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cDNA CLONING AND SEQUENCING OF HUMAN MILKBILE SALTSTIMULATED LIPASE
No full textWehave isolated and sequenced cDNAclones covering the entire coding sequence of
human milk bile salt-stimulated lipase (BSSL). The deduced amino acid sequence starts with a
21 residues leader peptide. The open reading frame continues with 722 amino acid residues.
The sequence contains in the C-terminal part a proline-rich repeat, 16 repeats of 11 amino
acid residues each. The mRNAwasestimated to be approximately 2500 nt from Northern blot.
The cDNAis 2432 bases long, which indicates that a near full-length copy of the transcript
have been isolated. Comparisons with other enzymes show that BSSL is a new memberofthe
supergene family of serine hydrolases. Not only is it closely related (in the N-terminal half
virtually identical) to lysophospholipase from rat pancreas and cholesterol esterase from bovine
pancreas, but also shows a high degree of homology to several esterases, e.g. acetyl choline
esterase. It contains the sequence which has been proposedto be the acetylcholine binding site
in acetyl choline esterase. In contrast, no such homologies could be found to typical lipases,
with the exception of the consensus sequence GXSXGtypical for serine hydrolases
PURIFICATION AND SUBSTRATE SPECIFICITIES OF LIPASES FROM GEOTRICHUM CANDIDUM
No full textTwo strains of the mould Geotrichum candidum (ATCC 34614 and CMICC 335426) each
produce two extracellular lipases. We have purified the four lipases to
electrophoretic homogeneity and each is a single species when analysed by
isoelectric focusing. The enzymes are glycosylated and have similar molecular
weights and isoelectric points. Peptide mapping and Western blotting shows that
the lipases are related and this has been confirmed by amino acid analysis. We
have examined the substrate specificities of the two lipases using triacylglycerols
and fatty acid methyl esters as substrates. One lipase is highly specific for the
release of cis-A9-fatty acids from these substrates. In contrast, the other 3
lipases are not specific, releasing both saturated and unsaturated fatty acids
LIPASE-CATALYZED HYDROLYSIS OF TRIGLYCERIDES FROM NEW OIL CROPS FOR OLEOCHEMICAL INDUSTRIES
No full textThe use of Rhizopus delemar lipase to specifically and mildly isolate industrially
interesting fatty acids from new agricultural oil seed crops was evaluated.
R. delemar-mediated hydrolysis of Crambe and Dimorphotheca oils was studied in an
emulsion system at room temperature. Crambe oil hydrolysis was also studied using lipase,
immobilized in a two-phase hollow-fiber membrane bioreactor. Progress of the hydrolysis
was monitored by GLC analysis.
Allowing the reaction to proceed in an oil - water emulsion gave a high yield of erucic
acid when using Crambe oil, and dimorphecolic acid when using Dimorphothecaoil. R.
delemar-mediated hydrolysis of Crambe oil in a membrane bioreactor also yielded high
proportions of erucic acid, albeit at a lower apparentrate.
It is concluded that lipases with 1,3-positional specificity can be used to produce high
concentrations of specialty fatty acids from novel vegetable oils and that immobilization of
these lipases in membrane bioreactors allows re-use of the enzyme, thus potentially
increasing cost-effectiveness of the reaction
The Physiology of Lipase Production by Pseudomonas species
No full text1. Lipase production by the Pseudomonasspecies was induced by
growth on Tween80.
2. Maximum activity was observed under Tween 80-limited
conditions.
3. Production of lipase in a Tween 80-limited continuous culture has
been optimised.
