2.1 Identification and Screening of Chitinolytic bacteria
Bacteria that produces chitinase have shown poor chitinolytic activity, sometimes requires additional hydrolytic enzyme or even microorganism to boost up their chitinolytic property (Anné et al., 2014). Studies on chitinase production by bacteria have described the role of actinomycetes in natural environments e.g. soil where it contributes to recycle nutrients (Gopalakrishnan, Pande, et al., 2011). They are therefore potential source of hydrolytic enzymes and thus antagonist to major phytopathogens (Jayamurthy et al., 2014). Therefore, this study aims at screening Actinomycetes as a potential source of chitinase enzyme.
Twenty-five actinobacterial cultures were checked for antagonistic activity against phytopathogenic fungus and among them ANU5a, ANU5b, CAI155a, CAI155b, CAI127, CAI140b, KAI27b and KAI90 were found to effectively inhibit fungal growth (Figure 2.1).
Among these eight antagonistic actinobacteria ANU5a broadly exhibited antifungal activity against all the phytopathogenic fungi tested. The difference in extent of inhibition from one organism to other could be attributed to the difference in efficiency and specificity of the antagonistic agent (enzyme/metabolite) produced by the culture (Table 2.1)
2.2 In vitro plant growth promotion of actinobacterial isolates
The preliminary screening results for PGP traits have been tabulated in Table 2.2. Almost all the isolate possesses one or the other PGP trait. IAA production was the best trait being observed in the test cultures. From the present investigation, 56% isolates generate IAA in the approximate amount of 19.75 - 55 µg/mL. The ability of bacteria to produce IAA depends on the availability of precursor molecule i.e. L-tryptophan. Root exudates are one of the important natural source of tryptophan that probably accelerate the auxin biosynthesis in the rhizosphere soil (Khamna et al., 2009).
Production of siderophore was also detected by only 52% of isolates (Table 2.2). The isolates on CAS agar formed an orange halo around the colonies. Most of them exhibited high ability to produce siderophores. Usually, soil microbes produces siderophores that bind to Fe3+ ions in the iron deficient environment. These siderophores bind to the iron molecule and transport it back to the microbial cell and make it available for growth (W. Lee et al., 2012). These siderophores can also be used by plants to source iron as macronutrient. A possible mechanism to control the phytopathogens also includes competition for iron, inhibition of phytopathogens through hydroxymate-type siderophores by streptomyces in plant rhizosphere soil is a good example for stimulating competition for iron in plant growth development (Khamna et al., 2009). Siderophore may increase the availability of minerals to soil bacteria that results into biosynthesis of antimicrobial compounds.
Among 25 cultures, 32% isolates were found to solubilize precipitated phosphate after 196h incubation (Figure 2.2). The bacteria could solubilize phosphorus either by organic acid and/or acid phosphatase production(Sharma et al., 2011). The action of organic acid synthesis by soil microbes is generally accepted for the mechanism involving mineral phosphate solubilization. Some of the examples of organic acids among phosphate solubilizers are glycolic, oxalic, lactic, fumaric, acetic, malonic, succinic acid (Sharma et al., 2011).
Therefore, among the above tested isolates, 3 isolates CAI 155a, CAI 155b, ANU 5b were showing maximum number of screened traits and ANU 5a exhibited almost all traits, which may promote plant growth directly or indirectly. Similar to our finding of multiple PGP activity, Streptomycetes have been reported to enhance plant growth (Khamna et al., 2009 , Ahmad, Ahmad, & Khan, 2008). While such reports from Indian subcontinent is very sparse.
2.3 Solvent tolerance on extracellular chitinase production
In order to screen for solvent tolerance, these four selected cultures were monitored for growth and chitinase activity on solvents flooded plates. Table 2.3, shows the solvent tolerance and chitinase production by 3 selected bacterial isolates in presence of iso-octane, n-Hexane, and iso-octane. Among these 3 isolates, ANU5a showed broad spectrum reponse toward solvent tolerance. However, growth of all isolates was inhibited Methanol, Acetone, Benzene, Toluene and Dichloromethane. Organic solvent tolerant chitinase in Streptomycetes is less reported than till date.
2.4 Selection of chitinolytic strain for chitinase production
The chitinolytic activity of the four selected isolates (CAI155a, CAI155b, ANU5a and ANU5b) were further confirmed by growing them in 100 mL BHM broth amended with 0.5% of α-chitin. Chitinase activity was measured by using Schales reagent. Figure 2.3 represents the chitinase activity of these 4 isolates.
Soil isolates ANU5a showed maximum chitinase activity as 0.032 Units/mL. Thus, ANU 5a was selected for further investigation.
2.5 16SrDNA identification of ANU5a
The sequencing results for ANU5a shares 99% sequence identity with 16S rDNA of Streptomyces cavourensis. We have submitted the sequence to NCBI GenBank with accession number KR061434. The analyzed sequence was subsequently used to construct a phylogenic tree to compare ANU5a and its genetically close relatives (Figure 2.4).
2.6 Influence of temperature and pH on ANU5a chitinase activity
The chitinase enzyme assay was performed at different pH (5, 6 and 7) and temperature (30 ºC and 37 ºC) to determine the optimal pH and temperature for the chitinase activity. Streptomycetes sp. ANU5a chitinase exhibited optimal activity at 5.0 pH and 50 ºC temperature, respectively (Figure 2.5A, 2.5B).
2.7 Profile of chitinase production by ANU5a
The duration of incubation period offers the potential for the cost effective production of enzymes. Time course study was carried out for the chitinolyic Streptomycetessp. ANU5a. The production of extracellular chitinases was detected by withdrawing the sample from each production flask (with additional supplement A without additional supplement B) at time interval of 24 h.
