Lipid Acyl Hydrolase Activity Research Articles

A novel enzymatic approach based on the use of multi-enzymatic systems for the recovery of enriched protein extracts from potato pulp.

Ten commercial available multi-enzymatic systems have been explored for the efficient recovery of patatin and protease inhibitors from potato pulp. Their enzyme activity profile was characterized, where corresponding enzyme activity profile led to similar protein recovery yield. Of those assessed, Depol 670L (DEP) and Ceremix 2XL (CER) were efficient for the recovery of protein extract enriched with patatin (up to 60.0%) and protease inhibitors (up to 72.0%), respectively. The efficiency of DEP was significantly dependent on the interactive effect of enzymatic units and incubation time (p-value <0.015), while CER was found to be affected by the quadratric effect of both variables (p-value <0.022-0.052). The enriched patatin DEP-based protein extract possessed higher lipid acyl hydrolase activity emphasizing its preservation. The enriched protease inhibitors CER-based protein extract resulted in higher trypsin inhibiting activity, when compared to the industrial method. DEP and CER-based protein extracts were structurally characterized by peptide mass mapping.

Bioactive compounds in potatoes: Accumulation under drought stress conditions

Background: Potato (Solanum tuberosum) is a valuable source of bioactive compounds. Besides starch, crude fibre, amino acids (AAS), vitamins and minerals, the tubers contain diverse phenolic compounds. These phenolics and AAS confer anti-oxidant protection against reactive oxygen species, tissue damage, and diseases like atherosclerosis, renal failure, diabetes mellitus, and cancer. Climate change and drought stress may become a major risk for crop production worldwide, resulting in reduced access for those who depend on the nutritional value of this staple crop.Objective: The aim of this study is to determine the effect of drought stress on water, lipid soluble antioxidants, anthocyanins (Ac), soluble phenols, proteins, free AAS, peroxidase (POD) and lipid acyl hydrolase activity (LAH) in tuber tissue.Methods: The study was carried out on three potato genotypes comprising one yellow-fleshed cultivar and two purple breeding clones. The plants were grown in pots (from April to September) in a glasshouse with sufficient water supply and under drought stress conditions. After harvest, the tubers of both variants were analysed for antioxidants measured as ascorbic acid (ACE) and Trolox equivalent (TXE) using a photo-chemiluminescent method. Amounts of anthocyanins (Ac), soluble phenols, proteins, as well as POD and LAH activities were analysed using a UV photometer. Finally, free AAS were measured by HPLC.Results: The results revealed that drought stress significantly reduces tuber yield, but has no significant effect on antioxidants, Ac, soluble phenols and POD. Drought stress significantly increased the levels of soluble protein (P &lt; 0.0001) and LAH (P &lt; 0.001). Also, total amounts of Functional Foods in Health and Disease 2015; 5(3): 108-116 Page 109 of 116 free AAS were higher in the drought stressed tubers (+34.2%, on average) than in the tubers grown with a sufficient water supply. Above all, proline was elevated due to drought stress.Key words: anthocyanins, antioxidants, free amino acids, phenols, proteins, tuber quality

Potato protein isolates: Recovery and characterization of their properties

An imitation of industrial potato fruit juice (PFJ) was prepared, using Canadian variety of potatoes, and was characterized of being composed of 22.9% patatin, 53.3% protease inhibitors, and 23.7% high MW proteins. To isolate potato proteins from PFJ, several extraction techniques were explored including thermal/acidic combination, acidic, FeCl3, MnCl2, ethanol and (NH4)2SO4 precipitations, and carboxymethyl cellulose complexation. (NH4)2SO4 precipitation led to the highest yield (98.6%) and to the recovery of protein isolates enriched in patatin with high resolubility. FeCl3 precipitation resulted in the highest purification factor (6.2) and isolates with the lowest relative proportion of high MW proteins (<4.6%); however, its optimal isolate showed a wide minimum solubility pH range of 3.0–6.0. FeCl3 and MnCl2 were identified as the best precipitating agents for the enrichment of isolates with >15kDa protease inhibitors. Trypsin inhibiting activities of protease inhibitors were highly preserved upon protein isolation than the chymotrypsin ones. Acidic-based protein isolate showed the highest specific lipid acyl hydrolase activity of patatin towards o-nitrophenyl butyrate, whereas FeCl3-based one exhibited the highest activity towards 4-nitrophenyl laurate.

