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Çàïðîñ:BACILLUS SUBTILIS LIPASE[All text]
Results:18
1. Chem Biol. 2008 Aug 25;15(8):782-9.

Loop grafting of Bacillus subtilis lipase A: inversion of enantioselectivity.

Boersma YL, Pijning T, Bosma MS, van der Sloot AM, Godinho LF, DrÃge MJ, Winter
RT, van Pouderoyen G, Dijkstra BW, Quax WJ.

Department of Pharmaceutical Biology, Groningen University Institute for Drug
Exploration (GUIDE), University of Groningen, A. Deusinglaan 1, 9713 AV
Groningen, The Netherlands.

Lipases are successfully applied in enantioselective biocatalysis. Most lipases
contain a lid domain controlling access to the active site, but Bacillus subtilis
Lipase A (LipA) is a notable exception: its active site is solvent exposed. To
improve the enantioselectivity of LipA in the kinetic resolution of
1,2-O-isopropylidene-sn-glycerol (IPG) esters, we replaced a loop near the
active-site entrance by longer loops originating from Fusarium solani cutinase
and Penicillium purpurogenum acetylxylan esterase, thereby aiming to increase the
interaction surface for the substrate. The resulting loop hybrids showed
enantioselectivities inverted toward the desired enantiomer of IPG. The
acetylxylan esterase-derived variant showed an inversion in enantiomeric excess
(ee) from -12.9% to +6.0%, whereas the cutinase-derived variant was improved to
an ee of +26.5%. The enantioselectivity of the cutinase-derived variant was
further improved by directed evolution to an ee of +57.4%.

PMID: 18721749 [PubMed - indexed for MEDLINE]


2. J Mol Biol. 2008 Oct 24;382(5):1184-94. Epub 2008 Aug 5.

Structural basis of poly(3-hydroxybutyrate) hydrolysis by PhaZ7 depolymerase from
Paucimonas lemoignei.

Papageorgiou AC, Hermawan S, Singh CB, Jendrossek D.

Turku Centre for Biotechnology, University of Turku and Abo Akademi University,
BioCity, Turku 20521, Finland. tassos.papageorgiou@btk.fi

The crystal structure of poly(3-hydroxybutyrate) (PHB) depolymerase PhaZ7
purified from Paucimonas lemoignei was determined at 1.90 A resolution. The
structure consists of a single domain with an alpha/beta hydrolase fold in its
core. The active site is analogous to that of serine esterases/lipases and is
characterized by the presence of a catalytic triad comprising Ser136, Asp242, and
His306. Comparison with other structures in the Protein Data Bank showed a high
level of similarity with the Bacillus subtilis lipase LipA (RMSD, 1.55 A).
Structural comparison with Penicillium funiculosum PHB depolymerase, the only PHB
depolymerase whose structure is already known, revealed significant differences,
resulting in an RMSD of 2.80-3.58 A. The two enzymes appear to utilize different
types of solvent-exposed residues for biopolymer binding, with aliphatic and
hydroxyl residues used in P. funiculosum PHB depolymerase and aromatic residues
in PhaZ7. Moreover, the active site of P. funiculosum PHB depolymerase is
accessible to the substrate in contrast to the active site of PhaZ7, which is
buried. Hence, considerable conformational changes are required in PhaZ7 for the
creation of a channel leading to the active site. Taken together, the structural
data suggest that PhaZ7 and P. funiculosum PHB depolymerase have adopted
different strategies for effective substrate binding in response to their diverse
substrate specificity and the lack of a substrate-binding domain.

PMID: 18706425 [PubMed - indexed for MEDLINE]


3. Appl Microbiol Biotechnol. 2008 Sep;80(3):437-45. Epub 2008 Jul 15.

Efficient secretion of Bacillus subtilis lipase A in Saccharomyces cerevisiae by
translational fusion to the Pir4 cell wall protein.

Mormeneo M, Andrÿs I, Bofill C, DÃ-az P, Zueco J.

