Pregastric Esterase Research Articles

Altering the Chain Length Specificity of a Lipase from Pleurotus citrinopileatus for the Application in Cheese Making.

In traditional cheese making, pregastric lipolytic enzymes of animal origin are used for the acceleration of ripening and the formation of spicy flavor compounds. Especially for cheese specialities, such as Pecorino, Provolone, or Feta, pregastric esterases (PGE) play an important role. A lipase from Pleurotus citrinopileatus could serve as a substitute for these animal-derived enzymes, thus offering vegetarian, kosher, and halal alternatives. However, the hydrolytic activity of this enzyme towards long-chain fatty acids is slightly too high, which may lead to off-flavors during long-term ripening. Therefore, an optimization via protein engineering (PE) was performed by changing the specificity towards medium-chain fatty acids. With a semi-rational design, possible mutants at eight different positions were created and analyzed in silico. Heterologous expression was performed for 24 predicted mutants, of which 18 caused a change in the hydrolysis profile. Three mutants (F91L, L302G, and L305A) were used in application tests to produce Feta-type brine cheese. The sensory analyses showed promising results for cheeses prepared with the L305A mutant, and SPME-GC-MS analysis of volatile free fatty acids supported these findings. Therefore, altering the chain length specificity via PE becomes a powerful tool for the replacement of PGEs in cheese making.

Flavour development in dairy cream using fish digestive lipases from Chinook salmon ( Oncorhynchustshawytscha) and New Zealand hoki ( Macruronusnovaezealandiae)

Digestive lipases from Chinook salmon and New Zealand hoki were evaluated as flavour modifying agents in dairy products. Cream was incubated either with fish lipase or commercially available lipases used in dairy flavour development – calf pregastric esterase (Renco™ PGE) and microbial lipase (Palatase® 20,000 L). The fish enzymes were more similar to calf PGE in terms of the total amount and types of fatty acids released over the course of the reaction. Like the pregastric esterase, the fish enzymes released mainly short chain fatty acids. The highest specificity was towards the key dairy product flavour and odour compounds, butanoic and hexanoic acids. The odour intensity of hexanoic acid produced by the salmon lipase, as measured by SPME–GC–MS, was similar to that produced by both Palatase® and PGE. Free fatty acid composition, together with sensory characteristics of lipase-treated creams, demonstrated the potential for flavour enhancement in dairy products using fish lipases.

Characterization of a Lipase from Sporidiobolus pararoseus 25-A Which Produces Cheese Flavor

A lipase from a Sporidiobolus pararoseus strain was purified from culture filtrate by (NH4)2SO4 precipitation, and DEAE-Toyopearl 650M, Butyl-Toyopearl 650M, and Toyopearl HW-55 chromatography. The purified enzyme appeared as a single band with a molecular mass of 37 kDa by SDS-PAGE. The optimum temperature and pH were approximately 60°C and 6.0, respectively. The specificity toward triglyceride was similar to that of pregastric esterase Lipase PGET. Addition of the lipase during the mozzarella cheese-making process produced a strong cheese flavor. Taken together, the lipase produced by S. pararoseus is considered a potential replacement of pregastric esterase in the dairy industry.

Purification and biochemical characterization of a turkey preduodenal esterase

A preduodenal esterase was purified to homogeneity from turkey ( Meleagris gallopavo) pharyngial tissue. Pure turkey pregastric esterase (TPrE) was obtained after anion exchange chromatography (DEAE-cellulose), Sephacryl S-200 gel filtration, anion exchange chromatography (Mono-Q sepharose) and affinity chromatography (Blue-Gel Affi Gel). The pure enzyme has an apparent molecular mass of 50 kDa, as determined by SDS/PAGE analysis. The optimum pH and temperature for enzyme activity using tributyrin as substrate were 8.5 and 48 °C, respectively. Under these conditions, the specific activity measured was 650 U/mg. No significant lipolytic activity was found when was tested on triolein or liprocil as substrates or with monolayer dicaprin with TPrE. In contrast, TPrE displayed a maximal activities of 800, 680 and 520 U/mg with vinyl acetate, vinyl propionate and vinyl butyrate, respectively. This enzyme retained 75% of its maximal activity when incubated for 30 min at pH 4 and 50 °C, but it was completely inactivated after an incubation for 10 min at 60 °C. The TPrE N-terminal amino acid sequence showed similarities to the N-terminal sequence of a thioesterase from mallard duck, but no similarity with known preduodenal lipases was found.

