Lipase Secretion Research Articles

Influencia de la alimentacion sobre el ritmo circadiano de las enzimas digestivas en el cultivo de juveniles de la langosta de agua dulce Cherax quadricarinatus (Parastacidae)

We analyze in juvenile Cherax quadricarinatus fattening stage, the circadian rhythm of total protein levels, activity of proteases, lipases and amylases, and their possible change as a function of morning (8 h) and evening (17 h) feeding. In general, the activity of digestive enzymes of juvenile C. quadricarinatus showed no circadian pattern of secretion of lipases except where there was an increase towards the evening-night and a decrease towards the early hours of the morning. The total protein levels, the activity of proteinases and amylases of juveniles of C. quadricarinatus registered in the midgut gland remained virtually unchanged in all experiments. However, a non-significant tendency to diminish the proteinase activity levels after the feeding pulse was observed, with a return to previous enzymatic levels after 3 h. In contrast, the amylase activity did not show an observable effect by the feeding schedule. The study of the patterns of the digestive enzyme secretion and their possible changes, could be used as a tool to establish the most favorable times of the day for juveniles feeding.

Influence of feeding on the circadian rhythm of digestive enzymes in cultivated juveniles of the freshwater crayfish Cherax quadricarinatus (Parastacidae)

We analyze in juvenile Cherax quadricarinatus fattening stage, the circadian rhythm of total protein levels, activity of proteases, lipases and amylases, and their possible change as a function of morning (8 h) and evening (17 h) feeding. In general, the activity of digestive enzymes of juvenile C. quadricarinatus showed no circadian pattern of secretion of lipases except where there was an increase towards the evening-night and a decrease towards the early hours of the morning. The total protein levels, the activity of proteinases and amylases of juveniles of C. quadricarinatus registered in the midgut gland remained virtually unchanged in all experiments. However, a non-significant tendency to diminish the proteinase activity levels after the feeding pulse was observed, with a return to previous enzymatic levels after 3 h. In contrast, the amylase activity did not show an observable effect by the feeding schedule. The study of the patterns of the digestive enzyme secretion and their possible changes, could be used as a tool to establish the most favorable times of the day for juveniles feeding.

Candida albicans VMA3 Is Necessary for V-ATPase Assembly and Function and Contributes to Secretion and Filamentation

The vacuolar membrane ATPase (V-ATPase) is a protein complex that utilizes ATP hydrolysis to drive protons from the cytosol into the vacuolar lumen, acidifying the vacuole and modulating several key cellular response systems in Saccharomyces cerevisiae. To study the contribution of V-ATPase to the biology and virulence attributes of the opportunistic fungal pathogen Candida albicans, we created a conditional mutant in which VMA3 was placed under the control of a tetracycline-regulated promoter (tetR-VMA3 strain). Repression of VMA3 in the tetR-VMA3 strain prevents V-ATPase assembly at the vacuolar membrane and reduces concanamycin A-sensitive ATPase-specific activity and proton transport by more than 90%. Loss of C. albicans V-ATPase activity alkalinizes the vacuolar lumen and has pleiotropic effects, including pH-dependent growth, calcium sensitivity, and cold sensitivity. The tetR-VMA3 strain also displays abnormal vacuolar morphology, indicative of defective vacuolar membrane fission. The tetR-VMA3 strain has impaired aspartyl protease and lipase secretion, as well as attenuated virulence in an in vitro macrophage killing model. Repression of VMA3 suppresses filamentation, and V-ATPase-dependent filamentation defects are not rescued by overexpression of RIM8, MDS3, EFG1, CST20, or UME6, which encode positive regulators of filamentation. Specific chemical inhibition of Vma3p function also results in defective filamentation. These findings suggest either that V-ATPase functions downstream of these transcriptional regulators or that V-ATPase function during filamentation involves independent mechanisms and alternative signaling pathways. Taken together, these data indicate that V-ATPase activity is a fundamental requirement for several key virulence-associated traits in C. albicans.

