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.
Saccharomyces cerevisiae contains a membrane-bound mitochondrial nuclease. The enzyme was purified nearly 500-fold from sphaeroplasts of the organism by differential centrifugation, differential solubilization, heparin-agarose chromatography, and gel filtration. A final specific activity of 98 mumol min-1 (mg of protein)-1 was obtained. The enzyme required further purification to achieve homogeneity. Two peaks of activity were obtained after gel filtration with apparent molecular weights of 140000 and 57000. Otherwise, these two components have nearly identical characteristics. Without detergent the enzyme is insoluble and has very low activity. Zwittergent 3-14 or Triton X-100 in the presence of KCl could be used to solubilize and activate the enzyme. A number of other detergents were much less effective in solubilizing or activating the nuclease. The enzyme requires Mg2+ for activity, and this can be replaced to some degree by Mn2+ but not by Ca2+ or Zn2+. It is most active at pH 6.5-7.0 and degrades the substrate to small oligonucleotides with 5'-phosphate ends. The relative rates of hydrolysis were 100 for poly(A), 31 for ssDNA, 19 for RNA, 2.1 for dsDNA, and less than or equal to 0.2 for poly(C). Under the assay conditions used the enzyme appears to constitute about 90% of the total nuclease activity of the cell. The enzyme is unstable, especially at neutral and alkaline pH.
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
The effect of medium composition on the production of four types of extracellular enzymes by Lysobacter enzymogenes was investigated. The nuclease, RNAase, alkaline phosphatase and proteases were produced in good yield after growth in tryptone broth. Much higher yields of the proteases, but low yields of the other three enzymes, were obtained using a skim milk/yeast extract or a chemically defined medium. The addition of NHI, HPOi-, ribonucleosides, Mg2+ or Mn2+ to tryptone broth reduced the production of some of the enzymes rather specifically. Of 12 monovalent and divalent metal ions tested, Mg2+ and Mn2+ had the greatest effect. Mg2+ at concentrations greater than about 0.1 mM inhibited the production of the nuclease, RNAase and the phosphatase but increased the proteases two-to threefold. Mn2+ at concentrations greater than about 0.01 mM inhibited production of the three enzymes more severely but did not stimulate protease synthesis. The extracellular enzymes produced with or without added Mg2+ or Mn2+ were analysed by PAGE and the activities associated with the cells and a shock fluid were determined. In addition, the effect of adding Mg2+ or Mn2+ to a growing, extracellular enzyme-producing culture was determined. The results suggest that the nuclease, RNAase and phosphatase are produced by a different mechanism than the proteases and that the metal ions interfere specifically with their production rather than with their release or by causing inhibition.
Lysobacter enzymogenes produces an alkaline phosphatase which is secreted into the medium. The gene for the enzyme (phoA) was isolated from a recombinant lambda library. It was identified within a 4.4-kb EcoRI-BamHl fragment, and its sequence was determined by the chain termination method. The structural gene consists of an open reading frame which encodes a 539-amino-acid protein with a 29-residue signal sequence, followed by a 119-residue propeptide, the 281-residue mature phosphatase, and a 110-residue carboxy-terminal domain. The roles of the propeptide and the carboxy-terminal peptide remain to be determined. A molecular weight of 30,000 was determined for the mature enzyme from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid sequence was compared with sequences available in the current protein data base, and a region of the sequence was found to show considerable homology with sequences in mammalian type 5 iron-containing purple acid phosphatases.The secretion of proteins through the prokaryotic cell envelope has been a major subject of study over the past two decades. Early research concentrated mainly on secretion by gram-positive organisms, since it was presumed that gram-negative bacteria secrete few proteins. However, it is now clear that many gram-negative organisms are able to secrete proteins to the periplasm, the outer membrane, and the medium (14). Extracellular proteins appear to cross the cytoplasmic membrane by a similar mechanism in grampositive and in gram-negative bacteria. They are synthesized as precursers with N-terminal signal sequences which are required for translocation through the cytoplasmic membrane. The signal peptide is subsequently cleaved by a peptidase to release the protein (26). There appear to be different mechanisms for the secretion of extracellular proteins to the outer membrane and the extracellular milieu. The subject has been reviewed extensively (14,22,25,26). It is clear that more work is required to determine the factors involved in the correct targeting of extracellular proteins, especially in gram-negative bacteria.Members of the genus Lysobacter are gram-negative, gliding bacteria which produce a number of extracellular enzymes. For references, see reference 40. Epstein and Wensink (7) have cloned and sequenced the gene for the 19.8-kDa a-lytic protease from Lysobacter enzymogenes. It codes for a polypeptide chain that is twice the size of the mature protease. It is synthesized with a propeptide which inhibits its activity until the enzyme is released from the cell (32). Other microbial proteases also are synthesized with an N-terminal extension (25).The characterization and comparison of genes for extracellular enzymes from an organism such as L. enzymogenes might help to determine the mechanism of the targeting process. The organism produces two extracellular phosphatases. One, a 25-kDa enzyme, is secreted into the medium, and the other, a 69-kDa phosphatase, is associated with the outer membrane (39,41). This report describes the cl...
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