INTRODUCTION:

Marine biology is a major field of science which involves the study of marine organisms, their behavior and interactions with the environment. Marine sciences are important as growing population stresses the ability of our society to produce food, water and hence advances in technology combined with demand will improve our ability to derive food and drinking water. The field also encompasses the use of marine microorganisms either partially or completely to make or to modify products, or to develop microorganisms for specific uses. Therefore, marine organisms have been proved to have many beneficial bioactivities such as production of industrial enzymes.

Enzymes act as biocatalysts which enhance the rate of reactions, modern technology involving production of large number of enzymes which has got high importance in industrial applications. Protease is one of the major constituents and it constitutes about 1-5% of the total protein content¹.

Proteases are called as peptidyl peptide hydrolases which have the property of hydrolyzing peptide bonds in proteins². Protease constitutes about 60% of enzyme production in the market and hence proves to be industrially important³.

Proteases are classified into 3 major groups based on the level of hydrogen ion concentration i.e., acid, alkaline and neutral proteases⁴. Protease enzymes having a pH of 2.0-5.0 are called acid proteases, those enzymes having a pH of 7.0-8.0 constitutes neutral proteases and alkaline proteases have a pH above 8.0⁵. Proteases provide the advantage of being grown and cultivated easily and hence microbial production is easier in comparison to other microbial sources⁶. Protease enzymes can produce eco-friendly products and hence they play an important role in modern biotechnology industries⁷. The microbial proteases lack pathogenicity and hence used for enormous applications in industries.

Protease enzyme has applications like in protein structure studies and in manufacturing industries like food, monogastric animal feed, detergents, grain wet milling, paper and pulp etc.⁸ Proteases are used for a group of industrial enzymes and other applications like detergents, cheese making, de-hairing, baking, waste management and silver recovery⁹. Among all the proteases produced alkaline proteases are the most important due to their high thermal and pH stability¹⁰. In textile industry proteases play an important role in removing the stiff and dull gum layer in order to achieve improved luster and softness¹¹. The use of protease is also extended in manufacture of biopharmaceutical products such as contact lenses and enzymatic deriders¹².

Marine habitats are being used widely to discover bioactive molecules such as antibiotics, enzymes, vitamins, drugs, bio-emulsifiers etc.¹³. Marine bacteria are having the capacity to survive in extreme habitats and produce novel metabolites like enzymes, vitamins, pigments, single cell proteins in comparison to terrestrial bacteria. Hence, due to these unique features they are becoming more important in industrial and medical fields¹⁴. Now a days there is extraordinary development in technologies for production of microbial products in order to meet the needs of food processing, pharma and textile industries¹⁵.

Though enzymes from various sources were being used for this purpose, microbial enzymes are now considered to be the most useful ones due to various reasons¹⁶. Out of the 4000 enzymes that are known to be used in these industries, most of them are having either bacterial or fungal origin. This is due to immense diversity and the ease of production of enzymes by microorganisms¹⁷. The purpose of the current study is to isolate, identify, characterize and to carry out applications of protease enzyme from marine bacteria isolated from mangrove sediment samples.

MATERIALS AND METHODS:

Sample collection:

The mangrove sediment samples were collected from Sundarbans (21.5631° N, 88.2595° E), West Bengal, India in January 2018. It was then brought in sterile condition and stored in Marine Biotechnology laboratory, Vellore Institute of Technology, Vellore, at 4° C for further use.

Isolation and cultivation of marine bacteria:

Serial dilution and spread plate technique were used. Seven tubes were prepared with 9ml of sterile sea water. Sediment sample (1gm) was serially diluted (1:10). Then, 100µl of diluted sample was plated on each plate using spread plate technique. Media used for plating was Zobell Marine Agar. The medium was prepared with 50% marine water (1:1 marine water and distilled water). Standard concentration of media was 65g in 1000ml and then plates were incubated at 37°C for 24 h.

Identification of microorganism:

Bacteria were identified by microscopic examination (Gram staining) and biochemical tests according to Bergey’s Manual of Systemic Bacteriology viz. indole test, methyl red test, Vogues-Proskauer test, citrate utilization test, urease test, nitrate reduction test, catalase and oxidase test in order to identify the organism at genus level.

