Biocatalysis and Agricultural Biotechnology 6 (2016) 219–226

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Biocatalysis and Agricultural Biotechnology

journal homepage: www.elsevier.com/locate/bab

Characterization of extracellular agarase production by Acinetobacter

junii PS12B, isolated from marine sediments
T. Leema Roseline, N.M Sachindra n
Department of Meat and Marine Sciences, CSIR-Central Food Technological Research Institute, Mysore 570020, India

art ic l e i nf o a b s t r a c t

Article history: A marine, gram negative, rod shaped bacterium that degrades agar was isolated from the east coast of
Received 8 February 2016 India and was identified to be Acinetobacter junii PS12B based on 16S rRNA gene sequencing. The effect of
Received in revised form different culture conditions, namely pH of the medium, time and temperature of incubation and the agar
21 April 2016
concentration in the medium, on the agarase production by the strain, was evaluated. The agarase
Accepted 23 April 2016
Available online 25 April 2016
production was significantly (po0.05) affected by the culture conditions. The optimum conditions as
determined by response surface methodology were found to be a temperature of 35 °C, pH of 7.0 and
Keywords: time of fermentation of 33 h and agar concentration of 0.5%. Under the optimum conditions, the isolate
Agar produced 0.17 units of agarase per ml of the medium. Ammonium nitrate and sodium nitrate were the
Agarase
best nitrogen source in the medium for agarase production by the isolate. Supplementing the agar
Acinetobacter junii
containing minimal media with simple sugars like glucose and galactose was found to enhance agarase
Response surface methodology
production significantly by two fold. The isolate was also able to degrade carrageenan, which indicates its
role in utilization of marine polysaccharides for the production of bioactive oligosaccharides.
& 2016 Elsevier Ltd. All rights reserved.

1. Introduction for agarases and expression of these genes by molecular genetic

techniques.
Most of the marine microbes have evolved degradation en- Agarase producing bacteria has been isolated from different
zymes to use agar or agarose as an energy and carbon source environments. Most of the agarase producing isolates were found
(Pluvinage et al., 2013) Marine bacteria secrete a specific glycoside to be of marine origin. However, Feng et al. (2012) isolated an
hydrolases (GH) enzyme agarase to utilize algal cell wall poly- agarolytic Rhodococcus sp. from printing and dyeing wastewater.
saccharides as a carbon and energy source. Agarases are classified Agarolytic bacteria have also been isolated from terrestrial soil
based on the mode of action, into α-agarase (EC 3.2.1.158), which (Suzuki et al., 2003; Hosoda et al., 2006; Lakshmikanth et al.,
cleaves α-1, 3 linkages to produce agaro-oligosaccharides and β- 2006b). In recent years, several novel agarase producing strains
agarase (EC 3.2.1.81) which cleaves β-1, 4 linkages to produce were isolated from marine environments. Agarolytic Aliagarivorans
neoagaro-oligosaccharides. β-Porphyranases hydrolyze the β-(1, 4) marinus and Aliagarivorans taiwanensis (Jean et al., 2009), Simiduia
glycosidic bonds of the porphyran moieties (G-L6S) in agar and agarivorans (Shieh et al., 2008) were isolated from seawater.
produces oligosaccharides with reducing ends at G residue (He- Flammeovirga sp. MY04 (Han et al., 2012), Agarivorans sp. (Hu
hemann et al., 2010). Based on the amino acid sequence similarity, et al., 2009) isolated from marine sediments were found to pro-
duce β–agarase. Seaweeds were found to be one of the primary
β-agarases are found in four distinct glycoside hydrolases (GH)
sources of agarolytic bacteria in the marine environment (Lee
families in the CAZy database includes GH16, GH50, GH86, and
et al., 2013; Oh et al., 2010). Agarolytic bacteria have also been
GH118, whereas α-agarase belong to GH96. To date, a vast number
isolated from the gut of mollusks (Fu et al., 2008; Jung et al., 2012).
of agarolytic micro-organisms from taxonomically diverse genera,
Applications of agarases are well-known in the areas of food,
as well as agarases and their encoding genes have been well re-
pharmaceuticals, cosmeceuticals, and biotechnology. The neoa-
ported and summarized (Chi et al., 2012; Fu et al., 2010; Michel
garooligosaccharides have been considered to have high economic
et al., 2006). The approaches to increase the productivity of
value, because of their physiological and biological activity with-
agarases by agarolytic organisms would be to isolate hyper-pro-
out toxicity, as GRAS. These oligosaccharides especially neoagar-
ducers or mutants for agarases or by cloning the genes encoding otetraose and neoagarohexaose exhibit antioxidative activity,
scavenging hydroxyl free radicals and superoxide anion radicals
n
Corresponding author. and inhibiting lipid peroxidation (Wang et al., 2004; Wu et al.,
E-mail address: [email protected] (N. Sachindra). 2005). Additionally, neoagarooligosaccharides acts as a low-calorie

