Optimal preparation of immobilized liposome-bound cellulase for hydrolysis of insoluble cellulose in an external loop airlift bioreactor

Immobilized liposome-bound cellulase (ILC) was optimally prepared for the ILC-catalyzed hydrolysis of insoluble cellulose in an external loop airlift bioreactor. The liposomes with mean diameters of 200, 100, and 50 nm were used to prepare three kinds of ILCs, i.e., ILC(200), ILC(100) and ILC(50), respectively. The activity and stability of ILC(100) were examined with soluble cellulose (CMC) in addition to the insoluble substrate of cellulose powder (CC31) in a shaking flask as well as the airlift bioreactors. The experiments were carried out with 45 degrees C and pH 4.8 being found to be optimal for the activity. The activity of ILC(100) was stable in either airlift or shaking flask bioreactor during the five times repeated hydrolyses of CC31 corresponding to a total reaction time of 240 h. This confirmed that the cellulase molecules were covalently bonded to the liposomes covalently bound to the chitosan gel beads. Nevertheless, the activity of ILC(100) with CMC steadily decreased throughout the repeated reactions, suggesting an adverse effect of CMC on the ILC(100) activity. Among the three ILCs, ILC(50) was found to be the most stable and productive biocatalyst during the repeated hydrolyses of insoluble CC31 in the airlift bioreactor. More than 70% of the initial activity of ILC(50) was retained even after the six times repeated reactions for 288 h. Conversely, the ILC(200) was found to be the most unstable catalyst. Such a difference in stability among these ILCs was suggested to be caused by the difference in physical stability of their liposome membranes to the liquid shear stress due to the rising bubbles and circulating liquid as well as that in the amount of the cellulase molecules unstably incorporated in the membranes. ILC(50) was thus shown to have the most potential for an efficient hydrolysis of insoluble cellulose in a practical airlift bioreactor.     

Development of effective modified cellulase for cellulose hydrolysis process

Cellulase was modified with amphilic copolymers made of alpha-allyl-omega-methoxy polyoxyalkylene (POA) and maleic acid anhydride (MAA) to improve the cellulose hydrolytic reactivity and cellulase separation. Amino groups of the cellulase molecule are covalently coupled with the MAA functional groups of the copolymer. At the maximum degree of modification (DM) of 55%, the modified cellulase activity retained more than 80% of the unmodified native cellulase activity. The modified cellulase shows greater stability against temperature, pH, and organic solvents, and demonstrated greater conversion of substrate than native cellulase does. Cellulase modification is also useful for controlling strong adsorption of cellulase onto substrate. Moreover, cellulase modified with the amphiphilic copolymer displays different separation characteristics which are new. One is a reactive two-phase partition and another is solubility in organic solvents. It appears that these characteristics of modified cellulase work very effectively in the hydrolysis of cellulose as a total system, which constitutes the purification of cellulase from culture broth, hydrolysis of cellulose, and recovery of cellulase from the reaction mixture. (c) 1995 John Wiley & Sons, Inc.     

Detrimental effect of cellulose oxidation on cellulose hydrolysis by cellulase

To effectively convert complex and recalcitrant biomass carbohydrates to simple platform sugars useful for fuel and chemicals production, mechanical or chemical pre-treatments are often required to make the carbohydrates more accessible for enzymatic hydrolysis. Due to their harsh conditions, some pre-treatments might negatively affect enzymatic hydrolysis because of events such as cellulose oxidation. To study how oxidative modification may impact cellulose's reactivity toward hydrolysis by cellulases, we prepared three cellulose substrates by cupric ion and hypochlorite oxidations, and subjected the derived celluloses to hydrolysis by various cellobiohydrolases from glycoside hydrolase families 6 and 7, and one cellulolytic Hypocrea jecorina extracellular enzyme mixture. We observed a profound decrease of enzymatic hydrolysis on the oxidized celluloses. The effect was attributed to the interference, from oxidized functional groups in cellulose, on its binding/activation in the active pocket/tunnel of cellobiohydrolases. Potential implication of the observed effect from cellulose oxidation on pre-treatment optimization and cellulase improvement was discussed.     

