a study of enz variations in algae

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International Journal of Biotechnology and Biochemistry

ISSN 0973-2691 Volume 6 Number 6 (2010) pp. 889899

Research India Publications

http://www.ripublication.com/ijbb.htm

Enzymatic Variations Among Different Species of

Marine Macroalgae from Okha Port,

Gulf of Kutch, India

J.I. Nirmal Kumar1, Sudeshna Chakraborty

2,Rita N. Kumar

3,

Manmeet Kaur Amb4

and Anubhuti Bora5

1Head, Professor,

2Lecturer,

3Head and

4,5Research Scholar

1,4,5P .G. Department of Environmental Science and Technology,

Institute of Science & Technology for Advanced Studies & Research (ISTAR),

Vallabh Vidyanagar 388 120, Gujarat, India2,3

Department of Biological and Environmental Sciences ,

N.V. Patel College of Pure & Applied Sciences, Vallabh Vidyanagar 388 120,

Gujarat, India1E-mail- [email protected],

2E-mail- [email protected],

3E-mail- [email protected],

4 E-mail- [email protected]

5E-mail- [email protected]

Abstract

The objective of the present investigation was to find enzymatic variation

between species of same classes of maroalgae. For the investigation hydrolytic

enzymes such as amylase and protease along with some respiratory enzymes

such as peroxidase, polyphenoloxidase, succinate dehydrogenase and nitrate

reductase were studied quantitatively in eighteen marine macroalgae

belonging to the three classes of marine algae i.e. Chlorophyceae,

Phaeophyceae and Rhodophyceae, collected from Okha coast, Gulf of Kutch,Western India during second week of January, 2009. During the present work

it was recorded that the hydrolytic enzymes such as amylase and protease

showed a range of 202 -74 mg maltose. g-1

FW hr-1

and 8.5 - 5.5 g. tyrosine

g FW-1

hr-1

respectively. While the respiratory enzymes such as peroxidase,

polyphenoloxidase, succinate dehydrogenase and nitrate reductase showed a

range of 13.2 - 4.0 units.mg-1

FW, 0.23-0.74 units.mg-1

FW, 0.001-0.015

units.mg-1

FW and 75-243 mol NO2. g-1

FW respectively which can be

correlated among different species .

Keywords: Marine macroalgae, Environmental factors, Amylase, Protease,

Peroxidase, Polyphenoloxidase, Succinate dehydrogenase, Nitrate reductase


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890 Nirmal Kumar J.I. et al

IntroductionEstuaries and adjacent coastal areas are very different in terms of water circulation

patterns, morphology, anthropogenic pressures etc. These water bodies are subjected

to several environmental factors which directly or indirectly affect the life forms

present in them. In coastal areas salinity, dissolved oxygen, pH, turbidity, nutrients

and chlorophyll are usually the key parameters responsible for the maintenance of

adequate conditions for reproduction, growth and survival of species. The physiology

of the biotic community in the coastal regions is greatly affected by the change in the

environmental conditions (UNEP, 2004). In marine ecosystems, macroalgae are

ecologically and biologically important which provide medicinal constituents,

nutrition, reproduction and an accommodating environment for other living organisms

(McClanahan et al. 2002). The aquatic organisms are very sensitive to changes in the

quality of water and pollutants. Marine organisms are susceptible to a variety ofdynamic environmental stresses that influence survivorship and distribution (Ross &

Alstyne, 2007). Thus, they provide important information about the environmental

conditions in which they survive. The macroalgae present in the marine ecosystem are

used as an indicative species for the marine system.

With respect to the present context the biochemical status of eighteen marine from

different species were studied and the results suggested that the algae which are

abundantly available in this ecosystem also have considerable potential of

carbohydrates, amino acids, proteins, phenols and lipids for their use as food and in

pharmaceutical industry as a source in preparation of nutrient supplements, medicine

and fine chemicals (Kumar et al, 2009 a).Also study on different pigments such as

Chlorophyll, Carotenoid and phycoerythrin content revealed that marine macroalgalspecies and their concentrations vary with different divisions (Kumar et al, 2009 b).

