INTRODUCTION:

Serratia marcescens is a effective bacterium that for degrades chitin, a 1, 4 - β-linked polymer of N-acetyl-β-D- glucosamine (GlcNAc)¹. Chitinase by S. marcescens involves at least four enzymes and a chitin-binding protein². Studies of the enzymology and the structures of the enzymes provide insight in how a natural set of chitinolytic enzymes may be built-up^3,4. S. marcescens chitinases may find applications as biocontrol agents against fungi and insects^5,6.

The potential of chitinase as a biopesticides against agriculturally harmful fungi and insects. Due to an increasing sensibility and pressure of public and environmental agencies against the application of chemical-based pesticides and their long-lasting adverse effects on ecosystems and human health, has motivated the search for non-hazardous alternatives⁶. Biocontrol agents were considered as the substitute of chemical pesticides that are most trustworthy. These agents could be interpretations of bacteria, fungi, viruses, plant extracts or antibiotics^7,8,9,10.

Inhibition or killing of harmful pests by biocontrol agents is environmentally safe and without creating any soil pollution⁸. In last two decades, researchers have observed for their anti-insects and antifungal biocontrol activities, which was received by chitinase. This research critically concentrated on the successful studies (inside and outside of laboratory) had been done for the evaluation of biocontrol potential of chitinases from different sources.

This pilot work designed to identify the effective strain of Serratia marcescens that can be used as biocontrol agent as the enzyme produced could interact with the chitin of fungi and degrade it.

MATERIAL AND METHODS:

Bacterial strain:

The bacterial strain were isolated from the prawn culture farms near Chennai (12°48'30.8"N 80°14'50.6"E), India, the organisms were purified, screened and characterized for the maximum estimation of the chitinase enzyme¹¹.

Chitinase production

The chitinase activity was assayed using DNS method, by measuring the reducing end group N-acetyl glucosamine (GlcNAc) using 1% colloidal chitin (w/v) as substrate. The standard reaction mixture consisting of 0.1 ml of suitably diluted enzyme and 0.1 ml of 1% (w/v) colloidal chitin (pH 4.5) was incubated at 50 C for 30 min. After incubation, the reaction was terminated by addition of 0.3 ml dinitro salicylic acid (DNS) reagent and heated in boiling water for 10 min. Then, the samples were rapidly cooled to room temperature by adding 0.25 ml of water and centrifuged at 8000 rpm for 10 min. At 540nm, the absorbance of the supernatant was measured¹².

Production media:

About 10% inoculum of both the isolates was added in separate flasks, each containing 100 ml synthetic media for checking the chitinase production¹³.

Synthetic media contained (g/L) was prepared by adding KH₂PO₄ – 0.7, NH₄NO₃ –0.1, NaCl– 0.2, MgSO₄.7H₂O– 0.15, CuSO₄.5H₂O – 0.2, NH₄Cl–0.1, Chitin– 10 in 100 ml of Distill water. Flasks were incubated at 30°C in an orbital shaker at 150 rpm. After every 24 hours, samples were taken up to 144 hours, centrifuged to get cell free supernatants, which were sterilized through 0.2 μm filter paper¹⁴.

Enzyme activity:

The chitinase activity was assayed using DNS method¹¹, by measuring the reducing end group N-acetylglucosamine (GlcNAc) using 1% colloidal chitin (w/v) as substrate. The standard reaction mixture consisting of 0.1 ml of suitably diluted enzyme and 0.1 ml of 1% (w/v) colloidal chitin (pH 4.5) was incubated at 50 C for 30 min. After incubation, the reaction was terminated by addition of 0.3 ml dinitrosalicylic acid (DNS) reagent and heated in boiling water for 10 min. Then, the samples were rapidly cooled to room temperature by adding 0.25 ml of water and centrifuged at 8,000 rpm for 10 min. The absorbance of the supernatant was measured at 540 nm. One unit of chitinase activity was defined as the amount of enzyme that liberates 1mol of N-acetyl glucosamine per minute under described conditions

Protein estimation:

An estimation of protein present in the sample was done using Lowry’s method¹⁵.

Chitinase purification:

Ammonium sulphate precipitation:

Solid ammonium sulfate was added to the 20ml of the enzyme at 10-100% saturation and allowed to stand for 4 hours. Precipitate was obtained at 60%, 70% and 80%. The precipitates were resuspended in 2ml of 50Mm of acetate-buffer.

Dialysis:

A small dialysis bag (5cm) was taken and tie the bottom side and also check the leakage by pouring distilled water. The diluted enzyme was poured and the top side was tied. 500ml of 25mM acetate buffer (pH 8) was filled up and the membrane was submerged for overnight. The dialysis bag was removed and the enzyme was collected, the protein content was estimated by Lowry’s method and also its enzyme activity was checked¹⁶.