Gastric Lipase Secretion in Children with Gastritis

Gastric lipase is one of the prepancreatic lipases found in some mammalian species and in humans. Our knowledge of the hormonal regulation of gastric lipase secretion in children and adolescents is still very limited. The aim of this study was to compare the activity of human gastric lipase (HGL) in gastric juice in healthy adolescents and in patients with gastritis. The adolescents were allocated to three groups: the first including patients with Helicobacter pylori gastritis (HPG; n = 10), the second including patients with superficial gastritis caused by pathogens other than H. pylori (non-HPG; n = 14) and the control group including healthy adolescents (n = 14). Activity of HGL was measured in gastric juice collected during endoscopy. Plasma concentrations of cholecystokinin (CCK), glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) were measured in all adolescents. Activity of HGL in the non-HPG group was significantly lower than in the HPG group (p < 0.005) and the control group (p < 0.005). Mean plasma GIP levels in the control group were lower than in the non-HPG group (p < 0.003) and the HPG group (p < 0.01). We conclude that the regulation of HGL secretion by GLP-1 and CCK is altered in patients with gastritis. Moreover, GIP is a potent controller of HGL activity, both in healthy subjects and in patients with gastritis.

Leucine markedly regulates pancreatic exocrine secretion in goats

Four goats (30.1±1.3kg) with common bile duct re-entrant catheter and duodenal catheter were used to evaluate the effects of duodenal leucine infusion on pancreatic exocrine secretion and plasma parameters with two 4×4 Latin square design experiments. In the long-term infusion experiment, goats were fed twice daily [700g/day, dry matter (DM) basis] at 8:00 and 18:00hours and were duodenally infused with 0, 3, 6, 9g/day leucine for 14days. Pancreatic juice and jugular blood samples were collected over 1-h intervals for 6h daily from d 11 to 14days to encompass a 24-h day. In the short-term experiment, goats were infused leucine for 10h continuously at the same infusion rate with Experiment 1 after feed deprivation for 24h repeated every 10days. Pancreatic juice and blood samples were collected at 0, 1, 2, 4, 6, 8 and 10h of infusion. The results showed that the long-term leucine infusion did not affect pancreatic juice secretion, protein output, trypsin and lipase secretion and plasma insulin concentration, but linearly increased α-amylase secretion. No changes in pancreatic protein and lipase secretion were observed in the short-term infusion. Pancreatic juice and α-amylase secretion responded quadratically, with the greatest values observed in the 3 and 6g/day leucine respectively. Trypsin secretion linearly decreased, while plasma insulin concentration increased linearly with increased leucine infusion. The results demonstrated that duodenal leucine infusion dose and time dependently regulated pancreatic enzyme secretion not associated with the change in plasma insulin concentration.

Endothelial Heparanase Regulates Heart Metabolism by Stimulating Lipoprotein Lipase Secretion From Cardiomyocytes

After diabetes mellitus, transfer of lipoprotein lipase (LPL) from cardiomyocytes to the coronary lumen increases, and this requires liberation of LPL from the myocyte surface heparan sulfate proteoglycans with subsequent replenishment of this reservoir. At the lumen, LPL breaks down triglyceride to meet the increased demand of the heart for fatty acid. Here, we examined the contribution of coronary endothelial cells (ECs) toward regulation of cardiomyocyte LPL secretion. Bovine coronary artery ECs were exposed to high glucose, and the conditioned medium was used to treat cardiomyocytes. EC-conditioned medium liberated LPL from the myocyte surface, in addition to facilitating its replenishment. This effect was attributed to the increased heparanase content in EC-conditioned medium. Of the 2 forms of heparanase secreted from EC in response to high glucose, active heparanase released LPL from the myocyte surface, whereas latent heparanase stimulated reloading of LPL from an intracellular pool via heparan sulfate proteoglycan-mediated RhoA activation. Endothelial heparanase is a participant in facilitating LPL increase at the coronary lumen. These observations provide an insight into the cross-talk between ECs and cardiomyocytes to regulate cardiac metabolism after diabetes mellitus.

High-level expression and characterization of a chimeric lipase from Rhizopus oryzae for biodiesel production

