20 Extracellular Hydrolytic Enzymes Produced

by Moderately Halophilic Bacteria

E. Mellado, C. Sánchez-Porro, S. Martín, A. Ventosa

20.1
Introduction

High salinity is the main characteristic of hypersaline habitats, in which the

salt concentration is generally higher than that of seawater. Among the best-
studied hypersaline environments are the saline lakes (Dead Sea, Great Salt
Lake), salterns used for the production of salt and some saline soils. Except for
a few eukaryotic organisms such as the brine shrimp (Artemia salina) or the
photosynthetic flagellate Dunaliella, most organisms adapted to live in these
hypersaline environments are prokaryotic microorganisms belonging to the
groups of archaea and bacteria (Rodríguez-Valera 1993). The salt require-
ments divide these populations of prokaryotic halophilic microorganisms
into two predominant physiological groups: extreme halophiles, which grow
optimally in media containing 15–30 % NaCl, and moderate halophiles, which
are able to grow optimally in media containing between 3 and 15 % NaCl.
Highly saline environments are dominated by extremely halophilic archaea,
mostly halobacteria. However, in the intermediate salinities, the most abun-
dant microorganisms are the moderate halophiles, a heterogeneous group
which includes very different Gram-negative and Gram-positive bacterial
species, as well as some archaea (Ventosa et al. 1998).
Although halophilic microorganisms have attracted much attention in
recent years, most studies have been performed in halobacteria. However,
moderately halophilic bacteria represent an excellent model of adaptation to
frequent changes in extracellular osmolality and constitute an interesting
group of microorganisms from a biotechnological point of view (Ventosa et
al. 1998). Thus, many of them accumulate intracellular organic osmolytes
named “compatible solutes” which can be used as stabilizers of enzymes and
whole cells (da Costa et al. 1997; Ventosa et al. 1998; Nieto et al. 2000), and they
produce halophilic exoenzymes that could be of commercial interest and
could be used in biodegradation processes (Ventosa et al. 1998). They have the
advantage that most species are able to grow in a wide range of salinities, in
contrast to the more strict requirements of salt presented by halobacteria.

A. Ventosa (Ed.)
Halophilic Microorganisms
© Springer-Verlag Berlin Heidelberg 2004
286 E. Mellado et al.

Thus, their euryhaline response would permit their use in processes in which
the salt or metallic ion concentrations are variable, and change from very low
to almost salt saturation.
Information on halophilic bacterial hydrolase genes is mainly restricted to
archaea, where a variety of hydrolases have been characterized (Oren 1994;
Margesin and Schinner 2001). Since moderately halophilic bacteria are wide-
spread in halophilic environments, in recent years the isolation and charac-
terization of hydrolytic enzymes produced by these microorganisms have
acquired enormous interest. The running of industrial processes at high salt
concentrations requires the availability of hydrolases showing optimal activi-
ties at such elevated salinity. The addition of such enzymes in laundry and
dishwashing detergents has been of great importance. The treatment of agri-
cultural waste and wastes from food processing industries constitutes other
areas of interest for halophilic hydrolytic enzymes. The use of hydrolases such
as amylases, proteases, lipases, DNAses, pullulanases, cellulases and xylanases
isolated from moderately halophilic bacteria in these industrial processes
could have the advantage of their optimal activities at different values of salt
concentrations, required in some of the bioprocesses. Furthermore, hydroly-
sis of high-molecular-weight biopolymers by hydrolases is an essential first
step in the degradation of organic material in nature. In this chapter we
review the most important characteristics of the hydrolases from moderately
halophilic bacteria characterized in recent years, highlighting their potential
biotechnological applications.

