ENZYME ACTIVITY PROFILING OF EXO -β-1,4- GLUCANASE, ENDO-β-1,3- GLUCANASE AND PROTEASE IN STREPTOMYCES SPECIES FROM HIGHLAND, MIDLAND AND LOWLAND AREAS OF KERALA, INDIA

1. Division of Microbiology, Jawaharlal Nehru Tropical Botanical Garden and Research Institute, Palode, Thiruvananthapuram, 695562 Kerala, India. 2. Department of Biotechnology and Biochemical Engineering, SCT College of Engineering, Thiruvananthapuram, 695518 Kerala, India. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


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(NH 4 ) 2 SO 4 ,2g; CaCO 3 , 2g and Trace salt solution, 1ml). After incubation of 7 days at 28 0 C Streptomyces like isolates were selected randomly based on their morphology. Number of total Streptomyces colonies were evaluated and transferred to Yeast extract-malt extract agar (ISP-2) plates (Yeast extract, 2g; Malt extract, 10g and Dextrose, 10g) to check the purity and maintained by periodical sub culturing.

Semi-quantitative screening of Enzyme Activities:-
The Streptomyces strains were initially screened for detecting exo -β-1,4-glucanase activity on synthetic medium containing Avicel (Sigma, USA) as the sole carbon source. The composition of medium was as follows: Avicel,1%; yeast extract, 0.1%; Peptone,0.1%; KH 2 PO 4 ,0.1% and MgSO 4 .7H 2 O,0.05%. The strains were spot inoculated on Avicel agar plates and incubated for 5 days at 28 0 C. After incubation 0.1% congo red dye was spreaded over the plates and washed with 1 mol.L -1 NaCl, used as a destaining solution. Exo-β-1,4-glucanase production was indicated by the presence of a pale halo around the colonies. A semi quantitative approach was used for measuring the enzyme activity that is the enzymatic index (EI) of strains were calculated using the expression.
Protease production of Streptomyces isolates were screened on skimmed milk agar containing Casein enzyme hydrolysate, 5%; Yeast extract, 2.5 and Glucose, 1%. The plates were spot inoculated and incubated at room temperature for 5 days at 28 0 C. Positive isolates were detected by the appearance of clear halos around the colonies. The enzymatic index (EI) of strains were calculated.

Quantitative Enzyme Assays:-
The exo-β-1,4-glucanase assay was carried out by measuring reducing sugars by DNS method (Miller, 1959) liberated from 0.5% (w/v) Avicel which is dissolved in 100mM sodium-acetate buffer (pH 5.0) was used as substrate. 0.5 ml of filtrate was added to 1 ml of 0.5% Avicel and incubated at 30 0 C for 1hr in shaker incubator. 2 ml of DNS reagent was added and incubated in boiling water bath for 5 minutes. After adding 0.5 ml 40% Rochelle salt the absorbance was taken at 540 nm wavelength using visible spectrophotometer. One unit of exo-β-1,4-glucanase activity (U mL -1 ) was defined as the amount of enzyme that released 1μmol of reducing sugars as glucose equivalents from one ml of Avicel per minute under given assay conditions (Oliveira et al., 2014).
Endo-β-1,3-glucanase activity was estimated by measuring the release of reducing sugars in a reaction mixture of 0.5ml of enzyme and 1ml 0.2% (w/v) CM-curdlan (Megazyme, USA) in 100mM sodium-acetate buffer (pH 5.0) incubated at 30 0 C for 1hr and the remaining procedures were similar as exo-β-1,4-glucanase assay. Enzyme activity was expressed by 1μmol of reducing sugar released per minute under standard assay conditions. The protease enzyme activity was determined by a modified method of Rupali (2015). Casein is used as substrate for determination of protease activity. 1% (w/v) of casein in 50mM phosphate buffer (pH 7.0) was used as substrate and incubated with 0.5 ml enzyme solution at 37 0 C for 30 minutes. The reaction was stopped by adding 2 ml of 100 mM trichloroacetic acid. After centrifugation at 7000 rpm for 15 minutes, 0.5 ml supernatant was taken and added 2 ml 0.5 M Na 2 CO 3 and 0.5 ml diluted Folin phenol reagent, then kept it in incubation under dark conditions for 30 minutes. The absorbance was read at 660 nm. One unit of protease activity is defined as the amount of enzyme that liberated 1μmole of tyrosine per ml per minute under standard assay conditions.

Determination of Soil pH and Electrical Conductivity:-
Soil pH and electrical conductivity (EC) were determined in 1: 3.0 soil/water ratio by a combination glass electrode HI98129, Hanna Instruments.

