Engineering cellulase mixtures by varying the mole fraction of Thermomonospora fusca E5 and E3, Trichoderma reesei CBHI, and Caldocellum saccharolyticum beta-glucosidase

In this study, different mole fractions of pure Thermomonospora fusca E(5) and E(3), plus Trichoderma reesei CBHI were studied for reducing sugar production at 2 h, degree of synergism, and cellulose binding. In addition, the effects of introducing the Caldocellum saccharolyticum beta-glucosidase into this cellulase system were investigated. The cellulases used were purified to homogeneity. Avicel PH 102 (4% w/w solution in 0.05 sodium acetate pH 5.5 buffer) was the substrate. Reactions were run at 50 degrees C for 2 h using total cellulase concentrations of 8.3 or 12.2 muM. A bimixture of T. fusca E(3) and T. reesei CBHI was very effective in hydrolyzing microcrystalline cellulose (9.1% conversion). The addition of endoglucanase E(5) to the mixture only increased conversion to 9.8%. However, when both E(5) and beta-glucosidase were added, conversion increased to 14%. It was also observed that increasing total cellulase concentration beyond 8.3 muM did little to increase percent conversion of cellulose into glucose. The results of the binding studies indicate no competition for binding sites between the endo- and exocellulases. (c) 1993 John Wiley & Sons, Inc.     

Adsorption and synergism of cellobiohydrolase I and II of Trichoderma reesei during hydrolysis of microcrystalline cellulose

Hydrolysis of microcrystalline cellulose (Avicel) by cellobiohydrolase I and II (CBH I and II) from Trichoderma reesei has been studied. Adsorption and synergism of the enzymes were investigated. Experiments were performed at different temperatures and enzyme/substrate ratios using CBH I and CBH II alone and in reconstituted equimolar mixtures. Fast protein liquid chromatography (FPLC) analysis was found to be an accurate and reproducible method to follow the enzyme adsorption. A linear correlation was found between the conversion and the amount of adsorbed enzyme when Avicel was hydrolyzed by increasing amounts of CBH I and/or CBH II. CBH I had lower specific activity compared to CBH II although, over a wide concentration range, more CBH I was adsorbed than CBH II. Synergism between the cellobiohy-drolases during hydrolysis of the amorphous fraction of Avicel showed a maximum as a function of total enzyme concentration. Synergism measured as a function of bound enzyme showed a continuous increase, which indicates that by decreasing the distance between the two enzymes the synergism is enhanced. The adsorption process for both enzymes was slow. Depending on the enzyme/substrate ratio it took 30-90 min to reach 95% of the equilibrium binding. The amount of bound enzyme decreased with increasing temperature. The two enzymes compete for the adsorption sites but also bind to specific sites. Stronger competition for adsorption sites was shown by CBH I. (c) 1994 John Wiley & Sons, Inc.     

Activity studies of eight purified cellulases: Specificity, synergism, and binding domain effects

The activities of six purified Thermomonospora fusca cellulases and Trichoderma reesei CBHI and CBHII were determined on filter paper, swollen cellulose, and CMC. A simple method to measure the soluble and insoluble reducing sugar products from the hydrolysis of filter paper was found to effectively distinguish between exocellulases and endocellulases. Endocellulases produced 34% to 50% insoluble reducing sugar and exocellulases produced less than 8% insoluble reducing sugar. The ability of a wide variety of mixtures of these cellulases to digest 5.2% of a filter paper disc in 16 h was measured quantitatively. The specific activities of the mixtures varied from 0.41 to 16.31 mumol cellobiose per minute per micromole enzyme. The degree of synergism ranged from 0.4 to 7.8. T. reesei CBHII and T. fusca E3 were found to be functionally equivalent in mixtures. The catalytic domains (cd) of T. fusca endocellulases E2 and E5 were purified and found to retain 93% and 100% of their CMC activity, respectively, but neither cd protein could digest filter paper to 5.2%. When E2cd and E5cd were substituted in synergistic mixtures for the native proteins, the mixtures containing E2cd retained 60%, and those containing E5cd retained 94% of the original activity. Addition of a beta-glucosidase was found to double the activity of the best synergistic mixture. Addition of CBHI to T. fusca crude cellulase increased its activity on filter paper 1.7-fold. (c) 1993 John Wiley & Sons, Inc.     