http://dx.doi.org/10.1016/j.bcab.2016.04.007
1878-8181/& 2016 Elsevier Ltd. All rights reserved.
220 T. Leema Roseline, N.M Sachindra / Biocatalysis and Agricultural Biotechnology 6 (2016) 219–226

additive to improve food quality, inhibit the growth of bacteria, defined as the amount of enzyme that released 1 mmol of galactose
and slow down the degradation of starch (Giordano et al., 2006). equivalents from the substrate per minute under the specified
They also have a moisturizing effect on skin and a whitening im- assay condition. The specific activity was expressed in one mmol
pact on melanoma cells (Kobayashi et al., 1997; Ohta et al., 2004). galactose equivalents/min/mg protein. Protein concentration was
Because of these functional implications, agar-derived oligo- determined by Lowry's method using bovine serum (BSA) as the
saccharides have broad applications in the health-food, pharma- standard (Lowry et al., 1951). Galactose was used as a reference for
ceutical, and cosmetic industries potentially. preparing the standard curve.
Single-factor optimization can be eliminated by using response
surface methodology (RSM) which is used to explain the combined 2.3. Random amplification of polymorphic DNA (RAPD) analysis of
effects of all the factors in a biotechnological process (Anil kumar agarolytic isolates
and Suresh, 2014). Therefore, the use of RSM in biotechnological
processes is gaining much importance for the optimization of In order to determine the similarity between the strains, Ran-
enzyme production (Beg et al., 2003; Kumar and Satyanarayana, dom Amplification of Polymorphic DNA (RAPD) was performed
2004; Rao et al., 2006). Some statistical approaches have been using a universal M13 primer (5′-GAGGGTGGCGGTTCT-3′) for mi-
employed for agarase production by Agarivorans albus YKW-34 (Fu crobial typing (Schillinger et al., 2003). The amplified product was
et al., 2009), Pseudoalteromonas sp. JYBCL1 (Jung et al., 2012), and run on 1.8% agarose gel. The RAPD banding pattern was further
Streptomyces lividans (Park et al., 2014). analyzed using GeneSyss software (SYNGENE, UK) and the simi-
In the present work, a marine bacterial isolate identified as larity of the band profiles and the grouping of the RAPD-PCR
Acinetobacter junii PS12B was evaluated for agarase production patterns were calculated based on the Pearson's coefficient and
under different culture conditions. The strain was characterized by agglomerative clustering with unweighted pairs group matching
investigating the growth and production of agarase in the pre- algorithm (UPGMA), and the dendrogram was constructed using
sence of various parameters to exploit the organism for production GeneSyss software.
of agarase. Further, the combined effect of different culture con-
ditions was evaluated using RSM for optimization of enzyme 2.4. Biochemical and molecular characterization of the selected
production. agarolytic isolate

