Separation and characterization of cellulose from sugarcane tops and its saccharification by recombinant cellulolytic enzymes.

In the present study, the cellulose from sugarcane tops (SCT) was separated and characterized for its purity. Approximately, 85% (w/w) of total cellulose present in raw SCT was recovered by using alkaline method. The monosaccharide analysis of SCT cellulose by HPLC showed 91% D-glucose, 7.5% D-xylose and 1.5% D-arabinose residues. Surface morphology study of dried cellulosic fibers by FESEM exhibited the fibrous structure. The FTIR analysis of separated cellulose displayed the peaks corresponding to the peaks obtained from commercial cellulose, confirming its purity. The crystallinity index (CrI) of separated cellulose increased to 49% after delignification and xylan extraction from 36% of raw SCT. The typical TGA curve of separated SCT cellulose showed decomposition and mass reduction at 327 °C resulting in single decomposition peak in TGA analysis, confirming its purity. CHNS analysis supported the purity of separated cellulose by confirming absence of nitrogen and sulfur. The separated cellulose was hydrolyzed by recombinant endo-ß-1,4-glucanase (CtCel8A), cellobiohydrolase (CtCBH5A) from Clostridium themocellum and ß-1,4-glucosidase (HtBgl) from Hungateiclostridium thermocellum at pH 5.8, 50 °C for 24 h, resulting in the production of 188 mg/g of total reducing sugar (TRS). The separated cellulose from SCT can be utilized as an alternative substrate for commercialization and for bioethanol production.

Assessment of combination of pretreatment of Sorghum durra stalk and production of chimeric enzyme (ß-glucosidase and endo ß-1,4 glucanase, CtGH1-L1-CtGH5-F194A) and cellobiohydrolase (CtCBH5A) for saccharification to produce bioethanol.

Optimization of pretreatment and saccharification of Sorghum durra stalk (Sds) was carried out. The chimeric enzyme (CtGH1-L1-CtGH5-F194A) having ß-glucosidase (CtGH1) and endo ß-1,4 glucanase activity (CtGH5-F194A) and cellobiohydrolase (CtCBH5A) from Clostridium thermocellum were used for saccharification. Chimeric enzyme will save production cost of two enzymes, individually. Stage 2 pretreatment by 1% (w/v) NaOH assisted autoclaving + 1.5% (v/v) dilute H2SO4 assisted oven heating gave lower total sugar yield (366.6 mg/g of pretreated Sds) and total glucose yield (195 mg/g of pretreated Sds) in pretreated hydrolysate with highest crystallinity index 55.6% than the other stage 2 pretreatments. Optimized parameters for saccharification of above stage 2 pretreated biomass were 3% (w/v) biomass concentration, enzyme (chimera: cellobiohydrolase) ratio, 2:3 (U/g) of biomass, total enzyme loading (350 U/g of pretreated biomass), 24 h and 30 °C. Best stage 2 pretreated Sds under optimized enzyme saccharification conditions gave maximum total reducing sugar yield 417 mg/g and glucose yield 285 mg/g pretreated biomass in hydrolysate. Best stage 2 pretreated Sds showed significantly higher cellulose, 71.3% and lower lignin, 2.0% and hemicellulose, 12.2% (w/w) content suggesting the effectiveness of method. This hydrolysate upon SHF using Saccharomyces cerevisiae under unoptimized conditions produced ethanol yield, 0.12 g/g of glucose. Abbreviation: Ct-Clostridium thermocellum, Sds-Sorghum durra stalk, TRS-Total reducing sugar, HPLC-High performance liquid chromatography, RI-Refractive index, ADL-acid insoluble lignin, GYE-Glucose yeast extract, MGYP-Malt glucose yeast extract peptone, SHF-separate hydrolysis and fermentation, OD-Optical density, PVDF-Poly vinylidene fluoride, TS-total sugar, FESEM-Field emission scanning electron microscopy, XRD-X-ray diffraction, FTIR-Fourier transform infra-red spectroscopy and CrI-Crystallinity index.

Sulfadiazine degradation by combination of hydrodynamic cavitation and Fenton-persulfate: parametric optimization and deduction of chemical mechanism.

