Cellulase Hydrolysis Research Articles

Cloning of AA9 Polysaccharide Monooxygenase gene AN3860 into pEX2B for expression in Aspergillus oryzae

Polysaccharide monooxygenases (PMOs) catalyze oxidative degradation of recalcitrant carbohydrate chains in cellulose, starch, and chitin. In biofuel industry, the conversion of rich lignocellulose source to fermentable sugars by hydrolytic cellulases can be synergistically boosted by cellulose-active PMOs (AA9 PMOs) found in a vast number of fungi that grow on biomass. Aspergillus nidulans, a filamentous fungus, possess a dozen of PMO-encoding genes, but only the AN3860 is expressed at a high level when cultured with wheat straw as the sole carbon source. Bioinformatic analysis indicates that AN3860 belongs to type 3 AA9 PMO subfamily that is capable of hydroxylating both C1 and C4 of the glycosidic linkages. Therefore, AN3860 may be a potential enzyme to improve cellulose hydrolysis efficiency, which has not been characterized. In this study, we describe the AN3860 cloning into Aspergillus oryzae AUT1-PlD. To facilitate the purification of AN3860, we added a CBM20 tag to its C-terminal. The recombinant vector was designed and constructed successfully. Simultaneously, we have obtained the clone of A. oryzae carrying the target gene by the ATMT method. Further expression optimization and characterization of AN3860 by both activity assays and spectroscopic techniques are underway.

Peroxidase application reduces microcrystalline cellulose recalcitrance towards cellulase hydrolysis in model cellulose substrates and rooibos biomass

We have identified a HRP enzyme with microcrystalline cellulose activity, which has not yet been explored. The current study investigated the effect of HRP pretreatment on the microcrystalline cellulose substrates, Avicel and filter paper. SEM findings showed that HRP pretreatment catalysed the para-microcrystalline regions of Avicel, cracking and opening the pores on the surface. On filter paper, HRP removed the para-microcrystalline regions exposing fibres. Crystallinity index (CrI) analysis confirmed that HRP increased the CrI of Avicel from 49 % to 54.19 % and filter paper from 42 % to 47 %. The cellulose crystallite sizes increased from 45 to 47 nm at the 002 lattices in Avicel, suggesting a reduction of crystalline cellulose. In addition, endoglucanase displayed 1.15-fold increased activity on HRP-pretreated Avicel, confirming reduced crystalline cellulose. These findings showed that HRP pretreatment changed the structural and chemical properties of Avicel, i.e., loosening crystalline cellulose to make the substrate accessible to enzymes during hydrolysis. Finally, these findings were supported by rooibos microcrystalline cellulose modification post-HRP pretreatment, resulting in a 95 % yield of soluble sugars at 25 mg enzyme cocktail/g biomass.

Kinetic understanding of fiber surface lignin effects on cellulase adsorption and hydrolysis

Cellulase hydrolysis is strongly affected by adsorption, which is inhibited by the non-productive binding between cellulase and lignin. However, lignin-bonded cellulase has also been found to be active in hydrolysis. Thus, in this work, lignin effects on cellulase adsorption and hydrolysis were studied using fibers with various surface lignin contents and structures based on kinetics. The surface lignin was changed by mechanical refining. Cellulase adsorbed on lignin showed reactivity to hydrolyze cellulose nearby according to the kinetic results, which is, however, much weaker compared with that by cellulose-bonded cellulase. With the decrease of surface lignin, this reactivity is also decreasing. Both cellulase adsorption and hydrolysis were increasing at first and then decreasing with the decrease of surface lignin as well as the lignin re-adsorption. It was concluded that lignin plays an important role in cellulase adsorption and non-productive binding occurs more easily between cellulase and the re-adsorbed lignin, as well as the residual lignin. At a relatively high surface lignin level, lignin structural and distribution changes were found to have a better effect on cellulase hydrolysis compared with lignin removal.

