Quantification of cellulase activity using cellulose-azure

Despite wide application of cellulose-azure as a substrate for measuring cellulase activity, there is no quantification of hydrolysis rate or enzymatic activities using this substrate. The aim of this study was to quantify the hydrolysis rate in terms of product formation and dye released using cellulose-azure. The amount of dye released was correlated with the production of glucose and the enzyme concentrations. It is shown that the lack of correlation can be due to (1) repression of the release of the azure-dye when azure-dye accumulates, (2) presence of degradable substrates in the cellulase powder which inflate the glucose measurements and (3) the degradation of cellulose which is not linked to the dye in the cellulose-azure. Based on the lack of correlation, it is recommended that cellulose-azure should only be applied in assays when the aim is to compare relative activities of different enzymatic systems.     

Enzymatic activity of cellulase adsorbed on cellulose and its change during hydrolysis

Hydrolysis of pure cellulose Avicel has been carried out, using Meicelase from Trichoderma viride, where the enzymatic activity of cellulase adsorbed on cellulose and its changes during the hydrolysis were investigated. A rapid drop of the hydrolysis rate during the reaction, that is always observed in enzymatic hydrolysis of cellulose, could be explained by a decline of specific activity of adsorbed enzyme, and it was implied that the decline results from a loss of synergistic action between endoglucanase and exoglucanase. An empirical equation expresses the change of hydrolysis rate during the reaction and also shows that the change of the hydrolysis rate is caused by the decline of the specific enzymatic activity of adsorbed enzyme.     

Reactivities of cellulases from fungi towards ribbon-type bacterial cellulose and band-shaped bacterial cellulose

We have investigated the reactivities of various cellulases onribbon-type bacterial cellulose (BC I) and band-shaped bacterial cellulose (BCII) so as to clarify the properties of different cellulases. BC I waseffectively hydrolyzed by exo-type cellulases from different fungi from twicetofour times as much as BC II, but endo-type cellulases showed little differencein reactivity on those substrates. One of the endo-type cellulases, EG II fromTrichoderma reesei, degraded BC II more rapidly thanexo-type cellulases even in the production of reducing sugars. The degree ofpolymerization (DP) of BC II was rapidly decreased by endo-type cellulases atanearly stage, while exo-type cellulases did not cause the decrease of DP atthe initial stage, though the decrease of DP was observed after an incubation of24 h. All exo-type cellulases adsorbed on BC I and BC II,whileendo-type cellulases except for EG II adsorbed slightly on both substrates. Itwas interesting to observe EG II adsorbed on BC I but not on BC II. It issuggested that the adsorption of enzyme on cellulose is important for thedegradation of BC I, but not for BC II. It is proposed that the ratio of aspecific activity of each enzyme between BC I and BC II represents thedifference in the mode of action of cellulase. Furthermore, the K _RW value, which we can calculate from thedecrease of DP/reducing sugar produced, is effective for discriminating themode of action of cellulase, especially the evaluation of randomness in thehydrolysis of cellulose by endo- and exo-type cellulases.     

One‐step zymogram method for the simultaneous detection of cellulase/xylanase activity and molecular weight estimation of the enzyme

Here, we describe a zymographic method for the simultaneous detection of enzymatic activity and molecular weight (MW) estimation, following a single electrophoresis step. This involved separating cellulase and xylanase activities from bacteria and fungi, obtained from different sources, such as commercial extracts, crude extract and purified proteins, under denaturing conditions, by 10% polyacrylamide gel electrophoresis, using polyacrylamide gels copolymerized with 1% (w/v) carboxymethylcellulose or beechwood xylan as substrates. Then, enzymes were refolded by treatment with 2.5% Triton X‐100 in an appropriate buffer for each enzymatic activity, and visualized by Coomassie blue staining for MW estimation. Finally, Congo red staining revealed bio‐active cellulase and xylanase bands after electrophoretic separation of the proteins in the preparations. This method may provide a useful additional tool for screening of particular cellulase and xylanase producers, identification and MW estimation of polypeptides that manifest these activities, and for monitoring and control of fungal and bacterial cellulase and xylanase production.     

