J. Biol. Macromol., 20 (1), 9-16 (2020)
ArticlePurification and characterization of an alginate lyase
from marine bacterium Microbulbifer sp. KIT−19
Yoshihiro Hakamada1,2,*and Souta Syouyama1,2
1

Department of Applied Bioscience, College of Bioscience and Chemistry, Kanazawa Institute ofTechnology (7-1 Ohgigaoka, Nonoichi, Ishikawa 921-8501, Japan)

2

Genome Biotechnology Laboratory, Kanazawa Institute of Technology, (3-1 Yatsukaho, Hakusan,
Ishikawa 924-0838, Japan)

Received July 17, 2019; Accepted December18, 2019

Alginate-degrading bacteria were isolated from seawater and a-systematic classification was
performed using 16S rDNA sequence. The isolated strain was identified as Microbulbifer sp. strain
KIT-19. The properties of alginate lyase (ALG19) derived from the strain KIT-19 were investigated.
ALG19 was purified by various column chromatography techniques, with specific activity of 10.1
units/mg and 7.08-fold purity. When the substrate was sodium alginate, the optimum pH of thepurified ALG19 was 8.5 and optimum temperature was 40°C. The activity of the purified ALG19
increased by 2.34-fold in the presence of 100 mM Na+. Alginate is composed of β-D-mannuronic acid(M) and 1,4-linked α-L-guluronic acid (G) residues. The activity toward poly- M blocks and poly-G
blocks of purified ALG19 on the basis of relative activity scale were 123% and 91% respectively,
when the decomposed activity to sodium alginate was 100%. Since ALG19 was poly-M blocks specificalginate lyase, it was thought that ALG19 belongs to the polysaccharide lyase (PL)-7 family. ALG19
could be considered to be an endo-type enzyme because it lowers the kinematic viscosity of sodium
alginate from 23.5 to 1.65 cSt (mm2/s).
Key words: Alginate lyase, Purification, Characterization, Microbulbifer, poly-M blocks specificity

Abbreviations: DEAE, diethylaminoethyl; DNS, 3,5-dinitrosalicylic acid; SDS-PAGE, sodium
dodecyl sulfate polyacrylamide gel electrophoresis; LBG, locust bean gum; CMC, carboxymethylcellulose;poly-M,

polymannuronate;poly-G,

polyguluronate;PL,

polysaccharide

lyase;

EDTA,

ethylendiaminetetraacetic acid; cSt, centi stokes.
Introduction

to cellulose. The most abundant carbohydrate in

Seaweeds, especially the brown algae, arebrown algae is alginate, which accounts for 17-

attracting attention as an abundant biomass next

45% of the total biomass[1]. Alginates are linear*

Correspondence author: Yoshihiro Hakamada.

polysaccharides consisting of 1,4-linked α-L-

E-mail: yhakamada@neptune.kanazawa-it.ac.jp

guluronic acid (G) residues and its C5 epimer, β-9Characterization of alginate lyase from KIT-19 strain

D-mannuronic

acid

(M)

residues.

This

decomposing bacterium of L. japonica waspolysaccharides can form homopolymeric blocks

isolated from the seashore of Kanazawa Port inof G residues (G-blocks) or M residues (M-blocks)

Japan. The seawater was passed through aor heteropolymers of alternating M and G (MG -membrane filter (pore size, 0.45 μm, Merck,[2]

blocks) . Alginate lyases are alginate-degrading

Darmstadt, Germany.) and the filter paper was

enzymes, which catalyze the depolymerization of

soaked in a seawater-based medium (pH 8.0)

[3]

alginate .
In

containing 0.2% (w/v) NaNO3, 0.001% (w/v) iron

theenZYmes

citrate n-hydrate, 0.006% (w/v) KH2PO4, and 2%(CAZy) database, alginate lyases are classified into(w/v) alginic acid. Single colony isolation wasseven polysaccharide lyase (PL) families including

carried out on the same medium containing 2%families PL- 5, 6, 7, 14, 15, 17 and 18 (http://www.

