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Alginate lyase: Review of major sources and

classification, properties, structure-function
analysis and applications

Benwei Zhu & Heng Yin

To cite this article: Benwei Zhu & Heng Yin (2015) Alginate lyase: Review of major sources and
classification, properties, structure-function analysis and applications, Bioengineered, 6:3, 125-131,
DOI: 10.1080/21655979.2015.1030543

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Bioengineered 6:3, 125--131; May/June 2015; © 2015 Taylor & Francis Group, LLC
Alginate lyase: Review of major sources and classification, properties,
structure-function analysis and applications
Benwei Zhu1,2 and Heng Yin1,*
1
Dalian Institute of Chemical Physics; Chinese Academy of Sciences; Dalian, PR China; 2University of Chinese Academy of Sciences; Beijing, PR China
A lginate lyases catalyze the degrada-
tion of alginate, a complex copoly-
mer of a-L-guluronate and its C5 epimer
b-D-mannuronate. The enzymes have
Downloaded by [189.187.98.156] at 20:06 02 September 2017
been isolated from various kinds of
organisms with different substrate specif-
icities, including algae, marine mollusks,
marine and terrestrial bacteria, and some
viruses and fungi. With the progress of
structural biology, many kinds of alginate
lyases of different polysaccharide lyases
families have been characterized by
obtaining crystal structures, and the cata-
lytic mechanism has also been elucidated.
Combined with various studies, we sum-
marized the source, classification and
properties of the alginate lyases from dif-
ferent polysaccharide lyases families. The
relationship between substrate specificity
and protein sequence was also
investigated.
Introduction
Keywords: alginate lyase, applications,
classification, structure, substrate
Alginate is the most abundant polysac-
specificity
charide (about 40% of dry weight) of
*Correspondence to: Heng Yin; Email:
brown algae, which consists of b-D-man-
yinheng@dicp.ac.cn
nuronate (M) and a-L-guluronate (G) as
Submitted: 02/02/2015
monomeric units.1 These units are linked
Revised: 03/09/2015
in 3 kinds of different blocks, poly b-D-
Accepted: 03/10/2015 mannuronate (polyM), poly a-L-guluro-
http://dx.doi.org/10.1080/21655979.2015.1030543 nate (polyG) and the heteropolymer
(polyMG).2 Some bacteria can also syn-
Commentary to: Benwei Z, Haidong T, Yuqi Q, thesize alginates to protect them from det-
Qingsong X, Yuguang D, Heng Y. Characteri- rimental factors such as antibiotics and
zation of a newendo-type alginate lyase from
Vibrio sp. W13, International Journal of Biolog- drought.3,4 Commercial alginates are pro-
ical Macromolecules, 2015, 75: 330-337; duced by extraction from biomass of
Benwei Z, Lishuxin H, Haidong T, Yuqi Q, brown algae such as Laminaria hyperborea,
Yuguang D, Heng Y. Characterization of a Macrocystis pyrifera, Laminaria japonica
new endo-type polyM-specific alginate lyase and etc. Alginate oligosaccharides are
from Pseudomonas sp., Biotechnology Letters,
2015, 37: 409-415. depolymerization products of alginate by
alginate lyase or physicochemical method.
www.tandfonline.com Bioengineered
COMMENTARY
They have attracted increasing attention
due to their wide applications in food and
pharmaceutical industry.5-7 They can be
used as growth promoters for plants and
therapeutic agents such as anticoagulants
and tumor inhibitors.8-10 They can also
induce the cytokine production and regu-
late the blood sugar as well as lipid.11,12
Due to the high-efficiency and high-speci-
ficity, alginate lyases for mild degradation
have been the focus for various fields.
Alginate lyase can degrade alginate by
b-elimination of glycosidic bonds and
produce unsaturated oligosaccharides with
double bonds at the non-reducing end.13
A number of alginate lyases have been
identified, cloned, purified and character-
ized from various sources,14-18 including
marine and terrestrial bacteria, marine
mollusks and algae. They can be classed
into 2 groups due to their substrate specif-
icities,13 molecular weights19 and action
modes. Now alginate lyases, especially
endolytic alginate lyases, have been widely
used in the production of alginate oligo-
saccharides,20 the elucidation of the fine
structures of alginate21-23 and the prepara-
tion of protoplast of red and brown
algae.24-26 Furthermore, alginate lyase also
shows great potential application in treat-
ment of cystic fibrosis by degrading the
polysaccharide biofilm of bacterium.27-29
Source and Classification
Alginate lyases have been isolated from
various sources, including marine algae,
marine mollusks (Littorina spp., Haliotis
spp, Turbo cornutus.), and a wide range of
marine and terrestrial bacteria. In addi-
tion, some lyases have been isolated from
fungi and viruses (Table 1). The alginate
125
 Table 1. Alginate lyase of different PL families from different sources and their substrate specificities

