Introduction

Trichoderma belongs to Hypocrea (Ascomycota) (Savoie et al. 2001) and introduced by Persoon (1974). These fungal species are commonly present in all climatic zones (Błaszczyk et al. 2014) and love to live in soil, root, and foliar environment (Huang et al. 2011). Rapid growth with maximum laccase potential is a characteristic of Trichoderma species (Kubicek et al. 2003). These species act as bio control agents (Benitez et al. 2004), biopesticides and fungicides (Howell 2003), releasing antibiotics (Vinale et al. 2008) during plant–pathogenic fungal interaction (Mukherjee et al. 2012), resistive to biotic and abiotic stresses (Mastouri et al. 2012) and play a vital role in plant diversity (Druzhinina et al. 2011; Mathys et al. 2012).

Many kingdoms have been explored for laccase potential, but fungal laccase processed higher activities (Bhamare et al. 2016) as asomycetous laccase sensu stricto exhibit superb qualities than basidiomycetes (Cazares-Garcıa et al. 2013). Trichoderma species are called “aggressive biodegraders” due to lytic enzymes important for cell wall components (Strakowska et al. 2014; Ramı´rez-Cavazos et al. 2014), periplasmic space, and fungal conidial membrane (Pokorny et al. 2005).

This multicopper enzyme is constitutive and inducible in nature (Kiiskinen et al. 2004; Sharma et al. 2005) and reported molecular weight of laccase ranges from 50 to 97 kDa (Chandra et al. 2015). This is an attractive biocatalyst in industrial applications (Giardina et al. 2015). Oxidative coupling reactions are also catalyzed by fungal laccase (Rodríguez-Couto 2019). This is useful in biofuel production and textiles finishing (Abd El Monssef et al. 2016). This enzyme possess “Janus-faced” range of activities (Giardina et al. 2010) e.g., azodye oxidation (Gomi et al. 2011), biochemical bleaching of pulp (Kalyani et al. 2012), biosensors constructor (Bilal et al. 2019; Gomes and Rebelo 2003), delignification of plant products (Copete-Pertuz et al. 2019), producer of other enzymes (Wang et al. 2002), humification (Hatakka 2001), paper processing (Michael et al. 2005), phenolics, non-phenolics and miscellaneous degradation (Litwińska et al. 2019), pollutants detoxifier (Tortella et al. 2013), polymerization or depolymerization processes (Viswanath et al. 2014), steroid transformation (Priyadarsini et al. 2011), wine and water discoloration (Ikehata et al. 2004), xenobiotic compounds degradation (Gomi et al. 2011), use in nanotechnology (Chhaya and Gupta 2010) and pharmaceutical products (Priyadarsini et al. 2011) as well. The major applications in the field of medicine are anticancer drugs (Sekme et al. 2013), hormone derivatives, preparation of antiviral agents, and antioxidants preparation (Rocasalbas et al. 2013). Furthermore, laccase markers are used in immunoassays (Warsinke et al. 2000), and to design energy transformation systems (Chen et al. 2001) and detoxify organic pollutants (Lynch and Moffat 2005).

Few literatures have been reported on laccase production from Trichoderma species (Holker et al. 2002). Although this is an expensive industrial enzyme, a major problem encountered by industrialists is the low production level from its native hosts. This study was designed to screen Trichoderma sp. with minimum cost and maximum laccase under optimum conditions. The laccase and strongest candidate were identified by the construction of molecular phylogeny. These samples were packed and stored (4 °C) in germ-free polythene bags from different sites in Lahore, Punjab Pakistan.

Materials and methods

Isolation Trichoderma species

Soils of 5–10 cm depth were collected from the Sugar beet rhizosphere. These samples were packed and stored (4 °C) in germ-free polythene bags from different sites of Lahore, Punjab Pakistan.

Malt Extract Agar (MEA) medium was prepared with little modification in g/L by adding Malt Extract 20, Agar 20, MgSO4.7H2O 0.5, K2HPO4 1, Yeast Extract 4, glucose 20, and peptone 6 (Coll et al. 1993) at pH 5.5. Streptomycin (200 mg/L) was added as an antibacterial agent. Complete MEA medium was sterilized in an autoclave for 20 min at 121 ºC. This medium was used to obtain the fungal isolations. The serial dilution made to get the pure Trichoderma cultures on Petri plates. These plates were incubated at 30 °C. After 7–10 days, pure mycelium was cultivated and stored at 4 °C. The pure living cultures were deposited in Institute of Agricultural Sciences, University of the Punjab, Lahore (IAGS).

