Int.J.Curr.Microbiol.App.Sci (2016) 5(5): 127-137 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 5 Number 5 (2016) pp. 127-137 Journal homepage: http://www.ijcmas.com Original Research Article http://dx.doi.org/10.20546/ijcmas.2016.505.014 Screening of Potent Laccase Producing Organisms Based on the Oxidation Pattern of Different Phenolic Substrates Sheena Devasia* and A. Jayakumaran Nair Department of Biotechnology, University of Kerala, Kariavattom, Trivandrum, Kerala, India *Corresponding author ABSTRACT Keywords Arthrographis, bacterial laccase, Enterobacter cloacae, guaiacol, laccase, lignin, oxidoreductase. Article Info Accepted: 12 April 2016 Available Online: 10 May 2016 Soil samples were collected from different forest areas of Kerala and screened for laccase activity using guaiacol plate assay technique. An increased concentration of guaiacol was used for the isolation of resistant strains. Substrate oxidation studies were carried out and the potent organisms were taken for enzyme production studies. The organisms, isolated by the screening strategies, were found efficient laccase producers. The potent organisms were identified as Arthrographis sp. and Enterobacter cloacae. The enzyme production rate of Arthrographis was found to increase logarithmically and a maximum quantity of 53 U/ml was obtained and the bacterial strain, Enterobacter cloacae, gave a maximum laccase activity of 8U/ml. The difference in the pattern of substrate oxidation by laccase from different organisms is paid attention in the study. Introduction oxidases (LMCO) were predominantly described in fungi and plants, where they occur as multigene family with sometimes more than 10 different laccase genes. The basidiomycete Coprinopsis cinerea and the plant Arabidopsis thaliana have both 17 different genes (Hoegger et al., 2006). Laccases (benzenediol: oxygen oxidoreductase, EC 1.10.3.2) catalyze the oxidation of various aromatic, particularly phenolic substrates (eg. hydroquinone, guaiacol, 2,6-dimethoxyphenol or phenylene diamine), coupled to the reduction of molecular oxygen to water. Laccases as well as ascorbate oxidases (EC 1.10.3.3) and ceruloplasmins/ferroxidase (EC 1.16.3.1) usually contain several copper atoms in the catalytic centre. They belong to the enzyme superfamily of multicopper oxidases, which is a widely distributed protein family among prokaryotes and eukaryotes. However, laccases or laccase-like multicopper Properties of Laccases Current knowledge about the structure and physico-chemical properties of fungal proteins is based on the study of purified proteins. Up to now, more than 100 laccases have been purified from fungi and been more or less characterized. The laccase 127 Int.J.Curr.Microbiol.App.Sci (2016) 5(5): 127-137 molecule, as an active holoenzyme form, is a dimeric or tetrameric glycoprotein, usually containing four copper per monomer (Cu) atoms bound to three redox sites (Type 1, Type 2 and Type 3 Cu pair). The molecular mass of the monomer ranges from about 50 to 100 kDa with acidic isoelectric point around pH 4.0. An important feature is the high level of glycosylation (with covalently linked carbohydrate moieties ranging from 10– 50% of the total weight, depending on the species or the heterologous host), which may contribute to the high stability of the enzyme (Duran et al., 2002). Several laccase isoenzymes have been detected in many fungal species. More than one isoenzyme is produced in most white-rot fungi. Substrates and Products formed by Laccase Laccase has broad substrate specificity as well as the advantage of not requiring an addition of harmful hydrogen peroxide to the oxidative reaction. Because of complex structure of lignin, its biodegradation system is considered highly nonspecific. Lignolytic enzymes can degrade environmental pollutants that differ structurally (Dec and Bollag, 1994). The substrate oxidation pattern of laccase is tabulated in Table 1. Materials and Methods Soil samples were collected from different forest areas of Kerala and screened for laccase activity. The serially diluted soil samples were inoculated on Vogel’s Mineral Media (VMM) agar plates (pH 5.6) containing guaiacol (Di- methoxy phenol) (Sigma) as substrate (Vogel, 1956; Coll et al., 1993). The positive strains were purified and