Determination of Soil Organic Carbon and Soil Organic Matter:-
The soil organic carbon (SOC) content was estimated by dichromate oxidation method in which the oxidation of K 2 Cr 2 O 7 in a concentrated H 2 SO 4 medium and the excess dichromate was measured using (NH 4 ) 2 Fe(SO 4 ) 2 (Yeomans and Bremner, 1989). Soil organic matter (SOM) were determined according to Pribyl, 2010. Statistical analysis:-All part of experiments in this study were performed in triplicate. Data were presented as mean ± SD. The p values <0.05 were considered statistically significant. To determine the correlations between the parameters linear regression analysis (Pearson method) was performed.

Number of Streptomyces Isolates:-
Morphologically different 95 Streptomyces strains were isolated from 21 locations of three different land areas viz. highland, midland and low land. The number of strains showed a decreasing trend from highland to lowland ( Figure  2). Soils from natural ecosystem were observed higher microbial content when compared to agricultural ecosystems. Highland areas in Kerala mainly Western Ghats regions represents high levels of biological diversity, less unexplored and mostly covers with protected land mass. Due to the lack of anthropogenic activities, number of Streptomyces isolates are significantly high in these areas when compared with other two geographical landmasses such as midland and lowland areas. Taxonomically diverse Streptomyces species are found in forest soils, they are involved in recalcitrant biopolymer degradation. This leads to the prominence of Streptomyces in forest nutrient turnover (Bontemps et al., 2013). According to Zhou et al., 2016, rich plant biomass in paddy field expects high number of hydrolase producing organisms, however gets only very small fraction of isolates from the area. Application of the high level of mineral fertilization along with chemical weed control agents predominantly detriments the biological activity of the soil because of higher loss of micro-organisms due to rigorous chemical plant protection (Bielińska and Pranagal 2007). The number of isolates showing a decreasing trend from highland to midland to lowland. This study, clearly reveals the relation between ecological variations and Streptomyces population, hence number of Streptomyces isolates can be considered as a delicate indicator of environmental changes.

Screening of Enzyme Activities:-
All the 95 isolates were evaluated for semi-quantitative exo-β-1,4-glucanase, endo-β-1,3-glucanase and protease production. From the total 95 isolates 85 strains were shown exo-β-1,4-glucanase activity (equivalent to 89% of the strains evaluated) 60 shown endo-β-1,3-glucanase activity (equivalent to 63% of the strains evaluated) and only 61 shown protease activity (equivalent to 64% of the strains evaluated). This is based on the observation of growth and measurement of hydrolysis halo, used for calculation of the enzymatic index (EI). The pale halo around the exo-β-1,4-glucanolytic colonies corresponds to the zone of Avicel (specific substrate for exo-β-1,4-glucanase) degradation ( Figure 3a), blue colouration around endo-β-1,3-glucanolytic colonies indicating the zone of Pachyman (specific substrate for endo-β-1,3-glucanase) degradation ( Figure 3b) and appearance of clear zone around the colonies surrounded by white color background indicated the presence of protease activity, corresponds to the zone of protein degradation ( Figure 3c).

Quantitative Enzyme Assays:-
As a result of semi-quantitative primary screening, strains with high EI values were considered as potent enzyme producers and were selected for quantitative enzyme assays. 8 isolates from highland areas and 7 isolates from midland areas, both expressed EI values in and above 4.5 and 5 isolates from low land area with EI values in and above 3.5 were selected for exo-β-1,4-glucanase assay. Endo-β-1,3-glucanase assay was carried out with highland strains showed EI values in and above 4.5, midland strains with EI values in and above 4.0 and lowland strains with EI values in and above 3.0. Protease quantitative assay was done with selected isolates from highland with EI values in and above 3.0, midland and lowland with EI values in and above 2.0.
The quantitative assay clearly revealed that activity of exo-β-1,4-glucanase in highland was ranged between 95 to 76 Units.mL -1 , in midland between 70 to 58 Units.mL -1 and in lowland it was in between 48 to 52 Units.mL -1 . The exoβ-1,4-glucanase activity was recorded highest in highland areas and gradually decreased in low land areas ( Figure  4a). Here the activity was greatly affected by land use type. Endo-β-1,3-glucanase activity were ranged from 136 to 231 Units.mL -1 and protease activity was from 3 to 14 Units.mL -1 in all land types. So there was no significant variation in the activities of endo-β-1,3-glucanase ( Figure 4b) and protease (Figure 4c) in different land areas. The 5b 2442 significant variation of exo-β-1,4-glucanase activity from highland to lowland suggest the sensitivity of the enzyme as an ecological and a soil quality indicator, so it can be used as a prominent indicator of environmental changes.
Knowledge in relation with soil enzyme activities provides necessary information relating to soil fertility. Land disturbances can negatively affect soil fertility, may leads to the manual addition of soil fertilizers and weedicides, adversely affect soil microbial population. In other words soil functional diversity in some extent is controlled by microbial enzyme activities (Gonnety et al., 2012). Among the three enzyme activities explored, exo-β-1,4glucanase expressed great variation in their activities in three different geographical land areas. It displayed highest levels of activities in high land areas and the lowest activity was observed in lowland areas. Midland areas showed an activity in between highland and lowland. When considering Kerala's geographical land masses, the anthropogenic activities are increasing from highland to lowland. In this study, exo-β-1,4-glucanase activity decreased with increasing anthropogenic activities. This trend was not observed in endo-β-1,3-glucanase and protease. In the light of these observations we can consider exo-β-1,4-glucanase as a best sensitive indicator for environmental changes. According to Gao et al., 2010, depending on the type of land use, land management and type of enzyme, the absolute enzyme activities may vary in different land areas. Activity of soil enzymes is more related to physical and chemical properties, geology and land uses of soil. Improper land management is obvious in majority of agricultural systems, by the rotating cultivation of crops. Soil quality degradations due to frequent land use changes decline soil microbial biomass and enzyme activities in arable soils compared with natural vegetation (Mganga et al., 2015). Kerala's natural geographical land classification is a best example for changes in environment and land use types. Highland areas are mostly covered with natural vegetation. A combination of natural and agricultural land areas are seen in midlands. But almost all part of lowland areas are arable lands. In this study, the results evidently validates that the sharp declining of number of Streptomyces strains and activity of exo-β-1,4glucanase enzyme by changing the environment from natural vegetation to arable lands.

