Cellulase Hydrolysis Research Articles

Pretreatment with γ-Valerolactone/[Mmim]DMP and Enzymatic Hydrolysis on Corncob and Its Application in Immobilized Butyric Acid Fermentation.

Corncob is a widely available raw material with high carbohydrate and low lignin content. To improve corncob conversion to the fermentable sugars, a novel method encompassing pretreatment using the γ-valerolactone (GVL)/1-methyl-3-methylimidazolium dimethylphosphite ([Mmim]DMP) system integrated with cellulase hydrolysis was developed and optimized. It is confirmed that lignin was extracted efficiently after combined pretreatment and that the subsequent enzymatic saccharification efficiency could be significantly enhanced, resulting in the yield of 94.9% glucose from cellulose and 53.3% xylose from xylan, respectively. Furthermore, the above fermentable sugars were used as carbon source for Clostridium tyrobutyricum immobilized in macroporous Ca-alginate-lignin beads with the extracted lignin as the active ingredient to evaluate the fermentability of butyric acid. The results showed that high butyrate productivity of 0.47 g/L/h and yield of 0.45 g/g were obtained after 10 repeated batches of fermentation, demonstrating an effective process for the production of butyric acid from abundant corncob waste-biomass.

Research on the Cellulase Hydrolysis of Colocasia Antiquorumin in Producing Ethyl Alcohol

The cellulase hydrolysis experiment in the process of the ethyl alcohol generated by colocasia antiquorum was studied in this paper.Adopting the single factor variable method, three influencing factors (the amount of cellulase, hydrolysis temperature and hydrolysis time) were selected to carry out enzymatic saccharification sugar production tests of pretreated colocasia antiquorums. The results demonstrate that the sugar yield amounts to the maximum (165mg/g) when the enzyme dosage is 0.04g/g, the enzymatic hydrolysis temperature is 45°C, and the reaction time is 48h.

Xylo-oligosaccharides enriched yeast protein feed production from reed sawdust

The aim of this study was to convert the cellulose and hemicellulose, in reed sawdust from the pulp mills, into yeast protein and xylo-oligosaccharide, then functionalize xylo-oligosaccharide as yeast feed. Both synchronous saccharification and fermentation and separate hydrolysis and fermentation of cellulase and Candida utilis were investigated to produce protein feed. By optimizing the fermentation conditions, 6.1 g/L of protein with 76.1% (7.1 g/L) xylo-oligosaccharide as the sugar was obtained. The final glucan and xylan utilization efficiencies in reed sawdust were 85.45% and 91.03%, respectively. Xylo-oligosaccharide enriched yeast protein feed from reed sawdust was thus realized by pretreatment, enzymatic hydrolysis and synchronous saccharification and fermentation.

Enzymatic hydrolysis of cellulosic materials using synthetic mixtures of purified cellulases bioengineered at N-glycosylation sites.

Mutant forms of recombinant endoglucanase II (EG II, N194A), cellobiohydrolase I (CBH I, N45A) and cellobiohydrolase II (CBH II, N219A) from Penicillium verruculosum with enhanced cellulase activities, achieved by engineering of enzyme N-glycosylation sites in our previous studies, were used as components of the binary and ternary mixtures of cellulases in hydrolysis of Avicel and milled aspen wood. Using the engineered forms of the enzymes at a dosage of 10mg/g substrate resulted in significant boosting of the glucose release from cellulose in the presence of excess β-glucosidase relative to the performance of the corresponding wild-type mixtures at the same loading. The boosting effects reached 11-40% depending on the reaction time and substrate type. In hydrolysis of both cellulosic substrates by the binary mixtures of cellulases, all the enzyme pairs exhibited synergism. The magnitude of the synergistic effects (Ks) did not depend notably upon the induced mutations in the enzymes, and they were in the range of 1.3-1.8 for the combinations of EG II with CBH I (or CBH II), and 2.3-2.9 for the CBH I-CBH II pair. The results of this study should provide a basis for the development of a more effective fungal strain capable of producing cellulase cocktails with enhanced hydrolytic performance against lignocellulosic materials.

