Biotransformations

Dr. Ved Pal Singh is Senior Lecturer in the Department of Botany, University of Delhi. Vaidyanathan, Department of Biochemistry and UGC Center of Advanced Study, Indian Institute of Science, Bangalore 560012, India (6).

Microbial degradation of styrene S. Hartmans

Microbial degradation of vinyl chloride S. Hartmans

K Saxena, P. Sharmila and Ved Pal Singh

A reduction of the nitro substituent, under both aerobic and anaerobic conditions, appears to be a common enzymatic mechanism in the environment [5,29]. In the second route, the nitro substituent is removed directly as nitrite with the formation of catechol.

Figure 1. Microbial degradation of nitroaromatic compounds [23].---, steps demonstrated;__, steps postulated; A, nitroreductase; B, aniline oxygenase;

O2N QNO2

O2NQNH2

O2NQNO2

PNP degradation was accompanied by the disappearance of the characteristic yellow color in the medium, indicating nitrogen consumption when nitrophenols were tested as a nitrogen source. The identification of the latter compound as the only organic metabolite of 2,4-DNP suggested the involvement of a reduction mechanism in the degradation pathway.

Figure 3. Initial steps in 2,4-DNT degradation pathway [47].

COOH

Simultaneous oxidation of 2-nitrobenzoate and 2,3-dihydroxybenzoate by resting cells of Achromobacter suggests the involvement of an inducible dioxygenase in the degradation of 2-nitrobenzoate. The enzyme system of Rhodococcus butanica could be successfully adapted to the kinetic resolution of α-arylpropionitriles, resulting in the formation of (R)-.

Figure 7. Proposed pathway for the degradation of nitriles: R1, -phenyl;

Linuron

I Chlorpropham

In the decomposition of CIPC (isopropyl-N-3-chlorophenyl-carbamate), which was studied using an enzyme preparation from Pseudomonas sp., 3-chloroaniline was detected. Whringht and Forey reported that Penicillium sp. hydrolyzed 4-chloro-2-butynyl-N-3-chlorophenyl carbamate) (Barban) yielding 3-chloroaniline [202].

C _N -cH2c' OAK3

Metabolism of DMNA by Methylosinus trichosporium OB3b (MT OB3b) in the presence of different concentrations of DMNA. The sequence of the reaction of DMNA with MT OB3b has not been clearly understood.

Figure 15. Major aerobic biotransformation reactions of halogenated anilines: a, dimerization; b, acetylation; c, oxygenation

CELL CONSTITU ENTS

Hydroxy derivatives, aldehydes and nitrite were formed from N-nitrosomethyl-N.amylamine by rat liver microsomes and from purified cytochrome P-450 IIB1 [84]. Recently, an increased oxidation of DMNA in pericentral microsomes, following pyrazole induction of cytochrome P-450 2El, has been reported [85].

Demethylation

I-TCA I

I-DCA

The transformation of CT to CO 2 takes place in mixed cultures under methanogenic, denitrifying and sulfate-reducing conditions, but typically CF is also produced by a parallel pathway. No significant CT transformation was observed in nitrate and oxygen respiring E cultures. In these cultures, CF persisted, indicating that other CT transformation products were formed by parallel pathways and were not derived from CF [79].

The intermediate product of P-450 transformation is phosgene, and this compound is probably also the intermediate product produced by MMO reactions [15]. Use of DCM as a growth substrate has been demonstrated in enrichment cultures [93,94], and by pure cultures of Pseudomonas and Hyphomicrobium species [21,95,96].

Figure 5. Pathways for transformation for I~2-DCA (mgfmethanogens).

The first group decomposed monochloroacetate (MCAA) but had little effect on DCAA and none on TCAA. The third group decomposed DCAA, but not MCAA and TCAA [101]. These studies showed that an inducible dehalogenase enzyme system removed the halogen from the carbon chain by hydrolysis of the carbon-hydrogen bond. Reductions are usually mediated by reduced cofactors present in anaerobic environments; they persist faster in more reduced environments with highly halogenated compounds;.