Stimulatory effect of oxidized cellulose on cellulase formation by Trichoderma reesei

Crystalline cellulase has been electrochemically oxidized to yield preparations containing various different percentages of oxidized end-groups. These celluloses have been used as carbon sources for growth and cellulase production by Trichoderma reesei . A low content of oxidized end groups in the celluloses (0.1–0.65%) stimulated cellulase production but not growth, whereas higher contents (> 1%) where inhibitory to both. The cellulolytic enzyme system secreted under stimulated conditions contained the same proportion of individual cellulase enzymes (cellobiohydrolase I and II, endoglucanase I) as the control, indicating a general stimulatory effect of oxidized cellulose. Activity of cellulases against oxidized celluloses in vitro was not stimulated, and only slightly inhibitory at high degrees of oxidation. The data support a potential role of cellulose oxidation in regulating cellulase formation by T. reesei .     

Adhesion of cellulose to cell walls to Trichoderma reesei

Carbohydrate-binding components were shown to be present at the surface of Listeria monocytogenes by means of a panel of neoglycoproteins using direct agglutination. These lectin-like components bind on neoglycoproteins bearing D-glucosamine, L-fucosylamine, or para-amino-phenyl-alpha-D-mannopyrannoside residues. The interactions were inhibited by the carbohydrate moieties specific to the neoglycoproteins. The protein nature of the lectin-like components of L. monocytogenes was ascertained by the loss of carbohydrate-binding capacity following protease treatment.     

工业纤维素诱导里氏木霉RUT C-30产葡聚糖酶的条件

为了研究工业纤维素诱导里氏木霉RUT C-30产葡聚糖酶的最佳条件,根据单因素实验结果,以工业纤维素、(NH4)2SO4和生物素为实验因素,滤纸酶活为响应值,进行中心组合设计,建立一个二次多项式数学模型,进行响应面优化,寻找最优产酶结果.经过优化,选出工业纤维素、(NH4)2SO4和生物素的添加量分别为39.485 g/L、6.232 g/L和249.872 μg/L,最高的滤纸比酶活为6.298 U/mL,实验验证,滤纸比酶活为6.118 U/mL,与预测值相差了2.86%.     

Action of purified Trichoderma reesei cellulases on cotton fibers and yarn

In this work the possibility and potential of treating cotton fibers and yarns instead of fabrics with monocomponent cellulases was investigated. Different pretreatments on fibers were performed and tested in order to improve the accessibility of cotton to enzymatic modification. The enzymatic treatments were evaluated microscopically and by analysing the effects of treated fibers on spinnability, yarn evenness, tenacity and pilling. The accessibility of the cotton fibers for cellulases could be increased by different pretreatments. Steaming of fibers prior to enzymatic treatment was found to be an efficient way to increase hydrolysis levels. Cellulase treatments of carded yarns resulted in modification of yarn properties. Decrease in yarn hairiness was observed and the knitted fabric made of the treated yarn showed a lowered tendency towards pilling. In all cases endoglucanase activity rather than cellobiohydrolase activity was responsible for these modifications.     