modified plate assay for selecting high tolerance laccase producing strains was conducted using an increased concentration of guaiacol. In contrast to most enzymes, which are generally very substrate specific, laccases act on a surprisingly broad range of substrates, including diphenols, polyphenols, different substituted phenols, diamines, aromatic amines and benzenethiols. Furthermore, laccases with unusual and potentially useful properties have been isolated from ascomycetes. Laccase from Monocillium indicum was the first laccase to be characterized from as ascomycetes showing peroxidative activity (Thakker et al., 1992). Examples include the higher stability and activity at alkaline pH together with the high thermostability of the laccase produced by Melanocarpus albomyces (Kiiskinen et al., 2002), the high acid tolerance of intracellular laccase from Hortaea acidophila (Tetsch et al., 2006), and the high activity of laccase from Xylaria polymorpha at elevated concentrations of NaCl (Liers et al., 2007). It was postulated that laccase acts as a defense mechanism against oxidative stress (Fernandez-Larrea and Stahl, 1996). This protective function was partly attributed to the chelation of copper ions during synthesis of the laccase enzyme. Substrate Oxidation Studies by Laccase The substrate tolerance was studied by incorporating 0.02% of guaiacol, p-cresol, paminophenol, p-phenylene diamine, hydroquinone and tropolone in VMM agar plates. The fungal plates were incubated at 27±1C for 5 days and the bacterial plate was incubated for 48 h at 37C. The growth and oxidation pattern were noted. Organisms and Culture Conditions Preparation of Pre-inoculum Pre inoculum was prepared by inoculating VMM agar plates for fungi and VMM broth 128 Int.J.Curr.Microbiol.App.Sci (2016) 5(5): 127-137 for bacteria. Agar plugs from the outer circumference of a fungal colony stored on a SDA plate was used as the inoculum. The fungal strains were inoculated to VMM plates and were incubated for 6 days at 27±1C. The bacterial strain was inoculated from NA slant to VMM broth and was incubated for 24 h at 120 rpm, 37C. Tolerance Assay The KSF2 strain was subjected to substrate tolerance assay. 1mM concentration of the phenolic substrates (p-cresol, p-aminophenol and p-phenylene diamine, guaiacol, hydroquinone and tropolone) were added to VMM broth, KSF2 culture was inoculated and incubated at 27±1ºC at 120 rpm. The oxidation of the substrate and the tolerance pattern was noted in broth cultures. The laccase assay was conducted at regular intervals using ABTS as substrate Preparation of Inoculum Agar plugs (5 mm diameter) from the outer circumference of fungal colonies growing on VMM plate (8 days) was used as inoculum for fungal production media and 1% bacterial culture with 0.7 Abs at 600nm was used for inoculating the bacterial production media. The potent strains selected were sent to IMTECH, Chandigarh, India for identification. Results and Discussion Activity Based Secondary Screening of Organisms Pure Culture Isolation Laccase production was carried out in VMM broth, pH 5.6. The fungal cultures were incubated at 27±1C and the bacterial culture at 37C. The samples were collected in every 24 h and centrifuged at 10,000 rpm for 15 min. The supernatant was taken for enzyme assay. The enzyme assay was conducted using 2,2’-azino-bis (3ethylbenzothiazoline-6-sulphonic acid (ABTS) (Biogene, USA) as substrate. Soil samples were plated on to guaiacol containing medium. Oxidation polymerization of guaiacol to bisphenoquinone was visualized as reddish brown zones on VMM agar plates (Fig. 1). Efficient and potent organisms screened by this method were purified and inoculated to the respective agar plates to study the colony morphology (Fig. 2). Of the 41 isolated strains isolated by this technique, 4 strains were selected after the primary screening studies. Out of the 4 strains, 3 were fungi (KSF1, KSF2 and KSF3) and the other one was bacteria, KSB4. The assay mixture contained 2mM ABTS in 0.1M sodium citrate buffer, pH 3.0. Oxidation of ABTS was monitored by determining the absorbance increase at 420 nm (ε = 3.6x104 M-1cm-1) (Jia Li Dong, 2004). One unit of enzyme activity was defined as the amount of enzyme required to oxidize 1 μmol of ABTS per min. Assay was