微生物表面展示技术及其在环境污染治理中的应用

微生物表面展示技术是通过基因工程手段,将短的外源肽或蛋白质表达在微生物细胞表面,该技术可以应用于开发活的细菌疫苗、筛选抗体库、生产生物细胞吸附剂以及制备整细胞生物催化剂。通过金属高效结合肽的肽库筛选和微生物展示技术,将金属结合肽直接展示在微生物的表面,用于处理环境中的重金属污染,为环境中重金属污染的防治提供了一条崭新的途径。利用微生物表面展示技术制备整细胞催化剂,用于有毒有机污染物的处理,可以极大地加快污染物的降解速率。简要介绍了微生物表面展示技术及其在重金属污染治理和毒性有机污染物的脱毒等环境生物修复方面的最新研究进展。     

重金属结合肽（蛋白）的结构分析

金属离子与金属结合肽（蛋白）的相互作用与应用研究,一直是生物无机化学的重点和热点,也是分子间相互作用研究领域的难点。本研究利用ClustalX、BLAST等生物信息技术与方法对大量已知的重金属结合肽进行分析与数据挖掘。确定筛选获得的重金属结合肽常富含His,无Cys,无金属结合肽模式序列,进化不保守;部分氨基酸序列结构（如六肽）可在蛋白数据库中找到相似序列。序列特征主要为Zn^2＋相关的转录因子。本研究为重金属结合蛋白-重金属离子的相互作用分析简化为重金属结合肽-重金属离子的结构模拟与分析提供了重要的理论基础和研究手段。     

Ni2+高效结合肽的筛选与作用研究

利用噬菌体随机十二肽库和金属亲和层析对重金属Ni2 进行结合肽筛选。经4轮生物淘洗、噬菌体扩增和DNA测序,获得一组多肽序列。GenBank Blast分析未发现同源序列,Clustal W多重序列比对也未找到Ni2 金属结合肽结合基序,但可能含有多聚组氨酸(His)2-5。噬菌体单克隆金属离子螯合树脂的亲和力测定和反筛、抑菌解毒试验表明:展示有金属结合肽的噬菌体不仅对Ni2 具有高亲和力,而且对其它金属离子也有作用,Cu2 、Ni2 、Co2 、Zn2 等金属离子对金属结合肽的亲和力显著高于Cd2 和Cr2 ,展示金属结合肽的噬菌体对重金属Ni2 具有一定的耐受和解毒作用。显微形态学观察也显示金属结合肽与金属螯合树脂的作用。对于了解重金属与多肽的相互作用机理以及环境重金属修复等均具有重要意义和价值。     

土壤重金属污染的植物修复

土壤重金属污染的危害范围广泛,使用传统的物理和化学修复方法成本高,对环境扰动大,而利用植物修复的效果较为明显,易于操作.本文论述了土壤重金属污染的单一植物、植物与微生物联合、植物与化学方法相结合.的修复方法,着重介绍了重金属超富'集植物的研究和植物体内螯合肽(PCs)的合成.生物螯合剂的应用及土壤重金属污染的动物、植物和微生物的联合修复将是未来研究的热点.     

微生物展示技术在重金属污染生物修复中的研究进展

自然界对环境金属污染物的迁移和转化具有微妙而复杂的选择控制机理,生物修复技术以其投资少、效率高、可以原位处理低浓度有害污染物的特性而在环境治理中具有极大潜力。考虑传统的生物修复技术常常不能满足重金属治理的要求,基于重金属离子高效结合肽的微生物展示技术,有望在重金属生物修复中发挥重要作用。     