(w/v) agar. The microorganisms with the highest

cazy. org/). In the CAZy database presents aalginate lyase activity were selected. The isolatedclassification of these enzymes in families andstrain named KIT-19 was grown aerobically at

subfamilies based on amino acid sequence

30°C for 4 days. The 16S rDNA gene of strain KIT-

similarities, intended to reflect their structural

19 was amplified by PCR from genomic DNA andfeatures. Alginate lyases are also enzymaticallysequenced using BigDye terminator v3.1 cycle

classified into two groups based on their substratesequencing kit (Applied Biosystems, CA, USA).

specificity. The first group is the enzyme (ECThe obtained 16S rDNA gene sequence of strain

4.2.2.3) degrading poly-M-blocks, while the

KIT-19 was aligned automatically with closely

second group is the enzyme (EC 4.2.2.11)

related sequences retrieved from Aporon DB-BA

degrading poly-G-blocks. Most of the family of

5.0 (Techno Suruga Lab., Shizuoka, Japan),PL-7 alginate lyases specifically degrade poly-M-

GenBank, DDBJ and EMBL[6]. GenBank is a partblocks and poly-G-blocks, but some enzymesof the International of Nucleotide Sequencedegrade G and GM-blocks and M and GM-

Database Collaboration, which is also comprised[4],[5]blocksCarbohydrate-Active. In addition, the same bacteriumof DDBJ and EMBL (http://www.insdc.org/). A

produces several alginate lyases (PL CAZy,phylogenetic tree was constructed using Neighbor-

http://www. cazy. org/). This is considered tojoining method and a Kimura-2-parameter model

efficiently decompose the alginic acid polymer

in Aporon 2.0 (Techno Suruga Lab.).

which is normally difficult to decompose. In thisstudy we have isolated several marine bacteria

Enzyme purification

The dialyzed culture brothstrains from seawater at the seashore of Kanazawa

was applied to a Toyopearl DEAE-650M column

Port in Japan. One isolate was closely related to(2.5 × 20 cm; Tosoh, Japan) equilibrated with 20

marine bacterium Microbulbifer salipaludis basedmM Tris-HCl buffer (pH 9.0). After washing withon 16S ribosomal DNA. This report presents thethe same buffer, elution of proteins was performed

isolation and characterization of a purified alginate

with linear gradient of 0–0.5 M NaCl (400 mL) inlyase (ALG19) from genus Microbulbifer.

20 mM Tris-HCl buffer (pH 9.0). The fractions
containing ALG19 activity were pooled andMaterials and Methods

dialyzed solution was applied to a Toyopearl
DEAE-650M column (2.5 × 30 cm) equilibrated

Isolation of marine bacterium and identification ofwith 20 mM Tris-HCl buffer (pH 9.0) containing

strain KIT-19

0.1 M NaCl. The proteins were eluted with a 400

A marine bacterium as a10

Y. Hakamada and S. SyouyamamL linear gradient of 0.1–0.3 M NaCl in the same

point agarose, pectate, carrageenan, and laminaran[6]buffer . The fractions containing ALG19 activity

were examined. Carbohydrates (0.2% (w/v)) were

were pooled. All enzyme purification steps wereincubated with the purified enzyme at 40°C for 20

conducted at 4°C.

min in 0.1 M Tris-HCl buffer (pH 8.0) solutions,and reducing sugar concentration was determined

Enzyme assays

The ALG19 activity wasusing the DNS method. To measure the substrates

determined at 40°C for 15 min in 0.1 M Tris-HCl

specificities of the purified enzyme, M-blocks andbuffer (pH 8.0) containing 0.5% (w/v) sodium

G-blocks were prepared from hydrolysis of 5%

alginate as substrate. After terminating the reaction,(w/v) sodium alginate in 1 M oxalic acid at 98°C

the reducing sugar produced was determined by thefor 10 h. The insoluble material was separated into

3,5-dinitrosalicylic acid (DNS) method

[7]

with

two fractions by precipitation at pH 2.85. The

glucose as standard. One unit of enzymatic activity

soluble fraction at pH 2.85 was used as thewas defined as the amount of enzyme that

substrate of poly-M and the insoluble fraction was

produced 1 μmol of reducing sugar as glucose perused as poly-G [9]. Statistical analysis with the use

minute under the assay conditions.

of Tukey test was used for significant differencesof degradative activities for sodium alginate, M-

Characterization of alginate lyase

Toblocks and G-blocks.