20.2
Moderately Halophilic Bacteria as a Source
of Extracellular Enzymes

The pioneering studies of Onishi and Kamekura on enzymes produced by

moderately halophilic bacteria permitted the characterization of some extra-
cellular enzymes (Kamekura 1986). Although only few hydrolases from this
group of halophiles have been purified and characterized in detail, the pro-
duction of these enzymes seems to be widely distributed among this group of
extremophiles. A recent study of isolates obtained from hypersaline habitats
in southern Spain revealed a wide extent of diversity of moderately halophilic
bacteria endowed with the potential to hydrolyze a rather large range of struc-
turally non-related polymers. In contrast, the reference strains obtained from
culture collections tested showed a very limited activity as producers of these
enzymes (Sánchez-Porro et al. 2003a). As shown in Table 20.1, most isolates
were identified as members of the genera Salinivibrio (55 isolates), Bacillus-
Salibacillus (29 isolates), Halomonas (25 isolates), Chromohalobacter (2 iso-
lates), Salinicoccus (2 isolates) and Marinococcus (1 isolate). Combined
hydrolytic activities have been detected in a number of strains. The possibility
20 Extracellular Hydrolytic Enzymes Produced by Moderately Halophilic Bacteria 287

Table 20.1. Taxonomic identification of the 122 environmental isolates able to produce
different hydrolytic enzymes. (From Sánchez-Porro et al. 2003a, with permission)

Genus Amylase DNAse Lipase Protease Pullulanase Total

Salinivibrio 11 9 3 16 16 55
Halomonas 4 3 10 2 6 25
Chromohalobacter 0 0 2 0 0 2
Bacillus-Salibacillus 7 8 5 5 4 29
Salinicoccus 0 0 1 1 0 2
Marinococcus 0 0 1 0 0 1
Non-identified 2 1 1 2 2 8
Total 24 21 23 26 28 122

of obtaining a wide variety of moderate halophiles producing extremozymes

would be of invaluable help for biotechnological applications.
A nuclease from Micrococcus varians subsp. halophilus able to hydrolyze
RNA and DNA exonucleolytically producing 5¢-mononucleotides has been
characterized. The enzyme presents a molecular weight of 99,000 Da and the
enzyme production is maximal in media containing 15–20 % salt (Kamekura
and Onishi 1978, 1983). This enzyme has an interesting application in the pro-
duction of the flavoring agents 5¢-inosinic acid and 5¢-guanylic acid
(Kamekura et al. 1982).
Another nuclease from Bacillus sp. strain N23-2 has been characterized
(Onishi et al. 1983). This enzyme presents an optimal activity at 2–3 M NaCl
or KCl for the hydrolysis of DNA and RNA. The estimated molecular weight of
this nuclease is 138,000 Da. This moderately halophilic bacterium was able to
grow optimally at 15 % salts and was characterized as a new species of the
genus Bacillus, B. halophilus (Ventosa et al. 1989).

20.2.1
Glycosyl Hydrolases: Amylases

Henrissat (1991) proposed a classification for glycosyl hydrolases, based on

amino acid sequence data and, when available, on structural information.
Currently, more than 60 families of glycosyl hydrolases have been described.
Among the polysaccharides produced by plants, starch, xylane and pullulane
are the most abundant. Except xylanases, all other hydrolases able to degrade
these polymeric glucosidic substrates have been detected among the moder-
ately halophilic bacterial community present in different salterns from south-
ern Spain (Table 20.1). Although xylane is a very abundant polysaccharide in
nature, xylanase enzymes have not yet been detected, suggesting a poor
xylanolytic activity in the hypersaline environments studied.
288 E. Mellado et al.

