Monoacylglycerol lipase from marine Geobacillus sp. showing lysophospholipase activity and its application in efficient soybean oil degumming.

Enzymatic degumming is an essential refining process to improve oil quality. In this study, a monoacylglycerol lipase GMGL was derived from marine Geobacillus sp., and was found that not only took monoacylglycerol (MAG) as substrate, but also had activity toward lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE) and glycerolphosphatidylcholine (GPC). Binding free energy showed LPC and LPE could bind with enzyme stably as MAG. It presented great potential in the field of enzymatic degumming. The phosphorus content in crude soybean oil decreased from 680.50 to 2.01 mg/kg and the yield of oil reached to 98.80 % after treating with phospholipase A1 (Lecitase Ultra) combined with lipase GMGL. An ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was developed to identify 21 differential phospholipids between crude soybean oil and enzymatic treatment. This work might shed some light on understanding the catalytic mechanism of monoacylglycerol lipase and provide an effective strategy for enzymatic degumming.

Recombinant expression, characterization, and application of a phospholipase B from Fusarium oxysporum.

In this study, a gene encoding a putative lipase from Fusarium oxysporum was optimized via codon optimization and expressed in Pichia pastoris KM71. The gene product was identified as a phospholipase B (PLB). The engineered P. pastoris was further cultured in a 3.6-L bioreactor. After optimization of the induction conditions, this system produced 6.6mgmL-1 protein and 6503.8UmL-1 PLB activity in the culture medium. Efficient expression of this PLB in P. pastoris should reduce the costs of production and application. The purified enzyme, with a specific activity of 1170Umg-1, was optimally active at pH 5.0 and 55°C. The results of a degumming experiment performed using the recombinant PLB showed that the phosphorus content of a test oil was decreased from 75.88ppm to 3.3ppm in 2h under optimal reaction conditions. This study provides a basis for the industrial use of F. oxysporum PLB in oil degumming applications.

Lipid Regulation of Acrosome Exocytosis.

Lipids are critical regulators of mammalian sperm function, first helping prevent premature acrosome exocytosis, then enabling sperm to become competent to fertilize at the right place/time through the process of capacitation, and ultimately triggering acrosome exocytosis. Yet because they do not fit neatly into the "DNA--RNA-protein" synthetic pathway, they are understudied and poorly understood. Here, we focus on three lipids or lipid classes-cholesterol, phospholipids, and the ganglioside G(M1)--in context of the modern paradigm of acrosome exocytosis. We describe how these various- species are precisely segregated into membrane macrodomains and microdomains, simultaneously preventing premature exocytosis while acting as foci for organizing regulatory and effector molecules that will enable exocytosis. Although the mechanisms responsible for these domains are poorly defined, there is substantial evidence for their composition and functions. We present diverse ways that lipids and lipid modifications regulate capacitation and acrosome exocytosis, describing in more detail how removal of cholesterol plays a master regulatory role in enabling exocytosis through at least two complementary pathways. First, cholesterol efflux leads to proteolytic activation of phospholipase B, which cleaves both phospholipid tails. The resultant changes in membrane curvature provide a mechanism for the point fusions now known to occur far before a sperm physically interacts with the zona pellucida. Cholesterol efflux also enables G(M1) to regulate the voltage-dependent cation channel, Ca(V)2.3, triggering focal calcium transients required for acrosome exocytosis in response to subsequent whole-cell calcium rises. We close with a model integrating functions for lipids in regulating acrosome exocytosis.

Characterization of a novel thermophilic phospholipase B from Thermotoga lettingae TMO: applicability in enzymatic degumming of vegetable oils.

A novel phospholipase B (TLPLB) from Thermotoga lettingae TMO has been cloned, functionally overexpressed in Escherichia coli and purified to homogeneity. Gas chromatography indicated that the enzyme could efficiently hydrolyze both the sn-1 and sn-2 ester bonds of 1-palmitoyl-2-oleoyl phosphatidylcholine as phospholipase B. TLPLB was optimally active at 70 °C and pH 5.5, respectively. Its thermostability is relatively high with a half-life of 240 min at 90 °C. TLPLB also displayed remarkable organic solvent tolerance and maintained approximately 91-161 % of its initial activity in 20 and 50 % (v/v) hydrophobic organic solvents after incubation for 168 h. Furthermore, TLPLB exhibited high degumming activity towards rapeseed, soybean, peanut and sunflower seed oils, where the phosphorus contents were decreased from 225.2, 189.3, 85.6 and 70.4 mg/kg to 4.9, 4.7, 3.2 and 2.2 mg/kg within 5 h, respectively. TLPLB could therefore be used for the degumming of vegetable oils.

