ESTIMATION OF EXTRACELLULAR LIGNINOLYTIC ENZYMES FROM WILD AURICULERIA POLYTRICHA, HELVELLA SP. AND MORCHELLA SP

Neha Thakur, Astha Tripathi*, Sukrit Sagar, Pardeep Kumar, Nagina Devi, Shaveta, Isha Sharma, Monika and Jyoti Sharma. Applied Sciences and Biotechnology, Shoolini University. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

Three mushroom samples have been selected for extracellular ligninolytic enzyme activities i.e commercially cultivated Auricularia polytricha, wild Helvella sp. and wild Morchella sp. To investigate the extracellular ligninolytic enzymes, cultures were incubated over a period of 20 days in nutrient rich medium (NRM) and nutrient poor medium (NPM) under static conditions. Samples showed green and brown coloration in solid medium plates; it indicates the presence of laccase and Manganese peroxidase (MnP). Production of extracellular ligninolytic enzymes were observed in all three species in different pattern. The results of the present study allow us to conclude that wild Morchella sp. and Helvella sp. are good for production of ligninolytic enzymes in comparison to commercially cultivated Auricularia polytricha.
We have selected three mushrooms strains, Auricularia polytricha, Helvella sp. and Morchella sp. for their extracellular enzymatic studies. The interest of Auricularia polytricha could be easily identified by pilose upper surface which is strongly capitates with dark brown smooth hymenium [13]. Morchella species is one of the most readily recognized of all the edible mushrooms. On the other hand Helvella species they grow in grass as well as humid hardwoods, such as beech, and in hedges and on the talus of meadows. These all are wood decaying WRF produce ligninolytic enzymes.

MnP Spot Test:-
Fungus culture (previously cultured in 2% MEA agar for 8 days) were inoculated in the kirk medium containing 10g of glucose, 2g of KH 2 PO4, 0.5g of MgSO 4 .7H 2 O, 0.1g of CaCl 2 , 2.2g of 2,2-dimethylsuccinate, 0.5g of ammonium tartrate, 20g of agar-agar per liter of medium. For the production of MnP medium was supplemented with 0.04g of MnCl 2 .4H 2 O per liter of the medium. The pH was adjusted to 5.0 before autoclaving at 15 psi for 20 min at 120ºC [22]. Fungal growth was estimated in terms of diameter of fungal colony. Extracellular enzymatic activity was measured by the colour intensity of the medium. Plates were observed once a day for three consecutive weeks.

Extracellular Ligninolytic Enzyme assay:-
The production of extracellular enzymes were carried out in the NRM containing 2g of ammonium tartrate, 10g of glucose, 1 g of KH 2 PO 4 , 1g of yeast extract, 0.5g of MgSO 4 [8] . The pH was adjusted to seven before autoclaving at 15 psi and 120ºC for 20 min, 150 ml flasks containing 50 ml liquid medium were inoculated with 5-8 days old five mycelium bits (5mm in diameter). Three replicates of flaks of both medium were incubated in static condition in BOD incubator and shaking condition in a rotator shaker at 150 rev/min at 30ºC.
The cultures were harvested at the 5 th , 10 th , 15 th and 20 th day of incubation. Each sample was centrifuged (10,000x g for 10 min) at 4ºC. The supernatant of liquid culture was used for enzyme assay. The enzymatic reactions were carried out in triplicate and determined using a double beam systronics UV/VIS spectrophotometer. All the enzyme activities were measured at room temperature (20 ± 2ºC). The enzymatic activity was expressed as international units (U) defined as the amount of enzyme required to produce 1µmol product min -1 and expressed as Ul -1 . Protein concentration was determined following Bradford method [3]. Protein content in the sample was determined from standard curve and the amount of protein lµgml -1 was calculated. Laccase activity was measured following the oxidation of ABTS (ε420, 36,000M -1 cm-1). The assay mixture contained 100mM sodium acetate buffer pH 5 and 5mM ABTS [12]. LiP activity was measured by the oxidation of 2mM VA to veratraldehyde (ε 310, 9,300 M -1 c -1 ) in 100mM sodium tartrate buffer (pH3) in the presence of 0.4 mM H 2 O 2 (30%) [4]. MnP activity was determined by the production of a Mn +3 tartrate complex (ε 238, 6,500M -1 cm -1 ) from 0.1 mM MnSO 4 in 100mM sodium tartrate buffer pH 4.5 with 0.1 Mm H 2 O 2 (30%) [6].
Statistical Analysis:-All the experimental analysis was carried out in triplicates. The results are expressed as mean values and standard deviation (SD). The results were analyzed using one-way analysis of variance (ANOVA) followed by Tukey's HSD Test using SAV v.9.1.3 program. Differences at P>0.05 were considered to be significant.

