TIBTECH - OCTOBER 1987 [Vol.

5]

EI BDDDB// two main layers, an outer layer

of protein-mannan complex and
an inner glucan layer 5. Enzyme
systems for yeast cell lysis are, there-
fore, usually a mixture of several
different enzymes which include
Enzymatic lysis and one or more [3(1-3)glucanase, pro-
tease, [5(1-6)glucanase, mannanase
or chitinase. They act synergistically
disruption of microbial cells in the lysis of the cell wall but only
two are essential for whole cell
breakage; a specific wall-lytic pro-
B. A. Andrews and J. A. Asenjo tease to degrade the outer layer of
protein-mannan and a lytic [~(1-
The development of expression systems for large recombinant 3)glucanase to degrade the inner
proteins which cannot be secreted by the host microbial cells glucan layer.
necessitates the development of novel techniques for cell disruption. In both Gram-positive bacteria and
The use of enzyme systems which provide biological specificity to the Gram-negative bacteria, a peptido-
process of cell lysis and disruption shows tremendous promise as a glycan component is responsible for
the strength of the wall. In Gram-
method of controlled lysis and selective product release. positive bacteria peptidoglycans are
the major wall polymers and are
Enzymatic lysis and disruption of mechanical disruption to increase associated with teichoic acids and
microbial cells has found a number the selectivity of product release, to polysaccharides. Gram-negative bac-
of important applications (Fig. 1). increase the rate and yield of teria have a two layer wall structure:
These include the production of extraction, and to minimize product the inner, rigid peptidoglycan com-
specialty wall polysaccharides 1, pro- damage. The industrial use of en- ponent is covered by an outer layer or
duction of intracellular, membrane zymes for the release of specific cell outer membrane composed of pro-
bound or wall enzymes 2, production proteins is coming of age with the teins, phospholpids, lipoproteins
of yeast autolysates and products development of expression systems and lipopolysaccharides 6. Conse-
from subcellular structures 3 and the for large recombinant proteins that quently, a single enzyme can lyse
recovery of recombinant DNA pro- cannot be secreted by the cell. By Gram-positive bacteria but pretreat-
ducts such as surface antigen par- choosing an appropriate enzyme ment with a detergent (e.g. Triton
ticles manufactured in yeast 4. system, selective and sequential X-100) is usually necessary to remove
At present, the only industrial product release may be obtained. the outer membrane of Gram-
scale microbial cell breakage equip- This review summarizes the latest negative cells. Three types of bac-
ment is the high pressure homogen- developments and trends in the teriolytic enzyme have been isolated;
izer: other equipment, such as bead production and utilization of enzyme glycosidases which split poly-
mills are used mainly at bench and systems which lyse microbial cells. saccharide chains, acetylmuramyl-L-
pilot plant scale. However, these alanineamidases which cleave the
mechanical methods have draw- The substrates for lysis- the cell junction between polysaccharides
backs. They have no biological walls and peptides; and endopeptidases
specificity, they generate high temp- The cell walls of yeast and bacteria which split polypeptide chains.
eratures in the cell suspensions are distinctly different, hence, in
which impose great demands on general, lytic systems are specific Sources and properties of cell lyric
cooling equipment, they generate for particular groups of microorgan- enzymes
high shear stresses which can harm isms. Yeast cell walls (Fig. 2) have A large number of microorganisms
the molecules to be recovered, and -Fig. 1
they necessitate a large capital invest- Recombinant proteins
ment in specialized equipment.
Enzymes have none of these dis- Antibiotics
Lysed cell
advantages. They can be used on
their own for the release of intra- Specialized lipids
cellular soluble proteins, particulate Enzyme attack ~ Wall polysaccharides
inclusions and for wall and mem-
brane associated materials. Or they Pigments
can be used in conjunction with
Enzymes
B. A. Andrewsand J. A. Asenjo are at the
Biochemical Engineering Laboratory, Productsthatcanbeisolated ~ ~'~-'~ ~ Intracellular polymers
University of Reading, PO Box 226, after enzymatic lysis of microbial cells.
Reading RG6 2AP, UK.
~) 1987, Elsevier Publications, Cambridge 0166- 943087/$02.00

have been found to produce micro-
bial lytic enzyme systems 7-25 (Table
1). These organisms exhibit preda-
tory activity against yeast and other
microbial cells and have been iso-
lated from such diverse habitats as
decaying plant material (e.g. rice7),
brewery effluents 8, sewage plants 9,
estuaries 9, soil 1 and the human
mouth 11.
