Sequential pretreatment of sugarcane bagasse by alkali and organosolv for improved delignification and cellulose saccharification by chimera and cellobiohydrolase for bioethanol production.

Sequential pretreatments for sugarcane bagasse (scb) by NaOH followed by organosolv under mild conditions were evaluated for cellulose recovery and dilignification. The best-optimized sequential pretreatment of scb was obtained at 10% (w/v) of raw scb loading at 1% (w/v) NaOH (50 °C, 2 h) followed by treatment with organosolv (85%, v/v phosphoric acid, 50 °C, 1 h) with chilled acetone. This sequentially pretreated scb showed cellulose recovery, 66.1% (w/w) and delignification, 83.2% (w/w). NaOH or organosolv pretreated scb showed lower cellulose recovery 47.4% (w/w) or 54.5% (w/w) with lower delignification, 61% (w/w) or 56% (w/w), respectively. Pretreated solid residue of sequentially pretreated scb was enzymatically saccharified by chimera (ß-glucosidase and endoglucanase, CtGH1-L1-CtGH5-F194A) and cellobiohydrolase (CtCBH5A) cloned from Clostridium thermocellum. Enzymatic hydrolysate of best sequentially pretreated scb gave total reducing sugar (TRS) yield, 230 mg/g and glucose yield, 137 mg/g pretreated scb. Only organosolv pretreated scb gave TRS yield, 112.5 mg/g and glucose yield, 72 mg/g of pretreated scb. Thus, sequentially pretreated scb resulted in 37% higher enzymatic digestibility than only orgnaosolv pretreated scb. Higher enzymatic digestibility was supported by higher crystallinity index CrI (45%) than those obtained with only organosolv pretreated (38%) or raw scb (25%). Field Emission Scanning Electron Microscope (FESEM) and Fourier-transform infrared (FT-IR) analyses showed enhanced cellulose exposure in sequentially pretreated scb. Preliminary investigation of bioethanol production at small scale by separate hydrolysis and fermentation (SHF) of enzymatic hydrolysate from best sequentially pretreated scb by Saccharomyces cerevisiae gave maximum ethanol yield of 0.42 g/g of glucose. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-020-02600-y.

In vitro prebiotic potential, digestibility and biocompatibility properties of laminari-oligosaccharides produced from curdlan by ß-1,3-endoglucanase from Clostridium thermocellum.

Curdlan or laminarin, a ß-1,3-glucan was hydrolysed by ß-1,3-endoglucanase (CtLam81A) from Clostridium thermocellum to produce laminari-oligosaccharides. TLC analysis of hydrolysed curdlan showed the presence of laminari-oligosaccharides of the degree of polymerization, DP2-DP7. This mixture of laminari-oligosaccharides displayed prebiotic properties. Laminari-oligosaccharides showed an increase in the growth of probiotic bacteria such as Lactobacillus plantarum DM5 and Lactobacillus acidophilus, while they did not promote the growth of non-probiotic bacteria (Escherichia coli and Enterobacter aerogenes). Laminari-oligosaccharides showed higher prebiotic activity score of 0.92 ± 0.01 and 0.64 ± 0.08 for L. plantarum DM5 and L. acidophilus NRRL B-4496, respectively, similar to those shown by inulin. Laminari-oligosaccharides showed higher resistance or low digestibility against α-amylase, artificial gastric juice and intestinal fluid than inulin indicating their bioavailability to the probiotic bacteria present in the gastrointestinal tract of human. The probiotic bacteria consumed laminaribiose and laminariotriose more readily than higher laminari-oligosaccharides as carbon source for their growth. The in vitro cytotoxicity assay of laminari-oligosaccharides (1 mg/ml) on human embryonic kidney (HEK 293) cells showed that the cell viability was not affected even after 72 h indicating their biocompatible nature. All the results amply indicated that laminari-oligosaccharides can serve as potential prebiotic additives for functional food products.