Dual Enzymolysis Assisted by Acrylate or Phosphate Grafting: Influences on the Structural and Functional Properties of Jujube Residue Dietary Fiber.

Jujube residue is an abundant and low-cost dietary fiber resource, but its relatively lower hydration and functional properties limit its utilization as an ingredient of functional food. Thus, cellulase and hemicellulase hydrolysis, enzymatic hydrolysis assisted by phosphate grafting (EPG), and enzymatic hydrolysis assisted by acrylate grafting (EAG) were used to improve the functional properties of jujube residue dietary fiber (JRDF) in this study. The results evidenced that these modifications all increased the porosity of the microstructure of JRDF and increased the soluble fiber content, surface area, and hydration properties, but reduced its brightness (p < 0.05). Moreover, JRDF modified by enzymolysis combined with acrylate grafting offered the highest extractable polyphenol content, oil, sodium cholate, and nitrite ion sorption abilities. Meanwhile, JRDF modified via enzymolysis assisted by phosphate grafting showed the highest soluble fiber content (23.53 g∙100 g-1), water-retention ability (12.84 g∙g-1), viscosity (9.37 cP), water-swelling volume (10.80 mL∙g-1), and sorption ability of copper (II) and lead (II) ions. Alternatively, JRDF modified with cellulase hydrolysis alone exhibited the highest glucose adsorption capacity (21.9 g∙100 g-1) at pH 7.0. These results indicate that EPG is an effective way to improve the hypolipidemic effects of JRDF, while EAG is a good choice to enhance its hydration and hypoglycemic properties.

Integrating ball milling assisted enzymatic hydrolysis of bamboo cellulose for controllable production of xylo-oligosaccharides, monosaccharides and cellulose nanofibrils

Green bioconversion technology has been regarded as a fundamental solution to energy challenges and environmental issues. During bioconversion, a combination of mechanical and enzymatic treatment on lignocellulosic biomass can not only conformity with environmental concepts but also enable efficient conversion. In this study, a ball milling assisted enzymatic treatment was applied for full utilization of bamboo cellulose. In addition to the production of soluble saccharides, the residues that remained after enzymatic hydrolysis were collected for producing high-quality nanocellulose. Ball milling efficiently broke the hydrogen bonding interactions between glucan chains thus promoting the following xylanase and cellulase conversion. The highest Xylo-oligosaccharides (XOS) yield reached 21.6%. Meanwhile, xylanase facilitated cellulase hydrolysis by separation of hemicellulose on cellulose. With a low commercial cellulase loading of 5 FPU/g glucan, a maximum yield of 76.6% glucose and 53.3% xylose was reached. Finally, nanocellulose was extracted from the enzymatic hydrolyzate residue with a minimum width of 3.5 nm and an average size of 294.8 nm. The present study proposed an efficient combination of mechanical treatment assisted enzymatic digestion, which is not only environmentally friendly but also allows for complete bioconversion of bamboo cellulose.

Stabilization of emulsions prepared by ball milling and cellulase treated pomelo peel insoluble dietary fiber: Integrity of porous fiber structure dominates the stability

Emulsion gels from the pomelo peel insoluble dietary fiber (PIDF) were developed. The emulsification potentials of PIDFs subjected to various degrees of ball milling (M−PIDFs), cellulase hydrolysis (C-PIDF), and cellulase hydrolysis followed by ball milling (CM-PIDFs) were evaluated. Emulsions prepared by M−PIDFs for different lengths of ball milling time exhibited similar stability characteristics, confirming that M−PIDF emulsion stability might be determined by the three-dimensional structure formed by M−PIDF stacking and oil droplet capture. C-PIDF had characteristics resembling those of Pickering particles. CM-PIDF emulsions got destabilized with ball milling time prolongation. Interface tension and particle size of C/CM-PIDF decreased gradually during ball milling. Rheological and fluorescence microscopy results revealed that the intact internal crosslinking structure frameworks were disrupted in CM-PIDF emulsions. Therefore, intact fiber-based networks, rather than small particle size or low interfacial tension, determine the stability of PIDF emulsions. This study deepens the understanding of PIDF as a clean emulsifier.

