里氏木霉液体发酵产纤维素酶的研究

在摇瓶试验基础上,采用里氏木霉(Trichoderma reesei)HC-415菌株进行5L自控罐产纤维素酶深层发酵试验。在通气量为 0.2—0.6vvm、搅拌速度为 400r/min、发酵液pH控制在5.8—6.1的条件下,发酵液的羧甲基纤维素(CMC)酶酶活最高为325.0mg糖/ml,滤纸糖酶(FPA)酶活最高达17.9mg糖/ml。发酵周期为108h。所得冻干纤维素酶粉CMC酶活最高3111IU/g,FPA最高135IU/g ,对发酵液得率平均6.7g/L。酶活总收率CMC酶活平均78.2％,FPA酶活平均73.5％。     

Cellulase production by Trichoderma reesei

Summary A model is proposed for the enzyme production by Trichoderma reesei (QM 9414), which assumes control of the active enzyme transport through the cell membrane as a key parameter for the enzyme activity change in the culture filtrate. In a stirred tank reactor, continuous cultivation of the fungus was carried out in the dilution rate range of D=0.01–0.032 h^–1. After changing the dilution rate it took 3–4 weeks to attain a steady state in enzyme activity. Reducing sugars, dissolved protein, enzyme activity (filter-paper and glucosidase activities), cellulose and nitrogen content of the sediment, the elementary analysis of the cell and the composition of the outlet gas were all determined during cultivation. At a dilution rate of D=0.025 h^–1 all of these properties change due to derepression (for D<0.025 h^–1) or repression (for D>0.025 h^–1) of the enzymes which are responsible for the active transport of cellulases from the cell into the medium. The cellulase excretion causes a decrease of the yield coefficient of growth and a reduction of the nitrogen content of the cells.In a two-stage system the time to attain a steady state increases to 4–6 weeks. At low dilution rates the enzyme activity is only slightly higher in the second stage than in the first. At high dilution rates, at which the enzyme is not excreted into the medium in the first stage, enzyme activity can be increased considerably in the second stage.     

Effect of Colloidal Materials on Cellulase Production by Trichoderma reesei Rut-C30

The addition of positively charged colloidal materials to the growth medium markedly increased the concentration of cellulase enzymes produced by Trichoderma reesei Rut-C30. Filter paper activities of up to 4 and 13 IU/ml have been achieved by the addition of colloidal materials, using 3% lactose and 3% cellulose, respectively, as a substrate. The particles exert their effect by binding soluble sugars and slowing their uptake by the organism.     

Cellulase secretion by immobilized protoplasts of Trichoderma reesei

Protoplasts from Trichoderma reesei were immobilized in alginate and induced to produce cellulase (endoglucanase and β-glucosidase) enzymes. The specific activities of the synthesized enzymes were higher in immobilized protoplasts than in both free and immobilized mycelia. Immobilized protoplasts show an enhanced rate of exocellular β-glucosidase production compared to intact mycelia due to the lack of cell wall. The ratio of the exocellular/intracellular β-glucosidase was 5.9 for immobilized protoplasts and 0.32 for free mycelia.     

纤维素酶在木质纤维素生物质转化中的应用研究

选育得到纤维素酶高产菌株里氏木霉突变菌株(Trichoderma reesei) 813A,优化了其发酵产酶条件。利用该菌株所产纤维素酶对天然木质纤维素的水解糖化过程进行研究,确定了实验条件下最优的糖化条件(温度50℃, pH 4.5,酶浓度6～8 FPU/mL,底物浓度2%)。以玉米叶和杨树叶为天然纤维素原料,水解糖化率分别达到86.2％和56.0%。通过酿酒酵母(Saccharomyces cerevisiae)将糖化液转化为酒精,产乙醇浓度达到 5%～5.8%,转化率为79.4%～92.1%。     

培养条件对一株木霉产纤维素酶过程影响的研究

采用固态发酵和连续监测正交实验结果的方法,研究了培养温度、初始pH值、液料比和接种量对一株木霉(Trichodermasp.)发酵过程中微晶纤维素酶活、CMC酶活和滤纸酶活的影响及影响程度。指出液料比在整个发酵过程中是对产酶影响最大的因素,温度在发酵初期影响较大,初始pH和接种量的影响均不显著。总体看来,培养温度、初始pH值、液料比和接种量分别为30℃、4、7和5%是比较合适的。     

