INTRODUCTION:

Leiognathus fish and bacterial partnerships play a crucial role in both potentially dangerous infections and advantageous symbiotic microbes. Marine microorganisms have been shown to have a number of advantageous bioactivities, and synthesis of industrial enzymes¹. Marine microbial enzymes are having unique physiological and metabolic characteristics, efficient nutrient utilization in oligotrophic conditions, and other unique catalytic capabilities². Extra cellular enzymes found in bacteria are often able to break down a variety of insoluble nutrients, including protein, cellulase, and starch, and gives energy³. The most important group of industrial enzymes are proteases, which is having the share of approximately 65% of all industrial uses, including detergents to remove protein-based stains from clothings⁴.

Bacterial, fungal and yeast-derived proteinases are the most prominent among the various types of proteases. Temperature, incubation time, pH level, agitation, media components and inoculum volume play important roles in the production of microorganism’s proteases⁵. Therefore, it would be very important to have enzymes that show optimal activity at extremes of pH, temperature and salinity⁶. The objective of the current study is to investigate enzyme producing potent bacterial isolates such as Pseudomonas stutzeri LE01, Bacillus firmus LE02, Cytobacillus firmus LE03, and Bacillus licheniformis LE04 from a marine fish, Leiognathus equula. Additionally, optimization of protease production in different culture conditions has been studied.

MATERIALS AND METHODS:

Sample Collection:

The ponyfish, Leiognathus equula were collected from Annankovil landing centre (Lat 11°30’N; Lon 79°46’E), Parangipettai, Southeast cost of India, Tamilnadu and was aseptically brought to the Fish Genetics Laboratory at CAS in Marine Biology, Annamalai University.

Isolation of Bacterial Strains:

The gill and dorsal region of the fish was swabbed with sterile cotton plug and mixed with sterile distilled water. Serial dilution was made from this mixture to obtain different concentration of bacterial load. The dilution factors from 10¹– 10⁴ were made from all the dilutions, and 0.1ml was spread into Zobell Marine agar, (Hi-Media) petri plates. The plates were incubated at 37°C for 24hours. The strains were selected based on colony morphology and go for pure culture by streak plate method.

Bacterial Strains Identification:

The bacterial strains were identified based on colony morphology and Gram staining described in Bergey’s manual of systematic bacteriology⁷.The morphologically identified strains further confirmed by 16S-rRNA gene sequencing and BLAST search. The sequences were submitted to NCBI GenBank and got unique accession numbers.

Screening of Isolates for Extracellular Enzyme Production:

Amylase Production:

The amylase enzyme assay was carried out by preparing starch agar plates were autoclaved at 121°C for 15 minutes to get a sterile condition. All the four bacterial strains were streaked in each starch agar plate and incubated for 24hours at 37°C. The results were noted after the incubation period by using 1% iodine as an indicator to observe the zone of clearance.

Protease Production:

The protease enzyme assay was carried out in Skim Milk agar medium autoclaved at 121°C for 15 minutes to get a sterile condition. All the bacterial strains were single streaked at each agar plates separately and incubated at 37°C for 24hours. After which the resulted zone of clearance was noted.

Lipase Production:

Isolated bacterial strains were screened for lipase activity using Tributyrin Agar plate method (TBA). The Tributyrin agar media were prepared and sterilized at 121°C for 15 min. All the four bacterial strains were streaked in each starch agar plate and incubated for 24 hours at 37°C. The results were noted after the incubation period by using 1% iodine as an indicator for the zone of clearance.

Cellulase Production:

Colonies were plated on 1% Carboxy Methyl Cellulase (CMC) agar plates and the plates were flooded with the respective dyes and were incubated with the dye for 10-12 minutes and then indicator wash with congo red. These strains were washed with 1M NaCl solution. The diameter of the clear zone was measured to select the strain with the maximum cellulase production.

Optimization of Protease Production:

The potential bacterial strain, Bacillus firmus LE02 was subjected to the optimization for the extreme protease production. The optimization was done by changing the condition such as salinity, pH and carbon source.

Effect of Sodium Chloride Concentration:

The effect of salinity was assessed by using various concentration of NaCl (1%, 2%, 3%, 4% and 5%) in Skim Milk agar plates under sterile condition after autoclaving at 121°C for 15 minutes for maximum protease production. The results were noted after the incubation period of 24hours at 37°C.

