Download Tools in Biocatalysis

Tools in Biocatalysis
Enzyme Immobilisation on Silica and
Synthesis of Enantiopure Amines
Karim Engelmark Cassimjee
Licentiate thesis
Royal Institute of Technology
School of Biotechnology
Stockholm 2010
© Karim Engelmark Cassimjee 2010
Royal Institute of Technology
School of Biotechnology
AlbaNova University Centre
SE-106 91 Stockholm
Sweden
ISBN 978-91-7415-653-9
TRITA-BIO Report 2010:11
ISSN 1654-2312
Printed in Stockholm, April 2010
Universitetsservice US-AB
Drottning Kristinas väg 53 B
SE-100 44 Stockholm
Sweden
Abstract
This thesis presents two techniques in the field of biocatalysis:
An enzyme immobilisation method based on the His6-tag for
attachment on modified silica oxide beads, and it’s employment in
aqueous and organic medium for synthesis applications. The
method functions as a one step extraction and immobilisation
protocol.
An equilibrium displacement system which enables complete
conversion in reactions with ω-transaminases where isopropylamine
is the donor, a route for synthesis of pharmaceutically interesting
enantiopure amines.
Biocatalysis is predicted to be a paramount technology for an
environmentally sustainable chemical industry, to which every
newly developed method represents a small but important step. The
work done here is aimed to be a part of this development.
Sammanfattning
I denna avhandling presenteras två tekniker inom ämnet biokatalys:
En metod för immobilisering av His6-enzym på modifierad kiseloxid,
och användning av detta konstrukt för kemiska synteser i vatten
och organiska lösningsmedel. Detta system fungerar även som en
snabb extraherings- och immobiliseringsmetod.
Ett jämviksförskjutningssystem som möjliggör fullständig omsättning i reaktioner med ω-transaminaser där isopropylamin är aminodonator, en syntesväg för tillverkning av farmakologiskt intressanta
kirala aminer.
Biokatalys förutspås att bli en ovärderlig teknologi i en miljömässigt
hållbar kemisk industri, i vilken varje ny metod är en liten men dock
viktig del. Detta arbete är menat som en del i denna utveckling.
List of publications
This thesis is written based on the work in the following
publications, referred to by the index roman numerals:
I
Silica-immobilized His6-tagged enzyme: alanine racemase in
hydrophobic solvent
Karim Engelmark Cassimjee, Martin Trummer, Cecilia
Branneby and Per Berglund
2008, Biotechnology and Bioengineering, 99, 3, 712-716
II
HisSi-immobilization, a one step extraction and immobilization
method for enzymes
Karim Engelmark Cassimjee, Robert Kourist, Diana Lindberg,
Marianne Wittrup Larsen, Nguyen Hong Thanh, Mikael
Widersten, Uwe T Bornscheuer and Per Berglund
Submitted manuscript
III
Transaminations with isopropyl amine: Equilibrium
displacement with yeast alcohol dehydrogenase coupled to in
situ cofactor regeneration
Karim Engelmark Cassimjee, Cecilia Branneby, Vahak Abedi,
Andrew Wells and Per Berglund
2010, ChemComm, accepted manuscript
Contents
1 INTRODUCTION 1 1.1 Enzymes, natural catalysts 1 1.2 Enzymes used in this thesis 1.2.1 Geobacillus stearothermophilus alanine racemase (AlaR) 1.2.2 Arthrobacter citreus ω‐transaminase 1.2.3 Candida antarctica lipase B (CalB) 1.2.4 Bacillus subtilis esterase 2 (BS2), Bacillus subtilis lipase A (BslA), Pseudomonas fluorescence esterase (PFE) and Solanum tubersum epoxide hydrolase (StEH1) 1.2.5 Bacillus subtilis L‐alanine dehydrogenase (AlaDH), yeast alcohol dehydrogenase (YADH, Saccharomyces cerevisiae) and Candida boidinii formate dehydrogenase (FDH) 2 2 3 4 1.3 Biocatalysis 1.3.1 Enzyme Immobilisation 1.3.2 Enzymatic Synthesis of Enantiopure Amines 6 7 7 2 PRESENT INVESTIGATION 4 5 11 2.1 Objectives 11 2.2 Enzyme Immobilisation on modified TLC plates (Paper I) 12 2.3 Enzyme immobilisation and extraction with modified silica oxide beads and employment in organic solvent, HisSi‐immobilisation (Paper II) 15 2.4 Efficient Enzymatic Synthesis of Chiral Amines (Paper III) 18 3 CONCLUSIONS 21 3.1 Short summary of the publications 21 3.2 Discussion and future outlook 21 4 ACKNOWLEDGEMENTS 23 5 REFERENCES 25 1 Introduction
1.1 Enzymes, natural catalysts
An amino acid is a molecule which contains an amino group and a
carboxylic acid group. In nature such molecules are present in many
different versions, twenty of which are commonly used for the
formation of chains with subsequent folding which are crucial for
any living cell, proteins. Each amino acid residue contributes with a
distinct side chain with properties elegantly chosen by evolution.
The protein will spontaneously fold in an ordered manner to form a
biologically functional unit. The study of proteins has resulted in
many astounding findings, including observations of the vital
contributions proteins serve for the prevalence of life on earth. A
protein which is capable of catalysing a chemical reaction is called
an enzyme. Enzymes are the foundation of chemical life; they
uphold essentially all chemical reactions at a viable rate within a
living organism.[1]
Observed at macro scale proteins behave in unexpected ways. E.g.
with rising temperature a protein solution (such as egg white) will
turn into a semi solid (an omelette[2]) whereas other compounds
typically will melt and/or lower in viscosity. The reason is the
unfolding and subsequent aggregation of the protein chains. Looking
at proteins in micro scale also yields fascinating findings. An enzyme
is able to bind one or more molecules or atoms (substrates) and
create an environment which is favourable for a desired chemical
reaction. The manner of how this is done is specific for each distinct
enzyme and to some extent remains a matter of debate; theories of
near attack conformers (NAC[3]) and favourable electrostatic
interactions[4] present plausible explanations of function. Consensus
lies in the transition state theory, applicable for all types of
catalysis, which is based on the lowering of the activation energy of
a given reaction.
