酶分子体外定向进化的研究进展

分子体外定向进化是改造酶分子的新策略,它主要通过体外模拟自然进化机制,利用基因随机突变、重组和定向筛选技术,使进化过程朝着人们需要的方向发展。简要介绍了酶分子体外定向进化的发展历史,详细介绍了突变文库构建和筛选方法的最新研究进展及应用情况。     

DNA改组技术在酶工程中的应用

DNA改组技术是一种应用最广泛且简单有效的分子定向进化方法.它是一种高通量的随机突变筛选技术,可以对目的基因进行多次重组和选择,得到性质优良的突变文库.介绍了DNA改组技术的原理及其在酶工程中的应用.     

微生物菌种选育中的基因组改组技术及其应用进展

该文论述了基因组改组技术的产生和原理、方法和特点,以及该技术的应用、意义及其发展前景.基因组改组技术是首先对微生物菌株进行诱变,筛选出正向突变的菌株,然后通过原生质体"递推式融合"使这些正向突变的若干个菌株进行基因组重组,从中筛选出符合育种要求的重组子,从而在短时间内获得性状得到大幅度提高的菌株.     

蛋白质定向进化技术的研究进展

蛋白质定向进化技术是蛋白质分子改造的一个重要策略.重点介绍了易错PCR、DNA改组等对编码蛋白质的基因进行随机突变和重组的技术,以及构建突变体库和高通量筛选的方法,并探讨了定向进化技术在蛋白质工程中的应用及前景.     

酶学方法第68卷(重组DNA)

本书介绍研究遗传工程、分子生物学方面很重要的重组DNA的研究方法及最新技术。内容包括重组DNA技术,用于重组DNA研究的酶学,DNA的合成、分离与提纯,用于重组DNA无性繁殖的载体和宿主,DNA无性繁殖系的筛选,无性繁殖基因表达的检验与分析方法。     

重组DNA技术对环境的影响

引言本文旨在介绍并讨论重组DNA技术对环境的潜在影响。此外还要讨论传统生物技术的应用和影响,因为它不仅具备一些与新技术相匹敌的效应,而且有助于洞悉现代基因操作的潜在影响。生物体中DNA的突变或者改变往往导致其表型的变化。突变可以是自发的,也可以通过辐射或化学药物加以诱导,使频率增高。遗传改造生物体的传统方法是先诱变,再筛选优越的性状。重组DNA技术带来的遗传改变可以进一步发展,且比传统方法更迅速,更专一。重组DNA技术刺激了数百家公司的发展,后者都希望在开发该技术的过程中获利。由于该领域的发展极其迅速,所以,客观、迅速地评价其对个人和环境的潜在风险是势在必行的。     

Red同源重组在大肠杆菌基因组修饰中的应用

Red同源重组技术发展迅速,已广泛应用于大肠杆菌基因组修饰,在点突变、基因敲除、序列整合等方面发挥着重要作用。简要综述了Red同源重组的重组机制和操作策略等研究进展,并介绍了Red同源重组在大肠杆菌基因组减小及多基因代谢途径优化方面的应用情况。     

酶的定向进化策略

定向进化技术已成为改造酶分子的一种有效策略,是在体外模拟自然进化进程,通过随机突变和（或）体外重组产生基因的多样性．再经筛选（或选择）获得所需性能大幅提高的酶。该文介绍近几年来酶定向进化技术研究的最新进展以及一些成功实例。     

利用DREAM设计和同源重组进行一步定点突变

目的：建立基于DREAM设计和同源重组的简便、快速定点突变方法。方法：设计两条包含突变的反向PCR（inverse PCR）引物,使其5'端互补从而产生同源重组,同时使用DREAM设计方案在上述引物中引入限制性内切酶位点以便突变子筛选。用能扩增长片段的高保真耐热 DNA聚合酶扩增全长的质粒DNA,直接转化大肠杆菌。转化到细菌中的全长质粒DNA PCR产物可利用其末端同源序列发生同源重组而环化。利用引入的酶切位点方便地进行突变子的筛选。结果：我们用该方法成功地对长度大于7 kb的质粒进行了定点突变。结论：本定点突变无需任何突变试剂盒和特殊的试剂,只需一步反应即可完成;利用DREAM设计使克隆筛选简便可靠,高保真耐热DNA聚合酶可保证多数突变子克隆不发生意外突变,而该酶扩增长片段的能力使该方法适合于大多数质粒不经亚克隆直接突变。     