Screening for protease enzyme production:

a. Primary screening:

Enzyme producing bacterial isolates were screened by using the plate assay technique and involved the usage of skim milk agar medium. Isolated bacterial isolates were inoculated by line inoculation method on skim milk agar plates and incubated at 37°C for 24 hrs. Clear Zone formations were seen around the area of inoculation. Zone forming isolated were considered for secondary screening.

Production of Enzyme:

Bacterial colonies were inoculated in 20ml Nutrient Broth (NB). Nutrient Broth was prepared in conical flasks by using 50% marine water (1:1 marine and distilled water). Flasks were incubated in shaker incubator at 37°C for 24 h. After growth was observed the cultures were centrifuged at the rate of 10,000rpm for 10 min and the crude enzyme thus obtained from this was used for further analysis.

Estimation of Enzyme Activity (Secondary screening):

Enzyme activity was estimated by Protease assay using casein as substrate. In brief following protocol was used. First, 0.5ml of the obtained crude enzyme was mixed with freshly prepared 0.5% casein solution in 0.05M glycin-NaOH buffer (which had a pH adjusted to 9). It was then subjected to pre incubation at 45°C for 30 min. Further the mixture is then incubated at 45°C for 30 min and 2ml of 10% of TCA solution was added to terminate the reaction. Reaction mixture was centrifuged at 10,000 rpm for 10 min at 4°C. Absorbance was read at 660nm using UV-Visible spectrophotometer. Enzyme liberated was measured by 1ug of tyrosine per minute under standard assay conditions¹⁸.

Estimation of total protein content:

Total protein content was estimated by Lowry’s method. Preparation of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0ml of standard protein solution was done. Total volume was made up till 1ml using distilled water. Alkaline-copper sulfate solution of 4ml was added. Incubation was done at room temperature for 10 min. Then, 0.5ml of FCR reagent was added. Incubation was done for 30min. Absorbance of Blue-Purple color formed was measured at 660nm⁹.

Antibacterial activity by well diffusion method:

The antibacterial activity was performed by well diffusion method against seven pathogens P1, P2, P3, P4, P5, P6 and P7. Muller-Hinton agar plates were prepared and sterilized at 121°C for 30mins, after solidification the organism was swabbed onto the surface of plates. The wells were punctured using sterile cork borer and 100µl of extract was added into the wells and incubated at 37°C for 24-48h¹⁹.

Removal of blood stain:

Purified proteinase enzyme was used as a Detergent to remove the blood stain. The blood-stained cloth pieces were placed on different petri plates and following pattern was made and noted for observations.

Petri Plate 1: Plate containing blood stained cloth+ distilled water.

Petri Plate 2: Plate containing blood stained cloth+ distilled water + detergent.

Petri plate 3: Plate containing blood stained cloth + distilled water + detergent + partially purified enzyme

Petri plate 4: Plate containing blood stained cloth + distilled water + partially purified enzyme.

RESULTS AND DISCUSSION:

Identification of organism:

Bacteria were identified as Grams negative rod-shaped organism based on Grams staining.

Fig. 1: Gram staining

The above-mentioned biochemical tests were performed and results were obtained as follows:

Table 1: Biochemical tests

Biochemical tests	S13	M17	V19
Catalase test	+	+	+
Oxidase test	-	-	-
Indole test	-	-	-
Methyl red test	+	+	+
VP test	-	-	-
Citrate test	-	-	-
Motility	Non Motile	Non Motile	Non Motile

The table1 shows the following results of biochemical tests of V19, M17 and S13. From the table all the three bacterial isolates showed that non-motile, catalase positive and methyl red positive.

Antibacterial activity:

Table 2: Antibacterial Activity

Pathogen	Positive Control	Zone of Inhibition (MM)
Pathogen	Positive Control	V19	M17	S13
S.aureus	Vancomycin	2mm	-	-
B.cerues	Streptomycin	2mm	-	-
E.coli	Ciprofloxacin	3mm	-	-
K.pneumoniae	Amphicillin	3mm	-	-
P.aeroginosa	Ciproflaxicin	-	2mm	-
Salmonella	Streptomycin	2mm	-	-
Streptococcus	Streptomycin	4mm	-	-

Fig. 2: Antimicrobial activity

From the table 2 and Fig.2a-2e we can see that crude extract V19 culture shows more resistance towards the bacterial pathogens when compared to extracts M17 and S13. The crude extract from the bacterial isolates S13 did not show any zone of inhibition, so it is not resistant to the bacterial pathogens.

