The role of electrostatic interactions in the mechanism of peptide bond hydrolysis by a Ser-Lys catalytic dyad

General-base catalysis in the active site of serine proteasesis carried out by the imidazole side chain of a histidine. Duringformation of the transition state, an adjacent carboxylic acidgroup stabilizes the positive charge that forms on the general-basecatalyst and as a result contributes several orders of magnitudeto the catalytic efficiency of these enzymes. In the recentlydiscovered family of self-cleaving proteins exemplified by theLexA repressor of Escherichia coli, instead of the imidazoleof a histidine, the active-site general-base catalyst was foundto be the -amino of a lysine. The considerably higher capacityof the lysine side chain for proton acceptance raises interestingquestions concerning the role of electrostatic interactionsin the mechanism of proton transfer by this highly basic group.The negative charge elimination studies described here and theireffects on the k_max and pK of LexA self-cleavage are consistentwith a model in which electrostatic interactions between anacidic side chain and the general-base catalyst form a barrierto proton transfer. The implications are that the -amino group,unlike the imidazole group, is capable of effecting proton transferwithout the intervention of a countercharge.     

Insights into the Structure of the Highly Glycosylated Ffase from Rhodotorula dairenensis Enhance Its Biotechnological Potential

Rhodotorula dairenensis β-fructofuranosidase is a highly glycosylated enzyme with broad substrate specificity that catalyzes the synthesis of 6-kestose and a mixture of the three series of fructooligosaccharides (FOS), fructosylating a variety of carbohydrates and other molecules as alditols. We report here its three-dimensional structure, showing the expected bimodular arrangement and also a unique long elongation at its N-terminus containing extensive O-glycosylation sites that form a peculiar arrangement with a protruding loop within the dimer. This region is not required for activity but could provide a molecular tool to target the dimeric protein to its receptor cellular compartment in the yeast. A truncated inactivated form was used to obtain complexes with fructose, sucrose and raffinose, and a Bis-Tris molecule was trapped, mimicking a putative acceptor substrate. The crystal structure of the complexes reveals the major traits of the active site, with Asn387 controlling the substrate binding mode. Relevant residues were selected for mutagenesis, the variants being biochemically characterized through their hydrolytic and transfructosylating activity. All changes decrease the hydrolytic efficiency against sucrose, proving their key role in the activity. Moreover, some of the generated variants exhibit redesigned transfructosylating specificity, which may be used for biotechnological purposes to produce novel fructosyl-derivatives.     

CD59活性位点相关性基因突变的构建与表达

构建了野生型和突变型CD59重组质粒,建立了高效真核表达系统,探讨了W40位点的生物学活性。采用基因点突变技术使CD59W40基因位置缺失（突变1,M1）及C39W40K41→W39W40W41（突变2,M2）,重叠延伸PCR（overlap extension PCR）定点诱变扩增突变基因,重组入真核表达质粒pIRES,利用阳离子脂质体（Lipfectamine2000）将重组质粒转染中国仓鼠卵巢细胞（CHO）进行表达。酶切鉴定及序列测定证实成功构建了pIRES-MICD59、pIRES—M2CD59和pIRES-WTCD59,突变基因约500bp。G418筛选出了CHO转染细胞的稳定细胞克隆,免疫荧光、ELISA检测筛选MICD59、M2CD59和WTCD59蛋白高表达株,连续传代30代有高表达;补体溶细胞反应显示与野生型CD59相比,突变型M1CD59失去对补体的抑制功能,而M2CD59抗补体活性略增高。证实CD59的W40位点对其功能具有重要作用,封闭此位点可提高补体活性,有望用于肿瘤治疗。     

常压室温等离子体诱变选育高产核黄素枯草芽孢杆菌

该研究以BS120作为出发菌株,通过常压室温等离子体诱变(atmospheric and room temperature plasma,ARTP)技术进行诱变处理,第一轮以40 mg/L 8-氮鸟嘌呤为筛选拮抗物进行筛选,得到核黄素产量和得率分别提升61. 60%和58. 12%的菌株BSG1。第二轮诱变以300 mg/L寡霉素为筛选拮抗物进行筛选,筛选获得菌株BSG3,核黄素产量和得率较BS120分别提升83. 59%和78. 76%。将核黄素操纵子表达质粒pMX45转入BSG3中,得到菌株BSG5,核黄素产量达到(4 467. 08±99. 47) mg/L,得率为(42. 56±1. 25) mg/g葡萄糖,较BS120分别提高140. 94%和120. 52%,展现了良好的核黄素发酵性能和遗传稳定性。     

Key Residues Affecting Binding Affinity of Sirex noctilio Fabricius Odorant-Binding Protein (SnocOBP9) to Aggregation Pheromone

