Kwaliteit en kwantiteit

Rede Wageningen, 18 December 195

Biodegradation of chlorinated unsaturated hydrocarbons in relation to biological waste-gas treatment

The original goal of the research described in this thesis was to develop a biological process for the removal of vinyl chloride from waste gases. The gaseous and carcinogenic vinyl chloride is used to produce the plastic polyvinyl chloride (PVC). During this production process waste gases containing vinyl chloride are generated. As a microorganism capable of growth on vinyl chloride as the sole carbon and energy source had been isolated it was envisaged that it might be possible to remove vinyl chloride from waste gases with a biological process.Besides the original strain, Mycobacterium L1, three additional vinyl chlorideutilizing strains were isolated subsequently. All strains were tentatively identified as Mycobacterium aurum.The first step in vinyl chloride metabolism in strain L1, was shown to be the oxidation of vinyl chloride to the corresponding epoxide, chlorooxirane, by alkene monooxygenase. Chlorooxirane is also the product of vinyl chloride oxidation in the human liver and is responsible for the carcinogenic properties of vinyl chloride. Alkene monooxygenase is also present in Mycobacterium E3 after growth on ethene. Extracts from strain E3 could be fractionated yielding two fractions which upon combination exhibited alkene monooxygenase activity, indicating that the enzyme consists of at least two components. One fraction was inhibited by acetylene, indicating it contained the oxygenase component of alkene monooxygenase, whereas the other fraction contained significant reductase activity. The corresponding fractions could also be obtained from extracts of vinyl chloride- grown cells of strain L1, Alkene monooxygenase appeared to be similar to the soluble three-component methane monooxygenases. These enzymes also oxidize alkenes to the corresponding epoxides.The capacity of alkene monooxygenase to oxidize vinyl chloride to the mutagenic and toxic chlorooxirane was exploited to generate and select monooxygenase mutants of the ethene-utilizing strain E3. As long as cells exhibit monooxygenase activity they produce chlorooxirane from vinyl chloride and are hampered in their growth. However, when monooxygenase activity is lost, due to a mutation, these cells are no longer inhibited by the presence of vinyl chloride. Using this technique a mutant of strain E3 no longer capable of growth on ethene was isolated. Subsequently, it was shown that this mutant lacks the reductase component of alkene monooxygenase. Growth of the mutant on epoxyethane (oxirane) resulted in synthesis of the other alkene monooxygenase component(s). Extracts of such cells could be used to detect and subsequently purify the reductase component of alkene monooxygenase.During growth of strain L1, on vinyl chloride in chemostat cultures it became evident that the original mineral salts medium was not optimal. The addition of extra iron to the medium resulted in an enhanced vinyl chloride consumption. Chemostat cultures were also used to determine to what levels vinyl chloride could be removed. This type of experiment was also done with the Xanthobacter autotrophicus GJ10 isolated by D.B. Janssen (University of Groningen) to determine 1,2-dichloroethane removal from air. 1,2-Dichloroethane is the precursor in the major production process of vinyl chloride. The concentrations of both compounds in the air that had passed through the cultures were significantly higher than the maximal allowable concentrations in waste gases according to the German legislation (TA-Luft). Therefore the affinity of both strains towards the respective substrates is too low to apply them in waste-gas treatment.The enzyme transforming chlorooxirane appeared to be very unstable. After a short interruption in the supply of vinyl chloride to a culture of strain L1, inactivation took place upon restoring the vinyl chloride supply to the culture. This is probably caused by chlorooxirane accumulation due to an insufficient activity of the chlorooxirane transforming enzyme after such an interruption in the supply of vinyl chloride. The chlorooxirane subsequently inactivates cell components including alkene monooxygenase. Based on the observed inactivation and the relatively low affinity for vinyl chloride it was concluded that development of a process to remove vinyl chloride based on the application of Mycobacterium aurum L1, was not feasible.As the formation of chlorooxirane is one of the major drawbacks of strain L1, microorganisms were isolated on compounds structurally related to vinyl chloride. Using this approach we hoped to isolate strains which add a water molecule to the double bond of vinyl chloride, resulting in formation 2-chloroethanol or acetaldehyde. 3-Chloroacrylic acid and styrene were used as vinyl chloride analogues in enrichment cultures. The enrichment cultures with 3-chloroacrylic acid did indeed result in the isolation of bacteria which hydrated the double bond of 3-chloroacrylic as the initial step in the degradation pathway of this compound. Unfortunately these enzymes did not exhibit any activity with vinyl chloride as substrate. With styrene as carbon source a number of microorganisms were isolated. Almost all of these isolates oxidized the unsaturated alifatic moiety of styrene yielding styrene oxide (phenyloxirane). The flavine adenine dinucleotide- dependent styrene monooxygenase has a high substrate specificity, only oxidizing phenyl substituted alkenes. In contrast to alkene monooxygenase the enzyme appears to require only one component for activity.Besides vinyl chloride, biodegradation of methyl chloride, the simplest chlorinated hydrocarbon was studied. Methyl chloride-grown cells of the isolated Hyphomicrobium strain MC1 could dechlorinate methyl chloride only under aerobic conditions. Simple hydrolytic dehalogenation was therefore not taking place. No methane monooxygenase activity could be detected in methyl chloride-grown cells.To be able to study a bioreactor for the removal of a chlorinated hydrocarbon from air, dichloromethane was selected as model contaminant. Dichloromethane is applied on a large scale as a solvent and consequently is present in numerous industrial waste gases. The strains isolated and characterized by the group of Th. Leisinger (ETH Zürich), as well as a new isolate, were shown to have a much better affinity towards dichloromethane than the value published for the purified dehalogenase. Due to this high affinity these microorganisms can be applied to remove dichloromethane to concentrations well below the maximal allowable levels in waste gases.A 0.066 m 3trickle-bed bioreactor was studied for the elimination of dichloromethane from waste gases. The reactor was filled with a polypropylene packing material on which a biofilm developed. The air containing dichloromethane was forced through the reactor counter-current to a circulating aqueous phase. The aqueous phase is used to neutralize and remove the hydrochloric acid formed during dichloromethane degradation. The biological system was very stable and not sensitive to fluctuations in the dichloromethane supply.Dichloromethane elimination with the trickle-bed bioreactor was determined at various gas and liquid flows and dichloromethane concentrations. It was possible to simulate the observed dichloromethane elimination efficiencies surprisingly well with a model incorporating Michaelis-Menten kinetics, diffusion in the biofilm and mass-transfer resistance in the gaseous as well as the liquid phase.On the basis of the experimental results it was concluded that removal of dichloromethane is technically feasible with the trickle-bed reactor

