Biosensor for Asparagine Using a Thermostable Recombinant Asparaginase from Archaeoglobus fulgidus

Abstract

Asparaginase from the hyperthermophilic microorganism Archaeoglobus fulgidus was cloned and expressed in Escherichia coli as a fusion protein with a polyhistidine tail. After heat treatment to denature most of the native E. coli proteins, the enzyme was purified by an immobilized metal ion affinity chromatography method. The activity of the enzyme was determined by monitoring the change in ammonium concentration in solution. It was found that the enzyme is thermostable at temperatures as high as 85 degrees C. The KM for L-asparagine was 8 x 10(-5) and 5 x 10(-6) M at 37 and 70 degrees C, respectively. The catalytic activity for L-asparagine was 5-fold higher than for D-asparagine. The enzyme was immobilized in front of an ammonium-selective electrode and used to develop a biosensor for asparagine. The biosensor had a detection limit of 6 x 10(-5) M for L-asparagine. Unlike a sensor based on asparaginase from E. coli, the biosensor based on recombinant asparaginase from A. fulgidus demonstrated higher stability.

Simultaneous cell disruption and semi-quantitative activity assays for high-throughput screening of thermostable L-asparaginases

“…Compared with the L-asparaginases from mesophilic bacteria, the P. yayanosii CH1 thermophilic L-asparaginase exhibited higher enzyme activity and better thermal stability, although its specific activity was not as high as that of the L-asparaginases from other thermophilic bacteria. However, the thermostability of the P. yayanosii CH1 L-asparaginase was higher than that of some thermophilic bacteria, such as Archaeoglobus fulgidus and Thermococcus zilligii 12 , 30 . The enzyme exhibited high stability at both the working temperatures (70–95 °C) and storage temperatures (−20–37 °C).…”

Ratiometric fluorescent l‐arginine and l‐asparagine biosensors based on the oxazine 170 perchlorate‐ethyl cellulose membrane

“…. The K m values were 120 mM for the asparaginase from E. coli , 79 μM for recombinant asparaginase from E. carotovora , and 80 μM at 37°C and 5 μM at 70°C for recombinant asparaginase from Archaeoglobus fulgidus . The kinetic parameters of the asparaginase immobilized in the matrix of EC and PU were calculated from the ratio of two fluorescence intensities at λ em = 565 and 625 nm.…”

“…For the detection of l ‐Asn, a miniaturized fiber optic biosensor was developed by immobilizing l ‐asparaginase and absorptive pH sensitive indicator onto plastic chips of 5 mm . An ammonium‐selective electrode was used to develop a biosensor for l ‐Asn by immobilizing asparaginase in the front of the electrode . Recombinant asparaginase from a strain of Erwinia carotovora was used to assemble a microplate‐based biosensor by immobilizing the enzyme into the well of a microplate in a 96‐well format and based on the colorimetric measurement of ammonia formation using Nessler's reagent .…”

Homoserine and asparagine are host signals that trigger in planta expression of a pathogenesis gene inNectria haematococca

“…Purified inducer 1 (0.18 mol of asparagine equivalent in 400 l of water) was mixed with 2 units of asparaginase (Sigma). After incubation at 37°C for 1 h, the mixture was heated at 70°C for 4 min to denature the enzyme (22) and then fractionated by TLC as described above. The eluted fractions were assayed for pelD-inducing activity.…”