| Similar to many other herbivores feeding on algae, abalones use enzymes to digestpolysaccharides of algae they consume. Among them, cellulase plays a critical role in thedegradation of cellulose to oligosaccharide and glucose. During industrial processing of abalone,the viscera, which accounts for20-30%of the body weight, is usually discarded or processed aslow-valued feedstuff. It is a waste of resources and a burden to environmental protection.In the present study, using abalone viscera as raw material, a cellulase was purified to highhomogeneity with an easy-to-apply method and its characteristics identified, especially itsdegradation on seaweed polysaccharide was studied. Further, the purified cellulase was appliedto hydrolyze seaweed Porphyra haitanensis to produce oligosaccharides, which revealedfavourable antioxidant activities. Meanwhile, a gene coding cellulase was cloned and the spatialstructure was simulated and its polygenetic tree was established. This paper provides message forthe comprehensive utilization of abalone viscera and enriched the research on cellulase theory,including cellulase structure, and phylogenetic evolution.The purification of cellulase from the viscera of abalone (Haliotis discus hannai) wasperformed by a series of biochemical separation methods. SDS-PAGE and Native-PAGE bothrevealed a single band with molecular mass of45kDa under non-reducing condition. Peptidemass fingerprinting (PMF) analysis obtained103amino acid residues which were identical tocellulases from other abalone species. Optimum temperature of the cellulase was45°C, it isquite thermal stable. Optimum pH of the cellulase was5.5. The enzyme activity wassignificantly affected by divalent ions, especially by Mn2+. It was quite interesting to notice thatthe activity drastically increased to283%in the presence of5mM Mn2+. The enzyme was alsoactivated by Mg2+, Ba2+, Ca2+at different levels, while it was strongly inactivated by Cu2+, Zn2+,Fe2+, Sn2+.In the determination of substrate specificity, the purified enzyme rapidly hydrolyzedCMC-Na while didn’t degrade salicin, microcrystalline cellulose, soluble starch, sucrose, mycose,sodium alginate, agar, indicating that it belongs to a kind of endo-β-1,4-glucanases and itsspecificity is to endo-β-1,4-glycosidic bond. Although the present cellulase could hydrolyze P.haitanensis polysaccharide, while the relative hydrolysis activity was only40.4%, much lowerthan that to CMC-Na. CMC-Na was thus used as substrate to investigate the kinetic parametersof the purified enzyme. As a result, the Kmvalues for CMC-Na was determined as2.45mg/mL,the kcatvalues for CMC-Na was2.18s-1and the catalytic efficiency (kc at/K m) values for it was 0.89mg-1·s-1·mL. The purified cellulase was used to produce antioxidant oligosaccharide,including a,a-diphenyl-b-picrythydrazyl (DPPH) free radical and hydrogen peroxide scavengingactivity, reducing capacity and metal ion chelating ability. DPPH radical scavenging activity withan EC50(concentration of antioxidant required to quench50%of the stable free radical) value of6.89mg/mL. Oligosaccharides scavenged hydroxyl radical efficiently and the scavengingcapability was concentration dependent with EC50of6.51mg/mL. The EC50of Fe2+chelatingability was5.24mg/mL.Based on the result of PMF and conservative sequence of cellulases from different aquaticanimals, the primers were designed for cloning of cellulase from the viscera of abalone Haliotisdiscus hannai by RT-PCR,3’-RACE and5’-RACE. One cellulase gene was cloned containing1896bp, which could code594amino acids. The GenBank accession number was KC456160.1,and the protein accession number was AGI61069.1. The one quarter at the N terminal wasregarded as propeptide, which was lost in the purified cellulase. It was supposed that in thetranslation of cellulase, the propeptide was incised and cellulase with activity did not possess thissection. Quite possibly, the purified cellulase and the cloned cellulase were different forms in theabalone viscera. The spatial structure of the cellulase was simulated and its polygenetic tree wasestablished. According to polygenetic tree analysis, the cellulase was similar to aquatic animals,while different to these from microorganisms and plants, suggesting abalone cellulase isinherited from its ancestor but not microorganism and plant.Abalone is a complex bioreactor, among whose viscera abundant enzymes existed toparticipate in its metabolism. In the present study, a cellulase was purified from the viscera andcharacterized its enzymatic properties. Further, the gene encoding a cellulase was cloned andspatial structure was simulated. Based on its sequence, polygenetic tree was established. Theresearch provided message for the comprehensive utilization of resources of abalone viscera andenrich the theoretical research of cellulases. |
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