Affiliation:
1. Genencor International, Inc., South San Francisco, California 94080
Abstract
A method is described for generating and screening variants of the β-galactosidase from
Lactobacillus delbrueckii
subsp.
bulgaricus
sensitive to several environmental stresses, with potential application in the food industry. Chemical mutagenesis with hydroxylamine or methoxylamine was performed on the β-galactosidase gene carried on an
Escherichia coli
expression vector. Mutants sensitive to cold, heat, low pH, low magnesium concentration, and the presence of urea were isolated by screening for reduced color development on β-galactosidase indicator plates. The mutations responsible for three variant β-galactosidases were localized, and the base substitutions were determined by DNA sequencing. The amino acid alterations associated with one low-pH-sensitive (pHs) and two urea-sensitive (Us) variants correspond to P584L (pHs1), G400S/R479Q (Us26), and G167E/E168K/E363K/V492M (Us17), respectively. Mutant pHs1 is also heat, cold, low magnesium, and urea sensitive; Us26 is also cold sensitive; and Us17 is also low-pH sensitive.
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology
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