Endo-(1→3)-β-D-glucanase GI from marine mollusk Littorina sitkana: Amino acid sequence and ESIMS/MS-estimated features of transglycosylation and hydrolysis reactions in comparison to analogous enzyme LIV from Pseudocardium sachalinensis

Article abstract of DOI:10.1016/j.molcatb.2011.11.012

The cDNA encoding the glucanase GI from Littorina sitkana (formerly named Littorina kurila) was cloned and sequenced, and the enzyme was assigned to glycoside hydrolases family 16 (GHF16) on the basis of amino acid sequence similarity. Structural features of the products of transglycosylation reaction using 6-O-methyl-β-D-glucuronic acid as an acceptor were established. Using colisionally induced dissociation (CID) tandem electrospray ionization mass spectrometry (ESIMS/MS) it was shown that GI transfers the residues of glyconic parts of the substrate mainly at C3 and C4 positions of 6-O-methyl-β-D- glucuronic acid and strictly at C3 position of glucose residue. The semi-quantitative characteristics of simultaneously passing hydrolysis and transglycosylation reactions catalyzed by retaining endo-(1→3)-β-D- glucanases GI from L. sitkana and LIV from Pseudocardium sachalinensis (formerly named Spisula sachalinensis) have been obtained by ESIMS. Laminaran was used as a donor, while glycerol was employed as an acceptor. The significant distinctions of catalytic properties of LIV and GI were revealed.

Full text of DOI:10.1016/j.molcatb.2011.11.012

Journal of Molecular Catalysis B: Enzymatic 75 (2012) 73–79
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Endo-(1→3)-␤-d-glucanase GI from marine mollusk Littorina sitkana: Amino acid
sequence and ESIMS/MS-estimated features of transglycosylation and hydrolysis
reactions in comparison to analogous enzyme LIV from Pseudocardium
sachalinensis
Maria S. Pesentsevaa,∗, Svetlana N. Kovalchuka,b, Stanislav D. Anastyuka, Mikhail I. Kusaykina,
Victoria V. Sovaa, Valerii A. Rasskazova, Tatyana N. Zvyagintsevaa
a Pacific Institute of Bioorganic Chemistry FEB RAS, 690022 Vladivostok, Prospect 100 Let Vladivostoku 159, Russian Federation
b Far Eastern Federal University, 8, Sukhanova Str., 690950 Vladivostok, Russian Federation
a r t i c l e i n f o
a b s t r a c t
Article history:
The cDNA encoding the glucanase GI from Littorina sitkana (formerly named Littorina kurila) was cloned
and sequenced, and the enzyme was assigned to glycoside hydrolases family 16 (GHF16) on the basis of
amino acid sequence similarity. Structural features of the products of transglycosylation reaction using
6-O-methyl-␤-d-glucuronic acid as an acceptor were established. Using colisionally induced dissociation
(CID) tandem electrospray ionization mass spectrometry (ESIMS/MS) it was shown that GI transfers the
residues of glyconic parts of the substrate mainly at C3 and C4 positions of 6-O-methyl-␤-d-glucuronic
acid and strictly at C3 position of glucose residue. The semi-quantitative characteristics of simultaneously
passing hydrolysis and transglycosylation reactions catalyzed by retaining endo-(1→3)-␤-d-glucanases
GI from L. sitkana and LIV from Pseudocardium sachalinensis (formerly named Spisula sachalinensis) have
been obtained by ESIMS. Laminaran was used as a donor, while glycerol was employed as an acceptor.
The significant distinctions of catalytic properties of LIV and GI were revealed.
Received 25 June 2011
Received in revised form
11 November 2011
Accepted 15 November 2011
Available online 23 November 2011
Keywords:
Marine mollusk
Littorina sitkana
Pseudocardium sachalinensis
Hydrolysis
Endo-(1→3)-␤-d-glucanase
Transglycosylation
ESIMS/MS
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
embryogenesis. Some (1→3)-␤-d-glucanases have been isolated
from the eggs of the sea urchin Strongylocentrotus purpuratus [2],
Laminarinases or (1→3)-␤-d-glucanases belong to O-glycoside
hydrolases, key enzymes of carbohydrate metabolism. These
enzymes are widely distributed in various organisms such as
archaea, bacteria, fungi, yeasts, plants, insects, fishes, invertebrates,
and are involved in many physiological processes [1]. Complex
structure of substrates (mixed (1→3);(1→6)- or (1→3);(1→4)-␤-
d-glucans), as well as considerable differences in their molecular
masses, conformations and other characteristics determine the
diversity of catalytic properties of (1→3)-␤-d-glucanases and their
functions in living organisms. These enzymes were studied in
many respects, but our knowledge of their structure and mech-
anism of action is still incomplete, especially for enzymes from
marine organisms. The marine invertebrates were found to be the
rich sources of (1→3)-␤-d-glucanases. They take part in diges-
tion of dietary polysaccharides and play an important role in
marine mollusks Pseudocardium sachalinensis [3], Mizuhopecten
yessoensis [4], Chlamys albidus [5], Haliotis discus hannai [6],
Perna viridis [7]. Physico-chemical and catalytic properties of
enzymes from these animals were studied in details and amino-
acid sequences have been reported. All (1→3)-␤-d-glucanases of
marine invertebrates were classified as retaining endo-enzymes
and according to amino-acid sequence analysis were assigned to
the 16 family of glycoside hydrolases. A distinguishing feature of
the catalytic action of (1→3)-␤-d-glucanases from sea mollusks is
their high transglycosylating activity, which is successfully used in
the enzymatic synthesis of novel compounds and the transforma-
tion of natural glucans with the aim of increasing their biological
activity [8,9]. Previously we isolated endo-(1→3)-␤-d-glucanase
from the gastropodian mollusk Littorina sitkana and investigated
its basic catalytic properties [10]. In present work we report amino
acid sequence of endo-(1→3)-␤-d-glucanase from L. sitkana and
comparative investigation of transglycosylating properties of endo-
(1→3)-␤-d-glucanases from L. sitkana and P. sachalinensis by the
means of mass spectrometry. The comparative investigation of
∗
Corresponding author. Tel.: +7 4232 310 705; fax: +7 4232 310 705.