Effect of endoglucanase and cellobiohydrolase from Trichoderma reesei on cellulose microfibril structure

Abstract Cellobiohydrolase (CBH, EC 3.2.91) was purified to homogeneity from Trichoderma reesei culture fluids by means of preparative isoelectric focussing (IEF). Its isoelectric points was 4.2. The degradation product of crystalline cellulose (Avicel and cotton) was predominantly cellobiose. The action of purified endoglucanase (EG) and CBH on cellulose microfibrils was followed by transmission electron microscopy (TEM) observations after Pt-C shadowing of the specimen. EG pretreatment of microfibrils resulted in submicrofibril formation. Addition of CBH induced the conversion of submicrofibrils into heterogeneous cellulose clusters and into homogeneous cellulose plaques. One structural effect of CBH was the increase in accessible cellulose surface area, possibly providing intermolecular entrace of water molecules between adjacent cellulose chains. Plaque formation is interpreted as a visible CBH action on crystalline cellulose to form swollen water-insoluble cellulose intermediates.     

Formation of cross-fractures in cellulose microfibril structure by an endoglucanase-cellobiohydrolase complex from Trichoderma reesei

Abstract An endoglucanase-cellobiohydrolase from Trichoderma reesei culture fluids was purified by means of preparative isoelectric focusing. The cellulase complex had a common apparent isoelectric point (p I ) of 3.8. Beyond this p I , the electrophoretic mobilities of endoglucanase and cellobiohydrolase were different under conditions of titration curves. The effect of this endoglucanase-cellobiohydronalase complex on Sinapis cellulose microfibril ultranstructure was observed by transmission electron microscopy after metal shadowing of the specimen. By the action of this cellulase complex, the microfibril structure was converted into an amorphous form of cellulose. Moreover, the hydrolase complex induced visible cross-fractures within the cellulose microfibril structure. The mean cellulose microfibril lenght of 1.2 μm was reduced to 0.9 μm in the presence (12 h) of this cellulase complex by the formation of shorter microfibril fragments.     

2种纤维素酶水解效果比较及酶蛋白组分分析

比较了自产纤维素酶和商品纤维素酶的水解效果,并采用超滤、层析、SDS-PAGE相结合的方法分析2种纤维素酶蛋白组分的差异。里氏木霉以纸浆为C源合成的自产纤维素酶的水解得率高于商品纤维素酶,自产纤维素酶水解48h的得率为66.24%,商品纤维素酶的得率为52.19%。自产纤维素酶中存在着Cel6A酶组分和XYNⅡ酶组分,而商品纤维素酶中没有检测到这2种酶组分。自产纤维素酶和商品纤维素酶的Cel1A酶组分和Cel7A酶组分间存在着分布和含量上的差异。自产纤维素酶在相对分子质量(2.5～3.5)×104范围内存在着几条蛋白条带,而商品纤维素酶则是在相对分子质量3.5×104附近存在着几条蛋白条带。     

Complexity of cellulases from Trichoderma reesei with acidic isoelectric points: A two-dimensional gel electrophoretic study using immunoblotting

Antibody specific to Trichoderma reesei cellulase (65 kDa, isoelectric point, pI, 7.7) shows immuno-cross reactivity with acidic hydrolase complexes containing other cellulases, (pI_app. 3.4–4.5) when tested under conditions of 2D-electrophoresis (1^st dim. PAGIF, 2^nd dim. SDS-PAGE) together with Western blotting. Degradation pattern of ¹⁴C(U)-labeled G₁–G₅ of the 65 kDa cellulase was followed by a 2-directional oligodextrin mapping procedure.Using preparative IEF, homologous antigen portions were detected in cellulases present within acidic hydrolase complexes showing mainly identical molar weight (M_r 65 kDa and 57 kDa) but a range of charge (pI 3.4–4.5). The pattern of acidic cellulases as found after analytical 2D-electrophoresis was reconstituted by preparative IEF (pI_app. 2.7–5.1) followed by SDS-PAGE separation. Homogeneous fractions (upon IEF) gave up to 8 different polypeptides per complex upon SDS-PAGE (M_r 70−20 kDa). Charge heterogeneity of individual acidic hydrolase complexes upon IEF is discussed as one reason for ‘multiplicity’ of acidic cellulases.     

