The protoplast fusion of Aspergillus sojae was studied to develop a breeding system for the more desirable koji-molds in the production of shoyu (Japanese fermented soy-sauce) with special attention as to their enzyme productivities. Double-marker mutants, as to conidial color and nutritional requirement, were first derived from the parental strains selected from genealogically unrelated A. sojae cultures showing characteristic enzyme-productivities. Protoplasts of the mutants were obtained in high yields by using a combined enzyme-system composed of the enzyme preparation obtained from a culture filtrate of Bacillus circulans IAM 1165 and commercial chitinase (ICN). The protoplasts were fused in PEG 6000. Many of the fused products that grew on minimal regeneration plates were found to be heterokaryons. Stable heterozygous diploids with green conidia were induced from heterokaryons on UV treatment. One hundred and thirty diploid strains were obtained and their enzyme-productivities were assayed. The activities of both protease and glutaminase were distributed within the range of those of the parents, and in most strains, the sum of the two activities did not exceed those in the cases of the parents. Exceptionally, a few diploids showing well-balanced high productivity of both enzymes were also obtained.

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