Fragmentation of cellulose by the major Thermomonospora fusca cellulases, Trichoderma reesei CBHI, and their mixtures

In this study, the fragmentation activities of Thermomonospora fusca cellulases E(2), E(3), E(5), Trichoderma reesei CBHI, and their mixtures were measured to study synergism in fragmentation. Fragmentation studies revealed that only two pure cellulases, T. fusca E(2) and E(5) had significant fragmentation activity. T. fusca E(3) shows strong synergism in fragmentation both in the production of reducing sugars and in fragmentation with both T. fusca endoglucananses and with T. reesei CBHI. Most mixtures containing CBHI produced higher rates of fragmentation than comparable mixtures containing E(3). The highest rate and extent of reducing sugar formation and the highest fragmentation activity were obtained with a mixture of E(2), E(3), and CBHI. (c) 1992 John Wiley & Sons, Inc.     

Effects of pH and high ionic strength on the adsorption and activity of native and mutated cellobiohydrolase I from Trichoderma reesei

Cellobiohydrolase I (CBHI) is the major cellulase of Trichoderma reesei. The enzyme contains a discrete cellulose-binding domain (CBD), which increases its binding and activity on crystalline cellulose. We studied cellulase-cellulose interactions using site-directed mutagenesis on the basis of the three-dimensional structure of the CBD of CBHI. Three mutant proteins which have earlier been produced in Saccharomyces cerevisiae were expressed in the native host organism. The data presented here support the hypothesis that a conserved tyrosine (Y492) located on the flat and more hydrophilic surface of the CBD is essential for the functionality. The data also suggest that the more hydrophobic surface is not directly involved in the CBD function. The pH dependence of the adsorption revealed that electrostatic repulsion between the bound proteins may also control the adsorption. The binding of CBHI to cellulose was significantly affected by high ionic strength suggesting that the interaction with cellulose includes a hydrophobic effect. High ionic strength increased the activity of the isolated core and of mutant proteins on crystalline cellulose, indicating that once productively bound, the enzymes are capable of solubilizing cellulose even with a mutagenized or with no CBD. © 1995 Wiley-Liss, Inc.     

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.     

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.     

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 .     

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。     

The competitive inhibition of Trichoderma reesei C30 cellobiohydrolase I by guanidine hydrochloride

TheP-nitrophenylcellobiosidase (PNPCase) activity of Trichoderma reesei cellobiohydrolase I (CBH I) was competitively inhibited by concentrations of guanidine hydrochloride (Gdn HC1) that did not affect the tryptophan fluorescence of this enzyme. The K_m of CBH I, 3.6 mM, was increased to 45.4 mM in the presence of 0.14 M Gdn HCl, the concentration that was required to inhibit the enzyme by 50%. A similar concentration of lithium chloride and urea had little effect on the PNPCase activity of CBH I. Maximal inhibition was pH dependent, occurring in the range of pH 4.0 to 5.0, which is in the range for maximal activity. Analysis of the inhibition data indicated that 1.2 molecules of Gdn HCl combine reversibly with I molecule of CBH I. Other hydrolases and proteases were also inhibited by Gdn HCl. It is suggested that the inhibition of CBH I by Gdn HCl occurs as a result of the interaction between the positively charged guanidinium group of Gdn HCl and the carboxylate group of glutamic acid 126, postulated to be in the catalytic center of this enzyme.     

里氏木霉与黑曲霉混合发酵产纤维素酶及其水解特性

研究了利用里氏木霉和黑曲霉混合培养产纤维素酶,以黑曲霉孢子悬浮液的不同活化浓度及不同的活化时间来寻找2个菌种发挥最大协同作用的结合点以及所产纤维素酶的水解特性。以里氏木霉单一培养和黑曲霉单一培养为参照进行对比研究。底物为农林废弃物之一的玉米秸秆,经过蒸气爆破预处理后,用作产酶C源。结果表明:黑曲霉孢子悬浮液活化浓度为10个/mL,活化时间为12 h时,滤纸酶比酶活最高,达3.32 U/mL,高于里氏木霉单一培养的2.25 U/mL,β-葡萄糖苷酶比酶活达1.32 U/mL,高于里氏木霉单一培养的0.57 U/mL。为进一步验证混合菌产纤维素酶的水解效果,利用混合菌产纤维酶的酶液及里氏木霉产纤维素酶的酶液进行酶水解实验,当酶用量为20 U/g绝干纤维素,底物质量浓度为100 g/L条件下水解48 h,混合菌所产酶液酶解得率达70.00%,高于里氏木霉所产酶液的酶解得率63.05%。实验表明里氏木霉与黑曲霉混合培养产酶是可行的,并优于单一菌种培养。     

里氏木霉Trichoderma reesei产纤维素酶的发酵培养基碳氮源优化

研究C、N源对里氏木霉(Trichoderma reesei)生产纤维素酶的影响,采用单因素实验方法和中心复合方法对发酵培养基进行优化。单因素实验表明:黄豆饼粉、玉米芯、玉米浆对纤维素酶的影响显著。通过响应面优化,得到最优培养基C、N源的组成:黄豆饼粉32.21 g/L,玉米芯42.29 g/L,玉米浆4.45 g/L。优化条件下,摇瓶发酵7 d的比酶活达到(10.65±0.50)U/mL。     

Ionic strength effect on adsorption of cellobiohydrolases I and II on microcrystalline cellulose

Cellobiohydrolase I (CBH I) has a higher adsorption affinity (K _ad) and tightness (–H _a) for Avicel than cellobiohydrolase II (CBH II). The adsorption processes of CBH I and II were exothermic, and the degree of exothermy were larger with the increasing ionic strength. Entropy change of CBH I was larger than CBH II with increasing ionic strength. CBH I was more effective than CBH II for binding at a given ionic strength.     

