To obtain basic information on the sorption and recycling of gold from aqueous systems the interaction of Au(III) and Au(0) nanoparticles on S-layer proteins were investigated. The sorption of protein polymers was investigated by ICP-MS and that of proteinaceous monolayers by QCM-D. Subsequent AFM enables the imaging of the nanostructures.
In this publication the gold sorption behavior of surface layer (S-layer) proteins (Slp1) of Lysinibacillus sphaericus JG-B53 is described. These biomolecules arrange in paracrystalline two-dimensional arrays on surfaces, bind metals, and are thus interesting for several biotechnical applications, such as biosorptive materials for the removal or recovery of different elements from the environment and industrial processes. The deposition of Au(0) nanoparticles on S-layers, either by S-layer directed synthesis 1 or adsorption of nanoparticles, opens new possibilities for diverse sensory applications. Although numerous studies have described the biosorptive properties of S-layers 2-5, a deeper understanding of protein-protein and protein-metal interaction still remains challenging. In the following study, inductively coupled mass spectrometry (ICP-MS) was used for the detection of metal sorption by suspended S-layers. This was correlated to measurements of quartz crystal microbalance with dissipation monitoring (QCM-D), which allows the online detection of proteinaceous monolayer formation and metal deposition, and thus, a more detailed understanding on metal binding.
The ICP-MS results indicated that the binding of Au(III) to the suspended S-layer polymers is pH dependent. The maximum binding of Au(III) was obtained at pH 4.0. The QCM-D investigations enabled the detection of Au(III) sorption as well as the deposition of Au(0)-NPs in real-time during the in situ experiments. Further, this method allowed studying the influence of metal binding on the protein lattice stability of Slp1. Structural properties and protein layer stability could be visualized directly after QCM-D experiment using atomic force microscopy (AFM). In conclusion, the combination of these different methods provides a deeper understanding of metal binding by bacterial S-layer proteins in suspension or as monolayers on either bacterial cells or recrystallized surfaces.
