Objective: To identify and analyze 3D architecture of the mutational sites of susceptible genes in a pedigree with familial hypercholesterolemia-like phenotype (FHLP). Methods: This is a case series study. A pedigree with suspected familial hypercholesterolemia was surveyed. The proband admitted in Beijing Anzhen Hospital in April 2019. Whole-exome sequencing was performed to determine the mutational sites of susceptible genes in the proband. Polymerase chain reaction (PCR) sequencing was used to verify the pathogenic variant on proband's relatives. The structural and functional changes of the proteins were analyzed and predicted by Discovery Studio 4.0 and PyMol 2.0. Results: The patients in the pedigree showed abnormal lipid profiles, especially elevated levels of total cholesterol(TC). The genetic screening detected the c.1330C>T SNP in the exon 8 of lipase C (LIPC) gene, this mutation leads to an amino acid substitution from arginine to cysteine at position 444 (Arg444Cys), in the proband and proband's father and brother. In this family, members with this mutation exhibited elevated TC, whereas lipid profile was normal from the proband's mother without this mutation. This finding indicated that LIPC: c.1330C>T mutation might be the mutational sites of susceptible genes. The analysis showed that Arg444Cys predominantly affected the ligand-binding property of the protein, but had a limited impact on catalytic function. Conclusion: LIPC: c.1330C>T is a new mutational site of susceptible genes in this FHLP pedigree.
Humans ; Male ; Hyperlipoproteinemia Type II/genetics* ; Lipase/genetics* ; Lipids ; Mutation ; Pedigree ; Phenotype ; Proteins

Objective: To explore the relationship between extracellular enzymes activity and virulence of Candida glabrata clinical isolates based on the infection model of Galleria mellonella larvae. Methods: Using experimental research methods, 71 strains of non-repetitive Candida glabrata were collected from Qinghai Provincial People's Hospital from June 2021 to January 2022. Bovine serum protein agar medium, egg yolk agar medium, sheep blood agar medium, Tween-80 agar medium and triglyceride agar medium were used to detect the aspartyl protease activity, phospholipase activity, hemolysis activity, esterase activity and lipase activity of Candida glabrata. Median lethal concentration (LC₅₀) was calculated by using 1.25×10⁸ CFU/ml,2.50×10⁸ CFU/ml,3.75×10⁸ CFU/ml,5.00×10⁸ CFU/ml suspension of Candida glabrata ATCC2001 to infect Galleria mellonella larvae. Histopathological and etiological analysis was performed to determine whether the infection model was successfully established. The clinical isolates of Candida glabrata were configured to infect Galleria mellonella larvae with LC₅₀ concentration to detect the pathogenicity of Galleria mellonella larvae.Spearman test or Pearson test were used to analyze the correlation between the extracellular enzyme activity of Candida glabrata clinical isolates and the pathogenicity of Galleria mellonella larvae. Results: 71 strains of Candida glabrata isolated clinically were detected to have low hemolytic activity after 2 days of culture. Aspartyl protease was detected after 4 days of culture, among which 7 strains (9.86%), 19 strains (26.76%) and 45 strains (63.38%) showed low, medium and high aspartyl protease activity. After 7 days of culture, 71 strains did not detect phospholipase, esterase and lipase activities. Candida glabrata on Galleria mellonella larvae of LC₅₀=2.5×10⁸ CFU/ml Fungal spore were found in the intestinal tissue pathological section of Galleria mellonella larvae in the experimental group, and Candida glabrata was identified by the microbial Mass Spectrometry after culture, while no fungi were found in the pathological section and culture of the control group. Spearman test shows that, there was a linear positive correlation between aspartyl protease activity and the survival rate of Galleria mellonella larvae (r = 0.73, P<0.01), the difference was statistically significant.Pearson test shows that, there was no significant linear relationship between hemolytic activity and survival rate of Galleria mellonella larvae (r = 0.16, P = 0.34), the difference was not statistically significant. Conclusion: The clinical isolates of Candida glabrata in this study had aspartyl protease activity and low hemolytic activity, but no phospholipase, esterase and lipase activity. The activity of aspartyl aspartyl protease of Candida glabrata was positively correlated with the pathogenicity of Galleria mellonella larvae.
Animals ; Sheep ; Larva/microbiology* ; Virulence ; Candida glabrata ; Agar ; Moths/microbiology* ; Esterases ; Aspartic Acid Proteases ; Lipase

