In this report, we describe some of the characteristics of the Comamonas testosteroni B-356 biphenyl (BPH)-chlorobiphenyl dioxygenase system, which includes the terminal oxygenase, an iron-sulfur protein (ISP BPH ) made up of an ␣ subunit (51 kDa) and a ␤ subunit (22 kDa) encoded by bphA and bphE, respectively; a ferredoxin (FER BPH ; 12 kDa) encoded by bphF; and a ferredoxin reductase (RED BPH ; 43 kDa) encoded by bphG. ISP BPH subunits were purified from B-356 cells grown on BPH. Since highly purified FER BPH and RED BPH were difficult to obtain from strain B-356, these two components were purified from recombinant Escherichia coli strains by using the His tag purification system. These His-tagged fusion proteins were shown to support BPH 2,3-dioxygenase activity in vitro when added to preparations of ISP BPH in the presence of NADH. FER BPH and RED BPH are thought to pass electrons from NADH to ISP BPH , which then activates molecular oxygen for insertion into the aromatic substrate. The reductase was found to contain approximately 1 mol of flavin adenine dinucleotide per mol of protein and was specific for NADH as an electron donor. The ferredoxin was found to contain a Rieske-type [2Fe-2S] center (⑀ 460 , 7,455 M ؊1 cm ؊1) which was readily lost from the protein during purification and storage. In the presence of RED BPH and FER BPH , ISP BPH was able to convert BPH into both 2,3-dihydro-2,3-dihydroxybiphenyl and 3,4-dihydro-3,4-dihydroxybiphenyl. The significance of this observation is discussed.The enzymatic steps involved in the conversion of biphenyl (BPH) and chlorobiphenyls (PCBs) into, respectively, benzoate and chlorobenzoates have been elucidated in many bacteria (5,8,11,12,23,24,29,32,43). The first step (shown in Fig. 1) involved BPH 2,3-dioxygenase (dox), which transforms BPH into 2,3-dihydro-2,3-dihydroxybiphenyl. This enzyme is believed to determine the substrate selectivity of BPH-degrading bacteria (17).PCB-degrading bacteria have been divided into four groups based on substrate selectivity patterns (4). Pseudomonas sp. strain LB400 has a unique feature in being able to degrade ortho-substituted PCB congeners preferentially (17). Most other bacteria preferentially transform meta-and para-substituted congeners. In another study (submitted for publication) based on alignments of gene and gene product sequences, we determined the existence of two separate BPH dox lineages among gram-negative bacteria. Comamonas testosteroni B-356 BPH dox belongs to a distinct phylogenetic lineage together with Pseudomonas sp. strain KKS102 (11,24). This group is characterized by the fact that the gene encoding the BPH ferredoxin reductase (RED BPH ) is located outside the bph gene cluster and is phylogenetically unrelated to all other known bacterial RED BPH -encoding genes.The two members of the second lineage are strain LB400 (8) and Pseudomonas pseudoalcaligenes KF707 (40). The BPH dox of these strains has a distinct substrate selectivity pattern, suggesting that only minor factors determine this characteri...

