Performance in synthetic applications of a yeast surface display-based biocatalyst

Article abstract of DOI:10.1039/c5ra04039f

This work demonstrates the effectiveness of yeast surface display (YSD) as a scaffold for biocatalysts in hydrophobic, non-aqueous environments. Two lipases, Candida antarctica lipase B (CalB) and Photobacterium lipolyticum sp. M37 lipase (M37L), were immobilized independently by surface display on Saccharomyces cerevisiae. The two YSD biocatalysts were employed to synthesize esters of butanol and saturated fatty acids of varying length (8 to 16 carbons) in heptane. Effects of fatty acid chain length and temperature on the esterification reaction were examined. The YSD catalysts synthesized butyl decanoate in 10 repeated batches with little loss in activity. Compared to a commercial immobilized lipase (Novozym 435), the activity of both YSD lipases was lower on a mass loading basis, but higher when normalized on estimates of protein loading. Initial-rate kinetics of the butyl decanoate reaction were measured for the CalB-displaying yeast. Kinetics and apparent activity of M37L in the multi-batch experiments depend heavily on water concentration; kinetics for M37L could not be elucidated with initial-rate methods. The difference between CalB and M37L in water requirements illustrates a critical parameter for optimization of lipase activity in non-aqueous environments. The activity of both lipases in a completely hydrophobic environment is a step towards more economical biocatalysis of industrial esterification.

Full text of DOI:10.1039/c5ra04039f

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Performance in synthetic applications of a yeast
surface display-based biocatalyst
Cite this: RSC Adv., 2015, 5, 30425
J. M. Eby and S. W. Peretti*
This work demonstrates the effectiveness of yeast surface display (YSD) as a scaffold for biocatalysts in
hydrophobic, non-aqueous environments. Two lipases, Candida antarctica lipase
B (CalB) and
Photobacterium lipolyticum sp. M37 lipase (M37L), were immobilized independently by surface display on
Saccharomyces cerevisiae. The two YSD biocatalysts were employed to synthesize esters of butanol and
saturated fatty acids of varying length (8 to 16 carbons) in heptane. Effects of fatty acid chain length and
temperature on the esterification reaction were examined. The YSD catalysts synthesized butyl
decanoate in 10 repeated batches with little loss in activity. Compared to a commercial immobilized
lipase (Novozym 435), the activity of both YSD lipases was lower on a mass loading basis, but higher
when normalized on estimates of protein loading. Initial-rate kinetics of the butyl decanoate reaction
were measured for the CalB-displaying yeast. Kinetics and apparent activity of M37L in the multi-batch
experiments depend heavily on water concentration; kinetics for M37L could not be elucidated with
initial-rate methods. The difference between CalB and M37L in water requirements illustrates a critical
parameter for optimization of lipase activity in non-aqueous environments. The activity of both lipases in
a completely hydrophobic environment is a step towards more economical biocatalysis of industrial
esterification.
Received 6th March 2015
Accepted 24th March 2015
DOI: 10.1039/c5ra04039f
www.rsc.org/advances
Lipases are chiral catalysts and can catalyze the hydrolytic
resolution of racemic mixtures. In addition, absent water, they
Introduction
7,8
Esters are valuable commodities with applications in the can catalyze the formation of ester bonds through esterication
9,10
fragrance, personal care, ne chemical, and energy industries. of a carboxylic acid or trans-esterication of a donor ester.
They are most oen produced from crude alcohols and organic
Lipase activity requires some structural exibility. In nature,
11
acids or esters using an acidic or basic inorganic catalyst. lipases are activated by interfacial contact, which causes a
Production of vinyl acetate, for example, involves reaction small conformational change in the enzyme's tertiary struc-
ꢀ
1
12,13
temperatures in excess of 100 C and corrosive materials.
Downstream cleanup is required to remove the acid/base and are in an active conguration when water is removed,
ture.
Many enzymes are functional in organic solvents if they
14
2
,3
6,15
their byproducts, making purication a major cost driver.
including lipases. A small amount of water may be required
Aggressive removal of water and excess reagents is required to to hydrate the enzyme, but the amount is small enough to
achieve commercially relevant conversions, further increasing have little or no effect on the reaction equilibrium.
process complexity and cost. Methods utilizing supercritical
Lipases have demonstrated activity in organic solvents such
solvents and ionic liquids, though promising, are still many as hexane and heptane and aqueous solutions as free (soluble)
16
17
4,5
15,18,19
years away from being commercially viable.
