Enzymological investigations on the causes for the PSE-syndrome, I. Comparative studies on pyruvate kinase from PSE- and normal pig muscles

A fast breakdown of glycogen is observed in muscles of stress-susceptible pigs leading to pale, soft and exudative (PSE) meat. We report a comparative study of pyruvate kinase from muscles of normal and PSE-prone pigs. Compared with enzyme from normal muscle, pyruvate kinase isolated from PSE-muscle shows a five times lower K(m) for phosphoenol pyruvate and a more than ten times higher k (cat)K (m) value. The pH-dependency of the enzymatic activity is shifted to more acidic values for pyruvate kinase from PSE muscles. According to isoelectric focusing, pyruvate kinase from PSE muscle consists of three isoforms, while only two isoforms are detectable in pyruvate kinase preparations from normal pigs. The various isoforms were isolated by preparative isoelectric focusing and their steady-state properties were compared. Isoform 3, which is found only in PSE muscle, shows a 10-fold higher specific activity, a 30-fold lower K(m) value and a 100-fold increased k (cat)K (m) value for phosphoenol pyruvate compared to isoform 1. The presence of isoform 3 in PSE-muscle appears to be responsible for the high activity of this enzyme under the more acidic conditions prevailing in PSE-muscle. In vitro phosphorylation and dephosphorylation experiments using total enzyme and purified isoenzyme 1 suggest that isoforms 2 and 3 arise from isoform 1 by phosphorylation. Thus protein phosphorylation seems to be responsible for the shift in activity of pyruvate kinase, a key enzyme of glycolysis, under the acidic conditions of PSE-muscles.     

Purification,characterisation and application of α‐l‐rhamnosidase from Penicillium citrinum MTCC‐8897

An α‐l ‐rhamnosidase secreted by Penicillium citrinum MTCC‐8897 has been purified to homogeneity from the culture filtrate of the fungal strain using ammonium sulphate precipitation and cation‐exchange chromatography on carboxymethyl cellulose. The sodium dodecyl sulphate/polyacrylamide gel electrophoresis analysis of the purified enzyme gave a single protein band corresponding to the molecular mass 51.0 kDa. The native polyacrylamide gel electrophoresis also gave a single protein band confirming the enzyme purity. The K_m and V_max values of the enzyme for p‐nitrophenyl α‐l ‐rhamnopyranoside were 0.36 mm and 22.54 μmole min^?1 mg^?1, respectively, and k_cat value was 17.1 s^?1 giving k_cat/K_m value of 4.75 × 10⁴ m ^?1 s^?1. The pH and temperature optima of the enzyme were 7.0 and 60 °C, respectively. The purified enzyme liberated l ‐rhamnose from naringin, rutin, hesperidin and wine, indicating that it has biotechnological application potential for the preparation of l ‐rhamnose and other pharmaceutically important compounds from natural glycosides containing terminal α‐l ‐rhamnose and also in the enhancement of wine aroma.     

Isolation of cathepsin B from the muscle of silver carp (Hypophthalmichthys molitrix) and comparison of cathepsins B and L actions on surimi gel softening

Cathepsin B from silver carp muscle was purified to 263-fold by acid treatment, ammonium sulfate fractionation, followed by a series of chromatographic separations. The molecular mass of the purified enzyme was 29 kDa as determined by SDS-PAGE and immunoblotting. The purified enzyme was activated by dithiothreitol and cysteine while it was substantially inhibited by E-64, suggesting the purified enzyme belongs to the cysteine proteinase family. Optimal pH and temperature were 5.5 and 35 °C, respectively. The enzyme catalyzed the hydrolysis of Z-Arg-Arg-MCA with a parameter of K_m (90 μM) and K_cat (20.3 s⁻¹), but hardly hydrolyzed Arg-MCA. Analysis of surimi gel strength and microstructure showed that cathepsins B and L were capable of destroying the network structure of silver carp surimi gels, consequently causing gel softening. Cathepsin L might play an important role in the modori effect.     

Characterisation of thermostable trypsin and determination of trypsin isozymes from intestine of Nile tilapia (Oreochromis niloticus L.)