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Biphenyl (BPH) dioxygenase oxidizes BPH to 2,3-dihydro-2,3-dihydroxybiphenyl in Comamonas testosteroniBiphenyl dioxygenase (BPH dox) 1 catalyzes the first step of the bacterial BPH degradation pathway. The enzyme introduces molecular oxygen into the ortho-meta positions on one of the aryl rings to generate 2,3-dihydro-2,3-dihydroxybiphenyl. In a previous study (1), we have reported the purification and characterization of Comamonas testosteroni strain B-356-BPH dox system. The enzyme comprises three components which are: the terminal oxygenase, an iron-sulfur protein (ISP BPH ) made up of an ␣-subunit (M r ϭ 51,000) and a ␤-subunit (M r ϭ 22,000), encoded by bphA and bphE, respectively; a ferredoxin (FER BPH , M r ϭ 12,000) encoded by bphF; and a ferredoxin reductase (RED BPH , M r ϭ 43,000) encoded by bphG. FER BPH and RED BPH were found to be involved in electron transfer from NADH to ISP BPH (1). The Rieske center of the oxygenase component is then believed to receive the electron and pass it to a mononuclear Fe 2ϩ which activates molecular oxygen for insertion into the substrate (2, 3). The ISP BPH component has been purified from BPH-induced bacteria of strain B-356 (1) and from Pseudomonas sp. LB400 (4). Since active purified FER BPH and RED BPH were difficult to obtain from cell extracts of parental strains (1, 4), these enzyme components were purified from Escherichia coli recombinant clones using the His-bind QIAGEN system (1). Both His-tagged FER BPH and His-tagged RED BPH from strain B-356 were able to transfer electrons from NADH to B-356-ISP BPH . However, purification of the individual active ISP BPH ␣ and ␤ subunits has not yet been reported.Understanding the various factors that contribute to the strain selectivity pattern toward substrate should help the modeling of new mutants with increased ability to degrade BPH analogs such as polychlorinated biphenyls. The BPH dox reactivity pattern is a major determinant affecting the performance of bacterial polychlorinated biphenyl degraders. The BPH dox-congener selectivity pattern is partly affected by the position of attack on the aromatic ring. For example, the capacity of Pseudomonas sp. LB400 to preferentially degrade the ortho-substituted polychlorinated biphenyl congeners was attributed to its ability to oxygenate BPH at ring positions 3 and 4 in addition to 2 and 3. Haddock et al. (5) have recently shown that partially purified LB400-BPH dox was able to attack 2,2Ј,5,5Ј-tetrachlorobiphenyl (for which there is no available ortho-meta sites for oxygenation) in a 3,4-position suggesting that the same enzyme catalyzes both type of attacks. Using site-directed mutagenesis, Erickson and Mondello (6) have provided evidence that minor structural differences of the ISP BPH ␣ subunit component are responsible for major changes in the reactivity pattern of strain LB400-BPH dox.Although the substrate selectivity of strain B-356 distinguishes it from strain LB400, we have recently shown that strain B-356 dox is also able to oxygenate BPH at both 2,3-and 3...

The lack of a commercially available robust and inexpensive laccase is a major barrier to the widespread application of this enzyme in various industrial sectors. By using an efficient system developed in Streptomyces lividans, we have produced by homologous expression 350 mg L(-1) of a bacterial laccase with a high purity and without any extensive purification. This is the highest production yield reported in the literature for a bacterial laccase. The secreted enzyme achieved oxidation under a wide pH range depending on the substrate: 4.0 for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) and 9.0 for 2,6-dimethoxyphenol. Furthermore, this bacterial laccase was found to be quite resistant under various conditions. It withstands pH from 3.0 to 9.0, shows a great thermostability at 70 degrees C and was highly resistant toward conventional inhibitors. For instance, while the laccase of Trametes versicolor was completely inhibited by 1 mM NaN(3), the laccase of Streptomyces coelicolor was fully active under the same conditions. To assess application potential of this laccase, we have investigated its ability to decolourise Indigo carmine. This enzyme was able to rapidly decolourise the dye in the presence of syringaldehyde as a redox mediator.