Evaluation of pre-treatment methods for Lantana camara stem for enhanced enzymatic saccharification.

This study evaluates certain pre-treatment methods for Lantana camara stem for efficient conversion to fermentable sugars. The composition analysis of L. camara stem showed 66.8% (w/w) holocellulose, 34.9% (w/w) cellulose and 17% (w/w) hemicellulose. Comparative analysis of various chemical, physical or physico-chemical pre-treatments on L. camara stem was performed. Of all pretreatment methods used, pre-treatment with 1% (v/v) H2SO4 assisted autoclaving gave maximum total reducing sugar yield 132.7 mg/g (13.2 g/L) of raw biomass in pretreated hydrolysate. Major contribution to total reducing sugar was from hemicellulosic fraction, because total pentose sugar yield was 119.4 mg/g of raw biomass whereas, glucose released was only 10 mg/g of untreated biomass. The enzymatic saccharification of pre-treated L. camara stem by 1% (v/v) H2SO4 assisted autoclaving was performed with partially purified carboxymethylcellulase from Bacillus amyloliquefaciens SS35. Enzymatic saccharification at 30 °C for 48 h gave total reducing sugar yield, 63.3 mg/g of pre-treated biomass in the hydrolysate, while untreated biomass gave 43.3 mg/g of untreated biomass. The total sugar yield i.e. the sum of pre-treated biomass hydrolysate total reducing sugar (132.7 mg/g of raw biomass) and enzymatic hydrolysate total reducing sugar (63.3 mg/g of pre-treated biomass) was 196.0 mg/g of raw biomass, indicating the effectiveness of pre-treatment method. Field emission scanning electron microscopy, Fourier transform infrared and X-ray diffraction analyses displayed enhanced porosity, removal of non-cellulosic sugars and increased cellulose crystallinity, respectively, in pre-treated L. camara stem, showing the effectiveness of acid assisted autoclaving pre-treatment.

Antitumor effect of chondroitin AC lyase (PsPL8A) from Pedobacter saltans on melanoma and fibrosarcoma cell lines by in vitro analysis.

BACKGROUND: PGs are involved in cellular communication and cancer biology. The role of CS in melanoma and fibrosarcoma cell lines was explored by using chondroitin AC lyase (PsPL8A). METHODS: The proliferation of mouse fibroblast L929, human melanoma (SK-Mel 28) and fibrosarcoma (HT-1080) cell lines after treatment with chondroitin AC lyase (PsPL8A) was studied by MTT assay. The mode of cell death was studied by Annexin-V FITC using flow cytometry and fluorescence microscopy. The alteration in mitochondrial cell potential was studied by JC-1 dye using fluorescence microscopy and flow cytometry. RESULTS: Treatment of L929 cells with PsPL8A imparts no cytotoxicity and showed no alteration in proliferation with nearly 95-98% cell viability. An overall 58% and 59% inhibition of SK-Mel 28 and HT-1080 cell proliferation was observed with 1.3 µM of PsPL8A after 24 h of incubation. The PsPL8A (1.3 µM) treated SK-Mel 28 and HT-1080 cells showed significant green fluorescence with annexin-V FITC under fluorescence microscopy and 56.6% and 35.5% apoptosis, respectively by flow cytometry analysis. The results of fluorescence microscopy and flow cytometry of SK-Mel 28 and HT-1080 upon treatment with PsPL8A (1.3 µM) for 24 h, gave green fluorescence due to dissipation of mitochondrial potential with JC-1 dye. CONCLUSIONS: Chondroitin AC lyase (PsPL8A) displayed anti-tumor potential against human melanoma SK-Mel 28 and fibrosarcoma HT-1080 cell lines, while the mouse fibroblast L929 cells were unaffected.

The multi-ligand binding first family 35 Carbohydrate Binding Module (CBM35) of Clostridium thermocellum targets rhamnogalacturonan I.