Trypsin from intestinal extracts of Nile tilapia (Oreochromis niloticus L.) was characterised. Three-step purification – by ammonium sulphate precipitation, Sephadex G-100, and Q Sepharose – was applied to isolate trypsin, and resulted in 3.77% recovery with a 5.34-fold increase in specific activity. At least 6 isoforms of trypsin were found in different ages. Only one major trypsin isozyme was isolated with high purity, as assessed by SDS-PAGE and native-PAGE zymogram, appearing as a single band of approximately 22.39 kDa protein. The purified trypsin was stable, with activity over a wide pH range of 6.0–11.0 and an optimal temperature of approximately 55–60 °C. The relative activity of the purified enzyme was dramatically increased in the presence of commercially used detergents, alkylbenzene sulphonate or alcohol ethoxylate, at 1% (v/v). The observed Michaelis–Menten constant (K_m) and catalytic constant (K_cat) of the purified trypsin for BAPNA were 0.16 mM and 23.8 s⁻¹, respectively. The catalytic efficiency (K_cat/K_m) was 238 s⁻¹ mM⁻¹.     

Purification and functional characterisation of an α‐l‐rhamnosidase from Penicillium citrinum MTCC‐3565

An extracellular α‐l ‐rhamnosidase from Penicillium citrinum MTCC‐3565 has purified to homogeneity from its culture filtrate using ethanol precipitation and cation‐exchange chromatography on carboxymethyl cellulose. The purified enzyme gave a single protein band corresponding to molecular mass of 45.0 kDa in SDS‐PAGE analysis showing the purity of the enzyme preparation. The native PAGE analysis showed the monomeric nature of the purified enzyme. Using p‐nitrophenyl α‐l ‐rhamnopyranoside as substrate, K_m and V_max values of the enzyme were 0.30 mm and 27.0 μm min mg^?1, respectively. The k_cat value was 20.1 s giving k_cat/K_m value of 67.0 mm s^?1 for the same substrate. The pH and temperature optima of the enzyme were 8.5 and 50 °C, respectively. The activation energy for the thermal denaturation of the enzyme was 29.9 KJ mol^?1. The α‐l ‐rhamnosidase was able to hydrolyse naringin, rutin and hesperidin and liberated l ‐rhamnose, indicating that the purified enzyme can be used for the preparation of α‐l ‐rhamnose and pharmaceutically important compounds by derhamnosylation of natural glycosides containing terminal α‐l ‐rhamnose. The α‐l ‐rhamnosidase was active at the level of ethanol concentration present in wine, indicating that it can be used for improving wine aroma.     

Purification and characterization of a novel glucoamylase from Fusarium solani

Thermostable enzymes are currently being investigated to improve industrial processes of starch saccharification. A novel glucoamylase was purified to electrophoretic homogeneity from the culture supernatant of Fusarium solani on a fast protein liquid chromatographic system (FPLC). The recovery of glucoamylase after gel filtration on FPLC was 31.8% with 26.2-fold increase in specific activity. The enzyme had a molecular mass of 40 kDa by SDS-PAGE and 41 kDa by gel filtration. The glucoamylase exhibited optimum activity at pH 4.5. The K_cat and K_m were 441/min and 1.9 mg/ml, respectively, for soluble starch, specificity constant (K_cat/K_m) was 232. The enzyme was thermally stable at 50 °C and retained 79% activity after 60 min at this temperature. The half-life of the enzyme was 26 min at 60°C. The enzyme was slightly stimulated by Cu²⁺ and Mg²⁺ and strongly inhibited by Hg²⁺, Pb²⁺, Zn²⁺, Ni²⁺ and Fe³⁺.     

Identification and kinetic analysis of a functional homolog of elongation factor 3, YEF3 in Saccharomyces cerevisiae

Yeast and other fungi contain a soluble elongation factor 3 (EF-3) which is required for growth and protein synthesis. EF-3 contains two ABC cassettes, and binds and hydrolyses ATP. We identified a homolog of the YEF3 gene in the Saccharomyces cerevisiae genome database. This gene, designated YEF3B, is 84% identical in protein sequence to YEF3, which we will now refer to as YEF3A. YEF3B is not expressed during growth under laboratory conditions, and thus cannot rescue growth of YEF3A deletion strains. However, YEF3B can take the place of YEF3A in vivo when expressed from the YEF3A or ADH1 promoters. The products of the YEF3A and YEF3B genes, EF-3A and EF-3B, respectively, were expressed from the ADH1 promoter and purified. Both factors possessed basal and ribosomal-stimulated ATPase activity, and had similar affinity for yeast ribosomes (103 to 113 nm). K_m values for ATP were similar, but the K_cat values differed significantly. Ribosome-dependent ATPase activity of EF-3A was more efficient than EF-3B, since the K_cat and K_cat/K_m values for EF-3A were about two-fold higher; however, the difference in K_cat/K_m values between the two factors was small for basal ATPase activity. © 1998 John Wiley & Sons, Ltd.     

