酶的定向进化及其应用

综述了生物催化剂（酶）分子定向进化的产生、原理、方法及应用,深入阐述酶分子定向进化过程中的相关问题,重点介绍了易错PCR（Error-prone PCR）和DNA改组（DNA shuffling）等几种典型的酶定向进化方法与成功实例,展望了生物定向进化研究的发展前景。     

酶的定向进化研究及其在工业生物催化中的应用

综合概括了各种蛋白质改造与微生物代谢途径改造的非理性定向进化技术的原理、特点,介绍了这些技术在高性能工业生物催化剂改造中的应用。     

改进酶稳定性的定向进化技术

酶作为一种生物催化剂,以其独特的优良特性,在绿色化学和清洁生产中得到了广泛的应用。随着酶定向进化技术的建立和发展,通过定向进化改进酶稳定性的研究越来越多。详细综述了各种定向进化方法的特点及在提高酶稳定性方面的应用,并从结构和功能的角度进一步解释了相关机理。     

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.     

过氧化氢酶Ⅰ结构延伸突变改善酶热稳定性的初步研究

嗜热脂肪芽孢杆菌过氧化氢酶Ⅰ及其取代突变酶８８（２５）的延伸突变是指在酶蛋白在Ｃ末上连接一段随机肽链,从而改变了酶蛋白的结构,研究结果表明,这种延伸突变的方法非常有效地提高了酶的热稳定性,并且随机肽链的疏水性一与其对应的延伸突变体酶的热稳定性呈现一定的负相关性,即随机肽链的疏水性越高,对应的突变体酶热稳定性越低。     

综合生物塘水体过氧化氢酶的活性,组成及其与过氧化氢的关系

在我国,关于水体酶学的研究尚未见诸报道。本文讨论了湖北黄州综合生物塘不同净化功能单元中过氧化氢酶(CAT)的组成(即未处理水样和通过孔径为3.0μ的滤膜的过滤水样各自所表现的活性)、CAT活性的变化趋势及其与底物(H_2O_2)浓度的关系。     

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。     

几种定向进化技术的比较及文库构建策略

蛋白质定向进化在蛋白质工程中取得了令人瞩目的成就,其核心技术---随机突变库构建技术已成为近年来体外定向进化研究的热点。在概述定向进化基本原理基础上,对几种随机突变技术进行了介绍、分类和比较,并对突变库的特征及构建策略予以分析和描述 。     

大肠杆菌碱性磷酸酶的体外定向进化研究

大肠杆菌碱性磷酸酶（E.coli alkaline phosphatase, EAP, EC 3.1.3.1）是一个非特异性二聚体磷酸单酯酶. 采用易错聚合酶链反应(error prone PCR)的方法,在原有高活力突变株的基础上,对EAP远离活性中心催化三联体的区域进行定向进化,经两轮error prone PCR,获得催化活力较亲本D101S突变株提高3倍、较野生型酶提高35倍的进化酶4-186,并对该酶的催化动力学特征进行了分析. 进化酶基因的DNA测序表明4-186含两个有义氨基酸置换：K167R和S374C,二者既不位于底物结合位点,也不位于酶的金属离子结合位点.     

定向进化提高嗜热拟青霉J18耐热β-1,3-1,4-葡聚糖酶在酸性条件下的催化能力

应用定向进化技术提高了嗜热拟青霉Paecilomyces thermophila J18耐热β-1,3-1,4-葡聚糖酶(PtLic16A)在酸性条件下的催化能力.结合易错PCR和DNA改组的方法,构建了β-葡聚糖酶的突变体文库;利用刚果红染色法建立了阳性克隆的高通量筛选体系.筛选得到的突变酶PtLic 16AM1的反应最适pH由7.0变化至5.5,且保持了原有的耐热性和比酶活.突变酶的DNA序列中有4个点位发生突变,引发了4处氨基酸替换,分别是T58S、Y110N、G195E和D221G.结构模拟结果显示,发生突变的4个氨基酸位点中,Y110N位置靠近酶活性中心,而T58S、G195E和D221G则离酶活性中心较远,其中T58S、G195E可能对酶最适pH的变化起到了关键作用.     

A convenient and robust method for construction of combinatorial and random mutant libraries

Here we describe a convenient and robust ligase-independent method for construction of combinatorial and random mutant libraries. The homologous genes flanked by plasmid-derived DNA sequences are fragmented, and the random fragments are reassembled in a self-priming polymerase reaction to obtain chimeric genes. The product is then mixed with linearized vector and two pairs of flanking primers, followed by assembly of the chimeric genes and linearized vector by PCR to introduce recombinant plasmids of a combinatorial library. Commonly, it is difficult to find proper restriction sites during the construction of recombinant plasmids after DNA shuffling with multiple homologous genes. However, this disadvantage can be overcome by using the ligase-independent method because the steps of DNA digestion and ligation can be avoided during library construction. Similarly, DNA sequences with random mutations introduced by error-prone PCR can be used to construct recombinant plasmids of a random mutant library with this method. Additionally, this method can meet the needs of large and comprehensive DNA library construction.     

