Alteration of Chain Length Selectivity of Candida antarctica Lipase A by Semi-Rational Design for the Enrichment of Erucic and Gondoic Fatty Acids

Title: Alteration of Chain Length Selectivity of Candida antarctica

Lipase A by Semi-Rational Design for the Enrichment of Erucic

and Gondoic Fatty Acids

Authors: Katja Zorn, Isabel Oroz-Guinea, Herike Brundiek, Mark Dörr,

and Uwe Bornscheuer

This manuscript has been accepted after peer review and appears as an

Accepted Article online prior to editing, proofing, and formal publication

of the final Version of Record (VoR). This work is currently citable by

using the Digital Object Identifier (DOI) given below. The VoR will be

published online in Early View as soon as possible and may be different

to this Accepted Article as a result of editing. Readers should obtain

the VoR from the journal website shown below when it is published

to ensure accuracy of information. The authors are responsible for the

content of this Accepted Article.

To be cited as: Adv. Synth. Catal. 10.1002/adsc.201800889

Link to VoR: http://dx.doi.org/10.1002/adsc.201800889

10.1002/adsc.201800889

Advanced Synthesis & Catalysis

FULL PAPER

DOI: 10.1002/adsc.201((will be filled in by the editorial staff))

Alteration of Chain Length Selectivity of Candida antarctica

Lipase A by Semi-Rational Design for the Enrichment of Erucic

and Gondoic Fatty Acids

Katja Zorn^a1, Isabel Oroz-Guinea^a1, Henrike Brundiek^b, Mark Dörr^aand Uwe T.

Bornscheuer^a*

^aUniversity of Greifswald, Institute of Biochemistry, Dept. of Biotechnology & Enzyme Catalysis, Felix-Hausdorff-Str. 4,

17487 Greifswald, Germany

Fax: (+49) 3834 420 744367; phone: (+49) 3834 420 4367; email: uwe.bornscheuer@uni-greifswald.de

^bEnzymicals AG, Walther-Rathenau-Str. 49a, 17489 Greifswald, Germany.

¹Equal contribution.

Received: ((will be filled in by the editorial staff))

Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201######.((Please

delete if not appropriate))

Abstract. Biotechnological strategies using renewable

chain length p-nitrophenol fatty acid esters of the best hits

materials as starting substrates are a promising alternative to

found, as well as the analysis of the contribution of each

traditional oleochemical processes for the isolation of

amino acid change and the outcome of combining several

different fatty acids. Among them, long chain mono-

of the aforementioned residue alterations and, finally, (v)

unsaturated fatty acids are especially interesting in industrial

the selection and application of the most promising

lipid modification, since they are precursors of several

candidates for the fatty acid enrichment biocatalysis. As a

economically relevant products, including detergents,

result, enrichment of C22:1 from Crambe ethyl esters was

plastics and lubricants. Therefore, the aim of this study was

achieved either, in the free fatty acid fraction (wt, 78 %) or

to develop an enzymatic method in order to increase the

in the esterified fraction (variants V1, 77 %; V9, 78 % and

percentage of long chain mono-unsaturated fatty acids from

V19, 74 %). Concerning the enrichment of C20:1 when

Camelina and Crambe oil ethyl ester derivatives, by using

Camelina oil ethyl esters were used as substrate, the best

selective lipases. Specifically, the focus was on the

variant was the single mutant V290W, which doubled its

enrichment of gondoic (C20:1 cisΔ11) and erucic acid

content in the esterified fraction from approximately 15 %

(C22:1 cisΔ13) from Camelina and Crambe oil derivatives,

to 34 %. A moderately lower increase was achieved by V9

respectively. The pursuit of this goal entailed several steps,

and its two derived triple mutant variants V19 and V20

including: (i) the choice of a suitable lipase scaffold to serve

(27 %).

as a protein engineering template (Candida antarctica

lipase A); (ii) the identification of potential amino acid

Keywords: Biocatalysis; Candida antarctica lipase A; lipid

targets to disrupt the binding tunnel at the adequate location;

modification; long chain mono-unsaturated fatty acid

(iii) the design, creation and high-throughput screening of

enrichment; semi-rational protein design

lipase mutant libraries; (iv) the study of the selectivity

towards different

materials. In this context, plant oils are of great

Introduction

interest not only because they come from renewable

resources, but also due to the possibility of modifying

the oil composition to fit the desired necessities by

new plant breeding and gene modification

technologies^[3]. Regardless of the aforementioned

strategies, enrichment of a desirable FA from oil can

be further pursued by classical separation

technologies, like distillation or crystallization.

However, such classical techniques work with the

implementation of harsh conditions like high

Fatty acids (FA) and their alkyl-derivatives are

interesting building blocks for their application in

oleochemistry^[1]. Particularly, long-chain FA have

been used in industry for the production of

surfactants, erucamide as polymer building block,

and lubricants^[2]. In order to obtain these valuable

compounds, natural occurring oils can be utilized as

an environmentally friendly alternative to fossil raw

1

10.1002/adsc.201800889

Advanced Synthesis & Catalysis

pressure, high temperature and the usage of polluting

agents like organic solvents^[4]. Moreover, the

isolation in high yields and purities of a desired

compound employing these classical separation

technologies is often not sufficient for direct

downstream processing. An attractive alternative is

the application of enzymes for the enrichment of

certain FA, as this represents an eco-friendly and

often more efficient alternative^[5]. In this sense some

of the features that biocatalysts offer involve the

possibility of working under mild reaction conditions

or their high chemo-, stereo- and regioselectivity

when using both, natural as well as unnatural

substrates.

accomplish this task by engineering its tunnel-type

binding site^[15], even though the wild type enzyme

does not display hydrolytic preference for the

targeted FA^[16]

.

Results and Discussion

In order to achieve the isolation of erucic and

gondoic acids, a variant capable of efficiently discern

and selectively remove FA depending on their chain

length was required. This goal could be reached

following two strategies: (i) by preferentially

hydrolyzing FA longer than C18 and therefore

causing their accumulation as FFA; or to the contrary,

(ii) by hampering the ester bond cleavage of those FA,

thus increasing their concentration in the ester-bound

fraction as the shorter FA are released (Scheme 1).

Given the characteristics of both, the substrates and

the structure of the enzyme, the second approach was

chosen as it was deemed the most straightforward

strategy.

Unsurprisingly, the most employed enzymes in

lipid modification are lipases (EC 3.1.1.3). These

proteins belong to the α/β-hydrolase fold family and

naturally catalyze the hydrolysis of triacylglycerides

(TAG) into glycerol and free FA (FFA), displaying

particular chemo-, regio- and stereoselectivity^[6]. For

that reason the use of lipases has been widely

described for the enrichment of, for instance, long-

chain poly-unsaturated FA from fish oil^[7], erucic acid

from Cruciferae plant oils^[4,8], and trans-FA from

frying oils^[9]. Additionally, protein engineering has

revealed itself as an important tool to overcome the

limitations displayed by these enzymes, such as

reduced enzymatic efficiency and stability under

challenging industrial conditions, or to enhance the

enzyme selectivity towards different substrates. In

this sense, several examples of its implementation in

the improvement of selectivity were reported for

n<18

n≥18

n<18

n≥18

n<18

n≥18

lipases from Rhizopus oryzae^[10], Candida rugosa^[11]

Moesziomyces antarcticus (formerly:

,

Scheme 1. Possible approaches for enrichment of long

chain FA either as FFA (A) or esters (B) catalyzed by a

selective lipase. In this example fatty acid ethyl esters

derived from the plant oils were used as substrates.

Pseudozyma/Candida antarctica)^{[9b, 12]}. Although

both, directed evolution and rational design

methodologies can be applied, some lipases display

an a priori favorable feature from the (semi)rational

design point of view: the unique architecture of their

substrate binding site, which forms a tunnel-like

cavity that fits the acyl chain of the FA^[13]. Thus,

amino acids surrounding this region are interesting

targets for rational design approaches, since altering

these residues could modify the tunnel’s shape and,

therefore, vary the lipase FA selectivity. This concept

Semi-rational design

Selecting the target residues

The CAL-A structure exhibits a classic α/β hydrolase

fold, containing the catalytic triad (S184, D334 and

H366), as well as a well-defined lid domain, which

takes part in forming a long (~30 Å) substrate binding

pocket^[15]. Both, the binding pocket and the catalytic

site, are deeply buried in the enzyme and covered by

a motile flap^[17](Figure 1). Hence, to adopt the active

state, CAL-A has to undergo a conformational

change, in which the flap domain moves away, giving

the substrate access to the catalytic centre and the

binding site.

In order to create CAL-A variants able to

discriminate FA longer than C18, the structural

conformation of the acyl-binding tunnel was

analyzed. The aim was the identification of potential

amino acid targets located at a position where the

blocking of the binding tunnel would allow the

hydrolysis of fatty acids with 18 carbon atoms or

shorter, while preventing the hydrolysis of longer

acyl groups.

has been proven for lipase 1 from C. rugosa^[11]and

[9b, 12a, 14]

lipase A from M. antarcticus

for enhancing

selectivity towards medium chain or trans fatty acids.

Consequently, the aim of the present study is the

enrichment of mono-unsaturated long chain fatty

acids from plant oils by an improved lipase. More

specifically, we focused on gondoic and erucic acids,

which are present in high contents in Camelina sativa

and Crambe abyssinica seed oils, respectively.

Separation of these FA from the other FA present in

the oil is challenging, due to the high content of C18-

FA of different saturation levels, which are highly

similar in their physical properties to the mentioned

mono-unsaturated fatty acids (MUFAs). Within a

screening of commercially available lipases and the

knowledge about already described lipases in the

literature, CAL-A was chosen as best option to

2

10.1002/adsc.201800889

Advanced Synthesis & Catalysis

combinations, the 22c-trick^[19b]was chosen to obtain

three mutant libraries where only two positions were

saturated at the same time. Hence, the created

libraries were: library I (V238X and V286X),

library II (V238X and V290X) and library III

(V286X and V290X). This resulted in a drastic

reduction of the necessary screening effort to 1,450

clones per library. Moreover, further combinations of

the best amino acid substitutions observed could be

subsequently explored for a better blockage of the

binding pocket.

High-throughput screening

The three pET22-plasmid based mutant libraries

created were transformed into E. coli BL21 (DE3)

C41 and screened on the robotic platform LARA

(laboratory automation robotic assistant)^[20]. The

process started by inoculating each well of a 96-well

microtiter plate with a single colony obtained after

transforming the E. coli cells with the corresponding

mutant library. Subsequently, protein variants were

overexpressed in the microtiter plate wells, harvested

and the selectivity assay was performed. Fortunately,

when CAL-A variants were overexpressed, their

accumulation in the periplasm caused a self-lysis of

the cells resulting in the release of the lipase variant

Figure 1. CAL-A crystal structure (PDB: 3GUU) showing

the lipase in its inactive state. The docked C18:1 fatty acid

(purple) is used to illustrate its probable location in the

CAL-A wt binding tunnel (wireframe). The tunnel is

mainly formed by the six consecutive helices of the cap

domain (shown in dark red). Amino acid residues V238,

V286 and V290 (green) are facing the tunnel and placed

nearby to the last carbon (Cω) of the FA. The active site

residues are highlighted in blue.

to the supernatant as it was previously described^{[9b, 16]}

.