A Shortcut to the Optimization of Cellulase Production Using the Mutant Trichoderma reesei YC-108

Trichoderma reesei YC-108, a strain isolated by a kind of newly invented plate was found to over produce cellulase and it was then used as a cellulase producer. To get the maximum amount of cellulase, the combination of the medium ingredients, which has a profound influence on metabolic pathway was optimized using response surface methodology. The optimum composition was found to be 24.63 g/L wheat bran, 30.78 g/L avicel, and 19.16 g/L soya-bean cake powder. By using the optimized medium, the filter paper activity (FPA) increased nearly five times to 15.82 IU/mL in a 30 L stirred fermenter, carboxymethyl cellulase activity (CMCase) was increased from 83.02 to 628.05 IU/mL and the CMCase/FPA ratio was nearly doubled compared with the parent strain at initial medium.     

Cellulase and xylanase production by Trichoderma reesei Rut C-30

Summary When grown on cellulose or xylan, Trichoderma reesei (strain Rut C-30) produced extra-cellular enzymes which could hydrolyze both cellulose and xylan to their respective monosaccharides. At low O₂ saturation, -glucosidase activity is greatly reduced for cellulose-grown but not xylan-grown cells.     

里氏木霉及其纤维素酶高产菌株的研究进展

随着纤维素在能源、材料及化工等领域的广泛开发和应用,里氏木霉作为一种重要的产纤维素酶工业用菌种,越来越受到人们的广泛关注.为了提高其酶活,人们做了大量的工作,获得了一些相当好的突变株.对里氏木霉及其突变株的基因组进行研究,有助于人们理解其高效产酶的机制,同时也有利于构建其基因工程菌.介绍里氏木霉Trichoderma reesei 的背景及其部分高产纤维素酶突变株,并阐述近些年来对其突变株的基因组的研究进展.     

Stability of the Cellulase of Trichoderma reesei under use conditions

Enzyme stability studies have been reinvestigated under the conditions used for cellulose hydrolysis (pH 4.8, 50°C, 24 hr). The cellobiohydrolase (CBH) component as measured on Avicel is less stable than other enzymes of the cellulase complex, and is 60% inactivated by merthiolate (and other Hg compounds) under the above conditions. Endo-β-1,4-glucanase is much more stable, and more resistant to merthiolate and other compounds. Under unshaken conditions the Avicelase of the Rutgers strain C 30 shows greater stability to heat than that of other available strains. Biocides must be selected not only for their ability to prevent contamination, but also for their compatibility with cellulases. Tetracycline and chlortetracycline are inexpensive, effective in very low concentrations, have no harmful effect on the enzymes, and are compatible with the yeasts that subsequently grow on the sugar solutions to produce alcohol. Attempts have been made to stabilize the enzymes by chemical modification in such a way as to maintain their solubility. Glutaraldehyde treatment greatly increased the enzyme size, lowered the pI values, and gave a slight shift in the pH activity curve. There was, unfortunately, no increase in enzyme stability, and the activity of enzymes on solid celluloses was adversely affected. Shaking greatly reduced the hydrolysis of Avicel by Trichoderma reesei C 30 enzyme. The adverse effect was accompanied by a decrease in recoverable enzyme and protein.     

Cellulase production by Trichoderma reesei when cultured on xylose-based media supplemented with sorbose

The ability of L-sorbose to stimulate cellulase production In shake flask culture of Trichoderma reesei was examined in mineral salts media (initial pH 5.0) containing either 1.0% D-xylose, 1.0% cellulose, and/or 0.1, 0.3, or 0.5% L-sorbose. When sorbose was the only carbon source, growth was limited, little substrate was utilized, pH increased, and cellulase activity was not apparent. The other carbon sources promoted good growth, pH dropped sharply to 2.5-3.0, substrate was utilized rapidly, and cellulase activity was detected. After three weeks of fermentation, twice as much cellulase activity was detected in the medium containing only cellulose as the carbon source, as compared to xylose as the carbon source. Cellulase activity was higher when media contained xylose supplemented with sorbose compared to xylose as the only carbon source. At 0.3 and 0.5% levels of sorbose supplementation of xylose-based media, cellulase activity was similar to that in cellulose-based media.     

