• The Korean Journal of Mycology
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• v.27 no.6 s.93
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• pp.399-405
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• 1999

Intergeneric hybrids between Saccharomyccopsis fiburigera KCTC 7393 and Saccharomyces cerevisiae KCTC 7049 (tyr-, ura-) were obtained by nuclear transfer technique. Nuclei isolated from the wild type S. fiburigera strain were transfered into auxotrophic S. cerevisiae mutants and new strains showing an increased starch degrading capability were selected. Maximum production of protoplasts was obtained from the treatment with 0.1 % Novozym 234 at $30^{\circ}C$ for 90 min, and most effective osmotic stabilizer for the isolation of protoplasts was 0.6 M KCl at pH 5.8. The frequency of protoplast regeneration was 14.64% under the conditions. Genectic stability, conidial size, DNA content, and nuclear stain suggested that the fusants were aneuploidy. The specific activity of ${\alpha}-amylase$ was observed to increase about $1.2{\sim}1.9$ folds.

• Korean Journal of Microbiology
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• v.35 no.4
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• pp.315-321
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• 1999

This study has been performed to deveope a yeast strain having high ${\alpha}$-amylase production ability using nuclear transfer method. Hybrids formed between the strains of Saccharomyces fiburigera KCTC 7393 and Saccharomyces cerevisiae KCTC 7049 (tyr-, ura-)were obtained by nuclear transfer technique. Nuclei isolated from the wild type S. fiburigera strain were transfered into auxotrophic mutants S. cerevisiae and selected the hybrids showing an increased starch degrading capability were selected (MN-16). This transformant grew best and produced maximal ${\alpha}$-amylase activity on the medium containing 2% (V/V) soluble starch. ${\alpha}$-Amylase from MN-16 was purified electrophoretically homogenety and its properties were investigated. The enzyme was purified about 10.6 fold with an overall yield 9.7% from the culture medium by ammonium sulfate fractionation. DEAE-Sephacel column chromatography, and Sephacryl S-200 column chromatography. The purified enzyme showed a single band on SDS-polyacrylamide gel electrophoresis. The molecular weight of the ${\alpha}$-amylase was estimated to be 53,000 daltons by SDS-PAGE and by gel permeation chromatography on Sephacryl S-200. The purified enzyme showed the maximum activity at pH 5.5 and 40${\circ}C$. The km value for soluble starch was 2.5㎎/㎖. The enzyme activity increased in the presence of $Ca^{2+}, Co^{2+}, EDTA, Mg^{2+}, Mn^{2+}, Zn^{2+}$, but inhibited by $Cu^{2+}, Fe^{2+}$, and $Ni^{2+}$