Available online at www.sciencedirect.
com
Bio-based surfactants: enzymatic functionalization and
production from renewable resources
Jane W Agger and Birgitte Zeuner
Bio-based surfactants produced from renewable resources are
increasing in market demand. In this review, we focus on
enzymatic functionalization and coupling of carbohydrate-based
heads to fatty aliphatic chains as tails for the synthesis of bio-
based surfactants. We point to concrete examples of how
transferase, lipase, and glycoside hydrolase-catalyzed
esterification or glycoside formation can link a variety of mono-
and oligosaccharides with fatty acids. Similarly, enzymatic
reductive amination also leads to coupling. Another approach for
surfactant synthesis is enzymatic carbohydrate functionalization
before click chemistry coupling, and here LPMOs, oxidases, and
dehydrogenases are relevant. C6 or C1-oxidizing activities are
particularly important for converting nonionic surfactants into
highly demanded anionic counterparts.
Address
Technical University of Denmark, Department of Biotechnology and
Biomedicine, Søltofts Plads 221, DK-2800 Kgs. Lyngby, Denmark
Corresponding author: Jane W Agger (jaag@dtu.dk)
Current Opinion in Biotechnology 2022, 78:102842
This review comes from a themed issue on Chemical
Biotechnology
Edited by Bernd Nidetzky and Byung-Gee Kim
For complete overview of the section, please refer to the article
collection, “Chemical Biotechnology (2023)”
Available online 11th November 2022
https://doi.org/10.1016/j.copbio.2022.102842
0958–1669/© 2022 The Author(s). Published by Elsevier Ltd. This is
an open access article under the CC BY license (http://
creativecommons.org/licenses/by/4.0/).
Introduction
Surfactants are amphiphilic molecules of incredible,
chemical variety. With a hydrophilic ‘head’ and a hy-
drophobic ‘tail’, surfactants decrease interfacial tension
and facilitate interactions between molecules of dif-
ferent polarity by locating at the interface, for example,
water/oil mixtures [1,2•]. The specific properties largely
dictated by the hydrophilic head determine their appli-
cation. Surfactants typically divide into nonionic, an-
ionic, cationic, and zwitterionic surfactants. Common
surfactant applications include detergents, personal care,
lubricants, pharmaceuticals, bioremediation, and
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agriculture. Anionic and nonionic surfactants are the
most widely produced groups [1–4].
Most current surfactant production is fossil-based and
environmentally challenging. The products may exhibit
problematic health and safety issues in terms of tox-
icology, bioaccumulation, and low biodegradability [3].
While the petrochemical production is currently more
economically favorable, sustainable production of bio-
based surfactants has important advantages also in terms
of biodegradability and biocompatibility [1,2•]. Im-
portantly, consumer demands and political incentives
may soon change the traditional perception that surfac-
tant production from renewable feedstocks is merely a
way of upgrading agroindustrial side streams, and the
sheer volume of surfactant consumption suggests they
be produced renewably. Online reports (www.
marketsandmarkets.com) estimate a surfactant market
size in 2022 of 46 billion USD, whereas the market sizes
are estimated to be 17 billion USD for bio-based sur-
factants and 5.5 billion USD for biosurfactants. The
compound annual growth rates are 4.5%, 5.1%, and 5.6%
for these markets, respectively.
Bio-based surfactants include any surfactant produced
from biological feedstocks, such as carbohydrates, fats, or
proteins [1]; suggestions for specific molecules and their
renewable sources were recently reviewed [5]. Bio-based
surfactants are produced chemically, enzymatically, or in
cell factories; the latter coined as biosurfactants. Che-
mical synthesis involves harsh conditions and excessive
solvent use. Microbial cell factories are scalable and
largely sustainable, but major disadvantages include that
complex genetic manipulation is required to alter the
chemical product structure and that the output is a
mixture of related products. Biosurfactant production
was recently reviewed [2•], thus, this review focuses on
development of enzyme-catalyzed surfactant synthesis.
Enzymes catalyze synthesis under environmentally low-
impact reaction conditions and can readily utilize re-
newable feedstocks in highly selective reactions. En-
zymes are praised for their high selectivity compared
with chemical synthesis, yet it is often their promiscuity
that is exploited in synthetic reactions [6,7].
