Structural stability of lipase from wheat germ in alkaline pH

The present investigation shows the effect of alkalinepH on the structure-function relationship of lipase from wheat germ. There is a 70% decrease in lipase activity atpH 10.0, which decreases to 93% atpH 12.0 as compared to neutralpH activity (Rajendranet al. 1990). This change is shown to be as a result of loss ofa-helical structure with a concomitant increase in aperiodic structure. The results with fluorescence spectra and tyrosyl ionization indicate gradual exposure of aromatic side chains of tyrosine and tryptophan to the bulk solvent along with the structural changes. The enzyme is in an extended form at alkalinepH with a volume change of -1300 ml/mol as also indicated by increase in reduced viscosity to 12.5 ml/g and significant decrease in sedimentation coefficient. The kinetics of the reaction points to a cooperative pseudo first-order reaction as determined by stopped-flow kinetic analysis in the ultraviolet region. The inactivation mechanism appears to follow a two-step mechanism of a fast and a slow reaction.This paper was presented partly at the 58th Annual General Body Meeting of the Society of Biological Chemists (India), Izatnagar, Uttar Pradesh, India, 1989.     

Effect ofpH on the formation of Compounds II and III of horseradish peroxidase

The formation of Compounds II and III of horseradish peroxidase from Compound I and potassium ferrocyanide and from Compound II and excess hydrogen peroxide, respectively, was studied as a function ofpH at 25°C and a constant ionic strength of 0.11. The yield of Compound II obtained increases progressively with increase inpH; a mixture of Compounds I and II is produced at acidicpH. Pure Compound III is obtained at allpH values, but the highest yield is obtained atpH values between 6.0 and 7.0. The yield of p-670, formed when Compound III is allowed to stand for 60 min, decreases with increase inpH, while the decay of Compound III also decreases with increase inpH. Therefore p-670 is the decay product of Compound III.     

Potential βPP-processing proteinase activities from alzheimer's and control brain tissues

Fluorogenic peptide substrates designed to encompass the reportedα-secretory and amyloidogenic cleavage sites of the amyloid-β precursor protein (βPP) were used to analyze proteinase activities in brain extracts from control patients and those with Alzheimer's disease (AD). Activity against the secretory substrate atpH 7.5 in control and AD brains produced a major endopeptidase cleavage at the Lys687-Leu688 bond (βPP770 numbering), consistent with theβPP secretase cleavage. Activity in control brains against the amyloidogenic substrate atpH 7.5 produced one cleavage at the Ala673-Glu674 bond, two residues C-terminal to the amyloidogenic Met-Asp site. However, in three of four AD brains, the major cleavage was at the Asp-Ala bond, one residue from the amyloidogenic site. Both endopeptidase and carboxypeptidase activities in AD brains were lower than in control brains. Proteinase activities against the secretory substrate had a major optimum atpH 3.0–4.0 and another atpH 6.0–7.5. Proteinase activities against the amyloidogenic substrate had a major optimum at or belowpH 3.0 and another atpH 6.0. Using both substrates, activities at lowpH were higher in AD brains than in controls, while atpH above 6.5, activities in control brains were higher than in AD. These results indicate that the levels of proteolytic enzymes in AD brains are altered relative to controls.     

Association-dissociation and denaturation-renaturation of high-molecular-weight protein: carmin from safflower seed (Carthamus tinctorius L.) in alkaline solution

The effect of alkalinepH on the association, dissociation, and denaturation of carmin, the high-molecular-weight protein from safflower seed was investigated in thepH range 7–12, using various biophysical techniques. The results indicate that the multimeric protein carmin dissociates atpH 8.0 where denaturation has not set in. The association-dissociation of the protein can be represented schematically as 11S 7S 4S 2S. AbovepH 10, the protein undergoes simultaneous dissociation and denaturation. The denaturation process appears to be complete at pH 12.5. The protein undergoes conformational change and covalent modifications and cleavage during the denaturation process. A reversibility study shows that the process of dissociation is reversible to a large extent, whereas denaturation appears to be irreversible. These results are discussed in terms of association-dissociation, denaturation and alkaline-catalyzed covalent modifications and cleavage of seed proteins.     

