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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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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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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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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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Translocator protein and neurodegeneration: insights from Alzheimer’s disease

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Neural Regeneration Research 2025年第4期20卷 1090-1091页

作者： Arpit Kumar Pradhan rainer Rupprecht Gerhard Rammes Klinik für Anaesthesiologie und Intensivmedizin der Technischen Universität München Klinikum rechts der IsarMunichGermany Graduate School of Systemic Neurosciences Ludwig-Maximilians-Universität MünchenMartinsriedGermany Department of Psychiatry and Psychotherapy University RegensburgRegensburgGermany

The 18 kDa translocator protein(TSPO)located on the outer mitochondrial membrane regulates several key cellular processes including mitochondrial homeostasis,cholesterol transport,apoptosis,cell proliferation,and maintenance of mitochondrial health(Rupprecht et al.,2022,2023).TSPO is expressed in both peripheral organs and the central nervous system,with a more pronounced expression in tissues that produce *** main reason why TSPO has garnered so much attention is because it plays a key role in neurosteroidogenesis by transferring cholesterol from the outer to the inner mitochondrial membrane,which is the rate-limiting step in neurosteroid synthesis.A cholesterol-recognizing amino acid consensus domain has been identified in the cytosolic C terminus of the TSPO protein by both in vitro and site-directed mutagenesis experiments(Li et al.,2001).However,the role of TSPO in the process of neurosteroid synthesis has been challenged by several studies,particularly TSPO knockout models,which suggest that TSPO removal does not affect the phenotype or the system’s viability(Tu et al.,2014).However,ligands targeting TSPO have been shown to enhance levels of neurosteroids which suggests that neurosteroidogenesis is one of the major functional roles mediated by the TSPO protein.

关键词： cholesterol organs

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Friction between a temperature dependent viscoelastic body and a rough surface

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Friction 2016年第1期4卷 50-64页

作者： rainer HEISE Institute for Mechanics Technische Universit(a)t Berlin Straβe des 17.Juni 135 Berlin D-10623 Germany

In this study, we investigate the friction between a one-dimensional elastomer and a one-dimensional rigid randomly rough surface. Special emphasis is laid on the temperature dependence of the elastomer and its effect on the frictional behavior of the contact. The elastomer is modeled as a Kelvin body in a one-dimensional substitute model in the spirit of the method of dimensionality reduction. The randomly rough surface is a self-affine one-dimensional fractal. We provide a short discussion of a conical indenter pressed in a displacement controlled process into an elastomer. These analytical considerations are taken as a basis for the treatment of the randomly rough counter surface in contact to an elastomer with and without temperature dependent viscosity. We identify dimensionless quantities describing this process, introduce a thermal length scale, and give estimates for the coefficient of friction as function of velocity, indentation and thermal quantities.

关键词： elastomer friction temperature dependence coefficient of friction

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Stochastic Differential Games with Reflection and Related Obstacle Problems for Isaacs Equations

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Acta Mathematicae Applicatae Sinica 2011年第4期27卷 647-678页

作者： rainer BUCKDAHN Département de Mathématiques Université de Bretagne Occidentale6avenue Victor-le-GorgeuB.P.80929285 Brest cedexFrance Institute for Advanced Study Shangdong UniversityJinan 250100China

In this paper we first investigate zero-sum two-player stochastic differential games with reflection, with the help of theory of Reflected Backward Stochastic Differential Equations （RBSDEs）. We will establish the dynamic programming principle for the upper and the lower value functions of this kind of stochastic differential games with reflection in a straightforward way. Then the upper and the lower value functions are proved to be the unique viscosity solutions to the associated upper and the lower Hamilton-Jacobi-Bettman-Isaacs equations with obstacles, respectively. The method differs significantly from those used for control problems with reflection, with new techniques developed of interest on its own. Further, we also prove a new estimate for RBSDEs being sharper than that in the paper of E1 Karoui, Kapoudjian, Pardoux, Peng and Quenez （1997）, which turns out to be very useful because it allows us to estimate the LP-distance of the solutions of two different RBSDEs by the p-th power of the distance of the initial values of the driving forward equations. We also show that the unique viscosity solution to the approximating Isaacs equation constructed by the penalization method converges to the viscosity solution of the Isaacs equation with obstacle.

关键词： stochastic differential games value function reflected backward stochastic differential equations,dynamic programming principle Isaacs equations with obstacles viscosity solution

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Influence of slant of objective on image formation in optical microscopes

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Chinese Optics Letters 2008年第8期6卷 603-606页

作者： rainer Kning Physikalisch-Technische Bundesanstalt Braunschweig Bundesallee 10038116 BraunschweigGermany

A microscope image formation model based on scalar diffraction and Fourier optics has been developed, which takes a slant angle between the optical axis and the observed surface into account. The theoretical investigations of the imaging of line structures using this model show that reflection type microscopes are much stronger influenced by the slant angle than transmission type microscopes. In addition, the slant angle changes the image contrast and the image shape of a line structure, especially its edge. The larger the slant angle, the stronger the decrease of the image contrast, and the less steep the edge slope in both types of microscopes. Furthermore, the larger the numerical aperture of the objective, the less the effect of the slant angle on the line image shape.

关键词： line this

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