We discuss recent developments within functionaliza-
tion of saccharides and lipids that could find important
applications in the demanded future expansion of sus-
tainable production of bio-based surfactants from
Current Opinion in Biotechnology 78 (2022) 102842
2 Chemical Biotechnology
Figure 1
Examples the three most important classes of enzyme-catalyzed bio-based surfactants. Carbohydrate esters: (a) xylo-oligosaccharide laureate esters
[8]. Alkyl glycosides: (b) α-D-glucose-elongated dodecyl β-D-maltoside diuronic acid [9•] and (c) pentyl β-D-xylosides prepared directly from wheat bran
in a 1-pentanol/water mixture [10••]. Amides: (d) MEGAs [11••].
renewable resources. These technologies are also useful
in development of new building blocks for other bio-
based materials. We begin with a brief review of recent
developments in enzymatic linking of the hydrophobic
and hydrophilic surfactant moieties.
Linking surfactant head and tail enzymatically
The stability of the surfactant depends on the stability of
each component, but certainly also on the type of che-
mical coupling between the hydrophilic and hydro-
phobic moieties. Hence, ester-linked surfactants are
labile under alkaline conditions and therefore less ap-
plicable in detergents, while acetals are labile under
acidic conditions. Amides and ethers are more stable
than the former two [1]. The prevalent enzyme-cata-
lyzed linkages between the head and tail moieties in
surfactants include ester, amide, and glycoside linkages
(acetals, Figure 1).
From an industrial point of view, the robust hydrolases
are the most important enzymes in synthesis.
Traditionally, enzymatic formation of linkages between
the hydrophilic head and the hydrophobic tail in sur-
factants is catalyzed by the rather versatile lipases or the
Current Opinion in Biotechnology 78 (2022) 102842
Current Opinion in Biotechnology
more specific glycoside hydrolases (GHs). These hy-
drolases carry out synthetic reactions either through the
thermodynamically controlled reversed hydrolysis,
which generally requires low water content, or via the
kinetically controlled transfer reactions, which require
substrates with a bond that is broken during the first step
of the reaction [12,13]. In certain cases, proteases have
also been employed for surfactant synthesis, for ex-
ample, for synthesis of amino acid-based surfactants
[14,15]. As outlined below, other enzyme activities are
also entering the scene.
The promiscuity of lipases is exploited in enzymatic
surfactant synthesis, not least because lipases are stable
and activated at interfaces [16,17]. Indeed, ester-linked
surfactants are classic products of biocatalysis. Recent
interesting examples include synthesis of laureate esters
of C5 sugars derived from lignocellulosic biomass
[8,10••,18]. The reactions were catalyzed by the stable
workhorse-immobilized Candida antarctica lipase B
(Novozym 435), whose reaction conditions can be opti-
mized to ensure high selectivity toward primary alcohols
[10••,18]. In the absence of a primary alcohol on xylo-
oligosaccharides (DP2–4), O-4 of the non-reducing-end
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moiety was selectively esterified [8] (Figure 1a). An even
broader substrate specificity can be found among the
acyltransferases that may catalyze formation of both es-
ters, amides, carbonates, and carbamates in water, al-
though with a clear preference for short-chain acyl
donors [19]. Recently, the promiscuous acetyl trans-
ferase EstCE1 was engineered to accommodate glucose,
maltose, and maltotriose as acceptor substrates, enabling
the formation of short-chain sugar esters [20].
Alkyl glycosides are nontoxic, biodegradable, nonionic
surfactants, and their enzymatic synthesis from renew-
able carbohydrates is well established [12,21]: in an al-
cohol/water mixture, GHs catalyze reversed hydrolysis
and/or transglycosylation to link the reducing end of the
carbohydrate moiety (typically a monosaccharide) to the
primary alcohol on the solvent alkanol. Longer carbo-
hydrate moieties may increase alkyl glycoside bio-
compatibility [22]. Such compounds can either be
obtained with endo-acting GHs such as β-mannanase
(EC 3.2.1.78, GH5_7) that further facilitate the option of
using renewable biomass in the process [21] as also ex-
emplified with the xylanases in the commercial Cellic
Ctec2 mixture applied directly on wheat bran [10••]
(Figure 1c). Alternatively, the carbohydrate moiety of
the alkyl glycoside can be elongated, for example, with a
cyclodextrin glucanotransferase (CGTase, EC
2.4.1.19, GH13) [9•,22]. Creating anionic alkyl glyco-
sides expands the application range and may be
achieved through oxidation of the alkyl glycosides to
give uronic acid moieties [9•,23] (see ‘Enzyme-catalyzed
oxidation’; Figure 1b). Alternatively, galacturonic acid
could be sourced from (enzymatically) depolymerized,
demethoxylated pectin, yet only chemical alkyl glyco-
side synthesis has been reported [24]. The corre-
sponding enzymatic approach is hampered by the fact
that exo-acting polygalacturonases (EC 3.2.1.67/
82, GH28) employ an inverting mechanism not suited
for transglycosylation. The retaining GH138 rhamnoga-
lacturonan α-1,2-galacturonohydrolases potentially cata-
lyze transglycosylation, yet their highly specific substrate
(RG-II) is much less abundant than homogalacturonan
[25]. Lipase-catalyzed (trans)esterification of uronic
acids remains unexplored.
