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THE CEC BRIDGE LIPASE PROJECT
No full textThe Commission of the European Community is implementing several priority
actions specifically designed for improving the competitiveness of European
Biotechnology. One of these actions aims at the establishment of a Community
network for training and research and has been executed, since 1982, in the
framework of three successive Community programmes BEP, BAP and BRIDGE. These
activities are conducted via two different types of projects : N-projects and T-projects.
The T-projects, larger targeted projects, are aiming at bottlenecks resulting from
structural or scale. constraints. One of these, the T-project on "Characterisation of
lipases for industrial applications : three-dimensional structure and catalytic
mechanism"does go backto a successful transnational collaborative research effort on
phospholipases in the Biotechnology Action Programme by groups in Marseille (F),
Utrecht (NL) and Tübingen (FRG). The aim of the T-project should be to acquire so
much new knowledge about a sufficient number of these enzymes that it will be
possible to understand why they are lipases and how they function as such
CATALYTIC MECHANISM OF PHOSPHOLIPASE A2
No full textPhospholipases A2 (MW 14,000) specifically hydrolyse the 2-acyl linkage of
phosphoglycerides in a calcium dependent reaction. From the three-dimensional
structure of native bovine pancreatic phospholipase Az at 1.7 A resolution in
conjunction with biochemical evidence the architecture of the active site became
apparent and a mechanism for the hydrolysis of both monomeric as well as
aggregated phospholipids could be proposed. In this mechanism a water molecule
activated by His48 was suggested as the nucleophile.
The enzyme possesses an extended area around the entrance to the active site
that is involved in the binding of aggregated substrates. Pro-phospholipase can
not degrade these aggregated substrates and it appears that in pro-phospholipase
part of this binding area is flexible.
The three-dimensional structure of a phospholipase Az mutant complexed with
a substrate analogue shows that the substrate analogue is bound to the calcium
ion in the enzyme’s active site both by its phosphate group and by the carbonyl
oxygen of the fatty acid residue bound at the C2 atom of the glycerol backbone.
The sn-2 chain of the inhibitor makes extensive hydrophobic contacts with the
disulfide bond between residues 29 and 45, and the side chains of residues Leu2,
Phe5, lle9, Leu19, Phe22 and Tyr52. The hydroxyl group of Tyr69 is hydrogenbonded
to one of the oxygen atoms of the inhibitor’s phosphate group
PROBING THE INTERACTION OF PHOSPHOLIPASE A2 AND PHOSPHOLIPIDS WITH RECOMBINANT DNA TECHNIQUES
No full textThe primary structure of eighty phospholipases A> from different sources is known. These sequences
reveal a high degree of homology, and aboutthirty percent ofall residuesis fully conserved. In addition
about twentyfive percentof the aminoacids is substituted by amino acids with similar properties like charge
and/or polarity and hydrogen-bonding capacity. The use of recombinant DNAtechniques has proovento be
a powerful techniqueto study the role of amino acid side chains in porcine pancreatic phospholipase Ap.
The application of these techniques has madeit possible to create a deletion mutant with enhanced activity
and improved crystallisation properties. It was shown that Leu-31 is important for the orientation of the
substrate molecule and is probably also importantfor the shielding of the active site from excess water. Tyr-
69 was shownto be involved in the orientation of the substrate molecule through an interaction with the
phosphate group ofthe substrate. A numberof absolute conserved residues like Tyr-52 and Tyr-73 are
important for folding and/or stability of the protein. Theresults of the studies described here support the
mechanism of catalysis of phospholipase A2 that was proposed already ten years ago
INTRINSIC ACTIVITY AND CATALYTIC RESIDUES OF THE LIPASE FROM GEOTRICHUM CANDIDUM
No full textThe triacylglycerol hydrolase secreted by Geotrichum candidum reportedly prefers substrates
having fatty acids with a cis-double bondin A9-position. A simple and rapid two step methodfor the
purification of high amountsofthis lipase was developedstarting from the commercially available
"GC-4" lipase powder (Amano, Japan). The molecular massof the purified lipase was 61 kDa, the
carbohydrate content 8.8 %. The heterogeneity observedin isoelectric focusing was dueto variable
glycosylation of the core protein.
With the aim to bioengineer new product oils the lipase was immobilized to an anion exchanger
and usedascatalystin the acidolytic modification of subcutaneouscalf fat with isostearic acid. Reaction
rates and yields were lower in comparisonto catalysis by other immobilized microbiallipases.
With the immobilizate of Geotrichum candidumlipaseoleic acid waspreferentially replaced
in the triglycerides by isostearic acid, however. This unique substrate specificity of the enzyme thus
prevails in transesterification processes as well.