Enzymatic esterication is an alternative to traditional by immobilization, and immobilization methods oen have
and immobilized enzymes.
Catalyst recycling is facilitated
20
catalysis that has lower energy requirements (lower tempera- positive impacts on thermal and physical stability. Moreover,
tures and pressures), generates less waste (no excess reagents or puried enzyme cost is a barrier to industrial implementation
byproducts), and represents “greener” (biodegradable, non- of biocatalysis. Purication and immobilization account for as
toxic) catalysis. Lipases catalyze a wide range of hydrolytic and much as 80% of the cost of the enzyme, so reduction of these
synthetic reactions, and are widely used in industry. They costs signicantly lowers the barrier for wider industrial appli-
21
6
2
evolved as esterases, and readily cleave the ester bonds in cation. Yeast surface display (YSD) represents a high degree
triglycerides, oen the rst step in biodiesel production. of process integration, combining production, purication,
and immobilization of recombinant protein into a single
22,23
operation.
Surface display (SD) has been applied to several chemistries,
North Carolina State University, Department of Chemical and Biomolecular
Engineering, 911 Partners Way, Raleigh, NC 27695-7905, USA. E-mail: peretti@
ncsu.edu
24
including chiral resolution by hydrolysis,
cellulose
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25
23,26,27
degradation, and synthesis of esters.
However, reports of
SD-catalyzed syntheses are few, and many require reaction
27
28–31
times on the order of days or relatively polar reactants.
Here, we demonstrate the ability of two lipases—lipase B from
Candida antarctica (CalB) and lipase from Photobacterium lip-
olyticum M37 (optimized for yeast expression, ycM37L)—in
surface display systems to synthesize fatty acid esters from fatty
acids and alcohols. A systematic characterization of YSD esteri-
cation dependence on water content, fatty acid chain length,
and temperature was performed using Novozym 435 (N435) as a
reference standard, with an additional focus on the stability of
YSD activity in response to repeated usage. Despite surface
protein loading levels that are approximately three orders of
magnitude below that of Novozym 435, these YSD-catalyzed
reactions reach signicant conversion levels in a matter of hours.
Fig. 2 Synthesis of butyl decanoate with the YSD biocatalyst. Solutions
of butanol and decanoic acid (both 100 mM in heptane) were
combined with dry yeast biocatalyst (0.4% w/v). The reactions were
stirred in a temperature-controlled water bath for six hours at 200
rpm. The values represent the average of three experiments, with
Results
Temperature dependence of the synthesis reaction
The temperature optima for YSD biocatalyzed synthesis of butyl standard deviations (vertical bars).
dodecanoate (laurate; Fig. 1), butyl decanoate (caprate, Fig. 2),
and butyl octanoate (caprylate; Fig. 3) were determined. Equi-
molar solutions of butanol and either octanoic, decanoic, or formed, which suggests lower thermal stability than that of
ꢀ
dodecanoic acid were stirred at 30, 40, 60, or 80 C for six hours. CalB. This result is unsurprising given the psychrophilic nature
Conversion of the fatty acid to its butyl ester was determined at of the source organism.
the end point.
The N435 resin reached equilibrium within six hours at every
The optimum temperature for the CalB YSD biocatalyst was temperature tested, and no thermal deactivation was observed
ꢀ
near 60 C for octanoic and decanoic acid, but no clear in the single batches used here. While changes in the fatty acid
optimum appeared for dodecanoic acid. The conversion at concentration were observed in some of the untransformed
ꢀ
ꢀ
6
0 C was more than twice that at 30 C for octanoic acid, but EBY100 samples, no product was detected in any of the
constant for dodecanoic acid. A slight decrease in conversion at samples.
