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Bacterial Biocatalysts: Molecular Biology, Three-Dimensional Structures, and Biotechnological Applications of Lipases

01 Jan 1999-Annual Review of Microbiology (Annual Reviews 4139 El Camino Way, P.O. Box 10139, Palo Alto, CA 94303-0139, USA)-Vol. 53, Iss: 1, pp 315-351
TL;DR: Three-dimensional structures of bacterial lipases were solved to understand the catalytic mechanism of lipase reactions and will enable researchers to tailor new lipases for biotechnological applications.
Abstract: ▪ Abstract Bacteria produce and secrete lipases, which can catalyze both the hydrolysis and the synthesis of long-chain acylglycerols. These reactions usually proceed with high regioselectivity and enantioselectivity, and, therefore, lipases have become very important stereoselective biocatalysts used in organic chemistry. High-level production of these biocatalysts requires the understanding of the mechanisms underlying gene expression, folding, and secretion. Transcription of lipase genes may be regulated by quorum sensing and two-component systems; secretion can proceed either via the Sec-dependent general secretory pathway or via ABC transporters. In addition, some lipases need folding catalysts such as the lipase-specific foldases and disulfide-bond–forming proteins to achieve a secretion-competent conformation. Three-dimensional structures of bacterial lipases were solved to understand the catalytic mechanism of lipase reactions. Structural characteristics include an α/β hydrolase fold, a catalytic ...