A novel gene encoding xanthan lyase of Paenibacillus alginolyticus strain XL-1

Xanthan-modifying enzymes are powerful tools in studying structure-function relationships of this polysaccharide. One of these modifying enzymes is xanthan lyase, which removes the terminal side chain residue of xanthan. In this paper, the cloning and sequencing of the first xanthan lyase-encoding gene is described, i.e., the xalA gene, encoding pyruvated mannose-specific xanthan lyase of Paenibacillus alginolyticus XL-1. The xalA gene encoded a 100,823-Da protein, including a 36-amino-acid signal sequence. The 96,887-Da mature enzyme could be expressed functionally in Escherichia coli. Like the native enzyme, the recombinant enzyme showed no activity on depyruvated xanthan. Compared to production by P. alginolyticus, a 30-fold increase in volumetric productivity of soluble xanthan lyase was achieved by heterologous production in E. coli. The recombinant xanthan lyase was used to produce modified xanthan, which showed a dramatic loss of the capacity to form gels with locust bean gum

Characterization of a Mixed Methanotrophic Culture Capable of Chloroethylene Degradation

A consortium of methanotrophs cultured from the St. Joseph's aquifer in Schoolcraft, MI, was found to exhibit similar methane consumption rates as pure cultures of methanotrophs. The methanotrophic consortium resides within a portion of the aquifer contaminated with a mixed waste plume of perchloroethylene (PCE) and its reductive dechlorination products from natural attenuation, trichloroethylene (TCE), cis-dichloroethylene (c-DCE), and vinyl chloride (VC). Oxidation kinetics for TCE, c-DCE, and VC were measured for the mixed methanotroph consortium and compared to reported rate parameters for degradation of these chloroethylene compounds by pure methanotrophic cultures. The results demonstrate that the kinetics of chloroethylene oxidation by the Schoolcraft methanotroph population mimic the degradation rates of pure methanotrophic cultures that primarily express particulate methane monooxygenase (pMMO). Molecular and biochemical analyses confirmed that sMMO was not being expressed by these cells. Rather, using competitive reverse transcriptionpolymerase chain reaction, pmoA, a gene encoding one of the polypeptides of the pMMO was found at a level of (1.57 ± 0.10) × 10–17 mol pmoA mRNA/g wet soil in soil slurries and (2.65 ± 0.43) × 10–17 mol pmoA mRNA/μl in groundwater. No expression of mmoX, a gene encoding one of the polypeptides of the sMMO, was detected.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63398/1/ees.2005.22.177.pd

C1 compounds as auxiliary substrate for engineered Pseudomonas putida S12

The solvent-tolerant bacterium Pseudomonas putida S12 was engineered to efficiently utilize the C1 compounds methanol and formaldehyde as auxiliary substrate. The hps and phi genes of Bacillus brevis, encoding two key steps of the ribulose monophosphate (RuMP) pathway, were introduced to construct a pathway for the metabolism of the toxic methanol oxidation intermediate formaldehyde. This approach resulted in a remarkably increased biomass yield on the primary substrate glucose when cultured in C-limited chemostats fed with a mixture of glucose and formaldehyde. With increasing relative formaldehyde feed concentrations, the biomass yield increased from 35% (C-mol biomass/C-mol glucose) without formaldehyde to 91% at 60% relative formaldehyde concentration. The RuMP-pathway expressing strain was also capable of growing to higher relative formaldehyde concentrations than the control strain. The presence of an endogenous methanol oxidizing enzyme activity in P. putida S12 allowed the replacement of formaldehyde with the less toxic methanol, resulting in an 84% (C-mol/C-mol) biomass yield. Thus, by introducing two enzymes of the RuMP pathway, co-utilization of the cheap and renewable substrate methanol was achieved, making an important contribution to the efficient use of P. putida S12 as a bioconversion platform host