ꢀ
8
0 C suggests potential thermal deactivation.
The optimum temperature for the ycM37L YSD biocatalyst
Re-use of the YSD biocatalyst
ꢀ
was near 40 C, above which temperature a steady decrease in
A signicant aspect of the commercial application of enzyme
catalysts is robustness. To demonstrate this, the YSD
ꢀ
activity was observed. At 80 C, essentially no product was
Fig. 1 Synthesis of butyl dodecanoate with the YSD biocatalyst. Fig. 3 Synthesis of butyl octanoate with the YSD biocatalyst. Solutions
Solutions of butanol and dodecanoic acid (both 100 mM in heptane) of butanol and octanoic acid (both 100 mM in heptane) were
were combined with dry yeast biocatalyst (0.4% w/v). The reactions combined with dry yeast biocatalyst (0.4% w/v). The reactions were
were stirred in a temperature-controlled water bath for six hours at stirred in a temperature-controlled water bath for six hours at 200
200 rpm. The values represent the average of three experiments, with rpm. The values represent the average of three experiments, with
standard deviations (vertical bars). standard deviations (vertical bars).
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biocatalysts were subjected to ten butyl decanoate synthesis dry catalyst (at equilibrium with the ambient humidity). The
ꢀ
batch reactions at 40 C. The temperature was chosen to prevent water content and its impact on the esterication activity
thermal deactivation of ycM37L. The catalysts were separated needed to be titrated, so the butyl decanoate reaction was run
from the reactor medium by centrifugation and used in again with dry catalyst (freshly prepared, not equilibrated with
subsequent batches without further processing. Both YSD ambient humidity) and water was added to match the theoret-
catalysts produced the ester product in all batches (Fig. 4). The ical production at batches 2, 6, and 8 (Fig. 5). The specic
ycM37L biocatalyst went through an initial 2-batch activation conversion does not match the theoretical water content exactly,
period, aer which it maintained fairly steady activity through but a clear trend is visible. Therefore, the lag in activity for
the end of the trial. The CalB biocatalyst held at or above the ycM37L is due to a lack of water necessary for enzyme exibility.
activity of its rst batch through 8 batches, aer which a gradual The decrease in activity for ycM37L and CalB is the result of
decrease to 60% of the peak activity was observed. A similar accumulated water—produced by the reaction—partitioning to
gradual decline was observed in the nal two batches for the hydrophilic cell surface and dramatically reducing the
ycM37L (to 80% of maximum). N435 retained its activity with quality of mixing. In other experiments with 0.5% water added
little variation through ten batches. Some small decrease in the directly to the YSD catalyst, little (if any) dispersion was
fatty acid concentration in EBY100 trials was observed, but no observed, and the activity was uniformly low (data not shown).
product ester was formed.
Dispersion of the CalB and ycM37L yeast catalysts in the
reaction began changing visibly at the h batch. In the initial
batches, the cells had been well-suspended, resulting in a turbid
reaction medium with few visible particles. Aer ve batches,
decanoic acid were combined in equimolar solutions at
the cells began to aggregate and fall out of suspension without
vigorous mixing. This phenomenon is examined in more detail
in the Discussion. Most of the aggregates could be re-dispersed
Kinetics of butyl decanoate synthesis
Initial-rate kinetics of YSD-biocatalyzed butyl decanoate
ꢀ
syntheses in heptane were measured at 40 C. Butanol and
concentrations between 25 and 250 mM. The concentration of
decanoic acid was monitored by GC for 30–60 minutes. Initial
rate data (Fig. 6) were used to generate a Lineweaver–Burke plot
in fresh reagent solution, but this became more difficult as the
(
Fig. 7) for synthesis catalyzed by YSD CalB. The vmax for the
number of batches increased. A similar effect was observed in
experiments with water added. The changes in dispersion were
hypothesized to be the result of water produced by the reaction.
Incomplete mixing, as indicated by poor YSD dispersion, is
proposed to be the cause of the changes in activity observed in
ycM37L and CalB. No changes in dispersion or activity were
observed in yeast without displayed lipase.
À1
reaction is 1.9 mM min , with a K
m
near 70 mM.
The rate of synthesis catalyzed by YSD ycM37L was effectively
zero without water. That is, no decanoic acid was consumed
(Fig. 8A). When water was added, to 0.4% v/v as in Fig. 5, non-
zeroth-order behavior was observed (Fig. 8B). At all substrate
concentrations tested, a lag time of approximately 15 minutes
was observed where the rate was near zero. This addition of
water gives rise to behavior that is not consistent with standard
enzyme kinetic mechanisms.
