Original Paper |
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Acta Biochim Biophys |
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doi:10.1111/j.1745-7270.2006.00210.X |
Purification and
Characterization of Alginate Lyase from Marine Vibrio sp. YWA
Yuan-Hong WANG*, Guang-Li YU,
Xin-Min WANG, Zhi-Hua LV, Xia ZHAO, Zhi-Hong WU, and Wei-Shang JI
Marine
Drug and Food Institute, Key Laboratory of Marine Drugs of Shandong Province,
Key Laboratory of Marine Drugs, Ministry of Education, Ocean University of
China, Qingdao 266003, China
Received: April 10,
2006
Accepted: June 10,
2006
This work was
supported by the grants from the National High Technology Research and Development
Program of China (2002AA624020) and the Science and Technology Bureau of
Qingdao (05-2-HY-49)
*Corresponding
author: Tel, 86-532-82032064; Fax, 86-532-82033054; E-mail, [email protected]
Abstract Extracellular alginate lyase secreted by
marine Vibrio sp. YWA, which was isolated from decayed Laminaria
japonica, was purified by a combination of ammonium sulfate precipitation
and diethylaminoethyl-Sephacel column chromatography. The results show that the
molecular mass of alginate lyase was approximately 62.5 kDa, with an optimal pH and temperature at
pH 7.0 and 25 ºC, respectively. Km was
approximately 72.73 g/L. The activity of the enzyme was enhanced by EDTA and Zn2+,
but inhibited by Ba2+. The substrates specificity
analysis shows that it was specific for hydrolyzing poly-b–D-1,4-mannuronate in alginate.
Key words marine Vibrio; alginate; alginate lyase;
purification; characterization
Alginate is a linear polysaccharide composed of two monomers, b–D-1,4-mannuronate
(M) and a–L-1,4-guluronate (G), and arranged in three forms, poly b–D-1,4-mannuronate
(M)n, poly a–L-1,4-guluronate (G)n and
heteropolymeric random sequences (MG)n [1]. Alginate is mainly
produced by brown alga, and can also be secreted by some pathogenic bacteria,
such as Pseudomonas aeruginosa which is known to be a component of the
capsule-like biofilm responsible for chronic pulmonary infection and
respiratory difficulty in patients with cystic fibrosis [2]. Alginate-derived
oligosaccharides produced by alginate lyases have been shown as functional
oligosaccharins [3], such as bifidus factor [4] and elicitor of plant growth
[5].
Up to now, the alginate lyase is mainly purified from marine algae,
mollusk, microorganism and virus, and the hydrolysis mechanism of these enzymes
is usually to split the glycosidic bond through the b-elimination reaction and
to form an unsaturated double bond between C4 and C5 in non-reducing end shown strong absorption at 235 nm [6–12]. However,
the enzymatic property and the hydrolyzing site of the alginate vary among
different origins.
In this paper, a marine bacteria (Vibrio sp. YWA), which
could secrete alginate lyase, was screened and isolated from the decayed Laminaria
japonica, and its biochemical property of the purified alginate lyase was
reported.
Materials and Methods
Materials and instruments
Kelp (L. japonica) was collected from Qingdao seashore in May
2003 (Qingdao, China). Alginate (1%, 120 mPas) was purchased from Qingdao Algae
Industry Company (Qingdao, China). (M)n and (G)n were prepared according to Wang et al. [13] and Liu et al.
[14]. DEAE-Sephacel (90 mm) was from Amersham Pharmacia Biotech (Piscataway, USA). Biocard
700E perfusion chromatography (ABI Company, Foster City, USA), J2-MC
centrifuger (Beckman, Fullerton, USA), Unico spectrophotometer (UV-2102 PCS;
Dayton, USA), HPLC (Waters, Milford, USA) and standard protein marker (Promega,
Madison, USA) were perchased respectively. Other reagents used were of
analytical grade.
Screening and cultivation of
the zymogenic strain
The rotted part of the kelp was cut and put into sterile seawater,
and the suspension was diluted and added to
selective medium [10 g/L alginate, 30 g/L NaCl, 6 g/L K2HPO4, 3 g/L KH2PO4, 3 g/L (NH4)2SO4, 0.5 g/L MgSO4∙7H2O and 0.1 g/L FeSO4∙7H2O] in which the alginate was used as the only carbon source. One
week later, lines were drawn on the solid selective medium plate to culture the
strains, and five generations were passaged successively. The solution was
filtered for the second time after zymosis. The enzyme activity of each
obtained strain was tested at 235 nm, and the strains were stored at –80 ºC. The Vibrio
sp. YWA strain with the highest activity was cultured in the selective
medium.
