GBF seriesarticles from GBF monograph serieshttp://hdl.handle.net/10033/6222862024-03-29T07:07:11Z2024-03-29T07:07:11ZFLOW INJECTION ANALYSIS FOR THE ON-LINE DETECTION OF LIPASE; A TOOL FOR THE AUTOMATIZATION OF LIQUID CHROMATOGRAPHYErdmann, HelmutChemnitius, Gabriele C.Schmid, Rolf D.http://hdl.handle.net/10033/6237542024-03-28T05:07:45Z1991-01-01T00:00:00ZFLOW INJECTION ANALYSIS FOR THE ON-LINE DETECTION OF LIPASE; A TOOL FOR THE AUTOMATIZATION OF LIQUID CHROMATOGRAPHY
Erdmann, Helmut; Chemnitius, Gabriele C.; Schmid, Rolf D.
The application of a FIA-FPLC unit for the post-column on-line detection of lipase activity is
presented. As the developmentof techniques such as Fast Protein Liquid Chromatography (FPLC)
and High Performance Liquid Chromatography (HPLC) has greatly reduced the time required for
protein purification, fast identification of enzyme-containingfractions is desired. Rapid detection of
enzymeactivity can be achieved by coupling a flow injection analysis (FIA) system to the liquid
chromatography unit. Usually lipase activity is detected in the presence of emulsified substrates,
whose evendistribution in the FIA system causes problems. In orderto solve these problems, the
solubilized substrates S,O,O’-tributyryl-1-thiogycerol (TBTG) and 1,2-O-dilauryl-rac-glycero-3-
glutaric-resorufinester (BM) were applied to determine the relationship between lipase concentration
and FIA response. Lipase and its substrate were injected simultaneously into two carrier
streams which were mixed together before passing a thermostated reaction coil. The cleaved
productof the lipase substrate was detected photometrically. The BM-substrate turned out to be
most suitable for FIA applications. The FPLC-FIA unit using the BM-substrate has successfully
been applied for post-column on-line monitoring of lipase activity during different lipase purification
steps. FIA response showsa linear correlation to lipase activity up to lipase concentrations of 120
U/ml.
1991-01-01T00:00:00ZComparison of Lipase Activities by Different AssaysErdmann, HelmutVorderwülbecke, T.Schmid, Rolf D.Kieslich, Klaushttp://hdl.handle.net/10033/6237532024-03-28T05:10:42Z1991-01-01T00:00:00ZComparison of Lipase Activities by Different Assays
Erdmann, Helmut; Vorderwülbecke, T.; Schmid, Rolf D.; Kieslich, Klaus
Recently several quick assay procedures have been developed (1 to 15), and are saidto detect
lipase activity. The hydrolysis of Triglycerols and artificial substrates oftencan not explain
differences of whatis a lipase and whatis an esterase. We established a numberoflipase assays
for the estimation ofactivities for a large numberof different lipase preparations, as immobilized
pure enzymes, nearly homogeneous preparations and samples of industrial grade. We found
remarkable differences concerning the lipase activities measured with different assays and report
about characteristics and comparability. At last, we want to arise the question "do we look for
the enzymes we are interested in ?"
1991-01-01T00:00:00ZCOMPARATIVE ANALYSIS OF LIPASES IN VIEW OF PROTEIN DESIGNKordel, MarianneMenge, UlrichMorelle, GillesErdmann, HelmutSchmid, Rolf D.http://hdl.handle.net/10033/6237522024-03-28T05:10:23Z1991-01-01T00:00:00ZCOMPARATIVE ANALYSIS OF LIPASES IN VIEW OF PROTEIN DESIGN
Kordel, Marianne; Menge, Ulrich; Morelle, Gilles; Erdmann, Helmut; Schmid, Rolf D.
The consensus sequences containing the active serine residue of 21 lipases were examined for
structural properties by secondary structure prediction and hydrophobicity plots. Mostof the
G-X-S-X-G peptides were found to form a turn structure andto be buried,i.e. inaccessible to water.
The structural characters of a second serine containing consensus peptide describedin literature
were compared to those of the G-X-S-X-G sequences. In addition, we investigated,if a correlation
of the structural features of these peptides to the substrate specificity (regio specificity and fatty acid
specificity) can be found.
1991-01-01T00:00:00ZCHARACTERISTICS OF A NEW LIPASE FROM A Thermus sp BACTERIUMSilva, A. M. G. M.Cabral, J. M.Costa, M. S.Garcia, F. A. P.http://hdl.handle.net/10033/6237512024-03-28T05:10:03Z1991-01-01T00:00:00ZCHARACTERISTICS OF A NEW LIPASE FROM A Thermus sp BACTERIUM
Silva, A. M. G. M.; Cabral, J. M.; Costa, M. S.; Garcia, F. A. P.
Thirty strains of the genus Thermus, isolated from hot sprins in Portugal, were
screened for the secrection of lipases. In the end, the strain LFF1 received our attention for further
characterization. We report here some kinetic properties of the crude extracellular extract when used
in a reversed micellar system of AOT in isooctane for the hydrolysis of triolein. In common with
other lipases, this extract showed maxima at pH 7 and temperatures in the range 40-50 °C, but significant
residual activities were also observed at higher (up to 80 °C) and lower (downto -3.5 °C)
temperatures. The hydrolysis oftriolein in the micellar system followed an apparent Michaelis-
Menten kinetic mechanism with K,,(app)=7.1%(v/v) and V_.„(app)=55.5 mole/(ml.h.mg protein).
The specific activity of the extract decreased continuously with increasing concentrations of protein
encapsulated in the reversed micelles. The aqueousextract lost less than 9% of its activity when
stored at 4 °C for almost 3 months.
1991-01-01T00:00:00Z