Cellulase induction by lactose in Trichoderma reesei PC-3-7

In an attempt to clarify the function of lactose in cellulase induction, experiments were carried out on cellulase formation by lactose along with other sugars in a resting cell system of Trichoderma reesei PC-3-7, a hypercellulase-producing mutant. Although lactose alone induces little cellulase under the conditions used, a synergistic effect on cellulase formation was observed following the respective addition of sophorose, cellobiose or galactose to lactose. The lactose consumption was more rapid when these sugars were added than in their absence. Furthermore, following lactose addition 10 h after the beginning of cultivation in the presence of cellobiose, cellulase formation was initiated with only a little lag, and lactose consumption started immediately, being complete in 14 h. \-Galactosidase induction experiments suggested that the rapid consumption of lactose is possibly not dependent on lactose degradation by the enzyme. From these results, it is suggested that lactose may function as an inducer for cellulase formation if it is taken up in the mycelium of T. reesei PC-3-7, and that sophorose, cellobiose or galactose may induce a putative lactose permease. *** DIRECT SUPPORT *** AG903066 00005     

Cellulase production in continuous culture by Trichoderma reesei on xylose-based media

Trichoderma reesei (QM 9414) produced cellulase in continuous culture, on media containing xylose (1%) supplemented with sorbose (0.3%) to induce cellulase production. Maximum cell mass of 4.54 kg/m(3) occurred at pH 4.0 and a dilution rate of 0.0391 h(-1) where residual substrate was 0.43 kg/m(3), but no cellulase was produced. Maximum cellulase production of 0.69 FPU occurred at pH 3.5 and a dilution rate of 0.0110 h(-1), where cell mass production was 2.56 kg/m(3) and residual substrate was 0.15 kg/m(3). Monod kinetic constants, corrected for endogenous metabolism, were 0.091 h(-1), 0.469 kg/m(3), 0.00923 h(-1), and 0.470 kg cells/kg xylose at pH 3.5, for the maximum specific growth rate, Michaelis-Menten coefficient, endogenous metabolism coefficient, and yield coefficient, respectively. Specific growth rate fitted a maturation time model, which predicted decreasing maturation time with increasing pH.     

Cellulase secretion from a hyper-cellulolytic mutant of Trichoderma reesei Rut-C30

Two strains of Trichoderma reesei, wild type QM6a and mutant Rut-C30, were grown in meida containing an inducer, insoluble crystalline cellulose (Avicel PH101), as carbon source for 11 days. The cell growth, expressed as myceliar protein content, of Rut-C30 was 4–5 times higher than QM6a. The lack of ultrastructural disorganization, and absence of intracellular enzyme release into the growth medium, indicated that none of these two strains had undergone any significant autolysis during the entire growth phase. Cellulase activities, mainly endoglucanase, cellobiase and filter paper degrading activity (disc) were enhanced in Rut-C30 cells. A major change was observed in the endoglucanase activity which was 30 times higher in Rut-C30 than QM6a, whereas, both -glucosidase and disc activities were 3 times enhanced in Rut-C30 compared to QM6a. In addition to synthesis, cellulase secretion was also enhanced in Rut-C30. Both the organisms contained same amounts of intracellular marker enzyme activities (e.g., inosine diphosphatase, thiamine pyrophosphatase, alkaline phosphatase). Finally, the enahncement of secretory activity of Rut-C30 was correlated with the proliferation of rough endoplasmic reticulum (RER) and increased phospholipid content. It appears that Rut-C30 is not only a hypercellulolytic but also a hypersecretor mutant.     

Production of Cellulase byTrichoderma reesei on Waste Cellophane

Utilization of waste cellophane for production of extracellular cellulose-degrading enzymes byTrichoderma reesei was studied on laboratory-scale experiments using liquid media containing varnished or non-varnished cellophane disintegrated physically to different degrees. Enzyme activities were directly proportional to the degree of cellophane disintegration,i.e. its specific surface area. Hydrolytic activities of cellulase on 1 % milled cellophane reached approximately 50 % yield of the activities acquired on 1 % microcrystalline cellulose. Addition of Xylocel, concentrated prehydrolyzate of beech wood, added to the media in amounts corresponding to 0.2 to 1.0 % concentration of reducing groups, did not improve the yield of cellulase on milled cellophane. However, β-_D-xylanase activity was increased several times due to the presence of lower xylooligosaccharides serving as specific β-_D-xylanase inducers. No differences in occurrence of multiple forms of endo-l,4-β-_D-glueanases and endo-l,4-β-_D-xylanases and their isoelectric points were observed.     

