title: Plant cell calcium-rich environment enhances thermostability of recombinantly produced alpha-amylase from the hyperthermophilic bacterium Thermotoga maritime.
abstract: In the industrial processing of starch for sugar syrup and ethanol production, a liquefaction step is involved where starch is initially solubilized at high temperature and partially hydrolyzed with a thermostable and thermoactive alpha-amylase. Most amylases require calcium as a cofactor for their activity and stability, therefore calcium, along with the thermostable enzyme, are typically added to the starch mixture during enzymatic liquefaction, thereby increasing process costs. An attractive alternative would be to produce the enzyme directly in the tissue to be treated. In a proof of concept study, tobacco cell cultures were used as model system to test in planta production of a hyperthermophilic alpha-amylase from Thermotoga maritima. While comparable biochemical properties to recombinant production in Escherichia coli were observed, thermostability of the plant-produced alpha-amylase benefited significantly from high intrinsic calcium levels in the tobacco cells. The plant-made enzyme retained 85% of its initial activity after 3 h incubation at 100 degrees C, whereas the E. coli-produced enzyme was completely inactivated after 30 min under the same conditions. The addition of Ca(2+) or plant cell extracts from tobacco and sweetpotato to the E. coli-produced enzyme resulted in a similar stabilization, demonstrating the importance of a calcium-rich environment for thermostability, as well as the advantage of producing this enzyme directly in plant cells where calcium is readily available.
authors: Department of Horticultural Science, North Carolina State University, Raleigh, North Carolina 27695, USA.
authors: Monica C
authors: MC
authors: Santa-Maria
authors: Chung-Jung
authors: CJ
authors: Chou
authors: G Craig
authors: GC
authors: Yencho
authors: Candace H
authors: CH
authors: Haigler
authors: William F
authors: WF
authors: Thompson
authors: Robert M
authors: RM
authors: Kelly
authors: Bryon
authors: B
authors: Sosinski
mesh_terms: D002118:Calcium / Q000494:pharmacology*; D003067:Coenzymes / Q000494:pharmacology*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006358:Hot Temperature; D030821:Plants, Genetically Modified / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics; D000516:alpha-Amylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D003067:Coenzymes; D011994:Recombinant Proteins; D000516:alpha-Amylases; D002118:Calcium