Column chromatography:

The column was prepared using glass wool of 1cm and silica gel of 20cm and kept for two days and allow it to packed properly. After that washed the column with 25mM of Tris HCL buffer. Pour 2ml of enzyme solution and add 27ml of Tris HCL buffer from the top. Collect the 2ml of the sample in 21 different fractions. Then, the optical density at 280nm was taken in UV spectrophotometer. Then, the enzyme assay and protein estimation was performed.

SDS PAGE of chitinase:

Biocontrol study:

Biocontrol potential of chitinase derived from Serratia marcescens was evaluated against plant pathogenic fungi Alternaria alternta¹⁷.

Fungal strain:

Alternaria alternata was obtained from microbial type culture collection (MTCC), Chandigarh, Punjab, India. Pure culture was maintained on Potato dextrose agar slant.

Inoculum preparation:

100 ml Potato dextrose broth was prepared and sterilized by autoclaving. After sterilization, 0.1ml of fungal spore derived from slant culture by scrapping of the slant with sterile distilled water containing 0.1% Tween 80. Slurry thus obtained was filtered through Whatmann number one filter paper to remove mycelli debris and recollected filtrate was used as the source of inoculum¹⁸.

Dual plate assay:

100 ml of Potato dextrose broth was prepared, sterilized. The sterilized media was poured into the petriplate and allowed to solidify. 0.1ml of fungal spore suspension was incubated with equal volume of Serratia culture.

Filter sterilized crude chitinase enzyme. 0.1ml of ammonium sulphate precipitate for 1hour were used. After the incubation period, 0.1ml of respective treatment was spread plated. Seeded plates were incubated at 30◦C. After the incubation period, fungal colonies were counted and recorded¹⁹.

Inhibition efficacy was determined by the following formula:

Dry weight in control-Dry weight in treatment

Inhibition efficacy (%)=^{-------------------------------------------------------------------------}×100

Dry weight in control

Dual liquid assay:

Dual liquid assay carried out to determine the biocontrol potential of respective treatment against Alternaria alternata. In this assay, 100ml of Potato dextrose broth was prepared in 250ml conical flask sterilized by autoclaving. After sterilization, 0.1ml of spore suspension and 0.1ml of respective treatment was added. Flasks incubated at 30◦C for five days under static condition²⁰.

After the incubation period, the inoculated flask through Whatman filter paper (pre-weighted), kept in a Petri dish followed by incubation at 40^oC (1hour) to determine fungal mycelial weight.

Inhibitory efficacy calculated by the following formula:

Dry weigh in control-Dry weight in treatment

Inhibition efficacy (%)= ---------------------------------------------–x100

Dry weight in control

Fungal hypha fragmentation assay:

Biocontrol potential of respective treatment as described earlier was used. In this method. 0.1ml of Potato dextrose agar was added to the sterile glass slide kept in sterile Petri plate with moisture filter paper. A tuft of fungal mycelia was placed gently on the agar, followed by the addition of 0.1ml of respective treatment. The preparation was kept under room temperature for 30 minutes. After the incubation period, sterile cover slip was gently placed, Petri Plates were incubated at 28^oC for 5 days. After the incubation period, the slides were absorbed under confocal inverted fluorescent microscope. The hyphal fragmentation was recorded in the respective treatment.

Fragmentation efficacy was calculated by the following formula:

Number of hyphae in control -Number of hyphae in treatment

Fragmentation= ------------------------------------------------------- x100

efficacy (%) Number of hyphae in control

RESULTS AND DISCUSSIONS:

Isolation and Identification of organism:

On performance of serial dilution and spread plate technique, different bacterial colonies were observed on the Nutrient agar plates. The pure cultures were streaked on 1% chitin agar plate, showed colonies with promising growth.

Screening of chitinase producers:

The four isolates numbered as SU01, SU02, SU03, SU05 and were subculture in nutrient agar slants in duplicates and stored at 4°C. The colour of the colonies ranged from white to red. Each colony had a clear shape. The colony morphology study was followed by the chitin-hydrolyzing test to confirm the production of chitinase enzyme. All the four isolates subjected to screening by streaking on chitin agar medium when allowed to grow on 1% colloidal chitin demonstrated the zone of chitinolytic following 24 h of incubation at 37^oC.

Storage:

The isolated and confirm organism was grown in Nutrient agar. The strain was also persevered in glycerol stock at -20^oC for further use.

Inoculum Preparation:

Serratia marcescens SU05 maintained as stock culture on NA slants were revived. The growth condition was at room temperature for 2 days by inoculation in 100 ml of minimal media²¹ (NH₄NO₃ – 0.14 g, KH₂PO₄ – 0.2 g, CaCl₂ – 0.03 g, NH₄NO₃ – 0.14 g, K₂HPO₄ – 0.2 g, peptone – 0.7 g, FeSO₄ – 0.50 g MnSO₄ – 0.16 g ZnSO₄ –0.14 g in 100 ml distilled water) at 100 rpm for 48 hours.

