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Physical & Chemical properties

Water solubility

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Reference
Endpoint:
water solubility
Data waiving:
other justification
Justification for data waiving:
other:

Description of key information

The solubility of alpha-amylases are generally between 1 and 100 g/L in tap water (moderate salinity at pH 7 or just below). Increasing pH generally leads to higher solubility. 

Key value for chemical safety assessment

Water solubility:
100 g/L
at the temperature of:
25 °C

Additional information

The solubility of alpha-amylases in purified water at pH 7 is between 1 and 100 g/l. The degree of solubility at a given pH is depending on the conditions (temperature, the amino acid sequence and structure of the enzyme and other components in the system such as salts). The amino acid sequence and structure affect the polarity including the isoelectric point (pI) of the enzyme which are important factors for solubility, the difference in solubility is thus a reflection of the variation in the amino acid sequence. Enzymes generally have the lowest solubility when the pH is close to pI (+/- 1 pH unit) and the solubility increases when pH is shifting away from pI, as long as the pH is not denaturing the enzyme. The pI of industrial alpha-amylases ranges from 3.5 to 8.5. Also post translational modifications influence the solubility, where the most important is glycosylation that typically increases the solubility.

The influence of pH and salt concentration on protein stability has been investigated in the following publications (Carbonnaux et al., 1995; Green, 1932; Guilloteau et al., 1992; Hofmeister 1888). In a more recent study, the solubility of alpha-amylases is described (Faber, 2006). The alpha-amylases investigated here had pI of 5.5-6.1 and showed an increased solubility with increasing pH when analyzed for in the pH range 6 to 10. The study shows a ten to hundredfold increase in solubility at pH 10 compared to pH 6 (Faber 2006). The effects of anions and cations on protein solubility in general are described by the Hofmeister series (Hofmeister 1888) and this was observed also to be the case for alpha-amylases although no reversal of the Hofmeister series upon changing polarity of the protein net charge was observed by Faber (Faber 2006) indicating that these concepts are not directly applicable to not highly purified enzymes like for example industrial alpha-amylases produced by microorganisms. However, these concepts indicate that the solubility of proteins like enzymes is dependent on the conditions in a given environment.

The conclusion is that the water solubility differs between different alpha-amylases, due to difference in amino acid sequence and presence of post translational modifications. Water solubility is also highly dependent on the aqueous environment, i.e. pH, salts present, temperature and stabilizing agents, and it is thus not possible to give one water solubility value for all industrially produced alpha-amylases but only a range. Industrial enzymes are produced in submerged fermentation followed by downstream purification. The final product is a mixture of the enzyme, constituents from the fermentation and stabilizing agents that are added in the downstream processing. On this background the solubility data generated are based either on finished products or enzymes purified in buffer and salts.

 

References

Carbonnaux, C., Riès-Kautt, M., & Ducruix, A., (1995); Protein Science,4, 2123 -2128.

Faber, C., (2006); PhD-thesis ‘Measurement and Prediction of Protein Phase Behaviour and Protein-Protein-Interactions’ at the Center for Microbial Biotechnology, Biocentrum-DTU, Technical University of Denmark.

Green, A. A., (1932); Physical Chemistry of Proteins,10, 47-66.

Guilloteau, J. P., Riès-Kautt, M., & Ducruix, A., (1992); Journal of Crystal Growth, 122, 223-230.

Hofmeister, F., (1888); Archiv für experimentelle Pathologie und Pharmakologie,24, 247 -260.