IJCRR

IJCRR - 14(11), June, 2022

Pages: 11-17

Date of Publication: 03-Jun-2022

Chitinase Activity by Chitin Degrading Strain (Bacillus Salmalaya) in Shrimp Waste

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

Category: Healthcare

Abstract:Introduction: Chitin degradation by chitinase enzyme can be used on a large scale for bioremediation of seafood waste and is environmentally friendly. Objective: The main aim of this study was to screen the potential of the strain as a chitin degrading agent. Methods: In this present study, the cell-free supernatant of Bacillus salmalaya was determined for its protein concentration. Bacillus salmalaya139SI was isolated from agricultural soil and it was identified by staining technique and colony morphology. The production of chitinase by Bacillus salmalaya139SI was optimized under different concentrations of substrate, pH and temperature. Results: Strain 139SI exhibited strong hemolytic activity and the crude protein concentration of Bacillus salmalaya was 84.09mg/ mL with OD value 0.462. Strain 139SI were also screened on colloidal chitin agar medium supplemented with mineral salt. Chikinase production was determined by clear zones of hydrolysis produced after 7 days of incubation at 37\?C. The maximum chitinase production was observed in Brain Heart Infusion broth supplemented with 1.0% colloidal chitin at pH 7 and temperature 35\?C after four days of incubation. Chitinase activity was observed when high concentrations of crude extract of 139SI able to degrade shrimp shell by showing the degradation zone at day 4. Conclusion: From the results, we concluded that the Bacillus salmalayahas potential to be a biofunctional chitinase that could degrade complex polysaccharides present in the organic wastes and applicable in cleaning the environment..

Keywords: Shrimp, Chitinase, Hemolytic activity, Bacillus salmalaya, Chitin, Degradation

Full Text:

Introduction

Shrimp has constituted a primary phase of crustacean consumption in the present years. An increase in shrimp waste is unavoidable owing to the increased amount of consumption. Shrimp waste is considered an imperative source of chitin. A critical sum of shrimp waste is delivered in Asia essentially in Thailand and India.¹ Solid waste, consisting of the head, shell, and tail portions, accumulates owing to shrimp processing. The waste composed of the cephalothoraxes and exoskeleton ² accounts for 50-70% of the weight of the raw material and is usually discarded. The recycling of chitinous waste is extremely important to keep the carbon-nitrogen balance in the ecosystem.²

Chitin is a sugar-like polymer and is available at a low cost. It appears to be safe for use in humans in the long term and has very low toxicity. Chitin plays a protective role in many lower eukaryotes similar to that of cellulose in plants. Metabolism of chitin in synthesis and degradation is essential for different morphogenesis.² Chitin occurs as crystalline microfibrils in nature. It can be found as the main component in the construction processing of fungal cell walls, Mollusca radula, nematode eggshells, worm and arthropod exoskeletons, cephalopod beaks, and fish scales.³ The absence of chitin in vertebrates and plants makes the chitin metabolism potentially useful parasite-specific targets for chemotherapeutic attack.¹

Marine bacteria for survival in aquatic ecosystems rapidly catabolize chitin. Some bacteria are producing chitinases probably to hydrolyze the diversity of chitins found in nature. As stated by Okonkoet al. (2006) several strains of microorganisms have been selected or genetically modified to increase the efficiency with which they produce enzymes.⁴ Chitinases are a large and diverse group of enzymes with different molecular structures, substrate specificity, and catalytic mechanisms. Many bacteria, including Serratia and Bacillus, produce four different chitinases, whereas filamentous fungi produce up to 20 different chitinases. As a result, there has been an increase in demand for chitin derivatives produced by the action of chitinases on chitin polymer for a variety of industrial, clinical, and pharmaceutical applications.⁴

Bacteria are thought to be the primary mediators of chitin degradation in nature. Their importance can be seen in both soil and water systems. The rate of chitin hydrolysis in soil systems is related to the bacterial population and abundance.⁵ In addition, the degradation process also depends on factors such as temperature and pH. Thus, the degradation of chitin by the chitinase enzyme can be used as bioremediation of seafood waste at a large scale and eco-friendly to the environment.⁶

The main aim of the current study was to analyze and screen the potential strain as a chitin-degrading agent. Therefore, the specific objectives were to screen the potential of Bacillus salmalaya in hydrolysis activity, to optimize cultural conditions for the production of chitinases like concentration, pH, and temperature, and to screen the potential of the strain as chitin degrading agent on shrimp shell.