For the present study different hydrolytic and respiratory enzymes were taken into

consideration as the enzymes are a group of compounds which participate in specific

reaction of vital metabolic pathways and are greatly affected by any change in

environmental conditions and different groups. The hydrolytic enzymes such as

Protease (EC 3.4.21.92), Amylase (EC 3.21.1) and respiratory enzymes such as

Peroxidase (EC 1.11.1.7), Polyphenoloxidase (EC 1.14.18.1), Succinate

dehydrogenase (EC 1.3.5.1) and nitrate reductase (1.7.1.1) were studied in eighteen

different macroalge belonging to three different species such as Chlorophyceae,

Phaeophyceae and Rhodophyceae.

The present investigation is an attempt to study the biochemical changesassociated with different algal species in the marine ecosystem. Moreover the work

also emphasizes on the correlation between different algal species belonging to

different groups due to the presence of hydrolytic and respiratory enzymes. The

present result will also be helpful in finding phylogenetic relation between different

species.

Materials and MethodsFor the present study 18 different marine macroalgal species belonging to three

different divisions viz, Chlorophyceae, Phaeophyceae and Rhodophyceae were


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Enzymatic Variations Among Different Species of Marine Macroalgae 891

collected from the coast of Okha, Jamnagar (lat. 2228_N and long. 6905_E) Gulf of

Kutch, India during second week of January, 2009. The algal samples were collected

from different coastal areas. The algal samples belonging to Chlorophyceae are

collected from intertidal zones, Phaeophyceae from subtidal zones and Rhodophyceae

from intertidal to subtidal zones. These samples were brought to laboratory in ice

box and washed twice with distilled water and used for the quantification of different

enzyme activities such as Protease (EC 3.4.21.92), Amylase (EC 3.21.1), Peroxidase

(EC 1.11.1.7), Polyphenoloxidase (EC 1.14.18.1), Succinate dehydrogenase (EC

1.3.5.1) and nitrate reductase (1.7.1.1). For consideration an average reading of the

triplicates of each set was considered.

Sample preparation:- One gram of algal samples were weighed and homogenized in

0.1 M sodium phosphate buffer (pH 7.0) by using a pre-chilled pestle and mortar. Thehomogenate was centrifuged at 10,000 g for 20 minutes and the supernatant was used

as enzyme source for the assay of proteases, peroxidase, succinate dehydrogenase and

polyphenoloxidase activities which were measured by the standard methodologies.

Protease activity (Mukherjee & Dasgupta, 1977):- The reaction mixture comprised of

2ml buffer, 1ml casein and 1ml enzyme extract. The tubes were incubated at 37oC +

10oC for one hour. The activities were terminated by the addition of TCA at zero

time. All the tubes were centrifuged and the clear supernatant was used for the

estimation of free amino acids. To 1ml of supernatant, 3ml of 0.5M sodium hydroxide

was added, thoroughly mixed and 1ml of Folin and Ciocalteaus Phenol reagent was

added after five minutes. The tubes were allowed to develop color for 30 minutes andabsorbance was measured at 660nm. The enzyme activity was calculated using

Tyrosine as standard and values were expressed in g tyrosine liberated/g. fresh

weight/hour.

Peroxidase activity (Reuveni et al., 1992):- POD activity was determined

spectrophotometrically based on the oxidation of guaiacol in the presence of H2O2.

The assay mixture contained 0.1M potassium phosphate buffer (pH 7.5), 4mM

guaiacol as donor, 3mM H2O2 as substrate, and 0.1ml crude enzyme extract. The total

reaction was placed in quartz cuvette and the optical density was recorded at 30

seconds intervals for 3 min at 420nm. The level of enzyme activity was determined by

measuring the difference in optical density.