Antitumor Activity of Carboxymethyl Pachymaran with Different Molecular Weights Based on Immunomodulatory and Gut Microbiota.

Carboxymethyl pachymaran (CMP) was treated via high-temperature and cellulase hydrolysis to obtain HTCMP, HTEC-24, and HTEC-48. The chemical structure and in vivo antitumor activities of the four types of CMPs were investigated. Compared with CMP (787.9 kDa), the molecular weights of HTCMP, HTEC-24, and HTEC-48 were decreased to 429.8, 129.9, and 68.6 kDa, respectively. The viscosities and particle sizes of the CMPs could also decrease with the decline in the molecular weights. All the CMPs showed antitumor abilities, but HTEC-24 exhibited the best activity. In the animal study, when curing the spleen and thymus, CMPs displayed immunomodulatory effects by increasing the secretion of IFN-γ and IL2 in mice. The CMPs also exerted an antitumor ability by regulating the gut microbiota in tumor-bearing mice. Our results established a foundation to develop an antitumor drug with CMP.

Enhancement of emulsion stability and functional properties of hemp protein‐based dairy alternative by different treatments after cellulase hydrolysis

SummaryHemp milk is a plant‐based beverage emerging as a sustainable dairy alternative and it is essential to improve the emulsification properties and stability of hemp protein for increasing acceptability. This study demonstrates the preparation of protein‐rich hemp milk with enhanced emulsion stability and functional properties through cellulase hydrolysis of fibre components in hemp protein. The emulsions were prepared using the hemp dispersions obtained after cellulase treatment followed by mechanical treatment (ME) and in combination with either enzymatic (trypsin) (EE) or mild chemical (pH‐shift) treatment (PE). The influences of these treatments on the particle size distribution, zeta potential, flow behaviour, microstructure, flocculation, and coalescence properties of the emulsions are investigated. The emulsions (EE/PE) prepared with dispersions processed through tryptic or pH shift treatments showed improved stability as indicated by reduced flocculation and coalescence indices (8.9% and 10.8%, for EE and 11.8% and 13.2% for PE) as compared to only mechanically treated emulsion (ME) (37.5% and 15.3%). Furthermore, the enhanced stability of EE and PE samples is attributed to a reduction in particle size and enhanced zeta potential (EE: 45.1 mV &gt; PE: 38.7 mV &gt; ME: 34.1 mV).

Heterologous expression and characterization of a MoAA16 polysaccharide monooxygenase from the rice blast fungus Magnaporthe oryzae

BackgroundCellulose is an organic carbon source that can be used as a sustainable alternative for energy, materials, and chemicals. However, the substantial challenge of converting it into soluble sugars remains a major obstacle in its use as a biofuel and chemical feedstock. A new class of enzymes knowns as copper-dependent polysaccharide monooxygenases (PMOs) or lytic polysaccharide monooxygenases (LPMOs) can break down polysaccharides such as cellulose, chitin, and starch through oxidation. This process enhances the efficiency of cellulose degradation by cellulase. ResultsThe genome of the fungus Magnaporthe oryzae, the causal agent of rice blast disease, contains the MGG_00245 gene, which encodes a putative PMO referred to as MoAA16. MoAA16 has been found to be highly expressed in planta during the early stages of fungal infection. The gene was optimized for heterologous expression in Pichia pastoris, and its oxidative cleavage activity on cellulose was characterized by analyzing soluble oligosaccharide products using highperformance anion exchange chromatography (HPAEC-PAD). The reaction catalyzed by MoAA16 requires 2 electrons from an electron donor, such as ascorbic acid, and aerobic conditions. It primarily produces Glc1 to Glc4 oligosaccharides, as well as oxidized cellobionic and cellotrionic acids. MoAA16 has been observed to enhance cellulase hydrolysis on phosphoric acid swollen cellulose (PASC) substrate, resulting in the production of more monosaccharide products. ConclusionsOur findings reveal the successful heterologous expression of MoAA16 in P. pastoris and its cellulose-active PMO properties. These results highlight the potential of MoAA16 as a promising candidate for applications in biofuel production and chemical synthesis.How to cite: Nguyen HM, Le LQ, Sella L, et al. Heterologous expression and characterization of a MoAA16 polysaccharide monooxygenase from the rice blast fungus Magnaporthe oryzae. Electron J Biotechnol 2023. https://doi.org/10.1016/j.ejbt.2023.06.002.