强化植物修复重金属污染土壤的策略及其机制

重金属对生态环境、农业生产、人类健康等诸多方面造成重要危害。植物修复因其具有经济有效、绿色生态等优点,已经成为土壤重金属污染修复研究领域的热点。由于植物重金属毒害、修复耗时过长等因素致使植物修复技术受限于研究阶段而不能广泛应用于实践。采用科学合理的强化措施提高植物修复的效率可能是解决该矛盾的关键之一。讨论了根瘤菌、丛枝菌根真菌、溶磷微生物和内生真菌构建的微生物-植物共生系统在强化植物修复过程中的具体应用;概述了EDTA、EDDS等螯合剂在改变土壤中重金属可溶态,促进重金属从土壤向植株转运的重要作用;介绍了植物中编码金属转运蛋白、金属硫蛋白、植物螯合肽等与重金属转运和代谢相关的基因在植物修复领域的实际应用;归纳了上述强化策略主要机制为微生物促进植物生长、缓解重金属植物毒性以及提高了土壤中重金属生物利用度,从而促进重金属在富集植物中积累和植物生物量的增加;最后总结并展望了植物修复强化技术在今后研究的重点及存在的问题。综述植物修复技术采用的主要强化策略及其机制,旨在为利用植物修复技术治理土壤重金属污染提供重要参考。     

赤泥在重金属污染治理中的应用研究进展

尽管赤泥在重金属污染环境修复研究中的应用越来越受到重视,但赤泥给污染环境修复带来突破的同时也给环境带来风险.目前,在赤泥的重金属环境修复研究中,针对赤泥的修复效果和修复过程研究较多,而对赤泥的钝化机理研究不够深入,赤泥修复的环境安全风险评价才刚起步.本文对近年来国内外关于赤泥修复重金属污染的研究进行综述,并指出了赤泥在今后的重金属污染治理中应加强的方向:一是深入赤泥对重金属钝化机制的研究;二是探索有效的赤泥安全评价方法;三是联合赤泥修复和生物修复技术(植物和微生物)对重金属和有机物复合污染进行修复.     

微生物展示技术在重金属污染生物修复中的研究进展

自然界对环境金属污染物的迁移和转化具有微秒而复杂的选择控制机理,生物修复技术以其投资少,效率高,可以原位处理低浓度有害污染物的特性而在环境治理中具有极大潜力。考虑传统的生物修复技术常常不能满足重金属治理的要求,基于重金属离子高效结合肽的微生物展示技术,有望在重金属生物修复中发挥重要作用。     

Ni2+高效结合肽的筛选与作用研究

利用噬菌体随机十二肽库和金属亲和层析对重金属Ni2+进行结合肽筛选。经4轮生物淘洗、噬菌体扩增和DNA测序,获得一组多肽序列。GenBank Blast分析未发现同源序列,Clustal W多重序列比对也未找到Ni2+金属结合肽结合基序,但可能含有多聚组氨酸（His）2－5。噬菌体单克隆金属离子螯合树脂的亲和力测定和反筛、抑菌解毒试验表明:展示有金属结合肽的噬菌体不仅对Ni2+具有高亲和力,而且对其它金属离子也有作用,Cu2+、Ni2+、Co2+、Zn2+等金属离子对金属结合肽的亲和力显著高于Cd2＋和Cr2＋,展示金属结合肽的噬菌体对重金属Ni2+具有一定的耐受和解毒作用。显微形态学观察也显示金属结合肽与金属螯合树脂的作用。对于了解重金属与多肽的相互作用机理以及环境重金属修复等均具有重要意义和价值。     

重金属污染对昆虫生长发育的影响

重金属污染已经成为一个全球性的环境问题,对生物多样性和人类健康构成了严重威胁。作为全球生物多样性的重要组成部分,昆虫因重金属污染而受到的潜在影响同样引起了人们的普遍关注。过量重金属可对昆虫的生长发育产生影响。环境中的重金属可通过昆虫的呼吸、表皮和摄食等途径进入昆虫体内,进入昆虫体内的过量重金属不仅能引起昆虫细胞超微结构的变化和遗传物质的改变,还可诱导昆虫细胞凋亡并影响细胞的活力和增殖。但昆虫能将过量的重金属以金属颗粒形式储存在具消化、存储或分泌功能的器官中,也能将其转运至溶酶体中解毒。同时,金属结合蛋白和抗氧化酶在昆虫对重金属的解毒过程中具有重要作用。     

Staphylococcal surface display of metal-binding polyhistidyl peptides

Recombinant Staphylococcus xylosus and Staphylococcus carnosus strains were generated with surface-exposed chimeric proteins containing polyhistidyl peptides designed for binding to divalent metal ions. Surface accessibility of the chimeric surface proteins was demonstrated and the chimeric surface proteins were found to be functional in terms of metal binding, since the recombinant staphylococcal cells were shown to have gained Ni(2+)- and Cd(2+)-binding capacity, suggesting that such bacteria could find use in bioremediation of heavy metals. This is, to our knowledge, the first time that recombinant, surface-exposed metal-binding peptides have been expressed on gram-positive bacteria. Potential environmental or biosensor applications for such recombinant staphylococci as biosorbents are discussed.     