Soil pH and Electrical Conductivity (EC):
p H and EC are the most significant parameters for measuring soil quality. Soil samples collected from three land areas were showed slight variations in pH. Soils under natural vegetation was characterized with high pH that means they are low acidic soils when compared with agricultural vegetation. pH values significantly decreased from higher to lower land regions. pH in highland areas ranged from 7.2 to 6 and in midland areas it was in 6.1 to 5.0. The highest acidity was noted in lowland regions, where the soils collected from paddy fields ranged from 3.7 to 4.8. This may be due to the application of herbicides and heavy fertilization of soils over the years for agricultural practices. A significant positive correlation (r 2 = 0.724) was shown between soil pH values and number of Streptomyces strains (Figure 5a). Along with number of Streptomyces isolates pH values also showed a decreasing trend from highland to low land. Significantly very low pH was detected in lowland areas. Decrease in pH was another strong reason for lowering microbial content and soil enzymatic activity. An increase in the concentration of hydrogen ions in soil has a negative effect on its enzymatic activity (Acosta-Martinez and Tabatabai, 2000).
Highest EC was recorded in lowland soils, ranged in between 679 to 885 µs cm -1 . In midland and highland it was detected at a range of 447 to 635 µs cm -1 and 107 to 352 µs cm -1 . It gradually decreased from lowland to high land. Number of microbial isolates and activity may be affected by EC. A highly significant negative correlation (r 2 = 0.908) was shown in between number of Streptomyces isolates and soil EC values (Figure 5b). EC values indicate large accumulation of soluble salts in soils. Negative significant relationship between EC and number of isolates validates that increasing salinity and sodicity tremendously affect the soil Streptomyces community. With increasing salinity microorganisms tend to dehydrate, diminishes microbial growth and activity. Microbial biomass and enzyme activity decreased exponentially with increase in EC (Rietz and Haynes, 2003).
Soil organic carbon and soil organic matter:-SOC and SOM percentage was increased with decreasing altitudes. Highest SOC and SOM percentage was reported in lowland areas, unfortunately the area with very less estimated number of Streptomyces isolates ( Figure 6). SOC and SOM showed an increasing trend from high land to low land areas, whereas a decreasing trend was obtained in case of number of strains. 3.5 fold increase in SOC and SOM percentage were detected in lowland areas and 2 fold increase in lowland areas when compared with highlands. It may be expected that due to the loss of microbial load in agricultural lands by improper soil management practices reduces the rate of carbon mineralization (Chander et al.,1998). 2443

Conclusion:-
The study was conducted to observe how Streptomyces isolates and their enzyme activities fluctuate between the natural geographical land areas in Kerala, highland, midland and lowland. According to this study, large number of Streptomyces strains were isolated from highland areas, the inhabitant of natural forest, compared to other land areas. pH and EC were highly correlated with the obtaining results, that point out to soil quality. Cumulative anthropogenic activities such as change in land uses especially from forest to rigorous agriculture for sustaining livelihood cause severe consequence on soil quality. Among the three enzymes we have selected for this study, the enzyme exo-β-1,4-glucanase from paddy field isolates showed declining activity than high land and midland isolates. So we can use this enzyme as a most sensitive indicator of environmental changes. Soil Streptomyces biomass and enzyme activities are very sensitive to change in environment and are crucial for maintaining soil quality. In conclusion, unsuitable land use and depletion of nutrients due to the extensive practice of agricultural chemicals influenced soil enzyme activities and microbial dynamics and demonstrated poor soil quality.