Biochemical and molecular characterization of a lipase produced by Rhizopus oryzae

Abstract

A novel strain of Rhizopus oryzae WPG secretes a noninduced lipase (ROLw) in the culture medium; purified ROLw is a protein of 29 kDa, the 45 N-terminal amino acid residues were sequenced, this sequence is very homologous to Rhizopus delemar lipase (RDL), Rhizopus niveus lipase (RNL) and R. oryzae lipase (ROL29) sequences; the cloning and sequencing of the part of the gene encoding the mature ROLw, shows two nucleotides differences with RDL, RNL and ROL29 sequences corresponding to the change of the residues 134 and 200; ROLw does not present the interfacial activation phenomenon when using tripropionin or vinyl propionate as substrates; the lipase activity is maximal at pH 8 and at 37 degrees C, specific activities of 3500 or 900 U mg(-1) were measured at 37 degrees C and at pH 8, using olive oil emulsion or tributyrin as substrates, respectively; ROLw is unable to hydrolyse triacylglycerols in the presence of high concentration of bile salts; it is a serine enzyme as it is inhibited by tetrahydrolipstatin and was stable between pH 5 and pH 8.

Expression in Pichia pastoris X33 of His-tagged lipase from a novel strain of Rhizopus oryzae and its mutant Asn 134 His: purification and characterization

“…Rhizopus oryzae was cultivated as described previously (Ben Salah et al 2006). E. coli was cultivated in LuriaBertani medium.…”

“…In previous work Ben Salah et al (2006), have produced, purified to homogeneity from the culture medium, a lipase from a novel strain of Rhizopus oryzae WPG (ROLw) and showed that the amino acid primary sequence of the 29 kDa mature ROLw lipase was 99.3% identical to Rhizopus oryzae lipase ROL32 (Sayari et al 2005), RDL (Derewenda et al 1994) and 98.9% identical to 269 amino acids in Rhizopus niveus lipase (RNL; Kohno et al 1994).…”

“…The production of extracellular lipases by Rhizopus species has been known for many years (Kohno et al 1994;Beer et al 1998;Ben Salah et al 1994, 2006Sayari et al 2005). Many studies on fungal lipases have investigated their effects on food spoilage and flavour changes, particularly in the dairy industry, food production, pharmaceuticals and industrial synthesis of fine chemicals (Jaeger and Reetz 1998).…”

“…For Rhizopus lipases, the structure of Rhizopus niveus is available (Kohno et al 1996) and sequence homology with other lipase families is determined previously (Ben Salah et al 2006). Therefore, the regions implied in the recognition of the substrate have been identified in the fundamental sequence of ROL32 (Sayari et al 2005).…”

“…Many individually named isolates, such as Rhizopus oryzae, Rhizopus arrhizus, Rhizopus delemar, Rhizopus niveus and Rhizopus javanicus latterly recognized to be the same organism, in spite of some minor variations, were combined into Rhizopus oryzae (Schipper 1984). Rhizopus oryzae lipase, Rhizopus delemar lipase (RDL) and Rhizopus javanicus lipase have a substitution in the His134 and the Leu 234 by an Asn and a Leu, respectively (Ben Salah et al 2006).…”