Organism PL family EC Substrate specificity Reference

Pseudomonas sp. E03 5 4.2.2.3 polyM 47

Stenotrophomonas maltophilia 6 4.2.2.- polyM, polyG 55
Flavobacterium spS20 7 4.2.2.11 polyG 17
Halitos discus hannai 14 4.2.2.- polyM, polyG 15
Chlorella virusATCV-1 14 4.2.2.14 Alginate 40
Sphingomonas sp. A1 15 4.2.2.- polyM, polyG 16
Saccharophagus degradans 17 4.2.2.3 polyM, polyG 45
4.2.2.-
Pseudoalteromonas sp SM0524 18 4.2.2.3 polyM, polyG 41
4.2.2.11

lyases can be classified into 2 groups due bifunctional alginate lyases belong to PL- like cleft in a novel (a/a)-barrel structure as
to their substrate specificities, one is G 18 family, while other lyases are dispersed the catalytic active domain. The structure
block-specific lyase (polyG lyase, in other 6 families. According to the 3- presented the possibility that alginate mole-
EC4.2.2.11), and the other is M block- dimensional structures, the alginate lyase cules might penetrate into the cleft to interact
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specific lyase (polyM lyase, EC4.2.2.3).
This classification has been widely
accepted, but some enzymes show activi-
ties toward both polyM and polyG,30-33
which may degrade alginate more effec-
tively. In terms of the mode of action,
alginate lyase can be grouped into endo-
lytic and exolytic alginate lyase.13 Endo-
lytic alginate lyase cleaves glycosidic bonds
inside alginate polymer and releases unsat-
urated oligosaccharides (di-, tri-, and
tetra-saccharides) as main products,30
while exolytic alginate lyase can further
degrade oligosaccharides into mono-
mers.31-33 Based on the analysis of hydro-
phobic cluster of primary structures,
alginate lyases can be grouped into 7 fami-
lies of Polysaccharide Lyase (PL) family
(Table 2), PL-5, -6, -7, -14,-15, -17, and
-18.34 Most of endolytic bacterial alginate
lyases are assigned to PL-5 and PL-7. The
most exolytic alginate lyases are grouped
into PL-15 and PL-17 families. Most algi-
nate lyases from bacteria are assigned into
PL-5, -7, -15, and -17 families. The lyases
isolated from marine mollusks and viruses
are collected in PL-14 family. The
are grouped into 3 families,35 including
parallel b-helix family, (a/a)6 barrel fam-
ily, jelly-roll family. The alginate lyases of
PL-6 belong to parallel b-helix family,36
the PL-5 and -15 members are grouped
into (a/a) 6 barrel family,37,38 while -7
and -14 are assigned into jelly-roll fam-
ily.39,40 The bifunctional alginate lyases of
PL-18 are newly characterized and shared
a similar sandwich structure.41 Further-
more，the alginate lyases can also be
grouped into 3 types based on their
molecular masses: small (25–30 kDa),
medium-sized (around 40 kDa), and large
lyases (>60 kDa).
Structure and Mechanism
of Action
The three-dimensional structures of vari-
ous alginate lyases have been elucidated in
Figure 1. The structure of alginate lyase A1-
III from Sphingomonas sp. A1 has been
resolved complexed with a trisaccharide
product.37 The overall structure of A1-III is
abundant in helixes and has a deep tunnel-
with the catalytic site of A1-III. This is the
only alginate lyase with resolved structure in
PL-5. As for PL-7, there are 5 alginate lyases
with resolved structures.19,38,42-44 The over-
all structures of these alginate lyases are all
b-sandwich fold like structure with a large
active cleft covered by 2 short flexible loops.
The overall structure of alginate lyase from
Chlorella virus for PL-14 show 2 antiparallel
b-sheets with a deep cleft showing a b-jelly
roll fold.40 The alginate lyases of PL-17 are
known as exolytic alginate lyases and only
one has resolved structure with a combina-
tion of (a/a) barrel and b-sandwich fold.45
The arrangement of (a/a) helixes produces
an open barrel structure and its additional
helix serves to maintain rigidity of the barrel.
The b-sandwich fold domain is composed of
3 co-planer layers of antiparallel b-strands
arranged as sheets and further supported by 4
small helices near the top layer, which inter-
jectbetweenthe2domains.Thealginatelyase
Aly-SJ02 of PL-18 displays a b-jelly roll scaf-
fold composed mainly of 2 anti-parallels
b-sheets.41
Alginate lyase catalyzes the degradation
of alginate by b-elimination mechanism,

