Mass spectrometric evidence of covalently-bound tetrahydrolipstatin at the catalytic serine of Streptomyces rimosus lipase

We have recently detected that the lipase from Streptomyces rimosus belongs to a large but poorly characterised family of SGNH hydrolases having the αβα-fold. Our biochemical characterisation relates to the specific inhibition of an extracellular lipase from Streptomyces rimosus (SRL, 24.2 kDa, Q93MW7) by the preincubation method with tetrahydrolipstatin (THL). In high molar excess (THL/SRL = 590 at 25 °C, pH = 7.0) and after 2 h of incubation in an aqueous system, 56% of the enzyme inhibition was reached. Under the same conditions and in the presence of 50% (v/v) 2-propanol/water, 71% enzyme inhibition was obtained. Kinetic measurements are in agreement with pseudo-first-order kinetics. The nucleophilic attack of the catalytic serine residue 10 of SRL occurs via an opening of the β-lactone ring of tetrahydrolipstatin and formation of a covalent ester bond. The intact covalent complex of SRL-inhibitor was analysed by ESI and vacuum MALDI mass spectrometry and, furthermore, the exact covalent THL linkage was determined by vacuum MALDI high-energy collision-induced dissociation tandem mass spectrometry.     

Production of extracellular enzymes in the entomopathogenic fungus Verticillium lecanii

This study investigates the mechanisms as well as strategies for purification and characterization of potential enzymes involved in pathogenesis of entomopathogenic fungi. The test strain of Verticillium lecanii that was screened, during the present investigation, proved to be an efficient producer of protein and polysaccharide degrading enzymes (amylase, protease, and lipase), hence indicating versatility in biochemical mechanisms. Halo zones produced colony growth of V. lecanii on agar confirmed activity of protease, amylase and lipase enzyme by the V. lecanii isolate. Enzymatic Index (EI) observed were: Protease – 2.195, Amylase- 2.196, Lipase- 2.147. Spectrophotometric analysis of enzymatic activity of V.lecanii at five different pH – 3, 5, 7, 9, 11 revealed that highest proteolytic activity of the V. lecanii isolate was reported at pH 7 and 9 whereas proteolytic activity was minimum at acidic pH 3. Maximum amylolytic activity of V. lecanii on the 7^th day of inoculation was at pH 3 i.e. in an acidic environment in contrast to neutral pH 7. Maximum lipolytic activity of V. lecanii was found at pH 7. Since enzyme production in entomopathogenic fungi is specific and forms an important criterion for successful development as well as improvement of mycoinsecticides, hence a significant conclusion from the present analysis is the degree of variation in secretion of enzymes in test strain of Verticillium lecanii.     

A simple method of chymotrypsin concentration and purification from pancreas homogenate using Eudragit® L100 and Eudragit® S100

The condition of chymotrypsin (ChTRP)–Eudragit^® (Eu) insoluble complex formation was studied with the aim of applying this information to the separation of chymotrypsin from a crude filtrate of bovine pancreas homogenate. The optimal pH of the complex precipitation was 4.60 for ChTRP–Eudragit^® L100 and 5.40 for ChTRP–Eudragit^® S100. The polyelectrolyte concentration necessary for the commercial enzyme precipitation was lower than 0.1% (w/v). The complex formation was inhibited by NaCl for both polyelectrolytes. The method was applied to recover the enzyme from bovine homogenate; ChTRP was precipitated by Eudragit^® addition. The non-soluble complexes were separated by simple centrifugation and re-dissolved by a pH change to 8.20. The best conditions to recover ChTRP brought about a purification factor of around 4 and 90% yield.     