Eco-friendly methodology for efficient synthesis and scale-up of 2-ethylhexyl-p-methoxycinnamate using Rhizopus oryzae lipase and its biological evaluation

Lipase-mediated synthesis of phenolic acid esters is a green and economical alternative to current chemical methods. Octyl methoxycinnamate, an important UVB-absorbing compound, was synthesized by the esterification of p-methoxycinnamic acid with 2-ethyl hexanol using Rhizopus oryzae lipase. A molar ratio of 1:2 of p-methoxycinnamic acid and 2-ethyl hexanol was found to give an optimum yield using cyclo-octane (50 ml) as reaction solvent, at a temperature of 45 °C, and 750 U of lipase, resulting in a yield of 91.3 % in 96 h. This reaction was successfully scaled up to 400-ml reaction size where 88.6 %bioconversion was achieved. The synthesized compound was found to have superior antioxidant activity as compared to ascorbic acid. The synthesized compound also exhibited good antimicrobial activity against Escherichia coli, Klebsiella pneumonia, Salmonella typhi, Staphylococcus aures, Candida albicans (yeast), Aspergillus niger, Alternaria solani, and Fussarium oxysporum by well diffusion method in terms of zone of inhibitions (in mm).     

An investigation into the protective effect of various salts on the acid inactivation of human serum butyrylcholinesterase (EC 3.1.1.8)

Human serum butyrylcholinesterase (EC 3.1.1.8) loses 100% of its activity toward butyrylthiocholine in 60 min atpH 3.0. This deactivation is retarded by 1.37 M ammonium sulfate to a loss of 40% after 60 min atpH 3.0. Reneutralization experiments suggest that the mechanism for this acid inactivation does not exclusively involve hydrolysis of peptide bonds or protonation of the enzyme's active site. Studies with different anions and cations demonstrate that the order of their effectiveness as protective agent against acid inactivation closely follows the Hofmeister series. No relationship was found between catalytic activation or inhibition by salt and protection from acid inactivation. Ultraviolet difference studies at 288 nm with enzyme brought topH 2.7 frompH 8.0 in the presence and absence of 1.37 M ammonium sulfate demonstrated no change in UV absorbance with ammonium sulfate present and approximately a 0.15 ODU rise in absorbance in the absence of ammonium sulfate. These results suggest that acidicpH conditions result in deactivating stereochemical changes in the active site of butyrylcholinesterase and that certain anions and cations, according to the Hofmeister series, are able to protect the enzyme from acid inactivation by stabilizing the active conformation of its active site.     

A convenient test for lipase activity in aqueous-based solutions

We proposed a convenient and accurate method for the measurement of lipase activity in a uniform aqueous-based substrate solution. In this work, lipase from Candida rugosa was used as the model lipase to test its catalytic ability toward p-nitrophenyl palmitate (p-NPP), which was suspended in a mixture of p-NPP ethanol solution and buffer. An ultraviolet–visible spectrophotometer was used to efficiently measure the liberated p-nitrophenol without extraction or centrifugation. Several factors that affected lipase activity were investigated, such as the ratio of p-NPP ethanol solution to buffer, the concentrations of p-NPP and lipase, as well as the temperature, reaction time, pH and agitation rate. Additionally, enzyme catalytic parameters such as K_m, V_m and “activation energy” were also assessed. We determined the optimal conditions for lipase in this homogeneous system and demonstrated lipase's catalytic performance in this condition followed Michealis–Menten kinetics.     

Isolation and Purification of Staphylococcal Lipase

An extracellular lipase was isolated from the cell-free supernatant fluid of a 24-hr culture of Staphylococcus aureus grown in Trypticase Soy Broth at 37 C with continuous agitation. The purification was achieved by precipitation with alcohol followed by differential precipitation at pH 8.6 and 4.3. Subsequent purification with Sephadex G 200 and BioGel 300 yielded a preparation which showed a 350- to 450-fold increase in specific activity over the original cell-free supernatant fluid. The purified lipase was homogeneous over a BioGel 300 column and showed a single peak on electrophoresis in a Veronal buffer (pH 8.6, Γ/2 = 0.1). The electrophoretic mobility was -7.78 × 10^-5 cm² per v per sec.     

