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Lipase lid

Maria Parapouli, Athanasios Foukis, Panagiota-Yiolanda Stergiou, Maria Koukouritaki, Panagiotis Magklaras, Olga A Gkini, Emmanuel M Papamichael, Amalia-Sofia Afendra, Efstathios Hatziloukas
Background: Microbial lipases catalyze a broad spectrum of reactions and are enzymes of considerable biotechnological interest. The focus of this study was the isolation of new lipase genes, intending to discover novel lipases whose products bear interesting biochemical and structural features and may have a potential to act as valuable biocatalysts in industrial applications. Results: A novel lipase gene ( lipSm ), from a new environmental Stenotrophomonas maltophilia strain, Psi-1, originating from a sludge sample from Psittaleia (Greece), was cloned and sequenced...
December 2018: Journal of Biological Research
Valerio De Vitis, Cristina Nakhnoukh, Andrea Pinto, Martina L Contente, Alberto Barbiroli, Mario Milani, Martino Bolognesi, Francesco Molinari, Louise J Gourlay, Diego Romano
Microbial carboxylesterases are important biocatalysts that selectively hydrolyze an extensive range of esters. Here, we report the biochemical and structural characterization of an atypical carboxylesterase from Bacillus coagulans (BCE), endowed with high enantioselectivity toward different 1,2-O-isopropylideneglycerol (IPG or solketal) esters. BCE efficiently catalyzes the production of enantiopure (S)-IPG, a chiral building block for the synthesis of β-blockers, glycerophospholipids, and prostaglandins; efficient hydrolysis was observed up to 65 °C...
March 2018: FEBS Journal
Oscar Romero, Blanca de Las Rivas, David Lopez-Tejedor, Jose M Palomo
Tailor-made peptides were investigated for site-specific tag labeling of Geobacillus thermocatenulatus lipase (GTL). GTL was first genetically modified by introducing a unique cysteine on the lid site of the enzyme to produce two variants (GTLσ-A193C and GTLσ-S196C). Chemical modification was performed by using a small library of cysteine-containing peptides. The synthesized peptide-lipase biocatalysts were highly stable, more active, more specific, and more selective toward different substrates than unmodified GTL...
February 16, 2018: Chembiochem: a European Journal of Chemical Biology
Malihe Masomian, Azmiza Syawani Jasni, Raja Noor Zaliha Raja Abd Rahman, Abu Bakar Salleh, Mahiran Basri
A total of 97 amino acids, considered as the signal peptide and transmembrane segments were removed from 205y lipase gene using polymerase chain reaction technique that abolished the low activity of this enzyme. The mature enzyme was expressed in Escherichia coli using pBAD expression vector, which gave up to a 13-fold increase in lipase activity. The mature 205y lipase (without signal peptide and transmembrane; -SP/TM) was purified to homogeneity using the isoelectric focusing technique with 53% recovery. Removing of the signal peptide and transmembrane segments had resulted in the shift of optimal pH, an increase in optimal temperature and tolerance towards more water-miscible organic solvents as compared to the characteristics of open reading frame (ORF) of 205y lipase...
October 28, 2017: Journal of Biotechnology
Ximena Zottig, Fatma Meddeb-Mouelhi, David M Charbonneau, Marc Beauregard
Recent investigations of Aneurinibacillus thermoaerophilus strains have allowed identification of a unique solvent tolerant lipase, distinct from known lipases. This work reports the expression and purification of this lipase (LipAT) and the first characterization of its structure and temperature and pH-dependent behaviour. LipAT has a secondary structural content compatible with the canonical lipase α/β hydrolase fold, and is dimeric at neutral pH. The protein was folded from pH 5 to 10, and association into folded aggregates at pH 7 and 8 likely protected its secondary structures from thermal unfolding...
December 2017: Protein Journal
Ioannis Karageorgos, Vitalii I Silin, Nikolai Zvonok, John Marino, David R Janero, Alexandros Makriyannis
Human monoacylglycerol lipase (hMAGL) plays a key role in homeostatic tuning of the endocannabinoid signaling system and supports aggressive tumorogenesis, making this enzyme a promising therapeutic target. hMAGL features a membrane-associated lid domain that regulates entry of endocannabinoid lipid substrates into the hydrophobic channel accessing the active site, likely from the membrane bilayer. The present work applied simultaneous surface plasmon resonance and electrochemical impedance spectroscopy measurements to show that, in absence of the substrate, hMAGL can remove phospholipid molecules from the membrane and, thereby, disintegrate pre-formed, intact, tethered phospholipid bilayer membrane mimetics (tBLMs) composed of unsaturated phosphatidylcholines...
