Cellulase Enzymatic Hydrolysis Research Articles

Xylooligosaccharides and glucose preparation from sugarcane bagasse by a combination of acetic acid treatment and sequential xylanase and cellulase enzymatic hydrolysis

Acetic acid of 2.5–10% (m/v) can be exploited to assisted hydrolyze sugarcane bagasse for directly producing XOS, however, it simultaneously generated high amounts of byproducts, increasing the difficulty of products purification, and there are evidences that the ratio of active ingredients (xylobiose (X2) and xylotriose (X3)) was lower. In this study, a strategy of acetic acid pretreatment with post-xylanase treatment of sugarcane bagasse for preparing xylooligosaccharides (XOS) was put forward. Firstly, the acid hydrolysis with post-xylanase hydrolysis processes varied the parameters applying single-factor experiments design using the 2.5–10% (m/v) acetic acid at a temperature of 130–190 °C and a duration of 20–60 min. According to single-factor experiments, a three-level three-factor Box-Behnken Design with response surface methodology was performed to maximize the XOS yields. As a results, the maximum XOS yield was achieved at over 60.7% with a sequential operation of acid hydrolysis (3.6% acetic acid, 180 °C, 35 min) and xylanase hydrolysis (12 h), and the ratio of (X2 + X3)/XOS was over 70%. Moreover, the treatment with 3.6% acetic acid and xylonase also improved enzymatic hydrolysis yield to 82.1%. Overall, favorable outcomes suggest that the combined approach of acidic and enzymatic hydrolysis is cost-effective for XOS with desired degree of polymerization and glucose preparation.

Extraction and Compositional Analysis of Anthocyanins and Polysaccharides from Aronia Melanocarpa Fruit

We have examined the basic composition of anthocyanins and polysaccharides, the active components in Aronia melanocarpa fruit. To this end, microwave-assisted ethanol extraction and cellulase enzymatic hydrolysis were employed to extract anthocyanins and crude polysaccharides, respectively. Aronia melanocarpa fruit polysaccharides were separated and purified by DEAE-52 cellulose and Sephadex G-100 glucan gel column chromatography, followed by high-performance liquid chromatography. The results demonstrated that the optimal extraction parameters of anthocyanin from Aronia melanocarpa fruit were a 60% ethanol volume fraction, a liquid-to-solid ratio of 30:1, a microwave time of 60 s, and a microwave power of 210 W. The extracted anthocyanins reached 200.47 mg/100 g, while the yield of polysaccharides was 3.82%. Anthocyanins in Aronia melanocarpa fruit were mainly composed of cyanidin, delphinidin, and mallow pigment, among which cyanidin was the main component, with a content as high as 90.79%. There were two distinct fractions of polysaccharides. The fraction-I polysaccharides were mainly composed of rhamnose, galacturonic acid, glucose, galactose, and arabinose, with a molar ratio of 8.80: 8.97: 2.38: 27.46: 47.94. In contrast, the fraction-II polysaccharides were primarily composed of rhamnose, glucuronic acid, galactose, xylose, and arabinose, with a molar ratio of 1.67: 9.22: 8.47: 70.69: 6.16. The results of this study can provide a reference for the in-depth development of Aronia melanocarpa resources

Insights into key factors affecting bioconversion efficiency of rattan biomass: The supramolecular structural variations of cellulose

Global energy concerns urged us to search for sufficient utilization of biomass to renewable energy. Herein, rattan biomass displaying herbaceous species-like anatomy and hardwood-like chemical composition was used as model of lignocellulose to determine its recalcitrance inhibiting efficient bioconversion. Delignification and continuous mild alkaline treatments were applied for deconstruction of rattan cane (Calamus simplicifolius) followed by cellulase enzymatic hydrolysis. Cellulose supramolecular structural variations were proved to be the major reason for the enhanced hydrolysis in addition to the removal of lignin and hemicelluloses matrix. Lowered crystallinity (50–65 %) as well as swelled crystallite sizes (4.8–5.0 nm) during allomorphic transformation favored the enhanced hydrolysis, rather than the crystalline cellulose II. Moreover, well-distributed separation and fibrillation of cellulose elementary fibrils also contributed to glucose yield promotion. The study will provide new insights to the strategy to efficient bioconversion of lignocellulosic biomass.

