Monocomponent Endoglucanase Research Articles

Enzymatic pretreatment for cellulose nanofiber production: Understanding morphological changes and predicting reducing sugar concentration

Enzymatic pretreatment plays a crucial role in producing cellulose nanofibers (CNFs) before fibrillation. While previous studies have explored how treatment severity affects CNF characteristics, there remains a lack of suitable parameters to monitor real-time enzymatic processes and fully comprehend the link between enzymatic action, fibrillation, and CNF properties. This study focuses on evaluating the impact of enzyme charge (using a monocomponent endoglucanase) and treatment time on cellulose fiber morphology and reducing sugar generation. For the first time, a random forest (RF) model is developed to predict reducing sugar concentration based on easily measurable process conditions (e.g., stirrer power consumption) and fiber/suspension characteristics like fines content and apparent viscosity. Polarized light optical microscopy was found to be a suitable technique to evaluate the morphological changes that fibers experience during enzymatic pretreatment. The research also revealed that endoglucanases initially induce surface fibrillation, releasing fine fibers into the suspension, followed by fiber swelling and shortening. Furthermore, the effect of enzymatic pretreatment on resulting CNF characteristics was studied at two fibrillation intensities, indicating that a high enzyme charge and short treatment times (e.g., 90 min) are sufficient to produce CNFs with a nanofibrillation yield of 19–23 % and a cationic demand ranging from 220 to 275 μeq/g. This work introduces a well-modeled enzymatic pretreatment process, unlocking its potential and reducing uncertainties for future upscaling endeavors.

Alkalization of Kraft Pulps from Pine and Eucalyptus and Its Effect on Enzymatic Saccharification and Viscosity Control of Cellulose.

Bleached kraft pulps from eucalyptus and pine were subjected to cold caustic extraction (CCE) with NaOH (5, 10, 17.5, and 35%) for hemicelluloses removal and to increase cellulose accessibility. The effect of these changes was evaluated in enzymatic saccharification with the multicomponent Cellic CTec3 enzyme cocktail, and in viscosity reduction of pulps with the monocomponent Trichoderma reesei endoglucanase (EG). After CCE with 10% NaOH (CCE10) and 17.5% NaOH (CCE17.5), hemicellulose content lower than 1% was achieved in eucalyptus and pine pulps, respectively. At these concentrations, cellulose I started to be converted into cellulose II. NaOH concentrations higher than 17.5% decreased the intrinsic viscosity (from 730 to 420 mL/g in eucalyptus and from 510 to 410 mL/g in pine). Cellulose crystallinity was reduced from 60% to 44% in eucalyptus and from 71% to 44% in pine, as the NaOH concentration increased. Enzymatic multicomponent saccharification showed higher glucose yields in all CCE-treated eucalyptus samples (up to 93%) while only CCE17.5 and CCE35 pine pulps achieved 90% after 40 h of incubation. Untreated bleached pulps of both species presented saccharification yields lower than 70%. When monocomponent EG was used to treat the same pulps, depending on enzyme charge and incubation time, a wide range of intrinsic viscosity reduction was obtained (up to 74%). Results showed that eucalyptus pulps are more accessible and easier to hydrolyze by enzymes than pine pulps and that the conversion of cellulose I to cellulose II hydrate only has the effect of increasing saccharification of CCE pine samples. Viscosity reduction of CCE pulps and EG treated pulps were obtained in a wide range indicating that pulps presented characteristics suitable for cellulose derivatives production.

Single-Step Fiber Pretreatment with Monocomponent Endoglucanase: Defibrillation Energy and Cellulose Nanofibril Quality

The combination of enzymatic pretreatment of cellulose fibers followed by mechanical defibrillation has become a green and low-energy route to obtain cellulose nanofibrils (CNF). However, the variability in the properties of the as-produced CNF remains a major challenge that needs to be addressed for any application to be realized. Herein, we study the effect of monocomponent endoglucanase (EG) on the energy consumed in defibrillation as well as the physical properties of the obtained CNF. This single-step enzymatic pretreatment (0.5–25 EGU/g cellulose fibers for 1–3 h) reduces the defibrillation energy (by up to 50%) at nearly 100% yield to obtain CNF of a similar morphology, size, and crystallinity compared to CNF obtained in the absence of pretreatment. Under mild conditions (5.6 EGU/g for 1 h), aiming to minimize energy consumption while preserving rheological properties, EG pretreatment increased the water retention value, reduced the molecular weight, and promoted structural surface modification (amorphogenesis), without significant cellulose solubilization. In addition, the carbohydrate binding module of the EG was found to improve the interaction of the catalytic core with the substrate. The combination of the factors considered here boosts the effect of the enzyme even if used at low loadings, facilitating high-yield, more sustainable production of CNF.