Lipase of <Emphasis Type="Italic">Aspergillus niger</Emphasis> NCIM 1207: A Potential Biocatalyst for Synthesis of Isoamyl Acetate

Commercial lipase preparations and mycelium bound lipase from Aspergillus niger NCIM 1207 were used for esterification of acetic acid with isoamyl alcohol to obtain isoamyl acetate. The esterification reaction was carried out at 30°C in n-hexane with shaking at 120 rpm. Initial reaction rates, conversion efficiency and isoamyl acetate concentration obtained using Novozyme 435 were the highest. Mycelium bound lipase of A. niger NCIM 1207 produced maximal isoamyl acetate formation at an alcohol/acid ratio of 1.6. Acetic acid at higher concentrations than required for the critical alcohol/acid ratio lower than 1.3 and higher than 1.6 resulted in decreased yields of isoamyl acetate probably owing to lowering of micro-aqueous environmental pH around the enzyme leading to inhibition of enzyme activity. Mycelium bound A. niger lipase produced 80 g/l of isoamyl acetate within 96 h even though extremely less amount of enzyme activity was used for esterification. The presence of sodium sulphate during esterification reaction at higher substrate concentration resulted in increased conversion efficiency when we used mycelium bound enzyme preparations of A. niger NCIM 1207. This could be due to removal of excess water released during esterification reaction by sodium sulphate. High ester concentration (286.5 g/l) and conversion (73.5%) were obtained within 24 h using Novozyme 435 under these conditions.     

Evaluating aqueous two-phase systems for Yarrowia lipolytica extracellular lipase purification

In this study an aqueous two-phase system (ATPS) composed of polyethylene glycol (PEG) and potassium phosphate was tested for the purification of lipase from Yarrowia lipolytica IMUFRJ 50682. Ultrafiltration and precipitation with acetone and kaolin were also used as traditional comparison methods Ultrafiltration was a good method with a purification factor of 6.55, but protease was also purified in this extract. For the precipitation with acetone and kaolin lower values of lipase and protease activity were found in relation to the original crude enzyme extract. Under the best conditions of ATPS (pH 6 and 4 °C), the purification fold was greater than 40 and selectivity was almost 500. Lipase was recovered in the salty phase which makes it easier to purify it. The optimum pH and temperature ranges for purified lipase with this system was 6–7 and 35–40 °C, respectively. Lipase thermostability was increased in relation to crude extract after the purification with the PEG/phosphate buffer system for temperatures lower than 50 °C. All enzyme extracts showed good stability to a wide pH range. Y. lipolytca lipase was successfully purified by using ATPS in a single downstream processing step and presented good process characteristics after this treatment.     

基因拷贝数和甲醇浓度对重组毕赤酵母产华根霉脂肪酶的影响

将华根霉脂肪酶基因克隆到甲基营养型毕赤酵母中表达,以甲醇利用快型菌株为宿主,在7 L发酵罐水平对脂肪酶基因拷贝数分别为3、5、6的3株基因重组菌——XY RCL-3、XY RCL-5、XY RCL-6进行高密度发酵调控,同时研究了甲醇浓度对表达华根霉脂肪酶的影响。结果表明,XY RCL-5在相同条件下发酵产酶能力高于XY RCL-6和XY RCL-3,最适甲醇诱导浓度控制在0.1%±0.02%时,酶活可达到12 500 U/mL,菌体干重达到204 g/L,蛋白浓度也能达到8.02 g/L。     

Production,partial purification and characterization of organic solvent tolerant lipase from Burkholderia multivorans V2 and its application for ester synthesis

Burkholderia multivorans V2 (BMV2) isolated from soil was found to produce an extracellular solvent tolerant lipase (6.477 U/mL). This lipase exhibited maximum stability in n-hexane retaining about 97.8% activity for 24 h. After performing statistical optimization of medium components for lipase production, a 2.2-fold (14 U/mL) enhancement in the lipase production was observed. The crude lipase from BMV2 was partially purified by ultrafiltration and gel permeation chromatography with 24.64-fold purification. The K_m and V_max values for partially purified BMV2 lipase were found to be 1.56 mM and 5.62 μmoles/mg min. The metal ions Ca²⁺, Mg²⁺ and Mn²⁺ had stimulatory effect on lipase activity, whereas Cu²⁺, Fe²⁺ and Zn²⁺ strongly inhibited the lipase activity. EDTA and PMSF at 10 mM concentration strongly inhibited the lipase activity. Non-ionic and anionic surfactants stimulated the lipase activity. BMV2 lipase was proved to be efficient in synthesis of ethyl butyrate ester under non-aqueous environment.     