The strains were activated, and the monoclonal ones were transferred
into a 500-ml erlenmayer flask containing 50 ml culture solution. The strains
were agitated at 25 ºC for 12 h, and 2% of the total was transferred into a
500-ml erlenmayer flask containing 100 ml culture solution, with the same
conditions described as above. The culture solution was collected after
incubation for 48 h.
Isolation and purification of
alginate lyase
The fermented culture solution was centrifuged for 30 min at 10,000 g,
4 ºC, and the supernatant was collected. (NH4)2SO4 was added into the supernatant to a
concentration of 35%, then the mixture was stirred for 2 h and centrifuged at
10,000 g for 20 min. The supernatant was further adjusted to 65% (NH4)2SO4, stirred for 2 h at 4 ºC, and then
centrifuged. The precipitate was collected and dialyzed against 20 mM phosphate
buffer (pH 7.0) at 4 ºC for 24 h, and the crude enzyme solution was acquired.
The crude enzyme solution was applied to a DEAE-Sephacel column (1.6
cm´20 cm), then eluted with a linear
gradient of 0.1–0.8 mM NaCl in 10 mM phosphate buffer (pH 7.0) with a flow rate of
0.5 ml/min, and absorbance at 280 nm was monitored. The fractions were
collected and the activity of each fraction was measured separately. The active
fractions were combined and dialyzed against 10 mM PBS (pH 7.0) for 24 h and
stored at –20 ºC.
Determination of protein
concentration
Protein concentration was determined by the Folin-phenol method, using
bovine serum albumin as a standard protein [15].
Determination of alginate
lyase activity
Mix 0.8 ml of 200 mM phosphate buffer with 0.1 ml of 10 g/L sodium
alginate, then 0.1 ml enzyme solution was added. The mixture was kept at room temperature
for 30 min, and the absorbance of the reaction mixture was measured at 235 nm.
One unit of the enzyme activity is defined as the increase of 0.01 in
absorbance at 235 nm per minute.
SDS-PAGE of alginate lyase
The molecular mass of alginate lyase was determined by SDS-PAGE
[16].
Effects of temperature on
enzyme activity
Alginate lyase was incubated in a water bath at a series temperature
of 0, 15, 25, 30, 35, 40, 45 and 50 ºC for 1 h, respectively, then quickly
cooled to 0 ºC. The enzyme activity was evaluated again to determine its
optimal working temperature.
Effects of pH on enzyme
activity
Three kinds of pH buffer solutions, 200 mM Na2HPO4-citric acid (pH 2.2–8.0), Na2HPO4-NaHPO4 (pH 6.0–8.0) and 50 mM Glycine-NaOH
(pH 8.6–10.6) were selected, and all experiment conditions were set at 25 ºC
for 2 h. The enzyme activity was determined by incubating it in a water bath at
25 ºC for 2 h.
The determination of the
enzyme kinetics curve
The enzyme, different substrates and buffer were mixed at a
proportion of 0.2:0.2:1.6 (V/V/V), cultured at 25 ºC for
30 min, and the absorbance was measured at 235 nm. The concentrations of the
substrates (alginate) were set to 1, 2.5, 5, 7.5, 10, 15, 20 and 30 g/L,
respectively. The kinetics curves were drawn by Lineweaver-Burk
double-reciprocal plot method, and the Km was
determined.
Effects of EDTA and metal ions
on enzyme activity
Alginate (10 g/L) was dissolved in PBS buffer (pH 7.0), which contains
EDTA and a different kind of metal ions (Zn2+, Mg2+, Ba2+
and Ca2+), and the enzyme was added to the above solution and
incubated at 25 ºC for 1 h. The absorbance at 235 nm with or without metal ions
in above solution was measured and compared.
Determination of the
distribution of the molecular mass of the alginate oligosaccharide
The distribution of the molecular mass of the alginate-derived
oligosaccharides was determined by PAGE method with 5% stacking gel and 22% resolving
gel at 200 V for 2.5 h [17].
Substrate specificity of the
alginate lyase
Five microliters of 2 g/L (M)n or (G)n solution was mixed with 1 ml purified alginate lyase and reacted at
25 ºC for a different time. The absorbances of the solution at 0.3, 0.6, 1, 2,
4, 6, 12 and 18 h were measured.
