Carboxyl Ester Research Articles

Protonation of carboxyl groups in EuDOTA-tetraamide complexes results in catalytic prototropic exchange and quenching of the CEST signal.

The CEST properties of EuDOTA-tetraamide complexes bearing pendant carboxylate and carboxyl ethyl esters were measured as a function of pH. The CEST signal from the Eu3+-bound water molecule decreased in intensity between pH 8.5 and 4.5 while the proton exchange rates (kex) increased over this same pH range. In comparison, the CEST signal in the corresponding carboxyl ester derivatives was nearly constant. Both observations are consistent with stepwise protonation of the four carboxylic acid groups over this same pH range. This indicates that negative charges on the carboxyl groups above pH 6 facilitate the formation of a strong hydrogen-bonding network in the coordination second sphere above the single Eu3+-bound water molecule, thereby decreasing prototropic exchange of protons on the bound water molecule with bulk water protons. The percentage of square antiprismatic versus twisted square antiprism coordination isomers also decreased as the appended carboxylic acid groups were positioned further away from the amide. The net effect of lowering the pH was an overall increase in kex and a quenching of the CEST signal.This article is part of the themed issue 'Challenges for chemistry in molecular imaging'.

Isolation of a fluoroglycofen-degrading KS-1 strain and cloning of a novel esterase gene fluE.

The bacterium KS-1, capable of degrading fluoroglycofen, was isolated from sludge collected at a herbicide factory. The isolate was identified as Lysinibacillus sp. according to its phenotypic features and 16S rDNA phylogeny. KS-1 degraded 85.25% of the fluoroglycofen (50 mg L-1) within 3 days of incubation. The optimum temperature and pH for fluoroglycofen degradation were 30°C and 7.0, respectively. Furthermore, Zn2+ and Cu2+ could significantly decrease the degradation rate. Three degradation products, which appeared during KS-1-mediated fluoroglycofen metabolism, were identified as deethyl-fluoroglycofen, acifluorfen and decarboxylate-acifluorfen. The fluE gene, which encodes a novel esterase that catalyzes the cleavage of carboxyl ester bonds of fluoroglycofen, was cloned from the KS-1 strain. Sequence alignment reveals that FluE shares 30%-40% amino acid sequence identity with members of the hormone sensitive lipase family. FluE was expressed in Escherichia coli BL21 and purified by Ni-NTA affinity chromatography. Purified FluE could efficiently hydrolyze fluoroglycofen and short-chain p-nitrophenol esters. However, no lipolytic activity was observed with esters containing acyl chains longer than 10 carbon atoms, thereby indicating that this enzyme is an esterase.

Role of carboxyl ester lipase (CEL) in pancreatic disease: Functional characterization of CEL protein variants differing in C-terminal sequence

An improved quantum-behaved particle swarm optimization (IQPSO) algorithm is employed to determine aerosol size distribution (ASD). The direct problem is solved using the anomalous diffraction approximation and Lambert–Beer's Law. Compared with the standard particle swarm optimization algorithm, the stochastic particle size optimization algorithm and the original QPSO, our IQPSO has faster convergence speed and higher accuracy within a smaller number of generations. Optimization parameters for the IQPSO were also evaluated; we recommend using four measurement wavelengths and 50 particles. Size distributions of various aerosol types were estimated using the IQPSO under dependent and independent models. Finally, experimental ASDs at different locations in Harbin were recovered using the IQPSO. All our results confirm that the IQPSO algorithm is an effective and reliable technique for estimating ASD.