Structure of the Alkalohyperthermophilic Archaeoglobus fulgidus Lipase Contains a Unique C-Terminal Domain Essential for Long-Chain Substrate Binding

Several crystal structures of AFL, a novel lipase from the archaeon Archaeoglobus fulgidus, complexed with various ligands, have been determined at about 1.8 Å resolution. This enzyme has optimal activity in the temperature range of 70-90 °C and pH 10-11. AFL consists of an N-terminal α/β-hydrolase fold domain, a small lid domain, and a C-terminal β-barrel domain. The N-terminal catalytic domain consists of a 6-stranded β-sheet flanked by seven α-helices, four on one side and three on the other side. The C-terminal lipid binding domain consists of a β-sheet of 14 strands and a substrate covering motif on top of the highly hydrophobic substrate binding site. The catalytic triad residues (Ser136, Asp163, and His210) and the residues forming the oxyanion hole (Leu31 and Met137) are in positions similar to those of other lipases. Long-chain lipid is located across the two domains in the AFL-substrate complex. Structural comparison of the catalytic domain of AFL with a homologous lipase from Bacillus subtilis reveals an opposite substrate binding orientation in the two enzymes. AFL has a higher preference toward long-chain substrates whose binding site is provided by a hydrophobic tunnel in the C-terminal domain. The unusually large interacting surface area between the two domains may contribute to thermostability of the enzyme. Two amino acids, Asp61 and Lys101, are identified as hinge residues regulating movement of the lid domain. The hydrogen-bonding pattern associated with these two residues is pH dependent, which may account for the optimal enzyme activity at high pH. Further engineering of this novel lipase with high temperature and alkaline stability will find its use in industrial applications.     

An ultraviolet spectrophotometric assay for the screening of sn-2-specific lipases using 1,3-O-dioleoyl-2-O-α-eleostearoyl-sn-glycerol as substrate

In the present study, we propose a continuous assay for the screening of sn-2 lipases by using triacylglycerols (TAGs) from Aleurites fordii seed (tung oil) and a synthetic TAG containing the α-eleostearic acid at the sn-2 position and the oleic acid (OA) at the sn-1 and sn-3 positions [1,3-O-dioleoyl-2-O-α-eleostearoyl-sn-glycerol (sn-OEO)]. Each TAG was coated into a microplate well, and the lipase activity was measured by optical density increase at 272 nm due to transition of α-eleostearic acid from the adsorbed to the soluble state. The sn-1,3-regioselective lipases human pancreatic lipase (HPL), LIP2 lipase from Yarrowia lipolytica (YLLIP2), and a known sn-2 lipase, Candida antarctica lipase A (CALA) were used to validate this method. TLC analysis of lipolysis products showed that the lipases tested were able to hydrolyze the sn-OEO and the tung oil TAGs, but only CALA hydrolyzed the sn-2 position. The ratio of initial velocities on sn-OEO and tung oil TAGs was used to estimate the sn-2 preference of lipases. CALA was the enzyme with the highest ratio (0.22 ± 0.015), whereas HPL and YLLIP2 showed much lower ratios (0.072 ± 0.026 and 0.038 ± 0.016, respectively). This continuous sn-2 lipase assay is compatible with a high sample throughput and thus can be applied to the screening of sn-2 lipases.     

Exploring the active site cavity of human pancreatic lipase

Within the scope of improving the efficiency of pancreatic enzyme replacement therapy in cystic fibrosis, the feasibility of shifting the pH-activity profile of pancreatic lipase toward acidic values was investigated by site specific mutagenesis in different regions of the catalytic cavity. We have shown that introducing a negative charge close to the catalytic histidine induced a shift of the pH optimum toward acidic values but strongly reduced the lipase activity. On the other hand, a negative charge in the entrance of the catalytic cleft gives rise to a lipase with improved properties and twice more active than the native enzyme at acidic pH.     

