Sampling, dilution, and loading device-coupled high-performance liquid chromatography method for successive on-line analyses of major carbohydrate products in immobilized cellulase hydrolysate of paper cellulose.

A device with direct sampling, dilution, and sample loading has been utilized to couple a vertically hanging immobilized cellulase bioreactor with a high-performance liquid chromatography system to make a successful on-line analysis of the 3 major carbohydrate products: glucose, xylose, and cellobiose in the cellulase hydrolysate. The coupled analysis system for successive on-line monitoring of the enzymatic hydrolysis of paper cellulose was stable over a period of 96 h. A comparative study involving 4 different quantification methods for the 3 carbohydrates was conducted. The internal standard calibration method with on-line dilution was observed to be the best procedure in terms of accuracy, precision, limit of detection, and systematic error reduction. With this method, the analysis for glucose had 99.9% accuracy, RSD below 2.4%, and a 2.1 ppm detection limit. Matrix matching was found important in the preparation of standard calibration curves with refractive index detection.     

Cellulase production by Trichoderma reesei using sawdust hydrolysate

Sawdust hydrolysates were investigated for their ability to support cell growth and cellulase production, and for potential inhibition of Trichoderma reesei Rut C30. Simultaneous fermentations were conducted to compare the hydrolysate-based media with the controls having equivalent concentrations of glucose and Avicel cellulose. Six hydrolysates differing in the boiling durations in the hydrolysis procedure were evaluated. The hydrolysates were found to support cell growth and induce active cellulase synthesis. The maximum specific cellulase production rate was 0.046 filter paper units (FPU)/(g of cells · h) in the hydrolysate-based systems, much higher than that (0.017 FPU/[g of cells · h]) in the controls.     

纤维素酶降解纤维素机理的研究

纤维素是自然界中含量最多的一类碳水化合物,同时它也是地球上数量最大的可再生资源。纤维素酶是一种高活性生物催化剂,在纤维素类资源的利用方面发挥重要的作用。本文综述了纤维素、纤维素酶的分子结构和纤维素酶对纤维素的降解机理,影响酶解的主要因素以及提高酶解效率的主要措施,并对纤维素酶研究存在的问题以及今后的发展作了进一步展望。     

Use of Cellulase Inhibitors to Produce Cellobiose

The economics driving biorefinery development requires high value-added products such as cellobiose for financial feasibility. This research describes a simple technology for increasing cellobiose yields during lignocellulosic hydrolysis. The yield of cellobiose produced during cellulose hydrolysis was maximized by modification of reaction conditions. The addition of an inhibitor from the group that includes glucose oxidase, gluconolactone, and gluconic acid during cellulase hydrolysis of cellulose increased the amount of cellobiose produced. The optimal conditions for cellobiose production were determined for four factors; reaction time, cellulase concentration, cellulose concentration, and inhibitor concentration using a Box-Behnken experimental design. Gluconolactone in the cellulase system resulted in the greatest production of cellobiose (31.2%) from cellulose. The yield of cellobiose was 23.7% with glucose oxidase, similar to 21.9% with gluconic acid.     

Characterization and performance of immobilized amylase and cellulase

The performance of cellulase and amylase immobilized on siliceous supports was investigated. Enzyme uptake onto the support depended on the enzyme source and immobilization conditions. For amylase, the uptake ranged between 20 and 60%, and for cellulase, 7–10%. Immobilized amylase performance was assessed by batch kinetics in 100–300 g/L of corn flour at 65°C. Depending on the substrate and enzyme loading, between 40 and 60% starch conversion was obtained. Immobilized amylase was more stable than soluble amylase. Enzyme samples were preincubated in a water bath at various temperatures, then tested for activity. At 105°C, soluble amylase lost ∼55% of its activity, compared with ∼30% loss for immobilized amylase. The performance of immobilized cellulase was evaluated from batch kinetics in 10 g/L of substrate (shredded wastepaper) at 55°C. Significant hydrolysis of the wastepaper was also observed, indicating that immobilization does not preclude access to and hydrolysis of insoluble cellulose.     