Consequently, the crystal structure of CAL-A

(PDB: 3GUU) was used for docking studies with

oleic acid (C18:1cisΔ9), gondoic acid (C20:1

cisΔ11), and erucic acid (C22:1 cisΔ13). The

following residues facing the tunnel in the

surroundings of the oleic acid's last carbon (Cω) were

identified as possible hotspots: V238, V286, and

V290 (Figure 1). When in silico substitution of these

residues by bulkier amino acids was performed, it did

not result in a full blockage of the tunnel.

Nonetheless, synergistic effects (which are hard to

predict) at these positions appeared promising to

accomplish this goal.

This fact allowed us to use this culture fraction

instead of the cell free extract for the hydrolytic

activity measurements. Consequently, the supernatant

was used in three separate p-nitrophenol (pNP) assays

towards pNP-myristate (pNP-C14:0) as control

substrate, pNP-oleate (pNP-C18:1) and pNP-erucate

(pNP-C22:1) to determine the chain length selectivity

of each mutant. Activity of the different clones

towards these substrates was calculated. The

hydrolytic activity towards pNP-C18:1 and

pNP-C22:1 was compared to the activity towards

pNP-C14:0. Surprisingly the results showed a two-

fold higher (80%) activity towards pNP-C22:1

while the average activity towards pNP-C18:1 of the

CAL-A wt was around 30-40% of the activity

determined towards pNP-C14:0.

Mutagenesis strategy and library generation

In order to test this hypothesis experimentally,

saturation of each chosen position with all possible

amino acids should be performed. Furthermore, to

study the potential synergistic effects when altering

different amino acids, more than one position must be

saturated simultaneously in the created library.

However, saturation mutagenesis at all three positions

at the same time by using NNN degenerate primers

would lead to a huge library of ~800,000 clones to

cover 95% of possible amino acid combinations^[18]. A

more reasonable alternative would be to reduce the

number of generated clones by decreasing the number

of codons introduced, thus creating "small, but smart"

mutagenesis library. However, even when choosing

this approach the number of clones to be screened

would be 24,000 or 32,000, depending on the strategy

used (Table S1)^[19]. Therefore, to reach a compromise

between a library size experimentally feasible to be

screened and exploring all possible amino acid

Taking into account these results, in a first step to

define a variant as a positive hit, those whose activity

towards pNP-C22:1 was equal or inferior to their

activity towards pNP-C18:1 were chosen. Then, the

pNP-C22:1/pNP-C18:1 activity ratio was calculated

for all of them, resulting in

a

CAL-A wt

pNP-C22:1/pNP-C18:1 ratio of approximately 2.60.

To narrow down the number of positive clones, only

those displaying a lower ratio than 0.8 were

considered selective. The outcome was a pre-

selection of 107 clones from library I, 257 from

library II and 173 from library III. Confirmation of

their selectivity was achieved by repeating the

screening experiment only with the pre-selected

clones by quadruplicate measurements. This re-

screening allowed further restricting the number of

chosen clones further and only those mutants

displaying pNP-C22:1/pNP-C18:1 activity ratio

3

10.1002/adsc.201800889

Advanced Synthesis & Catalysis

lower than 0.5 (28 from Lib I, 59 from Lib II and 33

from Lib III) were sequenced. Non-surprisingly,

mutations on these clones encoded mostly for large,

uncharged amino acids that could block the tunnel.

the hydrolysis of pNP-FA esters shorter than C20 was

increased in all the cases. This shift in the substrate

preference was reflected in both activity ratios

calculated:

pNP-C20:1/pNP-C18:1

and

pNP-C22:1/pNP-C18:1 (Figure 3). The values

obtained were especially promising for the erucic

acid derivative, where the most reduced hydrolytic

activities were determined. It is noteworthy that

variants belonging to each one of the three created

libraries were present among the 15 most selective

clones. Moreover, as it is subsequently described,

when the number of variants was further narrowed,

this fact continued being true, indicating the impact

of all three chosen positions on the FA selectivity.

CAL-A variants chain length selectivity towards

pNP derivatives

Selectivity of the variants chosen from the mutant

libraries

From the sequenced clones, the 15 most selective

variants were chosen for further characterization

(Table S2). Therefore, the variants were

overexpressed in a small-scale culture (50 ml) and

purified by based on the His-tag present using

immobilized metal ion affinity chromatography

(IMAC). The activity of the purified variants was

determined spectrophotometrically towards a range of

different chain length pNP-FA esters, including

pNP-C14:0, -C18:1, -C18:2 (-linoleate), -C18:3

(-linolenoate), -C20:1, and -C22:1 (Figure 2).

3.0

pNP-C20:1/pNP-C18:1

pNP-C22:1/pNP-C18:1

2.5

2.0

1.5

1.0

0.5

0.0

pNP-C14:0

pNP-C18:1

pNP-C18:2

pNP-C18:3

pNP-C20:1

pNP-C22:1

14

12

10

8

wt

10 11 12 13 14 15

V1 V2 V3 V4 V5 V6 V7 V8 V9

V

CAL A variant

6

Figure 3. Activity ratios calculated for CAL-A variants:

pNP-C20:1/pNP-C18:1 (■), pNP-C22:1/pNP-C18:1 (■).

4

2

0

wt

V1 V2 V3 V4 V5 V6 V7 V8 V9

V10 V11 V12 V13 V14 V15

As the outcome of this selectivity investigation, four

variants were identified with a C22:1-over-C18:1

value lower than 0.1 (more than twenty-fold lower

than CAL-A wt: 2.5): V1 (CAL-A V238L, V290L),

V2 (CAL-A V238Y, V290N), V3 (CAL-A V238I,

V290M), and V9 (CAL-A Q286, V290W).

CAL-A variant

Figure 2. Chain length selectivity profile displayed by

each CAL-A variant towards pNP-C14:0 (■), -C18:1(■),

C18:2 (■), -C18:3 (■), -C20:1 (■) and -C22:1 (■).

Regarding to the variants discriminating between

C20:1-over-C18:1, as mentioned above also all of

them had a better selectivity than CAL-A wt

(pNP-C20:1/pNP-C18:1 ratio 1.52). In this case, the

ratios achieved ranged between 1.15 and 0.26,

standing out CAL-A V2 (CAL-A V238Y, V290N),

V3 (CAL-A V238I, V290M), V6 (CAL-A V238L,

V286H, V290M) and V12 (CAL-A V238L, V290N)

with pNP-C20:1/pNP-C18:1 ratio below 0.6. Taking

into account these results, variants V1, V2, V3, V9

and V12 were selected to further study their behavior

towards Crambe and Camelina oil derivatives.

CAL-A V6 was discarded as a candidate due to its

low activity compared to the other variants and its

low overexpression levels.

The chain length profile of CAL-A wt showed that

this lipase does not possess a pronounced chain

length specificity, i.e., CAL-A wt did not sharply

differentiate between pNP-FA esters with chain

length ranging from C14 to C22 (Figure 2). These

results match those previously published by our

group^[12a]. Interestingly, when compared to the wild

type enzyme, almost all CAL-A variants maintained

or increased their activity towards the pNP-FA esters

shorter than C18. In particular, CAL-A variants V2,

V3 and V14 presented up to six times increased

activity for the hydrolysis of pNP-C14:0 when

compared to CAL-A wt. The exceptions to this

observation were CAL-A variants V6 and V8 that –

although they exhibited a similar hydrolytic activity

towards pNP-C14:0– had notably decreased activity

towards longer chain pNP-FA derivatives (Figure 2).

Regarding to the effect of the amino acid

substitutions, the selectivity of the variants towards

Prior to perform the oil enrichment biocatalysis, a

better expression system was required. The successful

expression of CAL-A in Pichia pastoris was

4

10.1002/adsc.201800889

Advanced Synthesis & Catalysis

described in previous studies^[9b]. Hence, mutations

present in the variants corresponding gene were

transferred to a methanol inducible P. pastoris

overexpression vector, thus creating the matching

pPICZα-CALA-His mutant plasmids. The resulting

constructs were used to transform P. pastoris GS115

cells. Up to 10 colonies from each transformation

were then seeded in three different plates: YPDS

containing either, 100 µg/ml or 500 µg/ml of Zeocin,

and YPMS-tributyrin containing 100 µg/ml of

Zeocin. Among them, from each transformation the

colony displaying the bigger halo and better growth

was selected, meaning higher protein overexpression

level and Zeocin resistance, in the tributyrin and

YPDS-Zeocin (500 µg/ml) plates (which are usually

indication of multiple gene insertions, respectively.

CAL-A expression experiments were performed on a

small scale in shake flasks (50 ml). The variants were

secreted to the supernatant due to the α-factor signal

peptide. Subsequently, supernatants were desalted

and used to confirm their hydrolytic activities

towards different chain length pNP-FA esters (Figure

S1). As expected, the variants obtained displayed

similar ratios for pNP-C20:1/pNP-C18:1 or

pNP-C22:1/pNP-C18:1 as their E. coli overexpressed

counterparts. Additionally, after desalting an aliquot

from each supernatant, protein concentration was

measured, ranging between 1.6 and 2.1 mg/ml, (Table

S3) and secreted proteins were analyzed via SDS-

PAGE (Figure S2).

Moreover, modelling of variants V1, V2, V3, V9 and

V12 containing an oleic acid molecule inside the

binding pocket was performed. The analysis of the

structures indicated the possible changes that the

binding site could undergo in each variant due to the

corresponding amino acid substitutions (Figure 4). As

it was intended, in all five resulting models, the

tunnel was either cut or narrowed, explaining the

newly achieved selectivity.

Analysis of single mutations in the most selective

variants

To investigate the contribution of each amino acid

change present in the double mutants variants created

in the combinatorial approach, the corresponding

single mutants were generated and overexpressed in

P. pastoris in a similar manner as described before.

Dialyzed supernatants containing the corresponding

overexpressed variant were used to study their

pNP-FA chain length profile (Figure 5A). All single

mutants, with the exception of V238Y, showed

decreased activity ratios of pNP-C20:1/pNP-C18:1

and pNP-C22:1/pNP-C18:1 in comparison to the

wildtype ranging between 0.6-2.0 and 0.1-0.8,

respectively (Figure 5B). However, these decreased

ratios of the single mutants did not reach to the level

of their corresponding double mutants, with

exception of single mutant V290W. From this fact it

can be assumed that the distinct selectivity of the

remaining four double mutant variants (V1, V2, V3,

and V12) resulted from the synergistic effects derived

from the simultaneous change of both amino acids. It

is noteworthy that only mutant V290W reached

activity ratios comparable to the ones of its related

double mutant V9, suggesting its major contribution

for the selectivity created in this variant. While the

pNP-C22:1/pNP-C18:1 ratios of both, V9 and

V290W, have been reported as 0.1, the

pNP-C20:1/pNP-C18:1 ratio of V290W with 0.4 was

slightly worse in comparison to V9 with 0.3. As a

consequence, a priori synergistic effects of mutation

V286Q with V290W could improve the selectivity

towards C20:1 further.

