Hydrolysis Of Cellulosic Substrates Research Articles

Functional characterization of endoglucanase (CelB) isolated from lignocellulose-degrading microbial consortium for biomass saccharification

Culture-independent metagenomic gene discovery allows exploration of genetic resources from uncultured microorganisms. In this study, an endoglucanase gene ( celB ) was identified from metagenomic DNA derived from a structurally stable thermophilic microbial consortium isolated from sugarcane bagasse collection site. CelB endoglucanase was classified in glycosyl hydrolase family 8 with 99% sequence identity to the homolog from Clostridium josui . The recombinant CelB was expressed in Escherichia coli and the purified enzyme exhibited the maximum activity on carboxymethylcellulose (CMC) with a specific activity of 86.17 U/mg at 60 °C, pH 5.0. The recombinant CelB showed specific cleavage pattern by releasing cellobiose (C2) as the major product from cellooligosaccharides (C3 to C6) substrates while C2–C4 were produced from hydrolysis of polysaccharides . The enzyme effectively released reducing sugars with the highest yield from corn stover followed by rice straw and sugarcane bagasse. In addition, supplementation of CelB at 10 mg/g biomass to Accellerase® 1500 (1 mg/g biomass) led to an improved hydrolysis on alkaline-pretreated sugarcane bagasse with 47% increase in glucose yield compared with using Accellerase® 1500 alone. Overall, the enzyme is a potential candidate for cellooligosaccharide production and saccharification of lignocellulosic materials in bio-industries. • CelB was isolated from metagenome of stable cellulolytic consortium. • CelB was classified as a GH8 endoglucanase related to Clostidium josui. • Recombinant CelB expressed in E. coli showed optimal activity at 60 °C, pH 5.0. • C2–C4 were the major product from hydrolysis of cellulosic substrates. • Additive effect of CelB to Accellerase 1500 was demonstrated at low FPU.

Enzymatic hydrolysis of barley straw for biofuel industry using a novel strain of Trametes villosa from Paranaense rainforest

Agricultural practices generate lignocellulosic waste that can be bioconverted by fungi to generate value-added products such as biofuels. In this context, fungal enzymes are presented as an alternative for their use in the hydrolysis of cellulose to sugars that can be fermented to ethanol. The aim of this work was to characterize LBM 033 strain and to analyze its efficiency in the hydrolysis of cellulosic substrates, including barley straw. LBM 033 strain was identified as Trametes villosa by molecular techniques, through the use of the ITS and rbp2 markers and the construction of phylogenetic trees. The cell-free supernatant of T. villosa LBM 033 showed high titers of hydrolytic enzymatic activities, necessary for the hydrolysis of the holocellulosic substrates, hydrolyzing pure cellulose to cellobiose and glucose and also degraded the polysaccharides contained in barley straw to short soluble oligosaccharides. These results indicate that macro fungi from tropical soil environments, such as T. villosa LBM 033 can be a valuable resource for in-house, cost effective production of enzymes that can be applied in the hydrolysis stage, which could reduce the total cost of bioethanol production.

Cellobiose dehydrogenase from Volvariella volvacea and its effect on the saccharification of cellulose

Abstract A gene encoding cellobiose dehydrogenase (VvCDH) from Volvariella volvacea was successfully expressed in Pichia pastoris with codon optimization using its native signal sequence. VvCDH had optimum pH and temperature at 5.5 and 60 °C respectively and showed a broad range of pH stability between 5 and 8. Kinetic analysis showed that the best substrate is cellobiose and that the K m for celloolgosaccharides increases with substrate length. Moreover, lactose is also efficiently oxidized, but glucose and maltose are poor substrates. A large amount of gluconic acid was generated and the overall hydrolysis yield was increased when adding VvCDH to Trichoderma reesei D-86271 enzymatic cocktail during hydrolysis of cellulose substrates, indicating VvCDH involved in the enzymatic cellulose saccharification. VvCDH shows some different enzymatic properties from basidiomycetous CDHs and can be supplemented to T. reesei cellulase cocktail for commercial application.