A small amount of water is oen necessary for enzymatic
32
activation in organic solvents, but the trials were initiated with
Fig. 4 Synthesis of butyl decanoate with the YSD biocatalyst in
ꢀ
multiple batch reactions at 40 C. Solutions of butanol and decanoic
acid were combined (to 100 mM in heptane) with dry catalyst at 0.5% Fig. 5 Changes in apparent activity of the YSD biocatalyst at different
w/v. After stirring at 200 rpm for six hours, the catalyst was removed water concentrations. The activity of the biocatalyst in the synthesis of
from the reaction by centrifugation and the liquid fraction was butyl decanoate was measured with water added in concentrations
sampled. The catalyst was used in the subsequent batch without that matched the theoretical water production of selected batches
further treatment.
from the multi-batch synthesis experiments.
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0
Fig. 6 Plot of initial rate (V ) vs. decanoic acid concentration for YSD
CalB-catalyzed synthesis of butyl decanoate. Substrates (equimolar
butanol and decanoic acid) and biocatalyst (0.5% w/v) were equili-
brated at 40 C, combined, and stirred at 200 rpm. Values and error
bars are the averages and standard deviations, respectively, of three
samples.
Fig. 9 Synthesis of decanoate esters with various alcohols. Solutions
of alcohol and decanoic acid (both 100 mM in heptane) were
combined with dry catalyst, 0.4% w/v. Water was slowly added to 0.4%
v/v, unless otherwise indicated. *From Fig. 5, 0.5% w/v.
ꢀ
Table 1 Solubility of decanoate ester reactants in water
53
À1
ꢀ
Reactant
Solubility in water (g kg at 25 C)
1
1
1
-Butanol
-Hexanol
-Octanol
79
5.9
0.46a
Decanoic acid
0.15
a
ꢀ
Solubility at 20 C.
Fig. 7 Lineweaver–Burke plot of inverse initial rates vs. inverse
substrate concentrations from (Fig. 5). The linear least-squares
2
regression has an R of 0.98. The values for Vmax and K
m
were derived
from the equation of the trend line.
Fig. 10 Butyl decanoate synthesis with doubled catalyst loading at
ꢀ
4
0 C. Solutions of butanol and decanoic acid were combined (to 100
mM in heptane) with dry catalyst at 0.4% w/v (1Â) or 0.8% w/v (2Â).
Water was slowly added (to 0.5% v/v) and the reaction was stirred at
Fig. 8 Butyl decanoate synthesis with YSD ycM37L. (A) Without water,
200 rpm. Samples were taken after 6 and 24 hours.
the reaction was too slow to measure initial rates. Water was added to
0
.4% v/v, and equilibrium was reached within 24 hours. (B) Plot of initial
rate (V ) vs. decanoic acid concentration for YSD ycM37L-catalyzed
0
synthesis of butyl decanoate. For all trials, substrates (equimolar
butanol and decanoic acid) and biocatalyst (0.5% w/v) were equili-
brated at 40 C, combined, and stirred at 200 rpm. Where indicated,
results with hexanol and octanol suggest that a threshold of
solubility or diffusion has been reached. The solubility of each
component in water is presented in Table 1.
ꢀ
water was slowly added to 0.4% v/v to initiate the reaction.
The effects of catalyst loading on butyl decanoate synthesis
were determined. The conditions from batch 6 of the re-use
experiment (Fig. 4) were approximated and run in parallel
with identical conditions but with doubled catalyst loading
Effects of alcohol hydrophobicity and catalyst loading
To examine the effects of alcohol hydrophobicity on the ester-
ication reaction, decanoic acid was esteried with 1-hexanol
and 1-octanol (Fig. 9). While a general trend is observed—lower
conversion with longer alcohol—the similarity between the
(Fig. 10). Doubling the amount of catalyst increases the speed
with which the YSD biocatalyst converts the fatty acid by a factor
of at least 1.4 (ycM37L), up to a factor of 2 (CalB). The nal
30428 | RSC Adv., 2015, 5, 30425–30432
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equilibrium conversion appears unaffected, and no changes preference for fatty acids shorter than C12,
RSC Advances
39–41
although there are
were observed in the control or the N435 standard.