Carbohydrate Binding Modules (CBMs) targeting cellulose, xylan and mannan have been reported, however, a CBM targeting rhamnogalacturonan I (RG I) has never been identified. We had studied earlier a rhamnogalacturonan lyase (CtRGL) from Clostridium thermocellum that was associated with a family 35 CBM, Rgl-CBM35. In this study we show that Rgl-CBM35 displays binding with ß-d-glucuronic acid (ß-D-GlcpA), Δ4,5-anhydro-d-galactopyranosyluronic acid (Δ4,5-GalpA), rhamnogalacturonan I, arabinan, galactan, glucuronoxylans and arabinoxylans. Rgl-CBM35 contains a conserved ligand binding site in the loops known for binding ß-D-GlcpA and Δ4,5-GalpA moiety of unsaturated RG I and pectic-oligosaccharides. Mutagenesis revealed that Asn118 plays an important role in binding ß-D-GlcpA, Δ4,5-GalpA, sugarbeet arabinan and potato galactan at its conserved ligand binding site present in surface exposed loops. EDTA-treated Rgl-CBM35 showed no affinity towards ß-D-GlcpA and Δ4,5-GalpA underscoring Ca2+ mediated ligand recognition. Contrastingly, the EDTA-treated Rgl-CBM35 and its mutant N118A displayed affinity for sugarbeet arabinan and potato galactan. The curtailed affinity of Y37A/N118A and R69A/N118A double mutants towards sugarbeet arabinan emphasized the presence of a second ligand binding site. Rgl-CBM35 is the first CBM reported to primarily target RG I and also is the first member of family 35 CBM possessing at least two ligand binding sites.

Novel insights into the degradation of ß-1,3-glucans by the cellulosome of Clostridium thermocellum revealed by structure and function studies of a family 81 glycoside hydrolase.

The family 81 glycoside hydrolase (GH81) from Clostridium thermocellum is a ß-1,3-glucanase belonging to cellulosomal complex. The gene encoding GH81 from Clostridium thermocellum (CtLam81A) was cloned and expressed displaying a molecular mass of ~82 kDa. CtLam81A showed maximum activity against laminarin (100 U/mg), followed by curdlan (65 U/mg), at pH 7.0 and 75 °C. CtLam81A displayed Km, 2.1 ±â€¯0.12 mg/ml and Vmax, 109 ±â€¯1.8 U/mg, against laminarin under optimized conditions. CtLam81A activity was significantly enhanced by Ca2+ or Mg2+ ions. Melting curve analysis of CtLam81A showed an increase in melting temperature from 91 °C to 96 °C by Ca2+ or Mg2+ ions and decreased to 82 °C by EDTA, indicating that Ca2+ and Mg2+ ions may be involved in catalysis and in maintaining structural integrity. TLC and MALDI-TOF analysis of ß-1,3-glucan hydrolysed products released initially, showed ß-1,3-glucan-oligosaccharides degree of polymerization (DP) from DP2 to DP7, confirming an endo-mode of action. The catalytically inactive mutant CtLam81A-E515A generated by site-directed mutagenesis was co-crystallized and tetragonal crystals diffracting up to 1.4 Šresolution were obtained. CtLam81A-E515A contained 15 α-helices and 38 ß-strands forming a four-domain structure viz. a ß-sandwich domain I at N-terminal, an α/ß-domain II, an (α/α)6 barrel domain III, and a small 5-stranded ß-sandwich domain IV.

Low-resolution SAXS and comparative modeling based structure analysis of endo-ß-1,4-xylanase a family 10 glycoside hydrolase from Pseudopedobacter saltans comb. nov.

