Modification of Lactobacillus beta-glucuronidase activity by random mutagenesis.
The Lactobacillus gasseri ADH beta-glucuronidase gene, gusA, was cloned previously and found to exhibit excellent activity in acidic pH ranges, with maximal activity at pH 5.0. In contrast, activity was limited in neutral pH ranges of 6-7. In an effort to improve the activity of the reporter enzyme in neutral pH ranges, the gusA gene was cloned into the broad host range vector, pGK12, and subjected to random mutagenesis by passage through Epicurian coli mutator strain XL1-Red. Two mutant alleles, gusA2 and gusA3, were recovered that produced beta-glucuronidase with increased activity in neutral pH ranges. One of these, gusA3, was significantly more active in the pH range of 4-8 in both Escherichia coli and L. gasseri. Sequence analysis of gusA2 and gusA3 revealed single base pair changes that resulted in D524G and D573A substitutions, respectively. The modified GusA3 enzyme has expanded potential for use as a reporter enzyme in expression hosts that are not acidophilic, as well as lactic acid bacteria and other microorganisms that grow in acidifying environments.
Callanan
Michael J
MJ
Department of Food Science, North Carolina State University, Raleigh, NC 27695, USA.
Russell
William M
WM
Klaenhammer
Todd R
TR
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005798:Genes, Bacterial; D005966:Glucuronidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007778:Lactobacillus / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D014169:Transformation, Bacterial
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D005966:Glucuronidase