Role of manganese and veratryl alcohol in the ligninolytic system of Bjerkandera sp. strain BOS55

IntroductionLignin is a three dimensional hydrophobic plant polymer derived from the random coupling of phenylpropanoid precursors. The chemical and physical characteristics of lignin require a nonspecific, extracellular oxidative process for biodegradation. White rot basidiomycetes are the only group of organisms having an efficient extracellular ligninolytic system. These fungi produce peroxidases and laccases that are involved in the initial attack of lignin. The peroxidases work with H 2 O 2 which is also enzymatically produced by the fungi by different H 2 O 2 -producing oxidases. For their proper operation, peroxidases require appropriate cofactors which are the best substrates of the enzymes. Lignin peroxidase (LiP) uses the de novo produced secondary metabolite, veratryl alcohol (3,4-dimethoxybenzyl alcohol) as cofactor; whereas, the cofactor of manganese peroxidase (MnP) is the metal Mn(II) which occurs naturally in wood. The in vitro oxidation of lignin preparations by LiP or MnP is only feasible in the presence of veratryl alcohol or manganese, respectively. Additionally, simple aliphatic organic acid metabolites such as oxalate are involved in lignin degradation. On the one hand, oxalate is a good chelator of Mn(III) generated by MnP. This complex is relatively stable forming a low molecular weight diffusible oxidant capable of oxidizing phenolic lignin. Oxalate is also oxidized by ligninolytic enzymes in the presence of their cofactors generating reactive oxygen species that possibly participate in the oxidation of lignin.The low molecular weight cofactors also influence the physiological regulation of white rot fungi. Veratryl alcohol is known to increase LiP activities in white rot fungal cultures, although, veratryl alcohol itself does not induce lip gene transcription. Manganese is essential for the induction of mnp gene expression and MnP activity in various white rot fungi. In contrast, manganese lowers LiP production so that the highest LiP activities are measured under manganese deficiency. Nonetheless manganese has no direct repressive effect on lip gene transcription.Despite the great research efforts conducted so far on various white rot fungi, the true mechanism of lignin degradation is still not fully understood. However, it is becoming clear that low molecular weight cofactors are important catalytic and physiological regulating components of the ligninolytic system.This thesis was dedicated to study the interrelationship between low molecular weight cofactors and ligninolytic enzymes. Bjerkandera sp. strain BOS55 was used as the fungus of study, since it was found to be an outstanding producer of ligninolytic enzymes and a great variety of secondary aryl metabolites. Moreover, this strain was observed to be a good degrader of polycyclic aromatic hydrocarbons (PAH) and biobleacher of kraft pulp in various screenings. The main objective of the thesis was to gain insight into the role of veratryl alcohol and manganese in regulating the physiology and participating in the ligninolytic system of Bjerkandera sp. strain BOS55.Chapter 2In Chapter 2, it was demonstrated for the first time that manganese inhibits the biosynthesis of veratryl alcohol in white rot fungi. This explains at least in part the general observation that LiP production is lowered in the presence of manganese although manganese itself does not repress the transcription of lip genes. The ten-fold increase in veratryl alcohol biosynthesis caused by manganese deficiency stimulated the LiP titres by protecting the enzyme from inactivation by physiological levels of H 2 O 2 . Adding veratryl alcohol to manganese containing cultures of the fungus sustained high LiP titres similar to that found under manganese deficient conditions. Moreover, a good correlation was observed between the LiP titres and veratryl alcohol concentrations irrespective of whether veratryl alcohol was produced by the fungus or added exogenously.Chapter 3In Chapter 3, the mechanism resulting in manganese inhibition of veratryl alcohol biosynthesis was studied. Potential biosynthetic precursors of veratryl alcohol were added to manganese deficient and sufficient cultures of Bjerkandera sp. strain BOS55 in order to bypass the inhibited step. The addition of fully methylated precursors (veratrate, veratraldehyde) equally increased the production of veratryl alcohol irrespective of the manganese concentration. This observation indicated that the reduction of the benzylic acid to benzyl alcohol group is not inhibited by manganese. All the other known precursors such as phenylalanine, cinnamate, benzoate/benzaldehyde as well as the partially hydroxylated benzylic compounds (e.g. 3-hydroxybenzoate, 4-hydroxybenzoate, protocatechuate, vanillate, isovanillate) did increase the veratryl alcohol production in the presence of manganese but never as much as that under manganese deficiency. From these observations it was concluded that no single step along the biosynthetic pathway was inhibited by the presence of manganese. Instead, the availability of phenolic precursors is limited when manganese was added. From this study, we also learned that there are several alternative precursors resulting in increased veratryl alcohol production which can potentially originate from lignin degradation. In addition, it was demonstrated that many of the veratryl alcohol precursors (phenylalanine, cinnamate, benzoate, 4-hydroxybenzoate) enhanced the production of anisyl and chloroanisyl metabolites indicating the existence of common precursors in these biosynthetic pathways. Deuterium labelled benzoate and 4-hydroxybenzoate were converted to a broad spectrum of labelled aryl metabolites.Chapter 4In Chapter 4, the interrelationship between cofactors and peroxidases in cultures grown on natural substrates (beech wood and hemp stem wood