Enhanced Reactivity of Rhizopus oryzae Lipase Displayed on Yeast Cell Surfaces in Organic Solvents: Potential as a Whole-Cell Biocatalyst in Organic Solvents

Immobilization of enzymes on some solid supports has been used to stabilize enzymes in organic solvents. In this study, we evaluated applications of genetically immobilized Rhizopus oryzae lipase displayed on the cell surface of Saccharomyces cerevisiae in organic solvents and measured the catalytic activity of the displayed enzyme as a fusion protein with α-agglutinin. Compared to the activity of a commercial preparation of this lipase, the activity of the new preparation was 4.4 × 10⁴-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate and 3.8 × 10⁴-fold higher in an esterification reaction with palmitic acid and n-pentanol (0.2% H₂O). Increased enzyme activity may occur because the lipase displayed on the yeast cell surface is stabilized by the cell wall. We used a combination of error-prone PCR and cell surface display to increase lipase activity. Of 7,000 colonies in a library of mutated lipases, 13 formed a clear halo on plates containing 0.2% methyl palmitate. In organic solvents, the catalytic activity of 5/13 mutants was three- to sixfold higher than that of the original construct. Thus, yeast cells displaying the lipase can be used in organic solvents, and the lipase activity may be increased by a combination of protein engineering and display techniques. Thus, this immobilized lipase, which is more easily prepared and has higher activity than commercially available free and immobilized lipases, may be a practical alternative for the production of esters derived from fatty acids.     

Glycerol Ester Hydrolase Activity of Microbacterium thermosphactum

Microbacterium thermosphactum possesses a significant glycerol ester hydrolase (lipase, EC 3.1.1.3) activity and a weak but definite carboxylic ester hydrolase (esterase, EC 3.1.1.1) activity. Harvested whole cell preparations contained 53 units of lipase activity with tripropionin as the substrate. This activity decreased with an increasing chain length of fatty acid in the triglyceride to 13 units with trilaurin as the substrate and no activity with tripalmitin. Maximum lipase activity was found at a temperature of 35 to 37 C and at a pH of 7.1 to 7.3. Lipase activity was associated with three different protein peaks when the protein of cell-free extract was fractionated by polyacrylamide gel electrophoresis.     

Physicochemical and immunological characterization of the type E botulinum neurotoxin binding protein purified fromClostridium botulinum

Type E botulinum neurotoxin is produced byClostridium botulinum along with a neurotoxin binding protein which helps protect the neurotoxin from adversepH, temperature, and proteolytic conditions. The neurotoxin binding protein has been purified as a 118-kDa protein. Secondary structure content of the neurotoxin binding protein as revealed by far-UV circular dichroism spectroscopy was 19% α-helix, 50%β-sheets, 28% random coils, and 3%β-turns. This compared to 22% α-helix, 44%β-sheets, 34% random coils, and noβ-turns of the type E botulinum neurotoxin. The complex of the two proteins revealed 25%α-helix, 45%β-sheets, 27% random coils, and 3%β-turns, suggesting a significant alteration at least in theα-helical folding of the two proteins upon their interaction. Tyrosine topography is altered considerably (28%) when the neurotoxin and its binding protein are separated, indicating strong interaction between the two proteins. Gel filtration results suggested that type E neurotoxin binding protein clearly complexes with type E neurotoxin. The interaction is favored at lowpH as indicated by an initial binding rate of 8.4 min^?1 atpH 5.7 compared to 4.0 min^?1 atpH 7.5 as determined using a fiber optic-based biosensor. The neurotoxin and its binding protein apparently are of equivalent antigenicity, as both reacted equally on enzyme-linked immunosorbent assay to polyclonal antibodies raised against the toxoid of their complex.     

Characterization of an extracellular lipase and its chaperone from Ralstonia eutropha H16

Lipase enzymes catalyze the reversible hydrolysis of triacylglycerol to fatty acids and glycerol at the lipid–water interface. The metabolically versatile Ralstonia eutropha strain H16 is capable of utilizing various molecules containing long carbon chains such as plant oil, organic acids, or Tween as its sole carbon source for growth. Global gene expression analysis revealed an upregulation of two putative lipase genes during growth on trioleate. Through analysis of growth and activity using strains with gene deletions and complementations, the extracellular lipase (encoded by the lipA gene, locus tag H16_A1322) and lipase-specific chaperone (encoded by the lipB gene, locus tag H16_A1323) produced by R. eutropha H16 was identified. Increase in gene dosage of lipA not only resulted in an increase of the extracellular lipase activity, but also reduced the lag phase during growth on palm oil. LipA is a non-specific lipase that can completely hydrolyze triacylglycerol into its corresponding free fatty acids and glycerol. Although LipA is active over a temperature range from 10 °C to 70 °C, it exhibited optimal activity at 50 °C. While R. eutropha H16 prefers a growth pH of 6.8, its extracellular lipase LipA is most active between pH 7 and 8. Cofactors are not required for lipase activity; however, EDTA and EGTA inhibited LipA activity by 83 %. Metal ions Mg²⁺, Ca²⁺, and Mn²⁺ were found to stimulate LipA activity and relieve chelator inhibition. Certain detergents are found to improve solubility of the lipid substrate or increase lipase-lipid aggregation, as a result SDS and Triton X-100 were able to increase lipase activity by 20 % to 500 %. R. eutropha extracellular LipA activity can be hyper-increased, making the overexpression strain a potential candidate for commercial lipase production or in fermentations using plant oils as the sole carbon source.     

