Introduction

One of the major constraints for economic conversion of lignocellulosics agro-residues into fermentable sugars is the high cost of commercial cellulolytic enzymes [1]. The existing commercial enzyme cocktails derived from the filamentous mesophilic fungal strains such as Trichoderma reesei (teleomorph Hypocrea jecorina) [2], Penicillium decumbens [3], and Acremonium cellulolyticus [4] are catalytically inefficient and therefore thermophilic fungal strains such as Thermoascus aurantiacus [5], Myceliophthora thermophila owing to the thermostability and faster rate of reaction are considered better choice [6]. However, owing to high crystallinity of the acid pre-treated lignocellulosics used in existing commercial/demonstration level 2G ethanol plants, a high protein loading (mg/g of the substrate) is required for hydrolysis which make the bioconversion process cost intensive [7, 8]. Recent studies have shown that newly discovered copper dependent LPMOs (lytic polysaccharide monooxygenases) classified as AA9 can oxidatively fracture the crystalline structure of the recalcitrant biomass in synergy with hydrolytic enzymes for enhancing saccharification efficiency and thus reduces protein loading rates appreciably [9,10,11,12]. These copper dependent mono-oxygenases or perhaps peroxygenases [13] introduce a single oxygen atom from molecular oxygen [14] or oxygen obtained from H2O2 [15] which is now proposed as a preferred co-substrate. However, complex nature of the reaction, insolubility of one of the substrates and oxidative enzyme inactivation makes LPMOs kinetic studies challenging. Further, studies reveal that in spite of LPMOs having very low kinetic rates the cleavage of recalcitrant polysaccharide is highly efficient. LPMOs requires an external electron donor such as ascorbate [9], gallate, reduced glutathione, plant derived flavonoids, and lignin building blocks [16, 17] to return to its initial [Cu(I)] form. Apart from these organic compounds based electron donors, enzymes like cellobiose dehydrogenase (CDH), glucose dehydrohenase (GDH) and photosynthetic pigments [18] also act as natural electron sources for reducing Cu2+, to carry out oxidative breakage of β-1, 4-glucosidic bonds of recalcitrant polysaccharide. Based on their regio-selectivity, these LPMOs are further classified into three groups: Type-1, 2, and 3 which catalyze oxidative cleavage of β-1, 4 linkage of cellulose at C1, C4 and both C1-C4, respectively [17, 19, 20].

In this study, two type-3 AA9 family LPMOs, derived from thermophilic fungal strains S. thermophilum and M. cinnamomea, designated as PMO9D_SCYTH and PMO9D_MALCI, respectively, were cloned and expressed in the methylotrophic yeast Pichia pastoris strain X33. These LPMOs were further purified using anion exchange chromatography and functionally characterized. The H2O2 driven kinetics of PMO9D_SCYTH and PMO9D_MALCI against avicel as substrate is being reported for the first time. In addition, high thermostability of these LPMOs, as evident from their half-life (t1/2), make them interesting candidate for designing efficient enzyme cocktails. The enzyme cocktails comprising of recombinant LPMOs and commercial cellulase Cellic CTec2 were also evaluated for the hydrolysis of acid/alkali pretreated sugarcane bagasse and rice straw.

Materials and Methods

Microbial Strains and Sequence Information

To derive the AA9 genes for this study, two previously isolated thermophilic fungus S. thermophilum NAIMCC-F-03113 and M.cinnamomea NAIMCC-F-03115 (identified on the basis of ITS sequencing with accession number KJ563253 and KJ563258, respectively) and deposited with NBAIM (National Bureau of Agriculturally Important Microorganisms, Maunath Bhanjan, India), were used [21]. S. thermophilum and M. cinnamomea cultures were grown on yeast potato soluble starch (YpSs) agar medium. E. coli strain DH5α and Pichia pastoris strain X-33 (Invitrogen, Carlsbad, CA), as described in earlier work by Basotra [22], were used as respective hosts for sub-cloning and heterologous expression of PMO9D_SCYTH and PMO9D_MALCI proteins. Based on the N-terminal histidine and other conserved histidine in the poly-peptide chain sequence, two LPMOs with gene model ID Scyth2p4_002689 and Malci1p7_001555 were selected out of the total of twenty seven and eight AA9 encoding genes found in the genome sequences of S. thermophilum (CBS 625.91) and M. cinnamomea (CBS 343.55), respectively (Prof. Adrian Tsang, Concordia University).

