Lysyl oxidase from jumbo squid (Dosidicus gigas) muscle: purification and partial characterization

Abstract

Lysyl oxidase (LOX; E.C.1.4.3.13) was purified from jumbo squid muscle (Dosidicus gigas) with 1900-fold and yield 1.9%, and characterized for the first time. The purification procedure consisted of fractionation with urea and a combination of size-exclusion and anion-exchange chromatography. The enzyme had a molecular weight of 32 kDa, as estimated by SDS-PAGE. Using a specific LOX substrate (1,5-diaminopentane), its optimum activity was determined at pH 8.2 and 65°C. Activation energy (E a ) of the enzyme was 69.94 kJ K )1 mol )1 . The enzyme was strongly inhibited by b-aminopropionitrile fumarate (BAPN), a specific LOX inhibitor. Moreover, purified LOX was able to work at different temperatures (20-90°C) at pH 8.2. Although further research is needed, the results from this work suggest that based on LOX activity, this enzyme may be of practical use in preventing textural changes in jumbo squid during storage or processing.

Relationship between muscle texture and the crosslinking degree of collagen fibers from octopus ( Octopus vulgaris ), guitarfish ( Rhinobatos productus ), and cazon ( Mustelus lunulatus )

“…It was possible to obtain a crude extract of Lox from the three organisms (Table ), with a higher extraction yield in CT from octopus ( p > .05); however, these values were lower than those seen in jumbo squid (Torres‐Arreola et al, ), in which a total Lox yield of 6.74% protein was obtained from its arms. These findings might be associated with the crosslinking degree of the collagen fibers since a higher content of the enzyme leads to a greater formation of covalent bonds involving Pyr (Torres‐Arreola et al, ). Based on the SDS‐PAGE electrophoretic profile of those crude extracts (Figure ), it is suggested that the molecular mass of Lox extracts obtained from octopus, guitarfish and cazon could be around 30–34 kDa.…”

“…Regarding Lox activity, the highest total activity was detected in the octopus crude extract ( p < .05), with no differences between guitarfish and cazon ( p ≥ .05) (Table ), while the specific activity presented the opposite behavior, with octopus having the lowest values ( p < .05). All the evaluated species showed lower specific activity than those described for jumbo squid ( D. gigas ), of 4.1 × 10 –3 nm H 2 O 2 /mg protein in the mantle (Ramírez‐Guerra, Fimbres‐Romero, et al, ) and 4.23 nM H 2 O 2 /mg protein in the arms (Torres‐Arreola et al, ). These differences in activity can be attributed to the differences that tend to exist between marine species, size, habitat, or catch season (Morales et al, ), which affects the arrangement of the crosslinking of collagen fibers (Torres‐Arreola et al, ) and the high concentration of Pyr (Ramírez‐Guerra, Fimbres‐Romero, et al, ).…”

“…Based on the SDS‐PAGE electrophoretic profile of those crude extracts (Figure ), it is suggested that the molecular mass of Lox extracts obtained from octopus, guitarfish and cazon could be around 30–34 kDa. The mature Lox molecule isolated from jumbo squid ( D. gigas ) migrates as a single protein at 32 kDa in SDS‐PAGE (Torres‐Arreola et al, ). Guitarfish and cazon shared similar electrophoretic profiles to each other, which differed from that of octopus, mainly in the 30–40 kDa range, potentially indicating a different Lox concentration and/or molecular weight.…”

“…Guitarfish and cazon shared similar electrophoretic profiles to each other, which differed from that of octopus, mainly in the 30–40 kDa range, potentially indicating a different Lox concentration and/or molecular weight. In marine and terrestrial organisms, different Lox isoforms have been reported that can range from 25 to 40 kDa in the mature form (Palamakumbura & Trackman, ; Torres‐Arreola et al, ).…”

“…The Lox enzyme was extracted at 4°C, using the procedure described by Kagan and Cai () with slight modifications by Torres‐Arreola et al (). The Lox was extracted using 50 g muscle of each individual species, mixing and homogenizing with 500 ml of 0.4 M NaCl containing 16 mM potassium phosphate buffer, pH 7.8 for 24 hr, centrifuged at 8,500 rpm for 40 min.…”