Table 2. Characteristics of alginate lyases from different PL families

PL family Enzyme Source Substrate specificity Action mode Structure Products (DP) Reference

5 AlyA1-III Sphingomonas sp. A1 polyM endolytic (a/a)6 2–3 37

6 AlyMG Stenotrophomas maltophilia KJ-2 polyMG endolytic — 2–4 55
7 PA1167 Pseudomonas aeruginosa PAO1 polyM, polyG endolytic b-sandwich 2–4 39
14 vAL-1 Chlorella virus ATCV-1 polyM endolytic b-sandwich 2–4 40
15 Atu3025 Agrobacterium tumefaciens polyG, polyM exolytic (a/a)6 1 38
17 Alg17C Saccharophagus degradans 2–40 polyM, polyG exolytic (a/a)6 C b-sandwich 1 45
18 Aly-SJ02 Pseudoalteromonas sp SM0524 polyM, polyG endolytic b-sandwich 2–3 41

126 Bioengineered Volume 6 Issue 3

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Figure 1. The overall structures of alginate lyases from different families.41
breaking the glycosidic bond between
monomers and producing a resulted dou-
ble bond between the C4 and C5 carbons
of the sugar rings. During the elimination,
the 4-O-glycosidic bond is eliminated and
simultaneously yields oligosaccharides
containing 4-deoxy-L-erythro-hex-4-eno-
pyranosyluronic acid as the nonreducing
terminal moiety.11 With the 3-dimen-
sional structures of several alginate lyases
been resolved,41,45,46 the catalytic mecha-
nism have been elucidated to be 3 steps.
The first step is the removal of the nega-
tive charge on the carboxyl anion-essen-
tially neutralizing the charge by a salt
bridge (Histidine or Lysine) and then a
general base-catalyzed abstraction of the
proton on C5, where one residue may be
required as the proton abstractor and
another as the proton donor or the residue
act as both proton donor and acceptor.
Finally, a transfer of electrons from the
carboxyl group forms a double bond
between C4 and C5, resulting in the elim-
ination of the 4-O-glycosidic bond.
www.tandfonline.com
Substrate Specificity
Alginates are composed of 4 different
types of linkage such as M-M, M-G, G-
G, and G-M with various extent of each
linkage (Fig. 2). Alginate lyases are classi-
fied based on their dominant cleaving
action on the different types of substrates
as shown in Figure 147. The alginate lyases
preferring M-rich alginates are assigned
into polyM lyases (EC 4. 2. 2. 3), while
lyases preferring G-rich alginates are
grouped into polyG lyases (EC 4. 2. 2.
11). Although an alginate lyase may be
named as polyM lyase or polyG lyase, the
enzyme usually displays moderate to low
processivity for the other homopolymer.
This confusing variable may result from
the quality and purity of substrate used
for these determinations. These concerns
are currently being addressed by using the
substrate with high purity and highly sen-
sitive and accurate techniques to analyze
the structure of the products. There are
several reports of alginate lyases with
Bioengineered
specific substrate specificities, such as the
lyase from ATCC43367 which is reported
to cleave only M-M linkages.48 An algi-
nate lyase isolated from marine mollusk
Lambis sp displays 100% of activity for
polyG while 0% of activity for polyM.49
What is more, an alginate lyase from Kleb-
siella pneumonia showing activities toward
G-blocks and MG-blocks is modified with
cleavage specificity for G-G linkage by
site-mutagenesis.50 However, some algi-
nate lyases show activities on both polyM
and polyG and are regarded as bifunc-
tional lyases, such as alginate lyase AlySJ-
02 from Pseudoalteromonas sp. SM0524,
AlyPEEC from Pseudoalteromonas sp
IAM14594, Aly from Pseudoalteromonas
sp. 272, and AlyA from Pseudoalteromonas
atlantica AR06.
Relationship Between Substrate
Specificity and Structure
The alginate lyases of PL7 family
have been well investigated. There are
127
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Figure 2. The substrate specificity of alginate lyase and structures of degradation products. The polysaccharide alginate containing 3 kinds of blocks (M,
G, MG) is cleaved to produce a 4-deoxy-L-erythro-hex-4- enepyranosyluronate moiety (open triangle) at the newly formed non-reducing end of the
product.47
25 characterized alginate lyases in PL-7
family, and 5 of them have been investi-
gated for structure-function relationship
by obtaining crystal structures. The struc-
