D-泛解酸内酯水解酶的定向进化

易错PCR结合DNA改组方法向D-泛解酸内酯水解酶基因中引入突变,并构建突变体库。利用酶的催化特点和产物特性建立了基于平板初筛和高效液相复筛的两步法D-泛解酸内酯水解酶活性筛选系统。用该筛选系统以酶活力和pH稳定性为指标对突变体库进行筛选,最终获得一株酶活力高且在低pH条件下稳定性好的突变体Mut E-861。该突变体的酶活力是野生型酶的5.5倍。对突变体和野生型酶在pH 6.0和pH 5.0条件下的残余酶活进行对比,在这两种pH条件下,突变体酶的酶活残留分别为75%和50%,而野生型酶只能保持原来的40%和20%。通过软件对突变体Mut E-861酶基因和野生型酶基因进行分析对比,发现突变体Mut E-861酶基因发生了三处点突变,其中突变使两处氨基酸取代,另一处为沉默突变,未引起氨基酸的变化。     

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.     

大肠杆菌严谨型RNA聚合酶的筛选及体内转录活性测定

利用选择性培养基筛选大肠杆菌自然突变菌株,经噬菌体P1转导和蛋白质互补试验,发现一株突变体(LCH001)的突变基因发生在编码RNA聚合酶β′亚基的rpoC基因上,经DNA序列分析,发现突变位点发生在第3406个碱基上,由G变成了T,导致编码的氨基酸由甘氨酸(GGT)变成半胱氨酸(TGT)。体内转录试验表明该突变RNA聚合酶转录严谨型启动子控制基因的活性显著降低,其β-半乳糖苷酶的活性是野生型菌株的18%,而转录非严谨型启动子控制基因的活性显著提高,其β-半乳糖苷酶的活性约是野生型菌株的5倍。研究结果对探讨RNA聚合酶结构与功能的关系以及RNA聚合酶在细菌严谨反应过程中的作用具有重要意义。     

Hb D Punjab基因的鉴定——DNA 扩增在异常血红蛋白研究中的应用

本文应用作者设计的寡核苷酸引物和探针,通过聚合酶链反应(PCR)扩增β珠蛋白DNA序列,并通过限制酶EcoRI酶谱分析或寡核苷酸杂交,快速鉴定Hb D Punjab基因。应用这种技术先后对汉、藏、哈萨克等3个民族4个家系的Hb D Punjab基因进行了鉴定。     

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.     

四种鹿属动物的线粒体DNA差异和系统进化关系研究

用聚合酶链反应 ,从水鹿、坡鹿、梅花鹿和马鹿等四种鹿属动物中分别扩增出线粒体DNA的细胞色素 b基因片段 ,并测定得到 367bp的碱基序列 ,它们之间的序列差异在 4 .0 9%～ 7.0 8%之间。用 NJ法、最大简约法和最大似然法进行分子系统进化树的构建和分析并得出 ,水鹿与坡鹿、梅花鹿和马鹿约在 2 4 0～ 2 80万年前分化的 ,梅花鹿和马鹿大约在 1 60万年前左右分化。     

黑木耳ISSR-PCR反应体系的正交优化

采用正交试验设计的方法,对黑木耳ISSR-PCR(简单重复序列区间-多聚酶链反应)反应体系中的5种主要因素(Taq聚合酶,Mg2+,模板DNA,dNTP及引物)4个水平进行优化筛选,确立了适合黑木耳ISSR分析的优化反应体系(20μL),通过梯度PCR试验筛选得到相应引物的最佳退火温度。     

酿酒酵母Mbp1基因缺失突变株耐受性研究

为了研究Mbp1基因对工业酿酒酵母(Saccharomyces cerevisiae)耐受性的影响,在敲除野生型工业酿酒酵母MF1015菌株Mbp1基因的基础上,分析Mbp1基因缺失菌株(突变菌株)与野生型在乙醇耐受性、耐热性、细胞壁完整性、呼吸强度、海藻糖含量、过氧化氢酶(CAT)、过氧化物酶(POD)、超氧化物歧化酶(SOD)、乙醇脱氢酶(ADH)活性等方面的差异。结果表明:在含刚果红或SDS的平板上突变菌株的菌落比野生型小、菌体存活率比野生型低;突变菌株的乙醇耐受性和耐热性均下降,在乙醇浓度为9%的液体培养基中,突变菌株的最大OD600约为3.15,而野生型最大OD600值约为3.48;在30℃和37℃时突变菌株和野生型的生长趋势基本一致,而在40℃时,突变菌株最大OD600约为3.0,而野生型约为4.5;用9%乙醇处理后,突变菌株胞内海藻糖含量、过氧化氢酶、过氧化物酶、超氧化物歧化酶、乙醇脱氢酶酶活分别比野生型低49%、80%、24%、37%、73%,而未经乙醇处理的突变菌株和野生型酶活无明显差别。推测Mbp1基因有助于工业酿酒酵母适应外部不良环境。     

拟南芥生长素受体基因TIR1启动子的克隆及序列分析

以野生型拟南芥 (Arabidopsis Columbia)基因组DNA为模板,通过PCR扩增得到拟南芥生长素受体基因TIR1启动子2008片段,将该片断克隆到PGM-T载体上.序列分析表明,该启动子大小为2008bp,RNA聚合酶识别序列TATA-box,TIR1特异表达和增强序列CAAT-box皆完整,与已报道的序列比较仅有3个核苷酸发生改变,同源性为99.85%.将该启动子与GUS基因融合,构建成表达载体后,在拟南芥和烟草叶片中做瞬时表达,结果分析显示:拟南芥和烟草叶片中均有GUS 酶活性存在.     

