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ANALYSIS OF GLYCOSYLATION HETEROGENEITY IN IFN-¥ PRODUCED BY CHO CELLS DURING BATCH AND CONTINUOUS CULTURE.
No full textThe production of recombinant human interferon-x (IFN- X¥ ) by Chinese hamster
ovary (CHO) cells in serum-free medium was heterogeneous with up to twelve molecular
weight variants secreted. Using the enzyme N-glycanase to remove all the (N-linked)
oligosaccharides or tunicamycin to inhibit glycosylation, it was found that the
variation was due to both variable asparagine residue occupation (Asnyg and/or Asn-
100) by complex oligosaccharide and to truncation of the IFN-$ polypeptide due to
proteolytic cleavage at the carboxy-terminal end. Using neuraminidase treatment,
terminal sialic acid expression was found to be invariant. When the CHD cells were
grown in two litre batch suspension culture in serum-free medium further
heterogeneity was discovered, seen as a reproducible shift towards the secretion of
underglycosylated IFN-Y with time. This phenomenon was independent of glucose
concentration in the medium. The IFN- ¥ was produced during the exponential growth
phase and declined in line with the cell growth rate and glucose uptake rates. In
contrast, during glucose-limited chemostat culture at a constant dilution rate of
0.015h”l, the relative levels of each major glycosylation variant did not change
significantly, although the proportion of fully glycosylated IFN-¥ was still less
than that seen at the start of batch culture while non-glycosylated IEN- ¥
represented 15% of the total IFN- ¥ secreted
Title_Foreword_Contents_List of authors_Photo of participants
No full textLipases are a highly interesting class of enzymes, since they exhibit activity only at the waterlipid
interphase. They are also of high practical relevance, since they are key enzymesin fat
metabolism, and they can be utilized for various industrial applications such as detergents and
chiral synthesis. Progress in both basic understanding and industrial use is hampered, however,
by the lack in fundamental knowledge about lipase structure and function. As a result, the CEC
has decided to support a 3 years’ program onthe structure analysis and protein engineering of
lipases, in the framework of a BRIDGE program (see the following article of Dr. B.
Nieuwenhuis). As a starting point, a CEC-GBF Workshop underlying this monograph was held
in Braunschweig from Sept. 12-15, 1990, summarizing the present state of knowledge in this
important area of biochemistry. Fortunately, researchers from most groups active in this field
from around the world gathered at this workshop, as indicated in Fig. 1.
As a result, the meeting permitted for an up-to-date assessment of the area, on a global scale.
Since the contributions of all active participants were submitted in written form during the
workshop, this monograph gives an excellent survey on the field of lipase structure, mechanism
and cloning.
At this point, I would like to acknowledge the invaluable contribution of several individuals who
have greatly contributed to this endeavour. First, I would like to thank the two co-organizers and
co-editors of this monograph, Prof. Lilia Alberghina of the Universita degli Studi di Milano and
Dr. Robert Verger of the CNRS Marseille, for their most important contributions in the
invitation of the specialists, the preparation and the organization of the workshop. Special thanks
are due to the CEC, represented by the BRIDGE Lipase Project Leader, Dr. Benedict
Nieuwenhuis, for both financial support and organizational help. The German government,
through the GBF, its National Research Center for Biotechnology, has contributed strongly
through financial subsidies and indirect help, mainly in organization. On a more personal note, I
would like to acknowledge the assistance of Dr. Marianne Kordel in thefinal preparations of
the workshop, of many students and assistants in our group, and in particular of Ms. Birgit
Balster and Ms. Sylvia Lenk for taking care of the workshop secretariat and the preparation of
this monograph. Finally, the experienced andskillful help of Dr. Johann Heinrich Walsdorff as
the copy editor of this monograph is gratefully acknowledged
LIPASE CATALYSED RESOLUTION OF BICYCLIC BUILDING BLOCKS FOR LEUKOTRIENE SYNTHESIS: ADVANTAGES OF ORGANIC MEDIA
No full textThe bicyclic bromohydrins rac-la,b were conveniently resolved with lipases
from Pseudomonas (Amano P) and Candida cylindracea (Amano AY-30) using vinyl