Pretreatment of barley straw with acid and alkaline solutions to boost the efficiency of fermentable yield enzymatic degradation in the separated fermentation technique for ethanol production

Barley straw is a lignocellulose agricultural waste material that can be utilized as a raw material for ethanol production since it is easy to find, cheap, and has the potential to produce ethanol yield. This research aims to select the optimum pretreatment conditions to increase fermentable sugar production during enzymatic hydrolysis for bioethanol production. Initially, sulfuric acid at a concentration of 0.5, 1.0, 1.5, and 2.0% (v/v) pre-treated barley straw biomass was applied by autoclaving at 121℃ for 15 lb/in2 pressure for 15 min. Furthermore, acid-treated barley straw under the alkaline condition with calcium hydroxide concentrations of 1, 2, 3, and 4% (w/v) was autoclaved at 121℃ for 15 lb/in2 pressure for 15 min. Subsequently, the pretreatment of barley straw in the acetic and alkaline processes was compared. It was found that calcium hydroxide at a concentration of 2% (w/v) gave more sugar concentrations. Finally, combined acid and alkaline pretreatment with 2% of cellulase enzymatic hydrolysis had the highest total sugar concentration of 205.43 g/L and reducing sugar of 134.42 g/L, producing the highest ethanol yield (16.17 g/L) by 24 hours of fermentation.

High Solid and Low Cellulase Enzymatic Hydrolysis of Cardoon Stems Pretreated by Acidified γ-Valerolactone/Water Solution

Lignocellulosic biomass is a nonedible matrix that can be efficiently exploited as feedstock in an integrated biorefinery after a proper pretreatment. An organosolv pretreatment using an acidified γ-valerolactone (GVL)/water solution was proposed to improve the cellulose enrichment and enzymatic saccharification of cardoon (Cynara cardunculus L.) stems. At the optimal pretreatment condition (140 °C, 0.6 GVL/water, and 2.24% H2SO4), xylan was efficiently removed from the cardoon, and up to 50% of its content was recovered in the aqueous fraction, while 86% of the cellulose was retained in the solid fraction. The resulting cardoon pulp showed a cellulose content of 91.5% and an enzymatic digestibility of 100%. An overall glucose production of 37.17 g/100 g raw material (90% theoretical maximum) was obtained using high solid loading (20% w/w) and a high enzyme dosage (60 FPU/g cellulose). At a low enzyme dosage, glucose concentrations of 169 g/L and 210 g/L were achieved using 10 FPU/g cellulose and 20 FPU/g cellulose, respectively. Therefore, an organosolv pretreatment can be an effective process for producing cellulose-enriched pulp with enhanced enzymatic digestibility from cardoon stems, providing a promising option for green lignocellulosic biorefineries that aim to produce high concentrations of glucose with low cellulase addition.

Structure of corn bran hemicelluloses isolated with aqueous ethanol solutions and their potential to produce furfural

The alkali-soluble hemicelluloses extracted with 10% KOH solution from corn bran were further isolated with different concentrations of aqueous ethanol solutions. Herein 92.2% of the original hemicelluloses can be obtained and the cellulase enzymatic hydrolysis rate of the alkali treated corn bran can reach to 97.2%. The corn bran hemicelluloses were mainly glucuronoarabinoxylan, in which xylose (48.4–53.8%) and arabinose (27.8–33.2%) were the main components. More linear hemicelluloses with high molecular weight tended to be precipitated in low concentration aqueous ethanol solutions. Furthermore, the relationship between the structural features of these alkali-soluble corn bran hemicelluloses and their furfural yield was investigated in MIBK (methyl isobutyl ketone)/H2O biphasic system. Results showed that the hemicelluloses with high xylose content are benefit to the furfural production, and the highest furfural yield of 67.7% was obtained.

