Practical Biochemistry for Colleges

185 Pages·1989·25.386 MB·English

by  E. J. Wood (Eds.)

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PRACTICAL BIOCHEMISTRY
FOR COLLEGES
Edited by
Ε J WOOD
Department of Biochemistry,
University of Leeds,
Leeds, UK
PERGAMON PRESS
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Copyright © 1989 International Union of
Biochemistry
All Rights Reserved. No part of this publication may
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First edition 1989
Library of Congress Cataloging in Publication Data
Practical biochemistry for colleges / edited by
E. J. Wood. — 1st ed.
p. cm.
Includes index.
1. Biochemistry—Laboratory manuals.
I. Wood, Edward J., 1941- .
QP519.P68 1989 547'.007'8—del 9 88-38534
British Library Cataloguing in Publication Data
Practical biochemistry for colleges
1. Biochemistry. Experiments
I. Wood, E. J. (Edward J.)
574.19'2'0724
ISBN 0-08-036140-4
Printed in Great Britain by BPCC Wheatons Ltd, Exeter
Preface
IT IS generally accepted that biochemistry courses, even medical too. Many use inexpensive, familiar, readily available materials.
biochemistry courses, should have some practical component. Such experiments are comparatively easy to schedule within fixed
The declared aims of such laboratory practicals are (a) to illustrate time periods, but do offer the possibilities to teach experimental
the lecture course, and (b) to allow the students to develop their design or starting points for open-ended projects. If microcompu­
technical ability, not only at handling biological materials and ters are available, there are now plenty of simulation programs
laboratory equipment, but also of designing protocols and experi­ for enzyme kinetics, and in addition it is worth teaching students
mental strategies to solve problems. Biochemical educators have how to use spreadsheets to optimize experimental design.
attempted to achieve these aims, with varying degrees of success, Techniques of Biochemistry includes the routine methods of gel
and one of their longstanding problems has been how to design filtration, ion-exchange and affinity chromatography, as well as
"good" laboratory practicals. Since designing good laboratory electrophoresis. Students should develop technical skills as well
practicals is time-consuming, it is inevitable that people would as an appreciation of the problems of handling delicate biological
wish to share each other's practical experiments. Consequently, materials. Experience with techniques is important: one is fre­
from the very first issue of Biochemical Education, there has been quently told by students that SDS-polyacrylamide gel electro­
a welcome for published experiments that are "tried and tested", phoresis is a technique for purifying proteins, these students being
inexpensive to mount, and offer ways of achieving the above unable to distinguish between a very useful analytical technique
aims. and a practical preparation method.
This book contains a collection of practical biochemistry As far as possible the techniques offered are inexpensive and
experiments that have appeared in Biochemical Education over this reaches the extreme with the mini-slab gel electrophoresis kit
the years since Volume 1 (1972). All were submitted to the jour­ (P 75).
nal by active teachers of biochemistry wanting only to share suc­ The Metabolism section includes a rather motley collection of
cessful experiments that they have developed (painstakingly, and experiments. It is much more difficult to illustrate a whole meta­
by trial and error) for their particular course in universities and bolic pathway (or its discovery) than to put on an enzyme assay.
colleges around the world. It is hoped that others will find it useful Rather fewer experiments of the "metabolism" type seem to have
to have an edited collection of these experiments. been sent in to Biochemical Education over the years. Notably
It is not possible to achieve a complete coverage of all parts of there are no Warburg experiments, but also very few utilizing
biochemistry, nor is it feasible to present a set of experiments that mitochondria or chloroplasts, or even homogenates. There are
will exactly complement a given biochemistry course. There are obviously problems with using animals as well as difficulties in
many reasons for this and in any case most institutions have their carrying out experiments using cell culture techniques, although
own set of laboratory practicals, built up over the years. Never­ it has to be admitted that the latter are becoming increasingly
theless, the range of experiments in the present volume is fairly important in biochemical research.