N-alkanoyl-N-methylglucamides (MEGAs) are nonionic
surfactants, which are biodegradable, nontoxic, mild to
the skin, stable under alkaline conditions, and therefore
in high demand in detergent and pharmaceutical in-
dustries [26]. Recently, the optimization of the highly
selective amidation to form a series of pure MEGAs
(Figure 1d) was reported using the adenylation domain
of a carboxylic acid reductase (CAR, EC 1.2.1.30) in a
coupled reaction with a polyphosphate kinase for ATP
regeneration from AMP in aqueous medium [11••].
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Bio-based surfactants Agger and Zeuner 3
Although AMP use adds to the cost, the method out-
performs a previously established lipase-catalyzed sol-
vent-free method in terms of product purity and energy
consumption [26]. CARs exhibit a broad substrate spe-
cificity and accommodate longer fatty acids (C6–C18 in
reduction [27]; C8–C12 were tested in the amidation re-
action [11••]). In comparison, acyltransferases are gen-
erally limited to short-chain acyl donors [11••].
Nevertheless, recent advances in the use of chitin dea-
cetylases (CDAs, EC 3.5.1.41) to (re-)N-acylate GlcN,
GlcNAc [28], and a chitosan tetramer [29••] represent
an interesting starting point for future developments in
utilization of chitin and chitosan, for example, for bio-
based surfactants. Importantly, a fungal CDA (CE4)
catalyzed N-acetylation with propiolate, thus enabling a
subsequent click chemistry step (Figure 2b) [29••]. Al-
ternatively, environmentally benign chemical methods
such as electrochemical N-acylation could be further
developed [30].
Enzymatic substrate functionalization
Besides performing the coupling reaction, enzymes are
eminent at introducing site-specific chemical functiona-
lization, which enables surfactant synthesis via click
chemistry connections. In addition, oxidation can trans-
form a neutral surfactant to an anionic one, rendering it
possible to upgrade and engineer the vast pool of re-
newable carbohydrates for specific products. Relevant
resources could be cellulose, chitin, or hemicellulose-
derived, but also inexpensive ones such as lactose or
starch-derived maltodextrins, with the notion of inherent
competition for food production.
Click chemistry
In 2022, the Nobel Prize in Chemistry was awarded to
the inventors of click chemistry. The term covers a pool
of chemical reactions that couple smaller molecular
building blocks into new conjugates. The terminology
was introduced in 2001 [31] and has gained immense
popularity, especially for joining biomolecules into new,
innovative entities. The approach offers fast, reliable,
and highly selective coupling of carbons via heteroatom
bonds (C–X–C), creating an endless number of oppor-
tunities for combinatorial experiments with biomole-
cules [31]. An exhaustive thematic issue in Chemical
Reviews was recently dedicated to click chemistry [32•],
including aspects of lipid and carbohydrate chemistry.
Besides a list of requirements that define a click chem-
istry reaction [31], the important reactivity in terms of
high thermodynamic driving forces (typically > 20 kcal/
mol) characterizes these reactions as “spring-loaded” for
a single-reaction trajectory with high yields and purity.
Of particular interest for coupling carbohydrates are the
carbonyl-type of reactions with formation of ureas,
Current Opinion in Biotechnology 78 (2022) 102842
4 Chemical Biotechnology
Figure 2
Functionalities for efficient coupling. (a) Overview of functional groups for click chemistry coupling between head (hydrophilic carbohydrate) and tail
(hydrophobic aliphatic chain). (b) Example of CDA-catalyzed (ClCDA [29••]) functionalization with propiolate (reverse hydrolysis, where propiolate
functions as acceptor) of glucosamine/chitosan followed by Cu(I)-catalyzed click reaction with an azide-aliphate to form the canonical azide-alkyne
cycloaddition (CuAAC).
thioureas, oxime ethers, hydrazones, and amides
(Figure 2a).