Unidad de MicrobiologÃ-a. Facultad de Farmacia, Burjassot, Valencia, Spain.

Both the secretion and the cell surface display of Bacillus subtilis lipase A
(Lip A) in Saccharomyces cerevisiae was investigated using different domains of
the cell wall protein Pir4 as translational fusion partners. LipA gene minus its
leader peptide was fused inframe in two places of PIR4 to achieve cell wall
targeting, or substituting most of the PIR4 sequence, after the signal peptide
and the Kex2 processed subunit I of Pir4 to achieve secretion to the growth
medium. Expression of the recombinant fusion proteins was investigated in a
standard and a glycosylation-deficient strain of S. cerevisiae, grown in
selective or rich medium. Fusion proteins intended to be retained at the cell
wall were secreted to the growth medium, most likely as result of the degradation
of the Pir4 moiety containing the cell wall retention domain, giving low levels
of lipase activity. However, the fusion intended for secretion was efficiently
secreted in a percentage of close to 90% and remained stable even in rich medium
at high cell density cultures, yielding values of over 400 IU of lipase activity
per milliliter of cell supernatant. This is, to our knowledge, the first report
of the efficient production, as a secreted protein, of lipase A of B. subtilis in
baker's yeast.

PMID: 18626643 [PubMed - indexed for MEDLINE]


4. J Comput Chem. 2009 Jan 15;30(1):154-62.

Force-field parameters for the simulation of tetrahedral intermediates of serine
hydrolases.

Otte N, Bocola M, Thiel W.

Max-Planck-Institut fÃ?r Kohlenforschung, Kaiser-Wilhelm-Platz 1, D-45470 MÃ?lheim
an der Ruhr, Germany.

CHARMM force-field parameters are reported for the tetrahedral intermediate of
serine hydrolases. The fitting follows the standard protocol proposed for
CHARMM22. The reference data include ab initio (RHF/6-31G*) interaction energies
of complexes between water and the model compound 1,1-dimethoxyethoxide,
torsional profiles of related model compounds from correlated ab initio
(MP2/6-311+G*//B3LYP/6-31+G*) calculations, as well as molecular geometries and
vibrational frequencies from density functional theory (B3LYP/6-31+G*). The
optimized parameters reproduce the target data well. Their utility is
demonstrated by a QM/MM study of the tetrahedral intermediate in Bacillus
subtilis lipase A, and by classical molecular modeling of enantioselectivity in
Pseudomonas aeruginosa lipase and its mutants. Copyright 2008 Wiley Periodicals,
Inc.

PMID: 18566980 [PubMed - indexed for MEDLINE]


5. Chembiochem. 2008 May 5;9(7):1110-5.

A novel genetic selection system for improved enantioselectivity of Bacillus
subtilis lipase A.

Boersma YL, DrÃge MJ, van der Sloot AM, Pijning T, Cool RH, Dijkstra BW, Quax WJ.

Department of Pharmaceutical Biology, University of Groningen, Groningen, The
Netherlands.

In directed evolution experiments, success often depends on the efficacy of
screening or selection methods. Genetic selections have proven to be extremely
valuable for evolving enzymes with improved catalytic activity, improved
stability, or with altered substrate specificity. In contrast, enantioselectivity
is a difficult parameter to select for. In this study, we present a successful
strategy that not only selects for catalytic activity, but for the first time
also for enantioselectivity, as demonstrated by the selection of Bacillus
subtilis lipase A variants with inverted and improved enantioselectivity. A
lipase mutant library in an aspartate auxotroph Escherichia coli was plated on
minimal medium that was supplemented with the aspartate ester of the desired
enantiomer (S)-(+)-1,2-O-isopropylidene-sn-glycerol. To inhibit growth of less
enantioselective variants, a covalently binding phosphonate ester of the opposite
(R)-(-)-1,2-O-isopropylidene-sn-glycerol enantiomer was added as well. After
three selection rounds in which the selection pressure was increased by raising
the phosphonate ester concentration, a mutant was selected with an improved
enantioselectivity increased from an ee of -29.6 % (conversion 23.4 %) to an ee
of +73.1 % (conversion 28.9 %) towards the (S)-(+)-enantiomer. Interestingly, its
amino acid sequence showed that the acid of the catalytic triad had migrated to a
position further along the loop that connects beta7 and alphaE; this shows that
the position of the catalytic acid is not necessarily conserved in this lipase.

