The Screening, Characterization and Identification of Chitinolytic Actinomycetes Streptomyces sp. ANU5a.


 Streptomyces sp. ANU5a is a potential plant growth-promoting Rhizobacteria and a highly effective biocontrol agent. It produces Chitinases in broth media containing Chitin. Earlier reports on the chitinase production revealed the key importance of casein hydrolysate and yeast extract as additional sources of carbon and nitrogen to increase the yield. 16S rRNA-based method was used to identification of a Chitinolytic Actinomycete a Streptomyces sp. ANU5a was completed from Mangrove soil samples. Ammonium sulphate precipitation method was used for partial purification of Chitinases. The Chitinases have optimum activity at a temperature of 50-55°C and pH 5-6. Three protein bands of 20kDa, 67kDa, and 240kDa are resolved by Native-PAGE of Ammonium Sulphate fractions; the smallest band of 20kDa belongs to Chitinase. Enzyme activity for chitinase was recorded by zymogram analysis. Streptomyces sp. ANU5a was found to have notable antifungal activity on culture plates. We correlate the antimicrobial activity of Streptomycetes sp. ANU5a with Chitinase activity and other PGPR traits.


Introduction
Streptomycetes sp. in the phyla Actinomycetes are broadly identi ed for the commercial level production of secondary metabolites and enzymes in variety of applications viz. medicine, agriculture and environment sectors. Streptomyces spp.
produces several chitinolytic enzymes to decompose chitins, some examples of these bacterial species includes S. antibioticus, S. griseus, S. plicatus, S. lividans, S. aureofaciens and S. halstedii. Chitinase producing actinomycetes are getting attenention due to their plant promotion and biocontrol property. They are soil borne microorganism and this could be another reason for their exploited use in bio-production and agriculture. Chitinolytic enzymes have different applications in research fungal protoplast preparation that degrades cell wall of fungi or ophthalmic preparations in human health care. Chitinase containing hydrolytic enzyme cocktails are using in paper pulp industry for high quality product formulation. In addition to immense application of chitinase and chitinolytic enzymes in medical and bio processing industry, they have major role in agricultural applications like PGPR, Bio-control and transgenic plant development against phytopathogens. Fungi represents a major group of phytopathogens which are aggressive in nature and typically treated chemically using fungicides. Exhaustive usage of such fungicides results as a major contributor to environmental pollution, increased pathogenicity in plants and increased resistance in species of veterinary importance such as arthropods and helminths (Sanborn et al. 2002).
Additionally, these chemicals are proven for serious health hazards to humans and deleterious effect on helpful insects in agriculture. The important consortium of bacteria is also disturbed in the affected ecological niche. This is one of the reasons to facilitate the use of biological control in future generation agriculture strategies. To follow up advancements in this eld of research, chitinolytic bacteria or chitinases are proposed to be used as an alternative to chemical treatment for increased yield.
These kind of approaches in agriculture can have direct impact on environment and human health.
Over the past few years streptomyces and certain bacillus were investigated as good plant growth promoters. Bacteria that directly or indirectly increases the plant growth via different mechanisms are known as plant growth promoting bacteria (PGPB). A recent inclination towards these bacteria in production is promising to increase the crop yield. Other inputs to dramatically increase the crop yield over the past 50 years primarily includes agricultural fertilizers enriched in nitrogen and phosphorus. There are limitations in the sustainability of crops cultivated through conventional methods.
At present, there are only few examples of commercially available chitinases from bacteria. Bacillus, Serratia, and Streptomyces spp. produces chitinolytic enzymes with limitations in optimal activity due to acidic pH (Kielak et al., 2013). As development of new and worth enzyme technology, researchers around the world are looking for ideal enzyme producers, for that point of view they are screening millions of isolates and cultures and another side few are trying to develop new strain via recombinant DNA Page 3/23 technology or by mutagenesis. In few screening programs, researchers are trying to correlate lytic funtion of bacterial exolate with their hydrolytic enzyme activity. In doing so, many studies divulge that antifungal activity is associated with chitinases as well as antifungal metabolites like 2-furancarboxaldehyde (S. Y. . Advancement on biocontrol and plant growth promotion strategies will be bene cial for commercial and agricultural means. This particular area of research is needed for exploratory and sustainable development. Therefore, further investigation on this area will be a great achievement for the researchers in coming near future. In this study, we did optimisation in the culture conditions for better yield of chitinase, also the screening and characterisation of metabolites by the Streptomyces strains (Table 1) Colloidal chitin was prepared as described by Aida et al. (2014) with minor modi cation. A known amount of dried chitin was suspended in concentrated hydrochloric acid and kept for overnight at 4ºC, followed by precipitation of dissolved chitin by chilled distilled water and centrifuged at 10,000 rpm for 20 minutes at room temperature. The pellet was washed repeatedly with distilled water until the pH of the chitin suspension was neutralized.