This paper reports the degradation of the sulfadiazine (SDZ) drug with a hybrid advanced oxidation process (AOP) of heterogeneous α-Fe2O3/persulfate coupled with hydrodynamic cavitation. The major objectives of the study are parametric optimization of the process and elucidation of the chemical mechanism of degradation. The optimum conditions for maximum SDZ degradation of 93.07 ± 1.67% were as follows: initial SDZ concentration = 20 ppm, pH = 4, α-Fe2O3 = 181.82 mg/L, Na2S2O8 = 348.49 mg/L, H2O2 = 0.95 mL/L, inlet pressure = 0.81 MPa (8 atm), orifice plate configuration: hole dia. = 2 mm and number of holes = 4. Density functional theory (DFT) calculations revealed that the atoms of SDZ with a high Fukui index (f 0) were potentially active sites for the attack of •OH and [Formula: see text] radicals. Fukui index calculation revealed that atom 11 N has a higher value of f 0 (0.1026) for oxidation at the α-amine group of the sulfadiazine molecule. Degradation intermediates detected through LC-MS/MS analysis corroborated the results of DFT simulations. Using these results, a chemical pathway has been proposed for SDZ degradation.

Extraction and characterization of xylan from sugarcane tops as a potential commercial substrate.

Xylan is the major hemicellulose present in sugarcane stem secondary cell walls. Xylan is composed of xylose backbone with a high degree of substitutions, which affects its properties. In the present study, the xylan from sugarcane tops (SCT) was extracted and characterized. Compositional analysis of xylan extracted from SCT (SCTx) displayed the presence of 74% of d-xylose residues, 16% of d-glucuronic acid residues and 10% of l-arabinose. High performance size exclusion chromatographic analysis of SCTx displayed a single peak corresponding to a molecular mass of ∼57 kDa. The Fourier transform infrared spectroscopic analysis of SCTx displayed the peaks corresponding to those obtained from commercial xylan. FESEM analysis of SCTx showed the granular and porous surface structure. Differential thermogravimetric analysis (DTG) of SCTx displayed two thermal degradation temperatures (Td) of 228°C, due to breakdown of the side chains of glucuronic acid and arabinose and 275°C, due to breakdown of xylan back bone. The presence of arabinose and glucuronic acid as a side chains was confirmed by the DTG and thermogravimetric analysis. The CHNS analysis of SCTx showed the presence of only carbon and hydrogen supporting its purity. The recombinant xylanase (CtXyn11A) from Clostridium thermocellum displayed a specific activity of 1394 ± 51 U/mg with SCTx, which was higher than those with commercial xylans. The thin layer chromatography and electrospray ionization mass spectroscopy analyses of CtXyn11A hydrolysed SCTx contained a series of linear xylo-oligosaccharides ranging from degree of polymerization 2-6 and no substituted xylo-oligosaccharides because of the endolytic activity of enzyme. The extracted xylan from SCT can be used as an alternative commercial substrate and for oligo-saccharide production.

Two-Step Saccharification of the Xylan Portion of Sugarcane Waste by Recombinant Xylanolytic Enzymes for Enhanced Xylose Production.

Sugarcane bagasse (SB) and sugarcane trash (SCT) containing 30% hemicellulose content are the waste from the sugarcane industry. Hemicellulose being heterogeneous, more complex, and less abundant than cellulose remains less explored. The optimized conditions for the pretreatment of SB and SCT for maximizing the delignification are soaking in aqueous ammonia (SAA), 18.5 wt %, followed by heating at 70 °C for 14 h. The optimization of hydrolysis of SAA pretreated (ptd) SB and SCT by the Box-Behnken design in the first step of saccharification by xylanase (CtXyn11A) and α-l-arabinofuranosidase (PsGH43_12) resulted in the total reducing sugar (TRS) yield of xylooligosaccharides (TRS(XOS)) of 93.2 mg/g ptd SB and 85.1 mg/g ptd SCT, respectively. The second step of saccharification by xylosidase (BoGH43) gave the TRS yield of 164.7 mg/g ptd SB and 147.2 mg/g ptd SCT. The high-performance liquid chromatography analysis of hydrolysate obtained after the second step of saccharification showed 69.6% xylan-to-xylose conversion for SB and 64.1% for SCT. This study demonstrated the optimization of the pretreatment method and of the enzymatic saccharification by recombinant xylanolytic enzymes, resulting in the efficient saccharification of ptd hemicellulose to TRS by giving 73.5% conversion for SB and 71.1% for SCT. These optimized conditions for the pretreatment and saccharification of sugarcane waste can also be used at a large scale.

