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*

An extracellular lipase from Aureobasidium pullulans was obtained and purified with a specific activity of 17.7 U/mg of protein using ultrafiltration and a DEAE-Sepharose Fast Flow column. Characterization of the lipase indicated that it is a novel finding from the species A. pullulans. The molecular weight of the lipase was 39.5 kDa, determined by sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme exhibited its optimum activity at 40 °C and pH of 7. It also showed a remarkable stability in some organic solutions (30%, v/v) including n-propanol, isopropanol, dimethyl sulfoxide (DMSO), and hexane. The catalytic activity of the lipase was enhanced by Ca²⁺ and was slightly inhibited by Mn²⁺ and Zn²⁺ at a concentration of 10 mmol/L. The lipase was activated by the anionic surfactant SDS and the non-ionic surfactants Tween 20, Tween 80, and Triton X-100, but it was drastically inhibited by the cationic surfactant cetyl trimethyl ammonium bromide (CTAB). Furthermore, the lipase was able to hydrolyze a wide variety of edible oils, such as peanut oil, corn oil, sunflower seed oil, sesame oil, and olive oil. Our study indicated that the lipase we obtained is a potential biocatalyst for industrial use.
Ascomycota/enzymology* ; Calcium ; Catalysis ; Corn Oil/metabolism* ; Detergents/chemistry* ; Enzyme Stability ; Fungal Proteins/chemistry* ; Glucans/chemistry* ; Hexanes/chemistry* ; Hydrogen-Ion Concentration ; Hydrolysis ; Industrial Microbiology ; Lipase/chemistry* ; Manganese/chemistry* ; Olive Oil/metabolism* ; Peanut Oil/metabolism* ; Sesame Oil/metabolism* ; Substrate Specificity ; Sunflower Oil/metabolism* ; Surface-Active Agents ; Temperature ; Zinc/chemistry*

The storage and transportation of raw milk at low temperatures promote the growth of psychrotrophic bacteria and the production of thermo-stable enzymes, which pose great threats to the quality and shelf-life of dairy products. Though many studies have been carried out on the spoilage potential of psychrotrophic bacteria and the thermo-stabilities of the enzymes they produce, further detailed studies are needed to devise an effective strategy to avoid dairy spoilage. The purpose of this study was to explore the spoilage potential of psychrotrophic bacteria from Chinese raw milk samples at both room temperature (28 °C) and refrigerated temperature (7 °C). Species of Yersinia, Pseudomonas, Serratia, and Chryseobacterium showed high proteolytic activity. The highest proteolytic activity was shown by Yersinia intermedia followed by Pseudomonas fluorescens (d). Lipolytic activity was high in isolates of Acinetobacter, and the highest in Acinetobacter guillouiae. Certain isolates showed positive β-galactosidase and phospholipase activity. Strains belonging to the same species sometimes showed markedly different phenotypic characteristics. Proteases and lipases produced by psychrotrophic bacteria retained activity after heat treatment at 70, 80, or 90 °C, and proteases appeared to be more heat-stable than lipases. For these reasons, thermo-stable spoilage enzymes produced by a high number of psychrotrophic bacterial isolates from raw milk are of major concern to the dairy industry. The results of this study provide valuable data about the spoilage potential of bacterial strains in raw milk and the thermal resistance of the enzymes they produce.
Animals ; Bacteria/genetics* ; Bacterial Proteins/chemistry* ; Biofilms ; Cold Temperature ; Dairy Products ; Endopeptidases/chemistry* ; Enzyme Stability ; Food Microbiology ; Hot Temperature ; Lipase/chemistry* ; Milk/microbiology* ; Peptide Hydrolases/chemistry* ; Phospholipases/chemistry* ; RNA, Ribosomal, 16S/genetics* ; Raw Foods/microbiology* ; beta-Galactosidase/chemistry*

Pomegranate leaf (PGL) has a definite role in regulating lipid metabolism. However, pharmacokinetic results show the main active ingredient, ellagic acid, in PGL has lower oral bioavailability, suggesting that the lipid-lowering effect of PGL may act through inhibiting lipid absorption in the small intestine. Our results demonstrated that pomegranate leaf and its main active ingredients (i.e., ellagic acid, gallic acid, pyrogallic acid and tannic acid) were capable of inhibiting pancreatic lipase activity in vitro. In computational molecular docking, the four ingredients had good affinity for pancreatic lipase. Acute lipid overload experiments showed that a large dosage of PGL significantly reduced serum total cholesterol (TG) and triglycerides (TC) levels in addition to inhibiting intestinal lipase activity, which demonstrated that PGL could inhibit lipase activity and reduce the absorption of lipids. We also found that PGL could reverse the reduced tight-junction protein expression due to intestinal lipid overload, promote Occludin and Claudin4 expression in the small intestine, and enhance the intestinal mucosal barrier. In conclusion, we demonstrated that PGL can inhibit lipid absorption and reduce blood TG and TC by targeting pancreatic lipase, promoting tight-junction protein expression and thereby preventing intestinal mucosa damage from an overload of lipids in the intestine.
Animals ; Enzyme Inhibitors ; administration & dosage ; chemistry ; Humans ; Hyperlipidemias ; drug therapy ; enzymology ; metabolism ; Intestinal Absorption ; Intestine, Small ; metabolism ; Kinetics ; Lipase ; chemistry ; metabolism ; Lipid Metabolism ; Lythraceae ; chemistry ; Male ; Mice ; Mice, Inbred ICR ; Plant Extracts ; administration & dosage ; chemistry ; Plant Leaves ; chemistry ; Triglycerides ; metabolism