Virtually all microorganisms used
for production of lytic enzymes are
safe (class 1 classification in ATCC)
and non-pathogenic. The only strains
that do not fall into this category are
some strains of Staphylococcus
(class 2 in ATCC) and they are
considered only mildly pathogenic.
Conditions for use of microbial
cell-lytic enzyme systems have been
investigated by different authors 12.
The pH and temperature optima vary
considerably in the range pH 6-11
and 35-60C (Table 1).
For all the lytic systems, there is
considerable variation between the
molecular weights of the different
systems and their component en-
zymes. However, most are relatively
small (in the range of 10 to 30 kDa), a
property which is important in that it
makes them relatively easy to separ-
ate from large intracellular proteins
after cell lysis.
There is little information avail-
able on the effect of enzyme and
substrate concentration on microbial
cell-lyric enzymes. However, one
study 28 showed that the rate of pro-
tein release from whole yeast cells in
the presence of lytic enzymes was a
linear function of enzyme concentra-
tion and was used with substrate
concentrations up to 110 gl -~ dry
weight of yeast cells.
Lysis of yeast
The yeast-lytic enzymes produced
by different Arthrobacter sp. have
been extensively studied in batch
culture and found to be inducible (by
whole yeast cells and cell walls) and
subject to catabolite repression by
g l u c o s e 1 5 ' 2 6 ' 2 7 : [~(1-3)glucanase, pro-
tease and mannanase activities have
been detected. The cell wall degrad-
ing enzymes from different strains of
Oerskovia xanthineolytica have also
*Jeffries, T. W. (1976)PhD Dissertation,
Rutgers University, New Jersey, USA.
--Fig. 2
Opening in
mannoprotein layer
Exposed
glucanlayer
ofOuterwall f ~
yeast cell
Lysing yeast cell showing wall structure.
been purified and characterized 13'*.
Jeffries* found four different fi(1-3)-
glucanases with distinct action pat-
terns in batch culture with autolysed
yeast cells as inducer: Scott and
Schekman z3, using a different strain,
found two synergistic activities in
batch culture with yeast glucan as the
carbon source; an endo-lytic
glucanase and an alkaline protease.
In general, in the enzyme systems
which lyse yeast, the optimum pH
values for the constituent enzymes
are markedly different, for glucanase
it is usually neutral whereas for
protease it is alkaline in most cases.
Lysis of bacteria
Bacteriolytic enzymes tend to have
pH optima around 6 or 7. Optimum
temperature ranges from 30 to 60C
with most between 35 and 40C. At
temperatures 5-10C above optimum
temperature, the stability of the
enzyme is usually low and the rate of
denaturation is high. Most of the
enzymes are active, not on whole,
live cells but on pretreated cells (e.g.
lyophilised or heat killed) or on
isolated cell walls. Many enzymes
are specific for Gram-positive bac-
teria and few are active on Gram-
negative cells. However, the lytic
protease of Micromonospora was
active against lyophilised cells
of Serratia marcescens, Pseudo-
monas aeruginosa, E. coli and Bacil-
lus subtilis 23 and the enzyme pro-
duced by Streptomyces coelicolor
lyses cells of both Gram-positive and
Gram-negative bacteria 25. The lytic
protease from Bacillus subtilis can
lyse cells of E. coli apparently with-
out the need for pretreatment 24. The
enzyme system from Cytophaga sp.
has been used for lysis of E. coli cells
in the presence of detergent (un-
published results from the authors'
laboratory).
TIBTECH - OCTOBER1987[Vol.5]
Openinginglucanlayer
Plasmamembrane
surface
" ~
(TakenfromHunterandAsenjo5.)
Methods of production
The production of bacteriolytic
enzymes has been studied mainly for
possible use in food preservation and
investigation of bacterial cell wall
structures. Almost all of the work has
been done in batch culture and early
studies indicated that enzyme syn-
thesis was non-inducible 22'29. How-
ever, in these studies a complex
nitrogen and carbon source was used
(bean cake extract), a component of
which might have induced bacterio-
lyric activity.
The production of enzymes able to
lyse whole yeast cells has been
studied in batch and continuous
culture. The synthesis of the lyric
enzymes of Cytophaga 9497 in batch
culture appeared to be constitutive 3.