Polyphenoloxidase activity (Meena et al., 2001):- 200 l of extract was added with1.5ml of 0.2M sodium phosphate buffer (pH 7.4) in the cuvette as blank. Later on

200l of 0.01M catechol was added to start the reaction. As soon as the reaction was

started the readings were taken at 495nm at an interval of 10 seconds up to the

difference of 0.05. Time required (t) for increase in the absorbance to 0.05 was

recorded.

Succinate dehydrogenase activity (Copper & Beevers, 1969): - The enzyme mixture

consisted of 2ml of 0.2M sodium succinate, 1ml of phosphate buffer, 1ml of TTC


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(Triphenyl tetrazolium chloride) and 2ml of enzyme extract. The mixture was

incubated in a water bath at 30oC. At various time intervals, 7ml of acetone was added

to stop the reaction. The mixture was centrifuged at 2000 g for 30min and the

supernatant was measured at 460nm. Standard curve was plotted against sodium

sulphite.

Nitrate reductase activity (Sempruch et al., 2008):- The samples were crushed in a

mortar at 0 to 4c in 6 ml of cysteine buffer and centrifuged at 10,000rpm for 10

minutes. The supernatant was used as a crude enzyme preparation. The enzyme was

assayed in 2ml of reaction mixture containing 100mM potassium phosphate buffer

(pH 7.5), 30mM KNO3, 0.8 mM NADH, and 0.8 ml of enzymes extract. NADH was

omitted in the control tube. The reaction mixture was incubated for 30 min. at 30C;

1mL of a 1%(w/v) solution of sulphanilamide in 3 M HCl and 1 ml of a 0.02% (w/v)solution of Nepthyl Ethylene Diamine Dihydrochloride were added to the mixture;

and the A540 was measured.

Amylase activity (Bernfeld, 1955):-For the activity weighed samples were ground in

five to ten volumes of ice cold 10 mM CaCl2 overnight at 4oC and then centrifuged at

20,000g at 4oC for 20 min. The supernatant was used as an enzyme source. The

reaction mixture consists of 1ml of starch solution, 1 ml of extracted enzyme which

was incubated for 15 minutes at 27oC and the reaction was stopped by addition of 2ml

of DNS reagent. The mixture was heated in a boiling water bath for 5 minutes. While

the tubes were warm, 1ml of potassium sodium tartarate was added. The content was

cooled and the volume was made 10ml by adding 6ml of distilled water. Theabsorbance was recorded at 560nm and maltose was used as a standard.

Statistical Analysis: - For authentication of the present result, standard error of the

obtained data was carried out. Cluster analysis was also carried out to record

correlation between different species on the basis of presence of enzyme activity and

represented in the form of dendrogram.

Results and DiscussionThe variation of different enzymatic activities like Amylase, Protease, Peroxidase,

Succinate dehydrogenase, Polyphenoloxidase and Nitrate reductase of eighteenmarine macroalgae as per their class has been shown in Fig. 1.1 to 1.6. The cluster

analysis of the algal species (Fig. 2.1 to 2.6) showed a strong correlation among

several species.

Maximum amylase activity was recorded in Ulva lactusa showing 202 mgmaltose. g

-1FW hr

-1followed by Caulerpa racemosa (138 mg maltose. g

-1FW hr

-1)

whereas C. seertulariodes showed the activity of 120 mg maltose. g-1

FW hr-1

respectively. Similar observations were also made by Ducan et al (1956) where

maximum amylase activity was recorded in U. lactuca as compared to other species

of Phaeophyceae followed by Rhodophyceae (Fig 1.1). It had been found that the total

amylase activities of different species are correlated irrespective of their division. The


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amylase activity of Chactomorpha spp, Cladophora fascicularis and Dictyota

bartayresiana formed a cluster which shown the similar activities whereas Fucus spp,

Scinaria farcellata and Porphyra spp formed another cluster (Fig. 2.1).

