title: The role of N1 domain on the activity, stability, substrate specificity and raw starch binding of amylopullulanase of the extreme thermophile Geobacillus thermoleovorans.
abstract: In order to understand the role of N1 domain (1-257 aa) in the amylopullulanase (gt-apu) of the extremely thermophilic bacterium Geobacillus thermoleovorans NP33, N1 deletion construct (gt-apuΔN) has been generated and expressed in Escherichia coli. The truncated amylopullulanase (gt-apuΔN) exhibits similar pH and temperature optima like gt-apu, but enhanced thermostability. The gt-apuΔN has greater hydrolytic action and specific activity on pullulan than gt-apu. The k cat (starch and pullulan) and K m (starch) values of gt-apuΔN increased, while K m (pullulan) decreased. The enzyme upon N1 deletion hydrolyzed maltotetraose as the smallest substrate in contrast to maltopentaose of gt-apu. The role of N1 domain of gt-apu in raw starch binding has been confirmed, for the first time, based on deletion and Langmuir-Hinshelwood kinetics. Furthermore, N1 domain appears to exert a negative influence on the thermostability of gt-apu because N1 truncation significantly improves thermostability.
authors.affiliation: Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi, 110 021, India.
authors.forename: M
authors.initials: M
authors.lastname: Nisha
authors.forename: T
authors.initials: T
authors.lastname: Satyanarayana
mesh_terms: D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D056506:Geobacillus / Q000201:enzymology* / Q000235:genetics; D005936:Glucans / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D013213:Starch / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D005936:Glucans; D050505:Mutant Proteins; C009109:pullulan; D013213:Starch; D006026:Glycoside Hydrolases; C082507:amylopullulanase