Correlation between physicochemical characteristics of lignin deposited on autohydrolyzed wood chips and their cellulase enzymatic hydrolysis

Enzymatic hydrolysis is a method to generate biofuel from biomass, and autohydrolysis is a popular method to pretreat biomass prior to enzymatic hydrolysis. The primary aim of the present study was to determine the role of lignin produced in the autohydrolysis process on the enzymatic hydrolysis of biomass. The HSQC and 31P NMR analyses confirmed that β-O-4 of lignin was reduced, while β-5, β-β, and S/G-ratio of lignin were increased with intensifying the hydrolysis intensity.The increase in the hydrolysis intensity significantly enhanced the condensed and non-condensed phenolic OH group of lignin. Interestingly, the cellulase enzyme adsorbed more on lignin that had more phenolic content, and its association with lignin reduced its activity for hydrolyzing cellulose microcrystals. Strong negative correlations were observed between the enzymatic hydrolysis yield and the condensed S-OH (r2 = 0.978) and G-OH (r2 = 0.961) of lignin generated in the autohydrolysis process.

In-situ lignin drives lytic polysaccharide monooxygenases to enhance enzymatic saccharification

Recently low-molecular lignin was reported to activate lytic polysaccharide monooxygenases (LPMOs) to oxidize cellulose. However, whether lignin formed in cell wall can play the role as electron donor for LPMOs is still largely unknown due to the complex ultrastructure of lignocellulosic biomass. In this study, we presented a new strategy to elucidate in-situ lignin function in LPMOs reaction. A lignocellulosic mimicking model was used as substrate, which was equipped with a polysaccharide template of self-assembled bacterial cellulose film and synthesized lignin. Remarkably, it has been demonstrated that lignin polymer deposited on cellulose can reduce LPMOs in-situ for cellulose oxidation and then boost cellulose hydrolysis, and the cellulose conversion ratio of the mimicked lignocellulosic film was increased by 26.0%. More importantly, lignin in-situ might exceed the well-known reductant of ascorbic acid to drive LPMOs for cellulase enzymatic hydrolysis with equivalent cellulose oxidation efficiency and extremely lower H2O2 generation, avoiding the inactivation of enzymes. The maximum H2O2 yield from lignin-driven LPMO reaction was 75.8% lower than that from ascorbic acid-driven reaction. Therefore, by using the lignocellulosic mimicking model, we have elucidated the function of in-situ lignin in boosting enzymatic hydrolysis. Such understanding could significantly promote current utilization of LPMOs in lignocellulosic biorefinery.

Study on preparation of chito-oligosaccharide by cellulase hydrolysis

By using cellulase enzymatic hydrolysis preparation to obtain low molecular weight chito-oligosaccharide. The conditions for the hydrolysis of chitosan by cellulase were studied by single factor experiment. The effects of temperature, pH value, reaction time and enzyme dosage on the decomposition efficiency of chitosan were investigated.The conclusions from the experiment are as follows: the optimum conditions for the preparation of chito-oligosaccharide by cellulase hydrolysis are reaction temperature of 50 °C, pH of 4.8, enzymatic hydrolysis time of 3.5 h, the enzyme dosage of 50 U/mg, in this case its economic benefits is greatest.

Lignin prepared from different alkaline pretreated sugarcane bagasse and its effect on enzymatic hydrolysis

Sugarcane bagasse (SCB) was pretreated by sodium hydroxide (SH), alkaline ethanol (AE) and alkaline hydrogen peroxide (AHP), and the solid residue with similar lignin content after different pretreatment was selected. Enzymatic saccharification with different substrates was performed at 2%, 10%, and 20% (w/v) solid loading. After that, the lignin from different substrate was extracted and its structure was characterized. Furthermore, the adsorption capability of isolated lignin to cellulase and its effect on enzymatic hydrolysis were studied. The results showed that, as the substrate content increased from 2% (w/v) to 20% (w/v), the glucose yield after digestion of SH, AE, and AHP pretreated SCB reduced by 41.8%, 35.4%, and 28.7%, respectively. The inhibitory effect of different prepared lignin on the digestibility of Avicel was as follows, SH pretreated lignin (SHL) > cellulase enzymatic hydrolysis lignin (CEL) > AE pretreated lignin (AEL) > AHP pretreated lignin (AHPL), which exhibited positive correlation with its non-productive adsorption capability to cellulase. Lignin samples with low negative zeta potential, high hydrophobicity and high ration of syringyl (S) to guaiacyl (G) were unfavorable for the enzymatic saccharification of cellulose.