Cellulase Hydrolysis of Unsorted MSW

A recent development in waste management and engineering has shown that the cellulase can be used for the liquefaction of organic fractions in household waste. The focus of this study was to optimize the enzyme hydrolysis of thermally treated municipal solid waste (MSW) by the addition of surfactant. Concurrently, the enzyme performance was analysed on pure cellulose in a solution of MSW wastewater. Results showed no effect of surfactant addition to the hydrolysis media as measured by viscosity and particle size distribution. MSW treatment wastewater was found to contain a high amount of calcium, potassium, sodium, chloride and others that may affect cellulolytic enzymes. Cellulase performance showed no effect of adding the metal ion-chelating agent EDTA to the solution. The cellulases were stable, tolerated and functioned in the presence of several contaminants.     

Enhanced glucose production from cellulose using coimmobilized cellulase and β-glucosidase

β-Glucosidase was covalently immobilized alone and coimmobilized with cellulase using a hydrophilic polyurethane foam (Hypol®FHP 2002). Immobilization improved the functional properties of the enzymes. When immobilized alone, the K_m for cellobiose of β-glucosidase was decreased by 33% and the pH optimum shifted to a slightly more basic value, compared to the free enzyme. Immobilized β-glucosidase was extremely stable (95% of activity remained after 1000 h of continuous use). Coimmobilization of cellulase and β-glucosidase produced a cellulose-hydrolyzing complex with a 2.5-fold greater rate of glucose production for soluble cellulose and a four-fold greater increase for insoluble cellulose, compared to immobilized cellulase alone. The immobilized enzymes showed a broader acceptance of various types of insoluble cellulose substrates than did the free enzymes and showed a long-term (at least 24 h) linear rate of glucose production from microcrystalline cellulose. The pH optimum for the coimmobilized enzymes was 6.0. This method for enzyme immobilization is fast, irreversible, and does not require harsh conditions. The enhanced glucose yields obtained indicate that this method may prove useful for commercial cellulose hydrolysis.     

Inhibition Performance of Lignocellulose Degradation Products on Industrial Cellulase Enzymes During Cellulose Hydrolysis

This study examined the inhibition performance by the major lignocellulose degradation products, including organic acids, furan derivatives, lignin derivatives, and ethanol, on a broadly used commercial cellulase enzyme Spezyme CP (Genencor International, Rochester, NY, USA) to cellulose hydrolysis at both the well-mixing state (shaking flask) and the static state (test tube). The cellulase activity in the cellulase complex of Spezyme CP was assumed to be one single “cellulase”, and the apparent kinetic parameters of this cellulase enzyme were measured as an approximate index of the inhibitory effect to the industrial cellulase enzyme. The inhibition performance of these degradation products was compared and analyzed using the determined apparent kinetic parameters. All the degradation products strongly inhibit the cellulose hydrolysis by cellulase enzyme, and the inhibitions on cellulase were all competitive type. The order of the inhibition strength by the lignocellulose degradation products to cellulase is lignin derivatives > furan derivatives > organic acids > ethanol. This study gave a quantitative view to the enzymatic hydrolysis of lignocellulose under the inhibition performance of the lignocellulose degradation products and will help to understand the lignocellulose recalcitrance to enzyme hydrolysis.     