Following CDA-catalyzed N-acetylation of a chitosan
fragment with propiolate, copper(I)-catalyzed azide-al-
kyne cycloaddition (CuAAC, [33]) was recently accom-
plished (Figure 2b) [29••]. In this case, the objective
was fluorescent labeling of chitosan, yet we suggest that
this canonical click chemistry method could yield many
other interesting compounds.
Enzyme-catalyzed oxidation
C1 and/or C4-specific oxidation of lignocellulose or
chitin-derived oligosaccharides by lytic polysaccharide
monooxygenases (LPMOs) [36,37] can generate func-
tionalized carbohydrate moieties readily available for
click chemistry (Figure 3a). A C4-specific LPMO (EC
1.14.99.56, AA9) controlled a single and specific oxida-
tion event creating C4-oxidized cellotrimers to -penta-
mers directly from cellulose [38••]. Continuing with
site-specific oxidation by a cellobiose dehydrogenase
(EC 1.1.99.18, AA3_1) in a subsequent (or one-pot) re-
action introduced a carboxylic acid at the reducing end,
thus creating a bifunctional carbohydrate segment with
endless options for aminations or similar couplings
[38••]. Functionalization with LPMOs is also proposed
for other purposes such as preparation of cellulose na-
nocrystals and nanofibers [39,40].
Carbohydrate oxidases and dehydrogenases (AA3) also
catalyze site-specific oxidation. While LPMOs consume
Current Opinion in Biotechnology 78 (2022) 102842
Current Opinion in Biotechnology
H2O2 as a cosubstrate, carbohydrate oxidases often pro-
duce H2O2 by reducing molecular oxygen. Each carbo-
hydrate oxidase displays strong substrate preferences
[41], and generally introduce oxidations on C1, C2, or C6
(Figure 3a). Dehydrogenases de facto generate the same
functionalities as the oxidases, but the mechanism is
different and depends on a cofactor as electron acceptor
(NADP+, flavin, or cytochrome domains) [42].
The understanding of the importance of H2O2 as an
ingredient in a natural microbial environment for bio-
mass degradation is beginning to emerge [43]. For car-
bohydrate functionalization, carbohydrate oxidases may
have the advantage of providing reactive oxygen species
for even more oxidative chemistry. Whether carbohy-
drate dehydrogenases are better candidates depends on
other process conditions, keeping in mind that most AA3
dehydrogenases also display a certain level of oxidase
activity, rendering it challenging to avoid H2O2 for-
mation.
The AA5 copper radical oxidases (CROs) have lately
received revived attention for their activities to-
ward mono- and disaccharides [44•] and fatty alcohols
[45,46••] (Figure 3a-b). Deletion of a loop close to the
active site in an aryl alcohol oxidase (CgrAAO, EC
1.1.3.13, AA5_2) resulted in an unprecedented change of
the enzyme’s regioselectivity from strictly C6 to strictly
C1 [44•], demonstrating the power of protein en-
gineering in tailoring enzyme properties. Interestingly,
native CgrAAO can also oxidize hydroxyl-methyl furfural
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 Bio-based surfactants Agger and Zeuner 5

Figure 3

Current Opinion in Biotechnology

(a) Enzyme-catalyzed functionalization of carbohydrates (here exemplified as glucose/galactose). Catalyzing enzyme families indicated together with
reaction arrows. In many cases, the enzymatic action is limited to either R = H or R = longer saccharide chains, yet for certain enzymes, both mono-
and oligosaccharides are accommodated. The formation of C3-oxidation is a rare event and only scarcely reported for the glucose–methanol–choline
(GMC) family [34]. (b) Copper radical oxidase (aryl alcohol oxidase, CRO–AlcOx) catalyzed oxidation of fatty alcohols to fatty aldehydes. The reaction is
dependent on two co-enzymes, namely horseradish peroxidase (HRP) for forming the radical (CRO–AlcOx·) and catalase to maintain H2O2 levels
sufficiently low to avoid enzyme inactivation. Two reaction arrows indicate a cascade of reactions via a Cu+ intermediate [35•].

(HMF), the degradation product of hexoses inevitably
present in biorefineries, into 2,5-diformylfuran (DFF),
which is an important precursor for other valuable ap-
plications [47,48].
