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Biochemical and Molecular Analysis of Laccase Enzyme in Saprobic Fungus; Sordaria fimicola.

Byline: Muhammad Ishfaq, Nasir Mahmood, Idrees Ahmad Nasir and Muhammad Saleem

Abstract

Saprobic fungi play an important role in decomposition and thus contributing to the global carbon cycle. Sordaria fimicola strains collected from diverse environment were evaluated for their laccase enzyme activity, while Aspergillus niger was used as control fungus. In laccase assay, S. fimicola strain N6 collected from the station 6 located at the Northern Facing Slope (NFS) of the Evolution Canyon 1 (EC 1), showed the maximum laccase enzyme activity (1.1 A/min). In the next step, complete laccase gene (1.917 Kb) was amplified and sequenced from the biochemically efficient isolates of S. fimicola viz. S1, S2, SF13, N6 and N7 and on comparison with reported sequence of Neurospora crassa (Accession no. M18334.1) 245 point mutations, 65 amino acid and post-translational modifications (PTMs) changes were detected in the in silico translation proteins.

In phylogenetic analysis, S1, S2, SF13, N6 and N7 strains of S. fimicola were found in clade-I alone, while reference sequence of N. crassa (Accession no. M18334.1) was present in clade-II. The laccase gene sequences were submitted to NCBI data base under accession numbers KM282173, KM282174, KM282172, KM282175 and KM282176 for S. fimicola strains S1, S2, SF13, N6 and N7, respectively. The biochemical as well as molecular data of the study depicted that environmental stresses affected the specific genes by bringing in mutations, which may result in genomic diversity among the organisms and their frontier molecules such as proteins. The observed laccase enzyme activity of S. fimicola-a non-pathogenic fungus, was found even better and comparable to A. niger, a pathogenic fungus. Therefore, being a saprophytic with short life cycle S. fimicola can become a fungus of choice to produce laccase enzyme at large scale.

Keywords: Sordaria fimicola; Laccase assay; Laccase gene; Phylogenetic analysis

Introduction

Laccases (EC 1.0.3.2) are used as a biocatalyst in many industrial processes including biopulping, bioleaching and treatment of industrial waste water (Bourdais et al., 2012). Laccase has ability to oxidize compounds like polyphenols, cyclic diamines, methoxy substituted phenols and other compounds (Baldrian, 2006). Fungi secrete enzymes in the growth media and purification of these secreted enzymes is considered relatively easier than from bacterial or plant sources, in which enzymes mostly are retained inside the cell as described by different workers (Archer et al., 2008; Upadhyay et al., 2016). Due to higher oxidation reduction potential (+800 mV) as compared to bacteria and plant, the fungal laccases have more application in biotechnology and food industry (Minussi et al., 2002).

Saprophytic fungi are unable to produce their own food and depend upon their enzymatic system, which breaks the complex biopolymers into simple nutritional components and subsequently absorbed from their surroundings (Alexopolous et al., 1996). Therefore, saprophytic fungus has potential to produce different extracellular enzymes, including laccases (Tellez-Jurado et al., 2005). Laccase from Neurospora crassa is an inducible secretory enzyme and other fungi that have laccase activity include Trichoderma and Botryosphaeria (Pointing et al., 2005; Ishfaq et al., 2016a).

Genes encoding laccase enzymes have been studied in different filamentous fungi like Aspergillus niger, A. oryzae and Trichoderma reesei (Hoffmeister and Keller, 2007; Gomaa and Momtaz, 2015). Fungal species belong to Ascomycota have different laccase encoding genes which are involved in the oxidization of syringaldazine dye (Dedeyan et al., 2000). In the current research S. fimicola fungus was evaluated for its laccase enzyme potential, further laccase gene analysis was carried out in order to exploit them to produce laccase enzyme at large scale.

