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Pectinolytic enzymes of large rumen treponemes.

Large spiral organisms isolated from the rumen of cattle produced and released into the external environment a complex of pectinolytic enzymes, consisting mainly of poly(1,4-alpha-D-galacturonide) lyase (EC 4.2.2.2, formerly EC 4.2.99.3), most active at pH 8.0 to 9.0, and another enzyme acting at pH below 7.0, probably a poly(1,4-alpha-D-galacturonide) glycanohydrolase (EC 3.2.1.15). The mixture of enzymes degraded polygalacturonate to saturated and unsaturated monogalacturonates as the end products. A pectin pectylhydrolase (pectinesterase) (EC 3.1.1.11) was also present in the clarified cultures.

Animals↗

Carboxyl ester lipase (bile salt-stimulated lipase), colipase, lipase, and phospholipase A2 levels in pancreatic enzyme supplements.

BACKGROUND: Pancreatic lipolytic activity originates from lipase (LIP) and its cofactor colipase (COL), carboxyl ester lipase (CEL), and phospholipase A2 (PLA2). Yet there are few data on the levels of individual lipolytic enzymes in pancreatic enzyme supplements (PES). This study determines activity and immunoreactive mass in some commonly used PES and thus contributes to the understanding of the poor relationship between 'lipase dose' and clinical improvements. METHODS: Recommended doses of each PES were incubated at 37 degrees C for 2 h in a 1-mM Tris-maleate buffer, pH 7.0, containing 150 mM NaCl and 1 mM CaCl2. Aliquots for determinations of enzyme activities and for immunochemical mass were taken every half hour. For comparison a standard dose was defined as 10,000 declared lipase units. RESULTS: No simple parallelism between LIP, COL, CEL, and/or PLA2 activities was seen. The LIP contents ranged from 135% to 301% of the standard dose. None of the PES were short of COL (227%-504%). The variation in CEL was twentyfold, and in PLA2 sevenfold. Less variations were seen in the mass composition. There was considerable variation in activity to mass ratios (particularly for CEL), declared lipase units per recommended dose (6000-160,000), and cost (0.36-3.52 SEK). CONCLUSIONS: PES differ considerably in their content of lipolytic enzymes. CEL activities were relatively low and COL and PLA2 activities high compared with normal duodenal content. The manufacturing procedure can be improved to increase the lipolytic activity in PES in a broader meaning. It seems to be most important to increase the amount of CEL. From these in vitro data we advocate a more careful decision in the choice of PES for each patient, depending on the total clinical picture. Money can be saved without disadvantage to the patient.

Carboxylesterase↗

Purification by affinity chromatography of glucosidase I, an endoplasmic reticulum hydrolase involved in the processing of asparagine-linked oligosaccharides.

Trimming glucosidase I and II have been solubilized from crude calf liver microsomes and partially enriched by a fractionated extraction procedure applying different concentrations of nonionic detergent and salt. The pH optimum of both enzymes was found to be close to 6.2, which discriminates them from hydrolases of lysosomal origin acting on p-nitrophenyl glycosides with the highest rate at more acidic pH. Glucosidase I and II and the nonspecific alpha-glucosidase(s) were inhibited by 1-deoxynojirimycin with median inhibitory concentration of 3 microM, 20 microM, 12 microM, respectively. Discrimination between these enzymes was strongly enhanced by N-alkylation of 1-deoxynojirimycin and formed the basis for the design of the affinity ligand. Glucosidase I has been purified to homogeneity by affinity chromatography on AH-Sepharose 4B with N-carboxypentyl-1-deoxynojirimycin as ligand. Sodium dodecyl sulfate gel electrophoresis of the purified enzyme revealed a subunit molecular mass of about 85 kDa. The molecular mass of the native enzyme, determined by gel chromatography, was approximately equal to 320-350 kDa, pointing to the association of subunits to a tetramer. Glucosidase I is rather stable when stored at 4 degrees C in the presence of detergent (t 1/2 approximately equal to 20 days) and showed high specificity for the hydrolysis of the terminal (alpha 1,2)-linked glucose residue in the natural substrate Glc3-Man9-(GlcNAc)2.