E-mail address: mpesentseva@gmail.com (M.S. Pesentseva).
1381-1177/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2011.11.012

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M.S. Pesentseva et al. / Journal of Molecular Catalysis B: Enzymatic 75 (2012) 73–79
catalytic properties of enzymes is aimed for better understanding
of structure–function relationship and for finding the sources with
advanced biotechnical potential.
and NH4OH (2.5%, 50 ␮L) was added to the aliquots to terminate
the reaction. The samples were analyzed by ESIMS.
2.6. Mass spectrometric analysis
2. Experimental
MALDI-TOFMS spectra for determination of the molecular mass
of the enzymes were recorded with an ULTRAFLEX III MALDI-
TOF/TOF (Bruker, Germany) mass spectrometer with delayed ion
extraction and reflector modes, equipped with nitrogen laser
(337 nm), with accelerating voltage of 25 kV. Sinapinic acid (3,5-
dimethoxy-4-hydroxycinnamic) was used as the MALDI matrix
in the positive-ion mode at a concentration of 10 mg/mL in 1:1
acetonitrile–water. Sample preparation: 1 ␮L of the matrix solu-
tion was applied to a stainless steel plate and air dried, then 1 ␮L of
a sample solution in water (∼0.1 mg/mL) was applied as a second
layer. The mixture was air dried and then introduced into the mass
spectrometer.
2.1. Materials
Marine mollusks L. sitkana and P. sachalinensis were collected
in Posíeta Bay (northwestern part of the Sea of Japan) in August
2010 near the Marine Experimental Station of the Pacific Institute of
Bioorganic Chemistry. Digestive organs: liver of L. sitkana and crys-
talline styles of P. sachalinensis were isolated and frozen at −70 ◦C.
Laminaran from brown alga Laminaria cichorioides was isolated as
described [11]. Glycerol was purchased from Sigma–Aldrich, 6-O-
methyl-␤-d-glucuronic acid was synthesized as described [12].
ESIMS spectra were recorded with a 6510 LC Q-TOF (Agi-
lent, USA) mass spectrometer with a dual electrospray-ionization
source. Spectra were acquired in the positive-ion mode, with
pre-calibration with a standard “HP-mix” for positive-ion mode.
Capillary voltage was set to 3500 V, and the drying gas tempera-
ture was 325 ◦C. The fragmentor voltage was set to 215 V. Isolation
window for MS/MS experiments was set to 1.3 mass units. Colli-
sion energy was optimized between 10 and 45 V by fragmentation
abundance. The dried sample was dissolved in 1:1 acetoni-
trile/water (concentration of the sample ranged from approx. 0.001
to 0.5 mg/mL) and introduced into the mass spectrometer at flow
rate of 5 ␮L/min using a syringe pump, manufactured by KD Scien-
tific (USA).
2.2. Analytical methods
Protein concentration in solution was determined by the
method of Bradford [13] using BSA as a standard. The homogene-
ity and molecular masses of the enzymes were determined by
SDS–PAGE according to the method of Laemmli [14] and MALDI
mass spectrometry. The oligosaccharide composition was analyzed
by high performance liquid chromatography (HPLC) on an Agilent
1100 chromatograph under following condition: Asahipak NH2P-
50 4A column, acetonitrile and filtered distilled water (6:4) as the
mobile phase, flow rate of 0.5 mL/min and refractive index detector.
The retention times of products were determinated using glucose
(Sigma–Aldrich) and collection of laminarioligosaccharides (kindly
provided by our collaborators from PIBOC) as standards.
2.7. RNA isolation and cDNA sequencing
2.3. Purification of enzymes
Total RNA was isolated from whole L. sitkana by the guani-
dine thiocyanate/phenol/chloroform method [17]. cDNA was
synthesized from total RNA and subjected to 22 cycles of ampli-
fication (10 s at 95 ◦C, 20 s at 63 ◦C and 90 s at 72 ◦C) with a
SMART cDNA Amplification Kit (Clontech, USA) in accordance
with the manufacturer’s instructions. The cDNA was diluted
20-fold and 1 ␮L of this solution was used for PCR with
Gluc1: 5ꢀ-TGGCC(A/T)GC(C/T)AT(T/C)TGGATG-3ꢀ and Gluc2: 5ꢀ-
CCATT(C/T)GA(T/C) AA(A/G)CCATT-3ꢀ primers designed on the
basis of the peptide sequences WPAIWM and PFDKPF, conserved
for all endo-(1→3)-␤-d-glucanases GHF16. PCR was set up using
Encyclo PCR Kit (Evrogen, Russia) and amplification was carried out
for 35 cycles (10 s at 95 ◦C, 15 s at 58 ◦C, 1 min at 72 ◦C). Obtained
PCR product of 380 bp was cloned with InsTAclone PCR Cloning Kit
(Fermentas, Lithuania) and sequenced using ABI Prism Big Dye Ter-
minator 3.1 Cycle Sequencing Kit (Applied Biosystems, USA) on ABI
Prism 310 Genetic Analyzer.
Endo-1,3-␤-d-glucanase from the liver of L. sitkana was puri-
fied as described previously [10]. Endo-1,3-␤-d-glucanase from the
crystalline styles of P. sachalinensis was purified reported in [15].
Homogeneity of 1,3-␤-glucanases was established by SDS elec-
trophoresis in 15% PAGE under reducing conditions [14].
2.4. Determination of enzyme activity
A standard reaction mixture contained a 0.1% (w/v) substrate
solution (200 ␮L) and an enzyme solution (50 ␮L) in 0.025 M
sodium acetate buffer, pH 5.2. The incubation was carried out for
20 min at 37 ◦C. The activity was determined from an increase in
the amount of reducing sugars by the method of Nelson using glu-
cose as a standard [16]. A unit of enzyme activity was defined as
that amount of the enzyme required to catalyze the formation of
1 ␮mol of glucose per minute under standard conditions.