Enhanced cellulase production in fed-batch culture of Trichoderma reesei C30

The use of a fed-batch cultivation of the fungus Trichoderma reesei (C30) allows cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] production to occur under optimum conditions, and results in extremely high enzyme titres and productivities. Enzyme levels of 26 U ml^?1 at productivities >130 U l^?1 h^?1 have been achieved. These results are compared with the values obtained in two-stage continuous cultivation of the organism at optimum pH and temperature.     

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%.     

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。     

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%。     

Improved heterologous gene expression in Trichoderma reesei by cellobiohydrolase I gene （cbh1） promoter optimization

To improve heterologous gene expression in Trichoderma reesei, a set of optimal artificial cellobiohydrolase I gene （cbhl） promoters was obtained. The region from -677 to -724 with three potential glucose repressor binding sites was deleted. Then the region from -620 to -820 of the modified cbhl promoter, including the CCAAT box and the Ace2 binding site, was repeatedly inserted into the modified cbhl promoter, obtaining promoters with copy numbers 2, 4, and 6. The results showed that the glucose repression effects were abolished and the expression level of the glucuronidase （gus） reporter gene regulated by these multi-copy promoters was markedly enhanced as the copy number increased simultaneously. The data showed the great promise of using the promoter artificial modification strategy to increase heterologous gene expression in filamentous fungi and provided a set of optional high-expression vectors for gene function investigation and strain modification.     

红色荧光蛋白在丝状真菌里氏木霉中的表达

目的:建立红色荧光蛋白在里氏木霉中的表达方法,为深入研究里氏木霉中纤维素酶的合成机理打下基础。方法:采用PCR方法分离了里氏木霉纤维二糖水解酶Ⅰ(CBHⅠ)的启动子(Pcbh1)和终止序列(Tcbh1),将这两个片段与红色荧光蛋白(DsRed)的基因连接,得到Pcbh1-DsRed-Tcbh1表达盒。用此表达盒和质粒pAN7-1对里氏木霉QM9414的原生质体进行共转化,并用含100μg/ml潮霉素B的选择性平板进行筛选。结果:经筛选得到20个抗性转化子,在乳糖的诱导下有5个转化子可以表达红色荧光蛋白。对插入片段进行了扩增和序列测定,结果表明DsRed通过同源重组整合到了转化子的基因组DNA上,并处于cbh1启动子的下游。结论:通过cbh1启动子可以实现红色荧光蛋白在里氏木霉细胞内的稳定表达。     

红色荧光蛋白在丝状真菌里氏木梅中的表达

目的：建立红色荧光蛋白在里氏木霉中的表达方法,为深入研究里氏木霉中纤维素酶的合成机理打下基础。方法：采用PCR方法分离了里氏木霉纤维二糖水解酶Ⅰ（CBHI）的启动子（Pcbh1）和终止序列（Tcbh1）,将这两个片段与红色荧光蛋白（DsRed）的基因连接,得到Pcbh1-DsRed-Tcbh1表达盒。用此表达盒和质粒pAN7—1对里氏木霉QM9414的原生质体进行共转化,并用含100μg/ml潮霉素B的选择性平板进行筛选。结果：经筛选得到20个抗性转化子,在乳糖的诱导下有5个转化子可以表达红色荧光蛋白。对插入片段进行了扩增和序列测定,结果表明DsRed通过同源重组整合到了转化子的基因组DNA上,并处于cbh1启动子的下游。结论：通过cbh1启动子可以实现红色荧光蛋白在里氏木霉细胞内的稳定表达。     

Ultrasound mediated enzymatic hydrolysis of cellulose and carboxymethyl cellulose

A recombinant Trichoderma reesei cellulase was used for the ultrasound‐mediated hydrolysis of soluble carboxymethyl cellulose (CMC) and insoluble cellulose of various particle sizes. The hydrolysis was carried out at low intensity sonication (2.4–11.8 W cm^?2 sonication power at the tip of the sonotrode) using 10, 20, and 40% duty cycles. [A duty cycle of 10%, for example, was obtained by sonicating for 1 s followed by a rest period (no sonication) of 9 s.] The reaction pH and temperature were always 4.8 and 50°C, respectively. In all cases, sonication enhanced the rate of hydrolysis relative to nonsonicated controls. The hydrolysis of CMC was characterized by Michaelis‐Menten kinetics. The Michaelis‐Menten parameter of the maximum reaction rate V_max was enhanced by sonication relative to controls, but the value of the saturation constant K_m was reduced. The optimal sonication conditions were found to be a 10% duty cycle and a power intensity of 11.8 W cm^?2. Under these conditions, the maximum rate of hydrolysis of soluble CMC was nearly double relative to control. In the hydrolysis of cellulose, an increasing particle size reduced the rate of hydrolysis. At any fixed particle size, sonication at a 10% duty cycle and 11.8 W cm^?2 power intensity improved the rate of hydrolysis relative to control. Under the above mentioned optimal sonication conditions, the enzyme lost about 20% of its initial activity in 20 min. Sonication was useful in accelerating the enzyme catalyzed saccharification of cellulose. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1448–1457, 2013