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CLONING, SEQUENCING AND REGULATION OF THE LIPASE GENE FROM PSEUDOMONASSP. M-12-33
No full textThelipase gene from Pseudomonas sp. M-12-33 was cloned. The cloned DNA was 2.9 Kb in length, which was
essential for production of the lipase and contained two open reading frames, lipA and lipX. The lipA gene was supposed
to comprise 1092 nucleotides and give a preproprotein of 364 aminoacids which was then processed to a mature
lipase protein of 320 amino acids. On the other hand, the lipX gene was assumedto code a protein of 344 amino acids
concerned with someregulation of the enzyme production
CHARACTERISTICS OF A NEW LIPASE FROM A Thermus sp BACTERIUM
No full textThirty strains of the genus Thermus, isolated from hot sprins in Portugal, were
screened for the secrection of lipases. In the end, the strain LFF1 received our attention for further
characterization. We report here some kinetic properties of the crude extracellular extract when used
in a reversed micellar system of AOT in isooctane for the hydrolysis of triolein. In common with
other lipases, this extract showed maxima at pH 7 and temperatures in the range 40-50 °C, but significant
residual activities were also observed at higher (up to 80 °C) and lower (downto -3.5 °C)
temperatures. The hydrolysis oftriolein in the micellar system followed an apparent Michaelis-
Menten kinetic mechanism with K,,(app)=7.1%(v/v) and V_.„(app)=55.5 mole/(ml.h.mg protein).
The specific activity of the extract decreased continuously with increasing concentrations of protein
encapsulated in the reversed micelles. The aqueousextract lost less than 9% of its activity when
stored at 4 °C for almost 3 months
Biosensors for Choline, Choline Esters and Inhibitors of Choline Esterase
No full textA choline electrode has been developped by coupling immobilized choline oxidase to a
hydrogen peroxide electrode. The coimmobilization of cholinesterase permits the measurementof
acetylcholine. The excess of both enzymes results in diffusion limited electrode
response for both substrates, with relative sensitivities 1: 0.6 for choline if compared with
acetylcholine.
When
kinetically controlled bienzyme sensor with a low activity of cholinesteraseis used,
a dimished sensitivity is obtained for acetylcholine with an increased sensitivity for inhibitors,
such as NaF, butoxycarboxime,trichlorfon, or dimethoate. Potentially disposable
sensors for those inhibitors have been.constructed with cholinesterase coimmobilized with
choline oxidase in a gelatin membraneon a platinum electrode and with cholinesterase
immobilized in polyurethane on a thick-film metallized platinum electrode. The decreased
formation rate of thiocompoundsfrom thiocholinesters serves as measurefor the inhibited
enzymein the latter setup
HOW TO IMPROVE ENZYME ELECTRODES ?
No full textThe mathematical background of two-step amperometric enzyme electrodes is
presented
THICK FILM BIOSENSORS FOR ETHANOL AND UREA
No full textTwo types of thick film biosensors with different substrate materials were tested for the
preparation of amperometric devices and conductivity sensors. We present the determination of
alcohol by an amperometric sensor and of urea based on conductometric measurements. Alcohol
oxidase and urease were immobilized by crosslinking with glutaraldehyde.