Although numerous glycosyl hydrolases from non-halophilic micro-

orgnisms have been described according to Henrissat’s classification, only a
few amylases from moderately halophilic bacteria have been characterized so
far. The use of halophilic glycosyl hydrolases in bioprocesses presents the
advantage to obtain optimal activities at high salt concentrations.
Two different amylases from a moderately halophilic Acinetobacter sp. iso-
lated from sea-sand were purified and characterized (Onishi and Hidaka
1978). Both enzymes presented optimal activity at pH 7.0 in 1.5–3 % NaCl or
KCl at 50–55 °C. The purified amylases had a molecular mass of 55,000 and
65,000 Da, respectively, and the hydrolysis products were maltose and mal-
totriose. Nesterenkonia halobia, a moderately halophilic Gram-positive coc-
cus, described by Onishi and Kamekura (1972) as Micrococcus halobius and
reclassified by Stackebrand et al. (1995) as a member of the genus Nesterenko-
nia, showed amylase activity in the supernant. The purified amylase was opti-
mally active at pH 6–7 in 1.4–2 M NaCl or KCl and a temperature of 50–55 °C.
More recently, Mota et al. (1997) isolated six moderately halophilic strains,
classified as N. halobia, from ponds of a saltern located in Huelva (Spain)
which are able to degrade starch. However, the features of these amylases have
not been described to date.
Amylolytic activity has also been observed in Micrococcus varians subsp.
halophilus. The purified enzyme presented two components with molecular
masses of 86 and 60 kDa, with optimal activity at 4.5–6 % NaCl or KCl and pH
6–7. The optimal temperature for enzymatic activity is between 55 and 60 °C,
depending on the presence or absence of CaCl2 in the reaction buffer. The
salinity of the growth medium exerts a significant influence on the amylase
activity, being optimal when grown at 11 % NaCl (Kobayashi et al. 1986). Two
other Micrococcus isolates have been shown to produce amylases with optimal
activities at 6–12 % NaCl (Onishi 1972a,b; Khire 1994).
Recently, an amylolytic enzyme from the cytoplasmic fraction of Halo-
monas meridiana has been partially purified and characterized (Coronado et
al. 2000a). The enzyme is optimally active at 37 °C and pH 7.0, being relatively
stable under alkaline conditions. The enzyme exhibited maximal activity at
10 % NaCl, although activity at a salinity as high as 30 % salts was detected.
The main products resulting from the hydrolysis of starch were maltose and
maltotriose. The salinity of the growth medium strongly influenced the amy-
lase activity of H. meridiana, thus, for optimal amylase activity, H. meridiana
cultures should be grown in media with 5 % salts. The a-amylase is produced
constitutively at low levels and the addition of starch induces the synthesis of
higher levels of enzyme. Glucose, on the other hand, represses the synthesis of
a-amylase by catabolic repression.
The gene encoding this a-amylase (AmyH) was cloned by functional com-
plementation of a Tn1732-induced mutant deficient in extracellular amylase
activity (Fig.20.1; Coronado et al.2000b).AmyH encodes a 457-residue protein
with a molecular mass of 50 kDa assigned to family 13 of Henrissat’s classifica-
20 Extracellular Hydrolytic Enzymes Produced by Moderately Halophilic Bacteria 289

tion of glycosyl hydrolases (Henrissat and Bairoch 1993) and containing the
four highly conserved regions in amylase enzymes (Nakajima et al. 1986). As
shown in Table 20.2,the numbers of acidic residues present in the amylase from
the moderately halophilic bacterium H. meridiana as well as in other halophilic
enzymes are higher than those in non-halophilic enzymes. This fact is related
to the adaptations involved in halophilic organisms (Coronado et al. 2000b).
Due to the importance of the potential applications of amylases, this
enzyme has been cloned and expressed in the heterologous hosts Escherichia
coli and Halomonas elongata. This is the first enzyme from a moderately
halophilic bacterium that has been cloned and expressed in a non-halophilic
host. Apart from their biotechnological interest, the characterization of
genes encoding amylase activity will be of invaluable help in elucidating
their regulatory mechanisms, and the structure–function relationship of
extracellular enzymes with optimal activity at high salt concentrations.

Fig. 20.1. A Isolation of the DNA flanking the left-end of the transposon Tn1732 in the
amylase-defective mutant II of H. meridiana. B Restriction maps of the overlapping plas-
mids pMJC21 to 23, isolated by functional complementation of the Amy– mutant II.
Below,the common 10 kb HindIII region carrying the amylase synthesis gene of H. merid-
iana (amyH) cloned in two different vectors, and the plasmids pHS182 and pHS183 used
for sequencing, are shown. The ability of the plasmids to confer amylase activity to H.
meridiana mutant II, H. elongata and E. coli is indicated on the right (+/–, extracellular
amylase activity on SW-2 plates, or growth on M63 medium plus starch as the sole carbon
source; N.D. not determined). Restriction sites: B,BamHI; D, DraI; E, EcoRI; EV, EcoRV; H,
HindIII; P, PstI; Sc, SacI. (From Coronado et al. 2000b, with permission)
290 E. Mellado et al.