rich and should suggest ideas to those in charge of arranging Molecular Biology experiments are still in their infancy in lab­
practical classes. Experiments might be used directly or might be oratory classes. They are not too difficult to put on but tend to be
modified. The most useful feature of them all is that they have expensive, requiring the use of expensive enzymes and isotopical-
actually been used successfully with classes, and exact details are ly-labelled compounds. The use of biotin-Iabelled probes (p 157)
given for putting them on. All of the experiments have been re­ may remove some of the problems with using isotopes. Most
read by their authors (many of whom said they were still using molecular biology manipulations are in themselves, rather
them), in order to eliminate misprints and to update where appro­ simple : it is the strategy of a series of procedures that is important.
priate. We are extremely grateful to the authors for their kind This is likely to be a fertile area and of course there are a wide
permission to reprint their experiments and for their careful atten­ range of manuals now available.
tion in checking the descriptions of their experiments. The Model Building section includes a number of novel ideas,
At least some of the experiments that appeared in the very all of them requiring only inexpensive and readily available
early issues of Biochemical Education are still used or are usable. materials. Models are useful in helping students to get to grips
All were very carefully considered for inclusion, and a few have with the shapes and interactions of biomolecules. At one extreme
been excluded because things have changed so dramatically that there are the various molecular model-building systems (usually
they no longer have much relevance. In contrast, some experi­ of plastic) and at the other, increasing use of computer graphics
ments, especially in the field of molecular biology, would not have to illustrate molecules and their interactions. The present section
been conceivable 15 years ago. One of the problems of the rapidly offers an intermediate, inexpensive and entertaining diversion
growing disciplines of biochemistry and molecular biology is that from these: they should be accepted with their limitations.
there is so much that is new, both information and techniques, Finally, the Clinical Biochemistry section includes a selection
and yet it is not possible to discard the old. Consequently students of experiments of clinical relevance that might be expected to
today have more and more pressed upon them. Writers of Edi­ appeal more to the medical student because of the clinical interest
torials for the journal have commented on this repeatedly — but in the substance or enzyme measured. Teaching biochemistry to
have not come up with any solutions! medical students poses many problems and some schools now
The experiments collected have been grouped into sections for offer a minimum of practical exercises on the grounds that the
convenience and tidiness — the juxtapositions are fairly arbitrary practising doctor will never use a pipette to assay an enzyme in
but may be useful for instructors. The groupings are into experi­ his or her professional life. This is not necessarily the case in all
ments on: enzymes, techniques, metabolism, molecular biology, parts of the world, but we have to accept that the trend is to rely
molecular model building and clinical biochemistry. It is appro­ on reported laboratory findings and to have sufficient knowledge
priate to offer a few comments on each of these categories at this to interpret them as part of a diagnosis.
point. The other practical problems with running laboratory classes
Enzymes and Enzymology contains a wide range of experi­ for medical (or indeed any biochemistry) students is that, because
ments from the simple to the sophisticated with some purifications of the AIDS danger, taking blood or using any material of human
xi
origin is going to become more and more difficult to justify. In A large part of the excitement of modern biochemistry and
some of the experiments the authors have suggested alternatives, molecular biology is that of doing things in the laboratory and
but those running laboratories must constantly be aware of this finding out how living things work. It is hoped that this collection
possible hazard. of experiments will stimulate the imagination of teachers and
Although the authors of papers have very kindly looked care­ instructors so that they will more effectively inspire the young
fully at what eventually appeared in print in the journals, I remain people entering the profession.
