Engineering, 2013, 5, 376-380
http://dx.doi.org/10.4236/eng.2013.510B076 Published Online October 2013 (http://www.scirp.org/journal/eng)
Isolation and Characterization of an L-Amino Acid
Oxidase-Producing Marine Bacterium
Zhiliang Yu1*, Hua Qiao1, Juanping Qiu1, Peiya Xu1, Peng Li2
1College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou, China
2College of Marine Science and Technology, Zhejiang Ocean University, Zhoushan, China
Received 2013
ABSTRACT
One marine bacterial strain, R3, has been newly isolated from the intertidal zone of Dinghai sea area. Measurements of
a-keto acids and H2O2 existing in fermentation supernatant were carried out to show that R3 can produce L-amino acid
oxidase (LAAO) with a broad substrate specificity. Physiological and biochemical analysis showed that it can grow
great at the conditions with sodium chloride concentration of 1.5% - 3%, temperature of 15˚C - 35˚C and pH of 6 - 7. In
addition, molecular identification of 16S rDNA was performed to show that R3 was proximal to Pseudoalterom onas
spp. with the highest identity of 98.5% to Pseudoalteromonas ru bra. Therefore, it was designated as Pseudoalterom o-
nas sp. R3. Further studies are required to arrive at a better understanding of this LAAO and secure an application.
Keywords: LAAO; Pseudoalteromonas ; Physiological Property; Biochemical Property; Identification
1. Introduction
L-amino acid oxidase (LAAO; EC 1.4.3.2) is dimeric
flavoprotein, and each subunit contains a non-covalently
bound FAD molecule as cofactor. It is able to catalyze
the stereospecific oxidative deamination of L-amino ac-
ids to a-keto acids,
4
NH+
and H2O2. When H2O2 is not
degraded by catalase, it can cause a decarboxylation of
the a-keto acid to the corresponding carboxylic acid.
Diverse studies have indicated that LAAOs have broad
bioactivities such as inducing apoptosis [1], cytotoxicity
[2,3], edema [4], hemolysis [5], hemorrhage [6], induc-
ing or inhibiting platelet aggregation [7,8], parasite-kill-
ing activity [9], and antimicrobial activity [10]. LAAO
may act as defence or attack weapons via H2O2 [11] or as
ideal molecular mechanisms for the acquisition of nitro-
gen from diverse amino acid sources [12]. In addition,
researches have showed that LAAO affects the relation
of tumor cells with the immune system [13] and is in-
volved in violacein synthesis [14]. So far, LAAO was
found to be applied as catalysts in bio-transformation [15]
and as part of biosensors in determination of the different
forms (D- or L-) of free amino acids [16].
This enzyme has been widely found in nature includ-
ing snake venoms, insect drugs [17], sea hare, algae [18]
and terrestrial microorganisms [19]. In contrast, little is
known about LAAO from marine microorganisms. The
objec tive of th is study is to isolate and chara cterize LAAO-
producing marine microorganism. We believe that this
study lays the foundation for further investigation on en-
zymatic properties, structure, biological function and ap-
plicat i on of L A A Os f r om marine microorganisms.
2. Materials and Methods
2.1. Sample Collection and Isolation of
LAAO-Producing Marine Microorganism
The intertidal zone sludge samples (30.03˚N, 122.11˚E)
were collected from different locations at the Dinghai sea
area. From each location, sample was collected at 50 to
100 cm depth under the sea surface. These samples were
placed in special pre-sterilized plastic bottles and brought
to the laboratory in aseptic co ndition. Then, 10 g of each
sludge sample was subjected to 90 mL of sterilized dis-
tilled water and serially diluted (up to 10-6 dilution).