Methodology.

Bacillus salmalaya139SI

Bacillus salmalaya139SI was originally isolated from soil obtained from a private farm in Selangor, Malaysia (2.99917°N 101.70778°E).⁷

Chitin from shrimp shells

Practical grade, chitin powder (Sigma, USA) was used and modified to form colloidal chitin as it is more homogenous distribution in agar media and act as a primary carbon source to bacteria in a medium for analysis of chitinase activity.

Growth condition and Chitinase production.

A single colony of 139SI, from the BHI agar plate, was cultured in 1 L of Difco ™, USA.Brain–Heart Infusion (BHI) medium containing 5 g/L KCl, 3 g/L dextroses, 2.5 g/L Na₂HPO₄, 14.5 g/L gelatin, 6 g/L BHI, and 6 g/L peptic digest of animal tissue with additional 1% (w/v) chitin powder from shrimp shell as an inducer in a shaking incubator at 150 rpm for 72 h at 35°C.The incubation continued until the OD₆₀₀ equaled 1. The 7 days culture was centrifuged (8000 × g for 20 min) by using (SORVALL ST 16R) centrifuge and filtered through a sterile syringe filter with a pore size of 0.2µm (Minisart syringe filter) to remove all particles and dead microorganisms without any influence on their ingredients. The supernatant formed was a crude extract of the chitinase enzyme. The cell-free broth or supernatant was concentrated by freeze-drying and was stored at −20 °C.

Estimation of protein content

Purified and estimation enzymes were estimated by the following method of Lowry et al. (1951)⁸ using Bovine serum albumin as the standard.⁹

Preparation of colloidal chitin

Colloidal chitin was prepared from chitin powder (sigmaChemicals Company, USA) by the modified method of Hsu and Lockwood.¹⁰

Preparation of chitin agar medium

The chitin medium was prepared using moist form colloidal chitin based on the modified protocols combining elements from Hsu et al. and Ram?rez et al.^10,11

Plate screening of Chitinase activity

Bacillus salmalaya139SI colonies from the plates were sub-cultured on Brain heart infusion broth (BHI; Difco) and incubated at 30°C for 24 hours. Later, a few drops of culture was placed onto the surface of BHI agar and spread using a sterile spreader or sterile cotton swab. This was incubated at 30°C for overnight. After incubation, the confluent growth of the culture was inoculated onto chitin agar using spot inoculation. Plates were incubated at 30°C for about a week (7 days). Following incubation, the bacterial cultures were observed for the production of chitinase. On the chitin agar, the clear zone of inhibition was noted around the colonies and was calculated using a Vernier caliper in (cm) and after 3 days of incubation, the chitinolytic index was calculated. The chitinolytic index was calculated using the below equation:

Optimization of cultural condition

Effect of substrate concentrations on chitinase production

Bacillus salmalaya strain 139SI was cultivated in different concentrations (0.3, 0.5, 0.8, and 1.0 %) of colloidal chitin enhance with minimal medium to determine the optimum concentration of colloidal chitin for chitinase production.

Effect of pH value on the chitinase production

Bacillus salmalaya strain 139SI was grown at a different pH ranges of the culture medium from 4 to 10. HCL was used for pH 4; phosphate buffer was used for pH 7 and NaOH for pH 10 in a minimal medium containing 1.0% colloidal chitin to determine the optimum pH for chitinase production.

Effect of temperature on the chitinase production

To determine the optimum temperature for chitinase production, Bacillus salmalaya strain 139SI was grown in the culture medium containing 1.0% colloidal chitin and incubated at different temperatures of 25°C, 30°C, 35°C, 40°C and 45°C up to 4 days.

The reaction of crude chitinase on shrimp shell

A number of prawn shells were prepared and treated with different concentrations of chitinase of strain139SI. Then, shell was observed in a month to identify the ability of chitinase in degradation action under a microscope (OLYMPUS SZ40).

Results:

Hemolytic activity

Bacillus salmalaya strain 139SIshowed a positive result as appears clear zone around the bacterial colony accompanied by lightened yellowish discoloration of the medium as in figure 1a. The complete lysis exhibits that strain 139SI has strong Beta (β) hemolysis because it could produce hemolysin substance, which is bacterial protein breakdown of the hemoglobin of the red blood cells and disrupting the structure of the membrane or punching a hole through the membrane.