some reports of preference for shorter acyl donors in more polar
42
solvents. While it is oen referred to as C. antarctica lipase, its
behavior appears to differ from that of the wild-type CalB used in
this study. The difference may be attributable to protein loading
or surface charge of the support. The weak correlation between
longer aliphatic alcohols and lower esterication activity suggests
that turnover of the fatty acid is the rate-limiting step in the
esterication reaction. The relative hydrophobicity of the reac-
tants (Table 1) compared to the yeast surface implies a diffusion
limitation, the mechanism for which would take signicant effort
to clarify. An upper limit on the accessibility of longer fatty acids
to the active site might explain the plateau in CalB activity for
dodecanoic acid in Fig. 1.
Discussion
Comparison of catalyst protein loading
The YSD catalysts and Novozym 435 standard were used in the
esterication reactions with equal mass loadings. However,
apparent differences in activity, stability, and specicity may be
due, to a degree, to disparities in enzyme loading on the
respective supports. Protein loading for N435 is not available
33
from the manufacturer or vendors, but a published estimate of
0 mg enzyme per gram of catalyst will be considered for scale.
Previous estimates for the enzyme copy number in the Aga2
3
34
system were on the order of 50 000, which supports our
35
5
measurements (lit., 2–5 Â 10 ). Given the molecular weights of Temperature optima for ester synthesis
CalB and ycM37L (33 and 38 kDa, respectively), and assuming
The lipase-catalyzed esterication of fatty acids (Fig. 1–3) to their
36
20 billion cells per gram of dry weight, 70% of which are
respective butyl esters was carried out at temperatures from 30 to
35
expressing the lipases, the protein loading for the YSD system
was measured. The protein loading in the YSD system (37 mg per
gdw (g dry weight) for CalB and 19 mg per gdw for ycM37L) is
three orders of magnitude lower than in N435. If the YSD bio-
catalyst activity is only an order of magnitude lower (i.e., equi-
librium in ꢁ24 hours vs. 1–2 hours for N435 [data not shown]),
this would suggest that the lipase in the YSD system is more
efficiently accessed. The YSD system has a higher surface-to-
volume ratio, and most of the protein is exposed to the
solvent, rather than absorbed in a pore in the commercial resin.
Increasing the catalyst loading increases the rate of conversion,
as expected (Fig. 10). Expressing more protein on the surface of
the cell via YSD should, therefore, increase the specic activity
of the biocatalyst. The current system—a single-copy episomal
plasmid engineered for a-agglutinin fusion—is convenient for
the facility of its characterization, but other display systems or
hosts (e.g., Pichia pastoris) might yield higher activity. N435's
higher loading might be a buffer against thermal degradation
and protein desorption, which would be a benet, but excess,
inaccessible protein also adds expense.
ꢀ
80
C. For either fatty acid, the optimum temperature for
synthesis (i.e., that at which the highest conversion was reached
ꢀ
ꢀ
in six hours) was 60 C for the CalB biocatalyst and 40 C for
ycM37L, except in the butyl decanoate reaction without water,
ꢀ
where the apparent optimum temperature was 30 C. The
difference could be due to batch-to-batch variation in the amount
of ambient moisture absorbed, further demonstrating the
sensitivity of the system to water content. A relatively low Topt is
expected for ycM37L because it comes from a psychrophile;
ꢀ
however, the esterication optimum is 15 higher than that
38,43
previously reported for hydrolysis.
The ycM37L biocatalyst
also experiences a sharp decline in activity at higher tempera-
ꢀ
tures, losing 50% of its activity at 60 and producing no product
ꢀ
at 80 . The support (yeast cell) is expected to confer some thermal
stability, likely resulting in the observed Topt, but the enzyme is
still sensitive to thermal denaturation.
The YSD CalB biocatalyst also sees some loss of activity at the
highest temperature, but half as much as ycM37L (<25% vs.
5
0%). This agrees with previous reports of the thermal stability
10,19
of CalB in non-aqueous environments.
