Characteristics of Saccharomyces cerevisiae gal1 Delta and gal1 Delta hxk2 Delta mutants expressing recombinant proteins from the GAL promoter.

Galactose can be used not only as an inducer of the GAL promoters, but also as a carbon source by Saccharomyces cerevisiae, which makes recombinant fermentation processes that use GAL promoters complicated and expensive. To overcome this problem during the cultivation of the recombinant strain expressing human serum albumin (HSA) from the GAL10 promoter, a gal1 Delta mutant strain was constructed and its induction kinetics investigated. As expected, the gal1 Delta strain did not use galactose, and showed high levels of HSA expression, even at extremely low galactose concentrations (0.05-0.1 g/L). However, the gal1 Delta strain produced much more ethanol, in a complex medium containing glucose, than the GAL1 strain. To improve the physiological properties of the gal1 Delta mutant strain as a host for heterologous protein production, a null mutation of either MIG1 or HXK2 was introduced into the gal1 Delta mutant strain, generating gal1 Delta mig1 Delta and gal1 Delta hxk2 Delta double strains. The gal1 Delta hxk2 Delta strain showed a decreased rate of ethanol synthesis, with an accelerated rate of ethanol consumption, compared to the gal1 Delta strain, whereas the gal1 Delta mig1 Delta strain showed similar patterns to the gal1 Delta strain. Furthermore, the gal1 Delta hxk2 Delta strain secreted much more recombinant proteins (HSA and HSA fusion proteins) than the other strains. The results suggest that the gal1 Delta hxk2 Delta strain would be useful for the large-scale production of heterologous proteins from the GAL10 promoter in S. cerevisiae.

Optimizing lipases and related enzymes for efficient application.

Although numerous reactions have been performed using lipases and related enzymes (e.g. esterases and phospholipases), it is still a challenge to identify the most suitable biocatalyst and best reaction conditions for an efficient application. Frequently used methods such as immobilization and optimization of the reaction medium cannot be transferred from one reaction system or substrate to another. However, in the past few years, rational protein design and directed evolution have emerged as efficient alternative methods to optimize biocatalytic reactions.

Developments and trends in enzyme catalysis in nonconventional media.

The conventional notion that enzymes are only active in aqueous media has long been discarded, thanks to the numerous studies documenting enzyme activities in nonaqueous media, including pure organic solvents and supercritical fluids. Enzymatic reactions in nonaqueous solvents offer new possibilities for producing useful chemicals (emulsifiers, surfactants, wax esters, chiral drug molecules, biopolymers, peptides and proteins, modified fats and oils, structured lipids and flavor esters). The use of enzymes in both macro- and microaqueous systems has been investigated especially intensively in the last two decades. Although enzymes exhibit considerable activity in nonaqueous media, the activity is low compared to that in water. This observation has led to numerous studies to modify enzymes for specific purposes by various means including protein engineering. This review covers the historical developments, major technological advances and recent trends of enzyme catalysis in nonconventional media. A brief description of different classes of enzymes and their use in industry is provided with representative examples. Recent trends including use of novel solvent systems, role of water activity, stability issues, medium and biocatalyst engineering aspects have been discussed with examples. Special attention is given to protein engineering and directed evolution.