Orphan nuclear hormone receptor Small Heterodimer Partner plays a protective role in diet‐induced islet dysfunction

Our early study found that deficiency of small heterodimer partner (SHP, NR0B2) protected the mice from diet‐induced obesity due to increased fatty acid oxidation but exacerbated glucose tolerance due to impaired glucose‐stimulated insulin secretion upon a 6‐month western diet regimen. Current study explored potential roles of SHP in the development of glucose intolerance furthermore. We assessed islet function using illumina beadchip array and morphometric measurement of pancreatic sections. Beadchip array revealed that many exocrine specific genes (for example, CUB and Zona Pellucida‐Like Domains (cuzd1), pancreas specific transcription factor 1a (ptf1a), trypsin 4 (try4) and pancreatic carboxyl ester lipase (cel) etc.) were markedly down‐regulated in the islets of SHP null mice in either chow or western diet‐fed condition. However, expression of major genes involved in insulin secretion and islet function were similar between WT and SHP null mice. Results from quantitative real‐time PCR analysis agreed with majority of beadchip array results except Glut2, which was decreased in SHP null islets. Corroborating with impaired insulin secretion and lower Glut2 mRNA level, glucose‐stimulated Ca2+ influx was greatly diminished in SHP null islets. Islet size was also decreased in SHP null mice fed either diet. The observed diabetic phenotype and pancreatic exocrine insufficiency in whole body SHP−/−mice was recapitulated in the pancreas specific SHPKO (PSHPKO) mice, where SHP deletion is confirmed in both endocrine and exocrine tissues. Interestingly the lean phenotype was reproduced only in liver‐specific SHPKO mice but not in PSHPKO mice. Even though detailed underlying molecular mechanisms for the observed phenotypes by SHP deletion remain to be elucidated, current data suggest that SHP plays distinctive and tissue‐specific role in the development of obesity and diabetes.Support or Funding InformationThis work had been supported by the National Institutes of Health 344 Grant R01DK093774 (to YKL)

Stereochemistry of Endogenous Palmitic Acid Ester of 9-Hydroxystearic Acid and Relevance of Absolute Configuration to Regulation.

Lipids have fundamental roles in the structure, energetics, and signaling of cells and organisms. The recent discovery of fatty acid esters of hydroxy fatty acids (FAHFAs), lipids with potent antidiabetic and anti-inflammatory activities, indicates that our understanding of the composition of lipidome and the function of lipids is incomplete. The ability to synthesize and test FAHFAs was critical in elucidating the roles of these lipids, but these studies were performed with racemic mixtures, and the role of stereochemistry remains unexplored. Here, we synthesized the R- and S- palmitic acid ester of 9-hydroxystearic acid (R-9-PAHSA, S-9-PAHSA). Access to highly enantioenriched PAHSAs enabled the development of a liquid chromatography-mass spectrometry (LC-MS) method to separate and quantify R- and S-9-PAHSA, and this approach identified R-9-PAHSA as the predominant stereoisomer that accumulates in adipose tissues from transgenic mice where FAHFAs were first discovered. Furthermore, biochemical analysis of 9-PAHSA biosynthesis and degradation indicate that the enzymes and pathways for PAHSA production are stereospecific, with cell lines favoring the production of R-9-PAHSA and carboxyl ester lipase (CEL), a PAHSA degradative enzyme, selectively hydrolyzing S-9-PAHSA. These studies highlight the role of stereochemistry in the production and degradation of PAHSAs and define the endogenous stereochemistry of 9-PAHSA in adipose tissue. This information will be useful in the identification and characterization of the pathway responsible for PAHSA biosynthesis, and access to enantiopure PAHSAs will elucidate the role of stereochemistry in PAHSA activity and metabolism in vivo.

Lack of association between the pancreatitis risk allele CEL-HYB and pancreatic cancer.

CEL-HYB is a hybrid allele that arose from a crossover between the 3’ end of the Carboxyl ester lipase (CEL) gene and the nearby CEL pseudogene (CELP) and was recently identified as a risk factor for chronic pancreatitis. Since chronic pancreatitis is a risk factor for the development of pancreatic cancer, we compared the prevalence of the CEL-HYB allele in patients with pancreatic ductal adenocarcinoma to spousal controls and disease controls. The CEL-HYB allele was detected using Sanger and next generation sequencing. There was no significant difference in the prevalence of the CEL-HYB allele between cases with pancreatic ductal adenocarcinoma compared to controls; 2.6% (22/850) vs. 1.8% (18/976) (p=0.35). CEL-HYB carriers were not more likely to report a history of pancreatitis. Patients with pancreatic cancer are not more likely than controls to be carriers of the CEL-HYB allele.

