A-glucuronidases

Several extraction techniques have also been described that use enzymatic or chemical reactions to improve extraction efficiency. A technique that has been used to increase the overall recovery of the marker residue is enzymatic hydrolysis to convert specific phase II metabolites (glucuronides or sulfates) back into the parent residue. Cooper etal used a glucuronidase to increase 10-fold the concentration of chloramphenicol residues in incurred tissue. As an example of a chemical reaction, Moghaddam et al. used Raney nickel to reduce thioether bonds between benomyl and polar cellular components, and as a result achieved a substantially improved recovery over conventional solvent extraction. In choosing to use either of these approaches, thorough characterization of the metabolism in the tissue sample must be available. 

Plasma samples were mixed (0.01 pi) with 50 pi of 2 M acetate buffer (pH = 5) containing 10 mg/ml ascorbic acid and 20 pi of 500 U a-glucuronidase. The suspension was held at 37°C for 3h then extracted with 3 X 200 pi of ethyl acetate. The combined organic extracts were evaporated to dryness and redissolved in 100 pi of methanol-water (4 6, v/v). Separation was performed in a microbore ODS column (150 X 1mm i.d. particle... 

Xylans are heteropolysaccharides which are depolymerized by j8-l,4-D-endoxylanases. Due to the abundance and variety of substituents in native xylans, different accessory enzymes are also needed for the total hydrolysis of xylan. The knowledge of a-glucuronidase, a-arabinosidase, and acetyl xylan- and feruloyl esterases has increased considerably in recent years. In addition to acting in synergism with endoxylanases and )8-xylosidase for the complete hydrolysis of xylan, some of these accessory enzymes are also capable of changing the structure of polymeric xylans. 

The a-glucuronidase of T reesei also had an acidic isoelectric point (25). It had a molecular weight of about 70 kDa as estimated by gel chromatography and a pH-optimum at pH 6 with 4-O-methylgucurono-xylobiose as substrate. The very recently characterized a-glucuronidase of the thermophilic fungus Thermoascus aurantiacus (51) was a single polypeptide chain with a MW of... 

ENZYMES IN BIOMASS CONVERSION Table II. Microbial producers of a-glucuronidases... 

This requires hydrolysis, and fermentation or transformation steps. While enzymes degrading the heteroxy-lan are known as xylanases, they also require the additional actions of / -xylosidases, a-arabinosidases, a-glucuronidases and certain esterases for total hydrolysis. 

The xylanolytic enzyme system of Trichoderma reesei, a well-known producer of cellulolytic enzymes, is versatile and well suited for the total hydrolysis of different xylans. It consists of two major, specific and several non-specific xylanases, at least one / -xylosidase, a-arabinosidase and a-glucuronidase and at least two acetyl esterases. The hydrolysis of polymeric xylans starts by the action of endoxylanases. The side-groupcleaving enzymes have their highest activities towards soluble, short xylo-oligosaccharides, and make the substituted oligosaccharides again accessible for xylanases and / -xylosidase. 

Trichoderma (9-10). Much less is known about the concurrent production of the enzymes which cleave substituent groups of the xylan polymer. The presence of acetyl xylan esterases (11,12) and a-glucuronidases (13-15) in xylanolytic enzyme systems has only recently been pointed out. Although a-arabinosidases have mainly been studied as arabinan-degrading enzymes (16), they have also been shown to release arabinose from xylans (17). 

Figure 1. Fractionation of proteins in the culture filtrate of Trichoderma reesei according to their pi values Xyl, xylanase Ara, arabinosidase AE, acetyl esterase / X, /3-xylosidase aG, a-glucuronidase / G, / -glucosidase CBH, cellobiohydrolase EG, endoglucanase. Chromatofocusing was performed in a PBE-94 anion exchange resin (Pharmacia) with a pH-gradient created by ampholyte buffers (Pharmacia). Solid line, A dotted line, pH. (Reproduced with permission from ref. 24. Copyright 1988.)...

MMF is rapidly and completely cleaved to form MPA, the active metabolite, after oral administration. MPA is converted in the liver and kidney to an inactive glucuronide. However, certain tissues, such as the epidermis, express a glucuronidase that converts the inactive glucuronide back to the active agent. The half-life of MPA is approximately 18 hours 90 to 95% of the mycophenolate dose is excreted in the urine. 

The complete degradation of hemicellulose becomes more complex than that of cellulase, since substituent-hydrolyzing activities are also necessary. With heteroxylans, apart from endo-l,4- 3-xylanase, which catalyzes the hydrolysis of internal 3-l,4-xylan links and P-xylosidase, which catalyzes the hydrolysis of xylooligossacharides, mainly xylobiose into xylose, other enzymes must act to accomplish complete hydrolysis, such as acetyl xylan esterase, a-glucuronidase, and a-L-arabinofuranosidase (1). 

Unlike starch or cellulose, BSG hemicellulose has a complex structure, which is still present, in part, on its autohydrolysis products. Oligosaccharides from BSG hydrolysis consist mainly of branched arabino-xylo-glucurono oligosaccharides that are not highly acetylated when compared to other xylans, such as from Eucalyptus wood (31). The action of several enzymatic activities including endo-l,4-P-xylanase P-xylosidase and accessory activities such as acetyl xylanesterase, a-glucuronidase, and a-arabino-furanosidase is therefore required for the complete hydrolysis of OCL to monosaccharides. 

Besides these factors, the synthetic nitrophenyl derivatives used for estimating P-xylosidase, acetyl xylanesterase, and a-arabinofuranosidase activities may not provide suitable information to be applied to a real, complex substrate and/or additional enzymes (such as a-glucuronidases)... 

Tenkanen, M. and Siika-aho, M., An a-glucuronidase of Schizophyllum commune acting on polymeric xylan. J Biotechnol 2000, 78 (2), 149-161. 

Nagy, T., Nurizzo, D., Davies, G. J., Biely, P., Lakey, J. H., Bolam. D. N., and Gilbert, H. J., The a-glucuronidase, GlcA67A, of Cellvibrio japonicus utilises the carboxylate and methyl groups of aldobiouronic acid as important substrate recognition determinants. J Biol Chem 2003. 

Breast Milk Hyperbilirubinemia. This type of hyperbilirubinemia affects about 30% of breast-fed newborns. It is due to a-glucuronidase in breast milk, which hydrolyzes conjugated bilirubin in the intestine. The unconjugated bilirubin, being more lipophilic, is passively absorbed. The condition lasts for a few weeks and is treated by discontinuing breast feeding. 

The a-glucuronidases from the two bacterial species mentioned above have been suggested to have aspartyl residues as part of hydrogen-bonding networks that participate in proton donation to the departing aglycone moiety [62, 63]. 

---