Identification, DNA extraction, sequence alignment, and molecular phylogeny

A modified CTAB procedure was followed to extract the total genomic DNA from the specimens (Doyle and Doyle 1987). Nuclear ribosomal ITS regions were used to study the target species. Sequencing of PCR products (Bidriectional) was done by Macrogen Inc. South Korea. The consensus was generated from both ITS1F and ITS4 in BioEdit version 7.2.5 (Hall 1999) and then homology searches were performed at the National Center for Biotechnology Information (NCBI) Web site using BLAST. Dataset of ITS initial blast (BLAST) and literature accessions were downloaded from GenBank (Benson et al. 2017). Sequences were aligned and edited using ClustalX 2.1 (Larkin et al. 2007) and BioEdit (Hall 1999). Sequences retrieved from GenBank and the newly generated sequences were aligned with MAFFT v. 10 (http://mafft.cbrc.jp/alignment/server/index.html; (Katoh and Standley 2013). The alignment was set at 596 positions. These sequences were used to construct the phylogenetic tree by using the maximum-likelihood method. The phylogeny was performed on MEGA version 10.0 (Tamura et al. 2011). Bootstrap values were adjusted from 1000 replicates. This phylogenetic tree helps to find the exact phylogeny of the filamentous fungi of samples. Branches less than 50% bootstrap replicates were collapsed.

Screening of laccase producing Trichoderma

All collected fungal isolates were cultured on Petri plates containing sterilized MEA augmented with 0.04% guaiacol and 0.01% (w/v) chloramphenicol (to avoid bacterial growth) adjusted at pH 5.5. The purpose of this synthetic substrate was to identify those Trichoderma species excreted maximum laccase by formation of maroon brown color zone around its mycelia colony. These Petri plates were incubated at 28–30 °C for 72 h and then screened for the formation of reddish brown zones around the fungal colonies (Kalra et al. 2013).

Laccase production medium and inoculation in submerged culture

After identification of Trichoderma species, pure agar culture blocks (5 mm) were inserted into a flask (250 mL) containing the basal nutrient medium without agar. At 28 °C, 150 rpm, the flasks were incubated for 8 days. The fungal liquid culture with mycelia was centrifuged at 10, 000 rpm at 4 °C for 20 min followed by filtration using Whatman filter papers. The obtained extracellular fluid supernatant was used for further investigations containing crude laccase.

Assay of laccase activity

The above-mentioned supernatant comprised of crude laccase used for the determination of enzymatic activity by measuring the oxidation of guaiacol substrate (Gao et al. 2011). Sodium acetate (50 mM) buffer (pH 4.5) and guaiacol (2 mM) were used for quantification. The reaction mixture for quantification of laccase under spectrophotometer comprised 1 mL crude enzyme supernatant, 3 mL sodium acetate buffer and 1 mL guaiacol (Chefetz et al. 1998) vortexed for 30 s, after that, it was incubated for 10 min at 30 °C. After 30 s absorbance was measured at 465 nm (ε465 = 12,100 M−1 cm−1). Formula used for calculation was E.A = (A* V) / (t * €* v), where E.A = Enzyme Activity (U/mL), A = Absorbance at 465 nm, V = Total volume of reaction mixture (mL), v = enzyme volume (mL), t = Incubation time (min) and € = Extinction Coefficient (M−1 cm−1) (Gao et al. 2011). The data of laccase from all Trichoderma species were compared to commercial Trametes versicolor laccase (Sigma/38429) as a control.

Enzyme purification gel electrophoresis

Laccase purification was conducted by the method of Chefetz et al. (1998). Filtrate was centrifuged at 13,000 rpm for 20 min at 10 °C and supernatant subjected to ammonium sulfate precipitation. Precipitates were dialyzed and loaded to DEAE-Cellulose anion-exchange column, equilibrated by 10 mM sodium acetate buffer (pH 5.5). The fraction containing laccase was pooled, concentrated, and dialyzed overnight. The DEAE-purified sample was loaded on to the column by adding 3 mL fraction. This fraction was dialyzed and purity of the laccase with its molecular weight was determined by SDS-PAGE (Duran et al. 2003) and visualized by the staining gel with Coomassie Brilliant Blue R-250. The relative molecular mass was estimated using standard molecular weight markers [ribonuclease (15.4 kDa), chymotrypsin (25.0 kDa), ovalbumin (43.0 kDa), and bovine serum albumin (67.0 kDa)].

