title: Cloning, expression, and purification of insect (Sitophilus oryzae) alpha-amylase, able to digest granular starch, in Yarrowia lipolytica host.
abstract: Raw-starch-digesting enzymes (RSDE) are of major importance for industrial applications, as their usage greatly simplifies the starch processing pipeline. To date, only microbial RSDE have gained considerable attention, since only microbial production of enzymes meets industrial demands. In this study, α-amylase from rice weevil (Sitophilus oryzae), the major rice pest, was cloned and expressed in Yarrowia lipolytica Po1g strain. The enzyme was secreted into the culture medium, and the peak activity (81 AU/L) was reached after only 29 h of culturing in 5-L bioreactors. Through simple purification procedure of ammonium sulfate precipitation and affinity chromatography, it was possible to purify the enzyme to apparent homogeneity (25-fold purification factor, at 5 % yield). The optimal conditions for the α-amylase activity were pH 5.0 and a temperature of 40 °C. The α-amylase studied here did not show any obligate requirement for Ca(2+) ions. The recombinant α-amylase appeared to efficiently digest granular starch from pea, amaranth, waxy corn, and waxy rice.
authors.affiliation: Department of Biotechnology and Food Microbiology, Poznań University of Life Sciences, ul. Wojska Polskiego 48, 60-627, Poznań, Poland, celinska@up.poznan.pl.
authors.forename: Ewelina
authors.initials: E
authors.lastname: Celińska
authors.forename: Wojciech
authors.initials: W
authors.lastname: Białas
authors.forename: Monika
authors.initials: M
authors.lastname: Borkowska
authors.forename: Włodzimierz
authors.initials: W
authors.lastname: Grajek
mesh_terms: D000818:Animals; D003001:Cloning, Molecular; D003470:Culture Media; D053938:DNA Fragmentation; D004271:DNA, Fungal / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007313:Insecta / Q000201:enzymology*; D008855:Microscopy, Electron, Scanning; D012275:Oryza; D011994:Recombinant Proteins / Q000096:biosynthesis; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013213:Starch / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D025062:Yarrowia / Q000235:genetics / Q000378:metabolism*; D000516:alpha-Amylases / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D003470:Culture Media; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; C085480:PDR1 protein, S cerevisiae; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D013213:Starch; D000516:alpha-Amylases