Using an Inducible Promoter of a Gene Encoding Penicillium verruculosum Glucoamylase for Production of Enzyme Preparations with Enhanced Cellulase Performance

BackgroundPenicillium verruculosum is an efficient producer of highly active cellulase multienzyme system. One of the approaches for enhancing cellulase performance in hydrolysis of cellulosic substrates is to enrich the reaction system with β -glucosidase and/or accessory enzymes, such as lytic polysaccharide monooxygenases (LPMO) displaying a synergism with cellulases.ResultsGenes bglI, encoding β-glucosidase from Aspergillus niger (AnBGL), and eglIV, encoding LPMO (formerly endoglucanase IV) from Trichoderma reesei (TrLPMO), were cloned and expressed by P. verruculosum B1-537 strain under the control of the inducible gla1 gene promoter. Content of the heterologous AnBGL in the secreted multienzyme cocktails (hBGL1, hBGL2 and hBGL3) varied from 4 to 10% of the total protein, while the content of TrLPMO in the hLPMO sample was ~3%. The glucose yields in 48-h hydrolysis of Avicel and milled aspen wood by the hBGL1, hBGL2 and hBGL3 preparations increased by up to 99 and 80%, respectively, relative to control enzyme preparations without the heterologous AnBGL (at protein loading 5 mg/g substrate for all enzyme samples). The heterologous TrLPMO in the hLPMO preparation boosted the conversion of the lignocellulosic substrate by 10–43%; however, in hydrolysis of Avicel the hLPMO sample was less effective than the control preparations. The highest product yield in hydrolysis of aspen wood was obtained when the hBGL2 and hLPMO preparations were used at the ratio 1:1.ConclusionsThe enzyme preparations produced by recombinant P. verruculosum strains, expressing the heterologous AnBGL or TrLPMO under the control of the gla1 gene promoter in a starch-containing medium, proved to be more effective in hydrolysis of a lignocellulosic substrate than control enzyme preparations without the heterologous enzymes. The enzyme composition containing both AnBGL and TrLPMO demonstrated the highest performance in lignocellulose hydrolysis, providing a background for developing a fungal strain capable to express both heterologous enzymes simultaneously.

To understand the superior hydrolytic activity after polymorphic conversion from cellulose I to II from the adsorption behaviors of enzymes

The role of the cellulose ultrastructure on the relationship between cellulase binding and activity is not clear yet. In this article, a quartz crystal microbalance with dissipation (QCM-D) was employed to monitor the interactions between a given cellulase and the cellulose substrates with varied polymorphs of pure cellulose I and II and the intermediate state (I/II). Initially, cellulose nanocrystals (CNCs) with polymorphs of cellulose I, I/II and II were prepared and spin-coated on QCM sensors. The cellulose substrates’ crystallinity degree was examined by XRD, and morphology was detected by AFM. Then, a commercial cellulase from Trichoderma reesei was used to test the adsorption and hydrolysis of cellulose substrates with polymorphs of I, I/II and II, respectively. The results revealed that in the enzyme adsorption and desorption process at a temperature of 15 °C, CNC-II had the lowest adsorption capacity with a total adsorption mass of 179 ng cm−2 but the highest reversible binding ratio of 33.7%; for comparison, the values were 235 ng cm−2 versus 25.6% and 207 ng cm−2 versus 26.9% for CNC-I and -I/II, respectively. And the conformation of adlayers on CNC-I, -I/II and -II derived from the QCM data became softer and softer in turn. On the other hand, CNC-II exhibited the best enzymatic hydrolytic ability among three substrates when enzymatic hydrolysis experiments were conducted at 45 °C. The results indicated that polymorphic conversion from I to II changes the affinity between the enzyme and cellulose surface; CNC-II has the lowest affinity to the enzyme, but the softer conformation of the adsorbed enzyme layer, and the more reversible adsorption may facilitate its hydrolytic activity. This article gives a perspective from the adsorption dynamics and conformation of the adsorbed enzyme layer, helping to understand the superior hydrolytic activity of cellulose with polymorph II. Thus, there is a potential of polymorphic conversion in the reduction of enzyme dosage and cost in the enzymatic hydrolysis process.

Oxidative cleavage of some cellulosic substrates by auxiliary activity (AA) family 9 enzymes influences the adsorption/desorption of hydrolytic cellulase enzymes

The addition of Lytic polysaccharide monooxygenase (LPMO's) increases the desorption of exoglucanases during the hydrolysis of cellulosic substrates.

Cellulase Enzyme Activity of Bacillus Circulans from Larvae Cossus Cossus in Lignocellulosic Substrat

Cellulase are the enzymes hydrolyzing cellulosic biomass and are produced by the microorganism that grown over cellulosic matter. Bacterial Cellulases poses more advantage when compared to the cellulases from other sources. This study aims to determine the optimum conditions of cellulase production from the bacteria Bacillus Circulans CC2 and CC4 in comparison with isolates hydrolize rice straw. CC2 and CC4 isolated from larvae Cossus cossus. The testing to produce cellulase was done with various pH and temperature. The enzyme activity was tested using DNS Method. The results showed that the isolated CC2 and CC4 have the same optimum temperature of 70C on condition CMC 1%, medium pH 7,0. Crude extract cellulase isolates CC2 work optimally at pH 4 while the isolates CC4 work at pH 7. highest activity in the cellulase enzymes hydrolyze the substrate on cellulose powder with optimum activity at pH 4 CC4 isolates of 2.775 x 10-2 U/ mL, while the enzyme activity CC2 isolates 4.359 x 10-2 U/mL, p this occurs due to the enzymatic hydrolysis of cellulosic substrates with cellulase enzymes will outline the cellulose into glucose which is a simple form.