After dilution, about 100 µL of each diluted sample was
plated on different agar medium including PDA (potato
200 g/L, sucrose 10 g/L, sea salt 30 g/L), Gause’s Me-
dium NO.1 (soluble starch 20 g/L, NaCl 0.5 g/L,
K2HPO4 0.5 g/L, FeSO4 0.01 g/L, MgSO4∙7H2O 0.5 g/L,
KNO3 1 g/L, sea salt 30 g/L; pH 7.2-7.4), and MM me-
dium (yeast extract 3 g/L, peptone 5 g/L, sea salt 30 g/L;
pH 7.2 - 7.4), and separately incubated at 28˚C and 25˚C
for 2 - 7 days, as necessary. The isolated colonies were
purified by streak-plate technique.
LAAO activity was determined by measuring its fer-
mentation products includ ing H2O2 and a-keto acids. The
*Corresponding a uthor.

Z. L. YU ET AL.
377
production of H2O2 was measured by using Amplex Red
Hydrogen Peroxide/Peroxidase Assay kit (Invitrogen, USA).
The production of a-keto acids were spectrophotometri-
cally measured based on hydrazine assay, according to
Len Sikora’s me t hod [20].
2.2. Sodium Chloride Tolerance and Cultural
Condition
Different concentrations of sodium chloride (0%, 1.5%,
3%, 4.5%, 6%, 7.5%, 9%, 10.5% and 12%) were added
to the MM medium (without sea salt). The seed of iso-
lated LAAO-producer was planted into different MM
medium and incubated at 25˚C, 160 rpm for 24 hours,
and salt tolerance was tested. The growth of the LAAO-
producer on MM medium incubated at different temper-
ature (5˚C, 15˚C, 25˚C, 35˚C and 45˚C) and at different
pH (3, 4, 5, 6, 7, 8, 9, 10, 11 and 12) was also investi-
gated to determine its growth range of temperature and
pH.
2.3. Physiological and Biochemical
Characteriz ation
The ability of the isolate to utilize various carbon and n i-
trogen sources, and other physiological and biochemical
properties were studied by the method recommended in
“The Manual of Systematic Methods of Determinative
Bacterial”, with minor modification by adding 3% so-
dium chloride to each medium.
2.4. Sequencing Analysis
The genomic DNA was isolated by bacterial genomic
extraction method (EDTA treatment method). PCR reac-
tions were performed in 50 µL containing 37 μL ddH2O,
5 μL 10 × Easy Taq buffer, 4 μL 2.5 mM dNTPs, 100
nM primer 27F (5’-GAGTTTGATC CTGGCTCAG-3’),
100 nM primer 1527R (5’-AGAAAGGAGG T GATCC-
AGCC-3’), 1 ng genomic DNA, and 1 U Taq DNA po-
lymerase with denaturation at 94˚C for 5 minutes fol-
lowed by 30 cycles of 1 minute at 94˚C, 50 seconds at
55˚C, 90 seconds at 72˚C and a final 10-minutes exten-
sion at 72˚C. At the end of reaction, PCR product was
cooled to 4˚C to await further use. After size confirma-
tion on 1.0% agarose gel, the PCR DNA product was
sent to Sangon Biotech (Shanghai) Co. Ltd for sequenc-
ing of 16 S rDNA. After sequencing, the similarity and
homology of partial 16S rDNA were online analyzed us-
ing BLAST search via NCBI.
3. Results
3.1. Isolation of LAAO-Producing Strain
A total of 157 pure isolates were obtained from the inter-
tidal zone of Dinghai sea area located in Zhoushan, Zhe-
jiang province, China. Three domains including 32 acti-
nomycetes, 51 fungus and 74 bacteria were morphologi-
cally characterized. Out of 157 isolates subjected to screen-
ing process, one isolate (R3) showed the capability to
produce LA AO based on the below re sults.