Cloning of PMO9D_SCYTH and PMO9D_MALCI cDNA in Pichia pastoris X33

Induction of lignocellulolytic enzyme production in S. thermophilum and M. cinnamomea was carried out by growing the cultures at 40 ° C and 180 rpm for 48 h using CWR medium, (cellulose, wheat bran and rice straw) in ratio of 3:1:1 as described previously by Rai [23]. Total RNA was extracted from the 100 mg frozen mycelium in liquid nitrogen, using HiPurA™ RNA Mini-prep Purification Kit (HiMedia, India). Enrichment of mRNA containing Poly (A) from total RNA using maxi mRNA isolation kit (Invitrogen, USA) was done and used for the complementary DNA synthesis [24, 25]. Based on the genome sequence for S. thermophilum (CBS 625.91) and M. cinnamomea (CBS 343.55) (Prof. Adrian Tsang, Concordia University), specific primers for ORFs were designed (Table A1) and amplification of the pmo9d_scyth and pmo9d_malci genes was done using Q5® High Fidelity DNA Polymerase (New England Bio-labs, USA) by initial denaturation at 95 °C for 5 min, 36 cycles of denaturation at 95 °C for 45 s, annealing at 55 °C for 45 s, extension at 72 °C for 40 s, and final extension at 72 °C for 10 min. The size of amplified products was confirmed by agarose gel-electrophoresis (1% w/v agarose) and then purified from gel using Gene clean® Turbokit (MP). The purified products of pmo9d_scyth and pmo9d_malci were introduced into plasmid (pPICZαA) between KpnI and XbaI restriction sites (pPICZαA-PMO9D_SCYTH and pPICZαA-PMO9D_MALCI fusants). These fusion sets were transformed into E. coli DH5α by heat shock method, and positive transformants were screened by colony PCR and re-confirmed by checking the size of fall-out obtained by restriction enzyme digestion of the plasmids from positive transformants. The isolated plasmid from positive transformants was linearized using PmeI (New England Bio-labs) and introduced in the competent cells of Pichia pastoris X33 by electroporation (Invitrogen, Carlsbad, CA, USA) which were prepared on the same day [26] and plated onto YPDS (1% Yeast extract, 2% peptone, 2% dextrose, and 1 M sorbitol) medium containing 100 μg mL–1 zeocin as a selectable marker.

Screening, Production and Enzyme Activity Assay

To screen the positive transformants, the colonies obtained were transferred to 25 ml buffered minimal glycerol yeast (BMGY) complex media which comprised of 1 % (w/v) yeast extract, 2 % (w/v) peptone, 100 mM KH2PO4/KOH pH 6.0, 1.3 % (w/v) yeast nitrogen base, 4 × 10-5 % (w/v) biotin, and 1 % (v/v) glycerol) in a 250-ml baffled flask and incubated at 30 °C and 250 rpm until the culture reached an OD600 of 2 to 6 in 16 to 18 h. The cells were harvested by centrifugation at 10,000×g for 10 min and re-suspended in 50 ml buffered complex medium (BMMY) which comprised of 1 % (v/v) methanol instead of glycerol as the sole carbon source to a final OD600 of 1.0, and incubated at 30 °C and 250 rpm. Methanol was added to a final concentration of 1% (v/v) at every 24 h interval up to 120 h to maintain induction of AOX1 promoter. After 5 days, the cells were pelleted by centrifugation at 10,000×g for 10 min at 4 °C. The resultant culture extract was used for enzymatic assay against carboxymethyl cellulose (CMC) [27, 28] as substrate. Enzymatic assay was done by incubating the reaction mixture, containing 0.5 ml of suitably diluted enzyme and 0.5 ml of CMC (2% w/v) prepared in 50 mM sodium citrate buffer (pH 6.0), at 50 °C for 20 min and then the reaction was terminated by adding 3 ml of 3,5-Dinitrosalicylic acid (DNS, 1% w/v) and boiled at 100 ° C for 10 min and the resultant developed color, indicative of reducing ends, was read 540 nm [29]. One unit of enzyme activity was defined as the amount of enzyme that released 1 μmol of glucose equivalent per minute.