1-Deoxynojirimycin↗

On-target endoglycosidase digestion matrix-assisted laser desorption/ionization mass spectrometry of glycopeptides.

The digestion of glycopeptides with endoglycosidases can be used in the process of their structural characterization, and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) is often used to analyze the products of these digestions. In the currently accepted protocol for the endoglycosidase digestion of glycopeptides on the MALDI target, the target must be incubated at 37 degrees C, and an hour or more is needed for digestion. We have modified the procedure so that the process can be performed at room temperature in 5 to 15 min, and digestions are performed in the presence of a MALDI matrix. The endoglycosidases used for digestion were endoglycosidase H and peptide-N-glycosidase F. Glycopeptides from asialofetuin and endopolygalacturonase (EPG) II were used as standards because their glycan structures have been previously characterized. Glycopeptides with unknown glycan structures were also digested, including glycopeptides from pectate lyase, EPG I, and pectin methylesterase from Aspergillus niger.

Amidohydrolases↗

Arabinoxylan and mono- and dimeric ferulic acid release from brewer's grain and wheat bran by feruloyl esterases and glycosyl hydrolases from Humicola insolens.

An enzyme preparation from the thermophilic fungus Humicola insolens, Ultraflo L, was able to solubilise more than half of the biomass of brewer's grain and wheat bran, two agro-industrial co-products. While almost all of the ferulic acid was released in the free form, the majority of diferulates were released still attached to soluble feruloylated oligosaccharides, except for the 8,5' benzofuran form, which remained mostly in the residue. H. insolens also produced an esterase capable of releasing over 50% of p-coumaric acid present in wheat bran, but only 9% from the brewer's grain. The polysaccharide content in the residues after enzyme treatment comprised mostly cellulose and arabinoxylan, which suggests that part of the arabinoxylan in these residues is inaccessible to the xylanases of H. insolens. Differences in the solubilised arabinose-to-xylose ratio coupled to high free ferulate release suggest that the structure of feruloylated arabinoxylan in barley and wheat may differ.

Ascomycota↗

Effect of blanching time and salt concentration on pectolytic enzymes, texture and acceptability of fermented green beans.

Effect of blanching time, salt concentration and fermentation on texture of green beans (var. Tuf) was determined. Beans were blanched at 90 degrees C for 1, 10 or 20 minutes and fermented with brine containing 3.08 and 4.6% salt (corresponding to 1.2 and 1.8% salt in the finished product). The beans were assessed for texture mechanically using Universal Testing Machine (Model 1011, Instron). Sensory evaluation was carried out to assess the taste, texture and overall acceptability of the products. Pectolytic enzyme (pectinestarase and exopolygalacturonase) activities were determined in fresh and fermented beans. Increase in blanching time improved the texture significantly. Significant (p<0.05) decreases in shearing energy of the beans were observed in the first two days of fermentation and, thereafter, there were no significant (p>0.05) changes except for samples blanched for one minute. Salt concentration showed a small but significant effect on texture only in samples blanched for one minute and this was probably due to activation by salt of residual pectolytic enzymes in the beans. Most acceptable beans were those blanched for 20 minutes and fermented with 1.2% salt brine.

Carboxylic Ester Hydrolases↗

A simple method of lysosomal hydrolase assay in a single somatic cell and its application.

A new and simple assay method for lysosomal hydrolase in a single cultured skin fibroblast is described. Using 4-methylumbelliferyl-glycosides and ordinary laboratory equipment, the activity can be measured with the highest sensitivity of 0.5 pmol of 4-methylumbelliferone released from the substrates. Two systems were compared and the methods described proved reliable for the diagnosis of several variants of beta-galactosidase deficiency.

Fibroblasts↗