Search

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.

The acceptor specificity of Streptococcus mutans GS-5 glucosyltransferase-D (GTF-D) was studied, particular the specificity toward non-saccharide compounds. Dihydroxy aromatic compounds like catechol, 4-methylcatechol, and 3-methoxycatechol were glycosylated by GTF-D with a high efficiency. Transglycosylation yields were 65°50°and 75°respectively, using 40 mM acceptor and 200 mM sucrose as glucosyl donor. 3-Methoxylcatchol was also glycosylated, though at a significantly lower rate. A number of other aromatic compounds such as phenol, 2-hydroxybenzaldehyde, 1,3-dihydroxybenzene, and 1,2-phenylethanediol were not glycosylated by GTF-D. Consequently GTF-D aromatic acceptors appear to require two adjacent aromatic hydroxyl groups. In order to facilitate the transglycosylation of less water-soluble acceptors the use of various water miscible organic solvents (cosolvents) was studied. The flavonoid catechin was used as a model acceptor. Bis-2-methoxyethyl ether (MEE) was selected as a useful cosolvent. In the presence of 15øv/v) MEE the specific catechin transglucosylation activity was increased 4-fold due to a 12-fold increase in catechin solubility. MEE (10-30␟/v) could also be used to allow the transglycosylation of catechol, 4-methylcatechol, and 3-methoxycatechol at concentrations (200 mM) otherwise inhibiting GTF-D transglycosylation activity.

A variety of aromatic hydrocarbon-degrading bacteria expressing different oxygenases were tested for their ability to produce indigo from indole. Styrene-grown cells of Pseudomonas putida S12 and CA-3 expressing styrene mono-oxygenase produced indigo at rates of 4–8 nmol min–1 mg dry wt–1. Toluene-grown cells of P. putida F1 and naphthalene-grown cells of P. putida PpG7 expressing dioxygenases formed indigo at rates of 1.5 and 2.5 nmol min–1 mg dry wt–1, respectively.

Styrene metabolism in styrene-degrading Pseudomonas putida CA-3 cells has been shown to proceed via styrene oxide, phenylacetaldehyde, and phenylacetic acid. The initial step in styrene degradation by strain CA-3 is oxygen-dependent epoxidation of styrene to styrene oxide, which is subsequently isomerized to phenylacetaldehyde. Phenylacetaldehyde is then oxidized to phenylacetic acid. Styrene, styrene oxide, and phenylacetaldehyde induce the enzymes involved in the degradation of styrene to phenylacetic acid by P. putida CA-3. Phenylacetic acid-induced cells do not oxidize styrene or styrene oxide. Thus, styrene degradation by P. putida CA-3 can be subdivided further into an upper pathway which consists of styrene, styrene oxide, and phenylacetaldehyde and a lower pathway which begins with phenylacetic acid. Studies of the repression of styrene degradation by P. putida CA-3 show that glucose has no effect on the activity of styrene-degrading enzymes. However, both glutamate and citrate repress styrene degradation and phenylacetic acid degradation, showing a common control mechanism on upper pathway and lower pathway intermediates.

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.

By using styrene as the sole source of carbon and energy in concentrations of 10 to 500 microM, 14 strains of aerobic bacteria and two strains of fungi were isolated from various soil and water samples. In cell extracts of 11 of the bacterial isolates, a novel flavin adenine dinucleotide-requiring styrene monooxygenase activity that oxidized styrene to styrene oxide (phenyl oxirane) was detected. In one bacterial strain (S5), styrene metabolism was studied in more detail. In addition to styrene monooxygenase, cell extracts from strain S5 contained styrene oxide isomerase and phenylacetaldehyde dehydrogenase activities. A pathway for styrene degradation via styrene oxide and phenylacetaldehyde to phenylacetic acid is proposed.