Calf Pregastric Esterase Research Articles

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.

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.

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.

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.

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.

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.

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.

Purification and characterization of pregastric esterase from calf

Calf pregastric esterase (PGE) was purified from calf gullet tissues. The tissue was excised and lyophilized, and lipid materials were extracted with acetone and n-butanol at −20 °C. Proteins were extracted from the delipidated tissue with a buffer containing a chaotropic salt (NaSCN) to solubilize hydrophobically bound protein aggregates. Calf PGE precipitated from the crude extract at pH 5.0. The precipitated, solubilized proteins were subjected to anion-exchange chromatography on DEAE-Sephacel, and the enzymatic activity was eluted using a linear gradient from 0.10 to 0.50 m NaCl at pH 8.0. Fractions with high specific activities were then chromatographed twice using gel filtration on Sephadex G-100. The resultant enzyme was shown to be pure upon discontinuous electrophoresis in 12% polyacrylamide containing 0.1% sodium dodecyl sulfate (SDS-PAGE). From SDS-PAGE gel patterns, a molecular weight of 49,000 was determined. The amino acid composition of the enzyme allowed calculation of an “average hydrophobicity” (Bigelow index) of 1150 cal/residue. This indicates that calf PGE is relatively hydrophobic, being similar to proteins such as α-lactalbumin and bovine serum albumin in average hydrophobicity.

IN VITRO OBSERVATIONS ON FACTORS AFFECTING CALF PREGASTRIC ESTERASE ACTIVITY

Three in vitro experiments were conducted to determine the efficacy of calf pregastric esterase (PGE) for hydrolyzing various fats and the influence of various factors on its lipolytic activity. Calf PGE was effective in hydrolyzing butterfat and coconut oil, hydrolyzed smaller amounts of the other plant oils tested and had a very limited capacity for lard and the six grades of tallow studied. The esterase retained good lipolytic activity for butterfat over the pH range normally encountered in the calf abomasum after feeding liquid diets. Rennet clotting of reconstituted skim-milk at pH 6.1 reduced enzyme hydrolysis of butterfat by 30%, presumably due to fat occlusion in the clot. Lecithin, skim-milk powder, casein, and lactalbumin markedly increased PGE activity; Ca++ had no effect. The bile salts taurodeoxycholate, glycochenodeoxycholate and taurochenodeoxycholate markedly inhibited PGE lipolysis, whereas others (taurocholate, deoxycholate, cholate, glycocholate) had little or no effect.

Assay and Characterization of Pregastric Esterase

A recording pH stat has been adapted to the assay of pregastric esterase preparations. Assay substrate containing 5ml n-tributyrin, 0.5ml 10% soy lecithin in mineral oil, 0.6g acid casein, and 95ml water is emulsified in a blender. Enzyme inoculum is prepared via rehydration of 0.2 to 1.0g powder in 75–80ml 0.5 m NaCl solution for 20min at ambient temperatures. The suspension is made up to 100ml and 1ml is inoculated into 5ml of substrate on a pH stat, set to control at pH 6.20 and 40C. The resultant slope is a function of enzyme activity. This assay is more precise than currently used colorimetric tests. Calf pregastric esterase was found most stable at pH 5.5, using the pH stat. The enzyme was most active at 45C. Kid and lamb enzymes also had maximum activity at 42–45C. pH optima occurred at 5.3 to 5.5 on both tributyrin and milk fat substrates. Minor peaks occurred at pH 8.5 and 8.7 for calf and kid, respectively. Pregastric esterase has esterase characteristics when evaluated on solutions and emulsions of triacetin. The enzyme is not as active upon 1-mono- and dibutyrin as pancreatic, milk, or Aspergillus niger lipases.