determine the pH profile of alginate lyase activity,
reactions were performed at 40°C for 15 min in 0.1

Effect of various cations and EDTA

M sodium citrate-NaOH buffer (pH 3.0–5.0),

purified enzyme was incubated with various

sodium acetate buffer (pH 5.0–6.0), potassium

cations (each at 1 mM: Ca2+, Mg2+, Mn2+, Fe2+,

phosphate buffer (pH 6.0–8.0), Tris-HCl buffer

Ni2+, Zn2+, Na+ , and K+) at 30°C for 30 min, and(pH 8.0–9.0), and glycine-NaOH buffer (pH 9.0–

the effect of EDTA on alginate lyase activity was11.0) containing 0.1 M NaCl.The effect of

examined using EDTA at a final concentration of 5

temperature on ALG19 activity was determined

mM. The remaining activity in the samples wasusing 0.1 M Tris-HCl buffer (pH 8.0) at 10, 20, 30,

determined by measuring the activities of ALG1940, 50, 60, 70, and 80°C. Reactions were initiatedunder standard conditions in the presence of[6]by adding enzyme and progressed for 15 min .

Thevarious additional reagents. The activity obtained

To determine the viscosity reduction of the 2%

using the reaction measurement

without any(w/v) sodium alginate by the purified ALG19 (2.3

additional reagent was recorded as 100%.

µg), the enzyme reaction was performed at 40°C

Furthermore the enzyme activity was determinedfor 90 min in 0.1 M Tris-HCl buffer (pH 8.0).

with enzyme reaction mixtures containing various

Viscosity reduction of the 2% (w/v) sodium

concentrations of NaCl (0 – 400 mM). Alginate

alginate by treatment of the ALG19 was measured

lyase activity was measured after incubating theusing Ubbelohde SU-0001 viscometer (SIBATA,

enzyme mixtures at 40°C for 15 min in 0.1 M Tris-[8]

Saitama, Japan) .
Substrate specificity

HCl buffer (pH 8.0)
The hydrolytic activities

Thin-layer chromatography (TLC)

A reaction

towards various polysaccharides, sodium alginate,mixture composed of 0.2% (w/v) sodium alginate,glucomannan,(LBG),

0.1M Tris-HCl buffer (pH 8.0), and 0.26 units of

low-melting-enzyme was incubated at 40°C for 30min. After the

locustcarboxymethylcellulose

bean(CMC),

gum

11Characterization of alginate lyase from KIT-19 strain

reaction, hydrolysis products from sodium alginate

nucleotide position.

was separated on 0.2 mm Silica Gel 60 (Merck,
Darmstadt, Germany.) aluminum plates with n-

Purification of alginate lyase

butanol/acetic acid/water (1:1:1, v/v) as eluent.

The ALG19 was purified to homogeneity

Sugars were detected by spraying the TLC plates

from cultures of Microbulbifer sp. KIT-19 and thewith 5% (v/v) sulfuric acid in ethanol, followed bysubsequent two-step column chromatographic

heating at 200°C until color developed.

procedure. The molecular mass of the purified
enzyme was estimated to be approximately 29.2

Results and Discussion

kDa using SDS-PAGE analysis against molecularmass standards (Fig. 2). Approximately 7.08 fold

Description of Microbulbifer sp. KIT-19

purification to a specific activity of 10.1 U/mg

The almost complete 16S rDNA sequence

protein was obtained for the ALG19 activity when

(1,461 bp) of the strain KIT-19 was analyzed. The

measured at pH 8.0 at 40°C by the DNS method.

phylogenic tree constructed from the sequence data

Viscosity reduction of the 2% (w/v) sodium

showed that the strain KIT-19 appeared within thealginate

evolutionary

genus

Ubbelohde SU viscometer. As a result, theMicrobulbifer ( Fig. 1). The homologies between

kinematic viscosity of the enzyme reaction mixture

the KIT-19 16S rDNA sequence and the 16S rDNA

was 1.65 cSt (mm2/s). However, the kinematicsequences of strains Microbulbifer elongatus[10]viscosity of the reaction mixture without enzyme

radiationencompassingand Microbulbifer salipaludis[11]

98.3%,

phylogenetic

respectively.