The 3ꢀ- and 5ꢀ-terminal regions of the cDNA encoding GI
were amplified by PCR with specific primers designed on
the basis of 380 bp cDNA fragment sequence and adapter-
specific primer.36 Gene specific primers for 5ꢀ- and 3ꢀ-RACE
were Gluc5: 5ꢀ-GTGGTTGTCGCCTGCGGAGGTGCC-3ꢀ and Gluc3: 5ꢀ-
GGCCACACCTCAAACATCTGG-3ꢀ, respectively. Two obtained cDNA
fragments of 1080 bp and 400 bp were cloned and sequenced as
described above.
2.5. Preparation of hydrolysis and transglycosylation products
An enzyme solution (50 ␮L; 10−2 units) was added to 250 ␮L
of laminaran solution (5 mg/mL), and the mixture was incubated
at 37 ◦C. At definite intervals of 1, 5, 10, 20, 40 and 24 h, samples
(50 ␮L) were taken. The reaction was terminated by adding of 50 ␮L
NH4OH (2.5%). The hydrolysis products were analyzed by ESIMS.
Transglycosylation products for ESIMS analyses were obtained
by adding the enzyme solution (50 ␮L; 10−2 units) in buffer to
250 ␮L of a solution containing laminaran solution (5 mg/mL) and
acceptors: glycerol (1%, 5%, and 10%) or 6-O-methyl-␤-d-glucuronic
acid (1 mg/mL). The incubation was performed at 37 ◦C. At definite
intervals of 1, 5, 10, 20, 40 and 24 h, aliquots of 50 ␮L were taken,
2.8. Sequence analysis and comparisons
Sequence analysis was carried out using the NCBI-BLAST2
website (http://www.ebi.ac.uk/blastall/). Domain architectures
were identified by SMART tool (http://smart.embl-heidelberg.de).

M.S. Pesentseva et al. / Journal of Molecular Catalysis B: Enzymatic 75 (2012) 73–79
75
Multiple sequence alignment was performed with the ClustalW
(http://www.ebi.ac.uk/Tools/clustalw2/index.html).
Bacillus 1→3;1→4-␤-d-glucanase [21–23] and by analysis of
crystal structure of ␬-carrageenase from Pseudoalteromonas car-
rageenovora [24] and agarase from Zobellia galactanivorans [25].
All above mentioned enzymes belong to glycoside hydrolase fam-
ily 16 (http://www.cazy.org/GH16.html), possess common enzyme
mechanism and have similar all-antiparallel ␤-sheet jellyroll archi-
tecture. The second conserved motif, WLWPAIW, have been suggest
to be substrate binding site [26]. The role of the last tryptophan
residues in the WPAIW motif have been predicted to provide aro-
matic stacking interaction with glucose residue on subsite – 1 in
the endo-(1→3)-␤-d-glucanase BglF from Nocardiopsis sp. [20].
Another conserved tryptophan residues (Trp118 in BglF), which
interacts with glucose residue on subsite – 2, is observed in GI at
Trp146. It is worth mentioning that endo-(1→3)-␤-d-glucanases
of GHF16 are fairly rich in aromatic residues. Besides two above-
mentioned tryptophan residues, analysis of the multiple alignment
revealed Trp129, Trp131, Trp209, Trp283, Trp295, Trp301, Trp305,
Phe9, Phe252, Phe256, Phe271 and Phe294 (residues numbering
of GI), conserved for the majority of endo-(1→3)-␤-d-glucanase
GHF16, and five sites (Phe5, Trp19 Trp177, Trp205, and Phe270)
with homologous substitutions (Fig. 1). It could be speculated that
these aromatic residues (or some of them) participate in laminaran
binding beyond the active-site cleft. The important role of trypto-
phan, phenylalanine and tyrosine residues for binding of glucose
residues of polysaccharides was shown [21,24,25,27,28].
3. Results and discussion
Previously we isolated (1→3)-␤-d-glucanase GI from the liver
of the marine gastropodean mollusk L. sitkana and its general
properties were investigated [10]. Glucanase GI had character-
istic features of (1→3)-␤-d-glucanases from marine mollusks.
Molecular mass of GI as estimated by gel-permeation chromatog-
raphy and SDS–PAGE analysis was 32 and 40 kDa, respectively
and 39.3 kDa (data not shown) according to MALDI-TOFMS anal-
ysis. It exhibited a maximum activity at pH 5.4 and 40 ◦C. The
Km value for laminaran hydrolysis was 0.13 mg/mL. The (1→3)-
␤-d-glucanase GI had narrow substrate specificity and hydrolyzed
only (1→3)-␤-d-glycosidic bonds in mixed (1→3);(1→6)- and
(1→3);(1→4)-␤-d-glucans down to glucose and glucooligosac-
charides. This enzyme acted with retention of the anomeric
configuration and catalyzed a transglycosylation reaction using
laminaran as donor and p-nitrophenyl-␤-d-glucoside as acceptor.
GI was classified as the glucan endo-(1→3)-␤-d-glucosidase (EC
3.2.1.39) [10].
3.1. cDNA cloning and GI primary structure
The multiple alignments revealed that two histidine residues,
His176 and His203, are conserved for the endo-(1→3)-␤-d-
glucanases (Fig. 1). The importance of histidine residues for
catalysis was shown by its chemical modifications in endo-(1→3)-
␤-d-glucanases from M. yessoensis and T. molitor [4,29]. Localization
of His142 of the endo-(1→3)-␤-d-glucanase from Nocardiopsis sp.
in active site was demonstrated by Fibriansah [20]. Moreover,
hypothesis that the histidine and aspartic acid residues, conserved
in ␬-carrageenases, ␤-agarases and laminarinases of GHF16 and
located in catalytic cleft, participate in proton trafficking dur-
ing the deglycosylation step of the catalytic cycle, was suggested
by a close inspection of the three-dimensional structure of the
␬-carrageenase from P. carrageenovora [24]. Thus, His176 of GI
together with Glu 151, Asp153 and Glu156 seems to be involved
in catalytic cycle.
