Recovery and purification of oligosaccharides from copra meal by recombinant endo-β-mannanase and deciphering molecular mechanism involved and its role as potent therapeutic agent.

Abstract

Production of manno-oligosaccharides (MOSs) from pretreated and defatted copra meal (dFCO) hydrolysis was achieved by endo-mannanase. Structural characterization of dFCO by FT-IR and NMR exhibited resemblance with galactomannan. The time-dependent hydrolysis of dFCO by recombinant endo-β-(1 → 4)-mannanase of Clostridium thermocellum by TLC and HPAEC displayed the release of mannose and MOSs mannobiose and mannotriose. Purified MOSs yielded 40 % mannobiose and 18 % mannotriose confirmed by mass spectroscopy which showed mannobiose (m/z = 365) and mannotriose (m/z = 527). The homology based structural analysis of catalytic endo-mannanase (CtManT) showed the catalytic core composed of Glu181 and Glu300 acting as acid/base and Glu288 as a nucleophile during galactomannan hydrolysis. Sub-site mapping of CtManT exhibited two aglycone and four glycone sites at cleavage sites existing on either side of β-(1 → 4)-linkage of galactomannan. Isolated MOSs displayed potential prebiotic characteristics and supported higher growth of probiotic Lactobacillus acidophilus and Bifidobacterium infantis than with standard inulin. Moreover, MOSs displayed over 97 % tolerance to simulated gastric juice, intestinal fluid, and α-amylase proving its potential as a stable prebiotic over inulin. In vitro cytotoxicity assay of MOSs (500 µg/mL) on human epithelial colorectal adenocarcinoma cell line (HT-29) demonstrated 60 % decreased viability of cells after 48 h displaying anti-tumorigenic property.