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FLOW INJECTION ANALYSIS AND IN-LINE BIOSENSORS FOR BIOPROCESS CONTROL: A COMPARISON
No full textMiniaturization will unify the different approaches chosen for
the application of biosensors in bioprocess control. The most
versatile system, which in our opinion is flow injection
analysis will be the method of choice for the introduction of
biosensors in bioprocess control. A lot of experience will be
gained for the future development of miniaturized total
chemical analysis systems
PROTEIN O-GLYCOSYLATION AND SEXUAL AGGLUTININS IN THE YEAST S.CEREVISIAE
No full textThe features of protein O-glycosylation in yeast are summarized. - The a
and a-agglutinin of haploid S.cerevisiae cells have been purified and their
one to one interaction has been studied in vitro; their carbohydrate moieties
do not seem to be essential. Both the corresponding genes have been cloned and
sequenced
PROBING THE CARBOHYDRATE SIDE CHAINS OF RECOMBINANT TISSUE PLASMINOGEN ACTIVATOR
No full textThe glycosylation of recombinant tissue plasminogen activator derived
from transfected CHO cells was assessed using glycosyltransferases to
probe for terminal Gal and GlcNAc residues and solid-state lectin
binding assays. Analysis of the oligosaccharides released from each
glycosylation site by hydrazinolysis by Con A affinity chromatography/
anion exchange HPLC/ BioGel P4 gel filtration corroborated the
glycosyltransferase and lectin-binding assay data. Significant
undersialylation of the oligosaccharides of t-PA and less than 1% of
free terminal GlcNAc residues was indicated. The above assays could be
useful in monitoring glycosylation status during quality control in
recombinant glycoprotein production
THE STRUCTURE OF HUMAN PANCREATIC LIPASE SUGGESTS A LOCALLY INVERTED, TRYPSIN-LIKE MECHANISM
No full textFurthercrystallographic refinement of the structure of human pancreatic lipase hasled to
an improved model which has been used for modelling studies of the hydrolysis of
triglyceride substrates. In addition to the removalof the flap, further changesin the protein
structure around the active site appear necessary to formulate a stereochemically plausible
mechanism. A locally inverted trypsin-like mechanism is presently favoured and demands
relatively modest changes of the X-ray structure. Additional new findings include the
interpretation of the difference density for a butylboronic acid derivative and the location of
a Ca2+ bindingsite
CRYSTALLOGRAPHIC STUDIES OF THE PANCREATIC LIPASE/COLIPASE SYSTEM
No full textCrystals of a complex between porcine pancreatic lipase (50000 Da) andits cofactor,
colipase (11000 Da), have been obtained. These 1/1 complex crystals are
disordered, and diffract weakly. Horse pancreatic lipase has been crystallized in
space group P21242), (89A,97A,145A ; pH 6.0, PEG 8000 10%, 20°C) and a 2.3 A
native data set has been collected. One PCMBS derivative was not sufficient to
produce a good map,andwearestill looking for other derivatives. Two crystal
forms of human pancreatic lipase (purified from pancreas juice) have been obtained,
different from the one reportedin thelitterature. They crystallize in the same
conditions and space groupsare P2, and P1. Native data sets have beencollected
ENZYMEKINETICS OF LIPOLYSIS: Lipase inhibition by proteins
No full textApart from their general biological significance, lipolytic enzymes play an increasingly important role in biotechnology:
clinical medicine, pharmacology, nutrition, food andoil technology. Lipids constitute a large part of the biomassof the hearth
andlipolytic enzymes play an essential role in the metabolic turnoverofthese lipids. Phospholipids and glycolipids constitute
fundamental and universal structural elements of biological membranes. Onthe other hand, triglycerides constitute the main
energy reservoir of higher animals. Lipases are required in the lipid transfer from one organism to another.
Forthe biochemist, perhaps the most importantand fascinating aspectof lipolytic enzymesis the unique physicochemical
character ofthe reactions they catalyze. These enzymesare perfectly water soluble andact very efficiently on water insoluble
lipidic substrates which spontaneously self organize in water as monomolecular films, bilayers, liposomes, emulsions or
micelles. This catalysis is essentially occurring at the lipid/waterinterface. Conventional enzymekineticsis oflittle relevance
under these heterogeneous conditions. The development ofan "interfacial enzymology" would be of general interest forall
biological sciences because in nature most enzymatic reactions take place at membranes.