GI contains two cysteine residues, Cys82 and Cys90, which
are conserved for the majority invertebrate’ endo-(1→3)-␤-d-
glucanase and formed a disulfide bond connecting different sides of
the loop between ␤3- and ␤4-strands [4]. The exceptions are endo-
(1→3)-␤-d-glucanase from Antarctic springtail C. antarcticus and
from pinewood nematode B. xylophilus. They lack these cysteine
residues because of deletion of about 40 amino acid residues in the
corresponding region as well as of about 20 amino acids at C-end
of their molecules. Phylogenetic analysis showed that this endo-
(1→3)-␤-d-glucanases are clustered together with glucanases from
bacteria with high bootstrap values indicating that endo-(1→3)-␤-
d-glucanases from B. xylophilus and C. antarcticus are more closely
related to those from bacteria than from eukaryotes. This suggests
that the enzymes were acquired by horizontal gene transfer from
bacteria [30,31].
The nucleotide sequence of full cDNA encoding GI was estimated
by RT-PCR, 5ꢀ-, 3ꢀ-RACE methods. The obtained cDNA sequence of
1636 bp contains an open reading frame of 1017 bp encoding a
polypeptide of 442 amino acid residues (GeneBank accession num-
ber FJ641204). Analysis of the L. sitkana endo-(1→3)-␤-d-glucanase
deduced amino acid sequence by SMART tool identified the
domain of glycoside hydrolase family 16 (pfam00722 Glyco hydro-
16, E = 9.10e−09) located from Met149 to Lys374. The predicted
mature enzyme GI with calculated molecular mass of 36.7 kDa con-
sists of 322 amino acids residues. Molecular mass GI corrected by
MALDI mass spectrometry was 39.3 kDa. Probably, the molecule of
enzyme was highly derivatized due to post-translational modifica-
tions.
Sequence comparisons of GI deduced amino acids showed
similarity of 67%, 65%, 64%, 65%, 63%, 61% and 59% to endo-(1→3)-
␤-d-glucanases of surf clam P. sachalinensis (GenBank AAP74223),
scallops M. yessoensis (GenBank AY848857) and Ch. albidus (Gen-
Bank DQ093347), the mussel P. viridis (GenBank ACM68926), Pacific
abalone Haliotis discus hannai (GenBank BAH84971), the cotton
bollworm Helicoverpa armigera (GenBank ABU9862) and the yel-
low mealworm Tenebrio molitor (GenBank ACS36221), respectively.
A somewhat lower degree of similarity is observed with endo-
(1→3)-␤-d-glucanases from the Antarctic springtail Cryptopygus
antarcticus (GenBank ACD93221) and the pinewood nematode
Bursaphelenchus xylophilus (GenBank BAE48357) – 52% and 54%,
respectively. The multiple alignment of the endo-(1→3)-␤-d-
glucanase from L. sitkana with those of other invertebrate is shown
in Fig. 1.
Analysis of the GI primary structure revealed that the enzyme
belongs to glycoside hydrolase family 16. GI shares high (52–67%)
sequence similarity with known invertebrates’ endo-(1→3)-␤-
d-glucanases and contains two conserved motives, which are
characteristic for endo-(1→3)-␤-d-glucanases GHF16 (Fig. 1). One
of them, catalytic EIDIME motif, includes two conserved glu-
tamic acid residues, which catalytic importance has been proved
by mutational analysis of laminarinase from Rhodothermus mar-
inus [18] and by the crystal structure and mutational analysis
of the endo-(1→3)-␤-glucanase GHF16 from Nocardiopsis sp.
[19,20]. Also the crucial role of these Glu residues has been
suggested by the crystal structure and mutational analyses of a
The primary structure of GI enzyme from marine invertebrates
is one of the few examples of that. We believe that it is a valuable
contribution for the future investigations. Though finding relation-
ship between the primary structure and exact features of enzyme
is a complex task and requires separate investigations, the data,
presented below could partly enlighten this relationship.