BackgroundProduction of biodiesel from non-edible oils is receiving increasing attention. Tung oil, called “China wood oil” is one kind of promising non-edible biodiesel oil in China. To our knowledge, tung oil has not been used to produce biodiesel by enzymatic method. The enzymatic production of biodiesel has been investigated extensively by using Rhizopus oryzae lipase as catalyst. However, the high cost of R. oryzae lipase remains a barrier for its industrial applications. Through different heterologous expression strategies and fermentation techniques, the highest expression level of the lipase from R. oryzae reached 1334 U/mL in Pichia pastoris, which is still not optimistic for industry applications.ResultsThe prosequence of lipases from Rhizopus sp. is very important for the folding and secretion of an active lipase. A chimeric lipase from R. oryzae was constructed by replacing the prosequence with that from the R. chinensis lipase and expressed in P. pastoris. The maximum activity of the chimera reached 4050 U/mL, which was 11 fold higher than that of the parent. The properties of the chimera were studied. The immobilized chimera was used successfully for biodiesel production from tung oil, which achieved higher FAME yield compared with the free chimeric lipase, non-chimeric lipase and mature lipase. By response surface methodology, three variables, water content, methanol to tung oil molar ratio and enzyme dosage were proved to be crucial parameters for biosynthesis of FAME and the FAME yield reached 91.9±2.5% at the optimized conditions by adding 5.66 wt.% of the initial water based on oil weight, 3.88 of methanol to tung oil molar ratio and 13.24 wt.% of enzyme concentration based on oil weight at 40°C.ConclusionsThis is the first report on improving the expression level of the lipase from R. oryzae by replacing prosequences. The immobilized chimera was used successfully for biodiesel production from tung oil. Using tung oil as non-edible raw material and a chimeric lipase from R. oryzae as an economic catalyst make this study a promising one for biodiesel applications.

The effect of α-mating factor secretion signal mutations on recombinant protein expression in Pichia pastoris

The methylotrophic yeast, Pichia pastoris, has been genetically engineered to produce many heterologous proteins for industrial and research purposes. In order to secrete proteins for easier purification from the extracellular medium, the coding sequence of recombinant proteins is initially fused to the Saccharomyces cerevisiae α-mating factor secretion signal leader. Extensive site-directed mutagenesis of the prepro-region of the α-mating factor secretion signal sequence was performed in order to determine the effects of various deletions and substitutions on expression. Though some mutations clearly dampened protein expression, deletion of amino acids 57–70, corresponding to the predicted 3rd alpha helix of α-mating factor secretion signal, increased secretion of reporter proteins horseradish peroxidase and lipase at least 50% in small-scale cultures. These findings raise the possibility that the secretory efficiency of the leader can be further enhanced in the future.

BICHEMICAL ALTERATIONS INDUCED BY AMYLIN IN WISTAR RATS

Amylin is a peptide hormone synthesized in β cells of pancreas together with insulin in response to glucose and nutrients. Amylin controls food intake and body weight but the exact mechanism is still unknown. This study aimed to examine the effects of amylin injection on hormones, metabolites and cytokines expression in rats. Wistar rats were injected amylin Intraperitoneally (IP) (10 µg kg-1 twice daily) for 7 days. Plasma and liver samples were collected for blood measurements and RT-PCR analysis. Amylin treatments induced significant decrease in body weight and food intake in time dependent manner. Moreover, amylin significantly increased insulin, leptin and lipase secretion. A decrease in plasma triglycerides, cholesterol and LPL and increase in HDL levels was recorded. There are upregulation in the expression of IL-1β and TNF-α in amylin treated rats. The results collectively indicate that amylin has anti-obesity like effects through regulation of proteins and obesity related gene expression.

Improved secretion of Candida antarctica lipase B with its native signal peptide in Pichia pastoris

Secretion efficiency of the 85-amino acid Sacchromyces cerevisiae alpha signal peptide and the 25-amino acid Candida antarctica lipase B signal (nsB) peptide were compared. Three reporter proteins used for the study are C. antarctica lipase A (CalA), lipase B (CalB) and hGMCSF. The copy number of recombinant α-CalB and nsB-CalB clones was determined by qPCR and clones with equivalent gene copies were used for comparative analysis. About threefold increased CalB production corresponding to an activity of 480Uml−1 was obtained with its native signal peptide, whereas with the alpha signal peptide the maximum activity was 160Uml−1. Also, CalB was secreted as a mature protein with native N-terminus when fused to its own signal peptide, while unprocessed CalB with N-terminal extension was detected with the alpha signal peptide. Real time PCR analysis of CalB strains indicated that the difference in protein expression was not at the transcriptional level. The nsB signal sequence was also effective in secreting CalA enzyme and its secretion efficiency was on par with the alpha signal sequence. Further, hGMCSF fused inframe with the nsB signal peptide was also efficiently secreted into the medium. These results indicate that the nsB signal peptide can be a better alternative to alpha signal peptide for heterologous protein expression in Pichia pastoris.

