Carboxyl Ester Hydrolase Activity Research Articles

PET-degrading microorganisms isolated from residues of the SAV biosphere reserve and identification of carboxylic ester hydrolase activity during their growth in the presence of polymer

The “Sistema Arrecifal Veracruzano” (SAV) is a vital marine ecosystem; its resources are continually perturbed due to contamination by anthropogenic activities, with multiple contaminants such as plastics. In aquatic ecosystems, plastics are almost immediately coated by inorganic and organic matter, which is then colonized by microbes to form a biofilm on plastic surfaces. This work aimed to isolate and identify plastic-degrading microorganisms isolated mainly from plastic residues of the SAV biosphere reserve. Eight bacteria and three fungi were isolated from a biofilm in plastic residues from islands of SAV. All the bacteria and one fungus showed evidence of degrading PET, over 10% for two bacteria and 17% for the fungus. All fungi belong to the genus Aspergillus, and bacteria belong to the genera Aneurinibacillus, Bordetella, Bacillus, and Lysinibacillus. Aneurinibacillus migulanus and Aspergillus flavus showed the highest values for PET degradation. Carboxylic ester hydrolase (CEH) activity was detected in all crude extracts from fungi and bacteria growing with PET triturates as a carbon source; the maximum CEH in bacteria was 255 U mg-1 and 780 U mg-1 for fungi.

Origin, structure and functional transition of sex pheromone components in a false widow spider

Female web-building spiders disseminate pheromone from their webs that attracts mate-seeking males and deposit contact pheromone on their webs that induces courtship by males upon arrival. The source of contact and mate attractant pheromone components, and the potential ability of females to adjust their web’s attractiveness, have remained elusive. Here, we report three new contact pheromone components produced by female false black widow spiders, Steatoda grossa: N-4-methylvaleroyl-O-butyroyl-L-serine, N-4-methylvaleroyl-O-isobutyroyl-L-serine and N-4-methylvaleroyl-O-hexanoyl-L-serine. The compounds originate from the posterior aggregate silk gland, induce courtship by males, and web pH-dependently hydrolyse at the carboxylic-ester bond, giving rise to three corresponding carboxylic acids that attract males. A carboxyl ester hydrolase (CEH) is present on webs and likely mediates the functional transition of contact sex pheromone components to the carboxylic acid mate attractant pheromone components. As CEH activity is pH-dependent, and female spiders can manipulate their silk’s pH, they might also actively adjust their webs’ attractiveness.

Phylogenetic analysis and target gene prediction of miR477 gene family in grape

To understand the molecular characteristics of the miR477 gene family of grape (Vvi-miR477) and to predict its target genes, the Vvi-miR477 genes were identified from previous small RNA sequencing data, then phylogenetic analysis and prediction of target gene were conducted. The Vvi-miR477 family consists of two precursor sequences and three mature sequences. The miR477 family members were mostly 19-22nt in length. The sequence is relatively conservative. Vvi-MIR477a and Vvi-MIR477b are located on chromosomes 1 and 2, respectively. These precursor sequences can form the typical stable stem-loop structure. Their minimum folding free energy is −39.10 kcal/mol and −50.90 kcal/mol, respectively. The MIR477 family can be divided into three groups. The prediction of target genes showed that Vvi-miR477 targets 26S proteasome, DEAD-box, GRAS family protein, Protein Phosphatase 2C, etc. The GO function of target genes was mainly enriched to six categories. The catabolic process, carboxylic ester hydrolase activity is shown to be high. This study provided a theoretical basis for further exploration of the molecular mechanism of miR477 in grape berry ripening.

Proteomic analysis of liver proteins of mice exposed to 1,2-dichloropropane.