Reminiscence of phospholipase B in Penicillium notatum.

Since the phospholipase B (PLB) was reported as a deacylase of both lecithin and lysolecithin yielding fatty acids and glycerophosphocholine (GPC), there was a question as to whether it is a single enzyme or a mixture of a phospholipase A2 (PLA2) and a lysophospholipase (LPL). We purified the PLB in Penicillium notatum and showed that it catalyzed deacylation of sn-1 and sn-2 fatty acids of 1,2-diacylphospholipids and also sn-1 or sn-2 fatty acids of 1- or 2-monoacylphospholipids (lysophospholipids). Further, it also has a monoacyllipase activity. The purified PLB is a glycoprotein with m.w. of 91,300. The sugar moiety is M9 only and the protein moiety consists of 603 amino acids. PLB, different from PLA2, shows other enzymatic activities, such as transacylase, lipase and acylesterase. PLB activity is influenced by various substances, e.g. detergents, deoxycholate, diethylether, Fe(3+), and endogenous protease. Therefore, PLB might have broader roles than PLA2 in vivo. The database shows an extensive sequence similarity between P. notatum PLB and fungal PLB, cPLA2 and patatin, suggesting a homologous relationship. The catalytic triad of cPLA2, Ser, Asp and Arg, is also present in P. notatum PLB. Other related PLBs, PLB/Lipases are discussed.

Degumming of vegetable oils by a novel phospholipase B from Pseudomonas fluorescens BIT-18.

Pseudomonas fluorescens BIT-18 was isolated from soil near a vegetable oil factory and shown to produce a B-type phospholipase. The enzyme was partially purified by ammonium sulfate precipitation. Gas chromatography demonstrated that the enzyme preparation hydrolyzed both the 1- and 2-ester bonds of phosphatidylcholine. When degumming of soybean, rapeseed, and peanut oil was performed with this enzyme preparation, oils with phosphorous contents lower than 5mg/kg were obtained after 5h of enzyme treatment at 40°C. The enzyme preparation did not show lipase activity, thus free fatty acids were only generated from the phospholipids. Therefore, this novel phospholipase B is potentially useful for the refining of high-quality oils with attractive yields.

Secretory phospholipases A2 induce neurite outgrowth in PC12 cells through lysophosphatidylcholine generation and activation of G2A receptor.

We previously demonstrated that secretory phospholipase A2 (sPLA2) and lysophosphatidylcholine (LPC) exhibit neurotrophin-like neuritogenic activity in the rat pheochromocytoma cell line PC12. In this study, we further analyzed the mechanism whereby sPLA2 displays neurite-inducing activity. Exogenously added mammalian group X sPLA2 (sPLA2-X), but not group IB and IIA sPLA2s, induced neuritogenesis, which correlated with the ability of sPLA2-X to liberate LPC into the culture media. In accordance, blocking the effect of LPC by supplementation of bovine serum albumin or phospholipase B attenuated neuritogenesis by sPLA2 or LPC. Overproduction or suppression of G2A, a G-protein-coupled receptor involved in LPC signaling, resulted in the enhancement or reduction of neuritogenesis induced by sPLA2 treatment. These results indicate that the neuritogenic effect of sPLA2 is mediated by generation of LPC and subsequent activation of G2A.

Characterisation and expression of phospholipases B from the opportunistic fungus Aspergillus fumigatus.

The phospholipase B family (PLB) are enzymes sharing phospholipase (PL), lysophospholipase (LPL) and lysophospholipase-transacylase (LPTA) activities. They have been shown to be important virulence factors in several human fungal pathogens including Candida albicans and Cryptococcus neoformans. Aspergillus fumigatus, a human opportunistic fungal pathogen leading to a high rate of mortality in immunosuppressed patients is known to possess an extracellular phospholipase B activity. In this paper, we report the molecular characterisation of three PLB genes from A. fumigatus (afplb) using degenerate primers in PCR amplification and data from the A. fumigatus genome project. They are expressed at 37 degrees C, and two of them (afplb1 and afplb3) are induced by lecithin. They encode proteins of 633, 588 and 630 amino acids, respectively, presenting together a T-Coffee score of 81. They also possess the amino acid triad responsible for enzymatic activity in the mammalian cytosolic PLA2 and other fungal PLBs. AfPLB1 and afPLB3 are secreted with a cleaved signal peptide. The complete cDNA sequences were obtained by RACE-PCR for the two secreted afPLBs and probably account for the extracellular phospholipase activity previously reported in the culture media of A. fumigatus.