由于越来越多地使用黄金数的应用,如电子,催化剂,生物传感器,或医疗器械,这种贵金属的需求增长在过去几年的时间内6-9。黄金以及许多其他的贵金属和重金属释放到环境中,通过工业废水稀的浓度,通过采矿活动,以及废物处理7,8,10,虽然大多数环境的污染较重或贵金属是一个持续的过程主要是由于科技活动。这导致自然生态系统的一个显著干扰并且可能潜在地威胁人类健康9。了解这些负面结果促进寻求新的技术来去除污染的生态系统和改善金属的工业废水回收金属。像沉淀或离子交换行之有效的物理 – 化学方法不是那么有效,尤其是在高LY稀释溶液7,8,11。生物吸附,无论是与活的或死的生物质,是污水处理10,12有吸引力的选择。使用这样的生物材料可以减少有毒化学品的消耗量。许多微生物已经描述累积或固定金属。例如,Lysinibacillus球形的细胞(L.球形 )JG-A12显示高结合能力的贵金属,例如,钯(Ⅱ),铂(II),金(III)和其他有毒金属如铅(II)的或U(Ⅵ)4,13, 巨大芽孢杆菌的为铬(VI)14细胞, 酿酒酵母的铂(II)和Pd(II)的15,和小球藻俗为金细胞(Ⅲ)和U(Ⅵ)16 17。以前金属如金的结合(Ⅲ),钯(Ⅱ),和Pt(II)中也已报道了脱硫脱硫弧菌 18和L.球形 JG-B53 19,20。然而,没有人升微生物结合大量的金属及其应用为吸附材料有限12,21。此外,金属结合能力取决于不同的参数,例如,细胞组合物中,所用的生物成分或环境和实验条件(pH,离子强度,温度等)。分离的细胞壁片段22,23的研究,如膜脂质,肽聚糖,蛋白质,或其它部件,有助于理解该金属结合复合构成的全细胞8,21的处理。
该电池组件在这项研究集中于有S层蛋白。 S-层蛋白是许多细菌和古细菌的外细胞膜的部分,它们构成约15 – 20%的这些微生物的总蛋白质量的。作为第一接口的环境中,这些细胞的化合物强烈地影响细菌吸着性质3。 S-层蛋白分子量范围从40到几百kDa的的是在细胞内产生的,但外被组装在那里他们能够形成层上的脂膜或聚合细胞壁成分。一旦分离,几乎所有的S-层蛋白质具有的固有属性自发自组装在悬浮液中,在界面处,或在表面上形成的平面的或管状结构3。蛋白质单层的厚度取决于细菌,是一个范围为5内- 25纳米24。在一般情况下,所形成的S-层蛋白结构可具有一倾斜(P1或P2),正方形(P4),或六边形(P3或P6)对称性为2.5晶格常数至35nm 3,24。晶格的形成似乎是在许多情况下,依赖于二价阳离子和主要对Ca 2+ 25,26,拉夫,J。等。 S层基纳米复合材料为在基于蛋白质的工程化纳米结构的工业应用。 (编辑蒂亚娜Z.树丛和Aitziber L. Cortajarena)(施普林格,2016(提交))。尽管如此,完整的反应级联,尤其是二价阳离子如Ca 2+和Mg 2+单体折叠,单体-单体相互作用,晶格的形成,和不同的金属的作用,仍然没有完全了解。
革兰氏阳性菌株L.球形 JG-B53 27(从后新的进化分类球形芽孢杆菌改名),从铀矿开采废料堆“哈伯兰”(约汉格奥尔根斯塔特,萨克森,德国)4,28,29隔离。其功能S-层蛋白(SLP1)具有一个正方形晶格,116 kDa的30的分子量,并在活细菌细胞31的厚度≈10纳米。在以往的研究中, 在体外形成一个封闭的和稳定的蛋白质层的厚度为约10nm,在不到10分钟19达到了。相关应变L.球形 JG-A12,还从“哈伯兰”桩一个分离物,具有高的金属结合能力和其分离的S层蛋白已经显示出对等贵金属的Au具有高的化学和机械稳定性和良好的吸附速率(Ⅲ),铂(II),和Pd(II)的4,32,33。这种贵金属的结合是或多或少特异于一些金属和取决于官能团的聚合物的外和内表面蛋白,并在其孔中,离子强度的可用性,和pH值。由蛋白质有关的官能团为金属相互作用是COOH-,NH 2 – ,OH – ,PO 4 – ,SO 4 – , – SO-和。原则上,金属结合的能力打开了广泛的应用, 拉夫,J。等光谱 。 S层基纳米复合材料为在基于蛋白质的工程化纳米结构的工业应用。 (编辑蒂亚娜Z.树丛和Aitziber L. Cortajarena)(施普林格2016(提交))。 例如,作为用于去除或回收biosorptive组件溶解有毒或有价金属,合成或定期结构的金属纳米颗粒(纳米颗粒)的催化,以及其他生物工程材料,如生物传感层3,5,18,33的定义沉积模板。规则排列的NP阵列状的Au(0)-nps可用于主要应用范围从分子电子学和生物传感器,超高密度存储设备,和催化剂对CO氧化34-37。这样的应用程序以及这些材料的智能设计的发展,就必须的基本金属绑定机制有了更深的了解。
一个先决条件,例如生物基材料的发展是可靠的实施的生物分子和技术表面38,39之间的界面层的。例如,聚电解质组装有层-层(层层)技术40,41已被用作用于S层蛋白39的重结晶的界面层</SUP>。这样的接口提供了一个比较简单的方法,可重复和定量的方式进行蛋白质涂层。通过进行不同的实验有和没有修改与粘合剂的启动子,有可能做出关于涂层动力学,稳定性层,以及金属与生物分子19,42,拉夫,J。等人的相互作用的语句。 S层基纳米复合材料为在基于蛋白质的工程化纳米结构的工业应用。 (编辑蒂亚娜Z.树丛和Aitziber L. Cortajarena)(施普林格,2016年(提交))。然而,蛋白质的吸附和蛋白质表面相互作用的复杂的机制尚未完全了解。特别是在构象,图案方向和涂层密度信息仍下落不明。
石英晶体微量天平耗散监测(QCM-D)的技术已引起关注,在近年来作为用于研究蛋白质的吸附,涂层动力学的工具,和相互作用的亲正如事实上纳米尺度19,43-45。此技术允许质量吸附在实时的详细检测,并且可以作为对蛋白质晶格19,20,42,46-48蛋白自组装过程和功能分子偶合的一个指标。另外,QCM-D测量开来研究金属的互动过程与天然生物条件下,蛋白质层的可能性。在最近的研究中,在S-层蛋白质的与选定的金属,如铕的相互作用(Ⅲ),金(Ⅲ),钯(Ⅱ),和Pt(II)中已研究了的QCM-D 19,20。吸附的蛋白质层可以作为革兰氏阳性细菌的细胞壁的简化模型。这种单一组分的研究有助于金属的相互作用有更深的了解。然而,仅仅QCM-D实验不允许有关表面结构和金属蛋白的影响报表。其它技术是必需的,以获得这样的信息。一个POS上的结构特性sibility用于成像的生物纳米结构和获取信息是原子力显微镜(AFM)。