Materials and Methods

Procurement of Fungal Strains and Culturing

Strains of S. fimicola (Roberge ex Desm.) Ces. and De Not., used in the study was isolated from the EC 1, located at the Mount Carmel, Israel. Whereas, the EC I have two opposite slopes, the Southern Facing Slope (SFS) with dry and harsh environment, which is quite different in flora and fauna from the NFS with moist and lush green environment (Nevo, 1995). On the SFS station 1 and 2 are located, while station 6 and 7 are present on the NFS and from each station 15 sample of S. fimicola were included, while one S. fimicola strain SF13 has been isolated from the premises of the University of Illinois at Urbana-Champaign (UIUC), USA. Potato Dextrose Agar (PDA) medium and PD broth were used for the culture reviving and sub-culturing of S. fimicola.

Laccase Assay

Fungal filtrate was separated from fungal mass by using Whatman filter paper No. 1. Bradford assay (Bradford, 1976) was used to estimate total secretory proteins from fungal filtrate. Laccase enzyme activity of S. fimicola was evaluated by using syringaldazine dye and absorbance was taken at 525 nm. The reaction mixture components were 0.1 mL syringaldazine (0.1 mM), 0.85 mL sodium citrate (0.1 M, pH-5.0) and 0.05 mL of the fungal growth medium filtrate containing 50 ug of total proteins. Absorbance value was read at 525 nm by using UV spectrophotometer (UV 1800 SHIMADZU), while 0.85 mL of 0.1 M sodium citrate (pH 5.0) and 0.1 mL of 0.1 mM syringaldazine was used as blank. Fifteen continuous readings were taken for each fungal strain filtrate sample with 1 min time interval.

The growth medium filtrate if showed high absorbance at 525 nm in different time intervals then it indicated the high enzyme activity in that fungal strain (Kahraman and Gurdal, 2002).

DNA Extraction and Ribotyping

The manual DNA isolation was carried out by 1% CTAB method with some modifications (Saghai-Maroof et al., 1984). The quality of extracted DNA was checked by 1% agarose gel electrophoresis and quantification was carried out by taking absorbance at 260 nm using UV spectrophotometer (UV 1800 SHIMADZU). S. fimicola strains found efficient in laccase enzyme production were subjected to ribotyping by amplification of 431 bases long hypervariable (V4) region of 18S rRNA gene as described earlier (Machouart-Dubach et al., 2001).

Laccase Gene Amplification and Sequencing

S. fimicola strains found efficient in laccase assay were further subjected to laccase gene targeting. Different primers were designed by using the N. crassa laccase gene (Accession no. M18334.1) as reference (Table 1). Amplification conditions were optimized by using different melting temperatures and MgCl2 concentration (1.5-3.0 mM) during PCR, while all other parameters of reaction mixture were kept constant including Go Taq flexi(r) Buffer (1x), dNTPs mixture (0.2 mM), Go Taq(r) DNA Polymerase (2.5 U), 25 pmol of each upstream as well as downstream primers and template DNA (100 ng) was used in 50 uL reaction mixture.

Temperature cycling condition used were; initial denaturation at 94degC for 4 min, followed by 40 cycles of denaturation temperature at 94degC for 1 min, annealing temperature at 50-60degC for 1 min, primer extension at 72degC for 1 min and final extension temperature was 72degC for 7 min, followed by hold at 4.0degC, while lid temperature was adjusted to 105degC to stop the evaporation. After laccase gene amplification, the PCR products were subjected to 1% agarose gel electrophoresis. The amplified DNA bands of laccase gene were purified from the gel and quantified by using NanoDrop ND-1000 (SPECTRAmax Plus) and were got sequenced in both directions from the Core Sequencing Facility available at UIUC, USA.

Bioinformatics Tools

The sequencing results of laccase gene from different strains of S. fimicola were submitted to NCBI database after peaks analysis by Chromas software. The laccase gene sequences were aligned by using ClastalW software in order to determine sequence variations. For finding protein sequence variations, gene sequences were translated by using protein translation and ExPASy Protparam tools. The post-translational modifications (PTMs) potential was predicted for laccase protein in S. fimicola by using different servers like LysAcet and PredMod for Acetylation, BPS for Methylation, DISPHOS and YinOYang for Phosphorylation and for Glycosylation NetNGlyc 1.0 and YinOYang were used. Molecular Evolutionary Genetics Analysis (MEGA 6.0.5) software was used for phylogenetic analysis (Tamura et al., 2013).