体外突变新技术：DNAShuffling技术

体外DNAshuffling技术是一种快速、高效并且衫的分子定向进化技术,和以往的基因突变技术不同,它是一种高通量的突变筛选技术,可以对靶序列进行多次重组和选择,利用该技术建立的突变文库（嵌合文库）具有容量大、多样性好等优点,且更易于实现有益突变的积累。DNAshuffling技术已广泛应用于酶、药物蛋白及其它功能蛋白的改造,并取得了相当大的成功,显示了该技术在蛋白质改造和分子育种方面的潜在应用价值。     

Purification and characterization of cytosol-specific phosphoenolpyruvate carboxykinase from chicken liver

Phosphoenolpyruvate carboxykinase of chicken liver cytosol was purified to homogeneity by procedures including affinity chromatography with GTP as a ligand. The purified enzyme showed a molecular weight of 68,000 on gel electrophoresis in the presence of dodecyl sulfate. Comparative studies on this enzyme and its isozyme purified from chicken liver mitochondria were performed. As regards amino acid composition, the cytosolic enzyme was quite different from the mitochondrial enzyme, but was rather similar to rat liver cytosolic phosphoenolpyruvate carboxykinase. Specific activities of the cytosolic enzyme were 30-100% higher than those of the mitochondrial enzyme for oxaloacetate-CO2 exchange, oxaloacetate decarboxylation, and phosphoenolpyruvate carboxylation reactions, though the relative rates of the activities were similar, decreasing in the order given. Apparent Michaelis constants for oxaloacetate in the oxaloacetate decarboxylation reaction were 11.6 and 17.9 microM for the cytosolic and the mitochondrial enzyme, respectively, but the values for GTP, GDP, phosphoenolpyruvate, and CO2 in the oxaloacetate decarboxylation and phosphoenolpyruvate carboxylation reactions were 1.3-2.2 times higher for the cytosolic enzyme than for the mitochondrial enzyme. Thus, the fundamental catalytic properties of the chicken liver phosphoenolpyruvate carboxykinase isozymes were rather similar, despite the marked difference in amino acid compositions.     

Expression of the vanadium-dependent bromoperoxidase gene from a marine macro-alga Corallina pilulifera in Saccharomyces cerevisiae and characterization of the recombinant enzyme

The vanadium-dependent bromoperoxidase from the marine macro-alga Corallina pilulifera was heterologously expressed in Saccharomyces cerevisiae. The enzyme was purified and crystals in "tear drop" form were obtained. The catalytic properties of the recombinant enzyme were studied and compared with those of the native enzyme purified from C. pilulifera. Differences in thermal stability and chloroperoxidase activity were observed. The recombinant enzyme retained full activity after preincubation at 65 degrees C for 20 min, but the native enzyme was completely inactivated under the same conditions. The chlorinating activity of the native enzyme was more than ten times higher than that of the recombinant enzyme. Other properties, such as K(m) values for KBr and H(2)O(2), and optimal temperature and pH, were similar for each source of C. pilulifera bromoperoxidase.     

硫酸盐还原菌固态发酵产品酶活测定方法的建立

为建立生物质固载硫酸盐还原菌（SRB）产品酶活的检测方法,首先确定亚硫酸盐还原酶（SiR）作为SRB胞外酶表征其生物活性的可行性;其次对固态发酵产品制备粗酶液的各种条件进行探讨,研究浸泡介质、浸泡时间、超声功率、超声时间、预处理方式等单因素对酶活的影响;由正交实验确定粗酶液制备的最佳工况。实验结果表明,先浸提后超声处理的酶活值显著高于仅用超声、浸提的结果。为确保酶活值测定不受干扰,排除超声温度对结果的影响,确定实验最佳工况：2g固态发酵产品经磷酸盐缓冲液25mL、浸提60min,酶活值为1．0799U／g。生物质固载SRB酶活检测方法的建立为固态发酵产品活性评价、固态发酵条件优化、可渗透反应墙生化性能评估提供一定的理论依据。     