Estimation of protein content

Bacillus salmalaya strain 139SI produced 0.32g of chitinase crust. The amount might be slightly not exactly as we used an old freeze-drying machine. Thus, there were some errors or mistakes that happened during freeze drying for instance incomplete drying since the process of removing moisture not working well. Based on the results from the BSA standard graph (figure 1b), Bacillus salmalaya strain 139SI has a high amount of crude protein concentration which is 84.09 mg/mL with an OD value of 0.462. Therefore, it proved chitinase crust of strain 139SI was pure. That might be due to the chitinase crust being influenced by other substances.

Plate screening of chitinase activity

In the primary screening of chitinase activity as shown in figure 2, Bacillus salmalaya139SI produced a prominent zone of hydrolysis on the colloidal chitin agar. The clear zone around the spot inoculation was increasing in diameter until day 7 with chitinolytic index 7. This indicated strain 139SI has a chitinolytic activity to break down the chitin compounds in the medium.

Effect of substrate concentrations on chitinase production.

Among the four different concentrations tested, the results showed Bacillus salmalaya strain 139SI produce chitinase maximally at the concentration of 1% of colloidal chitin with absorbance value (1.845±0.092), followed by colloidal chitin at 0.8% (1.541±0.103), 0.5% (1.418±0.068). Beyond 0.5%, the substrate concentration decreased enzyme activity (Table 1). But above 0.5% of colloidal chitin concentration, chitinase production was significantly increased.

Effect of pH value and temperature on the chitinase production.

In order to evaluate the effect of pH of the media on the chitinase production, Bacillus salmalaya139SI were grown at different pH (4, 7, and 10). The optimal pH for chitinase production was examined when kept at 37°C. Among the tested pH, neutral condition pH 7 (1.318±0.029) supported the maximum chitinase production. In pH 4, chitinase production was 0.235±0.040 followed by pH 10 was 0.297±0.135 (Table 2). Chitinase production was relatively stable at pH 7. However, for alkaline and acidic conditions, it rapidly lost its chitinase production. In addition, from the observation of the broth culture medium, the culture broth for acidic and alkaline are slightly clear compared to pH 7 medium that are cloudier which indicates cell growth. This test, it showed an excessively high or low pH led to poor cell growth and chitinase production.

Data presented in table 3 clearly indicated incubation temperature affects many biological processes, including the growth rate and enzyme production. The organism exhibited good growth as well as chitinase production at 35°C with an average of 1.732±0.072. The incubation temperature 30°C (1.460±0.036) also found to influence the chitinase production. It has been observed that in both lower and higher temperatures chitinase production decreased.

The reaction of crude chitinase on shrimp shells

Five different concentrations of crude chitinase were tested on the shrimp shell to check the chitin degradation ability. Among the five concentrations, 200 mg/ml until 300 mg/ml of crude chitinase was more effective in degrading ability compared to shrimp shell treated with lower concentration. This is because the degradation activity can be seen as early as day 4 and more clearly seen in the following week. At the end of the month, all concentrations showed the potential of degradation activity (Figure 3). In this study, a high concentration of crude chitinase was used to get the result within a month and also high concentration will produce more effective enzyme action mechanism in hydrolyzing the chitin. Thus, this study showed the degradation of chitin-composed material and action mechanisms of the chitinase enzyme were associated with the volume of the treatment given on the shrimp shell.

Discussion:

Hemolytic or also referred to as hemolysis is the breakdown of the membrane of red blood cells by a substance known as hemolysin. A hemolysin is a group of bacterial proteins, which causes the lysis of the red blood cell (RBC) membrane in the growth substrate ¹². Many types of bacteria possess hemolytic proteins. These proteins act by desegregating into the membrane of RBC and disrupting the structure of the membrane.¹² Bacteria are differentiated based on their hemolytic properties. In the study by Dadrasnia and Salmah (2015), Bacillus salmalaya was found to be a potential degrader of crude oil waste. In the study, haemolytic activity for Bacillus salmalaya139SI was detected as the presence of a definite clear zone around the colony.⁷ In the other finding by the same authors, Bacillus salmalaya139SI also identified as biosurfactant bacteria. As is known, biosurfactant bacteria were all positive in hemolytic activity as it is an initial test that has always been used to identify biosurfactant-producing bacteria. Therefore, hemolytic activity appears to be a good criterion in screening in the search for surfactant-producing strains. As reported by Abu et al., (2018), the bacterial colonies that were streaked on blood agar medium exhibited g β-hemolytic activities and were found to have the potential to produce bioflocculant to remove organic matter.¹³ Thus, isolated Bacillus salmalaya139SI were represented as members of a novel species of the genus Paenibacillus based on the hemolytic activities⁷ and also the ability to bind efficiently and degrade animal or human hemoglobin that could provide an effective heme source to ensure its successful growth and proliferation in vivo.^13,14