纤维素酶的二步分离纯化新工艺

 以普通定性滤纸为底物 ,经碱处理后 ,研究其对纤维素酶的亲和吸附作用。结果表明 ,普通定性滤纸对纤维素酶具有比较强的特异性吸附作用 ,能够从粗酶液中分离出纤维素酶 ,再经POROS 2 0HQ阴离子交换柱纯化后即可得到电泳纯的纤维素酶。该法大大简化了传统的纤维素酶纯化工艺 ,所得的纤维素酶活力极高 ,比活达 35 0U/mg以上 ,滤纸一步吸附后纤维素酶的纯化倍数为 9 5 5 ,活性回收率在 10 %左右。纯化后的纤维素酶为内切 β 葡聚糖酶 ,相对分子质量为 6 0 0 0 0 ,最佳 pH为 4 0 ,最佳温度为 70℃。     

Effect of Bovine Serum Albumin (BSA) on Enzymatic Cellulose Hydrolysis

Bovine serum albumin (BSA) was added to filter paper during the hydrolysis of cellulase. Adding BSA before the addition of the cellulase enhances enzyme activity in the solution, thereby increasing the conversion rate of cellulose. After 48 h of BSA treatment, the BSA adsorption quantities are 3.3, 4.6, 7.8, 17.2, and 28.3 mg/g substrate, each with different initial BSA concentration treatments at 50 °C; in addition, more cellulase was adsorbed onto the filter paper at 50 °C compared with 35 °C. After 48 h of hydrolysis, the free-enzyme activity could not be measured without the BSA treatment, whereas the remaining activity of the filter paper activity was approximately 41 % when treated with 1.0 mg/mL BSA. Even after 96 h of hydrolysis, 25 % still remained. Meanwhile, after 48 h of incubation without substrate, the remaining enzyme activities were increased 20.7 % (from 43.7 to 52.7 %) and 94.8 % (from 23.3 to 45.5 %) at 35 and 50 °C, respectively. Moreover, the effect of the BSA was more obvious at 35 °C compared with 50 °C. When using 15 filter paper cellulase units per gram substrate cellulase loading at 50 °C, the cellulose conversion was increased from 75 % (without BSA treatment) to ≥90 % when using BSA dosages between 0.1 and 1.5 mg/mL. Overall, these results suggest that there are promising strategies for BSA treatment in the reduction of enzyme requirements during the hydrolysis of cellulose.     

Cellulase production and regulation byAgaricus bisporus

Cellulose is degraded during the growth of the cultivated mushroomA. bisporus on composted straw. At the time of sporophore enlargement, a marked increase in extracellular endocellulase activity occurs. A high level of enzyme activity is maintained during subsequent cropping cycles. Some of the factors affecting growth and production of extracellular endocellulase activity byA. bisporus cultured in simple defined liquid media have been examined. Endocellulase production by the fungus closely paralleled mycelial growth in cultures containing microcrystalline cellulose. The enzyme was induced by various celluloses and cellobiose. In the presence of a cellulose inducer, glucose and cellobiose repressed enzyme production. Endocellulase activity in culture filtrates was inversely related to cellulose concentration in the culture. Although the activity of free enzyme was low, in high concentrations of cellulose more cellulose was degraded. Evidence was obtained for the existence of two forms of endocellulase activity. One form adsorbed strongly to cellulose and was predominant in cultures low in cellulose. In cultures with a high cellulose content, a nonadsorbable form of the enzyme was more abundant. It is suggested that the pattern of cellulase activities produced whenA. bisporus is grown on different concentrations of cellulose is partly accounted for by its adsorption to the cellulose.     

Lactic acid production from cellulosic material by synergetic hydrolysis and fermentation

The hydrolysis process on corncob residue was catalyzed synergetically by the cellulase from Trichoderma reesei and the immobilized cellobiase. The feedback inhibition to cellulase reaction caused by the accumulation of cellobiose was eliminated efficiently. The hydrolysis yield of corncob residue was 82.5%, and the percentage of glucose in the reducing sugar reached 88.2%. The glucose in the cellulosic hydrolysate could be converted into lactic acid effectively by the immobilized cells of Lactobacillus delbrueckii. When the enzymatic hydrolysis of cellulose and the fermentation of lactic acid were coupled together, no glucose was accumulated in the reaction system, and the feedback inhibition caused by glucose was also eliminated. Under the batch process of synergetic hydrolysis and lactic acid fermentation with 100 g/L of cellulosic substrate, the conversion efficiency of lactic acid from cellulose and the productivity of lactic acid reached 92.4% and 0.938 g/(L·h), respectively. By using a fed-batch technique, the total concentration of cellulosic substrate and lactic acid in the synergetic process increased to 200 and 107.5 g/L, respectively, whereas the dosage of cellulase reduced from 20 to 15 IU/g of substrate in the batch process. The results of the bioconversion of renewable cellulosic resources were significant.     