四季豆幼苗中尿囊酸酰胺水解酶的一些特性

酰脲代谢在许多固氮豆科植物氮素代谢中起重要作用;尿囊酸的酰胺水解酶(EC3.5.3.9)分解尿囊酸成为脲基乙醇酸和CO2、NH3,脲基乙醇酸的酰胺水解酶进一步分解脲基乙醇酸产生乙醛酸和CO2、NH3.该文首次报告测定四季豆尿囊酸降解酶(分解尿囊酸的酶)的方法,酶反应基质需要盐酸苯肼存在.在四季豆干种子、幼苗根、茎和叶,均可测出尿囊酸降解酶活力.从四季豆幼苗分离出两个尿囊酸降解酶.一个分子量大于200 kD,另一个分子量为13.5 kD;小分子量的尿囊酸降解酶(没有脲基乙醇酸酰胺水解酶或脲酶活力)用于性质研究.酶反应产物分析表明,该酶是尿囊酸的酰胺水解酶.该酶反应的最适pH为8.5.Mn2 是该酶的金属辅助因子.Km为76μmol/L,Vmax为16.7 nKat/mg(=1 002 nmol min1mg1).乙醛酸和乙醇酸抑制该酶活力.赖氨酸残基和色氨酸残基是酶活力的必需基团;巯基和酪氨酸残基不是酶活力的必需基团.     

Enzymatic modification of vegetable protein: Immobilization of Penicillium duponti enzyme on reconstituted collagen and the use of the immobilized-enzyme complex for solubilizing vegetable protein in a recycle reactor

Penicillium duponti enzyme was immobilized on reconstituted collagen by macromolecular complication, impregnation, and covalent crosslinking techniques. The immobilization of the enzyme on collagen has a twofold purpose: (1) providing a protein microenvironment for the proteolytic enzyme; and (2) extending the useful life the enzyme once immobilized on the collagen matrix. Two types of collagen were used, one produced by the United States Department of Agriculture and the other produced by FMC. The USDA collagen contained unhydrolyzed telepeptide linkages and required pretreatment to reduce collagenaselike activity of the enzyme. Activity analysis of the immobilized enzyme complex showed that membranes with enzyme loading less than 10 mg enzyme/g of wet membrane in the reactor were dimensionally stable. The degree of crosslinking was an important parameter. Membranes with structural opening up to three times the initial dry thickness were found to be the maximum limit for controlled release of enzyme from the collagen membrane during enzymatic reaction. Higher activities and better stability of the enzyme in collagen membrane were found for covalent crosslinking of the enzyme to treated collagen films. The hydrolysis of soybean vegetable protein with the immobilized enzyme in a recycle reactor at enzyme loading of mg/g of wet membrane at 40°C, pH 3.4, produced 56.5% of soluble protein in 10h. The production is equivalent to 1.84 h total contact time between the substrate and the immobilized enzyme. The average productivity based on a stable enzyme activity and 20g of dry membrane was 329 mg of protein/g/mg of active enzyme immobilized. The productivity of the free enzyme in a batch reactor was 62.5 mg protein/h/mg enzyme.     

Heterologous expression and characterization of wild-type human cytochrome P450 1A2 without conventional N-terminal modification in Escherichia coli

In this study, wild-type human CYP1A2 without the conventional N-terminal modification (second codon GCT) or the truncation of the N-terminal hydrophobic region was functionally expressed in Escherichia coli. Its enzymatic properties were compared with N-terminally modified CYP1A2. Although modified CYP1A2 is almost all high-spin, some wild-type CYP1A2 shifted to low-spin. Spectral binding titrations with several ligands could be performed with wild-type enzyme, but not with modified enzyme. Kinetic parameters for several substrates were similar for the two CYP1A2 enzymes. However, the oxidation rates of phenacetin by modified enzyme were approximately 2-fold higher than those by wild-type enzyme. The intermolecular isotope effects were approximately 2 for phenacetin O-deethylation catalyzed by both enzymes. However, the wild-type enzyme, but not the modified enzyme, increased C-hydroxylation when O-deethylation rates were lowered by deuterium substitution. Molecular switching indicates that phenacetin rotates within the active site of wild-type enzyme and suggests a looser conformation in the active site of the wild-type enzyme than of the modified enzyme. These results reveal that the overall enzymatic properties of wild-type CYP1A2 enzyme are quite similar to those of modified CYP1A2, although its active site environment seems to differ from that of the modified enzyme.     