1,2-Dichloropropane (1,2-DCP) is recognized as the causative agent for cholangiocarcinoma among offset color proof-printing workers in Japan. The aim of the present study was to characterize the molecular mechanisms of 1,2-DCP-induced hepatotoxic effects by proteomic analysis. We analyzed quantitatively the differential expression of proteins in the mouse liver and investigated the role of P450 in mediating the effects of 1,2-DCP. Male C57BL/6JJcl mice were exposed to 0, 50, 250, or 1250ppm 1,2-DCP and treated with either 1-aminobenzotriazole (1-ABT), a nonselective P450 inhibitor, or saline, for 8h/day for 4weeks. Two-dimensional difference in gel electrophoresis (2D-DIGE) combined with matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF/TOF/MS) was used to detect and identify proteins affected by the treatment. PANTHER overrepresentation test on the identified proteins was conducted. 2D-DIGE detected 61 spots with significantly different intensity between 0 and 250ppm 1,2-DCP groups. Among them, 25 spots were identified by MALDI-TOF/TOF/MS. Linear regression analysis showed significant trend with 1,2-DCP level in 17 proteins in mice co-treated with 1-ABT. 1-ABT mitigated the differential expression of these proteins. The gene ontology enrichment analysis showed overrepresentation of proteins functionally related to nickel cation binding, carboxylic ester hydrolase activity, and catalytic activity. The results demonstrated that exposure to 1,2-DCP altered the expression of proteins related with catalytic and carboxylic ester hydrolase activities, and that such effect was mediated by P450 enzymatic activity.

Specific assay of carboxyl ester hydrolase using PEG esters as substrate

The bile salt-stimulated lipase or carboxyl ester hydrolase (CEH) is a non-specific enzyme secreted by the exocrine pancreas and mammary glands. Recently we demonstrated that PEG esters were good substrates for CEH as it exhibited the highest specific activity ever recorded for this enzyme on PEG-8 monocaprylate. The aim of this study was to develop a specific and sensitive method for assaying CEH in biological samples in which several lipases are contained. Eight different PEG-n mono and dicaprylates with ethylene oxide units ranging from n = 6 to 32 were tested using the pH-stat technique. PEG-20 dicaprylate was the best substrate to discriminate CEH from other digestive lipases. Since pancreatic lipase related-protein 2 (PLRP2) was also found to display a significant activity on PEG-20 dicaprylate, experiments were designed to optimize the specificity of the assay for CEH. The main parameters for increasing CEH activity, while reducing that of PLRP2, were the pH and the concentration of bile salts. A linear relationship between the enzyme activity and the mass of CEH was established. The specificity of this assay was validated using gastrointestinal fluids containing CEH, PLRP2, gastric and pancreatic lipases.

Variations in enzyme activity in stomach and pancreatic tissue and digesta in piglets around weaning

A study was performed to investigate the effect of weaning at 4 weeks of age on the activity of digestive enzymes in the stomach and pancreatic tissue and in digesta from 3 days prior to weaning to 9 days postweaning in 64 piglets. In stomach tissue the activity of pepsin and gastric lipase was determined. Pepsin activity declined abruptly after weaning but 5 days postweaning the weaning level was regained and in the gastric contents no change in pepsin activity was observed. Weaning did not influence the activity of gastric lipase. The activity of eight enzymes and a cofactor was measured in pancreatic tissue. The effect of weaning on the enzyme activity was highly significant for all enzymes except elastase. The activity of all enzymes remained at the weaning level during day 1–2 postweaning followed by a reduction of the activity. The activity of trypsin, carboxypeptidase A, amylase and lipase exhibited minimum activity 5 days postweaning. Trypsin activity increased to the preweaning level on day 7–9 whereas the activity of the others increased but did not reach the preweaning level. The activity of chymotrypsin, carboxypeptidase B and carboxyl ester hydrolase decreased during the entire experimental period. In digesta no effect of weaning was observed on the activity of amylase and trypsin. The activity of chymotrypsin was reduced after weaning in the proximal third of the small intestine and lipase and carboxyl ester hydrolase activity was reduced in the middle and distal parts of the small intestine after weaning. The present study shows that the activities of the digestive enzymes in the pancreatic tissue are affected by weaning. Even though the pancreatic secretion cannot be judged from these results they show that the enzymes respond differently to weaning. In general the activity of the digestive enzymes in pancreatic tissue is low on day 5 postweaning which in interaction with other factors may increase the risk of developing postweaning diarrhoea.