透明颤菌血红蛋白的DNA改组研究

为提高透明颤菌血红蛋白在限氧条件下促进宿主细胞生长的能力,首先通过易错PCR向透明颤菌血红蛋白基因中引入突变,再结合DNA改组对其进行改造。将改组基因置于透明颤菌血红蛋白天然启动子下游,转化大肠杆菌DH5α,构建改组文库。以限氧培养条件下菌体沉淀的颜色为指标进行试管初筛,再以限氧和极端限氧条件下菌体湿重为指标进行摇瓶复筛,最终得到一个高活性突变蛋白VHb'042506。该蛋白使宿主的菌体湿重在限氧和极端限氧条件下较原基因转化子分别提高了31.25%和58.75%。经测序和比对,该基因与原基因相比发生了11处碱基点突变,致氨基酸4处错义突变。CO差光谱实验显示该蛋白具有更强的特征吸收。     

Improved catalytic efficiency of Endo-β-1,4-glucanase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> BME-15 by directed evolution

Bacillus subtilis endo-β-1,4-glucanase (Cel5A) hydrolyzes cellulose by cleavage of the internal bonds in the glucose chains, producing new ends randomly. Using directed evolution techniques of error-prone polymerase chain reaction (PCR) and DNA shuffling, several Cel5A variants with improved catalytic activity had been screened from the mutant library, which contained 71,000 colonies. Compared with the wild-type enzyme, the variants (M44-11, S75 and S78) showed 2.03 to 2.68-fold increased activities toward sodium carboxymethyl cellulose (CMC), while the M44-11 also exhibited a wider pH tolerance and higher thermostability. Structural models of M44-11, S75, S78, and WT proteins revealed that most of the substitutions were not located in the strictly conserved regions, except the mutation V255A of S75, which was closed to the nucleophile Glu257 in the catalytic center of the enzyme. Moreover, V74A and D272G of M44-11, which were not located in the substrate binding sites and the catalytic center, might result in improved stability and catalytic activity. These results provided useful references for directed evolution of the enzymes that belonged to the glycoside hydrolase family 5 (GH5).     

蛋白质定向进化的研究技术及应用

蛋白质定向进化是在人类改造蛋白质的过程中产生的。蛋白质定向进化通常分三步进行,即随机诱变、体外重组和筛选。每一步都有多种研究技术。蛋白质定向进化大大加速了人类改造蛋白质的步伐,在实际应用研究和基础理论研究中都具有重要意义。     

Improved thermostability and the optimum temperature of Rhizopus arrhizus lipase by directed evolution

To expand the functionality of lipase from Rhizopus arrhizus (RAL) we have used error-prone PCR and DNA shuffling methods to create RAL mutants with improved thermostability and the optimum temperature. One desirable mutant with three amino acids substitution was obtained. The mutated lipase was purified and characterized. The optimum temperature of the mutant lipase was higher by 10 °C than that of the wild-type RAL (WT-RAL). In addition, the thermostability characteristic of the mutant was also improved as the result of directed evolution. The half-life (T_1/2) at 50 °C of the mutant exceeded those of WT-RAL by 12-fold. To confirm which substitution contributed to enhance thermostability and the optimum temperature for lipase activity, three chimeric lipases: chimeric lipase 1(CL-1; A9T), chimeric lipase 2 (CL-2; E190V) and chimeric lipase 3 (CL-3; M225I) from the WT-RAL gene were constructed. Each of the chimeric enzymes was purified and characterized. Amino acid substitution at position 190 was determined to be critical for lipase thermostability and the optimum temperature, while the residue at position 9 and 225 had only marginal effect. The mutational effect is interpreted according to a simulated three-dimensional structure for the mutant lipase.     

分段DNA shuffling: 一种大分子海藻糖合酶有效的定向进化方法

[目的]红色亚栖热菌(Meiothermus ruber)海藻糖合酶(Trehalose synthase,M-TreS)将麦芽糖转化生成海藻糖只需一步反应,且具有很好的热稳定性及pH耐受性,是潜在的工业生产海藻糖的酶源.为了提高该酶的性能,有必要对其进行定向进化.[方法]M-TreS基因(M-treS)大小为2 889bp.该蛋白质分子本身具有很大的进化空间,但是却不宜进行全长基因Shuffling.分段DNA shuffling是为大分子蛋白质(基因≥2 000 bp)的进化而设计的一种方法.该方法分为三步:(1)用两对引物分别扩增目的基因的上游片段和下游片段;(2)上下游片段各自进行Shuffling; (3)利用重叠延伸PCR连接上下游突变群,建立完整基因的突变文库.[结果]结合易错PCR,通过该方法经一轮进化获得一株酶活力是野生型1.6倍、催化效率是野生型2倍的突变株.序列分析表明,该突变株共有6个位点发生了氨基酸的替代,其中一个来自易错突变,2个来自同源重组,3个为随机突变.[结论]分段DNA shuffling是进化大分子蛋白质的有效方法.