Effect of cellulase on the pore structure of bead cellulose

An enzymatic treatment with cellulases fromTrichoderma viride was investigated in its effect on the pore structure of different types of bead cellulose. One objective of this study was to establish a suitable procedure for combined enzymatic treatment and solvent exchange that would restore the original pore structure which the beads had before drying without causing major losses in mechanical stability. Another aim was to further increase the accessible pore space and internal surface area for separation of large molecular weight compounds with regard to Chromatographic applications. Finally, an attempt was made to extend the findings for unsubstituted beads to the derivatives carboxymethyl (CM) and diethylaminoethyl (DEAE) cellulose beads. The enzymatically treated samples were characterized by microscopic methods and porosity measurements such as mercury porosimetry, nitrogen sorption and size exclusion chromatography. It was found that under controlled conditions the low-porosity surface layer of dried beads could be removed making the internal pore space accessible without reducing the resistance to deformation of the beads. Additionally, a shift in pore size distribution towards larger pores was observed. Supplementary swelling treatments in solvents of high swelling power could substantially restore the former porosity of the dried beads but did not enhance the accessibility to the cellulases to a considerable extent. Internal pore volume and surface area of the derivatives were dramatically increased in the case of DEAE upon enzymatic hydrolysis, however, at the expense of mechanical stability, whereas CM was found to be less affected.     

A boronate-modified renewable nanointerface for ultrasensitive electrochemical assay of cellulase activity

The saccharification of cellulosic biomass to produce biofuels and chemicals is one of the most promising industries for green-power production and sustainable development. Cellulase is the core component in the saccharification process. Simple and efficient assay method to determine cellulase activity in saccharification is thus highly required. In this work, a boronate-affinity surface based renewable and ultrasensitive electrochemical sensor for cellulase activity determination has been fabricated. Through boronate-sugar interaction, celluloses are attached to the electrode surface, forming the cellulose nano-network at the sensing interface. Cellulase degradation can lead to the variation of electrochemical impedance. Thus, electrochemical impedance signal can reflect the cellulase activity. Importantly, via fully utilizing the boronate-affinity chemistry that enables reversible fabrication of cellulose nano-network, a renewable sensing surface has been firstly constructed for cellulase activity assay. Thanks to interfacial diffusion process of electrochemical sensor, the product inhibitory effect in the cellulase activity assays can be circumvented. The proposed electrochemical sensor is ultrasensitive for label-free cellulase activity detection with a very simple fabrication process, showing great potential for activity screen of new enzymes in saccharification conversion.     

Production of cellulase on mixtures of xylose and cellulose

Cellulase production by the RUT-C30 mutant of the fungusTrichoderma reesei was studied on mixtures of xylose and cellulose. In mixed substrates, the lag phase of the growth cycle was shorter and reached the maximum of total productivity in a shorter time compared to growth on the single substrate, cellulose. A diauxic pattern of utilization of the two carbon sources was observed as well: Xylose was utilized first to support growth, followed by cellulose to induce the cellulase enzyme production and provide an additional carbon source for cellular metabolism. Of the various mixtures of xylose and cellulose used in batch enzyme production, a ratio of 30∶30 g/L of xylose to cellulose was optimal. This mixture produced the highest maximal enzyme productivity of 122 IFPU/L h, and its total productivity reached a maximum value of 55 IFPU/L h in less time than others. However, similar total productivities and higher enzyme titers were observed for growth on cellulose alone.     

Effect of a nonionic surfactant on Trichoderma cellulase treatments of regenerated cellulose and cotton yarns

It has been shown that some surfactants affect the hydrolysis of cellulose by cellulase. In this study, the effect of the surfactant Tween 20 on the hydrolysis of different cellulosic fibers was investigated and related to the cellulose fiber structure. It was found that this non-ionic surfactant enhanced the enzymatic saccharification of highly crystalline cellulosics such as Avicel, Tencel and cotton but not of cuprammonium rayon. The enhanced saccha-rification effected by the surfactant is attributed to inhibition of non-productive sorption of the endoglucanase of the cellulose surface which gives greater access to the cellulose chain ends by the exoglucanase. Although all three fibers lost tensile strength as a result of the enzymatic treatment, no further decrease was effected by the presence of the surfactant.     