Surprisingly, the single mutation of V238Y

showed

an

increased

activity

ratio

of

pNP-C22:1/pNP-C18:1 of 3.9, being nearly two-fold

higher than the wildtype. This opposite effect in

selectivity was reversed again by the synergistic

effects in the double mutant V2 (V238Y, V290N) to

a ratio of 0.4.

Figure 4. Binding pockets of CAL-A wt (A) and CAL-A

variants V1 (B), V2 (C), V3 (D), V9 (E) and V12 (F). The

docked C18:1 (purple) is used to illustrate the probable

location of the FA in CAL-A binding tunnel (wireframe).

The tunnel is mainly formed by the six consecutive helices

of the cap domain shown in red. Amino acid residues V238,

V286 and V290 (green) are facing the tunnel and placed

nearby to the Cω. The active site residues are highlighted

in blue. Note: Helix αD1 is removed for a better view onto

the binding tunnel.

Creation of triple mutants

As aforementioned, the main aim behind the creation

of three different libraries bearing two saturated

positions each was to explore the possible synergistic

effects of substituting more than one amino acid

5

10.1002/adsc.201800889

Advanced Synthesis & Catalysis

simultaneously. The study of the selectivity of the

single mutant and double mutant variants confirmed

the significance of the influence of these effects for

the reshaping of the tunnel architecture appropriately.

discrimination. Overexpression of lipase variants

V16-V20 was confirmed by SDS-PAGE (Figure S2)

and protein concentration was determined (Table S3),

prior to the analysis of the hydrolytic activity of the

supernatants (Figure 6).

7

A

pNP-C14:0

pNP-C16:0

pNP-C18:1

Table 1. Amino acid changes present in the triple

mutant CAL-A variants.

6

pNP-C18:2

pNP-C18:3

pNP-C20:1

pNP-C22:1

5

4

3

2

1

0

Amino acid

Triple

Mutants

V238

V286

V290

V16

(V1+V286Q)

L

Q

L

V17

(V2+V286Q)

Y

I

Q

N

M

W

V18

(V3+V286Q)

8I

wt

8L

0L

8Y

0N

0M

6Q

0W

23

29

23

29

28

V

29

V

CAL A variant

V19

(V9+V238L)

L

I

5

4

3

2

1

0

V20

(V9+V238I)

B

pNP-C20:1/pNP-C18:1

pNP-C22:1/pNP-C18:1

Comparing the generated triple mutants to the already

investigated variants, their tendency to hydrolyze the

longer FA was further decreased. Unfortunately, with

the exception of V16, also the activity towards C18

FA was partially affected, leading to lower specific

activities towards these substrates. Especially acute

was the aftermath in V20, which displayed a sharp

decrease in activity with substrates longer than

pNP-C14:0. These results indicate that an

accumulation of bulky residues at those positions in

the binding tunnel may cause a larger steric hindrance,

hence hampering as well the hydrolysis of FA shorter

8I

wt

8L

0L

8Y

0N

6Q

0M

0W

23

29

23

29

28

29

V

29

V

CAL A variant

than

C20.

Despite

this

fact,

the

Figure 5. (A) Chain length profile displayed by the

selected CAL-A variants (wt, V238L, V238I, V238Y,

V286Q, V290L, V290M, V290N and V290W)

overexpressed in P. pastoris towards pNP-C14:0 (■),

pNP-C16:0 (■), -C18:1 (■), -C18:2 (■), -C18:3 (■), -C20:1

(■) and -C22:1 (■). (B) Activity ratios calculated for the

aforementioned CAL-A variants: pNP-C20:1/pNP-C18:1

(■), pNP-C22:1/pNP-C18:1 (■).

pNP-C22:1/pNP-C18:1 activity ratio calculated for

the triple mutant variants was further diminished

when compared to their parental double mutant

variants. In this case the obtained values ranged from

0.25 to 0.04, while those obtained for the variants

with two altered amino acids varied between 0.4 and

0.06.

Similarly,

the

values

of

the

pNP-C20:1/pNP-C18:1 activity ratio of the new

variants were lowered (0.5-0.25) regarding to their

precursors (1.0-0.5), excluding V16 that remained

alike (1.4). Consequently, these variants were

selected as good candidates for further application in

gondoic and erucic FA enrichment. Only V17 was

excluded due to both, low overexpression yields and

low specific activity.

In the previous experiments it is clearly observed

how, with the exception of CAL-A V290W, the

discrimination between FA esters of a chain length

longer than C18 was increased when a pair of

residues was altered, compared to the single residue

modifications. Therefore, several combinations of the

amino acid substitutions from the best double variants

were investigated (Table 1). Ideally, these new

variants would lead to a better blockage of the

binding pocket, resulting in

a

sharper FA

6

10.1002/adsc.201800889

Advanced Synthesis & Catalysis

overexpression was monitored by measuring the

absorbance at 600 nm, the wet cell weight, and the

lipase activity towards pNP-C14:0. Additionally,

final protein concentration in the supernatant was

determined. Absorbance at 600 nm increased along

time from 1.2-2.7 (t₀) to 270-370 (t₁₆₂), depending on

the variant overexpressed. In a similar way, the final

wet cell weight achieved was approximately 0.2 g/ml

of culture for all clones. In contrast, both, lipase

concentration and hydrolytic activity strongly

depended on the variant studied. In this sense,

enzyme concentration in the dialyzed supernatant

ranged between 1.1 and 2.5 mg/ml, while the activity

towards pNP-C14:0 varied more than ten-fold (120.1

U/mg from V20 to 1,672.4 U/mg from V1)

(Table S4).

In addition, to verify that the amino acid changes

introduced did not affect the fermented variants

stability, their melting temperatures were analyzed

and compared to the wt (Table 2). As a result it could

be confirmed that, although the melting temperature

decreased up to 6.5 ºC for V2 and V19, in all cases it

remained above 82 ºC, indicating a high stability

towards thermal unfolding.

A

5

pNP-C14:0

pNP-C16:0

pNP-C18:1

pNP-C18:2

pNP-C18:3

pNP-C20:1

pNP-C22:1

4

3

2

1

0

wt

V16

V17

V18

V19

V20

CAL A variant

B

3.0

pNP-C20:1/pNP-C18:1

pNP-C22:1/pNP-C18:1

2.5

2.0

1.5

1.0

0.5

Table 2. Melting temperatures obtained for the CAL-A wt

0.0

and fermented variants.

wt

V16

V17

V18

V19

V20

CAL A variant

CAL-A

variant

Melting temperature

(ºC)

Figure 6. (A) Chain length profiles displayed by the

selected CAL-A variants (wt, V16, V17, V18, V19 and

V20) overexpressed in P. pastoris towards pNP-C14:0 (■),

pNP-C16:0 (■), -C18:1 (■), -C18:2 (■), -C18:3 (■), -C20:1

(■) and -C22:1 (■). (B) Activity ratios calculated for the

aforementioned CAL-A variants: pNP-C20:1/pNP-C18:1

(■), pNP-C22:1/pNP-C18:1 (■).

89.0

87.9

89.0

82.5

87.8

83.6

83.1

83.5

82.5

84.2

wt

V290W

V1

V2

V3

V9

V16

V18

V19

V20

High cell density expression of CAL-A variants

One of the main advantages of using P. pastoris as

host is that it can grow to very high cell densities,

thus, expressing and secreting recombinant proteins

accordingly, while only very few proteins from the

host cell are present in the fermentation broth^[21]

.

Lipase specificity on transesterified oil substrates

Taking advantage of this fact, large-scale production

of CAL-A wt and the most promising variants, i.e.

V290W, V1, V2, V3, V9, V16, V18, V19 and V20,

was performed to obtain sufficient amounts of protein

for the biocatalysis experiments.

Fermentation of the clones was a fed-batch

process where cells were inoculated and grown on

glycerol, followed by a fed-batch with glycerol (50%)

step and the subsequent induction phase involving

fed-batch with methanol. After 162 h the cultures

supernatant was used for FA enrichment. During this

process the cultures growth and protein

Besides discriminating towards different FA chain

lengths, lipases can display several types of

selectivities when acting on TAGs. For instance,

lipase regioselectivity, i.e. the ability to distinguish

between primary (sn-1,3) and secondary (sn-2) ester

functionalities in a triacylglycerol molecule, may

affect the hydrolysis pattern of these enzymes when

acting on determined oil^[22]. Formerly, CAL-A was

described as

a

sn-2 selective lipase^[23]

,

this

information was proven to be an artifact due to acyl

migration in the products of this regio-unspecific

7

10.1002/adsc.201800889

Advanced Synthesis & Catalysis

lipase^[24]. Nonetheless, lipase regioselectivity is an

important factor to take into account when

performing FA selective enrichment on an oil

substrate. For instance, C22:1 in Crambe oil is mostly

located on the sn-1,3 positions of the glycerol and the

remaining FA in this oil are prone to be located at the

sn-2 position^[25]. Interestingly, although some studies

showed that C22:1 enrichment could be achieved

utilizing sn-1,3 specific lipases, this strategy was less

successful than using lipases with natural selectivity

towards long-chain fatty acids like C. rugosa or

G. candidum lipase^[4,8b]. In order to analyze the

enrichment capacity of the newly created CAL-A

variants depending exclusively on the different chain

length of the FA present in the oils, FA ethyl ester

(EE) derivatives obtained from Crambe and

Camelina oils were used as substrates. Regarding

their composition, Crambe oil and, thus, its EE

derivatives are mainly composed of C22:1 (58.7 %)

and unsaturated FA of C18 (31.2 %) (Figure S3). On

the other hand, Camelina oil and its derived EE

contain primarily C18:3 (38.0 %) and similar

amounts of C18:1, C18:2, and C20:1 (15.5 % of

each) (Figure S4).

A

80

60

40

20

V9

Wt

V290W

V1

V2

V3

V16

V18

V19

V20

0

20

40

60

80

100

FFA [%]

B

80

60

40

20

Wt

V290W

V2

Biocatalysis using Crambe ethyl esters as substrate

0

20

40

60

80

100

When performing the enrichment biocatalysis with

the selected variants on the Crambe esterified FA

(EFA) fraction, CAL-A wt was also included as

control to verify the created selectivities. Depending

on their behavior the used biocatalysts could be

differentiated in two groups: (i) those which enriched

the desired C22:1 in EFA fraction as it was planned;

and (ii) those which caused an accumulation of the

FA in the FFA (Figure 7).

