In silico and Biochemical Approach for Isolated, Purified, and Characterized Protease Inhibitor from Limonia acidissima

Prathamesh Kale¹, Ashpak Tamboli², Javeed Manure³ and, Manjusha Dake^1*

¹Protein Biochemistry Lab, Dr. D. Y. Patil Biotechnology and Bioinformatics Institute, Dr. D. Y. Patil Vidyapeeth Tathawade, Pune, India.

²Sahyadri College of Pharmacy, Sangola, Solapur, India.

³Appasaheb Birnale College of Pharmacy, Sangli, India.

Corresponding Author E-maul:manjusha.dake@dpu.edu.in

Abstract: Proteases produced by various bacterial and fungal pathogens are associated with pathogenicity and cause septic hazards. Thereby inhibition of such proteases by protease inhibitors from natural sources is effective strategy to combat infectious diseases. Protease inhibitors originating from plant source are abundant in nature and play an important role in defense mechanism against virulent microbes. Many protease inhibitors can be isolated and purified from plant sources and formulated as therapeutic drugs to combat human diseases. In spite of their applications in biomedicines, it is necessary to explore the novel biochemical characteristics of protease inhibitors with improved efficacy. The objective of the current study was extraction, purification and biochemical characterization of protease inhibitor from L. acidissima. The protease inhibitor was isolated from L. acidissima by buffer extraction method and confirmed for anti-protease activity using standard trypsin assay. The inhibitor was found to be active between pH ranging from 5-9 with maximal activity at pH 7. The inhibitor showed thermal stability at a temperature of 50-60°C and retained 70% activity at 50°C for 2 hr. Inhibitor activity was enhanced by thermal stabilizers glycine, urea, Calcium chloride (CaCl₂₎, glycerol, and sucrose at 50°C; metal ions Cu²⁺ and Al³⁺ and detergents like Sodium dodecyl sulfate (SDS), Triton-X 100, Tween-80. Protease inhibitor was also stable in the presence of oxidizing and reducing agents Dimethyl sulfoxide (DMSO), Hydrogen peroxide (H₂O₂₎, β-mercaptoethanol, and Sodium Thioglycolate. Thus, optimization and characterization studies indicate that the isolated inhibitor is active under natural conditions. Protease inhibitor showed positive results for anticancer, anti-diabetic and anti-inflammatory activities indicating the therapeutic potential of inhibitor molecule. Further identification and separation of bioactive molecules from the buffer extract of L. acidissima was carried out by Flash chromatography followed by Liquid chromatography-high-resolution mass spectrometry (LC-HRMS) analysis that revealed the presence of Diisobutylphthalate as bioactive phytoconstituent. Docking studies performed revealed the anti-diabetic, anti-inflammatory, and anti-cancer potential of Diisobutylphthalate. Thus, in silico studies support the activity of our isolated protease inhibitor to ensure a promising future in the development of therapeutics.

Keywords: Biochemical activities; Bbiochemical characterization; Limonia acidissima; Molecular docking; protease inhibitor

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