Isolation and properties of AMP deaminase from jumbo squid (Dosidicus gigas) mantle muscle from the Gulf of California,Mexico

Adenosine monophosphate (AMP) deaminase was purified from jumbo squid mantle muscle by chromatography in cellulose phosphate, Q-Fast and 5′-AMP sepharose. Specific activity of 2.5 U/mg protein, 4.5% recovery and 133.68 purification fold were obtained at the end of the experiment. SDS–PAGE showed a single band with 87 kDa molecular mass, native PAGE proved a band of 178 kDa, whereas gel filtration detected a 180 kDa protein, suggesting the homodimeric nature of this enzyme, in which subunits are not linked by covalent forces. Isoelectric focusing of this enzyme showed a pI of 5.76, which agrees with pI values of AMP deaminase from other invertebrate organisms. AMP deaminase presented a kinetic sigmoidal plot with V_max of 1.16 μM/min/mg, K_m of 13 mM, K_cat of 3.48 μM.s⁻¹ and a K_cat/K_m of 267 (mol/L)⁻¹.s⁻¹. The apparent relative low catalytic activity of jumbo squid muscle AMP deaminase in the absence of positive effectors is similar to that reported for homologous enzymes in other invertebrate organisms.     

Isolation and properties of 5′-nucleotidase isolated from jumbo squid (Dosidicus gigas) mantle muscle from the Gulf of California,Mexico

The enzyme 5′-nucleotidase of jumbo squid (Dosidicus gigas) mantle was purified and its SDS–PAGE showed a single band of 33 kDa, whereas a protein with a molecular mass of 107 kDa was detected by gel filtration suggesting a homotrimeric nature of this enzyme. Subunits of the named enzyme were not linked by covalent bonds. Isoelectric focusing of this enzyme showed a pI of 3.6–3.8 and presented a hyperbolic kinetics with V_max of 1.16 μM/min/mg of protein, K_m of 1.49 mM, K_cat of 3.48 μM of P_ι s⁻¹ and K_cat/K_m relation of 356.52 ((mol/L)⁻¹ s⁻¹). Purified enzyme preferred AMP as substrate (by 6.7-folds) than IMP, showing a K_m of 6.34 mM, V_max of 0.19 μM/min/mg of protein a K_cat of 0.3388 mol of P_ι s⁻¹ and K_cat/K_m relation of 53.44 ((mol/L)⁻¹ s⁻¹). The low K_m in relation to purified AMP deaminase of the same organism suggested a high contribution of 5′-nucleotidase in AMP degradation in jumbo squid mantle.     

An α‐l‐rhamnosidase from Aspergillus awamori MTCC‐2879 and its role in debittering of orange juice

The extracellular α‐l ‐rhamnosidase has been purified by growing a new fungal strain Aspergillus awamori MTCC‐2879 in the liquid culture growth medium containing orange peel. The purification procedure involved ultrafiltration using PM‐10 membrane and anion‐exchange chromatography on diethyl amino ethyl cellulose. The purified enzyme gave single protein band in SDS‐PAGE analysis corresponding to molecular mass 75.0 kDa. The native PAGE analysis of the purified enzyme also gave a single protein band, confirming the purity of the enzyme. The K_m and V_max values of the enzyme for p‐nitrophenyl‐α‐l ‐rhamnopyranoside were 0.62 mm and 27.06 μmole min^?1 mg^?1, respectively, yielding k_cat and k_cat/k_m values 39.90 s^?1 and 54.70 mm ^?1 s^?1, respectively. The enzyme had an optimum pH of 7.0 and optimum temperature of 60 °C. The activation energy for the thermal denaturation of the enzyme was 35.65 kJ^?1 mol^?1 K^?1. The purified enzyme can be used for specifically cleaving terminal α‐l ‐rhamnose from the natural glycosides, thereby contributing to the preparation of pharmaceutically important compounds like prunin and l ‐rhamnose.     