This report presents a case of a male infant, aged 32 days, who was admitted to the hospital due to 2 days of bloody stools and 1 day of fever. Upon admission, venous blood samples were collected, which appeared pink. Blood biochemistry tests revealed elevated levels of triglycerides and total cholesterol. The familial whole genome sequencing revealed a compound heterozygous variation in the LPL gene, with one variation inherited from the father and the other from the mother. The patient was diagnosed with lipoprotein lipase deficiency-related hyperlipoproteinemia. Acute symptoms including bloody stools, fever, and bloody ascites led to the consideration of acute pancreatitis, and the treatment involved fasting, plasma exchange, and whole blood exchange. Following the definitive diagnosis based on the genetic results, the patient was given a low-fat diet and received treatment with fat-soluble vitamins and trace elements, as well as adjustments to the feeding plan. After a 4-week hospitalization, the patient's condition improved and he was discharged. Follow-up showed a decrease in triglycerides and total cholesterol levels. At the age of 1 year, the patient's growth and psychomotor development were normal. This article emphasizes the multidisciplinary diagnosis and treatment of familial hyperlipoproteinemia presenting with symptoms suggestive of acute pancreatitis, including bloody ascites, in the neonatal period.
Humans ; Infant ; Male ; Acute Disease ; Ascites ; Cholesterol ; Hyperlipoproteinemia Type I/genetics* ; Hyperlipoproteinemias ; Lipoprotein Lipase/genetics* ; Pancreatitis ; Triglycerides

Diacylglycerol (DAG) is an intermediate product in lipid metabolism and plays an important physiological role in human body. It is mainly prepared by hydrolyzing lipid with lipase. However, research on the detection method of 1, 2-diacylglycerol (1, 2-DAG) and 1, 3-diacylglycerol (1, 3-DAG) and catalytic specificity of lipase was not enough, which limits its wide application. To address these challenges, an efficient quantitative detection method was first established for 1, 2-DAG (0.025-0.200 g/L) and 1, 3-DAG (0.025-0.150 g/L) by combining supercritical fluid chromatography with evaporative light scattering detector and optimizing the detection and analysis parameters. Based on the molecular docking between Thermomyces lanuginosus lipase (TLL) and triolein, five potential substrate binding sites were selected for site-specific saturation mutation to construct a mutation library for enzyme activity and position specificity screening. The specificity of sn-1, 3 of the I202V mutant was the highest in the library, which was 11.7% higher than the specificity of the wild type TLL. In summary, the position specificity of TLL was modified based on a semi-rational design, and an efficient separation and detection method of DAG isomers was also established, which provided a reference for the study of the catalytic specificity of lipase.
Humans ; Diglycerides ; Molecular Docking Simulation ; Binding Sites ; Catalysis ; Lipase/genetics*