Results and Discussion:-Identification:-
The samples were identified as Helvella sp. and Morchella sp. on the basis of their micro and macroscopic features (Fig.1). In Himachal Pradesh there is a huge variation in climatic conditions due to the variations in altitude (450-6500 m). The climatic condition varies from hot and sub-humid tropical (450-900m). Mushrooms have extraordinary capacity to manage climatic conditions.

Spot Test of Extracellular Enzymes:-
With the agar plate screening, selected strains of wood colonizing WRF were tested for spot test. In comparison to the growth on MEA, strains grew noticeably slower on the selected agar media. In particular ABTS had inhibitory effects on fungal growth.

Laccase Spot Test:-
The extracellular ABTS-oxidizing activity of the both fungal strains into the modified Kirk medium is showed in Fig.2. Morchella sp. showed high ABTS-oxidizing activity in comparison to Auricularia polytricha and Helvella sp. The absence of extracellular ABTS-oxidizing activity does not necessarily imply the lack of capacity to produce these oxidative enzymes but could reflect a possible inhibition of their expression; the oxidative enzyme system is not homogeneous; its production and properties depend on the conditions and culture media. Fungal strains oxidized ABTS to the dark green ABTS cation radicals (ABTS) indicating the production of extracellular oxidoreductases [10]. It gave positive reaction immediately after inoculation, formed dark green zone around the mycelial bit and Auricularia polytricha and Helvella sp. showed green colour after 5 days of inoculation. The green colour preceded the fungal mycelium clearly demonstrating that extracellular mechanisms were responsible for the oxidation [10].

MnP Spot Test:-
All the three selected species Morchella sp., Auricularia polytricha and Helvella sp. showed positive reaction of extracellular MnP and formed brownish colour in medium plate (Fig. 2). Helvella sp. and Morchella sp. showed dark brown coloration in comparison to Auricularia polytricha however mycelial growth was noticeably slow in Morchella sp with MnCl 2 . MnP plates were evaluated after 3 weeks of incubation for the formation of brownish flecks of manganese oxide (MnO 2 ) caused by the action of MnP. MnP was thought to play a crucial role during the primary attack on lignin, because it generates highly reactive Mn 3+ which acts as a low molecular mass redox mediator and forms water soluble lignin fragments. Moreover, there are indications that MnP is even capable of mineralizing lignin up to carbon dioxide [10]. It was assumed that MnP plays an important role in mineralization of lignin.

