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Title:Genetic enhancement, regulation, cellular localization and purification of amylolytic enzymes produced by Clostridium acetobutylicum ATCC 824
Author(s):Annous, Bassam Akram
Doctoral Committee Chair(s):Blaschek, Hans-Peter M.
Department / Program:Biology, Genetics
Biology, Microbiology
Chemistry, Biochemistry
Discipline:Biology, Genetics
Biology, Microbiology
Chemistry, Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Biology, Genetics
Biology, Microbiology
Chemistry, Biochemistry
Abstract:Two types of mutants of Clostridium acetobutylicum were isolated. The amylolytic enzyme synthesis in C. acetobutylicum BA 101 and BA 105 was amplified and catabolite de-repressed, respectively, as compared to the parental ATCC 824 strain.
There was an 8.3-fold increase in total amylolytic activity when C. acetobutylicum ATCC 824 was grown in P2 medium containing starch. When this strain was grown on starch in the presence of glucose, total amylolytic activity decreased with increasing glucose concentration. Amylolytic enzyme production by C. acetobutylicum BA 101 was 1.8- and 2.5-fold higher than that of the ATCC 824 strain grown on starch or glucose, respectively. While there was no change in amylolytic activity of C. acetobutylicum BA 105 when grown on starch, this strain produced 6.5-fold more amylolytic activity on glucose relative to the wild type strain.
Amylolytic activity was primarily cell-associated (cell-bound and intracellular) when C. acetobutylicum ATCC 824 was grown on glucose or maltose and primarily extracellular when it was grown on dextrin or starch. The cell-associated amylolytic enzymes of this strain were intracellular (up to 71% of total activity). When grown in starch-based P2 medium, the intracellular amylolytic activity was 90% membrane-bound and 10% cytoplasmic. The amylolytic enzymes of C. acetobutylicum BA 101 and BA 105 were primarily extracellular on all carbohydrates tested.
Amylolytic enzymes purified from the culture supernatant of C. acetobutylicum ATCC 824 displayed only $\alpha$-amylase activity. The molecular weight of the purified $\alpha$-amylase (160-fold) was determined by SDS-PAGE to be 61 Kda. HPLC analysis of end-products of enzyme activity on various substrates indicated that the enzyme acted specifically in an endo-fashion on the $\alpha$-1,4-glucosidic linkages. Enzyme activity was optimal over a pH-range of 4.5-5.0 and temperature of 55$\sp\circ$C, but was rapidly inactivated at higher temperatures. Addition of calcium chloride (2-5 mM) increased $\alpha$-amylase activity by ca. 20%, while the addition of 19 $\mu$g/ml acarbose (a differential inhibitor of amylases) resulted in 50% inhibition.
C. acetobutylicum BA 101 and BA 105 produced up to 1.8-fold higher levels of butanol than the ATCC 824 strain when grown in starch-based (60 g/1) P2 medium containing yeast extract. (Abstract shortened with permission of author.)
Issue Date:1991
Rights Information:Copyright 1991 Annous, Bassam Akram
Date Available in IDEALS:2011-05-07
Identifier in Online Catalog:AAI9136529
OCLC Identifier:(UMI)AAI9136529

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