Anti-HBZ Monoclonal antibody, clone 4D5-2E6

In contrast to the well-characterized and more common maleylpyruvate isomerization route of the gentisate pathway, the direct hydrolysis route occurs rarely and remains unsolved. In Pseudomonas alcaligenes NCIMB 9867, two gene clusters, xln and hbz, were previously proposed to be involved in gentisate catabolism, and HbzF was characterized as a maleylpyruvate hydrolase converting maleylpyruvate to maleate and pyruvate. However, the complete degradation pathway of gentisate through direct hydrolysis has not been characterized. In this study, we obtained from the NCIMB culture collection a Pseudomonas alcaligenes spontaneous mutant strain that lacked the xln cluster and designated the mutant strain SponMu. The hbz cluster in strain SponMu was resequenced, revealing the correct location of the stop codon for hbzI and identifying a new gene, hbzG. HbzIJ was demonstrated to be a maleate hydratase consisting of large and small subunits, stoichiometrically converting maleate to enantiomerically pure D-malate. HbzG is a glutathione-dependent maleylpyruvate isomerase, indicating the possible presence of two alternative pathways of maleylpyruvate catabolism. However, the hbzF-disrupted mutant could still grow on gentisate, while disruption of hbzG prevented this ability, indicating that the direct hydrolysis route was not a complete pathway in strain SponMu. Subsequently, a D-malate dehydrogenase gene was introduced into the hbzG-disrupted mutant, and the engineered strain was able to grow on gentisate via the direct hydrolysis route. This fills a gap in our understanding of the direct hydrolysis route of the gentisate pathway and provides an explanation for the high yield of D-malate from maleate by this D-malate dehydrogenase-deficient natural mutant.

Asymmetric telechelic alpha-hydroxyl-omega-(carboxylic acid)-poly(epsilon-caprolactone) (HA-PCL), alpha-hydroxyl-omega-(benzylic ester)poly( epsilon-caprolactone) (HBz-PCL), and an asymmetric telechelic copolymer alpha-hydroxyl-omega-(carboxylic acid)-poly(epsilon-caprolactone-co-gamma-butyrolactone) (HA-PCB) were synthesized by ring-opening polymerization of epsilon-caprolactone (CL). CL and CL/gamma-butyrolactone mixture were used to obtain homopolymers and copolymer respectively at 150 degrees C and 2 hr using ammonium decamolybdate (NH4) [ Mo10O34] (Dec) as a catalyst. Water (HA-PCL and HA-PCB) or benzyl alcohol (HBz-PCL) were used as initiators. The three polylactones reached initial molecular weights between 2000 and 3000 Da measured by proton nuclear magnetic resonance (H-1-NMR). Compression-molded polylactone caplets were allowed to degrade in 0.5 M aqueous p-toluenesulfonic acid at 37 degrees C, and monitored upto 60 days for weight loss behavior. Data showed that the copolymer degraded faster than the PCL homopolymers, and that there was no difference in the weight loss behavior between HA-PCL and HBz-PCL. Caplets of the three polylactones containing 1%(w/w) hydrocortisone were placed in two different buffer systems, pH 5.0 with citrate buffer and pH 7.4 with phosphate buffer at 37 degrees C, and monitored upto 50 days for their release behavior. The release profiles of hydrocortisone presented two stages. The introduction of a second monomer in the polymer chain significantly increased the release rate, the degradation rate for HA-PCB being faster than those for HBz-PCL and HA-PCL. At the pH studied, only slight differences on the liberation profiles were observed. SEM micrographs indicate that hydrolytic degradation occurred mainly by a surface erosion mechanism. Copyright (C) 2009 John Wiley & Sons, Ltd.