Lipase Production Through Solid-State Fermentation using Agro-Industrial Residues as Substrates and Newly Isolated Fungal Strains

ABSTRACTExtracellular lipase production by Penicillium chrysogenum, Trichoderma harzianum and Aspergillus flavus was carried out through solid state fermentation using agro-industrial residues as substrates. For all three strains, the growth temperature was 29±1 °C, and 65% w (g/gds) moisture content. The effect of three factors on lipase production rate was investigated: initial pH (6.0 and 7.0), time of fermentation (72 h, 96 h and 120 h), and type of mixed substrate (wheat bran-olive oil, and wheat bran-castor oil cake). The process was optimized applying a mixed level factorial design. Fermentation time and pH were found to have positive effects on lipase production and secretion rates. However, the time effect was larger than initial pH. Type of substrate demonstrated minor effective importance than the other two factors, and Aspergillus flavus showed the larger lipase production among the three strains. Results indicated that the three fungal strains were able to grow and produce lipase in both culture mediums. The maximum lipase activity achieved was 121.35 U/gds by Aspergillus flavus, which was five and nine times the lipase produced by Trichoderma harzianum and P. chrysogenum respectively, at the same conditions. An initial neutral pH and 96 h of fermentation time were the optimum conditions for lipase production by Aspergillus flavus.

Lipase and Protease Double-Deletion Mutant of Pseudomonas fluorescens Suitable for Extracellular Protein Production

Pseudomonas fluorescens, a widespread Gram-negative bacterium, is an ideal protein manufacturing factory (PMF) because of its safety, robust growth, and high protein production. P. fluorescens possesses a type I secretion system (T1SS), which mediates secretion of a thermostable lipase (TliA) and a protease (PrtA) through its ATP-binding cassette (ABC) transporter. Recombinant proteins in P. fluorescens are attached to the C-terminal signal region of TliA for transport as fusion proteins to the extracellular medium. However, intrinsic TliA from the P. fluorescens genome interferes with detection of the recombinant protein and the secreted recombinant protein is hydrolyzed, due to intrinsic PrtA, resulting in decreased efficiency of the PMF. In this research, the lipase and protease genes of P. fluorescens SIK W1 were deleted using the targeted gene knockout method. Deletion mutant P. fluorescens ΔtliA ΔprtA secreted fusion proteins without TliA or protein degradation. Using wild-type P. fluorescens as an expression host, degradation of the recombinant protein varied depending on the type of culture media and aeration; however, degradation did not occur with the P. fluorescens ΔtliA ΔprtA double mutant irrespective of growth conditions. By homologous expression of tliA and the ABC transporter in a plasmid, TliA secreted from P. fluorescens ΔprtA and P. fluorescens ΔtliA ΔprtA cells was found to be intact, whereas that secreted from the wild-type P. fluorescens and P. fluorescens ΔtliA cells was found to be hydrolyzed. Our results demonstrate that the P. fluorescens ΔtliA ΔprtA deletion mutant is a promising T1SS-mediated PMF that enhances production and detection of recombinant proteins in extracellular media.

A Rare Case of GIST with Elevated Lipase and Amylase Levels and Its Effect on Treatment Options

Case Description: A 56-year-old Hispanic male presents with 3/10, nonradiating, diffuse upper abdominal pain of 2 weeks duration. The pain had no prandial or postural associations. He also complained of unintentional 30-pound weight loss in 1 month. Patient had no history of alcohol or drug abuse and was not on any medications. Abdominal exam was unremarkable. Laboratory results showed lipase level of 533 U/L and amylase level of 383U/L with rest of the metabolic profile being normal. Abdominal sonography revealed fatty infiltration of liver with normal gallbladder and intra/extra hepatic biliary system. Computerized tomography abdomen with contrast showed a 2.8 × 2.6 cm focal, hyperdense soft tissue mass abutting the lesser curvature of the stomach with mass effect. Esophagogastroduodenoscopy (EGD) showed a 2-3 cm sub mucosal lesion along the lesser curvature and a small, sliding hiatal hernia. An Endoscopic Ultrasound (EUS) confirmed an exophytic, well-circumscribed lesion 3.36 cm in diameter, arising from the muscularis propria of the gastric wall without an associated lymphadenopathy or invasion. FNAC showed groups of spindle cells negative for CD34 and CD117. Partial gastrectomy was performed and biopsy showed findings consistent with Gastrointestinal Stromal tumor (GIST). Discussion: Serum lipase is mainly derived from pancreatic acinar cells. Typically, higher cut off levels (> 3 times upper limit of normal) has higher specificity for acute pancreatitis. However, asymptomatic lipase and amylase elevation has been associated with diverse gastrointestinal etiologies. To date, GIST has not been reported to be associated with such high levels of lipase or amylase elevation. Gastric lipase has been localized to entire mucosa of the stomach with maximal activity in the fundic area. We propose that the mesenchymal tumor in this case is a direct source of these enzymes or produced a mechanical stimulus for gastric lipase secretion. However, whether or not GIST can be enzymatically active or not would warrant further studies. The normalization of both enzymes within 48 hours of surgery postulates a causative rather than a plausible association. Furthermore, since Imatinib or sunitinib, tyrosine kinase inhibitors are treatment options for metastatic or recurrent GIST tumors, these elevations in lipase and amylase levels may affect further treatment options.

