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Structural and conformational insights of a putative β-1,4-xylosidase (PsGH43F) of glycoside hydrolase family 43 from Pseudopedobacter saltans were investigated by computational and Circular Dichroism (CD) analyses. PsGH43F was cloned and expressed in E. coli BL21 (DE3) cells and the purified enzyme gave the size ~50 kDa on SDS-PAGE analysis. Multiple Sequence Alignment of PsGH43F sequence followed by superposition of modeled structure with homologous structures displayed the presence of three conserved catalytic amino acid residues, Asp33, Asp149 and Glu212. The secondary structure analysis by CD showed 2.72 % α-helix and 36.06 % β-strands. The homology modeled structure of PsGH43F displayed a 5-bladed β-propeller fold for catalytic module at N-terminal and a β-sandwich structure for CBM6 at the C-terminal. Ramachandran plot displayed 99.5 % of residues in the allowed regions. MD simulation of PsGH43F revealed the compactness and stability of the structure. Molecular docking studies of PsGH43F with xylo-oligosaccharides revealed its maximum binding affinity for xylobiose. MD simulation of PsGH43F-xylobiose complex confirmed the increased structural and conformational stability in presence of substrate. The Hydrodynamic diameter analysis of PsGH43F by DLS was in the range, 0.25-0.28 μm.

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.

The structure and molecular dynamics of α-L-arabinofuranosidase (PsGH43_12) of family 43 glycoside hydrolase, subfamily 12 from Pseudopedobacter saltans were studied. The modeled PsGH43_12 structure displayed 5-bladed β-propeller fold at N-terminal and β–sandwich fold at C terminal. Ramachandran plot showed 95.7% residues in favored and 3.3% in the generously allowed region and only 1% residues in the disallowed region. The secondary structure analysis of PsGH43_12 by circular dichroism revealed 2.7% α-helices, 30.33% β-strands and 66.97% random coils. Protein melting study of PsGH43_12 showed complete unfolding at 65°C and did not require any metal ion for its stability. Molecular docking analysis confirmed the involvement of active site residues Asp71, Asp180 and Glu247 in the catalysis, which was also confirmed by the site-directed mutagenesis of these residues. SAXS analysis displayed that PsGH43_12 is monomeric and a fully folded state in solution form. Guinier analysis gave the radius of gyration (Rg) 2.8 ± 0.09 nm. The maximum dimension and Rg of PsGH43_12 estimated from P(R) plot were 9.7 nm and 2.81 nm, respectively. The ab initio derived dummy model of PsGH43_12 displayed a bell-like shape. The ab initio derived dummy model superposed well with its comparative modeled structure except the N-terminal His6-tag region.

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-β-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.

Cellulosomes are highly complex cell-bound multi-enzymatic nanomachines used by anaerobes to break down plant carbohydrates. The genome sequence of Ruminococcus flavefaciens revealed a remarkably diverse cellulosome composed of more than 200 cellulosomal enzymes. Here we provide a detailed biochemical characterization of a highly elaborate R. flavefaciens cellulosomal enzyme containing an N-terminal dockerin module, which anchors the enzyme into the multi-enzyme complex through binding of cohesins located in non-catalytic cell-bound scaffoldins, and three tandemly repeated family 16 glycoside hydrolase (GH16) catalytic domains. The DNA sequence encoding the three homologous catalytic domains was cloned and hyper-expressed in Escherichia coli BL21 (DE3) cells. SDS-PAGE analysis of purified His6 tag containing RfGH16_21 showed a single soluble protein of molecular size ~89 kDa, which was in agreement with the theoretical size, 89.3 kDa. The enzyme RfGH16_21 exhibited activity over a wide pH range (pH 5.0–8.0) and a broad temperature range (50–70 °C), displaying maximum activity at an optimum pH of 7.0 and optimum temperature of 55 °C. Substrate specificity analysis of RfGH16_21 revealed maximum activity against barley β-d-glucan (257 U mg−1) followed by lichenan (247 U mg−1), but did not show significant activity towards other tested polysaccharides, suggesting that it is specifically a β-1,3-1,4-endoglucanase. TLC analysis revealed that RfGH16_21 hydrolyses barley β-d-glucan to cellotriose, cellotetraose and a higher degree of polymerization of gluco-oligosaccharides indicating an endo-acting catalytic mechanism. This study revealed a fairly high, active and thermostable bacterial endo-glucanase which may find considerable biotechnological potentials.