Purification and Characterization of PMO9D_SCYTH and PMO9D_MALCI

One-step purification of PMO9D_SCYTH and PMO9D_MALCI was done using anion exchange chromatography (UNOsphere™ Q cartridge; 5 ml; Bio-Rad, USA). Sample was prepared by precipitating the culture supernatant (200 mL) using 80% (v/v) acetone (< 4 °C), precipitates were harvested by centrifugation (8000×g for 30 min) and the resultant pellet was dissolved in 10 mL of buffer A (Tris-HCl; 50 mM; pH 8.0). A linear salt gradient from 0 to 100% buffer B (buffer A with 1 M NaCl) at a flow rate of 1 ml min–1 was used to elute the bound protein using AKTA prime fast protein liquid chromatography system (GE Healthcare, USA). The profiling of eluted fractions for activity against CMC was carried out in the presence of 1 mM ascorbic acid which was used as a reductant. Positive fractions were pooled and used for enzymatic assay and kinetic studies. The protein content in the culture supernatant and in the chromatographic fractions was determined using Lowry’s method [30]. The homogeneity of the protein present in the fractions was analyzed by 12% SDS-PAGE using previously reported protocol [31]. Further, the activity of these purified LPMOs was determined using 1 mM 2,6-dimethoxyphenol (2,6-DMP) as substrate in presence of 100 μM H2O2 and 20 μl of purified enzyme in 116 mM sodium phosphate buffer (pH 6.0). The increase in absorption at 469 nm was detected for 5 min to determine the peroxidase activity of LPMO as reported by Zhang [32].

Molecular Mass Determination and Zymography

The active fractions were concentrated using spin filters (GE Healthcare, USA) having molecular weight cutoff of 3 kDa and subjected to electrophoresis under denaturing conditions on 12% polyacrylamide gel using Mini-Protein II system (BioRad) along with Precision Plus Protein™ dual color standard molecular weight marker (BioRad) as a reference to estimate the molecular weight of the purified enzymes. For zymography, 150 μg of purified PMO9D_SCYTH and PMO9D_MALCI were mixed with dye and loaded (without heating and addition of reducing agent β-mercaptoethanol) on to 12% polyacrylamide gel co-polymerized with 0.2% CMC [33]. After electrophoresis, gels were washed with deionized water and kept in Triton X-100 (2.5%, v/v) for 1 h at 4 °C, then gels were washed with deionized water and kept in 50 mM sodium citrate buffer (pH 6.0) containing 10 mM ascorbic acid along with/without 50 μM H2O2, and then incubated overnight at 50 °C. After incubation, gels were stained with 0.1% Congo red solution under shaking for 30 min, and destaining was done using 1 M NaCl solution for 1 h. Clear halos on red background indicated to the active protein bands.

pH, Temperature and Thermostability Profiling Studies

The pH profiling of the purified LPMOs was carried out by measuring the enzyme activity between 3.0 and 12.0 using 50 mM sodium acetate (pH 3.0–5.0), 50 mM sodium citrate (pH 6.0), 50 mM sodium phosphate (pH 7.0–8.0), and 50 mM NaOH-Glycine (pH 9.0–12.0) as buffer systems. The temperature optima of the purified LPMOs was determined between 30 and 80 ° C using CMC (2% w/v) as substrate and 1 mM ascorbic acid as a co-substrate, prepared in 50 mM sodium phosphate buffer (pH 7.0) for PMO9D_SCYTH and 50 mM NaOH-Glycine buffer (pH 9.0) for PMO9D_MALCI, respectively. The stability of LPMOs was determined by incubating the purified LPMOs up to seven days at 60 °C and pH 5.0, 7.0, and 9.0 for PMO9D_SCYTH and up to 6 days at 50 °C and pH 5.0, 7.0, and 9.0 for PMO9D_MALCI, and subsequently assayed for residual enzyme activity.

Effect of Metal Ions on Enzyme Activity

To evaluate the effect of metal ions and other compounds, purified LPMOs were incubated for 30 min at room temperature in 5 mM solution of AlCl3, CaCl2, CaCO3, CoCl2, CuCl(I), CuCl2(II), CuSO4, dithiothreitol (DTT, C4H10O2S2), ethylene diamine tetra-acetic acid (EDTA, C10H16N2O8), FeCl3, FeSO4, KCl, lead acetate {Pb(C2H3O2)2}, lead nitrate (Pb(NO3)2), LiCl, MgCl2, MgSO4, MnCl2, MnSO4, mercuric sulphate (HgSO4), NaCl, N-bromosuccinide (NBS, C4H4BrNO2), sodium dodecyl sulfate (SDS, NaC12H25SO4), sodium tungstate (Na2WO4), ZnCl2, ZnSO4, and β-mercaptoethanol (C2H6OS), and subsequently assayed for residual enzyme activity.

Substrate Specificity Studies

The substrate specificity of the purified LPMOs was determined using following substrates: avicel, laminarin, lichenin, β-glucan (barley), beechwood xylan, birchwood xylan, oat spelt xylan, wheat arabinoxylan (both low viscosity and high viscosity), de-branched arabinan, larchwood xylan, xyloglucan, and rye arabinoxylan. LPMOs activity against these substrate was determined using 1.0% (w/v) of each substrate in 50 mM sodium phosphate buffer (pH 7.0) at 60 °C for PMO9D_SCYTH and in 50 mM NaOH-Glycine buffer (pH 9.0) at 50 °C for PMO9D_MALCI, incubated for 2 h and the amount of released reducing sugars was measured using DNS as described in section 2.3.

