Search

Abstract Phospholipase A1 (PLA1) is a kind of specific phospholipid hydrolase widely used in food, medical, textile. However, limitations in its expression and enzymatic activity have prompted the investigation of the phospholipase-assisting protein PlaS. In this study, we elucidate the role of PlaS in enhancing the expression and activity of PlaA1 through N-terminal truncation. Our research demonstrates that truncating the N-terminal region of PlaS effectively overcomes its inhibitory effect on host cells, resulting in improved cell growth and increased protein solubility of the protein. The yeast two-hybrid assay confirms the interaction between PlaA1 and N-terminal truncated PlaS (∆N27 PlaS), highlighting their binding capabilities. Furthermore, in vitro studies using Biacore analysis reveal a concentration-dependent and specific binding between PlaA1 and ∆N27 PlaS, exhibiting high affinity. Molecular docking analysis provides insights into the hydrogen bond interactions between ∆N27 PlaS and PlaA1, identifying key amino acid residues crucial for their binding. Finally, the enzyme activity of PLA1 was boost to 8.4 U/mL by orthogonal test. Study significantly contributes to the understanding of the interaction mechanism between PlaS and PlaA1, offering potential strategies for enhancing PlaA1 activity through protein engineering approaches.

Objective To identify and analyze the phospholipase A (PLA) gene family in Talaromyces marneffei (T. marneffei), providing the basis for further research on the potential pathogenic effect and mechanism of T. marneffei PLA. Methods The members of the PLA gene family in T. marneffei were genome-wide identified by using bioinformatics, and their gene structure, chromosome distribution, protein physicochemical property and structure, conserved motif, as well as phyletic evolution, were analyzed. The expression levels of PLA genes in T. marneffei under macrophage treatment were analyzed using the qRT-PCR method. Results A total of eight PLA genes were identified from the genome of T. marneffei, unevenly located on 5 chromosomes without any tandem duplications or fragment duplications. The amino acid residues of PLA proteins ranged from 577 to 1546, molecular weights from 65 120 to 170 690, and the isoelectric point from 4.47 to 9.28. All PLA proteins were hydrophilic, with all but PLA3 classified as unstable hydrophilic proteins. Phylogenetic analysis revealed that PLA was divided into two subfamilies: PLA2 and patatin-like phospholipase, consisting of 3 and 5 members respectively. Within the same subfamily, gene structures, protein-conserved motifs, and protein structures were relatively similar, whereas significant differences existed between different subfamilies. qRT PCR results showed that the expression levels of PLA3, PLA6, and PLA8 were upregulated in T. marneffei infected with mouse macrophages. Conclusions PLA3, PLA6, and PLA8 may be related to the survival and reproduction of T. marneffei in macrophages. [ABSTRACT FROM AUTHOR]

Phospholipase A1 (PLA1) is a kind of specific phospholipid hydrolase widely used in food, medical, textile. However, limitations in its expression and enzymatic activity have prompted the investigation of the phospholipase-assisting protein PlaS. In this study, we elucidate the role of PlaS in enhancing the expression and activity of PlaA1 through N-terminal truncation. Our research demonstrates that truncating the N-terminal region of PlaS effectively overcomes its inhibitory effect on host cells, resulting in improved cell growth and increased protein solubility of the protein. The yeast two-hybrid assay confirms the interaction between PlaA1 and N-terminal truncated PlaS (∆N27 PlaS), highlighting their binding capabilities. Furthermore, in vitro studies using Biacore analysis reveal a concentration-dependent and specific binding between PlaA1 and ∆N27 PlaS, exhibiting high affinity. Molecular docking analysis provides insights into the hydrogen bond interactions between ∆N27 PlaS and PlaA1, identifying key amino acid residues crucial for their binding. Finally, the enzyme activity of PLA1 was boost to 8.4 U/mL by orthogonal test. Study significantly contributes to the understanding of the interaction mechanism between PlaS and PlaA1, offering potential strategies for enhancing PlaA1 activity through protein engineering approaches. [ABSTRACT FROM AUTHOR]

The food enzyme with phospholipase A1 (phosphatidycholine 1‐acylhydrolase, EC 3.1.1.32) and lysophospholipase (2‐lysophosphatidylcholine acylhydrolase, EC 3.1.1.5) activities is produced with the genetically modified Aspergillus niger strain PLN by DSM. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. It is intended to be used for the production of refined edible fats and oils by degumming. Since residual amounts of total organic solids are removed during this process, dietary exposure was not calculated and toxicological studies were considered unnecessary for the assessment of this food enzyme. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no matches were found. The Panel considered that the risk of allergic reactions upon dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use. [ABSTRACT FROM AUTHOR]

This study investigated the incorporation of nervonic acid into the chemical structure of phosphatidylcholine via a lipase-catalyzed acidolysis reaction to obtain a functional phospholipid. Lipase immobilization was conducted, and Amberlite XAD7-HP was selected as a carrier to immobilize phospholipase A1 (PLA1) for subsequent experiments. The main acidolysis reaction parameters, including enzyme load, substrate ratio, temperature, and water content, were studied against the reaction time. The optimum reaction conditions obtained were enzyme load, 20%; reaction temperature, 55 °C; water content, 1%; and reaction time, 9 h. The maximum incorporation of nervonic acid into phosphatidylcholine was 48 mol%, with PC recovery at 61.6 mol%. The positional distribution of structured phosphatidylcholine shows that nervonic acid was found in the sn-1 position due to enzyme specificity and in the sn-2 position, possibly due to acyl migration. [ABSTRACT FROM AUTHOR]

The phospholipid and free fatty acid (FFA) composition of neuronal membranes plays a crucial role in learning and memory, but the mechanisms through which neuronal activity affects the brain's lipid landscape remain largely unexplored. The levels of saturated FFAs, particularly of myristic acid (C14:0), strongly increase during neuronal stimulation and memory acquisition, suggesting the involvement of phospholipase A1 (PLA1) activity in synaptic plasticity. Here, we show that genetic ablation of the PLA1 isoform DDHD2 in mice dramatically reduces saturated FFA responses to memory acquisition across the brain. Furthermore, DDHD2 loss also decreases memory performance in reward-based learning and spatial memory models prior to the development of neuromuscular deficits that mirror human spastic paraplegia. Via pulldown-mass spectrometry analyses, we find that DDHD2 binds to the key synaptic protein STXBP1. Using STXBP1/2 knockout neurosecretory cells and a haploinsufficient STXBP1+/− mouse model of human early infantile encephalopathy associated with intellectual disability and motor dysfunction, we show that STXBP1 controls targeting of DDHD2 to the plasma membrane and generation of saturated FFAs in the brain. These findings suggest key roles for DDHD2 and STXBP1 in lipid metabolism and in the processes of synaptic plasticity, learning, and memory. Synopsis: The mechanisms by which neuronal activity affects lipid metabolism in the brain remain little understood. This study identifies the phospholipase A1 isoform DDHD2 and the key synaptic protein STXBP1 as important regulators of lipid metabolism in synaptic plasticity and long-term memory in mice. DDHD2 produces saturated free fatty acids (FFAs) in response to long-term memory acquisition. DDHD2 is targeted to the synapse via an interaction with STXBP1. Ablation of DDHD2 in mice diminishes saturated FFA responses to memory acquisition and leads to progressive neuromuscular and cognitive/memory deficits. STXBP1-haploinsufficient mice similarly exhibit reduced saturated FFAs. Ablation of phospholipase D1 has little effect on saturated FFAs and long-term memory. The phospholipase A1 isoform DDHD2 and the key synaptic protein STXBP1 control lipid metabolism in learning and long-term memory. [ABSTRACT FROM AUTHOR]