A molecule is defined as chiral if it cannot be superimposed upon its
mirror image, like your left or right hand. A molecule is not chiral (or
achiral) if it has symmetry elements of the second kind, i.e. a mirror
1
plane, a centre of inversion, or a rotation-reflection axis.[5] The chiral
mirror versions of the otherwise identical compounds are named
enantiomers. In nature such molecules are predominantly existent
as one of the possible versions. Nineteen of the twenty amino acids
which are the common building blocks of proteins are chiral and are
with few exceptions present as the L-enantiomer, as opposed to the
D-enantiomer.[1] In organic chemistry the common way of denoting
the enantiomers are with an S (left handed) or R (right handed).[5]
When the substrate is chiral, the enzyme interacts differently with
the different enantiomers. The enzyme, composed of only L-amino
acid residues, is often specific for the enantiomeric version present
in the environment in which it was evolved. This is possible since
the enzyme is itself chiral and enantiopure. A lower or sometimes
virtually inexistent catalytic power for the other enantiomer is often
observed. The understanding of this is of paramount importance in
the production of pharmaceuticals since the enantiomeric version of
a pharmaceutically active compound is often of critical importance
for the function in the human body.[1, 6] Enzymes also exhibit other
forms of specificity; substrate specificity, chemospecificity and
regiospecificity. The use of enzymes in chemical synthesis can
provide the chemist with a means of creating enantiopure
compounds, performing specific reactions in the presence of other
reactive compounds and/or steering of reactions to specific groups,
in an environmentally compatible setting.[7] Enzyme catalysis has
proven to be a success story for life, of which the qualities remain to
be fully utilised in chemical engineering. Many enzymes are
presently being utilised in industry and academic research, but the
current exploitation does not by any means reach the full potential.
1.2 Enzymes used in this thesis
1.2.1 Geobacillus stearothermophilus alanine racemase (AlaR)
Alanine racemase (AlaR) is present in all bacteria; in this work the
gene from the thermophilic bacterium G. stearothermophilus was
used. Alanine racemase interconverts the enantiomers of the amino
acid alanine (see Figure 1). In nature it generally exists in the L-form
as all amino acids, and is by the enzyme converted into the D-form.
2
The D-alanine is thereafter combined with other compounds to form
peptidoglycan, the fabric of the stable bacterial cell wall.
O
H3N
AlaR
O
L-alanine
O
H3N
O
D-alanine
Figure 1: Racemisation of alanine catalysed by alanine racemase (AlaR). The interconversion of enantiomers is referred to as racemisation,
hence the name alanine racemase. Although it remains to be shown,
it is conceivable that this enzyme or a mutant thereof, may well be
able to racemise other compounds of pharmaceutical interest. If this
would be achieved a dynamic kinetic resolution[8] could be
constructed, a process in which one of the enantiomers is reacted
while continuously produced by the enzyme from the corresponding
enantiomer. Theoretically, this produces an enantiomerically pure
product with a yield of one hundred percent.
1.2.2 Arthrobacter citreus ω-transaminase
Transamination is the process in which an amine and a ketone react
to interchange their functional groups, producing the corresponding
ketone and amine (see Figure 2). This is common biochemistry and
is the manner in which an amino acid is produced from a ketoacid.
O
NH2
ω-TA
NH2
O
Figure 2: A transamination reaction where S‐methylbenzylamine and acetone is produced from acetophenone and isopropylamine, catalysed by an ω‐transaminase (ω‐TA). A cofactor is needed for the reaction, the transaminases utilises the
versatile PLP (pyridoxal-5’-phosphate) for this purpose. One can
portray the reaction as the amination of PLP which gives PMP
(pyridoxamine-5’-phosphate) which then donates the amino group to
another ketone, thereby restoring the PLP.[9] The transaminases
labelled with the Greek letter omega (ω) are able to accept aliphatic
ketones as their substrates, i.e. non-acids.[10] For a synthetic
chemist this property is rather interesting, together with the
enantiospecific nature of these enzymes. Typically only one amine
enantiomer is accepted as substrate or product, whereas the
standard chemical synthesis will produce the racemic mixture. In
3
this work an optimised mutant gene of A. citreus ω-transaminase
(Cambrex Karlskoga AB) was used.
Although ω-transaminases inherently are able to accept non-natural
substrates, many obstacles remain for the synthetic chemist to
conquer. E.g. the transamination reaction is an equilibrium (runs in
both ways) which is often shifted to the substrate side for the
reactions in question (represented by the unbalanced double arrow
in Figure 2), with low product yield as a consequence. Also, many
substrates and/or products tend to inhibit the enzyme which
practically stops the reaction at high concentrations. These are very
limiting factors in large scale synthesis.
1.2.3 Candida antarctica lipase B (CalB)
Candida antarctica lipase B (CalB), also known as Pseudozyma
antarctica lipase B (PalB)[11], is probably the most commonly used
enzyme in industry. It was isolated in Antarctica, hence the name. It
is thermostable and displays high enantioselectivity.[12] Lipases are
enzymes that catalyse the hydrolysis of the ester bonds in
triacylglycerols (fats), as a part of fat catabolism.[1] In vitro lipases
can be used for transacylation[13] if employment in non-aqueous
media is possible, for applications such as polymer synthesis[14, 15]
and resolution (enantiomeric purification) of chiral secondary
alcohols[16]. CalB has proven to be a stable and versatile catalyst in
many different solvents. Additionally, rational redesign (see section
1.3) of the enzyme has resulted in broadening of its reaction
range.[17-20]
1.2.4 Bacillus subtilis esterase 2 (BS2), Bacillus subtilis lipase
A (BslA), Pseudomonas fluorescence esterase (PFE) and Solanum
tubersum epoxide hydrolase (StEH1)
Bacillus subtilis esterase 2 (BS2), Bacillus subtilis lipase A (BslA),
Pseudomonas fluorescence esterase (PFE) and Solanum tubersum
epoxide hydrolase (StEH1) are all readily cloned and overexpressed
in Escherichia coli. Esterases and lipases essentially carry out the
same reaction, hydrolysis of ester bonds. Lipases however, function
in the interface between water and lipid (fat) which is necessary in
fat catabolism.[1] Use of these enzymes for transacylation requires
employment in non-aqueous media.