Evaluation of H2O2 as a Co-substrate for LPMOs Activity

The effect of H2O2 as an enhancer of LPMOs activity was determined with the modified method as described by Bissaro [15], in which the LPMO activity of PMO9D_SCYTH and PMO9D_MALCI was determined in the presence of 20-100 μM H2O2, using avicel (1% w/v) as substrate. The reaction was carried out using 50 mM sodium phosphate buffer (pH 7.0) at 60 °C for PMO9D_SCYTH and in 50 mM NaOH-Glycine buffer (pH 9.0) at 50 °C for PMO9D_MALCI, adding 1 mM ascorbic acid (after 5 min to the addition of H2O2) and incubating for 2 h, after incubation the reaction mixtures were centrifuged at 10,000×g for 10 min and syringe filtered (0.45 μm), released reducing sugars were determined using 1 ml of the resultant aliquot using DNS.

Determination of Enzyme Kinetic Parameters

The Lineweaver-Burke plots were used to determine Michaelis-Menten kinetic parameters (Km and Vmax) and kcat employing avicel instead of chitin used for AA11 by Kuusk [34]. The reactions were carried out in 50 mM sodium phosphate buffer (pH 7.0) at 60 °C for PMO9D_SCYTH and in 50 mM NaOH-Glycine buffer (pH 9.0) at 50 °C for PMO9D_MALCI in the presence of 1 mM ascorbic acid as a reductant and 60 μM H2O2 and 40 μM H2O2 as respective co-substrate for PMO9D_SCYTH and PMO9D_MALCI. The Kinect studies were also studied on the CMC using 50 mM sodium phosphate buffer (pH 7.0) at 60 °C for PMO9D_SCYTH and in 50 mM NaOH-Glycine buffer (pH 9.0) at 50 °C for PMO9D_MALCI, using 1 mM ascorbic acid as a reductant; and against 2,6-DMP as substrate, 100 μM H2O2, and 20 μl enzyme in 116 mM sodium phosphate buffer (pH 6.5, for maximum sensitivity).

High-resolution Mass Spectroscopy (HRMS) Studies

High-resolution mass spectroscopy (HRMS) analysis of oxidative oligosaccharides obtained during 96 h hydrolysis of avicel and CMC (2% w/v) in the presence of 1 mM ascorbic acid and H2O2 (60 μM H2O2 for PMO9D_SCYTH and 40 μM for PMO9D_MALCI), using 50 mM sodium phosphate buffer (pH 7.0) at 60 °C for PMO9D_SCYTH and in 50 mM NaOH-Glycine buffer (pH 9.0) at 50 °C for PMO9D_MALCI was done. The analysis of the products was carried out using Brucker microTOF Q II Mass spectrometer in +ve ESI mode with capillary voltage 4500 V and source temperature 180 °C. Sample of 100 μl prepared in combination of acetonitrile (3:7) was directly injected to the ion source of the spectrometer.

Alignment of Type-1 and Type-3 LPMOs

To study the evolutionary relationship of PMO9D_SCYTH and PMO9D_MALCI with other type-1 and type-3 LPMOs, the amino acid sequences of these two LPMOs were aligned with the LPMOs available in the database: http://www.cazy.org/AA9_characterized.html. The phylogenetic tree of PMO9D_SCYTH (gene model ID Scyth2p4_002689), PMO9D_MALCI (gene model ID Malci1p7_001555), NCU00836 (Neurospora crassa OR74A, GenBank No. EAA34466.1), NCU07760 (Neurospora crassa OR74A, GenBank No. EAA29018.1), PaLPMO9D (Podospora anserine S mat+, GenBank No. CAP66744.1), PaLPMO9E (Podospora anserina S mat+, GenBank No. CAP67740.1), LPMO9B (Gloeophyllum trabeum KUC 8013, GenBank No. AEJ35168.1), GtLPMO9A-2 (Gloeophyllum trabeum NBRC 6430, GenBank No. BAV57612.1), LPMO9A (Trichoderma reesei RUTC-30, GenBank No. CAA71999.1), PaLPMO9A (Podospora anserina S mat+, GenBank No. CAP73254.1), PsLPMOA (Pestalotiopsis sp. NCi6, GenBank No. ANB32141.1) and TaLPMO9A (Thermoascus aurantiacus, Gen Bank No. ACS05720.1) was constructed with MEGA X using neighbor-joining method [35]. Bootstrap values calculated from 1000 trees are shown at each node.