Biochemical and functional characterization of a novel thermoacidophilic, heat and halo-ionic liquids tolerant endo-β-1,4-glucanase from saline-alkaline lake soil microbial metagenomic DNA

The lignocellulosic biomass usually need pre-treatment with acid at high temperature or solved in ionic liquid (IL) before cellulases hydrolysis. Thus, thermoacidophilic, thermostable and IL-tolerant cellulases are greatly desired, but rarely reported. In this study, a novel endo-β-1,4-glucanase gene nmGH45 was directly cloned from saline-alkaline lake soil microbial metagenomic DNA and expressed in Pichia pastoris. Recombinant NMgh45 was active and stable at pH 3.0–9.0, with maximum activity at pH 4.5 and >80% residual activities at pH 3.0–11.0. It exhibited maximal activity at 60–70 °C and remaining >90% and 68% residual activities at 80 °C for 2 h and 90 °C for 1 h. Besides, NMgh45 retained >88% relative activities in 4 M NaCl, and was stable for 24 h. Significantly, it was highly IL-tolerant, remaining 92.8% and 43.8% residual activities in 10% and 20% 1-butyl-3-methylimidazolium chloride. Compared with other non-halo-tolerant counterparts, NMgh45 was enriched with acidic amino acids on the protein surface, and the conformation of NMgh45 was stable at high salinity condition based on molecular dynamic simulation. Finally, the productivity of recombinant NMgh45 was optimized at multi-levels. The thermoacidphilic and acid-alkaline-halo-tolerant properties make NMgh45 a promising enzyme for basic research and industrial applications.

Defining functional diversity for lignocellulose degradation in a microbial community using multi-omics studies

BackgroundLignocellulose is one of the most abundant forms of fixed carbon in the biosphere. Current industrial approaches to the degradation of lignocellulose employ enzyme mixtures, usually from a single fungal species, which are only effective in hydrolyzing polysaccharides following biomass pre-treatments. While the enzymatic mechanisms of lignocellulose degradation have been characterized in detail in individual microbial species, the microbial communities that efficiently breakdown plant materials in nature are species rich and secrete a myriad of enzymes to perform “community-level” metabolism of lignocellulose. Single-species approaches are, therefore, likely to miss important aspects of lignocellulose degradation that will be central to optimizing commercial processes.ResultsHere, we investigated the microbial degradation of wheat straw in liquid cultures that had been inoculated with wheat straw compost. Samples taken at selected time points were subjected to multi-omics analysis with the aim of identifying new microbial mechanisms for lignocellulose degradation that could be applied in industrial pre-treatment of feedstocks. Phylogenetic composition of the community, based on sequenced bacterial and eukaryotic ribosomal genes, showed a gradual decrease in complexity and diversity over time due to microbial enrichment. Taxonomic affiliation of bacterial species showed dominance of Bacteroidetes and Proteobacteria and high relative abundance of genera Asticcacaulis, Leadbetterella and Truepera. The eukaryotic members of the community were enriched in peritrich ciliates from genus Telotrochidium that thrived in the liquid cultures compared to fungal species that were present in low abundance. A targeted metasecretome approach combined with metatranscriptomics analysis, identified 1127 proteins and showed the presence of numerous carbohydrate-active enzymes extracted from the biomass-bound fractions and from the culture supernatant. This revealed a wide array of hydrolytic cellulases, hemicellulases and carbohydrate-binding modules involved in lignocellulose degradation. The expression of these activities correlated to the changes in the biomass composition observed by FTIR and ssNMR measurements.ConclusionsA combination of mass spectrometry-based proteomics coupled with metatranscriptomics has enabled the identification of a large number of lignocellulose degrading enzymes that can now be further explored for the development of improved enzyme cocktails for the treatment of plant-based feedstocks. In addition to the expected carbohydrate-active enzymes, our studies reveal a large number of unknown proteins, some of which may play a crucial role in community-based lignocellulose degradation.