Aims: This study was aimed to express Meyerozyma guilliermondii strain RT lipase using Komagataella phaffii X-33 expression system and its biochemical characterization and analyse the predicted structure of the product. Methodology and results: Meyerozyma guilliermondii strain RT obtained from the previous study was used as the source of RT lipase gene. Extracellular M. guilliermondii strain RT lipase expression has significantly been improved up to 56 U/mg at 24 h cultivation in Yeast extract-Peptone-Dextrose (YPD) medium containing (in w/v): 1% yeast extract, 2% peptone, 2% dextrose with 0.5% v/v methanol induction. Characterization of RT lipase showed optimum activity at 45 °C and pH 9. It exhibited stability in the alkaline pH range (8 to 10) and retained 50% of its residual activity at 30 °C for 30 min. Substrate specificity analysis revealed that it preferred short to medium-chain triacylglycerols (C2-C12) with the highest activity towards caprylic acid (C8). Pairwise alignment revealed three substitutions (S2L, S92L and S193L) present in non-CTG-clade hosts (K. phaffii). Homology modelling (YASARA) was used to predict the structures of RT lipase [wild type (wt) and recombinant (rc)]. Mutational analysis of the structures showed the differences in loops that might attribute to the reduction of the optimum temperature from 75 °C (wt) to 45 °C (rc). Conclusion, significance and impact of study RT lipase was successfully overexpressed extracellularly using K. phaffii expression system with 91.8-fold higher specific activity than the native host. The conceptual advances on the importance of codon optimization before expressing a protein from a CTG-clade species in a non-CTG-clade yeast have been highlighted and the effect of the rare codon usage in recombinant protein characteristics has been evident.
Candida ; Lipase--analysis

Autosomal recessive congenital ichthyosis (ARCI) is characterized by being born as collodion babies, hyperkeratosis, and skin scaling. We described a collodion baby at birth with mild ectropion, eclabium, and syndactyly. Whole exome sequencing showed a compound heterozygous variant c.[56C>A], p.(Ser19X) and c.[100G>A], p.(Ala34Thr) in the PNPLA1 gene [NM_001145717; exon 1]. The protein encoded by PNPLA1 acts as a unique transacylase that specifically transfers linoleic acid from triglyceride to ω-hydroxy fatty acid in ceramide, thus giving rise to ω-O-acylceramide, a particular class of sphingolipids that is essential for skin barrier function. The variant was located in the patatin core domain of PNPLA1 and resulted in a truncated protein which could disrupt the function of the protein. This case report highlights a novel compound heterozygous mutation in PNPLA1 identified in a Chinese child.
Humans ; Infant, Newborn ; Acyltransferases/genetics* ; Ceramides/metabolism* ; Collodion ; Ichthyosis, Lamellar/genetics* ; Lipase/metabolism* ; Mutation ; Phospholipases/genetics*

Humans ; Muscular Diseases/genetics* ; Cardiomyopathies/genetics* ; Lipid Metabolism, Inborn Errors/genetics* ; Mutation ; Lipids ; Muscle, Skeletal ; Acyltransferases/genetics* ; Lipase/genetics*

Proteus mirabilis lipase (PML) features tolerance to organic solvents and great potential for biodiesel synthesis. However, the thermal stability of the enzyme needs to be improved before it can be used industrially. Various computational design strategies are emerging methods for the modification of enzyme thermal stability. In this paper, the complementary algorithm-based ABACUS, PROSS, and FoldX were employed for positive selection of PML mutations, and their pairwise intersections were further subjected to negative selection by PSSM and G_REMLIN to narrow the mutation library. Thereby, 18 potential single-point mutants were screened out. According to experimental verification, 7 mutants had melting temperature (T_m) improved, and the ΔT_m of K208G and G206D was the highest, which was 3.75 ℃ and 3.21 ℃, respectively. Five mutants with activity higher than the wild type (WT) were selected for combination by greedy accumulation. Finally, the T_m of the five-point combination mutant M10 increased by 10.63 ℃, and the relative activity was 140% that of the WT. K208G and G206D exhibited certain epistasis during the combination, which made a major contribution to the improvement of the thermal stability of M10. Molecular dynamics simulation indicated that new forces were generated at and around the mutation sites, and the rearrangement of forces near G206D/K208G might stabilize the Ca²⁺ binding site which played a key role in the stabilization of PML. This study provides an efficient and user-friendly computational design scheme for the thermal stability modification of natural enzymes and lays a foundation for the modification of PML and the expansion of its industrial applications.
Enzyme Stability ; Lipase/chemistry* ; Molecular Dynamics Simulation ; Proteus mirabilis/metabolism* ; Solvents/chemistry*