3.2. Hydrolysis and transglycosylation
It has been known that endo-(1→3)-␤-d-glucanases from
sea mollusks, in comparison to other O-glycoside hydrolases

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M.S. Pesentseva et al. / Journal of Molecular Catalysis B: Enzymatic 75 (2012) 73–79
L.sitkana
----------------------GAVMFRDDFNGGGLDTNN---WNYEVS-MYGGMNWEFQVYTND-KSNVYTNNGKLFLKPTKTVDDPRWDENFLHSGVMDVAQIWG--YCTQSAQYGCHRE 93
--ATPSPTTASGTHAPTGEICSGDLIFEDEFDE--LDMQK---WNHEST-LGGGGNWEFEWYTNS-RYNSYTEDGFLYIKPTLLAD--ENGEDFLSSGQLDINGGSPADECTNPQWYGCART 113
----------------------NGLIFQEEFDS--FNLDI---WEHEMT-AGGGGNWEFEYYTNN-RSNSYVRDGKLFIKPTLTTD--KLGEGSLSSGTLDLWGSSPANLCTGNAWYGCSRT 91
----------------------GALIFADDFDS--FDLEK---WQHENT-LAGGGNWEFQYYNNN-RTNSFTHSGRLFIRPSLMSD--QFGEQFLSSGHLNVEGGAPADRCTNPQWWGCERT 91
T.molitor
S.purpuratus
H.armigera
P.sachalinensis ----------------------GTVVFRDDFNG-AFDPAG---WNYEVS-MYGGYNWEVQAYVPD-ARNIFTRNGHLFIKPTLTTDHPNYNDGNLNSATMDLTALYG--YCTNADRYGCIRE 92
C.albidus
------------------------AGFRDDFTT--WNPNN---YQIGVS-AWGGGNHEFQVYTPE-PSNLFVRDGSLYIKPTFTRDSRHFTDGNLYYGTMDLYHLWN--KCTQHDNNGCQKH 89
------------------------AGFRDDFTT--WDPSD---YQIEVS-AWGGGNHEFQVFTPE-PSNLFVRNGNLYIKPTFTRDSAHFNDGSLYYGTMDVNSLWH--RCTQHDNNGCHKQ 89
-----------------------AVVFRDDFHS--FNKGS---YKIECS-AWGGGNHEFQVYVNE-PSNLYARGGHFYLKPTLTTDDPRYDDNRLYHGQMKVTDFCGGGPCTQSANWGCDRT 92
----------------------GNTVFEDSFNSHQLNPKH---WHHEIT-CWGGGNGEFQMYTPE-AANTYIKNGVLYLKPTFTAD--KFGDDFFQHGVLDVKQQWG--SCTAAQDNGCRRQ 89
------------------------LDWEDEFNGGNLAD----RWNFELG-CNGWGNNELQCYTDNRGANARQEDGKLVISAVREWWGDGVNPD----------------------------- 64
----------------------GVIWQEDFNSLDTS-----KWNFETGNNNGWGNSELECYTTS-SNNVRVENGNLVIEARRENV-DGCS------------------------------- 61
M.yessoensis
P.viridis
H.discus
C.antarcticus
B.xylophilus
Nocardiopsis sp ----------------TESDMRATLVWSDEFDGPAGSAPDPANWNHETG-DHGWGNNELQNYTDSRANSALDGNGNLVITARQEADGG---------------------------------- 71
●
●
▼
▼
▼
▼
L.sitkana
---GKNGILPPVMSGKVKSKP--VLKYGTVEVRARIP-KGDWLWPAIWMLPR--DSHYGGWPRSGEIDIMESRGNVRASG-----HGVNEVSSTLHWGTSAGDNHYGQTTH--AKQAADW 198
--GTADNYLNPIKSARIRSLYSLSFKYGKVEVRAKLP-TGDWLWPAIWMLPR--WNQYSGWPISGEIDIMESRGNADLVNASGANIGSKLVSSTLHWGPAWNINMYMMTHVESSNPAG-F 227
--GSNDNLLNPIQSARLRTVESFSFKYGRLEVEAKLP-TGDWLWPAIWLLPK--HNGYGEWPASGEIDLVESRGNADIKDADGLSAGVDQMGSTMHWGPFWPLNGYPKTHAT-------- 198
--GSPSNILNPIKSARIRTVNSFSFRYGRVEVRAKMP-AGDWLWPAIWLMPA--YTTYGTWPASGEIDLVESRGNRAMT-FNGVHIGTHEAGSTLHYGPYPAMNGWERAHWIRRNTAG-Y 204
---GRNGILPPVMSGKIKSKK--TIRFGKVEARCRIP-RGDWIWPAIWMLPR--DSVYGGWPRSGEIDMMESRGNTVARDGSGHNHGVNEVG-HLHWDQMPVIIDSVRRT---GDLDGDW 200
SYGGNSEILPPVMSGKITTNF--AMTYGRVNVRAKIP-KGDWLWPAIWMLSR--DRSYGGWPRSGEIDIMESRGNTKAVLW-GQNSGVNYVASTLHWGPDFNNNRFQKTHGSKRKSGGAD 203
SYGGDSEILPPVMSGKITTNF--AMTYGRVNVRAKIP-KGDWLWPAIWMLPR--DWSYGGWPRSGEIDIMESRGNTKAILG-GQNSGVNYVASTLHWGPAYNHNAFAKTHASKRKYGGDD 203
AVNGN--VLNPIKSGKVTTNA--AIRYGTVTVRARIP-EGDWLWPAIWMLPR--DWKYGGWPRSGEIDLMEARCNSYMKCG-SNLEGVQEVASTLHWGPDAGQNRFYKTHGELDKGSGDW 204
---GA--QIPPIMSSKVFSVA--SITHGRVEVVAKIP-KGDWIWPAIWLLPPGWPWKYGAWPASGEIDIMESRGNVHLSEANGATQGVDRVLSTIHYGASPSQHRQQGDSK-TSKTGTTW 200
---------KEFTSARMTT--KANWLHGKFEMRARLP-KGKHLWPAFWMMPQ--NSEYGGWPRSGEIDITEYR-----------GQRPQQILGTLHFGAAPDNKGDVGTGE--RDFPIDF 157
-----------FTSGRIHTRGKFDFKYGTIEARIKLPNLANGLWPAFWMLG----AESNTWPDQGEMDIMEAG----VADAIAAGNVNKEILGTFHWSNN-GQHAQYGTN---YVAPNDL 158
-----------YTSARLTTQNKVQPQYGRVEASIQIP-RGQGIWPAFWMLGA--DFPNTPWPDSGEIDIMEN-----------IGREPHLVHGSLHGPGYFGGEPLTGSYM--HPQGWSF 164
T.molitor
S.purpuratus
H.armigera
P.sachalinensis
C.albidus
M.yessoensis
P.viridis
H.discus
C.antarcticus
B.xylophilus
Nocardiopsis sp
L.sitkana
SNSFHTWRLEWTHDHIATFVDNQQILRVTPPSGGFSELGHTS------N-IWAGND-KMAPFDKEFYAIFNVAVGGTNGFFPENW-DYGYPKPWSNTSPHAAQDWWNGRSKWESSWQGD- 308
DADWHNYQMTWTENDISFSIDDALLGTFAPPDGGFWEWGDLD--SSGFANPWRTSKSEMAPFDQEFYLLINLACGGMA-YFPDDV-TNPGGKPWSNTSPTASTDFWKGRDQWLPTWKLET 343
----------------KFYVDDELLLNVDPATG-FWDLGEFENDAPGIDNPWAYNPNKLTPFDQEFYLILNVAVGGVN-YFGDGL-TYTPAKPWSNDSPTASKDFWSDFNTWYPTWNGE- 298
NSNFHRYQLEWTPDYLRFSIDDLELGRVTPGNGGFWEFGGFNS-NPNIENPWRYG-SKMAPFDEKFYLIINLAVGGTNGFFPDGV-SNPNPKPWWNGSPTAATGFWNGRWGWLPTWNLGV 321