The alcohol sensor was fabricated by printing four working electrodes and the auxillary electrode
using a platinium paste and the reference electrode using a silver paste on a conventional thick
film Al,O,-substrate. The determination of alcohol is based on H,O,-measurement. Thelinear
range of this sensor without any additional membraneis limited to 0.8 mmol/l. Hence, real
samples required dilution prior to analytic. The results obtained for different beverages correlate
acceptably with values obtained by gas chromatography. The conductometric sensors were made
using "green-tape"-technique resulting in multi-layer-strips, which include four metal layers
printed in a silver-palladium paste. Urea is hydrolysed by urease to ammonia and carbon dioxide
resulting in an increase in conductivity. The analytical range of the urea sensors was 1 umol/l up
to 5 mmol/l with a linear range from 10 umol/l to 200 umol/l
IMPROVEMENT OF AN E. COLI FERMENTATION BY ON-LINE MONITORING OF GLUCOSE AND TOTAL AMINO ACIDS USING A FIBRE OPTIC FIA-SYSTEM
No full textAnew methodfor the determination of glucose and total amino acids using a fibre optic biosensor
in combination with a flow injection system (FIA) is presented. The biosensors are basedonfibre
optic oxygen optrodes which measure the oxygen consumptionvia dynamic quenching ofthe fluorescence
ofan indicator by molecular oxygen. Enzymecartridgeswith immobilized glucose oxidase (EC
1.1.3.4.) fromA. niger and amino acid oxidase (EC 1.4.3.2) from Crotalus adamanteus were integrated
into flow through chamberscovering oxygen optrodes. The FIA-system wasusedto dilute and buffer
the on-line sterilefiltered samples taken automatically from the fermenter by-pass loop. This detection
set-up was successfully used to improvethecell density of E. coli fermentations by controlled medium
supply. The fermentations were carried out for the production of recombinant protein
Enzymes and Antibodies for Biosensors - Studies at the GBF
No full textEnzymes and antibodies are key components of most biosensors and have also found wide application in
instrumental analysis, e.g. in flow injection analysis [1, 2]. Among the enzymes studied in enzyme
sensors, oxidases are of particular importance since they consume oxygen and form hydrogenperoxidetwo
species which can easily be monitored by electrochemical or photometric transducers. While many
oxidases with a wide range ofsubstrate specificity are commercially available, we have set out to further
expand this range by the methods of a) screening of suitable microbial enzymes,and b) by applying the
techniques of protein engineering.
So far, we have successfully screened a microbial amino acid oxidase, a microbial xanthine oxidase and
a microbial acyl-CoA oxidase with unusual substrate specificity. In another part of our work focussed on
the "protein design" of biosensor-related proteins, we have solved the structures of two glucose oxidases,
purified to homogeneity a thermophilic, hydrogen peroxide forming NADH oxidase, and purified and
crystallized a bacterial FMN-dependentluciferase. In the field of immunoreagents for biosensors, we have
set out to solve the structure of a pesticide-specific monoclonal antibody.
The status and the focus of these investigations will be discussed
DEVELOPMENT OF SURFACE ACOUSTIC WAVE SENSORS INCORPORATING LANGMUIRBLODGETT FILMS
No full textA sensitive surface acoustic wave (SAW) device has been developed by depositing phospholipids or stearic acid
onto the channel of the device. For this purpose the Langmuir-Blodgett (LB) technique has been used to deposit an
optimal numberof layers which was found to be between 20 and 40. Theaffinity of alcohols to phosphatidylcholine
coated channels increased with increasing chain length. The observed differences in mass loading, however, are
rather small, which meansthat electrostatic and Van-der-Waals interactions rather than specific effects determine
the binding
USE OF CRYSTALLINE BACTERIAL SURFACE LAYERS(S-LAYERS) FOR THE DEVELOPEMENTOF BIOANALYTICAL SENSORS
No full textThe developement of sensors for monitoring bioanalytical parameters
has attracted much attention in medical, biotechnological and
environmental applications. Although there are several different
detection principles available now there is still enough innovation
potential for developing alternative supports as immobilization matrices
for biologically active component(s). Aside from the well established
carriers such as synthetic polymer matrices, carbon, gold or platinum
electrodes crystalline bacterial surface layers (S-layers) are an
important alternative since they have shown an unsurpassed high and
accurate binding capacity for a broad spectrum of enzymes (1)
An Integrated Services Approach to Biological Sequence Databases
No full textDatabase users in molecular biology are faced with steadily increasing amounts of raw data,
multiple database providers and services. Here we describe the integration of a set of previously
isolated database services and demonstrate their accessibility through a uniform userinterface. A
multi-layered software architecture is applied to make different degrees of service integration
transparent to the user. We focus on the design of specialized gateways that integrate services
differing in temporal behavior and stateless or state dependent operation. Gateways may reside
on heterogeneousplatforms. A link layeris introducedto integrate individual query functions in
order to interrelate simple, complex and state dependent services through a common, unique
interface. It is possible to generate new complex services by a combination of multiple functions.
Wedescribe the application of the World Wide Web (WWW)as the implementation framework
of the interface layer. To assure interoperability of services, integrity of data resources must be
supervised. Consistency control is issued by a dedicated synchronization layer