Properties of elastase from Atlantic cod, a cold-adapted proteinase

Area of Science:

• Biochemistry
• Enzymology
• Marine Biology

Background:

• Elastases are serine proteases vital for protein digestion, with significant variations observed across species.
• Understanding fish enzymes provides insights into adaptations to diverse environmental conditions, particularly temperature.

Purpose of the Study:

• To purify and characterize intestinal elastase from Atlantic cod (Gadus morhua).
• To compare the biochemical and kinetic properties of cod elastase with porcine intestinal elastase.
• To investigate the functional adaptations of cod elastase in relation to its cold-water habitat.

Main Methods:

• Purification of Atlantic cod intestinal elastase using standard biochemical techniques.
• Characterization of enzyme properties including molecular mass (SDS-PAGE), isoelectric point, and stability (heat, pH).
• N-terminal amino acid sequencing for homology analysis.
• Enzyme inhibition studies using phenylmethylsulfonyl fluoride and other inhibitors.
• Kinetic analysis using Suc-Ala-Ala-Ala-p-nitroanilide substrate to determine turnover rate (kcat) and Michaelis constant (Km).
• Substrate specificity determination via digestion of oxidized B-chain of insulin and synthetic substrates.

Main Results:

• Atlantic cod intestinal elastase was purified with a molecular mass of 24.8 kDa and an isoelectric point above pI 9.3.
• The cod enzyme showed reduced heat and acidic pH stability compared to porcine elastase but exhibited N-terminal sequence similarity.
• Cod elastase displayed lower sensitivity to phenylmethylsulfonyl fluoride inhibition, with similar sensitivity to other inhibitors.
• The cod enzyme demonstrated a >2-fold higher turnover rate (kcat) and slightly higher Km values than porcine elastase, resulting in ~2-fold greater catalytic efficiency (kcat/Km).
• Optimal substrate digestion occurred at the carbonyl side of alanine residues, with activity also observed at valine and leucine residues.

Conclusions:

• Atlantic cod intestinal elastase is a distinct enzyme with biochemical properties adapted to its marine environment.
• The higher catalytic efficiency of cod elastase suggests an evolutionary adaptation for efficient nutrient assimilation in cold temperatures.
• Comparative analysis with porcine elastase highlights species-specific adaptations in enzyme function and stability.