized using a high hydrostatic pressure refolding technique. NCU06650 was solubilized without any additives at alkaline pH, and the addition of arginine or non-detergent sulfobetain (NDSB) significantly improved the process at acidic pH. In contrast, NCU09423 was solubilized only when NDSB was added at alkaline pH. Both enzymes displayed a Ca2+-dependent lipolytic activity toward E. coli membrane. Mass spectrometry analysis using the synthetic phospholipids as substrates demonstrated that both enzymes preferentially cleaved the sn-2 ester linkage of substrates and generated 1-acyl lysophospholipids, demonstrating that they are bona fide PLA(2). (C) 2015 Elsevier Inc. All rights reserved.