纤维素降解菌L-06的筛选、鉴定及其产酶条件的分析

从用于堆肥的水稻秸秆中初筛出一株高效纤维素降解菌L-06, 根据18S rRNA基因序列和菌株形态分析, 初步鉴定该菌为斜卧青霉(Penicillium decumbens)。研究了液体发酵培养基中氮源、碳源、表面活性剂、培养温度、起始pH以及接种量对该菌株各纤维素酶活力的影响。在最适条件下, 该菌的b-葡萄糖苷酶(BGL)、外切纤维素酶(CBH)于培养第3天酶活力分别达到1662 u/mL和2770 u/mL; 内切纤维素酶(EG)、滤纸糖化力(FPase)于培养第4天酶活力分别达到18064 u/mL和4035 u/mL。在产酶优化实验中, 该菌的EG和CBH在pH10的培养条件下分别保持了70%和43%的酶活性; 在50oC培养条件下EG和CBH分别保持了68%和59%的酶活性。表现出了耐热, 耐碱的特性。     

烟曲霉A-16产纤维素酶工艺优化及酶学特性北大核心CSCD

分离筛选高效降解稻草的菌株,研究菌株产纤维素酶工艺条件及酶学性质。采用刚果红染色法从腐败木质下的土壤中分离筛选到一株产纤维素酶菌株,结合菌株的形态特征和18S rDNA序列同源性比较进行鉴定;通过单因素试验和响应面分析法确定菌株最适产酶条件,并对纤维素酶的稳定性进行研究。分离纯化得到的菌株命名为烟曲霉(Aspergillus fumigatus A-16);响应面实验结果表明,最优产纤维素酶工艺参数为:稻草粉添加量7 g/100 mL,pH 6.0,温度65℃,发酵时间5 d;在此最优条件下,该菌产生的羧甲基纤维素酶(CMCase)和滤纸酶(FPA)活力分别为2 954.76 U/mL和1 086.37 U/mL;其总活力较优化前提高了26.4%。该纤维素酶的适宜反应温度为70℃,适宜pH 6.0。在80℃热处理90 min条件下酶活力可保持在80%以上,说明该酶热稳定性较好。同时,在pH 5.0-7.0范围内比较稳定,放置1 d后可保持70%以上的酶活力。该研究可为利用富含纤维素的生物质原料开发洁净能源及食品级葡萄糖资源提供了基础支撑。     

纤维素降解菌Aspergillus sp. YN1的产酶条件及酶学特性

从牛羊粪堆肥中筛选出一株纤维素降解菌Aspergillus sp.YN1,主要研究了液体发酵培养基中碳源、氮源、培养温度、起始pH、通气量以及接种菌龄对菌株YN1的羧甲基纤维素酶活(CMC酶活)及滤纸酶活的影响。研究结果表明,在优化条件下,该菌的CMC酶活、滤纸酶活在培养第3天分别达到0.53U/mL和0.15U/mL。在酶学特性研究中,菌株YN1的CMC酶的最适反应温度为70°C,最适反应pH4.0(酶促反应为30min)。用不同温度处理1h或不同pH处理2h,YN1的CMC酶在30°C?50°C或pH3.0?4.0之间仍可保持80%以上的酶活性,对热及酸表现出较高的稳定性。     

低温纤维素酶菌株CNY086选育及发酵培养基优化(Ⅱ)

自渤海湾海泥中分离21株低温纤维素酶产生菌。其中菌株CNY01为绿色木霉(Trichoderma viride), 酶活力为67.30 U/mL。以该菌株为出发菌株, 经UV、DES等诱变, 选育出高产突变菌株CNY086, 酶活力为92.17 U/mL。该突变菌株低温纤维素酶发酵具有遗传稳定性。通过单因素和正交实验确定突变菌株CNY086低温纤维素酶发酵最适培养基: 秸秆粉1.20%、麸皮0.70%、硫酸铵0.50%、磷酸二氢钾0.55%, 上述条件下CNY086菌株酶活力达到108.55 U/mL。     

大伏革菌产纤维素酶条件优化及高效菌株筛选

真菌胞外纤维素酶活可作为高效生防菌株筛选的指标之一,通过测定野生大伏革菌(Phlebiopsis gigantea)菌株在最优条件下所产生纤维素酶活力高低来达到筛选防治针叶树根腐病高效菌株的目的。以大伏革菌菌株液体培养过程中纤维素酶活力为指标,通过单因素和正交试验结合的方法,筛选出大伏革菌最适产纤维素酶条件为(g/L):葡萄糖30.00,蛋白胨5.00,磷酸二氢钾3.00,硫酸镁1.50,装液量120 mL/250 mL,接种量5%(V/V),初始pH为4.0。测定野生大伏革菌菌株纤维素酶活力的大小显示:08077号菌株纤维素酶活力最大,13025号菌株最小。研究得出08077号菌株比芬兰商业化生物制剂Rotstop-F具有更好的防治中国小孔异担子菌的潜能。     

