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

A rapid gas chromatography method was developed for determination of lipase activity using tributyrin as substrate. The standard curves of butyric acid hydrolyzed from tributyrin were linear in the range of 0.11-11.35 mmol L(-1). The recoveries of low, moderate and high concentrations of tributyrin were 90.3%, 104.6%, 89.4% with RSD of 3.01%, 4.50%, 6.64%, respectively. The incubation time was only 5 minutes which was less than with the half time of the conventional titrimetry and spectrophotometry. The optimum pH value was 7.5 and the optimum temperature was 32 degrees C. Based on the Lineweaver-Burk plots, the Michaelis-Menten constant was 0.25 mmol mL(-1). The effect of orlistat on the enzyme inhibiting activity was studied to prove the accuracy of this method. It was found that the half-inhibition concentration (IC50) of orlistat was 0.0485 mg mL(-1). The small total reaction volume, the simple treating procedures, the high accuracy and precision present the advantages of the new method.
Chromatography, Gas ; methods ; Lipase ; analysis ; metabolism ; Triglycerides ; metabolism

Lipases are widely used enzymes in biotechnology. Although they catalyze the same reaction, their sequences vary. Therefore, it is highly desired to develop a fast and reliable method to identify the types of lipases according to their sequences, or even just to confirm whether they are lipases or not. By proposing two scales based pseudo amino acid composition approaches to extract the features of the sequences, a powerful predictor based on k-nearest neighbor was introduced to address the problems. The overall success rates thus obtained by the 10-fold cross-validation test were shown as below: for predicting lipases and nonlipase, the success rates were 92.8%, 91.4% and 91.3%, respectively. For lipase types, the success rates were 92.3%, 90.3% and 89.7%, respectively. Among them, the Z scales based pseudo amino acid composition was the best, T scales was the second. They outperformed significantly than 6 other frequently used sequence feature extraction methods. The high success rates yielded for such a stringent dataset indicate predicting the types of lipases is feasible and the different scales pseudo amino acid composition might be a useful tool for extracting the features of protein sequences, or at lease can play a complementary role to many of the other existing approaches.
Amino Acids ; analysis ; chemistry ; Computational Biology ; Lipase ; chemistry ; classification ; Models, Chemical ; Sequence Analysis, Protein ; methods

We investigated the transesterification of crude cottonseed oil with methyl acetate to biodiesel, by using Lipozyme TL IM and Novozym 435 as catalysts. Results showed that the biodiesel yield significantly increased with the addition of methanol into the reaction system, and the highest biodiesel yield of 91.83% was achieved with the optimum conditions as follows: n-hexane as solvent, molar ratio of methyl acetate to oil 9:1, 3% methanol based on the oil mass to inhibit the creation of acetic acid, 10% Lipozyme TL IM and 5% Novozym 435 as catalyst based on the oil mass, reaction temperature 55 degrees C and reaction time 8 h. Additionally, we explored the kinetics of lipase-catalyzed crude cottonseed oil to biodiesel, and proposed a kinetic model.
Acetates ; metabolism ; Biofuels ; analysis ; Catalysis ; Cottonseed Oil ; chemistry ; metabolism ; Enzymes, Immobilized ; metabolism ; Lipase ; metabolism

BACKGROUND/AIMS: Non-pancreatic elevations of serum lipase have been reported, and differential diagnosis is necessary for clinical practice. This study aimed to evaluate the clinical efficacy of serum lipase subtype analysis for the differential diagnosis of pancreatic and non-pancreatic lipase elevation. METHODS: Patients who were referred for the serum lipase elevation were prospectively enrolled. Clinical findings and serum lipase subtypes were analyzed and compared by dividing the patients into pancreatitis and non-pancreatitis groups. RESULTS: A total of 34 patients (12 pancreatitis vs. 22 non-pancreatitis cases) were enrolled. In univariate analysis, the fraction of pancreatic lipase (FPL) in the total amount of serum lipase subtypes was statistically higher in patients with pancreatitis ([median, 0.004; interquartile range [IQR], 0.003 to 0.011] vs. [median, 0.002; IQR, 0.001 to 0.004], p = 0.04). Based on receiver operating characteristic curve analysis for the prediction of acute pancreatitis, FPL was the most valuable predictor (area under the receiver-operating characteristic curve [AUROC], 0.72; 95% confidence interval [CI], 0.54 to 0.86; sensitivity, 83.3%; specificity, 63.6%; positive predictive value, 55.6%; negative predictive value, 97.5%). In multivariate analysis, a cut-off value higher than 0.0027 for the FPL was associated with acute pancreatitis (odds ratio, 8.3; 95% CI, 1.3 to 51.7; p = 0.02). CONCLUSIONS: The results did not support that serum lipase subtype analysis could replace standard lipase measurement for the diagnosis of acute pancreatitis. However, the test demonstrated adequate sensitivity for use in triage or as an add-on test for serum lipase elevation.
Diagnosis ; Diagnosis, Differential* ; Humans ; Lipase* ; Multivariate Analysis ; Pancreatitis ; Prospective Studies ; ROC Curve ; Sensitivity and Specificity ; Treatment Outcome* ; Triage