Modification studies with reagents specific for amino acid residues presumedto be active in the
catalytic center showedthat the Geotrichum candidumlipase wasvery resistentto inhibition, as
only a carbodiimide and photooxydation rendered the lipase inactive. With the aim to enhance the
accessibility of the active center, reagents were then applied in the presence of 20 % isopropanol
and inhibition with the serine specific reagents phenylmethylsulfony!fluoride and benzene boronic
acid was achieved. Thedata indicate that a catalytic triad aspartic acid : histidine : serine may beinvolvedin
catalysis
THE BEHAVIOUR OF THE CANDIDA RUGOSALIPASEIN THE PRESENCE OF SOLUBLE SUBSTRATES
No full textNew data are presented for the reaction behaviour of the C. rugosalipase in the presence
of soluble substrates. The expected interpretation in terms of the formation of a dimeric
species ofthe enzyme has found a rival model in the adsorption of the lipase on the surface of
the reactor made of glass
CULTIVATION STRATEGIES OF REC LIPASE FROM STAPHYLOCOCCUS CARNOSUS
No full textIncreasing interest in lipases has led to the developement of new applications and production processes.
Scince manyyears it has been knownthat certain Staphylococci produce extracellular lipases (Arvidson
et al., 1971; Sztajer et al., 1987). Some species are pathogenic, while others have been extensively used
as starter cultures for food industry. In this work the formation of a lipase from S. carnosus TM 300
whichis generally recognized as safe was studied. The organism carrying a plasmid pLipPS1 with the
lipase gene from S. hyicus was constructed by Götz et al. (1985).
Lipase production from bacterial strains depends on environmental factor such as cultivation temperature,
composition of nitrogen, carbon and lipid sources, the concentration of inorganic salts and the
availability of oxygen. Additionaly, care must be taken to preserve the biological activity of the product.
When carrying out cultivations of S. carnosus TM 300::pLipPS1 in shaking flasks without baffles a
lipase production of more than 20 mg/l was obtained. In contrast to this result, the cultivation in a lab
scale stirred and aerated fermentor yielded muchless lipase. As shown in this paper, this discrepancy
originates from differences in cultivation conditions which maylead to lipase inactivation
AMPEROMETRIC IMMUNOSENSORS FOR BIOPROCESS CONTROL
No full textAmperometric enzyme immunosensors have been developed for
monoclonal antibodies (IgG), alpha-interferon and the pesticide
2,4 dichlorophenoxyacetic acid (2,4 D). Both in sensitivity and
measuring frequency the immunosensors are sufficient for practical
bio-reactor control. For pesticide monitoring, however , the
sensitivity should be increased dramatically
Flow-Injection Analysis Based on Biosensors to Improve the Automation and Control of Food Production Processes
No full textTrying to adaptflow-injections systems to industrial food production processes,
we started testing the parts (sampling, sample pretreatment, new flow-injection
systems). Now we are building up a first measurementunit
SIGNAL GENERATION AND EVALUATION IN FLUORESCENCE-BASED ON-LINE FIBER OPTIC BIOSENSORS
No full textThe signal generation, size and transmission of pre-steady-state and steady-state signals in
fluorescence based fiber optic biosensors for on-line use and their applicability in several flow
sensor configurations are investigated. In contrast to the theory, the signal for dynamic column
biosensorswith larger particles was foundto be onlyslightly higher than for a column sensorwith
smaller particles. This is due to interparticle interactions, which reduce the areathat is accessible
for mass transfer, on particles with a diameter below 100 pm. Additionally this system has shown
that dynamic biosensors with hydrolases can not be used for the determination of the end-pointof
hydrolytic processes, when the equilibrium of the substrate conversion remains below 100%. The
flow of optical power in fluorescent fiber optic sensors is analyzed, multiplicative noise is
identified as the dominantlimitation of sensor performance. Evanescent coupling is a promising
concept for fast biosensors. The observed nonlinear relation between absorbance and
concentration is explained as an optical effect, caused by multimode operation. The use of
special fibers with increased surface area gives higher sensitivity, reduced nonlinearity
DEVELOPMENT OF AN ENZYMATIC MULTICHANNEL FLOW INJECTION ANALYSIS SYSTEM FOR MONITORING MAMMALIAN CELL FERMENTATIONS
No full textA multichannel flow injection analysis system described earlier /1/, is adapted
to mammalian cell fermentations by increasing the sensitivity of the system. The
analysis is based on the conversion of the compounds by immobilized dehydrogenases,
resulting in the built up or consumption of NADH which is measured with a fluorescence-
detector. The method presented here has a linear glucose determination range
of 0.05 g/1 to 3.5 g/1 and a non-linear range for lactate of 0.05 g/1 to 2.7 g/l