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a1>3-GALACTOSYLTRANSFERASE : THE USE OF RECOMBINANT ENZYME FOR THE SYNTHESIS OF a-GALACTOSYLATED GLYCOCONJUGATES
No full textWe have reported the isolation and characterization of a bovine
cDNA clone containing the complete coding sequence for UDPGal:
Galß1>4G1cNAc «1>3-galactosyltransferase (Joziasse, D.H. et al.,
(1989) J. Biol. Chem. 264, 14290-14297). Insertion of this cDNA clone
into the genome of Autographa californica nuclear polyhedrosis virus
(AcNPV) and subsequent infection of Sf9 insect cells with recombinant
virus, resulted in high-level expression of enzymatically active
al>3-galactosyltransferase. The recombinant &1>3-galactosyltransferase
could be readily detergent solubilized and subsequently
purified by affinity-chromatography on UDP-hexanolamine-Sepharose.
The recombinant «1>3-galactosyltransferase showed the expected
preference for the acceptor substrate N-acetyllactosamine
(GalB1>4G1cNAc), and demonstrated enzyme kinetics identical to those
previously reported for affinity-purified calf thymus a1 >3-
galactosyltransferase
MODIFIED GLYCOSYLATION IN RECOMBINANT HUMAN TISSUE INHIBITOR OF METALLOPROTEINASES (TIMP), PRODUCED IN CHO CELLS GROWN IN THE PRESENCE OF GLYCOPROCESSING INHIBITORS
No full textTissue inhibitor of metalloproteinases (TIMP)is a glycoprotein of approximate M=30KDa, which has 2 Nlinked
glycosylation sites giving rise to heterogeneous glycosylation, and characterised by a multiple
bandpattern for the native protein on SDS-PAGE.
The present work reports the production of TIMP with modified glycosylation by the use of a range of
specific glycoprocessinginhibitors during cell culture. The inhibitors interfere with the normal trimming
pathway during N-glycosylation of glycoproteins.
Modified TIMPs showed greater homogeneity of glycosylation, higher iso-electric points, and similar
inhibitory activity of metalloproteinases, compared with native TIMP. Theinhibitors could be used over a
wide range of concentrations without having a significant effect on the production of TIMP,or on cell
growth.
This approach to modification of glycosylation may show significant advantagesin the production of
glycoproteinsfor crystallization studies over more conventional methods such as enzymic
deglycosylation, use of tunicamycin, or expression in coli
THE CRYSTAL STRUCTURE OF LIPASE FROM MUCOR MIEHEI
No full textThe crystal structure of lipase from the fungus Mucor miehei has been determined; it
has revealed the enzyme's main chain structure as well as the details of the
interactions madeby the individual sidechains. The enzymecontains a central 8 strand
6 sheet structure that extends acrossthe full depth of the molecule. Arranged across
this, in some of the segmentslinking the strands, are several helices which pack
against the sheet structure. There is an N terminal helix which appearsto sit at the
centre of the convex surface created by the ß sheet.
The serine (144) at the catalytic site has ben identified by chemical experiment.
Inspection of the structure at this serine showedit to be part ofa triad: asp... his ... ser,
equivalent at the active atomsto that seen in the serine proteases. Thereis no similarity
in the lipase main chain structure to those of the trypsin related or the subtilisin related
serine proteases - thus the appearanceofthe asp- his - ser triad is an example of an
independentsolution of these side chainsfor a catalytic reaction.
There is a small helix situated over the catalytic residues, effectively blocking them
from the surrounding solvent. This lid explains the inactivity of the enzyme in aqueous
conditions. The side chains on this helix are on one side polar and on the other nonpolar.
This suggests that underthe influence of the interface at a micelle the lid could
be destabilised by non-polar interactions andbe displaced, exposing the catalytic triad to
the lipid at the interface
CRYSTALLISATION AND PRELIMINARY CRYSTALLOGRAPHIC STUDIES OF LIPASES
No full textLipases from two Pseudomonas species have been crystallised in various forms
some of which appear suitable for a detailed structural analysis by X-ray
crystallography. All crystals were grown using the method of vapour diffusion by
hanging drop or sitting drop, at 4C or 15C. Different crystal forms tended to appear
at the two temperatures chosen for the crystallisation trials. The two lipases used
came from Pseudomonas Amano, and Pseudomonas Glumae. Crystals appeared under
different conditions for the two enzymes, but at least one crystal form of each
diffracted to greater than 3.0A. The work on P. Amano has as yet gone no further
than the ascertation of conditions for crystallisation to occur, but space groups
have been determined for three crystal forms of P. Glumae. The first crystals that
were tested proved to be trigonal, but were unsuitable for detailed investigation, the
two others were both possible candidates for high resolution studies. One form is
tetragonal, crystallising in space group P422, the other is orthorhombic, the crystals
growing in space group P222, It is on this second form that subsequent structural
analysis has been concentrated
Identification of the THL binding site on human pancreatic lipase
No full textPancreatic lipase is considered as a serine hydrolase that plays a key
function in dietary fat absorption by hydrolysing triglycerides into
diglycerides and subsequently into monoglycerides and free fatty acids.
Although it has been assumed [1,2] that porcine pancreatic lipase has two
functionally important sites: a catalytic site involving Serj109, and a
topographically distinct interfacial "recognition site" or " substrate binding
site" controlled by Serı52, the catalytic mechanism has not been
demonstrated experimentally thus far. On the other hand, the X-ray structure
of human pancreatic lipase [3] shows clearly that Serj52 forms a triad with
His263 and Asp 176 which givesa spatial superposition with the catalytic triad
of the serine protease trypsin (Fig.1). \
1102 esp
Recently Tetrahyrolipstatin exe
(THL), a_ selective and
irreversible inhibitor of er
pancreatic lipase has been used
to identify the THL binding site
on porcine pancreatic lipase.