Screening of chitinolytic actinobacteria
Pre-obtained Actinobacterial cultures of mangrove soil sediment were screened for their chitinolytic activity. Cultures were inoculated on colloidal chitin (0.1%, w/v) containing agar plate, followed by cultivation at 30ºC for 72 h. Individual cultures with clear zone resulting from hydrolysis of chitin were selected for further screening and characterization. In order to get better visibility, plates were stained with Calco ore White and observed under UV (Anil et al 2007). Clear zone against uorescent background could be considered as positive chitinase producer.

Siderophore production
Production of siderophore by the actinobacterial isolates was determined using Chrome Azurol S (CAS) as per the method of Schwyn and Neiland (1991) with minor modi cations. The Actinomycetes broth was deferrated before autoclaving using 8hydroxyquinoline (0.1% w/v) as a iron-chelating agent. Sterilized 10% (v/v) CAS dye solution was added to the sterilized Actinomycetes medium and mixed properly before pouring into petridish. Actinobacterial isolates were spot inoculated onto these plates and incubated for 96 h at 30°C. Siderophore production by bacterial isolates was indicated by an orange halo around the colony.

Antifungal activity
A co-culture technique (Trivedi et al 2008) was used to test the ability of actinobacterial isolates to inhibit the growth of several phytopathogenic fungi (Aspergillus avus MTCC 16404, Fusarium oxysporum NCIM 1072 and Magnaporthae oryzae). The cultures were spot inoculated on one side of oat meal agar plate and incubated at 30°C for 48 h after which each fungal strain was inoculated next to the bacterial culture and incubated at 30°C(Hrdý et al., 2020). The plates were incubated up to 5 days for monitoring antifungal activity.
1.5 Extracellular enzyme production 1.5.1 Amylase Bacterial isolates were inoculated on BHM containing (1%, w/v) starch and incubated at 30ºC for 48h. After incubation, plates were ooded with iodine solution. Positive culture exhibited clear zone around the colony against the blue black background due to hydrolysis of starch.

Cellulase
Culture were inoculated on on BHM containing (1%, w/v) CMC and incubated at 30 ºC for 72 h. After incubation, plates were ooded with (0.1%, w/v) Congo red for 1h at room temperature followed by destaining with 1M NaCl till suitable contrast is obtained. The clear zone around the culture indicated production of cellulase enzyme.

Protease
In order to screen for protease producers, BHM amended with (1%, w/v) gelatine agar plates were used. Cultures were spot inoculated and incubated for 72h at 30ºC. Then agar plates were ooded with 10mL of Frazeir's reagent (15% HgCl 2 in 2N HCl).
Clear zone around colony were marked as protease producers.

Lipase
Actinobacterial isolates producing lipase were screened using BHM agar plates amended with Tributyrine (1% v/v). Bacterial cultures were spot inoculated and incubated at 30ºC for 120 h. Zone of clearance around the colony indicated production of lipase enzyme.

Maintenance of isolates
The potential cultures were sub-cultured and maintained on Actinomycetes isolation agar slants and preserved in refrigerator at 4ºC.
1.6 Identi cation of bacterial isolate 1.6.1 16s rRNA based molecular identi cation of ANU5a Genomic DNA was extracted from the selected isolates according to Ausubel et al. (1997). PCR was done using 16S rRNA universal primers 8F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) (MWG Biotech Pvt. Ltd., Bangalore, India) using 20-25 ng template DNA and 30 µl of each PCR reaction for 35 cycles. The thermocycler was set up for each PCR cycle of initial denaturation at 94°C for 5 mins followed by denaturation at 94°C for 2 mins, annealing at 55°C for 1 min, extension at 72°C for 15 mins (BioRad PCR cycler, USA).Agarose Gel electrophoresis was done for PCR products using 1.2% w/v agarose gel in TAE buffer (1X). 1kb DNA ladder (Bangalore Genei, Bangalore, India) was used to compare the resolution of bands. EtBr stained gel was visualized in Gel Documentation system (Alpha-Inotech, USA). Agarose gel puri ed PCR products were used in sequencing by automated DNA analyzer 3730 using ABIPRISMs BigDye™ cycle sequencing kit (Applied Biosystems, FosterCity, CA). The sequencing results produced approximately 1400bp of sequences, sequence data was analysed using NCBI servers and submitted to NCBI Genbank.

Inoculum preparation
The inoculum was prepared by method of Butterworth (1984) with minor modi cation. Chitinolytic actinobacterium ANU5a was inoculated on Actinomycetes isolation agar plate and incubated at 30ºC for 48 h after which the spores were harvested using Tween 20 (0.1%, w/v) amended (20%, w/v) glycerol and stored at -20°C. Henceforth, this seed culture will be used as inoculum.

Pro le of chitinase production by ANU5a
The 250 ml Erlenmeyer asks containing 100 mL of BHM amended with (0.5%, w/v) chitin akes was inoculated with activated seed culture of ANU5a and incubated on orbital shaker (120 rpm) at 30°C. The aliquots of 2 mL were withdrawn after 24, 48, 96, 120 and 144 h of inoculation and monitored for chitinase activity.