Effect of pH:

The effect of pH was determined by using different pH (5.0, 6.0, 7.0, 8.0 and 9.0) adjusted in Skim Milk agar plates under sterile condition after autoclaving at 121°C for 15 minutes for maximum protease production. The results were noted after the incubation period of 24 hours at 37°C.

Effect of Carbon Source:

The effect of carbon source was determined by using different carbon sources (Glucose, Lactose, Fructose, Sucrose and Maltose) in Skim Milk agar plates under sterile condition after autoclaving at 121°C for 15 minutes for maximum protease production. The results were noted after the incubation period of 24hours at 37°C.

Estimation of Protease Concentration:

The optimum condition such as salinity 1%, pH 9 and lactose 1% was maintained in a single plate of Skim Milk agar to assess the amount of protease production. The concentration of protease produced by Bacillus firmus LE02 was tentatively analyzed by comparing the standard proteinase K (Hi-Media). The standard proteinase K (5µl) was poured into the well of Skim Milk agar plate and incubated for 24hours at 37°C and measured the zone of clearance.

RESULTS:

Isolation, Identification and Screening for Enzyme Production:

Four dominant colonies were isolated and were maintained in agar slants and stored at -4°C for further studies. The bacterial names were confirmed by the nucleotide BLAST result as Pseudomonas stutzeri LE01, Bacillus firmus LE02, Cytobacillus firmus LE03, and Bacillus licheniformis LE04. The sequence details and NCBI GenBank accession number is given in Table 1. The pure culture of Bacillus firmus LE02 is given in Fig.1.

Table 1. Name of the bacterial isolates

Strain code	GenBank Accession Number	Identified Bacteria
LE01	PP106510	Pseudomonas stutzeri
LE02	PP106518	Bacillus firmus
LE03	PP106527	Cytobacillus firmus
LE04	PP106579	Bacillus licheniformis

Fig. 1. Pure culture of Bacillus firmus LE02

Amylase Production:

The bacterial stains were screened for amylase production and amoung four bacterial strains, Bacillus firmus LE02 showed maximum production of 5mm of zone of clearance, followed by Bacillus licheniformis LE04 (4.5mm), Cytobacillus firmus LE03 (4.3mm) and Pseudomonas stutzeri LE01 (4mm).

Protease Production:

Among the four bacterial strains subjected to protease production, two strains showed positive for protease production and maximum was found in Bacillus firmus LE02 with the zone of clearance of 4 mm and followed by Cytobacillus firmus LE02 (2mm).

Lipase Production:

The lipase production was conducted for all the bacterial strains, only Bacillus firmus LE02 showed protease production with the zone of clearance of 3mm.

Cellulase Production:

Among all the four bacterial strains screened for cellulase production, Bacillus firmus LE02 and Cytobacillus firmus LE02 produced cellulase enzyme with maximum zone of clearance of 3mm and 4mm respectively.

Optimization of Protease Production:

Among all the four strains, the most potential Bacillus firmus LE02 strain was selected for optimization of protease production. Bacillus spp. are the most capable bacteria to produce industrial protease enzyme which account about 65% of industrial sectors as per previous reports.

Effect of Salinity:

The effect of sodium chloride was investigated by changing the NaCl concentration in the culture condition and the protease production was maximum at 1% NaCl concentration with maximum zone of clearance of 5 mm. The result is given in Fig 2.

Fig 2. Protease production by Bacillus firmus LE02 at different NaCl concentration

Effect of pH:

The effect of different pH for maximum production of protease was studied by changing the pH of the culture conditions and showed maximum protease production at pH 9.0 with maximum zone of clearance of 4mm. The result is shown in Fig 3.

Fig. 3. Protease production by Bacillus firmus LE02 at different pH

Effect of Carbon Source:

In this study the effect of different carbon source was used in the culture condition for maximum protease production and maximum production was at 1% lactose with zone of clearance of 4 mm. The result is given in Fig 4.

Fig. 4. Protease production by Bacillus firmus LE02 at different carbon source

Estimation of Protease Concentration:

The optimal condition such as salinity 1%, pH 9.0 and lactose 1% for maximum protease production was done. The results show that the concentration of protease produced by Bacillus firmus LE02 was 0.045mg/ml (Table 2).