The strain selected based on screening for the agarolytic ac-

2. Materials and methods tivity was characterized by performing various biochemical tests
such as gram staining, motility test, oxidase activity, catalase
2.1. Sampling, isolation, screening of agarolytic marine bacteria production, Methyl red-Voges Proskauer (MR-VP), oxidation and
fermentation of sugars. Utilization of glucose, mannitol, inositol,
Marine samples such as seaweeds, seawater, and sediment sucrose and 31 other sugars was assessed using Hi-carbo kit (Hi-
collected from the east coast of India (Rameshwaram, Mandapam Media Laboratories, India). Hydrolysis of esculin, gelatin and starch
and Tuticorin) were suspended in the sterilized minimal mineral were also performed using standard techniques. Growth in mi-
salts medium (MMS) (Lakshmikanth et al., 2006b) containing (g/L) neral salts media at different temperatures of 10 °C, 25 °C, 37 °C
K2HPO4 (1.2), KH2PO4 (0.3), MgSO4 (0.1), FeCl3 (0.1), NH4NO3 (1.0), and 50 °C, at different pH of 4.0, 7.0 and 10.0 were performed to
CaCl2 (0.1) and agar (1.0) (pH 7.0). After incubation at 37 °C for characterize the growth of organisms. Hemolytic activity of the
72 h, a loop full of the media was streaked on MMS media plates cultures was tested using blood agar. Morphological analysis of the
containing 1.5% agar. Following incubation, the colonies exhibiting culture isolate was carried out by scanning electron microscopy
an obvious clear zone around the colony or pit formation, in- (SEM) according to the method of McDougall et al. (1994). Briefly,
dicative of agar degradation were selected as agarolytic bacteria selected strain grown in Tryptone soya broth were centrifuged,
and purified by repeated streaking. Further confirmation of agar- washed thrice with phosphate buffer saline (pH 7.0) to remove
olytic activity was carried out by spot inoculation of purified cul- salts, fixed with glutaraldehyde (2%) and subjected to gradual al-
ture on MS agar plates and overlaying with Lugol's iodine after coholic dehydration. The processed samples were then analyzed
incubation for 24 h at 37 °C. The clear zone around the colony by SEM (Leo-435 VP, Leo Electron Microscope, Zeiss Ltd., and
indicates the agarolytic activity. Cambridge, UK).
For molecular characterization of the agarolytic isolates, geno-
2.2. Agarase activity measurement mic DNA was extracted from the bacterial strain using the stan-
dard DNA isolation protocol (Sambrook and Russell, 2001). For 16S
The isolated and purified cultures were inoculated to the en- rRNA gene sequencing and phylogeny analysis, 16S rRNA gene was
zyme production medium (Lakshmikanth et al., 2006b) containing amplified using the universal bacterial forward and reverse pri-
(g/L) K2HPO4 (0.38), MgSO4 (0.20), FeCl3 (0.05), NH4NO3 (1.0) mers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (51-
supplemented with 0.3% agar as a sole source of carbon. The pH GGTTACCTTGTTACGACTT-31) respectively. Each PCR mixture of
was adjusted to 7.0 before sterilization at 121 °C for 15 min. The 25 μl contained template DNA (20 ng/μl), 0.2 μM of each primer,
culture was incubated at 37 °C on the orbital shaker at 180 rpm. 0.25 mM of each deoxynucleoside triphosphate (dNTP), and 2.5 U
After 24 h of incubation, the production media was centrifuged at of Taq DNA polymerase in a final concentration of 10 mM Tris–HCl,
8000 rpm for 15 min at 4 °C and the supernatant collected was 50 mM KCl, and 1.5 mM MgCl2. PCR was performed under the
taken as a crude extracellular enzyme. following cycle conditions: an initial denaturation step at 94 °C for
Agarase activity was measured by the release of reducing su- 5 min and 35 cycles of denaturation at 94 °C for 30 s, annealing at
gars according to the 3, 5-dinitro salicylic acid (DNS) method 55 °C for 2 min, and extension at 72 °C for 2 mins, with a final
(Miller, 1959). Briefly, 1 ml of the crude enzyme solution was extension step at 72 °C for 10 min (Primus 25 Thermal cycler). The
mixed with 1 ml of substrate (0.25% agar in 20 mM Tris-Cl buffer, PCR product of was purified and sequenced at Amnion Biosciences,
pH 7.5), vortexed and incubated for 60 min at 37 °C. After in- Bangalore, India. The 16S rRNA gene sequences obtained was
cubation, 1 ml of DNS solution was added to the mixture and he- subjected to nBLAST for similarity identification. Multiple align-
ated in boiling water bath for 10 mins, cooled, and absorbance was ment and sequence similarity with that of available sequences of
measured at 540 nm (Hitachi U 2900). One unit (U) of activity was reference strains from GenBank database were analyzed using
 T. Leema Roseline, N.M Sachindra / Biocatalysis and Agricultural Biotechnology 6 (2016) 219–226 221