Medium optimization for chitinase production by ANU5a
The optimization of medium components for chitinase production by isolate ANU5a was done following one factor at a time approach. Here, the components were varied one at a time with the remaining factors held constant. The chitinase production by isolate ANU5a in BHM medium containing different chitinous source at (1%, w/v) as a sole carbon source viz. α-chitin, β-chitin, colloidal chitin (α and β). Similarly, 250ml Erlenmeyer asks containing 100 ml of (1%, w/v) α-chitin in BHM with varying nitrogen sources were inoculated with ANU5a and incubated at 30°C. The in uence of temperature (30°C and 37°C), pH (5 to 8) on chitinase production by isolate ANU5a was also investigated.
1.10 Analytical procedure: 1.10.1 Chitinase assay Chitinase assay was performed using modi ed colorimetric method for the estimation of N acetyl amino sugar as described by Schales (Schales and Schales 1945). One unit of chitinase activity was de ned as the amount of enzyme required to liberate 1ng of N-acetylglucosamine/mL.min under speci ed assay conditions.

SEM analysis of chitin degradation by ANU5a
The visualize the extent of polymer degradation, the degraded chitin akes were were immersed in iso-amylacetate, dried using lique ed carbon dioxide and coated with a layer of sublimated osmium tetroxide using an osmium plasma-coater (Itoh et al., 2013). The osmium plasma coated specimens were glued onto carbon-aluminium stub and viewed under Scanning Electron Microscope (JSM-6320F; JEOL Ltd., Tokyo, Japan). Scanning was performed using at 10 kV.

Zymogram analysis
Chitinase activity was detected in polyacrylamide gels containing chitin as described by Escott et al. (1998).

Identi cation 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.
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 e ciency and speci city of the antagonistic agent (enzyme/metabolite) produced by the culture (

In vitro plant growth promotion of actinobacterial isolates
The preliminary screening results for PGP traits have been tabulated in Table 2 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 Fe 3+ ions in the iron de cient environment. These siderophores bind to the iron molecule and transport it back to the microbial cell and make it available for growth (W. . 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 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 nding of multiple PGP activity, Streptomycetes have been reported to enhance plant growth (

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 ooded plates. Table 2.3, shows the solvent tolerance and chitinase production by 3 selected bacterial isolates in presence of isooctane, 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.

Selection of chitinolytic strain for chitinase production
The chitinolytic activity of the four selected isolates (CAI155a, CAI155b, ANU5a and ANU5b) were further con rmed 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.

16SrDNA identi cation 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).

In uence 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).

Pro le 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 ask (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.

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.

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 %.

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.
The pH stability of chitinase varies from organism to organism. The effect of different pH on chitinase production is depicted in who investigated that the chitinase from Streptomyces to be stable over a pH range of 4.0 to 10.

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.

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 ful ll the bacterial nitrogen demand much easily than chitin; it could be a reason for decrease in enzyme production at higher concentration.

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, signi cant improvement in yield of enzyme could be achieved by media optimization which is summarized in Table 2.4.

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 akes (Figure 2.10) using ESEM con rmed the degenerated native structure of the chitin akes Moreover, the microbial chitinases resulted in degradation of chitin ber which is visible as increased porosity of the substrate (Hao et al. 2016).

Native PAGE and Zymogram Analysis
The puri ed chitinase exhibited a three protein bands on Native-PAGE. The relative molecular weight of enzymes was approximately 20kDa. Further identi cation of the target band was carried out by zymogram analysis as shown in Figure 2.11.
After subjecting the partial puri ed 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.

Discussion
The association of decomposition of chitin and antifungal properties is described in earlier reports (Huang et al., 2005) Streptomyces griseus H7602 have considerable potential as a biocontrol in papper against root rot disease (Phytophthora capsici). Hoster, Schmitz, and Daniel (2005) also highlighted the role of puri ed chitinase from Streptomyces griseus MG3 strain as possible antifungal against the phytopathogenic fungi such as Aspergillus nidulans, Botrytis cinerea, Fusarium culmorum, Guignardia bidwellii and Sclerotia sclerotiorum. In a same way selected strain were studied for antifungal as well as chitinolytic activity. ANU5a was found to be an e cient strain, as well as possesses plant growth promotion traits along with its ability to secrete a range of extracellular hydrolytic enzymes. This strain was identi ed as Streptomyces sp. ANU5a. It would be worth considering this organism for further investigation with respect to its plant growth promoting and agronomic practices (Aam et al., 2010).  Plates showing extracellular enzyme production, (A) CMC agar plate for cellulase activity, (B) Tributyrin agar plate for lipase activity, (C) Starch agar plate for amylase activity, (D) Gelatin agar plate for protease activity.

Figure 3
Chitinase Activity of promising Actinomycetes isolates.

Figure 4
Page 20/23 Phylogenetic tree based on 16S rDNA gene sequences from strain ANU5a and other related organisms constructed using the neighbor-joining algorithm. (The tree was validated by bootstrap analysis (1,000 replications). Bar, 0.005 substitutions per 100 nucleotides.

Figure 5
Effect of pH (A) and temperature (B) on chitinase activity.

Figure 6
Effect of incubation period on chitinase (a) without additional supplement and (b) with additional supplements (Casein hydrolysate, yeast extract).

Figure 8
Effect of pH on chitinase production.

Figure 9
Effect of temperature on chitinase production.