基于易错PCR的黄曲霉毒素解毒酶体外分子定向进化

运用定向进化-易错PCR方法,提高黄曲霉毒素解毒酶的活力及稳定性,并结合辣根过氧化物酶 (HRP)-隐性亮绿 (RBG) 快速高通量筛选系统,构建了库容约为104的突变体库。经过两轮易错PCR,最终分别获得了耐高温70 ℃突变酶A1773、pH 4.0稳定性的突变酶A1476,pH 4.0和pH 7.5均表现稳定性的突变酶A2863,其酶活力比野生酶分别提高了6.5倍、21倍和12.6倍。经序列分析表明,发现突变酶A1773发生了Glu127Lys和Gln613Arg突变;突变酶A2863发生了Gly73     

GI双突变体GIK253RA198C的构建及其性质分析

以双引物法对葡萄糖异构酶（GI）基因进行定点突变,将突变体基因于大肠杆菌中表达,获得了GI双点突变体GIK253RA198C.研究K253R和A198C双点突变对GI的结构和性质的作用,结果表明GIK253RA198C的热稳定性明显下降,最适反应温度降低5℃.文章从结构和机制上解释了为何同是K253R突变,对SM33 GI和密苏里游动放线菌GI的热稳定性产生不同的影响,认为这是由于Lys253在两种GI结构的位置上存在微小差异,从而使引入的Arg对亚基间的相互作用产生了相反效应所引起.     

枯草蛋白酶E的定点突变及其对酶性质的影响

用定点突变的方法研究S221C/P225A,N118S/S221C/P225A,D60N/S221C/P225A和Q103R/S221C/P225A突变对蛋白酶活性,酯酶活性与蛋白酶活性之比的影响。结果表明：S221C/P225A突变使蛋白酶活性比枯草蛋白酶E低73000多倍,酯酶活性与蛋白酶活性之比是Subtiligase的3倍;N118S/S221C/P225A突变使蛋白酶活性和酯酶活性分别比S221C/P225A突变下降3.6倍和15倍,酯酶与蛋白酶活性之比下降4倍,同时增加变体酶的热稳定性;D60N/N118S/S221C/P225A突变使蛋白酶活性比N118S/S221C/P225A突变体下降15倍,但对酯酶活性几乎没有影响,酯酶与蛋白酶活性之比增加14倍,分别是S221C/P225A突变体和Subtiligase的3.3倍和10.3倍;但是,Q103R/N118S/S221C/P225A突变使蛋白酶活性比N118S/S221C/P225A突变体增加5倍,酯酶活性下降55倍,酯酶与蛋白酶活性之比下降1000倍。     

定点突变提高木聚糖酶Umxyn10A的热稳定性

木聚糖酶是微生物半纤维素降解体系中的一种关键酶,被广泛地应用在工业中的多个领域。本研究为了提高木聚糖酶Umxyn10A (ABL73-883.1)的热稳定性,将Umxyn10A与GH 10家族4种耐热木聚糖酶进行多序列同源比对以及三维结构的同源建模分析,选定了Umxyn10A第31位氨基酸位点进行定点突变,将氨基酸Ala (A)突变为Phe (F)。分别将Umxyn10A和Umxyn10AA31F在大肠杆菌中进行重组表达,分析2种重组酶的酶学特性,结果发现,Umxyn10AA31F的最适反应温度为85℃,较野生重组酶提高了5℃;在65℃下的半衰期为105 min,较野生重组酶(15 min)提高了6倍;在70℃下突变重组酶的半衰期为15 min,较野生重组酶(5 min)提高了2倍;与此同时突变重组酶的pH耐受区间较原酶也有一定的增大。结果表明,将第31位氨基酸位点Ala突变为Phe能够显著的提高Umxyn10A的热稳定性。     

F43Y及I354M,L358F定点突变对植酸酶热稳定性及酶活性的改善

对重组酵母PPNPm8的植酸酶phyAm基因进行PCR介导的定点突变,即将植酸酶43位的苯丙氨酸替换为酪氨酸(F43Y),将其354、358位的异亮氨酸、亮氨酸分别替换为甲硫氨酸和苯丙氨酸(I354M,L358F),得到了2个突变体PPNPm-1(F43Y)及PPNPm-2(I354M,L358F).含突变基因的重组表达载体pPIC9kphyAm-1,pPIC9kphyAm-2在毕赤酵母GS115中表达,对表达产物进行酶活性测定及热稳定性检测.结果表明:突变体PPNPm-1最适反应温度比未突变体PPNPm8上升了3℃,75℃处理10min,热稳定性提高15%,比活力提高11%;PPNPm-2最适反应温度未改变,热稳定性比PPNPm8仅提高3%,比活力降低6.5%.对突变前后的植酸酶空间结构进行比较预测,发现突变氨基酸Tyr43与空间位置相邻的Asn416之间形成氢键,增强了酶的热稳定性.     