Hydrolysis of tocopheryl and retinyl esters by porcine carboxyl ester hydrolase is affected by their carboxylate moiety and bile acids

The objective of this study was to examine the in vitro hydrolysis of vitamin E esters (α-tocopheryl acetate, α-tocopheryl succinate and α-tocopheryl nicotinate) by pancreatic carboxyl ester hydrolase (CEH) at the concurrent presence of different bile acids at different concentrations. The assay was performed by measuring the amount of α-tocopherol released by porcine pancreatic juice upon addition to different solutions of α-tocopheryl esters, which were dispersed in bile acid mixed micelles at 37°C, pH 7.4. The CEH activity was 10 U in the final assay, and the optimal concentration of cholate in this in vitro-system was determined to 30 mM for the hydrolysis of α-tocopheryl acetate. The hydrolysis of α-tocopheryl esters required presence of pancreatic juice and bile acids, and the results showed furthermore that the ability of pancreatic CEH towards hydrolysis of different α-tocopheryl esters increased with increasing lipophility, irrespective of the type or concentration of bile acid present in the assay. Likewise, retinyl palmitate was hydrolyzed at a faster rate than retinyl acetate. The structure of the bile acid influenced the rate of hydrolysis. Thus, cholate followed by glycodeoxy- and glycochenodeoxycholate were the most effective activators of CEH among the bile acids tested in this assay. The presence of γ-tocopherol or all- trans-retinyl acetate in the assay showed a non-competitive inhibition of the hydrolysis rate of α-tocopheryl acetate.

Development of digestive enzymes in pigs with emphasis on lipolytic activity in the stomach and pancreas.

The effect of age and weaning on the activities of digestive enzymes with emphasis on the lipolytic enzymes before and after weaning was investigated. The activities of amylase, chymotrypsin, trypsin, carboxyl ester hydrolase, pancreatic lipase, and colipase in pancreatic tissue and the activity of gastric lipase in the cardiac mucosa of the stomach in 45 pigs were response variables. The activity of trypsin was not affected by weaning and the rate of increase was similar during the whole experiment. The activities of chymotrypsin and amylase decreased at weaning (P < .05). After weaning the activity of chymotrypsin increased more slowly than before weaning (P < .001), whereas the rate of increase of amylase activity remained unchanged. Lipase, colipase, and carboxyl ester hydrolase activities decreased at weaning (P < .001), whereas gastric lipase activity increased at weaning (P < .01). The development of lipase, colipase, and carboxyl ester hydrolase activity decreased postweaning (P < .01), whereas gastric lipase activity increased before weaning and remained constant after weaning. Pancreatic lipase had a considerably higher capacity for hydrolyzing tributyrin, and the total activity of pancreatic lipase was up to 600 times higher than that of gastric lipase. The lipolytic enzymes displayed a non-parallel pattern of development, and we suggest that this reflects the importance of these enzymes during the suckling and postweaning phases, respectively. However, the significance of gastric lipase for the digestion of fat in pigs remains to be elucidated.

Novel bacterial isolates for the resolution of esters of tertiary alcohols

Resolution of a tertiary allylic terpene alcohol (‘linalool’) employed in flavor and fragrance applications was achieved via biocatalytic hydrolysis of its acetate ester using the carboxyl ester hydrolase activity of lyophilized bacterial cells. Several strains of the genera Rhodococcus, Nocardia, Arthrobacter and Mycobacterium were found to be active. In general, the enantioselectivity was low; only with Rhodococcus ruber SM 1792 (S)-linalool was obtained in 58% e.e. (E-value ~5).

Carboxylic ester hydrolase activity in hairless and athymic nude mouse skin.

