Purification and gene cloning of Alpha-amylase of Neurospora crassa
1988
Kang, I.K. (Lucky Research Inst., Taedok (Korea R.)) | Kim, M.S. | Yang, C.H. (Seoul National Univ., Seoul (Korea R.). Coll. of Natural Science)
Alpha-amylase (EC.3.2.1.1) of Neurospora crassa (ATCC 9279) was cloned in E. coli HB101 using shotgun method, and the enzymes isolated from both N. crassa and E. coli were compared. Chromosomal DNA isolated from the spores of N. crassa was partially digested with PstI restriction endonuclease and rejoined to pBR322 which had been digested with the same enzyme. The resulting recombinant DNA were introduced into E. coli HB101 which had competency by treating with CaCl2. As a result, about 8000 colonies which showed tetracycline resistance were selected and two of the colonies which had 13.5 Kb recombinant plasmid exhibited starch degrading activity on starch-containing plate when treated with D-cycloserine. Alpha-amylases from both N. crassa and E. coli were isolated by using ammonium sulfate precipitation, DEAE-cellulose ion exchange column chromatograph and Bio-Gel P150 gel filtration column. As a result, about 81.3 fold and 5.6 fold purifications in specific activities were obtained respectively, and specific activities of the gel filtrates were 6.1 u/mg and 85 u/mg respectively. The properties of both enzymes were compared and they showed quite 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 70deg C and optimal pH were about 6 and 10
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