Kinetic Analysis of the Digestion of Bovine Type I Collagen Telopeptides with Porcine Pepsin

Collagen is frequently digested using pepsin in industries to produce a triple helical collagen without the N‐ and C‐terminal telopeptides. However, kinetic analysis of this reaction is difficult because several Lys residues in the N‐ and in the C‐terminal telopeptides form covalent bonds, leading to multiple substrates species, and pepsin cleaves collagen at various sites in the N‐terminal and in the C‐terminal telopeptides, yielding different products. Here we performed kinetic analysis of the digestion of bovine type I collagen with porcine pepsin. The reaction could be monitored by SDS‐PAGE by measuring the intensity of the protein bands corresponding to the variant β11 chain. We obtained kinetic parameters relative to the decrease in the variant β11 chain upon digestion. At pH 4.0, the K_m and k_cat values increased with increasing temperature (30 to 65 °C), although the k_cat/K_m values were stable. Additional cleavage at the helical region was detected at 45 to 65 °C. At 37 °C, the K_m and k_cat values increased with decreasing pH, and the k_cat/K_m values at pH 2.1 to 4.5 were stable and higher than those at pH 5.0 and 5.5. No additional cleavage was detected at the examined pH. Thus, the optimal pH and temperatures for selective digestion of collagen telopeptides with pepsin are 2.1 to 4.5 and 30 to 40 °C, respectively. These results suggest that the method might be useful for the kinetic analysis of the digestion of collagen telopeptides with pepsin.     

Enzymological investigations on the causes for the PSE-syndrome, II. Comparative studies on glycogen phosphorylase from pig muscles

In order to investigate the cell biological causes for the fast breakdown of glycogen which is observed during the development of the PSE (pale, soft, exudative) syndrome in muscles of stress-susceptible pigs, muscle glycogen phosphorylase (GP) as a key enzyme in two isoforms, a and b, of the energy turnover was isolated from M. longissimus dorsi of normal and PSE-prone pigs of the German Landrace. GP b as well as GP a from normal and PSE-muscles exist in a dimeric form with a molecular weight of 97 000 D per subunit. The tendency for tetramerization of GP b increases in the presence of ATP, whereas the enzyme activity is simultaneously inhibited. The catalytic activities of GP a and GP b from both groups of animals show an optimum at pH 7.0. GP b can be activated to GP a by phosphorylation with the result of a 25% higher optimum specific activity in the case of normal and PSE-muscles. In interaction with glycogen and glucose-1-phosphate GP b follows the characteristics of a Michaelis-Menten kinetic, whereas the binding of AMP and phosphate proves to be allosteric. In comparison of the structural and kinetic characteristics of GP from normal as well as PSE-muscles no significant differences could be determined, indicating that GP does not belong to those factors which are triggering an accelerated energy turnover of ATP in muscles of stress-susceptible pigs.     

The trigger for PSE condition in stress-susceptible pigs

Tightly coupled mitochondria from M. longissimus dorsi (LD) muscle of different breeds of pigs showed variations in the rate of Ca²⁺ efflux during anaerobiosis. The initial fast efflux rate of Ca²⁺ could be used to relate the stress-susceptibility of the pigs used. Mitochondria from LD muscle of stress-susceptible (SS) Pietrain and Poland China pigs showed an anaerobic rate of Ca²⁺ efflux twice that of stress-resistant (SR) Large Whites. Mitochondria from crossbreds (Large White x Saddleback) showed a Ca²⁺ efflux rate intermediate between that of purebred SS and SR pigs. Halothane enhanced the rate of Ca²⁺ efflux only in SS pigs, and this effect could be eliminated by MgCl₂. The marked difference in the rate of Ca²⁺ efflux between SS and SR pigs, and the enhancement in the rate of Ca²⁺ efflux by halothane only in SS pigs, offers an ultimate explanation for the formation of PSE (pale, soft and exudative) muscle and malignant hyperthermia in SS pigs. The excess Ca²⁺ liberated from mitochondria of SS pigs is postulated to be the trigger for PSE meat and also the case of malignant hyperthermia.     