Understanding the Role of PNPLA3 Genetic Variants in Patients with Chronic Hepatitis C Infection

In 2008, in an attempt to identify genetic determinants of liver steatosis, Romeo et al. ran an independent genomewide association study on 2,121 patients enrolled in the Dallas Heart Study that had hepatic fat content assessed by proton magnetic resonance spectroscopy [1]. By concentrating just on nonsynonymous sequence variations the authors were able to discover a variation (rs738409 C[G) at position 148 in the patatin-like phospholipase-3 (PNPLA3) gene as the strongest determinant of steatosis in patients. The genetic polymorphism encodes an isoleucineto-methionine substitution. The PNPLA3 gene encodes a 481 amino acid protein of unknown function that belongs to the patatin-like phospholipase family; the progenitor of this family, patatin, has nonspecific lipid acyl hydrolase activity. Following this breakthrough discovery, several candidate gene studies have demonstrated that the G allele of PNPLA3 single nucleotide polymorphism (SNP) influences liver fat accumulation, is associated with disease severity in adult and pediatric populations with NAFLD, and ultimately increases the risk of NASH [2–5]. These results did not go unnoticed among colleagues working in the hepatitis C virus (HCV) field, as several studies were designed to assess the clinical role of the PNPLA3 polymorphisms in patients with chronic hepatitis C. The reasons for this interest are many fold. First, steatosis is known to negatively impact the natural history of HCV infection as it accelerates progression to cirrhosis [6, 7]. Second, liver steatosis has been shown to be a negative moderator of treatment outcome to interferon (IFN)-based therapies, with patients showing a fatty liver achieving lower sustained virological response rates across all HCV genotypes [8]. Third, with respect to the plethora of clinical trials currently investigating future potential anti-HCV drugs, the identification of a further genetic polymorphism able to influence treatment outcome would have an enormous impact in the design of future studies [9]. Among the first to demonstrate the impact of the PNPLA polymorphism in patients with HCV infection were the studies by Valenti et al. and Trepo et al. who, by analyzing large cohorts of HCV patients in Europe, almost simultaneously reported the G allele of rs738409 SNP to be associated not only with the presence of histologically determined liver steatosis, but also with the presence of cirrhosis and accelerated fibrosis progression [10, 11]. Ginanni-Corradini et al. further expanded these findings by reporting an increased rate of hepatocellular carcinoma (HCC) development in patients with the GG genotype of rs738409 in a cohort of 222 HCV patients [12]. Indeed by multivariate analysis the GG genotype was an independent factor associated with HCC development carrying a 2.23 odds ratio. Taken together these data rather unequivocally correlate the G allele of the PNPLA3 SNP with a worse prognosis of chronic hepatitis C. On the other hand the first studies were not able to solve if the G allele of the PNPLA3 SNP had an impact on IFNbased treatment efficacy. Indeed Valenti et al. were able to show a modest effect only in HCV-1 patients, with GG patients achieving a 10 % SVR rate compared to the 36 % obtained by the CG/CC counterpart, while Trepo et al. could not replicate these findings in their cohort. These discrepant results are not surprising given the extreme heterogeneity of the cohorts in terms of known moderators of treatment outcome such as disease severity, HCV A. Aghemo (&) A M Migliavacca Center for Liver Diseases, First Division of Gastroenterology, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Universita degli Studi di Milano, Milan, Italy e-mail: alessio.aghemo@policlinico.mi.it

Cultivar Variability of Patatin Biochemical Characteristics: Table versus Processing Potatoes (Solanum tuberosum L.)