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University of Groningen
Bacterial biocatalysts
Jaeger, K-E.; Dijkstra, B.W.; Reetz, M.T.
Published in:
Annual Review of Microbiology
DOI:
10.1146/annurev.micro.53.1.315
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Publication date:
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Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Jaeger, K-E., Dijkstra, B. W., & Reetz, M. T. (1999). Bacterial biocatalysts: Molecular Biology, Three-
Dimensional Structures, and Biotechnological Applications of Lipases.
Annual Review of Microbiology
,
53
(1), 315-+. https://doi.org/10.1146/annurev.micro.53.1.315
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Annu. Rev. Microbiol. 1999. 53:315–51
Copyright
c
° 1999 by Annual Reviews. All rights reserved
BACTERIAL BIOCATALYSTS: Molecular
Biology, Three-Dimensional Structures,
and Biotechnological Applications
of Lipases
K-E. Jaeger
Lehrstuhl Biologie der Mikroorganismen, Ruhr-Universit
¨
at, D-44780 Bochum,
Germany; e-mail: karl-erich.jaeger@ruhr-uni-bochum.de
B. W. Dijkstra
Laboratory of Biophysical Chemistry, Rijksuniversiteit Groningen, NL-9747
AG Groningen, The Netherlands; e-mail: bauke@chem.rug.nl
M. T. Reetz
Max-Planck-Institut f
¨
ur Kohlenforschung, D-45470 M
¨
ulheim an der Ruhr, Germany;
e-mail: reetz@mpi-muelheim.mpg.de
Key Words secretion, directed evolution, enantioselectivity
Abstract Bacteria produce and secrete lipases, which can catalyze both the hy-
drolysis andthesynthesis of long-chain acylglycerols. Thesereactions usuallyproceed
with high regioselectivity and enantioselectivity, and, therefore, lipases have become
very important stereoselective biocatalysts used in organic chemistry. High-level pro-
duction of these biocatalysts requires the understanding of the mechanisms underlying
gene expression,folding, and secretion. Transcription of lipase genes may be regulated
by quorum sensing and two-component systems; secretion can proceed either via the
Sec-dependent general secretory pathway or via ABC transporters. In addition, some
lipases need folding catalysts such as the lipase-specific foldases and disulfide-bond–
forming proteins to achieve a secretion-competent conformation. Three-dimensional
structures of bacterial lipases were solved to understand the catalytic mechanism of
lipase reactions. Structural characteristics include an
α/β hydrolase fold, a catalytic
triad consisting of a nucleophilic serine located in a highly conserved Gly-X-Ser-X-
Gly pentapeptide, and an aspartate or glutamate residue that is hydrogen bonded to a
histidine. Foursubstrate binding pocketswere identified fortriglycerides: an oxyanion
holeand three pockets accommodatingthe fattyacids boundat positionssn-1, sn-2, and
sn-3. The differences in size and the hydrophilicity/hydrophobicity of these pockets
determine the enantiopreference of a lipase. The understanding of structure-function
relationships will enable researchers to tailor new lipases for biotechnological applica-
tions. At the same time, directed evolution in combination with appropriate screening
0066-4227/99/1001-0315$08.00 315
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316 JAEGER
DIJKSTRA
REETZ
systems will be used extensively as a novel approach to develop lipases with high
stability and enantioselectivity.
CONTENTS
Introduction....................................................
316
Definition of a Lipase ............................................ 317
Screening for Lipase Activity ...................................... 318
Hydrolysis
.................................................... 318
Synthesis
..................................................... 318
Classification of Bacterial Lipases .................................. 319
Regulation of Lipase Gene Expression .............................. 322
Secretion and Folding ............................................325
ABC Exporters
................................................. 325
Secretion Across the Inner Membrane
................................ 325
Secretion Across the Outer Membrane
................................ 325
Folding Catalysts
............................................... 326
Three-Dimensional Structures of Bacterial Lipases .....................328
The Fold of Lipases
............................................. 329
The Catalytic Residues of Lipases
................................... 330
The Catalytic Mechanism
......................................... 331
Interfacial Activation
............................................ 333
Substrate Binding
............................................... 333
Structural Determinants of Enantiomeric Selectivity
......................335
Biotechnological Applications of Lipases ............................336
Lipases as Catalysts in Synthetic Organic Chemistry
......................336
Immobilization Techniques
........................................ 338
Directed Evolution of Enantioselective Lipases
.......................... 339
Conclusions and Future Directions.................................. 342
The authors dedicate this article to Professor Dr. Ulrich K. Winkler on the
occasion of his 70th birthday. Uli Winkler was among the first to isolate
bacterial lipases from Serratia marcescens and Pseudomonas aeruginosa
and to study their physiology and biochemical properties.
INTRODUCTION
Nearly 100 years ago, a landmark report was published by C Eijkmann (34) de-
scribing the following simple experiment: beef tallow spread on the ground of a
glass plate was overlaid with agar that was inoculated with different bacteria. After
3–4 days of incubation, Ca, Na, and NH
4
soaps had formed. Eijkmann concluded
that lipases had been produced and secreted by the bacteria, among them Bacillus
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BACTERIAL LIPASES 317
pyocyaneus (today named Pseudomonas aeruginosa), Staphylococcus pyogenes
aureus (S. aureus), B. prodigiosus (today Serratia marcescens), and B. fluorescens
(today P. fluorescens). B. coli communis (Escherichia coli) was found to be lipase
negative (34). Only a few lipase-producing bacteria were further characterized
(95), but research was intensified when it became generally accepted that lipases