tural basis for depolymerization of alginate
lyase has been elucidated.51 As shown in
Figure 3, the alginate lyases contain 3
highly conserved regions, (R/E) (S/T/N)
EL, Q (I/V) H, YFKAG (V/I) YNQ.
According to the crystal structures and the
sequence analysis of several PL family 7
lyases, those conserved regions form the
cavity composed of a jelly-roll b-sandwich
structure, and the cavity is assumed to
bind a suitable substrate. Thus, conserved
amino acids residues are thought to play
an important role in catalytic activity or
folding of the structure. It has been
reported that Y195, H119, Q117 and
R72 may be involved in the catalytic site
for ALY-1.52 Therefore, the amino acids
in catalytic and substrate binding sites
would be highly conserved depending on
the substrate specificity. The polyM-spe-
cific alginate lyases contain QVH in
128
conserved regions and polyG-specific
enzymes involve QIH in conserved
regions, while the polyMG lyases include
QIH in the conserved regions. In addi-
tion, the substitution of hydrophobic resi-
dues in the isoleucine site of domain QIH
could have enormous influence on the
high affinity to polyG block. For this per-
spective, the substrate specificity is deter-
mined by the isoleucine of QIH domain
and valine of QVH domain. The isoleu-
cine of QIH domain is confirmed to be
indispensible for reorganization of the
polyG or G-G bond.53 So, if an alginate
lyase with unknown substrate specificity
contained isoleucine or valine in Q (I/V)
H region, this enzyme could degrade
polyM or polyG and polyMG, respec-
tively. The region YFKAG (V/I) YNQ has
been reported from various alginate lyases
with different specificities, indicating that
this region may be of functional or struc-
tural importance for alginate degradation
independent of substrate preference. Con-
sidering that the alginate lyase totally lost
Bioengineered
activity with deletion of this region, the
results suggest that the region may be
needed to catalyze alginate degradation.
Functionality and Promising
Application of Alginate Lyase
Depolymerized alginate produced by
enzymatic hydrolysis with low DP pos-
sesses various kinds of biological activi-
ties.8-14 Oligomeric alginates obtained
from lyase degradation of alginate act as
oligosaccharines and regulate physiologi-
cal processes in plants and microorgan-
isms, such as promoting the growth of
Bifidobacterium spp., enhancing the ger-
mination and shoot elongation in plants.
The oligosaccharides exhibit many physio-
logical activities, such as antitumor, stimu-
lating production of cytokines, regulating
the blood lipids and sugars. The efficient
alginate lyases w are key tools for produc-
tion of functional oligosaccharides from
alginate.
Volume 6 Issue 3
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Figure 3. Multiple alignment of amino acid sequences of alginate lyases of PL 7 family. The conserved residues were overlaid with red and 3 highly con-
served regions-(R/E) (S/T/N) EL, Q (I/V) H, YFKAG (V/I) YNQ were boxed in blue.

The alginate lyases with different sub-
strate specificities are invaluable tools for
determining sequence of substrate and
preparing the oligosaccharides with spe-
cific structures. Alginate lyases possess dif-
ferent substrate specificities depending on
the differences in amino acids and distrib-
uting of monosaccharide residues in sub-
strate. Various enzymes could recognize 4
different types of linkages such as M-M,
M-G, G-M, and G-G. The alginate lyases
of PL-5 family prefer to degrade polyM
substrate. The AlgA from Pseudomonas sp
E03 hydrolyzes the polyM substrate in an
endolytic manner and releases a range of
oligomannuronate with low DP.11 The
AlxM from Photobacterium sp.
ATCC43367 is also a polyM-specific algi-
nate lyase.48 And a polyM lyase from Sar-
gassum fluitans specifically depolymerize
polyM.54 The alginate lyases specific for
G-G linkage have not been described
except for a mutant enzyme with cleavage
specificity for G-G linkage.14 This G-G
linkage specific enzyme is constructed by
mutating the gene encoding Klebsiella
pneumonia AlyA, which cleaves both G-G
and G-M linkages. The enzyme isolated
from Klebsiella pneumonia shows activity
only toward G-G linkage.50 The