Chitinase activity in production medium without any additional source other than chitin and BHM showed maximum activity at 72 h of incubation and after which the activity declined. Whereas, the maximum activity for chitinase in production medium B supplemented with additional nutritional sources was detected to be at 120 h of incubation and after which the activity declined. Figure (2.6A) represents that Streptomycetes sp. ANU 5a secretes chitinase with maximum activity of 110 U/mL in production medium B, whereas Figure (2.6B) exhibited 34 Units/mL in production medium A. This study indicates, additional nutritional sources are required for better enzyme production.
2.8 Optimization of chitinase production
Optimization of medium and production parameter for chitinase production by selecting the best nutritional and environmental condition is important to increase the chitinase yield (Saadoun et al., 2009). The traditional method of “optimizing one factor at a time” technique was used in this study. This method is determined by varying one factor while keeping the other factor at a constant level.
The aim of this investigation was to optimize the chitinase production medium for Streptomyces sp. ANU 5a. The cultural characters were optimized by amending with different concentration of chitin, various carbon and nitrogen sources, optimum pH, temperature and amino acid source supplementation were studied.
2.9 Effect of different chitin substrate on chitinase production by Streptomyces ANU5a
A study pertaining to the physiological requirement of any culture enables one to manipulate conditions so as to maximize product formation. There are difference in the performance of various Actinomycetes on the sources of carbon and energy depending upon their physiology and preference for substrate. The production of chitinase is inducible and affected by nature of substrate used in production. Therefore, the choice of an appropriate inducing substrate is of great importance. These solid substrates serve as support and/or nutrient source. As shown in Figure 2.7, different chitin substrate materials like α chitin, β chitin, colloidal chitin as well as varying concentration of α chitin was tested for their successful utilization as substrate. Among different substrate tested with variable concentration, α chitin showed maximum enzyme activity with 0.5g % (w/v) chitin concentration. The activity of chitinase was found to be maximum i.e 100 U/ml. As α chitin gave maximum activity for the enzyme, hence it was used for further experiment with their particular concentration 0.5g %.
2.10 Effect of pH on chitinase production by Streptomyces ANU5a
The hydrogen ion concentration has a marked effect on enzyme production. This may be due to stability of extracellular enzyme at this particular pH and the rapid denaturation at lower or higher pH values, which ultimately lowers the enzyme activity. Chitinases are fairly stable over broad pH range (Saber et al., 2015).
The pH stability of chitinase varies from organism to organism. The effect of different pH on chitinase production is depicted in Figure 2.8. Among the different pH checked, the maximum enzyme production (61U/ml) was observed on 7th day at pH 7 which declined with further increasing initial pH. Similar results were observed by Sowmya et al. (2012) and Thiagarajan et al., 2011), who investigated that the chitinase from Streptomyces to be stable over a pH range of 4.0 to 10.
2.11 Effect of temperature on chitinase production by Streptomyces ANU5a
The selection of temperature depends on the optimum growth temperature of the selected culture. As enzymes are primary metabolites, so their production is mainly related to growth. More the growth more will be the enzymes produced. The production of enzyme at different temperature 30ºC and at 37ºC was studied. As depicted in Figure 2.9 the optimum temperature for chitinase production is at 30 ºC. It is found to be in mesophilic range. The chitinase was found to maximum as 42 U/mL at 30 ºC supplemented with α chitin.
2.12 Effect of nitrogen source on chitinase production by Streptomyces ANU5a
The requirement of nitrogen source in the production medium of chitinases has been reported to vary considerably by various researchers. Production of chitinases is sensitive to nitrogen sources and nitrogen level in the medium. Few reports on nitrogen supplement in chitinase production medium indicates inhibitory effect (Suresh & Chandrasekaran, 1998). In this study yeast extract and casein hydrolysate were used to optimize nitrogen sources for maximum chitinase production. BHM broth supplemented with varying concentration (0.05g % to 0.2 g %) of yeast extract and casein hydrolysate were used. As shown in Fig.2.10 A & B, the enzyme was found to be enhanced when 0.05g % yeast extract and 0.2g % casein hydrolysate were applied as external source of nitrogen.
Present study indicates higher concentration of yeast extract and casein hydrolysate has inhibitory effect on chitinase production. They may contribute to fulfill the bacterial nitrogen demand much easily than chitin; it could be a reason for decrease in enzyme production at higher concentration.
2.13 Effect of media component on chitinase production
In this study, different media component were examined for maximum enzyme production. Result of this experiment as depicted in table (2.4) indicates; followed by addition of different media component into chitinase production medium, enzyme production was found to increase than without co-substrates. With one factor at a time approach, significant improvement in yield of enzyme could be achieved by media optimization which is summarized in Table 2.4.
2.14 ESEM Analysis
Chitin degradation and cell morphology of Streptomycetes sp. ANU5a were analysed by using Environmental Scanning Electron Microscope (ESEM). The morphological examination of the degraded chitin flakes (Figure 2.10) using ESEM confirmed the degenerated native structure of the chitin flakes Moreover, the microbial chitinases resulted in degradation of chitin fiber which is visible as increased porosity of the substrate (Hao et al. 2016).
2.15 Native PAGE and Zymogram Analysis
The purified chitinase exhibited a three protein bands on Native-PAGE. The relative molecular weight of enzymes was approximately 20kDa. Further identification of the target band was carried out by zymogram analysis as shown in Figure 2.11.
After subjecting the partial purified enzyme of the Streptomyces sp ANU5a to zymogram analysis, three bands were exhibited (240,67 & 20 kDa), in which two bands were showing chitinase activity. The size of the chitinase activity bands exhibited in the zymogram is relatively small compared to that observed by Saadoun et al., (2009). Who reported the molecular mass of chitinase from Streptomyces (strain 242) to be 55kDa to 97 kDa.