Physicochemical and functional properties of coconut (Cocos nucifera L) cake dietary fibres: Effects of cellulase hydrolysis, acid treatment and particle size distribution

Effects of cellulase hydrolysis, acid treatment and particle size distribution on the structure, physicochemical and functional properties of coconut cake dietary fiber (DCCDF) were studied. Results showed that both the cellulase hydrolysis and acid treatment contributed to the structural modification of DCCDF as evident from XRD, FT-IR and SEM analysis. Moreover, the cellulase hydrolysis enhanced soluble carbohydrate content, water holding capacity (WHC) and swelling capacity (WSC), α-amylase inhibition activity (α-AAIR), glucose dialysis retardation index (GDRI) and cation-exchange capacity (CEC) of DCCDF; but it had undesirable effects on colour, oil holding capacity (OHC) and emulsifying capacity (EC). On other hand, acid treatment decreased the WHC, WSC and GDRI, but improved the colour, CEC, OHC and emulsion stability of DCCDF. Furthermore, the WHC, WSC and EC of DCCDF increased as the particle size reduced from 250 to 167 μm, while the GDRI, OHC, α-AAIR and emulsion stability decreased with decreasing particle size.

The composition of accessory enzymes of Penicillium chrysogenum P33 revealed by secretome and synergistic effects with commercial cellulase on lignocellulose hydrolysis

Herein, we report the secretome of Penicillium chrysogenum P33 under induction of lignocellulose for the first time. A total of 356 proteins were identified, including complete cellulases and numerous hemicellulases. Supplementing a commercial cellulase with increasing dosage of P33 enzyme cocktail from 1 to 5 mg/g substrate increased the release of reducing sugars from delignified corn stover by 21.4% to 106.8%. When 50% cellulase was replaced by P33 enzyme cocktail, release of reducing sugars was 78.6% higher than with cellulase alone. Meanwhile, glucan and xylan conversion was increased by 37% and 106%, respectively. P33 enzyme cocktail also enhanced commercial cellulase hydrolysis against four different delignified lignocellulosic biomass. These findings demonstrate that mixing appropriate amount of P33 cocktail with cellulase improves polysaccharide hydrolysis, suggesting P33 enzymes have great potential for industrial applications.

Physico-chemical oxidative cleavage strategy facilitates the degradation of recalcitrant crystalline cellulose by cellulases hydrolysis

BackgroundEfficient enzymatic conversion of recalcitrant crystalline cellulose is critical for enabling cost-effective industrial conversion of cellulosic biomass to biofuels and chemicals. Fully understanding enzyme digestion mechanism is paving a new way to design efficient process for biomass conversion. Accordingly, a continuing drive is inspiring to discover new routes to promote crystalline cellulose disruption.ResultsHerein, a physico-chemical oxidative cleavage strategy of irradiation oxidation/post-reduction (IOPR) was employed to treat crystalline cellulose I to cleave glycosidic bonds association with some new oxidized and reduced chain ends, thus boosting downstream degradation by cellulases from Trichoderma reesei. The hydrolysis performance of treated crystalline cellulose was conducted with either T. reesei Cel7A (TrCel7A) alone, or a cellulase enzyme mixture (90% Celluclast 1.5 L, 10% β-glucosidase). 81.6 and/or 97% of conversion efficiency have been reached for 24-h and 48-h cellulase hydrolysis, respectively. The high efficient conversion of crystalline cellulose after IOPR is mainly attributed to generating some new chain ends, which are identified by MAIDI-TOF–MS and HPLC. Furthermore, the nanoscale architectures of crystalline cellulose before and after IOPR are systematically investigated by XRD, EPR, ATR- FTIR, GPC, and XPS techniques. Together with TEM images, the results reveal a fascinating digestion mechanism of “peel-off” and “cavity-formation” paradigms toward degrading crystalline cellulose by cellulase mixtures after IOPR treatment.ConclusionsThis encouraging results show that the proposed IOPR approach will become a potential competitive alternative to current biomass pretreatment. It opens a new avenue toward the implementation of pretreatment and the design of enzyme cocktails in lignocellulosic biorefinery.

Adsorption activity of coconut (Cocos nuciferaL.) cake dietary fibers: effect of acidic treatment, cellulase hydrolysis, particle size and pH.