CLUSTAL X. The phylogenetic tree was constructed using the MEGA 2.7. Effect of carbon and nitrogen sources on production of agarase
6 program (Biodesign Institute, Tempe, AZ, USA) by the method of
neighbour-joining (Kumar et al., 2008). Different inorganic (ammonium chloride, ammonium nitrate,
sodium nitrate) and organic (yeast extract, peptone, casein hy-
drolysate) nitrogen sources were tested at a concentration of 0.1%
2.5. Effects of culture conditions on agarase production by selected
in the minimal salt medium. Different carbon sources were tested
isolate
by supplementing the MS medium containing ammonium nitrate
(0.1%) and agar (0.3%) with monosaccharide's (glucose, fructose
The effect of incubation time (24, 48, 72, 96 and 120 h), tem-
and galactose), disaccharides (ribose, raffinose, sucrose) and cel-
perature (25, 30, 37, and 50 °C), pH (4 to 10) and the initial con-
lulose at 0.1% level. Optimized culture conditions were employed
centration of agar in the fermentation medium (0.1–0.3% w/v) was
during the study.
evaluated by culturing the selected organism under specific con-
ditions, by varying one parameter and keeping the other condi-
2.8. Statistical analysis
tions constant (24 h, 37 °C, pH 7.0 and 0.3% agar). The growth of
the organism was determined by centrifuging 2 ml of culture
Statistical analysis was performed for each experiment by
suspension, suspending the cell pellets in 2 ml physiological saline
analysis of variance (ANOVA) techniques using the statistical
and measuring the absorbance of the suspension at 600 nm. To software STATISTICA (StatSoft, 1999). All experiments were con-
check multiple polysaccharide degrading activity of the selected ducted in triplicate, and the mean values are reported.
strain, the isolate was inoculated into MS medium containing Κ-
carrageenan, ι-carrageenan, λ-carrageenan, chitosan, alginate, and
agar. After 24 h of incubation, the enzyme activity of the culture 3. Results
supernatant was assayed using the same polysaccharide as the
substrate. 3.1. Screening, isolation and selection of agarolytic bacteria