Presence of two DNA polymerases in Tetrahymena pyriformis.

Two DNA polymerases were detected in Tetrahymena pyriformis, strain GL. One (enzyme I) was sensitive to N-ethylmaleimide, while the other (enzyme II) was insensitive. The molecular weight of the enzymes, as determined by glycerol gradient centrifugation analysis, were approximately 130,000 and 70,000, respectively. Optimal concentration of MgCl2 was 10mM for enzyme I and 18mM for enzyme II. KCl inhibited enzyme I but stimulated enzyme II. Poly (dA-dT) served effectively as a template for enzyme I, while poly(dA).(dT)12-18 was an effective template for enzyme II. Enzyme I activity increased with cell growth and sharply declined after the cells reached the stationary phase. On the other hand, enzyme II activity appeared only at the end of log phase. In cells synchronized by starvation-refeeding technique enzyme I was markedly stimulated in correspondence to the rate of DNA synthesis, whereas the level of enzyme II activity changed to lesser extent. By ethidium bromide treatment, only enzyme I activity was induced.     

Characterization and partial purification of two enzymes transferring N-acetylglucosamine to dolichyl monophosphate and ribonuclease A

Two N-acetylglucosamine (GlcNAc) transferases which catalyze the incorporation of GlcNAc into GlcNAc-P-P-dolichol (dolichol enzyme) and into bovine pancreatic ribonuclease A (RNAseA enzyme) were solubilized from the rat liver microsomes in a non-ionic detergent, Triton X-100. Both enzyme activities were adsorbed on activated CH-Sepharose 4B, and could be eluted with a linear KCl gradient. Two enzyme activities were separated by this column with the dolichol enzyme eluting before the RNAseA enzyme. A 49-fold and 136-fold purification was achieved for the dolichol and the RNAseA enzyme, respectively. The addition of exogeneous dolichyl phosphate resulted in a 3-5-fold stimulation of the purified dolichol enzyme, but did not affect the purified RNAseA enzyme. The addition of RNAseA stimulated only the RNAseA enzyme. Whereas, tunicamycin could inhibit only the dolichol enzyme. The purified dolichol enzyme had a Km of 14 X 10(-6) M for UDP-GlcNAc and the reaction was saturated with about 0.25 M dolichyl phosphate. The purified RNAseA enzyme had a Km of 4.55 X 10(-6) M for UDP-GlcNAc and was saturated with about 0.36 mM RNAseA. The pH optima and the metal ion requirement for the two enzymes were different. These results suggest that because of the different properties of these two enzymes they may have distinct functions regarding the core glycosylation of N-linked glycoproteins. It is well established that the dolichol enzyme catalyzes the formation of the first dolichol-linked intermediate GlcNAc-P-P-dolichol, whereas according to the present finding, the RNAseA enzyme may catalyze the transfer of GlcNAc directly from UDP-GlcNAc into acceptor protein.     

The effects of fasting on glucose-6-phosphatase of mouse liver and kidney.

Glucose-6-phosphatase levels were measured in livers and kidneys of control mice and mice fasted for 24, 48, and 72 h, respectively. The enzyme was assayed with and without deoxycholate supplementation. When assayed in the absence of deoxycholate, significant increases in liver enzyme levels was observed after fasting for 48 and 72 h, respectively. For the kidney enzyme, a significant increase was observed only after 48 h fasting. When the enzyme was assayed in the presence of 4.8 mmol/l deoxycholate, fasting-induced increases of liver enzyme levels were further potentiated, but no significant differences were observed in kidney enzyme levels between fasted and control mice.