Glycerol Ester Hydrolase Activity of Lactic Acid Bacteria

Seventeen strains of lactic acid bacteria were assayed for their glycerol ester hydrolase activity by using an improved agar-well technique, and eight strains by determining the activity in cell-free extracts using a pH-stat procedure. All cultures tested showed activity and hydrolyzed tributyrin more actively than they did tricaproin. The cell extract studies demonstrated that the cells contained intracellular esterases and lipases. The culture supernatant fluid was without activity. The lipase and the esterase differed in their relative activity to each other in the different extracts and in the ease by which they could be freed from the cellular debris. It is suggested that the lipase of these organisms is an endoenzyme and the esterase an ectoenzyme.     

Nutritional Requirements of Microbacterium thermosphactum

Microbacterium thermosphactum requires cysteine, α-lipoate, nicotinate, pantothenate, p-aminobenzoate, biotin, and thiamin for aerobic growth in glucose-mineral salts medium. Glucose cannot be replaced by Casamino Acids or acids of the tricarboxylic acid cycle as sole carbon and energy sources. The organism can also grow anaerobically in the minimal synthetic medium.     

The Growth of Microbacterium thermosphactum on Beef

Microbacterium thermosphactum grew aerobically at 5°C on beef when the pH was as low as 5.4. It did not grow anaerobically if the pH of the meat was below 5.8, but grew readily at pH 6.0 or higher. On vacuum-packaged beef of pH 5.8 or lower, growth was dependent not only on pH but also on the oxygen permeability of the packaging film. Above pH 5.9 growth occurred with all the films tested.     

Energetics of growth of Microbacterium thermosphactum at low temperatures

Microbacterium thermosphactum was grown at 5°C and 9°C in glucose-limited continuous cultures. The end products of glucose metabolism were L-lactate and ethanol, and these compounds accounted for 86–92% of the glucose utilized. With input glucose concentrations less than 3 mM Y _glu ^Max was found to be 40–43, Y _ATP ^Max 20–21 and m _s 0.1–0.2. These values are almost identical to those found previously for cultures at 25°C and show that this psychrotroph grows with a very high energetic efficiency over a wide range of temperatures. With a higher (but still limiting) input glucose concentration of 5.6 mM at 9°C, cellular efficiency declined as there was a marked reduction in Y _glu. This decrease was accounted for in mathematical terms by an increase in m _s to 0.7, whilst Y _glu ^Max and Y _ATP ^Max remained high at 38 and 19 respectively.     

Identification of the Major Prostaglandin Glycerol Ester Hydrolase in Human Cancer Cells

Prostaglandin glycerol esters (PG-Gs) are produced as a result of the oxygenation of the endocannabinoid, 2-arachidonoylglycerol, by cyclooxygenase 2. Understanding the role that PG-Gs play in a biological setting has been difficult because of their sensitivity to enzymatic hydrolysis. By comparing PG-G hydrolysis across human cancer cell lines to serine hydrolase activities determined by activity-based protein profiling, we identified lysophospholipase A2 (LYPLA2) as a major enzyme responsible for PG-G hydrolysis. The principal role played by LYPLA2 in PGE₂-G hydrolysis was confirmed by siRNA knockdown. Purified recombinant LYPLA2 hydrolyzed PG-Gs in the following order of activity: PGE₂-G > PGF_2α-G > PGD₂-G; LYPLA2 hydrolyzed 1- but not 2-arachidonoylglycerol or arachidonoylethanolamide. Chemical inhibition of LYPLA2 in the mouse macrophage-like cell line, RAW264.7, elicited an increase in PG-G production. Our data indicate that LYPLA2 serves as a major PG-G hydrolase in human cells. Perturbation of this enzyme should enable selective modulation of PG-Gs without alterations in endocannabinoids, thereby providing a means to decipher the unique functions of PG-Gs in biology and disease.     

Energetics of Microbacterium thermosphactum in glucose-limited continuous culture.

Microbacterium thermosphactum was grown at 25 degrees C in glucose-limited continuous culture under aerobic (greater than 120 microM oxygen) and anaerobic (less than 0.2 microM oxygen) conditions. The end products of the anaerobic metabolism of glucose were identified as L-lactate and ethanol. Together these compounds accounted for between 85 and 90% of the glucose utilized over the full range of growth rates studied. In addition, 4% of the glucose utilized was incorporated into cellular material. Under anaerobic conditions the molar growth yield was 40 g (dry weight) of cells per mol of glucose utilized, and the maintenance energy coefficient was 0.4 mmol of glucose utilized per g (dry weight) of cells per h. For cells grown under aerobic conditions in the corresponding values were 73 g/mol and 0.2 mmol/g per h, respectively. The molar growth yield with respect to adenosine 5'-triphosphate varied with the growth rate of the culture, and the true molar growth yield with respect to adenosine 5'-triphosphate was found to be 20 g/mol of adenosine 5'-triphosphate.     

Energetics of Microbacterium thermosphactum in glucose-limited continuous culture.

Microbacterium thermosphactum was grown at 25 degrees C in glucose-limited continuous culture under aerobic (greater than 120 microM oxygen) and anaerobic (less than 0.2 microM oxygen) conditions. The end products of the anaerobic metabolism of glucose were identified as L-lactate and ethanol. Together these compounds accounted for between 85 and 90% of the glucose utilized over the full range of growth rates studied. In addition, 4% of the glucose utilized was incorporated into cellular material. Under anaerobic conditions the molar growth yield was 40 g (dry weight) of cells per mol of glucose utilized, and the maintenance energy coefficient was 0.4 mmol of glucose utilized per g (dry weight) of cells per h. For cells grown under aerobic conditions in the corresponding values were 73 g/mol and 0.2 mmol/g per h, respectively. The molar growth yield with respect to adenosine 5'-triphosphate varied with the growth rate of the culture, and the true molar growth yield with respect to adenosine 5'-triphosphate was found to be 20 g/mol of adenosine 5'-triphosphate.     

Purification and Properties of a Glycerol Ester Hydrolase (Lipase) from Propionibacterium shermanii

An intracellular glycerol ester hydrolase (lipase) from Propionibacterium shermanii was recovered from cell-free extracts and purified by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography on diethylaminoethylcellulose. Maximum enzyme activity was observed at pH 7.2 and 47 C when an emulsion of tributyrin was used as substrate. The enzyme was stable between pH 5.5 and 8. Heating the enzyme solution at 45 C for 10 min resulted in a 75% decrease in activity. Maximum rate of hydrolysis of triglycerides was observed on tripropionin, followed in order by tributyrin, tricaproin, and tricaprylin. The lipase was strongly inhibited by mercury and arsenicals, but specific sulfhydryl reagents had little or no inhibiting effect on the enzyme activity. The enzyme also showed some esterase activity, but the hydrolysis of substrates in solution was small as compared to the hydrolysis of substrates in emulsion.     

A Note on the Spoilage of Prepacked Lamb Chops by Microbacterium thermosphactum

S ummary . The microflora developing at 5° on lamb chops, prepacked commercially, was found to consist largely of facultatively anaerobic, Gram positive, catalase positive, benzidine negative rods resembling Microbacterium thermosphactum. In contrast, beef steaks under the same conditions had a microflora made up predominantly of Gram negative bacteria.     

A Selective Medium for the Enumeration of Microbacterium thermosphactum in Meat and Meat Products

S ummary . A selective medium for the isolation and enumeration of Microbacterium thermosphactum is described and data is presented on its use in assessing the composition of the flora of meat and meat products.