IJCRR

IJCRR - 14(11), June, 2022

Pages: 31-36

Date of Publication: 03-Jun-2022

Analysis and Characterization of Chitinase in Bacillus salmalaya Strain 139SI

Author: Abdulkhaliq J. Alsalman, Arshad Farid, Mohammed Al Mohaini, Maitham A. Al Hawaj, Muhammad Muzammal, Muhammad Hashim Khan, Arezoo Dadrasnia, Yousef N. Alhashem, Shakira Ghazanfar, Eman M. Almusalami, T

Category: Healthcare

Abstract:Introduction: Chitinases are enzymes that hydrolyze the internal ?-1,4 glycosidic linkages of chitin, a significant structural component of arthropod exoskeletons and fungus cell walls. Objective: Main objective of current study was to determine the protein and to check the antimicrobial, antifungal activity of chitinase in Bacillus salmalaya strain 139SI. Methodology: Purified and estimation enzymes were quantified by the method of Lowry method. Antimicrobial action of chitinase was done using standard Disc Diffusion method while the hyphal extension inhibition assay was used to test the antifungal activity of pure chitinase strains 139SI, 140SI, and 141SI. Results: In this study, Bacillus salmalaya 139SIexhibited strong hemolytic activity and their protein concentration was measured as 56.43 mg/mL. In addition, strain 139SI had strong antifungal activity against phytopathogenic fungus including Fusarium sp., R. solani and Phytophthora sp. Strain 139SI had the capability in degrading the peptidoglycan component of cell walls of gram-negative bacteria such as Escherichia coli but not against the gram-positive, Staphylococcus aureus. Chitinase activity was observed when 200?l crude extract of 139SI able to degrade 0.09 g chitin of shrimp shell by breaking down shrimp shell structure and bonds of chitin effectively as early as 2 days or up to 7 days. Conclusion: Hence, based on the results, B. salmalaya 139SI has potential to be a novel biofunctional chitinase that could use as a biological agent in degrading the chitin component of fungal cell walls and shells waste of many kind of insect and crustaceans for solving future problem in the agricultural sector and fishery industry.

Keywords: Chitinases, B. Salmalaya, Biofunctional, Peptidoglycan, Antimicrobial, Antifungal activity

Full Text:

Introduction

Many diseases, affect agricultural crops, resulting in considerable productivity losses including fungal infections.¹ The cell wall of the fungus is the first structure that comes in contact with the host. Chitin is a homopolymer of N-acetylglucosamine (GlcNAc) units that makes up the major component of the fungal cell wall.² As chitin is a major cell wall component of most pathogenic fungi, chitinase-producing bacteria can break down chitin, which can be employed as a biological control of fungal infection in crops. Chitinase also has been proven in as a biocontrol agent in degrading chitin in the exoskeleton of white fly.³

Chitin is a crucial part in the construction of fungal cell walls, as well as other vertebrates (fish scales) and invertebrates (mollusks, nematodes, worms, arthropods, cephalopods, etc.).⁴ Chitinase is a hydrolytic enzyme and belongs to the glycosyl-hydrolase family that hydrolyzes glycosidic links in chitin and converts polymeric chitin to chitooligosaccharides.⁵

Furthermore, seafood processing businesses, notably those in the processing foods industry, exploit marine resources to meet humanity's needs, resulting in massive amounts of chitin-containing waste in coastal areas.⁶ Despite its gigantic abundance, utilization of chitinous waste such as crab, shrimp, and lobster is still in its primitive stage due to the crystallinity and insolubility of chitin itself. Chitinase is capable in breakage of bonds typical to that of chitin in shrimp therefore, the degradation of chitin crustacean waste by chitinase enzyme is one of the industrial interest because it can be used as bioremediation of seafood waste at a large scale and has green characteristics, not harm the environments.⁶ The main objective of this study was to analyze and characterize chitinase activities in B. salmalaya strain 139SI. To analyze chitinase activities in B. salmalaya 139SI, 140SI and 141SI strains and their relationship with chitinase enzyme purification and observe the potential of the strains against various types of fungi and bacteria. To test the capability of the strains in degrading chitin-composed material such as shrimp shells.

Materials and Methodology

Isolation and screening of the strain

Bacillus salmalaya 139SI was first discovered in soil from a private farm in Selangor, Malaysia.⁷

Growth condition and Chitinase production

A single colony of 139SI, 140SI and 141SI from a Brain Heart infusion(BHI) agar plate was inoculated and grown in 1 litre of Difco™, USA.BHI medium containing potassium chloride (5 g/L), dextrose (3 g/L), disodium hydrogen phosphate (2.5 g/L), gelatin (14.5 g/L), BHI (6 g/L), and peptic digest of animal tissue (6 g/L) plus 1 percent (w/v) chitin powder from shrimp shell as inducer was shaken in a shaking incubator at 150 rpm for 72 hours at 35 °C. The 72-hour culture was centrifuged (8000×g for 15 minutes) and filtered through a Whatman no.1 filter using a SORVALL ST 16R centrifuge. The cell-free broth or supernatant was concentrated by freeze-drying (at the Microbiology laboratory, near PASUM) and was stored at −20 °C.