The first group included almost all newly

generated variants: V1, V3, V9, V16, V18, V19 and

V20 (Figure 7A). These lipases showed their

preference for the hydrolysis of FA shorter C20, thus

achieving enrichments of C22:1 in the EFA ranging

from 67.7 to 78.3 %. Especially high enrichment

levels were achieved by V1 (77.2 %), V9 (78.3 %),

V19 (73.7 %), and V20 (72.4 %). On the other hand,

an increase of the C22:1 concentration in the FFA

fraction was possible by using CAL-A wt. This result

was surprising since, although a higher activity for

the hydrolysis of C22:1 when compared to the

hydrolysis of C18:1 was previously observed while

FFA [%]

C

80

60

40

20

V9

Wt

V290W

V1

V2

V3

V16

V18

V19

V20

55

60

65

70

75

80

85

C22:1 [%] in enriched fraction

Figure 7. Content of C22:1-EE in the Crambe EFA (A) or

FFA (B) fraction over content of FFA as mean averages of

triplicates displayed with their errors, as well as their

theoretical recovery of C22:1 (C), which is defined as the

amount of C22:1 present in the enriched fraction (either

FFA or EFA) in comparison to the total amount of C22:1

in the sample (in both FFA and EFA), over the C22:1

content in the most enriched fraction.

testing the pNP-derivatives, leading to

a

pNP-C22:1/pNP-C18:1 ratio of ~2.5, it was not

anticipated to have such a large impact when using

the complex EFA mixture (Figures 3, 5, 6 and S1).

Nevertheless, an enrichment of C22:1 in the FFA

fraction could be observed accomplished by the wt

enzyme up to a concentration of 78.1 % (Figure 7B).

It is noteworthy to mention that these results

demonstrate that it was possible to completely

engineer the inversion of the CAL-A wt selectivity.

Additionally, even though variants V290W and V2

displayed pNP-C22:1/pNP-C18:1 ratios quite low

8

10.1002/adsc.201800889

Advanced Synthesis & Catalysis

(0.1 and 0.4, respectively), these lipases also showed

some enrichment of C22:1 in the FFA fraction, but

only up to a concentration of 62.3 %.

40

30

20

10

0

A

V3

V9

V16

V18

V19

V20

Wt

V290W

V1

V2

V3

Regarding to the hydrolysis yields accomplished,

even though the amount of enzyme present to reach a

sufficient degree of substrate hydrolysis (10-40%)

was estimated (Figure S5), maximal FFA contents

obtained significantly varied after 48 h, ranging from

40.4 % for V1 to 81.4 % for V290W. Especially,

when examining V9 and its corresponding triple

mutants V19 and V20, a similar slope can be

observed leading to the assumption that similar

maximal values of enriching C22:1 can be attained by

reaching a higher degree of hydrolysis. However, the

steepest slope was observed for V1 as well as the

highest theoretical recovery of 81.3 % (Figure 7C).

This value is defined as the amount of C22:1 present

in the enriched fraction (either FFA or EFA) in

comparison to the total amount of C22:1 in the

sample (in both FFA and EFA). In comparison, the

theoretical recovery for the wt only reached 62.3 %

and for V9 as far as 45.7 %.

The results achieved in this work are comparable

to those described in previous studies using C. rugosa

and G. candidum lipases as biocatalysts^[4]. However,

it must be taken into account that, to the best of our

knowledge, the proteins used in the aforementioned

examples were commercial samples composed of a

mixture of several isoenzymes in different

proportions. Since modifications of the fermentation

conditions lead to different lipase amounts and

(iso)enzymatic profiles, reproducibility of the results

could hence be challenging^[26]. On the other hand,

CAL-A wt is a well-studied lipase with great

potential in biocatalysis due to its high level of

overexpression, stability and the possibility of

immobilization^{[21, 27]}. Therefore, the described CAL-

A variants represent a very attractive alternative for

C22:1 enrichment from plant oils.

V9

0

20

40

60

80

100

FFA [%]

V3

V9

V16

V18

V19

V20

B

Wt

V290W

V1

V2

V3

80

60

40

20

V9

10

15

20

25

30

35

40

C20:1 [%] in enriched fraction

Figure 8. Content of C20:1-EE in the Camelina EFA (A)

fraction over content of FFA as mean averages of

triplicates displayed with their errors, as well as their

theoretical recovery of C20:1 (B), which is defined as the

amount of C20:1 present in the enriched fraction (either

FFA or EFA) in comparison to the total amount of C20:1

in the sample (in both FFA and EFA), over the C20:1

content in the most enriched fraction.

a concrete structural reason for this reduced activity

towards C20:1 but not towards C22:1 could not be

identified by molecular modeling studies (Figure S6).

Regarding V1 and its derivative V16 the data analysis

detected a similar behavior in enrichment, producing

a slight decrease of gondoic acid in the EFA fraction.

This fact indicated that for these two variants the

chain length selectivity cut-off was generated

between C20 and C22. These results are endorsed by

the results obtained in the pNP-FA selectivity

Biocatalysis using Camelina ethyl esters as substrate

Interestingly, when analyzing the results for the

reactions with Camelina EFA, the same long chain

FA enrichment behavior for the enzyme variants

could not be observed. In this case, the best results

for the enrichment of C20:1 in the EFA fraction were

obtained with the variants derived from CAL-A

V290W. The double, V9, and triple mutants, V19 and

V20, enriched C20:1 from a concentration of 15.4 %

to approx. 27 % (Figure 8A). Interestingly, the

highest enrichment, 34.1 %, was obtained with the

single mutant V290W. However, the theoretical

recovery determined for the best four variants was

moderate reaching 52-59 % (Figure 8B). It is

certainly intriguing that variant V290W was not able

to enrich C22:1 in the Crambe EFA, but it enriched

C20:1 in the Camelina EFA. As it is explained below,

experiment

pNP-C20:1/pNP-C18:1

where

both

ratios

variants

decreased

show

in

comparison to CAL-A wt but close to 1, which means

similar rates in hydrolysis for both substrates (Figure

3 and 6). Concerning V2, V3 and V18, only a minor

enrichment was produced in the EFA, not

representing more than 5 % on any of these cases.

Finally,

despite

the

pNP-C22:1-

and

9

10.1002/adsc.201800889

Advanced Synthesis & Catalysis

pNP-C20:1/pNP-C18:1 ratios obtained for CAL-A wt

were quite similar (2.2 and 2.4, respectively), no

significant C20:1 enrichment was observed for

CAL-A wt in the FFA fraction.

The different performance of the variants can be

explained due to two non-exclusive reasons: (i)

although Camelina and Crambe samples are

comparable concerning the FA types present, there is

a great difference in their concentrations. Thus, while

in both oils FA chain lengths range between C14 to

C24, the concentration of the desired FA is dissimilar,

representing 15.4 % (C20:1) and 58.7 % (C22:1),

respectively. This may be especially significant for

CAL-A wt, as crowding effects could play a role in

the conversion of the long-chain EFA by this enzyme.

Hence, in the Crambe EFA it is more likely that the

enzyme meets a molecule of C22:1-EE in comparison

to contacting a molecule of C20:1-EE using

Camelina EFA as substrate. Secondly, (ii) in the

robotic screening the assay was directed towards the

identification

of

clones

with

lower

pNP-C22:1/pNP-C18:1 ratio. As a consequence,

variants discerning shorter FA than erucic and not

gondoic acid were favored (“You get what you screen

for”^[28]). Consequently, V1 was identified with a ratio

of 0.4 and V16 was derived from it with an improved

ratio of 0.3, while they are not able to discriminate

towards C20:1.

Figure 9. Elucidation of the CAL-A FA binding tunnel

structure with (A) CAVER tunnel computation and

(B-E) the results of the molecular dynamics studies of

C22:1 binding to CAL-A wt. The main tunnel is shown

from different angles. Highlighted in the red circle is the

cisΔ13 double bond of the C22:1 molecule. The FA

(purple) was previously docked in the CAL-A crystal

structure (3GUU) displaying the binding tunnel

(wireframe), the cap domain (red), the catalytic residues

(blue) and the hotspots for protein engineering (green).

Bioinformatic analysis of changes in the tunnel

structure

When analyzing the main FA binding tunnel of CAL-

A there are striking features to mention. The tunnel is

located in the cap domain of the enzyme^[15]and can

reach a length of up to 36.1 Å (Figure 9). The

beginning of the tunnel is buried inside the protein

scaffold at the active site, which is connected to

smaller tunnels important as solvent or small ion

channels (Figure 9A). The main part of the tunnel

(33.7 Å) has a radius ≥1.5 Å, being large enough to

fit the cross section of a FA molecule, i.e. a

carboxylic acid with a long aliphatic chain containing

as cross section units C₂H₄with a radius of approx.

1.44 Å. In close proximity of the active site a minor

appendix leaves the main acyl-binding tunnel. This

groove was investigated by Brundiek et. al. as an

alternative binding pocket able to bind medium chain

FA^[12a]. The major binding tunnel displays a linear

section of 15 Å in the beginning and takes then

several turns until it narrows down and is exposed to

the surface in the lid region.

the wt enzyme was striking. CAL-A wt preferred the

hydrolysis of C20:1 and C22:1 over C18:1. An

explanation for this was found in the tunnel

architecture, which allows the cis double bond to

adopt an angle closer to its natural conformation

(110°). While the cis double bond of C18:1 (cisΔ9) is

located in the linear part of the FA binding tunnel

(Table S5), the cis double bonds of C20:1 (cisΔ11)

and C22:1 (cisΔ13) suit with the first turn of the

tunnel (Figure 9 B-E).

When analyzing changes introduced to the FA

binding tunnel of CAL-A by its mutants, it can be

said that each variant displayed an at least by 10 Å

shortened tunnel (Table 3; Figure S6). However,

convincing structural reasons for the distinct

selectivities were not found neither for the distinct

chain-length selectivity of V1 and V16 between C20

and C22 FA, nor the decreased activity of V290W

towards C20:1, but not C22:1.

In the previous molecular modeling experiments it

was observed that the conformation of the tunnel

underlies a certain degree of freedom as well as the

binding of the FA molecule. However, the FA

selectivity towards different chain length of MUFAs,

.

10

10.1002/adsc.201800889

Advanced Synthesis & Catalysis

Table 3. Summary of the distance from the catalytic active

site to the first bottleneck determined for CAL-A wt, the

single mutant V290W, the double mutants V1-3, 9, and 12,

and the triple mutants V16-20. More specifically, the

tunnel graphs are displayed for the FA binding tunnel

calculated from the catalytic Ser185 with a minimal probe

radius of 0.6-0.7. Path lengths were calculated with a probe

radius of 1.50 Å using Caver Analyst 1.0.

Regarding to the FA enrichment process with

Crambe ethyl ester derivatives, it is noteworthy that

when the biocatalysis was mediated by CAL-A wt, up

to 78 % enrichment of C22:1 was observed in the

FFA fraction. On the other hand, a complete

inversion of this selectivity was achieved when the

best variants of the above were tested in the same

procedure. Hence, C22:1 was enriched in the EFA

fraction to 77 % with V1, 78 % with V9, and 74 %

with V19. Within these variants the best theoretical

yield of C22:1 was determined for V1 with 81 %. In

the case of using Camelina oil ethyl esters as

substrate, the lipases behaved differently. CAL-A wt

could not enrich C20:1 in neither, the EFA nor in the

FFA fraction, while the best variant was the single

mutant V290W, which more than doubled the content

of C20:1 in the EFA fraction to 34 %. The next best

catalysts comprised V9 and its two derived triple

mutant variants V19 and V20, which were able to

increase the percentage of C20:1 up to 27 %.