Analysis of amino acid sequence and phylogram

The protein sequencer ABI Procise 491HT was used to sequence the laccase band by the method of Matsudaira (1987).

Statistical analysis

Assays were carried out in triplicate, and the results were presented as mean ± standard deviation.

Results

In this study, 10 species of Trichoderma were evaluated for laccase activity. The colony characteristics were also determined (Fig. S1, Table 1). Trichoderma fungal colonies were screened for evaluation of laccase potential by guaiacol substrate. Due to the variation in substrates utilization, this extracellular protein gained commercial and industrial importance. Pure cultures of these species were identified on molecular basis using ITS markers. Maximum-likelihood analysis was applied to construct the phylogenetic tree to identify these species (Fig. S2). Purified fungal mycelium made a specific clade with its neighboring species with maximum bootstrap values confirmed robust tree topology. Phylogenetic analysis clearly facilitated in identification of filamentous species of Trichoderma.

Table 1 List of Laccase (U/mL) producing Trichoderma Species
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Out of ten, four species were able to oxidize guaiacol in shake flask than other Trichoderma species including T. cremeum, T. longipile, T. citrinoviride, and T. atroviride. Reddish brown color was produced by oxidation of guaiacol by extracellular laccase secretion in the plate media, but the incubation time and growth rate varied according to the individual colony. Laccase secretion potential in preliminary screening was in this order T. atroviride > T. cremeum > T. longipile and > T. citrinoviride. Amongst all species, Trichoderma atroviride was more efficient in laccase production confirmed by guaiacol enzyme assay (Fig. S3). This species also showed fast growth and dark brown coloration within 3 days of incubation followed by T. cremeum, T. longipile, and T. citrinoviride. The mycelium of these species was stored at 4 °C for farther analysis.

In this study, the optimum conditions were applied to find the strongest candidates and compared with commercial laccase as a control of Trametes versicolor laccase (5 U/mL). Trichoderma cremeum, T. longipile, and T. citrinoviride exhibited strong laccase activity in submerge shake flasks less than T. atroviride. The laccase activity from T. atroviride was 2.62 U/mL (Fig. 1) considered the strongest candidate in laccase production. Secondary productive species were T. cremeum (1.98 U/mL), T. longipile (1.84 /mL) and T. citrinoviride (1.65 U/mL) (Fig. 1). Literature cited that T. atroviride, T. harzianum and T. reesei were the strongest and competent laccase secreting candidates amongst Trichoderma species by the utilization of augmented media. But the laccase activities related to these species were low in this experimental work. The lowest value of laccase (U/mL) in the shake flask was demonstrated by T. beinartii (0.42 U/mL) (Fig. 1). Many of these species have not been explored till yet in literature. This study has been explored many laccase producing Trichoderma species (Fig. 1). The strongest candidate, T. atroviride was used for farther analysis.

Fig. 1
figure 1

Comparative evaluation of laccase activity (U/mL) from Trichoderma Species compared with commercial laccase (T. versicolor)

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The purification of laccase from Trichoderma atroviride was determined by 60% ammonium sulfate precipitation method. After the partial purification, the molecular weight was estimated by SDS-PAGE. Standard protein markers were used to quantify the purified TaLacc1. The molecular weight was ~ 57 kDa determined by SDS-PAGE (Fig. S4). The band position after staining facilitated in the quantification.

The previously reported literature distinguishes the laccase genes from the other copper blue oxidases in comparative analysis of laccase sequences. The length of amino acid sequences of T. atroviride laccase was near to the typical fungal laccases (510 aa). The calculated molecular weight of the protein sequence was nearly ~ 57 kDa.

The purified TaLacc1 of Trichoderma atroviride was identified by amino acids sequences and conducted by Blast analysis of the database in GenBank. A phylogram of T. atroviride was constructed to find the exact identity of laccase. The sequences of various laccases were obtained from the NCBI GenBank database used as queries to find the laccase genes of T. atroviride, which removed the doubts regarding laccase identity.

For the phylogenetic analysis of the putative sequences of laccase, multiple alignments were performed with CLUSTALW in BioEdit software. The alignments were manually adjusted. Based on the generated alignments, phylogenetic trees were constructed with MEGA version 10.0. Statistical significance was evaluated with a bootstrapping of 1000 repetitions. The phylogeny was confirmed by using the maximum likelihood method, and the alignments were differentially edited to corroborate the topology of the tree (Fig. 2).