Results

Among the sixty one S. fimicola strains evaluated for their laccase enzyme activity, the isolate N6 collected from the station 6 of the EC 1 showed the maximum laccase enzyme activity (1.1 A/min) (Fig. 1). The isolates N7 (1.0 A/min), S1 (0.95 A/min), IQ36.5 (0.9 A/min), S2 (0.85 A/min) and SF13 (0.82 A/min) were found to be second, third, fourth, fifth and sixth most efficient isolates for laccase production (Fig. 1).

Five S. fimicola strains with maximum laccase enzyme activity from different environment viz. S1, S2, N6, N7 and SF13, were shortlisted for laccase gene amplification and sequencing. As regards DNA isolation, a genomic DNA band of ~15 Kb was observed from S. fimicola strains by using 1% gel electrophoresis. After DNA extraction S. fimicola strains were confirmed by ribotyping for their purity of cultures. Ribotyping results showed that sequences of V4 domain of 18S rRNA gene were 100% similar with each other as well as with previously reported sequence of S. fimicola (Accession No AY545724.1). The sequences of V4 domain of S. fimicola strains S1, S2, N6, N7 and SF13 were submitted to NCBI data base under accession numbers KF487278, KF487279, KF487281, KF487282 and LM654514, respectively.

Table 1: Primers used to amplify laccase gene in S. fimicola

Sr. No.###Name###Sequence (5'-3')###Expected PCR Product (bp)

1###LACU-F###TCC AGA CTC GGA GGT GAA###798

2###LACU-R###GAA ATG CGA GTG GTA CCA C

3###LACM-F###GGC ATG CAC CAG CGC AAC###799

4###LACM-R###TGA TGG GGA TCG TGT TGC

5###LACL-F###GAA CAC CAA CAG CAT CGC###753

6###LACL-R###ACC AAG ACC AAC ACC AGC

After amplification, complete laccase gene including exons and introns were got sequenced from all five shortlisted strain of S. fimicola and compared with the reference gene sequence of N. crassa (Accession no. M18334.1) by using ClastalW. The deduced laccase proteins from all five strains of S. fimicola as well as in reference gene of N. crassa (Accession no. M18334.1) were consisted of 619 amino acids (Table 2; Fig. 2). The PTMs of the laccase proteins in S. fimicola strains from the opposite slopes of the EC 1 were compared with each other and these were found to be common among all the five strains of S. fimicola except for the acetylation by server predMod (Table 3). The phylogenetic analysis of Laccase gene using 12 nucleotides sequences available at NCBI, placed the S. fimicola strains in clade-I and N. crassa in clad-II (Fig. 3).

Discussion

Out of total sixty one strains of S. fimicola, the strain N6 collected from the station 6 of the mild NFS showed the maximum laccase enzyme activity, while S. fimicola strains IQ36.6, S2, N7 and S1 were found second, third, fourth and fifth most efficient strains for laccase enzyme activity. The average laccase enzyme activity of S. fimicola was found comparable to the A. niger as described earlier (Ishfaq et al., 2014). Laccase enzyme production has been reported from soil Ascomycota species (Levasseur et al., 2010). Laccases are distributed in Ascomycetes, Deuteromycetes and Basidiomycetes, being particularly abundant in many white rot fungi, which are involved in lignin metabolism (Gochev and Krastanov, 2007).

Ribotyping results of S1, S2, N6, N7 and SF13 revealed that sequences of hypervariable V4 domain of 18S rRNA gene were 100% similar with each other (Ishfaq et al., 2016b) as well as with previously reported sequence of S. fimicola (Accession No AY545724.1), which confirmed that S. fimicola strains were original and pure without any contamination. This was in line with the finding of other coworkers, who used 18S rRNA gene sequencing for species identification in different studies (Caetano-Anolles, 2002; Meyer et al., 2010).