Pterostilbene ameliorates tumor necrosis factor alpha–induced pancreatitis in vitro

BackgroundWe have previously demonstrated that pterostilbene, a compound in blueberries, exerts antiproliferative effects against pancreatic adenocarcinoma. However, little is known about the anti-inflammatory effects of pterostilbene in pancreatitis. Therefore, the aim of this study was to evaluate the effect of pterostilbene on inflammatory markers in an in vitro pancreatitis model. We hypothesized that pterostilbene would ameliorate the immediate inflammatory response in tumor necrosis factor alpha (TNF-α)–induced pancreatitis through downregulation of signal transducer and activator of transcription 3 (STAT3) and inhibition of TNF-α–induced secretion of lipase and proinflammatory cytokines interleukins (ILs) 1β and 6. MethodsAR42J acinar cells were pretreated with TNF-α to induce pancreatitis followed by 25 and 50μM pterostilbene for 15 and 30min. Secretion of lipase was quantified using a lipase assay and used as a marker of TNF-α–induced pancreatitis. Detection of STAT3, IL-1β, and IL-6 was performed using enzyme-linked immunosorbent assay. Analysis of variance and Tukey post hoc analysis were used for statistical analysis. ResultsTNF-α increased the secretion of lipase, IL-1β, and IL-6. Pterostilbene treatment inhibited TNF-α–induced secretion of lipase (P<0.01 and P<0.001), IL-1β (P<0.05), and IL-6 (P<0.05 and P<0.01). Inhibition of STAT3 by pterostilbene occurred with treatment doses of 25 and 50μM (P<0.001 and P<0.01). ConclusionsThe dietary compound pterostilbene exerts an anti-inflammatory effect in pancreatitis through downregulation of STAT3 and decreased the secretion of lipase, IL-1β, and IL-6. Pterostilbene's amelioration of pancreatitis in vitro makes it an advantageous anti-inflammatory agent. Further studies are necessary to determine pterostilbene's role as a protective or therapeutic agent in pancreatitis.

Identification of virulence markers in clinically relevant strains of Acinetobacter genospecies.

Nine Acinetobacter strains from patients and hospital environment were analyzed for virulence markers, quorum sensing signal production, and the presence of luxI and luxR genes. The strains had several properties in common: growth in iron limited condition, biofilm formation, and no active protease secretion. Significantly higher catechol production was determined in patient isolates (P < 0.03), but other invasiveness markers, such as lipase secretion, amount of biofilm, cell motility, antibiotic resistance, and hemolysin production, showed large variability. Notably, all members of the so-called A. calcoaceticus-A. baumannii complex, regardless of whether the source was a patient or environmental, secreted mediumto long-chain N-acyl homoserine lactones (AHL) and showed blue light inhibition of cell motility. In these strains, a luxI homologue with a homoserine lactone synthase domain and a luxR putative regulator displaying the typical AHL binding domain were identified.