responsible for any errors that have crept in since then. I would
very much appreciate hearing about them with a view to future
correction. Leeds, 1988 E. J. WOOD
xii
Some Properties of an Enzyme: a Demonstration Table 1 Demonstration of Alkaline Phosphatase Activity
Experiment with Alkaline Phosphatase
Tube Buffer Activator Inhibitor Dialysis
D F EVERED number Addition (ml) (ml) (ml) Bag (ml)
1 None 10 - - 2 enzyme
Department of Biochemistry
2 Activator 9 1 - 2 enzyme
Chelsea College (University of London)
London SW3 6LX, UK 3 Inhibitor 9 - 1 2 ml enzyme
+ inhibitor
4 Inhibitor and 8 1 1 2 ml enzyme
The requirements of a demonstration experiment are that it Activator + inhibitor
should be visible from the back row of a large lecture theatre. 5 Boiled enzyme 10 - - 2 ml enzyme
It should use cheap, readily available materials and apparatus. (Control) after boiling*
Furthermore, it should always work. If an experiment is to be
repeated by students it should be open-ended to suggest * The rest of the enzyme solution, after samples are taken for tubes 1 and
additional experiments. 2, is placed in a dialysis bag closed with double knots at each end. The
bag is placed in a boiling water bath for 10 min., cooled and then put
The present experiment illustrates the catalytic action of the
into tube 5.
hydrolytic enzyme, alkaline phosphatase, utilizing phenol-
phthalein diphosphate as substrate. Hydrolysis of this substrate
releases phenolphthalein which produces a red colour in the alka­ (2) the catalytic action of the enzyme is destroyed by heating and
line medium. This coloration can be seen by eye. (3) substrates and metal ion activators are mostly small molecules
that can pass through dialysis tubing.
Method
Buffer, pH 10: 5.8g Na2C03 and 3.8g NaHC03 dissolved in 11 Discussion
water. Enzyme, alkaline phosphatase, freshly prepared, 10 mg in The present experiment is unusual in that the inhibitor, citrate,
10 ml buffer. Commercial enzyme is suitable (BDH Ltd, Poole, exerts its i2nh+ibitory effect competively with the metal ion acti­
Dorset). Enzyme and inhibitor (alkaline phosphatase solution vator, Mg 2, a+nd not the substrate. The inhibition is removed by
containing 0.2M inhibitor): 4 ml enzyme solution containing adding Mg . This effect has b1een shown with alkaline p2hospha­
240 mg sodium citrate. Phenolphthalein diphosphate solution,
tase from human blood serum and calf small intestine. Further­
0.1M, freshly prepared, 280 mg in 5 ml buffer. Extract any free 3
more, the reaction has been used as an assay of alkaline
phenolphthalein with an equal volume of ethyl acetate with gentle
phosphatase in blood serum in medical laboratories.
mixing. Discard the ethyl acetate layer.
For class experiments the following are suggested:
Activator, 2M magnesium acetate, 2g tetrahydrate in 5 ml buffer.
(1) Determine the pH optimum of the enzyme by using buffers
Inhibitor, 2M sodium citrate solution, 3g in 5 ml buffer. of different known pH values. With acidic buffers it will be neces­
Set up five large test tubes (24 x 150 mm) as shown in Table 1. sary to add alkali to the contents of the dialysis bags at the end of
the experiment.
2 + 2 + 2+ 4
Results (2) Study the specificity of different metal ions for activating the
It should be found that the dialysis bags in tubes 2 and 4 are fully enzyme, e.g. substitute Zn , Co , etc for the Mg ions.
coloured. The bag in tube 5 is colourless. Tubes 1 and 3 show bags
which have a diminished coloration. With a stored or partially- References
hydrolysed sample of substrate magnesium phosphate may '2Steenson, Τ I and Evered, D F (1963) Lancet, ii, 792
precipitate in tube 2. 3Evered, D F and Steenson, Τ I (1964) Nature, 202, 491-492
4
These experiments demonstrate that: (1) enzymes are macromol- Huggins, C and Talalay, Ρ (1945) / Biol Chem 159, 399-410
ecules and, therefore, cannot pass through a dialysis membrane, Clark, Β and Porteous, J W (1965) Biochem J 95, 475-482
3
A Simple Laboratory Experiment to Demonstrate The simplest transamination reaction to study is that catalysed
Transamination by the enzyme GPT. This enzyme is obtained commercially at a
reasonable price and solvent systems are published for separat­
SUSAN DEWHURST and IAN SMALLMAN ing glutamate from alanine (substrate and product α-amino
acids) and pyruvate from 2-oxoglutarate (substrate and product
Brighton Centre for Advanced Biology α-οχο acids).