Genomic sequence of Sordaria macrospora was found most closely related to N. crassa (Nowrousian et al., 2010) than other sequenced filamentous fungi; therefore, in the current research, gene sequences of N. crassa were used to design the primers for the amplification of S. fimicola genes. The complete laccase gene including exons and introns were amplified from all five strains of S. fimicola and sequencing results were aligned with the reported laccase gene of N. crassa (Accession # M18334.1). In all five strains of S. fimicola, a total of 245 base substitutions were observed in the exonic region of laccase gene. As a result of base substitutions, a total of 65 amino acids changes were observed in all five strains of S. fimicola (Fig. 2).

These amino acids changes were common in all strains of S. fimicola with 100% prevalence except M(109)R amino acid shift which was present only in strain N6 and N7 with 40% prevalence (Table 2; Fig. 3) and this amino acid change may have impact on laccase enzyme activity of strains N6 and N7 as evident in biochemical assay performed for laccase enzyme. The sequence variations in laccase genes and their proteins were reported earlier in Ascomycetes fungi (Lyons et al., 2003). The laccase genes amplified and sequenced from Ganoderma lucidium, Phlebia brevispora and Trametes exhibited 65-74% nucleotide sequence homology (Galhaup et al., 2002). Laccase enzyme gene and protein isolation has been reported in different fungi, including A. niger and A. oryzae (Couto and Toca-Herrera, 2006, 2007).

Table 2: Analysis of laccase proteins derived from different strains of S. fimicola by ExPASy Protparam tool

Strains###No. of Amino acid.###Mol. Wt. of Protein (Da)###PI. of Protein###Gene length (Exons) (bp)###Dominated Amino acid (%)

RefE###619###68120.7###7.67###1857###Gly (G) 9.5

LACS1###619###67956.9###8.31###1857###Gly (G) 9.7

LACS2###619###67956.9###8.31###1857###Gly (G) 9.7

LACN6###619###67981.9###8.50###1857###Gly (G) 9.7

LACN7###619###67981.9###8.50###1857###Gly (G) 9.7

LACSF13###619###67956.9###8.31###1857###Gly (G) 9.7

Table 3: Predictions of PTMs in laccase proteins by using different servers

Servers used to predict PTMs###Serial no.###Laccase

###S1###N7###S2###N6

###Acetylation

###1###K38###K38###K38###K38

LysAcet###2###K104###K104###K104###K104

###1###K2###K2###K2

###2###Nil###K104###Nil

###3###Nil###K114###Nil

###4###K174###K174###K174

PredMod###5###K256###K256###K256

###6###K272###K272###K272###Nil

###7###K362###K362###K362

###8###K461###K461###K461

###9###K601###K601###K601

###10###K616###K616###K616

###Methylation

###1###K274###K274###K274###K274

BPS###2###K611###K611###K611###K611

###Phosphorylation

###1###T84###T84###T84###T84

DISPHOS###2###T88###T88###T88###T88

###1###S186###S186###S186###S186

YinOYang###2###S499###S499###S499###S499

###Glycosylation

###1###N139###N139###N139###N139

NetNGlyc 1.0###2###N282###N282###N282###N282

###1###T81###T81###T81###T81

YinOYang###2###S186###S186###S186###S186

The deduced laccase proteins consisted of 619 amino acids in all strains of S. fimicola as well in reference gene (Accession no. M18334.1) of N. crassa (Table 2); but N. crassa protein has different molecular weight (68120.7 Da) as compared to laccase proteins derived from strains of S. fimicola (Table 2). This difference in molecular weight is attributed to the amino acids substitutions at 65 different positions in all five samples of S. fimicola as compared to reference control protein (Fig. 2). This is also evidenced from the laccase protein molecular weight, where molecular weight of the S1, S2 and SF13 were same (67956.9 Dalton); while the molecular weight of N6 and N7 were same (67981.9 Dalton) (Table 2). The laccase protein from Cryphonectria parasitica was 591 amino acids long and has 57% similarity with N. crassa as reported earlier by Choi et al. (1992).