The ability of the
enzyme (CalB) to operate at higher temperatures makes it
advantageous in systems where the reactants are solid or are
insoluble at lower temperatures (for example, hexadecanoic
acid in heptane). However, in the three systems studied here,
ycM37L reaches an equal conversion at a lower temperature.
Intriguingly, N435 did not exhibit the same temperature
dependence. At temperatures that do not deactivate the enzyme,
the rate is such that the reaction reaches equilibrium within six
hours. At higher temperatures, though, a dip in activity is
expected. However, if N435 (purportedly CalB) only loses 25% of
Specicity for the fatty acid and alcohol
Both ycM37L and CalB appear to convert fatty acids with chain
lengths of C12 and shorter to their respective butyl esters with
35
equal ease. The preference (for CalB) persists at higher
temperatures (see Fig. 1 vs. 2 and 3). The water requirement for
ycM37L makes a similar analysis impossible (no water was
added), although some water was clearly present, likely absorbed
from the atmosphere. With acyl donor chains longer than
35
dodecanoic acid, the activity drops signicantly. Analysis of the
binding pocket of CalB reveals a channel that is the length of a
ꢀ
its activity at 80 C, it could conceivably reach equilibrium
before the chosen end point.
37
1
2-carbon chain. A similar analysis has not been performed on
the structure of M37L, and no study of its acyl donor size speci-
city for esterication could be found in the literature. However,
Re-use of the YSD biocatalyst

the prole for hydrolysis of p-nitrophenyl esters by M37L in an The lipase catalysts were applied to ten batches of the butyl
38
aqueous environment has been reported and follows a similar decanoate synthesis reaction (Fig. 4). The reaction temperature
trend, though the drop-off is more pronounced between C8 and was chosen to maximize conversion without denaturing ycM37L.
C12. Studies of the specicity of N435 suggest weak or no Both catalysts maintained activity through all batches, but the
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ycM37L biocatalyst took two batches to reach a stable activity controlled, and removal of some water from the reaction is
level. This effect can be ascribed to an initial lack of water recommended.
necessary for enzyme exibility, water that was produced by the
The stability of ycM37L in a nonaqueous environment has
reaction in sufficient quantities to fully activate the enzyme in not been reported, but cross-linked enzyme aggregates steadily
two batches. The effect could be reproduced by adding water to lost activity in hydrolytic assays containing p-nitrophenyl octa-
50
identical reactions starting with dry catalyst (Fig. 5).
noate. Applications of YSD to synthetic reactions have had
While a small amount of water is necessary for peak activity, mixed results. With CalB displayed on Saccharomyces, the
27
repeated use of the catalysts without drying resulted in accu- catalyst lost activity within 5 cycles in toluene. A different
mulation of water in and around the hydrophilic yeast cells. system—Rhizomucor lipase displayed on Pichia—had much
Wetted cells do not disperse evenly in the solvent (heptane); better stability in biodiesel reactions, only losing 20% of its
51
aggregation and precipitation of the cells were observed to activity aer ten batches. Notably, lamentous systems
gradually worsen, starting around batch 5. Eventually, the employing Aspergillus (but no CalB) had excellent stability,
catalyst clumps to a degree that it cannot be dispersed by producing methyl esters at more than 90% conversion aer
52
mechanical stirring, and the cells cling to the walls of the ten to 15 (ref. 26) batches. The YSD system described here
reaction vessel (glass vials). A concomitant decrease in observed compares favourably to other SD schemes in terms of activity
activity is the result. The negative control, which did not retention, even robust lamentous systems.
produce any product (or water), did not experience the same
problems with dispersion (Fig. 11). The acrylic resin-supported
lipase did not vary in the course of the ten batches. Because the
Kinetics of butyl decanoate synthesis
44
YSD has been applied broadly, as described elsewhere, but
characterization of YSD catalyst kinetics is absent in the liter-
ature. Given the strong dependence of kinetics on the immo-
resin is hydrophobic, it would not accumulate water in the same
way as the yeast, so none of the water-related problems with
mixing and activity were expected. Mitigating the effects of
water in the reaction would be critical for industrial imple-
mentation of the YSD biocatalyst. At the experimental scale,
equilibrating the catalyst to its peak water activity and adding
molecular sieves to scavenge free water would be plausible. At a
larger scale, catalyst drying between batches might be
necessary.