remain enzymatically active in organic solvents (166), making them ideal tools
for the organic chemist. The aim of this review is not to discuss every lipase
described in the literature but rather to present recent information on selected
and novel lipases. It covers the period from 1994, when a comprehensive re-
view on bacterial lipases appeared (58), until 1998. During this time a number of
novel lipases were cloned and characterized, considerable progress was made in
understanding the regulation of lipase gene expression, and detailed knowledge
became available concerning folding and secretion. Moreover, three-dimensional
structures of lipases were solved and used to explain functional characteristics.
These developments, as well as other aspects such as the important role of li-
pases in biotechnological applications, are discussed, with special emphasis on
enantioselective biotransformations. The enormous interest in lipases is reflected
by a rapidly growing number of excellent review articles and monographs cover-
ingmolecular biology, biochemicalcharacterization, three-dimensional structures,
and biotechnological applications of lipases from prokaryotic and eukaryotic ori-
gins (4, 46,58, 59, 66, 102,121,122, 125, 165).
DEFINITION OF A LIPASE
What exactly is a lipase? At present, there is no satisfying answer to this simple
question. Lipolytic reactions occur at the lipid-water interface where lipolytic sub-
strates usually form an equilibrium between monomeric, micellar, and emulsified
states. Until recently, two criteria have been used to classify a lipolytic enzyme
as a “true” lipase (EC 3.1.1.3): (a) It should be activated by the presence of an
interface, that is, its activity should sharply increase as soon as the triglyceride
substrate forms an emulsion. This phenomenon was termed “interfacial activa-
tion” (124). (b) It should contain a “lid” (see below), which is a surface loop of the
protein covering the active site of the enzyme and moving away on contact with
the interface (17, 24, 158). However, these obviously suggestive criteria proved
to be unsuitable for classification, mainly because a number of exceptions were
described of enzymes having a lid but not exhibiting interfacial activation (159).
Therefore, lipases are simply defined as carboxylesterases catalyzing the hydrol-
ysis (and synthesis) of long-chain acylglycerols (37). There is no strict definition
available for the term “long-chain, but glycerolesters with an acyl chain length of
10 carbon atoms can be regarded as lipase substrates, with trioleoylglycerol be-
ing the standard substrate. Hydrolysis of glycerolesters with an acyl chain length
of <10 carbon atoms with tributyrylglycerol (tributyrin) as the standard substrate
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318 JAEGER
DIJKSTRA
REETZ
usually indicates the presence of an esterase (62). It should be emphasized that
most lipases are perfectly capable of hydrolyzing these esterase substrates.
SCREENING FOR LIPASE ACTIVITY
Hydrolysis
Microbiologists generally want to use a simple and reliable plate assay allowing
the identification of lipase-producing bacteria. The most widely used substrates
are tributyrin and triolein, which are emulsified mechanically in various growth
media and poured into a petri dish. Lipase production is indicated by the formation
of clear halos around the colonies grown on tributyrin-containing agar plates (6)
and orange-red fluorescence visible on irradiation with a conventional UV hand
lamp at λ = 350 nm on triolein plates, which additionally contain rhodamine B
(74). Lipase activity in bacterial culture supernatants is determined by hydroly-
sis of p-nitrophenylesters of fatty acids with various chain lengths (C-10) and
spectrophotometric detection of p-nitrophenol at 410 nm. However, care must
be taken to interpret the results because these fatty acid monoester substrates are
also hydrolyzed by esterases. This problem can be overcome by using the triglyc-
eride derivative 1,2-O-dilauryl-rac-glycero-3-glutaric acid resorufin ester (avail-
able from Boehringer Mannheim Roche GmbH, Germany), yielding resorufin,
which can be determined spectrophotometrically at 572 nm or fluorometrically at
583 nm. A number of novel fluorogenic alkyldiacylglycerols were synthesized
and used for analysis of both lipase activity and stereoselectivity (167). A more
laborious but reliable method for identifying a “true” lipase is the determination of
fatty acids liberated from a triglyceride, usually trioleoylglycerol, by titration (62).
Automated equipment allows the parallel assay of a large number of samples. De-
termination of kinetics of lipolysis requires a tight control of the interfacial quality
achieved by using the monolayer technique: A lipid film is spread at the air/water
interface in a so-called “zero-order” trough consisting of a substrate reservoirand a
reaction compartment. Lipase-catalyzed hydrolysis of the lipid monolayer results
in changes of the surface pressure, which can be readjusted automatically by a
computer-controlled barostat (111). It should be emphasized that this technique
requires expensive equipment and experienced personnel.
Synthesis
Biotechnological applications of lipases prompt a demand for techniques to de-
termine their activity and, if relevant, stereoselectivity. A standard reaction is
the lipase-catalyzed esterification of an alcohol with a carboxylic acid, e.g. the
formation of octyl laurate from lauric acid and n-octanol reacted in an organic
solvent (114). The initial rate of ester formation can be determined by gas chro-
matography. No single method is available to determine the enantioselectivity of
a lipase-catalyzed organic reaction. Generally, the enantioselectivity of product
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Citations
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TL;DR: Various industrial applications of microbial lipases in the detergent, food, flavour industry, biocatalytic resolution of pharmaceuticals, esters and amino acid derivatives, making of fine chemicals, agrochemicals, use as biosensor, bioremediation and cosmetics and perfumery are described.