BACKGROUND/AIMS: Recent studies reported that 1g of gabexate mesilate (GM) was effective in preventing endoscopic retrograde cholangiopancreatography (ERCP)-related pancreatic damage. The aim of this study was to evaluate the effectiveness of low dose GM for the prevention of ERCP-related pancreatic damage. METHODS: This study was performed prospectively with 102 consecutive patients (68 for the GM group, 34 for the placebo group) who were scheduled for ERCP. Infusion of GM (500 mg) was started 30 minutes before ERCP and continued for 12 hours afterward. The serum amylase and lipase were measured before ERCP and 4, 8, and 24 houps after ERCP. RESULTS: The incidence of hyperenzymemia was 45.6% in the GM group and 55.9% in the control group (p=0.40). Acute pancreatitis was developed in only one patient who was given the placebo. Although difficult cannulation, visualization of the pancreatic duct, performance of therapeutic procedures, and longer total procedure time were associated with an increased incidence of hyperenzymemia, the incidence of pancreatic damage was not affected by the GM treatment in these conditions. CONCLUSIONS: Prophylactic treatment with 500 mg of GM has no advantage for the prevention of ERCP-related pancreatic damage. Considering the cost effectiveness, further studies are necessary to identify the patients at greatest risk fot acute pancreatitis.
Amylases ; Catheterization ; Cholangiopancreatography, Endoscopic Retrograde* ; Cost-Benefit Analysis ; Gabexate* ; Humans ; Incidence ; Lipase ; Pancreatic Ducts ; Pancreatitis ; Prospective Studies

Our previous work has indicated that enzymatic resolution of glycidyl butyrate are strongly affected by many factors, including concentration of substrate, amount of lipase, the temperature, pH, shaking speed and reaction time. In this study, Plackett-Burman design was undertaken to evaluate the effects of the six factors. By regression analysis, concentration of substrate, amount of lipase and the temperature were found to be important for enzymatic resolution of glycidyl butyrate. In the second phase of the optimization process, a response surfacemethodology (RSM) was used to optimize the above critical factors, and to find out the optimal concentration levels and the relationships between these factors. By solving the quadratic regression model equation using appropriate statistic methods, the optimal parameter of the variables were determined as: 0.499 mol/L glycidyl butyrate, 30.23 mg/g lipase and 29.68 degrees C. In the optimum condition, the value of enantiomeric excess(ee%) was 93.28%. Compared to 84.65% which was the maximum ee% under the non-optimized condition, this study has a significant advancement. The experimental data under various conditions have validated the theoretical values.
Butyrates ; chemistry ; Catalysis ; Enzyme Activation ; Epoxy Compounds ; chemistry ; Lipase ; metabolism ; Molecular Conformation ; Propanols ; chemistry ; Regression Analysis ; Stereoisomerism ; Substrate Specificity ; Temperature

OBJECTIVETo investigate the dynamic changes of lipoprotein lipase (LPL) expression in the hippocampus of epileptic rats and to study its effect on vitamin E levels in rats following status epilepticus (SE).

METHODSRat model of SE was induced by intraperitoneal injection of pilocarpine. The rats receiving an injection of normal saline were used as a control group. The expression of LPL in the hippocampal tissue was determined using immunofluorescent methods and the level of vitamin E was examined by the colormeric method 12 hrs, 24 hrs, 3 days, 7 days and 14 days after SE.