November 1, 2017: Analytical Biochemistry
Nathalie Willems, Mickäel Lelimousin, Jakob Skjold-Jørgensen, Allan Svendsen, Mark S P Sansom
Lipases naturally function at the interface formed between amphiphilic molecules and the aqueous environment. Thermomyces lanuginosus lipase (TLL) is a well-characterised lipase, known to exhibit interfacial activation during which a lid region covering the active site becomes displaced upon interaction with an interface. In this study, we investigate the effect the amino acid sequence of the lid region on interfacial binding and lid dynamics of TLL. Three TLL variants were investigated, a wild-type variant, a variant containing an esterase lid region (Esterase), and a Hybrid variant, containing both wild-type lid residues and esterase lid residues...
March 2018: Chemistry and Physics of Lipids
Norhayati Yaacob, Nor Hafizah Ahmad Kamarudin, Adam Thean Chor Leow, Abu Bakar Salleh, Raja Noor Zaliha Raja Abd Rahman, Mohd Shukuri Mohamad Ali
The alkaline cold-active lipase from Pseudomonas fluorescens AMS8 undergoes major structural changes when reacted with hydrophobic organic solvents. In toluene, the AMS8 lipase catalytic region is exposed by the moving hydrophobic lid 2 (Glu-148 to Gly-167). Solvent-accessible surface area analysis revealed that Leu-208, which is located next to the nucleophilic Ser-207 has a focal function in influencing substrate accessibility and flexibility of the catalytic pocket. Based on molecular dynamic simulations, it was found that Leu-208 strongly facilitates the lid 2 opening via its side-chain...
August 12, 2017: Molecules: a Journal of Synthetic Chemistry and Natural Product Chemistry
Sridhar S N C, Deendyal Bhurta, Dharmvir Kantiwal, Ginson George, Vikramdeep Monga, Atish T Paul
A series of novel diaryl substituted pyrazolyl 2,4-thiazolidinediones were synthesized via reaction of appropriate pyrazolecarboxaldehydes with 2,4-thiazolidinedione (TZD) and nitrobenzyl substituted 2,4-thiazolidinedione. The resulting compounds were screened in vitro for pancreatic lipase (PL) inhibitory activity. Two assay protocols were performed viz., methods A and B using p-nitrophenyl butyrate and tributyrin as substrates, respectively. Compound 11e exhibited potent PL inhibitory activity (IC50 =4.81µM and Xi50 =10...
August 15, 2017: Bioorganic & Medicinal Chemistry Letters
Laura Sams, Sawsan Amara, Almahdi Chakroun, Sébastien Coudre, Julie Paume, Jacqueline Giallo, Frédéric Carrière
The cDNA encoding human gastric lipase (HGL) was integrated into the genome of Pichia pastoris using the pGAPZα A transfer vector. The HGL signal peptide was replaced by the yeast α-factor to achieve an efficient secretion. Active rHGL was produced by the transformed yeast but its levels and stability were dependent on the pH. The highest activity was obtained upon buffering the culture medium at pH5, a condition that allowed preserving enzyme activity over time. A large fraction (72±2%) of secreted rHGL remained however bound to the yeast cells, and was released by washing the cell pellet with an acid glycine-HCl buffer (pH2...
October 2017: Biochimica et Biophysica Acta
Binquan Luan, Ruhong Zhou
Candida antarctica lipase B (CalB), resembling many other lipases structure-wise, contains a flexible lid that undergoes a surprisingly large conformational change when catalyzing hydrophobic substrates (e.g. triglycerides). Despite extensive and important applications in industry, it is so far still elusive whether CalB can be activated on a hydrophobic surface, like other lipases. From large-scale all-atom molecular dynamics simulations, we discovered an open state that strikingly shows a much wider and more stable entrance to the catalytic site than the one suggested by previous crystal structures...
June 21, 2017: Physical Chemistry Chemical Physics: PCCP
Poonam Syal, Ved Vrat Verma, Rani Gupta
Biodiesel, an environment friendly alternative for fuels, contains methyl esters of long-chain fatty acids. Our group has reported a methanol-stable YLIP9 from Yarrowia lipolytica MSR80 that shows poor catalysis of long-chain fatty acids. To shift its substrate specificity, residues within lid and binding pocket were identified for sequential mutations using YLIP2 as the template. Of the two point mutations (Glu116Leu and Ser119Val) introduced in the lid, the former mutation (YLIP9L1) increased the catalytic rate by ∼2-fold without any change in substrate specificity...
November 2017: International Journal of Biological Macromolecules
Jonathan Maiangwa, Mohd Shukuri Mohamad Ali, Abu Bakar Salleh, Raja Noor Zaliha Raja Abd Rahman, Yahaya M Normi, Fairolniza Mohd Shariff, Thean Chor Leow
The dynamics and conformational landscape of proteins in organic solvents are events of potential interest in nonaqueous process catalysis. Conformational changes, folding transitions, and stability often correspond to structural rearrangements that alter contacts between solvent molecules and amino acid residues. However, in nonaqueous enzymology, organic solvents limit stability and further application of proteins. In the present study, molecular dynamics (MD) of a thermostable Geobacillus zalihae T1 lipase was performed in different chain length polar organic solvents (methanol, ethanol, propanol, butanol, and pentanol) and water mixture systems to a concentration of 50%...