Although many important discoveries have been made regarding the jasmonate signaling pathway, how jasmonate biosynthesis is initiated is still a major unanswered question in the field. Previous evidences suggest that jasmonate biosynthesis is limited by the availability of fatty acid precursor, such as ⍺-linolenic acid (⍺-LA). This indicates that the lipase responsible for releasing α-LA in the chloroplast, where early steps of jasmonate biosynthesis take place, is the key initial step in the jasmonate biosynthetic pathway. Nicotiana benthamiana glycerol lipase A1 (NbGLA1) is homologous to N. attenuata GLA1 (NaGLA1) which has been reported to be a major lipase in leaves for jasmonate biosynthesis. NbGLA1 was studied for its potential usefulness in a species that is more common in laboratories. Virus-induced gene silencing of both NbGLA1 and NbGLA2, another homolog, resulted in more than 80% reduction in jasmonic acid (JA) biosynthesis in wounded leaves. Overexpression of NbGLA1 utilizing an inducible vector system failed to increase JA, indicating that transcriptional induction of NbGLA1 is insufficient to trigger JA biosynthesis. However, co-treatment with wounding in addition to NbGLA1 induction increased JA accumulation several fold higher than the gene expression or wounding alone, indicating an enhancement of the enzyme activity by wounding. Domain-deletion of a 126-bp C-terminal region hypothesized to have regulatory roles increased NbGLA1-induced JA level. Together, the data show NbGLA1 to be a major lipase for wound-induced JA biosynthesis in N. benthamiana leaves and demonstrate the use of inducible promoter-driven construct of NbGLA1 in conjunction with its transient expression in N. benthamiana as a useful system to study its protein function. [ABSTRACT FROM AUTHOR]

This study investigated the incorporation of nervonic acid into the chemical structure of phosphatidylcholine via a lipase-catalyzed acidolysis reaction to obtain a functional phospholipid. Lipase immobilization was conducted, and Amberlite XAD7-HP was selected as a carrier to immobilize phospholipase A1 (PLA1) for subsequent experiments. The main acidolysis reaction parameters, including enzyme load, substrate ratio, temperature, and water content, were studied against the reaction time. The optimum reaction conditions obtained were enzyme load, 20%; reaction temperature, 55 °C; water content, 1%; and reaction time, 9 h. The maximum incorporation of nervonic acid into phosphatidylcholine was 48 mol%, with PC recovery at 61.6 mol%. The positional distribution of structured phosphatidylcholine shows that nervonic acid was found in the sn-1 position due to enzyme specificity and in the sn-2 position, possibly due to acyl migration.

Context: Ethanolamine plasmalogens (EPls) and choline plasmalogens (CPls) are classes of ethanolamine ether phospholipids (ePE) and choline ether phospholipids (ePC), respectively. EPls play crucial roles in maternal and breastfed infant bodies and stimulate gonadotropin secretion by gonadotrophs. Aims: To estimate changes in and importance of plasma concentrations of EPls and CPls, utilising newly developed enzymatic fluorometric assays for ePE and ePC in postpartum Holstein cows. Methods: Plasma samples were collected from 3 weeks before expected parturition until approximately 8 weeks after parturition (16 primiparous and 38 multiparous cows) for analysis. Key results: Plasma concentrations of ePE and ePC, most of which are plasmalogens, declined before and increased after parturition and stabilised near the day of the first postpartum ovulation (1stOV). From weeks 2 to 3 after parturition, third-parity cows exhibited ePE concentrations that were higher than those of other parity cows. The days from parturition to 1stOV correlated with days from parturition to conception. On the day of 1stOV, milk yield correlated with plasma concentration of both ePE and ePC, while ePC concentration correlated negatively with milk fat percentage. At the early luteal phase after 1stOV, plasma ePE concentration correlated with plasma anti-Müllerian hormone concentration (r = 0.39, P < 0.01), and plasma ePC concentration correlated with plasma follicle-stimulating hormone concentration (r = 0.43, P < 0.01). Conclusion: The concentrations of ePE and ePC changed dramatically around parturition and 1stOV, and the concentrations correlated with important parameters for milk production and reproduction. Implications: The blood plasmalogen may play important roles in postpartum dairy cows. Plasmalogens have important roles in the physiological functioning of mothers and breastfeeding infants, and have novel roles in stimulating gonadotrophs. In Holstein dairy cows, the plasma concentrations of ether phospholipids, most of which are plasmalogens, dramatically changed around parturition and around the day of the first postpartum ovulation, and the concentrations correlated with important parameters for milk production and reproduction. Blood plasmalogen may play important roles in postpartum dairy cows. [ABSTRACT FROM AUTHOR]

ABSTRACT Phosphatidylglycerol (PG) is the major membrane phospholipid of Staphylococcus aureus and predominately consists of molecular species with ≥16-carbon acyl chains in the 1-position and anteiso 12(S)-methyltetradecaonate (a15) esterified at the 2-position. The analysis of the growth media for PG-derived products shows S. aureus releases essentially pure 2-12(S)-methyltetradecanoyl-sn-glycero-3-phospho-1′-sn-glycerol (a15:0-LPG) derived from the hydrolysis of the 1-position of PG into the environment. The cellular lysophosphatidylglycerol (LPG) pool is dominated by a15-LPG but also consists of ≥16-LPG species arising from the removal of the 2-position. Mass tracing experiments confirmed a15-LPG was derived from isoleucine metabolism. A screen of candidate secreted lipase knockout strains pinpointed glycerol ester hydrolase (geh) as the gene required for generating extracellular a15-LPG, and complementation of a Δgeh strain with a Geh expression plasmid restored extracellular a15-LPG formation. Orlistat, a covalent inhibitor of Geh, also attenuated extracellular a15-LPG accumulation. Purified Geh hydrolyzed the 1-position acyl chain of PG and generated only a15-LPG from a S. aureus lipid mixture. The Geh product was 2-a15-LPG, which spontaneously isomerizes with time to a mixture of 1- and 2-a15-LPG. Docking PG in the Geh active site provides a structural rationale for the positional specificity of Geh. These data demonstrate a physiological role for Geh phospholipase A1 activity in S. aureus membrane phospholipid turnover. IMPORTANCE Glycerol ester hydrolase, Geh, is an abundant secreted lipase whose expression is controlled by the accessory gene regulator (Agr) quorum-sensing signal transduction pathway. Geh is thought to have a role in virulence based on its ability to hydrolyze host lipids at the infection site to provide fatty acids for membrane biogenesis and substrates for oleate hydratase, and Geh inhibits immune cell activation by hydrolyzing lipoprotein glycerol esters. The discovery that Geh is the major contributor to the formation and release of a15-LPG reveals an unappreciated physiological role for Geh acting as a phospholipase A1 in the degradation of S. aureus membrane phosphatidylglycerol. The role(s) for extracellular a15-LPG in S. aureus biology remain to be elucidated.