Sirex noctilio Fabricius (Hymenoptera Siricidae) is a major quarantine pest responsible for substantial economic losses in the pine industry. To achieve better pest control, (Z)-3-decen-ol was identified as the male pheromone and used as a field chemical trapping agent. However, the interactions between odorant-binding proteins (OBPs) and pheromones are poorly described. In this study, SnocOBP9 had a higher binding affinity with Z3D (Ki = 1.53 ± 0.09 μM) than other chemical ligands. Molecular dynamics simulation and binding mode analysis revealed that several nonpolar residues were the main drivers for hydrophobic interactions between SnocOBP9 and Z3D. Additionally, computational alanine scanning results indicated that five amino acids (MET54, PHE57, PHE71, PHE74, LEU116) in SnocOBP9 could potentially alter the binding affinity to Z3D. Finally, we used single-site-directed mutagenesis to substitute these five residues with alanine. These results imply that the five residues play crucial roles in the SnocOBP9-Z3D complex. Our research confirmed the function of SnocOBP9, uncovered the key residues involved in SnocOBP9-Z3D interactions, and provides an inspiration to improve the effects of pheromone agent traps.     

产β-葡萄糖苷酶酿酒酵母菌株紫外诱变选育及酶学性质分析

为了得到高产胞外β-葡萄糖苷酶的酿酒酵母（Saccharomycaes cerevisiae）菌株,采用紫外线诱变技术对菌株MQFSM-3进行处理,研究了紫外诱变条件对菌株致死率的影响,并对突变菌株酶活及酶学性质进行评价。结果表明,出发菌株在辐照剂量为3500μJ/cm2的紫外灯下照射9 min,致死率为81.09%;通过初筛、复筛及多次传代培养,突变菌株UV-45产酶稳定,酶活提高41.64%,该酶最适温度为50℃,在20⁵0℃热稳定性良好;最适p H为5.5,在p H4.0⁶.5酶活力相对稳定。      

胶质芽孢杆菌HJ07的UV与NTG诱变育种及其对铝土矿浸矿效果

以从河南铝土矿样筛选出的一株胶质芽孢杆菌HJ07为出发菌株,对其进行紫外(UV)与亚硝基胍(NTG)诱变育种及铝土矿浸矿脱硅研究.分别通过紫外线照射120 s与采用质量浓度为600 mg·L^-1的亚硝基胍处理,出发菌株HJ07的致死率分别达到89%与90%,正突变率分别达到16.5%与18.7%.从突变菌株中筛选所得的两株菌种UV-2与NTG-5的生长代谢活性与脱硅能力明显比出发菌株高.在铝土矿浸出体系中,UV-2与NTG-5达到生长稳定期的时间比HJ07分别缩短了48 h与24 h,且生长稳定期具有更大的细菌浓度.浸矿12 d后,UV-2与NTG-5菌株浸出液中SiO2的质量浓度分别比HJ07提高了约25.6%与12.5%,且达到浸出终点的时间分别缩短了3 d和2 d.UV-2与NTG-5菌株较出发菌株HJ07具有更强的产酸与产胞外聚合物的能力.被UV-2菌株作用后的铝土矿表面的溶蚀程度更加显著,矿物表面形成了明显的菌胶团.     

柚苷酶高产菌株的选育及培养基优化

为了获得高产稳定的柚苷酶产生菌,以分离纯化出的36株黑曲霉菌为材料,利用常规筛选方法从中选出黑曲霉菌株M53,采用紫外线和Li Cl复合诱变,选育出一株柚苷酶高产突变株M53-7,并通过正交实验对该菌株液态发酵产酶的培养基组成进行优化。实验结果表明,正交优化后的培养基组成为蔗糖0.5%、豆饼粉3.0%、NH₄NO₃1.0%、K₂HPO₄0.1%,无机盐Ca Cl₂、Mg SO₄·7H₂O、Zn SO4在发酵培养基中的添加量分别为0.1%、0.5%、0.1%;采用最佳发酵培养基对突变菌株M53-7进行摇床发酵,其发酵液中柚苷酶活力可达到906.28 U/m L,且产酶性能稳定。      

麦芽三糖生成酶高产菌株的选育

以蛾微杆菌（Microbacterium imperiale）Mebl-012菌株为出发菌株,利用常压室温等离子体（atmospheric and room temperature plasma,ARTP）和紫外复合诱变,筛选麦芽三糖生成酶高酶活菌株。将不同ARTP致死率下的菌悬液进行了混合,均匀涂布筛选平板进行初筛,之后用摇瓶发酵进行复筛,最终筛选出了一株麦芽三糖生成酶高产突变菌株Microbacterium imperiale Metp-57,酶活达到241.32 U/m L,较出发菌株Mebl-012（产量为116.43 U/m L）提高了107.26%。以ARTP诱变过程中筛选出的高产菌株Metp-57为出发菌株,进行紫外诱变处理,得到高产突变株Metu-24,其麦芽三糖生成酶酶活可达到408.41 U/m L,较原始菌株Mebl-012提高了250.77%,且遗传性状稳定。突变株Metu-24较原始菌株Mebl-012的生长速率有明显提高。以本实验麦芽三糖生成酶水解淀粉,制备的麦芽三糖糖浆中麦芽三糖占主要成分,含量达到46.7%。