Plant Lipases Research Articles

Characterization of a GDS(L)-like hydrolase from Pleurotus sapidus with an unusual SGNH motif

The GDS(L)-like lipase from the Basidiomycota Pleurotus sapidus (PSA_Lip) was heterologously expressed using Trichoderma reesei with an activity of 350 U L−1. The isoelectric point of 5.0 was determined by isoelectric focusing. The novel PSA_Lip showed only 23.8–25.1%, 25.5%, 26.6% and 28.4% identity to the previously characterized GDSL-like enzymes phospholipase, plant lipase, acetylcholinesterase and acetylxylan esterase, from the carbohydrate esterase family 16, respectively. Therefore, the enzyme was purified from the culture supernatant and the catalytic properties and the substrate specificity of the enzyme were investigated using different assays to reveal its potential function. While no phospholipase, acetylcholinesterase and acetylxylan esterase activities were detected, studies on the hydrolysis of ferulic acid methyl ester (~ 8.3%) and feruloylated carbohydrate 5-O-transferuloyl-arabino-furanose (~ 0.8%) showed low conversions of these substrates. By investigating the hydrolytic activity towards p-nitrophenyl-(pNP)-esters with various chain-lengths, the highest activity was determined for medium chain-length pNP-octanoate at 65 °C and a pH value of 8, while almost no activity was detected for pNP-hexanoate. The enzyme is highly stable when stored at pH 10 and 4 °C for at least 7 days. Moreover, using consensus sequence analysis and homology modeling, we could demonstrate that the PSA_Lip does not contain the usual SGNH residues in the actives site, which are usually present in GDS(L)-like enzymes.

Plant lipases – molecular structure, role in ontogeny and biotechnological potential

Lipases are enzymes commonly found in microorganisms, fungi, plants and animals. Their main function in cell metabolism is the hydrolysis (lipolysis) of ester bonds between fatty acids and glycerol in mono-, di- and triacylglycerols. In plants, lipases play an important role in ontogeny, participating in both vegetative development and generative stages. These enzymes may also be a component of plant responses to biotic and abiotic stresses. Based on the similarity of the amino acid sequence and vacuolar localization of some plant lipases to yeast Atg15, we present a hypothesis about the participation of lipases in autophagy (precisely, in the degradation of the autophagic body) in plants. Despite the narrow substrate specificity and the type of reactions catalysed in cells, lipases find numerous biotechnological applications. The physicochemical features of lipases, which determine, for example, wide substrate specificity in vitro or high stability in a wide range of pH and temperature, make these enzymes the subject of applied research, and plant lipases show an increasing potential in this area of science and industry.

Evaluation of the influence of chemical and physical factors on mixtures of fungal and plant lipases

Lipases are biocatalysts that may have distinct biochemical characteristics depending on the source. The combination of lipases from different sources with complementary characteristics is a viable strategy for increasing the enzymatic activity. In this study, fungal (Aspergillus niger 01 - CBMAI 2084) and plant (orange frit – orange peel fragment) lipases were analyzed separately and together in different concentrations. In addition, we evaluated the influence of organic solvents and ultrasonic effect on lipase activity, as well as substrate specificity [p-nitrophenyl butyrate (pNPB), p-nitrophenyl-laurate (pNPL) and p-nitrophenyl-palmitate (p-NPP)] and enzymatic immobilization in different supports (celite, silica, aluminum oxide, Lewatti, calcium alginate and gelatin). Increased enzyme activity was observed in formulations with higher concentration of fungal extract. The combination of 50% fungal extract and 25% plant extract increased about 55% lipase activity, showed the highest absolute lipase activity among all combinations and was selected for the following study. Plant extract showed the highest lipase activity in the hydrolysis of p-NPB and p-NPL, however, fungal extract showed the highest hydrolytic activity on p-NPP. When applied to synthetic substrates, the combination of plant and fungal extracts showed less stability and activity in synthetic substrates than isolated extracts, however lipase activity increased after 20s on ultrasound bath. Regarding to immobilization techniques, the adsorption on silica was the most efficient for all lipase extracts.