The

using

ALG19

was

measuredby

was 23.5 cSt (mm2/s). These numerical values

were 99.5% andtree

represent the time required for the liquid to move asuggested that the strain KIT-19 belonged to thecertain distance, for example, 1.65 cSt (mm2/s)

Microbulbifer genus. Therefore, it was named as

represents a rapid decrease in sodium alginate

Microbulbifer sp. KIT-19.

viscosity. Endo-type enzymes have been reported
in various polysaccharide-degrading enzymes,
including alginate lyase, to cause a decrease in
substrate viscosity[12, 13, 14, 15]. This experimental
result suggested the possibility that ALG19 is an
endo-type enzyme.

Fig. 1.

Neighbor-Joining tree based on 16S rDNA

sequences of Microbulbifer sp. KIT-19 and otherbacteria. The unrooted tree was constructed by Neighbor
Joining analysis. Numbers at nodes indicate levels ofbootstrap support (%). Scale bar: 0.01 substitutions per

12

Y. Hakamada and S. Syouyama

Fig. 2.

optimum temperature at 40°C, similar to other

SDS-PAGE of purified ALG19. The protein

enzymes.

was stained with Coomassie brilliant blue (lane 1-4).
Lane 1, protein mass markers (in kDa); lane 2, culture
broth; lane 3, partial purified enzyme eluted from first
DEAE 650M column; lane 4, purified enzyme eluted
from second DEAE 650M column.

Effect of pH on the activity and stability of
ALG19
The alginate lyase activity was apparent overa narrow range of pH values, with more than 90%of the maximum activity detected between pH 8.0
and 9.0 (Fig. 3A). This pH profile was similar to
AlgMsp from Microbulbifer sp. 6532A[16]. The
purified ALG19 was a semi-alkaline enzyme, with
an optimum pH of 8.5.
To examine the stability of the ALG19 atdifferent pH values, the enzyme was preincubatedat 30°C for 30 min in various buffers (50 mM) andthe residual activities were assayed. The enzyme
was stable, with more than 80% of the originalactivity detected throughout the wide pH range
from 5.0 to 10 (data not shown).
Effect of temperature on the activity and stabilityof ALG19
The optimum temperature of ALG19 for thereaction with sodium alginate as substrate wasFig. 3.

detected at around 40°C and pH 8.0 in 0.1 M Tris-

purified ALG19 activity. The activity was measured in

HCl buffer (Fig. 3B). The thermal stability of the

0.1 M sodium citrate-NaOH buffer (pH 3.0–5.0, open

purified ALG19 was assayed in 0.1 M Tris-HCl

triangles), 0.1 M sodium acetate buffer (pH 5.0–6.0,

buffer (pH 8.0) after heating for 30 min at various

open circles), 0.1 M potassium phosphate buffer (pHtemperatures. The enzyme was stable up to 30°C,

6.0–8.0, closed circles), 0.1 M Tris-HCl buffer (pH 8.0–

and above this temperature the residual activity

9.0, closed triangles), and 0.1 M glycine-NaOH buffer

decreased gradually. More than 60% of the original(pH 9.0–11, closed squares) at 40°C for 40 min. The

activity remained even after heating at 40°C. In

relative activity was calculated assuming the activity at

general, the optimum temperature of the enzymes

a pH 8.5 (closed triangles) as 100%.

fro m v ar io u s m ar in e b acter ia, Vib rio sp .

temperature on ALG19 activity was determined by

JAM-A9m[17], Sphingomonas sp. ZH0[18]

Effects of (A) pH and (B) temperature on the

The effect of, andincubating the reaction mixtures at 10, 20, 30, 40, 50, 60,, are around70, and 80°C in 0.1 M Tris-HCl buffer (pH 8.0).

30°C to 40°C. The present enzyme had an

Reactions were initiated by adding ALG19 (32 ng/μL)

Saccharophagus sp. Myt-1

[19]

13

Characterization of alginate lyase from KIT-19 strain

Substrate specificity

and terminated after 15 min. The relative activity was

The enzyme exhibited highest activity on

calculated assuming the activity at 40°C as 100%.

sodium alginate, although glucomannan, LBG,
Effect of metal ions and EDTA

CMC,

low-melting-point

agarose,pectate,Purified ALG19 was incubated with various

carrageenan, and laminaran were not hydrolyzed[6].

cations (each at 1 mM) at 30°C for 30 min, andIn addition, we used poly M- and poly-G-blocks,

residual activity was then assayed by the DNS

which were prepared following the method of

method. The various metal ions, K+, Mg2+, andHaug et al.[9], and sodium alginate as substrates.