Length

[Å]

Length

[Å]

Length

[Å]

Variant

33.65

20.85

21.65

19.09

22.26

22.24

22.23

20.42

20.31

21.91

Wt

V1

V2

V16

V17

V18

V19

V20

19.33

V290W

-

V3

V9

V12

Therefore, the generated

variants have

demonstrated their capability to enrich long chain

mono-unsaturated FA, utilizing as starting substrate

complex mixtures of FA ester derivatives obtained

from renewable sources. In addition, different lipase

variants could be chosen in a practical situation to

best fit the process requirements, depending on the

substrates used, the required purity and the desired

recovery of FA. Moreover, further optimization of the

biocatalysis reaction conditions would probably lead

to both, better recovery yields and higher level of

enrichment. Thus, they represent promising

candidates for their application in industrial lipid

modification as a more environmentally friendly

alternative to traditional oleochemical processes.

Conclusions

The work presented herein describes the semi-

rational protein design of CAL-A selectivity to

enhance the enrichment of mono-unsaturated long

chain FA from Camelina and Crambe seed oil ethyl

ester derivatives.

Initially, molecular modelling on the CAL-A

structure allowed the identification of three possible

hotspots for site-directed saturation mutagenesis:

V238, V286, and V290. This led to the creation of

three different “small, but smart” libraries bearing

two saturated positions simultaneously: library I

(V238X and V286X), library II (V238X and V290X)

and library III (V286X and V290X). High-throughput

screening in LARA, and the subsequent

characterization of the pNP-FA ester hydrolysis

preference, allowed the identification of five CAL-A

variants, i.e. V1 (V238L, V290L), V2 (V238Y,

V290N), V3 (V238I, V290M), V9 (Q286, V290W)

and V12 (V238L, V290N), displaying significantly

Experimental Section

Materials

Unless stated otherwise all chemicals were purchased

from Fluka (Buchs, Switzerland), Sigma (Steinheim,

Germany), Merck (Darmstadt, Germany), VWR

(Hannover, Germany), or Carl Roth (Karlsruhe,

Germany). Primers were synthesized by Sigma

(Steinheim, Germany) and Thermo Fisher Scientific

(Waltham, MA, USA). Sequencing was performed by

Eurofins MWG Operon (Luxembourg, Luxembourg).

E. coli TOP10 strain was obtained from Invitrogen

(Carlsbad, CA, USA) and E. coli BL21 (DE3) C41

strain was purchased from Lucigen (Middelton,

USA). Plasmids pGAPZ and pPICZα were purchased

from Invitrogen (Carlsbad, USA).

lower

pNP-C20:1/pNP-C18:1,

pNP-C22:1/pNP-C18:1 ratios (between 0.6-1.3 and

0.4-0.06, respectively), in comparison to CAL-A wt

(2.4 and 2.2, respectively). Consequently, the

capability of these variants of accepting FA longer

than C18 was dramatically decreased. The fact that

amino acid changes present in all three positions

influenced the catalytic specificity confirmed the

suitability of the chosen site-directed mutagenesis

targets. Furthermore, analysis of single and triple

mutants derived from the aforementioned variants

highlighted the relevance of the synergic effects

achieved by altering more than one residue at the

same time. In general, when compared to their

parental double mutant variants, single mutant

variants displayed higher pNP-C20:1/pNP-C18:1,

pNP-C22:1/pNP-C18:1 activity ratios (between 0.4-

2.5 and 0.1-3.9, respectively), while in the triple

mutants these values were decreased (between 0.25-

1.4 and 0.04-0.25, respectively).

CAL-A cloning and overexpression in E. coli

The plasmid pET22b bearing the gene encoding

CAL-A from Moesziomyces antarcticus (Candida

antarctica) was previously cloned in our group^[12a]

.

E. coli C41(DE3) cells were transformed with

11

10.1002/adsc.201800889

Advanced Synthesis & Catalysis

pET22b(+)_ppro_CALA_His or the corresponding

derivatives by electroporation (MicroPulser™

electroporator). Similarly, in order to preserve them,

E. coli Top10 cells were transformed with pPICZα or

pGAPZα vector bearing the desired CAL-A wt /

mutant gene.

well as stored at −80 °C in YPD medium containing

30 % of glycerol for conservation and further use.

P. pastoris overexpression of CAL-A variants in

small scale: a freshly plated P. pastoris GS115

colony transformed with the desired CAL-A gene

was used to inoculate 5 ml BMGY medium (1 %

yeast extract, 2 % peptone, 100 mM potassium

phosphate (pH 6.0), 1.34 % YNB, 4x10^-5% biotin

and 1 % glycerol) with Zeocin (0.1 mg/ml) in a

250 ml Erlenmeyer baffled flask. The culture was

shaken overnight at 30 ºC and 180 rpm. Using 5 ml of

overnight culture 45 ml of BMGY media were

inoculated and kept at 30 °C and 180 rpm.

When P. pastoris clones were overexpressing the

desired protein under the control of the inducible

promoter (double and triple mutants), after 24 h the

BMGY cultures were centrifuged at 4500 g for

15 min and washed with 5 ml sterilized 50 mM

sodium phosphate buffer with 1 mM NaCl at pH 7.5.

The washed cells were centrifuged again and the cell

pellet was resuspended with 50 ml BMMY (1 %

yeast extract, 2 % peptone, 100 mM potassium

phosphate (pH 6.0), 1.34 % YNB, 4x10^-5% biotin

and 0.5 % methanol) containing Zeocin (0.1 mg/ml).

Subsequently, the culture mixture was incubated at

30 °C and 180 rpm for 96 h in a 250 ml Erlenmeyer

baffled flask. Every 24 h 250 µl of sterilized

methanol (0.5 % final culture volume) was added to

maintain the protein overexpression. After 96 h of

induction the culture was harvested by centrifugation

(20 min, 4500 rpm, 4 °C). The supernatant (~50 ml)

was collected, its hydrolytic activity towards

pNP-C14:0 measured and a SDS-PAGE run as

described below. Then the corresponding supernatant

was divided into aliquots and frozen at –80 °C.

Overexpression of CAL-A wt and the different

variants in E. coli C41 was achieved by growing a

colony bearing the plasmid in a 5 ml LB O/N culture

containing ampicillin (0.1 mg/ml) at 37 °C and

180 rpm. Depending on the experimental setup, 50 ml

of ZYM-5052 medium^[29]was inoculated with 1-2 %

volume of the E. coli O/N culture. After inoculation,

cultures were incubated at 37 °C and 180 rpm for 6 h

and, then, cultivated at 30 °C for 22 h. The cultures

were harvested at 3900 g for 60 min at 4 °C. The

supernatant (SN) was discarded and the cell pellet

was resuspended in 10 ml sodium phosphate buffer

(50 mM; pH 7.5) containing sodium chloride (1 mM).

Cell lysis was performed by ultrasonication

(Sonoplus HD2070 UW2070 KE76, Bandelin

electronic, Berlin, Germany) at 30 % power and

50 cycles for 5 to 20 min on ice. The suspension was

centrifuged at 3900 g for 30 min at 4 °C.

Subsequently, the supernatant, containing the cell

free extract (CFE), was separated from the cell pellet

and snap frozen with liquid N₂. The frozen samples

were stored at –80 °C until further usage.

CAL-A cloning and overexpression in P. pastoris

Prior to the electroporation of P. pastoris GS115

competent cells, the pPICZα (wt, V290W, double

mutants and triple mutants) or pGAPZα (wt and

single mutants) vector bearing the desired CAL-A

wt / mutant gene was purified from a 20 ml (LB-low

salt containing 25 µg/ml Zeocin) O/N culture of the

corresponding E. coli Top10 strain and digested using

PmeI or AvrII restriction enzyme to obtain the pure

linearized plasmid. After electroporation of the

GS115 competent cells with the linear plasmid (5-

10 µg), 1 ml of ice cold sorbitol 1 M was added. The

mixture was incubated at 30 ºC during 1 h without

shaking. Subsequently, 250 µl were seeded in YPDS

plates containing 1 % yeast extract, 2 % peptone, 2 %

dextrose, 1 M sorbitol, 2 % agar and 100 µg/ml

Zeocin. After 72 h incubation at 30 ºC the

transformed colonies grew.

Selection of the best P. pastoris clones: once

obtained the transformants, screening of the best

clones was performed by plating 10 µl from a 0.5 ml

YPD (1 % yeast extract, 2 % peptone, 2 % dextrose

and 100 µg/ml Zeocin) O/N culture in three different

plates:(i) an indicator plate supplemented with

tributyrin^[30]and YPDS plates containing either (ii)

100 or (iii) 500 µg/ml of Zeocin. These plates were

incubated during 96 h at 30 ºC. One clone from each

transformation was chosen based on the size of the

halo and the resistance towards Zeocin. The selected

clones were then used for lipase overexpression, as

When overexpressing variants under the control of

the constitutive promoter a similar protocol was

followed. However, since the constitutive promoter

needs no additional feeding with methanol for

induction, it was used YPD media instead of the

BMGY and BMMY media.

Fed-batch P. pastoris fermentation for the

overexpression of CAL-A variants V290W, V1, V2,

V3, V9, V16, V18, V19 andV20: a 250 ml

Erlenmeyer baffled flask with 50 ml of BMGY

medium was inoculated with a freshly plated

P. pastoris colony per variant. The flask was

incubated for ∼48 h at 30 °C on an orbital shaker at

180 rpm. Once the cell density reached OD₆₀₀50,

0.6 l of Basal Salts Medium (BSM was made as

described by Invitrogen in Pichia Fermentation

Process Guidelines) were seeded with 30 ml of the

shake flask culture in a 1 l fermenter. In the glycerol

batch phase the culture was grown until the glycerol

was completely consumed (24 h). Then, the glycerol

fed-batch phase was initiated by adding glycerol

50 % containing 1.2 % PTM1 salts at 5 ml/min/l

during 4 h to increase the cell biomass. After that

time, the feeding was stopped and the MeOH

induction was not started until all the remaining

glycerol was consumed (3 h). During the MeOH

fed-batch phase the methanol was introduced slowly

12

10.1002/adsc.201800889

Advanced Synthesis & Catalysis

to avoid cell death. Therefore, MeOH flow started on

1.5 ml/min/l and was increased up to 3 ml/min/l when

the culture was fully adapted to methanol utilization

and there methanol limitation occurred. The induction

phase continued during 96 h. The temperature was

kept constant at 30 °C and the pH was maintained

between 5.0-6.0 using NH₄OH (30 %) and H₃PO₄

(50 %). The airflow and the stirrer speed were

adjusted to maintain the dissolved oxygen (DO)

above 20 %. When the fermentation finished the

culture was centrifuged at 4500 rpm during 1 h and

the supernatant (SN) was collected and frozen at –80

ºC for later lyophilization. The fermentation process

was followed by taking 10 ml samples at least once a

day. Samples were analyzed for cell growth (OD₆₀₀

and wet cell weight) and hydrolytic activity of the SN

towards pNP-C14:0.

PROTEAN^(R)Tetra System apparatus (Bio-Rad) at

200 V for 40 min. Molecular weight marker was

purchased from either Carl-Roth (Roti®-Mark

Standard) or from Thermofisher (Unstained Protein

MW Marker). Proteins were stained with Coomassie

Brilliant Blue G-250 (Bradford).