Alterations in the Transcriptome of Rye Plants following the Microdochium nivale Infection: Identification of Resistance/Susceptibility-Related Reactions Based on RNA-Seq Analysis.

Microdochium nivale is a progressive and devastating phytopathogen that causes different types of cereal crop and grass diseases that are poorly characterized at the molecular level. Although rye (Secale cereale L.) is one of the most resistant crops to most of the phytopathogens, it is severely damaged by M. nivale. The recent high-quality chromosome-scale assembly of rye genome has improved whole-genome studies of this crop. In the present work, the first transcriptome study of the M. nivale-infected crop plant (rye) with the detailed functional gene classification was carried out, along with the physiological verification of the RNA-Seq data. The results revealed plant reactions that contributed to their resistance or susceptibility to M. nivale. Phytohormone abscisic acid was shown to promote plant tolerance to M. nivale. Flavonoids were proposed to contribute to plant resistance to this pathogen. The upregulation of plant lipase encoding genes and the induction of lipase activity in M. nivale-infected plants revealed in our study were presumed to play an important role in plant susceptibility to the studied phytopathogen. Our work disclosed important aspects of plant-M. nivale interactions, outlined the directions for future studies on poorly characterized plant diseases caused by this phytopathogen, and provided new opportunities to improve cereals breeding and food security strategies.

Hydrolysis of vegetable and microbial oils catalyzed by a solid preparation of castor bean lipase

Motivated by the search for inexpensive biocatalysts , this study aimed to investigate the potential of lipase from castor bean seeds for hydrolysis of high-acidity oils. Biochemical properties, kinetics, and thermal stability were investigated in reactions with vegetable (macaw pulp and kernel) and microalgal ( Dunaliella salina , Nannochloropsis gaditana , and Chlorella minutissima ) oils. Enzyme performance was optimal at pH 4.5 and 35 °C. Thermal stability tests revealed a half-life of 9.6 h at 35 °C and apparent V max and K m values of 181.82 U g −1 and 215.15 mmol L −1 , respectively. The rate of hydrolysis was influenced by oil type, fatty acid composition , and oil concentration in the reaction medium. Castor bean lipase showed higher specificity toward vegetable oils rich in polyunsaturated fatty acids . The highest hydrolysis percentage of macaw pulp oil (53.55 ± 2.03%) was obtained at an oil/water weight ratio of 1:4 after 3 h of reaction. Lower performances were achieved with microalgal oils (31–43%), attributed to the presence of pigments , particularly chlorophyll, which has a negative effect on biocatalyst activity. The highest hydrolysis percentage was obtained using D. salina oil (43%) in 4 h of reaction. N. gaditana and C. minutissima oils had a hydrolysis percentage of 34 and 31%, respectively. D. salina oil had a similar fatty acid composition to macaw pulp oil, with 37.5% oleic acid and 15.4% linoleic acid . Nevertheless, the hydrolysis percentage of N. salina oil was 10% lower than that of macaw pulp oil. The low-cost solid preparation of castor bean lipase showed satisfactory performance in catalyzing the hydrolysis of vegetable oils. This economically attractive strategy can be used to obtain free fatty acids from non-edible oils for the production of biodiesel by hydroesterification. • Plant lipases have emerged as a feasible low-cost alternative with high specificity. • Castor bean lipase showed higher specificity toward vegetable and microbial oils. • This attractive strategy can be used in reactions of hydroesterification.

Optimization of lipase extraction from pequi seed ( Caryocar brasiliense Camb.)

