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Improved degradation of azo dyes by lignin peroxidase following mutagenesis at two sites near the catalytic pocket and the application of peroxidase-coated yeast cell walls

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Frontiers of Environmental Science & Engineering 2021年第2期15卷 33-42页

作者： Karla IlićĐurđić Raluca Ostafe Olivera Prodanović AleksandraĐurđevićĐelmaš Nikolina Popović rainer fischer Stefan Schillberg Radivoje Prodanović University of Belgrade-Faculty of Chemistry Studentski trg 12-1611000 BelgradeSerbia Molecular Evolution Protein Engineering and Production facility(MEPEP) Purdue UniversityWest LafayetteIN 47907USA Institute of Molecular Biotechnology RWTH Aachen University52074 AachenGermany Institute for Multidisciplinary Research Kneza Višeslava 111030 BelgradeSerbia Departments of Biological Sciences and Chemistry Purdue UniversityWest LafayetteIN 47907USA Fraunhofer Institute for Molecular Biology and Applied Ecology IME 52074 AachenGermany

The enzymatic degradation of azo dyes is a promising alternative to ineffective chemical and physical remediation *** peroxidase(LiP)from Phanerochaete chrysosporium is a hemecontaining lignin-degrading oxidoreductase that catalyzes the peroxide-dependent oxidation of diverse molecules,including industrial *** enzyme is therefore ideal as a starting point for protein ***,we subjected two positions(165 and 264)in the environment of the catalytic Trp171 residue to saturation mutagenesis,and the resulting library of 104 independent clones was expressed on the surface of yeast *** yeast display library was used for the selection of variants with the ability to break down structurally-distinct azo dyes more *** identified mutants with up to 10-fold greater affinity than wild-type LiP for three diverse azo dyes(Evans blue,amido black 10B and Guinea green)and up to 13-fold higher catalytic ***,cell wall fragments displaying mutant LiP enzymes were prepared by toluene-induced cell lysis,achieving significant increases in both enzyme activity and stability compared to a whole-cell *** cell wall fragments retained their initial dye degradation activity after 10 reaction cycles each lasting 8 *** best-performing mutants removed up to 2.5-fold more of each dye than the wild-type LiP in multiple reaction cycles.

关键词： Bioremediation Enzyme immobilization Protein engineering Yeast surface display

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Expression of Recombinant Tryptophan Decarboxylase in Different Subcellular Compartments in Tobacco Plant

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Acta Botanica Sinica 2002年第3期44卷 314-317页

作者：王淼李秋荣 Stefano Di FIORE rainer fischer 中国科学院沈阳应用生态研究所沈阳110016 沈阳军区总医院沈阳110016 Botanical Institute of Rheinisch-Westflisch Technische Hochschule Aachen

The gene encoded for tryptophan decarboxylase (TDC), which is the key enzyme in terpenoil indole alkaloids pathway, was targeted to different subcellular compartments and stably expressed in transgenic tobacco (Nicotiana tabacum L.) plants at the levels detected by Western blot and tryptamine accumulation analysis. It was shown that the TDC was located in subcellular compartments, the chloroplasts and cytosol. The recombinant TDC targeted to chloroplasts and cytosol in tobacco plants was effectively expressed as soluble protein by Western blot analysis and enzymatic assay. The level of tryptamine accumulation in chloroplast was higher than that in cytosol and very low in vacuole and endoplasmic reticulum (ER) to be hardly detected by Western blot analysis. It was indicated that the highest amount of tryptamine was in chloroplasts, lower in endoplasmic reticula and the lowest in vacuoles as compared to those in wild type plants. The TDC targeted to different subcellular compartments of tobacco plants and its expression level were studied by different nucleotide sequences coding signal peptides at 5'-end of tdc gene in order to know the effects of the TDC in compartmentation on its functionality.

关键词： tryptophan decarboxylase alkaloids biosynthetic pathway Nicotiana tabacum subcellular compartment

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Expression of Strictosidine Synthase in Selected Subcellular Compartments of Tobacco Plant

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Acta Botanica Sinica 2002年第5期44卷 579-582页

Strictosidine synthase (STR) is a key enzyme involved in the biosynthesis of terpenoid indole alkaloids (TIA) by condensing tryptamine and secologanin into strictosidine. The transgenic tobacco plants targeting STR to subcellular compartments were established to express STR in chloroplast, vacuole and endoplasmic reticulum (ER) by the tobacco stable transformation. It was shown that STR was effectively expressed in the above subcellular compartments by Western blot analysis and STR enzymatic assay. In vitro , STR enzymatic assay was measured indirectly by fluorimetrically detecting depletion of tryptamine feeding on secologanin in the reaction mixture. The tryptamine were completely depleted by STR in the crude extract of leaves of transgenic tobacco plants targeting and expressing STR in the chloroplast, vacuole and ER, which ascertained the STR functionally targeted to the three subcellular compartments. To confirm STR correct targeting and expressing in chloroplast, the chloroplasts were isolated and the fractions of purified chloroplasts were analyzed by Western blot. The hypothesis of STR correct targeting to the chloroplast was tested. The results have implications on our understanding of the complex intracellular trafficking in metabolic intermediates of TIA biosynthesis.