Time-course microarray analysis for identifying candidate genes involved in obesity-associated pathological changes in the mouse colon.

BackgroundObesity is known to increase the risk of colorectal cancer. However, mechanisms underlying the pathogenesis of obesity-induced colorectal cancer are not completely understood. The purposes of this study were to identify differentially expressed genes in the colon of mice with diet-induced obesity and to select candidate genes as early markers of obesity-associated abnormal cell growth in the colon.MethodsC57BL/6N mice were fed normal diet (11% fat energy) or high-fat diet (40% fat energy) and were euthanized at different time points. Genome-wide expression profiles of the colon were determined at 2, 4, 8, and 12 weeks. Cluster analysis was performed using expression data of genes showing log2 fold change of ≥1 or ≤−1 (twofold change), based on time-dependent expression patterns, followed by virtual network analysis.ResultsHigh-fat diet-fed mice showed significant increase in body weight and total visceral fat weight over 12 weeks. Time-course microarray analysis showed that 50, 47, 36, and 411 genes were differentially expressed at 2, 4, 8, and 12 weeks, respectively. Ten cluster profiles representing distinguishable patterns of genes differentially expressed over time were determined. Cluster 4, which consisted of genes showing the most significant alterations in expression in response to high-fat diet over 12 weeks, included Apoa4 (apolipoprotein A-IV), Ppap2b (phosphatidic acid phosphatase type 2B), Cel (carboxyl ester lipase), and Clps (colipase, pancreatic), which interacted strongly with surrounding genes associated with colorectal cancer or obesity.ConclusionsOur data indicate that Apoa4, Ppap2b, Cel, and Clps are candidate early marker genes associated with obesity-related pathological changes in the colon. Genome-wide analyses performed in the present study provide new insights on selecting novel genes that may be associated with the development of diseases of the colon.Electronic supplementary materialThe online version of this article (doi:10.1186/s12263-016-0547-x) contains supplementary material, which is available to authorized users.

Understanding the activation mechanism of Thermomyces lanuginosus lipase using rational design and tryptophan‐induced fluorescence quenching

The attractiveness of lipases as industrial biocatalysts underlines the importance of understanding their molecular action in detail, helping future efforts to improve performance and applicability. Lipases represent a special class of carboxyl ester hydrolases, which act on long‐chained water insoluble acyl‐glycerols at the water‐lipid interface. This heterogeneous catalysis makes it very difficult to monitor the structural changes taking place in the enzyme during activation in its preferred milieu. In this review, we cover recent developments toward a deeper understanding of the interfacial activation phenomenon of lipases and discuss a selection of biophysical methods suitable for studying lipase activation. For many lipases, such as that from Thermomyces lanuginosus, a structural domain called the “lid” is directly involved in the activation process. This has been shown through alteration of the lid‐residue composition using rational design. The resulting lid‐mutants have highlighted the lid's role in governing enzymatic activity and interfacial activation. Moreover, a recently developed fluorescence‐based technique called the Tryptophan Induced Quenching method has proved to enable the direct measurement of lipase activation in water‐ethanol mixtures, and thus represents an intriguing method to gain further insight into the structural movements taking place at the water‐lipid interface.Practical applications: Lipases are important industrial biocatalysts widely used in industrial applications including detergent, leather, paper, fuel, fine chemicals, baking, cosmetics, and food. Knowledge of structural changes of lipase upon activation and identification of the amino acids involved in lipase activation is crucial for design of new variants with improved or new properties. Rational design of the lid‐region in lipases has an interesting potential for optimization of lipase function and adapting them to different environments. In order to leverage on the design of new lipase variants and their altered function, methods that enable direct measurement of lipase action directly in solution, in real time, are desired. In this context, fluorescence based methods have shown great potential and provides interesting perspectives for probing the delicate movements occurring in lipases upon activation.Interfacial activation of lipases and the lid movements studied by rational design, fluorescence quenching methods, and solvent effect – polarity driven activation.

