纤维二糖水解酶Ⅰ吸附结构域的新功能

从拟康氏木霉S-38中分离纯化得到的外切酶Ⅰ（CBHI）,经过木爪蛋白质酶的有限酶解后通过一系列的柱层析分离获得其吸附结构域（CBM）．利用CBM与纤维素在40％下作用24h后,利用红外光谱测定纤维素结构的变化,发现纤维素的氢键作用减弱,利用分子动力学模拟进一步确认实验的结果,并从纳米尺度上阐明了在CBM分子吸附作用于纤维素表面的过程中纤维素链间的氢键作用明显降低．本研究表明CBM分子不仅具有使CBHI分子定位于纤维素表面的作用,还会在吸附过程中导致纤维表面结构的破坏．本研究为CBHI分子催化结构域与吸附结构域分子间的协同模型提供了一重要证据．     

纤维二糖水解酶Ⅰ吸附结构域的新功能

从拟康氏木霉S-38中分离纯化得到的外切酶Ⅰ(CBHI),经过木爪蛋白质酶的有限酶解后通过一系列的柱层析分离获得其吸附结构域(CBM).利用CBM与纤维素在40℃下作用24h后,利用红外光谱测定纤维素结构的变化,发现纤维素的氢键作用减弱,利用分子动力学模拟进一步确认实验的结果,并从纳米尺度上阐明了在CBM分子吸附作用于纤维素表面的过程中纤维素链间的氢键作用明显降低.本研究表明CBM分子不仅具有使CBHI分子定位于纤维素表面的作用,还会在吸附过程中导致纤维表面结构的破坏.本研究为CBHI分子催化结构域与吸附结构域分子间的协同模型提供了一重要证据.     

微紫青霉CBHⅠ酶纤维素结合结构域在大肠杆菌中的分泌型表达及性质研究

为研究纤维素酶纤维素结合结构域的结构与功能 ,进而深入了解天然纤维素的生物降解机制和提高纤维素酶的生物工艺学价值 ,采用 PCR技术体外扩增了携带微紫青霉外切葡聚糖纤维二糖水解酶 ( CBH ) CBD编码区的 DNA片段 ,将 CBD编码区 DNA片段插入带有 Erwiniacarotovora pe1 B前导肽序列的大肠杆菌质粒 p KK- tac- new上进行了表达 .携带微紫青霉 CBDCBH 编码区的大肠杆菌重组菌株 DH5α( p KK- tac- new- 8)产生有活性的分泌型 CBDCBH 蛋白 .SDS-PAGE检测显示所产生的 CBDCBH 蛋白分子量约 1 0 .8k D.在 IPTG诱导下 ,该菌株所产生的CBDCBH 蛋白含量达 45.2 mg/L,且 90 %以上的 CBD蛋白分泌到培养物上清液中 .结晶纤维素 CF-1 1溶液经 CBDCBH 处理后 ,浊度比对照提高了 1 2 8.9% ,天然棉花纤维结构经 CBDCBH 处理后产生一定程度的非水解性降解作用 ,表明微紫青霉 CBDCBH 具有解聚天然结晶纤维素的作用 .     

康氏木霉纤维素酶转录因子ACEII与外切纤维二糖水解酶基因 I（cbh1）启动子的结合分析

ACEI、ACEII和Xyr1是康氏木霉中调控纤维素酶基因表达的转录因子。体外实验已证实ACEI和Xyr1可与cbh1启动子上的287bp序列（-304bp~-18bp）结合从而调控cbh1基因转录,但ACEII是否可与此序列结合仍未清楚。为进一步研究ACEII调控纤维素酶基因表达的机制,利用PCR技术扩增康氏木霉ACEII DNA结合区的基因序列,并使其在大肠杆菌中表达。凝胶迁移率移动试验表明ACEII DNA结合区不能与cbh1启动子的287bp序列结合。提示了康氏木霉cbh1基因在诱导表达时起调控作用的主要是Xyr1,而不是ACEII。这对阐明真菌纤维素酶基因表达调控的分子机制具有重要的意义。     