CRO-catalyzed oxidation of fatty alcohols into aldehydes
(fragrances) or completely to fatty acids [45,46••] is an
alternative strategy to obtain groups accessible for click
chemistry. While complete oxidation to carboxylic
acids is an unwanted spontaneous aldehyde hydration in
fragrance synthesis, this may even be desirable for bio-
based surfactant synthesis. All oxidase activities men-
tioned here require the concomitant removal of H2O2 to
sustain activity, for instance, by combining them with
catalase (EC 1.11.1.6) in catalytic amounts. AA5 CROs
also need stoichiometric amounts of peroxidase (EC
1.11.1.7) for their continued activation, turning them
into Cu(II)-radicals that can then perform the two-
electron oxidation of a primary alcohol [35•]. Despite
these complicating reaction requirements, fine-tuning of
conditions has shown that oxidation of fatty alcohols may
proceed to completion [46••]. The biocatalytic applic-
ability of CROs shows potentially high productivity and
improved oxygen binding in a case study on engineered
galactose oxidase (EC 1.1.3.9, AA5_2) [49•], indicating
that these enzymes are close to industrial relevance.
Classically oxidizing enzymes: laccase (EC
1.10.3.2, AA1) in combination with 2,2,6,6-trimethylpi-
peridine-1-oxyl (TEMPO) was explored for turning
nonionic alkyl glycosides into anionic surfactants
[9•,23,50]. Oxidation of octyl-β-D-glucoside and dodecyl
β-D-maltoside to their (di)uronic acid derivative was
achieved with 85% conversion into the desired product
by carefully optimizing the reaction conditions and
concentrations of substrates, enzyme, and mediator

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6 Chemical Biotechnology
[23,50]. Oxidation of longer α-D-glucose-elongated de-
rivatives suffered from depolymerization issues, while
subsequent CGTase-catalyzed elongation of the anionic
alkyl glycosides (Figure 1b) was more efficient (> 80%
conversion) [9•]. Thus, enzymes can provide anionic
surfactants either by oxidizing the entire alkyl glycoside
or by upstream oxidation of the carbohydrate before
linking head and tail.
Enzyme-catalyzed amination
Another highly wanted functionalization for click
chemistry is amination. Recently, the first fully enzy-
matic route from nonactivated carbohydrates to selec-
tively aminated sugars was demonstrated using enzyme
cascade systems [51••]. Using the previously described
galactose oxidase/horseradish peroxidase/catalase
system, oxidation at O-6 in Gal and GalNAc was
achieved. 6-aldo-D-galactose was subsequently aminated
with ω-transaminase (ω-TA, EC 2.6.1.18) and 1-pheny-
lethylamine to yield 6-amino-6-deoxy-D-galactose, yet
the corresponding conversion of 6-aldo-GalNAc could
not be fully confirmed. With pyranose dehydrogenase
(EC 1.1.99.29, AA3_2) coupled to laccase, Gal was oxi-
dized at O-2 and further aminated with the ω-TA [51••].
A one-pot enzyme cascade system was recently designed
to upgrade triglycerides, coconut oil, and soybean oil to
fatty amines [52•], which are important in production of
cationic surfactants. Following lipase-catalyzed hydrolysis
to yield free fatty acids, these were reduced by CAR
coupled with glucose dehydrogenase and polyphosphate
kinase for NADPH and ATP regeneration, respectively.
The resulting fatty aldehydes were aminated by ω-TA
using excess isopropylamine. Using pure triglycerides,
conversion yields > 95% were achieved [52•], yet more
optimization is required to ensure a sustainable and eco-
nomically feasible process directly from renewable oil
feedstocks. One important parameter to investigate is the
practical implications for surfactant properties when
making bio-based surfactants with a mixture of aliphatic
chains rather than homogeneous tail length.
Conclusions
Biocatalytic synthesis of bio-based surfactants from re-
newable feedstocks for environmentally benign pro-
duction is an area of many new prospects. The current
knowledge base is already widely evolved in terms of the
enzymatic toolbox, and future bio-based surfactants
therefore depend largely on the creativity of scientists in
their efforts to combine knowledge from different fields
in biocatalysis. Important aspects to keep in mind
though are that production costs cannot accelerate be-
yond a regular ‘high-bulk, low value’ production scheme,
and therefore process intensification is an exceptionally
important theme to invest in [53]. Aiming at renewable
feedstocks also implicates a range of techno-economical
Current Opinion in Biotechnology 78 (2022) 102842
evaluations in terms of feedstock availability, stability,
and pretreatment strategies. Bio-based surfactants will
continue to develop alongside cell factory-based bio-
surfactants, and the two approaches are equally justified
to meet the increasing market demands.
Conflict of interest statement
The authors declare that they have no known competing
financial interests or personal relationships that could
have appeared to influence the work reported in this
paper.
Data availability
No data were used for the research described in the ar-
ticle.
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