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Crystallization Studies on a Bacillus licheniformis Alpha-amylase

Proteins are important biological products with unique functions, annually produced at the hundreds of millions of dollars value on a worldwide basis. The application of crystallization for these materials primarily was led by structural biologists and crystallographers who are keen on obtaining large and well-ordered crystals for protein structure determination via X-ray diffraction. Usually for this, crystallization is done on a small scale by vapor diffusion using a supersaturated solution of the material. In the past decades, production crystallization has slowly received increasing attention for the large-scale recovery of proteins. Among the numerous products, an industrial enzyme (alpha-amylase) that is extensively involved in food processing and laundry products was chosen for examination due to the lack of relevant data in the literature and the potential industrial interest in crystallizing this material. The chosen alpha-amylase is a product of Genencor International (the Danisco division) and is derived from a genetically modified Bacillus licheniformis. In parallel to the underlying principles that govern the bulk crystallization of small molecules, the broad topics of investigation for this macromolecular material included determination of solubility, studies of nucleation thresholds, and investigation of crystal growth kinetics and special phenomena accompanying the crystallization process. All these studies were preceded by a series of characterization tests conducted for the material. On the whole, this study aimed to extend the existing fundamental knowledge of bulk crystallization for biological macromolecules. Specifically, it intended to enrich the solubility and crystallization kinetic data for the alpha-amylase. The experimental data of this study were all obtained at conditions in line with industrial practice, which included the use of moderate temperatures, mild pH conditions and simple inorganic salts ((NH4)2SO4, Na2SO4 and NaCl) in order for the findings to be transferred to the industry directly. In a 20 mM sodium phosphate buffer (with no added salts), alpha-amylase solubility increased with solvent temperature and had a minimum at pH between 6.4 and 7.1. A generalized equation (as a function of pH and temperature) was obtained to correlate the data. The three inorganic salts examined affected the alpha-amylase solubility in a different manner, both qualitatively and quantitatively. Evidently, the interaction effect of a salt varied with solution pH. This confirms the importance of studying solubility with the two or more condition parameters at the same time. With relevance to crystal growth, the metastable region of the material was relatively wide at (NH4)2SO4 and Na2SO4 concentrations corresponding to maximum solubility. For example, σSNT was 1.2  0.2 in solutions with 5 wt% ammonium sulfate at pH 7.0 and 25oC. A wide metastability range is useful for the practical operation of batch crystallizers as nucleation can be minimized. This range, however, diminished as the salt concentration increased beyond the maximum solubility points, imposing a limit on the range of salt concentration favorable for growth processes. In systems with no added salts at pH 7.0, the solution metastability was slightly higher at 10oC than at 40oC. This would suggest a future further examination of the salt system at a lower temperature, say of 10oC. To develop a batch crystallizer, the growth kinetic data of the material have to be known. Throughout the growth studies, the alpha-amylase crystals obtained were lozenge-shaped thin plates. Apparently, habit was not influenced by the crystallization conditions chosen. Similar to other proteins crystallized in bulk, the growth rate of alpha-amylase demonstrated a second-order dependence upon supersaturation. Importantly, the alpha-amylase demonstrated crystal growth rate dispersion (GRD) under all the conditions tested. To simplify the analysis of growth kinetic results, the seed crystals used were common history (CH) seeds whose growth rates are proportional to their sizes. The spread of growth rates (CV) was 0.54 for the unsieved CH seeds used. Due to GRD, growth rate coefficient data varied with crystal size. For instance, in solutions containing 5 wt% ammonium sulfate at pH 7.0 and 25oC, the growth rate coefficient for seed crystals initially at 20 m was 2.47 m/hr. This order of magnitude was equivalent to that of many other proteins. Although being small, industrial crystallization was feasible with these kinetics, as demonstrated by the sample design calculations included. To improve the design, it is recommended to further examine the solubility, metastability and growth kinetics of the above system at other temperatures to obtain a wider growth rate range. As the important phenomenon of growth rate dispersion has seldom been examined for protein and enzyme materials in the crystallization literature, this study is a significant contribution to this area.