I. INTRODUCTION

Marine fungi are either obligate or facultative in nature i.e. they can either exclusively grow in marine environment. The growth and the developmental conditions for the marine fungal species is very much different that the terrestrial ones. Therefore, they tend to produce enzymes with stability even at higher environmental parameters. They are a potential and the richest source of bioactive compounds and secondary metabolites. Due to the vast diversity of fungus in marine environment along with the production of potential bioactive compounds of industrial applications, they are promising targets for different enzyme screening programs when compared to their terrestrial equivalents^1-3.

For the past few years, the use of enzymes in various fields of application is of greater importance. Many of these enzymes of potential use are widely dispersed in nature. Of all the enzymes studied for such industrial application, the extracellular enzymes such as the fungal peroxidases are given more importance. Fungal peroxidases (lignin peroxidase, manganese peroxidase) and fungal laccases are the two main classes of extracellular enzymes that have been extensively studied and evaluated for their ability to remove toxic phenolic compounds from agricultural and industrial wastewater. These enzymes are also used in the study of degradation of intractable xenobiotics. Laccase is one such enzyme which has been studied extensively and is also exploited for its varied industrial application^4-6. This potential enzyme has the capability of soil bioremediation, pulp delignification, effluent and waste detoxification to name a few, by catalysing a wide array of substrates such as amines, diphenols, polyphenols and other electron rich compounds, using molecular oxygen with the production of water molecule⁷. They also engage in enzymatic conversion of intermediate compounds from lignin. Laccases are ligninolytic enzymes consisting of copper. Presence of copper helps in the catalysis of oxidation reactions of the substrates^8-10. They belong to protein family that include bilirubin oxidase, ascorbate oxidase and ceruloplasmin. These enzymes are believed to utilize a free radical catalysed reaction mechanism due to its strong oxidation potential in which the substrate forms an unstable free radical that further undergoes nonenzymatic reactions including hydration and disproportionation reactions¹¹. Laccases are known for their widespread and extensive applications including dye bleaching, ethanol production, food industry and paper and pulp processing. They are present in different higher species of plants where these enzymes are responsible for the lignin synthesis. Lignin is the structural component of the cell wall in plants^12-14. Laccase is also present in fungi, bacteria and certain insects. Most of the laccase enzymes studied so far belong to the fungal origin. They are secreted extracellularly during the secondary metabolism of certain fungal strains^15-18. Such extracellular secretion has been observed in organisms such as Phlebia radiata, Pleurotus ostreatus, Trametes versicolor and wood rotting fungi of genus Basidiomycete, Deuteromycetes, Ascomycetes etc. Some fungal sp. belonging to genus Zygomycetes and Chytridiomycetes do not produce laccase. Other organisms producing laccases such as Azospirillum lipoferum, Marinomonas mediterranea, Streptomyces griseus, E. coli, Bacillus subtilis etc. have been purified as well as characterized¹⁹.

Enzyme production when carried out in submerged fermentation yields maximum quantity than when compared to other mode of fermentation. Submerged fermentation involves the growth of microorganisms in different complex mediums. Enzyme production can be monitored and controlled. Maximum enzyme yield can be procured when the microorganism is optimised for the different environmental parameters such as temperature, pH, salt concentration, carbon and nitrogen sources etc.²⁰

Plate assay can be used as the primary screening method for the screening of laccase enzyme producers with polymeric dyes as the enzyme substrate. Tannic acid, guaiacol and ABTS serve as an effective indicator or the enzyme substrate for the production of laccase from the microorganisms. Fungi have been recognized as a potential source of laccase enzyme which can be exploited up to its molecular level for wide applications in the field of microbial biotechnology. In the present study, we focussed on the screening and the identification of the ligninolytic enzyme producing marine fungus by plate assay technique using guaiacol, tannic acid and ABTS as indicator substrates. The aim of this experiment was to optimize the fungal strain for the maximum production of the enzyme followed by the enzyme stability.

II. MATERIAL AND METHODOLOGY:

1) Chemicals:

Media and the chemicals used in the experiment were purchased from Hi Media Laboratories, Mumbai, India.

2) Sample collection:

The marine soil sample was collected from three different spots in Kovalam beach (8.4004˚N, 76.9787˚E), Kerala. The sediment sample was aseptically placed in clean sterile bags and was transported to the Microbial Biotechnology Laboratory, VIT, Vellore, Tamil Nadu, India for further isolation of marine fungi.

