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。     

Cellulase production on bagasse pretreated with hot water

Because pretreatment of biomass with hot water only in differential flow systems offers very digestible cellulose and potentially less inhibition by liquid hydrolysate, solids and liquid hydrolysate from bagasse pretreated with hot water were fed to a batch cellulase production system using the Rut C30 strain of Trichoderma reesei to determine the suitability of these substrates for cellulase production. The organism was found to be sensitive to inhibitors in the liquid hydrolysate but could be adapted to improve its tolerance. In addition, filtering of the material reduced inhibitory effects. The organism was also sensitive to some component in the solids, and they had to be washed heavily to achieve good growth and cellulase production rates. Even then, a lag was found before enzyme production would commence on pretreated solids whereas no such lag was experienced with Solka Floc. However, once enzyme production began, as high and even somewhat greater cellulase productivities were realized with washed pretreated solids. Adding lignin to Solka Floc delayed enzyme production, suggesting that lignin or other materials in the lignin solids could cause the lag observed for pretreated bagasse, but more studies are needed to resolve the actual reason for this delay.     

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.     

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.     

Active nuclear shuffling system using a swollen conidium of Trichoderma reesei

Cellulase hyperproducers of Trichoderma reesei can be constructed using autopolyploidization and haploidization techniques. To increase the efficiency of this method, the active nuclear shuffling system in a swollen conidium was effective. A dried mature green conidium of a model strain, T. reesei QM6a (IFO 31326), was swollen to make room for a larger autopolyploid nucleus. After colchicine treatment, a larger autopolyploid nucleus was produced in such a swollen conidium. Benomyl treatment of swollen conidia generated multiple smaller nuclei from one larger autopolyploid nucleus. Those smaller nuclei were transported through conidia to mycelia after germination. This system could contribute to increasing the efficiency of genetic shuffling.     

Dynamics of cellulase production by glucose grown cultures of Trichoderma reesei Rut-C30 as a response to addition of cellulose

An economic process for the enzymatic hydrolysis of cellulose would allow utilization of cellulosic biomass for the production of easily fermentable low-cost sugars. New and more efficient fermentation processes are emerging to convert this biologic currency to a variety of commodity products with a special emphasis on fuel ethanol production. Since the cost of cellulase production currently accounts for a large fraction of the estimated total production costs of bioethanol, a significantly less expensive process for cellulase enzyme production is needed. It will most likely be desirable to obtain cellulase production on different carbon sources—including both polymeric carbohydrates and monosaccharides. The relation between enzyme production and growth profile of the microorganism is key for designing such processes. We conducted a careful characterization of growth and cellulase production by the soft-rot fungus Trichoderma reesei. Glucosegrown cultures of T. reesei Rut-C30 were subjected to pulse additions of Solka-floc (delignified pine pulp), and the response was monitored in terms of CO₂ evolution and increased enzyme activity. There was an immediate and unexpectedly strong CO₂ evolution at the point of Solka-floc addition. The time profiles of induction of cellulase activity, cellulose degradation, and CO₂ evolution are analyzed and discussed herein.     

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.     

Profile of enzyme production by trichoderma reesei grown on corn fiber fractions

Corn fiber is the fibrous by-product of wet-mill corn processing. It typically consists of about 20% starch, 14% cellulose, and 30% hemicellulose in the form of arabinoxylan. Crude corn fiber (CCF) was fractionated into de-starched corn fiber (DSCF), corn fiber with cellulose (CFC) enriched, and corn fiber arabinoxylan (CFAX), and these fractions were evaluated as substrates for enzyme production by Trichoderma reesei. T. reesei QM9414 and Rut C-30 grew on CCF, DSCF, CFC, or CFAX and secreted a number of hydrolytic enzymes. The enzymes displayed synergism with commercial cellulases for corn fiber hydrolysis. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Polykaryon formation using a swollen conidium of Trichoderma reesei