Brighton College of Technology Our original procedure was to stop the enzyme reaction by
Applied Science Department adding ethanol, evaporate the incubation mixture to dryness, re-
dissolve the solids obtained in a small volume of water and apply
Brighton BN1 4FA, UK
to thin layer chromatograms. The results obtained were not
convincing because the control, to which no enzyme had been
added, contained a number of products, in addition to pyruvate,
The transfer of amino groups, commonly known as transami­ which could be visualised using semicarbazide and UV light.
8
nation, was first recognised as an enzymatic reaction by the These products were presumably formed as a result either of the
Russian biochemists Braunstein and Kritzmann in 1937. These heat instability of pyruvate or of a condensation reaction
workers demonstrated the enzymatic formation of glutamic acid between pyruvate molecules.
from 2-oxoglutarate and certain amino acids. However, it was Since the aim of the experiment was a qualitative demon­
1
not until 1950 that the broad scope of transaminations was stration of transamination it was decided not to stop the reaction
established. by the addition of ethanol. Hence, no increase in volume of the
In general terms transamination reactions can be summarised: incubation mixture would occur. This eliminated the need to
reduce the volume of the incubation mixture by heating prior to
., , ., aminotransferase chromatography. The omission of the heating step resulted in
α-amino acidx + α-οχο acid2 *
the control producing only one spot which corresponded to that
α-οχο acidi + α-amino acid2
produced by pyruvate. This result demonstrated clearly that only
in the presence of the enzyme GPT is pyruvate converted to 2-
The metabolic roles of transamination reactions are several and
oxoglutarate with the concomitant conversion of glutamate to
important and include, (a) amino acid synthesis, (b) amino acid
alanine:
degradation, (c) liaison between carbohydrate and amino acid
metabolism, (d) synthesis of several specific compounds, includ­ GPT
ing urea and -γ-aminobutyric acid. In view of the significance of pyruvate + glutamate 2-oxoglutarate + alanine
transamination in metabolism the inclusion of a simple labora­
tory experiment to demonstrate this reaction in undergraduate Materials
3
practical classes would reinforce lecture information. Glutamic pyruvic transaminase (GPT) from porcine heart (No
G-9880, 100 units in 0.5 cm ) was purchased from Sigma
Background Chemical Company, Fancy Road, Poole, Dorset. L-glutamic
When studying transamination reactions it is important to acid, sodium salt; DL-alanine, sodium pyruvate, ninhydrin and
demonstrate that two products are formed which are similar to, semicarbazide were purchased from BDH Ltd, Poole, Dorset as
but different from, the two substrates used. The formation of a were the Merck TLC plastic sheets, cellulose (without fluor­
new α-amino acid is easy to demonstrate using thin-layer escent indicator) 20 x 20 cm, layer 0.1mm, Art 5577. 2-
chromatography since suitable solvents are available for separat­ oxoglutaric acid was purchased from BCL, Bell Lane, Lewes,
2
ing amino acids. The separated amino acids can be subsequently East Sussex.
visualised using the ninhydrin reaction. Demonstration of the
3
formation of a new α-οχο acid is not so easy. Most methods rely Experimental
3
on the production of a 2,4-dinitrophenylhydrazone or similar (a) Enzymic reaction To the experimental tube add 1.5 cm
3 3 3
coloured derivative so that visualisation on chromatograms is not 0.01 M phosphate buffer pH 7.6, 0.5 cm 0.1 M glutamate,
3
a problem. In our hands, however, ascending and descending 0.5 cm 0.1 M pyruvate and 0.5 cm 1:100 dilution of GPT. The
paper chromatography of the 2,4-dinitrophenylhydrazone deriv­ enzyme is omitted from the control tube and replaced by 0.5 cm
atives of pyruvate and 2-oxoglutarate gave a number of spots. water. Both tubes are incubated at 37°C for 45 min.