The effects of acidic treatment, cellulase hydrolysis, particle size distribution and pH on the adsorption capacity of defatted coconut cake dietary fibers (DCCDF) were studied. The results demonstrated that cellulase hydrolysis could significantly improve the soluble dietary fiber content, water holding ability and adsorption ability of DCCDF on cholesterol, bile and nitrite ions. Acidic treatment enhanced the oil holding capacity and adsorption ability in cholesterol and nitrite ions. Moreover, the adsorption ability of DFs in cholesterol, nitrite and bile all increased with reduced particle size (250 to 167 μm), and DCCDF demonstrated a higher adsorption capacity at pH 2.0 than at pH 7.0. The change in adsorption capacity of DCCDF might be suitable for application in the food industry as a low-calorie and cholesterol lowering functional ingredient.

Culturable fungal diversity and cellulase production by mixed culture Aspergillus fungi from Sanya mangrove.

Fungi are the most suitable cellulase producers attributing to its ability to produce a complete cellulase system. 33 Genus, 175 Species fungi were isolated from Sanya mangrove, Hainan, China. Using congo red cellulose (CMC) medium, five fungi of cellulose-degrading were selected for further study. Molecular biology and morphological identification showed that all of these five fungi belong to Aspergillus fungi. The cellulase produced by these fungi were monitored during liquid state fermentation. The optimum conditions study for enzyme production illustrated that the highest activities appeared at pH 3.0, 35°C after fermentation for 3 days. Beyond that, the enzyme activity of mixed fungi is 11-26% higher than pure. The study demonstrated that mixed culture improved the hydrolysis of fungi cellulase.

Endoglucanase (EG) Activity Assays.

Cellulosic biomass, the most common organic compound of primary energy source on earth, is a network of interwoven biopolymers of plant cell walls. Degradation of cellulose is important for global carbon recycling. Moreover, biofuel, a renewable fuel whose energy can be derived from cellulosic biomass by enzymatic hydrolysis of cellulases. Among cellulases are endoglucases that act synergistically for subsequent hydrolytic reactions to break down the polymeric cellulose. However, in cellulolytic enzyme activity endoglucanase plays a prominent role in initiating and sustaining the hydrolytic process. Endoglucanase randomly cleaves the cellulose polymer into smaller sugar and oligomeric polysaccharides. Characterization and quantification of endoglucanase activity is important for industry and in the overall study of cellulose degradation. All assays including those for endoglucanase fall into two broad categories either qualitative or quantitative. Quantitative assays can tell if the enzyme is present, how much and its activity. Measurement can be done indirectly using a secondary colorimetric product like (1) molybdenum blue, (2) 3-amino-5-nitrosalicylic acid, (3) bicinchoninic acid (BCA), and (4) 2-cyanoacetamide or directly using an antibody in an ELISA. In this chapter, we discuss several common protocols for the measurement of endoglucanase activity.

Chemical, structural, and ultrastructural analysis of waste from the carrageenan and sugar-bioethanol processes for future bioenergy generation

Macroalgae and sugarcane biorefineries are designed to generate bioproducts for commercial use. Due to the high carbohydrate content of Kappaphycus alvarezii and sugarcane bagasse (SCB), biorefineries are aimed at generating such bioproducts as bioethanol, sucrose, carrageenan, and electricity production. Although there are several studies on SCB, the comparative study between residue carrageenan extraction and SCB has not yet been published. The samples were chemically characterized followed by structural, ultrastructural, and enzymatic hydrolysis analyzes. The content of protein in the wastes varied depending on the method used. Galactan (8.2%) and glucan (55.3%) were major polysaccharides in the residue carrageenan extraction, whereas in SCB, major polysaccharides were hemicellulose (25.2%) and glucan (38.2%). SCB was found to contain 24.5% of lignin, but the residue carrageenan extraction showed the presence of only 4.5% of insoluble aromatic compounds. ATR, NMR, and UV-Visible data on the residue carrageenan extraction revealed that the glucan fraction was similar to that in SCB. In contrast, the XRD analysis of SCB revealed a higher index of crystallinity than in the residue carrageenan extraction. The residue carrageenan extraction from the K. alvarezii does not show any type of recalcitrance against the enzymatic hydrolysis of cellulases, being hydrolyzed 100% in glucose. However, SCB showed a maximum conversion to glucose of 30%, requiring an additional pretreatment step. Thus, a biorefinery of K. alvarezii can be exploited not only to produce carrageenan but also to generate glucose for future bioenergy generation. An example would be the production of fourth generation bioethanol.

