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were compared with those of a commercial, nonrecombinant, CRL preparation containing the
various isoforms. According to Mileto et al. (1998), the lipase isoenzymes (CRLs) of the yeast C. rugosa share ca. 40% and 30% sequence homology with lipases of G. candidum and Yarrowia
lipolytica, respectively. The domain of sequence conservation occurs in the N-terminal half of
the protein. For the resolution of isoforms via heterologous expression, the lip 1 gene,
encoding the major CRL form, was expressed in C. maltosa —a related yeast with the same
codon usage as C. rugosa (Mileto et al., 1998). A recombinant lipase was thus produced and
secreted in an active form in the culture medium. Production of Pseudomonas lipases requires correct folding and secretion through the membrane. A controllable expression of the gene lip H, encoding a lipase-specific foldase, is important for overexpression of lipase in the homologous host E. coli (Reetz and Jaeger, 1998). Construction of appropriate His-tagged fusion proteins permitted overexpression, secretion, and one-step purification of lipase from culture supernatants of the homologous host P. aeruginosa.
An efficient expression system for the previously only weakly expressed thermophilic
lipase BTL-2 (B. thermoatenulatus Lipase II) has been developed for overexpression of the
lipase in E. coli (Rua et al., 1998). The gene was subcloned in the pCVT-EXP1 (pT1)
expression vector downstream of the temperature-inducible lambda promoter PL. Three
different expression vectors were constructed. The expression vectors pT1-BTL2 and pT1-pre
BTL 2 allowed comparable lipase expression levels of 7000–9000 U/g cells (Rua et al.,
1998). Using the expression vector pT1-Omp ABTL2, the soluble lipase production levels
were between 30,000 and 660,000 U/g cells, depending on the specific E. coli strain used to
express the gene (Rua et al., 1998).
In S. epidermidis RP62A, the lipase gene (geh SE1) on the chromosome is immediately
flanked by the ica AA0 BC operon, which is involved in biofilm formation (Simons et al.,
1998). This association has been claimed to suggest a possible role of lipase in staphylococcal
colonization of the skin. The DNA sequence and the deduced lipase sequence revealed that
geh SE1 is very similar to the lipase sequence of S. epidermidis strain 9 and is organized as a
preproenzyme. The part of geh SE1 coding for the mature lipase was cloned and overexpressed
as a fusion protein with an N-terminal histidine tag in E. coli (Simons et al., 1998).
The lipase was purified and was shown to be biochemically closely related to the lipase of S.
aurelis NCTC 8530 (Simons et al., 1998). van Kampen et al. (1998) used site-directed mutagenesis and domain exchange to investigate the role of C-terminal domains of S. hyicus lipase (SHL) and S. aureus lipase
(SAL) in substrate selectivity. A single point mutation coding for the substitution of Val for
Ser 356 in SHL yielded an enzyme that retained full lipase activity, but with more than 12fold
lower phospholipase activity. Starting with this S356V variant of SHL, the C-terminal 40
amino acids were replaced by the corresponding SAL sequence. The resulting change in
phospholipase/lipase activity ratio showed that in the C-terminal domain, Ser 356 mainly
determines the phospholipase activity (van Kampen et al., 1998). Rhizop. niveus lipase has a unique structure consisting of two noncovalently bound polypeptides (A-chain and B-chain). To improve this enzyme by protein engineering, Kohnoet al. (1999) developed a new expression system for producing the lipase in the yeast Saccharomyces cerevisiae. The efficient expression system used the strain ND-12 B and the multicopy plasmid pJDB 219.A thermophilic lipase of B. thermoleovorans ID-1 was cloned and sequenced by Cho et al. (2000). The lipase gene coded 416 amino acid residues and contained the conserved pentapeptide Ala–X–Ser–X–Gly, as do other Bacillus lipases. For expression in E. coli, the lipase gene was cloned in pET-22b(+) vector with a strong T7 promoter (Cho et al., 2000). The lipase activity was approximately 1.4-fold greater than the activity with the native promoter. Pignede et al. (2000) isolated the lip 2 gene from the lipolytic yeast Y. lipolytica. The gene encoded a 334-amino acid precursor protein. The secreted lipase was a 301-amino acid glycosylated polypeptide (Pignede et al., 2000). The lip 2p protein is processed by the KEX 2-like endoprotease encoded by XPR6
(Pignede et al., 2000). Deletion of the XPR6 gene resulted in the secretion of an active but less stable proenzyme. The proregion did not inhibit lipase secretion and activity and played an
essential role in the production of a stable enzyme. The overexpressing strains correctly
processed the gene, secreting 100-fold more activity than the wild type (Pignede et al., 2000).
A. oryzae produces at least three extracellular lipolytic enzymes, L1, L2, and L3. Of
these, the L3 lipase (a triacylglycerol lipase) gene (provisionally designated tglA) was
cloned (Toida et al., 2000). Nucleotide sequencing of the genomic DNA and cDNA
revealed that the L3 gene (tglA) had an open reading frame of 954 nucleotides, including
three introns of 47, 83, and 62 bp. The deduced amino acid sequences of the tglA gene
implied a protein of 254 amino acid residues, including a single sequence of 30 amino acids
that was homologous to a sequence of fungal cutinases. Three residues presumed to form
the catalytic triad, Ser, Asp, and His, were conserved (Toida et al., 2000). The cloned
cDNA of the tglA gene was expressed in E. coli to encode a functional triacylglycerol lipase
(Toida et al., 2000).
11. Concluding remarks
As discussed here, lipases are versatile enzymes that are used widely. Lipases are
becoming increasingly important in high-value applications in the oleochemical industry
and the production of fine chemicals. Lipases are capable of regioselective and stereoselective
biotransformations and allow resolution of racemic mixtures. Lipases with improved
properties are being produced by natural selection and protein engineering to further enhance
usefulness of these enzymes. Simultaneously, advances are being made in bioreactor and
reaction technologies for effectively using the lipases. Various kinds of immobilized enzyme
reactors and multiphase reaction systems have greatly influenced the processes that require
catalysis by lipases (Balca
˜
o et al., 1996; Bouwer et al., 1997; Giorno et al., 1995, 1997;
Malcata et al., 1991, 1992b; Xu et al., 2000, 2001; Xin et al., 2001). Lipase-based processing
has a promising future; however, the rate of progress is slow. Factors posing limitations
include a relatively high cost of lipases and a lack of enzymes with the optimal range of
catalytic specificities and properties required in the various applications.
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