Results
Screening of the zymogenic
strain
After zymogenic incubation, the bacteria which could degrade
alginate were selected. Under the transmission electron microscope, the length of
the flagellum was 3.5 times longer than that of the vibrios. The 16S rDNA
sequence and the physiological and biochemical characteristics analysis
indicated the stain belong to Vibrio sp. (data not shown).
Isolation and purification of
the alginate lyase
Crude enzyme precipitated from 65% (NH)2SO4 was separated by DEAE-Sephacel column. The enzyme activity of each
fraction was tested by the ultraviolet absorption method. The first peak, which
had the highest enzyme activity with 0.4 mM NaCl was collected and combined.
The data are listed in Table 1. The specific activity of the pure enzyme
is 5.25 times than that of the crude enzyme, and 332 times of that of the
zymogenic solution.
SDS-PAGE of the pure enzyme
The alginate lyase purified from (NH4)2SO4 precipitation and DEAE-Sephacel column was
shown as a single band (Fig. 1), and the molecular mass was
approximately 62.5 kDa.
Effects of temperature on
enzyme activity
The enzyme activity was measured
at different temperatures, and the data show that the optimal temperature was
25 ºC [Fig. 2(A)]. The thermal stability results show that the enzyme
was stable between 0 and 30 ºC [Fig. 2(B)], but its activity
decreased quickly when the temperature was higher than 30 ºC, and lost 90%
activity when the temperature was higher than 50 ºC.
Effects of pH on the enzyme
activity
The enzyme activity was determined at different pH values, and the highest
enzyme activity was observed in phosphate buffer at pH 6.8–7.2. The enzyme
activity decreased at pH>7.5 [Fig. 3(A)], and the enzyme activity
was relative stable at pH 6.0–7.5 [Fig. 3(B)].
Enzyme kinetic curve
Different concentrations of alginate were used as specific
substrates to determine the enzyme kinetic properties (Fig. 4). The
kinetic curve was drawn by the Lineweaver-Burk double-reciprocal plot method,
and the Km was calculated as 72.73 g/L, which indicates the alginate lyase has a high
activity for alginate hydrolysis.
Effects of EDTA and metal ions
on the enzyme activity
The effects of EDTA and metal ions on enzyme activity are shown in
Fig. 5. The enzyme activity of alginate was obviously increased in the
presence of EDTA, Zn2+, Mg2+ and Ca2+, but
obviously decreased in the presence of Ba2+.
Substrate specificity of the
enzyme
The distribution of the molecular mass of the alginate-derived
oligosaccharide was measured at different time intervals by PAGE. The results (Fig.
6) show that as time goes on, more and more bands appeared, indicating that
the content of the oligosaccharide was increasing. As the low polymerized
oligosaccharide could not be stained with Alcian blue, the silver staining
method was used to improve the sensitivity.
Degradation of the alginate
lyase on different substrates
The specility of alginate lyase produced by Vibrio sp. YWA
was evaluated with alginate, (M)n and (G)n, and the data are shown in Fig. 7. The results indicated the
alginate lyase has specific hydrolysis effect on (M)n but not
(G)n in alginate.
Discussion
The alginate lyase was purified to homogeneity by ammonium sulfate
precipitation. Thirty-five percent of the ammonium sulfate was used to
eliminate the non-active protein, and 65% of the ammonium sulfate precipitate
contained crude alginate lyase. The crude enzyme was eluted using DEAE-Sephacel
column by a linear gradient of 0.1–0.8 mM NaCl in phosphate buffer and a single
peak with high alginate lyase activity was obtained. The purified enzyme
displayed a single band in SDS-PAGE and its molecular mass was approximately
62.5 kDa.
The study on the substrate specificity has shown that the alginate
lyase had a higher hydrolysis activity to (M)n than
that to (G)n, and this result was confirmed by carbohydrate electrophoresis
analysis in Fig. 7. So the enzyme was ascribed to mannuronate lyase.
Based on the characterization study of the alginate lyase from Vibrio
sp. YWA, this alginate lyase is significantly different from others (such
as alginate lyases from abalone, Haliotis discus hannai, bacterium Sphingomonas
sp.) in molecular weight and optimal pH and temperature [1820]. Further study on
the DNA sequence will provide new information on the structure-activity
relationship study of alginate lyase. The new alginate lyase from Vibrio sp.
YWA will be useful for the production of alginate-derived oligosaccharides
and also as a tool for the fine structure analysis of alginate [22].