4
An interesting feature with Bacillus subtilis esterase (BS2) is its
promiscuous activity towards amides.[21] Also, it is active towards
tertiary alcohols.[22-24] These traits make the enzyme attractive for
use in synthesis applications, and interesting for exploring the
possibility of use in organic solvent. Some activity in 1-propanol has
been shown with this enzyme, but none in non-polar solvents.
Bacillus subtilis lipase A (BslA) is a rather typical small lipase[25],
which has never been employed in non-aqueous environments.
Pseudomonas fluorescence esterase (PFE) was in other works
selected for engineering, and subsequently used for synthesis of
attractive compounds.[26-28] Still, it remains to be successfully
immobilised and employed in organic solvent.
Solanum tuberosum epoxide hydrolase 1 (StEH1) converts epoxides
to diols, and is of synthetic interest because of the enantioselectivity.[29, 30] Large scale employment is hindered by the low
solubility of the substrates, aromatic epoxides, in water. Activity in
an organic solvent may well enable industrial use.
1.2.5 Bacillus subtilis L-alanine dehydrogenase (AlaDH), yeast
alcohol dehydrogenase (YADH, Saccharomyces cerevisiae) and
Candida boidinii formate dehydrogenase (FDH)
Several enzymes were in this thesis utilised for analysis and reaction
engineering purposes.
Bacillus subtilis L-alanine dehydrogenase (AlaDH) specifically
oxidises L-alanine to pyruvate with concomitant production of the
spectrophotometrically detectable reduced form of nicotine adenine
dinucleotide (NADH from NAD+). This can be used in an activity
assay for alanine racemase.[31]
Yeast alcohol dehydrogenase (YADH, Saccharomyces cerevisiae) is
selective for oxidation/reduction of small alcohols and ketones with
NAD+/NADH as cofactor.[32] Ethanol and acetaldehyde are the best
substrates, methanol and formaldehyde and also isopropanol and
acetone can be converted. Extremely low or virtually no activity has
been observed for other alcohols or ketones.
5
Candida boidinii formate dehydrogenase (FDH) is an enzyme which
is used for cofactor regeneration. In the exothermic oxidation
reaction of converting formate to carbon dioxide the enzyme reduces
NAD+ to NADH. Cofactors are relatively expensive compounds and
need to be regenerated so as to keep the total amount at a low level
for an industrially feasible process.[33]
1.3 Biocatalysis
Enzymatic synthesis refers to any chemical process in which an
enzyme is involved in the production of a given compound,
synonymous with biosynthesis (in vivo). The term also refers to the
in vitro use of enzyme catalysis in synthetic chemistry or
biocatalysis; the production of chemicals such as pharmaceuticals,
cosmetics, polymers, household reagents etc. This is a fairly new
application of biotechnology. Biocatalysis together with other uses of
natures tools for industrial production of materials, chemicals or
energy form white biotechnology, an important and growing field.[34,
35]
Classic synthetic chemistry is generally associated with organic
solvents (i.e. non-aqueous environments), high temperatures and
metal catalysts. Numerous efficient processes have been developed
in this field, and it is undoubtedly the foundation of today’s
chemical industry. Still, nature has been performing advanced
chemical transformations in aqueous environments at moderate
temperature by employment of enzymes since the beginning of life.
The harnessing, mimicking and modification of these natural
processes remain an engineering challenge, but with great promise
of reward by an environmentally sustainable chemical industry.[36-38]
The modification of enzymes for adaptation to the unnatural
environment present in a chemical synthesis is often considered
necessary, for which many strategies have been developed. Often the
compounds in the process are poor substrates, or a more heat stable
or solvent tolerable enzyme is desired. By using an experimentally
determined structure of the enzyme (determined by X-ray
chrystallography or NMR) the enzyme engineer can locate specific
amino acid residues suitable for mutation, this is called rational
redesign. When such attempts are unsuccessful, or a structure is
6
unavailable, the amino acid chain can be altered in a randomised
fashion and a library of mutants can be screened for the desired new
properties. An improved clone can be further randomised and a new
library can be screened. The iterative process is repeated for the
potential discovery of the required enzyme. This strategy is called
directed evolution. In the work presented here the conditions were
engineered to enable the enzyme to function and/or several enzymes
were used to form an enzymatic cascade reaction to obtain an
efficient synthesis.
1.3.1 Enzyme Immobilisation
The employment of an enzyme in a synthetic chemical process may
be facilitated if the enzyme in question can be immobilised. The
advantages of immobilising the enzyme include: the initiation and
discontinuation of the reaction is easily accomplished by simple
addition and removal of the catalyst, reusability can be ensured if
the enzyme is sufficiently stable, the absence of protein in the
product can be assured and reaction condition promiscuity[39] (e.g.
the activity of an enzyme in non-aqueous conditions) can be
induced. The latter is mainly a result of structural stabilisation,
hindrance of aggregation and unfolding of the enzyme. There is no
general immobilisation method valid for all enzymes and applicable
for synthesis applications, though many methods have been
developed. E.g. enzyme molecules can be chemically cross-linked to
form what is called a cross-linked enzyme crystal or aggregate (CLEC
or CLEA), which create a material to the most part consisting of
enzyme, hence giving a high activity to volume ratio. More
straightforward methods typically include the single or multipoint
binding of enzyme to a support material. This can be done in both
reversible and irreversible ways.[40-42] None of the methods are
general in the sense that they are directly useable for any enzyme of
choice. The positioning of the active site (the binding spot of the
substrates), surface properties (i.e. hydrophobicity or reactive amino
acid residues) and flexibility are factors that are crucial for the
immobilised enzyme to function.
1.3.2 Enzymatic Synthesis of Enantiopure Amines
Amines are common motifs in pharmaceuticals. Such molecules are
also prevalent as active entities in cells. In mammals e.g. dopamine,
epinephrine (adrenaline) and serotonin are amines present as signal
7
substances. Synthesis of a wide range of enantiopure amines are of
great interest for the pharmaceutical industry. The use of enzymes,
ω-transaminases (section 1.2.2), for this purpose has been shown to
be effective[43-47] although with many obstructing parts that need to
be addressed.
