Occasionally, the use of an enzyme inhibitor may be necessary to suppress
undesired side reactions due to further metabolism. You should be aware that the
majority of them (in particular serine hydrolase inhibitors) are extremely toxic and
must be handled with the utmost care, like (for example) osmium tetraoxide.
Preservation of Enzyme Stability
The following processes lead to rapid enzyme deactivation and thus should be avoided:
• Dilution of enzyme solutions: Enzymes are not happy in very dilute solutions
and are therefore more stable at higher protein concentrations.
• Additives serving as enzyme stabilizers (such as polyhydroxy compounds,
carbohydrates, inactive filler proteins, etc.) should not be removed.
• Enzyme purification: It is an unfortunate, but commonly encountered fact that
pure proteins are generally less stable than crude preparations.
• Freezing and thawing: If required, freezing should be performed fast via shockfreezing (by dropping the protein solution or cell suspension into liquid nitrogen). Slow freezing leads to the formation of large ice crystals, which tend to
damage proteins. The addition of polyhydroxy compounds (e.g., glycerol, sorbitol) serving as cryo-protectant is strongly recommended.
• Very high concentrations of substrate and/or product.
• Highly charged (inorganic) ions, such as Cr
3+ , Cr
6+ , Co
2+ , Ni
2+ , Fe
3+ , Al
3+
should be avoided. Alkali and halide ions are usually harmless if present at
low to moderate concentrations. Some enzymes are stabilized by Mg
2+ , Ca
2+ or
Zn
2+ ions at low concentrations.
• High ionic strength: Buffer concentrations should be low, i.e., 0.05–0.1 M.
• Extreme temperatures: The best temperature range for performing a biocatalytic
reaction is between 20 and 30
C. Proteins and whole cells should be stored at
0 to +4
C. Most lyophilized enzyme preparations and whole cells can be stored
at this temperature for many months without a significant loss of activity.
• Extreme pH: The optimal pH-range for the most commonly used enzymes is
7.0–7.5. If acid or base is emerging during the reaction, a proper pH must be
maintained by, e.g., addition of an acid or base scavenger; if possible, this should
occur in a continuous mode via an autotitrator.
• High shear forces, caused by sharp edges or rapid stirring, cause deactivation.
Thus gentle shaking on a rotary shaker is recommended. If stirring is required,
use an overhead stirrer with a glass paddle with round edges at low to moderate
speed rather than a magnetic stirring bar, which acts as mortar and grinds your
enzymes or cells to pieces.
• Metal surfaces: Some alloys can liberate traces of harmful metal ions; reactors
and components thereof made from glass or plastic are preferable.
• Precipitation of proteins – e.g., by addition of (NH 4 ) 2 SO 4 or a water-miscible
organic solvent, such as acetone or i-propanol – often leads to a significant
degree of deactivation. On the contrary, lyophilization is the milder method for
the isolation of proteins from aqueous solutions.
• Complete dehydration causes irreversible deactivation. Proteins should therefore
be able to keep their minimum of ‘structural water’ to retain their activity. In
general, this is safely achieved via lyophilization.
416
5 Appendix
undesired side reactions due to further metabolism. You should be aware that the
majority of them (in particular serine hydrolase inhibitors) are extremely toxic and
must be handled with the utmost care, like (for example) osmium tetraoxide.
Preservation of Enzyme Stability
The following processes lead to rapid enzyme deactivation and thus should be avoided:
• Dilution of enzyme solutions: Enzymes are not happy in very dilute solutions
and are therefore more stable at higher protein concentrations.
• Additives serving as enzyme stabilizers (such as polyhydroxy compounds,
carbohydrates, inactive filler proteins, etc.) should not be removed.
• Enzyme purification: It is an unfortunate, but commonly encountered fact that
pure proteins are generally less stable than crude preparations.
• Freezing and thawing: If required, freezing should be performed fast via shockfreezing (by dropping the protein solution or cell suspension into liquid nitrogen). Slow freezing leads to the formation of large ice crystals, which tend to
damage proteins. The addition of polyhydroxy compounds (e.g., glycerol, sorbitol) serving as cryo-protectant is strongly recommended.
• Very high concentrations of substrate and/or product.
• Highly charged (inorganic) ions, such as Cr
3+ , Cr
6+ , Co
2+ , Ni
2+ , Fe
3+ , Al
3+
should be avoided. Alkali and halide ions are usually harmless if present at
low to moderate concentrations. Some enzymes are stabilized by Mg
2+ , Ca
2+ or
Zn
2+ ions at low concentrations.
• High ionic strength: Buffer concentrations should be low, i.e., 0.05–0.1 M.
• Extreme temperatures: The best temperature range for performing a biocatalytic
reaction is between 20 and 30
C. Proteins and whole cells should be stored at
0 to +4
C. Most lyophilized enzyme preparations and whole cells can be stored
at this temperature for many months without a significant loss of activity.
• Extreme pH: The optimal pH-range for the most commonly used enzymes is
7.0–7.5. If acid or base is emerging during the reaction, a proper pH must be
maintained by, e.g., addition of an acid or base scavenger; if possible, this should
occur in a continuous mode via an autotitrator.
• High shear forces, caused by sharp edges or rapid stirring, cause deactivation.
Thus gentle shaking on a rotary shaker is recommended. If stirring is required,
use an overhead stirrer with a glass paddle with round edges at low to moderate
speed rather than a magnetic stirring bar, which acts as mortar and grinds your
enzymes or cells to pieces.
• Metal surfaces: Some alloys can liberate traces of harmful metal ions; reactors
and components thereof made from glass or plastic are preferable.
• Precipitation of proteins – e.g., by addition of (NH 4 ) 2 SO 4 or a water-miscible
organic solvent, such as acetone or i-propanol – often leads to a significant
degree of deactivation. On the contrary, lyophilization is the milder method for
the isolation of proteins from aqueous solutions.
• Complete dehydration causes irreversible deactivation. Proteins should therefore
be able to keep their minimum of ‘structural water’ to retain their activity. In
general, this is safely achieved via lyophilization.
416
5 Appendix