Extracellular Quantitative Protein Determination:-
The Bradford assay is a protein determination method that involves the binding of Coomassie brilliant blue G-250 dye to proteins [3]. When the dye binds to protein, it is converted to a stable un-protonated blue form [23]. This is anionic form of dye. In Auricularia polytricha maximum protein concentration was observed on 15 th day in NRM and minimum concentration was observed on 5 th day NPM (3.57 µg/ml). In Halvella sp. maximum protein concentration was observed on 15 th day in NPM (16.0 µg/ml) and minimum concentration was observed on 20 th day in NPM (2.89 µg/ml). In Morchella sp. maximum protein concentration was measured on 15 th day in NPM (14.53 µg/ml) and minimum activity was observed on 10 th day in NPM (2.3 µg/ml). In all three selected varieties protein concentration was higher on 15 th day in both medium.
Extracellular Ligninolytic enzyme assay:-Laccase: Laccase was first detected in the Japanese lac tree Toxicodendron verniciflua. Later, it was found in certain other plants and fungi, but is also found in molds, black yeasts and some bacteria [17]. Laccase has been identified as one of the enzyme that plays a major role in lignin degradation. Laccase only attacks phenolic subunits of lignin, but its substrate range can be extended to non-phenolic subunits by the inclusion of a mediator [17]. In Auricularia polytricha maximum laccase activity was observed on 10 th day in NRM (63.54UL -1 ) and minimum was observed on 20 th day in NRM (47.21UL -1 ). In Helvella sp. maximum laccase activity was observed on 10 th day in NPM (73.17UL -1 ) and minimum was observed on 20 th day in NPM (43.86UL -1 ) respectively. In Morchella sp. maximum laccase activity was observed on 10 th day in NRM (67.3UL -1) and minimum was observed in NRM on 20 th day (48.63UL -1 ). It was witnessed that supplementation and incubation conditions also affected the enzymatic 972 activity. A few WRF gave good laccase activity under high nitrogen condition and medium supplemented with Cu but in the absence of Cu activity was not found and under low nitrogen like Volvariella volvacea, Piloderma byssinum. [5] AAO:-The lignin degradation system of WRF is mainly composed of laccase, LiP and MnP. It is considered that, these lignin degrading enzymes do not functions but mutually interact with each other as well as with other oxidases, such as AAO. AAO activity was described for the first time in the fungus Polystictus versicolor (a synonym of Trametes versicolor) in 1960 [9]. Since then, AAO has been detected and characterized in other white-rot basidiomycetes including Pleurotus species, Bjerkandera adusta and some ascomycetous fungi [14]. AAO is the main oxidase enzyme is all selected varities. In Auricularia polytricha maximum AAO activity was observed on 10 th day in NRM (546.4 UL -1 ) and minimum was observed on 20 th day in NPM (395.75UL -1 ). In Helvella maximum AAO activity was observed on 10 th day in NPM (509 UL -1 ) ;and minimum was observed on 20 th day in NRM (369.2UL -1 ). In Morchella sp. maximum AAO activity was observed on 5 th day in NRM (550.42UL -1 ) and minimum activity was observed on 10 th day in NPM (468.5UL -1 ).
LiP:-One of the best known ligninolytic enzymes is LiP, which was discovered a little earlier than MnP [15]. LiP is a glycoprotein that contains one mole of iron protoporphyrin IX as a prosthetic group. LiP catalyzes the oxidation of non-phenolic aromatic compounds like veratryl alcohol. In Auricularia polytricha maximum LiP activity was observed on 15 th day in NRM (44.56UL -1 ) and minimum activity was observed on 5 th day in NRM (1.0 UL -1 ). In Helvella sp. maximum LiP activity was observed on 15th day in NPM (51.38UL -1 ) and minimum activity was observed on 5 th day under static conditions (1.0UL -1 ). In Morchella sp. maximum LiP activity was observed on 15 th day in NPM (55.29 L -1 ) and minimum activity was observed on NPM in 20 th day (4.29UL -1 ).

MnP:-
MnP is a second group of extracellular enzymes secreted by WRF. MnP operate by oxidizing Mn 2+ to chelated Mn 3+, which acts as a diffusible oxidant at locations remote from the enzyme active site. MnP is widely distributed in WRF, including P. chrysosporium, P. radiata, Nematoloma frowardi, P. eryngii and B. adusta [14] and this peroxidase described for the first time in B. adusta [14]. In Auricularia polytricha maximum MnP activity was observed on 15 th day in NPM (46.4UL -1 ) and minimum activity was observed on 5 th day in NPM (1.04 UL -1 ). In Helvella sp. maximum MnP activity was observed on 15 th day in NPM and minimum was observed on 5 th day in NRM (2.33UL -1 ). In Morchella sp. maximum MnP production was observed on 15 th day in NRM (57.32UL -1 ) and minimum activity was observed on 20 th day in NRM (9.73UL -1 ).
Mushrooms are recognized as important food items since ancient times. Their usage is being increased day by day for their significant role in human health, nutrition and disease. Fungi are ideal food because they have a fairly high content of protein (typically 20-30% dry matter as crude protein) which contains all of the essential amino acids [17]. For carrying out our study, we first selected three species two wild that is Morchella sp. and Helvella sp. and one commercially cultivated Auricularia polytricha. The extracellular laccase, AAO, LiP and MnP are thought to be involved in lignin degradation by WRF, which produce different combination of these extracellular enzymes. Proteins, Laccase and MnP production was maximum in Helvella sp. AAO (Aryl Alcohol Oxidase) production was maximum in Auricularia polytricha however Morchella sp. showed high LiP activity. In WRF laccase besides functioning as lignin degrading enzyme, it was also important in pigment production, polyphenol detoxification, fruiting body formation, sporulation and antimicrobial agent [7]. In general Laccase and MnP are more widely distributed among WRF than LiP [20]. Production of enzymes was good in Morchella and Helvella sp. in comparison to Auricularia polytricha (Table-1).