3) Isolation of marine fungi:

The soil sample was serially diluted upto 10^-7in sterile saline solution and plated on different complex media such as Potato Dextrose Agar (PDA), Malt Extract Agar (MEA) and Sabouraud Dextrose Agar (SDA). Chloramphenicol in 0.01% concentration was added to the plates to inhibit bacterial growth. The plates were incubated at 28˚C for one week. Morphologically distinct fungal colonies thus obtained were further sub-cultured on PDA and maintained at 4ºC until further use.

4) Primary screening:

The pure isolated cultures obtained were screened for the production of enzyme laccase using the plate assay technique with tannic acid, guaiacol and 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) as the indicator enzyme substrates at a concentration of 0.5%, 0.03% and 1mM respectively. Chloramphenicol acted as an antibacterial agent and was supplemented at a concentration of 0.01%. Post the incubation period of 5-7 days at 28ºC, the presence of dark red-brown coloured precipitates or hollow zone around the colony indicated the positive test for laccase activity with tannic acid and guaiacol as indicator substrates. The presence of dark blue-green coloured precipitates around the colony denoted positive test for laccase activity with ABTS as substrate. Control plates were maintained.

5) Enzyme assay for laccase:

Strains giving positive results for plate assay was assessed for enzyme activity. Guaiacol and ABTS were used as substrates for the laccase enzyme assay. Samples were withdrawn at a regular interval during the 10 days incubation. The enzyme extract was prepared by centrifuging pure cultures grown in Potato Dextrose Broth (PDB) at 8000 rpm for 15 min.

a) Guaiacol:

Due to oxidation of guaiacol by enzyme laccase intense brown colour is developed which can be read at 450nm. For substrate, sodium acetate buffer (10mM) at pH 5.0 and guaiacol (2mM) was used. The blank was made by addition of 1mL distilled water, 3 mL of sodium acetate buffer and 1mL guaiacol. The reaction mixture was made by adding 1mL of enzyme extract, 3 mL of sodium acetate buffer and 1mL guaiacol. The crude enzyme mixture was incubated at 28±2 ˚C for 15 min and its absorbance was read at 450 nm²².

b) ABTS:

ABTS is an efficient substrate for laccase enzyme assay. Due to oxidation of ABTS by enzyme laccase intense bluish green colour is developed which can be read often at 420nm. For substrate, sodium acetate buffer (concentration of 10mM) at pH 5.0 and ABTS (1mM) was used. The reaction mixture was made by adding 1mL of enzyme extract, 3 mL of sodium acetate buffer and 1mL ABTS. The blank was made by addition of 1mL distilled water, 3 mL of sodium acetate buffer and 1mL ABTS. The crude enzyme mixture was incubated at 28±2˚C for 15 min and the absorbance was read at 420nm.

Laccase enzyme activity was expressed as International Units (IU), wherein, 1 IU is the amount of laccase enzyme required to oxidize 1µmol of guaiacol or ABTS per min of the reaction. Enzyme activity in U/mL is calculated by the following formula:

EA = (A * V)/(t * e * v)

where, EA = Enzyme Activity

A= Absorbance at 450nm and 420nm for guaiacol and ABTS respectively

V= Total Volume of the reaction mixture

v=Crude enzyme volume

t= Total incubation time

e= Extinction Coefficient

6) Optimization of laccase enzyme activity:

Effect of temperature and pH on the reaction mixture during the oxidation of ABTS and guaiacol was studied. Enzyme mixture containing the buffer, substrates and the crude enzyme was subjected to different sets of temperature such as 25ºC, 30ºC, 35ºC, 40ºC and 45ºC. For the study of the effect of the varying pH on the reaction mixture, the pH of the buffer was adjusted to different values such as 3, 5, 7, 9 and 11 and incubated for 15 minutes. Post the incubation period, the absorbance of enzyme mixture is recorded spectrophotometrically. The optimum temperature and pH for the enzyme activity were detected.