Pyruvate and 2-oxoglutarate are the α-οχο acid substrate and (b) Demonstration of alanine formation A cellulose thin layer
product of the enzyme L-alanine:2-oxoglutarate aminotrans­ plate is spotted with 5 drops of each of the following solutions;
ferase, EC 2.6.1.2, commonly known as glutamic pyruvic 0.0167 M glutamate, 0.0167 M alanine, enzyme-containing
transaminase (GPT). The multiple spots produced by the 2,4- incubation mixture, and control. The chromatogram is run in
4
dinitrophenylhydrazone derivatives of these α-οχο acids are freshly prepared propanol:34% aqueous ammonia (7:3 v/v) for
probably due to the lack of specificity of the method. 2V2 h. After drying it is sprayed with 0.2% ninhydrin in acetone
Modifications to this method such as extractions into various and heated at 110°C for a few min.
solvents prior to chromatography and visualisation of the 2,4- (c) Demonstration of 2-oxoglutarate formation A cellulose thin
dinitrophenylhydrazones by spraying with 2% NaOH in 90% layer plate is spotted with 10 drops of each of the following
EtOH still gave a number of spots whose colours were not solutions; 0.0167 M pyruvate, 0.0167 M 2-oxoglutarate, enzyme
sufficiently distinctive. containing incubation mixture and control. The chromatogram is
l
For this reason an attempt was made to detect the α-οχο acid run in freshly prepared ethanol:34% aqueous ammonia:water
product on chromatograms without resorting to 2,4-dinitro­ (80:4:16 v/v) for 2/i h. After drying it is sprayed with 0.1% semi­
6
phenylhydrazone formation. Mono- and dicarboxylic acids can carbazide in 0.15% sodium acetate, re-dried and viewed under
be visualised by spraying with an acid-base indicator. The UV light of wavelength 254 nm.
application of a number of indicators including bromocresol
green, bromocresol blue and chlorophenol red to pyruvate and Results and Discussion
2-oxoglutarate spots on thin layers resulted in the clear detection The α-amino acid chromatogram shows the control containing
of 2-oxoglutarate but not pyruvate. However, spraying with only one spot with R f value corresponding to that of glutamate
semicarbazide followed by UV illumination at 254 nm caused whereas the enzyme-containing incubation mixture contains two
7
both pyruvate and 2-oxoglutarate to show up as blue-purple spots with Rf values corresponding to those of glutamate and the
spots on a turquoise background. product alanine.
5
The α-οχο acid chromatogram shows the control containing References
only one spot with R f value corresponding to that of pyruvate Mahler, H R and Cordes, Ε Η (1971) 'Biological Chemistry', (second
whereas the enzyme-containing incubation mixture shows two 2edition) Harper and Row Publishing, pp 790-793
spots with Rf values corresponding to those of pyruvate and the Randerath, Κ (1968) Thin Layer Chromatography, (second edition)
product 2-oxoglutarate. 3Academic Press, pp 110-115
This practical demonstrates that the transamination reaction Friedemann, Τ Ε (1957) Methods in Enzymology, Vol 3, pp 414-418,
between pyruvate and glutamate requires a transaminase 4Academic Press
enzyme. It also shows that the two products are similar to, but 5Seligson, D and Shapiro, Β (1952) Analyt Chem 24, 754-755
different from, the two substrates. The presence of substrates as
Smith, I and Seakins, J W Τ (1976) 'Chromatographic and Electro-
well as products in the enzyme-containing incubation mixture 6phoretic Techniques', Vol 1, (fourth edition) Heinemann, ρ 246
indicates that the reaction does not proceed to completion. Such
Myers, W F and Huang, Κ Y (1969) Methods in Enzymology, Vol 13,
an observation can lead to a discussion of (a) the meaning of, 7pp 431-434, Academic Press
and value for, the equilibrium constant of transamination
Umbarger, Η Ε and Magasanik, Β J (1952) / Amer Chem Soc 74,
reactions, and (b) the roles of transamination reactions in amino
84253-4255
acid anabolism and catabolism.
