紫红曲的原生质体紫外线诱变育种

对紫红曲原生质体进行不同时间紫外线照射诱变,实验表明,采用紫外线（ＵＶ）照射６０ｓ,可获得高产突变株,该突变株Ｍ１－１的红曲色素产量是亲本株的红曲色素产量的２．２７倍,说明原生质体进行诱变处理是简单易行的较好方法。     

用细小病毒H-1探测人细胞中的直接诱变与间接诱变途径

用细小病毒H-1为探针,可在人细胞中检测由紫外线照射诱导的三种不同致突变途径.直接突变产生于细胞的结构性复制(或修复)功能对受损DNA模板的复制;间接无靶突变和间接有靶突变分别产生于紫外线诱导的易错修复功能对完整模板和模板上的靶损伤的作用.在低剂量范围内,间接无靶突变在某些DNA毒剂诱导的细胞突变中起主要作用.某些DNA损伤,如烷化剂诱导的次级损伤(无碱基位损伤)可作为诱导性易错修复功能的靶损伤.     

γ-聚谷氨酸高产菌株的选育和发酵培养基的初步优化

以实验室保藏的一株枯草芽孢杆菌为出发菌株,采用紫外、亚硝基胍以及60Coγ射线对其进行复合诱变,获得一株γ-聚谷氨酸高产突变株,在基础培养基中产量是出发菌株的3.11倍,并且突变株经过传代10次,发酵能力稳定;通过正交试验对突变株的发酵培养基进行了初步优化,突变株在一组培养基上的γ-聚谷氨酸产量可达到33.81g/L,是优化前的1.11倍。该突变株的摇瓶发酵产量较高,值得深入开发研究。     

丝裂霉素诱变黑曲霉产植酸酶过程模型化

用均匀设计分析方案,进行丝裂霉素C对黑曲霉化学诱变菌丝体和原生质体诱导产植酸酶选育,并以诱变时间(X_1)和丝裂霉素C的浓度(X_2)建立回归方程．实验筛选出具有4株较高酶活的菌株,诱变菌丝体得到2株酶活是始发菌株183．89％和147．55％;诱变原生质体得到2株酶活是始发菌株395．52％和381． 98％．由响应面模型初步判断,在丝裂霉素C浓度为5～30 mg·L-1和诱变时间45～60 min时,丝裂霉素C 对黑曲霉诱变原生质体选育产植酸酶菌株的效果比对菌丝体诱变效果好．用均匀设计对丝裂霉素C诱变菌丝体和原生质体的效果进行回归分析,所得模型调整后的相关系数分别为89．02％和99．13％,模型拟合程度好,实验误差小,可以用此模型较好的对丝裂霉素C的黑曲霉菌丝体诱变进行分析和预测．     

VC二步发酵伴生菌苏云金芽孢杆菌的选育

苏云金芽孢杆菌经紫外线(UV)、亚硝基胍(NTG)及两者的复合诱变,获得1株高效伴生菌株UN-366.在pH 6.5~6.8的发酵培养基中与氧化葡萄糖酸杆菌混合发酵,UN-366的平均糖酸转化率提高了6.32%,古龙酸的质量浓度达到了83.7 g/L,经连续5代转接发酵实验,性状稳定.     

Salsolinol合成酶的克隆和表达

 Salsolinol 合成酶是一种催化多巴胺和乙醛生成Salsolinol 的酶,与帕金森病发病机制密切相关。研究发现Salsolinol 合成酶与泛素的氨基酸序列高度相似,只有4 个氨基酸位点有差异。本研究以泛素基因为模板,采用聚合酶链式反应技术对4 个位点进行定点突变,将突变基因片段克隆到载体pET30a-GST 上,构建pET30a-GST-Sal synthase 重组载体,转化BL21 后,IPTG 诱导重组菌表达融合蛋白,经亲和层析柱纯化。结果表明,实现目的位点的定点突变,获得Sal 合成酶基因,成功构建了GST-Sal synthase 原核表达质粒,在大肠杆菌中表达纯化后得到较高纯度的GST-Sal synthase 融合蛋白。     