The optimization of extraction conditions (time and volume) of lipase from pequi seed using ammonium sulfate and additives (tween 80 and calcium chloride) was studied. The results of statistical analysis showed significant effect of time and volume parameters on response variables enzymatic activity and protein recovery, where the highest activity of 1726.18 U was obtained in 3.0 ml of extraction solution for 25 min and 98.74% recovery in 2.5 ml for 15 min. The response surface indicated a better performance for both variables as time increased and volume decreased. The coefficients of determination and correlation graph validated the planning data. Thus, the optimal values of time and volume to obtain the greatest enzymatic activity in the lipase extraction process from pequi seed were fixed in 25 min and 3.0 ml of extraction solution composed of ammonium sulfate + tween 80. Practical applications An alternative plant lipase source obtained from pequi seed has been investigated for the first time. The results show that experimental conditions established in this work allow the efficient extraction of lipase, an enzyme with potential for application in several industrial segments.

Study of lipase producing gene in wheat – an in silico approach

BackgroundLipases (EC 3.1.1.3) catalyze the hydrolysis of oil into free fatty acids and glycerol forming the 3rd largest group of commercialized enzymes. Plant lipases grab attention recently because of their specificity, less production and purified cost, and easy availability. In silico approach is the first step to identify different genes coding for lipase in a most common indigenous plant, wheat, to explore the possibility of this plant as an alternative source for commercial lipase production. As the hierarchy organization of genes reflects an ancient process of gene duplication and divergence, many of the theoretical and analytical tools of the phylogenetic systematics can be utilized for comparative genomic studies. Also, in addition to experimental identification and characterization of genes, for computational genomic analysis, Arabidopsis has become a popular strategy to identify crop genes which are economically important, as Arabidopsis genes had been well identified and characterized for lipase. A number of articles had been reported in which genes of wheat have shown strong homology with Arabidopsis. The complete genome sequences of rice and Arabidopsis constitute a valuable resource for comparative genome analysis as they are representatives of the two major evolutionary lineages within the angiosperms. Here, in this in silico approach, Arabidopsis and Oryza sativa serve as models for dicotyledonous and monocotyledonous species, respectively, and the genomic sequence data available was used to identify the lipase genes in wheat. ResultsIn this present study, Ensembl Plants database was explored for lipase producing gene present in wheat genome and 21 genes were screened down as they contain specific domain and motif for lipase (GXSXG). According to the evolutionary analysis, it was found that the gene TraesCS5B02G157100, located in 5B chromosome, has 58.35% sequence similarity with the reported lipase gene of Arabidopsis thaliana and gene TraesCS3A02G463500 located in the 3A chromosome has 51.74% sequence similarity with the reported lipase gene of Oryza sativa. Homology modeling was performed using protein sequences coded by aforementioned genes and optimized by molecular dynamic simulations. Further with the help of molecular docking of modeled structures with tributyrin, binding efficiency was checked, and the difference in energies (DE) was −9.83 kcal/mol and −6.67 kcal/mol, respectively. ConclusionsThe present work provides a basic understanding of the gene-encoding lipase in wheat, which could be easily accessible and used as a potent industrial enzyme. The study enlightens another direction which can be used further to explore plant lipases.

Heterologous Expression and Characterization of Plant Lipase LIP2 from Elaeis guineensis Jacq. Oil Palm Mesocarp in Escherichia coli

In order to determine the potential of biochemical and structural features of Elaeis guineensis Jacq. oil palm mesocarp lipases, the LIP2 gene was isolated, expressed, purified and characterized through the Escherichia coli microbial recombinant system. Gene analysis of LIP2 revealed that it is composed of 1584 base pairs which are encoded in 528 amino acid residues with a molecular weight of around 57 kDa. LIP2 has distinctive lipolytic properties in terms of α/β fold and the catalytic triad for lipase. The LIP2 lipase was successfully expressed and purified from E. coli Rosetta (DE3) via affinity chromatography. The optimal temperature and pH for the lipase activity was 30 °C and a pH of 9, respectively. Stability was profoundly increased with the addition of metal ions (Ca2+, Mg2+, Mn+, and Ni+), along with organic solvents (ethanol and octanol). pNP myristate was the most suitable among all pNP esters. In biophysical characterization analysis, LIP2 has a thermal denaturing point at 66 °C, which mostly consists of random patterns (39.8%) followed by α-helix (30.3%), turns (23.8%) and β-sheet (6.2%). From the successful purification and characterization, the potential of oil palm mesocarp lipase was able to be further explored.