Production media:

About 10% inoculum of both the isolates was added in separate flasks, each containing 100 ml synthetic media for checking the Bacitracin production.

Synthetic media contained (g/L), KH₂PO₄ – 0.7, NH₄NO₃ – 0.1, NaCl – 0.2, MgSO₄.7H₂O – 0.15, CuSO₄.5H₂O – 0.2, NH₄Cl – 0.1, Chitin –10 After the incubation, the supernatant was used to estimate the quantity of chitinase present in the medium using DNS and protein assay²³.

Purification:

Ammonium sulphate precipitation:

Ammonium sulphate precipitation method for various concentration it is found that at 80% salt solution the precipitation of chitinase enzyme was found to be maximum (Figure - 1).

Figure 1: Different concentration of ammonium sulphate precipitate

Dialysis:

The enzyme obtain after dialysis is a partially purified enzyme.The activity of partially purified chitinase enzyme was subjected to DNS and Lowry’s method for estimation.

Table 1: Estimation of partially purified enzyme

ASSAY	TEST VALUE
DNS method	1.98
Lowry’s method	0.12

Silica gel chromatography:

The partially purified enzyme was subjected to silica gel chromatography and various fraction was obtain at the time interval of 2 minutes (Figure -2). On further estimation of purified sample showed maximum amount of chitinase was obtain at the time interval of 20 minutes²².

Lane 1: Protein marker

Lane 2: Ammonium sulphate precipitate

Lane 3: Purified enzyme

Figure 2: Silica gel chromatography and SDS PAGE

SDS PAGE of chitinase:

Molecular weight of pure enzyme was found to be 56 kDa (Figure -2).

Biocontrol potential:

Among the different treatment of tested fungi with different formulation, high inhibitory efficacy recorded in partially purified chitinase enzyme (Figure -3).

Figure 3: Inhibitory effect of Serratia marcescens on fungal colonies

Dual plate assay

Dual plate assay showed 20.0, 35.0, 45.0% of inhibition in Serratia marcescens cells, crude enzyme and ammonium sulphate precipitate respectively.

Dual liquid assay:

As in dual plate assay, distinct reduction in fungal mycelia recorded in enzyme treatment. Dry weight of fungal mycelia found to reduce at maximum level in ammonium sulphate precipitate treatment followed by culture supernatant. Less reduction observed in Serratia marcescens cell treatment. Mycelial dry weight of fungi in ammonium sulphate precipitate, culture supernatant and Serratia marcescens found to be 12.0, 30.0 and 55.0 mg respectively. Williams et al., 1956 estimated pigments using chromatographic methods²⁴.

Hyphal fragmentation test:

Biocontrol potential of chitinase against Serratia marcescens confirmed by hyphal fragmentation test. It showed in all treatment fungal hyphal fragmentation noticed among the different treatment. Complete fragmentation recorded in ammonium sulphate precipitate followed by crude enzyme. Serratia marcescens cells treatment also showed fungal fragmentation. Fragmentation efficacy found to be 40.0, 60.0, 85.0% in Serratia marcescens cells, crude enzyme and ammonium sulphate precipitate respectively (Figure 4).

Figure 4: Fungal hyphae degradation

Stereo microscopic image:

Biocontrol potential of chitinase was confirmed by stereo microscopic analysis of fungi treated with respective treatment that showed complete degeneration of fungal colonies into the fragments (Figure -5).

Figure 5: Stereo microscopic image of fungal culture (treatment) treated with crude chitinase enzyme

SUMMARY AND CONCLUSION:

Serratia marcescens is efficient of producing enzyme chitinase and degrade chitin at substrate level but still a lot more is required to be studied to harness it at commercial scale and utilized it for the mass scale degradation of chitin wastes generated in the seafood industries. In the following study out of the four isolates screened microorganisms, the isolate SU05 showed maximum chitinase activity and selected for further research. The isolate SU05 was identified as Serratia marcescens using the standard identification parameters like Gram staining followed by the biochemical assays. The crude enzyme is purified by Ammonium sulphate precipitation (the precipitate was obtain in (60% 70% and 80%), dialysis, silica gel (the peak is obtained at 2.04 mg/ml), Sephadex gel (the peak is obtained at 1.96). In addition, its molecular weight is determined by SDS PAGE was 56 kDa. Biocontrol potential of chitinase showed effective inhibition of the fungal organism by all the tested techniques that helpful in agriculture sector to prevent plant pathogenic fungal organism.

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Received on 22.10.2019 Modified on 29.01.2020

Research J. Pharm. and Tech. 2021; 14(5):2412-2416.

DOI: 10.52711/0974-360X.2021.00425