کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2020441 1542337 2014 7 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Bioprocess and downstream optimization of recombinant bovine chymosin B in Pichia (Komagataella) pastoris under methanol-inducible AOXI promoter
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Bioprocess and downstream optimization of recombinant bovine chymosin B in Pichia (Komagataella) pastoris under methanol-inducible AOXI promoter
چکیده انگلیسی


• We optimized the production and downstream of recombinant bovine chymosin expressed in Pichia pastoris under methanol-inducible AOXI promoter.
• We performed cell growth and recombinant bovine chymosin production in BSM with biodiesel-byproduct crude glycerol as a low cost carbon source.
• Biomass level at the beginning of methanol-induction phase influenced on cell growth and bovine chymosin expression.
• The specific growth rate impacted on heterologous chymosin expression during induction phase by methanol-exponential feeding fermentations.
• Recombinant chymosin purified by gel filtration chromatography was used to determinate optimal temperature and pH of milk-clotting activity.

A clone of the methylotrophic yeast Pichia pastoris strain GS115 transformed with the bovine prochymosin B gene was used to optimize the production and downstream of recombinant bovine chymosin expressed under the methanol-inducible AOXI promoter. Cell growth and recombinant chymosin production were analyzed in flask cultures containing basal salts medium with biodiesel-byproduct glycerol as the carbon source, obtaining values of biomass level and milk-clotting activity similar to those achieved with analytical glycerol. The effect of biomass level at the beginning of methanol-induction phase on cell growth and chymosin expression was evaluated, determining that a high concentration of cells at the start of such period generated an increase in the production of chymosin. The impact of the specific growth rate on chymosin expression was studied throughout the induction stage by methanol exponential feeding fermentations in a lab-scale stirred bioreactor, achieving the highest production of heterologous chymosin with a constant specific growth rate of 0.01 h−1. By gel filtration chromatography performed at a semi-preparative scale, recombinant chymosin was purified from exponential fed-batch fermentation cultures, obtaining a specific milk-clotting activity of 6400 IMCU/mg of chymosin and a purity level of 95%. The effect of temperature and pH on milk-clotting activity was analyzed, establishing that the optimal temperature and pH values for the purified recombinant chymosin are 37 °C and 5.5, respectively. This study reported the features of a sustainable bioprocess for the production of recombinant bovine chymosin in P. pastoris by fermentation in stirred-tank bioreactors using biodiesel-derived glycerol as a low-cost carbon source.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 104, December 2014, Pages 85–91
نویسندگان
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