The result showed that THL
binds to Serı52 of the lipase
covalently [4]. The same
attempt was also made for
human pancreatic lipase, the
data indicate strongly that ee
human and porcine pancreatic see
lipases share high :
similarity/identity not only in
their primary structure and
enzymatic characteristics, but
also in their catalytic
mechanism
CLONING, EXPRESSION AND CHARACTERIZATION OF CUTINASE, A FUNGAL LIPOLYTIC ENZYME
No full textA cutinase from the fungus Fusarium solani pisi has been overproduced in E. coli
by placing a phoA-signal/cutinase hybrid gene under the control of the tac promoter.
Due to its periplasmic location the recombinant enzyme can be easily purified in
large quantities. Assays using p-nitrophenylbutyrate suggest that the overproduced
and authentic enzyme are catalytically equivalent. The specific activities on
tributyrin (4000u/mg) and triolein (800u/mg) demonstrate the lipolytic nature of the
enzyme. The cutinase, however, differs from classical lipases in that no measurable
activation around the CMC of the tributyrin substrate is observed. We also provide
evidence that the recombinant enzyme is quite thermostable
FLOW INJECTION ANALYSIS FOR THE ON-LINE DETECTION OF LIPASE; A TOOL FOR THE AUTOMATIZATION OF LIQUID CHROMATOGRAPHY
No full textThe 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
FIFTEEN YEARS OF BIOSENSOR RESEARCH IN BERLIN-BUCH
No full textBiosensors for the determination of about 60 different substances,
such as low molecular weight metabolites, enzyme activities, inhibitors
and antigens, have been developed on the laboratory scale. The
biocomponents used comprise single and up to five coupled enzymes,
cell organelles, microorganisms, enzyme labelled antibodies and tissue
S:ices
BIOSENSORS BASED ON RECEPTORS : BIOSENSOR BASED ON A SUGAR TRANSPORT PROTEIN
No full textThe bacterial membrane protein lactose-permease transports lactose across the cell membrane
in symport with a proton. For application in a biosensor, the protein is reconstituted in
planar lipid membranes spread on a glass surface. In the space between the membrane and
the glass surface a pH-sensitive fluorescence dye is entrapped. When lactose is added to the
external medium, a change in the fluorescence signal is detected which permits to determine
the external lactose concentration.
Synthetic lipopeptides are immunologically active compounds with low molecular mass and
biophysically interesting properties. They have a potential for the immobilization of antigens on
transducer surfaces. Fluorescence-labelled and non-labelled lipopeptides can be obtained by solid
phase peptide synthesis. A new synthetic route for lipopeptides with C-terminallipid part is
described which allows the N-terminal coupling of marker molecules. An antigenic partial sequence
of a foot-and-mouth disease virus protein has been used to study the localisation of
antibody binding to the lipopeptide.
Lipid bilayers are examined by non-destructive spectral ellipsometry which yields information
about their stability, thickness and structure. Diode arrays allow the spectral fluorescence
and reflectance measurementsof antigen-antibody interactions. Binding of antibodies alters the
thickness of the active film and the refractive indices in the sensing interface. Both effects are
monitored by spectral interferometry as a new approach to label-free immuno-sensors.
Immunosensorsand biosensors based on transport proteins using electrical transducer principles
are discussed. Characteristic capacitance and voltage changes due to membrane coatings
on electrolyte/TagzOs/SiO2Si(ETOS) structures were investigated. The layer arrangement of
these structures is similar to those used in ourion sensitive field effects transistors (ISFETs)
H202-forming NADHoxidase from Thermus thermophilus HB8 for cofactor recycling in biosensor applications: molecular cloning ofthe gene and its expression in E.coli
No full textOxidoreductases represent a great potential for the construction of amperometric biosensors
for the measurement of clinically and biotechnologically important substrates. In many
enzymatic redox processes, NAD(P)t serves as cofactor and is consumed in stoichiometric
amounts. The consumption of the cofactor makes the application economically unfeasible.
Efficient recycling of the cofactor is therefore of great importance for the application of a lot
of oxidoreductases in biosensors. Some dehydrogenases have been usedfor cofactor recycling
in coupled enzyme reactions [1, 2]. However, additional substrates of these enzymes are
again required for this type of cofactor regeneration. An attractive alternative was suggested
by using the NAD(P)H oxidase (EC 1.6.99.3) which catalyzes the oxidation of NAD(P)H.This
enzymeuses dioxygen from air as a substrate and reducesit with the formation of hydrogen
peroxide [3]. It can be applied for the measurement ofsubstrates in amperometric enzyme
electrodes which are enzymatically coupled to NAD(P)*- reducing dehydrogenases.
The NAD(P)H oxidase from thermophilic bacteria is particularly interesting for the
development of amperometric biosensors, since the high stability of proteins promises enzyme
electrodes with a longer lifetime. We have recently reported the purification and some
properties of an NADH oxidase from Thermus thermophilus HB8 [4]. Since only minute
amounts of the NADH oxidase are present in T. thermophilus HB8cells, we have cloned the
NADHoxidase gene from T. thermophilus HB8 and efficiently expressed in E. coli and
purified the enzyme forits application in biosensors[5]