大黄欧文氏菌蔗糖异构酶控制产物特异性基序的定点突变

大黄欧文氏菌(Erwinia rhapontici)蔗糖异构酶催化蔗糖异构为异麦芽酮糖和海藻酮糖,具有一个可能控制产物特异性的325RLDRD329基序.本研究以定点突变方法对该基序的带电荷氨基酸进行突变,共构建R325D、R328A、R328D、R328Q和D329N 5个突变体.通过对突变体的酶学特性及突变体转化蔗糖的产物组成分析,结果显示所构建突变体的Km值上升约2~5倍,比活力下降至野生型SI比活力的11.8%~25.3%.HPLC分析显示Arg325和Arg328分别突变为Asp,导致产物中异麦芽酮糖/海藻酮糖的比例从6.93分别降至0.96和2.92,并伴随一个未知寡糖出现.Arg328突变为Ala和Gln同样导致反应产物中海藻酮糖比例上升,异麦芽酮糖比例下降.但是突变体D329N反应产物比例没有变化.以上结果表明325RLDRD329基序对大黄欧文氏菌蔗糖异构酶的酶活具有重要作用,并对酶的产物特异性产生影响.本研究结果将为该酶的作用机理研究奠定基础.     

米根霉α-淀粉酶热稳定性的理性设计

真菌α-淀粉酶被广泛应用于麦芽糖浆生产工业,但其热稳定性普遍较差,在制糖工艺中增加了由于酶活力损失而引起的追加生产成本。在充分研究了热稳定性对于真菌α-淀粉酶应用于工业生产的重要性的基础上,为提高米根霉α-淀粉酶(ROAmy)的热稳定性,基于酶蛋白B-factor分析和分子动力学模拟,利用重叠PCR技术分别对ROAmy中的3个氨基酸残基G128、K269和G393进行了单点突变及组合突变。结果表明,所获得的7个突变体均比原酶具有更好的热稳定性,其中效果最好的为组合突变体G128L/K269L/G393P,其在55℃下的热失活半衰期(t_(1/2))约为原酶的5.63倍。同时,该突变体的最适温度由50℃提高到了65℃,最大反应速率(V_(max))和催化效率(k_(cat)/K_m)分别提高了65.38%和99.86%。通过蛋白结构功能比较分析,发现氢键数目的增多或脯氨酸在特殊位置中的引入可能是突变体热稳定性得到提高的主要因素。     

扩展青霉脂肪酶K56R叠加突变对热稳定性的影响

目的:扩展青霉脂肪酶随机突变体ep8是一株热稳定性比野生型有所提高的突变体.获得热稳定性提高的优良菌株.方法:在ep8的基础上利用重叠延伸PCR构建叠加突变重组质粒pPIC3.5K-ep8一K56R,将该质粒电转毕赤酵母(Pichia paaoris)GS115进行异源表达.结果:该叠加突变脂肪酶在毕赤酵母中获得了活性表达.15%SDS-PACE结果分析表明突变脂肪酶PEL-ep8-K56R-GS分子量与野生型PEL-GS一致,约为28kDa.叠加突变脂肪酶在37℃时酶活为852U/mL、野生型为760u/mL、随机突变体为824u/mL,叠加突变体酶活相比野生型提高了21.1%,相比随机突变体提高了3.4%.热稳定性分析数据表明叠加突变脂肪酶Tm值为40.1℃、野生型为38.7℃、随机突变体为39.9℃,Tm值相比野生型提高了1.4℃,相比随机突变体提高了0.2℃.     

