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Biomedical subjects

P F Fox

Publications and source records attributed to P F Fox.

At least 73 records · Page 4Linked to original sources
PubMed1985

Isolation and characterization of heat stable proteinases from Pseudomonas isolate AFT 21.

Pseudomonas strain AFT 21 produced three heat stable extracellular proteinases in milk and nutrient broth at 7 or 21 degrees C, but the proportions depended on medium and cultivation temperature. The three proteinases were EDTA- and o-phenanthroline-sensitive metalloenzymes and were not inhibited by N-ethylmaleimide or phosphoramidon. Proteinases I and II showed maximum activity at pH 7-7.5 and proteinase III at pH 8.5. All three enzymes showed maximum activity at 45-47.5 degrees C, but had relatively high (19-27% of maximum) activity at 4 degrees C. They were unstable at 55 degrees C in phosphate buffer, pH 6.6, or synthetic milk ultrafiltrate (SMUF) containing 12 mmol Ca2+, but were stabilized by short preheating at 100 degrees C. They were extremely heat stable in both phosphate buffer and SMUF, pH 6.6, at 70-150 degrees C. Their D-values at 140 degrees C were 69, 54 and 80 s respectively. The Z-values for Pseudomonas AFT 21 proteinase III in phosphate buffer and SMUF were 29.7 and 30.3 degrees C respectively; the corresponding activation energies for inactivation were 8.7 x 10(4) J mol-1 and 9.2 X 10(4) J mol-1.

Animals↗
PubMed1983

Thermal stability of an extracellular proteinase from Pseudomonas fluorescens AFT 36.

A metalloproteinase, isolated from a shaken milk culture of Pseudomonas fluorescens AFT 36 by chromatography in DEAE and CM-cellulose and Sephadex G-150, was very unstable in 0.1 M-phosphate buffer, pH 6.6, being completely denatured above 70 degrees C in 1 min. It was also unstable in a Ca-containing buffer (synthetic milk salts, SMS) between 50 and 60 degrees C (minimum at 55 degrees C), but stability was very high above 80 degrees C in this buffer. D-values were determined at 10 degrees C intervals in the range 70-150 degrees C in SMS from which a Z value of 31.9 degrees C and an Ea of 8.82 X 10(4) J mol-1 were calculated; the half-life at 150 degrees C was 9 s. Instability at 55 degrees C was due to autolysis as evidenced by gel electrophoresis, gel filtration and increase in 2,4,6-trinitrobenzenesulphonic acid-reactive amino groups. The extent of inactivation experienced at 80 degrees C was inversely related to the rate of heating to 80 degrees C, i.e. length of time spent in the neighbourhood of 55 degrees C. Addition of increasing concentrations of caseinate substrate reduced inactivation of the enzyme at 55 degrees C, presumably due to substrate binding. Attempts to stabilize the enzyme at 55 degrees C by addition of EDTA or by adjusting the reaction pH to 4.2, at which the enzyme has little proteolytic activity, were unsuccessful, although autolysis was prevented. Unlike the proteinase from Ps. fluorescens MC 60, AFT 36 proteinase did not inactivate itself on cooling to 55 degrees C from 80, 100 or 150 degrees C, but did regain autolytic activity on cooling to below 50 degrees C to an extent dependent on the duration of holding at the lower temperature. It is suggested that on heating to approximately 55 degrees C, a conformational change occurs which renders the enzyme susceptible to proteolysis by still active enzyme; at higher temperatures the enzyme, although susceptible to autolysis, is inactive; an active conformation is restored on cooling to below 50 degrees C.

Animals↗
PubMed1983

Isolation and some properties of extracellular heat-stable lipases from Pseudomonas fluorescens strain AFT 36.

Aeration increased the growth and lipase production in milk by Pseudomonas fluorescens strain AFT 36, isolated from refrigerated bulk milk. A heat-stable lipase was isolated from a shaken milk culture of this microorganism by DEAE-chromatography and gel filtration in Sepharose 6B. The lipase-rich fraction from DEAE cellulose contained 3 lipases that were separated by gel filtration; only the principal lipase, which represented approximately 71% of total lipolytic activity, was characterized. The purified enzyme showed maximum activity on tributyrin at pH 8.0 and 35 degrees C; it had a Km on tributyrin of 3.65 mM and was inhibited by concentrations of substrate greater than approximately 17 mM. The enzyme was very stable over the pH range 6-9; it was relatively heat-labile in phosphate buffer in the temperature range 60-80 degrees C, where it was stabilized significantly by Ca2+. It was, however, very stable at 100-150 degrees C: the D values at 150 degrees C were approximately 22 s and 28 s in phosphate buffer and synthetic milk serum respectively; the corresponding Z values in the temperature range 100-150 degrees C were approximately 40 and approximately 42 degrees C and the Ea for inactivation were 7.65 X 10(4) J mol-1 and 6.97 X 10(4) J mol-1 respectively.

Animals↗
PubMed1982

Isolation and general characterization of a heat-stable proteinase from Pseudomonas fluorescens aft 36.