Largely, one can group the synthetic transamination reactions by
denoting them as being performed in synthesis mode or resolution
mode (see Figure 3). The latter is referring to a process in which the
racemate of an amine is used; one enantiomer is removed by the
enantiospecific transaminase leaving the other, which can be
extracted in enantiopure form. In this case the maximum yield is
fifty percent. In synthesis mode the enantiopure amine is
synthesised from the corresponding ketone, a reaction which in
theory can reach a yield of one hundred percent if it is run to
completion. However, as mentioned in section 1.2.2, in synthesis
mode the equilibrium most often favours the substrates.[10]
A Resolution mode NH3
O
NH2
ω-TA
O
NH2
B Synthesis mode
O
NH2
ω-TA
NH2
O
Figure 3: Examples of two different types of transamination reactions employed in industry. In A, the reaction denoted as run in resolution mode. This equilibrium reaction greatly favours the products. In B, the reaction in synthesis mode, as described in Figure 2. Here the equilibrium is strongly shifted towards the substrates. The impediment of the transamination reaction by substrate and/or
product inhibition has been tackled by means of directed evolution
with some success.[48-50] The problem with the unfavourable
equilibrium has been partly solved by removal of the products or
large excess of amino donor, thus shifting the equilibrium.[10, 51-58]
When using the industrial chemical isopropylamine as donor
acetone is produced beside the enantiopure amine. Acetone is rather
8
volatile and can be removed by distillation. But, in larger scale
distillation is not always feasible since it requires efficient mixing
and heating. Raising the temperature also requires a heat stable
enzyme.
9
10
2 Present Investigation
2.1 Objectives
As in all fields of research, and even more so in the rather uncharted
field of biocatalysis there is a need for method development. In this
thesis two methods are presented. The enzyme immobilisation
method and the equilibrium displacement system that is described
here were solutions to problems at hand in the current work.
Immobilised metal affinity chromatography (IMAC) is a technique
commonly used for purification of proteins with a His6-tag, six
histidine residues inserted at either terminal of the protein chain.
The structure formed by the histidine residues can be compared
with heme, the iron-ion binding molecule in red blood cells. The
His6-tag is an effective binder of metal ions. This enables separation
of the tagged protein from a solution by introduction of a matrix
containing e.g. nickel or cobalt ions. It is conceivable that these
features enable the use of the His6-tag immobilisation for synthesis
applications in a variety of solvents. In this work a procedure for
His6-tag immobilisation onto a modified silica support and
subsequent reaction engagement was evaluated. The use of this
protocol for enzyme immobilisation from crude cell extract was also
tested.
ω-transaminases are generally enantioselective and have a broad
substrate range (section 1.2.2 and 1.3.2). The synthetic chemist is
thereby provided with a catalyst for producing pharmacologically
interesting amines of high enantiopurity. However, the
transamination reaction comes with many bottlenecks as mentioned
in section 1.3.2. An alternative solution to the problem of the
unfavourable equilibrium in synthesis mode was constructed, where
isopropylamine is the amino donor. An enzymatic cascade reaction
where enzyme selectivity was used to form an equilibrium
displacement system is presented in this work.
11
2.2 Enzyme Immobilisation on modified TLC plates
(Paper I)
OH
OH +
OH
O
O Si
O
O
Si
O
OH
O
OH
NH2
O
NH2
Δ
OH
O
O
O
Si
OH
N
OH
O
O
O
Si
NH2
OH
1. Co(II)
2. His6-Enzyme
O
O
O
Si
N
His
O-
His
2+
Co
-O
His
His
His
His
Figure 4: Modification of silica oxide for subsequent binding of cobalt(II) and His6‐tagged enzyme. Thin layer chromatography (TLC) plates are frequently used as a
quick analysis method for separation and visualisation of different
compounds. They are usually assembled by grafting silica oxide on a
metal surface. Here, such plates were instead used as a carrier for
enzyme. After modification the plates were able to efficiently bind
cobalt ions, and thereafter His6-tagged enzyme. The plates were
treated with 3-aminopropyltriethoxysilane, then resacetophenone
(2’,4’-dihydroxyacetophenone). At this point the colour was yellow
(due to Schiff base formation, see Figure 6). Resacetophenone is an
efficient chelator where cobalt(II) could be bound, which turned the
12
O
H3N
O
AlaR
H3N
O
D-Ala
O
L-Ala
NAD+
AlaDH
NADH,H+
O
NH3 +
O
O
pyruvate
Figure 5: Spectrophotometric assay for the racemisation of D‐alanine. The produced NADH is detected at 340 nm.
colour into deep purple. The material is porous; the area on which
the cobalt binds greatly exceeds the apparent area of the flat
surface. The reaction scheme used for the modification of the silica
is described in Figure 4. Geobacillus stearothermophilus alanine
racemase was used as a test enzyme for the immobilisation method,
which also opens for further testing of this enzyme for synthetic
purposes. The enzyme was tethered to the modified plates and
tested for activity in different conditions. In aqueous environment
(100 mM CHES buffer, pH 9.1) the enzyme was active with retained
kinetic properties compared to the solubilised free enzyme. The
assay (see Figure 5) was based on the racemisation of D-alanine and
the selective oxidation of L-alanine by L-alanine dehydrogenase
(AlaDH), producing NADH which is spectrophotometrically
detectable at 340 nm. Since an assay for non-aqueous media could
not be found (i.e. the available substrates were insoluble in organic
solvent) the solvent tolerability of the immobilised enzyme was
evaluated by immersion in different media, drying, and thereafter
testing in aqueous buffer. It was found that the enzyme was more
tolerant towards the more hydrophobic solvent hexane than ethyl
acetate, possibly due to stripping of structural water molecules from
the enzyme causing unfolding and irreversible aggregation.[59, 60]
Table 1 shows the activity of the immobilised enzyme (tested in
aqueous buffer) after immersion in n-hexane, ethyl acetate, or dry
storage in 4 °C and -20 °C.