Protease assay:

Fig. 3: Graph for Protease assay

Protease assay was performed using tyrosin standard. OD values for tyrosin standard as well as the crude extract samples were taken using spectrophotometer at 660nm. Obtained values were plotted on a graph. As per the obtained values enzyme released was compared with per ug of tyrosin standard.

Blood stain removal:

Blood stains on the cotton cloth shown above at 0^th time.

The clothes were washed after 5 minutes as follows:

a. Washed with distilled water

b. Washed with distilled water +detergent

c. Washed with distilled water + crude extract

d. Washed with distilled water + crude extract+ detergent

The clothes were washed after 10 minutes as follows:

a. Washed with distilled water

b. Washed with distilled water +detergent

c. Washed with distilled water + crude extract

d. Washed with distilled water+crude extract+detergent

The clothes were washed after 15 minutes as follows:

a. Washed with distilled water

b. Washed with distilled water +detergent

c. Washed with distilled water + crude extract

d. Washed with distilled water+crude extract+detergent

The blood stain removal was performed for the bacterial isolate V19 and it was observed that the stain was removed efficiently in 15mins with the solution containing distilled water, detergent and crude extract. Similar research was carried out by Sarika Kedar Marathe (published in the journal of Genetic Engineering and Biotechnology) showed that the blood stain removal was completely removed in 20 min and the bacterial isolate V19 proved to be more efficient by removing the stain in 15mins.

DISCUSSION:

Proteases from microbial sources are preffered than the enzymes obtained from plant and animal sources since they possess all charcteristics desire for their biotechnological application.

Proteolytic bacteria are widespread in nature and are able to grow under various growth conditions, such as different temperatures, pH and ionic strength.

The bacteria that exist in marine sample represent very important and diversified enzymatic potential. But sufficient information is not much available on their role in industrial and hence the present study was carried to isolate and charcterize the potent protease positive bacteria from the marine sediment sample of Sunderbans (21.5631^оN, 88.2595^о E), West Bengal, India.

In this study 8 bacterial isolates were able to produce proteolytic enzymes. The results indicated that 3 proteolytic bacteria were dominant protease producer; it was identified as Bacillus sp. Formation of clear zone on skim milk agar medium around the isolated colonies was considered as indication of good growth with the ability of protease production. Protease production was further confirmed by performing Protease assay.

The antibacterial activity of 3 strains M17, V19 and S13 was done and V19 showed better activity by achieving the largest zone of inhibition against 6 out of 7 pathogens. The blood stain removal activity of the strain V19 proved to be more efficient by removing the stain in 15mins, which further lead to the confirmation that proteases have high activity against blood stain removal.

They observed maximal production when malt extract was utilized as nitrogen source detailed that ammonium sulfate was found to the best nitrogen source for protease production. In washing detergents the utilization of enzymes, for example, proteases in like manner about half of fluid detergent, 25% of powder detergent had practically all powder bleach added substances currently contain enzymes to help in breaking down of stains that are generally difficult to expel with traditional surfactants alone. They function as scissors to remove the stain piece by piece from the surface of the fabrics²⁰.

CONCLUSION:

Proteases are important enzymes obtained from marine organisms which can synthesize bioactive compounds. Screening was performed and maximum proteolytic activity was revealed. The extract of V19 strain showed largest zone of Inhibition against microbial pathogens and hence used for the blood stain removal application. The strain proved to be effective in removing the blood stain in 15mins and thus exhibited higher proteolytic property.

Marine natural products provide unique compounds that are not readily available from any other source. The discovery of novel secondary metabolites isolated from the marine actinomycetes has recently exceeded that of their terrestrial counterparts. In this context, our aim is directed towards isolating antibiotic producing marine actinomycetes. Enzyme screening is also an important tool for future generation. They are tools of nature that help provide everyday production in an environmentally cognizant mode. Recent economic use of enzymes, along with new applications, will prolong to play a vital role in maintaining and improving the quality of life for the next generations. In future, the potent microbial strain involved in antibacterial activity and enzyme production will be identified by DNA sequence.

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Received on 20.02.2020 Modified on 23.04.2020

Research J. Pharm. and Tech. 2021; 14(8):4236-4240.

DOI: 10.52711/0974-360X.2021.00735