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Biphenyl dioxygenase (BPH dox) oxidizes biphenyl on adjacent carbons to generate 2,3-dihydro-2,3-dihydroxybiphenyl inComamonas testosteroni B-356 and in Pseudomonassp. strain LB400. The enzyme comprises a two-subunit (α and β) iron sulfur protein (ISPBPH), a ferredoxin (FERBPH), and a ferredoxin reductase (REDBPH). B-356 BPH dox preferentially catalyzes the oxidation of the double-meta-substituted congener 3,3′-dichlorobiphenyl over the double-para-substituted congener 4,4′-dichlorobiphenyl or the double-ortho-substituted congener 2,2′-dichlorobiphenyl. LB400 BPH dox shows a preference for 2,2′-dichlorobiphenyl, and in addition, unlike B-356 BPH dox, it can catalyze the oxidation of selected chlorobiphenyls such as 2,2′,5,5′-tetrachlorobiphenyl on adjacent meta-paracarbons. In this work, we examine the reactivity pattern of BPH dox toward various chlorobiphenyls and its capacity to catalyze themeta-para dioxygenation of chimeric enzymes obtained by exchanging the ISPBPH α or β subunit of strain B-356 for the corresponding subunit of strain LB400. These hybrid enzymes were purified by an affinity chromatography system as His-tagged proteins. Both types, the chimera with the α subunit of ISPBPH of strain LB400 and the β subunit of ISPBPH of strain B-356 (the αLB400βB-356 chimera) and the αB-356βLB400 chimera, were functional. Results with purified enzyme preparations showed for the first time that the ISPBPH β subunit influences BPH dox’s reactivity pattern toward chlorobiphenyls. Thus, if the α subunit were the sole determinant of the enzyme reactivity pattern, the αB-356βLB400 chimera should have behaved like B-356 ISPBPH; instead, its reactivity pattern toward the substrates tested was similar to that of LB400 ISPBPH. On the other hand, the αLB400βB-356 chimera showed features of both B-356 and LB400 ISPBPH where the enzyme was able to metabolize 2,2′- and 3,3′-dichlorobiphenyl and where it was able to catalyze the meta-para oxygenation of 2,2′,5,5′-tetrachlorobiphenyl.

2,3-Dihydro-2,3-dihydroxybiphenyl-2,3-dehydrogenase (B2,3D) catalyzes the second step in the biphenyl degradation pathway. The nucleotide sequence of Comamonas testosteroni B-356 bphB, which encodes B2,3D, was determined. Structural analysis showed that the dehydrogenases involved in the bacterial degradation of aromatic compounds are related to each other and that their phylogenetic relationships are very similar to the relationships observed for dioxygenases that catalyze the initial reaction in the degradation pathway. The bphB sequence was used to produce recombinant active His-tagged B2,3D, which allowed us to describe for the first time some of the main features of a B2,3D. This enzyme requires NAD ؉ , its optimal pH is 9.5, and its native M r was found to be 123,000, which makes it a tetramer. These characteristics are very similar to those reported for the related enzyme cis-toluene dihydrodiol dehydrogenase. The K m value and maximum rate of metabolism for 2,3-dihydro-2,3-dihydroxybiphenyl were 73 ؎ 16 M and 46 ؎ 4 nmol min ؊1 g ؊1 , respectively. Compared with the cis-toluene dihydrodiol dehydrogenase, B2,3D appeared to be more substrate specific since it was unable to attack cis-1,2-dihydroxy-cyclohexa-3,5-diene.

Short-chain aliphatic esters are small volatile molecules that produce fruity and pleasant aromas and flavors. Most of these esters are artificially produced or extracted from natural sources at high cost. It is, however, possible to ‘naturally’ produce these molecules using biocatalysts such as lipases and esterases. A gene coding for a newly uncovered lipase was isolated from a previous metagenomic study and cloned into E. coli BL21 (DE3) for overexpression using the pET16b plasmid. Using this recombinant strain as a whole-cell biocatalyst, short chain esters were efficiently synthesized by transesterification and esterification reactions in organic media. The recombinant lipase (LipIAF5-2) showed good affinity toward glyceryl trioctanoate and the highest conversion yields were obtained for the transesterification of glyceryl triacetate with methanol. Using a simple cetyl-trimethylammonium bromide pretreatment increased the synthetic activity by a six-fold factor and the whole-cell biocatalyst showed the highest activity at 40°C with a relatively high water content of 10% (w/w). The whole-cell biocatalyst showed excellent tolerance to alcohol and short-chain fatty acid denaturation. Substrate affinity was equally effective with all primary alcohols tested as acyl acceptors, with a slight preference for methanol. The best transesterification conversion of 50 mmol glyceryl triacetate into isoamyl acetate (banana fragrance) provided near 100% yield after 24 hours using 10% biocatalyst loading (w/w) in a fluidized bed reactor, allowing recycling of the biocatalyst up to five times. These results show promising potential for an industrial approach aimed at the biosynthesis of short-chain esters, namely for natural flavor and fragrance production in micro-aqueous media.