高活力天冬氨酸酶的分离提纯及性质

采用基因工程技术构建具有较高活性的突变菌株 Ｔ３３， 以它为产酶菌株分离提取天冬氨酸酶， 并研究它的酶学特性． 结果表明， 分子量为 １９０ ０００． 最适反应 ｐ Ｈ＝ ８０， 最适反应温度为 ３７ ℃． 在ｐ Ｈ＝ ６０ 时， 该酶呈负协同效应； 在ｐ Ｈ＝ ８０ 时， 该酶呈正协同效应． 该酶α螺旋度为３６３％     

双水相体系纯化天冬氨酸酶、纤维素酶和脂肪酶

用聚乙二醇(PEG)/磷酸钾双水相抽提技术分别从天冬氨酸粗酶、商品纤维素酶和脂肪酶中分离纯化出天冬氨酸酶(Aspartase)、纤维素酶(Cellulase)和脂肪酶(Lipase)。研究了PEG分子量及浓度、磷酸钾浓度、蛋白质含量、pH、氯化钠浓度等因素对分配系数,酶活回收率和分离效果等参数的影响,并设计出双水相体系抽提三种酶的相组成系统。     

天冬氨酸酶的分离提纯及其变性复性的研究

以E.Coli J-3为原料,经菌体破碎、链霉素处理、热处理、硫铵分级、磷酸钙胶处理、DEAESephadex A-50柱层析、羟基磷灰石柱层析、Ultrogel AcA 34凝胶过滤、HPLC层析九个步骤,得到聚丙烯酰胺梯度凝胶电泳纯的天冬氨酸酶。并探讨了各种环境因素对变性复性程度的影响。结果表明,不同的变性、复性条件会诱导不同构象的产生。诱导酶的性质与天然酶不同。     

聚乙烯醇固定化细胞生产L－天门冬氨酸的研究

用聚乙烯醇作为主要载体，用共混、复合和加入事种助剂的方法，对固定天门冬氨酸酶的大肠杆菌，研究了固定化细胞的性质及固定化细胞的储存稳定性。探索了固定化细胞间隙和连续发酵生产Ｌ－天门冬氨酸的生产工艺．获得了间隙和连续发酵的生产能力及固定化细胞的半寿期．     

Stimulation of Erwinia sp. Fumarase and aspartase synthesis by changing medium components

The optimal concentrations of nutrient medium components, aeration conditions, and pH providing for maximum biomass yields, as well as fumarase and l-aspartase activities, during submerged cultivation of Erwinia sp. were determined. The data showed that different concentrations of carbon source (molasses) and pH of the nutrient medium were required to reach the maximum fumarase and l-aspartase activities. Calculations performed by application of the additive lattice model suggested that the combination of these optimized factors would result in 3.2-, 3.4-, and 3.8-fold increases as compared to the experimental means in Erwinia sp. biomass, and l-aspartase and fumarase activities, respectively. The conditions of the fumaric acid biotransformations into l-malic and l-aspartic acids were optimized on the basis of intact Erwinia sp. cells, a fumarase and l-aspartase producer. In the cases of fumarate transformation into l-malic acid and of fumarate transformation into l-aspartic acids, fumarase and l-aspartase activities increased 1.5- and 1.7-fold, respectively. The experimental data were consistent with these estimates to 80% accuracy. In comparison with the additive lattice model, the application of polynomial nonlinear model allowed the between-factor relations to be considered and analyzed, which resulted in 1.1-, 1.27-, and 1.1-fold increases in Erwinia sp. biomass and fumarase and l-aspartase activities for the case of cultivation. In the case of fumarate transformation into l-malic acid, this model demonstrated a 1.7-fold increase in fumarase activity, whereas during fumarate transformation into l-aspartic acid no significant change in aspartase activity was observed.     

Continuous production of L-aspartic acid

For the continuous production ofl-aspartic acid from fumaric acid and ammonia by the action of aspartase, the enzyme extracted fromEscherichia coli orE. coli cells having high aspartase activity were immobilized by various methods. In 1973 we succeeded in the industrial production ofl-aspartic acid usingE. coli cells immobilized with polyacrylamide gel. For the improvement of this process, we developed a novel technique using κ-carrageenan as the immobilizing matrix forE. coli cells. Further, EAPc-7 strain, having higher aspartase activity, was contracted from the parentE. coli by continuous cultivation with a definite medium. The aspartase activity was about seven times higher than that of the parent cells. In 1982 we changed from the conventional method to the improved method, using EAPc-7 strain immobilized with κ-carrageenan.     

天冬氨酸酶C端缺失突变酶基因的克隆和表达

利用多聚酶链反应（PCR）技术,从天冬氨酸酶基因的3'-端删除42个碱基,缺失突变基因经克隆、转化和筛选,得到一株有C端缺失14肽的天冬氨酸酶突变体表达的转化子．突变酶纯酶活性为野生型酶的1．21倍．圆二色谱和荧光光谱的研究表明,突变酶的结构比野生型酶松散或更具柔性,天冬氨酸酶C端14肽不是其功能所必需的．     

天冬氨酸酶基因工程菌质粒pXZ70的稳定性

将pSC101质粒上的Par区域（控制质粒分配的基因座）克隆到含有天冬氨酸酶基因的质粒pXZ70上，构建成重组质粒pZZ1，将pZZ1质粒转化入大肠杆菌JM109细胞中，得到一株质粒可稳定遗传的重组子细胞，培养100代后，含质粒菌数仍在90％以上．