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Title:Isolation and Biochemical Characterization of an Alpha-Amylase From Lactobacillus Amylovorus and Sequencing of the Corresponding Gene
Author(s):De Parasis, Joseph
Doctoral Committee Chair(s):Bruce M. Chassy
Department / Program:Food Science and Human Nutrition
Discipline:Food Science and Human Nutrition
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Biology, Microbiology
Abstract:In this study the enzyme $\alpha$-amylase ($\alpha$-1,4-D-glucan 4-glucanohydrolase, EC has been isolated and characterized from the extracellular media of Lactobacillus amylovorus DSM 20531. The gene coding for $\alpha$-amylase has been isolated by molecular cloning in Escherichia coli and the DNA sequence determined. The conditions for optimal production of $\alpha$-amylase were determined; maximum activities of $\alpha$-amylase were found at stationary phase after growth of Lactobacillus amylovorus on galactose and glucose containing media. It was found that the ratio in $\alpha$-amylase activity in galactose containing medium compared to glucose containing medium had a 5-fold increase in activity during the logarithmic phase of growth and decreased to 2-fold at the stationary phase of growth. The $\alpha$-amylase was purified to apparent homogeneity by ammonium sulfate fractionation, Octyl-Sepharose hydrophobic chromatography, Mono Q anion exchange chromatography, and Superose 6 gel filtration chromatography. The enzyme appeared as a homogenous 126 kDa protein in 10% SDS-PAGE gels. The enzyme was found to be stable between 25$\sp\circ$C and 55$\sp\circ$C, and the highest activity was observed at 42$\sp\circ$C. The gene encoding the $\alpha$-amylase from Lactobacillus amylovorus was found to express efficiently Lactobacillus casei. Even though the level of $\alpha$-amylolytic activity was higher in Lactobacillus casei, the pattern of apparent repression by glucose was similar to that observed in Lactobacillus amylovorus. The protein isolated from Lactobacillus casei had the same apparent molecular weight and biochemical characteristics as $\alpha$-amylase from Lactobacillus amylovorus. The complete sequence of the fragment containing the $\alpha$-amylase gene inserted into the plasmid pLPCR2 was determined. Putative promoter, ribosome binding site, and leader signal sequences were identified. The G + C content of the coding sequence for the $\alpha$-amylase gene was found to be 38%, a value within the range found for other lactobacilli strains. The sequence of the putative $\alpha$-amylase promoter from Lactobacillus amylovorus was found to be similar to the consensus sequence of promoters for both gram-positive and gram-negative bacterial $\alpha$-amylases. The ribosome-binding site sequence from Lactobacillus amylovorus was found to more closely resemble the sequence found in lactococci than from bacilli. (Abstract shortened by UMI.).
Issue Date:1998
Description:258 p.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1998.
Other Identifier(s):(MiAaPQ)AAI9904429
Date Available in IDEALS:2015-09-25
Date Deposited:1998

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