Xyloglucan adsorption for measuring the specific surface area on various never-dried cellulose nanofibers

Abstract In this paper, we explore xyloglucan adsorption to cellulose nanofibers as a method for the evaluation of their quality (i. e., the degree of disintegration) and the accessible surface area in the wet state and at low ionic strength. This method was shown to be capable of estimating the surface areas of 14 different cellulose nanofiber qualities from both hardwood and softwood with different pretreatments, including enzymatic hydrolysis using a monocomponent endoglucanase, TEMPO-mediated oxidation, and carboxymethylation. The cellulose surface measured using this method showed a correlation with the degree of disintegration expressed as transmittance for different concentrations of xyloglucan.

Specific Surface Area Increase during Cellulose Nanofiber Manufacturing Related to Energy Input

Softwood fibers pretreated with a monocomponent endoglucanase were used to prepare a series of cellulose nanofiber qualities using a microfluidizer and 2 to 34 MWh ton-1 of energy input. The specif ...

Morphological properties of nanofibrillated cellulose produced using wet grinding as an ultimate fibrillation process

Nanofibrillated cellulose (NFC) aqueous suspensions were produced from once-dried bisulfite softwood pulp using enzymatic or 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-mediated oxidation pretreatments, followed by wet grinding, as an ultimate fibrillation technique. Two commercial enzyme solutions: cellulase, with the major activity of endoglucanase and exoglucanase, and monocomponent endoglucanase, were compared to facilitate the nanofibrils isolation from cellulose fibers. The influence of their concentrations, as well as the other processing conditions, was analyzed. The morphology of the produced NFC was characterized using optical microscopy, atomic force microscopy (AFM), field emission gun-scanning electron microscopy (FEG-SEM), and morphological fiber analyzer (MorFi). Nanofibrils with a wide size distribution were produced. The average lateral dimensions of 12 ± 7 nm for the most disintegrated enzymatically hydrolyzed NFC and 4 ± 2 nm for TEMPO-oxidized NFC were determined from the AFM height images. The degree of polymerization (DP) decreased and the crystallinity index (CI) increased with an increase of the concentration of both enzyme solutions. TEMPO-oxidation did not have a significant impact on the cellulose CI; however, the DP was strongly affected. The monocomponent endoglucanase solution was found to have a better effect on the nanofibrils isolation rather than their depolymerization.

Enzyme-aided alkaline extraction of oligosaccharides and polymeric xylan from hardwood kraft pulp

In this paper we describe the effect of enzyme treatments on the production of polymeric xylan, oligosaccharides and hemicellulose lean pulp by alkaline extraction of bleached hardwood kraft pulp. Enzyme treatments were carried out before one or in between two subsequent alkaline extractions by purified Trichoderma reesei xylanase and endoglucanase II (Cel 5a) as well as by a commercial monocomponent endoglucanase (FibreCareR). Without enzyme pre-treatment 61% and 7% of the pulp xylan was extracted in high purity in the first and second alkaline stage, respectively. Higher molecular mass xylan was obtained in the second than in the first alkaline extraction. Xylanase treatment before alkaline extraction hydrolyzed up to 12% of xylan to xylooligosaccharides. According to our results, preparation of polymeric xylan, and/or oligosaccharides as well as hemicellulose lean pulp with cellulose content of 93–94%, is possible by enzyme-aided alkaline extraction process.