Recent work on the regulation of cell
lytic enzyme synthesis in Cytophaga
NCIB 9497 and Oerskovia xanthineo-
Iflica included both batch and
continuous culture studies 19'*. For
both strains, the synthesis of lytic
enzyme systems is inducible and
subject to catabolite repression by
glucose. At low dilution rates in
carbon limited continuous culture,
high ~(1-3)glucanase activities and a
high glucanase/protease ratio are
obtained in both strains, at high
dilution rates all enzyme activities
are similar to batch values. For both
systems, continuous culture pro-
vides a several fold increase in
enzyme concentration and product-
ivity over batch culture. This has
meant that a process for the produc-
tion of protein using yeast lyric
enzymes can be designed in which an
enzyme production fermenter of only
1.5-2 m 3 is required to lyse the yeast
cells produced in a 100 m 3 con-
tinuous culture fermenter. The med-
ium required for enzyme production
~Andrews, B. A. (1985) PhD Thesis,
University of London, UK.
TIBTECH- OCTOBER1987 [Vol. 5]
--Table 1
M i c r o b i a l lytic enzyme systems
Optimum
Optimum temperature
Source pH (C) Substrate Ref.
Oerskovia xanthineolytica/ Saccharomyces, 13-15
Arthrobacter luteus Candida, make an accurate cost comparison
A rth ro bacter GJ M - 1 Hansenula, with non-enzymic breakage methods
(Zymolyase/Lyticase) Pichia and on an industrial scale.
g lucanase 5-6.5 45-50 other yeasts The enzyme system from Cyto-
protease (alkaline) 9-10 35 phaga sp. has been efficiently used
whole yeast cell activity 7.5 30-35 for the lysis of bacterial cells. The
Oerskovia CK activity of the Cytophaga sp. system
glucanase with 35-40 S. cerevisiae 16 was approximately one order of
proteolytic activity 60 magnitude higher than that of other
whole yeast cell activity 9.0 35-40 bacteriolytic strains. Process design
Rhizoctonia sp. calculations similar to those carried
glucanase 5.5 55-60 Candida, 18 out for yeast cell lysis have shown
protease 6.5 40 Saccharomyces, that the enzyme production fer-
whole yeast cell activity 6.0 40 Hansenula menter w o u l d be 1.5-2% of the
Cytophaga NCIB 9497 9.0 45-55 S. cerevisiae, 19 volume of the cell production vessel
Bacillus, in batch enzyme production and
Corynebacteria, if continuous culture is used, only
E. coli 0.4-0.5%; in other words, a 42 1
Lysozyme fermenter could produce sufficient
(hen egg-white) 6-7 35 E. coli enzyme for a cell production unit of
M. lysodecticus 10 m 3 (Ref. 20).
and other
bacteria
Products of lysis
Cytophaga B-30 9.5 50 Staphilococcus a
Ideally, the cell debris would be
(Lysopeptase)
particulate and the protein product to
Staphylococcus sp. 6-7 37 M~rococcus 21 be recovered would be larger than the
luteus
lytic enzyme protein. Then cell
Streptomyces globisporius 6.5 50 M. lysodecficus 22 debris separation could be achieved
(N-acetylmuramidase) Streptococcus by using a centrifuge or microporous
(Mutanolysin) membrane filter and product purifi-
Micromonospora sp. Nr. 152 11 60 E. coil 23 cation and separation from the lytic
(lytic protease) S. marcescens enzyme would be achieved by a
P. aeroginosa
B. subtilis
'classic' chromatography sequence
(e.g. ion exchange followed by gel
Bacillus subtilis 797 7.8-8.5 30 E. coil 24 filtration). Some lytic enzymes have a
(lytic protease) strong affinity for yeast glucan and
aMiles Technical Information (1984)lysopeptidase. wall debris 2a, which w o u l d allow a
substantial fraction of the lytic
enzymes to be separated along with
the cell debris.
in this process is only 0.25% of the lysis of B. subtilis cells at a concen-
medium necessary for yeast produc- tration of 9-13 g 1-1 using the en- Process design
tion 31. Cloning the genes for the lytic zymes produced by Cytophaga sp. Tailoring enzyme systems for a
enzymes could result in a several fold With yeast cells, concentrations of particular use and manipulating
increase in activity of the enzyme up to 110 g 1-1 dry weight have been process conditions introduces a con-
systems in the producer strains. used in disruption reactors. With siderable degree of specificity to
lysozyme it was estimated that using cell disruption and product release.