2.6. Evaluation of combined effect of culture conditions on agarase From the marine samples, 65 bacterial strains were isolated,
production and six of the isolates had shown good agarolytic activity as de-
monstrated by zones of clearing around the colony after flooding
The combined effect of culture conditions was evaluated using with iodine, and were selected for further studies (Fig. 1(a)). The
three-level factorial design. The independent factors considered geographic details of selected isolates is given as supplementary
for optimization included temperature (X1; °C), time (X2; h), pH data. RAPD fingerprints of the potential agarolytic isolates are
(X3) and agar concentration (X4; % w/v), while the enzyme activity shown in Fig. 1(b) with distinct banding patterns. The number of
was the dependent factor. The detail of factors and the experi- bands varied between 3 and 9, with fragment size ranging be-
mental run design is presented in Table 1. The data was analyzed tween 250 and 2370 bp. According to dendrogram and cluster
for determining the response profile of different factors, and the analysis, isolates were grouped into two major, and each group
response surface plots were drawn using the software STATISTICA was subdivided into two sub-groups. The selected cultures (PS12B,
(StatSoft, 1999). PS18, AS, SAR, CS15M, MBW, SW17 groups) with banding patterns
as observed by RAPD profiles proves that the agarolytic cultures
Table 1
are diverse (Fig. 1(b)). Six selected isolates were screened for
Experimental design for studying the combined effect of different variables on agarase activity by submerged fermentation studies. The agarase
agarase production. production significantly (p o0.05) differed between isolates, with
isolate PS12B showing highest specific activity of 1.72 70.23 fol-
Run Temperature X1 Incubation time pH X3 Agar concentration
lowed by the isolate SAR 1.52 70.13, while the isolate SW17
number (°C) X2 (h) (% w/v) X4
(0.45 70.07) showed the lowest activity (Fig. 1(c)). Hence, isolate
1 20 6 4 0.1 PS12B was selected for further study.
2 20 6 7 0.5
3 20 6 10 0.3 3.2. Biochemical, physiological profile and phylogenic analysis of the
4 20 24 4 0.5
5 20 24 7 0.3
potential agarolytic strain PS12B
6 20 24 10 0.1
7 20 42 4 0.3 As the isolate PS12B showed highest agarase activity, the iso-
8 20 42 7 0.1 late was further characterized. Colonies produced on minimal
9 20 42 10 0.5
mineral salts medium at 37 °C for 2–4 days were circular, off-
10 35 6 4 0.5
11 35 6 7 0.3 white, opaque and rod-shaped. The isolate was a gram-negative,
12 35 6 10 0.1 catalase-positive and oxidase-negative. The isolate was able to
13 35 24 4 0.3 grow at temperatures between 25 and 40 °C, at a pH range of 5–10.
14 35 24 7 0.1 The isolate could not hydrolyze esculin, gelatin, and cellulose and
15 35 24 10 0.5
16 35 42 4 0.1
was non-hemolytic. Additionally, PS12B strain was able to utilize
17 35 42 7 0.5 the sugars, lactose, xylose. maltose, fructose, dextrose, galactose,
18 35 42 10 0.3 raffinose, trehalose, melibiose, sucrose, L-arabinose, mannose, in-
19 50 6 4 0.3 ulin, sodium gluconate, salicin, glucosamine, dulcitol, inositol,
20 50 6 7 0.1
sorbitol, mannitol, adonitol and unable to utilize the sugars α-
21 50 6 10 0.5
22 50 24 4 0.1 methyl-D-glucoside, rhamnose, cellobiose, melezitose, α-methyl-
23 50 24 7 0.5 D-mannoside, xylitol, ONPG, esculin, D-arabinose, citrate, mal-
24 50 24 10 0.3 onate, sorbose. Detailed physiological and biochemical character-
25 50 42 4 0.5 ization are provided as supplementary data. Phylogenetic analysis
26 50 42 7 0.3
27 50 42 10 0.1
(Fig. 2) based on 16S rRNA gene sequences revealed that agarolytic
strain PS12B closely matches with Acinetobacter junii., with 99%
222 T. Leema Roseline, N.M Sachindra / Biocatalysis and Agricultural Biotechnology 6 (2016) 219–226

Fig. 1. Screening of agarolytic isolates. a) Detection of agarolytic activity on agar plate, b) Dendogram drawn using RAPD profile of bacterial isolates, c) Agarolytic activity of
organism isolated from marine environment (mean 7 SD, n ¼ 3).

Fig. 2. Phylogenetic tree of Acinetobacter sp. PS12B associated with other members of the genus Acinetobacter using 16S rRNA sequence.

identity. 7.0, indicating that higher growth at pH 7.0 resulted in higher

enzyme production.
3.3. Effects of culture conditions on agarase production It is well known that temperature is one of the most critical
parameters that have to be controlled in the bioprocess. The effect
The optimal conditions for agarase production with respect to of temperature on the production of agarase by Acinetobacter junii
initial pH of the medium, temperature, concentration of agar and PS12B was clearly evident as the ideal temperature for increased
incubation time by the selected isolate Acinetobacter junii. PS12B agarase production was between 30 and 37 °C (Fig. 3(b)). The
were investigated using one-factor-at-a-time experiments. The pH enzyme production was highest at 30 °C (0.17 þ0.001 U/ml) fol-
of the growth medium had a significant (p o0.05) effect on en- lowed by at 37 °C (0.147 0.01 U/ml). The enzyme production was
zyme production, and significantly (p o0.05) higher enzyme pro- significantly (p o0.05) reduced when the growth temperature was
duction (0.16 þ0.007 U/ml) was observed at pH 7.0 compared to 25° or 50 °C. However, the growth was marginally higher at 37 °C
other pH (Fig. 3(a)). Growth of the organism was also highest pH compared to 30 °C, but was lowest at 25° and 50 °C. The
 T. Leema Roseline, N.M Sachindra / Biocatalysis and Agricultural Biotechnology 6 (2016) 219–226 223