具蛋白酶抗性的Armillariella tabescens β-甘露聚糖酶MAN47的分子定向改造

利用定点突变的方法提高Armillariella tabescens β-甘露聚糖酶MAN47的胰蛋白酶抗性。首先根据其氨基酸序列,找到胰蛋白酶的水解位点—赖氨酸(Lys, K)和精氨酸(Arg, R),再利用生物信息学软件获得酶分子结构中K和R与周围溶剂的接触程度,选定暴露程度最大的K280为候选突变位点,进行模拟突变,并分析突变前后的氢键键长和整体结构的变化。根据氢键键长的变化,确定突变体为K280N。对K280N设计突变引物,用重叠延伸PCR技术对MAN47野生型man基因进行突变,PCR产物与大肠杆菌-酿酒酵母穿梭表达载体PYCα连接,在大肠杆菌DH5α中扩增后转入酿酒酵母Saccharomyces cerevisiae,经人工肠液(pH 6.8 10mg/ml胰蛋白酶溶液)筛选,得到抗胰蛋白酶的最佳突变株。结果表明突变酶在用人工肠液处理180min后,其半衰期为173min,而野生型酶为99min,其他酶学性质与野生型酶基本一致。     

应用拉氏图信息提高短乳杆菌谷氨酸脱羧酶催化性能

谷氨酸脱羧酶(Glutamate decarboxylase,GAD)是用于催化L-谷氨酸脱羧合成γ-氨基丁酸(γ-aminobutyrate,GABA)的唯一酶,提高GAD的催化活力或热稳定性,有利于GABA的高效制备和生产。以热稳定性和活性为筛选目标,通过研究短乳杆菌GAD1407三维模拟结构的拉氏图,确定不稳定氨基酸残基位点K413,采用定点突变的方法构建该位点的突变体,并测定野生型酶和突变酶的热稳定性和活力。结果表明突变酶K413A和突变酶K413I分别在热稳定性和酶活力上获得了提高,突变酶K413A在50℃的半衰期为105 min,是野生酶的2.1倍;突变酶K413I热稳定性没有明显的提高,但其酶活力却得到了有效提高,约为野生型的1.6倍。因此,通过拉氏图提供的结构信息可为利用理性设计提高GAD活性和热稳定性提供指导。     

Spontaneous mutations in bacteria: chance or necessity?

Several investigators have recently reported that significant numbers ofappropriately adapted mutants can be induced in bacterial and yeast strains by exposing stationary phase cells to specific environmental challenges. The resulting mutants are said to be both selection-induced and demonstrably non-random in origin; if this interpretation is correct, it is in direct conflict with the conventional neo-Darwinian view, which is that spontaneous mutants are truly random in origin and arise without the intervention of any overtly adaptive forces. We believe that there are alternative ways of accounting for the appearance of many (and probably all) of the additional mutants which proponents of the adaptive mutation theory claim are observed only after they applied the appropriate selective pressure. Having reviewed the available evidence, we consider that most (if not all) of the sorts of mutants which are said to have been induced following exposure of stationary-phase cells to intense selective pressure are equally likely to have been generated during the operation of certain well-known, conventional (and essentially random) cellular DNA repair processes. Evidence in support of our view can be found in the mainstream literature on the origins of spontaneous mutations. We also note that some of the molecular models which have recently been proposed to explain the production of selection-induced mutations preferentially (or even only) in genes of adaptive significance may turn out to be of considerable interest in their own right, even although the mutants whose origins they were intended to explain may turn out to have arisen in a manner which is totally independent of the conditions used for their selection.     

A resolution of the mutation load paradox in humans

Current information on the rate of mutation and the fraction of sites in the genome that are subject to selection suggests that each human has received, on average, at least two new harmful mutations from its parents. These mutations were subsequently removed by natural selection through reduced survival or fertility. It has been argued that the mutation load, the proportional reduction in population mean fitness relative to the fitness of an idealized mutation-free individual, allows a theoretical prediction of the proportion of individuals in the population that fail to reproduce as a consequence of these harmful mutations. Application of this theory to humans implies that at least 88% of individuals should fail to reproduce and that each female would need to have more than 16 offspring to maintain population size. This prediction is clearly at odds with the low reproductive excess of human populations. Here, we derive expressions for the fraction of individuals that fail to reproduce as a consequence of recurrent deleterious mutation () for a model in which selection occurs via differences in relative fitness, such as would occur through competition between individuals. We show that is much smaller than the value predicted by comparing fitness to that of a mutation-free genotype. Under the relative fitness model, we show that depends jointly on U and the selective effects of new deleterious mutations and that a species could tolerate 10's or even 100's of new deleterious mutations per genome each generation.     