Lipases of germinating jojoba seeds efficiently hydrolyze triacylglycerols and wax esters and display wax ester-synthesizing activity

BackgroundSimmondsia chinensis (jojoba) is the only plant known to store wax esters instead of triacylglycerols in its seeds. Wax esters are composed of very-long-chain monounsaturated fatty acids and fatty alcohols and constitute up to 60% of the jojoba seed weight. During jojoba germination, the first step of wax ester mobilization is catalyzed by lipases. To date, none of the jojoba lipase-encoding genes have been cloned and characterized. In this study, we monitored mobilization of storage reserves during germination of jojoba seeds and performed detailed characterization of the jojoba lipases using microsomal fractions isolated from germinating seeds.ResultsDuring 26 days of germination, we observed a 60–70% decrease in wax ester content in the seeds, which was accompanied by the reduction of oleosin amounts and increase in glucose content. The activity of jojoba lipases in the seed microsomal fractions increased in the first 50 days of germination. The enzymes showed higher activity towards triacylglycerols than towards wax esters. The maximum lipase activity was observed at 60 °C and pH around 7 for triacylglycerols and 6.5–8 for wax esters. The enzyme efficiently hydrolyzed various wax esters containing saturated and unsaturated acyl and alcohol moieties. We also demonstrated that jojoba lipases possess wax ester-synthesizing activity when free fatty alcohols and different acyl donors, including triacylglycerols and free fatty acids, are used as substrates. For esterification reactions, the enzyme utilized both saturated and unsaturated fatty alcohols, with the preference towards long chain and very long chain compounds.ConclusionsIn in vitro assays, jojoba lipases catalyzed hydrolysis of triacylglycerols and different wax esters in a broad range of temperatures. In addition, the enzymes had the ability to synthesize wax esters in the backward reaction. Our data suggest that jojoba lipases may be more similar to other plant lipases than previously assumed.

Investigation on enzymatic activity of rubber seed as source of plant lipase

Rubber seed is a non-edible seed that is abundantly available and considers agricultural wastes. A potential lipase from rubber seed was examined based on the enzymatic activity and its application in the hydrolysis reaction. The enzymatic activity characterization study was determined based on p-nitrophenol release in the hydrolysis reaction. The initial evaluation showed that temperature and pH significantly influence the reaction. The optimum condition based on enzymatic activity for rubber seed was found at 30 ℃ and pH 8. The rubber seed lipase extract was then used in enzymatic hydrolysis reactions of rubber seed oil, palm oil, and canola oil. The highest FFA percentage of 63% was found from the rubber seed oil. The results indicate that rubber seed extract has shown its potential enzymatic activity. However, further studies need to be done to apply this rubber seed in various lipase catalysed reactions.

Less explored plant lipases: Modeling and molecular dynamics simulations of plant lipases in different solvents and temperatures to understand structure-function relationship

Lipase is a versatile enzyme found in microorganisms, animals and plants. It has applications in a wide variety of fields ranging from the food industry to the pharmaceutical. For these applications, mainly microbial lipases are exploited in great detail. On the other hand lipases from the plant source have been characterized to a much lesser extent. Although many plant lipase sequences have been reported in UniProtKB, till date there is no report on the crystal structure of any plant lipase. In view of very limited availability of structural information on plant lipases, in this study, we modeled the three-dimensional structure of seven plant lipases and studied the conformational changes under four different solvents at two different temperatures. Most lipases have a lid domain and its movement is implicated in the interfacial activation of lipases. Among the 56 conditions tested in this study, some lipases at certain condition exhibit the lid domain movement thus implying the functional importance. Laborious purification and minimal yield are the likely reasons for poor characterization of plant lipases. In this scenario, the results of computational studies on plant lipases under different environmental conditions will provide useful data for subsequent in vitro functional studies.

