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. 2017 Dec 15:17:49-54.
doi: 10.1016/j.btre.2017.12.004. eCollection 2018 Mar.

Purification and biochemical characterization of an Aspergillus niger phytase produced by solid-state fermentation using triticale residues as substrate

Alberto A Neira-Vielma  1   2 , Cristóbal N Aguilar  1 , Anna Ilyina  3 , Juan C Contreras-Esquivel  1 , María das Graça Carneiro-da-Cunha  2 , Georgina Michelena-Álvarez  4 , José L Martínez-Hernández  3
Affiliations

Purification and biochemical characterization of an Aspergillus niger phytase produced by solid-state fermentation using triticale residues as substrate

Alberto A Neira-Vielma et al. Biotechnol Rep (Amst). .

Abstract

In this study, an extracellular phytase produced by Aspergillus niger 7A-1, was biochemically characterized for possible industrial application. The enzyme was purified from a crude extract obtained by solid-state fermentation (SSF) of triticale waste. The extract was obtained by microfiltration, ultrafiltration (300, 100 and 30 kDa) and DEAE-Sepharose column chromatography. The molecular weight of the purified enzyme was estimated to be 89 kDa by SDS-PAGE. The purified enzyme was most active at pH 5.3 and 56 °C, and retained 50% activity over a wide pH range of 4 to 7. The enzymatic thermostability assay showed that the enzyme retained more than 70% activity at 80 °C for 60 s, 40% activity for 120 s and 9% after 300 s. The phytase showed broad substrate specificity, a Km value of 220 μM and Vmax of 25 μM/min. The purified phytase retained 50% of its activity with phosphorylated compounds such as phenyl phosphate, 1-Naphthyl phosphate, 2-Naphthyl phosphate, p-Nitrophenyl phosphate and Glycerol-2-phosphate. The inhibition of phytase activity by metal ions was observed to be drastically inhibited (50%) by Ca++ and was slightly inhibited (10%) by Ni++, K+, and Na+, at 10 and 20 mM concentrations. A positive effect was obtained with Mg++, Mn++, Cu++, Cd++ and Ba++ at 25 and 35% with stimulatory effect on the phytase activity.

Keywords: Animal feed; Aspergillus niger; Biochemical characterization; Phytase; Solid-state fermentation.

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Figures

Fig. 1
Fig. 1
SDS-PAGE analysis of phytase from A. niger: A, – molecular weight markers bands; B, – purified phytase.
Fig. 2
Fig. 2
Zymogram of phytase from A. niger, on a non-denaturing electrophoretic gel.
Fig. 3
Fig. 3
Effect of temperature on the activity of A. niger purified phytase: A, – screening to determine the best temperature range; B, – optimum temperature estimation.
Fig. 4
Fig. 4
Thermostability profile of A. niger purified phytase at 80 °C.
Fig. 5
Fig. 5
Effect of pH on the A. niger phytase: A,– screening to determine the best pH range; B, – optimum pH).
See this image and copyright information in PMC

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