Several reports can be referred, on laccase genes in Ascomycota such as Gaeumannomyces graminis (Edens et al., 1999), Magnaporthe grisea (Iyer and Chattoo, 2003) and Mauginella (Palonen et al., 2003). Laccase is monomers having a molecular mass in the range of 40000-130000 Da with a covalently linked carbohydrate content of 10-25% in fungi and 20-45% in plants (Claus, 2003). As regard isoelectric point (pI) value of laccase proteins, N6 and N7 strains showed 8.50, while strains S1, S2 and SF13 showed 8.31; while the (pI) value of reference control laccase protein was 7.67 (Table 2). However, this finding was found in contrary to an earlier report of Terraza et al. (2007), according to which (pI) of fungal laccase was in range of 4-5.

As regards PTMs, these were common among all strains of S. fimicola except for the acetylation, which was predicted in N7 strain at K104 and K114 (Table 3). Ceriporiopsis subvermispora has a single laccase gene and its multiple isoforms are formed by the process of PTMs possibly by glycosylation and phosphorylation as reported by different coworkers (Larrondo et al. 2003; Feng et al., 2015).

The Phylogenetic analysis placed the S1, S2, SF13, N6, and N7 in the clade-I along with S. macrospora (XM003349355.1) depicting no or negligible intraspecific variation between Sordaria spp., while reference sequence of N. crassa (Accession no. M18334.1) was found in clade-II (Fig. 3). The fifty specific laccase gene sequences from different organisms including fungi were compared by multiple sequence alignment and results of the phylogenetic analysis reported diversity of the derived proteins (Satpathy et al., 2013). The protein coding genes have been used to study the phylogenetic analysis among Ascomycetes and Zygomycetes fungi as reported earlier by several co-workers (Diezmann et al., 2004; Tanabe et al., 2004). The biochemical and molecular analysis revealed the laccase activity of S. fimicola strains better and comparable to A. niger. Therefore, being a non-pathogenic with a short life cycle S. fimicola can be used as an alternative fungus for A. niger to produce laccase on a larger scale.

Acknowledgements

The work was financially supported by Higher Education Commission (HCE), Islam Abad, under International Research Support Initiative Program and by the Special research grant given by the Vice Chancellor, University of the Punjab (New campus) Lahore. We gratefully acknowledge Dr. Youfu "Frank" Zhao, Crop Sciences Department, University of the Illinois at Urbana-Champaign, USA, for his help.

References

Alexopolous, C.J., C.W. Mims and I.M. Blackwel, 1996. Introductory Mycology, 4th ed. John Wiley and Sons, Inc. New York, USA

Archer, D.B., I.F. Connerton and D.A. MacKenzie, 2008. Filamentous fungi for production of food additives and processing aids. Adv. Biochem. Eng. Biotechnol., 111: 99-147

Baldrian, P., 2006. Fungal laccases-occurrence and properties. FEMS Microbiol. Rev., 30: 215-242

Bourdais, A., F. Bidard, D. Zickler, V. Berteaux-Lecellier, P. Silar and E. Espagne, 2012. Wood utilization is dependent on catalase activities in the filamentous fungus, Podospora anserine. PloS ONE, 7: e29820

Bradford, M.M., 1976. Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72: 248-254

Caetano-Anolles, G., 2002. Tracing the evolution of RNA structure and the rooting of the universal tree of the life. Nucleic Acids Res., 30: 2575-2587

Choi, G.H., G.L. Thomas and L.N. Donald, 1992. Molecular analysis of the laccase gene from the chestnut blight fungus and selective suppression of its expression in an isoelectric hypovirulent strain. Mol. Plant-Microbe In., 5: 119-128

Claus, H., 2003. Laccases and their occurrence in prokaryotes. Arch. Microbiol., 179: 145-150