所呈现的研究的目的是调查的金(Au(III)和Au(0)-nps)到S层蛋白质的吸附,在L的特定SLP1 球形 JG-B53。 5.0使用ICP-MS和使用QCM-D固定的S-层 – 悬浮的蛋白质上一批规模2.0的pH范围内进行了实验。此外,关于晶格稳定性金属盐溶液的影响进行了研究与随后AFM研究中。这些技术的组合,有助于更好地理解在体外金属的相互作用过程中为更多地了解关于结合特定金属的亲和力对整个细菌细胞活动的工具。这些知识不仅是适用的过滤材料的发展为金属环保的回收和再保护的关键源49,同时也为高度有序的金属纳米颗粒关于各种技术应用的阵列的发展。
在这项工作中研究了Au构成的结合S-层蛋白使用的不同的分析方法的组合进行了研究。特别是,Au构成的结合是非常有吸引力的,不仅对于Au中从采矿水域或处理溶液的回收,而且还用于材料,例如,感觉表面的结构。对于在Au相互作用的研究(金(Ⅲ)和Au(0)-nps)悬浮和重结晶SLP1的单层,该蛋白质必须被隔离。因此,本研究表明培育成功的革兰氏阳性细菌菌株L的球形 JG-B53?…
The authors have nothing to disclose.
目前的工作是部分由BMWI和BMBF项目“Aptasens”(BMBF / DLR 01RB0805A)资助的IGF-项目“S-筛”(490 ZBG / 1)资助。特别感谢的Tobias J.半滑舌鳎的宝贵帮助,在原子力显微镜的研究和埃里克V.斯通读取稿件为以英语为母语。此外,该论文的作者要感谢艾琳里特和萨布丽娜Gurlit(从研究所的资源生态为ICP-MS测定的援助),曼加沃格尔,南希·昂格尔,卡伦E. Viacava和亥姆霍兹研究所生物技术组佛莱堡的资源技术。
equiment and software | |||
Bioreactor, Steam In Place 70L Pilot System | Applikon Biotechnology, Netherlands | Z6X | Including dO2, pH sensors of Applikon Biotechnology and BioXpert software V2 |
Noninvasive Biomass Monitor BugEye 2100 | BugLab, Concord (CA), USA | Z9X | — |
Spectrometer Ultrospec 1000 | Amersham Pharmacia Biotech, Great Britain | 80-2109-10 | Company now GE Healthcare Life Sciences |
MiniStar micro centrifuge | VWR, Germany | 521-2844 | For centrifugation of cultivation samples |
Research system microscope BX-61 | Olympus Germany LLC, Germany | 037006 | Microscope in combination with imaging software |
Cell^P (version 3.1) | Olympus Soft Imaging Solutions LLC, Münster, Germany | — | together with microscope |
Powerfuge Pilot Separation System Serie 9010-S | Carr Centritech, Florida, USA | 9010PLT | For biomasse harvesting |
T18 basic Ultra Turrax | IKA Labortechnik, Germany | 431-2601 | For flagella removal and sample homogenization |
Sorvall Evolution RC Superspeed Centrifuge | Thermo Fisher Scientific, USA | 728411 | Used within protein isolation |
Mobile high shear fluid processor, M-110EH-30 Pilot | Microfluidics, Massachusetts, USA | M110EH30K | Used for cell rupture |
Alpha 1-4 LSC Freeze dryer | Martin Christ Freeze dryers LLC, Osterode, Germany | 102041 | — |
UV-VIS spectrophotometry (NanoDrop 2000c) | Thermo Fisher Scientific, USA | 91-ND-2000C-L | For determination of protein concentration |
Mini-PROTEAN vertical electrophoresis chamber | Bio-Rad Laboratories GmbH, Munich, Germany | 165-3322 | For SDS-PAGE |