2017: PeerJ
Gang Chen, Jing Tang, Ming Miao, Bo Jiang, Jian Jin, Biao Feng
The changes of lid movement and catalysis behavior of Rhizopus chinensis lipase under high hydrostatic pressure treatment was studied. Molecular dynamics simulation showed that the lipase lid under pressure was partially opened at below 200MPa but got more closed at over 400MPa. The interfacial activation changed little at pressure below 400MPa but became marginal with the pressure increased to 500MPa. The lipase hydrolysis ability by high pressure treatment underwent a course of initial increasing then reducing with maximum activity obtained at 200MPa and 40°C...
October 2017: International Journal of Biological Macromolecules
Jose M Palomo
This chapter describes the rational design and synthesis of semisynthetic lipases by site-directed incorporation of tailor-made peptides on the lipase-lid site to improve its activity, specificity, and enantioselectivity in specific biotransformations. Cysteine was genetically introduced at a particular point of the oligopeptide lid of the enzyme, and cysteine-containing peptides, complementary to the amino acid sequence on the lid site of Geobacillus thermocatenulatus lipase (BTL), were covalently attached on the lid of two different cysteine-BTL variants based on a fast thiol-disulfide exchange ligation followed by desulfurization...
2017: Methods in Enzymology
Joyeeta Mukherjee, Munishwar Nath Gupta
Use of biodiesel as an alternative to non-renewable sources of energy has become an attractive option in recent years. The enzymatic synthesis of biodiesel by transesterification of fats/oils with an alcohol is a much more sustainable route than the chemical method. However, cost effectiveness of the enzymatic route is a major barrier in its commercialization. In this work, a high activity biocatalyst design of Thermomyces lanuginosus lipase is made by dually bioimprinting it with substrate and a surfactant (which is believed to open up the lid covering the active site of the lipase) during precipitation of the lipase in organic solvent...
June 2016: Biotechnology Reports
Faez Iqbal Khan, Dongming Lan, Rabia Durrani, Weiqian Huan, Zexin Zhao, Yonghua Wang
Lipases are important industrial enzymes. Most of the lipases operate at lipid-water interfaces enabled by a mobile lid domain located over the active site. Lid protects the active site and hence responsible for catalytic activity. In pure aqueous media, the lid is predominantly closed, whereas in the presence of a hydrophobic layer, it is partially opened. Hence, the lid controls the enzyme activity. In the present review, we have classified lipases into different groups based on the structure of lid domains...
2017: Frontiers in Bioengineering and Biotechnology
Daniel Kübler, Anna Bergmann, Lukas Weger, Kim N Ingenbosch, Kerstin Hoffmann-Jacobsen
Detergents are commonly applied in lipase assays to solubilize sparingly soluble model substrates. However, detergents affect lipases as well as substrates in multiple ways. The effect of detergents on lipase activity is commonly attributed to conformational changes in the lid region. This study deals with the effect of the nonionic detergent, poly(ethylene glycol) dodecyl ether, on a lipase that does not contain a lid sequence, lipase A from Bacillus subtilis (BSLA). We show that BSLA activity depends strongly on the detergent concentration and the dependency profile changes with pH...
February 6, 2017: Journal of Physical Chemistry. B
Laura Riccardi, Jose M Arencibia, Luca Bono, Andrea Armirotti, Stefania Girotto, Marco De Vivo
Human monoacylglycerol lipase (MAGL) is a membrane-interacting enzyme that generates pro-inflammatory signaling molecules. For this reason, MAGL inhibition is a promising strategy to treat pain, cancer, and neuroinflammatory diseases. MAGL can hydrolyze monoacylglycerols bearing an acyl chain of different lengths and degrees of unsaturation, cleaving primarily the endocannabinoid 2-arachidonoylglycerol. Importantly, the enzymatic binding site of MAGL is confined by a 75-amino-acid-long, flexible cap domain, named 'lid domain', which is structurally similar to that found in several other lipases...
May 2017: Biochimica et Biophysica Acta
E Mateos-Diaz, S Amara, A Roussel, S Longhi, C Cambillau, F Carrière
Structural studies on lipases by X-ray crystallography have revealed conformational changes occurring in the presence of surfactants/inhibitors and the pivotal role played by a molecular "lid" of variable size and structure depending on the enzyme. Besides controlling the access to the enzyme active site, the lid is involved in lipase activation, formation of the interfacial recognition site (IRS), and substrate docking within the active site. The combined use of surfactants and inhibitors has been critical for a better understanding of lipase structure-function relationships...
2017: Methods in Enzymology
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