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TL;DR: Novel biotechnological applications have been successfully established using lipases for the synthesis of biopolymers and biodiesel, the production of enantiopure pharmaceuticals, agrochemicals, and flavour compounds.

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TL;DR: The latest trend in lipase research is the development of novel and improved lipases through molecular approaches such as directed evolution and exploring natural communities by the metagenomic approach.
Abstract: Lipases, triacylglycerol hydrolases, are an important group of biotechnologically relevant enzymes and they find immense applications in food, dairy, detergent and pharmaceutical industries. Lipases are by and large produced from microbes and specifically bacterial lipases play a vital role in commercial ventures. Some important lipase-producing bacterial genera include Bacillus, Pseudomonas and Burkholderia. Lipases are generally produced on lipidic carbon, such as oils, fatty acids, glycerol or tweens in the presence of an organic nitrogen source. Bacterial lipases are mostly extracellular and are produced by submerged fermentation. The enzyme is most commonly purified by hydrophobic interaction chromatography, in addition to some modern approaches such as reverse micellar and aqueous two-phase systems. Most lipases can act in a wide range of pH and temperature, though alkaline bacterial lipases are more common. Lipases are serine hydrolases and have high stability in organic solvents. Besides these, some lipases exhibit chemo-, regio- and enantioselectivity. The latest trend in lipase research is the development of novel and improved lipases through molecular approaches such as directed evolution and exploring natural communities by the metagenomic approach.

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Cites background from "Bacterial Biocatalysts: Molecular B..."

  • ...…1993,1994; Pandey et al. 1999; Abdou 2003 Streptomyces exfoliatus Arpigny and Jaeger 1999 Sulfolobus acidocaldarius Jaeger et al. 1999 Vibrio chloreae Jaeger et al. 1999 T ab le 2 C om m er ci al ba ct er ia l lip as es , so ur ce s, ap pl ic at io ns an d th ei r in du st ri al su pp lie rs . n.…...

    [...]

  • ...…3.1.1.3) have emerged as key enzymes in swiftly growing biotechnology, owing to their multifaceted properties, which find usage in a wide array of industrial applications, such as food technology, detergent, chemical industry and biomedical sciences (Jaeger et al. 1994, 1999; Pandey et al. 1999)....

    [...]

  • ...…activity and thus have a very diverse substrate range, although they are highly specific as chemo-, regio- and enantioselective catalysts (Jaeger et al. 1994, 1999; Jaeger and Reetz 1998; Kazlauskas and Bornscheur 1998; Pandey et al. 1999; Beisson et al. 2000; Gupta and Soni 2000; Jaeger…...

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  • ...…marcescens Matsumae et al. 1993,1994; Pandey et al. 1999; Abdou 2003 Streptomyces exfoliatus Arpigny and Jaeger 1999 Sulfolobus acidocaldarius Jaeger et al. 1999 Vibrio chloreae Jaeger et al. 1999 T ab le 2 C om m er ci al ba ct er ia l lip as es , so ur ce s, ap pl ic at io ns an d th ei…...

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  • ...However, physical para- S. epidermidis Simons et al. 1996; Jaeger et al. 1999 S. haemolyticus Oh et al. 1999 S. hyicus Jaeger et al.1999; Van Kampen et al.2001 S. warneri Pandey et al.1999; Van Kampen et al.2001 S. xylosus Pandey et al.1999; Van Kampen et al.2001 Serratia marcescens Matsumae et al.…...

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TL;DR: The α/β hydrolase fold is a typical example of a tertiary fold adopted by proteins that have no obvious sequence similarity, but nevertheless, in the course of evolution, diverged from a common ancestor.

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TL;DR: This contribution provides a summary of the biochemical and molecular biological characteristics of e-PHA depolymerases and focuses on the intracellular mobilization of storage PHA by intracesllular PHA depolemerases (i-P HA depolyMERases) of PHA-accumulating bacteria.
Abstract: Polyesters such as poly(3-hydroxybutyrate) (PHB) or other polyhydroxyalkanoates (PHA) have attracted commercial and academic interest as new biodegradable materials. The ability to degrade PHA is widely distributed among bacteria and fungi and depends on the secretion of specific extracellular PHA depolymerases (e-PHA depolymerases), which are carboxyesterases (EC 3.1.1.75 and EC 3.1.1.76), and on the physical state of the polymer (amorphous or crystalline). This contribution provides a summary of the biochemical and molecular biological characteristics of e-PHA depolymerases and focuses on the intracellular mobilization of storage PHA by intracellular PHA depolymerases (i-PHA depolymerases) of PHA-accumulating bacteria. The importance of different assay systems for PHA depolymerase activity is also discussed.

605 citations

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"Bacterial Biocatalysts: Molecular B..." refers background in this paper

  • ...Human pancreatic lipase (163) and the lipase from the fungusRhizomucor miehei (14, 28) were the first X-ray structures of lipases elucidated....

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