Glucoamylase Producing Recombinant Strains of Aspergillus niger Obtained by Intraspecific Protoplast Fusion

S. GARGOVA, G.VANCHEVA and I. STOILOVA
Higher Institute of Food and Flavour Industries, Department of Biotechnology, BG-4002 Plovdiv, Bulgaria

Abstract

GARGOVA, S., G. VANCHEVA and I. STOILOVA, 2002. Glucoamylase producing recombinant strains of Aspergillus niger obtained by intraspecific protoplast fusion.Bulg. J. Agric. Sci., 8: 71–78

Protoplast isolation and regeneration was carried out in two strains of Aspergillus niger – MV 92 and MN 76 using well-characterized morphological and phenotypical markers. Protoplast production from hyphae of A. niger was optimized and it was established that a maximum yield of protoplasts was achieved after treating with 2% Helicase on 18-hour mycelium in the presence of 0.8M NaCl and for a duration of 4h. Isolated protoplasts were capable of a high regeneration frequency when 0.6M sucrose was used as an osmotic stabilizer. An intraspecific protoplast fusion was successfully carried out. Diploids derived from heterokaryons were segregated to stable haploid recombinant strains. In the recombinant strain A. niger 5C 2.5 fold higher activity of glucoamylase in the culture filtrate was shown as compared to the more productive MN 76 parent strain, and the time of cultivation was decreased by 24 hours.

Key words: Aspergillus niger, protoplasts, regeneration, fusion, glucoamylase