Hydrolysis of Pretreated Substrates Using Cocktails of PMO9D_SCYTH and PMO9D_MALCI with Cellic CTec 2

The hydrolysis enhancing potential of PMO9D_SCYTH and PMO9D_MALCI was determined by constituting a cocktail with Cellic CTec2 (Novozymes) as benchmark cellulase. The saccharification was carried out using alkali and acid treated sugarcane bagasse and rice straw as substrate [22]. Saccharification was performed in 5 ml glass vial containing 1 ml reaction mixture prepared using acetate buffer (50 mM, pH 5.0), 70 mg of pretreated substrate (7% substrate loading) and 100 μl of suitably diluted Cellic CTec2 (10 mg protein/gds substrate) as benchmark control. To analyze the synergistic effect, 10 μl of benchmark enzyme was replaced with PMO9D_SCYTH (21 μg purified protein/gds) and PMO9D_MALCI (45 μg purified protein/gds). The same set of reactions was also carried out in presence of ascorbic acid (1 mM) and H2O2 (60 μM H2O2 for PMO9D_SCYTH and 40 μM for PMO9D_MALCI). The enzyme hydrolysis was carried for 96 h at 50 °C and released glucose and reducing sugars were quantified using glucose oxidase peroxidase kit (Span Diagnostic, India) and DNS method, respectively.

Results and Discussion

Cloning and Expression of PMO9D_SCYTH and PMO9D_MALCI in Pichia pastoris X33

The genome wide analysis of data shows that S. thermophilum and M. cinnamomea harbors twenty-seven and eight AA9 genes, respectively, from which two LPMOs with gene model ID Scyth2p4_002689 from S. thermophilum and Malci1p7_001555 from M. cinnamomea were selected for cloning and expression. Open reading frames (ORFs) encoding AA9 proteins were amplified using cDNA as template and designated as PMO9D_SCYTH and PMO9D_MALCI {705 bp; Fig A1 (a) and 540 bp, Fig A1 (b), respectively}. The amplicon was cloned in-frame with the secretion signal (S. cerevisiae α-factor) into the expression vector pPICZαA under the control of AOX1 promoter. The resultant plasmid was transformed into P. pastoris X33 by electroporation and plated onto YPDS/zeocin medium and incubated at 30 °C for 72 h. Thirty two of the resultant transformants of each clone were screened for the expression of AA9 on BMMY medium with 1% methanol (added at 24 h intervals) under shaking conditions for 120 h and culture extracts were assayed for LPMO activity using CMC as substrate. Due to the presence of alcohol oxidase in the host strain in P. pastoris X33, determination of LPMOs activity was erroneous using Amplex Red reagent (Thermo Fisher Scientific, U. S.) which detects H2O2 as futile by-product in the reaction [36]. To overcome this problem, the screening of the clones harboring LPMO gene was carried out using CMC as substrate. The AA9 enzymes which were previously classified as GH61 exhibiting weak endoglucanase activity against CMC as substrate has been used to characterize purified recombinant GH61 from Trichoderma reesei [28], previously. The maximal expressed LPMO activity of PMO9D_SCYTH (402.2 U/L) and PMO9D_MALCI (743.5 U/L) was found in two clones (#21 and #6) (Fig A2), respectively; and were chosen for further purification and characterization studies.

Characteristics of Purified LPMOs

The recombinant PMO9D_SCYTH and PMO9D_MALCI were purified by anion exchange chromatography. The purity of active fractions was analyzed by SDS-PAGE, PMO9D_SCYTH and PMO9D_MALCI was found to be ~ 27 kDa and ~ 20 kDa (Fig. 1), respectively, which were slightly larger than the predicted size (24.0 kDa for PMO9D_SCYTH and 16.9 kDa for PMO9D_MALCI after signal peptide processing) determined using ExPASy compute based pI/Mw software tool. This larger size of the recombinant proteins may be ascribed to the presence of c-myc epitope and 6X His-tags at the C-terminus that accounts for ~2.7 kDa. Both the LPMOs were examined for the presence of glycosylation site (NetNGlyc 1.0 Server, DTU Bioinformatics, Technical University of Denmark (http://www.cbs.dtu.dk/services/NetNGlyc/) and no N-glycosylation site was found in both the LPMOs (threshold = 0.5). However, differential glycosylation has been reported in few of the recently reported LPMOs (MtLPMO9B and MtLPMO9C) from Myceliophthora thermophila [16]. Previously, LPMO proteins (http://www.cazy.org/AA9_characterized.html) with molecular weight ranging from 22 kDa to 36 kDa have been reported. The activity of the LPMO PMO9D_SCYHT and PMO9D_MALCI determined using 2,6-DMP as substrate was found to be 99.6 and 47.73 U g-1, respectively, which was found to be higher as compared to previously reported LPMOs, Neurospora crassa LPMO9C 32.3 U g-1, LPMOF 2.2 U g-1, and Myriococcum thermophilum LPMO (Myrth2p4_000359) 30.9 U g-1 [37]. Zymograms of PMO9D_SCYTH and PMO9D_MALCI (Fig. A3) showed more prominent halos against red background in the gel incubated in presence of H2O2, suggesting the boosting effect of H2O2 on the activity of LPMOs [15, 33].