LAAO is able to catalyze the stereospecific oxidative
deamination of L-amino acid to a-keto acid. As carbonyl
derivative, a-keto acid can be easily measured by sensi-
tive method using 2,4-dinitrophenylhydrazine (DNP) which
can react with carbonyl group to generate dinitro-ph e -
nylhydrazone with a brown-red color and characteristic
absorbance maxima at 520 nm. As shown in Figure 1(c),
R3 fermentation supernatant without L-amino acid sub-
strate showed flaxen and OD520 was very low. Similarly,
a mixture of R3 fermentation supernatant treated at 95˚C
for 5 minutes and substrate L-Phe (Figure 1(d)) also
showed flaxen with low OD520 value. In contrast, me-
dium with L-Phenylpyruvic acid (Figure 1(a)) and R3
fermentation supernatants with L-Phe (Figure 1(b)) both
had brown-red color with high OD520. All these results
indicate that R3 with L-amino acid substrate can generate
a-keto acid. Therefore, R3 can produce LAAO.
LAAO is able to catalyze L-amino acid to release
H2O2 which can be detected using Amplex Red Hydro-
gen Peroxide/Peroxidase Assay kit. Our results showed
that, for either R3 fermentation supernatant without sub-
strate L-Met or R3 fermentation supernatant treated at
95˚C for 10 minutes followed by addition of L-Met, no
H2O2 was detected. In contrast, huge amount of H2O2
(1.84 mmol/L) was detected in R3 fermentation superna-
tant with L-Met, indicating that R3 can use L-Met to re-
lease H2O2. Therefore, R3 can produce LAAO.
3.2. Morphological Characteristics
On MM media plate, R3 displayed a red color and round
form with smooth. Based on the Gram-staining, R3 was
identified as a gram-negative strain with the size of about
3.15 μm × 1.05 μm (15 × 100).
(a) (b) (c) (d)
Figure 1. Detection of a-keto acids. (a) Positive control: Phe-
nylpyruvi c acid with MM medium (OD520 0.202); (b) L-Phe
with R3 (OD520 0.145); (c) R3 without L-Phe (OD520 0.038);
(d) Treatment of B3 at 95˚C for 10 minutes followed by
adding of L-Phe (OD520 0.051).

Z. L. YU ET AL.
378
To determine its substrate specificity, 16 common L-
amino acids were selected as substrates for oxidation re-
action. It was found that almost 11 out of 16 substrates
can be effectively used by R3 to generate H2O2 based on
Amplex Red Hydrogen Peroxide/Peroxidase Assay kit as
shown in Figure 2. L-Leu gave the highest activity, fol-
lowed by L-Lys, L-Tys, L-Asn, L-Gln, L-Met, L-cystine,
L-Arg, L-Trp, β-Val and L-Glu. On the other hand, the
other substrates showed comparatively low activity. These
results indicate that LAAO of R3 has a broad substrate
specificity.
3.3. Sodium Chloride Tolerance and Dependence
of Temperature and pH
To assess NaCl tolerance of R3, different concentrations
of NaCl from 0% to 12% were added to MM medium.
Figure 3 showed that R3 needed NaCl for growth and
cannot survive in NaCl-free medium. In addition, it can
grow the best with 1.5% - 3% of NaCl. Further increase
of NaCl concentration inhib ited its growth. All these fin-
dings totally agree with the fact that R3 was isolated
from the sea area. As expected, both temperature (Figure
4) and pH (Figure 5) were very important factors for R3
growth. R3 can grow great at temperature between 15˚C
and 35˚C and the optimal temperature was 25˚C. With
the either decrease or increase of temperature, the growth
became worse. At either 5˚C or 45˚C, there was almost
no growth. Regarding pH, R3 can tolerate basic condi-
tion much better than acidic one. The best pH for R3
growth was 6 - 7. At pH lower than 4, there was almost
no growth.
3.4. Physiological and Biochemical Tests
In the physiological and biochemical tests (Table 1), R3
showed positive results (+) on starch hydrolysis test, in-
dole test, utilization citr ate test and oxidase test, and neg-
ative results (−) on lysine decarboxylation enzyme test,
Figure 2. Substrate specificity of R3 LAAO.
Figure 3. Effect of temperature on the R3 growth.