Determination of protein melting temperature

Measurement of the melting temperature for the

CAL-A variants overexpressed in high cell density

cultures were performed using a Prometheus NT.48

equipment

(NanoTemper

Inc.,

Germany).

Approximately 4 μl of each sample containing

1 mg_prot/ml was aspirated into standard-treated glass

capillaries (NanoTemper Inc., Germany). Capillaries

were loaded into a 48-capillary sample holder and

thermostated inside the instrument at 20 ºC for

~ 5 min before measurements. Excitation power was

set at 10 %. Intrinsic fluorescence was measured at

330 and 350 nm in presence of a heat gradient

ranging from 20 to 95 ºC (ΔT=0.1 ºC/min).

CAL-A purification

The presence of the C-terminal His-tag enabled a

one-step purification procedure of CAL-A by IMAC.

The fraction containing the soluble protein (25 ml)

was applied to 5 ml of Co⁺²–IDA agarose column

(Roti^®garose-His/Co Beads; Roth, Karlsruhe,

Germany) pre-equilibrated with 50 mM sodium

phosphate buffer containing 300 mM NaCl and

30 mM imidazole, pH 7.0. The column was washed

with equilibrating buffer (50 ml) in order to remove

the unbound material. The desired protein was eluted

with 10 ml of the same buffer containing 300 mM

imidazole. Subsequently, in order to desalt and

concentrated the resulting protein solution two

methods could be used: (i) concentration of the eluted

fraction by centrifugal filter units, Vivaspin 20

(10 kDa cut off, Satorius AG, Göttingen, Germany),

to 1 ml. This volume was subsequently washed five

times with washing buffer (5 mM sodium phosphate

buffer, pH 7.5). Alternatively, (ii) dialysis with

ZelluTrans dialysis membranes (10 kDa cut off, Roth,

Karlsruhe, Germany) was performed, followed by

lyophilisation.

Hydrolysis of pNP-esters of different chain length

CAL-A hydrolytic activity was determined by

following the initial hydrolysis rate of pNP esters

spectrophotometrically during

Infinite M2000 Pro)

5

at

min (TECAN

λ=410 nm

(ε=10,998 M⁻¹cm⁻¹) in a 96-well microtiter plate.

The assays were performed in phosphate buffer

(50 mM; pH=7.5) containing NaCl (1 mM),

isopropanol (10% v/v), Triton X-100 (0.5% v/v), the

fatty acid derivative (1 mM) and the enzyme.

Reactions were initiated by the addition of the

substrate and the Triton X-100 dissolved in

isopropanol. As the standard substrate for measuring

enzyme activity pNP-C14:0 was chosen. One unit of

lipase activity was defined as the amount of enzyme

releasing 1 μmol pNP per minute. All measurements

were performed at least in triplicates.

Chain length selectivity was investigated by

measuring the hydrolytic activity towards several

p-nitrophenyl (pNP) esters, including pNP-myristate

Determination of protein concentration

(C14:0),

(C18:1),

-palmitate

(C16:0),

(C18:2),

-oleate

Protein concentration was determined by the

bicinchoninic acid (BCA) assay (Pierce™ BCA

Protein Assay Kit", ThermoFisher Scientific,

Waltham, USA) with bovine serum albumin as a

standard. Absorbance of all samples was measured in

triplicates (λ = 562 nm) (Tecan Infinite M2000 Pro,

Tecan Group Ltd., Männedorf, Switzerland).

-linoleate

-linolenate

(C18:3), -gondoate (C20:1), and -erucate (C22:1).

Determination of protein concentration

pNP-C14:0 and pNP-C16:0 were purchased from

Fluka, while the FA esters derived from C18:1

(cisΔ9), C18:2 (cisΔ9, cis-Δ12), C18:3 (cisΔ9,

cisΔ12, cisΔ15), C20:1 (cisΔ11) and C22:1 (cisΔ13)

acids were synthesized in one-gram scale using the

corresponding acid choride derivatives (NuCheck,

USA) as described by Brundiek et al. (2012)^[31]. To a

250 ml round-bottom flask, with magnetic stirring,

under an inert atmosphere (N₂), anhydrous ZnCl₂

SDS-PAGE

Sodium dodecyl sulfate polyacrylamide gel

electrophoresis (SDS-PAGE) was performed using a

12.5% and 5 % acrylamide in the resolving and

stacking gels, respectively. Gels were run on a Mini-

13

10.1002/adsc.201800889

Advanced Synthesis & Catalysis

(0.54 g, 3.96 mmol), anhydrous dichloromethane

(30 ml), and pNP (1.0 g) were added. The

corresponding acid chloride (1.0 g) was added

dropwise by a syringe, and the reaction mixture was

refluxed until complete conversion was observed by

thin layer chromatography (~1 h). The reaction

mixture was then cooled to room temperature in an

ice bath. Afterwards, 30 ml of water were added to

the reaction mixture to, subsequently, decant the

organic layer. The aqueous phase was extracted with

diethyl ether (Et₂O, 2×30 ml). The combined organic

layers were further washed with NaHCO₃(2×30 ml)

and, then, dried with anhydrous Na₂SO₄. After

filtration and evaporation in vacuo this yielded a dark

brown product. Purification was performed by flash

chromatography (hexane/Et₂O, 10:1) to afford the

pure pNP-FA derivative as a clear, beige oil. The

identity of all compounds synthesized was confirmed

Bioinformatic tools

Molecular modelling was performed using YASARA

(www.yasara.org) based on the 2.1 Å-resolution X-

ray structure of molecule A of CAL-A (PDB:

3GUU). Structural refinement was carried out by a

molecular dynamics simulation in water. Docking

experiments with the refined structure were

performed using oleic, gondoic, and erucic acid as

ligands. The simulation cell was arranged including

the catalytic residues and the whole binding tunnel of

CAL-A. For the docking experiment the

dockrunensemble macro from YASARA was used.

The binding of the docked FA was further

investigated for CAL-A wt and V290W by molecular

dynamic simulations with an Amber14 forcefield

utilizing the mdrun macro of YASARA under

physiological conditions and a frame rate of 250 ps.

Snapshots were evaluated concerning the FA binding

of the tunnel and the tunnel architecture.

1

by H NMR spectroscopy (300 MHz; dilute solution

in CDCl₃) (Figure S7).

1

- pNP-C18:1 (0.83 g, 62% yield): H-NMR (300

Further characterization of the tunnel was

performed with the software CAVER Analyst 1.0^[32]

(www.caver.cz). Tunnels were computed starting

from the catalytic Ser184 with a maximal distance of

5 Å and a solvent radius of 0.7 Å.

MHz; dilute solution in CDCl₃): δ=8.27 (2H, d,

J=9.25 Hz, 2×Ar-H), 7.27 (2H, d, J=9.25 Hz, 2×Ar-

H), 5.43–5.30 (2H, m, -HC=CH–), 2.60 (2H, t,

J=7.55 Hz, α-CH₂), 2.11–1.91 (4H, m, H₂C–

HC=CH–CH₂), 1.83–1.69 (2H, m, β-CH₂), 1.49–1.19

(20H, m), and 0.88 (t, J=6.61 Hz, CH₃).

1

- pNP-C18:2 (1.14 g, 85% yield): H-NMR (300

Generating CAL-A mutant libraries

MHz; dilute solution in CDCl₃): δ=8.26 (dd, J=9.25

Hz, 2H, 2×Ar-H), 7.27 (dd, J=9.25 Hz, 2H, 2×Ar-H),

5.45-5.27 (m, 4H, 2×-HC=CH–), 2.78 (t, J=6.0 Hz,

2H =CH–CH₂–HC=), 2.59 (t, J=7.45 Hz, 2H, α-CH₂),

2.11–1.99 (m, 4H, H₂C–HC=CH–CH₂), 1.82–1.70

(m, 2H, β-CH₂), 1.40–1.24 (m, 14H), and 0.89 (t,

J=6.61 Hz, 3H, CH₃).

Three different CAL-A mutant libraries were

generated to perform saturation mutagenesis

simultaneously at two positions with a "small, but

smart" focused library^[19b]. Saturation positions for

each library were: V238X and V286X for library I;

V238X and V290X for library II; and V286X and

V290X for library III. CAL-A libraries I and II were

generated via MegaWhop with specific primers for

each variant to produce megaprimers (MP1 and

MP2)^[33](Table S6). The plasmid containing the

CAL-A gene (100 ng) was mixed with 10x Buffer B

(5 µl), dNTPs (2 µl), 25 mM MgSO₄(1 µl), 0.5 µl

OptiTaq, forward primer 16.21 (1 µl, Lib I+II), the

corresponding reverse primer 16.19 (Lib I) or 16.22

(Lib II) (1 µl), and distilled water (38.5 µl). The PCR

program included the following temperature steps:

95 °C for 2 min; subsequently 30 cycles of the

following: 95 °C for 15 s, 55 °C for 30 s, 72 °C for

42 s; and finally a holding step at 72 °C for 10 min.

The PCR product was purified (Nucleo Spin^®Gel

and PCR Clean-up, Macherey & Nagel, Düren,

Germany) and used in the optimized protocol for the

MegaWhop as megaprimer. The plasmid containing

the CAL-A gene (100 ng) was mixed with 10x Pfu

buffer (5 µl), dNTPs (1 µl), purified megaprimer

(5 µl), undiluted PfuPlus polymerase (0.25 µl), and

distilled water (37.75 µl). The following program was

used: hold 94 °C for 2 min, 25 cycles of the following

three steps: 94 °C for 30 s, 55 °C for 30 s, and 72 °C

for 437 s. Finally hold at 72 °C for 10 min. The

template DNA was digested with 1 µl of DpnI

(10 U/µl solution) for 3 h at 37 °C and 20 min at

80 °C.

1

- pNP-C18:3 (0.98 g, 74% yield): H-NMR (300

MHz; dilute solution in CDCl₃): δ=8.26 (dd, J=9.25

Hz, 2H, 2×Ar-H), 7.27 (dd, J=9.25 Hz, 2H, 2×Ar-H),

5.54-5.19 (m, 6H, 3×-HC=CH–), 2.81 (t, J=5.71 Hz,

2H =CH-CH₂-HC=), 2.60 (t, J=7.42 Hz, 2H, α-CH₂),

2.14–1.97 (m, 4H, H₂C–HC=CH–CH₂), 1.82–1.68

(m, 2H, β-CH₂), 1.41–1.24 (m, 13H), and 0.98 (t,

J=7.4 Hz, 3H, CH₃).

1

- pNP-C20:1 (1.17 g, 89% yield): H-NMR (300

MHz; dilute solution in CDCl₃): δ=8.27 (dd, J=9.20

Hz, 2H, 2×Ar-H), 7.27 (dd, J=9.20 Hz, 2H, 2×Ar-H),

5.37-5.32 (m, 2H, -HCOCH–), 2.60 (t, J=7.45 Hz,

2H, α-CH₂), 2.06–1.96 (m, 4H, H₂C–HC=CH–CH₂),

1.80–1.65 (m, 2H, β-CH₂), 1.37–1.25 (m, 24H), and

0.88 (t, J=6.60 Hz, 3H, CH₃).

