β-Glucuronidases for Comprehensive Glucuronide Hydrolysis | IMCSzyme®

No. IMCSzyme is a purified beta-glucuronidase product with no esterase activity. Other natural products as crude extracts will typically have esterase, which can cleave the acetyl group from 6-MAM and convert it to morphine. The presence of esterase can be tested with an esterase-sensitive substrate, like Calcein-AM, as shown in our publication in Journal of Analytical Toxicology.

Hydrolysis of glucuronidated benzodiazepines is one of the easier classes of substrates for glucuronidases. IMCSzyme® has been reported by Morris et al. to achieve near complete hydrolysis of glucuronidated benzodiazepines in 5 minutes at room temperature.

While urine samples routinely have a pH range from 4 to 9, we recommend customers use the included hydrolysis buffer. A buffer is used to adjust the sample pH into the recommended range. For IMCSzyme®, use RHB to adjust the pH to 7 to 8. For IMCSzyme® RT, the RT Buffer (RTB) will adjust the pH to 5.5 to 6.5.

Yes, incubating in a water bath or a heating block will benefit from a lower temperature due to differences in heat transfer efficiencies.

For IMCSzyme®:

• When using a water bath or heating block, incubate at 45 °C for 30 minutes.
• When using a convection oven, heat transfer is slower, hence setting the temperature from 55 to 60 °C and incubate for 30 minutes.

IMCSzyme® RT does not require heating and is able to effectively hydrolyze samples at room temperature. Heating of IMCSzyme® RT does not increase hydrolysis efficiency.

We do not recommend diluting the enzyme and incubating longer. At a lower enzyme concentration, the stability of the enzyme in the reaction will become more of a factor. Urine samples are known to contain inhibitors that can inactivate the efficiency of any enzyme over time. If the enzyme concentration is too low, there is a strong likelihood that the reaction would not go to completion during the longer incubation, as the enzyme would be inactivated before the time expires. This could result in a false negative result. At the suggested enzyme concentration and shorter incubation time, the enzyme can withstand a hostile sample long enough to complete hydrolysis.