A hyper-thermostable α-amylase from Pyrococcus furiosus accumulates in Nicotiana tabacum as functional aggregates

Alpha amylase breaks down starch and produces simple sugars, which makes it an essential enzyme in the textile, food and brewing industries. Commercially available alpha-amylase is mostly produced from bacterial (Bacillus) or fungal (Aspergillus) cultures. A more useful alpha amylase is one which is very stable at very high temperatures, and compatible with high temperature industrial processes. Such an enzyme from the bacterial strain Pyrococcus furiosus (PFA) expressed in E.coli forms insoluble inclusion bodies and thus is not feasible for industrial applications. We expressed PFA in tobacco and found that plant-produced PFA forms functional aggregates with an accumulation level up to 3.4 g/kg fresh weight in field conditions. The aggregates are functional without requiring refolding and therefore have potential to be applied as homogenized plant tissue without extraction or purification. PFA can also be extracted from plant tissue upon dissolution in a mild reducing buffer containing a detergent. Like the enzyme produced in P. furiosus and in E. coli, plant produced PFA preserves its activity at high temperature and has a long shelf life when stored in dried leaf tissue. With tobacco's large biomass and high yield, hyper-thermostable alpha-amylase was produced at a scale of 42 kg per hectare. We therefore conclude that tobacco may be a suitable bioreactor for industrial production of active thermostable alpha amylase.