Substitution of Val72 residue alters the enantioselectivity and activity of Penicillium expansum lipase

Error-prone PCR was used to create more active or enantioselective variants of Penicillium expansum lipase (PEL). A variant with a valine to glycine substitution at residue 72 in the lid structure exhibited higher activity and enantioselectivity than those of wild-type PEL. Site-directed saturation mutagenesis was used to explore the sequence-function relationship and the substitution of Val72 of P. expansum lipase changed both catalytic activity and enantioselectivity greatly. The variant V72A, displayed a highest enantioselectivity enhanced to about twofold for the resolution of (R, S)-naproxen (E value increased from 104 to 200.7 for wild-type PEL and V72A variant, respectively). In comparison to PEL, the variant V72A showed a remarkable increase in specific activity towards p-nitrophenyl palmitate (11- and 4-fold increase at 25 and 35?°C, respectively) whereas it had a decreased thermostability. The results suggest that the enantioselective variant V72A could be used for the production of pharmaceutical drugs such as enantiomerically pure (S)-naproxen and the residue Val 72 of P. expansum lipase plays a significant role in the enantioselectivity and activity of this enantioselective lipase.     

Characterization of Neutral Lipase BT-1 Isolated from the Labial Gland of Bombus terrestris Males

Background

In addition to their general role in the hydrolysis of storage lipids, bumblebee lipases can participate in the biosynthesis of fatty acids that serve as precursors of pheromones used for sexual communication.

Results

We studied the temporal dynamics of lipolytic activity in crude extracts from the cephalic part of Bombus terrestris labial glands. Extracts from 3-day-old males displayed the highest lipolytic activity. The highest lipase gene expression level was observed in freshly emerged bumblebees, and both gene expression and lipase activity were lower in bumblebees older than 3 days. Lipase was purified from labial glands, further characterized and named as BT-1. The B. terrestris orthologue shares 88% sequence identity with B. impatiens lipase HA. The molecular weight of B. terrestris lipase BT-1 was approximately 30 kDa, the pH optimum was 8.3, and the temperature optimum was 50°C. Lipase BT-1 showed a notable preference for C8-C10 p-nitrophenyl esters, with the highest activity toward p-nitrophenyl caprylate (C8). The Michaelis constant (K_m) and maximum reaction rate (V_max) for p-nitrophenyl laurate hydrolysis were K_m = 0.0011 mM and V_max = 0.15 U/mg.

Conclusion

This is the first report describing neutral lipase from the labial gland of B. terrestris. Our findings help increase understanding of its possible function in the labial gland.     

Formation of isoaspartate 99 in bovine and porcine somatotropins

Asparagine 99 in bovine (BST) and porcine somatotropins (PST) was converted to an isoaspartate residue during incubation at neutral or alkalinepH. Isoaspartate 99 BST or isoaspartate 99 PST was resolved from the normal somatotropin by reversed-phase high-performance liquid chromatography (HPLC). The altered peptide of residues 96–108 which contains isoaspartate 99 was detected by tryptic peptide mapping of the modified BST or PST. Amino acid sequencing, amino acid analysis, mass spectrometry, and co-elution with a chemically synthesized peptide containing isoaspartate 99 were used to demonstrate the existence of isoaspartate in the modified peptides. Peptide bond cleavage between Asn 99 and Ser 100 also occurred during incubation of BST and PST at neutral or alkalinepH. This chemically cleaved product was resolved on reversed-phase HPLC from both the isoaspartate 99 and normal somatotropin molecules.     

Lipases are soluble and active in glycerol carbonate as a novel biosolvent

Glycerol carbonate was synthesized as biosolvent for the development of soluble enzymatic system. The effects of various reaction parameters on activity and stability of lipases were investigated using the transesterification of ethyl butyrate with n-butanol as a model reaction. Enzymatic activity in glycerol carbonate was compared with that in water and in conventional organic solvents with different ionizing and dissociating abilities. The pK_a value of trichloroacetic acid and transesterification activities of Candida antarctica lipase B and Candida rugosa lipase in glycerol carbonate are similar to those in water, indicating that ionizing and dissociating powers are capable of satisfactorily predicting the biocompatibility of organic solvents for soluble enzymatic systems.     

