OBJECTIVE: To explore the clinical phenotype and genetic features of two children with MEGDEL syndrome due to variants of the SERAC1 gene. METHODS: Two children who had presented at the Fujian Medical University Union Hospital respectively on July 14, 2020 and July 28, 2018 were selected as the study subjects. Clinical features and results of genetic testing were retrospectively analyzed. RESULTS: Both children had featured developmental delay, dystonia and sensorineural deafness, along with increased urine 3-methylglutaric acid levels. Magnetic resonance imaging revealed changes similar to Leigh-like syndrome. Gene sequencing revealed that both children have harbored pathogenic compound heterozygous variants of the SERAC1 gene, including c.1159C>T and c.442C>T in child 1, and c.1168C>T and exons 4~9 deletion in child 2. CONCLUSION Children with MEGDEL syndrome due to SERAC1 gene variants have variable clinical genotypes. Delineation of its clinical characteristics and typical imaging changes can facilitate early diagnosis and treatment. Discovery of the novel variants has also enriched the spectrum of SERAC1 gene variants.
Humans ; Retrospective Studies ; Metabolism, Inborn Errors ; Hearing Loss, Sensorineural/genetics* ; Dystonia ; Carboxylic Ester Hydrolases

With the decrease of forest timber resources, the recycling of waste paper has received increasing attention. However, the stickies produced in the process of waste paper recycling may negatively affect the production of recycled paper. The biological decomposition of stickies, which has the advantages of high efficiency, high specificity and pollution-free, is achieved mainly through the enzymatic cleavage of the ester bond in the stickies components to prevent flocculation. Cutinase is a serine esterase that can degrade some components of the stickies. Previous research indicated that the anchor peptide tachystatin A2 (TA2) is able to bind polyurethane. In this study, the cutinase HiC derived from Humicola insolens was used to construct a fusion protein HiC-TA2 by megaprimer PCR of the whole plasmid (MEGAWHOP). The enzymatic properties and the degradation efficiency of the fusion protein on poly(ethyl acrylate) (PEA), a model substrate of stickies component, were determined. The results showed that the degradation efficiency, the size decrease of PEA particle, and the amount of ethanol produced by HiC-TA2 were 1.5 times, 6.8 times, and 1.4 times of that by HiC, respectively. These results demonstrated that TA2 improved the degradation efficiency of HiC on PEA. This study provides a useful reference for biological decomposition of stickies produced in the process of recycled paper production.
Carboxylic Ester Hydrolases/genetics* ; Fungal Genus Humicola ; Polyurethanes

Cutinase can degrade aliphatic and aromatic polyesters, as well as polyethylene terephthalate. Lack of commercially available cutinase calls for development of cost-effective production of efficient cutinase. In this study, eight cutinase genes were cloned from Sclerotinia sclerotiorum. The most active gene SsCut-52 was obtained by PCR combined with RT-PCR, expressed in Escherichia coli BL21 and purified by Ni-NTA affinity chromatography to study its characteristics and pathogenicity. Sscut-52 had a total length of 768 bp and 17 signal peptides at the N terminals. Phylogenetic analysis showed that its amino acid sequence had the highest homology with Botrytis keratinase cutinase and was closely related to Rutstroemia cutinase. Sscut-52 was highly expressed during the process of infecting plants by Sclerotinia sclerotiorum. Moreover, the expression level of Sscut-52 was higher than those of other cutinase genes in the process of sclerotia formation from mycelium. The heterologously expressed cutinase existed in the form of inclusion body. The renatured SsCut-52 was active at pH 4.0-10.0, and mostly active at pH 6.0, with a specific activity of 3.45 U/mg achieved. The optimum temperature of SsCut-52 was 20-30 ℃, and less than 60% of the activity could be retained at temperatures higher than 50 ℃. Plant leaf infection showed that SsCut-52 may promote the infection of Banlangen leaves by Sclerotinia sclerotiorum.
Ascomycota/genetics* ; Carboxylic Ester Hydrolases ; Cloning, Molecular ; Phylogeny

A new method to express oligomerized feruloyl esterase (FAE) in Pichia pastoris GS115 to improve the catalytic efficiency was developed. It was realized by fusing the foldon domain at the C-terminus of FAE, and the fusion protein was purified by histidine tag. Fusion of the feruloyl esterase with the foldon domain resulted spontaneously forming a trimer FAE to improve the catalytic performance. The oligomerized FAE and monomeric FAE were obtained by purification. The apparent molecular weight of the oligomerized FAE was about 110 kDa, while the monomeric FAE about 40 kDa, and the optimum temperature of the oligomerized FAE was 50 °C, which is the same as the monomeric one. The optimal pH of the oligomerized FAE is 5.0, while the optimal pH of the monomer FAE is 6.0. When compared with the monomeric ones, the catalytic efficiency (kcat/Km) of the oligomerized FAE increased 7.57-folds. The catalytic constant (kcat) of the oligomerized FAE increased 3.42-folds. The oligomerized FAE induced by foldon have advantages in the catalytic performances, which represents a simple and effective enzyme-engineering tool. The method proposed here for improving the catalytic efficiency of FAE would have great potentials for improving the catalytic efficiency of other enzymes.
Carboxylic Ester Hydrolases ; metabolism ; Catalysis ; Molecular Weight ; Pichia ; genetics ; metabolism ; Polymerization ; Protein Engineering ; Substrate Specificity