7) Optimization of potential fungal strain:

The PDB broths were adjusted to different sets of environmental parameters such as pH (3, 5, 7, 9 and 12), temperature (4˚C, 28˚C, 37˚C and 45˚C), carbon source (glucose, dextrose, starch and sucrose at concentration of 2% (w/v)), nitrogen source (thiourea, peptone, yeast extract and sodium nitrate at concentration of 2% (w/v)), mineral salts (MgSO₄, ZnSO₄, CuSO₄ and KH₂PO₄ at concentration of 0.5% (w/v)), amino acids (tryptophan, tyrosine, glutamine and arginine at a concentration of 0.5% (w/v)), NaCl (concentration; 3%, 5%, 7% and 9%), aeration (static and rotary shaker) and light conditions (light and dark). The flasks were incubated for 5-7 days. pH was measured before and after the treatment. Post the incubation period, the total mycelial biomass is observed.

8) Antimicrobial activity:

Antimicrobial activity was performed using the Kirby Baur well diffusion method against Gram-positive organisms (Proteus mirabilia and Staphylococcus aureus) and Gram-negative organisms (Enterococcus sp. and Klebsiella sp.)²³

9) Identification:

Morphological characterization was done using microscopic observation by LPCB staining. The genomic DNA was extracted from potential fungal isolate. The PCR amplification of 28s rRNA gene of the isolates was done using 1.5μL of 5'-TACTACCACCAAGATCT-3' and 5'-ACCCGCTGAACTTAAGC-3' as forward and reverse primers respectively, 5μL of deionized water, and 12μL of Taq Master Mix (Taq DNA polymerase supplied in 2X Taq buffer, 0.4mM dNTPs, 3.2mM MgCl₂ and 0.02% bromophenol blue). The amplification reaction was followed by initial denaturation of the DNA at 94˚C for 5 min and further annealing of the DNA at 51˚C for 30 s leading to final extension using MgCl₂ with a final concentration of 1.5mM at 72˚C. The PCR product was sequenced using the primers. Sequencing reactions were performed using ABI PRISM® BigDyeTM Terminator Cycle Sequencing Kits with AmpliTaq® DNA polymerase (FS enzyme) (Applied Biosystems). The phylogenetic position of the potential fungal strain was determined by performing a nucleotide sequence database search using the BLAST program from National Centre for Biotechnology Information (NCBI) GenBank. The nucleotide sequencing result was submitted to the GenBank NCBI and accession number was obtained. Further neighbour joining phylogenetic tree was constructed using Mega 5.0 software.

III. RESULT AND DISCUSSION:

1. Isolation of fungus from marine soil sample:

The soil sample was serially diluted in sterile saline and aliquots from 10^-4 to 10^-7 were taken for plating on PDA, SDA and MEA. A total of 12 marine fungal strains were isolated and maintained in PDA at 4ºC until further use. The fungal strains were designated as FL1- FL12 as shown in Figure 1.

Figure 4: Strains fl2, fl3, fl4, fl6, fl7 and fl12 showing positive test for plate assay with abts.

3. Enzyme assay for laccase:

Potential strains giving positive result for plate assay was subjected to the enzyme activity (FIGURE 6). Enzyme assay was done using guaiacol and ABTS as substrates. Strain FL6 showed best laccase activity amongst the rest of the 5 strains (FIGURE 5). The enzyme activity has been tabulated in TABLE 1. Maximum activity of 4.75 * 10^-7U/mL and 14.7 * 10^-6U/mL was observed on day 6 when subjected to enzyme assay with guaiacol and ABTS as substrates.

Figure 5: Strain FL6 with the maximum laccase enzyme production

FIGURE 6: Laccase enzyme activity of strain FL6 during 12 days of incubation with ABTS and guaiacol as substrate.

Table 1: Laccase enzyme activity with guaiacol as substrate (left) and ABTS as substrate (right)

Incubation (d)	Enzyme activity (U/mL) (Guaiacol)
3	4.70 *10^-7
6	4.75 *10^-7
9	0.98 *10^-7
12	0.04 *10^-7

Incubation (d)	Enzyme activity (U/mL) (ABTS)
3	13.8 *10^-6
6	14.7 *10^-6
9	0.03 *10^-6
12	0.009 *10^-6

4) Antimicrobial activity:

Strain FL6 showed best enzymatic activity for both laccase and lignin peroxidase. Furthermore, the antimicrobial activity was performed for the potential strain FL6. Kirby Baur well diffusion method was used. Results indicate that the strain FL6 showed strong inhibition towards Gram-negative than the Gram-positive microorganisms (TABLE 3, FIGURE 8). Gram-negative bacteria have additional outer membrane over thin layer of peptidoglycan; which consist of asymmetric distribution of lipids and phospholipids. Gram-positive bacteria have thick layer of peptidoglycan and teichoic acid. The asymmetric distribution in outer membrane makes Gram-negative bacteria more susceptible to fungal strain ⁽²³⁾.