References
1 Ji MH. Seaweed Chemistry. Beijing: Science
Press 1997
2 Batten JC, Matthew DJ. Cystic fibrosis. In: Hodson
ME, Norman AP, Batten JC eds. The Respiratory System. London: Oxford University
Press 1983
3 Darvill A, Bergmann C, Cervone F, De Lorenzo
G, Ham KS, Spiro MD, York WS et al. Oligosaccharins involved in plant
growth and host-pathogen interactions. Biochem Soc Symp 1994, 60: 89–94
4 Akiyama H, Endo T, Nakakita R, Murata K,
Yonemoto Y, Okayama K. Effect of depolymerized alginates on the growth of
bifidobacteria. Biosci Biotechnol Biochem 1992, 56: 355–356
5 Yonemoto Y, Tanaka H, Yamashita T, Kitabatake
N, Ishida Y, Kimura A, Murata K. Promotion of germination and shoot elongation
of some plants by alginate oligomers prepared with bacterial alginate lyase. J
Ferment Bioeng 1993, 75: 68–70
6 Iwamoto Y, Araki R, Iriyama K, Oda T, Fukuda
H, Hayashida S, Muramatsu T. Purification and characterization of bifunctional
alginate lyase from Alteromonas sp. strain No. 272 and its action on
saturated oligomeric substrates. Biosci Biotechnol Biochem 2001, 65: 133–142
7 Matsubara Y, Kawada R, Iwasaki K, Oda T, Muramatsu
T. Extracellular poly(a–L-gulurnoate) lyase
from Corynebaterium sp.: Purification, characteristics, and
conformational properties. J Protein Chem 1998, 17: 29–36
8 Takeshita S, Sato N, Igarashi M. A highly
denaturant-durable alginate lyase from a marine bacterium: Purification and
properties. Biosci Biotechnol Biochem 1993, 57: 1125–1128
9 Miyazaki M, Obata J, Iwamoto Y.
Calcium-sensitive extracellular poly(a–L-gulurnoate) lyase
from a marine bacterium Pseudomonas sp. strain F6: Purification and some
properties. Fish Sci 2001, 67: 956–964
10 Tomoko S, Shigeki Y, Isao K. Some properties
and cation mode of (1-4-a–L-guluronide) lyase
form Enterobacter cloacae M-1. Carbohydr Res 1997, 304: 125–132
11 Song K, Yu WG, Han F, Han WJ, Li JB. Purification
and characterization of alginate lyase from marine bacterium Vibrio sp.
QY101. Acta Biochim Biophys Sin 2003, 35: 473–477
12 Li JB, Yu WG, Han F, Han WJ, Song K.
Purification and characterization of a novel alginate lyase from marine Vibrio
sp. QY102. Wei Sheng Wu Xue Bao 2003, 43: 753–757
13 Wang Y, Yu GL, Zhao X. Preparation and
structural characterization of several oligoguluronic acids. Gao Deng Xue Xiao
Hua Xue Xue Bao 2005, 26:179–183
14 Liu B, Wang CY, Zhang HR. Preparation and
identification of series of oligomannuronates. Gao Deng Xue Xiao Hua Xue Xue
Bao 2006, 27: 485–487
15 Li JW, Xiao NG, Yu RY. Principles and Methods
of Biochemistry Experiments. Beijing: Beijing House of Peking University 1979
16 Laemmli UK. Cleavage of structural proteins
during the assembly of the head of bacteriophage. Nature 1970, 227: 680–685
17 Yu GL, Chi LL, Guan HS. Application of PAGE on
the analysis of marine acidic oligosaccharides. Zhong Guo Hai Yang Yao Wu 2001,
20: 7–11
18 Teotia S, Khare SK, Gupta MN. An efficient
purification process for sweet potato b-amylase by affinity
precipitation with alginate. Enzyme Microb Technol 2001, 28: 792–795
19 Shimizu E, Ojima T, Nishita K. cDNA cloning of
an alginate lyase from abalone, Haliotis discus hannai. Carbohydr Res
2003, 338: 2841–2852
20 Hashimoto W, Miyake O, Ochiai A, Murata K.
Molecular identification of Sphingomonas sp. A1 alginate lyase (A1-IV‘)
as a member of novel polysaccharide lyase family 15 and implications in
alginate lyase evolution. J Biosci Bioeng 2005, 99: 48–54
21 Zhang ZQ, Yu GL, Guan HS, Zhao X, Du Y, Jiang
X. Preparation and structure elucidation of alginate oligosaccharides degraded
by alginate lyase from Vibro sp. 510. Carbohydr Res 2004, 339:
1475–1481