Properties of epoxide hydrolase from the yeast Rhodotorula glutinis

Epoxide hydrolases are ubiquitous enzymes that can be found in nearly all living organisms. Some of the enzymes play an important role in detoxifying xenobiotic and metabolic compounds. Others are important in the growth of organisms like the juvenile hormone in some insects. The role of these enzymes in some organisms is still not fully understood.Epoxides are highly reactive valuable intermediates used by the pharmaceutical industry. Enantiopure epoxides are of high value in the production of pharmaceuticals like pain-killers or protease-inhibitors. There are a number of ways to produce enantiopure epoxides, but nowadays an environmentally friendly manner has a high preference. One such environmentally friendly method is the use of the epoxide hydrolases. These enzymes are able to enantioselectively hydrolyze one epoxide-enantiomer to its vicinal diol. By this so-called kinetic resolution, it is possible to obtain both the epoxide and diol enantiopure. Enantiopure diols are also of high value in the fine and pharmaceutical chemistry.The first goal of this project is achieved: the epoxide hydrolase from Rhodotorula glutinis has been isolated and purified. The second goal (optimization of the reaction conditions) has been performed but it is still favorable to further optimize them. Initial experiments of enzyme stability towards temperature and pH has been performed with crude enzyme extracts, not with the purified enzyme. With respect to the third goal (a suitable method for the isolation and separation of epoxide and diol), the use of a recently described membrane reactor is recommended. The performance of this reactor, however, has not been verified for the EH studied.The enzyme is partially characterized. The EH was found to be a membrane associated enzyme. Whether or not it is actually a membrane bound enzyme (and how many times it passes the membrane) is still unknown. The enzyme consists of two (most probably) identical subunits with a molecular mass of 45 kDa. Amino acid analysis revealed that the enzyme belongs to thea/b-hydrolase fold family, because the characteristic catalytic histidine motive (G H F) can be found in the amino acid sequence. The N-terminal sequence, however, could not be detected. The amount of purified enzyme was too low to establish its the three-dimensional structure.With partially purified enzyme sample, the specific activity and enantioselectivity could be enhanced when detergents were added. Non-ionic detergents had the largest positive effects, e.g. the specific activity for 1,2-epoxyhexane and styrene oxide was enhanced three and eight times, respectively. In the same way, the enantioselectivity for 1,2-epoxyhexane and styrene oxide could be enhanced over 10 and nearly 5 times, respectively. In addition, non-ionic detergents had an enzyme stabilizing effect. Anionic detergents had a very clear negative effect: enzyme activities were reduced to 20%.Another method investigated to influence the stability, the activity and the enantioselectivity consists of polymerizing the epoxide hydrolase in a network. The enantioselective conversion of (±)-1,2-epoxyoctane was reversed from a preference for ( R )-1,2-epoxyoctane to ( S )-1,2-epoxyoctane when the enzyme had been imprinted with ( S )-1,2-epoxyoctane prior to co-polymerization. This is the first time that the above mentioned method was successfully performed with a membrane-associated enzyme of thea/b-hydrolase fold family to which EH belongs. The half-life of the immobilized and imprinted biocatalyst was enhanced at least 7-fold. Most remarkable was that washing the immobilized EH with HCl, followed by washing it with buffer, resulted in about 50% of the residual activity, while native EH completely lost its activityThe effect of increasing epoxide amounts (up to 10 mmol per 10 mL of water, leading to phase separations) on both the activity and enantioselectivity has been studied, including the effect of detergents on such two-phase enzymatic conversions. It appeared that cell-free extracts without detergents gave the highest activity at 10 mmol epoxide per 10 mL of water added, without loss of enantioselectivity as compared to 1 mmol epoxide per 10 mL of water emulsions.It is recommended either to use whole cells (overexpressing EH or not) in a bioreactor to produce enantiopure epoxides and diols in large quantities or to use an immobilized-imprinted enzyme polymer for the conversion of smaller quantities of epoxides in an enantioselectivity of your choice. Further investigations to improve both methods (whole cells, the enzyme properties and the immobilization and imprinting procedures) are required to optimize this type of conversions for practical applications

Understanding Human Carboxylesterase 2

Dissertation presented to obtain the Ph.D degree in Engineering and Technology Sciences, BiotechnologyThe first barrier oral drugs and prodrugs encounter prior to reaching an organism’s systemic circulation is the gastrointestinal (GI) tract, specifically the intestine, which is the primary section for absorption. Therefore, it is fundamental to understand the permeability of the therapeutic agent as well as its potential metabolism by human enterocytes, since biotransformation may result in the inactivation of the therapeutic agent or, to the contrary, in the formation of more therapeutically active metabolites. Carboxylesterases (CESs), phase I metabolising enzymes, are important in the metabolism of several drugs and prodrugs with amide, ester, or thioester functional groups. After cytochrome (CYP) P450s and UDP-glucuronosyltransferases (UGTs), CESs are the most relevant enzymes for the metabolism of therapeutic agents.(...

