Classification of Lactococcus lactis cell envelope proteinase based on gene sequencing, peptides formed after hydrolysis of milk, and computer modeling.

Lactococcus lactis strains depend on a proteolytic system for growth in milk to release essential AA from casein. The cleavage specificities of the cell envelope proteinase (CEP) can vary between strains and environments and whether the enzyme is released or bound to the cell wall. Thirty-eight Lc. lactis strains were grouped according to their CEP AA sequences and according to identified peptides after hydrolysis of milk. Finally, AA positions in the substrate binding region were suggested by the use of a new CEP template based on Streptococcus C5a CEP. Aligning the CEP AA sequences of 38 strains of Lc. lactis showed that 21 strains, which were previously classified as group d, could be subdivided into 3 groups. Independently, similar subgroupings were found based on comparison of the Lc. lactis CEP AA sequences and based on normalized quantity of identified peptides released from αS1-casein and ß-casein. A model structure of Lc. lactis CEP based on the crystal structure of Streptococcus C5a CEP was used to investigate the AA positions in the substrate-binding region. New AA positions were suggested, which could be relevant for the cleavage specificity of CEP; however, these could only explain 2 out of 3 found subgroups. The third subgroup could be explained by 1 to 5 AA positions located opposite the substrate binding region.

Interactions between Zygosaccharomyces mellis and Wallemia sebi in diluted molasses.

The yeast Zygosaccharomyces mellis and the mould Wallemia sebi were isolated from the same sample of crystalline sugar. Interactions between these fungi were investigated using a diluted molasses medium (water activity 0.89, pH 6.0) as a model system for the syrup film covering the surface of moist crystalline sugar. Single and mixed cultures of Z. mellis and W. sebi were incubated at 25 degrees C for 400 h. Our results show that the growth of Z. mellis in single culture was limited by available glucose and fructose, and that W sebi was able to invert sucrose to glucose and fructose in both single and mixed culture. Furthermore, the presence of W. sebi in the mixed culture increased the maximum specific growth rate of Z. mellis from 0.074 to 0.19 h(-1) and the growth yield of Z. mellis from 7.3 x 10(6) to 5.4 x 10(7) cfu/ml. These results indicate that the ability of W. sebi to invert sucrose may stimulate the growth of Z. mellis. Finally, the presence of Z. mellis inhibited the ability of W. sebi to invert sucrose: W. sebi was able to invert 1.0 g sucrose/l per h in single culture but only 0.6 g sucrose/l per h in mixed culture. As predicted by Raoults law, this corresponded to a reduction in the water activity of the growth medium from 0.890 to 0.850 in single culture, and to 0.865 in mixed culture.

An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains.

To select a Saccharomyces cerevisiae reference strain amenable to experimental techniques used in (molecular) genetic, physiological and biochemical engineering research, a variety of properties were studied in four diploid, prototrophic laboratory strains. The following parameters were investigated: 1) maximum specific growth rate in shake-flask cultures; 2) biomass yields on glucose during growth on defined media in batch cultures and steady-state chemostat cultures under controlled conditions with respect to pH and dissolved oxygen concentration; 3) the critical specific growth rate above which aerobic fermentation becomes apparent in glucose-limited accelerostat cultures; 4) sporulation and mating efficiency; and 5) transformation efficiency via the lithium-acetate, bicine, and electroporation methods. On the basis of physiological as well as genetic properties, strains from the CEN.PK family were selected as a platform for cell-factory research on the stoichiometry and kinetics of growth and product formation.