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。     

植物群落的演替理论

演替是植被动态的中心问题.与演替有关的理论有接力植物区系学说、初始植物区系学说、促进忍耐和抑制学说、生活史对策演替学说、资源比率学说、Odum-Margelef生态系统发展理论、McMahon系统概念模型、变化镶嵌体稳态学说、演替的尺度等级系统观点. 文章论述了演替的分类、演替的进程、演替机制、顶极群落与非平衡态、植物群落的线性与线性系统、演替的细胞学和遗传学特征、演替与进化、演替的研究方法等.     

Protein engineering: opportunities and challenges

The extraordinary properties of natural proteins demonstrate that life-like protein engineering is both achievable and valuable. Rapid progress and impressive results have been made towards this goal using rational design and random techniques or a combination of both. However, we still do not have a general theory on how to specify a structure that is suited to a target function nor can we specify a sequence that folds to a target structure. There is also overreliance on the Darwinian blind search to obtain practical results. In the long run, random methods cannot replace insight in constructing life-like proteins. For the near future, however, in enzyme development, we need to rely on a combination of both.     

Employing directed evolution for the functional analysis of multi-specific proteins

Multi-specific proteins located at the heart of complex protein–protein interaction (PPI) networks play essential roles in the survival and fitness of the cell. In addition, multi-specific or promiscuous enzymes exhibit activity toward a wide range of substrates so as to increase cell evolvability and robustness. However, despite their high importance, investigating the in vivo function of these proteins is difficult, due to their complex nature. Typically, deletion of these proteins leads to the abolishment of large PPI networks, highlighting the difficulty in examining the contributions of specific interactions/activities to complex biological processes and cell phenotypes. Protein engineering approaches, including directed evolution and computational protein design, allow for the generation of multi-specific proteins in which certain activities remain intact while others are abolished. The generation and examination of these mutants both in vitro and in vivo can provide high-resolution analysis of biological processes and cell phenotypes and provide new insight into the evolution and molecular function of this important protein family.     

蛋白质体外进化建库技术研究

蛋白质体外进化技术是蛋白质工程发展的一个里程碑,也是改造蛋白质的一种有效工具。它不仅具有重要的应用价值,而且有助于蛋白质结构与功能的研究。通过蛋白质体外进化技术已成功地改造了许多蛋白质,有些已应用于工农业生产。体外进化技术分为两步:建库和筛选。本文主要对蛋白质体外进化策略及对体外随机突变技术、DNA重组技术、利用活细胞自身修复系统构建突变文库等几种定向进化突变文库建立技术进行了介绍与论述,同时还对蛋白质体外进化技术的应用及与其它学科结合的研究前景进行了分析,为获得具有改进功能或全新功能的蛋白质提供理论基础。     

Vegetation dynamics: patterns in time and space

This paper introduces the collection of contributions in this special volume on temporal and spatial patterns of vegetation dynamics. First, it is pointed out that the dynamics of any piece of vegetation, large or small, is always dependent on the degree of isolation of that piece towards its environment. Then ten types of island situation are treated ranging from very much to very little isolated: remote species-rich oceanic islands, remote species-poor islands, young big islands near a continent, small off-shore islands, emerging islands, isolated hills, landscape islands, isolated patches of vegetation, and gaps in stands of vegetation.Also, eight forms of vegetation dynamics are treated, ranging from short-term to long-term changes and involving larger and larger units: individuals, patches, communities, landscapes and vegetation regions. The forms of dynamics are fluctuation, gap dynamics, patch dynamics, cyclic succession, regeneration succession, secondary succession, primary succession, and secular succession. Each form of dynamics may occur under varying degrees of isolation.The general conclusion is that processes and patterns of vegetation dynamics cannot be generalized in any simple manner. The 20 papers collected in this volume, divergent as they are, express the complexity of vegetation dynamics.     

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。     

Performance of a recombinant strain of<Emphasis Type="Italic"> Streptomyces lividans</Emphasis> for bioconversion of penicillin G to deacetoxycephalosporin G

We examined the performance of Streptomyces lividans strain W25 containing a hybrid expandase (deacetoxycephalosporin C synthase; DAOCS) gene, obtained by in vivo recombination between the expandase genes of S. clavuligerus and Nocardia lactamdurans for resting-cell bioconversion of penicillin G to deacetoxycephalosporin G. Strain W25 carried out a much more effective level of bioconversion than the previously used strain, S. clavuligerus NP1. The two strains also differed in the concentrations of FeSO₄ and α-ketoglutarate giving maximal activity. Whereas NP1 preferred 1.8 mM FeSO₄ and 1.3 mM α-ketoglutarate, recombinant W25 performed best at 0.45 mM FeSO₄ and 1.9 mM α-ketoglutarate. Electronic Publication     

嗜热酯酶APE1547催化活性的定向进化研究

对来源于嗜热古菌Aeropyrum pernix的酯酶(APE1547)催化活性进行定向进化研究。利用APE1547特殊的稳定性,建立了准确的高通量高温酯酶筛选方法。对第一代随机突变库筛选获得了催化活性较野生型提高1.5倍的突变体M010,序列分析表明其氨基酸突变为R526S。从第二代突变库中筛选出的总活力提高5.8倍突变体M020,突变位点为R526S/E88G/A200T/I519L,其比活力与M010一致,但表达量比野生型提高约4倍。对M020酶学性质表征发现,其最适pH为8.5,比野生型向碱性偏移0.5;活性中心残基酸性基团的解离常数(pK1)由野生型的7.0提高至7.5。晶体结构分析表明,突变位点R526距离活性中心较近,将其突变为Ser降低了活性中心的极性,抑制了催化残基His的解离,使酸性基团的解离常数升高。     

