کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
8359440 1542293 2018 6 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
High-level expression, purification, and enzymatic characterization of a recombinant Aspergillus sojae alkaline protease in Pichia pastoris
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
High-level expression, purification, and enzymatic characterization of a recombinant Aspergillus sojae alkaline protease in Pichia pastoris
چکیده انگلیسی
An alkaline protease (Ap) was cloned from Aspergillus sojae GIM3.33 via RT-PCR technique. A truncated Ap without the signal peptide was successfully expressed in the Pichia pastoris KM71 strain. The following describes the optimal process conditions for the recombinant engineering of a strain expressing a recombinant Ap (rAp) in a triangular flask: inoculum concentration OD600 value 20.0 in 40 mL working volume (in 500 mL flasks), methanol addition (1.0%; volume ratio), 0.02% biotin solution (60 μL), and YNB primary concentration (13.0 g/L). Under these conditions, the protease activity of rAp in the fermentation broth reached 400.4 ± 40.5 U/mL after induction for three days. The rAp was isolated and purified, and its enzymatic characteristics were tested. Its optimal pH was 10.0, and it remained stable in a pH range of 7.0-10.0. Its optimal temperature was 45 °C and it retained >50% activity at 40 °C for 60 min. The rAp activity was significantly inhibited by PMSF, Zn2+ and Fe2+ and the rAp had a broad substrate specificity for natural proteins and synthetic peptide substrates, and preferred substrates at P1 position with large hydrophobic side-chain groups. Compared to Papain (8.7%) and Alcalase (12.2%), the degree of hydrolysis of rAp to soy protein isolate was 16.5%; therefore, rAp was a good candidate for the processing of food industry byproducts.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 148, August 2018, Pages 24-29
نویسندگان
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