13
Table 1: Remaining enzyme activity after immersion in solvent or cold storage. Remaining enzyme activity (%)
Time n‐hexane
ethyl acetate
4 °C
‐20 °C 15 min 100
10
100
100 1 h 100
0
100
100 4 days 53
0
100
100 4 months 0
0
100
100 This practical technique, based on the well established His6-tag, was
aimed as a first step towards a general immobilisation method for a
wide range of enzymes for synthetic applications.
Figure 6: Photograph of the modified silica oxide plates prepared for binding metal ions and thereafter His6‐
tagged enzyme.
14
2.3 Enzyme immobilisation and extraction with
modified silica oxide beads and employment in
organic solvent, HisSi-immobilisation (Paper II)
Section
2.2
describes the modification of TLC plates.
The leap to applying
the same scheme for
silica oxide microbeads is not very
long.
The
same
method as depicted
in Figure 4 was used Figure 7: Photographs after synthesis and washing of and a chelating pow- the modified silica oxide beads. On the left, the yellow der was obtained powder obtained after binding of resacetophenone, which
effectively the colour is a result of the Schiff base formation. On the right, the powder now blue after adsorption of bound cobalt(II) and
cobalt ions. His6-tagged enzyme.
This method was named HisSi-immobilisation. Figure 7 shows
photographs of the silica oxide powder after modification (yellow)
and binding of cobalt(II) (blue).
O
R1
O
R2
HO
O
R3
R1
O
R3
HO
R2
CalB: R1: n‐heptyl R2: ethyl R3: n‐hexyl BS2, PFE: R1: methyl R2: vinyl R3: 1‐phenylethyl BslA: R1: ethyl R2: methyl R3: propyl Figure 8: A general transacylation reaction. The details of the reactions are specified as definitions of the R‐groups for the different cases. The enzymes C. antarctica lipase B (CalB), B. subtilis esterase 2
(BS2), B. subtilis lipase A (BslA), P. fluorescence esterase (PFE) and
S. tubersum epoxide hydrolase (StEH1) were cloned with a His6-tag,
overexpressed in E. coli and bound to the constructed carrier matrix.
The initial tests with immobilised CalB in aqueous buffer showed
activity, as with the alanine racemase in paper I. HisSi-immobilised
CalB effectively hydrolysed 4’-nitrophenylacetate in MOPS-buffer at
pH 7.0. The possibility of using enzyme immobilised in this way for
15
chemical synthesis in organic solvent was then evaluated. The lipases and esterases were directly employed in organic solvent for
O
OH
StEH1
OH
Figure 9: Hydrolysis by StEH1 of S,S‐trans‐stilbene oxide to form meso‐hydrobenzoin. transacylation reactions (see Figure 8). CalB was used for the
acylation of 1-hexanol. With the BS2 and PFE an enantioselective
acylation of the secondary alcohol 1-phenylethanol was tested. The
epoxide hydrolase, StEH1, was used for catalysis of the reaction in
Figure 9. The powder with bound StEH1 was mixed with water to
create a paste which was used to cover the inside of a reaction
vessel; then the substrate, trans-stilbene oxide, was dissolved in
organic solvent (cyclohexane) and added. This created a two phase
system with high substrate concentration in the liquid phase. The
results of the different reactions are summarised in table 2.
Table 2: A Summary of the results from employment of HisSi‐immobilised enzyme in organic solvent. The figures show the maximum conversion achieved after 24 h. CalB
BS2
BslA
PFE
StEH1
Conversion 100%
20%
6%
5%
3% cyclohexane n‐hexane cyclohexane toluene water/cyclohexane Solvent CalB, an enzyme known for its stability in organic solvents, was
active and gave complete conversion in the chosen reaction. The
transacylation reaction was the synthesis of hexyloctanoate from
ethyloctanoate and hexanol in cyclohexane.
The 20% conversion with BS2 is rather surprising since this enzyme
never has been successfully employed in any organic solvent. The
other lipase and the esterases were inactivated with time in the
organic solvents tested. However, low conversions with enantioselectivity could be achieved (the measurement of the enantiomeric
excess, the ee (%), was not exact at these low conversions due to the
signal-to-noise ratio in the gas chromatograph). It is conceivable
that the polar surface of the silica oxide results in unfolding of the
enzyme when submersed in non-polar solvent. Still, the existence of
activity and enantioselectivity in this rather unoptimised system
16
with these bacterial enzymes is promising. For the StEH1 the
conversion was very low, but detectable. Assuming that the formed
diol can be adsorbed to the silica the actual conversion could be
higher; the detectable amount in the solvent corresponded to a
conversion of 3%.
To further simplify the handling, using the modified silica oxide
beads for binding of enzyme from a crude extract was tested. CalB
was expressed and obtained as a periplasmic preparation from E.
coli to which the powder was added and gently stirred. After
separation the powder could be directly used for synthesis. This
enables the chemist to obtain a usable catalyst without extensive
purification and/or lyophilisation. The yield of immobilised enzyme
was measured by an active site titration. The yield in this case
describes the fraction of active enzyme on the beads compared to the
loss of activity in the solution from which it was immobilised. In
other words, the yield in this case refers to the eventual loss of
activity upon immobilisation. The immobilised CalB had a yield of
58%.
17
2.4 Efficient Enzymatic Synthesis of Chiral Amines
(Paper III)
A transamination equilibrium reaction in synthesis mode with
isopropylamine as donor produces acetone as a by-product; removal
of the acetone and thus shifting of the equilibrium was performed
with YADH This alcohol dehydrogenase is selective for small alcohols
and ketones. The amine acceptor is most often a larger ketone,
hence unaffected by the presence of YADH..