关键词： Nicotiana tabacum strictosidine synthase subcellular compartment expression

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四甲基氯化铵在PCR扩增小麦基因中的关键作用

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遗传 1997年第2期19卷 1-4页

作者：廖玉才李和平 rainer fischer 华中农业大学作物遗传改良国家重点实验室德国亚琛技术大学植物及分子遗传研究所

利用高简并性引物，用ＰＣＲ法从小麦ＤＮＡ或ｃＤＮＡ中合成小麦几丁质酶基因、葡聚糖酶基因和苯丙氨酸解氨酶基因片段。在ＰＣＲ反应中添加四甲基氯化铵（ＴＭＡＣｌ）是合成这些特异基因片段的关键。合成的ＰＣＲ片段都经末端补齐和... 详细信息

利用高简并性引物，用ＰＣＲ法从小麦ＤＮＡ或ｃＤＮＡ中合成小麦几丁质酶基因、葡聚糖酶基因和苯丙氨酸解氨酶基因片段。在ＰＣＲ反应中添加四甲基氯化铵（ＴＭＡＣｌ）是合成这些特异基因片段的关键。合成的ＰＣＲ片段都经末端补齐和磷酸化后用于克隆。核酸序列分析证实，这些ＰＣＲ产物分别与用于设计ＰＣＲ引物的基因具有高度的同源性。

关键词：四甲基氯化铵小麦基因扩增聚合酶链反应

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小麦苯丙氨酸解氨酶基因和几丁质酶基因转录起始点的鉴定

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遗传 1997年第5期19卷 10-13页

作者：廖玉才李和平 rainer fischer 华中农业大学作物遗传改良国家重点实验室德国亚琛技术大学植物及分子遗传研究所

本文应用改良的引物延伸法，鉴定了一个小麦的苯丙氨酸解氨酶和几丁质酶基因的转录起始点。这种方法只需少量同位素，ＳｅｐｈａｄｅｘＧ－２５柱和严格的杂交条件。利用两种基因的特定引物及总ＲＮＡ进行的引物延伸反应产物在电泳的凝... 详细信息

本文应用改良的引物延伸法，鉴定了一个小麦的苯丙氨酸解氨酶和几丁质酶基因的转录起始点。这种方法只需少量同位素，ＳｅｐｈａｄｅｘＧ－２５柱和严格的杂交条件。利用两种基因的特定引物及总ＲＮＡ进行的引物延伸反应产物在电泳的凝胶上均呈现一条十分清楚的带，表明这两种多拷贝小麦基因的转录分别起始于一个核苷酸。小麦苯丙氨酸解氨酶基因的转录起始点为５′－ＣＴＧＣＣＧＡＧＴ－３′序列中的第２个Ｃ；而小麦几丁质酶基因转录起始点为５′－ＡＴＣＡＣＣＡＧＣ－３′序列中的第２个Ａ。它们都分别位于各自基因ＴＡＴＡ盒的下游。

关键词：小麦苯丙氨酸解氨酶基因几丁质酶基因转录

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Improved degradation of azo dyes by lignin peroxidase following mutagenesis at two sites near the catalytic pocket and the application of peroxidase-coated yeast cell walls

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Frontiers of Environmental Science & Engineering 2021年第2期 14-23页

作者： Karla Ili? ?ur?i? Raluca Ostafe Olivera Prodanovi? Aleksandra ?ur?evi? ?elma? Nikolina Popovi? rainer fischer Stefan Schillberg Radivoje Prodanovi? University of Belgrade-Faculty of Chemistry Molecular Evolution Protein Engineering and Production facility (MEPEP) Purdue University Institute of Molecular Biotechnology RWTH Aachen University Institute for Multidisciplinary Research Departments of Biological Sciences and Chemistry Purdue University Fraunhofer Institute for Molecular Biology and Applied Ecology IME