Protein determination

Purified and estimation enzymes were quantified by the method of Lowry method.⁸ Bovine serum albumin was used as standard. Firstly, Lowry reagent or alkaline copper sulfate solution was prepared by adding and mixing 50 ml solution A (2% sodium carbonate was added into 0.1 M NaOH) with 1 ml of solution B (0.5% copper sulfate was added into 1% sodium potassium tartrate solution). Then, Standard Protein Solution (BSA) was prepared by dissolving 200 mg of BSA into 100 ml of distilled water and diluting this stock BSA into 100 ml of distilled water. Finally, the solution was prepared and its absorbance was measured at 700 nm using the NV203 spectrophotometer.

In the test tubes, different dilutions of BSA solutions were prepared by mixing stock BSA solution (1 mg/ ml) and water as shown in table 1. The BSA concentration ranges from 0.05 to 1 mg/ml. 5 mL alkaline copper sulfate reagent was added to these various dilutions. After incubating for 10 minutes at room temperature, added 0.5 ml of reagent FolinCiocalteau solution (reagent solutions) to each tube and incubated again for 30 minutes or until the solution becomes blue. Each of the test tubes has a final volume of 6.5 ml. A standard calibration curve was created by plotting the absorbance against the protein concentration graph.

Assay of purified chitinase for antifungal 

The hyphal extension inhibition assay was used to test the antifungal activity of pure chitinase strains 139SI, 140SI, and 141SI.⁹

Antimicrobial action of chitinase

The antimicrobial action of Chintinase was done using the standard Disc Diffusion method.¹⁰

100ug/ml original extract of chitinase crust 139SI, 140SI and 141SI were dropped onto disc (6.0/mm). Then, they placed 25mm perimeter from each other onto bacteria culture along with positive and negative control dick. The inoculated plates were cooled to allow the bioactive compounds to diffuse into the agar, then incubated at 37°C and checked for inhibitory zones around the wells after 24, 48, and 72 hours. A ruler was used to measure inhibition zones in millimeters under and around discs.

Effect of crude chitinase prawn shell

Two prawn shell were prepared and treated with different concentrations of purified chitinase of strain 139. Prawn shell 1 was treated with 200ug/ml of strain 139SI, while prawn shell 2 was treated with 400ug/ml of strain 139SI. Then, shell structure was observed in days 1, 3,7,14,21,28 and 41 days to identify the ability of chitinase in degradation action under a 1000x magnification compound microscope (NIKON 261871).

Results

Chitinase production

Chitinase crust (in powder form) was produced and extracted from 25ml of bacterial supernatant of each strain of Bacillussalmalaya after being freeze-dried. Strain 141SI produced a large amount which is 1.19g compared with strain 1140SI (1.06g) and 139SI (0.89g), respectively.

Enzyme purification

The concentration of chitinase of each strain plotted against absorbance at 700nm on the BSA standard graph is highlighted in figure 1. The concentration of purified protein of strain 139SI was 87.86 Mg ml ^-1 and its absorbance was 0.464 O.D. While, concentrations of purified protein of strain 140SI and 141SI were 82.70 Mg ml ^-1 and 56.43 Mg ml ^-1 and their absorbance were 0.435 O.D and 0.288 O.D, respectively. Based on the result, the regularity of purified enzyme chitinase in each sample can be simplified as shown; 139SI>141SI>140SI.

Antifungal

Based on Figure 2(a & b), the results showed that 100ug/ml chitinase could inhibit the growth of R. solani. The mean and standard error of the inhibition zone of strain 139SI within 2 days was 15 ± 9.90, while strain 140SI and 141 SI were 11.5 ± 4.95 and 13.5 ± 6.36, respectively. The inhibitory indices of all samples have a certain regularity against R. solani, which is 139SI> 141SI > 140SI.

Based on Figure 2(c & d), the results showed that 100μg/ml of all purified chitinase could inhibit the mycelial growth of Phytophthora sp. The mean and standard error of the inhibition zone of strain 139SI within 2 days was 19 ± 10.61, while strain 140SI and 141 SI were 16.5 ± 10.61 and  16.5 ± 7.78, respectively. The inhibitory indices of all samples have a certain regularity against Phytophthora sp. mycelial growth which is 139SI> 141SI > 140SI.