This is an inexpensive practical which does not require the use Dawson, R M C, Elliott, D C, Elliott, W H and Jones Κ M (1986) 'Data
for Biochemical Research' (third edition) Clarendon Press, Oxford, ρ
of sophisticated equipment. It can be used with classes of any
50
size. Furthermore, it can be completed within one 4-hour
practical session at the end of which students obtain 'take away'
results.
6
Simple Visual Demonstrations of the Catalytic Activity where the bubbles are discharged, leaving the pellets to fall back
of Immobilized Cells and Enzymes down to the bottom of the flask again. Thus at any one time pellets
can be seen rising and falling in the flask. Furthermore, after some
PETER S J CHEETHAM* and CHRISTOPHER BUCKEf period of continuous use the liquid smells strongly of ethanol,
and, if nutrients are included in the sucrose solution, growth of
*PPF International Ltd the cells occurs in situ causing the pellets to darken in colour, and
Ashford, TN24 OLT, UK even crack on occasions.
and Alternatively, the pellets can be packed into a chromatography
column and sucrose solution pumped slowly up the column. Reac­
f Polytechnic of Central London
tion is indicated by the generation of bubbles of CO2 from the
115 New Cavendish Street,
pellets, which pass up and out of the column. The rate of reaction
London W1M 8JS, UK can be quantified by measuring the volume of gas collected by the
downwards displacement of water. The ethanol concentration in
the eluate can be assayed by using an ethanol test-kit (Boehringer-
Much of the work carried out in biotechnology uses various forms Mannheim), or by the method of Bonnidisen, 6 which depends on
of immobilized enzymes as catalysts because of the great variety measuring the amounts of ΝADH formed by the oxidation of the
of reactions they catalyse, their high catalytic activities and stereo- ethanol by alcohol dehydrogenase, using semicarbazide to react
specificities, and the mild conditions under which they operate. with the acetaldehyde formed. The immobilized cells have an
However, before industrial application can be made enzymes or initial activity of approximately 10 mg of ethanol produced per
cells possessing enzyme activities must usually be immobilized so gram of cells per hour, and the stability of the immobilized cells
as to ensure their easy recovery after reaction has been com­ is such that after 80 h continuous use the column has lost half of
pleted. Then the immobilized biocatalyst may be employed in its original activity.
continuous reactors so allowing re-use of the enzyme(s), and pre­
venting contamination of the product by the enzyme or cells. Enzyme Immobilization
Immobilization may be defined as any technique which severely For a demonstration of immobilized enzyme activity, freeze-dried
limits the free diffusion of cells or enzyme molecules. Methods of catalase Sigma is dissolved in water to give a 10% (dry wt/v)
immobilization include covalent binding and adsorption to solid solution and pellets formed as described above. The final concen­
supports, entrapment or encapsulation in solid supports; or aggre­ tration of enzyme in the pellets is about 2% (dry wt/v). When the
gation of the cells or enzymes. 12 ,Techniques for the immo­ freshly formed pellets are placed in a dilute H2O2 solution (about
bilization of cells and enzymes to solid supports are of particular 0.3%, w/v), bubbles of oxygen are evolved immediately, demon­
interest to biochemists wishing to study enzyme kinetics and the strating catalase activity. Bubbles continue to be generated until
tertiary structure of proteins,1 and are acquiring analytical appli­ the substrate is exhausted, but production is resumed upon
cations especially in the form of enzyme electrodes. 1 However, addition of fresh H2O2. However, after several hours' incubation
methods of immobilization attract interest chiefly from bio­ the catalase activity is found to be chiefly in the bulk solution and
chemists microbiologists and chemical engineers interested in the not associated with the pellets showing that the catalase has not
commercial application of enzymes. Here simple methods for the been permanently immobilized.
