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 Chakraborty2, Rita N. Kumar3, Manmeet Kaur Amb4 and Anubhuti Bora5 Head, Professor, 2Lecturer, 3Head and 4,5Research Scholar 1,4,5 P .G. Department of Environmental Science and Technology, Institute of Science & Technology for Advanced Studies & Research (ISTAR), Vallabh Vidyanagar 388 120, Gujarat, India 2,3 Department of Biological and Environmental Sciences , N.V. Patel College of Pure & Applied Sciences, Vallabh Vidyanagar 388 120, Gujarat, India 1 E-mail- istares2005@yahoo.com, 2E-mail- nfils09@gmail.com, 3 E-mail- nfils09@gmail.com, 4E-mail- istares2005@yahoo.com 5 E-mail- istares2005@yahoo.com
1

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-1FW, 0.23-0.74 units.mg-1FW, 0.001-0.015 units.mg-1FW 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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Introduction
Estuaries 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 of dynamic 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 macroalgal species 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 changes associated 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 Methods

For the present study 18 different marine macroalgal species belonging to three different divisions viz, Chlorophyceae, Phaeophyceae and Rhodophyceae were

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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. The homogenate 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 and absorbance 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 with 1.5ml 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. The absorbance 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 Discussion

The variation of different enzymatic activities like Amylase, Protease, Peroxidase, Succinate dehydrogenase, Polyphenoloxidase and Nitrate reductase of eighteen marine 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 mg maltose. g-1 FW hr-1 followed by Caulerpa racemosa (138 mg maltose. g-1 FW 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-1which is much higher followed by other species belonging to Phaeophyceae and Rhodophyceae and shown range between 5 to 7.3 g tyrosine. g1 FW 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 to recognize 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 of Chactomorpha 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 proteases and 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-1FW) 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 by ROS 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-1FW than other two groups (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 analysis showed 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 highly variable; 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

Figure 1.3

Figure 1.4

Figure 1.5

Figure 1.6

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Cluster Analysis of Different Enzymes Among Different Species

Amylase Activity
6

Protease Activity
5

4
4

Distance

Distance
1 2 9 7 10 13 15 17 16 18 3 8 4 11 5 14 6 12

1
0

Algal Species

0 1 6 2 3 4 5 11 8 7 10 9 13 18 15 17 16 12 14

Algal Species

Figure 2.1
Peroxidase Activity
6 5 4

Figure 2.2
Polyphenoloxidase Activity
6 5 4

Distance

3 2 1 0 1 6 3 4 5 2 7 14 16 8 15 11 9 10 13 12 17 18

Distance

3 2 1 0 1 12 14 4 18 8 15 7 11 10 13 16 9 17 2 3 5 6

Algal Species

Algal Species

Figure 2.3
Succinate Dehydrogenase Activity
6

Figure 2.4
Nitrate Reductase Activity
5

Distance
1 7 2 5 8 3 11 12 6 9 4 10 13 14 16 15 18 17

Distance

0 1 4 15 2 17 12 14 6 13 9 11 3 5 16 18 8 7 10

Algal Species

Algal Species

Figure 2.5

Figure 2.6

Where,

Enzymatic Variations Among Different Species of Marine Macroalgae Sr.No Name of the Species Chactomorpha spp Cladophora fascicularis Ulval lactusa Caulerpa racemosa Caulerpa seertulariodes Valoniopsis pachynema Sargassum ilicifolium S. polycustum Dictyota bartayresiana Fucus spp Padina gymnospora Porphyra nesnamesis Scinaria farcellata Champia compressa Porphyra spp Liagora erecta Acanthophora delibi Lamour Soliera robusta Division Chlorophyceae Chlorophyceae Chlorophyceae Chlorophyceae Chlorophyceae Chlorophyceae Phaeophyta Phaeophyta Phaeophyta Phaeophyta Phaeophyta Rhodophyta Rhodophyta Rhodophyta Rhodophyta Rhodophyta Rhodophyta Rhodophyta

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Acknowledgements
The authors are thankful to University Grants Commission for providing financial support for the present work.

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