对硝基苯纤维二糖苷（PNPC）结合于外切纤维素酶I（CBHI）时分子构型和构象变化的研究

以固定浓度的外切纤维素酶I（1,4-β-D-glucan cellobiohydrolase I,CBHI）,对不同浓度的对硝基苯纤维二糖苷（p-nitrophenol-cellobioside,PNPC）在4℃下进行结合．以紫外线光谱、荧光光谱的变化和等温滴定量热法（isothermal titration calorimitry,ITC）分别确定酶的饱和结合位点数（saturation binding point,SBP）．然后,对在4℃和SBP条件下,结合时形成的“PNPC-CBHI”复合物进行PNPC分子构型变化和CBHI分子构象变化的分析．结果表明,在水解反应不能进行的条件下,CBHI结合PNPC的反应是一个由负焓变（-△H）控制的不可逆的放热过程．结合可导致PNPC的构型转变为PNP（对硝基苯）分子构型,而CBHI的构象变化可循环发生．“PNPC-CBHI”复合物与CBHI和PNPC之间不存在可逆性的平衡过程,在酶分子不断循环进行的结合／解离过程中,由CBHI构象变化提供的能量,使PNPC分子不断转化为PNP分子构型,结合复合物解离出PNP与纤维二糖后,再生的CBHI才可与PNPC结合．并对这一结果应用于解释酶催化机理的普适性进行了讨论．     

微紫青霉CBHI酶的CBD蛋白编码区在大肠杆菌中的亚克隆及表达

对插入质粒pUC18－181上的微紫青霉（Penicilliumjanthinellum）CBHI酶的cDNA基因进行一系列DNA体外操作,包括进行序列定向缺失,最后将两末端修饰为平端后进行连接使质粒环化。用得到的产生序列定向缺失的重组质粒转化大肠杆菌JM109。利用CBD能吸附到结晶纤维素上的特性,从随机选取的24个缺失转化子中筛选到一株含CBD编码区的转化子JM109（pUC18C）,所表达的CBD融合蛋白分子量为21kD．JM109（pUC18C）所产生的LacZ-CBD融合蛋白可通过对纤维素的吸附-解吸附过程一步纯化。其IPTG诱导的pNPC酶活力为零,表明该菌已不再具有CBHI酶活力。     

Domain Structure and Conformation of a Cellobiohydrolase from Trichoderma pseudokiningii S-38

A cellobiohydrolase (CBH) with a molecular mass of 66 kD was purified from Trichoderma pseudokiningii S-38. Papain digestion produced a 59- to 60-kD core domain with 54% of intact activity on crystalline cellulose and with full activity against soluble substrates. Digestion products also included two small peptides with molecular mass of about 3–4 kD, which are heavily glycosylated and difficult to purify; the mixed peptides displayed the capacity to disorganize the cellulose fiber. The sequencing results indicated that the intact enzyme had a blocked N-terminal and there was a 10-amino-acid sequence in the N-terminal of the core protein of Ser-Gly-Thr-Ala-Val-Thr-Cys-Leu-Ala-Asp. Fluoresence and circular dichroism properties indicated that the core protein has an independent conformation and is conformationally similar to intact enzyme, suggesting that the spectroscopic properties of the intact enzyme come from the core protein.     

Investigation of the function of mutated cellulose-binding domains of Trichoderma reesei cellobiohydrolase I.

The function of the cellulose-binding domain (CBD) of the cellobiohydrolase I of Trichoderma reesei was studied by site-directed mutagenesis of two amino acid residues identified by analyzing the 3D structure of this domain. The mutant enzymes were produced in yeast and tested for binding and activity on crystalline cellulose. Mutagenesis of the tyrosine residue (Y492) located at the tip of the wedge-shaped domain to alanine or aspartate reduced the binding and activity on crystalline cellulose to the level of the core protein lacking the CBD. However, there was no effect on the activity toward small oligosaccharide (4-methylumbelliferyl beta-D-lactoside). The mutation tyrosine to histidine (Y492H) lowered but did not destroy the cellulose binding, suggesting that the interaction of the pyranose ring of the substrate with an aromatic side chain is important. However, the catalytic activity of this mutant on crystalline cellulose was identical to the other two mutants. The mutation P477R on the edge of the other face of the domain reduces both binding and activity of CBHI. These results support the hypothesis that both surfaces of the CBD are involved in the interaction of the binding domain with crystalline cellulose.     