RESULTSLPL was expressed in the control and SE groups. In the SE group, the LPL expression began to increase 24 hr after SE (P<0.05), reached a peak 3 days after SE (P<0.01), and kept at a high level 7 days after SE (P<0.01). By 14 days, the LPL expression was reduced to the level similar to the control group. The level of vitamin E began to decline 12 hrs after SE (P<0.01), and decreased to a nadir 24 hrs after SE (P<0.01). At 3 and 7 days after SE, the levels of vitamin E were still significantly lower than the controls (P<0.05). By 14 days, the vitamin E level increased to the level similar to the control group.

CONCLUSIONSThe over-expression of LPL in the hippocampus may play an important role in the oxidative stress mechanisms following SE by regulating the uptake of vitamin E.

Animals ; Fluorescent Antibody Technique ; Hippocampus ; metabolism ; Lipoprotein Lipase ; analysis ; physiology ; Male ; Oxidative Stress ; Rats ; Rats, Sprague-Dawley ; Status Epilepticus ; metabolism ; Vitamin E ; analysis ; metabolism

Estrogen-related receptor a (ERRalpha) is a key regulator for energy metabolism and adipogenesis. However, its role in lipolysis is unknown. To study the function of ERRalpha in lipolysis, primary cultured differentiated porcine adipocytes were treated by a specific inverse agonist XCT790 or infected with adenoviral vector expressed ERRalpha for 48 h, in the absence and/or presence of specific protein kinase A (PKA) inhibitor or extracellular signal-related kinase (ERK) inhibitor. Then, we measured the triglyceride (TG) content and the glycerol release into the culture media to analysis the effect of ERRalpha on lipolysis; Further, we analyzed the expression of PPARgamma, perilipin A, p-perilipin A, HSL and ATGL with Western blotting. Here, we found that ERRalpha significantly increased adipocytes differentiation, TG accumulation and glycerol release. Separately or simultaneously block the PKA and ERK pathway do not significantly altered the effect of ERRalpha on glycerol release. ERRalpha significantly up-regulated the proteins expression of PPARgamma, perilipin A, HSL and ATGL, while the p-perilipin A protein level was not significantly changed. These findings imply that ERRalpha could increase lipolysis via up-regulating HSL and ATGL, thereby to supply more FFA as substrate for a larger turnover of cellular triglyceride pool during adipocytes differentiation.
Adipocytes ; cytology ; metabolism ; Animals ; Animals, Newborn ; Cells, Cultured ; Glycerol ; analysis ; Lipase ; metabolism ; Lipolysis ; physiology ; Receptors, Estrogen ; metabolism ; physiology ; Sterol Esterase ; metabolism ; Swine ; Triglycerides ; analysis

Chylomicronemia is a severe type of hypertriglyceridemia characterized by chylomicron accumulation that arises from a genetic defect in intravascular lipolysis. It requires urgent and proper management, because serious cases can be accompanied by pancreatic necrosis or persistent multiple organ failure. We present the case of a 1-month-old infant with chylomicronemia treated by plasmapheresis. His chylomicronemia was discovered incidentally when lactescent plasma was noticed during routine blood sampling during a hospital admission for fever and irritability. Laboratory investigation revealed marked triglyceridemia (>5,000 mg/dL) with high chylomicron levels. We therefore decided to perform a therapeutic plasmapheresis to prevent acute pancreatitis. Sequence analysis revealed a homozygous novel mutation in exon 4 of GPIHBP1: c.476delG (p.Gly159Alafs). Glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1) stabilizes the binding of chylomicrons near lipoprotein lipase and supports lipolysis. Mutations of GPIHBP1, the most recently discovered gene, can lead to severe hyperlipidemia and are known to make up only 2% of the monogenic mutations associated with chylomicronemia. The patient maintains mild hypertriglyceridemia without rebound after single plasmapheresis and maintenance fibrate medication so far. Here, we report an infant with chylomicronemia due to GPIHBP1 mutation, successfully treated by plasmapheresis.
Chylomicrons ; Exons ; Fever ; Humans ; Hyperlipidemias ; Hypertriglyceridemia ; Infant* ; Infant, Newborn* ; Lipolysis ; Lipoprotein Lipase ; Multiple Organ Failure ; Necrosis ; Pancreatitis ; Plasma ; Plasmapheresis* ; Sequence Analysis