Sites in the AAV5 capsid tolerant to deletions and tandem duplications

Gene therapy vectors based on adeno-associated virus (AAV) have shown much promise in clinical trials for the treatment of a variety of diseases. However, the ability to manipulate and engineer the viral surface for enhanced efficiency is necessary to overcome such barriers as pre-existing immunity and transduction of non-target cells that currently limit AAV applications. Although single amino acid changes and peptide insertions at select sites have been explored previously, the tolerance of AAV to small deletions and tandem duplications of sequence has not been globally addressed. Here, we have generated a large, diverse library of >10⁵ members containing deletions and tandem duplications throughout the viral capsid of AAV5. Four unique mutants were identified that maintain the ability to form viral particles, with one showing improved transduction on both 293T and BEAS-2B cells. This approach may find potential use for the generation of novel variants with improved and altered properties or in the identification of sites that are tolerant to insertions of targeting ligands.     

基于易错PCR技术的黏质沙雷氏菌脂肪酶基因LipA的定向进化

利用易错PCR技术对黏质沙雷氏菌脂肪酶基因LipA进行定向进化,经过筛选最终获得一个比活力比野生酶提高了425 U/mg的突变体ep1,测序分析ep1有5个氨基酸发生了突变,与野生酶相比ep1的最适pH值由原来的8.5降低为7.5,Tm值提高3℃,Km值由原来的40 mg/mL降低为12.5 mg/mL.对其三维结构进行分析,推测酶学性质的改变可能与处在活性中心右前方双螺旋发卡结构上的158A、和处在下部β卷曲折叠拐角处的S375G的突变有关.     

Enhanced thermostability and tolerance of high substrate concentration of an esterase by directed evolution

A newly isolated enantioselective esterase from Pseudomonas fluorescens KCTC 1767, which is currently considered as a biocatalyst for the production of a commercially valuable (S)-ketoprofen, has revealed a low structural and thermal stability. In order to enhance the stability, directed evolution was attempted on this enantioselective esterase by successive steps of an error prone and staggered extension PCR. After the second round of evolution, the best mutant 6–52 with enhanced thermal stability was selected and analyzed. DNA sequence analyses of 6–52 revealed that the three amino acid residues (L120P, I208V, and T249A) were changed and the mutation L120P was presumed as a structurally important residue due to its presence in all positive variants. The purified mutant 6–52, when incubated at 50 and 55 °C for 2 h, remained its activity over 30 and 10%, respectively, whereas there were no detectable activities in wild-type enzyme. The analysis of 6–52 in the presence of 15% ethanol showed 1.8-fold increase in the activity, compared to that of wild-type enzyme. The K_m and V_max values of 6–52 were estimated to be slightly increased, leading to 1.2-fold-higher the catalytic efficacy k_cat/K_m than that of wild-type enzyme. Additionally, the mutant 6–52 was more resistant to high substrate concentrations than that of wild-type enzyme.     

Succession following the catastrophic eruption of Ksudach volcano (Kamchatka, 1907)

Ksudach Volcano, southern Kamchatka Peninsula, erupted in 1907 and impacted over 2000 km² of forests with air-fall pumice deposits. We identified three impact zones. In Zone I, deposits deeper than 100 cm destroyed all vegetation. Two early successional stages occur, a lichen-dominated desert and isolated patches of a pioneer herb stage. Zone II is defined by pumice deposits 30 to 100 cm deep. Deposits of 70 to 100 cm destroyed all vegetation, but left scattered snags. Here primary succession dominates recovery, but its rate varies. Isolated trees survived in deposits of 30 to 70 cm and primary and secondary successional stages form a complex mosaic termed an intermediate succession. In Zone II, the primary stages found in Zone I are joined by a dwarf shrub-herb stage and a secondary birch forest stage. Zone III occurs where thinner deposits permitted some vegetation to survive in all locations. Secondary succession dominates in deposits of 10 to 30 cm. Trees suffered damage, but survived deposits of 20 to 30 cm, while other vegetation layers were eliminated. Deposits of 10 to 20 cm eliminated mosses and lichens and but only reduced the number of dwarf shrubs and herbs. Deposits of less than 10 cm damaged herb, moss and lichen layers but did not eliminate any species. All sampled vegetation remains in a pre-climax state, having yet to recover fully from earlier eruptions. Reconstructed vegetation maps for before 1907 and for ca. 1925 are compared to the map of vegetation in 1994. Based on degree of soil formation, vegetation recovery and colonization rates at different pumice depths, and the current vegetation, we estimate that full recovery of the soil-vegetation system will take more than 2000 years.     

微生物酶分子改造研究进展

近年来,越来越多的酶蛋白已经采用重组微生物反应器进行高效生产。为了改善酶蛋白的催化性能,提高其环境适应性,同时提高酶蛋白的表达量,降低生产成本,各种针对酶蛋白分子改造的基因工程技术已经得到大量的应用。综述了用于酶分子改造和进化的主要分子生物学方法,如定点突变、易错PCR、基因改组、密码子优化等技术及其应用成就。