PURIFICATION OF THERMAMYLASE IN MULTICOMPARTMENT ELECTROLYZERS WITH ISOELECTRIC MEMBRANES - THE PROBLEM OF PROTEIN SOLUBILITY

The main alpha-amylase from Bacillus licheniformis (called thermamylas e because of its resistance to high temperatures, 90 degrees C) has be en subjected to purification by isoelectric focusing in multicompartme nt electrolyzers with isoelectric membranes. The enzyme tended to prec ipitate, producing severe smears in proximity of its pI value (7.18). Solubility could not be ameliorated by any of the known means typicall y adopted in isoelectric focusing and compatible with enzyme activity, such as addition of neutral and zwitterionic surfactants (e.g., Nonid et, lamidopropyl)dimethylammonio]-1-propane-sulfonate, up to 2%), mixe d hydro-organic solvents (glycerol, ethylene glycol, propylene glycol) and addition of zwitterions unable to form micelles, such as taurine. However, addition of sugars, notably saccharose, sorbitol, and, to a lesser extent, sorbose, greatly improved protein solubility in the pI proximity. The improvement was dramatic if these sugars were admired w ith 0.2 M taurine; Additionally, the increment of solubility (which oc curred when reaching a level of 40% of the different sugars) was accom panied by a large pI shift, typically reducing the pI value by as much as 0.4 pH units (e.g., from a pI of 7.18 in the absence of additives to a pI of 6.80 in presence of a mixture of 40% sucrose and 0.2 M taur ine, the best solubilizer in all the series investigated). This appare nt pI shift was not due to a change of pH gradient caused by the prese nce of additives, since pH measurements in the absence as well as pres ence of additives gave identical results. The results are explained by the theory of Timasheff and Arakawa on stabilization of protein struc ture by solvents: sugars (at ca. 1 M concentration) and zwitterions su ch as taurine belong to class I stabilizers, characterized by negative binding to proteins and by increasing the surface tension of water. A s a result, the protein is in a state of ''superhydration'' which migh t prevent binding to Immobilines in the gel matrix and might alter som e pKs on the protein surface. In solutions of 40% saccharose and 0.2 M taurine, thermamylase could be successfully purified to a single isoe lectric and isoionic band in the multicompartment electrolyzer.