Combined iron (III) chloride/sodium citrate with enzymatic hydrolysis for xylo-oligosaccharides and monosaccharides production from poplar

Currently, the production of xylo-oligosaccharides (XOS) from lignocelluloses by chelating system hydrolysis has not been investigated. Herein, iron (III) chloride/sodium citrate (IC/SC) chelating system hydrolysis and xylanase hydrolysis were used to produce XOS from poplar. Then, the delignification of IC/SC-hydrolyzed poplar was performed by p-toluenesulfonic acid (p-TsOH) pretreatment to increase the accessibility of cellulase. The results demonstrated that 42.3% of XOS with an extremely low by-product (xylose/XOS = 0.11) was produced from poplar by 50 mM IC/SC hydrolysis (molar ratio of 1:1, 170 °C, 60 min) and xylanase hydrolysis. The second step IC/SC hydrolysis and xylanase hydrolysis of poplar increased the yield of XOS to 51.3%. Finally, the glucose yield of p-TsOH-pretreated poplar (60% p-TsOH, 70 °C, 30 min) was greatly increased from 37.5% to 83.8% by cellulase hydrolysis with Tween 80 addition. The novel strategy proposed in this work was feasible for XOS and monosaccharides production from poplar.

Brassica juncea leaf cuticle contains xylose and mannose (xylomannan) which inhibit ice recrystallization on the leaf surface.

Conjugated sugars showed antifreeze activity in the cuticle by ice recrystallization inhibition rather than thermal hysteresis, enhancing freezing capacity at the surface of B. juncea leaves. Antifreeze biomolecules play a crucial role in mitigating the physical damage from frost by controlling extracellular ice crystal growth in plants. Antifreeze proteins (AFPs) are reported from the apoplast of different plants. Interestingly, there is no report about antifreeze properties of the cuticle. Here, we report the potential antifreeze activity in the Brassica juncea (BJ) leaf cuticle. Nano LC-MS/MS analysis of a cuticle protein enriched fraction (CPE) predicted over 30 putative AFPs using CryoProtect server and literature survey. Ice crystal morphology (ICM) and ice recrystallization inhibition (IRI) analysis of ABC supernatant showed heat and pronase-resistant, non-protein antifreeze activities as well as hexagonal ice crystals with TH of 0.17°C and IRI 46%. The ZipTip processed ABC supernatant (without peptides) had no effect on TH activity, confirming a non-protein antifreeze molecule contributing to activity. To understand the origin and to confirm the source of antifreeze activity, cuticular membranes were isolated by pectinase and cellulase hydrolysis. FTIR analysis of the intact cuticle showed xylose, mannose, cellulose, and glucose. Xylanase and cellulase treatments of the ZipTip processed ABC supernatant led to an increase in sugar content and 50% loss in antifreeze activity. UV spectroscopy and NMR analysis supported the finding of FTIR and enzyme hydrolysis suggesting the contribution of xylose and mannose to antifreeze activity. By TLC analysis, conjugated sugars were found in the cuticle. This work has opened up a new research area where the antifreeze capacity needs to be established with regard to complete characterization and mechanism of action of the antifreeze carbohydrates (conjugated sugars) on the leaf surface.

Dietary fiber extraction from citrus peel pomace: Yield optimization and evaluation of its functionality, rheological behavior, and microstructure properties.

