Purification and characterization of a digestive cysteine proteinase from the American lobster (Homarus americanus)

Abstract

A new cysteine proteinase was isolated from the digestive juice of the American lobster (Homarus americanus). The enzyme was purified by a combination of affinity and ion-exchange chromatography and gel filtration. The cysteine proteinase accounted for 80% of the proteolytic activity in the lumen of the hepatopancreas. The most potent heavy-metal inhibitors were Hg, Cu, and Ag ions. Inhibition by organic proteinase inhibitors, including E-64 [L-trans-epoxysuccinyl-leucylamido-(4-guanidino)butane] and activation of the enzyme by 2-mercaptoethanol and dithiothreitol are characteristic of cysteine proteinases. Several similarities to papain are noted and include the N-terminal sequence, of which 22 of the first 28 amino acids are identical. Some notable differences are the higher Mr of 28,000 compared with 23,350 for papain, and the low isoelectric point (pI 4.5) of the lobster enzyme. The effects of pH and temperature on catalytic activity of the lobster proteinase were studied with benzyloxycarbonylalanine p-nitrophenyl ester as the substrate. The kcat./Km value was effectively temperature-independent between 10 and 60 degrees C. The pH-activity profile for the lobster enzyme revealed four apparent protonation states, of which only two are active.

Complementary Proteomic and Biochemical Analysis of Peptidases in Lobster Gastric Juice Uncovers the Functional Role of Individual Enzymes in Food Digestion

“…In addition, Leu, Tyr, Thr and Glu were generally found in the P1 position and Ala and Nle at P1ʹ. Cathepsin L1 has low P1 preference for Ile, Val and Pro which is consistent with previous studies using p-nitrophenyl esters of benzyloxycarbonyl amino acids (Laycock et al 1989) .…”

“…We identified six peptidases in H. americanus gastric juice of which four had been previously reported and partially characterized (Laycock et al 1989;Laycock et al 1991;Rojo et al 2010b) . The role of these individual enzymes in overall protein digestion has not been elucidated and therefore we utilized a set of 124 diverse peptides as a synthetic protein source to uncover the substrates specificity.…”

“…N-terminal sequencing of these proteins identified them as cathepsin L1, a cysteine peptidase (GenBank X63567) and cathepsin D1, an aspartic acid peptidase (GenBank EU687261), respectively. Cathepsin L1 was shown to have sensitivity to E-64, optimum activity at pH 5.0 and a preference for cleaving on the C-terminal side of lysine residues (Laycock et al 1989). Cathepsin D1 was sensitive to pepstatin A, had optimum activity at pH 4.0 and hydrolyzed an internally quenched fluorescent substrate between a pair of phenylalanine resides (Rojo et al 2013).…”

“…Since then, proteins with amylase (Wojtowicz and Brockerhoff 1972), chitinase and chitobiase specificity (Lynn 1990) have been isolated and partially characterized; however the proteins responsible for these activities have not been determined. Two peptidases capable of degrading casein and hemoglobin have also been isolated from H. americanus gastric juice (Laycock et al 1989;Laycock et al 1991;Rojo et al 2010a;Rojo et al 2010b;Rojo et al 2013). N-terminal sequencing of these proteins identified them as cathepsin L1, a cysteine peptidase (GenBank X63567) and cathepsin D1, an aspartic acid peptidase (GenBank EU687261), respectively.…”

“…When directly compared to bovine cathepsin D, the lobster enzyme showed higher catalytic efficiency below 25 °C and underwent faster loss of activity at 60 °C (Rojo et al 2013). In addition, cathepsin L1 had lower thermal stability when compared to the cysteine peptidases papain but had constant catalytic efficiency (kcat/Km) between 10 and 60 °C (Laycock et al 1989). These observations indicate that H. americanus peptidases exhibit cold-adapted properties relative to the enzymes isolated from mesophiles (Rojo et al 2013).…”