Fig. 1
figure 1

SDS–PAGE of purified LPMOs. Lane W1, standard protein marker in the order of increasing molecular mass in kDa (Precision Plus Protein™ Dual Color Standard molecular weight marker, BioRad). Lane W3, purified PMO9D_SCYTH (~ 27 kDa). Lane W4, purified PMO9D_MALCI (~ 20 kDa)

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Optimum pH and Temperature, and Thermostability

The purified PMO9D_SCYTH was optimally active at pH 7.0 and was appreciably active over a wide range of pH (5.0–9.0). On the other hand, PMO9D_MALCI was optimally active at pH 9.0 and showed appreciable activity at pH 4.0–9.0 (Fig. 2a). The optimal activity for PMO9D_SCYTH and PMO9D_MALCI was found at 60 °C and 50 °C, respectively (Fig. 2b); the enzyme activity declined below and over these temperatures. The purified PMO9D_SCYTH showed stable activity at 60 °C (t1/2 = 23.37 h, pH 5.0; t1/2 = 60.58 h, pH 7.0), whereas, PMO9D_MALCI showed stable activity at 50 °C (t1/2 = 134 h, pH 5.0; t1/2 = 144 h, pH 7.0) (Fig 2c, d). Temperature and pH profiling suggest that both of these LPMOs have a significant potential in 2G ethanol biorefineries for both acid and alkali pretreated substrates. Also, the enzyme stability at high temperature is beneficial for efficient saccharification [4, 6].

Fig. 2
figure 2

Effect of pH (a) and temperature (b) on the activity of purified LPMOs. Thermostability was checked at three different pH 5.0, 7.0, and 9.0 for PMO9D_SCYTH (at 60 °C, c) and PMO9D_MALCI (at 50 °C, d). Bars represent mean ± SE (n = 3)

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Effect of Metal Ions

Both the enzymes showed the maximum positive modulation of activity in the presence of Mn2+ ion (157 and 165% residual activity of PMO9D_SCYTH and PMO9D_MALCI, respectively) (Fig. 3), Langston [38] demonstrated that the addition of Mn2+ shows positive effect on the enzymatic activity of the LPMOs from thermophilic fungus Thermoascus aurantiacus. In the case of PMO9D_SCYTH, metal ions/compounds, Al3+, Ca2+, Co2+, Cu(I), Cu(II), Pb(II), Li1+, Mg2+, Hg2+, W2+, Zn2+, DTT, and 2-Mercaptoethanol showed enhanced activity, whereas Cu(I), K1+, Li1+, Mg2+, Mn2+, Na1+, SDS, and 2-Mercaptoethanol elevated the PMO9D_MALCI activity slightly. In presence of N-bromosuccinimide significant loss in the enzyme activity (11% residual) was observed in PMO9A_SCYTH indicating the presence of tryptophan residues at the active site of enzyme [39], whereas PMO9A_MALCI retained 72% residual activity suggesting the absence of tryptophan in its active site. LPMOs are known metalloenzymes containing Cu2+/1+ ion at their active site and utilize a copper-oxyl, oxygen rebound mechanism [14] for their attack on crystalline cellulose. Further, LPMOs have been shown to bind Ca2+, Mg2+, Fe2+, Co2+, and Zn2+ at their active site which leads to the altered dynamics of substrate interaction as determined by NMR [40].

