caa a22 4500 605499101 CHVBK 20210128100549.0 cr unu---uuuuu 210128e20150301xx s 000 0 eng 10.1007/s00253-014-6314-2 doi (NATIONALLICENCE)springer-10.1007/s00253-014-6314-2 Cloning, expression, and purification of insect ( Sitophilus oryzae ) alpha-amylase, able to digest granular starch, in Yarrowia lipolytica host [Elektronische Daten] [Ewelina Celińska, Wojciech Białas, Monika Borkowska, Włodzimierz Grajek] 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 (81AU/L) was reached after only 29h 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 pH5.0 and a temperature of 40°C. The α-amylase studied here did not show any obligate requirement for Ca2+ ions. The recombinant α-amylase appeared to efficiently digest granular starch from pea, amaranth, waxy corn, and waxy rice. The Author(s), 2014 Amylase nationallicence Granular starch nationallicence Rice weevil nationallicence Yarrowia lipolytica nationallicence Recombinant protein expression nationallicence Celińska Ewelina Department of Biotechnology and Food Microbiology, Poznań University of Life Sciences, ul. Wojska Polskiego 48, 60-627, Poznań, Poland aut Białas Wojciech Department of Biotechnology and Food Microbiology, Poznań University of Life Sciences, ul. Wojska Polskiego 48, 60-627, Poznań, Poland aut Borkowska Monika Department of Biotechnology and Food Microbiology, Poznań University of Life Sciences, ul. Wojska Polskiego 48, 60-627, Poznań, Poland aut Grajek Włodzimierz Department of Biotechnology and Food Microbiology, Poznań University of Life Sciences, ul. Wojska Polskiego 48, 60-627, Poznań, Poland aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/6(2015-03-01), 2727-2739 0175-7598 99:6<2727 2015 99 253 https://doi.org/10.1007/s00253-014-6314-2 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-014-6314-2 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Celińska Ewelina Department of Biotechnology and Food Microbiology, Poznań University of Life Sciences, ul. Wojska Polskiego 48, 60-627, Poznań, Poland aut NATIONALLICENCE 700 1- Białas Wojciech Department of Biotechnology and Food Microbiology, Poznań University of Life Sciences, ul. Wojska Polskiego 48, 60-627, Poznań, Poland aut NATIONALLICENCE 700 1- Borkowska Monika Department of Biotechnology and Food Microbiology, Poznań University of Life Sciences, ul. Wojska Polskiego 48, 60-627, Poznań, Poland aut NATIONALLICENCE 700 1- Grajek Włodzimierz Department of Biotechnology and Food Microbiology, Poznań University of Life Sciences, ul. Wojska Polskiego 48, 60-627, Poznań, Poland aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/6(2015-03-01), 2727-2739 0175-7598 99:6<2727 2015 99 253