The enzymatic degradation of azo dyes is a promising alternative to ineffective chemical and physical remediation *** peroxidase(LiP) from Phanerochaete chrysosporium is a hemecontaining lignin-degrading oxidoreductase that catalyzes the peroxide-dependent oxidation of diverse molecules,including industrial *** enzyme is therefore ideal as a starting point for protein ***,we subj ected two positions(165 and 264) in the environment of the catalytic Trp171 residue to saturation mutagenesis,and the resulting library of 10～4 independent clones was expressed on the surface of yeast *** yeast display library was used for the selection of variants with the ability to break down structurally-distinct azo dyes more *** identified mutants with up to 10-fold greater affinity than wild-type LiP for three diverse azo dyes(Evans blue,amido black 10 B and Guinea green) and up to 13-fold higher catalytic ***,cell wall fragments displaying mutant LiP enzymes were prepared by toluene-induced cell lysis,achieving significant increases in both enzyme activity and stability compared to a whole-cell *** cell wall fragments retained their initial dye degradation activity after 10 reaction cycles each lasting 8 *** best-performing mutants removed up to 2.5-fold more of each dye than the wild-type LiP in multiple reaction cycles.

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A New Kind of Atomic Force Microscopy Scan Control Enabled by Artificial Intelligence:Concept for Achieving Tip and Sample Safety Through Asymmetric Control

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Nanomanufacturing and Metrology 2024年第2期7卷 50-59页

作者： Johannes Degenhardt Mohammed Wassim Bounaim Nan Deng rainer Tutsch Gaoliang Dai Physikalisch-Technische Bundesanstalt Bundesallee 10038116 BrunswickGermany Institut Für Prozessmess-und Sensortechnik Technische Universität Ilmenau EhrenbergstraBe 2998693 IlmenauGermany Institute of Production Metrology TU Braunschweig SchleinitzstraBe 2038106 BrunswickGermany

This paper introduces a paradigm shift in atomic force microscope(AFM)scan control,leveraging an artificial intelligence(AI)-based *** contrast to conventional control methods,which either show a limited performance,such as proportional integral differential(PID)control,or which purely focus on mathematical optimality as classical optimal control approaches,our proposed AI approach redefines the objective of control for achieving practical *** presented AI controller minimizes the root-mean-square control deviations in routine scans by a factor of about 4 compared to PID control in the presented setup and also showcases a distinctive asymmetric response in complex situations,prioritizing the safety of the AFM tip and sample instead of the lowest possible control *** development and testing of the AI control concept are performed on simulated AFM scans,demonstrating its huge potential.

关键词： Atomic force microscopy Artificial intelligence Deep reinforcement learning Optimal control

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Predicting potential invasion risks of Leucaena leucocephala(Lam.)de Wit in the arid area of Saudi Arabia

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Journal of Arid Land 2024年第7期16卷 983-999页

作者： Haq S MARIFATUL Darwish MOHAMMED Waheed MUHAMMAD Kumar MANOJ Siddiqui H MANZER Bussmann W rainer Department of Ethnobotany Institute of BotanyIlia State UniversityTbilisi 0162Georgia The Centre of Excellence on Sustainable Land Management(CoE-SLM) Indian Council of Forestry Research and EducationDehradun 248006India Department of Botany and Microbiology College of ScienceKing Saud UniversityRiyadh 11451Saudi Arabia Department of Botany State Museum for Natural HistoryKarlsruhe 76133Germany

The presence of invasive plant species poses a substantial ecological impact,thus comprehensive evaluation of their potential range and risk under the influence of climate change is *** study uses maximum entropy(MaxEnt)modeling to forecast the likelihood of Leucaena leucocephala(Lam.)de Wit invasion in Saudi Arabia under present and future climate change *** the MaxEnt modeling,we integrated climatic and soil data to predict habitat suitability for the invasive *** conducted a detailed analysis of the distribution patterns of the species,using climate variables and ecological *** focused on the important influence of temperature seasonality,temperature annual range,and precipitation *** distribution modeling used robust measures of area under the curve(AUC)and receiver-operator characteristic(ROC)curves,to map the invasion extent,which has a high level of accuracy in identifying appropriate *** complex interaction that influenced the invasion of *** was highlighted by the environmental parameters using Jackknife ***,the actual geographic area where *** was found in Saudi Arabia was considerably smaller than the theoretical maximum range,suggesting that it had the capacity to expand *** MaxEnt model exhibited excellent prediction accuracy and produced reliable results based on the data from the ROC *** and temperature were the primary factors influencing the potential distribution of ***,an estimated area of 216,342 km^(2)in Saudi Arabia was at a high probability of invasion by *** investigated the potential for increased invasion hazards in the future due to climate change scenarios(Shared Socioeconomic Pathways(SSPs)245 and 585).The analysis of key climatic variables,including temperature seasonality and annual range,along with soil properties such as clay composition and nitrogen content,unveiled their substanti

关键词： area under the curve invasive species invasion risks climate change MaxEnt model