O
NH2
NH2
ω-TA
O
O C O
NADH,H+
YADH
FDH
NAD+
OH
-O
O
Figure 10: A transamination reaction with isopropylamine as donor and acetophenone as acceptor, catalysed by an ω‐transaminase (ω‐TA). The unfavourable equilibrium is displaced by selectively removing the formed acetone with yeast alcohol dehydrogenase (YADH, S. cerevisiae) and regenerating the cofactor NADH with C. boidinii formate dehydrogenase (FDH). This is an example of utilisation of enzyme selectivity for practical
use in an enzymatic cascade reaction. The cofactor NADH was
regenerated with FDH, producing carbon dioxide which effectively
drives the reaction to completion. Figure 10 shows such a displaced
transamination reaction where the acceptor is acetophenone, the
desired product in this case is (S)-methylbenzylamine.
The gene for an A. citreus ω-transaminase variant (Cambrex
Karlskoga AB) was cloned and successfully overexpressed in E. coli
BL21(DE3), and purified by IMAC. The use of purified enzyme
facilitated the measurement of kinetic parameters and the influence
of excess concentration of the cofactor PLP. It was found that
exceeding the concentration beyond that of the enzyme was
detrimental to the reaction. Figure 11 shows the activity versus the
excess concentration of PLP, it is clear that the activity is greatly
decreased with increasing amount of cofactor. Since the enzyme
concentration is usually very low, in the micromolar range, this
effect is of importance. Already at millimolar concentrations the PLP
will practically completely inhibit the proceeding of the reaction.
18
Rate (U/mg)
1
0.8
0.6
0.4
0.2
0
0
200
400
[PLP]/[E] Figure 11: The graph shows the lowering in activity as a result of excess [PLP], i.e. adding more PLP than enzyme. The reason for this effect remains unknown; it is plausible that PLP
(which is an aldehyde) can function as a good amine acceptor and
thereby inhibit the reaction with the desired acceptor. It should be
noted that the figure shows initial rates, the complete progress of
the reaction with different concentrations of PLP was not evaluated.
[acetophenone] mM
A balanced ratio of PLP to enzyme was obtained by dissolving an
excess of PLP with the purified enzyme solution and then changing
the buffer. Assuming that no enzyme dissociates from the PLP
during this process this provides an enzyme ready for use with the
correct amount of cofactor. No cofactor was thereafter added in the
synthesis reactions.
2
Equilibrium reaction
Displaced reaction
1
0
0
6
12
18
Time (h)
Figure 12: Comparison of a displaced versus a non‐displaced transamination reaction, followed spectrophotometrically by the consumption of acetophenone. 19
The equilibrium displacement system was verified with acetophenone as the amine acceptor. Though it is a poor substrate, it was
chosen because the reaction can easily be followed spectrophotometrically.[61] Figure 12 shows a comparison of the displaced versus the
non-displaced reaction as the consumption of acetophenone over
time. The displaced reaction continues towards completion while the
non-displaced stops at equilibrium conversion. Here the initial
concentration of acetophenone was low, only 1.8 mM, hardly a
useable large scale process. This concentration was chosen to keep
the starting absorbance at a reasonable level and to have a practical
equilibrium conversion point for comparison. Even with this large
excess of amine donor, 564 mM of isopropylamine was used, the
conversion (without displacement) only reached 82%. The amount of
formed product, (S)-methylbenzylamine, and the enantiomeric
excess was measured with high pressure liquid chromatography
(HPLC). Indeed, the displaced reaction had virtually reached
completion and the product amine was enantiomerically pure.
The displacement system was explored for other amino acceptors.
Table 3 shows the results; with 20 mM substrate concentration the
displaced reactions also reached completion.
Table 3: Results from synthesis reactions of four different amines, with
and without the displacement system. Product amine eep (%) Conversion (%) No displacement Conversion (%) with displacement NH2
a
>99 b
>99 c
>99 c
>99 >99.9 82 >99.9 63 >99.9 89 >99.9 68 a
NH2
b
O2N
NH2
c
NH2
HO
c
OH
a
b
1.8 mM acetophenone. Conversions were calculated based on formed product. 20 mM 4‐nitroacetophenone (two phase system as saturation is reached at ~2 c
mM). Conversions were calculated based on formed product. 20 mM ketone substrate. Conversions were calculated based on consumed substrate. 20
3 Conclusions
3.1 Short summary of the publications
Largely, this thesis describes two new tools in the field of
biocatalysis:
Paper I and II: An employment expansion of the His6-tag
for use in synthesis applications is presented. By using
modified silica oxide for binding cobalt(II) and thereafter
His6-enzyme a versatile carrier matrix was created, which
is also applicable as a one step extraction and
immobilisation matrix. This provides a rapid and facile
means of obtaining an enzyme catalyst in a suitable form
for use in synthesis applications.
Paper III: An equilibrium displacement system for
shifting the otherwise unfavourable equilibrium reaction
with ω-transaminases in synthesis mode for production
of pharmaceutically important enantiopure amines was
devised. Isopropylamine as donor is preferably used by
the industrialist. The formed acetone in such a reaction
is selectively removed by yeast alcohol dehydrogenase, in
the presence of larger ketones. With cofactor regeneration
where carbon dioxide is irreversibly produced the
equilibrium can be fully shifted to the desired products.
3.2 Discussion and future outlook
The immobilisation method which has been devised here is based on
the use of silica oxide, not a cost efficient chemical. Still the process
is suitable for evaluation and small scale synthesis. The same idea
can be employed with another carrier, e.g. porous glass or an
alternative material that can be tailored into a chelator. Further
development of the technique is needed, what is shown here is the
facile usage of the His6-tag for enzyme extraction and immobilisation
for synthesis applications. The method includes the use of cobalt(II),
which is toxic. Use of other metal ions, e.g. iron(II), will offer a more
environmentally sustainable method should they provide enough
affinity. Cobalt(II) was the only tested ion in the experiments here.
21
Many times the practical threshold of obtaining, purifying and
immobilising the protein catalyst deters the organic chemist from
starting the experiments. Another catalyst might be the final choice,
from which the entire process is later optimised. This method could
be the basis for facilitating the use of enzymes rather than other less
environmentally sustainable alternatives.
The synthesis of enantiopure amines is a topic into which much
research effort is currently invested. Already biocatalytic synthesis of
enantiopure amines is employed in industry, still with room for a lot
of improvement and further development. The work done here is an
example of how an efficient synthesis can be made; another tool in
the box. A plethora of pharmaceuticals and fine chemicals contain
chiral amines, and more could be made with increased availability of
methods of their production.