Importance of cellulase cocktails favoring hydrolysis of cellulose

Depolymerization of lignocellulosic biomass is catalyzed by groups of enzymes whose action is influenced by substrate features and the composition of cellulase preparation. Cellulases contain a mixture of variety of enzymes, whose proportions dictate the saccharification of biomass. In the current study, four cellulase preparation varying in their composition were used to hydrolyze two types of alkali-treated biomass (aqueous ammonia-treated rice straw and sodium hydroxide-treated rice straw) to study the effect on catalytic rate, saccharification yields, and sugar release profile. We found that substrate features affected the extent of saccharification but had minimal effect on the sugar release pattern. In addition, complete hydrolysis to glucose was observed with enzyme preparation having at least a cellobiase units (CBU)/carboxymethyl cellulose (CMC) ratio (>0.15), while a modified enzyme ratio can be used for oligosaccharide synthesis. Thus, cellulase preparation with defined ratios of the three main enzymes can improve the saccharification which is of utmost importance in defining the success of lignocellulose-based economies.     

Column cellulose hydrolysis reactor: An efficient cellulose hydrolysis reactor with continuous cellulase recycling

Summary The use of a column cellulose hydrolysis reactor with continuous enzyme recycling was demonstrated by incorporating a continuous ultrafiltration apparatus at the effluent end of the column reactor. Using this setup, over 90% (w/v) cellulose hydrolysis was achieved, resulting in an average sugar concentration of 6.8% (w/v) in the effluent stream. The output of the system was 1.98 g of reducing sugar/l/h with a ratio of 87% (w/v) of the reducing sugars being monomeric sugars. Batch hydrolysis reactors were less effective, resulting in 57% (w/v) of the cellulose being hydrolyzed. The output of the batch reactor was 1.33 g of reducing sugar/l/h with similar product concentrations and percentage of monomeric sugars. The ratio of reducing sugar/filter paper unit of cellulase activity for the column method was 69.1 mg/U as compared to only 21.2 mg/U for the batch reactor.     

Synergistic proteins for the enhanced enzymatic hydrolysis of cellulose by cellulase

Reducing the enzyme loadings for enzymatic saccharification of lignocellulose is required for economically feasible production of biofuels and biochemicals. One strategy is addition of small amounts of synergistic proteins to cellulase mixtures. Synergistic proteins increase the activity of cellulase without causing significant hydrolysis of cellulose. Synergistic proteins exert their activity by inducing structural modifications in cellulose. Recently, synergistic proteins from various biological sources, including bacteria, fungi, and plants, were identified based on genomic data, and their synergistic activities were investigated. Currently, an up-to-date overview of several aspects of synergistic proteins, such as their functions, action mechanisms and synergistic activity, are important for future industrial application. In this review, we summarize the current state of research on four synergistic proteins: carbohydrate-binding modules, plant expansins, expansin-like proteins, and Auxiliary Activity family 9 (formerly GH61) proteins. This review provides critical information to aid in promoting research on the development of efficient and industrially feasible synergistic proteins.     

Cellulose hydrolysis and cellulase adsorption after pretreatment of cellulose materials

A number of cellulosic materials were chemically and physically treated before being incubated with cellulase from Penicillium funiculosum. The most effective pretreatment for maximum increase in enzyme adsorption and rate of saccharification was a combination of homogenisation-ultrasonification-NaOH (10% w/v) treatment.     

Characterization of dicarboxylic acids for cellulose hydrolysis

In this paper, we show that dilute maleic acid, a dicarboxylic acid, hydrolyzes cellobiose, the repeat unit of cellulose, and the microcrystalline cellulose Avicel as effectively as dilute sulfuric acid but with minimal glucose degradation. Maleic acid, superior to other carboxylic acids reported in this paper, gives higher yields of glucose that is more easily fermented as a result of lower concentrations of degradation products. These results are especially significant because maleic acid, in the form of maleic anhydride, is widely available and produced in large quantities annually.     