Selectivity Aspects of Lipolysis of Milkfat (Butteroil) by Immobilized Pregastric Esterases from Kid Goats and Lambs

ABSTRACTPregastric esterases derived from the salivary tissues of kid goats and lambs were immobilized by physical adsorption in a continuous flow, hollow‐fiber reactor and subsequently used to hydrolyze anhydrous milkfat, thereby producing lipolyzed butteroils. These 2 esterases were used in separate trials conducted at temperatures of 40 °C, 45 °C, and 50 °C and pH values of 5.0, 6.0, and 7.0. High‐performance liquid chromatography (HPLC) was used to determine the relative proportions of the different fatty acid residues released as functions of the reactor space time for the indicated operating conditions. For both esterases, the rates of the lipolysis reactions were fastest at 40 °C and a pH of 6.0. The rates of release of the native individual fatty acid residues were modeled using expressions of the generalized Michaelis‐Menten form based on a ping pong bi bi mechanism. Nonlinear regression analyses indicated that for both esterases, the experimental data were consistent with the form of the ping pong bi bi mechanism for which the rate controlling step is deacylation of the enzyme.

Purification and characterization of a pregastric esterase from a hygienized kid rennet paste.

Rennet pastes obtained by maceration of gastric tissues from suckling kids are used traditionally to produce some artisanal cheeses in Spain. Besides milk-clotting function, rennet pastes provide proteolytic activity and lipolytic system, essentially pregastric, necessary in the development of piquant flavor typical of these cheeses. A simple and reproducible procedure allows us to obtain a standardized rennet paste that posses the desired activity and is of good microbiological quality. Concomitantly, a kid pregastric esterase (KPGE) was purified to homogeneity. The purification procedure was based on an aqueous extract of hygienized rennet paste (HRP), which was chromatographed on DEAE-Sepharose Fast Flow then adsorbed on phenyl superose followed by a re-chromatography on the same column. The final enzymatic preparation, where the overall activity recovery was 3%, showed a molecular mass of 53 kDa. The highest activity was determined on p-nitrophenyl butyrate, but marked hydrolysis was also detected on beta-naphthyl caprylate. In contrast, low activity on tributyrin (substrate under emulsion form) was detected, thus confirming the esterase character of purified enzyme.

Lipolysis of Butter Oil by an Immobilized Kid Goat Pregastric Esterase

ABSTRACT: A kid goat pregastric esterase immobilized in a continuous flow, hollow‐fiber reactor was employed to hydrolyze anhydrous milkfat, producing lipolyzed butter oil. The effects of temperature and pH on the rate of lipolysis were monitored by titration of the effluent stream for total acidity. The overall rate of hydrolysis was fastest at 40 °C and pH 6.0. The reaction rate was modeled by an expression of the general Michaelis‐Menten form based on a ping pong bi bi mechanism. Nonlinear regression analyses indicated that the experimental data were consistent with the form of the ping pong bi bi mechanism that assumes that the rate‐controlling step is acylation of the enzyme.

Production of lipolyzed butteroil by a calf pregastric esterase immobilized in a hollow fiber reactor: III. Effect of glycerol

Lipolysis of butter oil in a hollow fiber reactor containing an immobilized calf pregastric esterase was studied at 40 degrees C, a pH of 6.0, and glycerol concentrations of 0, 150, and 500 g/L in the buffer solution. The concentrations of 10 fatty acid species in the lipolyzed product were determined using high-performance liquid chromatography. The rate of loss of enzyme activity and the relative selectivities of this esterase depended on the glycerol concentration. By contrast, the overall rate of release of fatty acids was not affected by the glycerol concentration. Loss of enzyme activity was modeled using first-order kinetics. The models for deactivation and reaction kinetics were fit simultaneously to the data. The model was successful in describing the rates of release of all 10 fatty acid species for a range of space times from 0 to 25 h. The parameters of the model were tested for dependence on glycerol concentration.

Lipolysis of Butter Oil by Immobilized Lamb Pregastric Esterase: I. Uniresponse Kinetics—pH and Temperature Effects

Lamb pregastric esterase, immobilized by physical adsorption on microporous polypropylene in a hollow fiber reactor, has been employed to effect the continuous hydrolysis of the triglycerides in butter oil. Experimental data were obtained at temperatures from 35 to 45°C and pH values from 5.5 to 6.5. The overall rate of hydrolysis was fastest at 40°C and a pH of 6.0. Nonlinear regression methods were employed to determine the kinetic parameters of rate expressions based on a generic Ping-Pong Bi Bi mechanism. The best nonlinear fit of the data was consistent with a mechanism that assumes that acylation of the enzyme is the rate-limiting step in the hydrolysis reaction.