acetate as acyl donor and solvent. Both enantiomers could be obtained with
>96% e.e
FORMALDEHYDE ANALYSIS BY AN ENZYMATIC FIA-SYSTEM
No full textThe method for formaldehyde analysis presented here is based on the
enzymatical reaction of formaldehyde with the coenzyme nicotinamide adenine
dinucleotide (NADt) by catalysis of the enzyme formaldehyde dehydrogenase
(FADH). The enzyme is immobilized on an epoxy resin. Formaldehyde is
measured by electrochemical detection of NADH, using a redox dye as
mediator. The analysis is carried out ina Flow-Injection-Analysis System (FIA)
with an electrochemical wall-jet detector
DEVELOPMENTOF A BIOSENSOR FOR THE DETERMINATION OF CATECHOLAMINESIN URINE
No full textThis paper describes an amperometric biosensor for the determination of catecholamines
in urine. An amperometric planar oxygen electrode is combined with an enzyme
membrane containing immobilized catecholoxidase. This enzyme reacts with
catecholamines under oxygen consumption being measured by the oxygen sensor. The
decrease in oxygen concentrationis proportional to the concentration of catecholaminesin
the solution with excellent linearity in the physiological relevant range. No interferences
have been observed dueto the high specificity of the enzyme
FIBEROPTIC BIOSENSOR FOR LACTATE WITH REDOX DYES AS COENZYME
No full textIn this paper an optical sensor is described wich is based on a new enzymatic
methode for the determination of lactate. The enzyme used in this reaction is
cytochrome bo, which is able to take redox dyes as electron acceptors. The
decolourization of the dye gives information aboutthe lactate concentration
TOWARDS HOME-MONITORING AND SCREENING OF PHENYLKETONURIA BY BIOSENSORS. Studies on Flow-Injection Analysis
No full textL-phenylalanine dehydrogenase (L-Phenylalanine : nAD* -
oxidoreductase (deaminating)) from Rhodococcus sp. M4 has been
immobilized on aminopropyl-CPG and incorporated into a FIA system
with fluorimetric detection of NADH. The system is amenable to
automation
LANGMUIR-BLODGETT-FILMS OF PHTHALOCYANINATO-POLYSILOXANE-POLYMERS AS A NOVEL TYPE OF CHEMFET-MEMBRANE
No full textThe formation of Langmuir-Blodgett films (LB-films) by substituted
phthalocyaninato-polysiloxane-polymers is a new principle in this
field of research since this material lacks the amphiphilic nature
which has been thought of being the basic requirement for successful
LB film production. Multilayers of the polymere were built up and
successfully transferred to Si/SiO, -substrates. They formed macroscopically
well-ordered, thermally and chemically stable films. In an
Electrolyte/Insulator/Silicon-configuration (EIS-system) they were
tested for their usability as CHEMFET-membranes. A long-time stability
of at least 3 months in contact with buffer solutions was observed. A
pH-sensitivity of 40-50 mV/pH was found with a permanent but irregular
baseline drift probably due to ionic migration in the membrane
The Potential of Surface Acoustic Wave Devices for the Selective Detection of Trace Amounts of Molecules
No full textChemical Sensors and Biosensors try to mimic the capability of living systems to detect very
small amounts of analyte molecules using specific host-guest type of reactions. Recent advances
in microelectronic technology have led to a new generation of sensor devices, the piezoelectric
microbalances, based on planar microfabrication techniques. They show a very high sensitivity
( up to femtograms ) for detecting molecules which adsorb to the surface of the device and
change its resonance frequency with mass loading. All chemical or biological microsensor
devices require a surface coating that will interact with the specific chemical or class of
chemicals or biomolecule to be detected. The majority of applications have made use of bulk
acoustic wave (BAW) devices, usually with an adsorptive coating to provide some degree of
selectivity towards the analyte of interest [Glassford]. The oscillation frequency of a BAW
device immersed in solution changes with the temperature of the solution, with the specific
gravity and conductivity as well as the microviscosity at the interfacial layer, which can be
changed by analyte binding
PROCESS ANALYSIS AND CONTROL BY ENZYME-FIA-SYSTEMS
No full textThe analysis of glucose, lactate, amino acids, maltose, lactose,
sucrose, disaccharides in presence and absence of glucose as well as
glutamine by enzyme-FIA systems and their use in the bioprocess analysis
and control are presented