The nucleotide sequence and initial characterization of pyruvate decarboxylase from the yeast Hanseniaspora uvarum

We have isolated a pyruvate decarboxylase (PDC) gene from the yeast Hanseniaspora uvarum using the Saccharomyces cerevisiae PDC1 gene as a probe. The nucleotide sequence of this gene was determined and compared to PDC genes from yeast and other organisms. The H. uvarum PDC gene is more than 70% identical to the S. cerevisiae PDC isozymes and possesses a putative thiamine diphosphate binding site. The PDC enzyme was purified and partially characterized. The H. uvarum PDC was very similar to other known PDCs; the K_m for pyruvate was 0·75 mM, and the enzyme is a homotetramer with subunits of M_r = 57 000. The sequence has been submitted to GenBank under Accession No. U13635.     

Relationship between rate of post mortem pH fall and ageing of Longissimus muscle in pietrain pigs

Twenty-six pietrain pigs exhibiting wide variability in pH₄₅ (pH value 45 min post mortem) values were used to assess the mechanical behaviour of cooked and raw Longissimus muscle at 24 h and 8 days post mortem. Rheological behaviour was studied in compression under strain conditions which allow specific determination of myofibrillar resistance. The muscles were divided into three groups on the basis of pH₄₅ values: (Severe pale, soft and exudative (PSE), pH₄₅ = 5.49 ± 0.03 (n = 9); PSE pH₄₅ = 5.78 ± 0.04 (n = 10); Normal, pH₄₅ = 6.35 ± 0.05 (n = 7). The three groups did not differ significantly for ultimate pH (pH_u = 5.57 ± 0.02 for the 26 muscles). A lower mechanical resistance of raw samples was found in the Normal group, regardless of the time post mortem. Although this difference was not significant, a significant relationship was found between this trait and pH₄₅ at both times post mortem (r = ?0.43, P < 0.05, at 24 h and r = ?0.44, P < 0.05, at 8 days post mortem). However, no significant effect of pH₄₅ was found on the mechanical resistance of cooked muscles. The relative decrease in myofibrillar strength after an 8-day ageing period was not affected by pH₄₅ but this was due to the fact that the three groups exhibited different starting points. Mechanical resistance of raw and cooked muscles at 24 h post mortem were significantly related to pH_u (r = ?0.55, P < 0.01, and r = ?0.49, P < 0.05, respectively). The present results show that after an 8-day ageing period, PSE muscles are not fully aged.     

Purification and characterization of a phytate-degrading enzyme from germinated oat (Avena sativa)

A phytate-degrading enzyme (myo-inositol hexakisphosphate phosphohydrolase) has been purified about 5,400-fold from germinated oat seedlings to apparent homogeneity. The molecular mass of the native monomeric enzyme was estimated to be about 67 kDa. Optimal pH for degradation of phytate was 5.0 and the optimal temperature 38 °C. Kinetic parameters for the hydrolysis of Na-phytate are K_M 30 µM and k_cat 356 s⁻¹ at 35 °C and pH 5.0. The oat phytase exhibits a broad affinity for various phosphorylated compounds and hydrolyses phytate in a stepwise manner. The first hydrolysis product was identified as D /L -l(1,2,3,4,5) P₅. © 1999 Society of Chemical Industry     

Purification and characterisation of carrot (Daucus carota L) pectinesterase

A pectinesterase isoform with an alkaline isoelectric point of over 8.66 was detected in crude extracts of carrot. The enzyme was purified by ion exchange and molecular exclusion chromatography. The molecular weight of the isoform was 25 kDa, determined in native conditions by filtration through Sephadex G‐75 SF. The enzyme showed a high affinity for its substrate, with K_m and V_max values of 0.031 mg ml^?1 and 6.77 units respectively for apple pectin. The pectinesterase activity exhibited an optimum around pH 7.4 and was activated by metallic ions, with optimum activities at NaCl concentrations between 130 and 330 mM and at CaCl₂ concentrations between 15 and 50 mM . The enzyme was activated most by Ca²⁺ and exhibited a greater tolerance of high concentrations of Na⁺. Comparison of its heat stability with other pectinesterases of vegetable origin indicated that the purified isoform was very thermolabile, being rendered inactive by heating for 5 min at 70 °C. The enzyme was inhibited by high concentrations of polygalacturonic acid and competitively inhibited by D ‐galacturonic acid, with a K_i value of 1 mM . Copyright © 2003 Society of Chemical Industry     

Isolation and characterisation of trypsin from sardinelle (Sardinella aurita) viscera