Mn2+ had a slight inhibitory effect. In addition,

The purified ALG19 exhibited the highest activity

ALG19 was inhibited by Fe2+, losing 92% activity.

toward poly- M-blocks (123%) on the basis of

Ca2+, Ni2+ and Zn2+ could inhibit the enzyme

relative activity scale, when the decomposed

activity between 20 to 30% as shown in Table 1.

activity to sodium alginate was 100%. The AGL19

Among the investigated metal ions, none had

activity on the poly G-blocks (91%) was less thanactivating effects on the activity of ALG19.

that of sodium alginate (100%) (Fig. 4). This poly

However, when the relative activity was calculated

M-block specific enzyme activity of ALG19 wasby assuming that the activity in 0 mM NaCl was

similar to the alginate lyase belonging to family

100 %, the alginate lyase activities at various NaCl

PL-7 from Microbulbifer sp. 6532A[16], Vibrio sp.

concentrations of 25 mM, 50 mM, 100 mM, 200

JAM-A9m[17], Vibrio sp. O2[21] and Pseudomonas

mM, and 400 mM were 175, 188, 234, 213, andaeruginosa[22]. Furthermore, these alginate lyases

190%, respectively. The purified ALG19 was acontaining AlgMsp from Microbulbifer sp. 6532A

salt-activated enzyme, similar to several alginate

were activated in Na+. On the other hand,alginate

lyases previously reported[5, 16, 20]. The effect of 10

lyases from Microbulbifer sp. Q7specificallymM EDTA on the enzyme activity of ALG19 was

degrade poly-G-blocks[23], and AlgL17 fromnegligible. Based on our results, for ALG19,

Microbulbifer sp. ALW1 is a poly-M-specificmonovalent metal ion such as Na+ was needed foroligoalginate lyase [24]. AlgL17 from Microbulbifer

its optimal activity but divalent metal ions such as

sp. ALW1 was classified into the family PL-17.

Ca2+ and Mg2+ were not essential.

Alginate lyases from genus Microbulbifer areclassified in families PL-7, 14 and 17. The family
members of PL-14 and PL-17 are mainly exooligoalginate

lyasesandvarioustypes

ofoligoalginate lyases, respectively.Since the aminoacid sequence of ALG19 is unknown, the family
cannot be classified properly. However, because
ALG19 is a poly-M-block-specific alginate lyase,and poly-M-block-specific alginate lyases are
currently classified only in family PL-7 (PL CAZy,
http://www. cazy. org/), it is possible that ALG19
belongs to family PL-7

14

Y. Hakamada and S. Syouyama

Fig. 4.

Substrate specificity of the purified ALG19

Fig. 5.

TLC analysis of degradation products offor alginate, poly-M-blocks and poly-G-blocks. Various

sodium alginate with the purified ALG19. The numbers

substrates were incubated with the purified enzyme aton the horizontal axes on the graphs indicate the reaction

40°C for 20 min in 0.1 M Tris-HCl buffer (pH 8.0)

time. Lane M, standard oligosaccharides; M1, mannose;

solutions, and reducing sugar concentration was

M2, mannobiose; M3, mannotriose; M4, mannotetraose;determined using the DNS method. The error bars

M5, mannopentaose; M6, mannohexaose. Lane C,

represent the average of three independent experiments.

control without enzyme.

The error bars are means ±S.D.
Tukey tests, *P<0.05, **P>0.05, ***P<0.05

References[1]

Analysis of hydrolysis products by TLC

Mabeau, S. and Kloareg, B. (1987) J. Exp.
Bot., 38, 1573-1580.

For digestion of 0.2% (w/v) sodium alginate,[2] Gacesa, P. (1988) Carbohydr. Polym., 8, 161-

a 1.0 mL reaction mixture in 0.1 M Tris-HCl buffer

182.