Cellulosomes are highly complex cell-bound multi-enzymatic nanomachines used by anaerobes to break down plant carbohydrates. The genome sequence of

The cellulosomal enzyme, RfGH51/2, of Ruminococcus flavefaciens contains an N-terminal module, a family 5 glycoside hydrolase GH5_4 with a putative endoglucanase activity, while C-terminal domain is a putative endo-mannanase (GH5_7). The two putative catalytic modules are separated by family 80 carbohydrate binding module (CBM80) having wide ligand specificity. The putative endo-mannanase module, GH5_7 (RfGH5_7), was cloned, expressed in Escherichia coli BL-21(DE3) cells and purified. SDS-PAGE analysis of purified RfGH5_7 showed molecular size ~ 35 kDa. Substrate specificity analysis of RfGH5_7 showed maximum activity against locust bean galactomannan (298.5 U/mg) followed by konjac glucomannan (256.2 U/mg) and carob galactomannan (177.2 U/mg). RfGH5_7 showed maximum activity at optimum pH 6.0 and temperature 60 °C. RfGH5_7 displayed stability in between pH 6.0 and 9.0 and thermostability till 50 °C. 10 mM Ca2+ ions increased the enzyme activity by 33%. The melting temperature of RfGH5_7 was 84 °C that was not affected by Ca2+ ions or chelating agents. RfGH5_7 showed, Vmax, 389 U/mg and Km, 0.92 mg/mL for locust bean galactomannan. TLC analysis revealed that RfGH5_7 hydrolysed locust bean galactomannan predominantly to mannose, mannobiose, mannotriose and higher degree of polymerization of manno-oligosaccharides indicating an endo-acting catalytic mechanism. This study revealed a highly active and thermostable endo-mannanase with considerable biotechnological potential.

The production of bioenergy from wastes attracts worldwide attention to overcome energy crisis and increasing pollution (Thakur et al., Microbial fermentation and enzyme technology, Taylor and Francis Group, Boca Raton, FL, 257–268, 2020). Lignocellulosic biomass can serve as an alternative source for bioenergy production. Thermostable enzymes can hydrolyze the lignocellulosic biomass and produce reducing sugars, which can be fermented to produce bioethanol by using fermenting microbes. Clostridium thermocellum is a gram-positive, anaerobic and rod-shaped, thermophilic microorganism having great potential applications. It can directly transform lignocellulosic biomass into valuable products such as acetate, ethanol, formate, and lactate. Clostridium thermocellum expresses a multi-enzyme complex bound to scaffoldin proteins called cellulosome that contains cellulolytic, hemicellulolytic, and other carbohydrate degrading enzymes. The thermophilic enzymes possess wide applications in several industries for producing sustainable green products. This chapter evaluates the production and properties of recombinant thermostable cellulases, hemicellulases, and pectinases from C. thermocellum, their structure, and applications in different industrial processes.

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.

α-l-Arabinofuranosidase (PsGH43_12) of family 43 glycoside hydrolase and subfamily 12 from Pseudopedobacter saltans was cloned, over-expressed and biochemically characterized. PsGH43_12 displayed molecular mass, ~ 65 kDa. It exhibited activity in pH (5–9) and temperature range (35–55 °C) with maxima at pH 6.5 and 50 °C. PsGH43_12 gave 88.7 U/mg specific activity against rye arabinoxylan and 78.9 U/mg against wheat arabinoxylan. PsGH43_12 displayed Km and Vmax, 3.02 mg/ml and 103 µmole/min/mg, respectively, against rye arabinoxylan and 2.17 mM and 100.7 µmole/min/mg, respectively, against pNP-α-l-arabinofuranoside. 10 mM Mg2+ or Ca2+ ions enhanced PsGH43_12 activity by 54% or 33%, respectively. PsGH43_12 hydrolyzed rye arabinoxylan and released only l-arabinosyl moiety as main product, confirming its specificity towards α-l-arabinofuranoside. The regioselective analysis by NMR showed that PsGH43_12 belongs to type III α-l-arabinofuranoside. The synergistic behavior of PsGH43_12 in saccharification of mild alkali pretreated finger miller stalk (FMS) along with xylanase (CtXyn11A) from Clostridium thermocellum and xylosidase (BoGH43) from Bacteroides ovatus gave twofold higher total reducing sugar (TRS) yield. TLC analysis of pretreated FMS hydrolysed by CtXyn11A and BoGH43 showed xylooligosaccharides and xylose. Addition of PsGH43_12 to above combination gave mostly xylose and arabinose confirming their synergistic behavior and displaying their applicability in hydrolysis of hemicellulosic biomass.

