北京棒杆菌天冬氨酸激酶M372I-T379S双突变体的构建及其酶学性质

为构建高酶活力天冬氨酸激酶(aspartokinase, AK), 并削弱或解除Lys(lysine)反馈抑制作用突变体, 通过定点突变和高通量筛选技术构建突变体M372I,T379S和M372I-T379S, 对野生型（WT）和突变体分别进行诱导表达、 纯化及酶学性质表征. 结果表明： 突变体M372I,T379S和M372I-T379S AK与WTAK相比, V_max分别提高了13.77,15.02,15.60倍, K_m和n值均降低； 最适pH值分别升高为8.0,8.5,8.5, 且半衰期分别延长了1.0,0.9,2.3 h； M372I-T379S AK最适温度为30 ℃, 比WT AK高2 ℃； 当浓度为1~10 mmol/L时, 突变体均削弱或部分解除了抑制剂Lys的反馈抑制作用.     

法夫酵母人工诱变及突变株筛选的研究

用硫酸二乙酯对法夫酵母进行了诱变，并用薄层色谱法及高效液相色谱法对突变株进行了筛选鉴定,当诱变剂浓度为1％、诱变温度为20℃时，法夫酵母诱变处理的最适时间约为5min．根据视觉差异分离二苯胺平板上长出的法夫酵母菌落，用薄层色谱法初筛，并用液相色谱法鉴定出了虾青素高产菌株、虾青索低产菌株、类胡萝卜素组成缺陷菌株．     

静电辅以激光对酿酒酵母菌的诱变效应研究

利用高压静电场辅以激光对甘蔗糖密工业生产用酿酒酵母菌进行处理.把酵母菌糖密酒精进行发酵,利用气相色谱仪对产生的乙醇含量进行分析.实验结果表明,与单一高压静电场和单一激光作用相比,高压静电场辅以激光对酿酒酵母菌具有明显的生物刺激作用.初步找到乙醇含量具有较大变化的高压静电场和激光变异菌株;将高压静电场辅以激光变异菌株的乙醇脱氢酶同工酶的比较,证实了高压静电场辅以激光对酿酒酵母菌的诱变作用明显.     

灰树花紫外诱变育种研究

应用原生质体紫外诱变技术,对灰树花菌株Gr1进行了紫外诱变处理.经过粗筛和精筛后,从50株诱变株中选出两株多糖含量和产量明显优于原始菌株的突变株Gr1a和Gr1g,其产量分别为1.74%和1.62%,多糖质量含量(w)分别为11.67%和12.01%.经过摇瓶试验以及发酵试验,两株诱变菌株Gr1a和Gr1g菌丝得率和多糖含量都很稳定,表明所得突变株是比原始菌株更优秀的稳定高产、高多糖含量的新菌株.     

Aspergillus oryzae 3.951白孢突变株的选育及其特性的研究

为改进酱油风味,作者于1984年选择了Aspergillus oryzae 3.951为出发菌株,经亚硝基胍、紫外线复合诱变处理,选育出白孢突变株,再经谷氨酰胺酶酶活筛选,获得Asp.白孢1号突变株。此菌株形态、生理特性与出发菌株相近,菌丝生长快,粗壮,浓厚,孢子白色,产孢较迟较少。蛋白酶、糖化酶酶活与出发菌株相近。谷氨酰胺酶酶活比出发菌株高34—50％,制曲后,未检出黄曲霉毒素B_1。     

禾黑芽枝霉及其诱变体的果胶酶生产特性

经连续六代紫外幅射诱变和筛选，获得能连续六代稳定高果胶酶活性的突变体ＧＢＩＢＵ７研究了该突变体的果胶酶形成特性。实验结果表明，丰富的多糖，无机氮源及适量的阴离子去污剂培养基有利于突变体ＧＢＩＢＵ７提高果胶酶活性。     

Screening of conformationally constrained random polypeptide libraries displayed on a protein scaffold

The selection of novel proteins or enzymes from random protein libraries has come to be a major objective in current biology, and these enzymes should prove useful in various biological and biomedical fields. New technologies such as in vitro selection of proteins in cell-free systems have high potential to realize evolu tionary molecular engineering of proteins. This review highlights an application of insertional mutagenesis of proteins to evolutionary molecular engineering. Random sequence proteins are inserted into the surface of a host enzyme which serves as a scaffold to display random protein libraries. Constraints on random polypeptide conformations owing to the proximity of N- and C-termini on the scaffold would result in greater screening efficiency of libraries. The scaffold enzyme is also used as a probe for monitoring the hill climbing of random sequence proteins on a fitness landscape and navigating rapid protein folding in the sequence space. Received 9 October 1997; received after revision 6 January 1998; accepted 19 January 1998     

Agrobacterium tumefaciens-mediated genetic transformation of the phytopathogenic fungus Penicillium digitatum

Agrobacterium tumefaciens-mediated transformation （ATMT） system was assessed for conducting insertional mutagenesis in Penicillium digitatum, a major fungal pathogen infecting post-harvest citrus fruits. A transformation efficiency of up to 60 transformants per 106 conidia was achieved by this system. The integration of the hph gene into the fungal genome was verified by polymerase chain reaction （PCR） amplification and sequencing. These transformants tested were also shown to be mitotically stable. Southern blot analysis of 14 randomly selected transformants showed that the hph gene was randomly integrated as single copy into the fungal genome of P. digitatum. Thus, we conclude that ATMT of P. digitatum could be used as an alternatively practical genetic tool for conducting insertional mutagenesis in P. digitatum to study functional genomics.     

蛋白质定点诱变技术的进展

蛋白质定点诱变技术是研究基因表达、蛋白质结构与功能关系，蛋白质的稳定性及分子进化等的重要工具．已发展了大量诱变质粒DNA特殊碱基的有效操作程序，所有的方法都按在体外用带有预先设计诱变部位的寡核苷酸与己知序列靶基因序列错配异源杂交、合成定点诱变的DNA完成的．对具有代表性的Hutchison法、Kunkel法、Promega法、Deng和Nickoloff法进行了综述，并讨论了它们的优点和局限性．     

梁料脱色菌—无花果曲霉（Aspergillus ficuum）的紫外诱变育种

染料脱色菌无花果曲霉（Aspergillus ficuum）经紫外线诱变后，筛选出变株P11在33℃、120r／min振荡培养条件下，3d内以对染料直接耐酸大红4BS的脱色率为88．54％比出发菌株提高了13％，经斜面传代10次，脱色能力保持稳定。