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Journal of King Saud University - Science
Volume 34 (3), 2022, 101829

Purification, characterization and studies of a novel cysteine protease inhibitor from Juglans regia: Implications as a potential biopesticide

Ashraf Ali Khana,1, Abu Bakr Ahmad Fazilib,1, Sheraz Ahmad Bhatc, Waseem Feeroze Bhata

Govt. Medical College Doda, Jammu and Kashmir, 182202, India.



To isolate and characterize a novel phytocystatin from walnut and investigate it for biopesticide development.


A battery of methodology was employed. Initially, phytocystatin was extracted and purified from walnut using ammonium sulfate saturation (60–80%), followed by gel filtration chromatography on the Sephacryl S-100 HR column. Further characterization studies including pH and temperature stability, molecular weight, secondary structure, protease inhibitory assay and antimicrobial activity were carried using various techniques viz: spectroscopy, electrophoresis, and circular dichroism (CD) techniques.


Thiol protease inhibitor from walnut (WCPI) was isolated and purified with high (71.4%) yield and 184-fold purification. The molecular weight of the purified inhibitor was found to be around 11.2 kDa. Kinetic tests revealed that the inhibitor competitively inhibited papain and other cysteine proteases such as ficin and bromelain. It also exhibited significant antimicrobial activity against bacterial species.


Walnut cysteine protease inhibitor (WCPI) from Juglans regia (Kashmiri walnut) was isolated to homogeneity and had features of other members of the phytocystatin family. It demonstrated potential antimicrobial activity and may serve as an initial step towards developing enhanced pest control methods based on natural molecules.

Keywords: Cystatin, SDS-PAGE, Chromatography, Antimicrobial activity.

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