(pH 8.0) and 0.34 units of enzyme was incubated[3] Gacesa, P. (1992) Int. J. Biochem., 24, 545-552.

at 40°C for 0–60 min. Hydrolysis products released[4]Zhu, B., Tan, H., Qin, Y., Xu, Q., Du, Y., andfrom sodium alginate by 0.34 units of purified

Yin, H. (2015) Int. J. Biol. Macromol., 75,

ALG19 contained oligoalginate, based on TLC

330-337.

analysis. The degree of polymerization (DP) 2-4 of[5]

Kobayashi, T., Uchimura, K., Miyazaki, M.,oligoalginate was equivalent to the degradation

Nogi,

products from sodium alginate at each timepoint

Extremophiles., 13, 121-129.

(Fig.5). This result revealed that ALG19 alginate

[6]

Y.,

and

Horikoshi,

K.

(2009)

Hakamada, Y., Ohkubo, Y., and Ohashi, S.

lyase-mediated degradation of alginate produced(2014) Biosci. Biotechnol. Biochem., 78,

DP 2-4 of oligoalginate as the most abundant722-728.

product.

Thischaracteristicof ALG19

is[7]

considered to be a property suitable for recovering

Miller, G. L. (1959) Anal. Chem., 31, 426428.

DP 2-4 of oligoalginate.

[8]

Ubbelohde, L. (1937) U.S.Patent, 2,091,896.

[9]

Haug, A., Larsen, B., and Smidsrod, O.
(1966) Acta Chem. Scand., 20, 183-190.

15

Characterization of alginate lyase from KIT-19 strain[10] Sun, C., Chen, Y., Zhang, X., Pan, J., Cheng,(2004) J. Biol. Chem., 279, 31863-31872.

H., and Wu, M. (2014) Mar. Genom., 18, 83-[23] Yang, M., Yu, Y., Yang,

85.

S., Shi, X., Mou,

H., and Li, L. (2018) Frontiers in Microbiol.,[11] Yoon, J.-H., Kim, I.-G., Shin, D.-Y., Kang, K.

9, 2894.

H., and Park, Y.-H. (2003) Int. J. System.

[24] Jiang, Z., Guo,Y., Wang, X., Li, H., Ni, H.,

Evolut. Microbiol., 53, 53-57.

Li, L., Xiao, A., and Zhu, Y. (2019) Protein[12] Manns, D., Nyffenegger, C., Saake, B., andExpr. Purif., 161, 17-27.

Meyer, A. S. (2016) RSC Adv., 6, 4539245401.

Communicated by Yasukawa Kiyoshi[13] Boyce, A. and Walsh, G. (2018) Mol. Biol.
Reports, 45, 2201-2211.
[14] Rozhkova, A., M., Miarzlou, D., Bashirova,
A.V., Zorov, I. N., Korotkova, O. G.,
Shashkov, I. A., and Sinitsyn, A. P. (2018)
Moscow University Chemistry Bulletin, 73,
53-57.
[15] Mangan, D., Cornaggia, C., Liadova, A.,
Draga, A., Ivory, R., Evans, D. E., andMcCleary, B. V. (2018) J. Cereal Science, 84,
90-94.
[16] Swift, S. M., Hudgens, J. W., Heselpoth, R.
D., Bales, P. M., and Nelson, D. C. (2014)
PLOS ONE. 9, e112939,.
[17] Uchimura, K., Miyazaki, M., Nogi, Y.,
Kobayashi, T., and Horikoshi, K. (2010)
Mar. Biotechnol., 12, 526-533.
[18] He, M., Guo, M., Zhang, X., Chen, K., Yan
J., and Irbis, C. (2018) J. Biosci. Bioengine.,
126, 310-316.
[19] Sakatoku, A., Tanaka, D., and Nakamura, S.
(2013) J. Microbiol. Biotechnol. 23, 872877.
[20] Xiao, L., Han, F., Yang, Z., Lu, X. Z., and Yu,
W.

G.

(2006)World

J.

Microbiol.

Biotechnol., 22, 81-88.
[21] Kawamoto, H., Horibe, A., Miki, Y., Kimura,T., Tanaka, K., Nakagawa, T., Kawamukai,
M.,

and

Matsuda,H.

(2006)Mar.

Biotechnol., 8, 481-490.
[22] Yamasaki, M., Moriwaki, S., Myake, O.,
Hashimoto, W., Murata, K., and Mikami, B.

16