Citrus fruits were widely used in processing and production, generating a large amount of peel pomace and a low utilization rate, resulting in substantial economic losses and environmental risks. It was important to extract compounds from citrus peel pomaces and find suitable preparation methods to improve their yield and physicochemical properties. Grapefruit peel pomace (GP) and navel orange peel pomace (OP) were used as raw materials in this study to prepare green and edible soluble dietary fiber (SDF) and insoluble dietary fiber (IDF). Analysis was done on the effects of solid-liquid ratio, cellulase hydrolysis time, cellulase dosage, and ultrasonic time on dietary fiber (DF) yield. To obtain the best DF preparation conditions, we used range analysis, variance analysis, and orthogonal experimental design. We also analyzed the structural, physicochemical, and rheological characteristics of SDF and IDF. According to the study's findings, SDF and IDF showed a loose and expansive structure with reduced particle size, higher specific surface area, and noticeably better physical and chemical properties after treating GP and OP with ultrasound-assisted composite enzyme method. Both SDF solution and IDF suspension were discovered through rheological analysis to be non-Newtonian pseudoplastic fluids, which was advantageous for expanding their applications in the field of food packaging. In conclusion, DF prepared using the ultrasound-assisted composite enzyme method was an excellent source of edible packaging materials, offering a benchmark for the recycling of other citrus peel wastes and ultimately paving the way for new methods of recycling citrus waste.

Synergistic study of cellulose enzymatic factors and cellulase adsorption properties under bioinformatics

Abstract Cellulose enzymatic saccharification is a key link in converting lignocellulose to fuel ethanol via a sugar platform. In this paper, the maximum information coefficient (MIC) algorithm is proposed to detect linear correlations and nonlinear correlations between bioinformatics variables from the application of bioinformatics. Meanwhile, to address the problems of excessive grid division and low statistical efficacy of MIC algorithm, BackMIC, an optimization algorithm using quiz control number of segments, is proposed to terminate the network search using the chi-square test. Then, cellulase hydrolysis and adsorption experiments were designed and performed, and the experimental data were analyzed based on the BackMIC algorithm. The optimum activity temperature of different cellulases was around 50°C, while the hydrolysis rate of cellulases of different treated raw materials increased and then decreased with the increase of temperature, and basically, the maximum was achieved in the temperature range of 50~55°C. In the pH test, the optimum temperature of cellulase hydrolysis was concentrated at 4.4~4.5. In the cellulase adsorption experiments, the enzymatic activity of steam-popped wheatgrass was adsorbed in the following order: FPA&gt; C1&gt; CMCase&gt; β-Case. The study of cellulase hydrolysis based on bioinformatics reveals the synergistic relationship between cellulase hydrolysis and adsorbed properties, which provides a theoretical basis for reducing the ineffective adsorption of cellulose and the development of enzymatic strengthening technology. The theoretical basis for reducing the ineffective adsorption of cellulose and the development of enzymatic enhancement technology can help reduce the cost of lignocellulose enzymatic digestion.

Delignification of switchgrass for xylo-oligosaccharides production using sorbic acid hydrolysis

Organic acid as a class of hydrolysis catalysts shows great potential in the production of xylo-oligosaccharides (XOS) from lignocelluloses. However, sorbic acid (SA) hydrolysis for XOS production from lignocellulose has not been reported and it was still unclear how lignin removal affected XOS production. Herein, two factors affecting the XOS production from switchgrass by SA hydrolysis were explored: hydrolysis severity factor (Log R0) and lignin removal. Benefiting from the lignin removal (58.4%) in switchgrass, a desired XOS yield of 50.8% with low by-products was obtained from delignified switchgrass by 3% SA hydrolysis at Log R0 = 3.84. Under these conditions, 92.1% of glucose was obtained by cellulase hydrolysis with Tween 80 addition. From a mass balance perspective, 10.3 g of XOS and 23.7 g of glucose could be produced from 100 g switchgrass. This work proposed a novel strategy for XOS and monosaccharides production from delignified switchgrass.