The isolation and characterization of lipase from Carica papaya latex using zwitterion sodium lauroyl sarcosinate as agent

Most of industrial lipases are derived from microbial sources, following by a wide variety of plants. Among plant lipases, lipase from Carica papaya latex has been the focus of intense and growing research due to low cost, easy acceptance by consumers and its unique characteristics. This enzyme has been successfully applied for lipid modification and synthesis of some organic compounds. However, research for its molecular structure has been limited due to the difficulty to isolate the enzyme from the latex matrix. In this study, we suggested a modified approach using sodium lauroyl sarcosinate to solubilize the latex, then the protein was precipitated by ammonium sulphate. We also carried out the characterization of the lipase obtained from Carica papaya latex. The results showed that freeze-drying the fresh latex could improve significantly lipase activity of latex powder in comparison with sun-drying or oven-drying. The zwitterion sodium lauroyl sarcosinate could solubilize nearly 50% of the latex and the achieved supernatant exhibited great lipase activity. There was no need to use an organic solvent to delipidate the latex prior to solubilization with sodium lauroyl sarcosinate due to possible denaturation of enzymes. The proteins which were fractionally precipitated with 50 - 60%, 60 - 70% and 70 - 80% ammonium sulphate saturation showed lipolytic activity. The fraction from 50 - 60% saturation with the greatest mass was subjected to ion exchange chromatography, SDS electrophoresis and kinetic parameter determination. The results showed the presence of two proteins with molecular mass ranging from 35 kDa to 55 kDa and both presented lipase activity. The Km and Vmax of the lipase fraction from 50 - 60% saturation was 1.12 mM and 1.2 x 10-6 mM.min-1.mL-1 respectively. So, the freeze-drying of papaya latex could help to preserve its lipase activity and the usage of sodium lauroyl sarcosinate could improve the isolation of the lipase from the papaya latex and pave the way for research on the molecular structure of Carica papaya latex lipases.

Cloning of lipase cDNA from Oleaeuropaea cv. Gemlik leaves and expression analysis in response to cold stress

The cDNA encoding lipase enzyme was cloned from Olea europaea var. europaea cv. Gemlik leaves using the RACE method. The full nucleotide sequence was 1546 bp long and encoded a putative protein of 412 amino acid long protein. The amino acid sequence showed identity to Triacylglycerol Lipase 1 sequences from different plants. A phylogenetic tree was constructed showing relations with various plant lipases. Multiple sequence alignment analysis of the putative protein sequence revealed that the enzyme contains conserved GXSXG pentapeptide motif, and the catalytic triad was predicted as Ser-182, His-379, and Asp-350 with bioinformatics tools. Real-time PCR was used to determine relative expression levels of lipase in leaves in response to cold stress.

Plant Lipases: A Potential Candidate for Commercial Exploitation

Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) catalyze the hydrolysis and the synthesis of esters formed from glycerol and long-chain fatty acids by acting at explicit position on the glycerol backbone of a lipid substrate. In view of the increasing understanding of lipases and their many applications in high-value syntheses and as bulk enzymes, these enzymes are having an increasing impact on bioprocessing. Lipases occur ubiquitously in nature, but only microbial lipases are presently commercially significant on industrial and therapeutic point of view. On the other hand recently plant originated lipase has provided a great alternative to microbial lipase due to the specificity, low cost, availability and ease of purification. Despite many applications of plant lipases, there are still challenges and limitations which create a gap between the small scale use and commercial exploitation of novel lipases. This review article gives an overall idea about the occurrence of various plant lipases along with different assay, purification, inhibition and immobilization process of these lipases with a specific reference to rice bran lipase.

