Glutaminase (Escherichia coli)

Inspired by the porous and fibrous structure of commercially available bamboo, herein we created an L-glutaminase enzyme reactor based on bamboo sticks. The enzyme was immobilized onto the bamboo sticks through a glutaraldehyde modification to achieve covalent bonding. The enzymatic hydrolysis efficiency of the prepared L-glutaminase@bamboo sticks based porous enzyme reactor was evaluated by chiral ligand exchange capillary electrochromatography using L-glutamine as the substrate. L-glutaminase@bamboo exhibited improved enzymatic hydrolysis performances, including high hydrolysis efficiency (maximum rate V_max: two fold higher than the free enzyme), prolonged stability (14 days) and good reusability. L-Glutaminase@bamboo sticks also expanded application capability in pharmaceutical industry in enzyme inhibitor screening. These excellent properties could be attributed to the micropores of bamboo sticks, which led to the fast enzymatic kinetics. The results suggest that the pores of bamboo sticks played an important role in the proposed enzyme reactor during the hydrolysis of L-glutamine and L-glutaminase inhibitor screening.

A chiral ligand exchange capillary electrochromatography (CLE-CEC) protocol was designed and implemented for D,L-amino acids enantioseparation with poly(maleic anhydride-styrene-methacryloyl-L-arginine methyl ester) as the coating. The block copolymer was synthesized through the reversible addition fragmentation chain transfer reaction. In the constructed CLE-CEC system, poly (methacryloyl-L-arginine methyl ester) moiety of the block copolymer played the role as the immobilized chiral ligand and Zn (II) was used as the central ion. Key factors, including pH of buffer solution, ratio of Zn (II) to ligands, the mass ratio of monomers in the block copolymer, which affect the enantioresolution were investigated. Comparing with the bare capillary, the CLE-CEC enantioresolution was enhanced greatly with the coating one. 5 Pairs of D,L-amino acids enantiomers obtained baseline separation with 5 pairs partly separated. The mechanism of enhancement enantioresolution of the developed CLE-CEC system was explored briefly. Further, good linearities were achieved in the range of 25.0 µM-5.0 mM for quantitative analysis of D-glutamine (r² = 0.997) and L-glutamine (r² = 0.991). Moreover, the proposed CLE-CEC assay was successfully applied in the kinetics study of glutaminase by using L-glutamine as the substrate.