Fig. 3
figure 3

Effect of Metal ions on the activity of purified LPMOs. Bars represent mean ± SE (n = 3)

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Substrate Specificity

The PMO9D_SCYTH and PMO9D_MALCI showed catalytic preference against cellulosic polysaccharides as substrate. These two LPMOs (PMO9D_SCYTH and PMO9D_MALCI) recognized CMC as preferred substrate releasing 24 and 44 m mol-1 h-1 reducing ends, respectively, which was much higher when compared 11 m mol-1 h-1 observed during homologous expression of T. reesei GH61 against CMC [28]. The observed high activities against avicel as substrate (Fig. 4) also indicate their catalytic efficacy against crystalline form of cellulose (0.36 and 0.17 U mg-1 specific activity of PMO9D_SCYTH and PMO9D_MALCI, respectively) was comparable to (0.08–0.3 U mg-1) observed for different configurations of LPMOs (PvLPMO9A), derived from Penicillium verruculosum, against avicel [41]. Both LPMOs exhibited minor catalytic activity against lichenan, debranched arabinan and barley-β-glucan (containing alternate β-1,3 and 1,4-linkages). Of the two, LPMOs (PMO9D_MALCI) was more versatile as it recognized both hexose and pentose sugar containing polysaccharides (beechwood xylan and rye arabinoxylans) whereas, PMO9D_SCYTH was active against β-1,4 and β-1,3 linked hexose polysaccharides in addition to larchwood xylan which is a galacto glucomannan that consists of a backbone of randomly distributed β-1,4-linked D-mannose and D-glucose units with α-1,6-linked D-galactose branches attached to mannose units [42]. Previously, NcLPMO9C from Neurospora crassa [43] and PaLPMO9H from Podospora anserine [44] have been shown to be active against cellulose, small soluble cello-oligosaccharides, (1-3, 1-4)-β-D-glucan in addition to xyloglucans and to lesser extent against glucomannan.

Fig. 4
figure 4

Substrate specificity of the purified LPMOs. B-β-G: β-glucan (barley), Beech WX: beechwood xylan, Birch WX: birchwood xylan, OSX: oat spelt xylan, WAX: wheat arabinoxylan (both low viscosity and high viscosity), DB: arabinan debranched arabinan, LWX : larchwood xylan, XG: xyloglucan, and RAX: rye arabinoxylan. Bars represent mean ± SE (n = 3)

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H2O2 as a Co-substrate for LPMOs Activity and Kinetic Parameters

The results in Fig. 5 show that H2O2 act as a co-substrate for LPMOs. LPMOs abstract oxygen from H2O2 during their action on crystalline cellulose to introduce a nick either at C1 or C4 position as reported before. The activity of PMO9D_SCYTH and PMO9D_MALCI was increased by H2O2 addition optimally at 60 μM (2.7-folds) and 40 μM (1.6-folds), respectively. Similarly, Bissaro [15] found 26-fold more soluble oxidized products in presence of 200 μM H2O2, for LPMO derived from Streptomyces coelicolor (ScLPMO10C), when compared to the reaction without H2O2. However, they observed progressive and rapid enzyme inactivation at higher H2O2 concentration. The Michaelis-Menten kinetic parameters (Km and Vmax) and kcat/km using Lineweaver-Burke plots were determined against avicel, CMC and 2,6-DMP (Table 1). The catalytic efficiency (kcat/Km) of PMO9D_SCYTH and PMO9D_MALCI was found to be 6.58 × 10-3 and 1.79 × 10-3 mg-1 ml min-1 (H2O2 driven avicel hydrolysis); 1.52 × 10-1 and 2.62 × 10-2 mg-1 ml min-1 (using CMC); and 3.57 × 10-1 and 1.03 × 10-1 mM-1 min-1 (using 2,6-DMP), respectively.

Fig. 5
figure 5

Effect of H2O2 addition on the activity of purified LPMOs. Ascorbic acid (Asc A) was added at 1 mM final concentration. Bars represent mean ± SE (n = 3)

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Table 1 Kinetic parameters
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High-resolution Mass Spectroscopy (HRMS) Analysis of Hydrolysates

The MS analysis of the hydrolysates obtained by hydrolysis of avicel and CMC showed presence of sodium adducts of the products with m/z of 365, 527, 689, 851, 1013, 1175, and presence of single (M + 16) and double oxidized (M + 32) as well as unoxidized cello-oligosaccharides with degree of polymerization (DP) primarily ranging between DP2 to DP7 (Table 2, Fig. A5). The observed double oxidized products suggested that both the LPMOs belong to type-3 class of AA9; the MS analysis of avicel hydrolysis in the presence of H2O2 also suggests that the rate of oxidation of LPMOs is increased in the presence of H2O2. The observed m/z ratio of corresponding peaks were similar to the pattern obtained by Bennati-Granier [45] for the LPMOs (PaLPMO9A and PaLPMO9H) derived from Podospora anserine cleaving cellulose at both the C1 and C4 glycosidic positions.