The food enzyme phospholipase A1 (phosphatidylcholine 1‐acylhydrolase; EC 3.1.1.32) is produced with the genetically modified Aspergillus oryzae strain NZYM‐PP by Novozymes A/S. The genetic modifications do not give rise to safety concerns. The food enzyme was considered free from viable cells of the production organism and its DNA. It is intended to be used in milk processing for cheese production. Dietary exposure to the food enzyme–total organic solids (TOS) was estimated to be up to 0.012 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 575.1 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 47,925. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no matches were found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood for this to occur is low. The Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use. [ABSTRACT FROM AUTHOR]

• UM2 can switch phospholipase and lipase activities through temperature variations. • UM2 exhibited strong hydrolysis ability towards different phospholipids. • UM2 was effectively used for crude oils with unique phospholipid composition. The limited availability of phospholipase A 1 (PLA 1) has posed significant challenges in enzymatic degumming. In this study, a novel PLA1 (UM2) was introduced to address this limitation, which had a unique thermo-responsive ability to switch phospholipase and lipase activities in response to temperature variations. Remarkably, UM2 displayed an unprecedented selectivity under optimized conditions, preferentially hydrolyzing phospholipids over triacylglycerols—a specificity superior to that of commercial PLA 1. Moreover, UM2 demonstrated high efficiency in hydrolyzing phospholipids with a predilection for phosphatidylcholine (PC) and phosphatidylethanolamine (PE). A practical application of UM2 on crude flaxseed oil led to a dramatic reduction in phosphorus content, plummeting from an initial 384.06 mg/kg to 4.38 mg/kg. Broadening its industrial applicability, UM2 effectively performed enzymatic degumming for other distinct crude vegetable oils with a unique phospholipid composition. Collectively, these results highlighted the promising application of UM2 in the field of oil degumming. [ABSTRACT FROM AUTHOR]

Abstract The food enzyme phospholipase A1 (phosphatidylcholine 1‐acylhydrolase; EC 3.1.1.32) is produced with the genetically modified Aspergillus oryzae strain NZYM‐PP by Novozymes A/S. The genetic modifications do not give rise to safety concerns. The food enzyme was considered free from viable cells of the production organism and its DNA. It is intended to be used in milk processing for cheese production. Dietary exposure to the food enzyme–total organic solids (TOS) was estimated to be up to 0.012 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 575.1 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 47,925. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no matches were found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood for this to occur is low. The Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

The food enzyme phospholipase A1 (phosphatidylcholine 1-acylhydrolase; EC 3.1.1.32) is produced with the genetically modified Aspergillus oryzae strain NZYM-LJ by Novozymes A/S. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. The food enzyme is intended to be used in baking processes. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.09 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 957.3 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, results in a margin of exposure of at least 10,600. A search for similarity of the amino acid sequence of the food enzyme to known allergens was made and one match was found. The Panel considered that, under the intended conditions of use the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood for this to occur is considered to be low. Based on the data provided, the Panel concludes that this food enzyme does not give rise to safety concerns under the intended conditions of use. [ABSTRACT FROM AUTHOR]

Six kinds of Mo‐basing nanomaterials (MoO3, MoO3@Ru, Mo‐PDA, MoPC, MoP, CNT@MoS2) were successfully synthesized, which were employed as carriers to immobilize phospholipase A1 (PLA1) for the hydrolysis of phospholipids (PLs). PLA1 was immobilized by a simple adsorption‐precipitation‐cross‐linking to form an "enzyme net" covering on nanoparticles. The greatest advantage of these nanoparticles was their strong hydrophilic surface. It not only permitted their dispersion in the aqueous phase, but also showed the strong affinity for PLs in the organic phase, because amphiphilic PLs had the polar head group and higher hydrophilicity than other oils components. Michaelis–Menten analysis revealed that higher catalytic activity and enzyme−substrate affinity were observed in several immobilized PLA1 than its free form. MoO3 was confirmed to be the best candidate for carrier. The highest specific activity of MoO3‐immobilized PLA1 reached 43.1 U/mg, which was about 1.8 times higher than that of free PLA1 (24.4 U/mg). In addition, the stability and recycling were also enhanced. The robust immobilized PLA1 was prepared in this work, showing great potential for the enzymatic degumming. [ABSTRACT FROM AUTHOR]

Abstract The food enzyme phospholipase A1 (phosphatidylcholine 1‐acylhydrolase; EC 3.1.1.32) is produced with the genetically modified Aspergillus oryzae strain NZYM‐LJ by Novozymes A/S. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. The food enzyme is intended to be used in baking processes. Dietary exposure to the food enzyme–total organic solids (TOS) was estimated to be up to 0.09 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 957.3 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, results in a margin of exposure of at least 10,600. A search for similarity of the amino acid sequence of the food enzyme to known allergens was made and one match was found. The Panel considered that, under the intended conditions of use the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood for this to occur is considered to be low. Based on the data provided, the Panel concludes that this food enzyme does not give rise to safety concerns under the intended conditions of use.

A new epoxy-based polymerizing/compositing synchronic process was carried out in 3D structure of a melamine sponge. The cured polymer formed a porous and wave-shaped coating on the surface of skeleton in a uniform way. The obtained composite sponge was used as a microflow support for the immobilization of phospholipase A 1 (PLA 1 , from Thermomyces lanuginosus/Fusarium oxysporum) by co-adsorption/cross-linking with polyethylenimine (PEI). The loading amount and the specific activity of immobilized PLA 1 under the optimal immobilization conditions were 45.7 mg/g support and 9.1 U/mg protein. The PLA 1 -immobilized microflow supports were placed in a columnar reactor, in which transesterification was operated in continuous circulation way. The reaction produced DHA/EPA-incorporated phosphatidylcholine, the hydrolysis side reaction could be inhibited by removal of free water in the reaction medium. Under optimized conditions the incorporation percentage of DHA/EPA and PC yield of 54.3% and 82.4% within 18 h at 55 ℃, respectively. After 10 cycles, the immobilized PLA 1 retained 71.2% of the original activity. The use of microflow support has an advantage on easier scaling up the reactor size towards much larger productivity and higher efficiency. • The novel microflow support showed advantages on easier scaling up, mass transfer and multiphase dispersion. • The immobilized PLA 1 catalyzed transesterification on microflow supports in a pump-driving columnar reactor. • Inhibition of hydrolysis optimized the reaction to produce DHA/EPA-rich phosphatidylcholine in high efficiency. [ABSTRACT FROM AUTHOR]

Animal venoms are a rich source of novel biomolecules with potential applications in medicine and agriculture. Ants are one of the most species-rich lineages of venomous animals. However, only a fraction of their biodiversity has been studied so far. Here, we investigated the venom components of two myrmicine (subfamily Myrmicinae) ants: Myrmica rubra and Myrmica ruginodis. We applied a venomics workflow based on proteotranscriptomics and found that the venoms of both species are composed of several protein classes, including venom serine proteases, cysteine-rich secretory protein, antigen 5 and pathogenesis-related 1 (CAP) superfamily proteins, Kunitz-type serine protease inhibitors and venom acid phosphatases. Several of these protein classes are known venom allergens, and for the first time we detected phospholipase A1 in the venom of M. ruginodis. We also identified two novel epidermal growth factor (EGF) family toxins in the M. ruginodis venom proteome and an array of additional EGF-like toxins in the venom gland transcriptomes of both species. These are similar to known toxins from the related myrmicine ant, Manica rubida, and the myrmecine (subfamily Myrmeciinae) Australian red bulldog ant Myrmecia gullosa, and are possibly deployed as weapons in defensive scenarios or to subdue prey. Our work suggests that M.rubra and M. ruginodis venoms contain many enzymes and other high-molecular-weight proteins that cause cell damage. Nevertheless, the presence of EGF-like toxins suggests that myrmicine ants have also recruited smaller peptide components into their venom arsenal. Although little is known about the bioactivity and function of EGF-like toxins, their presence in myrmicine and myrmecine ants suggests they play a key role in the venom systems of the superfamily Formicoidea. Our work adds to the emerging picture of ant venoms as a source of novel bioactive molecules and highlights the need to incorporate such taxa in future venom bioprospecting programs. [ABSTRACT FROM AUTHOR]