定向进化技术改良β-糖苷酶的低聚糖合成性能

对携带糖苷酶基因pBBGly的质粒pBtac2进行易错PCR定向进行研究,以改良其合成低聚糖催化性能。来源于Thermus thermophilus的耐高温β-糖苷酶,通过一轮易错PCR随机突变和限制性酶切再连接介导的体外基因重组,获得了两株高转糖基活性的突变酶:N339TF401S和F401S。它们能以天然糖类为糖基受体,低聚糖的合成得率分别为48%和62%,是野生型酶的6~8倍,而底物的水解活力只有野生型酶的0.08%和1.3%。另外,在突变酶的酶反应中,糖基受体对供体的水解反应有显著的促进作用,而野生型酶没有此特征。并且它们的催化水解动力学特征也明显区别于野生酶,水解反应初始浓度对速度关系呈一条直线。     

Functional promoter analysis using an approach based on an in vitro evolution strategy

In vitro evolution imitates the natural evolution of genes and has been very successfully applied to the modification of coding sequences, but it has not yet been applied to promoter sequences. We propose an alternative method for functional promoter analysis by applying an in vitro evolution scheme consisting of rounds of error-prone PCR, followed by DNA shuffling and selection of mutant promoter activities. We modified the activity in embryogenic sugarcane cells of the promoter region of the "Goldfinger" isolate of banana streak virus and obtained mutant promoter sequences that showed an average mutation rate of 2.5% after applying one round of error-prone PCR and DNA shuffling. Selection and sequencing of promoter sequences with decreased or unaltered activity allowed us to rapidly map the position of one cis-acting element that influenced promoter activity in embryogenic sugarcane cells and to discover neutral mutations that did not affect promoter function. The "selective-shotgun" approach of this promoter analysis method immediately after the promoter boundaries have been defined by 5' deletion analysis dramatically reduces the labor associated with traditional "linker-scanning" deletion analysis to reveal the position of functional promoter domains. Furthermore, this method allows the entire promoter to be investigated at once, rather than selected domains or nucleotides, increasing the prospect of identifying interacting promoter regions.     

Directed evolution of alpha-aspartyl dipeptidase from Salmonella typhimurium.

Model-free approaches (error-prone PCR to introduce random mutations, DNA shuffling to combine positive mutations, and screening of the resultant mutant libraries) have been used to enhance the catalytic activity and thermostability of alpha-aspartyl dipeptidase from Salmonella typhimurium, which is uniquely able to hydrolyze Asp-X dipeptides (where X is any amino acid) and one tripeptide (Asp-Gly-Gly). Under double selective pressures of activity and thermostability, through two rounds of error-prone PCR and three sequential generations of DNA shuffling, coupled with screening, a mutant pepEM3074 with approximately 47-fold increased enzyme activity compared with its wild-type parent was obtained. Moreover, the stability of pepEM3074 is increased significantly. Three amino acid substitutions (Asn89His, Gln153Glu, and Leu205Arg), two of them are near the active site and substrate binding pocket, were identified by sequencing the genes encoding this evolved enzyme. The mechanism of the enhancement of activity and stability was analyzed in this paper.     

通过DNA改组技术获得高活性β-葡萄糖苷酸酶

β 葡萄糖苷酸酶是在植物转基因中广泛应用的报告基因 .以质粒pBI12 1中的GUS基因为基础 ,利用DNA改组方法 ,经DNaseⅠ降解 ,PrimerlessPCR ,PrimerPCR对GUS基因进行了突变和改组 ,然后将改组的GUS基因连接到原核表达载体pG2 5 1中 ,构建了库容为 10 8的突变体库 .经过活性的筛选 ,得到活性提高的克隆 ,再以此为基础 ,经过新的改组、筛选得到活性大幅度提高的克隆GUS2 4 .基因测序显示 ,GUS2 4与GUS基因之间的同源性为 99 7% ,共有 6个核苷酸位点发生了改变 ,分别是 :379位的A突变为G ,396位的T突变为C ,711位的G突变为A ,95 8位T突变为C ,990位的T突变为C ,1649位的A突变为G .核苷酸序列推导的氨基酸序列显示 ,3个氨基酸发生了突变 ,12 7位的Ser突变为Gly ,32 0位的Trp突变为Arg ,5 5 0位的Asn突变为Ser.X gluc染色检测和荧光测活结果显示GUS2 4基因表达的 β 葡萄糖苷酸酶基较GUS基因表达产物活性提高 3倍     