Characterization of direct cellulase immobilization with superparamagnetic nanoparticles

Abstract

Methods of cellulase immobilization on magnetic particles via glutaraldehyde binding were studied. The binding was confirmed by transmission electronic microscopy (TEM), X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR) and vibrating sample magnetometry (VSM). Samples analyzed by TEM and XRD showed that the magnetic particles with or without bound cellulase were all nanosized particles with a mean diameter of 11.5 nm, and the binding process did not cause significant changes in particle size and structure. Analysis by FTIR showed that the binding of cellulase to the magnetic nanoparticles might be via covalent bonding between residual amine groups on Fe₃O₄ nanoparticles and amine groups of the cellulase. The VSM analysis showed that magnetic nanoparticles with or without bound cellulase were all superparamagnetic. The immobilized cellulase had a wider pH and temperature range and improved storage stability compared with the free enzyme. Determination of the Michaelis constants revealed that the immobilized cellulase had a greater affinity for the cellulosic substrate than the free enzyme. The immobilized cellulase showed better performance on hydrolysis of steam-exploded corn stalks than of bleached sulfite bagasse pulp.     

Pre-steady-state kinetics for hydrolysis of insoluble cellulose by cellobiohydrolase Cel7A

The transient kinetic behavior of enzyme reactions prior to the establishment of steady state is a major source of mechanistic information, yet this approach has not been utilized for cellulases acting on their natural substrate, insoluble cellulose. Here, we elucidate the pre-steady-state regime for the exo-acting cellulase Cel7A using amperometric biosensors and an explicit model for processive hydrolysis of cellulose. This analysis allows the identification of a pseudo-steady-state period and quantification of a processivity number as well as rate constants for the formation of a threaded enzyme complex, processive hydrolysis, and dissociation, respectively. These kinetic parameters elucidate limiting factors in the cellulolytic process. We concluded, for example, that Cel7A cleaves about four glycosidic bonds/s during processive hydrolysis. However, the results suggest that stalling the processive movement and low off-rates result in a specific activity at pseudo-steady state that is 10-25-fold lower. It follows that the dissociation of the enzyme-substrate complex (half-time of ~30 s) is rate-limiting for the investigated system. We suggest that this approach can be useful in attempts to unveil fundamental reasons for the distinctive variability in hydrolytic activity found in different cellulase-substrate systems.     

Substrate heterogeneity causes the nonlinear kinetics of insoluble cellulose hydrolysis

Nonlinear kinetics are commonly observed in the enzymatic hydrolysis of cellulose. This nonlinearity could be explained by any or all of the following three factors: enzyme inactivation, product inhibition, or substrate heterogeneity. In this study, four different approaches were applied to test the above hypotheses using two Thermomonospora fusca endocellulases, E2 and E5. The lack of stimulation of cellulase activity by beta-glucosidase rules out the possibility of product inhibition as a cause of the observed nonlinearity. The results from the other three approaches all provide strong evidence against enzyme inactivation and strong evidence for substrate heterogeneity as the cause of the nonlinear kinetics. The most direct evidence for substrate heterogeneity is that pretreatment of swollen cellulose with either E2cd or E5cd gave a product that was hydrolyzed at a much (3- to 4-fold) slower rate than untreated swollen cellulose even though the initial treatment degraded only 15-18% of the substrate. Furthermore, the activation energy of E2 catalyzed hydrolysis of swollen cellulose increased from 10 kcal/mol for the initial rate to 29 kcal/mol for hydrolysis after 24% digestion.     

Kinetics of solka floc cellulose hydrolysis by trichoderma viride cellulase

A product inhibition model is developed to describe the hydrolysis of cellulose by the Trichoderma viride enzyme system. It is assumed that noncompetitive inhibition by cellobiose dominates the reaction kinetics. Experiments show that this is indeed a reasonable assumption for initial cellulose concentrations of up to 15 g/liter and at hydrolysis extents up to 65′. Kinetic parameters were determined for the noncompetitive inhibitionmodel in batch experiments with durations of up to 1.5 hr. These parameterswere then used in predicting reaction progress for up to 10 hr. Cellobiose was added to the reaction mixture at the onset of some runs and againreliable predictions were obtained for up to 8 hr of hydrolysis. Finally reaction was carried out in a membrane reactor whereby the product cellobiose was being continuously removed and again reasonable predictability was obtained with a higher net reaction rate.     