Phospholipase A1 (PLA1) is an enzyme that cleaves an ester bond at the sn-1 position of glycerophospholipids, producing a free fatty acid and a lysophospholipid. PLA1 activities have been detected both extracellularly and intracellularly, which are well conserved in higher eukaryotes, including fish and mammals. All extracellular PLA1s belong to the lipase family. In addition to PLA1 activity, most mammalian extracellular PLA1s exhibit lipase activity to hydrolyze triacylglycerol, cleaving the fatty acid and contributing to its absorption into the intestinal tract and tissues. Some extracellular PLA1s exhibit PLA1 activities specific to phosphatidic acid (PA) or phosphatidylserine (PS) and serve to produce lysophospholipid mediators such as lysophosphatidic acid (LPA) and lysophosphatidylserine (LysoPS). A high level of PLA1 activity has been detected in the cytosol fractions, where PA-PLA1/DDHD1/iPLA1 was responsible for the activity. Many homologs of PA-PLA1 and PLA2 have been shown to exhibit PLA1 activity. Although much has been learned about the pathophysiological roles of PLA1 molecules through studies of knockout mice and human genetic diseases, many questions regarding their biochemical properties, including their genuine in vivo substrate, remain elusive. [ABSTRACT FROM AUTHOR]

Phospholipase is an enzyme that hydrolyzes various phospholipid substrates at specific ester bonds and plays important roles such as membrane remodeling, as digestive enzymes, and the regulation of cellular mechanism. Phospholipase proteins are divided into following the four major groups according to the ester bonds they cleave off: phospholipase A1 (PLA1), phospholipase A2 (PLA2), phospholipase C (PLC), and phospholipase D (PLD). Among the four phospholipase groups, PLA1 has been less studied than the other phospholipases. Here, we report the first molecular structures of plant PLA1s: AtDSEL and CaPLA1 derived from Arabidopsis thaliana and Capsicum annuum, respectively. AtDSEL and CaPLA1 are novel PLA1s in that they form homodimers since PLAs are generally in the form of a monomer. The dimerization domain at the C-terminal of the AtDSEL and CaPLA1 makes hydrophobic interactions between each monomer, respectively. The C-terminal domain is also present in PLA1s of other plants, but not in PLAs of mammals and fungi. An activity assay of AtDSEL toward various lipid substrates demonstrates that AtDSEL is specialized for the cleavage of sn-1 acyl chains. This report reveals a new domain that exists only in plant PLA1s and suggests that the domain is essential for homodimerization. [ABSTRACT FROM AUTHOR]

Cotton is the most important crop for the production of natural fibres used in the textile industry. High salinity, drought, cold and high temperature represent serious abiotic stresses, which seriously threaten cotton production. Phospholipase A S has an irreplaceable role in lipid signal transmission, growth and development and stress events. Phospholipase A can be divided into three families: PLA1 , PLA2 and pPLA. Among them, the PLA1 family is rarely studied in plants. In order to study the potential functions of the PLA1 family in cotton, the bioinformatics analysis of the PLA1 family was correlated with cotton adversity, and tissue-specific analysis was performed. Explore the structure-function relationship of PLA1 members. It is found that the expression of GbPLA1-32 gene is affected by a variety of environmental stimuli, indicating that it plays a very important role in stress and hormone response, and closely associates the cotton adversity with this family. Through further functional verification, we found that virus-induced GbPLA1-32 gene silencing (VIGS) caused Gossypium barbadense to be sensitive to salt stress. This research provides an important basis for further research on the molecular mechanism of cotton resistance to abiotic stress. [ABSTRACT FROM AUTHOR]

Phospholipases have been used in different food processes, but mainly on degumming vegetable oils. More recently, the use of these enzymes has been extended to the manufacture of bread and dairy products. However, little is known on phospholipases effect on lecithin-based emulsions and how the emulsion size contributes to such effect. This work aimed to explore the effect of phospholipase type A1 (PLA1) on lecithin-based emulsions with different droplet size distribution. The PLA1 was able to hydrolyze lecithin phospholipids aggregated in (oil-in-water) emulsions, generating different lysophospholipids. The larger the particle size in the emulsion, the higher the enzymatic activity of PLA1. According to theoretical calculations, the lysophospholipids had higher hydrophile-lipophile balance (HLB) and had a lower critical packing parameter (p) than phospholipids. In consequence, the emulsions having more lysophospholipids were more prone to flocculate and to coalescence.

N-acyl-phosphatidylethanolamine (NAPE)-hydrolyzing phospholipase D (NAPE-PLD) is a zinc metallohydrolase enzyme that converts NAPEs to bioactive N-acyl-ethanolamides. Altered NAPE-PLD activity may contribute to pathogenesis of obesity, diabetes, atherosclerosis, and neurological diseases. Selective measurement of NAPE-PLD activity is challenging, however, because of alternative phospholipase pathways for NAPE hydrolysis. Previous methods to measure NAPE-PLD activity involved addition of exogenous NAPE followed by TLC or LC/MS/MS, which are time and resource intensive. Recently, NAPE-PLD activity in cells has been assayed using the fluorogenic NAPE analogs PED-A1 and PED6, but these substrates also detect the activity of serine hydrolase-type lipases PLA1 and PLA2. To create a fluorescence assay that selectively measured cellular NAPE-PLD activity, we synthesized an analog of PED-A1 (flame-NAPE) where the sn-1 ester bond was replaced with an N-methyl amide to create resistance to PLA1 hydrolysis. Recombinant NAPE-PLD produced fluorescence when incubated with either PED-A1 or flame-NAPE, whereas PLA1 only produced fluorescence when incubated with PED-A1. Furthermore, fluorescence in HepG2 cells using PED-A1 could be partially blocked by either biothionol (a selective NAPE-PLD inhibitor) or tetrahydrolipstatin (an inhibitor of a broad spectrum of serine hydrolase-type lipases). In contrast, fluorescence assayed in HepG2 cells using flame-NAPE could only be blocked by biothionol. In multiple cell types, the phospholipase activity detected using flame-NAPE was significantly more sensitive to biothionol inhibition than that detected using PED-A1. Thus, using flame-NAPE to measure phospholipase activity provides a rapid and selective method to measure NAPE-PLD activity in cells and tissues.