visual demonstration of the catalytic activities of whole cells and If a permanently-immobilized enzyme is required two
enzymes are described, involving the generation of C0 2 and O2 by approaches are possible. The pellets containing catalase formed
immobilized yeast cells and catalase respectively. with higher alginate concentrations of up to 8% (w/v) can be dried
to approximately 30% of their original volume in a stream of cold
Methods air so as not to denature the enzyme, drying having the effect of
Cells and enzymes are immobilized using a cheap, simple, quick decreasing both the size of the pellets and the size of the pores
and versatile method we have developed for cells, sub-cellular in the pellet preventing leakage of enzyme. Secondly, glucose
organelles and high molecular weight enzymes, by entrapping oxidase (BDH), which has a mol wt of just over 150 000, does not
them in calcium alginate gel pellets. 3-4'5 Alginate is a complex leak even from undried pellets formed from low concentrations
polysaccharide extracted from seaweeds which has the useful of alginate when immobilized as above. Its activity when supplied
property of being gelled by calcium and other divalent and tri- with a dilute glucose solution can be assayed by the rate of disap­
valent cations, its most important use being as a thickening agent pearance of the substrate as measured by a Boehringer glucose
in the food industry. The gel is stable, strong and inert, and pos­ kit, or qualitatively by using dip sticks.
sesses a microporous structure. Most importantly, little enzyme If required, pellets of immobilized catalase or glucose oxidase
denaturation occurs during immobilization presumably because can be used in columns as for the yeast cells, and the activity
no heat, free radicals of pH changes are generated. measured by the volume of oxygen produced or by the amount
of glucose which has been consumed.
Cell Immobilization
Yeast cells (Saccharomyces cerevisiae), obtained as dried pelleted Discussion
cells are rehydrated and stirred into a 1.5% (dry wt/v) aqueous The above experiments describe simple, cheap and versatile
sodium alginate solution (BDH) at room temperature until evenly methods of demonstrating the enzyme activities of immobilized
mixed. The cell suspension is then extruded drop-wise from a cells and enzymes, and are dependent on the non-denaturing
syringe fitted with a needle, or pumped dropwise into a stirred nature of the immobilization method used. For more advanced
bath of 0.1 M CaCb, from a height of about 10 cm. Each drop students, these methods could be used for detailed investigations
forms a spherical alginate gel pellet which becomes sufficiently of methods of immobilization and measurement of the activity,
gelled for use after about 10 min incubation in the CaCb solution. stability and substrate specificity of the enzymes and cells. For
The concentration of entrapped cells used can be as high as 95% instance, the optimal pH, temperature, substrate concentration
(wet wt/v). The immobilized yeast cell pellets are then suspended and flow rate could be determined by the effects of these par­
in a dilute sucrose solution (approximately 10%, w/v) and after a ameters on the amounts of gas produced. Likewise, the effects of
lag period of a few hours, which is the time required for the the size of the pellets and the rate at which they are shaken can
sucrose to diffuse to the cells and to be metabolized, CO2 is gener­ be used to estimate the effect of internal and external diffusional
ated by the cells. This CO2 collects as bubbles around the pellets restrictions. The effect of immobilization or operational use on
so causing them to rise to the surface of the sucrose solution, the viability of the cells can be estimated by dissolving away the
7
3
alginate with phosphate-buffered saline followed by plating out Kierstan, Β and Bucke, C (1977), Biotechnology and Bioengineering, 19,
4
and counting of the released cells. 387-397
Cheetham, P S J (1979), Enzyme and Microbial Biotechnology, 1,
5
References 183-188
Mosbach, Κ (1976), "Methods in Enzymology" Vol 44, Academic Press, Cheetham, P S J, Blunt, Κ W and Bucke, C (1979) Biotechnology and
2 6
London and New York Bioengineering, 22, 2155-2168
Cheetham, P S J (1980), in "Topics in Enzyme and Fermentation Bio­ Bonnidsen, R (1963), in "Methods in Enzymic Analysis", Bergmeyer,
technology", Wiseman, A (ed), John Wiley & Sons, New York, Vol 4, H V (ed), Academic Press, New York, p. 285
pp 189-238
8