Biochemical characteristics of patatin proteins purified by ion-exchange and affinity chromatography from tubers of 20 potato cultivars were studied to evaluate their genotype differences with respect to utility groups, table potato cultivars (TPCs) and processing potato cultivars (PPCs). Both groups of cultivars showed similar values of protein content in dry matter (3.98-7.39%) and of patatin relative abundance (5.40-35.40%). Three mass levels (∼40.6, 41.8, and 42.9 kDa) of purified patatins were found by MALDI-TOF MS within all cultivars. Differences among mass levels corresponding with the mass of sugar antenna (∼1.2 kDa) confirmed the previous concept of different glycosylation extentsin patatin proteins. It was showed that the individual types of patatin varying in their masses occur in the patatin family in a ratio specific for each of the cultivars, with the lowest mass type being the major one. Electrophoretic analyses demonstrated wide cultivar variability in number of patatin forms. Especially 2D-PAGE showed 17-23 detected protein spots independently on the utility group. Specific lipid acyl hydrolase (LAH) activity of purified patatins from the individual tested cultivars varied between 0.92 and 5.46 μmol/(min mg). Patatin samples within most of the TPCs exhibited higher values of specific LAH activity than samples of PPCs. It may be supposed that individual patatin forms do not have similar physiological roles.

Molecular characterization, expression and chromosomal localization of porcine PNPLA3 and PNPLA4

Patatin-like phospholipases (PNPLAs) comprise a protein family whose members contain a conserved patatin-like domain with lipid acyl hydrolase activity. The cloning and characterization of full-length cDNAs of PNPLA3 and PNPLA4 in pigs is reported and, for the first time, an alternative splicing variant of porcine PNPLA4, which skips exon 5 of the normal transcript, was identified. Subsequently, tissue expression analysis by quantitative PCR indicated that porcine PNPLA3 was mainly expressed in both adipose and liver whereas porcine PNPLA4 was abundant in liver. Porcine PNPLA3 and PNPLA4 were assigned respectively to chromosome 5p11-p15 and Xp24.

A Sequence Variation (I148M) in PNPLA3 Associated with Nonalcoholic Fatty Liver Disease Disrupts Triglyceride Hydrolysis

Obesity and insulin resistance are associated with deposition of triglycerides in tissues other than adipose tissue. Previously, we showed that a missense mutation (I148M) in PNPLA3 (patatin-like phospholipase domain-containing 3 protein) is associated with increased hepatic triglyceride content in humans. Here we examined the effect of the I148M substitution on the enzymatic activity and cellular location of PNPLA3. Structural modeling predicted that the substitution of methionine for isoleucine at residue 148 would restrict access of substrate to the catalytic serine at residue 47. In vitro assays using recombinant PNPLA3 partially purified from Sf9 cells confirmed that the wild type enzyme hydrolyzes emulsified triglyceride and that the I148M substitution abolishes this activity. Expression of PNPLA3-I148M, but not wild type PNPLA3, in cultured hepatocytes or in the livers of mice increased cellular triglyceride content. Cell fractionation studies revealed that ∼90% of wild type PNPLA3 partitioned between membranes and lipid droplets; substitution of isoleucine for methionine at position 148 did not alter the subcellular distribution of the protein. These data are consistent with PNPLA3-I148M promoting triglyceride accumulation by limiting triglyceride hydrolysis.

Chemical Composition and Nutritional Value of Protein Concentrates Isolated from Potato (Solanum tuberosumL.) Fruit Juice by Precipitation with Ethanol or Ferric Chloride

Effects of protein precipitators, ethanol and ferric chloride, on yield, resolubility, chemical composition and nutritional value of protein concentrates isolated from industrial potato fruit juice (PFJ) were studied. Optimum precipitating concentrations of ethanol and ferric chloride in PFJ were 4 M (23.1% v/v) and 20 mM (2% w/v), resulting in yield of 69% and 86.5% of total protein, respectively. Contents of total glycoalkaloids and potassium in both protein concentrates were significantly lower (P < 0.05) as compared with contents in PFJ dry matter. Both protein concentrates exhibited high nutritional value; values of essential amino acid index (EAAI) were 81.7% and 82.7%, respectively. Fraction of patatin proteins (39-43 kDa) represented with EAAI value of 86.1% the nutritionally improving protein component. Lipid acyl hydrolase activity of patatin family was not negatively affected by cooled ethanol precipitation. It can be thus suggested that biological and enzymatic activities of this protein family are utilizable after this type of precipitation.