Table 20.2. Amino acid composition, charge, and theoretical isoelectric point of several
amylases and one serine protease from halophilic and non-halophilic microorganisms.
(From Coronado et al. 2000b, with permission)

Microorganism Extracellular Acidic Basic Net pI

enzyme residues residues charge
(%) (%)

Halomonas meridianaa Amylase 12.4 5.5 –32 4.65

Alteromonas haloplanktis Amylase 7.9 8.4 +2 8.12
Aeromonas hydrophila Amylase 9.5 8.6 –2 6.43
Bacillus sp. Amylase 7.2 6.2 –14 6.20
Thermomonospora curvata Amylase 9.2 7.6 –10 6.04
Streptomyces griseus Amylase 9.7 7.8 –11 5.63
Natronococcus sp.a Amylase 22.6 6 –81.5 4.12
Natrialba asiaticaa Serine protease 11.5 4.7 –49 4.11
a Halophilic microorganisms.

On the other hand, moderate halophiles could serve as cell hosts for the
production of heterologous proteins of biotechnological importance. The
physiological properties of this group of bacteria, such as their simple nutri-
tional requirements or their ability to grow optimally at high salt concentra-
tions preventing the contamination risks, make them interesting hosts for the
industrial production of enzymes (Ventosa and Nieto 1995; Ventosa et al.
1998). In this sense, the heterologous expression of the Bacillus licheniformis
a-amylase gene in the moderate halophiles H. meridiana and H. elongata has
been reported (Coronado et al. 2000b). The recognition and correct process-
ing of the signal peptide of the Bacillus a-amylase by Halomonas lead to the
potential use of species of this halophilic genus for the expression and secre-
tion of enzymes.
Moreover, an extracellular a-amylase gene from the hyperthermophilic
archaeon Pyrococcus woesei has been expressed in the moderate halophile
Halomonas elongata, under the control of a native H. elongata promoter.
Although the pyrococcal a-amylase was not released to the extracellular
medium, the thermal stability, metal ion interactions, optimal temperature
and pH values for the recombinant a-amylase isolated from the halophilic
host were comparable with those of the native pyrococcal enzyme (Frillingos
et al. 2000).

20.2.2
Proteases

Proteases constitute an important group of enzymes with the potential ability

to catalyze the hydrolysis of proteins. Proteases assist the hydrolysis of large
20 Extracellular Hydrolytic Enzymes Produced by Moderately Halophilic Bacteria 291

polypeptides into smaller peptides and amino acids. The most commercially
important field of application for hydrolytic proteases is their addition to
detergents, which are used mainly in household and industrial laundry and in
household dishwashers. A large number of additional applications have been
described for proteases, including leather treatment, bioremediation pro-
cesses or preparation of drugs in the pharmaceutical industry. Before the
studies performed by our research group, only an extracellular protease pro-
duced by an unidentified moderately halophilic bacterium, designated
Pseudomonas sp. strain A-14, was purified. The molecular weight of this
enzyme was estimated to be 12,000 Da, the optimum pH for activity was 8.0,
and the enzyme presented maximal activity at 18 % NaCl concentration (Van
Qua et al. 1981).

A Relative protease activity

120

100

80
(%)

60

40

20

0
5.5 6 6.5 7 7.5 8 8.5 9 9.5 10

pH

B
Relative protease activity

120

100

80
(%)

60

40

20

0
25 35 45 55 65 75

Temperature (ºC)

Fig. 20.2. Effect of pH (A), temper- C

ature (B), and NaCl concentration
(C) on the activity of the protease
Relative protease activity

120

CP1 produced by the moderately 100

halophilic bacterium Pseudoal- 80

teromonas sp. strain CP76. The rel-
(%)

60
ative protease activity is defined as 40
the percentage of activity detected 20
with respect to the maximum pro- 0
tease activity detected in the enzy- 0 0.5 1 1.5 2 2.5 3 3.5 4
matic assay. (From Sánchez-Porro NaCl (M)
et al. 2003b, with permission)
292 E. Mellado et al.