An extracellular proteinase from Pseudomonas fluorescens, strain AFT 36, was isolated to homogeneity by chromatography on DEAE-cellulose and Sephadex G-150; a 230-fold increase in specific activity with a recovery of 53% was obtained. The enzyme was optimally active at pH 6.5 and 45 degrees C; activity declined rapidly at higher temperatures but significant activity persisted down to 4 degrees C. Activity was strongly inhibited by 10(-3) M EDTA and was partially restored by addition of Zn2+, Ca2+ or Co2+. The Km values on methylated casein and sodium caseinate were 18.2 and 7.1 mg/ml, respectively. The enzyme was very labile in phosphate buffer and in a mild salts buffer at 55 degrees C but was very stable in the latter at more than 80 degrees C.

Cations, Divalent↗
PubMed1982

Heat stability of milk: influence of cationic detergents on pH sensitivity.

The heat stability--pH profile of milk is shifted to more acidic values by anionic compounds such as SDS, lysolecithin and beta-lactoglobulin and to more alkaline values by cationic compounds such as quaternary ammonium compounds. Proline, which reduces the hydrophobicity of proteins, destabilized milk slightly. The results suggest that the heat stability of milk and the shape of the HCT-pH curve may be dependent on micellar charge effects.

Animals↗
PubMed1982

Heat stability of milk: influence of modification of lysine and arginine on the heat stability-pH profile.

Several dicarbonyl compounds (glyoxal, substituted glyoxals, diacetyl and 1, 2-cyclohexanedione) had a marked stabilizing effect on the heat stability of milk, especially in the presence of urea. These reagents are believed to modify arginine more or less specifically suggesting an important role for arginine residues in heat stability. In contrast, modification of lysine residues with dansyl chloride, acetic anhydride or cyanoborohydride had little effect on maximum heat stability although it did alter the HCT-pH profile. Since diacetyl is a natural constituent of fermented milks and cheese, it may be acceptable as an additive to increase the heat stability of milk.

Acetic Anhydrides↗
PubMed1981

Heat stability of milk: interrelationship between assay temperature, pH and agitation.

As determined by the standard subjective assay procedure, the minimum in the heat stability--pH curve of milk persisted down to at least 116 degrees C. However when samples were not agitated during heating the minimum became progressively less pronounced as the assay temperature was lowered and it disappeared at approximately 116 degrees C. Activation energies (Ea) for unagitated samples were approximately 30 Kcal/mole at pH 6.87 (maximum) and at pH 7.28, throughout the temperature range 116--145 degrees C and for the pH 6.95 (minimum) sample at 116--125 degrees C; however Ea for the pH 6.95 sample increased to approximately 100 Kcal/mole in the range 127--135 degrees C suggesting that some highly temperature-dependent reaction had occurred and caused premature coagulation at certain pH values, i.e. to a heat stability minimum. The stability of concentrated milk (20% total solids) was very low at pH values above 6.9, regardless of whether the samples were agitated or not during heating and the maximum/minimum in the heat stability--pH curve persisted down to at least 90 degrees C in both agitated and quiescent samples.

Animals↗
PubMed1979

Isolation and characterization of porcine beta-casein.

Porcine beta-casein was isolated by chromatography on DEAE-cellulose. The protein had a molecular weight of 24 900 as determined by gel filtration on Sephadex G-100 in guanidine-HCl. Its amino acid composition differed from bovine beta-casein especia-ly in respect to serine, alanine and leucine. In common with bovine beta-casein the N-terminal amino acid was arginine; the C-terminal was either alanine or valine, while the C-terminal of bovine beta-casein is valine. At any temperature porcine beta-casein was more sensitive to Ca2+ than bovine beta-casein, while at a fixed Ca2+ concentration porcine beta-casein aggregated at a lower temperature than bovine beta-casein. Porcine beta-casein was susceptible to hydrolysis by calf chymosin but the proteolytic specificity differed from that of calf chymosin on bovine beta-casein.

Amino Acids↗
PubMed1977

Isolation and characterization of sheep pepsin.

Sheep pepsin was isolated (approx. 120-fold purification) from aqueous abomasal homogenates by (1) pH fractionation, (2) chromatography on Sepharose 4B-poly-L-lysine columns and (3) gel filtration on Sephadex G-100. The enzyme had mol.wt. approx. 34000, N-terminal valine and C-terminal alanine. The amino acid composition of sheep pepsin was generally similar to that of pig and ox pepsins, with a very low content of basic residues and a high content of acidic and hydroxy-amino acids. The pH optimum for NN-dimethyl-casein and NN-dimethyl-haemoglobin as substrates was approx. 1.8. The Km and kcat. for NN-dimethyl-haemoglobin were 46micronM and 1100min-1 respectively, and for NN-dimethyl-casein the corresponding parameters were 50micronM and 420min-1. These values were generally similar to those for pig and ox pepsins. At the pH optimum of 4.6, the sheep pepsin was about 50% as active on benzyloxycarbonyl-L-histidyl-L-phenyl-alanyl-L-tryptophan ethyl ester as was pig pepsin. The pH optimum for the hydrolysis of N-acetyl-L-phenylalanyl-L-di-iodotyrosine by sheep, ox and pig pepsins was approx. 1.85.

Amino Acids↗