Figure 4. Effect of salt concentration on R3 growth.
Figure 5. Effect of pH on R3 growth.
half a solid agar test, gelatin hydrolysis test, H2S produc-
tion test, acetamide test, V-P test, methyl red test and me-
lezitose monohydrate test. It can use carbon sources in-
cluding galactose, glucose, lactose, fructose, maltose and
rhamnose and nitrogen sources including (NH4)2HPO4,
KNO3, arginine, methionine, glycine and tyrosine.
3.5. 16S rDNA Sequence Analysis
The partial 16S rDNA of R3 was amplified using univ er-
sal primers 27F and 1527R. After PCR amplification,
L-Leu
L-Lys
L-T yr
L-Asn
L-Gln L-Met
L-c ystine
L-Arg
L-Trp
L-Val
L-Glu L-Ser L-Pro
Beta-Ala
DL-Asp
L-Cys
0
20
40
60
80
100
Relative activity (%)
Substrates
05 10 15 20 25 30 35 40 45 50
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
OD（600nm）
Temperature（℃）
0.0 1.5 3.0 4.5 6.0 7.5 9.010.512.0
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
OD（600nm）
Concentration of sodium choride（％）
345678910 11 12 13
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
OD （ 600nm）
pH

Z. L. YU ET AL.
379
Table 1. Physiological and biochemical characteristics of the R3 isolate.
Ite ms R3 Carbon Sources R3 Nitrogen Sources R3
Lysine decarboxylation enzyme − Inositol − (NH4)2HPO4 +
Half a solid agar − Sorbitol − KNO3 +
Gelatin hydrolysis − Mannitol − Arginine +
Starch hydrolysis + Glucose + Methionine +
H2S production − Sucrose − Phenylalanine −
Indole test + Rhamnose + Glycine +
Methyl red test − Lactose + Tyrosine +
V-P test − Fructose +
Utilization citrate + Maltose +
Acetamide − Galactose +
Melezitose monohydrate −
Oxidase test +
around 1.4 Kb fragment was successfully obtained (data
not shown) and sequenced. Then the Blast search via
NCBI was carried out to evaluate the similarity and ho-
mology of R3 with other organisms. It was found that R3
had very high homology with Pseudoalteromonas spp.,
with the highest identity of 98.5% to Pseudoalteromonas
rubra. Therefore, we designated it as Pseudoalteromonas
sp. R3.
4. Discussion
LAAOs form a family of proteins with various enzymatic
properties, structure and biological function. Extensive
studies indicate that LAAOs have promising biotechno-
logical and medical applications. This enzyme is widely
distributed in nature including snake venoms, insect drugs,
sea hare, fungi, bacteria and algae. Unlike snake venom
LAAOs which have been widely and deeply investigated
to show broad bioactivities such as apoptosis, cytotoxic-
ity, edema, hemolysis, hemorrhage, platelet aggregation,
parasite-killing activity and antimicrobial activity, non-
snake venom LAAOs need to be further studied and their
functional role and application remain to be revealed.
Especially, very little is known about LAAOs from ma-
rine microorganism. In this study, we successfully iso-
lated an LAAO-producing marine bacterium from inter-
tidal zone of Dinghai sea area. Based on physiological
and biochemical tests together with molecular ana lysis, it
was designated as Pseudoalteromonas sp. R3. To arrive
at a better understanding and address the commercializa-
tion of LAAO from this isolate, future works are planned
to clone the gene coding for this Pseudoalteromonas sp.
R3 LAAO and to study its structure, biological and phy-
siological roles, relationship between function and struc-
ture, and mechanism of transcription in vivo.
5. Acknowledgements
This work was sup ported by Natural Science F oundation
of Zhejiang Province, China (Y5100153), Welfare Tech-
nology Applied Research Project of Zhejiang Province,
China (2011C23007), and Natural Science Foundation of
ZJUT (2010021 3) to Z.L. Yu.
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