Table 3: Antimicrobial activity of strain FL6 against Gram-positive organisms (Staphylococcus aureus and Enterococcus sp.) and Gram-negative organisms (Proteus mirabilis and Klebsiella sp.). Diameter is measured in cm.

Organisms	50	75	100	125
Organisms	Concentration (µL)
Staphylococcus aureus (+ve)	0.2	0.4	0.5	0.7
Enterococcus sp. (+ve)	0.7	0.9	1.0	1.2
Klebsiella sp. (-ve)	0.3	0.7	0.9	1.1
Proteus mirabilis (-ve)	0.5	0.8	1.0	1.3

FIGURE 8: Antimicrobial activity of strain FL6 against Gram-positive organisms (Staphylococcus aureus (A) and Enterococcus sp.(B)) and Gram-negative organisms (Proteus mirabilis (C) and Klebsiella sp.(D)).

5) Optimization of laccase enzyme activity:

The laccase enzyme activity increased with the increase in the temperature with the optimum temperature of 35ºC. The enzyme activity recorded at this temperature was 4.89 U/mL. It was observed that with further increase in the temperature, the enzyme activity reduced drastically. Similar results have been reported by several other researchers stating that the enzyme activity is optimum around 35-45˚C ⁽²⁴⁾. Effect of pH on the enzyme activity was also studied for the strain FL6. It was observed that the optimum pH value for the laccase enzyme activity was at pH 5. Activity was recorded as 4.2 U/mL when ABTS was used as the enzyme substrate. The enzyme activity decreased with the increase in the pH value. Researchers have reported the optimum pH value for enzyme activity as 4-6 ⁽²⁵⁾ (FIGURE 9).

FIGURE 9: Effect of varying temperature and pH on the laccase enzyme activity of strain FL6.

6) Optimization of fungal strain:

The strain was optimised in submerged fermentation under different sets of environmental parameters. The fungal growth was maximum at 37˚C and pH 5 with starch and yeast extract as the carbon and nitrogen source respectively. Adiveppa et al. ⁽²⁶⁾ in one such study has reported the growth of Coprinus sp. in an ascending fashion from pH 5 to pH 7 at 30˚C. Sivakumar et al. ⁽²⁷⁾ have reported the optimum growth of Ganoderma sp. at pH 6. This observation was in line with Holker et al. ⁽²⁸⁾ and Robles et al. ⁽²⁹⁾ who in their respective study observed the optimal pH range for fungal laccase production from 4.0 to 6.0. Temperature has also been an important factor in the growth of the organism. In one study, Periyasamy et al. ⁽³⁰⁾have reported the optimum condition for the growth and laccase enzyme production of Pleurotus sp.as 50˚C whereas, Sadhasivam et al. ⁽³¹⁾ found that the optimum enzyme activity was found at 35˚C. Palonen et al. ⁽³²⁾and Xu et al. ⁽³³⁾ indicated that laccase enzymes are stable at 30–50˚C and rapidly lose their activity when the temperature is raised above 60˚C. This result disagrees with that obtained by Kalra et al. ⁽¹⁵⁾ who observed that the optimum temperature for enzyme activity ranged in between 45–50˚C. When it comes to carbon source, Piscitelli et al. ⁽³⁴⁾ have reported that glucose is the best carbon source for the enzyme production. When the growth medium is supplemented with nitrogen source, the enzyme activity is also seen to be elevated. Adiveppa et al. ⁽²⁶⁾ has reported that amongst the nitrogen sources, yeast extract supports the maximum laccase enzyme production followed by peptone and ammonium sulphate.

Gogna et al. ⁽³⁵⁾ in one study have reported that the ammonium salts are the most commonly used nitrogen source for the fungal enzyme activity. In a study performed by a group of scientists, it was observed that laccase production by white-rot fungi is strongly affected by the presence of amino acids in the media. Various amino acids and their analogues have shown stimulatory and inhibitory effects on the enzyme production by Cyathus bulleri. In this study, when the culture medium is supplemented with arginine, tryptophan, tyrosine or glutamine, it stimulated laccase production. Out of this, glutamine favoured the maximum growth of the isolate.