Quantitative Assessment of the Enzymatic Degradation of Amorphous Cellulose by Using a Quartz Crystal Microbalance with Dissipation Monitoring

The systematic evaluation of the degradation of an amorphous cellulose film by a monocomponent endoglucanase (EG I) by using a quartz crystal microbalance with dissipation monitoring (QCM-D) identified several important aspects relevant to the study the kinetics of cellulose degradation by enzymes. It was demonstrated that, to properly evaluate the mechanism of action, steady state conditions in the experimental set up need to be reached. Rinsing or diluting the enzyme, as well as concentration of the enzyme, can have a pronounced effect on the hydrolysis. Quantification of the actual hydrolysis was carried out by measuring the film thickness reduction by atomic force microscopy after the enzymatic treatment. The values correlated well with the frequency data obtained by QCM-D measurement for corresponding films. This demonstrated that the evaluation of hydrolysis by QCM-D can be done quantitatively. Tuning of the initial thickness of films enabled variation of the volume of substrate available for hydrolysis which was then utilized in establishing a correlation between substrate volume and hydrolytic activity of EG I as measured by QCM-D. It was shown that, although the amount of substrate affects the absolute rate of hydrolysis, the relative rate of hydrolysis does not depend on the initial amount of substrate in steady state system. With this experimental setup it was also possible to demonstrate the impact of concentration on crowding of enzyme and subsequent hydrolysis efficiency. This effort also shows the action of EG I on a fully amorphous substrate as observed by QCM-D. The enzyme was shown to work uniformly within the whole volume of swollen film, however being unable to fully degrade the amorphous film.

Depolymerization of carboxymethylcellulose in a hydro-alcoholic medium by a mono-component endocellulase

Carboxymethylcellulose (CMC) is a cellulose ether with a wide range of applications and markets. In order to tailor the molecular weight and the rheological profile of CMC, the industrial production process often implies a difficult to control depolymerization step with high concentrations of hydrogen peroxide or other oxidizing agents at alkaline pH. Because CMC dissolved in an aqueous buffer at concentrations above 3–4% (w/w) generates extremely high and unmanageable viscosities, we have developed an alternative process of enzymatic depolymerization with a selected, recombinant mono-component endocellulase for the preparation of a highly concentrated aqueous solution of low molecular weight CMC, from a 240–300 kDa CMC dispersed as a slurry in a hydro-alcoholic reaction medium, followed by solvent recovery. Wood cellulose from different tree sources and a few commercially available cellulases, either of natural origin or recombinant, were screened. The recombinant, mono-component endoglucanase Cel12A from T. reesei was selected for CMC hydrolysis carried out in isopropanol. At the end of the reaction, the enzyme is thermally inactivated and the solvent recovered by distillation. This novel industrial process yields a stable aqueous solution of a low viscosity, ready-to-use 35% (w/w) CMC, which is finding increasing new practical applications.

Optimization of Treatments for the Conversion of Eucalyptus Kraft Pulp to Dissolving Pulp

Eucalyptus wood is known worldwide for its use in the production of kraft pulps and dissolving pulps. In our previous study, the feasibility of using a eucalyptus kraft pulp as a dissolving pulp was investigated. It was demonstrated that a kraft pulp subjected to a sequence of treatments that included enzymatic treatments using a xylanase and a monocomponent endoglucanase and, an alkali extraction, accomplished the requirements of a commercial dissolving pulp in terms of cellulose reactivity and hemicellulose content. Nevertheless, the low degree of polymerization and the presence of cellulose II showed that the combination of treatments had to be optimized. As a result, the parameters involved in the sequence of treatments were examined as well as their influence in the production of dissolving pulps. Furthermore, new sequences were tested. It was noticed that by decreasing the concentration of alkali in the alkali extraction stage, no cellulose II formed at short reaction times (10 min.) and that the degree of polymerization was increased. On the other hand, the cellulose reactivity and the hemicellulose content were barely affected. Moreover, a high pulp consistency (10%) led to an inhomogeneous mixture and the stirring effect was slightly noticeable.

Exploring enzymatic treatments for the production of dissolving grade pulp from different wood and non-wood paper grade pulps 10th EWLP, Stockholm, Sweden, August 25–28, 2008