English

Cellulases are enzymes that act on the hydrolysis of cellulosic substrates and comprise a complex capable of hydrolyzing cellulose and producing glucose. This conversion can be performed by an enzymatic complex found in secretions of such microorganisms as fungi and bacteria. The challenge is to transform this conversion into a quicker and more cost-effective process. The objective of this study was to evaluate the activity of the cellulase produced by the fungus Pycnoporus sanguineus (L.) Murrill, strains D1-FB, D3-FB, D5-FB and D10-FB, isolated from Baccharis dracunculifolia D. C. (Asteraceae), in the production of cellulolytic enzymes. This study was conducted using sugarcane bagasse enriched with carboxymethylcellulose at 1% as a substrate. The material was kept in an incubator at a temperature ranging from 25 to 45°C for 43 days, with the cellulase activity being quantified every seven days. The indirect quantification method was employed to quantify reducing sugars that were determined by reaction with Domain Name System (DNS). After evaluation, it was observed that the fungus strain P. sanguineus (L.) (D10-FB) presented the highest cellulase activity, with values of 16.32±2.65 IU/g of fermented substrate after 29 days of fermentation, using sugarcane bagasse as the substrate, at a temperature of 30°C and pH 5.5. Key words: Cellulase, sugarcane bagasse, cellulose, Pycnoporus sp.

Characterization of modular bifunctional processive endoglucanase Cel5 from Hahella chejuensis KCTC 2396

Cel5 from marine Hahella chejuensis is composed of glycoside hydrolase family-5 (GH5) catalytic domain (CD) and two carbohydrate binding modules (CBM6-2). The enzyme was expressed in Escherichia coli and purified to homogeneity. The optimum endoglucanase and xylanase activities of recombinant Cel5 were observed at 65 °C, pH 6.5 and 55 °C, pH 5.5, respectively. It exhibited K m of 1.8 and 7.1 mg/ml for carboxymethyl cellulose and birchwood xylan, respectively. The addition of Ca(2+) greatly improved thermostability and endoglucanase activity of Cel5. The Cel5 retained 90 % of its endoglucanase activity after 24 h incubation in presence of 5 M concentration of NaCl. Recombinant Cel5 showed production of cellobiose after hydrolysis of cellulosic substrates (soluble/insoluble) and methylglucuronic acid substituted xylooligosaccharides after hydrolysis of glucuronoxylans by endo-wise cleavage. These results indicated that Cel5 as bifunctional enzyme having both processive endoglucanase and xylanase activities. The multidomain structure of Cel5 is clearly distinguished from the GH5 bifunctional glycoside hydrolases characterized to date, which are single domain enzymes. Sequence analysis and homology modeling suggested presence of two conserved binding sites with different substrate specificities in CBM6-2 and a single catalytic site in CD. Residues Glu132 and Glu219 were identified as key catalytic amino acids by sequence alignment and further verified by using site directed mutagenesis. CBM6-2 plays vital role in catalytic activity and thermostability of Cel5. The bifunctional activities and multiple substrate specificities of Cel5 can be utilized for efficient hydrolysis of cellulose and hemicellulose into soluble sugars.

Unique Contribution of the Cell Wall-Binding Endoglucanase G to the Cellulolytic Complex in Clostridium cellulovorans

The cellulosomes produced by Clostridium cellulovorans are organized by the specific interactions between the cohesins in the scaffolding proteins and the dockerins of the catalytic components. Using a cohesin biomarker, we identified a cellulosomal enzyme which belongs to the glycosyl hydrolase family 5 and has a domain of unknown function 291 (DUF291) with functions similar to those of the surface layer homology domain in C. cellulovorans. The purified endoglucanase G (EngG) had the highest synergistic degree with exoglucanase (ExgS) in the hydrolysis of crystalline cellulose (EngG/ExgS ratio = 3:1; 1.71-fold). To measure the binding affinity of the dockerins in EngG for the cohesins of the main scaffolding protein, a competitive enzyme-linked interaction assay was performed. Competitors, such as ExgS, reduced the percentage of EngG that were bound to the cohesins to less than 20%; the results demonstrated that the cohesins prefer to bind to the common cellulosomal enzymes rather than to EngG. Additionally, in surface plasmon resonance analysis, the dockerin in EngG had a relatively weak affinity (30- to 123-fold) for cohesins compared with the other cellulosomal enzymes. In the cell wall affinity assay, EngG anchored to the cell surfaces of C. cellulovorans using its DUF291 domain. Immunofluorescence microscopy confirmed the cell surface display of the EngG complex. These results indicated that in C. cellulovorans, EngG assemble into both the cellulolytic complex and the cell wall complex to aid in the hydrolysis of cellulose substrates.