Effect of pH on the production of lipolyzed butter oil by a calf pregastric esterase immobilized in a hollow-fiber reactor: I. uniresponse kinetics.

A calf pregastric esterase immobilized in a hollow-fiber reactor was employed to hydrolyze milkfat, thereby producing a lipolyzed butteroil. The reaction kinetics can be modeled by a two-parameter model of the general Michaelis-Menten form based on a ping-pong bi-bi mechanism; the rate of enzyme deactivation can be modeled as a first-order reaction. The initial concentration of accessible glyceride bonds, [G](O), was estimated by complete saponification of the substrate butteroil as 2400 mM. An extra sum of squares test indicated that not only the parameters of the kinetic generalized Michaelis-Menten model, but also the deactivation-rate constant varied significantly with pH. The optimum pH, for lypolysis is near 6.0 at a temperature of 40 degrees C because at this pH the rate of deactivation of the esterase is minimized.

Effect of pH on the production of lipolyzed butter oil by a calf pregastric esterase immobilized in a hollow-fiber reactor: II. Multiresponse kinetics.

The lipolysis of butter oil in a hollow-fiber reactor containing an immobilized calf pregastric esterase was studied at 40 degrees C and at pH values of 4.0, 5.0, 6.0, and 7.0. The concentrations of ten fatty acid species in the lipolyzed product were determined using high-performance liquid chromatography (HPLC). The relative specificity of this esterase depended on pH. Three mathematical models derived from a generalized Michaelis-Menten mechanism were tested for their ability to describe the rates of release of individual specific fatty acids. Loss of enzyme activity was modeled using first order kinetics. The models for deactivation and reaction kinetics were fit simultaneously to the data. The parameters of the model were also tested for dependence on pH. The model was successful in describing the rates of release of all ten fatty acid species for a range of space times and pH values.

Lipolysis of Butteroil over an Immobilized Calf Pre-Gastric Esterase: Variation of Rate and Product Distribution with Temperature

Calf pre-gastric esterase immobilized in a hollow fiber reactor catalyzed the hydrolysis of butteroil from 22 to 40°C, with the fastest rate at 30°C, but variations in the relative rates of release of individual fatty acid residues with temperature led to differences in product composition with concomitant adverse effects on flavor profiles. The hydrolyzates produced at 30°C were characterized by less desirable flavors than those prepared at either 22 or 40°C.

Calf Pregastric Esterase Catalyzed Hydrolysis of 4-Nitrophenylalkanoates: pH and Temperature Effects

A commercial extract from the tongue and epiglottal region of suckling calf was partially purified to yield a calf pregastric enzyme with esterase activity against 4-nitrophenylalkanoate esters (C2-C12) at 370C, pH 7.2. The Km against 4-nitrophenylacetate was 0.023 mM, however, when 4-nitro-phenyldodecanoate was used as the substrate, the Km was 1.06 AM. The maximum activity was achieved at ca. 1.6 AM, which is similar to its critical micelle concentration. The reactivity is dependent upon pH. A pK of 7.23 which was obtained in the pH range 5.5-9.0 is indicative of a single ionizable residue. The activity dependence upon temperature of the partially purified enzyme was determined within the range of 25°C to 48°C and Eyring parameters were calculated.

Fatty acid specificity of immobilized kid goat pre-gastric esterase: Effects of pH

Kid goat pre-gastric esterase immobilized in a hollow fiber reactor was used to hydrolyse butteroil at buffer pH values of 5.0, 6.0 and 7.0. Overall hydrolysis proceeded fastest at pH 6.0, but changes in volatile fatty acid ratios with pH suggest that the same enzyme:substrate system can produce different flavor profiles, e. g., at pH 6.0 the relative rate of production of undesirable soaplike flavors is minimized.

Inhibition studies on calf pregastric esterase: the enzyme has no functional thiol group.

Pregastric esterase (PGE) (EC 3.1.1.3) was purified to homogeneity from calf pharyngeal tissue. The enzyme had an apparent molecular mass of 50 kDa, as determined by SDS/PAGE. The serine-binding reagent diethyl p-nitrophenyl phosphate was a potent inhibitor of PGE. This is in accordance with the claim that a functional serine residue is necessary for the lipolytic activity of lipases. PGE was not inhibited by the thiol reagents 5,5'-dithiobis(2-nitrobenzoic acid) or 4,4'-dithiopyridine. A partial inhibition with dodecyldithio-5-(2-nitrobenzoic acid) was observed, but the same degree of inhibition was caused by the non-esterified fatty acid C(12:0). PGE shows a great sequence similarity to gastric lipases. Gastric lipases have three cysteine residues, and two of these form a disulphide bridge. Blocking the remaining free cysteine with thiol reagents inactivates the gastric lipases. The fact that PGE is not inhibited by thiol reagents indicates that PGE has no functional free thiol group. The PGE cDNA codes only for two cysteines, and their involvement in the formation of a disulphide bridge was demonstrated.