Abstract The feasibility of producing dissolving grade pulps for viscose production from different fiber raw materials, such as paper grade pulps from wood and non-woody plants, was investigated. Commercial dried bleached hardwood kraft pulps from eucalypt, and bleached non-wood soda/AQ pulps from flax, hemp, sisal, abaca, and jute, were subjected to enzymatic and chemical pre-treatments in order to improve the accessibility and reactivity of cellulose and to reduce their hemicellulose content. A commercial monocomponent endoglucanase and a commercial xylanase were employed for biochemical treatment. The chemical treatment consisted of cold alkaline extraction. The effects of these pre-treatments on pulps were studied by reactivity, according to Fock's method, and viscosity measurements, determination of hemicellulose content, and recording of molecular weight distributions. The results were compared to those of commercial bleached eucalypt dissolving pulp. Eucalypt and sisal pulps showed high improvement in reactivity, reaching levels near or even higher than that of the eucalypt dissolving pulp (65%–70%), and a low hemicellulose content (2%–4%), when both were submitted to a sequence of treatments consisting of an initial xylanase treatment followed by cold alkaline extraction, and a final endoglucanase treatment. However, the viscosity decreased considerably. A uniform and narrow molecular weight distribution was observed in both eucalypt and sisal pulps after this sequential pre-treatment.

Pretreatment of Bleached Cotton Fibers with Whole and Monocomponent Cellulases for Nonwoven Applications

Enzymatic treatment of fabrics has been carried out in the past to enhance the aesthetic properties, which has often resulted in the reduction of fabric strength. The objective of the current investigation was to develop process recipes for the production of soft and strong nonwoven fabric from enzymatically treated cotton fibers. Two different enzymatic solutions, the whole cellulase and Cellulose Binding Domain (CBD)-free monocomponent endoglucanase, were selected for this purpose. The latter was used as potential enzyme solution that could reduce the fiber strength loss during hydrolysis. This paper discusses the changes in reducing power, protein adsorption, surface properties and single fiber tenacity of bleached cotton fibers and compares with those obtained with raw cotton treated at identical conditions. The results observed in this study showed that bleached cotton was less susceptible to damage and weakening than raw cotton fibers. Thus, the aggressive whole cellulase is more suitable for treatment of bleached cotton fibers, whereas CBDfree endoglucanase is sufficient for treatment of raw cotton fibers.

Effect of cellulase pretreatment of raw and bleached cotton fibers on properties of hydroentangled nonwoven fabrics

AbstractThis study was undertaken to investigate the effect of enzymatic pretreatment of cotton (polysaccharides) fibers on the properties of resulting nonwoven fabric. Enzymatic treatment is known to improve the esthetical properties of fabrics but will likely lead to a reduction in strength. In the case of nonwovens the strength loss can be even more drastic as cellulase may attack bonded areas of the fabric. In this work, raw and bleached cotton fibers were treated with enzyme solutions prior to fabric formation to avoid possible damage to the bonded areas and improve strength retention. These fibers were first modified with commercially available whole cellulases and monocomponent endoglucanase enzyme solutions. Then they were formed into a fabric and bonded via hydroentangling. Parameters such as bending modulus, fabric tenacity, fiber strength, length and reducing power were measured for each sample. The pretreatment of cotton fibers prior to fabric formation showed that the resulting nonwovens could be stronger and more drapeable than the same fabric composed of untreated fibers. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 2007

Improved Accessibility and Reactivity of Dissolving Pulp for the Viscose Process: Pretreatment with Monocomponent Endoglucanase

A high accessibility is an essential prerequisite for a homogeneous substitution of cellulose material. In this study, chemical and enzymatic pretreatments to increase the accessibility of cellulose materials have been investigated. Dissolving pulp has been treated with a monocomponent endoglucanase. Fock's method, a microscale process similar to the viscose process, showed an increase in cellulose yield. Simultaneously, the viscosity decreased. To clarify whether the increase in reactivity was due solely to the decrease in the degree of polymerization, the dissolving pulp was also subjected to acid hydrolysis. At a given viscosity level, the enzymatic pretreated pulp had a higher reactivity than the pulp subjected to acid hydrolysis. To achieve 100% reactivity, according to Fock, the acid-treated pulp showed a lower molecular weight compared to the enzymatic-treated pulp. A monocomponent endoglucanase can thus be used to increase the reactivity and accessibility of dissolving pulp in the viscose process.

Monocomponent endoglucanase treatment increases the reactivity of softwood sulphite dissolving pulp

Softwood dissolving pulp was treated with a commercial monocomponent fungal endocellulase. The reactivity of the pulp for the production of rayon and cellulose derivatives as determined with the Fock method increased drastically with relatively low amounts of enzyme, and the yield loss and decrease of viscosity were moderate. The mechanism behind the increased reactivity is discussed.