Polaina J., MacCabe A.P. (ed.). Industrial Enzymes. Structure, Function and Applications
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350 JOSÉ DA CRUZ FRANCISCO ET AL.
water on the enzyme. Colombié and co-workers (Colombie et al., 1998) have
proposed controlling thermodynamic water activity in the fixed bed reactor during
continuous esterification of oleic acid with ethanol in order to maintain a high and
constant conversion. In continuous esterification in scCO
2
the water released as a
reaction product is continuously removed by the scCO
2
stream keeping the necessary
water activity for the enzyme almost unchanged over the reaction process. While the
water removal drives the esterification to completion, for transesterification this is
not so. In transesterification reactions water is produced. Water has some solubility
in scCO
2
(Wiebe et al., 1941; King et al., 1983; Francisco et al., 2003b) therefore
in continuous transesterification the enzyme is dried by the scCO
2
and consequently
inactivated. This problem has been solved by saturating glyceryltrioleate with water
prior to the interesterification with caprylic acid (Bister, 2004). The addition of
water during esterification reactions has been earlier reported earlier for batch and
continuous trials in both n-hexane and scCO
2
(Dumont et al., 1991; Dumont et al.,
1992; Bernard and Barth, 1995).
The effect of flow rate on the product quality of a reaction was illustrated by
Gunnlaugsdottir and Sivik (Gunnlaugsdottir and Sivik, 1997) for the continuous
lipase catalyzed transesterification of cod liver oil with ethanol. In a continuous
reaction by selectively extracting the desired product or by-product, the higher the
flow rate, the higher the recovery of the ethyl esters. Such behavior is due to the
fact that the reaction mixture is exposed to a higher volume of extraction fluid for
a set period of time.
The continuous transesterification of natural occurring vegetable oils to obtain
structured oils faces other challenges. Because natural occurring oils are mixtures
of different triacylglycerols the range of products yielded is wide and the overall
yield of the desired compounds is reduced. On the other hand, the purification
of the reaction product is rendered complex. One possibility to overcome such
constrain is to develop a reaction/fractionation process in a system with a series of
cyclone separator downstream or perform the fractionation in a fixed bed column
with reflux. The use of a series of cyclone separator system was advanced by Knez
and collaborators (Knez and Habulin, 1992 and 1994; Knez et al., 1995). This
represents one of the present limitations and challenges in the use of scCO
2
for
producing structured lipids and the challenge.
7. ScCO
2
VERSUS ORGANIC MEDIUM
The choice for the scCO
2
as the reaction medium will still be a matter of debate.
scCO
2
offer the same advantages for lipase catalysis as organic solvents namely the
solubilization of the hydrophobic lipid substrates, simple recovery of immobilized
lipase as well as the reversal of hydrolysis in favor of synthesis (Martins et al., 1994;
Gunnlaugsdottir and Sivik, 1997). scCO
2
will also support reactions thermodynam-
ically unfavorable in aqueous media (e.g. synthesis of esters and amides) (Martins
et al., 1994). Besides the advantageous scCO
2
properties pointed out earlier in this
chapter, this solvent allows combined processes reaction/extraction and fractionation
USE OF LIPASES IN SYNTHESIS OF STRUCTURED LIPIDS 351
of the products. Some drawbacks should be noted. For instance, the synthesis of
geranyl acetate was performed in scCO
2
by immobilized M. miehei lipase catalyzed
transesterification of geraniol and propyl acetate at 14MPa and 40
C. The yield of
geranyl acetate was found to be 30% after three days (Chulalaksananukul et al.,
1993). In a previous work however, the yield of the same reaction in hexane was
85% (Chulalaksananukul et al., 1992) after the same period of time. The great
difference in the reaction yield in both media is attributed to reactants and product
solubility effect (Halling, 1990). When the solvent is less polar the solvation of
the more polar OH and COOH group decline relatively faster than that of the
ester group. scCO
2
is more polar than n-hexane, thus in case of esterification the
equilibrium is favored in n-hexane where the product is less soluble.
In general the use and exploration of scCO
2
as a reaction medium will have to
be considered for each case of interest. More effort is needed until the time when
commercial plants can be constructed for structured lipids production, especially in
continuous processes in scCO
2
.
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SECTION D
NUCLEIC ACIDS ENZYMES
SECTION D
NUCLEIC ACIDS ENZYMES
CHAPTER 21
RESTRICTION AND HOMING ENDONUCLEASES
KRZYSZTOF J. SKOWRONEK AND JANUSZ M. BUJNICKI
∗
Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular
and Cell Biology, Warsaw, Poland
∗
iamb@genesilico.pl
1. INTRODUCTION: HISTORY AND BIOLOGY OF RESTRICTION
AND HOMING
1.1. Restriction-Modification Systems
In the early 1950s it was observed that certain bacteria ‘restricted’ the growth of
bacteriophages previously propagated on different strains. In the early 1960s, it
was found that the restriction is due to the cleavage of the bacteriophage DNA
by endonucleases that are specific for certain sequences and sensitive to covalent
modification of bases in the phage genome. Some of these ‘restriction endonu-
cleases’ (REases) produced discrete DNA fragments upon cleavage, a property very
useful for analyzing and recombining the DNA. This opened the door to the rapid
development of numerous genetic engineering techniques as well as prompted the
search for more REases with novel recognition sequences (reviewed early by Arber
and Linn (1969) and in the recent historical perspective (Roberts, 2005)). Thus
far, more than 3000 REases with over 250 different sequence specificities have
been isolated from various organisms (Roberts et al., 2005). The characteristics of
REases have been comprehensively reviewed in a book edited by Alfred Pingoud
(2004).