Effect of cellulase adsorption on the surface and interfacial properties of cellulose

The surface properties of several purified cellulose (Sigmacell 101, Sigmacell 20, Avicel pH 101, and Whatman CF 11) were characterised, before and after cellulase adsorption. The following techniques were used: thin-layer wicking (except for the cellulose Whatman), thermogravimetry, and differential scanning calorimetry (for all of the above celluloses). The results obtained from the calorimetric assays were consistent with those obtained from thin-layer wicking – Sigmacell 101, a more amorphous cellulose, was the least hydrophobic of the analysed celluloses, and had the highest specific heat of dehydration. The other celluloses showed less affinity for water molecules, as assessed by the two independent techniques. The adsorption of protein did not affect the amount of water adsorbed by Sigmacell 101. However, this water was more strongly adsorbed, since it had a higher specific heat of dehydration. The more crystalline celluloses adsorbed a greater amount of water, which was also more strongly bound after the treatment with cellulases. This effect was more significant for Whatman CF-11. Also, the more crystalline celluloses became slightly hydrophilic, following protein adsorption, as assessed by thin-layer wicking. However, this technique is not reliable when used with cellulase treated celluloses.     

离子液体介质中纤维素酶降解纤维素的研究进展

以不可再生资源为原料和能源进行的传统加工工业正面临着资源日益枯竭的现实,所以对可再生资源的研究势在必行。在各种可再生资源中,纤维素生物质是唯一可再生的碳资源,具有取之不尽用之不竭的物质基础,被普遍认为将会部分替代或补充不可再生资源。但由于纤维素的超分子结构,传统的工艺很难将其降解转化,离子液体作为一种新型的绿色溶剂,不仅能够很好地溶解纤维素,同时也是纤维素酶解反应的良好溶剂。综述了国内外离子液体对纤维素溶解、再生以及降解的近期研究成果,分析了其中存在的问题,提出了离子液体降解纤维素的发展方向。     

EPR characterization of cellulose triacetate fibers used for enzyme immobilization

EPR studies of a nitroxide spin label and of the nitroxide spin-labeled albumin entrapped in cellulose triacetate fibers were carried out. The EPR spectra have shown that within the fiber only two phases are present: a liquid one of medium viscosity trapped inside microcavities, and a polymeric one surrounding them. After entrapment, spin-labeled albumin is distributed mainly in the liquid phase, though a not negligible amount of it remains within the polymeric matrix. The EPR studies have shown that, after the standard procedure of drying, the albumin is almost completely precipitated, but about 85% of it returns to solution when the fiber is again placed in the solution. The behavior of the albumin dissolved inside the microcavities toward denaturating agents and pH change, and that of the free albumin in solution is similar; the minor differences noticed indicate a second-order interaction between the fiber and the protein.     

A spectrophotometric method for determination of the solubilizing activity of cellulase complexes

Summary A standardized spectrophotometric method for determination of the solubilizing activity of microbial cellulase complexes has been developed. It is based on the release of coloured compounds from microcrystalline cellulose (Avicel SF^®) dyed with Levafixbrillantrot E-2B^®.

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Eine spektrophotometrische Methode zur Bestimmung der solubilisierenden Aktivität des Cellulase-Komplexes (Kurze Mitteilung)

Zusammenfassung Es wurde eine standardisierte spektrophotometrische Methode zur Bestimmung der Aktivität des Cellulase-Komplexes von Mikroorganismen ausgearbeitet. Die Methode beruht auf der Möglichkeit, mit Levafixbrillantrot E-2 B^® gefärbte mikrokristalline Cellulose (Avicel SF^®) als Substrat zu verwenden.

     

Enzyme-catalyzed,gas-phase reactions

UV-49, the mutant ofPenicillium funiculosum, showed higher production of cellulase although the specific activity with regards to filter paper activity was unaltered (0.7 U/mg). Fractionation of culture filtrates by isoelectric focusing indicated the presence of three endoglucanases and two β-glucosidases in the parent strain, whereas one endoglucanase and one β-glucosidase for UV-49. The thermostability of activity towards filter paper, CM-cellulose and ρ-nitrophenyl-β-D-glucoside of the parent strain was about 30–50% more than the corresponding activities of the mutant. The saccharification of bagasse with the enzymes from parent (66%) and mutant (62%) was comparable. However, the recovery of enzyme from residual cellulose was 10–20% less in case of the mutant. The formation of higher oligosaccharides in the hydrolysates of UV-49 on prolonged incubation indicated the presence of transglycosylation activity in the enzyme complex. In contrast the parent strain produces glucose; the desired end product of the practical saccharification.     