The cellulolytic fungus, Trichoderma has oval and mononucleate conidia. When these conidia are incubated in a liquid medium, they begin to swell and their shape becomes spherical followed by an increase in inner space. In such swollen conidia, it is possible to produce a larger autopolyploid nucleus using a mitotic arrester compared with the case of the original conidia. In this study, polykaryon formation was attempted using these swollen conidia. Dried mature green conidia of Trichoderma reesei QM6a (IFO 31326) were incubated in Mandel's medium in order to swell. The swollen conidia were treated with a mitotic arrester, colchicine, for autopolyploidization. After autopolyploidization, polykary on formation was carried out using the swollen conidia. After the treatment, multiple smaller nuclei whose diameter was almost the same as that of the original strain were generated from an autopolyploid nucleus in a swollen conidium. A cellulase hyperproducer without decrease in growth rate could be selected using such swollen conidia.     

Modification of hardwood dissolving pulp with purifiedTrichoderma reesei cellulases

Hardwood dissolving pulp was treated with purified Trichoderma reesei endoglucanases and cellobiohydrolases. Endoglucanases were more efficient in hydrolysing pulp carbohydrates than were the cellobiohydrolases at the same protein dosage. Endoglucanases also lowered the viscosity and improved the alkaline solubility more dramatically. There was a clear correlation between the alkaline solubility and viscosity, and therefore the solubility could only be improved by lowering the viscosity of the pulp. At the same degree of cellulose degradation, endoglucanase II was found to be most effective in reducing the viscosity and thus improving the solubility. Cellobiohydrolases had a less pronounced effect on the viscosity or solubility.     

Expression of Trichoderma reesei exo-cellobiohydrolase I in transgenic tobacco leaves and calli

Expression of Trichoderma reesei exo-cellobiohydrolase I (CBHI) gene in transgenic tobacco was under the control of CaMV 35S promoter. In transgenic leaf tissues, CBHI activity up to 66.1 μmol/h/g total protein was observed. In transgenic calli, the highest CBHI activity was 83.6 μmol h/g total protein. Protein immunoblot analysis confirms the presence of CBHI enzyme in both transgenic calli and leaf tissues. CBHI expression levels accounted for about 0.11% and 0.082% of total protein in transgenic leaf tissues and calli, respectively, Furthermore, expression of CBHI gene did not affect normal growth and development of transgenic plants.     

Exopolysaccharide production by a marine cyanobacterium Cyanothece sp.

The adsorption and the hydrolytic action of purified cellulases of Trichoderma reesei, namely, cellobiohydrolase I (CBH I), endoglucanase II (EG II), and their core proteins, on steam-pretreated willow were compared. The two enzymes differed clearly in their adsorption and hydrolytic behavior. CBH I required the cellulose-binding domain (CBD) for efficient adsorption and hydrolysis, whereas EG II was able to adsorb to steam pretreated willow without its CBD. Absence of the CBD decreased the hydrolysis of cellulose by EG II, but the decrease was less pronounced than with CBH I. A linear relationship was observed between the amount of enzyme adsorbed and the degree of hydrolysis of cellulose only for CBHI. EG II and EG II core appeared to be able to hydrolyze only 1 to 2% of the substrate regardless of the amount of protein adsorbed.     

Use of measurement uncertainty analysis to assess accuracy of carbon mass balance closure for a cellulase production process

Closing carbon mass balances is a critical and necessary step for verifying the performance of any conversion process. We developed a methodology for calculating carbon mass balance closures for a cellulase production process and then applied measurement uncertainty analysis to calculate 95% confidence limits to assess the accuracy of the results. Cellulase production experiments were conducted in 7-L fermentors using Trichoderma reesei grown on pure cellulose (Solka-floc), glucose, or lactose. All input and output carbon-containing streams were measured and carbon dioxide in the exhaust gas was quantified using a mass spectrometer. On Solka-floc, carbon mass balances ranged from 90 to 100% closure for the first 48 h but increased to 101 to 135% closure from 72 h to the end of the cultivation at 168 h. Carbon mass balance closures for soluble sugar substrates ranged from 92 to 127% over the entire course of the cultivations. The 95% confidence intervals (CIs) for carbon mass balance closure were typically ±11 to 12 percentage points after 48 h of cultivation. Many of the carbon mass balance results did not bracket 100% closure within the 95% CIs. These results suggest that measurement problems with the experimental or analytical methods may exist. This work shows that uncertainty analysis can be a useful diagnostic tool for identifying measurement problems in complex biochemical systems.     