conducted at regular intervals. The laccase production rate and the optimum day of enzyme production were noted for each of the selected organisms. Statistical model was constructed based on enzyme activity and day of incubation. Modified Plate Assay for High Tolerance Laccase Producing Strains Potent DMP tolerant laccase producing organisms were isolated by this technique. The concentration of guaiacol in the plates was increased to 0.04% to find the tolerance of the organisms. All the organisms could grow in the increased concentration of 129 Int.J.Curr.Microbiol.App.Sci (2016) 5(5): 127-137 guaiacol and oxidize the substrate. The fungal colonies were incubated for 5 days at 27°C and the bacterial colony was incubated for 48 h at 37°C. The bacterium was found to be less tolerant to the increased concentration of the substrate as after incubation period the viability of the organism decreased to considerable extends (Fig.3). Substrate Oxidation Isolated Strains Pattern of optimum day of enzyme production. The quantity of enzyme produced by the strains, enzyme production pattern and the optimum day of enzyme production by each organism also differed. The results are tabulated in Table 3 and Fig. 6. The enzyme production rate of KSF2 was found to increase logarithmically till 102nd day of inoculation. From this secondary screening method KSF2 and KSB4 were screened as the most potent strains for laccase production. the Out of the six compounds selected for the study guaiacol was oxidized by all the selected strains. KSF1 could grow in the presence of p-cresol, p-aminophenol and hydroquinone but could not oxidize the compounds. KSF2 could grow in the presence of all substrates except tropolone and could oxidize p-phenylene diamine. KSF3 could not grow in the presence of any of the substrates. KSB4 and KSF2 could grow on all substrates except tropolone and could oxidize p-phenylene diamine and hydroquinone. Results are tabulated in Table 2. The oxidation reaction is presented in Fig. 4. The level of tolerance was directly proportional to the amount of laccase produced by the organisms. Results indicate that the laccase production is high in the exponential growth phase; the activity appears to be closely correlated with biomass production. Different strains can produce different laccases, each with its own unique features. Environmental factors influence the ability of fungi to produce high titers of laccase, and different strains react differently to these conditions. Screening of strains capable of producing high concentrations of enzyme and then to optimize the conditions for laccase production is important in the industrial production of the enzymes. Statistical Model Construction Based on Enzyme Activity and Day of Incubation The potent strain, KSF2 was inoculated to the VMM agar plates supplemented with the assay substrates (guaiacol, catechol and ABTS) to confirm the best substrate for laccase assay. The plates are incubated at 27±1ºC for 5 days. It was observed that the laccase of KSF2 was more reactive to ABTS (Fig. 5). Different statistical models were tried to find the correct fitted model to explain the enzyme activity when day was set as the variable (Fig. 7). The model for which the coefficient of determination (R2) is maximum, was selected as the best suited model. It was observed that the cubic model was best suited to explain the enzyme activity by the isolated strains. Activity Based Secondary Screening of Organisms The tolerance of the organism in SmF is tried for its application studies. In 1mM concentration of the substrates KSF2 was able to grow in broths supplemented with paminophenol and p-phenylene diamine and The organisms were inoculated into VMM broth for laccase production and ABTS assay was conducted every 24 h to find the 130 Int.J.Curr.Microbiol.App.Sci (2016) 5(5): 127-137 control. There was a decrease in laccase activity observed in the presence of phenolic compounds. was also able to oxidize them. paminophenol was not oxidized by the organism in plate assay method. The enhanced production of laccase in shaking condition attributed to the oxidation of paminophenol. Morphological and Identification of Organisms All the other substrates inhibited the growth of the organism in broth culture. The oxidation of the two