OBJECTIVE: To assess the effect of early abdominal puncture drainage (APD) on autophagy and Nrf-2/HO-1 pathway in rats with severe acute pancreatitis (SAP) and explore the possibile mechanism. METHODS: Thirty-two male SD rats were randomly divided into sham-operated (SO) group, SAP group with retrograde injection of 4% sodium taurocholate, APD group with insertion of a drainage tube into the lower right abdomen after SAP induction, and APD + ZnPP group with intraperitoneal injection of 30 mg/kg ZnPP 12 h before APD modeling. Blood samples were collected from the rats 12 h after modeling for analysis of amylase and lipase levels and serum inflammatory factors. The pathological changes of the pancreatic tissue were observed with HE staining. Oxidative stress in the pancreatic tissue was detected with colorimetry, and sub-organelle structure and autophagy in pancreatic acinar cells were observed by transmission electron microscopy. The expressions of autophagy-related proteins and Nrf-2/HO-1 pathway were detected using RT-PCR and Western blotting. RESULTS: Compared with those in SAP group, the rats with APD treatment showed significantly alleviated pathologies in the pancreas, reduced serum levels of lipase, amylase and inflammatory factors, lowered levels of oxidative stress, and activated expressions of Nrf-2/HO-1 pathway in the pancreas. The ameliorating effect of ADP was significantly inhibited by ZnPP treatment before modeling. APD obviously reversed mitochondrial and endoplasmic reticulum damages and p62 accumulation induced by SAP. CONCLUSION APD treatment can suppress oxidative stress and repair impaired autophagy in rats with SAP by activating the Nrf-2/HO-1 pathway, thereby reducing the severity of SAP.
Acute Disease ; Amylases/blood* ; Animals ; Autophagy ; Drainage ; Heme Oxygenase (Decyclizing) ; Lipase/blood* ; Male ; NF-E2-Related Factor 2 ; Oxidative Stress ; Pancreas/pathology* ; Pancreatitis/surgery* ; Punctures ; Rats ; Rats, Sprague-Dawley

Aims: Every year, an estimated 25 million tons of waste oil are produced worldwide, and the generation of waste oil is one of the biggest global environmental problems. The incorporation of oil as a substrate for lipase production has been studied and shown to have a positive impact on its production. Burkholderia sp. is one of the major lipase-producing bacteria with their ability in bioremediation of oil-contaminated soil. This study aims to compare the production of lipase by Burkholderia cenocepacia ST8 using waste cooking oil and unused cooking oil as feedstock. Methodology and results: The effect of different types of waste cooking oil (sunflower oil and palm oil) and concentration (1-3%) of waste cooking oil, agitation speed (100-400 rpm) and initial dissolved oxygen concentration (10-50%) on lipase production by B. cenocepacia ST8 under batch fermentation mode were investigated. The major fatty acids of which had been consumed were determined using gas chromatography. Results showed that 2% (v/v) of single used sunflower cooking oil produced the highest lipase activity of 138.86 U/mL with a productivity of 2.10 U/mL/h; agitation speed of 300 rpm produced the highest lipase activity of 183.56 U/mL with a productivity of 3.06 U/mL/h while 30% initial concentration of dissolved oxygen produced a lipase activity of 176.45 U/mL with a productivity of 2.94 U/mL/h. Oleic acid and linoleic acid were found to be the most consumed by B. cenocepacia ST8 among other fatty acids. Conclusion, significance and impact of study This study shows that 2% (v/v) single used sunflower cooking oil was the better type and optimum concentration of carbon source for the production of lipase by the fermentation of B. cenocepacia under 300 rpm and 30% initial concentration dissolved oxygen. The incorporation of 2% (v/v) single used sunflower cooking oil may be a great alternative to reduce the cost for the production of lipase as well as reducing the amount of waste oil generation.
Lipase ; Burkholderia cenocepacia ; Waste Management ; Biodegradation, Environmental