BACKGROUND: In Tunisia, sardinelle (Sardinella aurita) catches totalled about 13 300 t in 2002. During processing, solid wastes including heads and viscera are generated, representing about 30% of the original raw material. Viscera, one of the most important by‐products of the fishing industry, are recognised as a potential source of digestive enzymes, especially proteases with high activity over a wide range of pH and temperature conditions. This paper describes the purification procedure and some biochemical characterisation of trypsin from S. aurita viscera. RESULTS: Trypsin from the viscera of sardinelle (S. aurita) was purified by fractionation with ammonium sulphate, Sephadex G‐75 gel filtration, Sepharose mono Q anion exchange chromatography, ultrafiltration and a second Sephadex G‐75 gel filtration, resulting in a 5.42‐fold increase in specific activity and 6.1% recovery. The molecular weight of the purified enzyme was estimated to be 24 kDa using size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified enzyme showed esterase‐specific activity on N‐α‐benzoyl‐L ‐arginine ethyl ester (BAEE) that was four times greater than its amidase‐specific activity on N‐α‐benzoyl‐DL ‐arginine‐p‐nitroanilide (BAPNA). The optimal pH and temperature for enzyme activity were pH 8 and 55 °C respectively using BAEE as a substrate. The trypsin kinetic constants K_m and k_cat on BAPNA were 1.67 mmol L^?1 and 3.87 s^?1 respectively, while the catalytic efficiency k_cat/K_m was 2.31 s^?1 L mmol^?1. CONCLUSION: Trypsin was purified from sardinelle (S. aurita) viscera. Biochemical characterisation of S. aurita trypsin showed that this enzyme can be used as a possible biotechnological tool in the fish‐processing and food industries. Copyright © 2008 Society of Chemical Industry     

Substrate specificity of a recombinant d-lyxose isomerase from Providencia stuartii for monosaccharides

The specific activity and catalytic efficiency (k_cat/K_m) of the recombinant putative protein from Providencia stuartii was the highest for d-lyxose among the aldose substrates, indicating that it is a d-lyxose isomerase. Gel filtration analysis suggested that the native enzyme is a dimer with a molecular mass of 44 kDa. The maximal activity for d-lyxose isomerization was observed at pH 7.5 and 45 °C in the presence of 1 mM Mn²⁺. The enzyme exhibited high isomerization activity for aldose substrates with the C2 and C3 hydroxyl groups in the left-hand configuration, such as d-lyxose, d-mannose, l-ribose, d-talose, and l-allose (listed in decreasing order of activity). The enzyme exhibited the highest activity for d-xylulose among all pentoses and hexoses. Thus, d-lyxose was produced at 288 g/l from 500 g/l d-xylulose by d-lyxose isomerase at pH 7.5 and 45 °C for 2 h, with a conversion yield of 58 % and a volumetric productivity of 144 g l^− 1 h^− 1. The observed k_cat/K_m (920 mM^− 1 s^− 1) of P. stuartiid-lyxose isomerase for d-xylulose is higher than any of the k_cat/K_m values previously reported for sugar and sugar phosphate isomerases with monosaccharide substrates. These results suggest that the enzyme will be useful as an industrial producer of d-lyxose.     

The effects of mixing,transport and duration of lairage on carcass characteristics in commercial bacon weight pigs

On arrival at the factory commercial crossbred pigs were subjected to various treatments: (i) penned in producer lots; (ii) mixed with pigs from another producer and (iii) mixed and subjected to an additional transport of 1-1.5h duration. Groups of pigs were killed on the day of arrival or after overnight lairage. Carcass parameters including pH₁ (pH 45 min post mortem) and pH_u (pH 20 h post mortem) values of the Longissimus dorsi (LD) and adductor muscles were taken post mortem. Mixing and transport had no statistically significant effect on any of the carcass parameters measured, although overnight lairage resulted in significant decreases in hot carcass weight, killing-out percentage and liver weight. The incidence of carcasses classified as pale soft and exudative [PSE (muscles with a pH₁ of 5.9 or less)] or dark firm dry [DFD (muscles with a pH_u of 6.2 or greater)] was high after both normal and overnight lairage. pH₁ and pH_u values of the LD and adductor muscles were not significantly affected by mixing or transport. Statistically significant differences in the incidence of DFD between treatment groups were observed. In general the incidence of DFD increased more after overnight lairage in pigs penned in producer lots, than in pigs penned in mixed producer lots.