Effect of cellulase mole fraction and cellulose recalcitrance on synergism in cellulose hydrolysis and binding

Elucidating the molecular mechanisms that govern synergism is important for the rational engineering of cellulase mixtures. Our goal was to observe how varying the loading molar ratio of cellulases in a binary mixture and the recalcitrance of the cellulose to enzymatic degradation influenced the degree of synergistic effect (DSE) and degree of synergistic binding (DSB). The effect of cellulose recalcitrance was studied using a bacterial microcrystalline cellulose (BMCC), which was exhaustively hydrolyzed by a catalytic domain of Cel5A, an endocellulase. The remaining prehydrolyzed BMCC (PHBMCC) was used to represent a recalcitrant form of cellulose. DSE was observed to be sensitive to loading molar ratio. However, on the more recalcitrant cellulose, synergism decreased. Furthermore, the results from this study reveal that when an exocellulase (Cel6B) is mixed with either an endocellulase (Cel5A) or a processive endocellulase (Cel9A) and reacted with BMCC, synergism is observed in both hydrolysis and binding. This study also revealed that when a "classical" endocellulase (Cel5A) and a processive endocellulase (Cel9A) are mixed and reacted with BMCC, only limited synergism is observed in reducing sugar production; however, binding is clearly increased by the presence of the Cel5A.     

Assessment of methods to determine minimal cellulase concentrations for efficient hydrolysis of cellulose

Summary The enzyme loading needed to achieve substrate saturation appeared to be the most economical enzyme concentration to use for hydrolysis, based on percentage hydrolysis. Saturation was reached at 25 filter paper units per gram substrate on Solka Floc BW300, as determined by studying (a) initial adsorption of the cellulase preparation onto the substrate, (b) an actual hydrolysis or (c) a combined hydrolysis and fermentation (CHF) process. Initial adsorption of the cellulases onto the substrate can be used to determine the minimal cellulase requirements for efficient hydrolysis since enzymes initially adsorbed to the substrate have a strong role in governing the overall reaction. Trichoderma harzianum E58 produces high levels of -glucosidase and is able to cause high conversion of Solka Floc BW300 to glucose without the need for exogenous -glucosidase. End-product inhibition of the cellulase and -glucosidase can be more effectively reduced by employing a CHF process than by supplemental -glucosidase.Offprint requests to: C. M. Hogan     

Effect of ethanol and yeast on cellulase activity and hydrolysis of crystalline cellulose

The process of bioconverting lignocellulosic materials into ethanol in the simultaneous saccharification and fermentation system depends upon the activity of Penicillium decumbens cellulase. The influence of both ethanol and the yeast on this cellulase activity has been studied and it has been found that ethanol in concentrations between 1% and 7% inhibits the enzymatic hydrolysis of crystalline cellulose but the inhibition is reversible. At ethanol concentrations between 1% and 9%, the activity of β-glucosidase increases with increasing ethanol concentration. Yeast has no effect on the enzymatic activity.     

Kinetic modeling of cellulose hydrolysis with first order inactivation of adsorbed cellulase

Enzymatic hydrolysis involves complex interaction between enzyme, substrate, and the reaction environment, and the complete mechanism is still unknown. Further, glucose release slows significantly as the reaction proceeds. A model based on Langmuir binding kinetics that incorporates inactivation of adsorbed cellulase was developed that predicts product formation within 10% of experimental results for two substrates. A key premise of the model, with experimental validation, suggests that V(max) decreases as a function of time due to loss of total available enzyme as adsorbed cellulases become inactivated. Rate constants for product formation and enzyme inactivation were comparable to values reported elsewhere. A value of k(2)/K(m) that is several orders of magnitude lower than the rate constant for the diffusion-controlled encounter of enzyme and substrate, along with similar parameter values between substrates, implies a common but undefined rate-limiting step associated with loss of enzyme activity likely exists in the pathway of cellulose hydrolysis.     