Action of Lipases of Staphylococcus aureus on Milk Fat

The activity of the lipase(s) of two strains of coagulase-positive Staphylococcus aureus was determined in milk fat incubated at 15, 22, and 30 C for 8 days. Total fat hydrolysis was measured by acid degree values (ADV). Neutral lipids were separated into component groups on a Florisil column. Free fatty acids were determined by temperature-programmed gas-liquid chromatography. The ADV were 25 to 50% greater at 22 than at 15 C and 4 to 7 times greater at 30 than at 22 C. The lipases liberated as much as 0.48 g of fatty acids per gram of fat during 8 days at 30 C. The enzyme showed a predilection for the palmitic acid-glycerol bond. Addition of fatty acids C₁₄ to C₁₈ inclusive to inoculated sterile skim milk caused inhibition of S. aureus as follows: (i) complete at 0.05 and 0.10% concentration of C₁₀ and (ii) partial at 0.05 and complete at 0.10% concentration of C₈. The samples showing inhibition were negative for peptonization, coagulase, and change in pH. Addition of oleic and stearic acid to sterile skim milk inoculated with S. aureus resulted in an increase in nonprotein nitrogen, and the C₄ to C₁₂ acids caused a decrease in protease activity.     

Lipase production by Bacillus stearothermophilus S-203 in shake flasks

Fermentation conditions for the production of lipase by Bacillus stearothermophilus S-203 have been established in 300 ml baffled shake flasks. The optimum medium consists of yeast extract 0.3%, ammonium acetate 0.5%, cotton-seed soil 0.4%, sucrose 0.5%, Tween 40 0.4%, phosphate buffer 0.04 M pH 6.9, and other inorganic salts. The optimum temperature is 45–55 °C, volume per flask 50 ml, shaking speed 300 rpm, and incubation time 24 hours. Under these conditions the yield of lipase was 59.5 units per ml.     

Scanning electron microscopic studies of lipase-catalysed esterification catalysis for the synthesis of stearoyl lactate and p-cresyl laurate

The state of three lipases, two from Rhizomucor miehei and one from porcine pancreas, employed in the esterification reactions leading to the preparation of food additive esters were investigated by scanning electron microscopy (SEM). The lipases employed in the synthesis of stearoyl lactic acid and p-cresyl laurate in 10 ml solvent at 40–60 °C in shake-flask experiments and 150 ml in non-polar solvents at 50–60 °C in bench-scale level experiments were compared. All three lipases, which were subjected to high temperatures and non-polar solvents for a prolonged period of incubation of 72–120 h, showed decrease in the compactness when compared to unused lipase. The presence of buffer preserved the activity and compactness and the absence of the same reduced the amount of enzyme per unit area on the support. R. miehei lipase samples subjected to reaction in presence of 0.0004 ml of 0.1 M buffer/mg enzyme preparation at different pH values (4.0–9.0) showed a decrease in compactness of the enzyme on the surface which correlated to an increase in esterification activity. An increase in volume of buffer (0.0002–0.003 ml/mg enzyme preparation) in the reaction mixture at pH 7.0 showed a decrease in compactness and also a reduction in activity. The studies indicate that a compromise between pH and volume of buffer can lead to variation in the extent of adsorption, distribution and activity, enabling the achievement of maximum conversions in the esterification reactions.     

A magnetically separable biocatalyst for resolution of racemic naproxen methyl ester

Candida rugosa lipase (CRL) was encapsulated via the sol–gel method, using 5, 11, 17, 23-tetra-tert-butyl-25,27-bis(2-aminopyridine)carbonylmethoxy-26, 28-dihydroxy-calix[4]arene-grafted magnetic Fe₃O₄ nanoparticles (Calix-M-E). The catalytic activity of encapsulated lipase (Calix-M-E) was tested both in the hydrolysis of p-nitrophenyl palmitate (p-NPP) and the enantioselective hydrolysis of racemic naproxen methyl ester. The present study demonstrated that the calixarene-based compound has the potential to enhance both reaction rate and enantioselectivity of the lipase-catalyzed hydrolysis of racemic naproxen methyl ester. The encapsulated lipase (Calix-M-E) had great catalytic activity and enantioselectivity (E > 400), as well as remarkable reusability as compared to the encapsulated lipase without supports (E = 137) for S-Naproxen.