sawdust) was studied. Beech wood and hemp stem wood substrates, which contain 6 and 25 mg kg -1dry wood of soluble manganese, respectively were favourable for MnP production. Many studies in the past have failed to demonstrate the presence of LiP on natural substrates even in fungi having lip genes. Surprisingly, Bjerkandera sp. strain BOS55 produced very high LiP titres on the wood substrates. The high LiP activity observed suggested that this enzyme may have an important role during wood decay. The significant LiP production in spite of the presence of manganese can be explained by the very high production of veratryl alcohol throughout the incubation. The fact that veratryl alcohol was produced in high amounts although soluble manganese was always present apparently contradicts the findings presented in Chapter 2 . However, the high veratryl alcohol production can be explained by the presence of lignin degradation products such as 4-hydroxybenzoate, protocatechuate, vanillate, isovanillate entering the biosynthetic pathway of veratryl alcohol as was suggested in Chapter 3.Chapter 5Bjerkandera sp. strain BOS55 is a good MnP producer. In Chapter 5, the conditions for optimal MnP production were examined. The highest production was observed in nitrogen rich medium with 0.2 to 1 mM manganese at a pH value of 5.2 and at a temperature of 30 oC. Two interesting phenomena were discovered while studying the physiology of MnP production. Firstly, significant MnP production was also observed in the absence of manganese which previously only has been shown to be the case for Pleurotus spp. However, unlike Pleurotus spp., MnP production in cultures of Bjerkandera sp. strain BOS55 was enhanced in response to increasing Mn levels. Secondly, it was demonstrated that the addition of various organic acid metabolites significantly increased the MnP titres under manganese sufficient conditions. The best results were obtained with glycolate.Chapter 6In Chapter 6, the study on the MnP production was continued in order to better understand why MnP is produced in the absence of manganese and to elucidate the induction mechanism under manganese deficient conditions. In the absence of manganese, oxalate and related organic acids, such as glycolate or glyoxylate were found to induce MnP production. The stimulatory effect of organic acids on MnP production was demonstrated to be due to the increased production of MnP proteins. Additionally, it was shown that the acids induced mnp gene transcription (unpublished data).The major MnP isozyme produced in the absence of Mn and in the presence of glycolate was purified and characterized. Like other MnP isozymes, this enzyme was able to efficiently oxidize Mn. However, unlike other MnP isozymes, it was also able to directly oxidize veratryl alcohol and 1,4-dimethoxybenzene with a very high affinity in the absence of manganese. These nonphenolic substrates are typical substrates of LiP. Methoxyphenols and aromatic amines could also be oxidized in the absence of manganese. The optimal pH for the manganese independent oxidation of all the substrates tested was 3.0 similar to that observed for LiP isozymes. On the other hand, the oxidation of Mn(II) and consequently the manganese dependent oxidation of phenolic substrate reached the highest rate at pH 4.5 as described for many MnP isozymes. The kinetic values in terms of turnover number and affinity for Mn(II) and veratryl alcohol oxidation were similar to those found for other MnP and LiP isozymes. Therefore, the Bjerkandera MnP could be best described as a hybrid enzyme between MnP and LiP, having a binding site for Mn(II) as well as for methoxy aromatics/phenols. This conclusion is supported by the finding that Mn(II) at concentrations greater than 0.1 mM severely inhibited veratryl alcohol oxidation by the enzyme; whereas Mn(II) has no effect on LiP. The fact that this enzyme can oxidize Mn(II) as well as directly oxidize veratryl alcohol and other aromatic amines and phenols clarifies the physiological relevance of the occurrence of this MnP under Mn deficient and sufficient conditions.ConclusionsIn conclusion, this thesis has resulted in new insights into the key regulatory role of manganese in lignin degradation such as the repressive effect of manganese on the production of aryl metabolites. The addition of manganese at concentrations as low as 33μM completely changed the pattern of peroxidases and aryl metabolites production compared to that under manganese deficiency. Additionally, it was observed that MnP is purposefully produced and regulated by organic acids in the total absence of manganese. Under these conditions, a novel type of MnP-LiP hybrid isozyme was isolated which was functional under any manganese regime using either veratryl alcohol or Mn(II) as a cofactor. Under natural conditions, such an enzyme would have considerable physiological significance since soluble manganese is known to be leached out or become oxidized to insoluble MnO 2 during fungal attack, possibly resulting in manganese deficient areas in wood. In manganese deficient areas, veratryl alcohol biosynthesis is stimulated and the enzyme can use this secondary metabolite as an alternative to manganese.Future research should elucidate the newly discovered role of organic acids in regulating MnP in white rot fungi. The physiological significance of this regulation may be due to the role of organic acids as an important source of reduced oxygen radicals. The oxidative stress resulting from radicals may be the signal for MnP gene expression as has been shown to be the case with H 2 O 2 . The radicals are also required for extensive degradation of lignin. Additionally, the presence of organic acids may serve as an early warning for the upcoming presence of bioavailable manganese. The fungal organic acid metabolites are well known for their ability to solubilize insoluble MnO 2 deposits in fungal attacked wood.</p