Oxidations are usually mediated by nonspecific oxygenases; they move faster in more oxidizing environments with less halogenated compounds; and they usually give alcohols and organic acid reaction products. Reductions and oxidations of compounds with more than 2 halogens per carbon atom are usually cometabolic, requiring prior or simultaneous consumption of a growth substrate or energy for a sustainable transformation.

Aerobic biodegradation of polycyclic and halogenated aromatic compounds

Complete mineralization of chlorinated polycyclic aromatic compounds, such as dibenzo-p-dioxins, dibenzofurans and biphenyls, requires the presence of two sets of genes, one set for the degradation of the polycyclic aromatic structure and a second set for the degradation of monocyclic chlorinated aromatics. . During the degradation of chlorinated dibenzofurans by bacterial strains, chlorinated salicylates will accumulate, and during the degradation of chlorinated dibenzo-p-dioxins, chlorinated phenols will accumulate. The degradation of these halogenated aromatics is discussed in another chapter of this book.

There are reports, for example, on the aerobic degradation of the tricyclic PAHs, phenanthrene and anthracene [18–20], and the tetracyclic PAHs, pyrene [21,22] and fluoranthrene [23,24] by Gram-negative and Gram- positive soil bacteria. In 1964, the metabolic sequence of enzymatic reactions leading to the degradation of naphthalene was first presented by Davies and Evans [29].

In most cases, the salicylate derived from naphthalene is oxidized to catechol by the action of the salicylate 1-hydroxylase. Most of the catabolic pathways for aromatic compounds are inducible, and the same is true for naphthalene catabolism. Activation of the nab genes from plasmid NAH7 requires both an inducer and the product of a regulatory gene.

The genes of the upper pathway leading to the formation of salicylate represent one module. An oxygenolytic cleavage of the ether bond occurs as the initial enzymatic step in the course of the bacterial attack on dibenzo-p-dioxin and dibenzofuran.

Figure 2. Catabolic pathway for degradation of dibenzo-p-dioxin by bacteria.

HOOCh(OH

Four of these ORFs show some homology to the components of the toluene dioxygenase from Pseudomonas putida F1 [ 70 ], and the benzene dioxygenase from another Pseudomonas putida isolate [ 71 ]. Other chlorinated metabolites can either be toxic, inhibit other enzymes of the degradation pathway, or accumulate [90]. 105], although some examples exist for the involvement of reductive dehalogenation in the aerobic degradation of chlorinated compounds.

Each of the three enzymes involved in hydrolytic dehalogenation of 4-CBA was individually subcloned, purified and characterized [120]. Sequence comparison of the DNA region encoding the 4-CBA dehalogenase from Arthrobacter sp.

Figure 4. Catabolic pathways for degradation of chlorinated benzoic acids

Microbial degradation of halogenated aromatics

Microorganisms play a very important role in maintaining stable concentrations of environmental chemicals, and these activities ensure the smooth functioning of the carbon cycle in nature. Even after three decades of continuous information growth in the field of microbial genetics, the genetic, biochemical and molecular mechanisms of transformation and mineralization of halogenated compounds in the biosphere by the microbial population are still poorly understood. Such widespread catabolic versatility may reveal principles not yet encountered in the intensively studied metabolic pathways of E.coli, Salmonella, and other genera and species.

Since excellent recent reviews of the microbial degradation of halogenated aromatics are available [5-8], a brief description of the degradation of some important chloroaromatics will be presented using available enzymological studies. In addition, they have found use in the production of wrapping paper, carbon paper, inks, paints, tires and many other products.

Polychlorinated biphenyls (PCBs)

139 to have effective means of treating production waste and a thorough understanding of the fate of these chemicals in the environment. The transcriptional start site of the bph operon (KF707) has been determined by reverse transcriptase mapping. Assessment of the genotoxicity of azo dyes thus requires studies in which the dyes are reduced (typically using dithionite reduction) and the genotoxicity of the reduction products is assessed [11].

Another approach to this problem is the use of a Salmonella/microsome assay modified with flavin mononucleotide (FMN), in which FMN is incorporated into the assay to reduce azo linkages [12]. Initially, the bacterial consortium was grown aerobically in the presence of the azo dye Mordant Yellow 3 (V) without biodegradation of the dye.