Genetic improvement of xylose metabolism by enhancing the expression of pentose phosphate pathway genes in Saccharomyces cerevisiae IR-2 for high-temperature ethanol production.

The pentose phosphate pathway (PPP) plays an important role in the efficiency of xylose fermentation during cellulosic ethanol production. In simultaneous saccharification and co-fermentation (SSCF), the optimal temperature for cellulase hydrolysis of lignocellulose is much higher than that of fermentation. Successful use of SSCF requires optimization of the expression of PPP genes at elevated temperatures. This study examined the combinatorial expression of PPP genes at high temperature. The results revealed that over-expression of TAL1 and TKL1 in Saccharomyces cerevisiae (S. cerevisiae) at 30°C and over-expression of all PPP genes at 36°C resulted in the highest ethanol productivities. Furthermore, combinatorial over-expression of PPP genes derived from S. cerevisiae and a thermostable yeast Kluyveromyces marxianus allowed the strain to ferment xylose with ethanol productivity of 0.51g/L/h, even at 38°C. These results clearly demonstrate that xylose metabolism can be improved by the utilization of appropriate combinations of thermostable PPP genes in high-temperature production of ethanol.

Synergistic action between extracellular products from white-rot fungus and cellulase significantly improves enzymatic hydrolysis

ABSTRACTWith a set of perfect extracellular lignocellulolytic enzymes, white-rot fungus has been recognized as playing an important role in the degradation of lignocellulose materials, which leads to the possibility of creating a composite enzymatic system with high hydrolysis efficiency in vitro. Echinodontium taxodii is a promising white-rot fungus for biologic pretreatment. In this study, we extracted the extracellular products of E. taxodii under solid-state fermentation conditions, mixed the extracellular products with cellulase to build a composite enzymatic system, and systematically evaluated the effect of this system on the hydrolysis of acid-pretreated and raw maize stovers. We found that the extracellular products from E. taxodii could significantly improve the hydrolysis efficiency of cellulase, with a synergistic action between the extracellular products and cellulase. Corn stovers treated with extracellular products were suitable for the enzymatic hydrolysis of cellulase. Furthermore, we found that pure proteins from the extracellular products were not sufficient to generate synergistic action. This finding suggests that non-protein substances may also be involved in the synergistic action between the extracellular products and cellulase.

Recyclable magnetic carboxymethyl chitosan/calcium alginate – cellulase bioconjugates for corn stalk hydrolysis

The use of cellulase hydrolysis of straw to produce fermentable sugars has many application prospects. However, cellulase is very expensive, which hampers its industrial applications. To improve cellulase’s catalytic activity and reduce the enzyme cost, magnetite carboxymethyl chitosan/calcium alginate – cellulase bioconjugate (MCCCB) was synthesized via an improved hydrothermal method, molecular self-assembly technology, physical absorption, embedding and covalent bonding. Its loading capacity was 3.95mg/mL, and the catalytic activity increased to 267.18%. We decreased the release rate, improved the reusability, and enhanced the stability of MCCCB. Corn stalk hydrolysis also greatly improved, and the overall yield of fermentable sugars increased by 698.26%. All of these results indicate that MCCCB could significantly improve the efficiency of cellulase, greatly reduce the cost of enzyme, and effectively promote the production of fermentable sugars.