Couto, S.R. and J.L. Toca-Herrera, 2006. Industrial and biotechnological applications of laccases: a review. Biotech. Adv., 24: 500-513

Couto, S.R. and J.L. Toca-Herrera, 2007. Laccase production at reactor scale by filamentous fungi. Biotech. Adv., 25: 558-569

Dedeyan, B., A. Klonowska, S. Tagger, T. Tron, G. Iacazio, G. Gil and J.L. Petit, 2000. Biochemical and molecular characterization of a laccase from Marasmius quercophilus. Appl. Environ. Microbiol., 66: 925-929

Diezmann, S., C.J. Cox, G. Schonian, R.J. Vilgalys and T.G. Mitchell, 2004. Phylogeny and evolution of medical species of Candida and related taxa: a multigenic analysis. J. Clin. Microbiol., 42: 5624-5635

Edens, W.A., T.Q. Goins, D. Dooley and J.M. Henson, 1999. Purification and characterization of a secreted laccase of Gaeumannomyces graminis var. tritici. Appl. Environ. Microbiol., 65: 3071-3074

Galhaup, C., H. Wagner, B. Hinterstoisser and D. Haltrich, 2002. Increased production of laccase by the wood-degrading Basidiomycete Trametes pubescens. Enzyme Microbial. Technol., 30: 529-536

Feng, B.Z., P.Q. Li1, L. Fu and X.M. Yu, 2015. Exploring laccase genes from plant pathogen genomes: a bioinformatics approach. Genet. Mol. Res., 14: 14019-14036

Gochev, V.K. and A.I. Krastanov, 2007. Fungal laccases. Bulg. J. Agric. Sci., 13: 75-83

Gomaa, O.M. and O.A. Momtaz, 2015. Copper induction and differential expression of laccase in Aspergillus flavus. Braz. J. Microbiol., 46: 285-292

Hoffmeister, D. and N.P. Keller, 2007. Natural products of filamentous fungi: enzymes, genes and their regulation. Nat. Prod. Rep., 24: 393-416

Ishfaq, M., N. Mahmood, I.A. Nasir and M. Saleem, 2014. Molecular and biochemical screening of local Aspergillus niger strains efficient in catalase and laccase enzyme production. Int. J. Agric. Biol., 16: 177-182

Ishfaq, M., N. Mahmood, Q. Ali, I.A. Nasir and M. Saleem, 2016a. Biochemical and molecular studies of various enzymes activity in fungi. Mol. Plant Breed., 7: 1-16

Ishfaq, M., N. Mahmood, I.A. Nasir and M. Saleem, 2016b. Biochemical and molecular analysis of superoxide dismutase in Sordaria fimicola and Aspergillus niger collected from different environments. Pol. J. Environ. Stud., in press

Iyer, G. and B.B. Chattoo, 2003. Purification and characterization of laccase from the rice blast fungus, Magnaporthe grisea. FEMS Microbiol. Lett., 227: 121-126

Kahraman, S.S. and I.H. Gurdal, 2002. Effect of synthetic and natural culture media on laccase production by white rot fungi. Bioresour. Technol., 22: 2-7

Larrondo, L.F., M. Avila, L. Sala, D. Cullen and T. Vicun, 2003. Heterologous expression of laccase cDNA form Ceriporiopsis subvermispora yields copper-activated apoprotein and complex isoform patterns. Microbiology, 149: 1177-1182

Levasseur, A., M. Saloheimo, D. Navaroo, M. Andberg, P. Pontarotti, K. Kruus and E. Record, 2010. Exploring laccase-like multicopper oxidase genes from the ascomycete Trichoderma reesei: a functional, phylogenetic and evolutionary study. BMC Biochem., 24: 11-32

Lyons, J.I., S.Y. Newell, A. Buchan and M.A. Moran, 2003. Diversity of ascomycete laccase gene sequences in a southeastern US salt marsh. Microb. Ecol., 45: 270-281