The Unique Binding Mode of Cellulosomal CBM4 from Clostridium thermocellum Cellobiohydrolase A

The crystal structure of the carbohydrate-binding module (CBM) 4 Ig fused domain from the cellulosomal cellulase cellobiohydrolase A (CbhA) of Clostridium thermocellum was solved in complex with cellobiose at 2.11 Å resolution. This is the first cellulosomal CBM4 crystal structure reported to date. It is similar to the previously solved noncellulosomal soluble oligosaccharide-binding CBM4 structures. However, this new structure possesses a significant feature—a binding site peptide loop with a tryptophan (Trp118) residing midway in the loop. Based on sequence alignment, this structural feature might be common to all cellulosomal clostridial CBM4 modules. Our results indicate that C. thermocellum CbhA CBM4 also has an extended binding pocket that can optimally bind to cellodextrins containing five or more sugar units. Molecular dynamics simulations and experimental binding studies with the Trp118Ala mutant suggest that Trp118 contributes to the binding and, possibly, the orientation of the module to soluble cellodextrins. Furthermore, the binding cleft aromatic residues Trp68 and Tyr110 play a crucial role in binding to bacterial microcrystalline cellulose (BMCC), amorphous cellulose, and soluble oligodextrins. Binding to BMCC is in disagreement with the structural features of the binding pocket, which does not support binding to the flat surface of crystalline cellulose, suggesting that CBM4 binds the amorphous part or the cellulose “whiskers” of BMCC. We propose that clostridial CBM4s have possibly evolved to bind the free-chain ends of crystalline cellulose in addition to their ability to bind soluble cellodextrins.     

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.     

Synergistic activity of Paenibacillus sp. BP-23 cellobiohydrolase Cel48C in association with the contiguous endoglucanase Cel9B and with endo- or exo-acting glucanases from Thermobifida fusca

Cellobiohydrolase Cel48C from Paenibacillus sp. BP-23, an enzyme displaying limited activity on most cellulosic substrates, was assayed for activity in the presence of other bacterial endo- or exocellulases. Significant enhanced activity was observed when Cel48C was incubated in the presence of Paenibacillus sp. BP-23 endoglucanase Cel9B or Thermobifida fusca cellulases Cel6A and Cel6B, indicating that Cel48C acts synergistically with them. Maximum synergism rates on bacterial microcrystalline cellulose or filter paper were obtained with a mixture of Paenibacillus cellulases Cel9B and Cel48C, accompanied by T. fusca exocellulase Cel6B. Synergism was also observed in cell extracts from recombinant clone E. coli pUCel9-Cel48 expressing the two contiguous Paenibacillus cellulases Cel9B and Cel48C. The enhanced cellulolytic activity displayed by the cellulase mixtures assayed could be used as an efficient tool for biotechnological applications like pulp and paper manufacturing.     

Stabilization of Crystalline Acid Carboxypeptidase from Penicillium janthinellum by Nonionic Surfactants,and Inhibition of Enzyme Activity by Anionic Compounds

The crystalline acid carboxypeptidase from Penicillium janthinellum IFO-8070 was stabilized by the addition of nonionic surfactants, such as Triton X-100, Brij 35, Span 40, and Tween 20. In the presence of these stabilizers, extremely diluted enzyme (0.3 μg/ml of 50 mm sodium acetate buffer, pH 3.7) was almost completely stable after 2 days incubation at 25°C. About 35% and 20% of the enzyme activities were activated by the addition of Triton X-100 and Brij 35, respectively. Triton X-100 completely retarded inactivation at freezing (?15°C). On the other hand, anionic surfactants of SLS and LBSA, and cationic surfactant of cetyltrimethylammonium bromide strongly inactivated the enzyme. The inhibition of the fatty acid series was roughly proportional to the molecular weight of the inhibitor. Di-, and Tri-carboxylic acids also inhibited the enzyme activity.