The use of Tween 20 in a sensitive turbidimetric assay of lipolytic enzymes

Tigerstrom, Richard G. Von; Stelmaschuk, Sheilah

doi:10.1139/m89-079

Cited by 49 publications

(36 citation statements)

References 11 publications

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“…The times of production of the P . aeruginosa enzymes during the cell growth cycle seem to be similar to those observed with L. enzymogenes and we noted recently that the two P. aeruginosa enzymes also are very active with Tween 20 as the substrate (von Tigerstrom & Stelmaschuk, 1989).…”

Section: R G V O N Tigerstrom a N D S Stelmaschuksupporting

confidence: 77%

“…Esterase activities were determined routinely by a turbidimetric assay using Tween 20 as the substrate (von Tigerstrom & Stelmaschuk, 1989). The assay solution contained 20 m-Tris/HCl, 1.8% (v/v) Tween 20, 3 mM-CaCl,, pH 8.5.…”

Section: Chemicalsmentioning

confidence: 99%

Section: Chemicalsmentioning

confidence: 99%

“…This step, and subsequent solubilization from the particulate fraction with Zwittergent 3-14, have been used to partially purify the enzyme (von Tigerstrom & Stelmaschuk, 1989). The particulate nature of the enzyme and the solubilization with detergent indicated that the esterase might be membraneassociated.…”

Section: Localization Of the Cell-associated Esterasementioning

confidence: 99%

“…The rate of increase of ODso0 was linear up to ODso0 1.2 and the rate of increase was essentially zero without added enzyme source. The spectrophotometric esterase assay with p-nitrophenyl palmitate (pNPP) (Winkler & Stuckman, 1979) and the titrimetric assay with olive oil and tributyrin have been described previously (von Tigerstrom & Stelmaschuk, 1989).Cell-free extracts and cells incubated with 0.2% (w/v) Zwittergent 3-14 for 30 min at 37 "C were used as sources of the cell-associated esterase and the culture supernatants as the source of the supernatant esterase. These were diluted with 20 mM-Tris/HCl, pH 8.0, if necessary.…”

mentioning

confidence: 99%

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Localization and Characterization of Lipolytic Enzymes Produced by Lysobacter enzymogenes

Tigerstrom

Stelmaschuk

1989

Microbiology

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Lipolysis by Lysobacter enzymogenes is due to the production of two major extracellular esterases. These were investigated using a turbidimetric assay with Tween 20 as the substrate. One esterase is cell-associated and found in the particulate fraction of cell-free extracts, the other esterase is secreted into the culture medium. In batch culture with 0.8% yeast extract as the medium the particulate esterase was produced mainly during the exponential growth phase and its yield was increased about twofold by the addition of olive oil and other substrates. The supernatant esterase was produced mainly after the exponential growth phase and was induced six-to tenfold by olive oil. PAGE, combined with activity staining using Tween 20 as the substrate, indicated that the two enzymes have different electrophoretic mobilities. Intact cells expressed 87 % of the particulate cell-associated esterase activity. The enzyme was released from cells or from the particulate fraction by incubation with 0.2% Zwittergent 3-14. Tween 20 (2%, v/v) or 1 mM-EDTA released little of the enzyme from whole cells. Separation of the inner and outer membranes of L. enzymogenes showed that the particulate esterase was localized in the outer membrane. Both esterases were very active with Tween 20 as the substrate, but they hydrolysed other compounds at very different rates. The relative activities with Tween 20, p-nitrophenyl palmitate, tributyrin and olive oil were 100,3, 11 and 0 for the particulate esterase and 100, 73, 0 and 28 for the secreted esterase. In addition to these two extracellular esterases, L. enzymogenes contains a hydrolase in the cytoplasm which is most active with tributyrin. INTRODUCTIONThe genus Lysubacter contains Gram-negative gliding bacteria which are noted for the production of many extracellular enzymes and for their role in biodegradation (Christensen & Cook, 1978;Reichenbach & Dworkin, 1981;Rosenberg & Varon, 1984). All species of the genus, except two strains of Lysobacter brunescens, were found to hydrolyse Tween when tested on solid media containing this detergent (Christensen & Cook, 1978). A number of the extracellular enzymes produced by Lysubacter species have been characterized (Jackson & Matsueda, 1970;Whitaker, 1970;Hedges & Wolfe, 1974;von Tigerstrom, 1980von Tigerstrom, , 1981von Tigerstrom, , 1984von Tigerstrom & Stelmaschuk, 1986, 1987, but the enzymes responsible for the lipase activity have not been investigated.Preliminary experiments indicated that L. enzymogenes produces two extracellular Tweendegrading hydrolases: one associated with the cells, the other secreted into the culture medium. Although the secreted enzyme can use olive oil as a substrate and might be called a lipase (Jensen, 1983), both enzymes prefer water-soluble substrates and for this reason we call the enzymes esterases rather than lipases (Sugiura, 1984). This paper describes the production of the two esterases by L . enzymogenes under different culture conditions, the major differences between the two enzymes and the localizat...

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Section: R G V O N Tigerstrom a N D S Stelmaschuksupporting

confidence: 77%

Section: Chemicalsmentioning

confidence: 99%

Section: Chemicalsmentioning

confidence: 99%

Section: Localization Of the Cell-associated Esterasementioning

confidence: 99%

mentioning

confidence: 99%

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Localization and Characterization of Lipolytic Enzymes Produced by Lysobacter enzymogenes

Tigerstrom

Stelmaschuk

1989

Microbiology

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Methods for lipase detection and assay: a critical review

Beisson¹,

Tiss

Rivière

et al. 2000

Eur. J. Lipid Sci. Technol.

332

169

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Structural and enzymatic characterization of NanS (YjhS), a 9‐O‐Acetyl N‐acetylneuraminic acid esterase from Escherichia coli O157:H7

et al. 2011

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There is a high prevalence of sialic acid in a number of different organisms, resulting in there being a myriad of different enzymes that can exploit it as a fermentable carbon source. One such enzyme is NanS, a carbohydrate esterase that we show here deacetylates the 9 position of 9-O-sialic acid so that it can be readily transported into the cell for catabolism. Through structural studies, we show that NanS adopts a SGNH hydrolase fold. Although the backbone of the structure is similar to previously characterized family members, sequence comparisons indicate that this family can be further subdivided into two subfamilies with somewhat different fingerprints. NanS is the founding member of group II. Its catalytic center contains Ser19 and His301 but no Asp/Glu is present to form the classical catalytic triad. The contribution of Ser19 and His301 to catalysis was confirmed by mutagenesis. In addition to structural characterization, we have mapped the specificity of NanS using a battery of substrates.

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The use of Tween 20 in a sensitive turbidimetric assay of lipolytic enzymes

Cited by 49 publications

References 11 publications

Localization and Characterization of Lipolytic Enzymes Produced by Lysobacter enzymogenes

Localization and Characterization of Lipolytic Enzymes Produced by Lysobacter enzymogenes

Methods for lipase detection and assay: a critical review

Structural and enzymatic characterization of NanS (YjhS), a 9‐O‐Acetyl N‐acetylneuraminic acid esterase from Escherichia coli O157:H7

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