Fig. 3. Effect of a) pH, b) temperature, c) the time course and d) concentration of agar on the growth and production of agarase enzyme (mean 7 SD, n ¼3).

incubation period was found to affect the enzyme production, as

increased fermentation period above 24 hrs resulted in significant
(p o0.05) reduction enzyme production even though the growth
increased after 24 h (Fig. 3(c)). The agarase activity was highest at
0.177 0.005 U/ml after 24 h of the fermentation that reduced
when incubated further. The decrease in enzyme production at
longer incubation periods even with increased growth may prob-
ably be due to the degradation of the enzyme by proteases, as the
isolate was also found to be proteolytic (Data not shown).
The initial concentration of agar plays a crucial role in the
production of agarase as the strain utilizes only agar as the sole
energy source, and agarase enzyme was induced by agar in the
fermentation medium. It is evident that the increase in agar con- Fig. 4. Multiple polysaccharide degrading activity of Acinetobacter junii. PS12B
(mean7 SD, n ¼3).
centration significantly (p o0.05) increased the agarase activity
reaching higher activity of 0.13 þ0.002 U/ml at 0.3% agar con-
centration tested and increased production coincided with in- 3.4. Combined effect of growth conditions on agarase production
creased growth (Fig. 3(d)). However, as the culture medium be-
comes more viscous when the concentration of agar increased, it One factor at a time experiment indicated that maximum
would be difficult to recover the crude enzyme. agarase production was affected by pH of the medium, time and
The activity of the isolate Acinetobacter junii PS12B to break temperature of incubation and agar concentration of the medium.
down other marine polysaccharides was evaluated by growing the Hence, this study was carried out to evaluate the combined effect
organism in minimal medium containing different polysaccharides of these variables on agarase production by Acinetobacter junii
and enzyme activity was determined as mentioned Section 2. The PS12B using response surface methodology to arrive at the opti-
isolate showed significantly (p o0.05) higher specific activity mized condition for maximum agarase production. Among the
(1.90 þ0.10 U/mg) with respect to agarase (Fig. 4). However, the variables screened, pH of the medium was identified as the most
isolate also exhibited carrageenase activity as it could break down significant (p r0.05) variables affecting the production of agarase.
all the three types (kappa, iota, and lambda) of carrageenan. The The interaction of four variables was determined by constructing
activity was lowest with respect to alginate lyase and chitosanase three-dimensional surface plots (Fig. 5(a)–(d) which compare the
activity. The organism also could degrade the agar containing red effect of two variables on agarase production, keeping the other
seaweed as it showed enzyme activity of 0.37þ 0.12 U/mg when two variables at the center of the level. In Fig. 5(a), agarase pro-
red seaweed was used as carbon source in the growth medium duction was plotted against concentration of agar and pH, which
and as the substrate in the enzyme assay. indicated that enzyme production increased with increase in pH
224 T. Leema Roseline, N.M Sachindra / Biocatalysis and Agricultural Biotechnology 6 (2016) 219–226

Fig. 5. Response surfaces plots of effect of a) agar concentration and pH, b) temperature and time, c) pH and time and d) time and agar concentration on agarase production
(other variables kept at the center level).