Surface morphology and cellulose structure of Napier grass pretreated with the ionic liquid 1-ethyl-3-methylimidazolium acetate combined with either water or dimethyl sulfoxide as a co-solvent under microwave irradiation

The combination of the ionic liquid (IL) 1-ethyl-3-methylimidazolium acetate with either water or dimethyl sulfoxide (DMSO) as a co-solvent was applied for the pretreatment of Napier grass under microwave irradiation at 150 °C for 1 h in order to enhance the subsequent cellulase enzymatic hydrolysis of the cellulose to sugars via increased enzyme accessibility. The influence of the IL/water and IL/DMSO (at different IL/DMSO (v/v) ratios) pretreatment on the structural changes of the Napier grass was examined using X-ray diffraction, Fourier-transform infrared spectroscopy, scanning electron microscopy, and thermogravimetric analyses. The transformation of cellulose I in the IL/DMSO pretreated Napier grass was clearly observed. The results indicated that cellulose structural changes occurred and a higher yield of fermentable sugars was obtained from the subsequent enzymatic hydrolysis for the pretreated Napier grass samples with a high crystallinity index.

Enhanced heterologous expression of Trichoderma reesei Cel5A/Cel6A in Pichia pastoris with extracellular co‐expression of Vitreoscilla hemoglobin

AbstractBACKGROUNDThe deficiency of family 5 endoglucanase (Cel5A) and family 6 cellobiohydrolase (Cel6A) has become a key limiting factor on cellulase enzymatic hydrolysis in bioprocessing of cellulosic biomass. To improve the production of Trichoderma reesei Cel5A / Cel6A, a Vitreoscilla hemoglobin (VHb) gene was tried to co‐express extracellularly for the first time with Cel5A / Cel6A in Pichia pastoris GS115.RESULTSNewly constructed recombinant of co‐expressing Cel5A / Cel6A extracellularly with VHb was consistent with the single expression at some key variables of culture condition, i.e. inoculum size, initial pH, culture temperature and methanol concentration. Comparing their single expression, the CMCase activity of co‐expressed Cel5A and Cel6A enzymes enhanced by 40% and 30%, respectively. With high‐cell‐density fed‐batch (HCDFB) fermentation, the co‐expressed Cel5A enzyme activity was 366.8 U mL‐1 with 4.3 g L‐1 protein content and the Cel6A enzyme activity reached 1.3 U mL‐1 with 2.23 g L‐1 protein content. The two co‐expressed enzyme activities were enhanced by 35% and 20%, respectively, compared with the single expression.CONCLUSIONVHb protein capable of binding oxygen can be successfully co‐expressed extracellularly with other target proteins. The co‐expression of VHb with recombinant Cel5A / Cel6A is efficient at improving oxygen‐limited condition and thus enzyme production in both shake‐flask and HCDFB fermentation. © 2017 Society of Chemical Industry

Facile, room-temperature pre-treatment of rice husks with tetrabutylphosphonium hydroxide: Enhanced enzymatic and acid hydrolysis yields

Aqueous solutions of tetrabutylphosphonium hydroxide have been evaluated as pretreatment media for rice husks, prior to sulphuric acid hydrolysis or cellulase enzymatic hydrolysis. Varying the water:tetrabutylphosphonium hydroxide ratio varied the rate of delignification, as well as silica, lignin and cellulose solubility. Pre-treatment with 60wt% hydroxide dissolved the rice husk and the regenerated material was thus heavily disrupted. Sulphuric acid hydrolysis of 60wt%-treated samples yielded the highest amount of glucose per gram of rice husk. Solutions with good lignin and silica solubility but only moderate to negligible cellulose solubility (10–40wt% hydroxide) were equally effective as pre-treatment media for both acid and enzymatic hydrolysis. However, pre-treatment with 60wt% hydroxide solutions was incompatible with downstream enzymatic hydrolysis. This was due to significant incorporation of phosphonium species in the regenerated biomass, which significantly inhibited the activity of the cellulase enzymes.