Length of Variable Numbers of Tandem Repeats in the Carboxyl Ester Lipase (CEL) Gene May Confer Susceptibility to Alcoholic Liver Cirrhosis but Not Alcoholic Chronic Pancreatitis.

BackgroundCarboxyl-ester lipase (CEL) contributes to fatty acid ethyl ester metabolism, which is implicated in alcoholic pancreatitis. The CEL gene harbours a variable number of tandem repeats (VNTR) region in exon 11. Variation in this VNTR has been linked to monogenic pancreatic disease, while conflicting results were reported for chronic pancreatitis (CP). Here, we aimed to investigate a potential association of CEL VNTR lengths with alcoholic CP.MethodsOverall, 395 alcoholic CP patients, 218 patients with alcoholic liver cirrhosis (ALC) serving as controls with a comparable amount of alcohol consumed, and 327 healthy controls from Germany and the United Kingdom (UK) were analysed by determination of fragment lengths by capillary electrophoresis. Allele frequencies and genotypes of different VNTR categories were compared between the groups.ResultsTwelve repeats were overrepresented in UK ACP patients (P = 0.04) compared to controls, whereas twelve repeats were enriched in German ALC compared to alcoholic CP patients (P = 0.03). Frequencies of CEL VNTR lengths of 14 and 15 repeats differed between German ALC patients and healthy controls (P = 0.03 and 0.008, respectively). However, in the genotype and pooled analysis of VNTR lengths no statistical significant association was depicted. Additionally, the 16–16 genotype as well as 16 repeats were more frequent in UK ALC than in alcoholic CP patients (P = 0.034 and 0.02, respectively). In all other calculations, including pooled German and UK data, allele frequencies and genotype distributions did not differ significantly between patients and controls or between alcoholic CP and ALC.ConclusionsWe did not obtain evidence that CEL VNTR lengths are associated with alcoholic CP. However, our results suggest that CEL VNTR lengths might associate with ALC, a finding that needs to be clarified in larger cohorts.

A Carboxyl Ester Lipase (CEL) Mutant Causes Chronic Pancreatitis by Forming Intracellular Aggregates That Activate Apoptosis

Patients with chronic pancreatitis (CP) frequently have genetic risk factors for disease. Many of the identified genes have been connected to trypsinogen activation or trypsin inactivation. The description of CP in patients with mutations in the variable number of tandem repeat (VNTR) domain of carboxyl ester lipase (CEL) presents an opportunity to study the pathogenesis of CP independently of trypsin pathways. We tested the hypothesis that a deletion and frameshift mutation (C563fsX673) in the CEL VNTR causes CP through proteotoxic gain-of-function activation of maladaptive cell signaling pathways including cell death pathways. HEK293 or AR42J cells were transfected with constructs expressing CEL with 14 repeats in the VNTR (CEL14R) or C563fsX673 CEL (CEL maturity onset diabetes of youth with a deletion mutation in the VNTR (MODY)). In both cell types, CEL MODY formed intracellular aggregates. Secretion of CEL MODY was decreased compared with that of CEL14R. Expression of CEL MODY increased endoplasmic reticulum stress, activated the unfolded protein response, and caused cell death by apoptosis. Our results demonstrate that disorders of protein homeostasis can lead to CP and suggest that novel therapies to decrease the intracellular accumulation of misfolded protein may be successful in some patients with CP.

Evaluation of the production of exopolysaccharides by two strains of the thermophilic bacterium Rhodothermus marinus

The thermophile Rhodothermus marinus produces extracellular polysaccharides (EPSs) that forms a distinct cellular capsule. Here, the first data on EPS production in strains DSM4252T and MAT493 are reported and compared. Cultures of both strains, supplemented with either glucose, sucrose, lactose or maltose showed that the EPS were produced both in the exponential and stationary growth phase and that production in the exponential phase was boosted by maltose supplementation, while stationary phase production was boosted by lactose. The latter was higher, resulting in 8.8 (DSM4252T) and 13.7mg EPS/g cell dry weight (MAT493) in cultures in marine broth supplemented with 10g/L lactose. The EPSs were heteropolymeric with an average molecular weight of 8×104Da and different monosaccharides, including arabinose and xylose. FT-IR spectroscopy revealed presence of hydroxyl, carboxyl, N-acetyl, amine, and sulfate ester groups, showing that R. marinus produces unusual sulfated EPS with high arabinose and xylose content.