An alternative splicing form of phosphatidylserine-specific phospholipase A1 that exhibits lysophosphatidylserine-specific lysophospholipase activity in humans.

Phosphatidylserine-specific phospholipase A1 (PS-PLA1), which acts specifically on phosphatidylserine (PS) and 1-acyl-2-lysophosphatidylserine (lyso-PS) to hydrolyze fatty acids at the sn-1 position of these phospholipids, was first identified in rat platelets (Sato, T., Aoki, J., Nagai, Y., Dohmae, N., Takio, K., Doi, T., Arai, H., and Inoue, K. (1997) J. Biol. Chem. 272, 2192-2198). In this study we isolated and sequenced cDNA clones encoding human PS-PLA1, which showed 80% homology with rat PS-PLA1 at the amino acid level. In addition to an mRNA encoding a 456-amino acid product (PS-PLA1), an mRNA with four extra bases inserted at the boundary of the exon-intron junction was detected in human tissues and various human cell lines. This mRNA is most probably produced via an alternative use of the 5'-splicing site (two consensus sequences for RNA splicing occur at the boundary of the exon-intron junction) and encodes a 376-amino acid product (PS-PLA1DeltaC) that lacks two-thirds of the C-terminal domain of PS-PLA1. Unlike PS-PLA1, PS-PLA1DeltaC hydrolyzed exclusively lyso-PS but not PS appreciably. Any other phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), and their lyso derivatives were not hydrolyzed at all. These data demonstrated that PS-PLA1DeltaC exhibits lyso-PS-specific lysophospholipase activity and that the C-terminal domain of PS-PLA1 is responsible for recognizing diacylphospholipids. In addition, human PS-PLA1 gene was mapped to chromosome 3q13.13-13.2 and was unexpectedly identical to the nmd gene, which is highly expressed in nonmetastatic melanoma cell lines but poorly expressed in metastatic cell lines (van Groningen, J. J., Bloemers, H. P., and Swart, G. W. (1995) Cancer Res. 55, 6237-6243).

Ectopic epididymal expression of guinea pig intestinal phospholipase B. Possible role in sperm maturation and activation by limited proteolytic digestion.

Guinea pig intestinal phospholipase B is a calcium-independent phospholipase hydrolyzing sequentially the acyl ester bonds at sn-2 and sn-1 positions of glycerophospholipids, promoting the formation of sn-glycero-3-phosphocholine from phosphatidylcholine. This 140-kDa glycoprotein from the brush border membrane of differentiated enterocytes contributes to lipid digestion as an ectoenzyme. The cDNA coding for guinea pig phospholipase B was revealed to be the homologue of AdRab-B, an mRNA appearing in rabbit upon intestine development. The sequence predicts a polypeptide of 1463 amino acids displaying four homologous repeats, two of them containing the lipase consensus sequence GXSXG. A 5-kilobase transcript was particularly abundant in mature ileal and jejunal enterocytes but was also detected in epididymis, where phospholipase B displayed a higher molecular mass (170 kDa versus 140 kDa in intestine), with no obvious evidence for enzyme activity. Trypsin treatment of phospholipase B immunoprecipitated from epididymal membranes reduced its size to 140 kDa, coinciding with the appearance of a significant phospholipase A2 activity. The same results were obtained in COS cells transfected with phospholipase B cDNA. Since sn-glycero-3-phosphocholine present at high concentrations in seminal plasma mainly stems from epididymis, this suggests a possible role of phospholipase B in male reproduction. This novel localization also unravels a mechanism of phospholipase B activation by limited proteolysis involving either trypsin in the intestinal lumen or a trypsin-like endopeptidase in the male reproductive tract.

cDNA cloning and expression of a novel family of enzymes with calcium-independent phospholipase A2 and lysophospholipase activities.

