Modification of Lactobacillus beta-glucuronidase activity by random mutagenesis

Area of Science:

• Enzyme Engineering
• Molecular Biology
• Microbiology

Background:

• The Lactobacillus gasseri ADH beta-glucuronidase (gusA) gene shows optimal activity at acidic pH (5.0) but limited function at neutral pH (6-7).
• Reporter enzymes are crucial for monitoring gene expression in various microbial hosts.
• The need exists for reporter enzymes functional across a wider pH spectrum.

Purpose of the Study:

• To engineer the Lactobacillus gasseri ADH gusA gene for enhanced activity in neutral pH ranges.
• To develop a more versatile reporter enzyme for diverse microbial applications.

Main Methods:

• Random mutagenesis of the gusA gene using the Epicurian coli XL1-Red mutator strain.
• Cloning of the mutated gusA gene into the broad host range vector pGK12.
• Enzyme activity assays across different pH ranges in Escherichia coli and Lactobacillus gasseri.
• Sequence analysis of mutant alleles to identify genetic alterations.

Main Results:

• Two mutant alleles, gusA2 and gusA3, were identified with increased beta-glucuronidase activity at neutral pH.
• The gusA3 mutant exhibited significantly enhanced activity in the pH range of 4-8.
• Sequence analysis revealed single base pair changes leading to D524G and D573A amino acid substitutions in gusA2 and gusA3, respectively.

Conclusions:

• The engineered GusA3 enzyme demonstrates broadened pH activity, making it suitable for non-acidophilic hosts.
• This modified enzyme expands the utility of beta-glucuronidase as a reporter in various microbial systems, including lactic acid bacteria.
• The study provides a valuable tool for gene expression studies in diverse bacterial environments.