Based on Figure 2 (e &f), the results showed that 100μg/ml of each purified chitinase could inhibit the growth of Fusarium sp. The mean and standard error of the inhibition zone of strain 139SI within 2 days was 15±11.31 while strain 140SI and 141 SI were  11.5±7.78 and  13.5±9.19, respectively. The inhibitory indices of all samples have a certain regularity against Fusarium sp. mycelial growth which is 139SI> 141SI > 140SI.

Overview of the mean and standard error of purified chitinase in inhibiting the mycelial growth of three different types of phytopathogenic fungi; R.solani, Phytophthora sp., Fusarium sp. are shown in Table 2.

Antibacterial

100ug/ml of purified chitinase of each strain 139SI, 140SI and 141SI had been tested against S. aureus (gram +ve) and E. coli (gram -ve). Based on the observation after 24 hours, all purified chitinase were resistant towards S. aureus while, strain 139SI was more sensitive compared to strains 140SI and 141SI against E. coli (Figure 3 a & b).

Effect of crude chitinase on prawn waste

Two prawn shell were treated with different concentrations of chitinase 139SI. Prawn shell that was treated with 200ug/ml of chitinase was started to degrade at day 7 and their mass. While, prawn shell treated with 400ug/ml of chitinase was degraded as early as day 3 and their shell part obviously shrunken at day 7 (figure 4 a, b,c,d & e).

Discussion:

Enzyme purification or protein measurement Lowry assay was used to measure the amount of purified chitinase concentration in each strain. The quantity of protein was calculated using a BSA standard curve, and the advantages of this test include its sensitivity and, most significantly, accuracy.¹¹ Crust chitinase of each sample was diluted twice before being measured at 700nm. Based on the result obtained from BSA standard graph (figure1), strain 139SI has the highest amount of purified chitinase enzyme concentration which is, 87.86 Mg ml^-1 at 0.464 O.D. Strain 140SI contained 82.70 Mg ml^-1 of chitinase enzyme concentration at 0.435 O.D. While strain 141SI only has 56.43 Mg ml^-1 of purified chitinase at 0.288 O.D even though, it produced the highest amount of chitinase crust (powder form) in the previous assay (freeze-drying assay). Hence, it proved that the chitinase crust of 141SI was not pure compared to 139SI. The reason might be due to the chitinase crust being dominated by other substances. Increased concentration of crude extracts will be accompanied by the enhancement of cell viability.⁷ Meanwhile, the highest concentration of chitinase enzyme will lead to increases in performance, especially against various types of fungi, and bacteria and the ability in degrade chitin-composed material.

Pathogenic fungi can cause disease in humans and animals and contamination and severe damage to crops, which lead to huge economic losses.¹² Significant growth retardation in the mycelial growth of R. solani, Fusarium sp. and phytophthora sp. by strain 139SI was observed. 139SI showed strong antifungal activity against all these types of fungi as strong as positive control inhibits fungal growth. While the zone of inhibition was less around the wells when applied with strain 141SI and followed with strain 140SI. The antifungal activity indices of all samples have a certain regularity, which is 139SI> 141SI > 140SI. Results above demonstrated inhibitory rates and antifungal activity increase with the rise of the concentration of purified chitinase. As the 139SI has the highest concentration of chitinase enzyme (87.86 mg/ml), hence it is more efficient in inhibiting all chitin-containing fungi. Antifungal activities must be along with hyphae distortion, heavy vacuolization, and swelling and lysing hyphae.¹³ It can be inferred that the over-expression of recombinant chitinase protein or enzyme has a strong and considerable antifungal effect.

Among all those three types of pathogenic fungi, 139SI was more sensitive towards phytophthora sp. and less effective against Fusarium sp. especially after 24 h incubation. Figure 2 depicts the inhibition zone of all pure chitinases, including 139SI, which ranges from 6 to 7 mm only after 24 hours. Therefore, the degree of inhibition is proportional to the amount of chitin in the cell wall of the target fungus. Chitinolytic enzymes, as well as the genes that code for them, could be used to create transgenic microorganisms with improved biocontrol capabilities and could be used to control fungal plant pathogens.¹³ The appearance of unambiguous zones of inhibition confirmed and identified antibacterial activity. Purified chitinase was used against two species of bacteria (S. aureus and E. coli), and microorganisms were considered positive for bioactive substances if an inhibitory zone of at least 8 mm wide was detected around the disc.¹⁴ Because B. salmalaya is a gram-positive bacterium, it showed no inhibition zone or antibacterial activity against S. aureus which is also a gram+ve bacteria and causes a wide range of infections ranging from skin infections to life-threatening diseases.¹⁴ Penicillin was employed as a positive control, with a 27mm inhibition zone.