固定化纤维素酶对预处理纤维素原料水解效果的影响

以壳聚糖为载体用交联法制备固定化纤维素酶,考察固定化纤维素酶对蒸爆、球磨、超声波、喷淋、高温预处理玉米秸秆纤维素原料的酶解效果.结果表明:物料经蒸爆预处理后酶水解效率最高可以达到95%,球磨预处理水解效率次之,达到60%.用电镜和FT-IR对处理前后秸秆结构进行表征分析,证明预处理对物料的物理结构及化学组成有一定的影响.蒸爆法和球磨法可以使物料致密的天然结构彻底破坏,从而增加物料的比表面积;蒸爆预处理可以使纤维素内部氢键和官能团改变,使物料更易于酶解.     

Adsorption of cellulase on cellulose: Effect of physicochemical properties of cellulose on adsorption and rate of hydrolysis

In the cellulase-cellulose reaction system, the adsorption of cellulase on the solid cellulose substrate was found to be one of the important parameters that govern the enzymatic hydrolysis rate of cellulose. The adsorption of cellulase usually parallels the rate of hydrolysis of cellulose. The affinity for cellulase varies depending on the structural properties of cellulose. Adsorption parameters such as the half-saturation constant, the maximum adsorption constant, and the distribution coefficient for both the cellulase and cellulsoe have been experimentally determined for several substrates. These adsorption parameters vary with the source of cellulose and the pretreatment methods and are correlated with the crystallinity and the specific surface area of cellulose substrates. The changing pattern of adsorption profile of cellulase during the hydrolysis reaction has also been elucidated. For practical utilization of cellulosic materials, the cellulose structural properties and their effects on cellulase adsorption, and the rate of hydrolysis must be taken into consideration.     

Kinetic studies of enzymatic hydrolysis of insoluble cellulose: (II). Analysis of extended hydrolysis times

The kinetics of enzymatic hydrolysis of pure insoluble cellulose by means of unpurified culture filtrate of Trichoderma reesei was studied, emphasizing the kinetic characteristics associated with the extended hydrolysis times. The changes in the hydrolysis rate and extent of soluble protein adsorption during the progress of reaction, either apparent or intrinsic, were investigated. The hydrolysis rate declined drastically during the initial hours of hydrolysis. The factors causing the reduction in the hydrolysis rate were examined; these include the transformation of cellulose into a less digestible form and product inhibition. The structural transformation can be partially explained by changes in the crystallinity index and surface area. The product inhibition was caused by the deactivation of the adsorbed soluble protein by the products, which essentially represents the so-called "un-competitive" inhibition. The kinetics of beta-glucosidase were also studied. The result has shown that the action of beta-glucosidase is competitively inhibited by glucose. It has been found that the integrated form of the initial rate expression cannot be used in predicting the progress of reaction because the digestibility of cellulose changes drastically as the hydrolysis proceeds, and that the rate expression for enzymatic hydrolysis of cellulose cannot be simplified or approximated by resorting to the pseudo-steady-state assumption. A mechanistic kinetic model of cellulose hydrolysis should include the following major influencing factors: (1)mode of action of enzyme, (2) structure of cellulose, and (3) mode of interaction between the enzyme and cellulose molecules.     

Enzymatic hydrolysis of cellulose: Evaluation of cellulase culture filtrates under use conditions

Culture filtrates from three mutant strains of Trichoderma reesei grown on lactose and on cellulose were compared under use conditions on four cellulose substrates. Cellulose culture filtrates contained five to six times as much cellulase as lactose culture filtrates. Unconcentrated cellulose culture filtrates produced up to 10% sugar solutions from 15% cellulose in 24 h. Specific activity in enzyme assays and efficiency in saccharification tests were low for enzymes from all the mutants. Over a wide range the percent saccharification of a substrate in a given times was directly proportional to the logarithm of the ratio of initial concentrations of enzyme and substrate. As a result of this, dilute enzyme is more efficient than concentrated enzyme, but if high sugar concentrations are desired, very large quantities of enzyme are required. Since the slopes of these plots varied, the relative activity of cellulase on different substrates may be affected by enzyme concentration.