Table 2 Mass spectroscopy analysis of non-oxidized and oxidized products produced by PMO9D-SCYTH and PMO9D_MALCI during hydrolysis of avicel and CMC
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Alignment of Type-1 and Type-3 LPMOs

The phylogenetic tree, generated using different type-1 and type-3 LPMOs from the database (http://www.cazy.org/AA9_characterized.html), showed that LPMO PMO9D_SCYTH derived from Scytalidium thermophilum clustered with type-3 LPMO of Pestalotiopsis sp. NCi6 (PsLPMOA) supported by high bootstrap value. On the other hand, LPMO from Malbranchea cinnamomea (PMO9D_MALCI) classified as type-3 LPMO, showed deep rooted branch that separated from S. thermophilum clade indicating to an earlier and distinct phylogenetic origin (Fig. 6). We have previously reported xylanases from Malbranchea to be highly alkaline active and thermostable [39] and the phylogenetic tree generated on the basis of ITS sequences also showed that this fungus belonging to order onygenales formed a separate and distinct branch [45, 46]. In addition, the HRMS product profile of the LPMOs from these fungi against avicel and carboxymethyl cellulose (CMC) also supports them to be type-3 LPMOs.

Fig. 6
figure 6

Showing the evolutionary history between PMO9D_SCYTH, PMO9D_MALCI and other already characterized LPMOs, inferred using neighbor-joining method. The optimal tree with the sum of branch length of 10.95368035 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. This analysis involved 12 amino acid sequences. All ambiguous positions were removed for each sequence pair. There were a total of 351 positions in the final dataset. Evolutionary analyses were conducted in MEGA X

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Hydrolysis of Pretreated Substrates Using Cocktails of PMO9D_SCYTH and PMO9D_MALCI with Cellic CTec 2

To evaluate the importance of LPMOs in the formulation of enzyme cocktails, the hydrolysis studies of acid and alkali treated bagasse and rice straw was carried out (at 7% solids loading) by formulating the enzyme cocktails by replacing commercial enzyme Cellic CTec2 (benchmark) with purified LPMO in 9:1 ratio. Supplementing the combination of Cellic CTec2 with PMO9D_SCYTH increased the glucan conversion for acid or alkali treated bagasse (by 39.4 and 19.4%) and rice straw (by 25.9 and 17.4%), respectively, when compared to using Cellic CTec2 alone. The cocktail containing Cellic CTec2 and PMO9D_MALCI enhanced the glucan conversion of acid or alkali treated bagasse (by 29.7 and 24.3%) and rice straw (by 22.7 and 29.2%), respectively, (Fig. 7). The amount of total reducing sugars released after the hydrolysis of acid or alkali treated bagasse enhanced by 18.9 and 17.5% and rice straw by 28.7 and 22.1%, respectively, using enzymes ratio Cellic CTec2:PMO9D_SCYTH (at 9:1 ratio). Similar experiments using Cellic CTec2:PMO9D_MALCI (at 9:1 ratio) enhanced the reducing sugar content for acid or alkali treated bagasse (by 21.3 and 23.6%) and acid or alkali treated rice straw (by 28.8 and 13.6%), respectively. Previously, 31 % higher glucose yields from steam-exploded (SE) birch was obtained by replacing 15 % of the protein in the Celluclast/Novozym 188 blend with LPMO (TaLPMO9A) derived from Thermoascus aurantiacus [47]; an ∼ 2.1-fold increase in sugar yield was observed when LPMO (AN3046) derived from Aspergillus nidulans was mixed with other hydrolases (endoglucanase and cellobiose dehydrogenase) during degradation of sorghum stover [48]; 21 and 18% increase in reducing sugars during hydrolysis of pretreated oak and kenaf, respectively, was obtained when LPMO (GtGH61) from Gloeophyllum trabeum was used with GtXyl10G or GtCel5B [49].

Fig. 7
figure 7

Percent increase of glucose released (a) and total reducing sugars (b) released after saccharification (96 h) of differently treated bagasse and rice straw using cocktails containing Cellic CTec2 and recombinant LPMOs (9:1 ratio). AcBg acid-treated bagasse, AlBg alkali-treated bagasse, AcRs acid-treated rice straw, AlRs, alkali-treated rice straw. Bars represent mean ± SE (n = 3)

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Conclusion

The observed high catalytic efficiency and thermostability of these newly discovered LPMOs from S. thermophilum and M. cinnamomea make them suitable candidates for formulation of potent lignocellulolytic cocktails. These LPMOs can be considered for expression in thermophilic fungal platform to further realize their full potential.