Lipase activity in Thermus thermophilus HB8: Purification and characterization of the extracellular enzyme

In this study, the lipolytic activity of Thermus thermophilus HB8 was examined. The addition of various oils increased the production of extracellular lipolytic activity, while a combination of olive oil and glucose increased both extracellular and intracellular lipolytic activity. The oxygen transfer rate had a significant influence on both biomass and production of extra- or intra-cellular lipolytic activity. The formation of white halos due to the hydrolysis of oleic acid ester (Tween 80) in agar plates containing Nile Blue and the formation of Ca2+-oleate indicated the secretion of lipase. When the cell-free supernatant of cells grown in basal reach medium or the corresponding intracellular extract were electrophoresed under denatured and renatured conditions, using α-naphthyl acetate and Fast Blue RR, major bands at 56 kDa or 62 and 32 kDa were observed, respectively. The 56 kDa extracellular enzyme was partial purified and characterized. Its peak of activity occurred at 80°C and pH 7.0, while the T1/2 was 1 h at 100°C. The Km of the partial purified enzyme was 1 mM and the Vmax was 0.044 U/mL/min when using p-nitrophenyl laurate as substrate. The presence of Ca2+ and Hg2+ stimulated lipase activity, whereas Zn2+, Co2+, or EDTA inhibited lipase activity. The highest activity was observed in the presence of coconut oil and p-nitrophenyl laurate (pNPL). Purified lipase was the most stable in the presence of various organic solvents, such as pentanol, chloroform and n-dodecane. Because of the superior thermostability and stability in the presence of organic solvents of T. thermophilus extracellular lipase, this lipase holds great promise for use in industrial applications.

Regulation of pancreatic exocrine secretion in goats: differential effects of short‐ and long‐term duodenal phenylalanine treatment

Four yearling goats (31.2 ± 2.5 kg), surgically fitted with common bile duct reentrant and duodenal catheter, were used in two 4 × 4 Latin square design experiments to investigate the effects of duodenal infusion of phenylalanine for different times on pancreatic exocrine secretion (PES). In experiment 1 (the long-term experiment), goats were duodenally infused with 0, 2, 4 or 8 g/day phenylalanine for 14 day. Pancreatic juice and jugular blood samples were collected over 1-h intervals for 6 h daily from day 11 to day 14 to encompass a 24-h day. In experiment 2 (the short-term experiment), goats were infused with phenylalanine for 10 h continuously at the same infusion rate as experiment 1 after feed deprivation for 24 h repeated every 10 day. Pancreatic juice and blood samples were collected at 0, 1, 2, 4, 6, 8 and 10 h of infusion. The volume and pH of pancreatic juice were measured, and a 5% subsample was composited and frozen until analysis of enzyme activities. Plasma was frozen until analysis of insulin and cholecystokinin (CCK). In experiment 1, pancreatic juice, α-amylase secretion and plasma CCK concentration responded quadratically (p < 0.05), with the top value observed at the 2 g/day phenylalanine. Trypsin secretion had a quadratic response (p < 0.05), with secretion increasing up to 4 g/day phenylalanine and decreasing thereafter. Phenylalanine linearly decreased pancreatic protein and lipase secretion (p < 0.05). The results of correlation analysis showed significant correlations (p < 0.05) between plasma CCK concentration and secretion of α-amylase and trypsin. However, the short-term phenylalanine infusion did not influence (p > 0.05) pancreatic juice, protein, α-amylase, lipase, trypsin secretion and plasma CCK concentration. These results indicate PES of ruminants is stimulated by phenylalanine and is potentially mediated by CCK in the long-term duodenal infusion treatment, but is not influenced by phenylalanine in the short-term duodenal infusion treatment.

Enhancing functional production of a chaperone-dependent lipase in Escherichia coli using the dual expression cassette plasmid