The carboxylic ester hydrolase activity was compared in athymic nude mouse skin and hairless mouse skin with respect to hydrolytic ability, heat inactivation, pH optima, and substrate specificity. Five aliphatic 5'-esters of 5-iodo-2'-deoxyuridine (IDU) were incubated with skin homogenate preparations, and the effect of linear chain length and branching of the ester substituent on hydrolysis rate was evaluated. The ester hydrolase activity was three times higher in athymic mouse skin relative to hairless variety. In both mice skin preparations maximum hydrolysis rates were obtained with the valeryl ester followed by butryl, isobutyryl, propionyl and pivaloyl derivatives. Kinetic studies, however, revealed that higher ester hydrolase activity (Vmax) in athymic mouse skin is also associated with higher Km values, while the carboxylic ester hydrolases from these two different strains of mice have similar biochemical properties with respect to heat inactivation and pH optima. Athymic mouse skin resembled hairless mice skin in terms of cholinesterase content. A significant fraction (70-80%) of ester hydrolyzing activities in both strains of mice skin resulted from cholinesterases (true and/or pseudo). The remaining activity was attributed to different ester cleaving enzymes in the two strains of mice. Carbonic anhydrases and arylesterases contributed to the ester hydrolyzing activity of the athymic and normal hairless mice skins, respectively. Product inhibition by the regenerated hydrolytic product, free IDU, was also noticed which resulted in incomplete conversion of rapidly hydrolyzable 5'-esters such as the valeryl and butyryl derivatives.

Boophilus microplus: characterization of enzymes introduced into the host.

A number of enzymes, presumably secreted by larvae of B. microplus under natural feeding conditions, have been investigated in the skin of previously unexposed calves 4 h after infestation at the attachment site. Carboxylic ester hydrolase activity was demonstrated in the dermis, immediately adjacent to the mouthparts, or in the attachment cone, depending on substrate and reaction pH. The carboxylic ester hydrolase acting on naphthol AS-D acetate (2-acetoxy-3-naphthoic-O-toluidide) at pH 7-1 was characteristically found in the dermis and not in the attachment cone. The use of specific inhibitors showed that this enzyme was primarily a B-esterase or carboxylesterase with possibly a small portion of C-esterase or acetylesterase. It is postulated that carboxylic ester hydrolase could contribute to the dilation observed in the subepidermal capillaries adjacent to the attachment sites of unexposed animals, through the formation of plasma kinins. Other enzymes demonstrated in the dermis, adjacent to the mouthparts, were triacylglycerol lipase, as an aggregated deposit, and small amounts of aminopeptidase (microsomal) and monophenol monooxygenase. Aminopeptidase (microsomal) was also demonstrated in the attachment cone or adjacent epidermis, according to the substrate used. No activity was found in the host tissue, in association with the attachment site, for either alkaline or acid phosphatase, acetylcholinesterase or cholinesterase, peroxidase or amine oxidase (flavin-containing), despite the intense histochemical reaction for the latter in the tissues of larvae.

HISTOCHEMICAL DEMONSTRATION OF ACID β‐GLUCURONIDASE AND CARBOXYLIC ESTER HYDROLASE ACTIVITY IN SENSITIZED LYMPHOCYTES DURING LYSIS OF ALLOGENEIC FIBROBLASTS

Sensitized spleen cells were incubated with 51Cr‐labelled target fibroblasts. The interaction of lymphoid cells and target cells was stopped by fixation in formalin vapour. Target cell lysis was evaluated by the release of 51Cr and the cultures were stained for acid β‐glucuronidase and carboxylic ester hydrolase. The sensitized lymphoid cells aggregated around cultured donor‐type, fibroblasts and showed a marked staining for β‐glucuronidase activity within a six‐hour incubation period. Spleen cells of unimmunized mice were negative or weakly positive for β‐glucuronidase. Staining for carboxylic ester hydrolase activity was observed only in spleen cells of allo‐immunized mice. The staining was less intense as compared with the staining for β‐glucuronidase. These observations add further support to the suggestion that lysosomal hydrolases might have a bearing on cell‐mediated target cell injury.