H o w are lyric enzymes used? 4000-5000 U 1-1, good protein ex- Since the pH and temperature optima
The cells to be lysed are usually traction could be obtained from of the crucial enzymes needed for cell
harvested from the fermenter by bacterial cells in one hour. Com- breakage can be very different, pH or
centrifugation, ultrafiltration or mercial enzyme preparations contain temperature changes could provide,
microporous filtration. They are 14 000-22 000 U g-1. for example, high protease activity in
mixed in a lysis reactor with the lytic The only lytic enzyme available on the early part of the lysis reaction and
enzymes and buffer. Typically, the a commercial scale for the industrial high glucanase and low protease
enzyme will be added as a crude disruption of cells is lysozyme activities at a later stage 32. Protease
supernatant at a concentration of 3 - (active only on bacterial cells and inhibitors (e.g. mannan) could also be
30% v/v (which corresponds to 0.12- specific activity as stated above); used to control the process35~ Even-
1.2 g protein 1-1 in the enzyme other bacteriolytic and yeast lytic tually, it might be possible to
reaction) or as low as 0.4% v/v if the enzymes (e.g. Zymolyase) are only improve control by using genetic
enzyme is produced in continuous available as laboratory reagents, so manipulation to develop an organism
culture 15. These values are for the with present data it is not possible to to produc e its own inducible lytic
 TIBTECH- OCTOBER1987[Vol. 5]
- Fig. 3

1
M ] Lytic
enzymes E

~-'~ ,~,Cellsr~ ,~, r ~ - [ ,~, r~ ,~, Organelle

enzyme system: this option is being I-erm' ~ /~ ~ ~ 1 ~ ~ , 1 . ) - ~ 1 ~-~ ~ product
explored.
Yeast cells have a double layer wall Protein particle
and by choosing an enzyme system Ex.Prod. Wall Cytoplasmic
with a higher glucanase or higher enzymes enzymes
lytic protease or a combination
of different glucanases (producing
glucan oligomers of different sizes), Process for the lysis of microbial cells including sequential disruption for
the rate of release of final product and selective product release. A, wall lysis in osmotic support; B, protoplast
disruption; C, lysis of organelles or protein particles. RM, raw materials; I,
byproducts as well as its quality and inoculu[;n; Ferm, fermenter; Ex. Prod., extracellular product; E, enzyme of
composition can be engineered. Ex- reagent used to lyse organelle or renaturation of protein. (Taken from Asenjo
amples of this are the release of and Andrews12).
invertase from cell walls, the pro-
duction of protein-flee glucan and
the production of glucan oligomers shown in Table 2. These include a selectively release products from the
with pre-specified characteristics 12. several fold increase in yield in cell is important.
If the enzymes of the yeast lytic preparation of a soluble glucan It appears that continuous culture
systems are purified it should be polysaccharide (12-15 fold with is highly advantageous for the pro-
possible to first treat cells with lytic bakers yeast) 1, the extraction of duction of lytic enzyme systems both
protease then remove or inactivate lipids a4 and the extraction of human to overcome catabolite repression
the protease and degrade the wall serum albumin made by genetically and to design systems with desired
with lytic glucanase 12. In osmotic engineered yeast cells 33. Other im- enzyme composition: enzyme con-
solution the intracellular osmotic portant applications of cell lytic centrations can be increased many
pressure would not break the peri- enzyme systems are cell killing for fold and the profile of enzyme
plasmic membrane thus allowing the microbial containment and the selec- components can be manipulated.
degradation and depolymerization of tive lysis of mixed microbial popula- Lytic systems with extremely high
glucan only. Gentle agitation, or tions which cannot be achieved by activities can thus be obtained mak-
another means of protoplast break- mechanical means. ing large scale application of this
age, would then allow release of technology feasible in the near
intracellular material (Fig. 3) 12. It has Conclusions future. Specific enzyme activities of
been found that in most cases wall Enzymatic methods of cell lysis lytic systems are high and thus
lytic proteases are very specific with can be highly specific in terms of the should mean that the cost of large
little or no activity on intracellular microorganism lysed and the product scale enzymatic lysis is very reason-
proteins 33. released. These two variables will able. Cloning of the genes for lytic
determine the activity profile of a enzymes in producer strains should
Applications lytic enzyme system to be used in a further decrease the cost of this
Bacteriolytic enzymes are already particular application. Mechanical technology. Lytic enzyme technology
used commercially on a large scale techniques for cell disruption are shows great promise for the isolation
for the release of intracellular and effective but highly non specific. The of high value subcellular fractions of
membrane bound enzymes and anti- concept of a biochemical cell refinery some large intracellular recombinant
biotics 12. Some of the applications of where enzymatic, physico-chemical proteins that cannot be secreted by
microbial lytic enzyme systems are and/or genetic techniques are used to the cell, and for other specialized
applications.