In 2004, Stoykovet al.¹⁵ mentioned that the expression of the inducer can generate of a signal and increase in the production of chitinase. It also has been reported by Sato and Araki, (2008) showed the significance of medium composition for the production of chitinase from Bacillus cereus by supplementing the medium directly with chitin.¹⁶ The culture medium was incubated until reached OD value equaled 1 because it was the highest value culture density (cell number) of the strain with a constant growth rate, thereby exhibiting a maximum chitinase production. In the study by Salmah and Dadrasnia (2015), the maximum production of biosurfactant was also measured until the OD value equaled to 1 and was found to be a potential degrader of crude oil waste.⁷

In the study by Budi et al., (2000), Paenibacillus species have been reported to have a high activity of cell wall degrading enzymes and chitinase, making this species commonly applied as biocontrol agents.³ To give comprehensive proof, Kumar et al., (2012) mentioned that different species of Bacillus have been reported to produce chitinase and the result from their findings, Bacillus amyloliquefaciens SM3 produced the highest chitinolytic activity compared to other isolated chitinolytic bacteria.¹⁷ However, in the study by ¹⁸ he reported different strains will give different abilities to secrete extracellular degradative enzymes.

Results of this research found that Bacillus salmalaya strain 139SI has the potential to be a mediator of chitin degradation and may be useful for biotechnological applications and the production of transgenic microorganisms with superior biocontrol capabilities. Chitin degradation could therefore be explored as a general model for understanding microbial degradation of biopolymers in the biosphere.

In this study, a high concentration of crude chitinase was used to get the result within a month, and also high concentration will produce a more effective enzyme action mechanism in hydrolyzing the chitin. Thus, this study showed the degradation of chitin-composed material and action mechanisms of the chitinase enzyme were associated with the volume of the treatment given on the shrimp shell.

In the study by Sorokulova et al., (2009), they reported that the B. cereus strain performed better in shell waste decomposition and was used for large-scale fermentation in 12 L of 10% shrimp shell waste broth.¹⁹ The similar report supported by Abirami et al., 2016 observed that Bacillus licheniformisSSCL10 rapidly degrade the shrimp shell completely within 12 days while another isolate of Bacillus subtilis took more days for degradation activity.²⁰ From the result obtained in this study, chitinase from Bacillus salmalaya139SI has efficiency in hydrolytic activity and it can also be used for the degradation of other chitin materials. Therefore, chitin degradation needs to be explored as a general model for understanding microbial degradation of biopolymers in the biosphere.

Conclusion

Chitinase is ubiquitous proteins that are widely distributed among all kingdoms of life. Chitinase as the name indicates is involved in the breakdown of chitin. The result concluded that Bacillus salmalaya are novel mesophilic bacterial strains that have strong hemolytic activity showing the best ability to produce a huge amount of chitinase in a short time. This enzyme may also be useful in the management of seafood waste industries. Colloidal chitin as the sole source of carbon can prove to be economical in terms of fermentation expenditure. Neutral pH along with a temperature around 35°C facilitates the highest yield. This work revealed that strain 139SI was also effective in hydrolyzing chitin medium and degrading shrimp shells at concentrations of 200, 250, and 300mg/ml of crude chitinase. Bacillus salmalaya139SI makes it a potential candidate for the bioremediation of seafood waste at a large scale. Strain 139SI performance was increased since it has the highest concentration of protein chitinase enzyme. However, much attention and research are needed for multiple potential applications in the future such as nanobiotechnology applications involving drug and gene delivery or in agriculture, food, and environmental protection.

Acknowledgments: The authors gratefully acknowledge Izzah Hazwani Binti Razali (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 or self-funding.

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

Authors’ Contribution: All authors contributed equally.

Figure 1 (a) Red arrow showed a clear zone produced by pure colonies of 139SI after 24h incubated at 37°C under aerobic conditions. Bacillus salmalaya strain 139SI has strong hemolytic activity on the blood agar medium (b) figure showing the concentration of chitinase of strain 139SI plotted against absorbance at 700nm on BSA standard graph. The crude protein concentration of Bacillus salmalaya139SI was 84.09 mg/mL and its absorbance was 0.462 O.D.

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