Acacia Xylan as a Substitute for Commercially Available Xylan and Its Application in the Production of Xylooligosaccharides.

Over the past two decades, birchwood and beechwood xylans have been used as a popular substrate for the characterization of xylanases. Recently, major companies have discontinued their commercial production. Therefore, there is a need to find an alternative to these substrates. Xylan extraction from Acacia sawdust resulted in 23.5% (w/w) yield. The extracted xylan is composed of xylose and glucuronic acid residues in a molar ratio of 6:1 with a molecular mass of ∼70 kDa. The specific optical rotation analysis of extracted xylan displayed that it is composed of the d-form of xylose and glucuronic acid monomeric sugars. The nuclear magnetic resonance analysis of extracted xylan revealed that the xylan backbone is substituted with 4-O-methyl glucuronic acid at the O2 position. Fourier transform infrared analysis confirmed the absence of lignin contamination in the extracted xylan. Xylanase from Clostridium thermocellum displayed the enzyme activity of 1761 U/mg against extracted xylan, and the corresponding activity against beechwood xylan was 1556 U/mg, which confirmed that the extracted xylan could be used as an alternative substrate for the characterization of xylanases.

Extraction, characterization of xylan from Azadirachta indica (neem) sawdust and production of antiproliferative xylooligosaccharides.

Xylan extracted from neem sawdust gave 22.5%, (w/w) yield. The extracted xylan was composed of xylose and glucuronic acid at a molar ratio of 8:1 and with a molecular mass, ~66 kDa. FTIR and NMR analyses indicated a backbone of xylan substituted with 4-O-methyl glucuronic acid at the O-2 position. FESEM analysis showed a highly porous and granular surface structure of xylan. A thermogravimetric study of xylan showed thermal denaturation at 271 °C. The hydrolysis of xylan by recombinant endo-ß-1,4-xylanase produced a mixture of xylooligosaccharides ranging from degree of polymerization 2-7. Xylooligosaccharides inhibited cell growth of human colorectal cancer (HT-29) cells but did not affect the mouse fibroblast cells confirming its biocompatibility. Western blotting, DNA laddering and flow cytometric analysis displayed inhibition of HT-29 cells by xylooligosaccharides. FLICA staining and mitochondrial membrane potential analyses confirmed the activation of the intrinsic pathway of apoptosis. The study amply indicated that the xylooligosaccharides produced from neem xylan could be potentially used as an antiproliferative agent.

Development of bi-functional chimeric enzyme (CtGH1-L1-CtGH5-F194A) from endoglucanase (CtGH5) mutant F194A and ß-1,4-glucosidase (CtGH1) from Clostridium thermocellum with enhanced activity and structural integrity.

Site-directed mutagenesis of ß-1,4-endoglucanase from family 5 glycoside hydrolase (CtGH5) from Clostridium thermocellum was performed to develop a mutant CtGH5-F194A that gave 40 U/mg specific activity against carboxymethyl cellulose, resulting 2-fold higher activity than wild-type CtGH5. CtGH5-F194A was fused with a ß-1,4-glucosidase, CtGH1 from Clostridium thermocellum to develop a chimeric enzyme. The chimera (CtGH1-L1-CtGH5-F194A) expressed as a soluble protein using E. coli BL-21cells displaying 3- to 5-fold higher catalytic efficiency for endoglucanase and ß-glucosidase activities. TLC analysis of hydrolysed product of CMC by chimera 1 revealed glucose as final product confirming both ß-1,4-endoglucanase and ß-1,4-glucosidase activities, while the products of CtGH5-F194A were cellobiose and cello-oligosaccharides. Protein melting studies of CtGH5-F194A showed melting temperature (Tm), 68 °C and of CtGH1, 79 °C, whereas, chimera showed 78 °C. The improved structural integrity, thermostability and enhanced bi-functional enzyme activities of chimera makes it potentially useful for industrial application in converting biomass to glucose and thus bioethanol.