Adaptive mutation inEscherichia coli strain FC40

Mutations can arise in static populations of cells that are subjected to nonlethal selective pressure, a phenomenon that has been called ‘adaptive mutation’. This phenomenon has been extensively studied in FC40, a strain ofEscherichia coli that cannot metabolize lactose (Lac⁻) but that reverts to lactose utilization (Lac⁺) when lactose is its sole energy and carbon source. The adaptive Lac⁺ mutations arise by two mutational processes: a recombination-dependent process that is highly active on the episome carrying the Lac⁻ allele, and an unknown process that affects the whole genome. Most of the Lac⁺ mutations are due to the first process, which also produces nonselected mutations on the F′ episome. However, about 10% of the Lac⁺ mutations arise in a subpopulation of cells that experience a period of transient hypermutation. Although minor contributors to any one type of mutation, the hypermutators account for nearly all cases of multiple mutations. The evolutionary implications of these results are: (i) DNA synthesis associated with recombination may be an important source of spontaneous mutation, particularly in cells that are not actively growing; (ii) the efficient mutational mechanism that occurs on the episome could result in the horizontal transfer of new alleles among species that carry and exchange conjugal plasmids; and (iii) a subpopulation of transient hypermutators could be a source of multiple mutations that would allow for rapid adaptive evolution under adverse conditions.     

The origin of adaptive mutants: Random or nonrandom?

Several recent reports have claimed that adaptive mutants in bacteria and yeast are induced by selective conditions. The results of these reports suggest that mutants can arise nonrandomly with respect to fitness, contrary to what has been widely accepted. In several cases that have received careful experimental reexamination, however, the detection of seemingly nonrandom mutation has been explained as an experimental artifact. In the remaining cases, there is no evidence to suggest that cells have the capacity to direct or choose which genetic variants will arise. Instead, current models propose processes by which genetic variants persist as mutations only if they enable cell growth and DNA replication. Most of these models are apparently contradicted by experimental data. One model, the hypermutable state model, has recently received limited circumstantial support. However, in this model the origin of adaptive mutants is random; the apparent nonrandomness of mutation is merely a consequence of natural selection. The critical distinction between the origin of genetic variation (mutation) and the possible consequence of that variation (selection) has been neglected by proponents of directed mutation.     

木霉突变体T1010变异特性分析

木霉突变体T1010与出发菌株实验比较,其生长速度、产孢量、菌丝重量分别提高13．62％、48．52％、29．03％;T1010对培养液中氨态氮的吸收率比出发菌株仍2提高37．09％,T1010对无机磷的吸收率比出发菌株提高15．17％,T1010对K’吸收率比出发菌株高16．49％,在pH值为7．34的培养液中培养3d后,T1010的pH值为5．77,出发菌株的pH值为6．67;突变体T1010对培养液中葡萄糖的吸收能力比出发菌株提高19．64％,突变体T1010利用培养液中的氨基酸量较少,比出发菌株降低18．26％。拮抗作用结果显示,T1010对病菌瓜萎蔫镰刀菌抑制作用、覆盖作用分别比出发菌株提高87．45％、57．38％。     

SPONTANEOUS MUTATION PARAMETERS FOR ARABIDOPSIS THALIANA MEASURED IN THE WILD

Mutations are the ultimate source of genetic diversity and their contributions to evolutionary process depend critically on their rate and their effects on traits, notably fitness. Mutation rate and mutation effect can be measured simultaneously through the use of mutation accumulation lines, and previous mutation accumulation studies measuring these parameters have been performed in laboratory conditions. However, estimation of mutation parameters for fitness in wild populations requires assays in environments where mutations are exposed to natural selection and natural environmental variation. Here we quantify mutation parameters in both the wild and greenhouse environments using 100 25th generation Arabidopsis thaliana mutation accumulation lines. We found significantly greater mutational variance and a higher mutation rate for fitness under field conditions relative to greenhouse conditions. However, our field estimates were low when scaled to natural environmental variation. Many of the mutation accumulation lines have increased fitness, counter to the expectation that nearly all mutations decrease fitness. A high mutation rate and a low mutational contribution to phenotypic variation may explain observed levels of natural genetic variation. Our findings indicate that mutation parameters are not fixed, but are variables whose values may reflect the specific environment in which mutations are tested.     

