Alkaline Extracellular Protease Produced by Saccharomycopsis lipolytica CX161-1B

Abstract

Saccharomycopsis lipolytica CX161-1B, a strain suitable for genetic studies, when grown at neutral pH produced a single alkaline extracellular protease, lower levels of acid extracellular protease(s) and no neutral extracellular protease. The alkaline protease was purified to homogeneity (as determined by polyacrylamide gel electrophoresis) by ultrafiltration, gel filtration and DEAE-cellulose chromatography. The molecular weight of the enzyme was estimated by gel filtration to be 27000-30000, and the isoelectric point was pH 5.7. The purified enzyme had an alkaline pH optimum (pH 9-10). It was completely inhibited by phenylmethylsulphonyl fluoride, reversibly inhibited by EDTA, partially inhibited by o-phenanthroline, and not inhibited by dithiothreitol, N-ethylmaleimide or 4-hydroxymercuribenzoic acid, indicating that it is a serine protease. The content of sulphur amino acids was determined, and the purified protease contained no more than 1.8% carbohydrate as determined by the phenol-sulphuric acid method. The N-terminal amino acid sequence (25 residues) was determined; the N-terminal amino acid was alanine.

Isolation and characterization of the TRP1 gene from the yeast Yarrowia lipolytica and multiple gene disruption using a TRP blaster

“…The yeast strains were routinely grown in YPD (1% yeast extract, 2% bacto-peptone and 2% glucose) at 28 • C (Y. lipolytica) or 30 • C (S. cerevisiae). Synthetic complete (SC) medium was composed of 0.67% yeast nitrogen base without amino acids (Difco), 2% glucose and drop-out amino acid mixture including all amino acids required (Ogrydziak and Mortimer, 1977). YM used for mating contained 0.3% yeast extract, 0.3% malt extract, and 0.5% bacto-peptone and CSM used for sporulation was composed of 0.67% yeast nitrogen base (Difco) and 1.5% sodium citrate (Barth and Gaillardin, 1996).…”

Dipeptidyl aminopeptidase processing and biosynthesis of alkaline extracellular protease from Yarrowia lipolytica

“…Skim milk (SKM) plates for detection of production of AEP by Y . lipolytica were prepared as described previously (Ogrydziak & Mortimer, 1977 ;Enderlin & Ogrydziak, 1993). SKM plates for detection of production of AEP by S. cerevisiae were prepared in a similar manner except for the addition of 0 1 '/o peptone, 50 mg His and Lys l-l, 100 mg Leu l-l, and 1 % (w/v) either galactose or glucose.…”

“…SKM plates for detection of production of AEP by S. cerevisiae were prepared in a similar manner except for the addition of 0 1 '/o peptone, 50 mg His and Lys l-l, 100 mg Leu l-l, and 1 % (w/v) either galactose or glucose. Extracellular protease activity secreted by S. cerevisiae strains was measured using the A,,, casein hydrolysis method (Ogrydziak & Scharf, 1982).…”

“…The dimorphic yeast Yarrowia lipolytica secretes high levels of a 32 kDa subtilisin-like alkaline extracellular protease (AEP) (Tobe et al, 1976;Ogrydziak & Scharf, 1982;Matoba et al, 1988). AEP has been used as a reporter molecule for in uiuo studies of functions of the signal recognition particle of Y .…”

A cytochemical study of acid carbohydrates on the surface of Candida lipolytica grown in tween 80-containing medium

“…Surfactants offer a potentially valuable model system for the study of extracellular metabolites production in lower eukaryotes and potential use in industry. In addition, genetic analysis is possible due to the discovery of its sexual cycle (12,37,38,43,44,49,60,62).…”