ACEI of Trichoderma reesei Is a Repressor of Cellulase and Xylanase Expression

We characterized the effect of deletion of the Trichoderma reesei (Hypocrea jecorina) ace1 gene encoding the novel cellulase regulator ACEI that was isolated based on its ability to bind to and activate in vivo in Saccharomyces cerevisiae the promoter of the main cellulase gene, cbh1. Deletion of ace1 resulted in an increase in the expression of all the main cellulase genes and two xylanase genes in sophorose- and cellulose-induced cultures, indicating that ACEI acts as a repressor of cellulase and xylanase expression. Growth of the strain with a deletion of the ace1 gene on different carbon sources was analyzed. On cellulose-based medium, on which cellulases are needed for growth, the Δace1 strain grew better than the host strain due to the increased cellulase production. On culture media containing sorbitol as the sole carbon source, the growth of the strain with a deletion of the ace1 gene was severely impaired, suggesting that ACEI regulates expression of other genes in addition to cellulase and xylanase genes. A strain with a deletion of the ace1 gene and with a deletion of the ace2 gene coding for the cellulase and xylanase activator ACEII expressed cellulases and xylanases similar to the Δace1 strain, indicating that yet another activator regulating cellulase and xylanase promoters was present.     

Cellulase production on glucose-based media by the UV-irradiated mutants of Trichoderma reesei

From 22,791 mutants of a cellulase hyper-producing strain of Trichoderma reesei (Hypocrea jecorina), ATCC66589, as the parent, we selected two mutants, M2-1 and M3-1, that produce cellulases in media containing both cellulose and glucose. The mutation enabled the mutants to produce cellulases, which were measured as p-nitrophenyl β-d-lactopyranoside-hydrolyzing activities, in media with glucose as a sole carbon source, although M2-1 exhibited different sensitivities to glucose from M3-1. When the mutants were grown for 8 days on a medium with cellulose as a sole carbon source, the filter-paper-degrading activities (FPAs) per gram of cellulose were 257 and 281 U for M2-1 and M3-1, respectively, values that were 1.1–1.2 times higher than that of the parental strain. Cellulase production by M2-1 and M3-1 on a medium with a continuously fed mixture of glucose and cellobiose resulted in 214 and 210 U of FPA/gram carbon sources, respectively, whereas less efficient production (140 U of FPA/gram carbon source) was achieved by the parental strain. The improved cellulase productivity of the mutants allows us to use glucose as a carbon source for efficient on-site production of cellulases with quality/quantity-controlled feeding of soluble carbon sources and inducers.     

Cellulase production by Trichoderma reesei (RUT-C30) in fed-batch culture

Summary Fed-batch cultures of Trichoderma reesei RUT-C30 attained quasi-steady state conditions, in respect of biomass concentration and enzyme production rate, commensurate with a specific cell maintenance coefficient of 0.029 g cellulose.g biomass.^–1h^–1 and specific cellulase production rate of between 9.6 and 11.9 IU (filter paper activity).g biomass.^–1h^–1. A maximum enzyme yield of 57 IU.m1^–1 at an overall productivity of 201 IU.L.^–1h^–1 resulted from a cellulose feed rate of 1.0g.L.^–1h^–1.     

碱与微波联合处理蔗渣对里氏木霉发酵产纤维素酶的影响

以蔗渣为原料,采用碱和微波辐射联合处理后用于里氏木霉纤维素酶的液态发酵。采用单因素试验与正交试验确定了最佳的处理条件为:0.30 mol/L的NaOH溶液浸泡,微波功率160 W,处理5 min。在此条件下得到的单位能耗的酶活净增值最高。后续发酵结束后,酶活较未经处理的蔗渣发酵后所得酶活有显著提高。其中,β-葡萄糖苷酶活、滤纸酶(FPase)活、羧甲基纤维素酶(CMCase)活分别提高了81.3%,88.2%,154.5%。