Electrostatic Patch Effect in Cylindrical Geometry. III. Torques

We continue to study the effect of uneven voltage distribution on two close cylindrical conductors with parallel axes started in our papers [1] and [2], now to find the electrostatic torques. We calculate the electrostatic potential and energy to lowest order in the gap to cylinder radius ratio for an arbitrary relative rotation of the cylinders about their symmetry axis. By energy conservation, the axial torque, independent of the uniform voltage difference, is found as a derivative of the energy in the rotation angle. We also derive both the axial and slanting torques by the surface integration method: the torque vector is the integral over the cylinder surface of the cross product of the electrostatic force on a surface element and its position vector. The slanting torque consists of two parts: one coming from the interaction between the patch and the uniform voltages, and the other due to the patch interaction. General properties of the torques are described. A convenient model of a localized patch suggested in [2] is used to calculate the torques explicitly in terms of elementary functions. Based on this, we analyze in detail patch interaction for one pair of patches, namely, the torque dependence on the patch parameters (width and strength) and their mutual positions. The effect of the axial torque is then studied for the experimental conditions of the STEP mission.Comment: 28 pages, 6 Figures. Submitted to Classical Quantum Gravit

Electrostatic Patch Effect in Cylindrical Geometry. I. Potential and Energy between Slightly Non-Coaxial Cylinders

We study the effect of any uneven voltage distribution on two close cylindrical conductors with parallel axes that are slightly shifted in the radial and by any length in the axial direction. The investigation is especially motivated by certain precision measurements, such as the Satellite Test of the Equivalence Principle (STEP). By energy conservation, the force can be found as the energy gradient in the vector of the shift, which requires determining potential distribution and energy in the gap. The boundary value problem for the potential is solved, and energy is thus found to the second order in the small transverse shift, and to lowest order in the gap to cylinder radius ratio. The energy consists of three parts: the usual capacitor part due to the uniform potential difference, the one coming from the interaction between the voltage patches and the uniform voltage difference, and the energy of patch interaction, entirely independent of the uniform voltage. Patch effect forces and torques in the cylindrical configuration are derived and analyzed in the next two parts of this work.Comment: 26 pages, 1 Figure. Submitted to Classical and Quantum Gravit

Az Istállós-kői-barlang ásatásának folytatása és a Bükki Aurignaci kérdése = Continuation of Excavating Istállós-kő Cave and Problem of Bükk Aurignacian