TABLLE 4: pH before and after subjecting to different environmental parameters along with the dry weight of the isolate FL6.

Environmental parameters	pH before treatment	pH after treatment	Dry weight (g/L)
Temperature	pH before treatment	pH after treatment	Dry weight (g/L)
4˚C	8.0	8.5	5.6
8˚C	8.1	8.9	96
37˚C	8.1	8.8	46.2
45˚C	7.9	8.3	5.3
pH
3	3	4.1	15.28
5	5	5.4	99.45
7	7	8.6	59.6
9	9	9.7	34
12	12	11.5	16
Salt concentration
1% NaCl	7.7	7.6	11
3% NaCl	7.8	7.9	13.8
5% NaCl	7.8	7.6	13.6
7% Nacl	7.8	6.9	13.3
Carbon source
Sucrose	7.8	7.9	74.7
Maltose	8.0	8.3	79.72
Dextrose	7.9	8.3	65.4
Starch	7.9	8.2	101
Nitrogen source
Sodium nitrate	7.8	10.2	34.6
Yeast extract	8.7	11.2	61.74
Peptone	8.5	10.9	23
Thiourea	8.1	8.1	8.3
Amino acid
Arginine	8.0	10.3	29
Tryptophan	8.2	8.6	36.9
Tyrosine	8.1	9.4	63.4
Glutamine	8.2	9.9	75.7
Mineral salt
ZnSO₄	7.4	7.5	3.15
MgSO₄	7.5	8.5	27
CuSO₄	6.6	6.3	2
KH₂PO₄	7.5	7.8	41.3
Light condition
Light	7.4	8.2	16.5
Dark	7.9	8.4	16.4
Aeration
Static	8.1	6.9	2.4
Rotatory shaker	7.4	8.1	17.15

Amino acids may act as an inducer for laccase production by many white-rot fungi ⁽³⁶⁾. Sun et al.⁽³⁷⁾, in one study have reported that all the six amino acids (alanine, histidine, glycine, arginine, aspartate, and phenylalanine) at a concentration of 1 mM, increases the catalytic ability of the enzyme production by Ganoderma lucidum strain 7071-9.

7) Identification of the potential fungal strain:

Identification of fungal strain FL6 was done with the help of Lactophenol Cotton Blue staining and 28s rRNA sequencing. Aseptate conidia with conidiospores were observed under 100x oil immersion. Further round blastoconidium were observed on a very basic conidiophore followed by hyphae disarticulating into arthrospores. The potential fungal strain was capable of producing laccase enzyme under optimum environmental conditions was identified as belonging to the genus Neurospora. The strain FL6 was identified as belonging to genus Neurospora using 28s rRNA gene sequence analysis. The phylogenetic determination was done followed by submission in NCBI database. The accession number obtained was MK114121 and maximum i.e. 99% similarity was observed with Neurospora crassa QR74A. The phylogenetic tree (FIGURE 13) obtained using Mega 5.0 software shows the related species and confirms that it belongs to Neurospora genus.

FIGURE 12:Spore chain morphology of genus Neurospora observed under 100x magnification of light Microscope

FIGURE 13: Phylogenetic tree of Neurospora crassa AJAS1 nucleotide sequences and reference sequences retrieved from NCBI Gen Bank constructed through the neighbour joining method.

IV. CONCLUSION:

From the present study it can be concluded that the strain Neurospora crassa AJAS1 is a potential source for laccase enzyme production. The fungal strain was capable of oxidising the phenolic substrates such as ABTS and guaiacol along with substrate such a tannic acid. The optimisation of the enzyme as well as the isolate gave the maximum enzyme activity of 4.89U/mL. Furthermore, this strain can be used for different industrial applications.

V. ACKNOWLEDGEMENT:

The authors would like to express their gratitude to the management of VIT, Vellore.

VI. CONFLICT OF INTEREST:

The authors declare no conflict of interest regarding the publication of this article.

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Received on 27.11.2018 Modified on 25.08.2020

Research J. Pharm.and Tech 2021; 14(12):6592-6600.

DOI: 10.52711/0974-360X.2021.01140


Figure 2: Strains fl2, fl3, fl4, fl6 and fl12 showing positive test for plate assay with tannic acid.	Figure 3: Strains fl2, fl3, fl7 and fl12 showing positive test for plate assay with guaiacol.