The lyophilized yeast cells are hygroscopic, as evidenced by
the difference in activity between freshly prepared catalyst and
catalyst that has been repeatedly exposed to the ambient
humidity in the lab (“dry” trial, Fig. 5). The hydrolytic activity of
the two batches of catalyst was the same within 5%, (data not
shown). To achieve and maintain optimum activity, therefore,
the water activity of the enzyme needs to be carefully
45
bilization support, comparisons to studies with free enzyme
offer little insight. CalB immobilized on polypropylene had Km
values for octanoic acid and octanol (2.5 mM and 2.1 mM,
54
respectively ) that were much lower than the 70 mM K
m
reported here. It is unlikely that the kinetic parameters derived
from these experiments reect the intrinsic properties of the
enzyme. Rather, this apparent K
lipase that is limited by its environment (the cell surface). Based
on the relative insensitivity to alcohol length, the K value likely
m
captures the effectiveness of a
m
reects slow diffusion of the fatty acid. However, a recent
kinetic study of N435 demonstrated a higher inhibition
constant for the fatty acid in synthesis of hexyl octanoate—in
55
addition to lower specicity for octanoic acid —which might
suggest that dead end decanoic acid–enzyme complexes play a
part here.
CalB does not require additional water, so the kinetics
observed in “dry” reactions are free of the inuence of lipase
“lid” opening or closing, provided the enzyme is hydrated, and
that the amount of water produced by the reaction is negligible.
Since ycM37L is apparently inactive without adding water, even
a small addition (0.4% v/v is ꢁ220 mM) dramatically changes
the observed activity. While the yeast cells are hygroscopic and
can be expected to absorb a signicant portion of the water
added, the effects of the additional water were too complex to
represent by zeroth-order initial rate experiments. At low
substrate concentrations, it is possible that hydrolysis is
competing with the ester synthesis reaction, and the individual
reactants' effects could not be deconvoluted from the cumula-
tive rate.
Fig. 11 State of the catalyst after 10 batches of butyl decanoate
synthesis. YSD biocatalysts displaying CalB (A) and ycM37L (B) aggre-
gate and fall out of suspension after several batches. Control yeast
cells (C) do not produce product or visibly change in their dispersion.
At the beginning of the experiment, both YSD biocatalysts looked like
the control. The N435 standard (D) does not undergo any visible
changes.
Experimental
Strains and media
The host for the recombinant production and display of CalB is
4
6
S. cerevisiae EBY100. Yeast are propagated in 2Â YAPD media
30430 | RSC Adv., 2015, 5, 30425–30432
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À1
À1
(
L
20 g L yeast extract, 100 mg L adenine hemisulfate, 40 g briey cooled in an aluminum bead bath before sampling. For
À1
À1
47
ꢀ
peptone, 40 g L glucose ) at 30 C. Yeasts carrying the repeated-use experiments, the liquid was aspirated with a pipette
surface display construct are propagated in synthetic complete and any suspended catalyst was removed by centrifugation
À1
(
SC) media lacking tryptophan, containing glucose (20 g L ) for (17 000g for 15–30 s). The supernatant was decanted and fresh
À1
outgrowth and galactose (20 g L ) for induction. Plasmid DNA reactant solution was used to re-suspend the cells.
is screened and stored in a lab strain of Escherichia coli DH5a.
Bacteria are grown in LB containing ampicillin at 37 C.
Substrates were prepared for the kinetics experiments by
diluting equal volumes of 0.500 M solutions of butanol and
decanoic acid in heptane. The substrates and biocatalyst were
48
ꢀ
ꢀ
incubated separately at the reaction temperature (40 C) for 15
Cloning
minutes before combining. The reactions were stirred at 200
rpm. Water was added, if necessary, with an automatic pipette
Cloning materials were purchased from New England Biolabs.
Construction of the yeast surface display vectors has been
(Viao) dispensing at the slowest setting to reduce droplet size.
35,46
described elsewhere.