1

- pNP-C22:1 (1.04 g, 81% yield): H-NMR (300

MHz; dilute solution in CDCl₃): δ=8.25 (dd, J=9.30

Hz, 2H, 2×Ar-H), 7.27 (dd, J=9.30 Hz, 2H, 2×Ar-H),

5.37-5.32 (m, 2H, -HC=CH–), 2.60 (t, J=7.45 Hz,

2H, α-CH₂), 2.06–1.96 (m, 4H, H₂C–HC=CH–CH₂),

1.81–1.70 (m, 2H, β-CH₂), 1.45–1.21 (m, 28H), and

0.88 (t, J=6.60 Hz, 3H, CH₃).

14

10.1002/adsc.201800889

Advanced Synthesis & Catalysis

The CAL-A library III was generated via

v/v), the fatty acid derivative (1 mM, pNP-C14:0,

pNP-C18:1 or pNP-C22:1) and 45 µl of SN sample

for each reaction. Reactions were initiated by the

addition of the substrate and the Triton X-100

dissolved in isopropanol. The final reaction volume

was 200 µl. One unit of lipase activity was defined as

the amount of enzyme releasing 1 µmol p-nitrophenol

per minute.

QuikChange™ with specific primers^[34](Table S6).

The plasmid containing the CAL-A gene (100 ng)

was mixed with 10x Buffer B (5 µl), dNTPs (2 µl),

25 mM MgSO₄(1 µl), 0.5 µl OptiTaq, forward

primer 16.20 (1 µl), reverse primer 16.19 (1 µl), and

distilled water (38.5 µl). The PCR program included

the following temperature steps: hold at 95 °C for

2 min, afterwards 10 cycles of the following: hold at

95 °C for 15 s, 55 °C for 30 s, 72 °C for 437 s.

Subsequent cycles (25) are prolonged by their

elongation time by 20 s per cycle. Finally hold at

72 °C for 10 min. The template DNA was digested

with 1 µl of DpnI (10 U/µl solution) for 3 h at 37 °C

and 20 min at 80 °C.

Creation of pPICZα-CALA-His vectors containing

the desired mutations

In order to overexpress in P. pastoris the enzyme

CAL-A wt and its most interesting variants,

QuikChange reactions were performed using as

template pPICZα-CALA-His / pGAPZα-CALA-His

vector or the corresponding derivative. For this

purpose, specific primers were designed to insert the

desired mutation (Table S7 and S8). The reactions

mixture included the CAL-A plasmid template

(100 ng, 1 µl), 10x Buffer B (5 µl), dNTPs (2 µl),

25 mM MgSO₄(1 µl), 0.5 µl OptiTaq, forward

primer (1 µl), reverse primer (1 µl), and distilled

water (q.s. 50 µl). The PCR program included the

following temperature steps: a denaturation step at

95 ºC during 2 min; then 20 cycles of amplification

including denaturation at 95 °C for 1 min, annealing

at 50 °C for 1.5min, and elongation at 72 °C for

2min, and, finally, temperature was hold at 72 °C for

10 min. The template DNA was then digested with

1 µl of DpnI (10 U/µl solution) for 3 h at 37 °C and

20 min at 80 °C. The PCR-products were first

transformed into electrocompetent E. coli TOP10

cells as described above. When the presence of the

desired mutation in the CAL-A gene was confirmed

by sequencing, a 30 % glycerol stock of the strain

was frozen at -80 ºC and 20 ml of LB-ls containing

25 µg/ml Zeocin were inoculated for plasmid

The PCR-products were first transformed into

electrocompetent E. coli TOP10 cells and, after

plasmid

purification,

transformed

into

electrocompetent E. coli C41 (DE3) cells, following

the protocol previously described.

Screening of CAL-A variants using the robotic

platform LARA

High-throughput screening of the CAL-A variant

libraries was performed in the "laboratory automation

robotic assisant" (LARA)^[20]. For this purpose, 5,000

colonies transformed with each of the mutant libraries

previously obtained were used to inoculate master

MTP (M-MTP) containing 220 µl per well of LB

with ampicillin (0.1 mg/ml). Utilizing the QPix

420 Colony Picking System (Molecular Devices,

USA) each well was inoculated with only one colony.

The so prepared M-MTPs were incubated at 37 ºC

and 700 rpm during 5 h and, subsequently, stored at

4 ºC O/N until later usage. These plates served as a

backup for the different libraries.

Overexpression of the CAL-A library variants was

achieved in microtiter plates (OE-MTP) containing

230 µl/well of auto-induction ZYM-5052 medium^[29]

per well and inoculated using the corresponding

grown master MTP. Before inoculation M-MTPs

were revitalized by incubation for 2 h at 37 ºC. After

inoculation they were incubated at 37 ºC and 700 rpm

for 6 h. For better folding at the protein expression

stage, the temperature was the temperature was

changed to 30 ºC for another 22 h. OD_600nmwas

monitored during the process to verify the growth of

the cells. The supernatants were transferred to a fresh

plate and stored at 4 ºC until performing the

hydrolytic activity measurements after centrifugation

of the OE-MTPs at 4 ºC and 4500 rpm.

purification

and

subsequent

P. pastoris

transformation (see Transformation of P. pastoris

section).

Fatty acid enrichment reactions

First, the right amount of lipase for the EFA

enrichment reactions was tested by checking the rate

of hydrolysis with 1, 5, 10, or 50 U of enzyme after

4 h reaction. The reaction solution contained 5 g

Crambe ethyl esters or Camelina ethyl esters

(Solutex, Zaragoza, Spain), 2 g gum arabic, and

100 ml buffer 50 mM sodium phosphate with 1 mM

NaCl at pH 7.5. The emulsification was achieved by

shearmixing at 24,000 rpm for 2 min (Ultra Turrax

T25 basic, IKA Labortechnik, Staufen, Germany).

Subsequently, 250 µl of the emulsion were dispensed

in 1.5 mL polypropylene tubes for each time sample.

The reaction was started by adding either buffer, or

the corresponding Us of enzyme per reaction vessel.

Enzyme solutions were prepared using the

lyophilised supernatants of the fermentation of

Hydrolytic activity reaction conditions in LARA

were similar to the conditions usually employed for

hydrolytic activity measurements outside the robot.

Initial hydrolysis rate of p-nitrophenyl esters was

followed spectrophotometrically for 5 min at λ=410

nm (ε=10.84 mM⁻¹cm⁻¹) at 37 °C in a 96-well

microtiter plate. The assay was performed in sodium

phosphate buffer (50 mM; pH=7.5) containing NaCl

(1 mM), isopropanol (10% v/v), Triton X-100 (0.5%

15

10.1002/adsc.201800889

Advanced Synthesis & Catalysis

P. pastoris GS115 strains overexpressing CAL-A

wild type and variants V290W, 1, 2, 3, 9, 16, 18, 19

and 20. Prior their use, enzyme solutions were

dialyzed towards distilled, deionized water and their

activity was determined towards pNP-myristate as

previously described. Reaction vessels were shaken at

1400 rpm and 37 °C. Reactions were stopped by

adding 25 µl 4 N HCl. Samples were then vortexed

and stored at –20 °C until extraction. After extraction

of the samples, as described underneath, and

subsequent evaporation of the solvent, samples were

dissolved in 500 µL n-hexane and 2 µL were loaded

in TLC, as described later, to check for a sufficient

rate of hydrolysis (10-40 %).

Long chain fatty acid enrichment biocatalysis

reactions were prepared in the same way as described

above. Reactions were started by adding the

corresponding amount of enzyme (5 or 10 U) and

taking time samples after 0; 0.5; 1; 2; 4; 8; 24; and

48 h. All reactions were carried out in triplicates.

treated with 50 µl 2,2-dimethoxypropane and freshly

prepared anhydrous 500 µl 2% methanolic hydrogen

sulfate. The samples were shaken at 14000 rpm in a

glass vial at 60 °C for 4 h. After the reaction, samples

were extracted twice with 300 µl n-hexane and the

collected organic phases were dried over anhydrous

Na₂SO₄. The organic layer was analyzed via GC

(GC-2010, Shimadzu, Duisburg, Germany).

For GC-analysis of fatty acid methyl and ethyl

esters (FAME and EFA) a BPX-70 column (SGE

0.25 mm diameter, 0.25 µm film thickness, 60 m

length; SGE Analytical Science, Victoria, Australia)

was used. Analysis of 1 µl of sample was carried out

with a flow rate of 0.52 ml/min and a temperature

program starting at (i) 180°C for 33 min, followed by

heating step (ii) to 230°C at 10 °C/min and a final

holding step for 5 min. Injector and detector

temperatures were set at 240 °C and 290 °C,

respectively.

Firstly, the samples were analyzed by peak

identification of the different FAME's to a standard

FAME-Mix

(Sigma

CRM47885,

Steinheim,

Thin-layer chromatography

Germany) or to single EFA standards, respectively

(Figures S3 and S4). The peaks were normalized to

their standards ethyl-tridecanoate (EFA) or methyl-

tridecanoate (H₂SO₄/MeOH method) and only the

FAME peaks were treated with their theoretical

Thin-layer chromatography (TLC) was used to

preliminarily check the rate of hydrolysis of the EFA.

For this purpose, 4 h time samples were extracted

twice with 500 µL diethylether by vortexing for 1

min and centrifuging at 16200 g during 1 min. The

upper organic layer was collected. The joined organic

phases were dried with anhydrous Na₂SO₄by

vortexing for 1 min and centrifuging at 16200 g for

5 min. The organic solvent was then evaporated

under a stream of nitrogen. Subsequently, the sample

was dissolved in 500 µL n-hexane and 2 µL of this

mixture was spotted on a silica TLC plate (Silica gel

on TLC Al plate, Sigma-Aldrich). This plate was

response correction factors (Eq. 1) (RCF, Table 4)^[37]

.

퐴

푅퐶퐹

푃푒푎푘

푐_푃푒푎푘

=

∗ 푐_{푆푡푎푛푑푎푟푑}

∗

(Eq. 1)

퐴

푅퐶퐹

푆푡푎푛푑푎푟푑

The EFA or FFA amount of the samples was

calculated by comparing the EFA amount determined

by the EFA analysis towards the whole FA amount

determined by the H₂SO₄/MeOH method (Eq. 2).

developed with

hexane:diethyl

a

solvent composed of n-

ether:glacial

acetic

acid

푐

푝_푁퐸퐹퐴= (1 − ^퐹퐴퐸퐸) ∗ 100%

(Eq. 2)

(79:20:1 v/v/v)^[35]. For visualization, the TLC plate

was stained with potassium permanganate solution

(0.1 g/L KMnO₄, 0.2 g/L CH₃COOH, 0.5 g/L

NaHCO₃).

푐

퐻

푆푂

4

2

The theoretical yield of enriched FA was

determined by comparing the total amount of this FA

in the sample to its amount present in its enriched

fraction (Either FFA or EFA) (Eq. 3).