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Animal venoms are a rich source of novel biomolecules with potential applications in medicine and agriculture. Ants are one of the most species-rich lineages of venomous animals. However, only a fraction of their biodiversity has been studied so far. Here, we investigated the venom components of two myrmicine (subfamily Myrmicinae) ants: Myrmica rubra and Myrmica ruginodis. We applied a venomics workflow based on proteotranscriptomics and found that the venoms of both species are composed of several protein classes, including venom serine proteases, cysteine-rich secretory protein, antigen 5 and pathogenesis-related 1 (CAP) superfamily proteins, Kunitz-type serine protease inhibitors and venom acid phosphatases. Several of these protein classes are known venom allergens, and for the first time we detected phospholipase A1 in the venom of M. ruginodis. We also identified two novel epidermal growth factor (EGF) family toxins in the M. ruginodis venom proteome and an array of additional EGF-like toxins in the venom gland transcriptomes of both species. These are similar to known toxins from the related myrmicine ant, Manica rubida, and the myrmecine (subfamily Myrmeciinae) Australian red bulldog ant Myrmecia gullosa, and are possibly deployed as weapons in defensive scenarios or to subdue prey. Our work suggests that M.rubra and M. ruginodis venoms contain many enzymes and other high-molecular-weight proteins that cause cell damage. Nevertheless, the presence of EGF-like toxins suggests that myrmicine ants have also recruited smaller peptide components into their venom arsenal. Although little is known about the bioactivity and function of EGF-like toxins, their presence in myrmicine and myrmecine ants suggests they play a key role in the venom systems of the superfamily Formicoidea. Our work adds to the emerging picture of ant venoms as a source of novel bioactive molecules and highlights the need to incorporate such taxa in future venom bioprospecting programs.

Phospholipase A1 (PLA1) is an enzyme that cleaves an ester bond at the sn-1 position of glycerophospholipids, producing a free fatty acid and a lysophospholipid. PLA1 activities have been detected both extracellularly and intracellularly, which are well conserved in higher eukaryotes, including fish and mammals. All extracellular PLA1s belong to the lipase family. In addition to PLA1 activity, most mammalian extracellular PLA1s exhibit lipase activity to hydrolyze triacylglycerol, cleaving the fatty acid and contributing to its absorption into the intestinal tract and tissues. Some extracellular PLA1s exhibit PLA1 activities specific to phosphatidic acid (PA) or phosphatidylserine (PS) and serve to produce lysophospholipid mediators such as lysophosphatidic acid (LPA) and lysophosphatidylserine (LysoPS). A high level of PLA1 activity has been detected in the cytosol fractions, where PA-PLA1/DDHD1/iPLA1 was responsible for the activity. Many homologs of PA-PLA1 and PLA2 have been shown to exhibit PLA1 activity. Although much has been learned about the pathophysiological roles of PLA1 molecules through studies of knockout mice and human genetic diseases, many questions regarding their biochemical properties, including their genuine in vivo substrate, remain elusive.

Phospholipase is an enzyme that hydrolyzes various phospholipid substrates at specific ester bonds and plays important roles such as membrane remodeling, as digestive enzymes, and the regulation of cellular mechanism. Phospholipase proteins are divided into following the four major groups according to the ester bonds they cleave off: phospholipase A1 (PLA1), phospholipase A2 (PLA2), phospholipase C (PLC), and phospholipase D (PLD). Among the four phospholipase groups, PLA1 has been less studied than the other phospholipases. Here, we report the first molecular structures of plant PLA1s: AtDSEL and CaPLA1 derived from Arabidopsis thaliana and Capsicum annuum, respectively. AtDSEL and CaPLA1 are novel PLA1s in that they form homodimers since PLAs are generally in the form of a monomer. The dimerization domain at the C-terminal of the AtDSEL and CaPLA1 makes hydrophobic interactions between each monomer, respectively. The C-terminal domain is also present in PLA1s of other plants, but not in PLAs of mammals and fungi. An activity assay of AtDSEL toward various lipid substrates demonstrates that AtDSEL is specialized for the cleavage of sn-1 acyl chains. This report reveals a new domain that exists only in plant PLA1s and suggests that the domain is essential for homodimerization.

In this study, we maximized the reactivity of phospholipids hydrolysis with immobilized industrial-class phospholipase A1 (PLA1) at the desired water content in the water-in-oil (W/O) microemulsion phase. The optimal hydrophobic-hydrophilic condition of the reaction media in a hydrophobic enzyme reaction is critical to realize the maximum yields of enzyme activity of phospholipase A1. It was attributed to enzymes disliking hydrophobic surroundings as a special molecular structure for reactivity. Immobilization of PLA1 was successfully achieved with the aid of a hydrophobic carrier (Accurel MP100) combination with the treatment using glutaraldehyde. The immobilized yield was over 90% based on simple adsorption. The hydrolysis reaction was kinetically investigated through the effect of glutaraldehyde treatment of carrier and water content in the W/O microemulsion phase. The initial reaction rate increased linearly with an increasing glutaraldehyde concentration and then leveled off over a 6% glutaraldehyde concentration. The initial reaction rate, which was predominantly driven by the water content in the organic phase, changed according to a typical bell-shaped curve with respect to the molar ratio of water to phospholipid. It behaved in a similar way with different glutaraldehyde concentrations. After 10 cycles of repeated use, the reactivity was well sustained at 40% of the initial reaction rate and the creation of the final product. Accumulated yield after 10 times repetition was sufficient for industrial applications. Immobilized PLA1 has demonstrated potential as a biocatalyst for the production of phospholipid biochemicals. [ABSTRACT FROM AUTHOR]

A two-stage enzymatic hydrolysis method, in which phospholipase A1 (PLA1) was added after phospholipase A2 (PLA2) was added for a certain time, was successfully carried out to prepare glycerylphosphorylcholine (GPC) from soybean concentrated phospholipid. Effects of reaction variables on hydrolysis reaction were optimized using response surface methodology, and the optimal conditions were as follows: PLA2 load of 1.25%, PLA1 load of 0.70%, substrate concentration of 13%, reaction temperature of 41°C, and stirring rate of 680 rpm. Under the optimal conditions, the GPC yield reached 83.07%, which is close to the predicted value by the fitted model. This paper not only provides an efficient and low-cost method to prepare GPC, but also improves the high-value utilization of soybean concentrated phospholipid. [ABSTRACT FROM AUTHOR]

The food enzyme with phospholipase A 1 (phosphatidycholine 1-acylhydrolase, EC 3.1.1.32) and lysophospholipase (2-lysophosphatidylcholine acylhydrolase, EC 3.1.1.5) activities is produced with the genetically modified Aspergillus niger strain PLN by DSM. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. It is intended to be used for the production of refined edible fats and oils by degumming. Since residual amounts of total organic solids are removed during this process, dietary exposure was not calculated and toxicological studies were considered unnecessary for the assessment of this food enzyme. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no matches were found. The Panel considered that the risk of allergic reactions upon dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use., Competing Interests: If you wish to access the declaration of interests of any expert contributing to an EFSA scientific assessment, please contact interestmanagement@efsa.europa.eu., (© 2024 European Food Safety Authority. EFSA Journal published by Wiley‐VCH GmbH on behalf of European Food Safety Authority.)

