Cloning, expression, characterization and role of the leader sequence of a lipase from Rhizopus oryzae

• Published in: Biochimica et biophysica acta 1998-08, Vol.1399 (2), p.173-180
• Main Authors: Beer, H.Dietmar, McCarthy, John E.G., Bornscheuer, Uwe T., Schmid, Rolf D.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; amino acid sequences; Base Sequence; Cloning; Cloning, Molecular; Enzyme Stability; Escherichia coli - enzymology; Expression; Gene Expression; Genes, Fungal; Leader sequence; Lipase; Lipase - biosynthesis; Lipase - chemistry; Lipase - genetics; Molecular Sequence Data; nucleotide sequences; Protein Folding; Protein Precursors - biosynthesis; Protein Precursors - genetics; Protein Sorting Signals - chemistry; Protein Sorting Signals - genetics; Rhizopus - enzymology; Rhizopus - genetics; Rhizopus oryzae; Sequence; triacylglycerol lipase
• ISSN: 0167-4781; 0006-3002; 1879-2634; 1878-2434
• DOI: 10.1016/S0167-4781(98)00104-3

A lipase from Rhizopus oryzae DSM 853 (ROL) was cloned from a chromosomal gene bank, sequenced and overexpressed in Escherichia coli. ROL and its precursors ProROL and PreProROL were purified and their pH and temperature profile was determined. In contrast to ROL, ProROL and PreProROL had considerably higher thermostability and a slightly higher pH optimum. Moreover, it could be demonstrated by in vitro experiments that the natural leader sequence of ROL is able to inhibit the folding supporting properties of the prosequence, resulting in a retardation of folding. In addition, there is strong evidence that all different lipase forms derived from Rhizopus sp. described in the literature are a result of different proteolytic processing and originate from the same gene.