Biocatalysis as a research area is in a sense in a battle of proving its
worth and value as opposed to established methods in organic
chemistry. New successful methods are needed to show that
enzymes can perform as catalytic tools that are in deed industrially
feasible, to open for more investment. Biocatalysis is predicted to be
a vital part in meeting the increasing environmental responsibility
put on the chemical industry.
22
4 Acknowledgements
I would like to express my sincere gratitude to my supervisor, Prof.
Per Berglund, for kindness, patience, scientific teaching and
pedagogic approach, and for providing an open and very stimulating
work environment. Thanks for letting me test my crazy ideas! Maybe
I have some more in store…
Prof. Karl Hult, my teacher in biochemistry (among other things)
since I started at KTH, has always kept the main focus on teaching
and learning more, a great scientist and humanist. I never stop
learning from you. Thanks!
My office mates! Anders, thanks for rocking! And for welcomed
musical breaks after office hours. Magnus (now Doc!), thanks for
listening to my grunts and providing yours, and all the funny signs
in the lab. Cecilia Hedfors, thanks for always smiling and explaining
techs and methods.
Ol’ members of the biocat group, thanks for lunches and coffee
breaks! Maria, thanks for the great shift-work and for being a great
travelling companion! Marianne, I’m still angry that you made me
run Midnattsloppet and missing it yourself, but I like you anyway.
Thanks for teaching me about Danes and beer. Linda, you’re the
most awesome physicist to ever become chemist! Thanks for
enlightening me with more doubts about the world. Per-Olof, since
HPR, no more sterilisations of fridges and unwilling alcohol
intoxications. Mats, thanks for good advice and discussions!
Mohamad, thanks for being bearded like me!
My great Erasmus students! You make coming to work a true joy!
Carla, there’s no salsa like chimichurri for microwaved meat balls.
Valentina, I hope you find a bicycle wherever you go! Silvia, enzymes
are great, especially ADH!
Co-authors (here, there, AstraZeneca, Cambrex, and elsewhere, not
named now) and all the people on the floors 1, 2 and 3, and “the
other” buildings, thanks for helping out! Oliver, amasing that you
always have a minute to spare, thanks a lot! Lotta, you are the
queen of f-n everything, the one and only! Ela, without you I would
23
still be looking for my socks in the tumble dryer, thanks, you are
great!! Erik, or should I say Markoolio, or DaddyCool? Since Bio-00
until Dr, let’s do it! The same goes for Jens, who knows about
loosing keys and finding couches, let’s do it! Henrik, sing it baby!
Harry, you always have time for a good explanation, thanks.
Christian, the duke is always ready!! Gea, you make a great
Japanese curry! (Yes, I do appreciate a good curry.) Johanna, the lab
could not survive without your cakes! Fredrika, you will conquer the
world with science, or run around it ten times. Cortwa, you’re
almost as nutty as me, smiling Cortwa! Nomchit, great Christmas
curry! (I love curry!) Johan, thanks for understanding the enjoyment
of living in the HPLC-room. Felicia, Panthera leo Leijon of the
Felidae? Qi, thanks to you I have a UV-detector, great! Gustav, the
MASSive man, thanks for all your efforts! Marcus, thanks for talks
and help in dark moments! Martin, AlaR to ω-TA, PLP for ever man!
Anton, there’s no other way to party, the dude! Fahmi, we shall
cooperate! Either in chemistry or at re-orient. Ernesto, I can
exchange some enzyme for Spanish lessons, ok?
All my friends! I love you guys and gals! No names here, but you
already know who you are! (Elsewhere, and at AlbaNova) You helped
out a lot with my work by talking about completely different things
than science. Thanks!!
I thank my mom for always being a great support and
understanding everything except science. I thank dad for cooking
the best curry on the planet, and feeding me after late work days.
(There’s nothing in the world as good as curry!)
Julia! My wonderful crazy cat!! Thanks for this year, and all the
years to come. I love you!
Financial support from The Swedish Governmental Agency for
Innovation Systems (VINNOVA) is gratefully acknowledged. EUCOST Action CM0701 is acknowledged for travel funds and for the
opportunity to join the COST Training School in Cascade
Chemoenzymatic Processes – New Synergies between Chemistry and
Biochemistry - in Italy 2009. KTH is acknowledged for travelling
scholarships in 2006, 2007 and 2008 that enabled conference
participation.