In a recent study, the protease CP1 produced by the moderately halophilic

bacterium Pseudoalteromonas sp. strain CP76 has been purified and charac-
terized in detail (Sánchez-Porro et al. 2003b). The enzyme is a homodimer
with a subunit size of 38 kDa. The enzyme is moderately thermophilic, pre-
senting optimal activity at 55 °C, at pH 8.5. An interesting feature of this pro-
tease is its salt tolerance over a wide range of NaCl concentrations (0–20 %
NaCl; Fig. 20.2). These characteristics make the protease CP1 interesting for
its application in biotechnological processes. The protease activity was inhib-
ited by EDTA, PMSF and Pefabloc. No significant inhibition was detected with
E-64, bestatin, chymostatin or leupeptin. According to these results and the
sequencing of the amino terminal region of the purified enzyme, the protease
CP1 has been classified as a serine metalloprotease. In order to improve the
production of the protease CP1 for industrial applications, the growth condi-
tions of Pseudoalteromonas sp. CP76 for optimal protease activity were stud-
ied. The production was optimal in a saline medium containing 7.5 % NaCl,
supplemented with sucrose, fructose and glycerol, whereas activity decreased
drastically when the organism was grown in the presence of maltose and
ammonium chloride. This study constitutes the first report on the purifica-
tion and in-depth characterization of a proteolytic enzyme from a moderately
halophilic microorganism. Further studies on the cloning, sequencing and
molecular characterization of this enzyme are in progress.

20.3
Future Prospects

Hypersaline environments constitute an excellent opportunity for harvesting

useful industrial enzymes. Although studies concerning both aspects, the
molecular characterization of enzymes from halobacteria and the halophilic
adaptations of these proteins, have been performed during last decade, the
studies of extracellular enzymes produced by moderately halophilic bacteria
are still in their infancy. Some of the most interesting hydrolase classes for use
in industrial biotransformations have been detected in this group, but only a
few extracellular enzymes have been characterized for future use at the indus-
trial level. The isolation and molecular characterization of new enzymes from
this group of extremophiles is a fascinating area of research for the next
decade.
The classical and cumbersome approach to isolate new hydrolytic enzymes
is to screen a wide variety of microorganisms for the desired hydrolytic activ-
ity. The enzymes and the corresponding genes are then recovered from the
identified microorganisms. However, it has been estimated that a large per-
centage (between 90–99 %) of microorganisms in nature cannot be cultivated
using standard techniques (Amann et al. 1995). An alternative approach to
exploit the diversity of halophilic environments for detection of hydrophilic
20 Extracellular Hydrolytic Enzymes Produced by Moderately Halophilic Bacteria 293

enzymes is to isolate DNA without culturing the organisms present and to

clone directly functional genes from environmental samples. An important
goal in the future will be to compare the hydrolase genes of cultured and
uncultured bacteria and to examine the relationship between the phylogeny
of these hydrolase genes. Genes encoding hydrolases may be particular inter-
esting examples of non-essential genes in uncultured bacteria.
Esterases and lipases are the most widely used biocatalysts in fine chemical
applications due to the advantages of these reactions for the production of
optically pure compounds. The characterization of these enzymes from mod-
erately halophilic bacteria will permit the performance of the aforementioned
catalytic reactions in a wide range of salt concentrations.
The availability of halophilic hydrolases in significant amounts for biopro-
cessing evaluations could be improved in the future using recombinant tech-
niques; on the other hand, the construction of new recombinant enzymes by
site-directed mutagenesis of specific residues in order to increase the activity
and/or stability will allow the use of these enzymes in different commercial
applications. In the last decade, diverse genetic tools for moderate halophiles
have been developed (Ventosa et al. 1998), facilitating their genetic manipula-
tion that will make possible the development of economical bioprocesses of
industrial interest.
The identification of the different intrinsic structural factors contributing
to the stability of moderately halophilic enzymes to the harsh environment in
which they must function and the elucidation of the mechanisms by which
osmotic solutes stabilize enzymatic activity will contribute to the molecular
understanding of halophilic activity of enzymes from this group of
extremophiles.
Furthermore, the resolution of the crystal structures of some of the mod-
erately halophilic enzymes characterized will help the understanding of the
specific interactions between salt ions, water molecules and the polypeptides,
which determine the strategy of haloadaptation of this group of proteins.

Acknowledgements. We thank the Spanish Ministerio de Ciencia y Tecnología (PB98-

1150 and 1FD97-1162) and Junta de Andalucía for financial support. C. Sánchez-Porro
and S. Martín were supported by fellowships from the Spanish Ministerio de Ciencia y
Tecnología and Junta de Andalucía, respectively.

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