Extraction, purification, and biochemical characterization of serine protease from leaves of Abrus precatorius

ABSTRACT

A protease from fresh leaves of Abrus precatorius was purified using two classical chromatography techniques: ion-exchange (DEAE-Sepharose) and Gel filtration (Sephadex G-75). The purified protease showed a molecular weight of ∼ 28 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum pH and temperature for the purified protease was 8 and 40°C, respectively. The purified protease was stable throughout a wide temperature range from 10 to 80°C and pH from 2 to 12. Protease activity was inhibited in the presence of Co²⁺, Ni²⁺, Hg²⁺, and Zn²⁺ while its activity has increased in the presence of Ca²⁺ and Mg²⁺. The protease was highly specific to casein when compared to its specificity for gelatin, bovine serum albumin, hemoglobin, and defatted flour of Ricinodendron heudelotii. Its V_max and K_m determined using casein as a substrate were 94.34 U/mL and 349.07 µg/mL respectively. Inhibition studies showed that this purified protease was inhibited by both phenylmethane sulfonyl fluoride and aprotinin which are recognized as competitive inhibitors of serine proteases.

KEYWORDS:

• Abrus precatorius
• ion exchange
• protease
• purification

Abbreviations::

• PMSF: phenylmethane sulfonyl fluoride
• DEAE: diethylaminoethyl
• BSA: bovine serum albumin

Acknowledgments

We are thoroughly thankful to Department of Biotechnology Government of India (DBT), and TWAS, The Academy of Sciences for the developing World for their intellectual and material contributions.

Additional information

Funding

SK thanks the SERB for financial assistance under the Fast Track Scheme (SB/FT/LS-190/2012). MA Vijayalakshmi acknowledges the financial assistance from the Department of Science & Technology (DST), Ministry of Science and Technology.