High-level expression of a polypeptides encoded by a large segment of the envelope gene of HIV-1 in E. coli by directed evolution

A random mutagenesis library of gp120-801 (a large segment of the envelope protein gene of HIV-1) was constructed using error-prone PCR and DNA shuffling methods, and one mutant of gp120-801 was selected from this library using a green fluorescent protein (GFP) fusion vector. After being cloned into pEX31b and expressed in E. coli, the expressed fusion protein reached to 15% of total bacterial proteins for the mutant but was just 1–2% for the wild type.     

Improvement of alkaline lipase from Proteus vulgaris T6 by directed evolution

To expand the functionality of lipase from Proteus vulgaris (PVL) we have used error-prone PCR and DNA shuffling methods to create PVL mutants with improved lipase activity. One desirable mutant with three amino acids substitutions was obtained. The mutated lipase was purified and characterized. The activity of the mutant lipase EF3.3 was 3.5 times higher than that of the wild-type (WT-PVL). The mutational effect is interpreted according to a simulated three-dimensional structure for the mutant lipase. Amino acid substitution at position 102 was determined to be critical for lipase activity, while the residue at positions 197 and 229 had only marginal effect.     

利用雨生红球藻表达系统高通量筛选活力提高的阿特拉津氯水解酶突变子

阿特拉津氯水解酶定向改造的关键是开发一种廉价的、表型改变明显的高通量筛选方法。利用高错误倾向PCR和DNA洗牌相结合的突变方法,对来源于假单胞菌ADP和节杆菌AD1的阿特拉津氯水解酶基因进行随机突变,以雨生红球藻为受体、以阿特拉津为选择压力对突变文库进行高通量筛选。筛选到的12个突变子序列分析显示,突变均为点替换,位点分散在全基因上,是在高错误倾向PCR及DNA洗牌过程中逐渐累积形成的。酶活力分析显示,突变子的酶活力均高于野生株,在添加1.0 mg/L阿特拉津培养液中的活力是野生株的1.9~3.6倍,在添     

Combinatorial engineering to enhance amylosucrase performance: construction, selection, and screening of variant libraries for increased activity

Amylosucrase is a glucosyltransferase belonging to family 13 of glycoside hydrolases and catalyses the formation of an amylose-type polymer from sucrose. Its potential use as an industrial tool for the synthesis or the modification of polysaccharides, however, is limited by its low catalytic efficiency on sucrose alone, its low stability, and its side reactions resulting in sucrose isomer formation. Therefore, combinatorial engineering of the enzyme through random mutagenesis, gene shuffling, and selective screening (directed evolution) was started, in order to generate more efficient variants of the enzyme. A convenient zero background expression cloning strategy was developed. Mutant gene libraries were generated by error-prone polymerase chain reaction (PCR), using Taq polymerase with unbalanced dNTPs or Mutazyme™, followed by recombination of the PCR products by DNA shuffling. A selection method was developed to allow only the growth of amylosucrase active clones on solid mineral medium containing sucrose as the sole carbon source. Automated protocols were designed to screen amylosucrase activity from mini-cultures using dinitrosalicylic acid staining of reducing sugars and iodine staining of amylose-like polymer. A pilot experiment using the described mutagenesis, selection, and screening methods yielded two variants with significantly increased activity (five-fold under the screening conditions). Sequence analysis of these variants revealed mutations in amino acid residues which would not be considered for rational design of improved amylosucrase variants. A method for the characterisation of amylosucrase action on sucrose, consisting of accurate measurement of glucose and fructose concentrations, was introduced. This allows discrimination between hydrolysis and transglucosylation, enabling a more detailed comparison between wild-type and mutant enzymes.     

Reducing mutational bias in random protein libraries

The success of protein optimization through directed molecular evolution depends to a large extent on the size and quality of the displayed library. Current low-fidelity DNA polymerases that are commonly used during random mutagenesis and recombination in vitro display strong mutational preferences, favoring the substitution of certain nucleotides over others. The result is a biased and reduced functional diversity in the library under selection. In an effort to reduce mutational bias, we combined two different low-fidelity DNA polymerases, Taq and Mutazyme, which have opposite mutational spectra. As a first step, random mutants of the Bacillus thuringiensis cry9Ca1 gene were generated by separate error-prone polymerase chain reactions (PCRs) with each of the two polymerases. Subsequent shuffling by staggered extension process (StEP) of the PCR products resulted in intermediate numbers of AT and GC substitutions, compared to the Taq or Mutazyme error-prone PCR libraries. This strategy should allow generating unbiased libraries or libraries with a specific degree of mutational bias by applying optimal mutagenesis frequencies during error-prone PCR and controlling the concentration of template in the shuffling reaction while taking into account the GC content of the target gene.     

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.