A T046892 OTKA pályázat kutatási programja keretében 2004-2005-ben sikerült tovább folytatni az Istállós-kői-barlang, 2005-2006-ban pedig a Herman Otto-barlang revíziós ásatását elvégezni. A program keretében lehetőség nyílt a teljes Bükki-Aurignacien kultúra techno-tipológiai és komplex rétegtani-őskörnyezettani, kronológiai és paleoetnológiai feldolgozására. Szerencsés módon William Davies (University of Southampton) és Marcel Otte (Universite de Liege) C14 programjával együttműködve, több új radiometrikus kormeghatározást elvégezni a Bükki-Aurignacien kultúrával kapcsolatban. Így sor került a különleges jelentőségű Miskolc-Tapolcai-kőfülke Aurignacien koponyaleletének C14 kormeghatározására is. Az eredményeket több konferencián és publikációban mutattuk be. | The number T46892 research program funded by the OTKA, included further excavations and revisions of two main sites: the Istallós-kő (2004-2005) and the Otto Herman Cave (2005-2006). The detailed research program on the Aurignacien Culture in the Bükk Mountains, involved processing all the available data; such as the techno-tipological and the complex stratigraphical-paleoenvironmental, the chronological and the paleoethnological. Working closely with other research programs led by William Davies (University of Southampton) and Marcel Otte (Universite de Liege) we were able to perform many new C14 radiometric dating. Among others, the extremely significant one was done on the occipital bone found in Miskolc-Tapolca's Rockshelter. We have presented the results and conclusions in many conferences and in publications

High resolution computed tomography detection of alimentary factors related to arthropathies in inflammatory bowel diseases

Alimentary factors of enterogen arthropathies were studied, in particular the early morphological detection, to prevent the disease progression by nutritional prescriptions. Authors compared the method of high resolution computed tomography (HRCT) with conventional X-ray and nuclear medicine investigations. The results showed the superiority of HRCT to detect cartilage erosions and ligamental calcifications in the sacroiliac joint and lumbar facet joint. The importance of nutritional factors is briefly discussed. The need for high quality and high quantity food of patients suffering from inflammatory bowel diseases (IBD) is well known. Elemental diet is effective for remission of IBD. Folic acid intake is extremely important. Total parenteral nutrition in acute active disease (Crohn's fistulas) has not been proved to effect IBD. Elimination of whole protein as a possible luminal factor for long term application can help, if elementary amino acids are in the diet. Eicosapentaenoic acid and docosahexaenoic acid (major components of fish oils) have beneficial effects. Polyunsaturated fatty acids in the diet can decrease the inflammation. Antioxidants, glutamine are essential in the diet. Removal of fat is effective to get remission. Alimentary complication in IBD patients can be osteoporosis, so there is a need for regular bone densitometry. Conclusion of the study suggests that HRCT offers more sensitive detection of enterogen arthropathy related changes. The predictive value of this diagnostic method is accurate enough to advise restrictive and/or supplemental diets of IBD patients.  Dietary therapy allows circumvention of the adverse side-effects of repeated courses of steroids

The Laser Astrometric Test of Relativity Mission

This paper discusses new fundamental physics experiment to test relativistic gravity at the accuracy better than the effects of the 2nd order in the gravitational field strength. The Laser Astrometric Test Of Relativity (LATOR) mission uses laser interferometry between two micro-spacecraft whose lines of sight pass close by the Sun to accurately measure deflection of light in the solar gravity. The key element of the experimental design is a redundant geometry optical truss provided by a long-baseline (100 m) multi-channel stellar optical interferometer placed on the International Space Station. The geometric redundancy enables LATOR to measure the departure from Euclidean geometry caused by the solar gravity field to a very high accuracy. LATOR will not only improve the value of the parameterized post-Newtonian (PPN) parameter gamma to unprecedented levels of accuracy of 1 part in 1e8, it will also reach ability to measure effects of the next post-Newtonian order (1/c^4) of light deflection resulting from gravity's intrinsic non-linearity. The solar quadrupole moment parameter, J2, will be measured with high precision, as well as a variety of other relativistic. LATOR will lead to very robust advances in the tests of fundamental physics: this mission could discover a violation or extension of general relativity, or reveal the presence of an additional long range interaction in the physical law. There are no analogs to the LATOR experiment; it is unique and is a natural culmination of solar system gravity experiments.Comment: 8 pages, 2 figures, invited talk given at the Second International Conference on Particle and Fundamental Physics in Space (SpacePart'03), 10-12 December 2003, Washington, D