sBackgroundThe lipase subfamilies I.1 and I.2 show more than 33% homology in the amino acid sequences and most members share another common property that their genes are clustered with the secondary genes whose protein products are required for folding the lipase into an active conformation and secretion into the culture medium. In previous studies, the lipase (LipA) and its chaperone (LipB) from Ralstonia sp. M1 were overexpressed in E. coli and the lipase was successfully refolded in vitro. The purpose of this study was to enhance the production of the active lipase LipA from Ralstonia sp. M1 in the heterologous host E. coli without in vitro refolding process, using two-plasmid co-expression systems and dual expression cassette plasmid systems.ResultsTo produce more active lipase from Ralstonia sp. M1 in E. coli without in vitro refolding process but with the help of overexpression of the chaperone (LipB1 and LipB3 corresponding to 56-aa truncated and 26-aa truncated chaperone LipB), six different expression systems including 2 two-plasmid co-expression systems (E. coli BL21/pELipABa + pELipB1k and BL21/pELipABa + pELipB3k) and 4 dual expression cassette plasmid systems (BL21/pELipAB-LipB1a, BL21/pELipAB-LipB3a, BL21/pELipA-LipB1a, and BL21/pELipA-LipB3a) were constructed. The two-plasmid co-expression systems (E. coli BL21/pELipABa + pELipB1k and BL21/pELipABa + pELipB3k) produced the active lipase at a level of 4 times as high as the single expression cassette plasmid system E. coli BL21/pELipABa did. For the first time, the dual expression cassette plasmid systems BL21/pELipAB-LipB1a and BL21/pELipAB-LipB3a yielded 29- and 19-fold production of the active lipase in comparison with the single expression cassette plasmid system E. coli BL21/pELipABa, respectively. Although the lipase amount was equally expressed in all these expression systems (40% of total cellular protein) and only a small fraction of the overexpressed lipase was folded in vivo into the functional lipase in soluble form whereas the main fraction was still inactive in the form of inclusion bodies. Another controversial finding was that the dual expression cassette plasmid systems E. coli BL21/pELipAB-LipB1a and E. coli/pELipAB-LipB3a secreted the active lipase into the culture medium of 51 and 29 times as high as the single expression cassette plasmid system E. coli pELipABa did, respectively, which has never been reported before. Another interesting finding was that the lipase form LipA6xHis (mature lipase fused with 6× histidine tag) expressed in the dual expression cassette plasmid systems (BL21/pELipA-LipB1a and BL21/pELipA-LipB3a) showed no lipase activity although the expression level of the lipase and two chaperone forms LipB1 and LipB3 in these systems remained as high as that in E. coli BL21/pELipABa + pELipB1k, BL21/pELipABa + pELipB3k, BL21/pELipAB-LipB1a, and BL21/pELipAB-LipB3a. The addition of Neptune oil or detergents into the LB medium increased the lipase production and secretion by up to 94%.ConclusionsOur findings demonstrated that a dual expression cassette plasmid system E. coli could overproduce and secrete the active chaperone-dependent lipase (subfamilies I.1 and I.2) in vivo and an improved dual expression cassette plasmid system E. coli could be potentially applied for industrial-scale production of subfamily I.1 and I.2 lipases.

Purification and characterization of a secretory lipolytic enzyme, MgLIP2, from Malassezia globosa

Malassezia globosa is a lipid-dependent yeast that is found on the human skin and is associated with various skin disorders, including dandruff and seborrhoeic dermatitis (SD). Despite its important role in skin diseases, the molecular basis for its pathogenicity is poorly understood. The current hypothesis is that dandruff and SD are linked to fatty acid metabolism and secretory lipolytic enzymes, which hydrolyse sebaceous lipids and release irritating free fatty acids. A previous genomic analysis of M. globosa identified a family of 13 homologous genes predicted to encode secreted lipases. We have also reported that M. globosa had significantly higher extracellular lipase activity compared with other species. To identify the major secretory lipases of this yeast during its growth, we successfully purified and characterized an extracellular lipase MgLIP2. Based on MALDI-TOF MS, the peptide mass fingerprint of a tryptically digested protein MgLIP2 corresponded to ORF MGL_4054 of M. globosa. This lipase showed high esterase activity against 4-nitrophenyl palmitate and 1-naphthyl palmitate but not 1-naphthyl acetate. This enzyme had optimal activity at 30 °C and pH 5.0. Furthermore, the activity significantly increased in the presence of Triton X-100 and was partially inhibited by PMSF but was unaffected by univalent and divalent metal ions.

Lipase from Aeromonas caviae AU04: Isolation, purification and protein aggregation

Abstract An extracellular thermostable lipase producing bacteria was isolated and identified as Aeromonas caviae AU04. The optimum temperature and pH for lipase secretion was found to be 30 °C and pH 7.0, respectively. The enzyme was purified 3.3-fold with 28.7% recovery by ammonium sulphate precipitation and hydrophobic interaction chromatography. The enzyme showed a strong tendency to aggregate in solution. The purified enzyme did not penetrate into stacking gel and also eluted in the void volume of the column in gel permeation chromatography. The aggregated lipase was partially solubilised by the addition of non-ionic detergent Triton X-100. The optimum temperature and pH for activity of the enzyme was found to be 60 °C and pH 7.0, respectively. The purified lipase was found to be stable in the presence of hydrophobic organic solvents with log P value more than 2.0. Incubation of enzyme with 1-hexane and toluene increased the lipase activity two fold. The thermostability and tolerance to organic solvents makes it an attractive and promising biocatalyst for enzyme mediated synthesis.