Branched Fatty Acid Esters of Hydroxy Fatty Acids Are Preferred Substrates of the MODY8 Protein Carboxyl Ester Lipase.

A recently discovered class of endogenous mammalian lipids, branched fatty acid esters of hydroxy fatty acids (FAHFAs), possesses anti-diabetic and anti-inflammatory activities. Here, we identified and validated carboxyl ester lipase (CEL), a pancreatic enzyme hydrolyzing cholesteryl esters and other dietary lipids, as a FAHFA hydrolase. Variants of CEL have been linked to maturity-onset diabetes of the young, type 8 (MODY8), and to chronic pancreatitis. We tested the FAHFA hydrolysis activity of the CEL MODY8 variant and found a modest increase in activity as compared with that of the normal enzyme. Together, the data suggest that CEL might break down dietary FAHFAs.

Cholesteryl octanoate breath test. Preliminary studies on a new noninvasive test of human pancreatic exocrine function.

A new breath test for noninvasive assessment of pancreatic exocrine function in humans was developed. The test is based on the hydrolysis of cholesteryl-[1-14C]octanoate by pancreatic carboxyl ester lipase (cholesterol esterase) with subsequent absorption and hepatic metabolism of the liberated octanoate to 14CO2. The rate at which 14CO2 appears in breath appeared to be proportional to the rate of hydrolysis. The substrate is administered as a gum acacia stabilized emulsion of vegetable oil (18 g) containing cholesteryl octanoate (2 g; 4.4 microCi) dispersed in a 500-ml isotonic meal. Tests were performed in 6 healthy volunteers and 11 patients with pancreatic disease with varying degrees of steatorrhea. In healthy subjects, 14CO2 output was rapid with peak output occurring at 60-90 min in all subjects; cumulative output in 4 h averaged 30%. Duplicate studies indicated that the time-course of 14CO2 recovery was reproducible. The pattern of 14CO2 output in patients with pancreatic disease varied widely. Patients without steatorrhea (fecal fat less than or equal to 7 g/day) or with mild steatorrhea (fecal fat 7-11 g/day) had normal or near normal patterns of 14CO2 output, whereas patients with moderate or severe steatorrhea (fecal fat greater than 11 g/day) expired 14CO2 at a rate one-third to one-tenth that of the healthy volunteers. Addition of pancreatic enzyme supplementation to the test meal increased 14CO2 output in 6 of 6 patients with moderate or severe steatorrhea, suggesting that the activity of pancreatic carboxyl ester lipase was rate limiting in these patients. In an additional study in a healthy volunteer, 14CO2 and 13CO2 were measured simultaneously in breath after ingestion of a test meal containing cholesteryl-[1-13C]octanoate and 14C-octanoate. 14CO2 was expired more rapidly than 13CO2, suggesting that hydrolysis of the substrate may also be rate limiting in healthy volunteers. These studies indicate that severe pancreatic exocrine dysfunction can be detected with a simple breath test in 60-90 min.

Oncogenic mutations and dysregulated pathways in obesity-associated hepatocellular carcinoma.