The structure and biophysical properties of endo ß-1,4-xylanase (PsGH10A) of family 10 glycoside hydrolase were characterized. The modeled PsGH10A structure showed classical (ß/α)8-barrel fold. Ramachandran plot displayed 99.1% residues in favored and 0.3% in the generously allowed region and only 0.6% residues in disallowed region. The secondary structure analysis of PsGH10A by CD revealed 31.75% α-helices 20.0% ß-strands and 48.25% random coils. Protein melting study of PsGH10A showed complete unfolding at 60°C and did not require any metal ion for its stability. Structural superposition and docking analysis confirmed the involvement of Glu156 and Glu263 residues in catalysis. SAXS analysis displayed that PsGH10A is monomeric in nature showing fully folded state in solution form. Guinier analysis gave the radius of gyration (Rg) 2.23-2.29nm. Kratky plot indicated that the protein is fully folded globular shaped and flexible in solution form. The ab initio derived dummy model of PsGH10A displayed chicken thigh like shape. The ab initio derived dummy model superposed well with its comparative modeled structure except the N-terminal His-tag region.

SAXS and homology modelling based structure characterization of pectin methylesterase a family 8 carbohydrate esterase from Clostridium thermocellum ATCC 27405.

Pectin methylesterase (CtPME) from Clostridium thermocellum of family 8 carbohydrate esterase (CE8) belongs to pectin methylesterase super family (E.C.3.1.1.11). BLAST analysis of CtPME showed 38% sequence identity with PME from Erwinia chrysanthemi. Multiple sequence alignment of CtPME with other known structures of pectin methylesterase revealed the conserved and semi-conserved amino acid residues. Homology modelling of CtPME structure revealed a characteristic right handed parallel ß-helices. The energy of modelled structure was minimized by using YASARA software. The Ramachandran plot of CtPME shows 83.7% residues in non-glycine and non-proline residues in most-favorable region, 13.8% in additional allowed region and 1.4% in generously allowed region, indicating that CtPME has a stable conformation. The secondary structure of CtPME predicted using PSI-Pred software and confirmed by the circular dichroism (CD) showed α-helices (3.1%), ß-sheets (40.1%) and random coils (56.9%). Small Angle X-ray Scattering (SAXS) analysis demonstrated the overall shape and structural characterization of CtPME in solution form. Guinier analysis gave the radius of gyration (Rg) 2.28 nm for globular shape and 0.74 nm for rod shape. Kratky plot gave the indication that protein is fully folded in solution. The ab initio derived dummy atom model of CtPME superposed well on modelled CtPME structure.

Molecular Cloning, Expression and Characterization of Pectin Methylesterase (CtPME) from Clostridium thermocellum.

Many phytopathogenic micro-organisms such as bacteria and fungi produce pectin methylesterases (PME) during plant invasion. Plants and insects also produce PME to degrade plant cell wall. In the present study, a thermostable pectin methylesterase (CtPME) from Clostridium thermocellum belonging to family 8 carbohydrate esterase (CE8) was cloned, expressed and purified. The amino acid sequence of CtPME exhibited similarity with pectin methylesterase from Erwinia chrysanthemi with 38% identity. The gene encoding CtPME was cloned into pET28a(+) vector and expressed using Escherichia coli BL21(DE3) cells. The recombinant CtPME expressed as a soluble protein and exhibited a single band of molecular mass approximately 35.2 kDa on SDS-PAGE gels. The molecular mass, 35.5 kDa of the enzyme, was also confirmed by MALDI-TOF MS analysis. Notably, highest protein concentration (11.4 mg/mL) of CtPME was achieved in auto-induction medium, as compared with LB medium (1.5 mg/mL). CtPME showed maximum activity (18.1 U/mg) against citrus pectin with >85% methyl esterification. The optimum pH and temperature for activity of CtPME were 8.5 and 50 °C, respectively. The enzyme was stable in pH range 8.0-9.0 and thermostable between 45 and 70 °C. CtPME activity was increased by 40% by 5 mM Ca2+ or Mg2+ ions. Protein melting curve of CtPME gave a peak at 80 °C. The peak was shifted to 85 °C in the presence of 5 mM Ca2+ ions, and the addition of 5 mM EDTA shifted back the melting peak to 80 °C. CtPME can be potentially used in food and textile industry applications.