A new thermostable rhizopuspepsin: Purification and biochemical characterisation. (January 2022)

• Record Type: Journal Article
• Authors: Chinmayee, C.V.; Martin, Asha; Gnanesh Kumar, B.S.; Singh, Sridevi Annapurna
• Is Part Of: Process biochemistry. Volume 112(2022)
• Page Start: 18
• Page End: 26
• Publication Date: 2022-01
• Subjects: Fungi -- Aspartic protease -- Purification -- Thermostability -- Substrate-specificity; Biochemical engineering -- Periodicals; Biotechnology -- Periodicals; Biochemistry -- periodicals; Biotechnology -- periodicals; Chemical Engineering -- periodicals; Génie biochimique -- Périodiques; Biotechnologie -- Périodiques; Biochemical engineering; Biotechnology; Periodicals; 660.63
• DOI: 10.1016/j.procbio.2021.11.007
• Languages: English
• ISSNs: 1359-5113

Abstract

Graphical abstract: Highlights: A new thermostable aspartate protease was purified from R. azygosporus . Optimum protease activity was observed at 52 °C and pH 3–4. Amino terminal sequencing and mass spectrometry analysis confirmed the identity of enzyme. Protease is an endopeptidase with broad substrate specificity and retains activity at 85 °C. 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".