Comparative transcriptomics and proteomics of p-hydroxybenzoate producing Pseudomonas putida S12: novel responses and implications for strain improvement

A transcriptomics and proteomics approach was employed to study the expression changes associated with p-hydroxybenzoate production by the engineered Pseudomonas putida strain S12palB1. To establish p-hydroxybenzoate production, phenylalanine-tyrosine ammonia lyase (pal/tal) was introduced to connect the tyrosine biosynthetic and p-coumarate degradation pathways. In agreement with the efficient p-hydroxybenzoate production, the tyrosine biosynthetic and p-coumarate catabolic pathways were upregulated. Also many transporters were differentially expressed, one of which—a previously uncharacterized multidrug efflux transporter with locus tags PP1271-PP1273—was found to be associated with p-hydroxybenzoate export. In addition to tyrosine biosynthesis, also tyrosine degradative pathways were upregulated. Eliminating the most prominent of these resulted in a 22% p-hydroxybenzoate yield improvement. Remarkably, the upregulation of genes contributing to p-hydroxybenzoate formation was much higher in glucose than in glycerol-cultured cells

Characterization of Cg10062 from Corynebacterium glutamicum: Implications for the Evolution of cis-3-Chloroacrylic Acid Dehalogenase Activity in the Tautomerase Superfamily†

A 149-amino acid protein designated Cg10062 is encoded by a gene from Corynebacterium glutamicum. The physiological function of Cg10062 is unknown, and the gene encoding this protein has no obvious genomic context. Sequence analysis links Cg10062 to the cis-3-chloroacrylic acid dehalogenase (cis-CaaD) family, one of the five known families of the tautomerase superfamily. The characterized tautomerase superfamily members have two distinctive characteristics: a P-cc-p structure motif and a catalytic amino-terminal proline. Pro-1 is present in the Cg10062 amino acid sequence along with His-28, Arg-70, Arg-73, Tyr-103, and Glu-114, all of which have been implicated as critical residues for cis-CaaD activity. The gene for Cg10062 has been cloned and the protein overproduced, purified, and subjected to kinetic and mechanistic characterization. Like cis-CaaD, Cg10062 functions as a hydratase: it converts 2-oxo-3-pentynoate to acetopyruvate and processes 3-bromopropiolate to a species that inactivates the enzyme by acylation of Pro-1. Kinetic and (1)H NMR spectroscopic studies also show that Cg10062 processes both isomers of 3-chloroacrylic acid at low levels with a clear preference for the cis isomer. Pro-1 is critical for the dehalogenase and hydratase activities because the PIA mutant no longer catalyzes either reaction. The presence of the six key catalytic residues and the hydratase activity coupled with the absence of an efficient cis-CaaD activity and the lack of isomer specificity implicate factors beyond this core set of residues in cis-CaaD catalysis and specificity. This work sets the stage for in-depth mechanistic and structural studies of Cg10062, which could identify the additional features necessary for a fully active and highly specific cis-CaaD. Such results will also shed light on how cis-CaaD emerged in the tautomerase superfamily because Cg10062 could be characteristic of an intermediate along the evolutionary pathway for this dehalogenase

Functional Genomic mRNA Profiling of Colorectal Adenomas:Identification and in vivo Validation of CD44 and Splice Variant CD44v6 as Molecular Imaging Targets

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide. High adenoma miss rates, especially seen in high-risk patients, demand for better endoscopic detection. By fluorescently \u27highlighting\u27 specific molecular characteristics, endoscopic molecular imaging has great potential to fulfill this need. To implement this technique effectively, target proteins that distinguish adenomas from normal tissue must be identified. In this study we applied in silico Functional Genomic mRNA (FGmRNA) profiling, which is a recently developed method that results in an enhanced view on the downstream effects of genomic alterations occurring in adenomas on gene expression levels. FGmRNA profiles of sporadic adenomas were compared to normal colon tissue to identify overexpressed genes. We validated the protein expression of the top identified genes, AXIN2, CEMIP, CD44 and JUN, in sporadic adenoma patient samples via immunohistochemistry (IHC). CD44 was identified as the most attractive target protein for imaging purposes and we proved its relevance in high-risk patients by demonstrating CD44 protein overexpression in Lynch lesions. Subsequently, we show that the epithelial splice variant CD44V6 is highly overexpressed in our patient samples and we demonstrated the feasibility of visualizing adenomas in ApcMin/+ mice in vivo by using a fluorescently labeled CD44v6 targeting peptide. In conclusion, via in silico functional genomics and ex vivo protein validation, this study identified CD44 as an attractive molecular target for both sporadic and high-risk Lynch adenomas, and demonstrates the in vivo applicability of a small peptide drug directed against splice variant CD44v6 for adenoma imaging