Biodegradation of mycotoxins : tales from known and unexplored worlds

Exposure to mycotoxins, secondary metabolites produced by fungi, may infer serious risks for animal and human health and lead to economic losses. Several approaches to reduce these mycotoxins have been investigated such as chemical removal, physical binding, or microbial degradation. This review focuses on the microbial degradation or transformation of mycotoxins, with specific attention to the actual detoxification mechanisms of the mother compound. Furthermore, based on the similarities in chemical structure between groups of mycotoxins and environmentally recalcitrant compounds, known biodegradation pathways and degrading organisms which hold promise for the degradation of mycotoxins are presented

Effects of aflatoxin B₁ (AFB₁) on hepatic gene expression in pigs and turkeys

The objective of the present study was to evaluate the efficacy of curcumin (CMN), an antioxidant supplied by turmeric (Curcuma longa) powder to ameliorate the adverse effects of aflatoxin B1 (AFB1) on performance of pigs and, to identify changes in gene expression in liver of pigs fed aflatoxin (AF). Twenty crossbred weanling pigs were weighed, ear-tagged, and assigned to each of four dietary treatments, which included: 1) basal diet (BD) containing no AFB1 or CMN; 2) BD + 1.0 mg AFB1/kg of diet; 3) BD + 100 mg curcumin (CMN)/kg of diet, and; 4) BD + 100 mg CMN/kg of diet + 1.0 mg AFB1/kg of diet. Aflatoxin reduced (P < 0.05) body weight gain (BWG), feed intake (FI) and feed efficiency of pigs. The addition of CMN to the diet contaminated with AFB1 improved feed efficiency (P < 0.05) but not BWG and FI. At the end of three week treatment period, livers were collected and microarray analysis was conducted to identify pathways that control growth, development, coagulation, immune function, metabolism, detoxification, and antioxidant status in liver of pigs. Genes with an adjusted permutation Fs test (false discovery rates) values less that 5% and fold change greater than 2.0 were considered differentially expressed across samples. Changes in expression were determined using microarray technique and results were validated using quantitative real time PCR (RTqPCR). Six genes were chosen for validation of expression using RT-qPCR, including TNF receptor superfamily, member 6 (FAS), glutathione S-transferase theta 1 (GSTT1), cyclin G1 (CCGN1), proteasome activator subunit 1 (PSME1), proteasome activator subunit 3 (PSME3), and cytochrome P450-2A19 (CYP2A19). There were no differences in the expression of the genes among the treatments except for GSTT1 and CYP2A19 that shifted the expression (down to up, and up to down regulation, respectively) with the addition of CMN in the diet contaminated with AFB1. Results demonstrate that pigs fed 1.0 mg AFB1/kg feed for 21 days had reduced growth performance associated with altIncludes bibliographical references (pages 138-152)

Influence of dietary factors on tansy ragwort toxicosis in rats and horses

Several experiments were conducted to investigate the effect of dietary factors such as amino acids, B vitamins, butylated hydroxyanisole (BHA) and phenobarbital on pyrrolizidine alkaloid toxicosis in rats and horses. The influence of St. John's wort, bracken, comfrey, and alfalfa on tansy ragwort toxicity was also examined. Effects of dietary tansy ragwort (Senecio jacobaea), comfrey (Symphytum officinale), bracken (Pteridium acquilinum) and alfalfa (Medicago sativa) on hepatic drug-metabolizing enzymes in rats were measured. Tansy ragwort and bracken increased (P<0.05) the activity of glutathione transferase and epoxide hydrolase. Comfrey and alfalfa increased (P<0.05) the activity of aminopyrine Ndemethylase. Feeding bracken or St. John's wort (Hypericum perforatum) in conjunction with tansy ragwort did not influence chronic toxicity of tansy ragwort as assessed by rat survival time. Dietary tansy ragwort resulted in increased (P<0.05) hepatic copper levels; the other plants did not affect copper levels. The results do not suggest any major interaction in the toxicity of tansy ragwort with bracken or St. John's wort. Dietary supplementation of rats with branched chain amino acids (BCAA: leucine, isoleucine, valine) did not alter their susceptibility to chronic poisoning by tansy ragwort (Senecio jacobaea), which contains hepatotoxic pyrrolizidine alkaloids (PA). Phenobarbital in the diet, which alters liver microsomal enzyme activity, also did not alter susceptibility to PA poisoning. A combination of BHA, cysteine and BCAA did increase (P<.05) survival time of rats fed tansy ragwort. Dietary BHA and cysteine increased the survival times of rats injected with the PA monocrotaline, with evidence that addition of vitamin B₁₂ and folic acid improved the effectiveness of this treatment. In a chronic feeding trial with tansy ragwort, a combination of BHA and cysteine increased (P<.05) the survival times of rats, showing protective activity against PA poisoning. A mixture of B-complex vitamins, or vitamin B₁₂-folic acid, was not effective in improving the response. Dried tansy ragwort, which contains pyrrolizidine alkaloids, was fed as 10% of a complete diet to horses, with and without a mixture of additives. The additives provided a dietary supplement equivalent to 1% cysteine, 0.75% BHA, 200 ppb vitamin B₁₂ and 5 ppm folic acid. The additives did not alter tansy ragwort toxicity, as assessed by survival time, liver histology, bromosulphalein clearance rate and serum gamma glutamyl transpeptidase activity. In horses fed tansy ragwort, bromosulphalein clearance rate was a sensitive indicator of liver damage, and declined to a level of about 20% of control values. Gamma glutamyl transpeptidase levels showed considerable variability but in general were elevated in animals fed tansy ragwort. Liver iron and copper levels were elevated, and liver zinc declined in tansy ragwort-fed horses

