Appraisal of antioxidant, anti-hemolytic and DNA shielding potentialities of chitosaccharides produced innovatively from shrimp shell by sequential treatment with immobilized enzymes

Highlights

• Immobilization of crude protease and chitinase on agar beads for repeated use.

• Generation of chitosaccharides (CS) from shrimp shell using immobilized enzymes.

• CS were characterized as an admixture; degree of polymerization up to 18.

• CS has potent antioxidant activities; inhibit oxidative lysis of RBC membrane.

• CS protects protein and DNA from oxidative damage; act as DNA shielding agent.

Abstract

Chitosaccharides (CS) of varied size were prepared from shrimp shell through sequential catalysis, using crude protease and chitinase enzymes immobilized on agar beads. In the optimized state, immobilization yield and activity yield for protease were 84% and 62%, and for chitinase were 75% and 57%, respectively. Immobilized protease and chitinase treatment improved CS yields (101 μg/ml) and retained 63% and 52% of activities after 10 reuses, respectively. Stronger radical-scavenging activity (RSA) of CS against ABTS, DPPH and hydroxyl radical was noted with EC₅₀ values 19.1, 26.4 and 29.6 μg/ml, respectively. Peroxyl and superoxide RSAs of 96.8% and 88.6% were noticed at 70 μg/ml of CS. Singlet oxygen quenching, reducing power and ferrous ion-chelating activities of CS were also pronounced. CS reasonably reduced oxidative damage of DNA, protein and RBC by inhibiting H₂O₂ and AAPH radicals. Reversible CS-DNA condensation leads to DNA stabilization without changing its conformation and advocates its employment in gene therapy.

Introduction

Chitin is a polymer of β (1–4) linked N-acetylglucosamine (NAG). Among the natural resources, crustacean shells have the highest chitin content (Halder et al., 2013, Kandra et al., 2012). Chitin and its semi-deacetylated form, chitosan, have versatile properties which are the factors underlying the current interest in their commercial exploitation (Majeti and Kumar, 2000, Qin, 1993). Chitosaccharides (CS), the oligomeric/monomeric form of chitin/chitosan have lower viscosity, more hydrophilicity, smaller molecular sizes and are absorbed more easily than the polymeric precursors in vivo, and hence are applied more widely in health-care, food and medicine than the polymeric precursors (Kim & Rajapakse, 2005). Despite such dynamic relevance, the high production cost of CS awkwardly restricts its practical uses. In India, more than 300 seafood-processing industries annually produce more than 10⁵ tons of chitinous shrimp shells as waste which are discarded through ocean dumping, incineration and land filling (Halder et al., 2013, Kandra et al., 2012, Philip and Nair, 2006). Utilization of these wastes for CS generation could be appropriate, in terms of commerce, resource mobilization and pollution abatement. In this aspect, enzymatic production of CS from chitinous biowaste through chitinase (E.C.3.2.1.14) treatment will be an important and indispensable aspect of crustacean waste management in view of several incompatibilities of conventional physicochemical techniques. However, major obstacles of industrial application of chitinase are lack of long term operational stability, difficulties during recovery of products and reuse of the enzyme. In this respect, immobilization of chitinase could provide an excellent remedy for fulfillment of the said lacuna.

Formation of free radicals, e.g. reactive oxygen species (ROS), is an unavoidable consequence during aerobic respiration. They have been implicated in the pathogenesis of a wide spectrum of diseases and aging processes due to their detrimental effect on bio-macromolecules such as DNA, protein and lipid (Kim and Rajapakse, 2005, Uttara et al., 2009). To overcome these crises, a recent practice is the supplementation of diets containing adequate antioxidants, which can break the oxidative sequence by delaying/preventing oxidant generation, competing for existing radicals and removing them from the reaction, thus protecting biomolecules from damage. In recent years, there has been an increasing interest in finding natural antioxidants from different low cost natural sources for dietary and pharmacological uses.

In our previous study, during fermentation of shrimp shell by chitinolytic Aeromonas hydrophila SBK1, concomitant production of chitinase and chitosaccharides was achieved (Halder et al., 2013). In this connection, an attempt has been made to access the saccharification potentiality of immobilized chitinase of the said organism from shrimp shell waste after protease pretreatment, and subsequent testing of the biopharmacological utilities of generated chitosaccharides.