PMID: 18383241 [PubMed - indexed for MEDLINE]


6. Biochim Biophys Acta. 2008 Feb;1784(2):302-11. Epub 2007 Nov 12.

Structural basis for the remarkable stability of Bacillus subtilis lipase (Lip A)
at low pH.

Rajakumara E, Acharya P, Ahmad S, Sankaranaryanan R, Rao NM.

Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad, 500 007, India.

Understanding the structural basis of altered properties of proteins due to
changes in temperature or pH provides useful insights in designing proteins with
improved stability. Here we report the basis for the pH-dependent thermostability
of the Bacillus subtilis lipase (Lip A) using spectroscopic and X-ray
crystallographic studies. At pH values above 7, lipase denatures and aggregates
when heated at temperatures above 45 degrees C. However, at pH below 6 lipase
denatures upon heating but the activity and its native structure is completely
recovered upon cooling. In order to obtain the structural basis of this unusual
stability of lipase, we determined high-resolution crystal structures of the
lipase in two different crystal forms at pH 4.5 and 5. These structures show
linear oligomerization of lipase using only two types of dimeric associations and
these inter-molecular interactions are completely absent in several crystal forms
of wild-type and mutant proteins obtained at basic pH. In accordance with the
crystallographic studies, spectroscopic investigations reveal an invariant
secondary structure in the pH range of 4-10. Quaternary organization of lipase at
low pH resulted in changes in the tryptophan environment and binding of
1-anilino-8-naphthalene sulfate (ANS) at low pH. Low pH stability of the lipase
is not observed in the presence of sodium chloride (>0.2 M) indicating the
importance of ionic interactions at low pH. Inter- and intra-molecular ionic
interactions that occur at pH below 6.0 are proposed to trap the molecule in a
conformation that allows its complete refolding upon cooling.

PMID: 18053819 [PubMed - indexed for MEDLINE]


7. Curr Opin Biotechnol. 2006 Dec;17(6):606-10. Epub 2006 Oct 17.

High-throughput screening for enhanced protein stability.

Bommarius AS, Broering JM, Chaparro-Riggers JF, Polizzi KM.

School of Chemical & Biomolecular Engineering, 315 Ferst Drive, Georgia Institute
of Technology, Atlanta, GA 30332-0363, USA. andreas.bommarius@chbe.gatech.edu

High thermostability of proteins is a prerequisite for their implementation in
biocatalytic processes and in the evolution of new functions. Various protein
engineering methods have been applied to the evolution of increased
thermostability, including the use of combinatorial design where a diverse
library of proteins is generated and screened for variants with increased
stability. Current trends are toward the use of data-driven methods that reduce
the library size by using available data to choose areas of the protein to
target, without specifying the precise changes. For example, the half-lives of
subtilisin and a Bacillus subtilis lipase were increased 1500-fold and 300-fold,
respectively, using a crystal structure to guide mutagenesis choices. Sequence
homology based methods have also produced libraries where 50% of the variants
have improved thermostability. Moreover, advances in the high-throughput
measurement of denaturation curves and the application of selection methods to
thermostability evolution have enabled the screening of larger libraries. The
combination of these methods will lead to the rapid improvement of protein
stability for biotechnological purposes.

PMID: 17049838 [PubMed - indexed for MEDLINE]


8. Chembiochem. 2006 Jan;7(1):149-57.

Directed evolution of Bacillus subtilis lipase A by use of enantiomeric
phosphonate inhibitors: crystal structures and phage display selection.