Investigating Extra Hepatic Steroid And Eicosanoid Metabolizing Enzymes In Cattle

Steroid and eicosanoid metabolism occurs in two phases and primarily within hepatic tissues, but localized metabolism has been examined in several extra-hepatic tissues in humans and rodents. Phase I of metabolism is performed by Cytochrome P450s (CYP) that add hydroxyl groups to the carbon ring structure which is further metabolized by phase II UDP-glucuronosyltransferase (UGT). The overall objectives of the following experiments were to: 1) determine the amount of extra-hepatic steroid metabolism within reproductive tissues of cattle across the estrous cycle; 2) determine the amount of extra-hepatic steroid metabolism and an oxylipin profile within reproductive tissues of cattle based on pregnancy status; and 3) determine the amount of endometrial blood perfusion in cattle using a novel laser Doppler technique. Activity of CYP1A was found within corpora lutea (CL) tissues of both pregnant and non-pregnant cattle, but not within endometrial tissues. Endometrial perfusion, measured using a novel laser Doppler technique, was also validated by measuring angiogenic factors in close proximity to the location of perfusion. A positive correlation (r = 0.28; P = 0.04) was observed between endometrial perfusion and nitrite concentration, an angiogenic factor. Endometrial blood perfusion was affected by the proximity to the CL, but not by the proximity of the dominant follicle. In addition, UGT was categorized across the estrous cycle and the activity was dependent upon the proximity of the CL. Oxylipins, including eicosanoids, were also profiled in CL of cattle that were non-pregnant and pregnant with 5 out of 39 oxylipins differentially expressed. The activity and oxylipin products of steroid and eicosanoid enzymes were not correlated with serum or luteal progesterone. Through these experiments, we have verified that there is localized metabolism of steroids and eicosanoids within reproductive tissues of cattle as well as fetal tissues. Also, we have achieved a full oxylipin profile of non-pregnant and pregnant cattle CL with five oxylipins contained in various amounts between pregnancy status

Genetic characterization of Rhodococcus rhodochrous ATCC BAA-870 with emphasis on nitrile hydrolysing enzymes

Includes abstract.Includes bibliographical references.Rhodococcus rhodochrous ATCC BAA-870 (BAA-870) had previously been isolated on selective media for enrichment of nitrile hydrolysing bacteria. The organism was found to have a wide substrate range, with activity against aliphatics, aromatics, and aryl aliphatics, and enantioselectivity towards beta substituted nitriles and beta amino nitriles, compounds that have potential applications in the pharmaceutical industry. This makes R. rhodochrous ATCC BAA-870 potentially a versatile biocatalyst for the synthesis of a broad range of compounds with amide and carboxylic acid groups that can be derived from structurally related nitrile precursors. The selectivity of biocatalysts allows for high product yields and better atom economy than nonselective chemical methods of performing this reaction, such as acid or base hydrolysis. In order to apply BAA-870 as a nitrile biocatalyst and to mine the organism for biotechnological uses, the genome was sequenced using Solexa technology and an Illumina Genome Analyzer. The Solexa sequencing output data was analysed using the Solexa Data Analysis Pipeline and a total of 5,643,967 reads, 36-bp in length, were obtained providing 4,273,289 unique sequences. The genome sequence data was assembled using the software Edena, Velvet, and Staden. The best assembly data set was then annotated automatically using dCAS and BASys. Further matepaired sequencing, contracted to the company BaseClear® BV in Leiden, the Netherlands, was performed in order to improve the completeness of the data. The scaffolded Illumina and mate-paired sequences were further assembled and annotated using BASys. BAA-870 has a GC content of 65% and contains 6997 predicted protein-coding sequences (CDS). Of this, 54% encodes previously identified proteins of unknown function. The completed 5.83 Mb genome (with a sequencing coverage of 135 X) was submitted to the NCBI Genome data bank with accession number PRJNA78009. The genome sequence of R. rhodochrous ATCC BAA-870 is the seventh rhodococcal genome to be submitted to the NCBI and the first R. rhodochrous subtype to be sequenced. An analysis of the genome for nitril