title: Characteristics and applications of recombinant thermostable amylopullulanase of Geobacillus thermoleovorans secreted by Pichia pastoris.
abstract: The 3'-deleted amylopullulanase gene from the extreme thermophile Geobacillus thermoleovorans (Gt-apuΔC) was expressed extracellularly in Pichia pastoris under both methanol-inducible AOX1 and constitutive GAP promoters. The expression of the gene (Gt-apuΔC) was higher under GAP promoter (36.2 U ml-1, α-amylase; 33.5 U ml-1, pullulanase) than that under AOX1 promoter (32.5 and 28.6 U ml-1). The heavily glycosylated Gt-apuΔC from the recombinant P. pastoris displays higher substrate specificity, thermal stability and starch saccharification efficiency than that expressed in Escherichia coli. The enzyme hydrolyses maltotriose and maltotetraose unlike that expressed in E. coli. The enzyme action on wheat bran liberates maltose and glucose without detectable amount(s) of maltooligosaccharides. The sugars released from wheat bran (glucose and maltose) could be fractionated by ultrafiltration, as confirmed by TLC and HPLC analysis. This is the first report on the production of recombinant amylopullulanase extracellularly in P. pastoris.
authors.affiliation: Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi, 110021, India.
authors.forename: M
authors.initials: M
authors.lastname: Nisha
authors.affiliation: Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi, 110021, India. tsnarayana@gmail.com.
authors.forename: T
authors.initials: T
authors.lastname: Satyanarayana
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004043:Dietary Fiber / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D056506:Geobacillus / Q000737:chemistry* / Q000201:enzymology; D005947:Glucose / Q000737:chemistry* / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008320:Maltose / Q000737:chemistry* / Q000378:metabolism; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013045:Species Specificity; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D004043:Dietary Fiber; D011994:Recombinant Proteins; D008320:Maltose; D006026:Glycoside Hydrolases; C082507:amylopullulanase; D005947:Glucose
keywords: Geobacillus thermoleovorans; Maltooligosaccharides; Pichia pastoris; Recombinant amylopullulanase; Starch saccharification; Wheat bran