upto 7.0 and then reduced, while increase in agar concentration obtained when ammonium nitrate (0.14 þ0.006 U/ml) or sodium
resulted in a marginal increase in enzyme production. The rise in nitrate (0.13 þ0.006 U/ml) was used as nitrogen source in the
temperature also increases enzyme production, but at high-tem- medium (Fig. 6(a)). Substitution of ammonium nitrate with other
perature agarase production reduces. Time also showed a minimal nitrogen sources (yeast extract peptone or casein) decreased the
effect on agarase production. The optimum levels of the four in- agarase production significantly (p o0.05), indicating that Acine-
dependent variables determined from the response profile (pro- tobacter junii PS12 B is not able to efficiently utilize complex ni-
vided as supplementary data) for maximum agarase production of trogen source in the medium for agarase production.
0.17 units, was found to be 0.5% (w/v) of agar, pH of 7.0 and in- Several reports have indicated that different carbon sources
cubation at 35 °C for 33 h. Optimized conditions were found to have different influences on extracellular enzyme production by
very close to the conditions obtained during one factor at a time various strains. Therefore, the effect of agar and agar with different
experiment. sugars like glucose, galactose, sucrose, agarose, ribose, raffinose
and cellulose on agarase production by Acinetobacter junii PS12B
3.5. Influence of nitrogen and carbon sources on agarase production was examined. Combinations of agar with monosaccharide's,
glucose, and galactose, significantly p o0.05) increased agarase
As optimization experiment did not yield higher enzyme pro- production by more than 2 fold (Fig. 6(b)). Cellulose had an ad-
duction, in order to increase the enzyme production, the effect of verse effect on agarase production.
addition of different carbon sources and nitrogen sources to the
minimal medium under optimal conditions on enzyme production 4. Discussion
was evaluated. The evaluation of effect of substituting ammonium
nitrate with other nitrogen sources on agarase production in- Numerous species of bacterial genera have been reported to be
dicated that the significantly higher agarase production was producers of agarase. Most of the reports on agarolytic bacteria are
 T. Leema Roseline, N.M Sachindra / Biocatalysis and Agricultural Biotechnology 6 (2016) 219–226 225