SHAMHTYRLDWTIDHIQVFVDNRHIMNIPQSRKVFGSLEDLV------DPIFGAVEPKAAPFDKQFYLILNVAIAGTNGFFPDNW-TYDQQKPWFSNSPTELQDFWNARFQWLQTWHGD- 312
WHGWHTYSLDWTAGHIVTYVDNVEIMRITTPSQSFWGWGAFS------GNNIWASGGKNAPFDKPFHLILNVAVGG--DFFADG--DYDVPKPWGGHNPMR--SFWEARHSWENTWKGD- 310
WHGWHTYSLDWTADHIITYVDNVEMMRINTPSQSFWGWGGFD------GNNIWASGGKNAPFDKPFHLILNVAVGG--DYFGNG--EYDVPKPWGNHNPMR--SFWEARHSWEHTWQGD- 310
ADGFHTYKLEWDANHIRVTVDGRQILYVSTPGNSYWGWGGFG------GSNPWAGGGRDAPFDQYFQLILNVAVGG--QYFPQGC-AYNHPRPWHDGSPRQEAEFYEKKSEWLSTWHGE- 314
ADSFHTYSVDWTAGHIRMDIDNQPVMAWTTPSQGYWSYSHQS------GTNVWSQGGNDAPFDGKMSLILNVAVGATNGYFQDSWHNTPHAKPWKNNSPTAMMDFWKSKQQWQSTWHGE- 313
SADFHTFGLDWSPDSMQWLLDDQVYHTESLQRNFWDGV----------------YNQNGSPFDKNFFIILNLAVGG--NFFGGEPFDPSESDGWAKN----------------------- 236
TTDFHVYKLTWSQSEIKMFIDDIQYMVFDITP--------------------------LPVFQKNFYVLLNLAVGG--NFPN--IHDAGAVTAPLPG----------------------- 225
ADTFHTFAVDWRPGSITWSVDGVAYQTYTSADTR----------------------GNPWVFDQPFFMILNVAVGG--DWPG-----YPDGSTQFPQ----------------------- 232
T.molitor
S.purpuratus
H.armigera
P.sachalinensis
C.albidus
M.yessoensis
P.viridis
H.discus
C.antarcticus
B.xylophilus
Nocardiopsis sp
L.sitkana
----KVAMEIDYIEMRYL----------- 322
D---TAAFKIDYVKIWAL----------- 358
----EAAMQVNYVRVYXEPGQTTYXLRDR 323
NDGQDASLQVDYVRIWAL----------- 339
----DVAMECDYVEMTQY----------- 326
----EVAMVVDYIEMIPH----------- 324
----EVALVIDYIEMIPH----------- 324
----RAAMVIDYVEMVQA----------- 328
----DVAMKVKSVKMIQY----------- 327
------TFEVEYVKKWTWN---------- 249
------QMLVDYIKVTQ------------ 236
------EMRVDYVRVYELG---------- 245
T_molitor
S.purpuratus
H.armigera
P.sachalinensis
C.albidus
M.yessoensis
P.viridis
H.discus
C.antarcticus
B.xylophilus
Nocardiopsis sp
Fig. 1. Alignment of invertebrates’ and bacterial endo-(1→3)-␤-d-glucanases sequences. Conserved residues are highlighted in black (strictly conserved) or in grey (homol-
ogous replacements). Triangles above the sequences indicate catalytic residues; circlets indicate substrate-binding tryptophan residues of the enzymes active site.
display high transglycosylation activity [7,32,33]. Transglycosy-
lating enzymes are traditionally considered to be good tools for
the enzymatic synthesis of new biologically active carbohydrates
[9]. In general, enzyme-catalyzed synthesis of oligosaccharides is
a useful method because it allows the formation of well-defined
oligosaccharides selectively in the absence of any protecting
groups. Previous study has been shown that the purified endo-
(1→3)-␤-d-glucanase GI possessed transglycosylating activity [10].
Transglycosylating properties were studied also for glucanases LIV
(from P. sachalinensis), L0 (from Ch. albidus), LV (from M. yessoensis)
and glucanase from P. viridis with some aryl glycosides, glycerol and
d-glucose as acceptor [32,33]. Early studies have been shown that
glycerol is a better acceptor than water and other alcohols but it
is less effective than glucose, p-nitrophenyl- and methylglycosides
for endo-(1→3)-␤-d-glucanases (LIV, L0, LV and glucanase from P.
viridis) [7,32]. It is interesting that glycerol as acceptor has different
action upon enzymatic reactions: it increases the rate of reaction
and changes the ratio of hydrolysis products for LIV. Probably, these
characteristics might be connected to the structure of active center
of the enzyme.
group), since structural features of these compounds could be elu-
cidated with a negative-ion CID ESIMS/MS [34]. Transglycosylation
products were detected by ESIMS for both acceptors. It should
be noted, that neutral substances (products of hydrolysis, built
up of glucose and transglycosylation products, built up of glu-
cose and glycerol) showed fragment ions from the glycosidic bonds
cleavage only. Thus, identification of glycosylation sites could only
be possible by CID ESIMS/MS analysis of GlcA-labeled products,
which were prepared by removing methyl groups from GlcAOMe-
products by treatment with aqueous ammonia as described for
deacetylation procedure [35]. Surprisingly, intensities of the ions
of GlcA-containing products were higher in the positive-ion mode.
