Molecular characterization of intergeneric hybrid between Aspergillus oryzae and Trichoderma harzianum by protoplast fusion.

Abstract

Protoplast fusion between Aspergillus oryzae and Trichoderma harzianum and application of fusant in degradation of shellfish waste. The filamentous chitinolytic fungal strains A. oryzae NCIM 1272 and T. harzianum NCIM 1185 were selected as parents for protoplast fusion. Viable protoplasts were released from fungal mycelium using enzyme cocktail containing 5 mg ml(-1) lysing enzymes from T. harzianum, 0.06 mg ml(-1) β-glucuronidase from Helix pomatia and 1 mg ml(-1) purified Penicillium ochrochloron chitinase in 0.8 mol l(-1) sorbitol as an osmotic stabilizer. Intergeneric protoplast fusion was carried out using 60% polyethylene glycol as a fusogen. At optimum conditions, the regeneration frequency of the fused protoplasts on colloidal chitin medium and fusion frequency were calculated. Fusant showed higher rate of growth pattern, chitinase activity and protein content than parents. Fusant formation was confirmed by morphological markers, viz. colony morphology and spore size and denaturation gradient gel electrophoresis (DGGE). This study revealed protoplast fusion between A. oryzae and T. harzianum significantly enhanced chitinase activity which ultimately provides potential strain for degradation of shellfish waste. Consistency in the molecular characterization results using DGGE is the major outcome of this study which can be emerged as a fundamental step in fusant identification. Now it is need to provide attention over effective chitin degradation to manage shrimp processing issues. In this aspect, ability of fusant to degrade shellfish waste efficiently in short incubation time revealed discovery of potential strain in the reclamation of seafood processing crustacean bio-waste.