Catalytic and Operational Stability of Acidic Proteases from Monterey Sardine (Sardinops sagax caerulea) Immobilized on a Partially Deacetylated Chitin Support

Abstract

Recovery of valuable compounds from by‐products generated from seafood processing represents an opportunity to produce value‐added products. Sardine acidic proteases were immobilized on partially deacetylated chitin through ionic linkages, using sodium tripolyphosphate as a support activating agent. Maximal enzyme activities of free and immobilized proteases were detected at pH 2, at which the immobilized form had higher stability than the free enzymes. Optimum temperatures for free and immobilized proteases were 40 and 50C, respectively, while thermal stability of immobilized proteases was greater than that of free proteases. Reusability studies showed that after a second catalytic cycle, the immobilized enzyme maintained about 40% of the initial activity. The immobilization process enhanced the long term storage stability of sardine proteases compared to free enzymes. Results suggest that the immobilized enzyme could be used as a biotechnological aid for food processing when low pH and temperature around 50C are needed. Practical Applications Fish waste constitutes a serious environmental problem due to high disposal costs. Besides, this waste is commonly discarded or used for low value processes. Therefore, this study proposes the immobilization of sardine (Sardinops sagax caerulea) acidic proteases from fish viscera waste, on a partially deacetylated chitin support extracted from shrimp (Penaeus spp.) head. This research suggests that seafood protease immobilization onto chitinous supports presents an opportunity to improve the stability of these enzymes and consequently enhance potential application of these valuable biocatalysts as biotechnological aid, mainly for food processing and other value added products.

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“…Also, the decrease in the specific activity may be due to the diffusional limitation of the substrate and product flow. Although GA creates very strong bonds between enzymes and carriers, it changes the structure of some enzymes during the binding process, causing loss of their activity (Salazar-Leyva et al, 2017).…”

“…Pervez et al (2017) reported that immobilization decreases the inhibition of enzymes either by stabilizing the enzyme structure or by eliminating the inhibitor. Moreover, multipoint covalent attachment between the enzyme molecule and an activated carrier makes the structure of the enzyme more rigid, consequently decreasing conformational changes which can be induced by extreme pH (Salazar-Leyva et al, 2017). Narwal et al (2016) suggested that titratable acidity of milk should be between 0.19 to 0.25 % lactic acid equivalents (which correspond to pH from 5.0 to 6.5) at the time of adding an enzyme.…”

“…As seen in Figure 3, immobilization increases the optimum temperature by 10°C which is probably a result of the thermal stability enhanced by immobilization. Covalent binding of enzyme onto chitosan enhanced the optimum temperature and stability of the biocatalysts for thermal inhibition (Salazar-Leyva et al, 2017). Hosseini and Varidi (2021) reported that free and immobilized rennet showed the highest activity at the same temperature (40 °C), and at higher temperatures there is a sharp decrease in the activity of the free enzyme compared to the immobilized enzyme.…”

“…Since there is no universal support suitable for all enzymes and all applications, it is important to examine different supports using different methods of enzyme immobilization. Chitosan is one of natural polymer (polysaccharides) derived from chitin by deacetylation process and has excellent biocompatibility, no toxicity, cheapness, high mechanical strength, and a susceptibility to chemical modifications (Salazar-Leyva et al, 2017;Yandri et al, 2020). One common approach for enzyme immobilization on chitosan is through multipoint covalent binding between the functional groups present on the surface of an activated chitosan by cross-linking agents such as GA and the surface functional groups of the enzyme protein.…”

Kinetic, Catalytic and Thermodynamic Properties of Immobilized Milk Clotting Enzyme on Activated Chitosan Polymer and its Application in Cheese Making

“…Chitosan is one of natural polymer (polysaccharides) derived from chitin by deacetylation process and has excellent biocompatibility, no toxicity, cheapness, high mechanical strength, and a susceptibility to chemical modi cations (Cahyaningrum and Sianita 2014;Salazar-Leyva et al 2017). One common approach for enzyme immobilization on chitosan is through multipoint covalent binding between the functional groups present on the surface of an activated chitosan by cross-linking agents such as glutaldehyde and the surface functional groups of the enzyme protein.…”