Available online at www.sciencedirect.com Chemical Engineering and Processing 47 (2008) 1007–1017 Factors affecting the solubility of Bacillus halmapalus -amylase Cornelius Faber a,b , Timothy J. Hobley b,∗ , Jørgen Mollerup c , Owen R.T. Thomas b,d , Svend G. Kaasgaard a a Novozymes A/S, Krogshøjvej 36, DK-2880 Bagsværd, Denmark b Center for Microbial Biotechnology, BioCentrum-DTU, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark c Department of Chemical Engineering, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark d Department of Chemical Engineering, School of Engineering, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK Received 1 June 2006; received in revised form 12 November 2006; accepted 6 February 2007 Available online 1 March 2007 Abstract A detailed study of the solubility of recombinant Bacillus halmapalus -amylase has been conducted. A semi-purified preparation from a bulk crystallisation was chos en that contained six isoforms with pI-values of between 5.5 and 6.1. The solubility was strongly affected by pH and could be reduced approximately 200-fold at pH 6 as compared to pH 10, leaving only 0.1 mg/mL in solution. Solubility could also be dramatically manipulated using salts. The choice of anions was found to be more important than of the cations, and the lowest solubility was found using sodium sulphate. For the anions, solubility followed the order expected from the Hofmeister series, however, a more complex behaviour was seen for the cations. With the exception of lithium, their efficiency to influence the solubility was reversed to what was expected. The polydispersity of the solution was reduced by salt addition and zeta potential measurements indicated a shift in pI caused by lithium. Possible explanations for the observations are discussed, extending our present understanding of how salts affect the solubility of proteins, one that to date is primarily based on experiments with lysozyme. © 2007 Elsevier B.V. All rights reserved. Keywords: Anions; Cations; Crystallisation; Hofmeister series; Polydispersity; Proteins; Retrograde; Salts; Zeta potential 1. Introduction The crystallisation of biological macromolecules is more dif- ficult than for many inorganic or small organic molecules, and with few exceptions, is only just starting to emerge as a versa- tile operation for large-scale protein recovery. One of the most important examples of industrial-scale crystallisation for pro- tein recovery and purification is the production of insulin [1]. The crystallisation of some bulk enzymes also plays an impor- tant role in their purification and concentration [2]. Perhaps more importantly, due to continued optimisation of the strains used for production of bulk enzymes, improved fermentation methods and demands for even higher product strengths, there is con- siderable risk of operating above the solubility limit, even when crystallisation is not desired. Whether crystallisation is desirable ∗ Corresponding author. Tel.: +45 45 25 27 06; fax: +45 45 88 41 48. E-mail address: th@biocentrum.dtu.dk (T.J. Hobley). or not in an industrial process, it is very important to know which factors control the solubility and crystallisation behaviour of the proteins. Unfortunately, literature on protein crystallisation is primarily orientated towards the use of micro-crystallisation techniques, where the goal is to obtain a few crystals suitable for structure analysis by X-ray diffraction. The availability of detailed solubility data, or at the least information on how various precipitants (e.g. presence of cer- tain salts) affect the solubility phase diagrams, would lead to better prediction of the crystallisation behaviour and, thus to better process control. However, comprehensive solubility data is only available for very few proteins, most notably lysozyme of high purity. The crystallisation of ovalbumin [3], and of micro- bial lipase from clarified concentrated fermentation broths [2] are among the few published works on bulk crystallisation in the presence of impurities. Although the knowledge of solubil- ity properties in industrial processes was regarded as important in both works, only little data on solubility was provided. Fur- thermore, whilst Hofmeister [4] proposed a series ranking the 0255-2701/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.cep.2007.02.015