Most REases recognize short sequences (4–8 bp) in the DNA and in the presence
of Mg
2+
ions they cleave it in both strands, yielding fragments with a 3
-OH and a
5
-phosphate. Some REases produce 3
or 5
overhangs (single stranded extensions
at the ends), while others yield blunt ends. Typically, they cleave DNA at the target
sites several orders of magnitude more readily than at any alternative sequence.
REases are typically accompanied by DNA methyltransferases (MTases) with the
same or very similar (sometimes slightly broader) specificity. The MTase uses
357
J. Polaina and A.P. MacCabe (eds.), Industrial Enzymes, 357–377.
© 2007 Springer.
358 SKOWRONEK AND BUJNICKI
S-adenosylmethionine (AdoMet) as a methyl group donor to specifically modify
a particular adenine or cytosine in the common target sites in the DNA, thereby
preventing the cleavage of these sites by the REase. Methylation provides protection
of the ‘self’ DNA against the destruction. On the other hand, it prompts the
destruction of the foreign DNA without the familiar modification pattern, e.g. the
invading phage DNA (Fig. 1).
Some REases (including those that lead to the discovery of the restriction
phenomenon) are not inhibited by methylation – conversely, they attack only methy-
lated DNA (see below for the classification). Genetically linked REase and MTase
genes form the so-called restriction-modification (RM) systems, which occur ubiqui-
tously in Bacteria and Archaea and in some viruses that infect lower Eukaryota
(Roberts et al., 2005). Closely related RM genes appear in phylogenetically distinct
Figure 1. Mechanism of action of RM systems. A) Protection of the self DNA by MTase (white circle,
MT), which methylates target sites (indicated in white) and cleavage of the unmethylated sites by
REase (black ‘pacman’, RE). B) Hypothetical mechanism of ‘selfish transposition’ (Kobayashi, 2001):
Disturbed expression of both RM proteins leads to a situation when the diminished MTase activity is
insufficient to protect all sites in the genome, and the unmethylated sites are attacked by the remaining
REase. If the cleavage is not very extensive, breaks may be repaired and if the entire RM system is
reinserted properly at the new location, the expression of MTase may resume to restore the global
methylation pattern and to save the cell from death. If the RM system is not properly restored, REase
destroys the genome of the host
RESTRICTION AND HOMING ENDONUCLEASES 359
organisms and in different genomic contexts, and are often associated with mobile
genetic elements such as plasmids, viruses, transposons and integrons, which
suggests that they have been frequently transmitted by horizontal gene transfer
(Jeltsch and Pingoud, 1996; Kobayashi et al., 1999). The comparison of closely
related bacterial genomes also suggests that, at times, RM genes themselves behave
as mobile elements and cause genome rearrangements (Kobayashi, 2001). It is
commonly believed that RM systems have been maintained by the prokaryotic cells
to defend them from infection by foreign DNAs and to maintain the species identity
(review: (Jeltsch, 2003)). An alternative hypothesis suggests that RM systems
behave as selfish genetic elements that are maintained because they form parasitic
toxin-antitoxin systems whose loss leads to cell death (review: (Kobayashi, 2001).
A third hypothesis suggests that RM systems are beneficial ‘evolution genes’
that serve to increase the genetic diversity, e.g. by inducing genome rearrange-
ments (review: (Arber, 2000)). Thus, RM systems can be regarded as intracellular
parasites at the verge of mutualism, which can confer advantage in certain contexts
(Rocha et al., 2001).
1.2. Homing
The discovery of the phenomenon called ‘homing’ dates back to the early 1970s and
the observation of a significant polarity of recombination for markers of an rRNA
gene in genetic crosses between yeast mitochondria, which in the 1980s was shown
to be due to the action of an intron-encoded ‘homing endonuclease’ (HEase) (see
(Dujon, 2005) for a historical perspective and other articles in a recently published
book (Belfort et al., 2005) for comprehensive reviews of fundamental studies and
applications of HEases). Briefly, homing is a gene conversion process where a
mobile sequence is copied and inserted into a cognate allele that lacks this sequence.
It is initiated by a site-specific double-strand cut in the target allele, catalyzed by
a nuclease encoded within that mobile sequence (reviews: (Belfort et al., 1995;
Gimble, 2000)). HEases are most often encoded within protein-encoding genes,
by introns that self-splice at the RNA level (both group I and group II, and in
Archaeal introns) or inteins that self-splice at the protein level, but they can also
be free-standing, occurring between genes. Homing is widespread and HEases or
their homologs have been found in all phyla.
HEases usually recognize very long DNA sequences (14–40 bp). Unlike REases,
they exhibit significant tolerance to substitutions in the target site and effectively
recognize whole families of sequences. Nonetheless, the size of the target sites
makes them very rare – e.g. the historic I-SceI HEase has only one such site in the
whole yeast genome of ∼13,000 kb. Compared to REases, HEases pose relatively
little threat to the integrity of the host DNA, because of the relative rareness of their
target sites. Therefore, HEases do not require the assistance of MTases to protect
the self DNA by reversible methylation against the multiple rounds of cleavage.
Instead, intron- and intein-encoded HEases irreversibly disrupt the target site by
inserting the DNA of its mobile element. The insertion sites of free-standing HEases