Characterization of a novel Lytic Polysaccharide Monooxygenase from Malbranchea cinnamomea exhibiting dual catalytic behavior.

A novel Lytic Polysaccharide Monooxygenase (LPMO) family AA9 (PMO9A_MALCI) protein from thermophilic fungus Malbranchea cinnamomea was cloned and expressed in Pichia pastoris. The expressed protein was purified to homogeneity using ion exchange and hydrophobic interaction chromatography. SDS-PAGE analysis showed PMO9A_MALCI to be ~27 kDa protein. High performance anion exchange chromatography and mass spectrometry confirmed that purified protein was active against an array of cellulosic (avicel, carboxy methyl cellulose) and hemicellulosic (birch wood xylan, wheat arabinoxylan and rye arabinoxylan) substrates, releasing both oxidized and unoxidized cello-oligosaccharide and xylo-oligosaccharide products respectively. Presence of double oxidized products during mass spectrometric analysis as well as in-silico analysis confirmed that the expressed protein belongs to Type 3 LPMO family. Molecular dynamic simulations further confirmed the sharing of common amino acid residues conserved for catalysis of both cellulosic and hemicellulosic substrates which further indicates that both substrates are equally preferred. Enzymatic cocktails constituted by replacing a part of commercial cellulase CellicCTec2 with PMO9A_MALCI (9:1/8:2) led to synergistic improvement in saccharification of acid and alkali pretreated biomass. This is the first report on heterologous expression of LPMO from M. cinnamomea, exhibiting catalysis of cellulose and pure xylan.

Producing methane, methanol and electricity from organic waste of fermentation reaction using novel microbes.

Residual solid and liquid streams from the one-pot CRUDE (Conversion of Raw and Untreated Disposal into Ethanol) process were treated with two separate biochemical routes for renewable energy transformation. The solid residual stream was subjected to thermophilic anaerobic digestion (TAD), which produced 95 ±â€¯7 L methane kg-1 volatile solid with an overall energy efficiency of 12.9 ±â€¯1.7%. A methanotroph, Methyloferula sp., was deployed for oxidation of mixed TAD biogas into methanol. The residual liquid stream from CRUDE process was used in a Microbial Fuel Cell (MFC) to produce electricity. Material balance calculations confirmed the integration of biochemical routes (i.e. CRUDE, TAD, and MFC) for developing a sustainable approach of energy regeneration. The current work demonstrates the utilization of different residual streams originated after food waste processing to release minimal organic load to the environment.

Expression of catalytically efficient xylanases from thermophilic fungus Malbranchea cinnamomea for synergistically enhancing hydrolysis of lignocellulosics.

In this study, two xylanase genes (GH10 and GH11) derived from Malbranchea cinnamomea, designated as XYN10A_MALCI and XYN11A_MALCI, respectively, were expressed in Pichia pastoris X33. The maximum level of xylanase expression was found to be 24.3U/ml for rXYN10A_MALCI and 573.32U/ml for rXYN11A_MALCI. The purified recombinant rXYN11A_MALCI was stable at 70°C and catalytically active against a variety of substituted (arabinoxylans) as well as unsubstituted xylans. The hydrolytic potential of recombinant xylanases for enhancing the hydrolysis of acid/alkali pretreated lignocellulosics (rice straw and bagasse) by the commercial cellulase Cellic CTec2 was assessed which revealed that both rXYN10A_MALCI and rXYN11A_MALCI act synergistically with commercial cellulases and resulted in 1.54 and 1.58 folds improved hydrolysis of acid treated rice straw and alkali treated rice straw using cocktail comprising of Cellic CTec2 and XYN11A_MALCI (8:2 ratio) when compared to Cellic CTec2 alone at same protein loading rate of (∼5.7mg/g biomass).

Purification and characterization of beta-glucosidase from Melanocarpus sp. MTCC 3922