--Table 2
New and future developments
Present and potential applications o f microbial lyric enzymes
should focus on understanding the
Ref. mechanism by which whole micro-
bial cells and subcellular fractions
Preparation of protoplasts, cell fusion and transformation of yeast 26 are enzymatically cleaved. This will
Production of intracellular enzymes 2 allow the optimization of product
Pretreatment to increase yeast digestibility 18 extraction particularly in those cases
Preparation of soluble glucan polysaccharide 1
Alkali extraction of yeast protein 18 where protein secretion cannot be
Pretreatment for Dyno-mill mechanical breakage of cells 18 obtained. It will also result in the
Extraction of specialized lipids from yeast 34 design of new processes for the
Production of yeast extracts 18 specific extraction of intracellular
Food preservation 35 proteins.
Extraction of pigments from red yeast 17
Release of recombinant proteins e.g. human serum albumin 33 References
Ethanol recovery from spent brewers yeast a 1 Jamas, S., Rha, C. K. and Sinskey, A. J.
Lysis of caries inducing microorganisms 36 (1986) Biotechnol. Bioeng. 28, 769-
aKrauss (1985) pers. commun. 784
2 Er-A1, Z., Klein, D., Buttat, E. and
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Zorner, E. (1984) Third Eur. Cong.
Biotechnol. (Vol. I), 1-629
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4 Valenzuela, P., Colt, D., Medina-
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Burke, R. L., Bull, P., Urdea, M. S. and
Graves, P. V. (1985) Bio/Technology,
3, 323-326
5 Hunter, J. B. and Asenjo, J. A. (1986)
in Separation, Recovery and Purifi-
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(Asenjo, J. A. and Hong, J., eds), pp. 9-
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6 Schnaitman, C. A. (1971) J. Bacteriol.
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7 Kobayashi, R., Miwa, T., Yamamoto,
S. and Nagasaki, S. (1980) J. Ferment.
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9 Reichenbach, H. and Dworkin, M.
(1981) in The Prokaryotes (Starr,
M. P., Stolp, H., Truper, H. G., Balows,
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M. P., Stolp, H., Truper, H. G., Balows,
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Newman, M. G., Socransky, S. S.,
Savitt, E. D., Propas, D. A. and
Crawford, A. (1976) J. Periodontology,
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Bacteriol. 142, 414-423
14 Kitamura, K. (1982) Agric. Biol.
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16 Obata, T., Iwata, H. and Namba, Y.
(1977) Agric. Biol. Chem. 41, 2387-
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V. E., Laurenova, G. I., Kislukhina, 32 Liu, L. C., Prokopakis, G. J. and
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J.B. and LeCorre, S. (1985) Process Noyes Data Corporation
[] [] [] [] [] [] [] [] [] [] []
Expression, glycosylation
and secretion of fungal
hydrolases in yeast
Hajirne Yoshizumi and Toshihiko Ashikari
Although there are now some systems for transfer and expression of
fungal genes, none gives expression at a level useful for production.
There are useful expression systems in yeast, however, and these have
been used to express genes coding for fungal extracellular hydrolases.
This review examines how properties of the genes and gene product
affect production and secretion of the enzyme in yeast. Similar
considerations apply to expression of other heterologous genes in
yeast and in other hosts.
Various h y d r o l a s e s used in i n d u s t r y f i l a m e n t o u s fungi has r e c e n t l y pro-
p r o d u c e d b y filamentous fungi offer gressed and some gene transfer
attractive targets for industrial gene systems for filamentous fungi have
technologists. R e c o m b i n a n t DNA b e e n r e p o r t e d 1-3. These have not yet
t e c h n o l o g y is e x p e c t e d to i m p r o v e r e a c h e d the stage of practical applica-
the p r o d u c t i v i t y , stability and sub- tions. On the other h a n d m a n y
strate specificity of such enzymes. foreign proteins of various origins
A l t h o u g h the m o l e c u l a r genetics of have already b e e n e x p r e s s e d in
the yeast, Saccharomyces cerevisiae,
H. Yoshizumi and T. Ashikari are at some of t h e m at a high level 4-7. Genes
the Laboratories of Applied Micro- of fungal extracellular proteins have,
biology, Research Center, Suntory Ltd., therefore, b e e n i n t r o d u c e d into yeast
1-1-1 Wakayama-dai, Shimamoto-eho, and their e x p r e s s i o n and secretion
Mishima-gun, Osaka, Japan. studied. In this article, w e r e v i e w the
(~) 1987, Elsevier Publications, Cambridge 0166-9430/87/$02.00