Abstract The food enzyme phospholipase A1 (phosphatidycholine 1‐acylhydrolase EC 3.1.1.32) is produced with the genetically modified Aspergillus niger strain NZYM‐FP by Novozymes A/S. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. The phospholipase A1 food enzyme is intended to be used for degumming of fats and oils. Since residual amounts of Total Organic Solids (TOS) are removed by the purification steps applied during degumming, dietary exposure estimation was not considered necessary. Genotoxicity tests did not raise safety concerns. The repeated dose 90‐day oral toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no‐observed‐adverse‐effect level (NOAEL) of 1,356 mg TOS/kg body weight (bw) per day, the highest dose tested. Similarity of the amino acid sequence to those of known allergens was searched and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions upon dietary exposure cannot be excluded, but the likelihood for this to occur is considered to be low. Based on the data provided and the removal of TOS during the degumming of fats and oils, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Objectives: Ethanolamine ether phospholipids (ePE) and choline ether phospholipid (ePC) are present in human serum or plasma. Decreases in ether phospholipids (plasmalogens) in serum (plasma) have been reported in several diseases such as Alzheimer's disease, Parkinson's disease, metabolic syndrome, schizophrenia. Therefore, need for assay of ether phospholipids in plasma may increase in the future. Nowadays, measurement of the ether phospholipids in human plasma seem to depend on tandem mass spectrometry (LC/MS/MS), but a system for LC/MS/MS is too expensive for most of ordinary clinical laboratories, moreover, use and maintenance of the system are time consuming. Design and methods: Phospholipase A1 (PLA1) hydrolyzes ester (acyl) bond at the sn-1 position of glycerophospholipids, but it does not act on ether bond at the sn-1 position. We confirmed by a HPLC method that treatment of plasma with PLA1 causes complete disappearance of all diacyl phospholipids, but ether phospholipids remain intact. On the basis of these observations, we developed an enzymatic assay method for ePE and ePC in human plasma by use of a fluorescence plate reader. Results: The amount of ePE in human plasma measured by the enzymatic method was well correlated to that by LC/ESI-MS method (R2 > 0.94), but the correlation of ePC between the two methods was bit poorer (R2 > 0.77) than that of ePE. Conclusion: The enzymatic method may be applied to assay of ether phospholipids (ePE and ePC) not only in human plasma but also to assay of ePE and ePC in the other tissues.

The food enzyme phospholipase A1 (phosphatidycholine 1‐acylhydrolase EC 3.1.1.32) is produced with the genetically modified Aspergillus niger strain NZYM‐FP by Novozymes A/S. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. The phospholipase A1 food enzyme is intended to be used for degumming of fats and oils. Since residual amounts of Total Organic Solids (TOS) are removed by the purification steps applied during degumming, dietary exposure estimation was not considered necessary. Genotoxicity tests did not raise safety concerns. The repeated dose 90‐day oral toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no‐observed‐adverse‐effect level (NOAEL) of 1,356 mg TOS/kg body weight (bw) per day, the highest dose tested. Similarity of the amino acid sequence to those of known allergens was searched and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions upon dietary exposure cannot be excluded, but the likelihood for this to occur is considered to be low. Based on the data provided and the removal of TOS during the degumming of fats and oils, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use. [ABSTRACT FROM AUTHOR]

Phospholipases have been used in different food processes, but mainly on degumming vegetable oils. More recently, the use of these enzymes has been extended to the manufacture of bread and dairy products. However, little is known on phospholipases effect on lecithin-based emulsions and how the emulsion size contributes to such effect. This work aimed to explore the effect of phospholipase type A1 (PLA1) on lecithin-based emulsions with different droplet size distribution. The PLA1 was able to hydrolyze lecithin phospholipids aggregated in (oil-in-water) emulsions, generating different lysophospholipids. The larger the particle size in the emulsion, the higher the enzymatic activity of PLA1. According to theoretical calculations, the lysophospholipids had higher hydrophile-lipophile balance (HLB) and had a lower critical packing parameter (p) than phospholipids. In consequence, the emulsions having more lysophospholipids were more prone to flocculate and to coalescence. [ABSTRACT FROM AUTHOR]

Phospholipase A1 (PlaA) plays a pivotal role in diverse applications within the food and biochemical medical industries. Herein, we investigate the impact of the accessory protein encoded by plaS from Serratia marcescens on PlaA activity in Escherichia coli. Notably, PlaS demonstrates the ability to enhance PlaA activity while concurrently exhibiting inhibitory effects on the growth of E. coli BL21 (DE3). Our study revolves around probing the inhibitory action of PlaS on E. coli BL21 (DE3). PlaS exhibits a propensity to heighten both the permeability of outer and inner cell membranes, leading to concomitant reductions in membrane fluidity and surface hydrophobicity. This phenomenon is validated through scanning electron microscopy (SEM) analysis, which highlights PlaS's capacity to compromise membrane integrity. Moreover, through a comprehensive comparative transcriptomic sequencing approach, we identify four down-regulated genes (galM , ybhC , ldtC , and kdpB) alongside two up-regulated genes (rbsB and degP). These genes are intricately associated with processes such as cell membrane synthesis and modification, energy metabolism, and transmembrane transport. Our investigation unveils the intricate gene-level mechanisms underpinning PlaS-mediated growth inhibition and membrane disruption. Consequently, our findings serve as a significant reference for the elucidation of membrane protein mechanisms, shedding light on potential avenues for future exploration. • The accessory protein PlaS located on cell membranes • PlaS damage the integrity of cell membrane and inhibit the growth of E. coli. • PlaS affect the permeability, fluidity and hydrophobicity of E. coli. • Comparative transcriptomic analysis the mechanism of PlaS [ABSTRACT FROM AUTHOR]

In this study, four kinds of phospholipase A1-metal (Al/Co/Cu/Mn) hybrid nanostructures were prepared for enhancing the stability of the free PLA1. The formed hybrid complexes were characterized by scanning electron microscope (SEM), Fourier infrared spectroscopy (FTIR), and X-ray diffraction (XRD). The stability and substrate specificity of immobilized enzymes were subsequently determined. After immobilization, the temperature tolerance of PLA1–metal hybrid nanostructures was enhanced. The relative activity of PLA1–Al/Co/Cu hybrid nanostructures remained above 60% at 50 °C, while that of free enzyme was below 5%. The thermal transition temperature measured by differential scanning calorimetry (DSC) was found to increase from 65.59 °C (free enzyme) to 173.14 °C, 123.67 °C, 96.31 °C, and 114.79 °C, referring to PLA1–Cu/Co/Al/Mn hybrid nanostructures, respectively. Additionally, after a storage for fourteen days at 4 °C, the immobilized enzymes could exhibit approximately 60% of the initial activity, while the free PLA1 was inactivated after four days of storage. In brief, using Co2+, Cu2+, Al3+, and Mn2+ as the hybridization materials for immobilization could improve the catalytic properties and stability of the free PLA1, suggesting a promising method for a wider application of PLA1 in many fields such as food, cosmetics, and the pharmaceutical industry.