Briey, the gene for wild-type CalB was
The samples were analyzed on a Shimadzu GC2010 equipped
amplied by PCR from a pET vector. The gene for ycM37L was
optimized for yeast expression and synthesized by GENEWIZ,
and amplied by PCR from their pUC vector. The PCR products
were digested with BamHI and NheI and ligated into similarly
digested vectors.
with an autosampler and a DB-5 capillary column (30 m  0.25
mm  0.25 mm). One mL of each sample was injected at a 30 : 1
split ratio with helium as the carrier gas. The injector and
ꢀ
detector were held at 300 C. The oven temperature was held at
ꢀ ꢀ ꢀ ꢀ
À1
9
5
0 C for 2 minutes, ramped to 160 C at 20 min , to 250 C at
ꢀ
À1
ꢀ
ꢀ
À1
min , and to 300 C at 25 min . The concentration of the
Production of the biocatalyst
fatty acid was determined by comparing the FID peak area to
49
Yeast are made electrocompetent in-house, transformed by standard curves prepared in heptane.
electroporation, and recovered in YAPD containing 0.5 M
sorbitol. Putative transformants are screened and selected with
Conclusions
uorescently activated cell sorting by means of a C-terminal c-myc
epitope in the fusion protein. Cultures of the sorted cells are Biocatalysts based on yeast surface display of ycM37L and CalB
grown in SC (glucose) to an optical density of ꢁ5 in baffled asks can effectively catalyze the synthesis of fatty acid esters of
ꢀ
ꢀ
at 30 C. The cells are harvested by centrifugation (4500 g, 4 C, 30 aliphatic alcohols. In six-hour batches at relatively mild
ꢀ
minutes) and re-suspended in SC (galactose with 0.1% glucose) temperatures (30–60 C), the YSD catalyst's activity is 20–50%
in a 2 L fermenter at 20 C. The fermenter is stirred at 250 rpm that of a commercial lipase (N435), despite enzyme loadings
with 4 L min sparged air. Aer 18 hours, the cells are harvested that are three orders of magnitude lower. The two lipases
by centrifugation (3000 g, 4 C, 60 minutes) and washed with exhibit similar specicity for acyl donors of C12 and shorter.
cold, distilled water. Cells were suspended in distilled water, The optimum temperature for synthesis with ycM37L is 40 C;
frozen, and dried. Lyophilization took place at ambient temper- with CalB, 60 C. Both biocatalysts demonstrated long-term,
ꢀ
À1
ꢀ
ꢀ
ꢀ
ature in a SpeedVac (Thermo Fisher) for >8 hours.
multi-batch stability in the synthesis of butyl decanoate, and
the need for control of water concentration in the reaction was
evident. This system represents an inexpensive, green alterna-
tive to conventional synthesis methods.
Chemicals
Media components are biology grade and were purchased from
Fisher (sugars and yeast nitrogen base) or Sigma-Aldrich (amino
acids). Heptane, lauric acid, pentanol, and hexanol were
purchased from VWR. The other fatty acids and butanol were
purchased from Fisher. Butanol and octanol were purchased
from Sigma-Aldrich. All organics were GC grade or better.
Lipase from C. antarctica immobilized on acrylic resin was
purchased from Sigma-Aldrich.
Acknowledgements
The authors gratefully acknowledge Sonia Herrero from
Professor Margo Daub's lab at NCSU for donating a lab strain of
E. coli; Professor Balaji Rao's lab for donating the pCT-EGFP
vector, the stocks for EBY100, and time on the cell sorter; and
Kerri Cushing for donating the pET22-CalB vector. This work
(
JE) was supported in part by NSF award 0832498 through the
Esterication reactions
Center for BioEnergy Research and Development, by the GAANN
Dry catalyst was weighed into clean 1.5 dram glass vials. Solu- Interdisciplinary Doctoral Program in Molecular Biotechnology,
tions of one fatty acid and one alcohol (1 mL of each, both 200 award P200A090129-11, and by the Eastman Chemical Center of
mM in heptane) were added to the vials. To reduce pipetting Excellence. The authors declare no conicts of interest.
errors and deviation due to evaporation, the reactant solutions
were dispensed at room temperature. The vials were sealed
and moved to a magnetically stirred heat block with digital
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