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COmbination of Targeted temperature management and Thrombectomy after acute Ischemic Stroke(COTTIS):a pilot study

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Stroke & Vascular Neurology 2024年第3期9卷 258-267页

作者： Jürgen Bardutzky rainer Kollmar Forat Al-Rawi Johann Lambeck Mohammad Fazel Christian Taschner Wolf-Dirk Niesen Department of Neurology and Neurophysiology Medical Center-University of FreiburgFaculty of MedicineUniversity of FreiburgFreiburgGermany Neurology and Neurointensive Care Darmstadt HospitalDarmstadtGermany BrainCool AB LundSweden Department of Neuroradiology Medical Center-University of FreiburgFaculty of MedicineUniversity of FreiburgFreiburgGermany

Background To evaluate the feasibility and safety of a fast initiation of cooling to a target temperature of 35℃by means of transnasal cooling in patients with anterior circulation large vessel occlusion(LVO)undergoing endovascular thrombectomy(EVT).Methods Patients with an LVO onset of<24hour who had an indication for EVT were included in the *** cooling(RhinoChill)was initiated immediately after the patient was intubated for EVT and continued until an oesophageal target temperature of 35℃was *** was maintained with surface cooling for 6-hour postrecanalisation,followed by active rewarming(+0.2℃/hour).The primary outcome was defined as the time required to reach 35℃,while secondary outcomes comprised clinical,radiological and safety *** Twenty-two patients(median age,77 years)were included in the study(14 received additional thrombolysis,4 additional stenting of the proximal internal carotid artery).The median time intervals were 309min for last-seen-normal-to-groin,58min for door-to-cooling-initiation,65min for door-to-groin and 123min for *** target temperature of 35℃was reached within 30min(range 13–78min),corresponding to a cooling rate of 2.6℃/*** recanalisation,86%of the patients had a body temperature of≤35℃.The median National Institutes of Health Stroke Scale at admission was 15 and improved to 2 by day 7,and 68%of patients had a good outcome(modified Rankin Scale 0–2)at 3 *** complications included asymptomatic bradycardia(32%),pneumonia(18%)and asymptomatic haemorrhagic transformation(18%).Conclusion The combined application of hypothermia and thrombectomy was found to be feasible in sedated and ventilated *** events were comparable to those previously described for EVT in the absence of *** effect of this procedure will next be evaluated in the randomised COmbination of Targeted temperature management and Thrombectomy after acute Ischemic Stro

关键词： maintained continued initiated

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Small mammal community composition impacts bank vole (Clethrionomys glareolus) population dynamics and associated seroprevalence of Puumala orthohantavirus

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Integrative Zoology 2024年第1期19卷 52-65页

作者： Felicitas Maria BUJNOCH Daniela REIL Stephan DREWES Ulrike MROSENFELD rainer G.ULRICH Jens JACOB Christian IMHOLT Julius Kühn-Institute Federal Research Centre for Cultivated PlantsInstitute for Epidemiology and Pathogen DiagnosticsRodent ResearchMünsterGermany University of Münster Institute for Evolution and BiodiversityMünsterGermany Friedrich-Loeffler-Institut Federal Research Institute for Animal HealthInstitute of Novel and Emerging Infectious DiseasesGreifswald-Insel RiemsGermany

Rodents are important reservoirs for zoonotic pathogens that cause diseases in *** is hypoth-esized to be closely related to pathogen prevalence through multiple direct and indirect *** example,the presence of non-host species can reduce contact rates of the main reservoir host and thus reduce the risk of transmission(“dilution effect”).In addition,an overlap in ecological niches between two species could lead to increased interspecific competition,potentially limiting host densities and reducing density-dependent pathogen transmission *** this study,we investigated the relative impact of population-level regulation of direct and indirect drivers of the prevalence of Puumala orthohantavirus(PUUV)in bank voles(Clethrionomys glareolus)during years with high *** compiled data on small mammal community composition from four regions in Germany between 2010 and *** equation modeling revealed a strong seasonality in PUUV control mechanisms in bank *** abundance of shrews tended to have a negative relationship with host abundance,and host abundance positively influenced PUUV seroprevalence,while at the same time increasing the abundance of competing non-hosts like the wood mouse(Apodemus sylvaticus)and the yellow-neckedfield mouse(Apodemusflavicollis)were associated with reduced PUUV seroprevalence in the *** results indicate that for PUUV in bank voles,dilution is associated with increased interspecific *** pressures leading to the decline of Apodemus *** a specific habitat could lead to the amplification of mechanisms promoting PUUV transmission within the host populations.

关键词： bank vole biodiversity Puumala orthohantavirus small mammals

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