substrates was visualized in broths and the laccase assay was conducted to study the effect of the presence of these compounds on enzyme production. The ABTS assay was conducted for the media with p-phenolic compounds. The culture was inoculated to modified VMM minimal media and was kept as The Ascomycetes strain KSF2 was identified as Arthrographis sp. by IMTECH, Chandigarh, India and was deposited with reference MTCC 8880. It has branched, hyaline conidiophores, thallic and arthric conidiogenesis and hyaline single celled conidia in dry chains (Fig 8). The bacterial strain KSB4 was identified as Enterobacter cloacae by IMTECH, Chandigarh, India and was deposited with reference MTCC 9145. Biochemical Table.1 Substrate oxidation pattern of laccase Substrate Product Colour Catechol Hydroquinone Pyrogallol 2,6 dimethoxy phenol Guaiacol ABTS Syringaldazine Catechin o-benzoquinone o-benzoquinone Purpurogallin 3,5,3’,5’ tetramethoxy diphenoquinone Biphenoquinone ABTS+ Quinones o-quinone Yellow Yellow Yellow Yellow Brown Blue Purple Yellow λmax (nm) 450 248 450 468 470 420 525 390 εmax (M-1cm-1) 2,211 17,252 4,400 35,645 26,600 36,000 65,000 4,019 Table.2 Oxidation Pattern of Phenolic Compounds by the Screened Organisms Substrates guaiacol p-cresol p-aminophenol p-phenylene diamine hydroquinone tropolone Growth/ Oxidation Pattern KSF2 KSF3 +/+ +/+ +/-/+/-/+/+ -/+/-/-/-/- KSF1 +/+ +/+/-/+/-/- 131 KSB4 +/+ +/+/+/+ +/+ -/- Int.J.Curr.Microbiol.App.Sci (2016) 5(5): 127-137 Table.3 Enzyme Activity of the Isolated Strains Organism Optimum day KSF1 KSF2 KSF3 KSB4 31 102 15 5 Enzyme activity (U/L) 155 52,764 15 8,317 Fig.1 Plate Assay of Laccase Fig.2 Cultural Characteristics of Isolated Strains on Agar Media 132 Int.J.Curr.Microbiol.App.Sci (2016) 5(5): 127-137 Fig.3 Modified Plate Assay for Laccase Fig.4 Oxidation of the Phenolic Compounds by the Potent Strains Fig.5 Oxidation of the Assay Substrates by KSF2 133 Int.J.Curr.Microbiol.App.Sci (2016) 5(5): 127-137 Fig.6 Laccase Production by the Strains Fig.7 Statistical Models Based on Enzyme Production Pattern Fig.8 Lactophenol Cotton Blue Staining of Slide Culture The selected potent strains can grow in the presence of guaiacol, p-cresol, paminophenol, p-phenylene diamine and hydroquinone explains their remarkable potential for application in bioremediation and wastewater treatment, especially in detoxification of phenolic wastes. The organisms, isolated by the screening strategies, were found efficient laccase producers. The enzyme production rate of Arthrographis KSF2 was found to increase 134 Int.J.Curr.Microbiol.App.Sci (2016) 5(5): 127-137 logarithmically and a maximum quantity of 53U/ml was obtained on the 102nd day of inoculation. The bacterial strain, Enterobacter cloacae KSB4, was also found promising with a maximum laccase production of 8U/ml. Laccases from ascomycetes and bacteria has been paid only less attention, although they have unusual and potential biotechnological use, hence from the primary and secondary screening studies conducted the ascomycetes, Arthrographis KSF2 and the proteobacteria, Enterobacter cloacae KSB4 were selected for further studies. measurement is required. As laccases oxidize various types of substrates, several different compounds have been used as indicators for laccase production. The traditional screening reagents tannic and gallic acid (Harkin and Obst, 1973) have nowadays mostly been replaced with synthetic phenolic reagents, such as guaiacol and syringaldazine (Nishida et al., 1988; De Jong et al., 1992) or with the polymeric dyes Remazol Brilliant Blue R (RBBR) and Poly R-478 (Barbosa et al., 1996; D’Souza et al., 1999; Raghukumar et al., 1999). RBBR and Poly R-478 are decolourized by lignindegrading fungi (Gold et al., 1988; Barbosa et al., 1996), and the production of ligninolytic enzymes is observed as a colourless halo around microbial growth. With guaiacol a positive reaction is indicated by the formation of a reddishbrown halo (Nishida et al., 1988), while with tannic and gallic acid the positive reaction is a dark-brown coloured zone (Harkin and Obst, 1973). These studies show that novel laccase producers can be discovered from environmental samples by very simple plate-test screening methods. 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