Epidemiological studies showed that obesity and its related non-alcoholic fatty liver disease (NAFLD) promote hepatocellular carcinoma (HCC) development. We aimed to uncover the genetic alterations of NAFLD-HCC using whole-exome sequencing. We compared HCC development in genetically obese mice and dietary obese mice with wild-type lean mice fed a normal chow after treatment with diethylnitrosamine. HCC tumor and adjacent normal samples from obese and lean mice were then subjected to whole-exome sequencing. Functional and mechanistic importance of the identified mutations in Carboxyl ester lipase (Cel) gene and Harvey rat sarcoma virus oncogene 1 (Hras) was further elucidated. We demonstrated significantly higher incidences of HCC in both genetic and dietary obese mice with NAFLD development as compared with lean mice without NAFLD. The mutational signatures of NAFLD-HCC and lean HCC were distinct, with <3% overlapped. Eight metabolic or oncogenic pathways were found to be significantly enriched by mutated genes in NAFLD-HCC, but only two of these pathways were dysregulated by mutations in lean HCC. In particular, Cel was mutated significantly more frequently in NAFLD-HCC than in lean HCC. The multiple-site mutations in Cel are loss-of-function mutations, with effects similar to Cel knock-down. Mutant Cel caused accumulation of cholesteryl ester in liver cells, which led to induction of endoplasmic reticulum stress and consequently activated the IRE1α/c-Jun N-terminal kinase (JNK)/c-Jun/activating protein-1 (AP-1) signaling cascade to promote liver cell growth. In addition, single-site mutations in Hras at codon 61 were found in NAFLD-HCC but none in lean HCC. The gain-of-function mutations in Hras (Q61R and Q61K) significantly promoted liver cell growth through activating the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/3-phosphoinositide-dependent protein kinase-1 (PDK1)/Akt pathways. In conclusion, we have identified mutation signature and pathways in NAFLD-associated HCC. Mutations in Cel and Hras have important roles in NAFLD-associated hepatocellular carcinogenesis.

No Association Between CEL–HYB Hybrid Allele and Chronic Pancreatitis in Asian Populations

A hybrid allele between the carboxyl ester lipase gene (CEL) and its pseudogene, CELP (called CEL-HYB), generated by nonallelic homologous recombination between CEL intron 10 and CELP intron 10', was found to increase susceptibility to chronic pancreatitis in a case-control study of patients of European ancestry. We attempted to replicate this finding in 3 independent cohorts from China, Japan, and India, but failed to detect the CEL-HYB allele in any of these populations. The CEL-HYB allele might therefore be an ethnic-specific risk factor for chronic pancreatitis. An alternative hybrid allele (CEL-HYB2) was identified in all 3 Asian populations (1.7% combined carrier frequency), but was not associated with chronic pancreatitis.

The Benzyl Ester Group of Amino Acid Monomers Enhances Substrate Affinity and Broadens the Substrate Specificity of the Enzyme Catalyst in Chemoenzymatic Copolymerization

The chemoenzymatic polymerization of amino acid monomers by proteases involves a two-step reaction: the formation of a covalent acyl-intermediate complex between the protease and the carboxyl ester group of the monomer and the subsequent deacylation of the complex by aminolysis to form a peptide bond. Although the initiation with the ester group of the monomer is an important step, the influence of the ester group on the polymerization has not been studied in detail. Herein, we studied the effect of the ester groups (methyl, ethyl, benzyl, and tert-butyl esters) of alanine and glycine on the synthesis of peptides using papain as the catalyst. Alanine and glycine were selected as monomers because of their substantially different affinities toward papain. The efficiency of the polymerization of alanine and glycine benzyl esters was much greater than that of the other esters. The benzyl ester group therefore allowed papain to equally polymerize alanine and glycine, even though the affinity of alanine toward papain is substantially higher. The characterization of the copolymers of alanine and glycine in terms of the secondary structure and thermal properties revealed that the thermal stability of the peptides depends on the amino acid composition and resultant secondary structure. The current results indicate that the nature of the ester group drastically affects the polymerization efficiency and broadens the substrate specificity of the protease.

Open and closed states of Candida antarctica lipase B: protonation and the mechanism of interfacial activation

Lipases (EC 3.1.1.3) are ubiquitous hydrolases for the carboxyl ester bond of water-insoluble substrates, such as triacylglycerols, phospholipids, and other insoluble substrates, acting in aqueous as well as in low-water media, thus being of considerable physiological significance with high interest also for their industrial applications. The hydrolysis reaction follows a two-step mechanism, or “interfacial activation,” with adsorption of the enzyme to a heterogeneous interface and subsequent enhancement of the lipolytic activity. Among lipases, Candida antarctica lipase B (CALB) has never shown any significant interfacial activation, and a closed conformation of CALB has never been reported, leading to the conclusion that its behavior was due to the absence of a lid regulating the access to the active site. The lid open and closed conformations and their protonation states are observed in the crystal structure of CALB at 0.91 Å resolution. Having the open and closed states at atomic resolution allows relating protonation to the conformation, indicating the role of Asp145 and Lys290 in the conformation alteration. The findings explain the lack of interfacial activation of CALB and offer new elements to elucidate this mechanism, with the consequent implications for the catalytic properties and classification of lipases.