However, fragment ions from cross-ring cleavages were observed,
providing information on the linkage types (Fig. 2). In this way, by
analyzing oligosaccharides with longer DP by ESIMS/MS it has been
shown that GI transfers residues of glyconic parts of the substrate
mainly at C3 and C4 positions of the GlcA and strictly at C3 of Glc
residues. Thus, disaccharides Glc-(1→3)-GlcA; Glc-(1→4)-GlcA and
trisaccharides Glc-(1→3)-Glc(1→3)-GlcA; Glc-(1→3)-Glc(1→4)-
GlcA were found and characterized in the products of reaction.
The further study of the features of transglycosylation reac-
tion catalyzed by (1→3)-␤-d-glucanase GI from the liver of marine
gastropodean mollusk L. sitkana using glycerol as acceptor was
performed in comparison with (1→3)-␤-d-glucanase LIV from
the crystalline styles of marine bivalve mollusk P. sachalinensis.
LIV is one of the first enzymes isolated from marine mollusks
and its kinetics and catalytic mechanism were studied by various
The features of transglycosylation reaction catalyzed by GI
were established by the means of mass spectrometry. Two accep-
tors were chosen: 6-O-methyl-␤-d-glucuronic acid (GlcAOMe) for
structural analysis, glycerol for semi-quantitative analysis and lam-
inaran as a donor. As GI was unable to transfer glucose residues
to GlcA directly, GlcAOMe compound was chosen as an accep-
tor in order to obtain GlcA-labeled products (by removing methyl

M.S. Pesentseva et al. / Journal of Molecular Catalysis B: Enzymatic 75 (2012) 73–79
77
Table 1
Yield (%) of the products of laminaran hydrolysis by endo-(1→3)-␤-d-glucanases GI
and LIV detected by HPLC. The degree of laminaran hydrolysis is about 20%.
Enzymes
Productsa (%)
Glc
Glc2
Glc3
Glc4
Glc5
G-I
L-IV
44
27.2
17.2
26.8
22.2
26.0
9.0
10.8
7.6
9.2
a
Glc, Glc2, Glc3, Glc4, Glc5 – glucose and glucobi-, tri-, tetra- and pentaose, respec-
tively.
hydrolysis) of both GI and LIV enzymes (Fig. 3A, 3B). Mass spec-
tra showed the peaks of the following ions: [Glc+Na]+ at m/z 203,
[Glc2+Na]+ at m/z 365, [Glc3+Na]+ at m/z 527, [Glc4+Na]+ at m/z
689, [Glc5+Na]+ at m/z 851, [Glc6+Na]+ at m/z 1013, [Glc7+Na]+ at
m/z 1175, [Glc8+Na]+ at m/z 1337.0. An analysis of the composi-
tion of the products at different stages of the reaction showed that
the type of action of LIV and GI is characteristic for endo-enzymes
from marine sources [39]. From ESIMS one can directly deduce the
products of primary and secondary hydrolysis [7], since laminaran
from L. cichorioides is known to contain large percent of manni-
tol residues, located at the reducing end [11]. Thus, by observing
“+2 Da” satellite signals it was confirmed that GI (Fig. 3A) accu-
mulated Glc3, Glc4, Glc5 and LIV (Fig. 3B) accumulated Glc3, Glc4,
Glc5, Glc7 as products of primary attack. Probably, LIV has more
extensive substrate-binding center. Both enzymes also produced
glucose from the first minutes of hydrolysis (data not shown).
Hence, probably, GI and LIV attacked mannitol-free reducing ends
of polysaccharide first. The tendency of LIV to attack substrate near
the reducing end was shown earlier [39]. ESIMS data was confirmed
by HPLC (Table 1). Both (1→3)-␤-d-glucanases are classified as
endo-enzyme of smaller-oligosaccharide-producing type accord-
ing to previous study [40].
To calculate kinetic parameters of transglycosylation reaction
for both LIV and GI enzymes, laminaran was used as a donor,
while glycerol was employed as an acceptor. The optimal con-
centration of laminaran was found to be 5 mg/mL. The enzymatic
activities at various concentrations using 0.1, 0.5 and 1.0 M glyc-
erol as the acceptor were investigated by ESIMS [4, 7, 10, and 33].
ESIMS showed the ions of hydrolysis (see above) and transglycosy-
lation reactions products: [GlyGlc+Na]+ at m/z 277.1, [GlyGlc2+Na]+
at m/z 439.4, [GlyGlc3+Na]+ at m/z 601.6, [GlyGlc4+Na]+ at m/z
763.8, [GlyGlc5+Na]+ at m/z 925.4, [GlyGlc6+Na]+ at m/z 1088.1,
[GlyGlc7+Na]+ at m/z 1250.3, [GlyGlc8+Na]+ at m/z 1412.4 (Fig. 4).
The results of investigation for LIV enzyme have shown that the rate
of hydrolysis is higher that the rate of transglycosylation reaction
(Fig. 4A). Contrary results were obtained for GI enzyme (Fig. 4B),
when the speed of hydrolysis reaction was higher only at low con-
centration of the glycerol.
Fig. 2. A positive-ion mode CID ESIMS/MS of transglycosylation products, obtained
under action of endo-1,3-␤-glucanases GI (10−2 U) on laminaran (5 mg/mL) as a
donor and GlcAOMe (1 mg/mL) as acceptor: square indicates ion [GlcGlcA-H+2Na]+
at m/z 401.063.
methods in detail [32,36]. Amino acid sequence of LIV is a second
such example of (1→3)-␤-d-glucanase from animal source [3].
Products of laminaran hydrolysis by GI and LIV were analyzed
by ESI mass spectrometry. These methods provide wide dynamic
range, high precision and ultimate sensitivity with some restric-
tions. There is a problem of detection of the monosaccharide ions,
such as [Glc+Na]+ and ionization ability of ESIMS seems to fall with
increasing DP [37,38]. Though the ESIMS method does not allow
carrying out accurate quantitative measurements, if the substances
are close on molecular weights and have a similar structure, it
is possible to measure a ratio between the intensities of the ions
and thus calculate kinetic parameters. Furthermore, use of HPLC
combined with mass spectrometry allows having total information
about hydrolysis products as well as transglycosylation products.