VersaDoc Imaging System 3000 | Bio-Rad Laboratories GmbH, Munich, Germany | 1708030 | Used for imaging of SDS-PAGE gels |
ICP-MS Elan 9000 | PerkinElmer, Waltham (MA), USA | N8120536 | For determination of metal concentration |
Zetasizer Nano ZS | Malvern Instruments, Worcestershire United Kingdom | ZEN3600 | For determination of nanoparticle size |
Q-Sense E4 device | Q-Sense AB, Gothenburg, Sweden | QS-E4 | ordered via LOT quantum design (software included with E4 platform) |
Q-Soft 401 (data recording) | Q-Sense AB, Gothenburg, Sweden | ||
Q-Tools 3 (data evaluation and modelling) | Q-Sense AB, Gothenburg, Sweden | ||
QCM-D flow modules QFM 401 | Q-Sense AB, Gothenburg, Sweden | QS-QFM401 | ordered via LOT quantum design |
QSX 303 SiO2 piezoelectric AT-cut quartz sensors | Q-Sense AB, Gothenburg, Sweden | QS-QSX303 | ordered via LOT quantum design |
Ozone cleaning chamber | Bioforce Nanoscience, Ames (IA), USA | QS-ESA006 | ordered via LOT quantum design |
Atomic Force Microscope MFP-3D Bio AFM | Asylum Research, Santa Barbara (CA), USA | MFP-3DBio | AFM measurements and imaging software |
Asylum Research AFM Software AR Version 120804+1223 | Asylum Research, Santa Barbara (CA), USA | — | imaging software included in Cat. No. MFP-3DBio |
Igor Version Pro 6.3.2.3 Software | WaveMetrics, Inc., USA | — | imaging software included in Cat. No. MFP-3DBio |
BioHeater | Asylum Research, Santa Barbara (CA), USA | Bioheater | Sample heater for AFM measurements |
Biolever mini cantilever, BL-AC40TS-C2 | Olympus Germany LLC, Germany | BL-AC40TS-C2 | Prefered cantilever for AFM measurements |
WSxM 5.0 Develop 6.5 (2013) | Nanotec Electronica S.L. , Spain | freeware | Software for AFM analysis |
Name | Company | Catalog Number | Comments |
Detergents and other equiment | |||
Calcium chloride Dihydrate (CaCl2 ∙ 2H2O) | Merck KGaA | 1.02382 | — |
acidic acid, 100 %, p.A. | CARL ROTH GmbH+CO.KG | 3738.5 | Danger, flammable and corrosive liquid and vapour. Causes severe skin burns and eye damage. |
Antifoam 204 | Sigma-Aldrich Co. LLC. | A6426 | For foam suppression |
bromophenol blue, sodium salt | Sigma-Aldrich Co. LLC. | B5525 | — |
Coomassie Brilliant Blue R (C45H44N3NaO7S2) | CARL ROTH GmbH+CO.KG | 3862.1 | — |
Deoxyribonuclease II from porcine spleen | Sigma-Aldrich Co. LLC. | D4138 | Typ IV , 2,000-6,000 Kunitz units/mg protein |
Ethanol, 95% | VWR, Germany | 20827.467 | Danger, flammable |