Functional analysis of a class I patatin gene SK24-1 in microtuber formation of transgenic potatoes

Expression of a class I patatin cDNA clone, SK24-1, in Escherichia coli revealed that the cDNA clone possessed lipid acyl hydrolase (LAH) activity. Transformed potato plants were obtained via Agrobacterium-mediated transformation using the chimeric constructs containing the sense and antisense cDNA under the control cauliflower mosaic virus 35S (CaMV 35S) promoter. In some sense transformed plants, both sense patatin RNA and LAH activity were increased and further resulted in a significant increase of percentage of plantlets that formed microtubers and numbers of microtubers per plantlet in vitro. All antisense plants displayed a reduction in LAH activity. Both sense and antisense RNA could be detected in antisense plants, but transcripts of antisense RNA resulted in a reduction of endogenous sense RNA. Moreover, expression of antisense cDNA in some antisense transformed plants led to a significant decrease in the number of microtubers formed. These results suggest that SK24-1 was involved in regulating microtuber formation. Key words: Patatin, potato, Escherichia coli, sense RNA, antisense RNA

Novel thermophilic and thermostable lipolytic enzymes from a Thailand hot spring metagenomic library

Functional screening for lipolytic enzymes from a metagenomic library (origin: Jae Sawn hot spring, Thailand) resulted in isolation of a novel patatin-like phospholipase (PLP) and an esterase (Est1). PLP contained four conserved domains similar to other patatin-like proteins with lipid acyl hydrolase activity. Likewise, sequence alignment analysis revealed that Est1 can be classified as a family V bacterial lipolytic enzyme. Both PLP and Est1 were expressed heterologously as soluble proteins in E. coli and exhibited more than 50% of their maximal activities at alkaline pH, of 7–9 and 8–10, respectively. In addition, both enzymes retained more than 50% of maximal activity in the temperature range of 50–75 °C, with optimal activity at 70 °C and were stable at 70 °C for at least 120 min. Both PLP and Est1 exhibited high V max toward p-nitrophenyl butyrate. The enzymes had activity toward both short-chain (C 4 and C 5) and long chain (C 14 and C 16) fatty acid esters. The isolated enzymes, are therefore, different from other known patatin-like phospholipases and esterases, which usually show no activity for substrates longer than C 10. We suggest that PLP and EstA enzymes are novel and have a; b potential use in industrial applications.

Transformation of potato using an antisense class I patatin gene and its effect on microtuber formation

AbstractAn antisense class I patatin gene under control of the CaMV 35S promoter was introduced into potato (Solanum tuberosum) cultivar E-potato 3 using the Agrobacterium tumefaciens system. PCR amplification and PCR–Southern blot analysis indicated that the antisense class I patatin gene had been integrated into the potato genome. Northern hybridization analysis showed that the antisense gene transcribed normally in the transgenic potato plants and resulted in a reduction of endogenous class I patatin mRNA. Total soluble protein content and lipid acyl hydrolase activity of microtubers, derived from transformed plants, decreased by a maximum of 36.4% and 31.4%, respectively, compared with control plants. The expression of this antisense gene also resulted in reductions of the plantlets forming tubers, tubers per plantlet and the effective tubers (≥50 mg) of the transformed plants.