Thereare at least 3 major reasonsfor using lipid monolayers as substratesfor lipolytic enzymes:
1. The technique is highly sensitive and very little lipid is needed to obtain kinetic measurements.
2. A rather common observation reported by many authors working onthekinetics oflipolytic enzymesis the presence of a
lag period in the hydrolysis of both emulsions, liposomes, micelles and monolayers. Such studies should preferably be done
on monolayers ofshort-chain lipids where the perturbing influence of increasing amounts of reaction products can be
minimized.
3. Probably the most important reason fundamentally is the possibility of varying the "quality of interface" determined by the
nature ofthe lipids forming the monolayer, the orientation of the molecules, molecular and charge density, water structure,
fluidity, etc..
LIPASE KINETICS AT THE TRIACYLGLYCEROL-WATER INTERFACE
No full textA new method,-called the "oil-drop method", was developed and adapted to studing the rate of enzymatic reactions, using
long chain triacylglycerols, the main physiological substrates of digestive lipases. The method is based on the variations versus
time in theoil/waterinterfacial tension (Yo/w) resulting from the accumulation of water insoluble lipolytic products on the
surface of a drop (1). Measurements were carried out with pure Porcine Pancreatic Lipase (PPL). This method was also used to
detect Human Gastric Lipase (HGL)at low pH,since difficulties were encountered in earlier studies when measuring lipolytic
activity under acidic conditions (2).
The lipolytic kinetics under high hydrostatic pressure (800 and 1200 bars) were also investigated with the oil-drop method,
and a specific two-fold increase in lipase activity was found to have occured.
A newprototype is being developed for automatically analyzing the oil-drop profile is being developed in order to improve
the data acquisition rate andthe accuracy of the measurements
LIPOPROTEIN LIPASE - THE MOLECULE AND ITS INTERACTIONS
No full textIt is clear from the primary sequences that lipoprotein lipase (LPL)is related
to hepatic lipase and to pancreatic lipase. Consideration of sequence homologies
and biochemical evidence suggests that the folded structure of LPL may also be
similar to that for pancreatic lipase. Both enzymes require small proteins for
function in their physiological environment, apolipoprotein CII and colipase,
respectively. There is however, no homology between the activators, which
probably evolved separately. The lipases are very efficient with turnover numbers
for triglyceride hydrolysis in the physiological environment of over 1000 sec" To
probe their mode of action we have compared hydrolysis of triglycerides and
phospholipids in mixed liposomes and in emulsions. Binding oflipase to the two
types of particles was similar. Yet, triglyceride hydrolysis was more than 10 times
more rapid and the ratio of triglyceride to phospholipid hydrolysis was more than
50-fold higher with the emulsion droplets than with the liposomes. This suggests
that the route of substrate entry into the active site is from below, and that the
lipase stays at the interface for several rounds oflipolysis. This would seem to fit
well with the x-ray structure of pancreatic lipase, which shows the active site at
the bottom of a cleft.
Lipoprotein lipase (LPL) is member of a protein family which also includes
hepatic lipase and pancreatic lipase. The three lipases are engaged in different
aspects oflipid transport and serve basically similar functions. They hydrolyze
triglycerides to fatty acids and monoglycerides which can be taken up by cells for
use in metabolic reactions, or be packaged for further transport
MOLECULAR CLONING OF A LIPASE AND OF A LIPASE-RELATED GENE FROM Candida cylindracea
No full textThe screening of genomic libraries from Candida cylindracea with
synthetic oligonucleotides has allowed us to isolate two lipaserelated
sequences. They have been characterized by restriction
mapping and partially sequenced. One of them contains a lipase
coding sequence
PRODUCTION OF LIPASE BY SPOROTRICHUM (CHRYSOSPORIUM) THERMOPHILE APINIS IMMOBILIZED IN ALGINATE AND POROUS GLASS BEADS
No full textSpores and mycelia of Sporotrichum (Chrysosporium) thermophile Apinis
were immobilized in alginate and porous glass beads for lipase production
in batch replacement culture and a column reactor. Young mycelia immobilized
in alginate produced higher levels of lipase than free cells or spores immobilized
in alginate or glass beads. Alginate beads were successfully used in
batch culture for more than 300 h without any signs of breakage and little
cell washout. Porous glass beads proved a better carrier for spore derived
inocula but activity levels were low. Crude lipase of S. thermophile was
stable upto 90°C both in free and immobilized state and thus requires further
characterization