A Fractal-Like Kinetic Equation to Investigate Temperature Effect on Cellulose Hydrolysis by Free and Immobilized Cellulase

According to fractal-like theory in the heterogeneous system, a cellulase-catalyzed kinetic equation that contained two parameters (rate constant k and fractal dimension h) was deduced. The equation described directly the mathematical relationship between reducing sugar concentration and hydrolytic time, and accurately fitted the experimental data of free/immobilized cellulase at 37, 40, 44, 47, and 50?°C (R (2)?>?0.99). The fitted h value is estimated as a constant (0.6148) in these tested temperatures. The fitted k value increased with temperature increase, and the relationship agreed with Arrhenius equation (R (2)?>?0.98). The fractal-like equation could predict accurately the experimental data at low temperature 34?°C for free/immobilized cellulase and high temperature 53?°C for immobilized cellulase, but the prediction at 53?°C for free cellulase was not accurate enough due to its lower stability than immobilized cellulase. The application of fractal-like theory in cellulase kinetics is successful.     

提高纤维素酶水解效率和降低水解成本

在我国可大量转化乙醇的是纤维质材料。纤维质材料转化乙醇的关键问题是纤维质转化为糖的过程,提高纤维素酶转化效率的方法有:(1)对纤维质材料进行预处理;(2)研究纤维素酶的最适作用条件;(3)纤维素酶的重复利用;(4)合理的发酵工艺等。本文分析了纤维素的结构以及纤维素酶的作用方式,总结了目前研究较多的几种纤维质材料预处理方法,及其对纤维素酶水解率的影响,并对研究纤维素酶的最适作用条件、纤维素酶的重复利用以及合理的发酵工艺进行了综述和分析。     

Immobilized Biomaterials—Techniques and Applications

The immobilization of enzymes, cofactors and whole cells offers the potential of economical exploitation of biomaterial activity. Enzymes or cells can be immobilized by binding to carriers through adsorption or covalent linkages or by entrapment in a polymer matrix or microencapsulation. The development of immobilized biomaterial systems requires the involvement of biochemical, kinetics and reactor design principles. Examples of commercial scale applications of immobilized biomaterial systems are the production of a fructose-rich syrup from starch and the synthesis of L -amino acids by selective hydrolysis of N-acyl-D ,L -amino acids.     

Bioconversion of mixed solids waste to ethanol

A mixed solids waste (MSW) feedstock, comprising construction lumber waste (35% oven-dry basis), alm ond treeprunings (20%), wheat straw (20%), office waste paper (12.5%), and newsprint (12.5%), was converted to ethanol via dilute-acid pretreatment followed by enzymatic hydrolysis and yeast fermentation. The MSW was pretreated with dilute sulfuricacid (0.4% w/w) at 210°C for 3 min in a 4-L stea mexplosion reactor, then washed with water to recover the solubilized hemicellulose. The digestibility of water-washed, pretreated MSW was 90% in batch enzymatic hydrolysis at 66 FPU/g cellulose. Using an enzyme-recycle bioreactor system, greater than 90% cellulose hydrolysis was achieved at a net enzyme loading of about 10 FPU/g cellulose. Enzyme recycling using mebrane filtration and a fed-batch fermentation technique is a promising option for significantly reducing the cost of enzyme in cellulose hydrolysis. The hexosesugars were readily fermentable using a Saccharomyces cerevisiae yeast strain that was adapted to the hydrolysate. Solid residue after enzyme digestion was subjected to various furnace experiments designed to assess the fouling and slagging characteristics. Results of these analyses suggest the residue to be of a low to moderate slagging and fouling type if burned by itself.     