Fatty acid quantification of different oil fractions

Reaction samples were extracted with 250 µl

diethylether containing 2 mM tridecanoate ethyl ester

and 2 mM tripentadecanoate (serving as internal

standards), by vortexing for 1 min and centrifuging at

16 200 g during 1 min. The upper organic layer was

collected. Each sample was extracted two more times

with 500 µl pure diethylether. The joined organic

phases were dried with anhydrous Na₂SO₄by

vortexing for 1 min and centrifuging at 16200 g for

5 min. The organic solvent was then evaporated

under a stream of nitrogen.

The residual oil was resuspended in 1 ml

anhydrous n-hexane and split in two aliquots for

direct analysis of the EFA (EFA) and treatment with

the methanolic hydrogensulfate method for

esterification of the total FA^[36]. For the methanolic

hydrogensulfate method, the 500 µl samples were

푛

푌

퐹퐴

=

^{퐹퐴 푖푛 퐸퐹퐴 표푟 퐹퐹퐴}∗ 100%

(Eq. 3)

푛

퐹퐴(퐻 푆푂

)

4

2

Table 4. RCF for flame ionization detectors to convert to

weight percent methyl ester^[37]

.

FA

RCF

1.06

1.03

1.02

1.00

0.99

FA

RCF

0.99

0.98

0.97

FA

RCF

0.97

0.96

C13:0

C15:0

C16:0

C18:0

C18:1

C18:2

C18:3

C20:0

C20:1

C20:2

C20:3

C22:0

C22:1

C24:0

C24:1

16

10.1002/adsc.201800889

Advanced Synthesis & Catalysis

Acknowledgements

[12]

a) H. B. Brundiek, S. K. Padhi, R. Kourist, A.

Evitt, U. T. Bornscheuer, Eur. J. Lipid Sci.

Technol. 2012, 114, 1148-1153; b) A. O.

Magnusson, J. C. Rotticci-Mulder, A.

Santagostino, K. Hult, ChemBioChem 2005,

6, 1051-1056.

J. Pleiss, M. Fischer, R. D. Schmid, Chem.

Phys. Lipids 1998, 93, 67-80.

A. Svendsen, J. Vind, S. A. Patkar, K. Borch,

Patent WO2008040738 A1, 2008.

D. J. Ericsson, A. Kasrayan, P. Johansson, T.

Bergfors, A. G. Sandstrom, J. E. Backvall, S.

L. Mowbray, J. Mol. Biol. 2008, 376, 109-

119.

O. Kirk, M. W. Christensen, Org. Process

Res. Dev. 2002, 6, 446-451.

The COSMOS project has received funding from the

European Union’s Horizon 2020 research and

innovation program under grant agreement No

635405. We are also grateful to Novozymes

(Bagsvaerd, Denmark) for providing samples of CAL-

A.

[13]

[14]

[15]

References

[1]

a) U. Biermann, U. Bornscheuer, M. A.

Meier, J. O. Metzger, H. J. Schäfer, Angew.

Chem. Int. Ed. 2011, 50, 3854-3871; b) U.

Schörken, P. Kempers, Eur. J. Lipid Sci.

Technol. 2009, 111, 627-645; c) G. M.

Borrelli, D. Trono, Int. J. Mol. Sci. 2015, 16,

20774-20840.

[16]

[17]

[18]

[19]

M. Widmann, P. B. Juhl, J. Pleiss, BMC

Genomics 2010, 11, 123-130.

[2]

[3]

E. C. Leonard, Ind. Crop. Prod. 1993, 1, 119-

123.

M. T. Reetz, D. Kahakeaw, R. Lohmer,

ChemBioChem 2008, 9, 1797-1804.

a) L. Tang, H. Gao, X. Zhu, X. Wang, M.

Zhou, R. Jiang, BioTechniques 2012, 52,

149-158; b) S. Kille, C. G. Acevedo-Rocha,

L. P. Parra, Z. G. Zhang, D. J. Opperman, M.

T. Reetz, J. P. Acevedo, ACS Synth. Biol.

2013, 2, 83-92.

M. Dörr, M. P. C. Fibinger, D. Last, S.

Schmidt, J. Santos-Aberturas, D. Böttcher, A.

Hummel, C. Vickers, M. Voss, U. T.

Bornscheuer, Biotechnol. Bioeng. 2016, 113,

1421-1432.

J. Pfeffer, S. Richter, J. Nieveler, C. E.

Hansen, R. B. Rhlid, R. D. Schmid, M.

Rusnak, Appl. Microbiol. Biotechnol. 2006,

72, 931-938.

R. V. Muralidhar, R. R. Chirumamilla, R.

Marchant, V. N. Ramachandran, O. P. Ward,

P. Nigam, World J. Microb. Biot. 2002, 18,

81-97.

a) C. Lu, J. A. Napier, T. E. Clemente, E. B.

Cahoon, Curr. Opin. Biotechnol. 2011, 22,

252-259; b) J. A. Napier, I. A. Graham, Curr.

Opin. Plant Biol. 2010, 13, 330-337.

C. Tao, B. B. He, T. ASAE 2005, 48, 1471-

1479.

K. Zorn, I. Oroz-Guinea, H. Brundiek, U. T.

Bornscheuer, Prog. Lipid Res. 2016, 63, 153-

164.

[4]

[5]

[20]

[6]

[7]

L. Casas-Godoy, S. Duquesne, F. Bordes, G.

Sandoval, A. Marty, Methods Mol. Biol. 2012,

861, 3-30.

a) J. A. Kralovec, S. Zhang, W. Zhang, C. J.

Barrow, Food Chem. 2012, 131, 639-644; b)

G. P. McNeill, R. G. Ackman, S. R. Moore, J.

Am. Chem. Oil Soc. 1996, 73, 1403-1407; c)

T. Hoshino, T. Yamane, S. Shimizu, Agric.

Biol. Chem. 1990, 54, 1459-1467.

[21]

[22]

[8]

[9]

a) G. P. McNeill, P. E. Sonnet, J. Am. Chem.

Oil Soc. 1995, 72, 213-218; b) N. Volkova, X. [23]

Li, L.-H. Zhu, P. Adlercreutz, Sustain. Chem.

E. Rogolska, C. Cudrey, F. Ferrato, R.

Verger, Chirality 1993, 5, 24-30.

Proc. 2016, 4; c) T. N. B. Kaimal, R. B. N.

Prasad, T. Chandrasekhara Rao, Biotechnol.

Lett. 1993, 15, 353-356; d) P. E. Sonnet, T. A.

Fogila, S. H. Feairheller, J. Am. Chem. Oil

Soc. 1993, 70, 387-391.

a) R. Borgdorf, S. Warwel, Appl. Microbiol.

Biotechnol. 1999, 51, 480-485; b) H. B.

Brundiek, A. S. Evitt, R. Kourist, U. T.

Bornscheuer, Angew. Chem. Int. Ed. 2012, 51,

412-414.

[24]

a) H. P. Heldt-Hansen, M. Ishii, S. A. Patkar,

T. T. Hansen, P. Eigtved, Vol. 389, 1989, pp.

158-172; b) D. Briand, E. Dubreucq, P.

Galzy, Eur. J. Biochem. 1995, 228, 169-175.

H. Grynberg, H. Szczepanska, J. Am. Chem.

Soc. 1966, 43, 151-152.

P. Domínguez de María, J. M. Sánchez-

Montero, J. V. Sinisterra, A. R. Alcántara,

Biotechnol. Adv. 2006, 24, 180-196.

J. Müller, M. A. Sowa, B. Fredrich, H.

Brundiek, U. T. Bornscheuer, ChemBioChem

2015, 16, 1791-1796.

[25]

[26]

[27]

[10]

[11]

a) R. D. Joerger, M. J. Haas, Lipids 1994, 29,

377-384; b) R. R. Klein, G. King, R. A.

Moreau, M. J. Haas, Lipids 1997, 32, 123-

130.

J. Schmitt, S. Brocca, R. D. Schmid, J. Pleiss,

Protein Eng. 2002, 15, 595-601.

[28] a) L. You, F. H. Arnold, Protein Eng. 1996, 9,

77-83; b) C. Schmidt-Dannert, F. H. Arnold,

Trends Biotechnol. 1999, 17, 135-136.

17

10.1002/adsc.201800889

Advanced Synthesis & Catalysis

[29]

[30]

[31]

F. W. Studier, Protein Expr. Purif. 2005, 41,

207-234.

[34]

[35]

L. Zheng, U. Baumann, J. L. Reymond,

Nucleic Acids Res. 2004, 32, e115.

M. Bertram, C. Manschot-Lawrence, E.

Flöter, U. T. Bornscheuer, Eur. J. Lipid Sci.

Technol. 2007, 109, 180-185.

a) S. Strelkov, M. von Elstermann, D.

Schomburg, Biol. Chem. 2004, 385, 853-861;

b) M. Tai, G. Stephanopoulos, Metab. Eng.

2013, 15, 1-9.

W. W. Christie, Lipid analysis: Isolation,

seperation, identification and structural

analysis of lipids, Vol. 15, Third ed., The oil

press lipid library, Shepperton (Middlesex,

UK), 2003.

G. Kouker, K.-E. Jaeger, Appl. Environ.

Microbiol. 1987, 53, 211-213.

H. Brundiek, S. Sass, A. Evitt, R. Kourist, U.

T. Bornscheuer, Appl. Microbiol. Biotechnol.

2012, 94, 141-150.

E. Chovancova, A. Pavelka, P. Benes, O.

Strnad, J. Brezovsky, B. Kozlikova, A. Gora,

V. Sustr, M. Klvana, P. Medek, L.

Biedermannova, J. Sochor, J. Damborsky,

PLoS Comput. Biol. 2012, 8, e1002708.

K. Miyazaki, in Methods in Enzymology, Vol.

Volume 498 (Ed.: V. Christopher), Academic

Press, 2011, pp. 399-406.

[36]

[37]

[32]

[33]

18

10.1002/adsc.201800889

Advanced Synthesis & Catalysis

FULL PAPER

Alteration of Chain Length Selectivity of

Candida antarctica Lipase A by Semi-Rational

Design for the Enrichment of Erucic and

Gondoic Fatty Acids

Candida antarctica

lipase A

RENEWABLE

MATERIALS:

CRAMBE AND

CAMELINA OILS

SEMI-RATIONAL

DESIGN

Adv. Synth. Catal. Year, Volume, Page – Page

≥ C20

Erucic acid  78 %

Gondoic acid  34 %

Katja Zorn¹, Isabel Oroz-Guinea¹, Henrike

Brundiek, Mark Dörr and Uwe T. Bornscheuer*

OLEOCHEMICAL

PROCESSES

≤ C18

19

Article Doi

Alteration of Chain Length Selectivity of Candida antarctica Lipase A by Semi-Rational Design for the Enrichment of Erucic and Gondoic Fatty Acids

DOI: 10.1002/adsc.201800889

Source and publish data:

Authors:

Article abstract of DOI:10.1002/adsc.201800889

Full text of DOI:10.1002/adsc.201800889

Products guided by the article

R&D Labs maybe for 6562-50-1

Relevant to this article

Hot Product