Optimization of cold-adapted lysozyme production from the psychrophilic yeast Debaryomyces hansenii using statistical experimental methods.

UNLABELLED: Statistical experimental designs were employed to optimize culture conditions for cold-adapted lysozyme production of a psychrophilic yeast Debaryomyces hansenii. In the first step of optimization using Plackett-Burman design (PBD), peptone, glucose, temperature, and NaCl were identified as significant variables that affected lysozyme production, the formula was further optimized using a four factor central composite design (CCD) to understand their interaction and to determine their optimal levels. A quadratic model was developed and validated. Compared to the initial level (18.8 U/mL), the maximum lysozyme production (65.8 U/mL) observed was approximately increased by 3.5-fold under the optimized conditions. PRACTICAL APPLICATION: Cold-adapted lysozymes production was first optimized using statistical experimental methods. A 3.5-fold enhancement of microbial lysozyme was gained after optimization. Such an improved production will facilitate the application of microbial lysozyme. Thus, D. hansenii lysozyme may be a good and new resource for the industrial production of cold-adapted lysozymes.

Salt-dependent expression of ammonium assimilation genes in the halotolerant yeast, Debaryomyces hansenii.

Debaryomyces hansenii is adapted to grow in saline environments, accumulating high intracellular Na(+) concentrations. Determination of the DhGDH1-encoded NADP-glutamate dehydrogenase enzymatic activity showed that it increased in a saline environment. Thus, it was proposed that, in order to overcome Na(+) inhibition of enzyme activity, this organism possessed salt-dependent mechanisms which resulted in increased activity of enzymes pertaining to the central metabolic pathways. However, the nature of the mechanisms involved in augmented enzyme activity were not analyzed. To address this matter, we studied the expression of DhGDH1 and DhGLN1 encoding glutamine synthetase, which constitute the central metabolic circuit involved in ammonium assimilation. It was found that: (1) expression of DhGDH1 is increased when D. hansenii is grown in the presence of high NaCl concentrations, while that of DhGLN1 is reduced, (2) DhGDH1 expression in Saccharomyces cerevisiae takes place in a GLN3- and HAP2,3-dependent manner and (3) salt-dependent DhGDH1 and DhGLN1 expression involves mechanisms which are limited to D. hansenii and are not present in S. cerevisiae. Thus, salt-dependent regulation of the genes involved in central metabolic pathways could form part of a strategy leading to the ability to grow under hypersaline conditions.

Response of bacteria and fungi to high-pressure stress as investigated by two-dimensional polyacrylamide gel electrophoresis.

In an attempt to generalize previous observations (Jaenicke et al., Appl. Environ. Microbiol. 1988, 54, 2375-2380) and to find a convenient model system for studies of the pressure response, we tested the suitability of Escherichia coli and Thermotoga maritima (bacteria), and of five different eukaryotic species including the filamentous fungi Asteromyces cruciatus and Dendryphiella salina, and the marine yeasts Debaryomyces hansenii, Rhodosporidium sphaerocarpum, and Rhodotorula rubra. Using two-dimensional polyacrylamide gel electrophoresis, detailed investigations on the pressure response were carried out with E. coli and Rhodosporidium sphaerocarpum. In the former organism, major pressure response proteins could not be detected, although there are significant differences in expression of some proteins as well as some minor components that are found in all of the high pressure cell extracts but not in extracts from cultures grown at atmospheric pressure. In Rhodosporidium sphaerocarpum, no change in protein expression patterns was observed between 0.1 and 20 MPa. However, approaching the limit of viability of 50 MPa, additional protein spots became detectable at 45 MPa. This finding correlates with the observation of abnormal growth forms of the organism at this pressure (Lorenz, R. et al. manuscript in preparation).

Killer toxin of Hanseniaspora uvarum.

The yeast Hanseniaspora uvarum liberates a killer toxin lethal to sensitive strains of the species Saccharomyces cerevisiae. Secretion of this killer toxin was inhibited by tunicamycin, an inhibitor of N-glycosylation, although the mature killer protein did not show any detectable carbohydrate structures. Culture supernatants of the killer strain were concentrated by ultrafiltration and the extracellular killer toxin was precipitated with ethanol and purified by ion exchange chromatography. SDS-PAGE of the electrophoretically homogenous killer protein indicated an apparent molecular mass of 18,000. Additional investigations of the primary toxin binding sites within the cell wall of sensitive yeast strains showed that the killer toxin of Hanseniaspora uvarum is bound by beta-1, 6-D-glucans.