Asian Journal of Microbiology, Biotechnology & Environmental Sciences Paper

Vol 5 Issue 3, 2003; Page No.(359-366 )

INTRASPECIFIC AUXOTROPHIC HYBRIDIZATION IN ASPERGILLUS NIGER AND PENICILLIUM DIGITATUM BY PROTOPLAST FUSION FOR STRAIN IMPROVEMENT IN VERBENOL PRODUCTION

RENU AGRAWAL, SMITHA C.V. RAO, C.N. GAYATHRI AND RAMYA PREMNATH

Abstract

Different auxotrophic fusant strains were produced from A. niger and P. digitatitzu in order to produce enhanced amounts of verbenol. Various auxotrophs were obtained by using a chemical mutagen ethyl methane sulphonate (EMS) and ultraviolet (UV) irradiation. The protoplasts of different auxotrophic strains of A. ?tiger and P. digztatum obtained under optimized conditions were fused in order to obtain a hyper performer. The protoplasts isolated were fused in equal numbers using polyethylene glycol 4000 at a concentration of 20% along with (0.01 M) CaC12 at pH 7.5 and 0.7 M KC1 as osmotic stabilizer. These were incubated for 10 min at 30°C. The regenerated stable f-usants were studied for the biotransformation. After growing the biomass for 20 h in PDB (pH 6.5; growing medium), biotransformation was performed in phosphate buffer (production medium; 0.05 M, pH 7.0) and incubated with alpha pinene for 6h at 30°C on a rotary shaker at 120 rpm. The biotransformation efficiency was studied in all the cases and the fusants were characterized morphologically, nutritionally, ploidy and biotransformation efficiency. The fusant between UV and EMS auxotrophs of P. digitatum was found to be the best with 51.73% conversion efficiency producing 93.1 ig/100 mg biomass as compared to the wild type which produced only 27 1.ig/100 mg biomass with a conversion efficiency of only 15% which is almost three and a half fold increase over the wild type culture. Morphologically the fusant was totally different from the parent as the colony size was smaller (0.7 cm dia) as compared to the wild type (1.0 cm dia) culture. The colour of the fusant colonies was greenish grey with red pigmentation to the medium where as the wild type colony was grey in colour.

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