4. Lipase has been purified to homogeneity, and someofits physicochemical
properties determined
COMPARATIVE ANALYSIS OF LIPASES IN VIEW OF PROTEIN DESIGN
No full textThe 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
BIOSENSING OF ORGANIC PEROXIDES
No full textReagentless bulk-type biosensors for hydroperoxides are described,
where horseradish peroxidase is incorporated into a graphite-epoxy
material. The principle of measurement is based on the faesibility
of HRP to facilitate an fast heterogeneous electron transfer to
carbon material, thus arrising an electrocatalytic current in
dependence on the peroxides at a favourable low potential of - 25
mV. The electrodes are mechanically robust, polishable, and durable,
having a life time of more than 40 days
Potentiometric Immunoassay (PIA)- Mixed Potential Shift by an Antigen/Antibody Reaction
No full textThis paper presents a method for the direct potentiometric detection of a specific antigen/antibody
reaction. The method is based on a principle called "modulation of a mixed potential" at an ionselective
electrode (ISE). The ion-selective membrane of an ISE is covered with antibody resp.
antigen molecules. A high sensitivity is obtained when the mixed potential is especially unstable
which is in general the case if a mixed potential between cations andanionsis built up across the
phase boundary ofthe ion-selective membrane towards the sample solution. If the antigen binds to
its specific antibody, a directly detectable shift results in the mixed potential. If the
"immunoelectrode" is completed by a reference electrode, the corresponding changes of the cell
voltage vs. concentration of antigen resp. antibody follows a typical immunological calibration
curve. Since no labeled antigen Tesp. antibody molecule is needed, the necessary ISE is simple to
construct andthe instrumentation is quite teasonable, PIA will be a very attractive method
INVESTIGATIONS ON GLUCOSE BIOSENSORS USING LANGMUIR-BLODGETT TECHNIQUESFOR THE IMMOBILIZATION OF DERIVATIZED GLUCOSE OXIDASE
No full textAnovel method for the detection of glucose using modified glucose oxidase on Langmuir-Blodgett
films is presented. A new polymer Langmuir-Blodgett film was used to immobilize modified glucose
oxidase together with an oxygen sensitive fluorescent indicator. Five different glucose oxidase
preparations were investigated: native, degtycosylated, N-palmitoy!-modified, periodate oxidized and
y-carbodiimide treated. The oxygen consumption in the prescence of glucose was measured via
dynamic quenchingof the fluorescence of the oxygen sensitive indicator
MONITORING AND CONTROL OF BIOTECHNOLOGICAL PRODUCTION PROCESSES BY BIO-FET-FIA-SENSORS
No full textSingle and multisensor Field Effect Transistors FET with a pH-sensitive
Si/SiO2/SizN4/TazOs-gate and reference electrode (for single semsor) were
developed and used for manufacturing the following BIO-FETs: for glucose analysis:
Glucose Oxidase-FETs (GOD-FET), for penicillin G and V analysis: Penicillin G
Amidase- and Penicillinase-FETs, for urea analysis: Urease-FET, and for cephalosporin
C analysis: Cephalosporinase-FET.
The GOD-FETswereintegrated into the FIA-system of Eppendorf (EVA) and used
for monitoring the glucose concentration in microbial cultivation and production processes
with recombinant_Escherichia coli K12 MF, recombinant Escherichia coli
JM103, Saccharomyces cerevisiae H620, and Candida boidinii. Urease-FET-FIA was
used to monitor the urea concentration in a simulatedcultivation of Cephalosporium
acremonium and UREASE-FET-FIA and GOD-FET-FIA for the monitoring of urea
and glucose concentrations in simulated Saccharomyces cerevisiae cultivations
BATCH INJECTION ANALYSIS FOR HIGH-SPEED BIOSENSING
No full textThe principles of a new, non-flow, injection technique, termed batch injection analysis
(BIA), for high speed biosensing are described. BIA is based on the reproducible injection of
small samples toward a nearby detector, which is immersedin a large-volume stirred bulk
solution. Passage of the sample zone over the detector surface results in sharp peak
readouts, similar to those of flow injection analysis (FIA). Sample throughputs and size, the
sensitivity, detection limits and reproducibility, are also similar to those of FIA. In addition, the
need for pumps and connecting tubingsis eliminated. Becauseof the limited solution
handling capability, BIA relies on the use of specific or reactive sensing surfaces. In
particular, the inherent specificity of biosensors makes them extremely attractive for the BIA
operation. Such characteristics and advantagesareillustrated for the use of enzymes or
tissues, coupled to amperometric, potentiometric or thermal BIA detectors. The high-speed
and simple BlA/biosensing operation holds a great promisefor clinical screening and
bioprocess monitoring