Glucose (main component due to HPLC, but its intensity is
lowered in MS due to the restrictions, mentioned above) and
glucooligosaccharides with different degree of polymerization
(DP) were found in the reaction products (20% of laminaran
Rel.Int.,%
100
Rel.Int.,%
100
A
B
527,17
527,17
75
75
689,23
689,23
50
50
365,11
365,11
851,29
203,02
200
851,28
25
25
1175,40
1200
203,01
1013,35
1000
1013,34
1337,45
1400
1175,40
1337,46
0
0
200
400
600
800
1000
1200
1400
400
600
800
Counts vs. Mass-to-Charge (m/z)
Counts vs. Mass-to-Charge (m/z)
Fig. 3. ESIMS of the products of laminaran hydrolysis by (1→3)-␤-d-glucanases GI (A) and LIV (B). The degree of substrate hydrolysis is about 20%.

78
M.S. Pesentseva et al. / Journal of Molecular Catalysis B: Enzymatic 75 (2012) 73–79
Rel.Int.,%
100
Rel.Int.,%
A
B
100
- hydrolysis products
- transglycosylation products
75
50
75
50
25
25
0
0
0
200
400
600
800
1000
0
200
400
600
800
1000
Counts vs. Mass-to-Charge (m/z)
Counts vs. Mass-to-Charge (m/z)
Fig. 4. ESIMS of transglycosylation and hydrolysis products, obtained under action of endo-1,3-␤-glucanases GI (A) and LIV (B) (10−2 U) on laminaran (5 mg/mL) as a donor
and glycerol (1.0 M for GI and 0.5 M for LIV) as acceptor.
200000
180000
160000
140000
120000
100000
80000
200000
180000
A
B
160000
140000
120000
100000
80000
60000
40000
20000
60000
40000
20000
0
0
0
0,2
0,4
0,6
0,8
1
1,2
0
0,2
0,4
0,6
0,8
1
1,2
Glycerol, M
Glycerol, M
Fig. 5. The products of transglycosylation (dotted line) and hydrolysis (line) reactions catalyzed by endo-1,3-␤-glucanases GI (A) and LIV (B) (10−2 U) at various concentrations
of glycerol (0.1, 0.5 and 1.0 M). These plots were calculated from ESIMS spectra as total intensity basis products of reactions (glucose and oligosaccharides with DP 2–5) in
initial stage of reaction.
GI
LIV
This observation was investigated more deeply. The plot (Fig. 5)
is based on the ESIMS spectral data of transglycosylation reaction
products (glucose and oligosaccharides with DP 2–5), which were
recorded at various concentrations of glycerol at the initial stage of
reaction (first minutes). Total intensity was calculated as a sum
of the intensities of the ions of glucose and oligosaccharides. It
was found that total amount of transglycosylation reaction prod-
ucts increased with increasing of glycerol concentration. For GI
enzyme reaction still ran at 1.0 M of glycerol concentration. For LIV
enzyme it was found that amount of transglycosylation products
increased up to 0.5 M of glycerol concentration but decreased when
the glycerol concentration was higher. According to the dynamics
of transglycosylation and hydrolysis products accumulation for LIV
we concluded that enzyme partly inactivated with increasing of
glycerol concentration. Probably, the affinity of the glycerol to the
catalytic center of LIV was higher. To confirm the influence of glyc-
erol concentration on the hydrolysis reaction we used independent
method (Nelson) for registration of the yield of reaction prod-
ucts (Fig. 6). The data shows that glycerol suppresses hydrolysis
of laminaran by GI enzyme while the transglycosylation products
are accumulated when the rate of hydrolysis by LIV has a linear
decrease. The ratio of Ktr,gly (transfer to glycerol) to Khydr (transfer
to water) can be estimated from the experiment with glycerol as
1,6
1,4
1,2
1
0,8
0,6
0,4
0,2
0
0
0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9
1
1,1
Glycerol, M
Fig. 6. The effect of glycerol on hydrolysis of laminaran (1 mg/mL) by 10−2 U of
endo-1,3-␤-glucanases GI and LIV (registration of products by Nelson method).
4. Conclusion
Both (1→3)-␤-d-glucanases GI and LIV were assigned to 16 fam-
ily of glycoside hydrolases. They showed high sequence similarity,
they hydrolyzed substrate according to endo-type with retention
of the anomeric configuration. Despite the same specificity, both
enzymes have significant differences in hydrolysis products and
the transglycosylation and hydrolysis reactions, passing at the same
time. The use of mass spectrometry allowed us to simultaneously
study the dynamics of hydrolysis and transglycosylation reactions
and to elucidate the structure of oligosaccharides and oligosides,
synthesized by the enzymes. Unfortunately, some limitations of the
MS technique do not allow identifying which hydroxyl groups are
Ktr,gly
Khydr
Itr,gly
[H2O]
Ihydr [Glycerol]
˛ =
=
·
were Itr,gly and Ihydr are the maxima of total intensity of transgly-
cosylation and hydrolysis products, respectively. The coefficients
were calculated: at 0.5 M of glycerol for LIV ˛ was 80 and for 1 M
glycerol for GI ˛ was 225. It must be noted that the value ˛ for LIV
identified by ESIMS is close to the value ˛ = 50 calculated in a former
study [33].

M.S. Pesentseva et al. / Journal of Molecular Catalysis B: Enzymatic 75 (2012) 73–79
79
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This work was supported by the program of the Russian
Academy of Science “Molecular and Cellular Biology”, the grant of
the Russian Foundation for Basic Research 09-04-00761-a and the
Russian Federation Government Grant, contract №11.G34.31.0010.
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