In mammals, glucuronidation is one of the principle means of detoxifying or inactivating compounds using the UDP glucuronyl transferase system. Compounds are conjugated by the glucoronyl transferase system to form glucuronides, which are then secreted in urine or into the lower intestine in bile. Furthermore, microorganisms in the gut, such as Escherichia coli, have evolved to utilize the excreted β-glucuronides as a carbon source. The β-glucuronidase (BGUS) enzyme catalyzes the hydrolysis of a wide variety of β-glucuronides. Thus, BGUS enzyme activity been reported in those organisms that utilize glucuronidation as a detoxification pathway, as well as in some of their endogenous microbe populations. All vertebrates and many mollusks, as well as certain bacteria, exhibit BGUS enzyme activity, whereas insects and plants that utilize a different detoxification pathway typically do not exhibit BGUS enzyme activity.
Given the key role of glucuronidation in detoxification of compounds, the BGUS enzyme has been used for detection of drugs in bodily samples, such as to detect the presence of illicit drugs in bodily samples of criminal suspects. For example, a bodily sample can be tested for the presence of a suspected drug by detecting the hydrolysis of the glucuronide form of the drug by BGUS.
Commercially available preparations of BGUS enzyme, for use for example in drug testing, include crude extract forms of the E. coli, snail and abalone versions of the enzyme. While these preparations are effective in hydrolyzing glucuronides, they typically include other proteins in addition to the BGUS, which may interfere with enzyme activity. Moreover, importantly, their level of enzyme activity is such that they typically require at least several hours (e.g., three hours or more) to analyze a sample. Including sample preparation time and analysis time, this means that evaluation of a drug sample typically can take at least two days using currently commercially available BGUS preparations. Furthermore, currently commercially available BGUS preparations are more efficient at neutral pH but are less efficient at lower pH (e.g., below 6.8).
Accordingly, there is a need for BGUS enzymes with enhanced activity that are more efficient for use in drug testing.
USPTO Backgrounds
USPTO Backgrounds