DrÃge MJ, Boersma YL, van Pouderoyen G, Vrenken TE, RÃ?ggeberg CJ, Reetz MT,
Dijkstra BW, Quax WJ.

Dept. of Pharmaceutical Biology, University of Groningen, A. Deusinglaan 1, 9713
AV Groningen, The Netherlands.

Phage display can be used as a protein-engineering tool for the selection of
proteins with desirable binding properties from a library of mutants. Here we
describe the application of this method for the directed evolution of Bacillus
subtilis lipase A, an enzyme that has important properties for the preparation of
the pharmaceutically relevant chiral compound 1,2-O-isopropylidene-sn-glycerol
(IPG). PCR mutagenesis with spiked oligonucleotides was employed for saturation
mutagenesis of a stretch of amino acids near the active site. After expression of
these mutants on bacteriophages, dual selection with (S)-(+)- and (R)-(-)-IPG
stereoisomers covalently coupled to enantiomeric phosphonate suicide inhibitors
(SIRAN Sc and Rc inhibitors, respectively) was used for the isolation of variants
with inverted enantioselectivity. The mutants were further characterised by
determination of their Michaelis-Menten parameters. The 3D structures of the Sc
and Rc inhibitor-lipase complexes were determined and provided structural insight
into the mechanism of enantioselectivity of the enzyme. In conclusion, we have
used phage display as a fast and reproducible method for the selection of
Bacillus lipase A mutant enzymes with inverted enantioselectivity.

PMID: 16342303 [PubMed - indexed for MEDLINE]


9. Chembiochem. 2005 Jun;6(6):1062-7.

Multiplex-PCR-based recombination as a novel high-fidelity method for directed
evolution.

Eggert T, Funke SA, Rao NM, Acharya P, Krumm H, Reetz MT, Jaeger KE.

Institut fÃ?r Molekulare Enzymtechnologie, Heinrich-Heine-UniversitÃ?t DÃ?sseldorf,
Forschungszentrum JÃ?lich, 52426 JÃ?lich, Germany. t.eggert@fz-juelich.de

A new and convenient method for the in vitro recombination of single point
mutations is presented. This method efficiently reduces the introduction of novel
point mutations, which usually occur during recombination processes. A multiplex
polymerase chain reaction (multiplex-PCR) generates gene fragments that contain
preformed point mutations. These fragments are subsequently assembled into
full-length genes by a recombination-PCR step. The process of multiplex-PCR-based
recombination (MUPREC) does not require DNase I digestion for gene-fragmentation
and is therefore easy to perform, even with small amounts of target DNA. The
protocol yields high frequencies of recombination without creating a wild-type
background. Furthermore, the low error rate results in high-quality variant
libraries of true recombinants, thereby minimizing the screening efforts and
saving time and money. The MUPREC method was used in the directed evolution of a
Bacillus subtilis lipase that can catalyse the enantioselective hydrolysis of a
model meso-compound. Thereby, the method was proved to be useful in producing a
reliable second-generation library of true recombinants from which better
performing variants were identified by using a high-throughput electrospray
ionization mass spectrometry (ESI-MS) screening system.

PMID: 15880674 [PubMed - indexed for MEDLINE]


10. Appl Environ Microbiol. 2005 Apr;71(4):1899-908.

Genes involved in SkfA killing factor production protect a Bacillus subtilis
lipase against proteolysis.

Westers H, Braun PG, Westers L, Antelmann H, Hecker M, Jongbloed JD, Yoshikawa H,
Tanaka T, van Dijl JM, Quax WJ.

Department of Pharmaceutical Biology, University of Groningen, Hanzeplein 1, P.O.
Box 30 001, 9700 RB Groningen, the Netherlands.