The present study was designed to synthesize and evaluate a series of benzylisoquinoline derivatives. These compounds were synthesized by Bischler-Napieralski cyclization to yield 1-benzyl-3,4-dihydroisoquinolines, and the products were obtained by reductions. All these compounds were identified by MS, (1)H NMR and (13)C NMR. The inhibitory activities on pancreatic lipase and preadipocyte proliferation for the synthesized compounds and alkaloids from Nulembo nucifera were assessed in vitro. Most of the compounds showed inhibitory activities on both pancreatic lipase and preadipocyte proliferation. Particularly, compounds 7p-7u and 9d-9f exhibited significant inhibitory activity on pancreatic lipase while compounds 7c, 7d, 7f, 7g, 7i, and 7j potently inhibited the proliferation of 3T3-L1 preadipocytes. Our results provided a basis for future evaluation and development of these compounds as leads for therapeutics for human diseases.
Adipocytes ; cytology ; drug effects ; Benzylisoquinolines ; chemical synthesis ; chemistry ; pharmacology ; Cell Proliferation ; drug effects ; Enzyme Inhibitors ; chemical synthesis ; chemistry ; pharmacology ; Humans ; Lipase ; antagonists & inhibitors ; metabolism ; Structure-Activity Relationship

PURPOSE: Carbamate insecticides are potent cholinesterase inhibitors capable of causing severe cholinergic toxicity. Use of carbamate rather than organophosphate insecticides has been increasing. Compared with organophosphate poisoning, relatively few studies have investigated carbamate-associated acute pancreatitis. We investigated general characteristics and pancreatitis of carbamate poisoning and the predictors, among those readily assessed in the emergency department. METHODS: We performed a retrospective review of consecutive patients, aged over 18 years, who were admitted between January 2008 and April 2012 to an emergency department (ED) of an academic tertiary care center for treatment of carbamate poisoning. Patients who exhibited poisoning by any other material, except alcohol, were excluded. After application of exclusion criteria, patients were divided according to carbamate-induced pancreatitis and non-pancreatitis groups. RESULTS: A total of 41 patients were included in this study. Among these 41 patients, the prevalence of acute pancreatitis was 36.6% (15 patients). Initial blood chemistry tests showed a statistically higher glucose level in the pancreatitis group, compared with the non-pancreatitis group (222, IQR 189-284 vs. 137, IQR 122-175 mg/dL, P<0.05). Regarding clinical courses and outcomes, a significantly higher proportion of patients developed pneumonia [10 (66.7%) vs. 6 (23.1%), P<0.05] and had a longer hospital stay (7 days, IQR 6-12 vs. 5 days, IQR 2-11, P<0.05), but no difference in mortality, in the pancreatitis group vs. the non-pancreatitis group. In multivariate analysis, the initial glucose was showing significant association with the presentation of carbamate-induced acute pancreatitis (odds ratio 1.018, 95% confidence interval 1.001-1.035, P<0.05). CONCLUSION: Carbamate-induced acute pancreatitis is common, but not fatal. Initial serum glucose level is associated with acute pancreatitis.
Blood Glucose ; Carbamates ; Chemistry ; Cholinesterase Inhibitors ; Emergency Service, Hospital ; Glucose ; Humans ; Insecticides ; Length of Stay ; Lipase ; Mortality ; Multivariate Analysis ; Organophosphate Poisoning ; Pancreatitis* ; Pneumonia ; Poisoning* ; Prevalence ; Retrospective Studies ; Tertiary Care Centers

