Thomas Wartmann, Irene Kunze, Bui Minh Due, Renate Manteuffel, Gotthard Kunze. Institute ..... Buttner, R., Bode, R., Birnbaum, D. (1991): Comparative study of ...
Microbio!. Res. (1995) 150, 113 -120
Microbiological Research ©
Gustav Fischer Verlag Jena
Comparative biochemical, genetical and immunological studies of glucoamylase producing Arxula adeninivorans yeast strains Thomas Wartmann, Irene Kunze, Bui Minh Due, Renate Manteuffel, Gotthard Kunze Institute of Plant Genetics and Crop Plant Research, Corrensstr. 3, D -06466 Gatersleben, Germany Accepted: February 5, 1995
Abstract Seven Arxula adeninivorans strains originating from The Netherlands (CSIR 1136, CSIR 1138 and CBS 8244T), South Africa (CSIR 1147, CSIR 1148 and CSIR 1149) and Siberia (Ls3) were compared concerning their secretory glucoamylase. Within t4e spectrum of secretory polypeptides obtained by SDS-polyacrylamide gel electrophoresis, the glucoamylase corresponding polypeptide, could be identified by means of specific antibodies directed against the glucoamylase of strain Ls3.The molecular masses of the glucoamylases vary from 84 to 95 kDa. After endoglycosidase F treatment of the secretory proteins, the N-linked carbohydrate content for each glucoamylase could be quantified at about 25%. Glucoamylase activity staining of secretory proteins separated under nondenaturing conditions revealed one glucoamylase active protein for the Dutch strains and two active forms for the strains originating from South Africa and Siberia. Highest activities of glucoamylase can be measured at the end of the exponential growth phase for each strain. The Dutch strain CSIR 1138 secretes the highest activity. Optimum values for temperature and pH were determined and compared. By means of pulsed field gel electrophoresis and Southern analysis, the glucoamylase corresponding gene could be localized on the second of the four chromosomes of each yeast strain. Key words: Arxula adeninivorans protein secretion - yeast
glucoamylase -
Introduction One of the characteristics of the yeast Arxula adeninivorans is its inducible production and secretion of starch degrading enzymes. Buttner et al. (1987) purified and characterized the secretory glucoamyCorresponding author : G. Kunze
lase (1.4-IX-D-glucan glucohydrolase, EC 3.2.1.3.) of the yeast strain Ls3 . They have found that the strain secretes only one amylase into a growth medium containing starch or maltose as carbon source. The optimal temperature of the enzyme is 60 -70 °C depending on the substrate concentration used in the enzyme assay. The highest activity could be obtained in a weak acid pH range between 4.0 and 5.5. The molecular mass of the extracellular glucoamylase of strainLs3 is 225 kDa as determined by Sephadex G-200 gel filtration, 110 kDa by SDSpolyacrylamide gel electrophoresis (SDS-P AG E)and 76 kDa after deglycosylation using endoglycosidase H. The native secretory glucoamylase of the strain A. adeninivorans Ls3 consists of two subunits and contains about 30% carbohydrate (Buttner et al. 1987). These authors characterized one intracellular glucoamylase with a molecular mass of about 95 kDa and assume that this intracellular form is further glycosylated during secretion (Buttner et al. 1991). In this study we compared the secretory glucoamylase of the strain Ls3 originating from Siberia, with those of three other A. adeninivorans strains from The Netherlands (CSIR 1136, CSIR 1138, CBS 8244T) and three strains from South Africa (CSIR 1147, CSIR 1148, CSIR 1149). This analysis should show whether the known properties of glucoamylase of Ls3 can be generalized tQ other A. adeninivorans strains.
Materials and methods Strains and growth conditions. The following strains, given by the Division of the "Centraalbureau voor Schimmelcultuno:s