www.tandfonline.com Bioengineered 129

 polyMG-specific alginate lyase from Sten-
otrophomas maltophilia KJ-2 preferably
degrades the glycosidic bond in M-G link-
age than that in G-M linkage.55 The algi-
nate lyases have been used to analyze
alginate fine structure to understand how
chemical composition influences the phys-
ical properties of alginate. The combina-
tion of alginate lyases with different
substrate specificities has used for deter-
mining the sequence and block distribu-
tion of alginate.56
In addition, the enzymes with strict
substrate specificities could be used
for preparation alginate with specific
structures. The AlxMB can be used for
preparation of homopolymannuronate
blocks and strictly alternating sequences
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of mannuronic and guluronic acids due
to its strict substrate specificity toward
polyM blocks.57 A simple method for
preparation of polyG blocks has been
developed by using a polyG lyase from
Flavobacterium multivolum.58 The
enzyme could degrade both G- and
MG- blocks but M blocks. The gulur-
onan lyase from Klebsiella aeruginosa
has no action on polyM, but can read-
ily cleave G-G linkages in guluronan.
Based on the substrate specificity of this
enzyme, it has been used for determin-
ing the activity of C-5 epimerase.59
Recently, the alginate lyases have been
used to treat the cystic fibrosis in combi-
nation with antibiotics.27-29 When algi-
nate lyase is co-administered with
antibiotics such as gentamycin, the killing
efficiency of mucoid Pseudomonas aerugi-
nosa in respiratory tract increases. There-
fore, the enzymes are expected to become
useful agents for the treatment of bacterial
mucoid biofilm-dependent diseases. What
is more, the alginate lyases are also used to
prepare the protoplast of algae for gene
engineering and study Fucus cell wall
development. With the consumption of
fossil source, the alginate, being regarded
as a renewable source for the production
of bioethanol, has obtained increased
attention. The saccharification of alginate
for production of bioethanol requires syn-
ergistic effects of alginate lyases with
endo- and exo- action modes. The engi-
neered microbial platforms for direct pro-
duction of bioethanol from alginate have
been reported.60-62
130
In conclusion, the enzymes are impor-
tant tools in a broad spectrum of biological
roles and applications. Alginate lyases with
different substrate specificities can be used
to produce oligosaccharides with various
biological functionalities. Moreover, they
can also be used for determination of the
fine structure of alginate and the prepara-
tion of tailor-made alginate. Furthermore,
alginate lyases with activities toward acety-
lated alginate can be utilized for the treat-
ment of cystic fibrosis in combination with
antibiotics. Therefore, due to the various
activities and biological functionalities, algi-
nate lyases have been widely applied. And
with the continuing developments for
screening and characterizing the enzymes,
the applications of alginate lyases will
become increasing wider in the near future.
Conclusion
The alginate lyases have important
roles and biotechnological applications.
Especially, the enzymes were key tools for
production of oligosaccharides from algi-
nate and determination of the fine struc-
ture of alginate. In addition, the alginate
lyases play essential role in saccharification
of the acidic polysaccharides for produc-
tion of bioethanol with synergistic effect
of endo- and exo-type alginate lyases.
There were several hundred kinds of algi-
nate lyases from various sources have been
isolated and characterized. These enzymes
differed from each other in substrate spe-
cificities, properties (including activities
and structures) and degradation products.
With the progress of structural biology,
many kinds of alginate lyases of different
PL families have been characterized by
obtaining crystal structures, and the cata-
lytic mechanism has also been elucidated.
In future, screening and characterization
of novel enzymes with high activities and
broad substrate specificities would
enhance and expand the utilization of the
lyases to produce oligosaccharides with
novel structures and biological activities
for applications in various fields.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were
disclosed.
Bioengineered
Funding
The research in our lab mentioned in
this paper was supported by the National
Key Technology Support Program
(2013BAB01B01), Special Fund for
Marine Scientific Research (201305015-
2), National Natural Science Foundation
of China (31370811) and Development
Fund for Collaborative Innovation Center
of Glycoscience of Shandong University.
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