离子液体[BMIM]Cl预处理对微晶纤维素酶解的影响

以微晶纤维素为研究对象, 设计了离子液体1-丁基-3-甲基咪唑氯盐(1-butyl-3-methylimidazolium chloride, [BMIM]Cl)预处理微晶纤维素Avicel的实验方法以实现纤维素的高效酶解糖化. 在[BMIM]Cl中Avicel完全溶解, 经水洗沉淀得到再生纤维素, 回收后的离子液体可重复利用. 预处理后底物酶解的可溶性糖转化率在24 h时高达94.65%, 较之同样条件下未经预处理底物的酶解糖转化率(48.57%)有飞跃性提升. 进一步考察了离子液体预处理对纤维素结构及形态的影响, 结果表明: [BMIM]Cl预处理后Avicel氢键减弱; 结晶度明显下降, 结晶型态由纤维素I型转变为纤维素II型; 由规整的平行排布转变为疏松有孔的无序形貌. 正是离子液体预处理引起的纤维素微观与宏观结构性质的显著改变使得再生后纤维素酶解的可溶性糖转化率大幅提高.     

秸秆超(亚)临界水预处理与水解技术

秸秆的资源化特别是乙醇化技术由于其技术可行性和产物高值化受到了广泛关注。预处理与水解是乙醇化的关键过程。目前针对秸秆的转化已经开展了多种化学或生物技术的研究,其中超（亚）临界技术与传统技术相比显示了独特的优势,如更高的反应速率、不需催化剂、无产物抑制等。本文在总结秸秆传统预处理与水解技术的基础上,对秸秆超（亚）临界水预处理与水解的过程和机理,特别是超临界亚临界组合技术的研究现状、工艺及其相关研究的进展进行了综述和分析,并阐述了超临界亚临界组合技术首先在超临界水中打破纤维结构进行初级水解,再通过亚临界反应将初级水解产物低聚糖进一步水解为葡萄糖的基本原理。最后对超（亚）临界技术在秸秆资源化领域的研究和应用前景进行了展望。     

Pretreatment of yellow poplar sawdust by pressure cooking in water

The pretreatment of yellow poplar wood sawdust using liquid water at temperatures above 220°C enhances enzyme hydrolysis. This paper reviews our prior research and describes the laboratory reactor system currently in use for cooking wood sawdust at temperatures ranging from 220 to 260°C. The wood sawdust at a 6–6.6% solid/liquid slurry was treated in a 2 L, 304 SS, Parr reactor with three turbine propeller agitators and a proportional integral derivative (PID) controller, which controlled temperature within ±1°C. Heat-up times to the final temperatures of 220, 240, or 260°C were achieved in 60–70 min. Hold time at the final temperature was less than 1 min. A serpentine cooling coil, through which tap water was circulated at the completion of the run, cooled the reactor’s contents within 3 min after the maximum temperature was attained. A bottoms port, as well as ports in the reactor’s head plate, facilitated sampling of the slurry and measuring the pH, which changes from an initial value of 5 before cooking to a value of approx 3 after cooking. Enzyme hydrolysis gave 80–90% conversion of cellulose in the pretreated wood to glucose. Simultaneous saccharification and fermentation of washed, pretreated lignocellulose gave an ethanol yield that was 55% of theoretical. Untreated wood sawdust gave less than 5% hydrolysis under the same conditions.     