Site-directed mutagenesis reveals new and essential elements for iron-coordination of the sulfur oxygenase reductase from the acidothermophilic Acidianus tengchongensis

Previous study on refolding of sulfur oxygenase reductase (SOR) inclusion bodies from recombinant Escherichia coli showed that iron was critical to the activity of the SOR from Acidianus ambivalens. In this study, enzymatic assays showed that 2,2′-Dipyridyl, Tiron and 8-hydroxyquinoline, which are specific for chelating ferrous or ferric ions, strongly inhibited the activity of SOR from A. tengchongensis, suggesting that iron atom is essential for SOR activity. Alignment of several functionally identified SORs and SOR-like sequences from genome database revealed a conserved, putative iron binding motif, H⁸⁶-X₃-H⁹⁰-X _n -E¹¹⁴-X _n -E¹²⁹ (numbering according to the Acidianus tengchongensis SOR sequence). Three mutants of SOR were generated by site-directed mutagenesis of H₈₆, H₉₀ and E₁₂₉ into phenylalanine or alanine residue in this study. Circular dichroism spectrum determination indicated that there was no change of the secondary structures of mutant SORs, H⁸⁶F, H⁹⁰F and E¹²⁹A, but all mutants were completely inactive. Through determination of iron contents we found that SOR mutants of H⁸⁶F, H⁹⁰F and E¹²⁹A completely or partially lost iron, while mutants of C³¹S, C¹⁰¹S, and C¹⁰⁴S (generated in a previous study) did not. This result indicated that H⁸⁶, H⁹⁰ and E¹²⁹ but not C³¹, C¹⁰¹, and C¹⁰⁴ were involved in binding to iron atom. Based on this and previous studies, it is proposed that the conserved motifs, C³¹-X _n -C¹⁰¹-X₂-C¹⁰⁴ and H⁸⁶-X₃-H⁹⁰-X₂₃-E¹¹⁴-X₁₄-(E/D)¹²⁹, are respectively for sulfur and molecular oxygen binding and activation. These two conserved motifs are essential elements for the SOR activity. Supported by National Natural Science Foundation of China (Grant Nos. 30670018, 30621005) and State Key Basic Research and Development Program of China (Grant No. 2004CB719602)     

xyl-d二型乙醇脱氢酶(SADH)G198R突变体的研究

本研究通过设计简并引物及文献参考,PCR 克隆得到嗜热厌氧菌菌株 xyl-d 的SADH,并利用定点诱变技术将该酶的198位苷氨酸(Gly)突变为精氨酸(Arg)。将野生型和突变基因连接到原核表达载体pET28a,IPTG诱导表达,再经镍柱纯化,然后比较了两个蛋白分别以 NAD/NADH,NADP/NADPH 为辅因子、异丙醇或异丁醛为底物、55℃条件下的酶活。发现突变后蛋白的总体催化活性相对于野生型有所下降,其中突变蛋白对 NAD/NADH 的亲和性分别下降了约8倍和6倍,而且对NADPH 的亲和性下降也非常明显,但对NADP的亲和性变化却不大。     

链霉素抗性突变理性筛选链霉素高产菌株

以灰色链霉菌(Streptomyces griseus) CICC11002为出发菌株,采用紫外诱变并结合链霉素抗性筛选法育种,以期获得高产菌株.灰色链霉菌孢子悬液经90％紫外致死剂量诱变后,在含有链霉素最小抑制浓度(0.8μg/mL)的培养基平板上,分离得到105株链霉素抗性突变菌.其中链霉素产量高于出发菌株的有19株,正突变率高达18.1％,同时获得了产链霉素能力约为出发菌株2倍的突变株SM-55.