The species of Chlorophyceae also showed greater activity of protease from 7.3 to

8.5 g. tyrosine g FW-1

hr-1

which is much higher followed by other species belonging

to Phaeophyceae and Rhodophyceae and shown range between 5 to 7.3 g tyrosine. g-

1FW hr

-1. The proteases of Chlorophyceae have been implicated in degradation of

phycobiliproteins during photoacclimation and nutrient starvation. Induction of

proteases in response to nitrogen or light limitation has also been described in some

diatom and chlorophyte species (Fig 1.2). Responses to stress are often mediated at

the level of proteins. While stressful environmental conditions can induce synthesis of

specific proteins, they can also affect protein stability and turnover by increasing the

rate of proteolysis of specific proteins (Llorens et al, 2003). It is important torecognize that protease measurements in the present study were made on material

freshly collected from the field and thus, from an unspecified set of environmental

conditions. The protease activity ofChactomorpha spp and Valoniopsis pachynema

were similar compared to other species and also the protease activity of Cladophora

fascicularis and Ulva lactusa are similar (Fig. 2.2).The apparent ineffectiveness of

common protease inhibitors is not unique to proteases from marine phytoplankton

(Berges & Falkowski, 1996). The precise function of algal cell associated proteases

remains unknown. The cell surface aminopeptidase indentified by Martinez and Azam

(1993) appear to be constitutive because changes in nutrient enrichment did not alter

activities. In contrast, our preliminary results suggest that nutrient deprivation leads to

increase in cell associated protease levels and to the induction of specific proteasesand furthermore that transitions from light to continuous darkness may cause even

more dramatic increases.

The Chlorophycean species showed high range of peroxidase activity (10 to 13.5

units.mg-1

FW) as compared to the other species (Fig 1.3). The high rate of peroxidase

activity was recorded in Cladophora fascicularis. Among all the eighteen species the

peroxidase activity was recorded more similar in Chactomorpha spp, Valoniopsis

pachynema and Ulva lactusa (Fig. 2.3). Thus the results are corroborated by the

findings of Ross and Alstyne (2007) where maximum peroxidase activity was

registered in Cladophora glomerata compared to other species. It may be because of

the fact that the present algal species may be a stress tolerant which indirectly shows

higher peroxidase activity which help in preventing the oxidative damage caused byROS which is produced due to stressed conditions. Similar results were obtained by

Ross and Alstyne (2007) where U. lactusa plants showed higher peroxidase activity

when subjected to frequent environmental stresses. The present result may be

because of the fact that Chlorophyceae members are present in the intertidal zones

which is regularly exposed to physiological stresses removed H2O2 more efficiently

than subtidal which would encounter these stresses much less frequently (Ross &

Alstyne, 2007). The present result correlates with the findings of the other workers

where ROS production and the activities of antioxidant enzymes in individual

subjected to environmental stresses (Lu et al, 2006). Intra specific comparisons of

ROS production in marine macroalgae have found that ROS production is lower


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following stress in algae acclimated to stressful environments. Phenotypic plasticity in

antioxidant enzyme activities in response to a variety of stresses has been documented

in many higher plants (Apel & Hirt, 2004). There is also evidence that antioxidant

enzyme activities in marine algae are altered in response to changes in environmental

conditions (Collen & Davison, 2001).

The maximum activity of Polyphenoloxidase was recorded in the species of

Chlorophyceae followed by the species of Rhodophyceae and Phaeophyceae. Tolber,

(1973) emphasized that the higher activity of polyphenoloxidase is the catalyzation of

the molecular oxygen to mono and dihydroxy phenolic compounds of the cell

(Fig.1.4). The polyphenoloxidase activity was recorded parallel in Chactomorpha spp

and Porphyra nesnamesis (Fig 2.4).