Performance of Various Organic Solvents as Reaction Media in Plant Oil Lipolysis with Plant Lipase

Fatty acids are intermediate substances in synthesis of oleochemical products. Enzymatic technology of fatty acids production (also known as lipolysis) is now developing as potential substitution for the conventional production of fatty acid, i.e. thermal hydrolysis of triglyceride. It offers more economical process condition, low energy consumption, and minimal product degradation compared to the conventional process. This research aims to evaluate performance of various organic solvents as reaction media in lipolysis with plant latex lipase. Organic solvents observed were chloroform, n-hexane, diethyl ether, benzene, acetone, ethanol, methanol, n-heptane, and isooctane. Analysis of each organic solvent effect on lipolysis was described based on solvents properties. Conversion of lipolysis with organic solvents is 0,10-1,25 times fold compared to conversion of non-solvent lipolysis. We suggest that dielectric constant and viscosity are the two main organic solvent properties affecting lipase performance in lipolysis. Overall, n-hexane, n-heptane, and isooctane are recommended to be used as reaction media in lipolysis with plant lipase because their effects to degree of lipolysis are positive. Keywords: lipolysis; lipase; organic solvent; frangipani

Functional Expression of Plant Lipases: The Case of CpLip1 from Carica papaya.

Carica papaya latex is one of the most studied sources of plant lipases. However, the complexity of the matrix composition makes it difficult to isolate and purify the lipolytic enzymes present in Carica papaya latex. Therefore, diverse strategies have been developed to study the catalytic properties of these enzymes.Recently the first lipase from Carica papaya latex (CpLip1) has been successfully cloned and expressed in order to study their catalytic properties. In order to improve the catalytic properties and increase the potential for its use at industrial scale.In this chapter, a practical protocol to recombinant CpLip1 lipase is given.

Cloning, Sequence Analysis and Expression Patterns during Seed Germination of a Rapeseed (Brassica napus L.) G-x-S-x-G-motif Lipase Gene

Lipases catalyze the hydrolysis of ester bonds in triacylglycerides, generating glycerol and free fatty acids. These enzymes are encoded by extremely complex gene families, and appear to fulfil many different biological functions. Although they are present in all types of organisms, available information on plant lipases is still very limited, as compared to their bacterial and animal counterparts. A full-length clone, BnLIP, encoding a putative lipase, has been isolated by PCR amplification of Brassica napus genomic DNA, with oligonucleotide primers derived from the sequence of an Arabidopsis thaliana homologue. The clone included an open reading frame of 1581 bp encoding a polypeptide of 526 amino acids, with a calculated molecular mass of 59.5 kDa. Analysis of the deduced protein sequence, sequence alignment with homologous proteins from related plant species, and a phylogenetic analysis revealed that the BnLIP protein belongs to the ‘classical’ GxSxG-motif lipase family. RT-PCR assays indicated that the BnLIP gene is expressed specifically, but only transiently, during seed germination: the lipase mRNA was not present at detectable levels in ungerminated seeds, was detected only three days after seed imbibition, but its levels decreased rapidly afterwards. No expression was observed in roots, stems or leaves of adult plants. This expression pattern suggests that BnLIP is one of the lipases involved in the hydrolysis of triacylglycerides stored in rapeseed seeds, ultimately providing nutrients and energy to sustain seedling growth until photosynthesis is activated.

English

Industrial-scale processes currently developed make use of chemical catalysis processes that are highly efficient but require very complex product purification steps. Enzymatic catalysis through plant lipases as biocatalysts is an alternative which, in contrast to chemical catalysis processes, appeared simple to perform, and can be done at low investment cost. Although microbial lipases have been extensively studied, little research has been focused on the use of plant lipases namely plant latex lipases. The present article outlines the most advanced knowledge concerning plant latex characterization in order to show how plant latex can be a promising alternative to catalyze transesterification for biodiesel production. This paper provides an overview regarding the main aspects of latex, such as the reactions catalyzed, physiological functions, specificities, sources and their industrial applications. Key words: Plant latex, lipase, Transesterification, purification, biodiesel.

