A new thermostable rhizopuspepsin: Purification and biochemical characterisation

Chinmayee, C. V. and Asha, Martin and Gnanesh Kumar, B. S. and Sridevi Annapurna, Singh (2022) A new thermostable rhizopuspepsin: Purification and biochemical characterisation. Process Biochemistry, 112. pp. 18-26.

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Abstract

The new thermostable fungal aspartic protease was produced through solid-state fermentation from Rhizopus azygosporus (MTCC 10195). The protease was purified by a three-tandem steps of ammonium sulfate precipitation (25−65% saturation), ion exchange (DEAE sepharose CL 6B) chromatography, and gel filtration (Sephacryl S 200) chromatography. The optimum pH and temperature for protease activity were 4 and 52 ± 1.8 ◦ C, respectively. The enzyme was unusually thermostable, as it retained 50 % of its activity beyond 85 ◦ C after 20 min of incubation. The apparent molecular weight was 33.2 ± 2.3 kDa with a final specific activity of 86.6 U/mg. The enzyme was rich in β sheets (63 %) and was inhibited by pepstatin A, indicating that it is an aspartic protease. MS/MS analysis revealed the enzyme is an endopeptidase cleaving the C-terminus of Phe, Leu, Lys, Val, His, Glu, Met, and Tyr. Amino-terminal sequencing, coupled with in-gel trypsin digestion and MS analysis revealed that the enzyme was being reported for the first time with “experimental evidence at protein-level”.

Item Type:	Article
Uncontrolled Keywords:	Fungi, Aspartic protease, Purification, Thermostability, Substrate-specificity, rhizopuspepsin
Subjects:	500 Natural Sciences and Mathematics > 07 Life Sciences > 03 Biochemistry & Molecular Biology > 07 Enzyme Biochemistry
Divisions:	Protein Chemistry and Technology
Depositing User:	Somashekar K S
Date Deposited:	06 Mar 2025 05:48
Last Modified:	06 Mar 2025 05:48
URI:	http://ir.cftri.res.in/id/eprint/19209

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