Water Hyacinth as Carbon Source for the Production of Cellulase by <Emphasis Type="Italic">Trichoderma reesei</Emphasis>

Water hyacinth (Eichhornia crassipes), an aquatic weed common to the subtropic/tropical regions, was utilized as an inexpensive lignocellulosic substrate for production of cellulase by Trichoderma reesei. The effects of process parameters like substrate pretreatment, substrate concentration, initial medium pH, mode of inoculation, and incubation temperature on cellulase production were investigated. Under optimal conditions, a maximal cellulase activity of 0.22 ± 0.04 IU/ml (approximately 73.3 IU/g cellulose) was recorded at the end of 15-day incubation period. Specific activity of the enzyme was 6.25 IU/mg protein. Hydrolysis of 1% substrate (water hyacinth) using crude enzyme dosage of 1.2 IU/g water hyacinth showed 28.7% saccharification in 1 h. The observations in present study indicate that saccharification of cellulose from water hyacinth was significantly higher by laboratory-produced cellulase than the commercial blend.     

基于聚乙烯醇/Fe2O3纳米颗粒的纤维素酶固定化

以聚乙烯醇/Fe2O3磁性纳米颗粒为纤维素酶固定化载体, 通过反复冻融的方法成功地实现了纤维素酶固定化. 采用透射电镜、红外光谱仪、振动样品磁强度计对固定化酶复合体进行了表征, 结果显示, 固定化酶复合体为大小约1 μm的微凝胶团, 内含10 nm左右的Fe2O3纳米颗粒. 研究影响固定化因素后发现, 当pH为6, 固定化时间为11 h, 纤维素酶/PVA质量比为4, PVA/Fe质量比为50时, 固定化纤维素酶效果最好. 通过该方法固定后酶活回收率达42%, 酶水解效率显著提高, 经过5次反应后的固定化酶相对酶活力保留50%以上. 因此, 基于聚乙烯醇/Fe2O3纳米颗粒的纤维素酶固定有利于酶的循环使用并显著提高酶的使用效率, 是一种有效固定化纤维素酶的新方法.     

Production of Cellulase from Kraft Paper Mill Sludge by Trichoderma Reesei Rut C-30

Paper mill sludge is a solid waste material generated from pulping and papermaking operations. Because of high glucan content and its well-dispersed structure, paper mill sludges are well suited for bioconversion into value-added products. It also has high ash content originated from inorganic additives used in papermaking, which causes hindrance to bioconversion. In this study, paper mill sludges from Kraft process were de-ashed by a centrifugal cleaner and successive treatment by sulfuric acid and sodium hydroxide, and used as a substrate for cellulase production. The treated sludge was the only carbon source for cellulase production, and predominantly inorganic nutrients were used as the nitrogen source for this bioprocess. The cellulase enzyme produced from the de-ashed sludge exhibited cellulase activity of 8 filter paper unit (FPU)/mL, close to that obtainable from pure cellulosic substrates. The yield of cellulase enzyme was 307 FPU/g glucan of de-ashed sludge. Specific activity was 8.0 FPU/mg protein. In activity tests conducted against the corn stover and α-cellulose, the xylanse activity was found to be higher than that of a commercial cellulase. Relatively high xylan content in the sludge appears to have induced high xylanase production. Simultaneous saccharification and fermentation (SSF) was performed using partially de-ashed sludge as the feedstock for ethanol production using Sacharomyces cerevisiae and the cellulase produced in-house from the sludge. With 6% (w/v) glucan feed, ethanol yield of 72% of theoretical maximum and 24.4 g/L ethanol concentration were achieved. These results were identical to those of the SSF using commercial cellulases.     

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.