Development of a low-cost industrial recovery process to produce a novel hyperthermophilic alpha-amylase overexpressed as inclusion bodies

A novel hyperthermophilic archaeal alpha-amylase, HAA;The enzymes were found to precipitate at very low salt concentration (<1% w/v) possibly due to their high hydrophobicity. Cysteines in WT formed very stable intramolecular disulfide bonds but there was no evidence of intermolecular disulfide bond formation. The enzymes formed very stable soluble aggregates at ∼0.2 mum diameter without covalent bonding. Circular dichroism was used to monitor their temperature and/or guanidinium chloride induced unfolding. The studies showed that the 4C enzyme is less stable than the WT enzyme, especially under alkaline, high-temperature conditions, which can turn alpha-helical structures into beta-sheet structures. Their pH activity profiles were similar but the 4C enzyme is less active at acidic conditions. Apparently, the intramolecular disulfide bonds in the WT enzyme help to stabilize the protein under extreme pH and temperature conditions. In most other situations, the differences between the WT and 4C enzymes were insignificant.;New IB recovery processes were tested using alkaline (pH 11) or heat (70--80°C) to solubilize IB and recover active enzyme. The process could be repeated until all IB were dissolved and extracted from the host cells. Compared to the traditional processes, these new IB recovery processes use fewer chemicals and have no issues regarding cell disruption, waste treatment, toxicity from additives, and purification steps needed to remove additives. The alkaline process is particularly simple and effective since it is not overly sensitive to salts. The feasibility of this novel production process was demonstrated in 1000 L fermentation and 100 L recovery scale.