Improved enzymatic saccharification of bulrush via an efficient combination pretreatment

Glycerol (Gly) was selected as hydrogen-bond-donor for preparing ChCl-based DES (ChCl:Gly), and the mixture of ChCl:Gly (20 wt%) and NaOH (4 wt%) was utilized for combination pretreatment of bulrush at 100 °C for 60 min (severity factor LogRo = 1.78). The effects of DES pretreatment on the chemical composition, microstructure, crystal structure, and cellulase hydrolysis were explored. NaOH-ChCl:Gly could remove lignin (80.1%) and xylan (66.8%), and the enzymatic digestibility of cellulose reached 87.9%. The accessibility of bulrush was apparently increased to 645.2 mg/g after NaOH-ChCl:Gly pretreatment. The hydrophobicity and lignin surface area were reduced to 1.56 L/g and 417 m2/g, respectively. The crystallinity of cellulose was increased from 20.8% to 55.6%, and great changes in surface morphology were observed, which explained the improvement of enzymatic hydrolysis efficiency. Overall, DES combined with alkali treatment could effectively promote the removal of lignin and xylan in bulrush, thus the relative saccharification activity was greatly affected.

A sequential combination of advanced oxidation and enzymatic hydrolysis reduces the enzymatic dosage for lignocellulose degradation

High cellulase cost is a significant challenge to reducing the production cost of the lignocellulosic ethanol industry. Fenton reagents can oxidize and degrade organic matter through the advanced oxidation process. This study combined the Fenton reagent with cellulase to improve lignocellulose degradation efficiency and reduce the amount of enzyme dosage. Fenton reaction studies showed that the experimental group of 1000 mmol/L H2O2 + 10 mmol/L FeSO4 was the most suitable condition for coupled cellulase hydrolysis and excessive reagents might cause a decrease in the promotion effect. The solid loading analysis evidenced that the lignocellulose degradation can be facilitated by the Fenton reagent in the 15%–30% solids range. Treated with the Fenton reagent, the release of free water and the increase of specific surface area in the degradation process were enhanced. It's indicated that the amount of cellulase decreased by 76.2% under the combination of advanced oxidation and enzymatic hydrolysis. The techno-economic analysis of 300,000 t/a lignocellulosic ethanol showed that the combination reduced 26.96% annual costs. These results confirmed that the method of combining advanced oxidation with enzymatic hydrolysis is an effective way to improve the economics of industrial ethanol.

A feruloyl esterase/cellulase integrated biological system for high-efficiency and toxic-chemical free isolation of tobacco based cellulose nanofibers

Tobacco based cellulose nanofiber (TCNF) is a novel nanocellulose that has recently been used to replace undesirable wood pulp fibers in the preparation of reconstructed tobacco sheets (RTS). However, given the strict requirements for controlling toxic chemical content in tobacco products, there is a global interest in developing a green, efficient, and toxic-chemical free approach to isolate TCNF from tobacco stem as a bioresource. In this study, we propose a creative and environmentally friendly method to efficiently and safely isolate TCNF from tobacco stem pulp, which involves integrated biological pretreatment followed by a facile mechanical defibrillation process. Feruloyl esterase is used to pretreat the stem pulp by disrupting the ether and ester bonds between lignin and polysaccharide carbohydrates within the fiber wall, which effectively facilitates cellulase hydrolysis and swelling of the stem pulp fiber, as well as the following mechanical shearing treatment for TCNF isolation. The results demonstrate that TCNF obtained by the comprehensive feruloyl esterase/cellulase/mechanical process exhibit uniform and well-dispersed nanofiber morphology, higher crystallinity, and stronger mechanical properties than those of the control. The addition of 0.5 % TCNF can replace wood pulp by 18 wt% ~ 25 wt% in the production of RTS samples while maintaining their reasonable strength properties.