The world is currently facing two major challenges, the resource scarcity and overproduction of waste. Due to the uneven distribution and excess consumption of resources, the demand for energy is high and increasing day by day. On the other hand, the mounting of waste needs strategic waste management. In the environment, microbes present utilize organic waste by releasing enzymes, but the rate of degradation is much slower than the production of waste. Organic waste majorly constitutes agricultural waste or municipal solid waste that can be converted to bioenergy (methane, methanol, bioethanol and biobutanol). Cellulose and hemicellulose portion of the solid wastes are hydrolyzed by cellulases and hemicellulases into fermentable sugars like glucose, xylose and arabinose. Fermenting microbes further utilize these sugars for the production of bioethanol, and residual biomass used as compost in the agricultural field. Ethanol produced from cellulose and hemicellulose is environmental friendly fuel as it emits lower carbon monoxide. An overview of breakdown of cellulosic and hemicellulosic polysaccharides present in agro-waste and production of bioethanol with focus on recent developments is described.

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.

Polysaccharide-based biopolymers are widely exploited for various applications, such as sensing, textile, tissue engineering scaffolds, wastewater treatment, food industry, and drug delivery. Chitin is one such naturally occurring polymer found in many lower eukaryotes serving a protective role. It is the second most abundant organic compound in nature, after cellulose. Chitin and its derivatives, such as chitosan and chitooligosaccharides are extensively used for such applications due to their unique biochemical and biophysical properties such as biocompatibility, biodegradability, nontoxicity, ability to form films, ready availability, etc. The commercial production of chitin has been reported from crustacean shells, the traditional method other than fungal and insect sources. In this chapter, different sources for bioextraction, the properties and applications of chitin and chitosan-based polysaccharides, and the challenges encountered and future prospects will be discussed.

The effect of alloying additions, such as copper, carbon, and molybdenum with carbonyl iron powder, on the densification behavior, microstructural evolution, and mechanical properties of spark plasma sintered (SPS) compacts have been investigated in this work. The sintering temperature, pressure, and time during SPS were 1120°C, 30 MPa, and 5 min, respectively. Fe–2Cu–0.8C–0.6Mo was found to exhibit the highest density, hardness, and also tensile strength among all the compositions attempted. The microstructural examination of fractured surfaces of the sintered samples revealed the evidence of a mixed mode of fracture in all the alloy compositions.

The present study investigated the comparative study of machinability parameters on the quality characteristics of force (Fz) and roughness (Ra) during the dry machining Nimonic 263 alloy. Machinability experiments are carried out with two coated i.e. PVD (TiAlN), KC25 (TiN/Al2O3/TICN) for dry turning at four speeds (60, 90, 120 m/min, 150 m/min), two feed (0.05, 0.12, 0.18 and 0.25 mm/rev) and three cutting depth (0.4, 0.6, 0.8 1.0 mm). The experiments are planned based on Taguchi’s L16 orthogonal array. The optimal parameters combination of the machinability parameters on quality characteristics is determined using ‘smaller-the better” characteristics in Taguchi analysis. From the experimental results, the most significant factor in case of both Fz and Ra is feed rate and cutting speed during the turning of 263 Nimonic alloy for both coated carbide insert using variance test (ANOVA). Pearson correlation coefficient analysis is also calculated to find the correlation between the machining parameters and machinability attributes. Also the calculated Pearson correlation coefficient showed a robust relationship between the feed, cutting depth and force and roughness. In addition, the lower magnitude of cutting force and roughness is observed for dry in both inserts at low level of feed (0.05 mm/rev) and cutting depth (0.4 mm) with moderate levels of speed (120 m/min).