glycerine, p.A. | CARL ROTH GmbH+CO.KG | 3783.1 | — |
Gold(III) chloride trihydrate (HAuCl4 ∙ 3H2O) | Sigma-Aldrich Co. LLC. | 520918 | Danger |
Guanidine hydrochloride (GuHCl) | CARL ROTH GmbH+CO.KG | 0037.1 | — |
Hellmanex III | Hellma GmbH & Co. KG | 9-307-011-4-507 | — |
Hydrochloric acid (HCl) (37%) | CARL ROTH GmbH+CO.KG | 4625.2 | Danger; Corrosive, used for pH adjustment |
Lysozyme from chicken egg white | Sigma-Aldrich Co. LLC. | L6876 | Lyophilized powder, protein =90 %, =40,000 units/mg protein (Sigma) |
Magnesium chloride Hexahydrate (MgCl2 ∙ 6H2O) | Merck KGaA | 1.05833 | — |
Magnetic stirrer with heating, MR 3000K | Heidolph Instruments GmbH & Co.KG, Germany | 504.10100.00 | Standard stirrer within experiment |
NB-Media DM180 | Mast Diagnostica GmbH | 121800 | — |
Nitric acid (HNO3) | CARL ROTH GmbH+CO.KG | HN50.1 | Danger; Oxidizing, Corrosing |
PageRuler Unstained Protein Ladder | ThermoScientific-Pierce | 26614 | — |
Poly(sodium 4-styrenesulfonat) (PSS) | Sigma-Aldrich Co. LLC. | 243051 | Average Mw ~70,000 |
Polyethylenimine (PEI), branched | Sigma-Aldrich Co. LLC. | 408727 | Warning; Harmful, Irritant, Dangerous for the environment; average Mw ~25,000 |
Potassium carbonate anhydrous (K2CO3) | Sigma-Aldrich Co. LLC. | 60108 | Warning; Harmful |
Ribonuclease A from bovine pancreas | Sigma-Aldrich Co. LLC. | R5503 | Type I-AS, 50-100 Kunitz units/mg protein |
Sodium azide (NaN3) | Merck KGaA | 106688 | Danger; very toxic and Dangerous for the environment |
Sodium chloride (NaCl) | CARL ROTH GmbH+CO.KG | 3957.2 | — |
Sodium dodecyl sulfate (SDS) | Sigma-Aldrich Co. LLC. | L-5750 | Danger; toxic |
Sodium hydroxide (NaOH) | CARL ROTH GmbH+CO.KG | 6771.1 | Danger; Corrosive, used for pH regulation within cultivation and pH adjustment |
Spectra/Por 6, Dialysis membrane, MWCO 50,000 | CARL ROTH GmbH+CO.KG | 1893.1 | — |
Sulfuric acid (H2SO4) | CARL ROTH GmbH+CO.KG | HN52.2 | Danger; Corrosive, used for pH regulation within cultivation |
Tannic acid (C76H52O46) | Sigma-Aldrich Co. LLC. | 16201 | — |
TRIS HCl (C4H11NO3HCl) | CARL ROTH GmbH+CO.KG | 9090.2 | — |
Tri-sodium citrate dihydrate (C6H5Na3O7 ∙ 2H2O) | CARL ROTH GmbH+CO.KG | 3580.2 | — |
Triton X-100 | CARL ROTH GmbH+CO.KG | 3051.3 | Warning; Harmful, Dangerous for the environment |
VIVASPIN 500, 50.000 MWCO Ultrafiltration tubes | Sartorius AG | VS0132 | — |
β-mercaptoethanol | Sigma-Aldrich Co. LLC. | M6250 | Danger, toxic |