Pseudomonas aeruginosa, maximal agarase production was ob-

served at pH 8.0 even though growth could occur at a pH range of
5–11 (Lakshmikanth et al., 2006a). Bacillus megaterium isolated
from marine environment exhibited higher agarase production at
pH 6.6 (Khambhaty et al., 2008).
Pseudomonas aeruginosa (Lakshmikanth et al., 2006a) and
Rhodococcus sp. (Feng et al., 2012) were found to have maximum
agarase production at a growth temperature of 30 °C. The optimal
temperature for production of agarase by marine microorganisms
was reported to be in the range of 20–40 °C (Lakshmikanth et al.,
2006a, 2006b; Fu et al., 2009; Khambhaty et al., 2008; Van der
Meulen and Harder, 1975), except for thermophilic organisms such
as Halococcus sp. 197A with an optimum temperature of 70 °C
(Minegishi et al., 2013). Acinetobacter sp. isolated from terrestrial
soil also produced agarase within a short period of time of 16–18 h
(Lakshmikanth et al., 2006b). Similar results were obtained by
strain Agarivorans sp. HZ 105 (Hu et al., 2009) where an increase in
agar concentration from 0.1–0.25% induced higher production of
agarase. In contrast Khambhaty et al. (2008) observed that low
agar concentration of 0.03% is sufficient for induction of agarase
production. According to Jonnadula and Ghadi (2011), Micro-
bulbifer sp. CMC-5 could degrade multiple marine polysaccharides.
However, Acinetobacter sp., AG LSL-1 isolated from terrestrial soil
Fig. 6. Effects of different nitrogen sources (a) and carbon sources (b) on agarase was not able to utilize other polysaccharides (Lakshmikanth et al.,
production by Acinetobacter junii PS12b (mean7 SD, n ¼3). 2006b). Similarly, agarolytic marine isolate Vibrio sp. Strain JT0107
was also not able to degrade other polysaccharides (Sugano et al.,
from those isolated from the marine environment (Chi et al., 2012; 1993). Lakshmikanth et al. (2006b) reported that agarase secreted
Fu et al., 2010). Several marine bacteria are known to be agarolytic, by the Acinetobacter strain was inducible by agar and are not re-
examples being Pseudoalteromonas sp. (Jung et al., 2012), Bacillus pressed by other simple sugars when supplemented along with
cereus ASK202 (Kim et al., 1999), Janthinobacterium sp. SY12 (Shi agar in the medium.
et al., 2008), Micrococcus sp. GNUM-08124 (Choi et al., 2011), Ca- Currently, there is an immense interest in the scientific com-
tenovulum sp. X3 (Xie et al., 2013) Agarivorans sp. LQ48 (Long et al., munity around the world in exploiting novel micro-organisms.
2010), Bacillus megaterium (Khambhaty et al., 2008). Marine microorganisms, with their distinctive nature, differ much
Based on phylogenetic analysis, the strain PS12B found to be in many aspects from their terrestrial counterparts and are well-
Acinetobacter junii. Acinetobacter species are widely distributed in known to produce diverse spectra of novel bioactive substances. In
nature and the hospital environment. Tsai et al. (2012) showed the present study a potential agarolytic strain identified as Acine-
that A. junii is a bacterium of low virulence and low mortality tobacter junii PS12b. was isolated and evaluated for agarase pro-
rates. However, only certain Acinetobacter species are pathogenic duction under different conditions. The enzyme production was
to humans, causing disease mainly in neonates and im- influenced by the pH of the growth medium, time, and tempera-
munocompromised individuals. The genus Acinetobacter com- ture of incubation and agar concentration in the medium. Under
prises of more than 23 known species and 11 unknown species, optimized conditions of pH (7.0), incubation time (33 h), tem-
which are commonly gram-negative, rod-shaped, non-pigmented, perature (35 °C) and agar concentration (0.5%) the isolate pro-
heterotropic, strictly aerobic, polar-flagellated bacteria. There are duced 0.17 units of agarase per ml of the growth medium. Am-
several species of Acinetobacter from marine environment are monium nitrate or sodium nitrate was found to the best nitrogen
known to degrade oil, edible and mineral oils (Tanaka et al., 2010; source for enzyme production. Supplementation of minimal
Luo et al., 2013), and produce enzymes (Fu et al., 2014). Aciento- medium containing agar with simple sugars enhanced the enzyme
bacter junii PS12B isolated in the study was found to be a good production by 2 folds. The isolate was also exhibited activity to-
candidate for agarase production. Comparing with Acinetobacter wards degradation of other marine polysaccharides like carra-
sp. reported earlier for agarase isolated from soil environment, the
geenan, indicating its potential for exploitation of marine poly-
bacterium from the present study could thrive under different
saccharides for the production of the bioactive oligosaccharides.
conditions as the organism was isolated from marine environment
and could degrade marine polysaccharide such as carrageenan.
Only one species of Acinetobacter has been reported as agarase
Acknowledgements
producer from the terrestrial environment (Lakshmikanth et al.,
2006a). This is the first report on Acinetobacter sp. from marine
The authors wish to thank the Director, CSIR-CFTRI for en-
environment producing agarase enzyme.
couragement and for the facilities provided. First author thanks,
Culture conditions are known to influence the enzyme pro-
UGC, Govt. of India (Grant No. 1269/NET-June 2011) for the sup-
duction by microorganisms. According to Hu et al. (2009) the
port in the form of fellowship.
Agarivorans sp. HZ105 isolated from marine sediment could grow
at a wide pH range of 6–11. The isolate in this study, Acinetobacter
junii PS12B could grow at wide pH range from 5 to 9, indicating
that the strain is alkali tolerant in nature and was found to pro- Appendix A. Supporting information
duce higher amount of agarase at pH 7.0. Lakshmikanth et al.
(2006b) reported that Acinetobacter sp. isolated from terrestrial Supplementary data associated with this article can be found in
soil also produces a higher amount of agarase at pH 7.0. In case the online version at http://dx.doi.org/10.1016/j.bcab.2016.04.007.
226 T. Leema Roseline, N.M Sachindra / Biocatalysis and Agricultural Biotechnology 6 (2016) 219–226

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