A turbidimetric esterase assay was developed using a Tween 20 solution in the presence of CaCl2 and Lysobacter enzymogenes esterase (EC 3.1.1.1) as the enzyme source. The reaction was followed by measuring the increase in the optical density at 500 nm (OD500) due to the hydrolytic release of the fatty acids from Tween 20 and their precipitation as the calcium salts. Concentrations of 1.8% Tween and 3 mM CaCl2 were found to be optimal for the assay of 0.036 to 0.15 esterase units in a 4-mL reaction mixture over a 30-min period. The esterase reactions were linear with time at least up to 1.2 OD500 and the rate of increase in the OD500 was proportional to the enzyme concentration. Low initial reaction rates were seen with low esterase activity, presumably because of the limited solubility of the fatty acid - calcium salt in a 1.8% Tween solution. This turbidimetric method is much simpler and at least 36 times more sensitive than the titrimetric assay with Tween 20, and at least four times more sensitive than a spectrophotometric assay with p-nitrophenyl palmitate. This assay has been used to determine the activities of cell-associated and excreted esterases produced by Lysobacter enzymogenes and Pseudomonas aeruginosa, and of lipolytic enzymes from porcine liver, Chromobacterium viscosum, Candida cylindracea, and wheat germ.
The alkaline phosphatase (EC 3.1 .3.1) associated with the outer membrane of Lysobacter enzymogenes was solubilized from a crude membrane preparation with detergent and purified 219-fold using ion-exchange chromatography and gel filtration. The yield of the purified enzyme was 21 % and the specific activity was 438 units mg-I. The enzyme was most active at pH 8.5, readily hydrolysed 5'-, 2'-and 3'-ribose and deoxyribose nucleotides, glucose &phosphate, glycerophosphates and p-nitrophenylphosphate and was strongly inhibited by EDTA and 8hydroxyquinoline. The EDTA-inhibited enzyme could be reactivated to some extent by CoC12 and more effectively by ZnC12. The phosphatase was slightly activated by p-nitrophenylphosphate and from kinetic studies using this substrate two K,,, values, 0.56 x 1 0 -4~ and 3.4 x M, were estimated. The enzyme retained activity in the presence of SDS, unless it was heated, and had an apparent M , of 69000, as determined by SDS-PAGE. Gel filtration data suggested that the native enzyme might consist of at least two subunits. The properties of the enzyme were consistent with the view that it is held in the outer membrane by hydrophobic interactions. METHODS Chemicals.The sources of most of the chemicals used during the purification and characterization of the phosphatase have been reported (von Tigerstrom, 1984). In addition, DEAE-Sephacel and Sephacryl S-200 were Abbreviation: pNPP, p-nitrophenylphosphate. 0001-2825 0 1986 SGM
In addition to an excreted phosphatase, Lysobacter enzymogenes produces a distinctly different alkaline phosphatase which remains associated with the cells. It is the only major phosphatase contained in the cells. Little enzyme activity is found in whole cells using the standard assay with p-nitrophenyl phosphate as the substrate, but maximum activity can be obtained after toluene or detergent treatment, or mechanical disruption of the cells. The phosphatase is not released from the cells by osmotic shock, or by treatment with MgC12 or high LiCl concentrations. It is associated with the particulate fraction in cell-free extracts and detergents solubilize the enzyme from intact and broken cells. Separation of the inner and outer membranes of the cells by sucrose gradient centrifugation showed that most, if not all, of this phosphatase is located in the outer membrane.
Lysobacter enzymogenes ATCC 29487 (UASM 495) produces an outer-membrane-associated phosphatase and an excreted phosphatase. The cell-associated enzyme was compared to phosphatases of nine other Gram-negative gliding bacteria and to that of Escherichia coli. The other three species of the genus Lysobacter also produce a particulate, cell-associated phosphatase. Antiserum prepared against the phosphatase from the outer membrane of L. enzymogenes effectively precipitated the phosphatases of two other L. enzymogenes strains and the enzymes of L. antibioticus, L. brunescens and L. gummosus. Some inhibition of the enzyme by the antiserum also was observed. No significant reaction could be detected between the antiserum and the cell-associated phosphatases of species of Cytophaga johnsonae, 'C. compacta', Myxococcus xanthus, E. coli and the excreted phosphatase of L. enzymogenes. The results indicate that the four species of the genus Lysobacter are closely related despite their physiological differences and that the outer-membrane-associated phosphatases of these organisms have different structural characteristics than the phosphatases of the other Gram-negative bacteria that were used. Furthermore, differences in the amino acid compositions of the cell-associated and the excreted phosphatase of L. enzymogenes confirm the immunological results and are in agreement with the physical and chemical differences noted between the two enzymes.
The endonucleases from Neurospora crassa and Saccharomyces cerevisiae are not closely related antigenically. They also differ with respect to their activity at pH 8, their degree of hydrophobicity, and their sensitivity to elevated temperatures. However, the two nucleases have similar specific activities, are inhibited by EDTA, and have nearly identical substrate specificities. Since the enzymes also have the same mode of action and intracellular location, these similarities may indicate that they have the same physiological role despite their structural differences.
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