An Efficient Method for Cellulose Nanofibrils Length Shearing via Environmentally Friendly Mixed Cellulase Pretreatment

Cellulose nanofibrils (CNFs) have potential applications in the development of innovative materials and enhancement of conventional materials properties. This paper focused on the mixed cellulase hydrolysis with major activity of exoglucanase and endoglucanase on the cellulose length shearing. By the cooperation of two-step production route, including (1) enzymatic pretreatment using cellulase fromTrichoderma virideand (2) mechanical grinding twice, a shorter cellulose nanofiber was fabricated. The influence of enzymatic charge and hydrolysis time on cellulose fibers was analyzed by using scanning electron microscopy (SEM), Fourier Transform Infrared Spectrometer (FTIR), and X-ray diffractometer (XRD). SEM images revealed that the surface morphology change, effective diameter sharpening, and length shearing of cellulose fibers are as a result of cellulase hydrolysis. The XRD suggested that the cellulase acted on the amorphous regions more strongly than the crystalline domains during layer-by-layer hydrolysis. The enzymatic charge and hydrolysis time significantly affected the yields and hydrolysis products concentration. The enzymatic pretreatment assisted mechanical grinding could improve the uniformity of CNF and helped to obtain CNF with exact length according to the requirement for special applications.

Cellulase production from thermochemically pretreated <i>Chenopodium album</i>

Lignocelllulosic biofuel production is the area of focus of different researchers. Cellulase mediated saccharification of delignified biomass is the rate limiting stage in biofuel generation. For enzymatic hydrolysis cellulase is key enzyme. There are several researches going on for cost effective cellulose production. In the current research, Chenopodium album was subjected to dilute sodium hydroxide treatment on 120°C. Efficiency of pretreatment was evaluated through scanning electron microscopy (SEM), X-ray diffraction (XRD) and biochemical composition analysis. After pretreatment, pretreated biomass was rinsed with tap water and was subsequently kept at 70°C for complete water removal. The biomass after complete water removal was applied for cellulase production using Fusarium oxysporum. Cellulase production was maximised using statistical technique. Within two days, highest cellulase production of 1.51 IU/mL was attained. The above study can be useful for efficient cellulase production for cost effective biofuel production.

Use of membrane separation in enzymatic hydrolysis of waste paper

A three-stage process containing phosphoric acid pretreatment, enzymatic hydrolysis, and membrane filtration was performed on waste paper as a lignocellulosic material. In the first two stages, the effect of phosphoric acid concentration, enzyme loading, hydrolysis time, and substrate concentration on the amount of products was investigated. At the third stage using a proper membrane, the effect of substrate concentration and transmembrane pressure (TMP) on yield of the reducing sugars was studied. The novelty of the present study was to demonstrate the application of ultrafiltration membrane on the enzymatic hydrolysis process of waste paper. The reducing sugars concentration was determined by using the 3,5-dinitrosalicylic acid (DNS) reagent method. According to the results, a value of 0.5% was determined as the optimum concentration for phosphoric acid in the pretreatment stage. The reducing sugars yield was obtained as 67.4% in this concentration. Moreover, for the enzymatic hydrolysis of waste paper, the suitable amounts of cellulase enzyme loading and hydrolysis time were determined as 50 mg/g substrate and 48 h, respectively. In the filtration stage, increase of substrate concentration and decrease of TMP resulted in higher rejection of the reducing sugars. The experimental results revealed that the highest rejection was 19.2% at TMP of 3 bar and substrate concentration of 100 g/L.

Microstructure Properties and Cellulase Hydrolysis Efficiency of Hybrid Pennisetum with [Amim]Cl Pretreatment

The complex microstructure of lignocellulosic biomass restricts its conversion into bio-ethanol. In this study, the effects of an ionic liquid (IL) 1-allyl-3-methylimidazolium chloride ([Amim]Cl) pretreatment on the microstructure properties and cellulase hydrolysis efficiency of hybrid Pennisetum (P. americanum × P. purpureum, lignocellulosic biomass) were investigated. After the [Amim]Cl pretreatment, the bonds of lignin-carbohydrate complex (LCC) and C=O in xylan were destroyed and the content of inter-molecular H-bonds O(6)H…O(3’) decreased by 47.2%, while the content of intra-molecular H-bonds of O(2)H…O(6) and O(3)H…O(5) increased by 9.5% and 47.0%, respectively. The crystallinity and the crystallite size decreased by 20.8% and 42.22%, respectively, and the cellulose crystalline structure changed from cellulose crystalline I to cellulose crystalline II. The specific surface area increased from 0.15 to 10.11 m2/g after the [Amim]Cl pretreatment. The glucose recovery increased by 10.3 times after being pretreated with [Amim]Cl, compared with the unpretreated sample.