An integrated process for the coproduction of xylooligosaccharides and Bacillus subtilis biomass from poplar

Xylooligosaccharides (XOS) and Bacillus subtilis biomass, also regarded as essential prebiotics, have a wide application prospect in feed industry. However, very few studies have reported the coproduction of XOS and Bacillus subtilis from lignocellulosic materials. A XOS yield of 28.8% could be produced from poplar through acetic acid (AC) hydrolysis at 170 °C for 30 min. After the delignification of the AC-hydrolyzed poplar by hydrogen peroxide/AC (HPAC) solution, 101.7 g/L glucose and 20.7 g/L xylose were released from the HPAC-pretreated poplar by cellulase hydrolysis, which was used as a carbon source to culture B. subtilis YS01. The results showed that after 36 h of culture, the viable count of the strain was 6.2 × 108 CFU/mL, which was close to the viable count (7.1 × 108 CFU/mL) of the strain cultured using glucose solution as the sole carbon source. The strain B. subtilis YS01 completely metabolized the glucose and xylose. Overall, the results of the study proposed a promising strategy to efficiently produce XOS and B. subtilis YS01 from poplar, which could be used in feed industry.

A new strategy for cellulases application in high temperature industrial scenarios with syringic acid assisting

AbstractIn the process of bioethanol production, more stable and active cellulase in high temperature condition is required. In this study, syringic acid was applied in cellulase hydrolysis system. At 70°C, TvEG3 activity increased 201.36%, CtBglA activity decreased 72.79% by syringic acid. With syringic acid assisting, TvEG3 thermostability was improved, CtBglA thermostability was reduced. Syringic acid scarcely affected CtCBH. In hydrolysis system with the cellulases containing TvEG3, CtCBH, and CtBglA, the reducing sugar yield improved by 28.37% with syringic acid assisting. With the molecular dynamic simulation in syringic acid system, the backbone root‐mean‐square deviation (RMSD) and the residue root‐mean‐square fluctuation (RMSF) of TvEG3, CtCBH reduced, while the RMSD and RMSF of CtBglA increased. The reduction in the number of secondary structures, especially α‐helix, caused the structure of CtBglA in the presence of syringic acid to collapse at high temperature. More secondary structures in TvEG3 and more α‐helix in CtCBH in the presence of syringic acid make them more stable at high temperatures. These means syringic acid can stabilize TvEG3 and CtCBH structure, destabilize CtBglA structure at high temperature. In summary, this study not only provides insight into cellulase hydrolysis at high temperature with syringic acid assisting but also demonstrates the promoting mechanism of syringic acid.

Glucose Conversion for Biobutanol Production from Fresh Chlorella sorokiniana via Direct Enzymatic Hydrolysis

Microalgae, which accumulate considerable carbohydrates, are a potential source of glucose for biofuel fermentation. In this study, we investigated the enzymatic hydrolysis efficiency of wet microalgal biomass compared with freeze-dried and oven-dried biomasses, both with and without an acidic pretreatment. With the dilute sulfuric acid pretreatment followed by amy (α-amylase and amyloglucosidase) and cellulase hydrolysis, approximately 95.4% of the glucose was recovered; however, 88.5% was released by the pretreatment with 2% (w/v) sulfuric acid, which indicates the potential of the acids for direct saccharification process. There were no considerable differences in the glucose yields among the three kinds of materials. In the direct amy hydrolysis without any pretreatment, a 78.7% glucose yield was obtained, and the addition of cellulase had no significant effect on the hydrolysis to glucose. Compared with the oven-dried biomass, the wet biomass produced a substantially higher glucose yield, which is possibly because the cross-linked cells of the oven-dried biomass prevented the accessibility of the enzymes. According to the results, the fresh microalgal biomass without cell disruption can be directly used for enzymatic hydrolysis to produce glucose. The enzymatic hydrolysate of the wet microalgal biomass was successfully used for acetone–butanol–ethanol (ABE) fermentation, which produced 7.2 g/L of ABE, indicating the application potential of wet microalgae in the bioalcohol fuel fermentation process.