Lactate dehydrogenase in an interpolyelectrolyte complex. Function and stability

A new method for encapsulating enzymes by multilayer polyelectrolyte coating is proposed. The method consists in a stepwise adsorption of polyelectrolytes from solution onto protein aggregates formed by salting out the proteins in highly concentrated salt solutions. Polystyrene sulfonate and fluorescence-labeled polyalylamine were used for capsule formation. The size of lactate dehydrogenase aggregates covered by four layer pairs of electrolytes was 1-5 microns, as indicated by fluorescence microscopy. The catalytic characteristics and stability of pig muscle lactate dehydrogenase (EC 1.1.1.13) incapsulated in multilayer electrolyte complex obtained by this method were studied. It was found that the affinity of the substrate pyruvate for the enzyme in the polyelectrolyte complex (K(M)) did not essentially change as compared with the free enzyme. Incapsulated lactate dehydrogenase showed the following features that distinguish it from the free form: (1) the lifetime in diluted solutions increases from 30 min (without capsules) to 1-2 days (in capsules); (2) a higher stability to basic denaturation (up to pH 10); and (3) the absence of substrate inhibition of enzyme in the polyelectrolyte complex. The changes in the catalytic characteristics of incapsulated lactate dehydrogenase are discussed in terms of an increase in effective pK values of amino acid perturbed by polyelectrolyte coating of enzyme.     

Convenient one-step purification and immobilization of lipase using a genetically encoded aldehyde tag

To avoid the unwanted and random covalent linkage between the cross-linker and enzyme's active site in covalent immobilization, a genetically encoded “aldehyde tag” was introduced into recombinant lipase and applied for the one-step purification and covalent immobilization of this enzyme. The effects of the immobilization time, temperature and the amount of enzyme were investigated, and the thermo-stability of immobilized lipase was also examined. The specific activity and the k_cat/K_m of the immobilized lipase using aldehyde tag (IL-AT) were 2.50 and 3.02 fold higher, respectively, than those of the traditionally immobilized lipase using glutaraldehyde (IL-GA). The newly immobilized lipase also presented better thermo-stability than the traditionally immobilized one. The results show that the recombinant enzyme could be conveniently immobilized without glutaraldehyde and that the enzyme's active site was well protected. This is a new immobilization method able to avoid glutaraldehyde or 2,4,6-trichloro-1,3,5-triazine as an activating agent. The greener method without hazardous chemicals for the one-step purification and immobilization of an enzyme using a genetically encoded “aldehyde tag” can be exploited for numerous other enzyme purification and immobilization applications.     

Development of a bioautographic method for the detection of lipase inhibitors

An autobiographic method based on the thin layer chromatogram was developed by using the chemical system that comprises p-Nitrophenyl butyrate and bromothymol blue for detecting the lipase inhibitor. Lipase inhibitory zones were visualized as blue spots against the greenish yellow background. This method could able to detect the well known lipase inhibitor, orlistat up to the concentration of 1 ng which is better than the earlier method. This method could also able to detect the lipase inhibition activities from the un-explored species of Streptomyces. The developed method can be used not only for the screening of unknown samples for the lipase inhibitors but also for the purification of the lipase inhibitors from the unknown samples.     

Selective antibody precipitation using polyelectrolytes: A novel approach to the purification of monoclonal antibodies

We evaluated the potential for polyelectrolyte induced precipitation of antibodies to replace traditional chromatography purification. We investigated the impact of solution pH, solution ionic strength and polyelectrolyte molecular weight on the degree of precipitation using the anionic polyelectrolytes polyvinylsulfonic acid (PVS), polyacrylic acid (PAA), and polystyrenesulfonic acid (PSS). As we approached the pI of the antibody, charge neutralization of the antibody reduced the antibody–polyelectrolyte interaction, reducing antibody precipitation. At a given pH, increasing solution ionic strength prevented the ionic interaction between the polyelectrolyte and the antibody, reducing antibody precipitation. With increasing pH of precipitation, there was an increase in impurity clearance. Increasing polyelectrolyte molecular weight allowed the precipitation to be performed under conditions of higher ionic strength. PVS was selected as the preferred polyelectrolyte based on step yield following resolubilization, purification performance, as well as the nature of the precipitate. We evaluated PVS precipitation as a replacement for the initial capture step, as well as an intermediate polishing step in the purification of a humanized monoclonal antibody. PVS precipitation separated the antibody from host cell impurities such as host cell proteins (HCP) and DNA, process impurities such as leached protein A, insulin and gentamicin, as well as antibody fragments and aggregates. PVS was subsequently removed from antibody pools to <1 µg/mg using anion exchange chromatography. PVS precipitation did not impact the biological activity of the resolubilized antibody. Biotechnol. Bioeng. 2009;102: 1141–1151. © 2008 Wiley Periodicals, Inc.     