The primary energy requirement of almost all life-forms on earth is derived from plant sources. The plant cell wall (PCW) has a chemically complex structure composed of glycoproteins, aromatic compounds and carbohydrates. The carbohydrates include cellulose, hemicellulose and pectin. Cellulose has homogeneous structure, whereas hemicellulose and pectin are heterogeneous. Pectin constitutes as the most complex polysaccharide and is abundantly found in the middle lamella and primary cell wall of PCW. It is also proportionally the highest constituent of plant dry matter. The pectic substances are hydrolyzed by pectinolytic enzymes which are widely present in bacteria, fungi and plants. Pectinolytic enzymes are classified as de-polymerising and de-esterifying enzymes. The de-esterifying enzymes are the accessory enzymes which remove the side chains. The pre-action of de-esterifying enzymes removes the steric hindrance and provides better access to other de-polymerizing enzymes for complete degradation of pectin. This review describes the pectin structure and the structure, function and biochemical properties of recombinant pectinolytic enzymes from different microbial sources. The focus is on the specific role of individual pectinolytic enzymes in various industrial applications such as juice extraction, vegetable puree production, fibre retting, waste water treatment, pharmaceuticals, biomass hydrolysis and production of other value-added products. Thus, the enhanced use of pectinolytic enzymes in these growing industries holds a great potential for gaining insights about their efficient role in various sectors.

This study focuses on enzymatic saccharification of hemicellulose part of the pretreated Finger millet straw (FMS) for production of xylose. The variation in the carbohydrate composition of FMS was analysed when subjected to different pretreatments. The recombinant endo-1,4-β-xylanase (CtXyn11A) was most active on the FMS pretreated with 1% (w/v) NaOH combined with oven heating at 120 °C for 20 min, resulting in a total reducing sugar yield (TRS) of 32 mg/g pretreated biomass. The pretreatment aided in concentrating the holocellulose content from 69.3% of raw powdered FMS to 76.4%. The post-treatment solid biomass yield was 0.36 g/g raw biomass. The two-step optimization of hemicellulose saccharification from the above pretreated FMS with i) endo-1,4-β-xylanase (CtXyn11A) at 55 °C and ii) exo-1,4-β-xylosidase (BoGH43A) at 37 °C, both at pH 7.5 by Box-Behnken design yielded the TRS of 70 mg/g pretreated biomass. The percentage conversion of xylan to xylose by CtXyn11A and BoGH43A was 24.7%.

Cellulose, hemicellulose, pectin, and lignin are the major components of plant cell wall. Hemicellulose is the second most abundant carbohydrate polymer on the earth. Hemicelluloses are branched, hetero-polysaccharides formed by β-(1 → 4)-linked backbones of hexoses like glucose (xyloglucan), galactose (galactan), mannose (mannan) or pentoses like xylose (xylan), and arabinose (arabinan). Xylan contains the backbone of 1,4-linked-β-d-xylopyranose with various side-chain substitutions such as arabinose, acetic acid, glucuronic acid, ferulic, acid, and p-coumaric acid. l-arabinose side chain is found in hemicelluloses like arabinan, arabinoxylan, oat spelt xylan, and arabinogalactan. The extent of side-chain substitution depends on the source of the xylan, which makes its structure complex and hinders its enzymatic hydrolysis. α-l-arabinofuranosidase hydrolyzes arabinose side chain present at α-1,2-, α-1,3-, and α-1,5-positions in arabinoxylan, thus potentiating other xylanolytic enzymes to act efficiently on the backbone. Therefore, α-l-arabinofuranosidase has potential application in agro-industrial processes because of its functioning synergistically with other hemicellulases. α-l-arabinofuranosidases are used for improving bread quality, for wine flavor, for clarification of fruit juices, as supplement for feedstock for enhancing digestion, in the production of medicinal compounds, and in the production of oligosaccharide and modification of their side chains. This chapter presents a comprehensive overview of α-l-arabinofuranosidase, sources, production, and its applications in food processing.

The development of new food products, improvement in food quality, and ease of food production process is of prime concern with the growing world population and rapidly rising demand for functional foods. These concerns make it imperative, the use of various enzymes such as glycoside hydrolases, lipases, proteases, transglutaminases, etc., in the processing of food and food ingredients. Crops and fruits used in food and brewing industry contain considerable amount of xylan. Xylan is a branched heteropolysaccharide and its main chain is composed of xylose subunits linked by β-(1 → 4) glycosidic bonds and contains different substitutions in the side chain. Xylanase cleaves β-(1 → 4) glycosidic bonds in heteroxylan randomly and converts it into xylooligosaccharides. In the last decade, xylanase has received appreciable attention owing to its applications in various food processing industries such as cereal food processing for the improvement of gluten agglomeration, baking industry for the improved texture of bread and cookies, clarification of fruit juices, production of xylooligosaccharide or arabinoxylooligosaccharides as prebiotic food supplements. This chapter presents a comprehensive overview of xylanase, its sources, production, and applications in food production and processing, with a particular focus on recent developments.