Figure 5. Proposed pathways for the microbial degradation of 2,4-D.

AMARANTH S03H

161 However, substantial degradation occurred when the culture was made anaerobic; nearly stoichiometric amounts of the corresponding aromatic amines were formed. Further degradation did not occur until the culture was reactivated, after which the amines were broken down to intermediates of the Krebs cycle by strain BN6, a member of the bacterial consortium known to be able to mine a variety of aromatic mines. to be used as a single source of carbon, nitrogen and energy. In addition to Mordant Yellow 3 (V), Acid Yellow 21 (VI), Amaranth (VII), Tartrazine (VIII) and 4-Hydroxyazobenzene-4'-Sulfonic Acid (IX) were tested for their ability to be metabolized in this system .

Under anaerobic conditions, in the presence of 10 mM glucose, all the dyes tested, except Tartrin, were completely decolorized. Possibly the most successful effort, in this regard, focused on the use of a soil microorganism (Pseudomonas sp.) that can utilize 4,4'-Dicarboxyazobenzene (X) as its sole source of carbon and nitrogen [21].

ORANGE I AND CARBOXYLATED ORANGE I

Attempts were made to adapt continuous cultures of this isolate to use the more complex azo dyes, Orange I (XI) and Orange II (XH).

ORANGE II AND CARBOXYLATED ORANGE II

TROPAEOLIN 0 XVI

Although ~4C-azo dyes were degraded to ~4C02 in nonligninolytic cultures, it should be emphasized that the degree of degradation observed was always greater in ligninolytic cultures. These studies also showed that aromatic rings containing hydroxyl, amino, acetamido, and nitro substituents were degraded to a greater extent than unsubstituted aromatic rings. In other studies, it was shown that five ~4C-labeled azo dyes, Orange I (XI), Orange II (XII), Acid Yellow 9 (XXIV), 4-(3-Methoxy-4- Hydroxyphenylazo) Benzenesulfonic Acid (XXV) ) , and 4-(2-Sulfo-3'-Methoxy-4-Hydroxyazobenzene-4-Azo-Benzenesulfonic Acid Mono Sodium Salt (XXVI), all of which were synthesized from ∼4C-sulfanic acid as well as ∼4C-sulfanilic acid itself, are degraded to 14CO2 by P.

In contrast, Streptomyces chromofuscus was only able to degrade three of the five dyes to ~4CO2, and the extent of degradation was only 1.1 - 3.6%. Interestingly, lignin peroxidases have been shown to catalyze the initial oxidation of many environmentally persistent organic compounds [32,38–43] .

XXIV

4-(3-METHOXY-4-HYDROXYPHENYLAZO)BENZENESULFONIC ACID XXV

XXVI

When veratryl alcohol was added to these reaction mixtures, oxidation of both dyes by lignin peroxidase was rapid and extensive [45]. Apparently, veratryl alcohol is oxidized by compound II, which is then reduced to the resting state and can then participate in the second round of the catalytic cycle. It is well known that the addition of veratryl alcohol accelerates the lignin peroxidase-mediated oxidation of several other organic compounds that are not substrates (or are poor substrates) for this enzyme in the absence of veratryl alcohol.

Although controversial, this theory has gained new credibility from the observation that low steady-state levels of veratryl alcohol radical are indeed formed during oxidation of. Another explanation suggests that veratryl alcohol functions to protect lignin peroxidases from inactivation by hydrogen peroxide in the presence of a poorly reducing cosubstrate [38,49].

NaO3$~OH N

Although most research on fungal biodegradation of azo dyes has focused on the use of P. Superficial growth of fungi on NR films has been observed with negligible consumption of the rubber [10]. From the chemical structure of the oligomers, it was assumed that the strain of Nocardia cleaved NR at the double bond shown by a wavy line in the formula in Figure 1 [11].

The possible influence of crosslink density on the microbial degradation of vulcanized NR was obscured by the microbicidal activity of the curative. However, in the curing system of CBS-sulfur, it was observed that the resistance of the vulcanizates is in good correlation with the crosslink density, regardless of the content of sulfur or CBS (Figure 2) [42].

Figure 1. Schematic representation of natural rubber-cleaving reaction.