高产纤维素酶枯草芽胞杆菌S-16的筛选及其发酵工艺优化

利用刚果红鉴别培养基及基础液体筛选培养基进行菌种筛选,从新疆盐碱地分离得到的16株菌株中筛选获得一株产纤维素酶活力较高的菌株S-16,对该菌株进行16SrDNA鉴定,确定该菌为枯草芽胞杆菌(Bacillus subtilis)。对S-16发酵产纤维素酶的主要影响因素进行研究,分别考察了碳源、氮源、培养基初始pH和接种量等因素对发酵产纤维素酶的影响。结合单因素影响实验得到优化后的培养基配方为:羧甲基纤维素钠1.5%,酵母粉1%,NaCl 1%,MgSO_4·7H_2O 2‰,KH_2PO_4·3H_2_O 1‰。优化后的发酵条件为:初始pH为8,接种量1%,种龄8h,培养时间48h。经过发酵工艺优化,S-16产生的羧甲基纤维素酶活(CMCase)和滤纸酶活(FPase)分别达到4.64IU/mL和0.46IU/mL,与初始培养条件下的酶活相比分别提高了3.14倍和1.30倍。本研究得到的枯草芽胞杆菌S-16及其优化发酵工艺为秸秆的快速腐熟和高产纤维素酶的应用奠定了基础。     

Identification and optimal degradation conditions for cellulase-degrading enzyme of a methanol-utilizing and cellulase-producing bacterium

采用刚果红染色法,从废弃矿山周边土壤中筛选出一株产纤维素酶的甲醇利用细菌,命名为xt - 04.形态特征、生理试验及16S rDNA序列和gyrB序列分析表明,该菌株属于Bacillus methylotrophicus.为提高该菌所产纤维素酶的降解能力,首先通过单因子实验考察了底物CMC -Na浓度、反应温度及缓冲液pH值对纤维素酶活力的影响;然后采用响应面分析法对影响纤维素酶活力的3个单因子进行了优化.结果表明,单因素实验得出的适宜反应温度、缓冲液pH和底物浓度分别为70℃、5.0和2％ (20 mg/mL);响应面法得出的最高酶活力条件:反应温度、pH和底物浓度分别为66.1℃、4.81和19.01mg/mL.在最优条件下,酶活力达到17.85 U/mL,比优化前的酶活力12.84 U/mL提高了39.01％.因此,鉴于这种纤维素酶能耐受较高温度和酸性条件,该菌株所产纤维素酶可能在工业中具有良好的应用前景.     

斜卧青霉纤维素酶和木聚糖酶高产菌株的选育

以纤维素酶高产菌株斜卧青霉A50为出发菌株,通过紫外诱变原生质体获得1株木聚糖酶活力提高80%而纤维素酶活力没有改变的6号菌。蛋白质电泳和酶谱检测结果显示,纤维素酶谱基本无差别,而木聚糖酶谱显示6号菌比A50多了一条带。6号菌优化后的产酶培养基组成为:麸皮7%、葡萄糖0.1%,该条件下,纤维素酶活为19.7IU/mL,木聚糖酶活力为215.4IU/mL。     

一株耐低温纤维素酶高产菌株的筛选、鉴定和产酶的初步试验

从若尔盖高寒湿地距表层80cm处土壤中筛选出一株纤维素酶高产菌株XW-1。根据形态学、生理生化特征以及16SrDNA核酸序列分析结果表明,该菌属于缺陷短波单胞菌(Brevundimonas sp.)。对该菌产酶条件研究表明,XW-1在含0.5%CMC-Na条件下,20°C培养3d后出现最高酶活,达到15.6U/mL。对其酶学性质初步研究表明,该菌株所产纤维素酶的最适pH为6.0,最适反应温度为20°C,15°C时相对酶活达到80%,并且在5°C时,相对酶活仍能保持56%。     