Table 2. Comparison of standard proteinase K and protease of Bacillus firmus LE02

Standard/Sample	Concentration (mg/ml)	Zone of clearance (mm)
Standard Proteinase K	0.05	10
Bacillus firmus produced protease	0.045	9

DISCUSSION:

Microbes serves to be a potent source of many industrial enzymes replacing and utilized as an alternative source that replaces chemical approaches. The present study identified four bacterial strains as Pseudomonas stutzeri LE01, Bacillus firmus LE02, Cytobacillus firmus LE03, and Bacillus licheniformis LE04 responsible for producing enzymes. Protease, as a group of enzymes holds significant importance both in terms of their physiological functions and their extensive use in commercial applications⁸. The bacterial strain, Bacillus firmus LE02 and Cytobacillus firmus LE03 resulted positive for protease production. Similarly, Pant et al.⁹ reported that Bacillus substilis produced maximum protease production of 22mm zone of clearance. Similarly,Ananthi et al.¹⁰ isolated Bacillus cereus MSU from Sardinella longiceps produced maximum protease production with the zone of clearance of 17mm. The microbial lipases are highly significant enzymes, occupying a predominant position due to their importance and serving as noteworthy biocatalysts in organic media¹¹. The present study revealed that, Bacillus firmus LE02 as the only lipase producing bacteria with the zone of clearance of 3mm. Saravanakumar et al.¹² reported that, potential lipase producing bacterial isolate from the intestine of Microstomus kitt was found to be Bacillus substilis. During the optimization study, Bacillus firmus LE02 produced maximum protease production at 1% NaCl concentration. Similarly,Patel et al.¹³ isolated a haloalkaliphilic Bacillus sp. which produce maximum protease production at 2% NaCl concentration. Other than Bacillus sp., Lama et al.¹⁴ reported maximum protease production at 2% NaCl concentration by Salinivibrio sp. Maximum protease production in this study resulted at pH 9.0 which is in accordance with the result of Joshi¹⁵ as Bacillus firmus TAP5 produced maximum protease production at pH 9.0. Boominadhan et al.¹⁶ isolated Bacillus sp. resulted in maximum protease production at pH 9.0. Similarly, Olajuyigbe and Ajele¹⁷ reported that maximum protease production was found to be at pH 9.0 with selected bacterial strains such as Bacillus macerans IKBM-11, B. licheniformis IKBL-17 and B. subtilis IKBS-10. Among the five different carbon sources, 1% Lactose was the best carbon source for maximum protease production by Bacillus firmus LE02. Similarly, lactose was found to be a better carbon source for maximum protease production in the study of Karbalaei-Heidari et al⁶ . The concentration of protease produced by Bacillus firmus LE02 was found to be 0.045mg/ml. Pant et al.⁹ isolated Bacillus subtilis, a thermo tolerant bacterium showing maximum protease production concentration of 2.31g/ml with zone of clearance of 22mm. The study of Shafee et al.¹⁸ optimized protease production by Bacillus cereus strain 146 with maximum rate of production approximately 2.0 µg/mL/min.

CONCLUSION:

The microbial enzyme such as protease, amylase, lipase and cellulase are significant in converting complex substances to simpler ones and gaining high interest in the enzyme industry for large scale production. The study was an attempt to enumerate the industrial enzyme producing bacterial strains such as Pseudomonas stutzeri LE01, Bacillus firmus LE02, Cytobacillus firmus LE03, and Bacillus licheniformis LE04 from the pony fish, Leiognathus equula. The production of extracellular enzyme is directly influenced by physiochemical parameters and optimization of these parameters would make the conditions more potential for maximum enzyme production is the need of enzyme industries. The present study bring out the optimum condition by changing salinity, pH and carbon source for maximum protease production by Bacillus firmus LE02 which can be further studied by identification, purification and characterization of protease in large-scale culture for industrial applications.

ACKNOWLEDGEMENT:

The authors thank The Dean and The Director, CAS in Marine Biology, Annamalai University for providing encouragement and support. We also thank RUSA for providing chemicals in the form of research project (DRD/RUSA 2.0/R&I/Project Proposal/Filed 5/8/2021).

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Received on 09.05.2024 Revised on 12.09.2024

Accepted on 21.12.2024 Published on 02.05.2025

Available online from May 07, 2025

Research J. Pharmacy and Technology. 2025;18(5):2032-2036.

DOI: 10.52711/0974-360X.2025.00290

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