This study reports the purification and characterization of beta-glucosidase from a newly isolated thermophilic fungus, Melanocarpus sp. Microbial Type Culture Collection (MTCC) 3922. The molecular weight of beta-glucosidase was determined to be ~ 92 and 102 kDa with SDS PAGE and gel filtration, respectively, and pI of ~ 4.1. It was optimally active at 60 C and pH 6.0, though was stable at 50 C and pH 5.0 - 6.0. The presence of DTT, mercaptoethanol and metal ions such as Na+, K+, Ca2+, Mg2+and Zn2+ positively influenced the activity of beta-glucosidase but the activity was inhibited in the presence of CuSO4. beta-Glucosidase recognized pNP- beta-glucopyranoside (pNPG) as the preferred substrate, and showed very low affinity for pNP- beta-D-cellobioside. Km and Vmax for the hydrolysis of pNPG by beta-glucosidase was calculated as 3.3 mM and 43.68 ‘molmin-1mg protein-1, respectively and k cat was quantified as 4 x 10³ min-1. beta-Glucosidase activity was enhanced appreciably in the presence of alcohols (methanol and ethanol) moreover, purified beta-glucosidase showed putative transglycosylation activity that was positively catalyzed in presence of methanol as an acceptor molecule

Purification and characterization of two endoglucanases from Melanocarpus sp. MTCC 3922.

This study reports the purification and characterization of endoglucanases (EG I and EG II) from a newly isolated thermophilic fungus, Melanocarpus sp. MTCC 3922. The molecular weight of EG I and EG II as with SDS-PAGE and pI were approximately 40 and 50 kDa, and approximately 4.0 and 3.6, respectively. EG I and EG II were optimally active at 50 and 70 degrees C, and pH 6.0 and 5.0, respectively. EG I was active over a broad range of pH (5.0-7.0), whereas, loss of activity was observed as the temperature was increased from 50 to 80 degrees C. However, EG II was active over pH 4.0-6.0 and temperature 40-80 degrees C. The presence of mercaptoethanol and SDS inhibited the EG I activity but showed no negative effect on EG II. Both the endoglucanases showed higher activity against barley-beta-glucan as compared to CMC. Km values of EG I and EG II for barley-beta-glucan were lower than CMC. Turn over number (K(cat)) and catalytic efficiency (K(cat)/Km) values of both the endoglucanases were higher with barley-beta-glucan as substrate than CMC. EG I showed affinity for Avicel indicating the presence of cellulose binding domains (CBD) whereas, EG II was found to lack CBD.

Diversity of plant cell wall esterases in thermophilic and thermotolerant fungi.

Fourteen thermophilic and thermotolerant fungal strains isolated from composting soils produced plant cell wall-acting esterases in a medium containing corn cobs and oat spelt xylan. The concentrated and dialyzed protein extracts of these fungi were fractionated using isoelectric-focusing, gels sliced and eluted protein in each slice was assayed for esterase activity against p-nitrophenyl acetate. A total of 84 esterases detected on the basis of pI were found to show distinct preferential substrate specificities towards p-nitrophenyl acetate, p-nitrophenyl ferulate and p-nitrophenyl butyrate, and were putatively classified as acetyl esterases and esterases types I and II. None of the esterases were active against p-nitrophenyl myristate. In addition, these esterases were characterized as acid, neutral or alkaline active.

Two endoxylanases active and stable at alkaline pH from the newly isolated thermophilic fungus, Myceliophthora sp. IMI 387099.

Two extra-cellular endoxylanases (Xyl Ia and Ib) were purified to homogeneity from the newly isolated thermophilic fungus, Myceliophthora sp. IMI 387099. Xyl Ia and Ib, having a molecular mass of approximately 53 kDa and pI of 5.2 and 4.8, respectively, were optimally active at 75 degrees C and at pH 6.0. They were stable at pH 9.2 at 60 degrees C for 2 h, but less stable at pH 6.0 and above 50 degrees C. Mg+2, Zn+2, Ca+2, Co+2 and DTT increased their activity by 1.5-3.0-folds, while SDS and NBS completely inhibited their activity. Both xylanases were active on pNPX and pNPC, but their activity on pNPC was three times higher than that on pNPX. Xyl Ia was more active than Xyl Ib on pNP-alpha-L-Arap, while the latter preferred pNP-alpha-L-Araf. Both xylanases showed two to four times higher activity on rye and wheat arabinoxylans than on birchwood xylan, but Xyl Ib was more active than Xyl Ia on oat spelt xylan. Wheat insoluble pentosan was a good substrate for Xyl Ia, while Xyl Ib preferred wheat soluble arabinoxylan. Xyl Ia had lower Km and higher kcat/Km ratios than Xyl Ib towards all three xylans tested. Both xylanases degraded X4-X6 in an endo-fashion and catalysed hydrolysis and trans-xylosylation reactions. HPLC and LC/MS analysis showed that Xyl Ia and Ib released the unsubstituted X2-X6 as well as mono and di-methyl glucuronic acid substituted X3 and X2 from arabinoxylans.