Staphylococcal Surface Display of Metal-Binding Polyhistidyl Peptides

Recombinant Staphylococcus xylosus and Staphylococcus carnosus strains were generated with surface-exposed chimeric proteins containing polyhistidyl peptides designed for binding to divalent metal ions. Surface accessibility of the chimeric surface proteins was demonstrated and the chimeric surface proteins were found to be functional in terms of metal binding, since the recombinant staphylococcal cells were shown to have gained Ni²⁺- and Cd²⁺-binding capacity, suggesting that such bacteria could find use in bioremediation of heavy metals. This is, to our knowledge, the first time that recombinant, surface-exposed metal-binding peptides have been expressed on gram-positive bacteria. Potential environmental or biosensor applications for such recombinant staphylococci as biosorbents are discussed.     

金属结合蛋白基因及其在清除重金属污染中的应用

一些微生物和植物由于对毒性金属具有独特的抗性机制,使得利用它们来清除日益严重的环境污染已发展成为一种十分有效的技术——生物修复。研究表明,不同的金属结合蛋白（如MT 和PC）,在生物忍耐和降解过量重金属毒性机制中起重要作用。愈来愈多的MT 和PC基因被克隆,并已成功地应用于生物遗传转化,这些转基因生物在清除重金属污染方面已显示出潜在的应用价值。 Abstract:Heavy metal pollution has become a global environmental hazard.The use of microorganisms and plants for the decontamination of heavy metals is recognized as a low lost and high efficiency method for cleaning up metal contamination.It shows that various metal-binding proteins such as metallothioneins (MTs) or phytochelatines (PCs) play an important role in defense systems and detoxification to heavy metals in organisms.Many genes of MTs and PCs have been cloned and utilized successfully in genetically modified bacteria and plants for increasing remediation capacity.These transgenic organisms have been displayed a great potential in bioremediation and phytoremediation of heavy metals.     

Enhanced toxic metal accumulation in engineered bacterial cells expressing Arabidopsis thaliana phytochelatin synthase

Phytochelatins (PCs) are metal-binding cysteine-rich peptides, enzymatically synthesized in plants and yeasts from glutathione in response to heavy metal stress by PC synthase (EC 2.3.2.15). In an attempt to increase the ability of bacterial cells to accumulate heavy metals, the Arabidopsis thaliana gene encoding PC synthase (AtPCS) was expressed in Escherichia coli. A marked accumulation of PCs was observed in vivo together with a decrease in the glutathione cellular content. When bacterial cells expressing AtPCS were placed in the presence of heavy metals such as cadmium or the metalloid arsenic, cellular metal contents were increased 20- and 50-fold, respectively. We discuss the possibility of using genes of the PC biosynthetic pathway to design bacterial strains or higher plants with increased abilities to accumulate toxic metals, and also arsenic, for use in bioremediation and/or phytoremediation processes.     

Engineering of microorganisms towards recovery of rare metal ions

The bioadsorption of metal ions using microorganisms is an attractive technology for the recovery of rare metal ions as well as removal of toxic heavy metal ions from aqueous solution. In initial attempts, microorganisms with the ability to accumulate metal ions were isolated from nature and intracellular accumulation was enhanced by the overproduction of metal-binding proteins in the cytoplasm. As an alternative, the cell surface design of microorganisms by cell surface engineering is an emerging strategy for bioadsorption and recovery of metal ions. Cell surface engineering was firstly applied to the construction of a bioadsorbent to adsorb heavy metal ions for bioremediation. Cell surface adsorption of metal ions is rapid and reversible. Therefore, adsorbed metal ions can be easily recovered without cell breakage, and the bioadsorbent can be reused or regenerated. These advantages are suitable for the recovery of rare metal ions. Actually, the cell surface display of a molybdate-binding protein on yeast led to the enhanced adsorption of molybdate, one of the rare metal ions. An additional advantage is that the cell surface display system allows high-throughput screening of protein/peptide libraries owing to the direct evaluation of the displayed protein/peptide without purification and concentration. Therefore, the creation of novel metal-binding protein/peptide and engineering of microorganisms towards the recovery of rare metal ions could be simultaneously achieved.     