However, Succinate dehydrogenase activity was recorded rich in the species of

Rhodophyceae which shown the range of 0.004 to 0.01 units.mg

-1

FW than other twogroups (Fig 1.5). The enzyme activity was recorded analogues in Chactomorpha spp,

Sargassum ilicifolium and Cladophora fascicularis (Fig. 2.5).The change in succinate

dehydrogenase activity in different species may be because of the fact that change in

the enzyme significantly affects the rate of TCA functioning and also the balance

between photosynthesis, respiration and photorespiration in the cell (Popov et al,

2007).

Maximum activity of nitrate reductase (NR) was recorded in Fucus spp, while

poor activity was recorded in Caulerpa seertulariodes. Range of nitrate reductase in

Chlorophyceae was between 170.4 to 75.6 mol NO2. g-1

FW, followed by

Phaeophyta which showed the range between 334.8 to 132 mol NO2. g-1

FW and

Rhodophyta between 176.4 to 81.6 mol NO2. g-1

FW (Fig .1.6). The cluster analysisshowed similar nitrate reductase activity among Chactomorpha spp, Caulerpa

racemosa and Porphyra spp (Fig. 2.6).Nitrate reductase is one of the key enzymes

involved in fixation of atmospheric nitrogen which is sensitive to light, temperature

and oxygen. The synthesis and activation of NR is regulated primarily by the presence

or absence of NO3 (Solomonson & Barber 1990, Crawford 1995).

In the present investigation, it is revealed that the members of Chlorophyceae

showed greater activity of enzymes like amylase, protease, peroxidase and

polyphenoloxidase while Rhodophyceae shown higher activity of Succinate

dehyrogenase than that of the species of other groups investigated, while NR shown

higher activity in Pheophyceae followed by Rhodophyceae and Chlorophyceae. Our

results suggest that proteases from macroalgae are easily measurable and highlyvariable; it may be due to responses to different environmental conditions. The

differences in enzyme activities between different habitats suggest that enzyme

activity is phenotypically plastic and is being modified in response to environmental

cues and that localized selection is occurring. The differences in the enzyme activities

among the species and their respective groups are phenotypically dependent and are

being modified in response to environmental conditions (Apel and Hirt, 2004). The

present results also evident, that the marine macro algae are susceptible to a variety of

dynamic stresses, depth, light penetration, transpiration and hydro and geochemical

properties that influence survivorship, activity and distribution of enzymes (Sousa

2001).


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From the present work it is clear that different species of algae consists of

different types of enzymatic activities in the natural conditions which help them to

sustain the present ecological conditions. Some of these enzymes such as peroxidases,

succinate dehydrogenases and polyphenoloxidases act as the stress induced enzymes

which also indicate the protection of the algal species form unfavorable conditions.

The present work helps to find the phylogenetic relation between different species can

be found out.

Enzymatic Variation of Eighteen Species of Marine Macroalgae,

Belonging to Chlorophyceae, Phaeophyceae and Rhodophyceae

(Values are the Average Readings of Triplicate)

Figure 1.1 Figure 1.2




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Sr.No Name of the Species Division

Chactomorpha spp ChlorophyceaeCladophora fascicularis Chlorophyceae

Ulval lactusa Chlorophyceae

Caulerpa racemosa Chlorophyceae

Caulerpa seertulariodes Chlorophyceae

Valoniopsis pachynema Chlorophyceae

Sargassum ilicifolium Phaeophyta

S. polycustum Phaeophyta

Dictyota bartayresiana Phaeophyta

Fucus spp Phaeophyta

Padina gymnospora PhaeophytaPorphyra nesnamesis Rhodophyta

Scinaria farcellata Rhodophyta

Champia compressa Rhodophyta

Porphyra spp Rhodophyta

Liagora erecta Rhodophyta

Acanthophora delibi Lamour Rhodophyta

Soliera robusta Rhodophyta

AcknowledgementsThe authors are thankful to University Grants Commission for providing financial

support for the present work.

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