Improvement of enzyme productivities through mutation or haploidization of heterozygous diploids obtained by protoplast-fusion of Aspergillus sojae.

Abstract

A new breeding process for Aspergillus sojae involving protoplast fusion was studied to obtain more desirable koji-mo\&s for soy-sauce production, especially as to their enzyme productivities. A pair of double-marker mutants with characteristic enzyme-productivities, derived from genealogically unrelated A. sojae cultures, were fused by means of the protoplast fusion technique. The fusants were UV-treated, and stable heterozygous diploids were obtained. Further improvement of their enzyme-productivities was attempted using two approaches. Through the usual mutation of the heterozygous diploids, the activities ofprotease and glutaminase were simultaneously improved to a certain extent. Through the haploidization of the heterozygous diploids with benomyl or pfluorophenylalanine (FPA), some haploids considered phenotypically to be recombinants were segregated, in addition to a large number of haploids that had reverted to the original double marker mutants. Onscreening these haploid-recombinants, the authors obtained a few more excellent recombinants, as to their enzyme productivities, that is, strains producing as much protease as the hyper-protease producer and producing almost as much glutaminase as the hyperglutaminase producing parent. It has been shown that mutation or especially haploidization of heterozygous diploids produced by cell-fusion could be a very useful technique for the breeding of new industrial koji-mo\&s.

Koji Molds for Japanese Soy Sauce Brewing: Characteristics and Key Enzymes

“…In addition, using a mutant strain with double markers for conidial color and auxotrophy, a diploid strain exhibiting the intermediate enzyme productivity of its two parental strains was generated by protoplast cell fusion of A. sojae 2048 strain with high protease activity and A. sojae 2165 strain with high glutaminase activity [ 22 ]. These diploid strains were irradiated with UV and treated with benomyl for haploidization, thus obtaining strains with high protease and glutaminase activities [ 23 ].…”

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“…Ushijima and Nakadai 116 used mutation and protoplast fusion techniques for improving LGlutaminase production by A. sojae employed in shoyu fermentation. Mulchandani and Bassi, 17,117,118,119,120 and hybridoma culture media 121 by flow injection analysis.…”

“…An amperometric enzymes electrode probe with membrane immobilized L-Glutaminase was described by Villarta et al 117 for determination of serum glutamine levels of humans. Botre et al 118 used L-Glutaminase based biosensor for determination of glutamine and glutamate in pharmaceutical formulations.…”