Cellulose production based on hemicellulose hydrolysate from steam-pretreated willow

The production cost of cellulolytic enzymes is a major contributor to the high cost of ethanol production from lignocellulosics using enzymatic hydrolysis. The aim of the present study was to investigate the cellulolytic enzyme production ofTrichoderma reesei Rut C 30, which is known as a good cellulase secreting micro-organism, using willow as the carbon source. The willow, which is a fast-growing energy crop in Sweden, was impregnated with 1–4% SO₂ and steam-pretreated for 5 min at 206°C. The pretreated willow was washed and the wash water, which contains several soluble sugars from the hemicellulose, was supplemented with fibrous pretreated willow and used for enzyme production. In addition to sugars, the liquid contains degradation products such as acetic acid, furfural, and 5-hydroxy-methylfurfural, which are inhibitory for microorganisms. The results showed that 50% of the cellulose can be replaced with sugars from the wash water. The highest enzyme activity, 1.79 FPU/mL and yield, 133 FPU/g carbohydrate, was obtained at pH 6.0 using 20 g/L carbon source concentration. At lower pHs, a total lack of growth and enzyme production was observed, which probably could be explained by furfural inhibition.     

Production of cellulase/β-glucosidase by the mixed fungi culture of Trichoderma reesei and Aspergillus phoenicis on dairy manure

A cellulase production process was developed by growing the fungi Trichoderma reesei and Aspergillus phoenicis on dairy manure. T. reesei produced a high total cellulase titer (1.7 filter paper units [FPU]/mL, filter paper activity) in medium containing 10 g/L of manure (dry basis [w/w]), 2 g/L KH₂PO₄, 2 mL/L of Tween-80, and 2mg/L of CoCl₂. However, β-glucosidase activity in the T. reesei-enzyme system was very low. T. reesei was then cocultured with A. phoenicis to enhance the β-glucosidase level. The mixed culture resulted in a relatively high level of total cellulase (1.54 FPU/mL) and β-glucosidase (0.64 IU/mL). The ratio of β-glucosidase activity to filter paper activity was 0.41, suitable for hydrolyzing manure cellulose. The crude enzyme broth from the mixed culture was used for hydrolyzing the manure cellulose, and the produced glucose was significantly (p<0.01) higher than levels obtained by using the commercial enzyme or the enzyme broth of the pure culture T. reesei.     

Xylanase production by Trichoderma reesei rut C-30 on rice straw

Xylanase production of Trichoderma reesei Rut C-30 was examined at different initial pH values (4.8, 5.9, and 7.0) on rice straw in shake flasks, and in a fermentor, for the best pH condition. Enzyme performance was tested on ammonia-treated dwarf elephant grass. The maximum xylanase activities, 92 and 122 IU/mL, were obtained at pH 4.8 in the shake flasks and fermentor, respectively, in which good growth of the fungus was observed during the first 24 h and consumption of proteins dissolved from the rice straw caused the pH to rise later to values between 6.4 and 6.7 (optimal for xylanase production). The xylanases from T. reesei were as effective as Multifect XL, a commercial enzyme preparation, in hydrolyzing ammonia-treated elephant grass.     