Enzymatic production of L-α-glycerylphosphorylcholine (L-α-GPC) is difficult due to the limited solubility of phosphatidylcholine (PC) in the aqueous phase. Surfactants can be used to improve the solubility and the dispersibility of non-polar chemicals in the aqueous media. In this study, various surfactants were investigated to improve L-α-GPC enzymatic production using phospholipase A1 (PLA1) in the aqueous phase. The results showed that Tween 20 was the most effective surfactant for enhancing L-α-GPC concentration. With 20 g.L−1 of Tween 20, the optimal conditions of PC hydrolysis were determined to be enzyme loading of 0.64 g.L−1 and substrate concentration of 60 g.L−1 at 45 °C for 1 h. In addition, the fed-batch catalytic process of PC was conducted to avoid substrate inhibition and increase product accumulation, resulting in 112.56 g.L−1 of L-α-GPC from 360.00 g.L−1 PC with yield of 91.36% within 3 h. [ABSTRACT FROM AUTHOR]

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Enzymatic degumming is an effective approach to produce nutritional, safe, and healthy refined oil. However, the high cost and low efficiency of phospholipase limit the application of enzymatic degumming. In this study, an 879 bp outer membrane phospholipase A (A1) (OM-PLA1) gene encoding 292 amino acid residues was isolated from the genome of Serratia marcescens. The recombinant OM-PLA1 profile of appropriately 33 KDa was expressed by the engineered Pichia pastoris GS115. The OM-PLA1 activity was 21.2 U/mL with the induction of 1 mM methanol for 72 h. The expression efficiencies of OM-PLA1 were 0.29 U/mL/h and 1.06 U/mL/OD600. A complex of magnetic graphene oxide (MGO) and OM-PLA1 (MGO-OM-PLA1) was prepared by immobilizing OM-PLA1 with graphene oxide-based Fe3O4 nanoparticles by cross-linking with glutaraldehyde. The content of phosphorus decreased to 5.1 mg/kg rapeseed oil from 55.6 mg/kg rapeseed oil with 0.02% MGO-OM-PLA1 (w/w) at 50°C for 4 h. MGO-OM-PLA1 retained 51.7% of the initial activity after 13 times of continuous recycling for the enzymatic degumming of rapeseed oil. This study provided an effective approach for the enzymatic degumming of crude vegetable oil by developing a novel phospholipase and improving the degumming technology. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Macromolecular crowding promotes membrane binding of phospholipase A1. • Phospholipase A1 promotes vesicle fusion. • Macromolecular crowding accelerates vesicle fusion. • Vesicle fusion deactivates phospholipase A1. Abstract Cells contain high levels of macromolecular crowding; understanding how macromolecular crowding impacts the behaviour of biological systems can give new insights into biological phenomena and disease pathologies. In this study, we assess the effect of macromolecular crowding on the catalytic activity of the biomembrane binding protein phospholipase A 1 (PLA 1). Using 3D-printed equilibrium dialysis chambers we show that macromolecular crowding increases the binding of PLA 1 to lipid vesicles. However, using a mass spectrometry assay of the hydrolysis of 1,2-dioleoyl- sn -glycero-3-phosphocholine (DOPC) by PLA 1 we surprisingly find that macromolecular crowding decreases the reaction rate and causes early cessation of the catalytic activity of PLA 1. Using kinetic equilibrium modelling, we are able to estimate the effect of macromolecular crowding on the association and dissociation rate constants for PLA 1 binding to the lipid vesicles. These data, coupled with particle sizing measurements enable us to construct a model to explain the early cessation of catalytic activity of PLA 1 with increasing levels of macromolecular crowding. This model suggests that compositional changes in the membrane, due to PLA 1 action, lead to the formation of larger vesicles, which deactivate the protein. This process is more rapid in the presence of macromolecular crowding agents, suggesting that a more detailed understanding of the effects of macromolecular crowding on membrane dynamics is required to understand membrane interacting proteins in macromolecularly crowded environments. The implications of this discovery are significant given the wide range of roles of membrane fusion and fission in neurocognitive processes and the failure of these processes in neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Free phospholipase A 1 was immobilized on magnetic Fe 3 O 4 /SiOx-g-P(GMA) nanoparticles. • The motion law of nanomagnetic enzyme particles in fluidized bed was simulated. • Using optimal numerical simulation, soybean oil was degummed in a multistage process. • The optimized conditions improved significantly the enzymolysis reaction. Abstract Compared to traditional methods, the enzymatic degumming shows the advantages of a clear reaction substrate, mild reaction conditions, low energy consumption and reduced environmental pollution. Free phospholipase A 1 (PLA 1) was immobilized on magnetic Fe 3 O 4 /SiOx-g-P (GMA) nanoparticles to obtain a nanomagnetic immobilized PLA 1 with a particle size of 100.50 ± 1.3 nm and an activity of 2150 ± 25 U/g. The Eulerian-Lagrangian model with particles as discrete phase and liquid as continuous phase was selected. The motion law of nanomagnetic enzyme particles in liquid-solid magnetic fluidized bed was simulated. Under the conditions of a magnetic field intensity of 0.022 T and a flow rate of soybean crude oil of 0.0025 m/s, the distribution status of nanomagnetic enzyme in the magnetic fluidized enhanced the enzymolysis reaction. Using the main parameters obtained by numerical simulation, soybean crude oil was degummed in a multistage magnetic fluidized bed. Under the conditions of a magnetic enzyme dosage of 0.10 g/kg, a reaction temperature of 63 °C and a reaction system pH of 6.3, after 6.0 h of reaction, the residual phosphorus contents in the oil was 8.2 mg/kg, and the relative activity of the nanomagnetic enzyme was still 86%. The optimized conditions improved significantly the efficiency and stability of the enzymolysis reaction of nanomagnetic enzyme. [ABSTRACT FROM AUTHOR]

In this study, we maximized the reactivity of phospholipids hydrolysis with immobilized industrial-class phospholipase A1 (PLA1) at the desired water content in the water-in-oil (W/O) microemulsion phase. The optimal hydrophobic-hydrophilic condition of the reaction media in a hydrophobic enzyme reaction is critical to realize the maximum yields of enzyme activity of phospholipase A1. It was attributed to enzymes disliking hydrophobic surroundings as a special molecular structure for reactivity. Immobilization of PLA1 was successfully achieved with the aid of a hydrophobic carrier (Accurel MP100) combination with the treatment using glutaraldehyde. The immobilized yield was over 90% based on simple adsorption. The hydrolysis reaction was kinetically investigated through the effect of glutaraldehyde treatment of carrier and water content in the W/O microemulsion phase. The initial reaction rate increased linearly with an increasing glutaraldehyde concentration and then leveled off over a 6% glutaraldehyde concentration. The initial reaction rate, which was predominantly driven by the water content in the organic phase, changed according to a typical bell-shaped curve with respect to the molar ratio of water to phospholipid. It behaved in a similar way with different glutaraldehyde concentrations. After 10 cycles of repeated use, the reactivity was well sustained at 40% of the initial reaction rate and the creation of the final product. Accumulated yield after 10 times repetition was sufficient for industrial applications. Immobilized PLA1 has demonstrated potential as a biocatalyst for the production of phospholipid biochemicals.