Progress of diagnosis and treatment for maturity-onset diabetes of the young

Maturity-onset diabetes of the young(MODY), a monogenic form of diabetes, has more than 8 well-known subtypes: MODY1(hepatocyte nuclear factor-4A, HNF4A), MODY2(glucokinase, GCK), MODY3(hepatocyte nuclear factor-1A, HNF1A), MODY4(pancreatic and duodenal homeobox, PDX), MODY5(hepatocyte nuclear factor-1B, HNF1B), MODY6(neuronal differentiation 1, NEUROD1), MODY7(carboxyl ester lipase, CEL) and MODY8(insulin, INS). The molecular pathology, clinical manifestations and treatment of MODYs are different and reviewed. Key words: Maturity-onset diabetes of the young; Adolescence; Gene; Pathology

Application of termite hindgut metagenome derived carboxyl ester hydrolases in the modification of cephalosporin substrates

In the pharmaceutical industry, de-acetylated cephalosporins are highly valuable starting materials for producing semi-synthetic β-lactam antibiotics. In this study a fosmid metagenome library from termite hindgut symbionts was screened for carboxyl ester hydrolases capable of de-acetylating cephalosporins. Recombinant Escherichia coli clones with esterolytic phenotypes on tributyrin agar plates were selected and further tested for de-acetylating activity against Cephalothin and 7-aminocephalosporanic acid (7-ACA). Two clones displaying de-acetylating activity were sequenced and the corresponding two carboxyl ester hydrolase encoding genes (axeA and axeB) belonging to the carbohydrate esterase family 7 (CE7) were identified. The primary structure of both the axeA and axeB revealed the presence of G-X-S-X-G sequence motif and respective subunit molecular masses of 40kDa. In addition to de-acetylating cephalosporin based molecules, the two enzymes were also shown to be true esterases based on their preferences for short chain length fatty acid esters.

Absorption and cleavage of enalapril, a carboxyl ester prodrug, in the rat intestine: in vitro, in situ intestinal perfusion and portal vein cannulation models.

In recent years prodrug strategy has been used extensively to improve the pharmacokinetic properties of compounds exhibiting poor bioavailability. Mechanistic understanding of the absorption and the role of intestine and liver in the activation of oral prodrugs is crucial. Enalapril, a carboxyl ester prodrug, is reported to be metabolized by human carboxylesterase-1 (CES1) but not by carboxylesterase-2 (CES2) to its active metabolite enalaprilat. Further, it has been reported that the small intestines of both rat and human contain mainly CES2. The objective of this work was to understand whether enalapril remains unchanged as it is absorbed through the intestine into the portal circulation. This was evaluated using different intestinal preparations, an in situ intestinal perfusion experiment and a portal vein cannulated rat model. No turnover of enalapril was seen with commercial rat intestinal S9 and microsomes, but reasonable turnover was observed with freshly prepared rat intestinal and mucosal homogenate and S9. In the intestinal perfusion study, both enalapril and enalaprilat were observed in the mesenteric plasma with the data suggesting 32% hydrolysis of enalapril in the intestine. In the portal vein cannulated rat, about 51% of enalapril absorbed into intestine was converted to enalaprilat. Overall, it was demonstrated that even though enalapril has been shown to be a specific substrate for CES1, it is converted to enalaprilat to a significant extent in the intestine. Such experimental techniques can be applied by other scientific groups who are working on prodrugs to determine the region and extent of activation. Copyright © 2015 John Wiley & Sons, Ltd.