The Influence of “Enzymatic Hydrolysis of Cellulosic Substrates” on the Final Quality of Coated Fabrics

Finishing of textile fabrics used in directly coated fabrics plays an important role in the final quality of the products. The amount of surface fibres in the textile substrate is directly related to the amount of surface defects in the final coated fabrics. In this research work defuzzing was carried out on the selected fabrics by common industrial procedures, such as shearing and singeing. Biopolishing was carried out on loom state fabrics and defuzzed samples. The rate of hydrolysis was studied by measuring the weight loss as well as microscopical analysis. Quantitative analysis of the surface defects was carried out by image analysis. The results obtained show clearly the influence of defuzzing, particularly biopolishing, on the enhancement of the surface quality of PVC coated fabrics.

Evaluation of the enzymatic susceptibility of cellulosic substrates using specific hydrolysis rates and enzyme adsorption

Adsorption of cellulases to cellulose is a critical step in the hydrolysis of cellulosic substrates. However, the importance of adsorption in determining the hydrolysis rate is unclear. The accessibility to cellulases and specific hydrolysis rates were measured for various substrates. No correlation was found between the amount of enzyme adsorbed and the initial hydrolysis rate for different substrates. Specific hydrolysis rates were found to differ among substrates. Furthermore, both accessibility to cellulases and the specific hydrolysis rate of substrates were found to be changed by chemical and physical pretreatment of the substrate.

Modelling and simulation of cellulase adsorption and recycling during enzymatic hydrolysis of cellulosic materials

Characteristics of cellulase adsorption on cellulosic substrates and their recycling during the hydrolysis of cellulose were studied. On the basis of the experimental data, a comprehensive model was set up comprising of sub-models for the enzyme adsorption, enzymatic hydrolysis and recycling of enzymes. The model equations consist of non-linear systems of ordinary differential and algebraic equations. The model parameters were identified by means of the experimental results of Singh et al. [1] and Bader et al. [2]. The simulation results with the model corresponded well with the experimental data. Thus, the good agreement between simulated and measured process variables indicates that the model is suitable for description of cellulase adsorption and recycling during hydrolysis of cellulosic substrates.

One-dimensional model of the acid hydrolysis of cellulosic substrates in a solid-liquid cyclone reactor

The use of rotating flow in an annulus is investigated as a means of enhancing the yield of glucose and xylose in the acid hydrolysis of cellulosic slurries. A one-dimensional model of such a cyclone reactor is developed for flow cases, co-current and counter-current flow. For the case of 250°C, 1% w/w acid, the one-dimensional model indicates an increase in the maximum glucose yield from 48.1% in a plug flow reactor to 69.3% in a co-current cyclone reactor, and up to 81.0% in a countercurrent cyclone reactor. The corresponding xylose yields are 91.6% for co-current operation and 97.7% for countercurrent operation. In the co-current case the maximum glucose and xylose yields do not occur at the same location in the reactor; however, in the countercurrent case they do. Although product yields are dramatically improved over those obtained in a plug flow reactor, the product concentrations are lower than would typically be obtained in a plug flow reactor.

Saccharification of cellulose by the cellulolytic enzyme system of Thermomonospora sp. II. Hydrolysis of cellulosic substrates

AbstractA saccharification of cellulosic material using culture filtrate from the stationary phase of a culture of Thermomonospora sp. produced primarily cellobiose up to levels inhibitory to further saccharification, while the use of whole broth resulted in the production of glucose as well. Glucose production was enhanced and continued throughout the saccharification (24–36 hr) by several additions of cellobiase activity in the form of culture solids. Using Solka‐Floc as substrate, the “difference sugar” level (total soluble sugar minus glucose) rapidly rose to the same relatively stable concentration under various hydrolysis conditions, which was independent of the total sugar and glucose concentrations. A rapid hydrolusis rate was observed initially during saccharification, followed by a much slower rate of sugar production. Repeated centrifugation of the reaction mixture and replacement of the supernatant with fresh enzyme solution resulted each time in the reinitiation of a rapid hydrolysis rate. Saccharifications using A vicel microcrystalline cellulose, acid‐swollen cellulose, and cotton as substrates were also studied. A modified method of making phosphoric‐acid swollen cellulose is described. Saccharification of this substrate by culture filtrate and sequential additions of culture solids resulted in an inverse relationship between the attained glucose concentration and cellobiose‐cellotriose concentrations.