With the goal of improving cold chain logistics and real-time food quality prediction, here a novel time-temperature indicator (TTI) gel based on immobilized phospholipase A1/polyvinyl alcohol microspheres was developed and used to monitor the freshness of cold pork. The morphology, size, and effect of enzyme loading on the PVA microspheres were investigated. Five groups of TTIs were then prepared with the PVA microspheres, and their initial performance was screened in orthogonal experiments. Subsequently, the total volatile basic nitrogen (TVB-N) and 2-thiobarbituric acid (TBA) values from pork, as well as the total color difference of the prepared TTIs, were determined at 4, 15, and 25 °C, respectively. The activation energies (E a) were evaluated, and the most similar E a values were found for TTI, TVB-N, and TBA with 58.15, 59.30, and 57.02 kJ/mol, respectively. In addition, fluctuating temperature tests were designed to verify the performance of the TTI, and the equivalent temperatures of the TTI and pork freshness were determined to be 382.8 and 383.9 K, respectively. The TTI gel developed in this study enables visual monitoring of pork quality and has promising commercialization potential as it overcomes challenges with the cost and morphological stability of other enzyme based TTIs. • A novel attempt: applying polyvinyl alcohol microspheres to enzyme-based time-temperature indicator (TTI). • Success in reducing the cost of TTI: Phospholipase A1 has been mass produced. • Excellent visualization: TTI had a long response time span and distinct color changes. • Good match: TTI could accurately predict the level of pork spoilage. [ABSTRACT FROM AUTHOR]

Phospholipase A1 from Thai banded tiger wasp (Vespa affinis) venom also known as Ves a 1 plays an essential role in fatal vespid allergy. Ves a 1 becomes an important therapeutic target for toxin remedy. However, established Ves a 1 structure or a mechanism of Ves a 1 function were not well documented. This circumstance has prevented efficient design of a potential phospholipase A1 inhibitor. In our study, we successfully recruited homology modeling and molecular dynamic (MD) simulation to model Ves a 1 three-dimensional structure. The Ves a 1 structure along with dynamic behaviors were visualized and explained. In addition, we performed molecular docking of Ves a 1 with 1,2-Dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC) lipid to assess a possible lipid binding site. Interestingly, molecular docking predicted another lipid binding region apart from its corresponding catalytic site, suggesting an auxiliary role of the alternative site at the Ves a 1 surface. The new molecular mechanism related to the surface lipid binding site (auxiliary site) provided better understanding of how phospholipase A1 structure facilitates its enzymatic function. This auxiliary site, conserved among Hymenoptera species as well as some mammalian lipases, could be a guide for interaction-based design of a novel phospholipase A1 inhibitor.

Lipids from microorganisms are ligands of Toll like receptors (TLRs) and modulate the innate immune response. Herein, we analyze in vitro the effect of total lipid extracts from Trypanosoma cruzi amastigotes of RA and K98 strains (with polar biological behavior) on the induction of the inflammatory response and the involvement of TLRs in this process. We demonstrated that total lipid extracts from both strains induced lipid body formation, cyclooxygenase-2 expression and TNF-α and nitric oxide release in macrophages, as well as NF-κB activation and IL-8 release in HEK cells specifically through a TLR2/6 dependent pathway. We also evaluated the inflammatory response induced by total lipid extracts obtained from lysed parasites that were overnight incubated to allow the action of parasite hydrolytic enzymes, such as Phospholipase A1, over endogenous phospholipids. After incubation, these total lipid extracts showed a significantly reduced pro-inflammatory response, which could be attributed to the changes in the content of known bioactive lipid molecules like lysophospholipids and fatty acids, here reported. Moreover, analyses of total fatty acids in each lipid extract were performed by gas chromatography-mass spectrometry. Our results indicate a relevant role of T. cruzi lipids in the induction of a pro-inflammatory response through the TLR2/6 pathway that could contribute to the modulation of the immune response and host survival.

Phospholipase A1 (PLA1) immobilized in calcium alginate can effectively overcome the mass transfer resistance at the lipid-water interface making more room for the enzyme to separate itself from the products of reaction and to bind with the next available molecule at the interface. The reaction of an immobilized PLA1 hydrolase from Thermomyces lanuginosus was comparatively faster than of its free form. The rate of phospholipid hydrolysis by PLA1 was studied in calcium-rich and calcium-depleted environments; and the extent of phosphorus removed from the crude rice bran oil as well as the amount of free fatty acids produced during the reaction were used as indices for analysing the rate of enzymatic hydrolysis under standard conditions of pH, temperature, time of incubation and agitation. The immobilized PLA1 was found to be superior in removing phosphorus in the presencem of 10 mM bivalent calcium ions in a solution. As compared to a maximum of 72.52 % phosphorus removed by 0.01 kg of free enzyme per kg of oil, the same amount of immobilized PLA1 removed phosphorus from oil by 94.12 % under the same experimental conditions (pH=6, 60 °C, 1-hour incubation). Both the free PLA1 and its immobilized form had shown extended rates of hydrolysis in a calcium-rich environment. The mass fractions of free fatty acids produced by the free enzyme and by its immobilized form were 14.9 and 14.16 %, respectively, under the above experimental conditions. The removal of phosphorusfrom oil was accompanied by a signifi cant reduction in colour and restoration of iodine value to the desired level.

In this contribution, a new chromatographic method was applied to study the hydrolysis of non-polar lipids, i.e. triacylglicerols (TAG), diacylglycerols (DAG) and monoacylglycerols (MAG), when crude sunflower lecithin is treated with Lecitase® Ultra, an enzymatic preparation with phospholipase A1 (PLA1) activity. Results not only proved the enzyme lipase activity toward non-polar lipids in selected reaction conditions (aqueous system, T = 50 °C, pH = 5) but also suggested the occurrence of acyl-migration phenomenon observed by other authors in similar systems. Results showed that 1 h of reaction was enough to decrease the content of TAG in 54%, while DAG and MAG concentration increased from 0.4 to 3.5 and from 1.9 to 6.5 g/100 g of crude lecithin, respectively. Along the reaction, different contents of glycerides could be achieved, obtaining products with different composition which, in combination with the presence of phospholipids (PL) and/or lysophospholipids (LPL), could present specific emulsifying/stabilizing properties with a wide range of applications in food and pharmaceutical industry. [ABSTRACT FROM AUTHOR]

DDL1 encodes a mitochondrial phospholipase A1 involved in acyl chain remodeling of mitochondrial phospholipids and degradation of cardiolipin in Saccharomyces cerevisiae. The deletion of DDL1 leads to respiratory growth defects. To elucidate the physiological role of DDL1, we screened for genes that, when overexpressed, suppress the respiratory growth defect of the DDL1 deletion mutant. Introduction of COQ8, COQ9, or COQ5, which are involved in coenzyme Q (CoQ) synthesis, using a multicopy vector suppressed the respiratory growth defect of the DDL1 deletion mutant. In contrast, introduction of COQ8 using a multicopy vector did not accelerate the growth of the deletion mutants of TAZ1 or CLD1, which encode an acyltransferase or phospholipase A2, respectively, involved in the remodeling of cardiolipin. These results suggest genetic interactions between the mitochondrial phospholipase A1 gene and the genes involved in CoQ synthesis. [ABSTRACT FROM AUTHOR]

Hymenopteran insects are infamous for their sting, and their ability to cause severe anaphylaxis and in some cases death. This allergic reaction is a result of allergens present in the venom. Hymenopterans have many common venom allergens, the most widespread of which include phospholipase A1, phospholipase A2, acid phosphatase, hyaluronidase, serine protease and antigen 5. While there have been studies that look at the phylogenetic histories of allergens within closely related species, to our knowledge, this is the first study using evolutionary analyses to compare across Hymenoptera the types of selection that are occurring on allergens. This research examined the publicly available sequences of six different groups of allergens and found that allergens had diverged and formed closely related clades which share greater sequence similarities. We also analysed the patterns of selection and found that they are predominately under the influence of negative selection. [ABSTRACT FROM AUTHOR]