Both intrathyroidal parathyroid adenoma and acute pancreatitis from hyperparathyroidism are rare disorders. We report a case of acute pancreatitis from hyperfunctioning intrathyroidal parathyroid adenoma in a 40-year-old man with severe abdominal pain. Serum chemistry values show-ed high amylase, lipase, calcium and intact parathyroid hormone level, and abdominal CT revealed acute pancreatitis. A 7 mm lesion was detected inside the left upper pole of thyroid on neck ultrasonography and confirmed to be a parathyroid lesion based on fine needle aspiration cytology. After exploratory parathyroidectomy, symptoms subsided. In patients who present with acute pancreatitis, hyperparathyroidism should also be considered if risk factors such as alcohol ingestion, gallstone, previous endoscopic retrograde cholangiopancreatography, and abdominal trauma do not exist. Exploratory parathyroidectomy should be performed in a case of acute pancreatitis from primary hyperparathyroidism.
Abdominal Pain ; Adult ; Amylases ; Biopsy, Fine-Needle ; Calcium ; Chemistry ; Cholangiopancreatography, Endoscopic Retrograde ; Eating ; Gallstones ; Humans ; Hyperparathyroidism ; Hyperparathyroidism, Primary ; Lipase ; Neck ; Pancreatitis* ; Parathyroid Hormone ; Parathyroid Neoplasms* ; Parathyroidectomy ; Risk Factors ; Thyroid Gland ; Tomography, X-Ray Computed ; Ultrasonography

In Korea, the use of herbal remedies is a common cause of drug-induced liver injury. However, the occurrence of both acute pancreatitis and acute hepatitis after taking herbal remedies has rarely been reported. Herein, we report a case of concurrent acute pancreatitis and acute hepatitis associated with Ceramium kondoi ingestion. A 58-year-old woman was diagnosed with advanced gastric cancer 7 months ago. Total gastrectomy and adjuvant chemotherapy was performed without complications. The patient had been well until recently, when she presented with severe abdominal pain after ingestion of Ceramium kondoi for 4 weeks. The laboratory findings demonstrated elevated liver enzymes and lipase, and abdominal computed tomography revealed pancreas swelling with fat infiltration. The diagnosis was made based on the diagnostic criteria for drug induced pancreatitis and the Russel Uclaf Causality Assessment Method scale for drug-induced liver injury. After cessation of Ceramium kondoi, she showed clinical and biochemical improvement.
Abdominal Pain/etiology ; Acute Disease ; Drug-Induced Liver Injury/*diagnosis/enzymology ; Female ; Humans ; Lipase/metabolism ; Liver/*drug effects ; Middle Aged ; Pancreas/*drug effects ; Pancreatitis/*chemically induced/*diagnosis ; Plant Extracts/chemistry/*toxicity ; Rhodophyta/chemistry/metabolism ; Tomography, X-Ray Computed

Directed evolution was conducted to improve the thermostability of lipase from Rhizopus chinensis CCTCC M201021. Mutations were introduced by two rounds of error-prone PCR and mutant lipase was selected by fast-blue RR top agar screening. Two positive variants were selected in the first-round and four in the second-round screening process. Ep2-4 was proved as the most thermostable lipase and its DNA sequencing revealed three amino acid substitutions: A129S, P168L and V329A. Compared with the parent, its half-life at 60 degrees C was 5.4- times longer and T50 was 7.8 degrees higher. Purified lipase of Ep2-4 was characterized and the result shows that its thermostability improved without compromising enzyme activity. According to the mimicked protein structure, mutation A129S formed a hydrogen bond with Gln133 and improved the thermostability by increasing the hydrophilicity and polarity of protein; mutation P168L by forming a hydrophobic bond with the nearby Leu164.
Cloning, Molecular ; Directed Molecular Evolution ; methods ; Enzyme Stability ; genetics ; Hot Temperature ; Industrial Microbiology ; Lipase ; chemistry ; genetics ; Mutation ; Pichia ; genetics ; metabolism ; Polymerase Chain Reaction ; methods ; Protein Engineering ; methods ; Rhizopus ; enzymology

OBJECTIVECurrent study was conducted to investigate and compare the impact of temperature and pH on the activities of amylase, protease and lipase in alimentary tract of Whitmania pigra.

METHODThe responses of amylase, protease, and lipase activities were determined over a wide range of temperatures (7-52 degrees C) and pH gradient (2.2-11.2).

RESULTThe highest lipase activity was found under 37 degrees C, pH 8.2, and the highest amylase activity was detected under 37 degrees C, pH 5.2, while protease activity peaked at 42 degrees C, pH 3.2 or pH 9.2.

CONCLUSIONThe optimal temperature in alimentary tract of Wh. pigra for lipase and amylase was 37 degrees C, and the responding temperature for protease was 42 degrees C. The optimal pH value in alimentary tract of Wh. pigra for lipase and amylase was pH 8.2 and pH 5.2, respectively. While pH 3.2 or 9.2 seems to be both favorable for high protease activity.

Amylases ; chemistry ; metabolism ; Animals ; Digestive System ; chemistry ; enzymology ; Enzyme Stability ; Hydrogen-Ion Concentration ; Leeches ; chemistry ; enzymology ; Lipase ; chemistry ; metabolism ; Peptide Hydrolases ; chemistry ; metabolism ; Temperature