Mammalian carboxylesterases hydrolyze a wide range of xenobiotic and endogenous compounds, including lipid esters. Physiological functions of carboxylesterases in lipid metabolism and energy homeostasis in vivo have been demonstrated by genetic manipulations and chemical inhibition in mice, and in vitro through (over)expression, knockdown of expression, and chemical inhibition in a variety of cells. Recent research advances have revealed the relevance of carboxylesterases to metabolic diseases such as obesity and fatty liver disease, suggesting these enzymes might be potential targets for treatment of metabolic disorders. In order to translate pre-clinical studies in cellular and mouse models to humans, differences and similarities of carboxylesterases between mice and human need to be elucidated. This review presents and discusses the research progress in structure and function of mouse and human carboxylesterases, and the role of these enzymes in lipid metabolism and metabolic disorders.
Amino Acid Sequence ; Animals ; Carboxylic Ester Hydrolases ; chemistry ; genetics ; metabolism ; Humans ; Intracellular Space ; metabolism ; Lipid Metabolism ; Mice ; Polymorphism, Single Nucleotide ; Protein Domains

To express feruloyl esterase A from Aspergillus oryzae in Pichia pastoris expression system and study its hydrolysis function, explore the conditions and effects of purification for ferulic acid extracts by macroporos resin. Using the total RNA from A. oryzae CICC 40186 as the template, we amplified coding sequence AorfaeA encoding a mature feruloyl esterase A (AorFaeA) by RT-PCR technique. Then, the coding sequence AorfaeA was successfully expressed in Pichia pastoris GS115 mediated by an expression plasmid pPIC9K. The purified recombinant AorFaeA (reAorFaeA) showed one single band on SDS-PAGE with an apparent molecular weight of 39.0 kDa. The maximum activity of reAorFaeA to methyl ferulate, measured by high-performance liquid chromatography (HPLC), was 58.35 U/mg. Then, reAorFaeA was used to release ferulic acid from de-starched wheat bran in the presence of xylanase. The purification tests for ferulic acid from the enzymatic hydrolysate were carried out with preselected macroporous resins. The results showed that macroporous resin HPD-300 had much higher adsorption and desorption capacities. Ferulic acid could be quantitatively recovered by 50% of the eluent concentration at a flow speed of 1 mL/min. Under the purification condition, the recovery ratio of ferulic acid was 92%, and the content of ferulic acid was increased from 0.13% in the raw material to 10.55%. This work exploits the breakdown of ferulic acid by recombinant enzymeand provids a good strategy to its "green production".
Aspergillus oryzae ; enzymology ; Carboxylic Ester Hydrolases ; biosynthesis ; genetics ; Cloning, Molecular ; Coumaric Acids ; chemistry ; Electrophoresis, Polyacrylamide Gel ; Hydrolysis ; Molecular Weight ; Pichia ; genetics ; metabolism

Engineering the existing or manual assembling biosynthetic pathways involves two important issues: the activity and expression level of key enzymes in the pathway. Concerning the enzyme expression study, the conventional approach is to use strong promoter to initiate the overexpression of the target protein. The excessive expression of the target protein usually result in the intracellular accumulation of a large number of inactive inclusion bodies, thereby seriously affect the physiological state of the cell and the effective functioning of the relevant biological pathways. To solve this problem, we would like to design a molecular switch to precisely manipulate the expression level of key enzymes in the biosynthetic process, which has important practical value for the study of metabolic rhythm of the biosynthetic pathway and to promote the efficiency of the biosynthetic pathway. Based on the basic principles of quorum sensing existing in the bacterial community and combining the dynamic characteristics of the enzymatic catalysis, we first established cell-cell communication mechanisms mediated by signal molecule homoserine lactone (AHL) in the E. coli community and target protein EGFP was expressed under the control of the promoter P(lux1). In the process of cell growth, AHL accumulated to a certain concentration to start the expression of target gene egfp. At the different cell growth stages, AHL-degrading enzyme AiiA was induced and resulted in the degradation of AHL molecule in a controlled environment, thereby controlling the transcription efficiency of target gene egfp and ultimately achieve the precise control of the level of expression of the target protein EGFP. The detection of cell growth state, the mRNA level and protein expression level of the target gene showed the artificially designed molecular switch can control the level of expression of a target gene in a convenient and efficient manner with a spatial and temporal regulation of rigor. The molecular switch is expected to be widely used in the field of metabolic engineering and synthetic biology research areas.
Carboxylic Ester Hydrolases ; genetics ; Escherichia coli ; enzymology ; genetics ; physiology ; Gene Expression Regulation, Bacterial ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Metalloendopeptidases ; genetics ; Quorum Sensing ; genetics ; physiology