紫外线和60Co-γ联合诱变褐色高温单孢菌及产酶特性研究

本研究以褐色高温单孢菌(Thermomonospora fusca)为出发菌株,通过紫外线和60Co-γ射线联合诱变,获得了一株纤维素酶高产菌株AV8,CMC酶活力达到0.679 IU/mL,与出发菌株相比,其产酶能力提高3.53倍。通过对AV8产纤维素酶的培养条件进行测定,结果显示:产纤维素酶最适应温度为55℃;该菌的最适产CMC酶初始PH值为7.0,最适产FPA酶初始PH值为8.0;当培养到第7天时,产CMC酶达到高峰。当培养到第8天时产FPA酶达到高峰。     

Mode of action of cellulases on dyed cotton with a reactive dye

Cotton woven fabrics which were previously dyed with a reactive dye were treated with a commercial cellulase preparation. Dyeing with a reactive dye for cotton apparently inhibited the weight loss activity and saccharification activity of cellulase. In addition, dyed cotton was treated with highly purified cellulases which were exo-type cellulases (Cellobiohydrolase I (CBH I) and Cellobiohydrolase II (CBH II)) and endo-type cellulase (Endoglucanase II (EG II)). Exo-type cellulases were inhibited more than endo-type cellulase by dyeing in the case of saccharification activity. CBH I was severely inhibited by dyeing as compared with CBH II or EG II from the viewpoint of morphological changes in the fiber surface. Dyes on the cellulose substrates severely influenced CBH I in spite of the rare modification, because CBH I hydrolyzed cellulose with true-processive action. The change in the activity of each cellulase component on dyed cotton can affect the synergistic action of cellulases.     

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。     

pH控制下绿色木霉（Trichoderma viride）流加发酵生产纤维素酶

绿色木霉（Trichodermaviride）在pH控制发酵条件下,采用流加葡萄糖发酵策略,可显著提高综合滤纸酶活力（FPA）和内切酶（endo—β—1,4-glucanase,EG）、外切酶exo—β-1,4-glucanase,CBH）、纤维二糖酶（cellobiase,CB）酶活。在5L发酵罐中采用pH控制和流加葡萄糖工艺,可提高CB酶含量,改变酶组分之间的比例,使得FPA、EG、CB和CBH酶活分别达到50．0U／mL,210．0U／mL,4．0U／mL和2．5U／mL,比摇瓶发酵分别提高了6．7．4．2、19、2．5倍。     

A new strategy for cellulases application in high temperature industrial scenarios with syringic acid assisting

In the process of bioethanol production, more stable and active cellulase in high temperature condition is required. In this study, syringic acid was applied in cellulase hydrolysis system. At 70°C, TvEG3 activity increased 201.36%, CtBglA activity decreased 72.79% by syringic acid. With syringic acid assisting, TvEG3 thermostability was improved, CtBglA thermostability was reduced. Syringic acid scarcely affected CtCBH. In hydrolysis system with the cellulases containing TvEG3, CtCBH, and CtBglA, the reducing sugar yield improved by 28.37% with syringic acid assisting. With the molecular dynamic simulation in syringic acid system, the backbone root-mean-square deviation (RMSD) and the residue root-mean-square fluctuation (RMSF) of TvEG3, CtCBH reduced, while the RMSD and RMSF of CtBglA increased. The reduction in the number of secondary structures, especially α-helix, caused the structure of CtBglA in the presence of syringic acid to collapse at high temperature. More secondary structures in TvEG3 and more α-helix in CtCBH in the presence of syringic acid make them more stable at high temperatures. These means syringic acid can stabilize TvEG3 and CtCBH structure, destabilize CtBglA structure at high temperature. In summary, this study not only provides insight into cellulase hydrolysis at high temperature with syringic acid assisting but also demonstrates the promoting mechanism of syringic acid.     

Visualization of Trichoderma reesei Cellobiohydrolase I and Endoglucanase I on Aspen Cellulose by Using Monoclonal Antibody-Colloidal Gold Conjugates

Monoclonal antibodies (MAbs) specific for cellobiohydrolase I (CBH I) and endoglucanase I (EG I) were conjugated to 10- and 15-nm colloidal gold particles, respectively. The binding of CBH I and EG I was visualized by utilizing the MAb-colloidal gold probes. The visualization procedure involved immobilization of cellulose microfibrils on copper electron microscopy grids, incubation of the cellulose-coated grids with cellulase(s), binding of MAb-colloidal gold conjugates to cellulase(s), and visualization via transmission electron microscopy. CBH I was seen bound to apparent crystalline cellulose as well as apparent amorphous cellulose. EG I was seen bound extensively to apparent amorphous cellulose with minimal binding to crystalline cellulose.     

