Genetic engineering of white shochu-koji to achieve higher levels of acid-stable α-amylase and glucoamylase and other properties when used for Shochu making on a laboratory scale

Abstract

The genes for acid-stable α-amylase and glucoamylase were cloned from white shochu-koji, Aspergillus kawachii. Both genes were used to transform the parent strain of white shochu-koji which carried a dominant selective marker gene, amdS, that originated from A. oryzae. Three lines of transformants were identified that secreted about 6-fold more acid-stable α-amylase activity and about 7-fold more glucoamylase activity than the parent strain in liquid culture. In solid culture, all three transformants had 2-fold higher acid-stable α-amylase activity and 2.4-fold higher glucoamylase activity than the parent strain. When koji was prepared on a laboratory scale, acid-stable α-amylase activity was 5.7-fold higher and glucoamylase activity was 3.8-fold higher than when the parent strain was used. Shochu was produced with a koji ratio of 33% or 10% using one line of transformants on a laboratory scale. Even with a koji ratio of 10%, the weight of the mash obtained with the transformant decreased to almost the same extent as with a koji ratio of 33% and the parent strain. Levels of flavour compounds in shochu produced with koji of the transformant were higher than in the shochu prepared with koji of the parent strain. In particular, levels of isoamyl acetate and β-phenethyl acetate were as high as 12.9 mg/litre and 3.8 mg/litre, respectively.