Desorption of zinc by extracellularly produced metabolites of Trichoderma harzianum, Trichoderma reesei and Coriolus versicolor

AIMS: To determine the role of fungal metabolites in the desorption of metals. METHODS AND RESULTS: Desorption of Zn from charcoal by three different fungi was compared against metal desorption with reverse osmosis water, a 0.1% Tween 80 solution and a 0.1 mol l(-1) CaCl(2) solution. All three fungal filtrates desorbed three times more Zn than either 0.1% Tween 80 or 0.1 mol l(-1) CaCl(2). Metal chelator production in Trichoderma harzianum and Coriolus versicolor was constitutively expressed while chelator production in Trichoderma reesei was induced by Zn. The presence of Zn inhibited the production of metal chelators by C. versicolor. Only C. versicolor was found to produce oxalic acid (a strong metal chelator). All fungi caused a marked decrease in pH, although this was not enough to explain the increased desorption of the metals by the different fungal filtrates. CONCLUSIONS: Metal chelation via organic acids and proteins are the main mechanisms by which the fungal filtrates increase zinc desorption. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study explain why plants inoculated with T. harzianum T22 take up more metal from soil, than noninoculated plants while metabolites produced by fungi could be used for metal leaching from contaminated soils.     

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％。     

Inhibition of the Trichoderma reesei cellulases by cellobiose is strongly dependent on the nature of the substrate

The inhibition effect of cellobiose on the initial stage of hydrolysis when cellobiohydrolase Cel 7A and endoglucanases Cel 7B, Cel 5A, and Cel 12A from Trichoderma reesei were acting on bacterial cellulose and amorphous cellulose that were [(3)H]- labeled at the reducing end was quantified. The apparent competitive inhibition constant (K(i)) for Cel 7A on [(3)H]-bacterial cellulose was found to be 1.6 +/- 0.5 mM, 100-fold higher than that for Cel 7A acting on low-molecular-weight model substrates. The hydrolysis of [(3)H]-amorphous cellulose by endoglucanases was even less affected by cellobiose inhibition with apparent K(i) values of 11 +/- 3 mM and 34 +/- 6 mM for Cel 7B and Cel 5A, respectively. Contrary to the case for the other enzymes studied, the release of radioactive label by Cel 12A was stimulated by cellobiose, possibly due to a more pronounced transglycosylating activity. Theoretical analysis of the inhibition of Cel 7A by cellobiose predicted an inhibition analogous to that of mixed type with two limiting cases, competitive inhibition if the prevalent enzyme-substrate complex without inhibitor is productive and conventional mixed type when the prevalent enzyme-substrate complex is nonproductive.     

里氏木霉纤维素酶的分离纯化及酶学性质

通过(NH4)2SO4分级沉淀、HiPrep 26/10 Desalting凝胶色谱脱盐、Source 15 Q阴离子交换色谱技术,里氏木霉(Rut C-30)纤维素酶主要组分得以初步分开,再经过Source 15 S阳离子交换色谱、HiPrep Sephacryl S-100 HR凝胶过滤色谱、Superdex 75 PrepGrade凝胶过滤色谱进一步分离纯化,得到2个纯化的内切葡聚糖酶组分EGⅡ、EGⅠ和一个外切葡聚糖酶组分CBHⅠ;经过SDS-PAGE电泳鉴定为电泳纯,测得相对分子质量分别为5.22×104,5.62×104和6.90×104。EGⅡ的最适反应pH是5.6,最适反应温度为65℃;EGⅠ的最适反应pH是4.4,最适反应温度为55℃;以羧甲基纤维素(CMC)为底物时,EGⅠ、EGⅡ的米氏常数(Km)分别为2.20 mg/mL、3.38 mg/mL。CBHⅠ的最适反应pH是5.8,最适反应温度为60℃,以对硝基苯基-β-D-纤维二糖苷(PNPC)为底物时,米氏常数(Km)为0.12 mg/mL。     