To explore the enzymatic route of cefatrizine synthesis, alpha-amino acid ester hydrolase (AEH) gene was cloned from the whole genome of Xanthomonas rubrillineans, and expressed heterologously in Escherichia coli BL21 (DE3). The effects of temperature, pH and substrates' molar ratio upon the transformation yield of cefatrizine by purified recombinant AEH were investigated. The monomer of AEH was determined as 70 kDa by SDS-PAGE. The optimal pH and temperature reaction were (6.0 +/- 0.1) and 36 degrees C for cefatrizine synthesis. The transformation yield was 64.3% under 36 degrees C, pH (6.0 +/- 0.1), when the concentrations of two substrates were about 30 mmol/L (7-ATTC) and 120 mmol/L (HPGM x HCl), respectively, and the enzyme consumption was 22 U/mL. The results pave the way for optimization of the industrial enzymatic synthesis of cefatrizine.
Carboxylic Ester Hydrolases ; biosynthesis ; genetics ; Catalysis ; Cefatrizine ; metabolism ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Kinetics ; Recombinant Proteins ; biosynthesis ; genetics ; Xanthomonas ; enzymology

Carboxylesterases (CESs) play important roles in the metabolism of endogenous and foreign compounds in physiological and pharmacological responses. The aim of this study was to investigate the effect of dexamethasone at different doses on the expression of CES1 and CES2. Imidapril and irinotecan hydrochloride (CPT-11) were used as special substrates for CES1 and CES2, respectively. Rat hepatocytes were cultured and treated with different concentrations of dexamethasone. The hydrolytic activity of CES1 and CES2 was tested by incubation experiment and their expression was quantitated by real-time PCR. A pharmacokinetic study was conducted in SD rats to further evaluate the effect of dexamethasone on CESs activity in vivo. Western blotting was performed to investigate the regulatory mechanism related to pregnane X receptor (PXR) and glucocorticoid receptor (GR). The results showed that exposure of cultured rat hepatocytes to nanomolar dexamethasone inhibited the imidapril hydrolase activity, which was slightly elevated by micromolar dexamethasone. For CES2, CPT-11 hydrolase activity was induced only when dexamethasone reached micromolar levels. The real-time PCR demonstrated that CES1 mRNA was markedly decreased by nanomolar dexamethasone and increased by micromolar dexamethasone, whereas CES2 mRNA was significantly increased by micromolar dexamethasone. The results of a complementary animal study showed that the concurrent administration of dexamethasone significantly increased the plasma concentration of the metabolite of imidapril while the ratio of CPT-11 to its metabolite SN-38 was significantly decreased. PXR protein was gradually increased by serial concentrations of dexamethasone. However, only nanomolar dexamethasone elevated the level of GR protein. The different concentrations of dexamethasone required suggested that suppression of CES1 may be mediated by GR whereas the induction of CES2 may result from the role of PXR. It was concluded that dexamethasone at different concentrations can differentially regulate CES1 and CES2.
Animals ; Carboxylic Ester Hydrolases ; genetics ; Dexamethasone ; pharmacology ; Gene Expression ; drug effects ; immunology ; Male ; Rats ; Rats, Sprague-Dawley ; Receptors, Cytoplasmic and Nuclear ; metabolism

Human carboxylesterase 1 (HCE1), belonging to a multigene serine hydrolase family, is a major liver carboxylesterase responsible for the hydrolysis and metabolism of various xenobiotics. It also plays an important role in the transportation and metabolism of endogenous cholesterol ester and free fatty acid, and is closely associated with the pathogenesis of hepatocellular carcinoma. This review describes current developments in the molecular structure, the roles in drug, toxins and lipid metabolism, and the early diagnosis for hepatocellular carcinoma of human carboxylesterase 1.
Carboxylic Ester Hydrolases ; genetics ; physiology ; Carcinoma, Hepatocellular ; diagnosis ; Cholesterol Esters ; metabolism ; Fatty Acids, Nonesterified ; metabolism ; Humans ; Liver Neoplasms ; diagnosis ; Xenobiotics ; metabolism