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KMID : 0364819880260020073
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Korean Journal of Microbiology
1988 Volume.26 No. 2 p.73 ~ p.81
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Purification and gene cloning of α-Amylase of Neurospora crassa
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Kang, Il-Koo/강일구
Kim, Mi-Suk/Yang, Chul-Hak/김미숙/양철학
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Abstract
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1
a -Amylase (EC.3.2.1.1) of Neurospora crassa (ATCC 9279) was cloned in E. con HBI01 using shotgun method, and the enzymes isolated from both N. crassa and E. con were compared.
Chromosomal DNA isolated from the spores of N. crassa was partially digested with Pstd restriction endonudease and rejoined to pBR322 which had been digested with the same enzyme. The resulting recombinant DNA were introduced into E. con 10101 which bad competancy by treating with CaCl2. As the result, about *000 colonies which showed tetracycline resistance were selected and two of the cob onies which had 13.5 Kb recombinant plasmid exhibit starch degrading activity on starch-containing plate when treated with D-cycloserine.
a -Amylases from both N. crassa and E. con were isolated by using ammonium sulfate precipitation, DEAE-cellulose ion exchange column chromatography and Bio-Gel P150 gel filtration column. As the result, about 81.3 fold and 5.6 fold purification in specific activities were obtained respectively, and specific activities of the gel filtrates were 6.1 u / mg and *5 u / mg respectively.
The properties of both enzymes were compared and they showed quite the similar patterns in optimal temperature, optimal pH and had same molecular weight about 100,000 daltons on gel filtration method. Optimal temperatures for both enzymes were 70°C and optimal pH were about 6 and 10.
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