Lamb Pregastric Esterase Catalyzed Hydrolysis of 4-Nitrophenyl-Acetate and -Dodecanoate: pH, Temperature and Bile Salt Effects

Two fractions, F2 and F3, eluted by ion exchange chromatography of the commercial extract of lamb pregastric enzyme have esterase activity against 4-nitrophenylacetate, PNPA. Preheat treatment of fraction F3 at pH 7.2, 50°C for 15 min effectively removes its lipase activity against tributyrin while leaving the esterase component relatively unaffected. The esterase components in fractions F2 and F3 and the lipase component in fraction F3 have K m values against PNPA equal to 0.96, 2.1 and 2.7 mM, respectively. When 4-nitrophenyldecanoate, PNPDe, was used as substrate, the maximum activity was reached at its critical micelle concentration, 1.6 μM, for catalysis by all three enzymes. The reactivity is dependent upon pH and p K values of 6.7 and 8.4, 6.7 and 7.5, and 7.3 were determined from the lipase and esterase components in fraction F3, and the fraction F2 esterase, respectively. The dependence upon temperature of the activities of the esterase components against PNPA were determined within the range 25-47.5°C and Arrhenius parameters have been calculated. The presence of sodium taurocholate affected the activity of each enzyme to a small and differing extent.

Improving the continuous production of lipolyzed butteroil with pregastric esterases immobilized in a hollow fiber reactor

Kid and calf pregastric esterases were semi-purified by micro- and ultrafiltration of a crude tissue preparation. When the resulting solutions were utilized to immobilize these enzymes in a polypropylene hollow fiber reactor, the activities obtained when both techniques were employed were greater than those observed when only microfiltration was performed.

Immobilization of pregastric esterases in a hollow fiber reactor for continuous production of lipolysed butteroil

Lingual Upases from calf, lamb and goal were semi-purified by cold storage and microfiltralion, and subsequently immobilized by physical adsorption on hollow fibers fabricated of microporous polypropylene. The immobilized lipases were employed in a hollow fiber membrane reactor to effect the continuous hydrolysis of a butterfat fraction that remains liquid at 18°C. The goat lipase showed higher lipolytic activity than the other two, but all three enzymes had good selectivilies for short-chain fatty acids. No deactivalion was observed for any of the immobilized enzymes during 5 d of continuous operation. A process based on this technology offers a number of advantages for the commercial production of lipolysed butteroil.

The cDNA sequence encoding bovine pregastric esterase.

The polymerase chain reaction (PCR) was used to amplify specific parts of the gene encoding calf pregastric esterase (PGE). Primers based on conserved regions in human gastric lipase (HGL) and rat lingual lipase (RLL) were used to screen a cDNA library prepared from calf tongue tissue. This resulted in the cloning of the entire coding sequence for PGE, which exists as a mature 378-amino-acid (aa) polypeptide with a molecular mass of 42,960 Da. The PGE, HGL and RLL genes all share a high degree of identity at both the nucleotide and amino-acid sequence levels. Except for the Gly-Xaa-Ser-Xaa-Gly sequence containing the active site Ser, there is little identity with non-preduodenal lipases.

Fatty acid composition of the triglyceride and free fatty acid fractions in different cows-, ewes- and goats-milk cheeses

The fatty acid composition of the triglyceride fraction and the individual and total free fatty acids in different cows-, ewes- and goats-milk cheeses were analysed. The highest total free fatty acid levels were recorded in blue cheeses (Roquefort: 26.0 g/kg; Cabrales: 57.3 g/kg) and in the aged Manchego cheese (32.4 g/kg), followed by those in hard cheeses manufactured using unpurified rennet pastes containing pregastric esterases (Parmesan: 13.7 g/kg; Majorero: 20.8 g/kg). These samples had sharp flavours. The soft cheeses with surface flora presented moderate levels of free fatty acids (Camembert: 5.1 g/kg; de la Vera: 9.9 g/kg). The remaining cheeses considered (Mahon: 8.7 g/kg; Roncal: 8.2 g/kg; Tetilla: 5.8 g/kg; Idiazabal: 5.6 g/kg) also exhibited moderate levels of lipolysis. A comparison of the fattyacid profiles for the two fractions studied yielded higher contents for the short-chain fatty acids in the free fatty acid fraction, except in the blue and goats-milk cheeses.