Machouart-Dubach, C.L., F.D. Martine, I. Chauvin, L.E. Gall, G. Catherine, L. Frederick and D. Francis, 2001. Rapid discrimination among dermatophytes, Scytalidium spp. and other fungi with a PCR-Restriction Fragment Length Polymorphism Ribotyping method. J. Clin. Microbiol., 29: 685-690

Meyer, A., C. Todt, N.T. Mikkelsen and B. Lieb, 2010. Fast evolving 18S rRNA sequences from Solenogastres (Mollusca) resist standard PCR amplification and give new insights into mollusk substitution rate heterogeneity. BMC Evol. Biol., 10: 70

Minussi, R.C., G.M. Pastore and N. Duran, 2002. Potential applications of laccase in the food industry. Trends Food Sci. Technol., 13: 205-216

Nevo, E., 1995. Asian, African and European biota meets at "Evolution Canyon" Israel. Local tests of global biodiversity and genetic diversity patterns. Proc. R. Soc. Lond., 262: 149-155

Nowrousian, M., J.E. Stajich, M. Chu, I. Engh, E. Espagne, K. Halliday, J. Kamerewerd, F. Kempken, B. Knab, H.C. Kuo, H.D. Osiewacz, S. Poggeler, N.D. Read, S. Seiler, K.M. Smith, D. Zickler, U. Kuck and M. Freitag, 2010. De novo assembly of a 40 Mb eukaryotic genome from short sequence reads: Sordaria macrospora, a model organism for fungal morphogenesis. PLoS Genetics, 6: e1000891

Palonen, H., M. Saloheimo, L. Viikari and K. Kruus, 2003. Purification, characterization and sequence analysis of a laccase from the ascomycete Mauginiella sp. Enzyme Microb. Technol., 33: 854-862

Pointing, S.B., A.L. Pelling, G.J.D. Smith, K.D. Hyde and C.A. Reddy, 2005. ScreeningofBasidiomycetesandXylariaceousfungiforlignin peroxidase and laccase gene-specific sequences. Mycol. Res., 109: 115-124

Saghai-Maroof, M.A., K.M. Soliman, R.A. Jorgensen and R.W. Allard, 1984. Fungal DNA isolation. Proc. Natl. Acad. Sci., 81: 8014-8018

Satpathy, R., R. Behera, S.K. Padhi and R.K. Guru, 2013. Computational phylogenetic study and data mining approach to laccase enzyme sequences. J. Phylogenetic Evol. Biol., 1: 108

Tamura, K., G. Stecher, D. Peterson, A. Filipski and S. Kumar, 2013. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol. Biol. Evol., 30: 2725-2729

Tanabe, Y., M. Saikawa, M.M. Watanabe and J. Sugiyama, 2004. Molecular phylogeny of Zygomycota based on EF-1alpha and RPB1 sequences: limitations and utility of alternative markers to rDNA. Mol. Phylogenet. Evol., 30: 438-449

Tellez-Jurado, A., A. Arana-Cuenca, A.E. Gonzalez-Becerra and O. Viniegra-Gonzalez Loera, 2005. Expression of heterologous laccase by Aspergillus niger cultured by solid state and submerged fermentations. Enzyme Microbial. Technol., 38: 665-669

Terraza, C.A., L.H. Tagle, F. Concha and L. Poblete, 2007. Synthesis and characterization of new bi-functional monomers based on germarylene or silarylene units: 4,4'-(R1R2-silylene) bis (phenyl chloroformates) and 4,4'-(R1R2-germylene) bis (phenyl chloroformates). Desig. Monomers Polym., 10: 253-261

Upadhyay, P., R. Shrivastava and P.K. Agrawal, 2016. Bioprospecting and biotechnological applications of fungal laccase. Biotechnology, 6: 15
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Author:Ishfaq, Muhammad; Mahmood, Nasir; Nasir, Idrees Ahmad; Saleem, Muhammad
Publication:International Journal of Agriculture and Biology
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Date:Feb 28, 2017
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