Co-operative actions and degradation analysis of purified xylan-degrading enzymes from Thermomonospora fusca BD25 on oat-spelt xylan

AIMS: To determine and quantify the products from the degradation of xylan by a range of purified xylan-degrading enzymes, endoxylanase, beta-xylosidase and alpha-l-arabinofuranosidase produced extracellularly by Thermomonospora fusca BD25. METHODS AND RESULTS: The amounts of reducing sugars released from oat-spelt xylan by the actions of endoxylanase, beta-xylosidase and alpha-l-arabinofuranosidase were equal to 28.1, 4.6 and 7% hydrolysis (as xylose equivalents) of the substrate used, respectively. However, addition of beta-xylosidase and alpha-l-arabinofuranosidase preparation to endoxylanase significantly enhanced (70 and 20% respectively) the action of endoxylanase on the substrate. The combination of purified endoxylanase, beta-xylosidase and alpha-l-arabinofuranosidase preparations produced a greater sugar yield (58.6% hydrolysis) and enhanced the total reducing sugar yield by around 50%. The main xylooligosaccharide products released using the action of endoxylanase alone on oat-spelt xylan were identified as xylobiose and xylopentose. alpha-l-Arabinofuranosidase was able to release arabinose and xylobiose from oat-spelt xylan. In the presence of all three purified enzymes the hydrolysis products of oat-spelt xylan were mainly xylose, arabinose and substituted xylotetrose with lesser amount of substituted xylotriose. CONCLUSIONS: The addition of the beta-xylosidase and alpha-l-arabinofuranosidase enzymes to purified xylanases more than doubled the degradation of xylan from 28 to 58% of the total substrate with xylose and arabinose being the major sugars produced. SIGNIFICANCE AND IMPACT OF THE STUDY: The results highlight the role of xylan de-branching enzymes in the degradation of xylan and suggest that the use of enzyme cocktails may significantly improve the hydrolysis of xylan in industrial processes.     

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%。     

拟康氏木霉(Trichoderma pseudokoningii)UV III纤维素酶合成的诱导与阻遏

拟康氏木霉 (T .pseudokoningii)TH经紫外诱变获得一抗高浓度葡萄糖阻遏突变株UVIII,纤维素酶产量显著提高。研究表明 ,UVIII对诱导物的敏感性增加了 10 0倍 ,并且对葡萄糖的吸收能力明显下降 ,导致部分解除了葡萄糖阻遏作用 ,这可能都是该突变株产酶提高的原因     

拟康氏木霉(Trichoderma psudodoningii)UV III产纤维素酶液体发酵研究

以拟康氏木霉(Trichoderma psudodoningii)TH为出发菌株,经紫外诱变荻得一抗高浓度葡萄糖阻遏突变株uV III,其液体发酵最适产酶培养基为(w/V)豆皮粉3%,硝酸铵0.6%,磷酸二氢钠0.65%,硫酸镁0.25%,氯化钙0.15%,pH5.0;最佳发酵条件为30℃,125r/min发酵7d CMCase活力可达103.55 IU/mL,滤纸酶活可达5.51 IU/mL,β一葡萄糖苷酶活可达0 96IU/mL,分别比出发菌株TH提高了1.40、2.34、0.60倍.     

里氏木霉和鸡腿菇利用秸秆共发酵产木质降解酶

为了更好地利用农业废弃物,提高其综合利用率,减少传统化学方法及秸秆焚烧过程造成的环境污染,实验对鸡腿菇、黑曲霉和里氏木霉3株产木质纤维素降解酶系的菌株进行混合平板产酶筛选,结果显示鸡腿菇和里氏木霉平板培养相容性良好,且产酶量高。在相容性实验的基础上,对鸡腿菇和里氏木霉的最优产酶条件进行了研究。在最优条件下:鸡腿菇和里氏木霉接种比例按5:2,接种时间间隔为12h,26oC、150r/min下,发酵3d产漆酶活力达3267.2U/mL,比单独发酵提高106%。