Antioxidant and anti-lipases activities in vitro of Mentha viridis and Eucalyptus globulus extracts

Plants are a source of bioactive compounds such as antioxidants and enzyme inhibitors. In this work, effect of the Eucalyptus globulus and Mentha viridis extraction by different solvents on phenolics content, flavonoids content, condensed tannins content, antioxidant activity and inhibitory effect against two lipases from Aspergillus niger and olive was studied. Methanolic (MeOH) extract showed the strongest antioxidant and anti-lipase activities. High correlation between total phenolics content, total flavonoids content, antioxidant activity and lipases inhibitory action was observed. Kinetic analysis of lipases inhibition was performed with Linweaver-Burk, Dixon and Cornish-Bowden plots. These results revealed competitive inhibition mode by E. globulus and non competitive mixed inhibition mode by M. viridis against the two lipases. Inhibition constant (KI) values calculated with E. globulus MeOH extract were 0.271 and 0.188mg/mL for Aspergillus niger and olive lipases respectively. With M. viridis MeOH extract KI values were 0.303 and 0.421mg/mL for the fungal and the plant lipases respectively. The inhibitory concentration 50% (IC50) values determined with E. globulus MeOH extract were 0.69mg/mL for Aspergillus niger lipase and 1.29mg/mL for olive lipase. With M. viridis MeOH extract IC50 were 0.43 and 1.44mg/mL for Aspergillus niger and olive lipases respectively.

Effect of changes in lipid classes during wilting and ensiling of red clover using two silage additives onin vitroruminal biohydrogenation

SUMMARYAlthough forage lipid is generally rich in polyunsaturated fatty acids (PUFA), recovery of these fatty acids (FA) in milk and meat of ruminant origin is generally low, due to microbial biohydrogenation (BH) taking place in the rumen. Since lipolysis is a prerequisite for BH, the latter process is expected to be enhanced when (conserved) forages contain lower levels of esterified FA (particularly polar lipids; PL). However, this was not observed in former studies with red clover (Trifolium pratenseL.). Furthermore, red clover inclusion in the herbivore's diet was associated with decreased rumen BH as compared with other forages. Differences in plant lipase activity during wilting and ensiling has been attributed to changes in disappearance from the PL fraction, but a potential role of microbial lipasesin silohas not yet been elucidated. Therefore, the aims of the present study were to assess whether BH of red clover FA is linked with PL levels of the (conserved) starting material and to clarify the possible role ofin silomicrobial activity on PL disappearance. In order to obtain sufficient variation in forage PL and microbial activity, laboratory-scale silages were made by wilting and ensiling damaged or undamaged red clover using molasses or formic acid as ensiling additive, while perennial ryegrass (Lolium perenneL.) was used as a control. Distribution of lipids within three lipid fractions (PL, free FA and neutral lipids) in forages was determined and BH calculated after 24 hin vitrorumen incubation. Results indicated microbial lipases in silages did not enhance FA disappearance from the PL fraction. A gradual decrease of FA in the PL fraction upon conservation was found, both in red clover and ryegrass, irrespective of the degree of damage. In red clover PL losses started from the wilting phase, while substantial PL disappearance from ryegrass only started upon ensiling. Proportions of PUFA remaining in the PL fraction after wilting and ensiling of red clover were positively correlated with PUFA BH, while this effect was not observed for ryegrass. Red clover PUFA seemed to be partially protected against ruminal BH, while disappearance of FA from the PL fraction did not seem to be hampered. Results indicated the encapsulation mechanism as a consequence of protein-bound phenol formation induced by polyphenol oxidase is still the most probable hypothesis to explain red clover's increased flow of PUFA across the rumen.