24
5 References
[1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] D. Voet, J. G. Voet, C. W. Pratt, Fundamentals of Biochemistry: Life at the Molecular Level, 2nd ed., Wiley, 2006. T. Golson, B. Fink, The Farmstead Egg Cookbook, St. Martin's Press, 2006. T. C. Bruice, Chem Rev 2006, 106, 3119. A. Warshel, P. K. Sharma, M. Kato, Y. Xiang, H. Liu, M. H. Olsson, Chem Rev 2006, 106, 3210. M. Jones, Organic Chemistry, 2nd ed., W. W. Norton & Company, Inc., New York, 2000. Y. S. Ding, J. S. Fowler, Drug Dev Res 2003, 59, 227. N. Ran, E. Rui, J. Liu, J. Tao, Curr Pharm Des 2009, 15, 134. J. Steinreiber, K. Faber, H. Griengl, Chem Eur J 2008, 14, 8060. R. B. Silverman, The Organic Chemistry of Enzyme‐Catalysed Reactions, Academic Press, Elsevier Science, 2000. J. S. Shin, B. G. Kim, Biotechnol Bioeng 1999, 65, 206. T. Boekhout, J Gen Appl Microbiol 1995, 41, 359. J. Uppenberg, N. Öhrner, M. Norin, K. Hult, G. J. Kleywegt, S. Patkar, V. Waagen, T. Anthonsen, T. A. Jones, Biochemistry 1995, 34, 16838. T. Tanaka, E. Ono, M. Ishihara, S. Yamanaka, K. Takinami, Agric Biol Chem 1981, 45, 2387. M. Takwa, Y. Xiao, N. Simpson, E. Malmstrom, K. Hult, C. E. Koning, A. Heise, M. Martinelle, Biomacromolecules 2008, 9, 704. M. Eriksson, L. Fogelström, K. Hult, E. Malmström, M. Johansson, S. Trey, M. Martinelle, Biomacromolecules 2009, 10, 3108. M. Martinelle, K. Hult, Biochim Biophys Acta 1995, 1251, 191. M. Svedendahl, K. Hult, P. Berglund, J Am Chem Soc 2005, 127, 17988. C. Branneby, P. Carlqvist, A. Magnusson, K. Hult, T. Brinck, P. Berglund, J Am Chem Soc 2003, 125, 874. P. Carlqvist, M. Svedendahl, C. Branneby, K. Hult, T. Brinck, P. Berglund, ChemBioChem 2005, 6, 331. M. Svedendahl, P. Carlqvist, C. Branneby, O. Allner, A. Frise, K. Hult, P. Berglund, T. Brinck, ChemBioChem 2008, 9, 2443. 25
[21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] R. Kourist, S. Bartsch, L. Fransson, K. Hult, U. T. Bornscheuer, ChemBioChem 2008, 9, 67. M. Schmidt, E. Henke, B. Heinze, R. Kourist, A. Hidalgo, U. T. Bornscheuer, Biotechnol J 2007, 2, 249. R. Kourist, S. Bartsch, U. T. Bornscheuer, Adv Synth Catal 2007, 349, 1393. S. Bartsch, R. Kourist, U. T. Bornscheuer, Angew Chem Int Ed Engl 2008, 47, 1508. G. van Pouderoyen, T. Eggert, K. E. Jaeger, B. W. Dijkstra, J Mol Biol 2001, 309, 215. U. T. Bornscheuer, J. Altenbuchner, H. H. Meyer, Biotechnol Bioeng 1998, 58, 554. M. Schmidt, D. Hasenpusch, M. Kahler, U. Kirchner, K. Wiggenhorn, W. Langel, U. T. Bornscheuer, ChemBioChem 2006, 7, 805. S. Park, K. L. Morley, G. P. Horsman, M. Holmquist, K. Hult, R. J. Kazlauskas, Chem Biol 2005, 12, 45. E. J. de Vries, D. B. Janssen, Curr Opin Biotechnol 2003, 14, 414. B. van Loo, J. Kingma, M. Arand, M. G. Wubbolts, D. B. Janssen, Appl Environ Microbiol 2006, 72, 2905. K. Inagaki, K. Tanizawa, B. Badet, C. T. Walsh, H. Tanaka, K. Soda, Biochemistry 1986, 25, 3268. V. Leskovac, S. Trivic, D. Pericin, FEMS Yeast Res 2002, 2, 481. H. Zhao, W. A. van der Donk, Curr Opin Biotechnol 2003, 14, 583. M. Kircher, Biotechnol J 2006, 1, 787. W. Soetaert, E. Vandamme, Biotechnol J 2006, 1, 756. C. Khosla, P. B. Harbury, Nature 2001, 409, 247. F. H. Arnold, Nature 2001, 409, 253. A. Schmid, J. S. Dordick, B. Hauer, A. Kiener, M. Wubbolts, B. Witholt, Nature 2001, 409, 258. K. Hult, P. Berglund, Trends Biotechnol 2007, 25, 231. D. Brady, J. Jordaan, Biotechnol Lett 2009, 31, 1639. L. Cao, Curr Opin Chem Biol 2005, 9, 217. U. T. Bornscheuer, Angew Chem Int Ed Engl 2003, 42, 3336. M. Höhne, U. T. Bornscheuer, ChemCatChem 2009, 1, 42. D. Koszelewski, D. Pressnitz, D. Clay, W. Kroutil, Org Lett 2009, 11, 4810. D. Koszelewski, I. Lavandera, D. Clay, G. M. Guebitz, D. Rozzell, W. Kroutil, Angew Chem Int Ed Engl 2008, 47, 9337. K. O. Lidström, PharmaChem 2007, 6, 5. 26
[47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] [60] [61] D. Koszelewski, M. Göritzer, D. Clay, B. Seisser, W. Kroutil, ChemCatChem 2010, 2, 73. H. Yun, B. Y. Hwang, J. H. Lee, B. G. Kim, Appl Environ Microbiol 2005, 71, 4220. B. Y. Hwang, B. G. Kim, Enzyme Microb Technol 2004, 34, 429. A. R. Martin, R. DiSanto, I. Plotnikov, S. Kamat, D. Shonnard, S. Pannuri, Biochem Eng J 2007, 37, 246. M. Höhne, S. Kuhl, K. Robins, U. T. Bornscheuer, ChemBioChem 2008, 9, 363. M. D. Truppo, J. D. Rozzell, J. C. Moore, N. J. Turner, Org Biomol Chem 2009, 7, 395. D. Koszelewski, I. Lavandera, D. Clay, D. Rozzell, W. Kroutil, Adv Synth Catal 2008, 350, 2761. M. D. Truppo, N. J. Turner, J. D. Rozzell, Chem Commun (Camb) 2009, 2127. P. P. Taylor, D. P. Pantaleone, R. F. Senkpeil, I. G. Fotheringham, Trends Biotechnol 1998, 16, 412. G. Matcham, M. Bhatia, W. Lang, C. Lewis, R. Nelson, A. Wang, W. Wu, Chimia 1999, 53, 584. W. Wu, M. Bhatia, C. M. Lewis, W. Lang, A. L. Wang, G. W. Matcham, C12P 1300; C12P 1302; C12P 1304; C12P 1322; C12P 1324 ed., Celgro, US, 1999. Le Chatelier's principle, If a chemical system at equilibrium experiences a change in concentration, temperature, volume, or partial pressure, then the equilibrium shifts to counteract the imposed change and a new equilibrium is established. L. A. Gorman, J. S. Dordick, Biotechnol Bioeng 1992, 39, 392. L. Yang, J. S. Dordick, S. Garde, Biophys J 2004, 87, 812. S. Schätzle, M. Höhne, E. Redestad, K. Robins, U. T. Bornscheuer, Anal Chem 2009, 81, 8244. 27