While gram-negative bacteria such as E. coli were inhibited by 100ug/ml pure chitinase strain. Strains 139SI, 140SI, and 141SI had 12mm, 11mm, and 10mm inhibition zones against E. coli agar plate culture, respectively. As the concentration of pure protein in strain 139SI increased, the highest antibacterial activity was seen. 140SI, on the other hand, was less sensitive to E. coli due to a decrease in pure chitinase. Ampicillin was utilized as a positive control; however, it was shown to be resistant to E. coli. Resistance to β -lactam antimicrobial drugs in E. coli has been proven to be ineffective against pathogenic E. coli, which causes diarrhea, meningitis, and urinary tract infections, among other clinical syndromes.¹⁵

This study showed the degradation of chitin-composed material and action mechanisms of the chitinase enzyme was related to the volume of treatment given on prawn shells. The highest concentration of purified chitinase was given on prawn shell, hence the enzyme action mechanisms in hydrolyze chitin, a linear polymer of β-(1,4)-linked N-acetylglucosamine (NAG),¹⁶ was more effective. This fits the result obtained in this study where 400ug/ml of purified chitinase was more effective in degrading the chitin of the prawn shell as their cracked can be seen as early at day 3 and obviously shrunken at day 7 compared to the prawn shell that was treated with 200ug/ml chitinase, which their degrading part only appear at day 7 and shrunken, not obvious. The chitin found in the peritrophic matrix and the interior layers of exoskeletons of crustaceans such as prawns, shrimp, and crabs provide support for the muscle system as well as growth and development.¹⁷ Chitinase enzymes can directly degrade their chitin-containing structures by breaking down a typical bond that binds with chitin and as crustacean shells especially on prawn shell as a major carbon or nitrogen source for the production of chitinase.¹⁸ As a result, it is great for bioremediation and waste management, as well as releasing nutrients and keeping the carbon, nitrogen, and other biogeochemical cycles in check.¹⁹

Conclusion

Chitinase is an enzyme that aids in the breakdown of chitin-based materials. Among all the Bacillus salmalaya strains tested, 139SI, which has strong hemolytic activity, showed the best and strongest antifungal activity against three types of phytopathogenic fungi, including Fusarium sp., Phytophthora sp., and R. solani, suggesting that it could be used in the field to combat plant pathogenic fungi. This work revealed that strain 139SI also has capable in degrading peptidoglycan of gram-negative bacteria such as Escherichia coli and degrading prawn shell structure at a concentration of 400ug/ml of chitinase enzyme. Increased concentration of crude extracts will be accompanied by the enhancement of cell viability. As the 139SI has the highest concentration of purified chitinase enzyme (87.86 mg/ml), hence their performance was increased especially against various types of fungus, bacteria, and degrading chitin-composed material. However, a larger scale trial is needed in the future for the degradation of marine waste as chitinase enzymes have the potential to be used as a biocontrol agents in controlling plant diseases.

Acknowledgment

The authors gratefully acknowledge Nur Huza Aziera Binti Mohamad Huzairo (Institute of Biological Sciences, Faculty of Science, University of Malaya, Malaysia) for experimentation assistance and Institute of Biological Sciences, Faculty of Science, University of Malaya, Malaysia, for providing financial support and lab facility for this research work.

Source of funding: This research received no external funding.

Conflict of Interest: The authors declare no conflict of interest.

Authors’ Contribution: All authors contributed equally

Figure 2 (a) Effect of purified chitinase 139SI on mycelial growth of R. solani after 24h incubated. (b)  Effect of purified chitinase on mycelial growth of  R. solani after 48h incubated.(c) Purified chitinase against Phytophthora sp. After incubated 24 h. (d) Purified chitinase against Phytophthora sp. 48h. (e) Effect of purified chitinase on mycelial growth of Fusarium sp. after 24h incubated. (f) Effect of purified chitinase on mycelial growth of Fusarium sp. after 48h incubated.

Figure 3: (a) Agar plate is shown strain139SI,140SI and 141SI not sensitive to the tested gram-positive bacteria, S. aureus after incubated 24 h. (b) Strain 139SI has larger antibacterial zone inhibition (12mm) against gram-negative bacteria, E.coli, compared to both strains of 140SI(10mm) and 141SI (11mm) strain.

Figure 4. (a)Prawn shell 1 before treatment at 0 days. The red circle showed the original shape and structure before being treated and degrade by the chitinase enzyme. (b) The red circle showed the degradation area of prawn shell 1 after 7 days; reduction in shell prawn chitin material by 200ug/ml chitinase enzyme from the strain 139SI (c) Prawn shell 2, before treatment at day 0. Red circle showed the original shape and structure before treatment and degrade by chitinase enzyme strain139SI. (d) The red circle showed prawn shell 2 part was degraded by 400ug/ml purified chitinase strain139SI as early as 3 days after treatment. (e) Red circle showed prawn shell 2 part was obviously shrunken at day 7.

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