Display of lead-binding proteins on Escherichia coli surface for lead bioremediation

Cell surface display of heavy metal-binding proteins has been used to enhance the adsorption capacity of heavy metals and the engineered microbial cells can be potentially used for the bioremediation of heavy metals. In this study, the proteins PbrR, PbrR691, and PbrD from the Cupriavidus metallidurans strain CH34 were displayed on the extracellular membrane of Escherichia coli BL21 cells, with the N-domain of ice-nucleation protein as the anchor protein to achieve specific adsorption of lead ions (Pb²⁺) and bioremediation of lead in the soil. The localization of fusion proteins was confirmed by western blot analysis. We investigated the effects of fusion pattern, expression level, heavy metal concentration, and the presence of other heavy metal ions on the adsorption of Pb²⁺ by these engineered bacteria, and the optimal linker peptide (flexible linker) and inducer concentration (0.5 mM) were obtained. The engineered bacteria showed specific selectivity and strong adsorption capacity for Pb²⁺. The maximum Pb²⁺ adsorption capacity of strains displaying the three proteins (PbrR, PbrR691, and PbrD) were 942.1-, 754.3-, and 864.8-μmol/g cell dry weight, respectively, which was the highest reported to date. The engineered E. coli bacteria were also applied to Pb²⁺-contaminated soil and the detoxification effects were observed via the seed germination test and the growth of Nicotiana benthamiana in comparison with the control BL21, which provides the proof-of-concept for in situ remediations of Pb²⁺-contaminated water or soil.     

Genetic engineering of Escherichia coli for enhanced uptake and bioaccumulation of mercury.

Synthetic phytochelatins (ECs) are a new class of metal-binding peptides with a repetitive metal-binding motif, (Glu-Cys)(n)Gly, which were shown to bind heavy metals more effectively than metallothioneins. However, the limited uptake across the cell membrane is often the rate-limiting factor for the intracellular bioaccumulation of heavy metals by genetically engineered organisms expressing these metal-binding peptides. In this paper, two potential solutions were investigated to overcome this uptake limitation either by coexpressing an Hg(2+) transport system with (Glu-Cys)(20)Gly (EC20) or by directly expressing EC20 on the cell surface. Both approaches were equally effective in increasing the bioaccumulation of Hg(2+). Since the available transport systems are presently limited to only a few heavy metals, our results suggest that bioaccumulation by bacterial sorbents with surface-expressed metal-binding peptides may be useful as a universal strategy for the cleanup of heavy metal contamination.     

Metal-binding proteins and peptides in bioremediation and phytoremediation of heavy metals

The expression of metal-binding proteins or peptides in microorganisms and plants in order to enhance heavy metal accumulation and/or tolerance has great potential. Several different peptides and proteins have been explored. This review focuses on cadmium (Cd) because of the significant importance of this metal and because of its global presence in many food materials.     

Molecular design of the microbial cell surface toward the recovery of metal ions

The genetic engineering of microorganisms to adsorb metal ions is an attractive method to facilitate the environmental cleanup of metal pollution and to enrich the recovery of metal ions such as rare metal ions. For the recovery of metal ions by microorganisms, cell surface design is an effective strategy for the molecular breeding of bioadsorbents as an alternative to intracellular accumulation. The cell surface display of known metal-binding proteins/peptides and the molecular design of novel metal-binding proteins/peptides have been performed using a cell surface engineering approach. The adsorption of specific metal ions is the important challenge for the practical recovery of metal ions. In this paper, we discuss the recent progress in surface-engineered bioadsorbents for the recovery of metal ions.