Analysis of the interactions between Eudragit® L100 and porcine pancreatic trypsin by calorimetric techniques

Flexible-chain polymers with charge (polyelectrolytes) can interact with globular proteins with a net charge opposite to the charge of the polymers forming insoluble complexes polymer-protein. In this work, the interaction between the basic protein trypsin and the anionic polyelectrolyte Eudragit^® L100 was studied by using isothermal calorimetric titrations and differential scanning calorimetry. Turbidimetric assays allowed determining that protein-polymer complex was insoluble at pH below 5 and the trypsin and Eudragit^® L100 concentrations required forming the insoluble complex. DSC measurements showed that the T_m and denaturalization heat of trypsin increased in the polymer presence and the complex unfolded according to a two-state model. ΔH° and ΔS° binding parameters obtained by ITC were positives agree with hydrophobic interaction between trypsin and polymer. However, ionic strength of 1.0 M modified the insoluble complex formation. We propose a mechanism of interaction between Eudragit^® L100 and trypsin molecules that involves both hydrophobic and electrostatic interactions. Kinetic studies of complex formation showed that the interaction requires less than 1 min achieving the maximum quantity of complex. Finally, a high percentage of active trypsin was precipitated (approximately 76% of the total mass of protein). These findings could be useful in different protocols such as a protein isolation strategy, immobilization or purification of a target protein.     

A DIGESTIVE LIPASE OF Pieris brassicae L. (LEPIDOPTERA: PIERIDAE): PURIFICATION, CHARACTERIZATION, AND HOST PLANTS EFFECTS

The properties of a digestive lipase from the larval midgut of Pieris brassicae were studied by performing biochemical purification, characterization, effect of host plants, and extracted inhibitors. The purification process revealed a lipase with a purification fold of 42, recovery of 18.12%, molecular weight mass of 72.3 kDa, optimal pH at 11, and optimal temperature at 30°C, as well as stability at the optimal temperature for 12 h. The purified enzyme was inhibited by the ions Na(+) , Mn(+) , Fe(2+) , and Cu(2+) and the inhibitors SDS, EDTA, TTHA, and mercaptoethanol. Ca(2+) and Mg(2+) increased activity of the purified lipase, but urea, PMSF, EGTA, and DTC had no effect on enzymatic activity. Feeding of larvae on three host plants, Trepaeolus majus, Brassica olearcea var. alba, and B. olearcea var. rubra revealed the highest lipase activity on T. majus, but the two varieties of B. olearcea significantly decreased lipase activity. Extraction of a crude inhibitor from two varieties of B. olearcea demonstrated that the crude inhibitor inhibited the purified lipase up to 75%. The inhibitor changed the kinetic parameters of the enzyme by elevating the K(m) , as in competitive inhibition. The data suggest a possible role for plant lipase inhibitors in host plant resistance.     

Charge patch attraction and reentrant condensation in DNA-liposome complexes

We investigated the formation of complexes between cationic liposomes built up by DOTAP and three linear anionic polyions, with different charge density and flexibility, such as a single-stranded ssDNA, a double-stranded dsDNA and the polyacrylate sodium salt [NaPAA] of three different molecular weights. Our aim is to gain further insight into the formation mechanism of polyion-liposome aggregates of different sizes (lipoplexes), by comparing the behavior of DNA with a model polyelectrolyte, such as NaPAA, with approximately the same charge density but with a higher flexibility. We employed dynamic light scattering (DLS) and transmission electron microscopy (TEM) measurements, in order to explore both the hydrodynamic and structural properties of the aggregates resulting from polyion-liposome interaction and to present a comprehensive picture of the complexation process. The phenomenology can be summarized in a charge ratio-dependent scenario, where the main feature is the formation of large equilibrium clusters due to the aggregation of intact polyion-coated vesicles. At increasing polyion-liposome ratio, the size of the clusters continuously increases, reaching a maximum at a well-defined value of this ratio, and then decreases (“reentrant” condensation). The aggregation mechanism and the role of the polyion charge density in the complex formation are discussed in the light of the recent theories on the correlated adsorption of polyelectrolytes at charged interfaces. Within this framework, the phenomena of charge inversion and the reentrant condensation, peaked at the isoelectric point, finds a simple explanation.     