Strategies to enhance the enzymatic hydrolysis of pretreated softwood with high residual lignin content

Pretreatment of Douglas-fir by steam explosion produces a substrate containing approx 43% lignin. Two strategies were investigated for reducing the effect of this residual lignin on enzymatic hydrolysis of cellulose: mild alkali extraction and protein addition. Extraction with cold 1% NaOH reduced the lignin content by only approx 7%, but cellulose to glucose conversion was enhanced by about 30%. Before alkali extraction, addition of exogenous protein resulted in a significant improvement in cellulose hydrolysis, but this protein effect was substantially diminished after alkali treatment. Lignin appears to reduce cellulose hydrolysis by two distinct mechanisms: by forming a physical barrier that prevents enzyme access and by non-productively binding cellulolytic enzymes. Cold alkali appears to selectively remove a fraction of lignin from steam-exploded Douglas-fir with high affinity for protein. Corresponding data for mixed softwood pretreated by organosolv extraction indicates that the relative importance of the two mechanisms by which residual lignin affects hydrolysis is different according to the pre- and post-treatment method used.     

Immobilization of cellulase using polyurethane foam

Cellulase was covalently immobilized using a hydrophilic polyurethane foam (Hypol®FHP 2002). Compared to the free enzyme, immobilized cellulase showed a dramatic decrease (7.5-fold) in the Michaelis constant for carboxymethylcellulose. The immobilized enzyme also had a broader and more basic pH optimum (pH 5.5–6.0), a greater stability under heat-denaturing or liquid nitrogen-freezing conditions, and was relatively more efficient in utilizing insoluble cellulose substrates. High molecular weight compounds (Blue Dextran) could move throughout the foam matrix, indicating permeability to insoluble celluloses; activity could be further improved 2.4-fold after powdering, foams under liquid nitrogen. The improved kinetic and stability features of the immobilized cellulase combined with advantageous properties of the polyurethane foam (resistance to enzymatic degradation, plasticity of shape and size) suggest that this mechanism of cellulase immobilization has high potential for application in the industrial degradation of celluloses.     

Automated filter paper assay for determination of cellulase activity

Recent developments in molecular breeding and directed evolution have promised great developments in industrial enzymes as demonstrated by exponential improvements in β-lactamase and green fluorescent protein (GFP). Detection of and screening for improved enzymes are relatively easy if the target enzyme is expressible in a suitable high-throughput screening host and a clearly defined and usable screen or selection is available, as with GFP and β-lactamase. Fungal cellulases, however, are difficult to measure and have limited expressibility in heterologous hosts. Furthermore, traditional cellulase assays are tedious and time-consuming. Multiple enzyme components, an insoluble substrate, and generally slow reaction rates have plagued cellulase researchers interested in creating cellulase mixtures with increased activities and/or enhanced biochemical properties. Although the International Union of Pure and Applied Chemists standard measure of cellulase activity, the filter paper assay (FPA), can be reproduced in most laboratories with some effort, this method has long been recognized for its complexity and susceptibility to operator error. Our current automated FPA method is based on a Cyberlabs C400 robotics deck equipped with customized incubation, reagent storage, and plate-reading capabilities that allow rapid evaluation of cellulases acting on cellulose and has a maximum throughput of 84 enzyme samples per day when performing the automated FPA.     

Pretreatment of corn fiber by pressure cooking in water

The pretreatment of corn fiber using liquid water at temperatures between 220 and 260°C enhances enzymatic hydrolysis. This paper describes the laboratory reactor system currently in use for cooking of corn fiber at temperatures ranging from 200 to 260°C. The corn fiber at approx 4.4% solid/liquid slurry was treated in a 2-L, 304 SS, Parr reactor with three turbine propeller agitators and a Proportional-Integral-Derivative (PID), controller that controlled temperature within ±1°C. Heat-up times to the final temperatures of 220, 240, or 260°C were achieved in 50 to 60 min. Hold time at the final temperature was less than 10 s. A serpentine cooling coil, through which tap water was circulated at the completion of the run, cooled the reactor’s contents to 180°C within 2 min after the maximum temperature was attained. Ports in the reactor’s head plate facilitated sampling of the slurry and monitoring the pH. A continuous pH monitoring system was developed to help observe trends in pH during pretreatment and to assist in the development of a base (2.0M KOH) addition profile to help keep the pH within the range of 5.0 to 7.0. Enzymatic hydrolysis gave 33 to 84% conversion of cellulose in the pretreated fiber to glucose compared to 17% for untreated fiber.