Cascade Enzymatic Hydrolysis Coupling with Ultrafine Grinding Pretreatment for Sugarcane Bagasse Saccharification

The biorefinery process for sugarcane bagasse saccharification generally requires significant accessibility of cellulose. We reported a novel method of cascade cellulase enzymatic hydrolysis coupling with ultrafine grinding pretreatment for sugarcane bagasse saccharification. Three enzymatic hydrolysis modes including single cellulase enzymatic hydrolysis, mixed cellulase enzymatic hydrolysis, and cascade cellulase enzymatic hydrolysis were compared. The changes on the functional group and surface morphology of bagasse during cascade cellulase enzymatic hydrolysis were also examined by FT-IR and SEM respectively. The results showed that cascade enzymatic hydrolysis was the most efficient way to enhance the sugarcane bagasse saccharification. More than 65% of reducing sugar yield with 90.1% of glucose selectivity was achieved at 50 °C, pH=4.8 for 72 h (1200 r/min) with cellulase I of 7.5 FPU/g substrate and cellulase II of 5 FPU/g substrate.

Effect of SC-CO 2 pretreatment in increasing rice straw biomass conversion

Rice straw, pretreated with supercritical carbon dioxide (SC-CO2) at 10–30 MPa and 40–110 °C for 15–45 min at a 1:1 g/g liquid: solid ratio, was analysed for glucose yield from enzymatic hydrolysis, main chemical composition, and supermolecular structure. Rice straw that was pretreated with SC-CO2 at 30 MPa and 110 °C for 30 min had a final glucose yield of 32.4 ± 0.5%, compared with 27.7 ± 0.5% for unpretreated straw, after enzymatic hydrolysis with a mixture of cellulose and β-glucosidase. Unpretreated and pretreated rice straw differed in chemical composition, with SC-CO2 pretreatment removing some non-lignocellulose material. Scanning electron microscopy showed that pretreated rice straw had extensive anomalous porosity and lamellar structures. Also, SC-CO2 pretreatment rendered fibres relatively fluffy and soft, which enhanced cellulase enzymatic hydrolysis.

Factors Affecting Wood Dissolution and Regeneration of Ionic Liquids

Three wood species, eucalyptus grandis (E. grandis), southern pine (S. pine), and Norway spruce thermomechanical pulp (N. spruce TMP) were pretreated by dissolution in the ionic liquid (IL) 1-allyl-3-methylimidazolium chloride ([AMIM]Cl). The wood was regenerated from the ionic liquid in high yield and the recycling of the ionic liquid was nearly quantitative. The lignin contents and the efficiencies of cellulase enzymatic hydrolyses of the regenerated wood were examined offering an understanding into the IL pretreatment efficiency. The components that remained within the recycled ILs were qualitatively characterized by 31P NMR spectroscopy. Wood density, pulverization intensity, and the nature of the regeneration nonsolvents were investigated as factors affecting the overall process. An increase in the wood density decreased the efficiency of the pretreatment, whereas extended pulverization periods decreased the yield of the regenerated wood after the IL pretreatment,with more glucose being released during the enzymatic hydrolysis. The yield of wood after IL pretreatment using water as the regeneration nonsolvent was found to be much higher than that of using methanol. As the reuse cycles of IL increased the wood regeneration yield increased, while certain wood components enriched within the recycled IL. The efficiency of cellulase enzymatic hydrolysis on the regenerated wood decreased with increasing reuse cycles of the IL.

Atividade da celulase e β-galactosidase no estudo da firmeza da polpa de mamões 'golden' e 'gran golden'

O objetivo desse trabalho foi avaliar a ação das enzimas celulase e β-galactosidase em relação à perda de firmeza dessas cultivares de mamões 'Gran Golden' e 'Golden' devido a relatos de uma perda de firmeza diferenciada entre as cvs. Os frutos foram armazenados a 25ºC e analisados diariamente quanto à firmeza da polpa e à atividade enzimática da celulase e β-galactosidase durante 8 dias. Os resultados de firmeza da polpa e atividade enzimática foram submetidos às análises de correlação e regressão. No 4º dia pós-colheita os mamões 'Golden' apresentaram firmeza média de 60,6 N e os 'Gran Golden' 31,1 N e a um aumento da atividade da celulase e da β-galactosidase. Os dados gerados neste trabalho sugerem que as enzimas celulase e β-galactosidase atuam diferentemente no processo de perda de firmeza dos frutos das cultivares Goldene Gran Golden. Aantecipaçãonaperdade firmezade 'Gran Golden' pode estar relacionada com a maior atividade dessas enzimas.