Incapsulation of enzymes into polyelectrolyte nano- and microcapsules and the problem of the development of enzymatic microdiagnostics

The incapsulation of proteins into polyelectrolyte microcapsules (PE-microcapsules) has been studied with the aim to develop microdiagnostica for the presence of low-molecular-weight compounds in native biological fluids. The problem was solved using two enzymes: lactate dehydrogenase and urease. Polyelectrolyte microcapsules were prepared using two polyanions: polystyrene sulfonate (PSS) and dextran sulfate (DS), and two polycations: polyallylamine (PAA) and polydiallylmethylammonium (PDADMA). CaCO3 microspherulites with the incapsulated enzyme served as a "core" in the formation of polyelectrolyte microcapsules. It was shown that the main problem in the preparation of a polyelectrolyte microdiagnosticum is the selection of an oppositely charged pair of polyelectrolytes optimal for the active functioning of the enzyme. It follows from the results obtained that the best polyelectrolyte pairs for the formation of the envelope of a PE-microcapsule are PAA/DS and PAA/PSS for lactate dehydrogenase and PSS/PDADMA for urease. Taking into account these data, we designed enzyme-containing microcapsules with different polyelectrolyte compositions and different numbers of layers and studied their properties.     

Production of an acidic and thermostable lipase of the mesophilic fungus Penicillium simplicissimum by solid-state fermentation

The production of a lipase by a wild-type Brazilian strain of Penicillium simplicissimum in solid-state fermentation of babassu cake, an abundant residue of the oil industry, was studied. The enzyme production reached about 90 U/g in 72 h, with a specific activity of 4.5 U/mg of total proteins. The crude lipase showed high activities at 35–60 °C and pH 4.0–6.0, with a maximum activity at 50 °C and pH 4.0–5.0. Enzyme stability was enhanced at pH 5.0 and 6.0, with a maximum half-life of 5.02 h at 50 °C and pH 5.0. Thus, this lipase shows a thermophilic and thermostable behavior, what is not common among lipases from mesophilic filamentous fungi. The crude enzyme catalysed the hydrolysis of triglycerides and p-nitrophenyl esters (C4:0–C18:0), preferably acting on substrates with medium-chain fatty acids. This non-purified lipase in addition to interesting properties showed a reduced production cost making feasible its applicability in many fields.     

An extracellular solvent stable alkaline lipase from Pseudomonas sp. DMVR46: Partial purification,characterization and application in non-aqueous environment

The biosynthesis of esters is currently of much commercial interest because of the increasing popularity and demand for natural products among consumers. Biotransformation and enzymatic methods of ester synthesis are more effective when performed in non-aqueous media. In present study, an organic solvent stable Pseudomonas sp. DMVR46 lipase was partially purified by acetone precipitation and ion exchange chromatography with 28.95-fold purification. The molecular mass of the lipase was found to be ∼32 kDa. The partially purified lipase was optimally active at 37 °C and pH 8.5. The enzyme showed greater stability toward organic solvents such as isooctane, cyclohexane and n-hexane retaining more than 70% of its initial activity. The metal ions such as Ca²⁺, Ba²⁺ and Mg²⁺ had stimulatory effects on lipase activity, whereas Co²⁺ and Zn²⁺ strongly inhibited the activity. Also lipase exhibited variable specificity/hydrolytic activity toward different 4-nitrophenyl esters. DMVR46 lipase was further immobilized into AOT-based organogels used for the synthesis of flavor ester pentyl valerate in presence of organic solvents. The organogels showed repeated use of enzyme with meager loss of activity even upto 10 cycles. The solvent-stable lipase DMVR46 thus proved to be an efficient catalyst showing an attractive potency for application in biocatalysis under non-aqueous environment.