Fig. 2
figure 2

Phylogram of amino acids sequence of purified laccase of T. atroviride indicated by (Colors indicated different clades) (color figure online)

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Laccase of T. atroviride exhibited close phylogenetic relationships with other Trichoderma species. The reason behind is the protein included in phylogenetic analysis were chosen according to the same criteria of methodology. The ITS sequences of all Trichoderma species were deposited in GenBank (Table 1).

Discussion

Trichoderma–filamentous fungi are the largest group of microorganisms which plays an important role in the environment. These species were selected due to the rapid growth rate in the media rather than other filamentous fungi. Natural substrates (e.g. agricultural residues, wheat bran, and rice straw/husk) mimic the natural conditions under which the particular fungus grows (Brijwani et al. 2010) and prolonged cultivation period is required for production of maximum laccase (Shekher et al. 2011; Rodríguez et al. 2005; Pandey et al. 1999). In this study, preference was given to a synthetic substrate. Substrate spectra of numerous laccases from fungal origin have been reported. Experimental procedure and choice of compound present difficulty in data comparison (Endo et al. 2003; Smirnov et al. 2001; Xu 1996). This enzyme possesses overlapping range of substrate specificity and non-specific towards substrates form one type of laccase to another. Laccase oxidize ortho and paradiphenol, but usually more efficient towards paradiphenol (Wood 1980). Guaiacol is an ortho-substituted compounds typically referred as the best substrates for most laccases (Thurston 1994). Trichoderma species collected from Sugar beet rhizosphere, formed a brown-zone around and below colony on the Plate-containing guaiacol substrate, which was a characteristic laccase production (extracellular) on a solid medium of filamentous fungi (Thurston 1994; Madhavi and Lele 2009).

The major issue behind this study was the high cost of laccase with low production level. In this experiment, strong Trichoderma candidates were screened with high laccase concentrations by the optimized cultural conditions. Nutrient broth beneath the mycelium in submerge flasks enhanced the extracellular secretion of laccase.

Holker et al. (2002) identified extracellular laccase in Trichoderma sp., however, little information is available on intracellular laccases in Trichoderma as well as other fungal species (Assavaning et al. 1992). Pokorny et al. (2005) worked on periplasmic laccase (extracellular) activity of T. harzianum, T. atroviride, and T. viride during maturation of conidia. Laccase in conidia synthesizes pigments and other constituents that safeguard the cells from stress factors (UV light, enzymatic lysis, and temperature, etc.) (Chakroun et al. 2010).

Sadhasivam et al. (2009) worked on Trichoderma harzianum by augmented CuSO4 (1 mM) in a liquid medium. This yielded high laccase (4.36 U/mL) after the 4th day. T. reesei possessed 1.22 U/mL in liquid medium and worked well in acidic pH (4.5) and 27 °C temperature. Desai and Nityanand (2011) entitled T. harzianum the strongest candidate amongst all Trichoderma species, whereas, Holker et al. (2002) found maximum laccase activity in spores of T. atroviride and T. harzianum found in this study.

Trichoderma belongs to those fungi which comprised minimum number of laccase genes (Cázares-García et al. 2013). However, the results in the present study indicated that laccase genes were earlier reported in ascomycetes should be reviewed in the future, as it is not possible for all of them to code the laccases. Molecular weight of T. atroviride of this study was in agreement with the previously reported fungal laccases, having between 55 and 70 kDa. In Trichoderma, purified laccases from T. harzianum WL1 and T. atroviride CTM 10,476 presented glycosylated laccase of 79 kDa and 80 kDa, respectively (Chakroun et al. 2010; Sadhasivam et al. 2009) contrary kDa of this purified laccase. In literature during evolution, T. reesei lost maximum quantity of the genetic message, while retained in T. virens (Kubicek et al. 2011). The generated data during speciation, it is possible that T. atroviride and T. virens maintained total laccase genes as common ancestor has, while T. ressei and T. harzianum lost extracellular laccase genes (Cázares-García et al. 2013).

This genus has preserved limited functional laccase amino acids in the course of evolution. Mostly, the whole protein sequence of intracellular laccase is preserved, while the evolutionary patterns of extracellular laccase are variable (Cázares-García et al. 2013). So, the recognition of Trichoderma laccase is important to analyze the robust phylogenetic analyses of this enzymatic function.

In this study, many Trichoderma species were evaluated for laccase potential to find out the strongest candidate. Only T. atroviride a biotechnological workhorse of the genus Trichoderma has been reported as a strong laccase producing organism. In addition, phylogenetic and evolutionary analyses were also carried out to identify the Trichoderma species as well as laccase enzymes.