Cellobiohydrolase II is the main conidial-bound cellulase in Trichoderma reesei and other Trichoderma strains

Monoclonal antibodies have been used to determine the presence of cellobiohydrolases I and II (CBH I and II), and endoglucanase I (EG I) on the surface of conidia from Trichoderma reesei QM 9414 and RUT C-30, and 8 other Trichoderma species. For this purpose, proteins were released from the conidial surface by treatment with a non-ionic detergent (Triton X-100 and -octylglucoside), followed by SDS-PAGE/Western blotting and immunostaining. Both CBH I and II were clearly present, but — unlike in extracellular culture fluids from Trichoderma — CBH II was the predominant cellulase. In T. reesei EG I could not be detected. The higher producer strain T. reesei RUT C-30 exhibited a higher conidial level of CBH II than T. reesei QM 9414. In order to assess the importance of the conidial CBH II level for cellulase induction by cellulose, multiple copies of the chb2 gene were introduced into the T. reesei genome by cotransformation using PyrG as a marker. Stable multicopy transformants secreted the 2- to 4-fold level of CBH II into the culture medium when grown on lactose as a carbon source, but their CBH I secretion was unaltered. Upon growth on cellulose, both CBH I and CBH II secretion was enhanced. Those strain showing highest cellulase activity on cellulose also appeared to contain the highest level of conidial bound CBH II. CBH II was also the predominant conidial cellulase in various other Trichoderma sp. However, roughly the same amount of conidial bound CBH II was detected in all strains, although their cellulase production differed considerably.     

Cellobiohydrolase I (Cel7A) from Trichoderma reesei has chitosanase activity

Cellobiohydrolase CBH I (Cel7A) from the filamentous fungus Trichoderma reesei (TrCBHI), which is a member of glycoside hydrolase family (GHF) 7, was expressed in Aspergillus oryzae. We found that the recombinant enzyme showed significant chitosanase activity, as well as cellulase activity, and acted in an endo-type manner on soluble polymeric substrate. Furthermore, another GHF7 CBH I from Aspergillus aculeatus (AaCBHI) expressed in A. oryzae also had chitosanase activity, while endoglucanase EG I (Cel7B) from T. reesei had no activity towards chitosan. To our knowledge, this is the first report of GHF7 enzymes possessing chitosanase activity.     

Characterization of commercial Trichoderma reesei cellulase preparations by denaturing electrophoresis (SDS-PAGE) and immunostaining using monoclonal antibodies.

Fifteen different cellulase preparations from Trichoderma reesei, obtained either commercially or from pilot plants, were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting using monoclonal antibodies against two cellobiohydrolases (CBH I, CBH II), an endoglucanase (EG I), and beta-glucosidase. The staining patterns were compared with the activities of the preparations against filter paper (FPU), carboxymethylcellulose (CMC-ase), cellobiose (beta-glucosidase), and azocasein (protease). Variable amounts of proteolytic degradation products of CBH I, CBH II, and EG I were seen in most samples, and only half of them contained intact beta-glucosidase. The degree of proteolysis did not correlate with any significant difference in the respective activities of these preparations against filter paper cellulose or carboxymethylcellulose. In more than 50% of all cases a decreased beta-glucosidase activity and the absence of intact beta-glucosidase protein in Western blots was observed in preparations displaying high proteolytic activity.     

秸秆质外体蛋白对纤维素酶活力的影响

研究了玉米秸秆在储存过程中质外体蛋白含量的变化情况,及其对Penicilllumexpansum纤维素酶活力的影响。结果表明随着储存时间的延长,可抽提的玉米秸秆质外体蛋白的数量逐渐减少,与P.expansum纤维素酶的协同作用也随之减弱。储存玉米秸秆质外体蛋白没有内源性纤维素酶活力,而新鲜玉米秸秆有内源性EG活性,但它的稳定性较差,储存半年后基本降解或失活。储存秸秆质外体蛋白对FPA酶活力、棉花酶活力、β-葡萄糖苷酶活力有明显的增效作用,最大增效分别达到95.32%、102.06%和96.6%。而对CMCase却表现出抑制作用,最大抑制率为49.52%,质外体蛋白-EG-βG表现出明显的协同关系,而它对CBH酶活力没有影响。消除内源性EG的影响后,新鲜玉米秸质外体蛋白对FPA活力、棉花酶活力、β-葡萄糖苷酶活力的促进率,以及CMCase的抑制率均高于储存秸秆的质外体蛋白。可见质外体蛋白是天然纤维素酶解研究不可忽视的影响因素。