Small lipases of Bacillus species, such as LipA from Bacillus subtilis, have a
high potential for industrial applications. Recent studies showed that deletion
of six AT-rich islands from the B. subtilis genome results in reduced amounts of
extracellular LipA. Here we demonstrate that the reduced LipA levels are due to
the absence of four genes, skfABCD, located in the prophage 1 region. Intact
skfABCD genes are required not only for LipA production at wild-type levels by B.
subtilis 168 but also under conditions of LipA overproduction. Notably, SkfA has
bactericidal activity and, probably, requires the SkfB to SkfD proteins for its
production. The present results show that LipA is more prone to proteolytic
degradation in the absence of SkfA and that high-level LipA production can be
improved significantly by employing multiple protease-deficient B. subtilis
strains. In conclusion, our findings imply that SkfA protects LipA, directly or
indirectly, against proteolytic degradation. Conceivably, SkfA could act as a
modulator in LipA folding or as a protease inhibitor.

PMCID: PMC1082511
PMID: 15812018 [PubMed - indexed for MEDLINE]


11. FEBS Lett. 2005 Feb 14;579(5):1177-82.

A generic system for the Escherichia coli cell-surface display of lipolytic
enzymes.

Becker S, Theile S, Heppeler N, Michalczyk A, Wentzel A, Wilhelm S, Jaeger KE,
Kolmar H.

Abteilung fÃ?r Molekulare Genetik und PrÃ?parative Molekularbiologie, Institut fÃ?r
Mikrobiologie und Genetik, Georg-August-UniversitÃ?t GÃttingen, Grisebachstrasse
8, D-37077 GÃttingen, Germany.

EstA is an outer membrane-anchored esterase from Pseudomonas aeruginosa. An
inactive EstA variant was used as an anchoring motif for the Escherichia coli
cell-surface display of lipolytic enzymes. Flow cytometry analysis and
measurement of lipase activity revealed that Bacillus subtilis lipase LipA,
Fusarium solani pisi cutinase and one of the largest lipases presently known,
namely Serratia marcescens lipase were all efficiently exported by the EstA
autotransporter and also retained their lipolytic activities upon cell surface
exposition. EstA provides a useful tool for surface display of lipases including
variant libraries generated by directed evolution thereby enabling the
identification of novel enzymes with interesting biological and biotechnological
ramifications.

PMID: 15710409 [PubMed - indexed for MEDLINE]


12. J Mol Biol. 2004 Aug 27;341(5):1271-81.

Structural basis of selection and thermostability of laboratory evolved Bacillus
subtilis lipase.

Acharya P, Rajakumara E, Sankaranarayanan R, Rao NM.

Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India.

Variation in gene sequences generated by directed evolution approaches often does
not assure a minimalist design for obtaining a desired property in proteins.
While screening for enhanced thermostability, structural information was utilized
in selecting mutations that are generated by error-prone PCR. By this approach we
have increased the half-life of denaturation by 300-fold compared to the
wild-type Bacillus subtilis lipase through three point mutations generated by
only two cycles of error-prone PCR. At lower temperatures the activity parameters
of the thermostable mutants are unaltered. High-resolution crystal structures of
the mutants show subtle changes, which include stacking of tyrosine residues,
peptide plane flipping and a better anchoring of the terminus, that challenge
rational design and explain the structural basis for enhanced thermostability.
The approach may offer an efficient and minimalist solution for the enhancement
of a desired property of a protein.

PMID: 15321721 [PubMed - indexed for MEDLINE]


13. Acta Crystallogr D Biol Crystallogr. 2004 Jan;60(Pt 1):160-2. Epub 2003 Dec 18.

Crystallization and preliminary X-ray crystallographic investigations on several
thermostable forms of a Bacillus subtilis lipase.

Rajakumara E, Acharya P, Ahmad S, Shanmugam VM, Rao NM, Sankaranarayanan R.

Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India.