Previous studies have suggested that activation of calcium-independent PLA2 (CaIPLA2) is an early event in cell death after hypoxic injury in proximal tubule cells. An approximately 28-kD CaIPLA2 with preferential activity toward plasmalogen phospholipids has been recently purified from rabbit kidney cortex (D. Portilla and G. Dai, J Biol Chem 271, 15,451-15,457, 1996). Their report describes the cloning of a full-length rat cDNA encoding CaIPLA2, using sequences derived from the purified rabbit kidney cortex enzyme. In addition, cDNA from rabbit kidney that encode the rabbit homologue of the enzyme and a closely related isoform were isolated. The rat cDNA is predicted to encode an approximately 24-kD protein, and each cDNA contains the sequence G-F-S-Q-G, which fits the active site consensus sequence G-X-S-X-G of carboxylesterases. Several lines of evidence (DNA sequence comparison, Southern blot analysis, and examination of the expressed sequence tag database) show that CaIPLA2 enzymes are encoded by a multigene family in rats, mice, rabbits, and humans. Northern analysis of various tissues from the rat indicated that the CaIPLA2 gene is ubiquitously expressed, with highest mRNA abundance observed in the kidney and small intestine. The rat CaIPLA2 cDNA, when expressed in a baculovirus expression system, and the purified rabbit kidney cortex protein exhibit both CaIPLA2 and lysophospholipase activities. The cloned CaIPLA2 cDNA are expected to aid in understanding the role of CaIPLA2 in cell death after hypoxic/ischemic cell injury.

Effects of dietary n-6/n-3 ratios on lipid and prostaglandin E2 metabolism in rat gastric mucosa.

The effects of increased dietary n-3 polyunsaturated fatty acids on gastric mucosal lipid metabolism were studied in rats fed for 8 weeks with different combinations of fish and corn oils. Lipid composition, ex vivo prostaglandin E2 (PGE2) production and enzymatic activities involved in phospholipid metabolism and peroxisomal oxidative catabolism of fatty acids and PGE2 were examined. With dietary n-6/n-3 compositional ratios ranging between 75 and 3.3 it was observed that: (i) the arachidonic acid-to-eicosapentaenoic acid ratio (AA/EPA) fell from infinity to 3.1 and 5.1 in phosphatidylcholines (PC) and phosphatidylethanolamines (PE), respectively; (ii) ex vivo production of PGE2 was lowered by a factor of about 2; and (iii) gastric phospholipase A2 activity was enhanced by 32%. With dietary n-6/n-3 ratio lower than 3.3, stimulation of PGE2-CoA oxidase activity was observed whilst the PGE2 level remained constant. These data suggest that the fish oil-induced decrease in ex vivo PGE2 production is more closely related to a decrease in the membrane AA level than to an enhanced oxidative catabolism of PGE2.

Effect of dietary lipids on the lipid composition and phospholipid deacylating enzyme activities of rat heart.

Rats were fed lard-enriched (17%) or corn oil-enriched (17%) diets and were compared with rats fed a low fat (4.5%) diet. Cardiac protein, DNA, phospholipid (PL) and fatty acid (FA) compositions were analyzed. Neutral phospholipase A, lysophospholipase and creatine kinase activities in the membrane and cytosolic compartments were also investigated. No significant modification of cardiac protein, DNA nor PL was observed among the three groups. Some alterations appeared in the FA composition. A lard-enriched diet induced a significant increase of 22:5n-3 and 22:6n-3 in heart phosphatidylcholine (PC) and phosphatidylethanolamine (PE), whereas a linoleic acid-rich diet induced a specific increase of 22:4n-6 and 22:5n-6 in these two major PL. Compared to rats fed the low fat diet, membrane-associated phospholipase A activity, measured by endogenous hydrolysis of membrane PC and PE, showed a significant increase (+45%) for both PL in rats fed corn oil. However, the activity of membrane-associated phospholipases, measured with exogenous [1-14C]dioleoyl PC, was not different among the three groups of rats. Cytoplasmic activity was decreased in rats fed corn oil, and lysophospholipase and creatine phosphate kinase activities were not significantly affected by diet. FA modification of the long chain n-6 FA induced by corn oil may be responsible for the observed increase in phospholipase activity. Physiological implications are suggested in terms of membrane degradation and prostaglandin production.

Comparison of vespid venoms collected by electrostimulation and by venom sac extraction.

Venoms of three vespid species, yellow jacket, bald-faced hornet, and yellow hornet, obtained by either electrostimulation of venom sac extraction were compared with regard to their enzymatic activity, antigenicity, and allergenicity. Phospholipase a, phospholipase B, and hyaluronidase enzymatic activities were present in all six preparations. The activity of venom sac extracts lay in the range found in different batches of venoms obtained by electrostimulation for each species. Analysis of sera from vespid-sensitive patients in the radioallergosorbent test (RAST) with discs coupled with either venom sac extracts or venoms obtained by electrostimulation showed a good correlation of the results within all three species (r = 0.95). In RAST inhibition the potency of venom sac extracts and venom obtained by electrostimulation was similar for each species. Analysis of rabbit antisera to the six preparations revealed similar patterns in immunoelectrophoresis and identity reactions between the major antigens within each species. Tissue protein contamination was detected in all venom sac extracts but not in venoms obtained by electrostimulation.