Molecular cloning, expression, and functional characterization of a cystatin from pineapple stem

Area of Science:

• Biochemistry
• Molecular Biology
• Food Science

Background:

• Cysteine proteases are enzymes involved in protein degradation.
• Cystatins are natural inhibitors of cysteine proteases.
• Pineapple (Ananas comosus) is a source of potential bioactive compounds.

Purpose of the Study:

• To clone and over-express pineapple cystatin.
• To characterize the functional activity and stability of recombinant pineapple cystatin.
• To evaluate the potential application of pineapple cystatin in food processing.

Main Methods:

• Cloning of pineapple cystatin cDNA into an expression vector.
• Over-expression in Escherichia coli (E. coli).
• Purification using affinity chromatography (His-tag and papain-coupling).
• Functional activity assessed by reverse zymography and papain inhibition assays.
• Thermal stability determined by incubation at various temperatures.
• Application tested on minced fish muscle.

Main Results:

• Successful over-expression of both fusion and non-fusion pineapple cystatins in E. coli.
• Recombinant cystatins were soluble and functionally active against papain.
• High-affinity inhibition of papain with comparable K(i) values (1.18 x 10(-10) M and 9.53 x 10(-11) M).
• Recombinant cystatins exhibited thermal stability up to 60°C.
• Demonstrated inhibition of endogenous protease activity in fish muscle.

Conclusions:

• Pineapple cystatin can be effectively produced as a recombinant protein.
• Recombinant pineapple cystatins possess potent cysteine protease inhibitory activity and are thermally stable.
• Pineapple cystatins show promise as natural stabilizers in the food industry to prevent protein degradation during processing.