Bacillus subtilis lipase loses activity above pH 10.5 and below pH 6.0. However,
at low pH, i.e. below pH 5.0, the lipase acquires remarkable thermostability.
Activity was unaltered for 2 h at 323 K at pH 4.0-5.0, although at pH values
above 7.0 the activity was lost rapidly within minutes. Circular-dichroism
studies indicate significant changes in the tertiary structure of the lipase,
whereas the secondary-structural content remained unaltered. To elucidate the
structural basis of the enhanced thermostability, three different forms have been
crystallized at low pH along with three crystal forms of two thermostable mutants
obtained using a directed-evolution approach.

PMID: 14684916 [PubMed - indexed for MEDLINE]


14. J Biotechnol. 2003 Feb 27;101(1):19-28.

Binding of phage displayed Bacillus subtilis lipase A to a phosphonate suicide
inhibitor.

DrÃge MJ, RÃ?ggeberg CJ, van der Sloot AM, Schimmel J, Dijkstra DS, Verhaert RM,
Reetz MT, Quax WJ.

Department of Pharmaceutical Biology, University Centre for Pharmacy, University
of Groningen, Antonius Deusinglaan 1, NL-9713 AV Groningen, The Netherlands.

Phage display can be used as a protein engineering tool to select proteins with
desirable binding properties from a library of randomly constructed mutants.
Here, we describe the development of this method for the directed evolution of
Bacillus subtilis lipase A, an enzyme that has marked properties for the
preparation of pharmaceutically relevant chiral compounds. The lipase gene was
cloned upstream of the phage g3p encoding sequence and downstream of a modified
g3p signal sequence. Consequently, the enzyme was displayed at the surface of
bacteriophage fd as a fusion to its minor coat protein g3p. The phage-bound
lipase was correctly folded and fully enzymatically active as determined from the
hydrolysis of p-nitrophenylcaprylate with K(m)-values of 0.38 and 0.33 mM for the
phage displayed and soluble lipase, respectively. Both soluble lipase and lipase
expressed on bacteriophages reacted covalently with a phosphonate suicide
inhibitor. The phage does not hamper lipase binding, since both soluble and
phage-bound lipase have a similar half-life of inactivation of approximately 5
min. Therefore, we conclude that the Bacillus lipase can be functionally
expressed on bacteriophages as a fusion to the phage coat protein g3p. The
specific interaction with the suicide inhibitor offers a fast and reproducible
method for the future selection of mutant enzymes with an enantioselectivity
towards new substrates.

PMID: 12523966 [PubMed - indexed for MEDLINE]


15. Acta Crystallogr D Biol Crystallogr. 2002 Jul;58(Pt 7):1168-74. Epub 2002 Jun 20.

Alternate conformations observed in catalytic serine of Bacillus subtilis lipase
determined at 1.3 A resolution.

Kawasaki K, Kondo H, Suzuki M, Ohgiya S, Tsuda S.

Structural Biology Group, Research Institute of Biological Resources, National
Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1
Tsukisamu-Higashi, Toyohira, Sapporo 062-8517, Japan.

Bacillus subtilis extracellular lipase (BsL) has an exceptionally low molecular
weight (19.4 kDa) for a member of the lipase family. A crystallographic study was
performed on BsL in order to design and produce mutant BsL that will be more
suitable for industrial uses based on analysis of the three-dimensional
structure. Recently, the crystal structure of BsL has been determined at 1.5 A
resolution [van Pouderoyen et al. (2001). J. Mol. Biol. 309, 215-226]. In the
present study, a new crystal form of BsL which provides diffraction data to
higher resolution was obtained and its structure was determined at 1.3 A using
the MAD method. It was found that the active-site residue Ser77 has alternate
side-chain conformations. The O(gamma) atom of the first conformer forms a
hydrogen bond to the N(epsilon) atom of His155, a member of the catalytic triad.
In contrast, the second conformer is constructed with a hydrogen bond to the
side-chain atom of the adjacent His76. These two conformers presumably correspond
to the active and inactive states, respectively. Similar alternate conformations
in the catalytic serine residue have been observed in Fusarium solani cutinase
determined at 1.0 A resolution and Penicillium purpurogenum acetylxylan esterase
at 0.9 A resolution. In addition, a glycerol molecule, which was used as a
cryoprotectant, is found to be located in the active site. On the basis of these
results, a model for substrate binding in the reaction-intermediate state of BsL
is proposed.