Transcriptional regulation of a patatin-1 gene in potato

Patatin is an abundant glycoprotein in the tubers of potato plants that has a lipid acyl hydrolase activity. Fusions of the promoter of patatin genes that are highly expressed in tubers with the reporter gene encoding beta-glucuronidase (GUS) have shown that patatin transcription has a high degree of tuber specificity. Patatin transcription was also inducible in other organs of transgenic potato by growth on high concentrations of sucrose. Experiments were conducted to define regions of the patatin promoter that confered tuber specific expression and sucrose inducibility. Sequences between -40 and -400 bp and between -400 and -957 bp of the transcriptional start site were able to confer tuber-specific expression on a heterologous truncated promoter. The cell specificity of GUS transcription in the transformants indicated that organ specificity was possibly determined by source-sink relationships of sucrose, or a metabolite of sucrose, in the whole plant.

Expression of a Patatin-Like Protein in the Anthers of Potato and Sweet Pepper Flowers

Patatin, the major glycoprotein in potato tubers, is encoded by a multigene family. RNA and protein analyses reveal that a homologous mRNA and an immunologically cross-reacting protein can be found in potato flowers, which is similar to patatin in that it displays a lipid acyl hydrolase activity. The patatin-like protein found in flowers has a higher molecular weight than the authentic tuber patatin. Deglycosylation experiments show that this is not due to differences in the glycosylation pattern. Immunocytochemical analysis shows the patatin-like protein to be present only in the epidermal cell layer of the anther, the exothecium, and in petals of potato flowers. Furthermore, the fact that a patatin-like protein can be detected in a similar tissue in sweet pepper, another solanaceous plant, could give a clue concerning the evolutionary origin of patatin.

Expression of a Patatin-like Protein in the Anthers of Potato and Sweet Pepper Flowers.

Patatin, the major glycoprotein in potato tubers, is encoded by a multigene family. RNA and protein analyses reveal that a homologous mRNA and an immunologically cross-reacting protein can be found in potato flowers, which is similar to patatin in that it displays a lipid acyl hydrolase activity. The patatin-like protein found in flowers has a higher molecular weight than the authentic tuber patatin. Deglycosylation experiments show that this is not due to differences in the glycosylation pattern. Immunocytochemical analysis shows the patatin-like protein to be present only in the epidermal cell layer of the anther, the exothecium, and in petals of potato flowers. Furthermore, the fact that a patatin-like protein can be detected in a similar tissue in sweet pepper, another solanaceous plant, could give a clue concerning the evolutionary origin of patatin.

Characterization of the lipid acyl hydrolase activity of the major potato (Solanum tuberosum) tuber protein, patatin, by cloning and abundant expression in a baculovirus vector.

Patatin is a family of glycoproteins that accounts for 30-40% of the total soluble protein in potato (Solanum tuberosum) tubers. This protein has been reported not only to serve as a storage protein, but also to exhibit enzymic activity. By using a baculovirus system to express protein from the patatin cDNA clone pGM01, it was unambiguously shown that the patatin coded by this DNA has lipid acyl hydrolase and acyltransferase activities. The enzyme is active with phospholipids, monoacylglycerols and p-nitrophenyl esters, moderately active with galactolipids, but is apparently inactive with di- and tri-acylglycerols.

Lipid acyl hydrolase of patatin

Patatin, the major protein of potato tubers, was copurified with lipid acyl hydrolase (LAH). The common identity of LAH and patatin was demonstrated by sodium dodecyl sulfate denaturation studies, isoelectric focusing, and the inhibition of LAH activity by patatin antiserum.

Phospholipids and Lipid Acyl Hydrolase (Phospholipase) in Leaf Galls (Hymenoptera: Cynipidae of Black Oak [Quercus robor L.

Phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine and cardiolipin are the major phospholipids in young leaves of black oak (Quercus robor L.). Except for phosphatidylcholine, young, developing cynipid-galls on black oak leaves, i.e. the insect-transformed tissues, contain less phospholipid than normal leaf tissues. Lipid acyl hydrolase activity determined by the cleavage of free fatty acids from a labeled phospholipid substrate is higher in the tissue extracts from galls than from leaves. The increase in enzyme activity and the altered phospholipid composition are discussed in relation to expected membrane modifications and transport phenomena in insect-transformed tissues.