Effects of chemical and enzymatic treatments on the hydrolysis of swine wastewater

Slow degradation of swine wastewater mainly due to the particulate and recalcitrant organic matters is a main disadvantage of anaerobic digestion leading to very long solids retention time. Therefore, to accelerate hydrolysis known as the rate-limiting step of the overall digestion process, chemical treatment processes under various acidic and alkaline conditions as well as enzymatic treatment processes using cellulase and protease enzymes were tested for the hydrolysis of the swine wastewater. The effectiveness of various treatment processes was compared mainly by means of an increment of soluble organics in the treated swine wastewater. Among various treatment processes tested in this study, cellulase enzymatic treatment resulted in the most efficient hydrolysis of the swine wastewater. For the cellulase enzymatic hydrolysis, the observed hydrolytic constant value was 0.42 d(-1) and 26.6% of soluble organics in the swine wastewater increased within 12 hr. Compared to untreated swine wastewater, pre-treated swine wastewater by cellulase enzymatic process showed 10.7% higher anaerobic digestibility at the end of 20 d incubation and 29% higher initial methane production rate. These results further confirmed the transformation of particulate and recalcitrant organic compounds in the swine wastewater into soluble and relatively easily biodegradable organic products by the cellulase enzymatic hydrolysis.

Dissolution of Wood in Ionic Liquids

The present paper demonstrates that both hardwoods and softwoods are readily soluble in various imidazolium-based ionic liquids (ILs) under gentle conditions. More specifically, a variety of ionic liquids can only partially dissolve wood chips, whereas ionic liquids such as 1-butyl-3-methylimidazolium chloride and 1-allyl-3-methylimidazolium chloride have good solvating power for Norway spruce sawdust and Norway spruce and Southern pine thermomechanical pulp (TMP) fibers. Despite the fact that the obtained solutions were not fully clear, these ionic liquids provided solutions which permitted the complete acetylation of the wood. Alternatively, transparent amber solutions of wood could be obtained when the dissolution of the same lignocellulosic samples was attempted in 1-benzyl-3-methylimidazolium chloride. This realization was based on a designed augmented interaction of the aromatic character of the cation of the ionic liquid with the lignin in the wood. After dissolution, wood can be regenerated as an amorphous mixture of its original components. The cellulose of the regenerated wood can be efficiently digested to glucose by a cellulase enzymatic hydrolysis treatment. Furthermore, completely acetylated wood was found to be readily soluble in chloroform, allowing, for the first time, detailed proton nuclear magnetic resonance (NMR) spectra and NMR diffusion measurements to be made. It was thus demonstrated that the dissolution of wood in ionic liquids now offers a variety of new possibilities for its structural and macromolecular characterization, without the prior isolation of its individual components. Furthermore, considering the relatively wide solubility and compatibility of ionic liquids with many organic or inorganic functional chemicals or polymers, it is envisaged that this research could create a variety of new strategies for converting abundant woody biomass to valuable biofuels, chemicals, and novel functional composite biomaterials.

Surface and pore structure modification of cellulose fibers through cellulase treatment

AbstractThe surface and pore structure of cellulose fibers have a significant impact on the properties and performance in applications. Cellulase enzymatic hydrolysis of cellulose fibers can result in changes to the surface and pore structure, thus providing a useful tool for fiber modification. This research characterizes these changes using various test methods such as fiber dimension, water retention value (WRV), hard‐to‐remove (HR) water content, freezing and nonfreezing bound water content, polymer adsorption, and crystallinity index. For a high‐dosage cellulase treatment (600 U/g dry solid), the fiber length was significantly decreased and the fibers were “cut” in the cross direction, not in the axial direction. The swelling capacities as measured by the WRV and HR water content increased for the high‐dosage treatment. Three independent measurements (nonfreezing bound water, polymer adsorption, and crystallinity index) are in good agreement with the statement that the amorphous regions of cellulose fibers are a more readily available substrate relative to crystalline regions. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 103: 3833–3839, 2007