PMID: 12077437 [PubMed - indexed for MEDLINE]


16. Biotechnol Bioeng. 2002 May 5;78(3):339-45.

Engineering of baker's yeasts, E. coli and Bacillus hosts for the production of
Bacillus subtilis Lipase A.

SÃ?nchez M, Prim N, RÃ?ndez-Gil F, Pastor FI, Diaz P.

Department of Microbiology, Faculty of Biology, University of Barcelona, Av.
Diagonal 645, 08028-Barcelona, Spain.

Lipases are versatile biocatalists showing multiple applications in a wide range
of biotechnological processes. The gene lipA coding for Lipase A from Bacillus
subtilis was isolated by PCR amplification, cloned and expressed in Escherichia
coli, Saccharomyces cerevisiae and Bacillus subtilis strains, using pBR322,
YEplac112 and pUB110-derived vectors, respectively. Lipase activity analysis of
the recombinant strains showed that the gene can be properly expressed in all
hosts assayed, this being the first time a lipase from bacterial origin can be
expressed in baker's S. cerevisiae strains. An important increase of lipase
production was obtained in heterologous hosts with respect to that of parental
strains, indicating that the described systems can represent a useful tool to
enhance productivity of the enzyme for biotechnological applications, including
the use of the lipase in bread making, or as a technological additive. Copyright
2002 Wiley Periodicals, Inc.

PMID: 11920450 [PubMed - indexed for MEDLINE]


17. J Mol Biol. 2001 May 25;309(1):215-26.

The crystal structure of Bacillus subtilis lipase: a minimal alpha/beta hydrolase
fold enzyme.

van Pouderoyen G, Eggert T, Jaeger KE, Dijkstra BW.

Laboratory of Biophysical Chemistry, University of Groningen, The Netherlands.

The X-ray structure of the lipase LipA from Bacillus subtilis has been determined
at 1.5 A resolution. It is the first structure of a member of homology family 1.4
of bacterial lipases. The lipase shows a compact minimal alpha/beta hydrolase
fold with a six-stranded parallel beta-sheet flanked by five alpha-helices, two
on one side of the sheet and three on the other side. The catalytic triad
residues, Ser77, Asp133 and His156, and the residues forming the oxyanion hole
(backbone amide groups of Ile12 and Met78) are in positions very similar to those
of other lipases of known structure. However, no lid domain is present and the
active-site nucleophile Ser77 is solvent-exposed. A model of substrate binding is
proposed on the basis of a comparison with other lipases with a covalently bound
tetrahedral intermediate mimic. It explains the preference of the enzyme for
substrates with C8 fatty acid chains.

PMID: 11491291 [PubMed - indexed for MEDLINE]


18. Appl Environ Microbiol. 1994 May;60(5):1670-3.

Genetic analysis and overexpression of lipolytic activity in Bacillus subtilis.

Dartois V, Coppÿe JY, Colson C, Baulard A.

Laboratoire de Gÿnÿtique Microbienne, Universitÿ Catholique de Louvain, Belgium.

The previously cloned Bacillus subtilis lipase gene (lip) was mapped on the
chromosome and used in the construction of a B. subtilis derivative totally
devoid of any lip sequence. Homologous overexpression was performed in this
strain by subcloning the lip open reading frame on a multicopy plasmid under the
control of a strong gram-positive promoter. A 100-fold overproducing strain was
obtained, which should facilitate purification of the secreted protein.
Furthermore, the delta lip strain BCL1050 constitutes an ideal host for the
cloning of heterologous lipase genes.

PMCID: PMC201535
PMID: 8017946 [PubMed - indexed for MEDLINE]