PERSPECTIVE: SPONTANEOUS DELETERIOUS MUTATION

Mildly deleterious mutation has been invoked as a leading explanation for a diverse array of observations in evolutionary genetics and molecular evolution and is thought to be a significant risk of extinction for small populations. However, much of the empirical evidence for the deleterious-mutation process derives from studies of Drosophila melanogaster, some of which have been called into question. We review a broad array of data that collectively support the hypothesis that deleterious mutations arise in flies at rate of about one per individual per generation, with the average mutation decreasing fitness by about only 2% in the heterozygous state. Empirical evidence from microbes, plants, and several other animal species provide further support for the idea that most mutations have only mildly deleterious effects on fitness, and several other species appear to have genomic mutation rates that are of the order of magnitude observed in Drosophila. However, there is mounting evidence that some organisms have genomic deleterious mutation rates that are substantially lower than one per individual per generation. These lower rates may be at least partially reconciled with the Drosophila data by taking into consideration the number of germline cell divisions per generation. To fully resolve the existing controversy over the properties of spontaneous mutations, a number of issues need to be clarified. These include the form of the distribution of mutational effects and the extent to which this is modified by the environmental and genetic background and the contribution of basic biological features such as generation length and genome size to interspecific differences in the genomic mutation rate. Once such information is available, it should be possible to make a refined statement about the long-term impact of mutation on the genetic integrity of human populations subject to relaxed selection resulting from modern medical procedures.     

A comprehensive model of mutations affecting fitness and inferences for Arabidopsis thaliana

As the ultimate source of genetic variation, spontaneous mutation is essential to evolutionary change. Theoretical studies over several decades have revealed the dependence of evolutionary consequences of mutation on specific mutational properties, including genomic mutation rates, U, and the effects of newly arising mutations on individual fitness, s. The recent resurgence of empirical effort to infer these properties for diverse organisms has not achieved consensus. Estimates, which have been obtained by methods that assume mutations are unidirectional in their effects on fitness, are imprecise. Both because a general approach must allow for occurrence of fitness-enhancing mutations, even if these are rare, and because recent evidence demands it, we present a new method for inferring mutational parameters. For the distribution of mutational effects, we retain Keightley's assumption of the gamma distribution, to take advantage of the flexibility of its shape. Because the conventional gamma is one sided, restricting it to unidirectional effects, we include an additional parameter, rho, as an amount it is displaced from zero. Estimation is accomplished by Markov chain Monte Carlo maximum likelihood. Through a limited set of simulations, we verify the accuracy of this approach. We apply it to analyze data on two reproductive fitness components from a 17-generation mutation-accumulation study of a Columbia accession of Arabidopsis thaliana in which 40 lines sampled in three generations were assayed simultaneously. For these traits, U approximately/= 0.1-0.2, with distributions of mutational effects broadly spanning zero, such that roughly half the mutations reduce reproductive fitness. One evolutionary consequence of these results is lower extinction risks of small populations of A. thaliana than expected from the process of mutational meltdown. A comprehensive view of the evolutionary consequences of mutation will depend on quantitatively accounting for fitness-enhancing, as well as fitness-reducing, mutations.     

SPONTANEOUS MUTATION ACCUMULATION IN MULTIPLE STRAINS OF THE GREEN ALGA,CHLAMYDOMONAS REINHARDTII

Estimates of mutational parameters, such as the average fitness effect of a new mutation and the rate at which new genetic variation for fitness is created by mutation, are important for the understanding of many biological processes. However, the causes of interspecific variation in mutational parameters and the extent to which they vary within species remain largely unknown. We maintained multiple strains of the unicellular eukaryote Chlamydomonas reinhardtii, for approximately 1000 generations under relaxed selection by transferring a single cell every ～10 generations. Mean fitness of the lines tended to decline with generations of mutation accumulation whereas mutational variance increased. We did not find any evidence for differences among strains in any of the mutational parameters estimated. The overall change in mean fitness per cell division and rate of input of mutational variance per cell division were more similar to values observed in multicellular organisms than to those in other single‐celled microbes. However, after taking into account differences in genome size among species, estimates from multicellular organisms and microbes, including our new estimates from C. reinhardtii, become substantially more similar. Thus, we suggest that variation in genome size is an important determinant of interspecific variation in mutational parameters.     

Induction of somaclonal variation by tissue culture and cytogenetic analysis in Oryza sativa L.

Protocols were developed for plant regeneration from callus induced in mature embryos of rice. Somaclonal variation was scored by genome mutation, chromosome mutation and plasmon mutation in R₀, R₁ and R₂ plant progenies. The frequency of haploids and diploids appeared in the ratio of 20:33. Variation in the chromosome number in callus cells was found to be high and age dependent. Different types of chlorophyll deficient mutants including albinos appeared in R₂ plant progeny where gene mutation frequency was the highest (52.4 %). The results revealed that a high frequency of somaclonal variation is possible to generate by tissue culture techniques. This revised version was published online in July 2006 with corrections to the Cover Date.