Effect of acetic acid and furfural on cellulase production of Trichoderma reesei RUT C30

Because of the high temperature applied in the steam pretreatment of lignocellulosic materials, different types of inhibiting degradation products of saccharides and lignin, such as acetic acid and furfural, are formed. The main objective of the present study was to examine the effect of acetic acid and furfural on the cellulase production of a filamentous fungus Trichoderma reesei RUT C30, which is known to be one of the best cellulase-producing strains. Mandels’s mineral medium, supplemented with steam-pretreated willow as the carbon source at a concentration corresponding to 10 g/L of carbohydrate, was used. Four different concentration levels of acetic acid (0–3.0 g/L) and furfural (0–1.2 g/L) were applied alone as well as in certain combinations. Two enzyme activities, cellulase and β-glucosidase, were measured. The highest cellulase activity obtained after a 7-d incubation was 1.55 FPU/mL with 1.0 g/L of acetic acid and 0.8 g/L of furfural added to the medium. This was 17% higher than that obtained without acetic acid and furfural. Furthermore, the results showed that acetic acid alone did not influence the cellulase activity even at the highest concentration. However, β-glucosidase activity was increased with increasing acetic acid concentration. Furfural proved to be an inhibiting agent causing a significant decrease in both cellulase and β-glucosidase production.     

Improved Cellulase Production by <Emphasis Type="Italic">Trichoderma reesei </Emphasis>RUT C30 under SSF Through Process Optimization

The major constraint in the enzymatic saccharification of biomass for ethanol production is the cost of cellulase enzymes. Production cost of cellulases may be brought down by multifaceted approaches which includes the use of cheap lignocellulosic substrates for fermentation production of the enzyme, and the use of cost efficient fermentation strategies like solid state fermentation (SSF). The current study investigated the production of cellulase by Trichoderma reesei RUT C30 on wheat bran under SSF. Process parameters important in cellulase production were identified by a Plackett and Burman design and the parameters with significant effects on enzyme production were optimized for maximal yield using a central composite rotary design (CCD). Higher initial moisture content of the medium had a negative effect on production whereas incubation temperature influenced cellulase production positively in the tested range. Optimization of the levels of incubation temperature and initial moisture content of the medium resulted in a 6.2 fold increase in production from 0.605 to 3.8 U/gds of cellulase. The optimal combination of moisture and temperature was found to be 37.56% and 30 °C, respectively, for maximal cellulase production by the fungus on wheat bran.     

Thermal Degradation of Cellulose Nanocrystals Deposited on Different Surfaces

Thermal degradation of cellulose nanocrystals deposited on flat solid surfaces was monitored by AFM coupled with analysis of obtained images using image processor. The nanocrystals deposited on TiO₂ substrate showed different degradation patterns compared to those deposited on the nanosized layer of amorphous cellulose. The degradation was complete within 20 minutes at 300 °C. The nanocrystal deposited on amorphous cellulose resisted the heat treatment up to 120 minutes. Visual comparison and analysis of the AFM images clearly demonstrated the impact of temperature on the degradation rate of the nanocrystals deposited on TiO₂ substrate.     

β-Glucosidase production by Trichoderma reesei

The hydrolysis of cellulose to the water-soluble products cellobiose and glucose is achieved via synergistic action of cellulolytic proteins. The three types of enzymes involved in this process are endoglucanases, cellobiohydrolases, and β-glucosidases. One of the best fungal cellulase producers is Trichoderma reesei RUT C30. However, the amount of β-glucosidases secreted by this fungus is insufficient for effective cellulose conversion. We investigated the production of cellulases and β-glucosidases in shake-flask cultures by applying three pH-controlling strategies: (1) the pH of the production medium was adjusted to 5.8 after the addition of seed culture with no additional pH adjustment performed, (2) the pH was adjusted to 6.0 daily, and (3) the pH was maintained at 6.0 by the addition of Tris-maleate buffer to the growth medium. Different carbon sources—Solka Floc 200, glucose, lactose, and sorbitol—were added to standard Mandels nutrients. The lowest β-glucosidase activities were obtained when no pH adjustment was done regardless of the carbon source employed. Somewhat higher levels of β-glucosidase were measured in the culture filtrates when daily pH adjustment was carried out. The effect of buffering the culture medium on β-glucosidase liberation was most prominent when a carbon source inducing the production of other cellulases was applied.