Takashi Ohshiro

MICROBIAL DESULFURIZATION OF DIBENZOTHIOPHENE IN THE PRESENCE OF HYDROCARBON

The bacterium, Rhodococcus erythropolis H-2, which can utilize dibenzothiophene (DBT) as a sole source of sulfur in the presence of hydrocarbon, was isolated from soil samples. When this strain was cultivated in a medium containing 0.27 mM DBT and 40% n-tetradecane, DBT was metabolized stoichiometrically to 2-hydroxybiphenyl within 1 day. This strain grew in the presence of n-octane and longer-carbonchain hydrocarbons, but not with n-hexane, styrene, p-xylene, cyclooctane or toluene. DBT degradation proceeded in the resting cell system with lyophilized cells of this strain. The addition of n-tetradecane enhanced the reaction rate, the optimal concentration being 40%. DBT degradation occurred in the reaction mixture even in the presence of 70% n-tetradecane, whereas at concentrations above 80% n-tetradecane suppressed the degradation.

Regulation of dibenzothiophene degrading enzyme activity of Rhodococcus erythropolis D-1

Abstract The regulation of dibenzothiophene (DBT) degrading activity of Rhodococcus erythropolis D-1 was examined. The enzymatic activity involved in DBT degradation of the strain D-1 was found in cell-free extracts of cells grown not only with DBT as a sole sulfur source but also with its analogs, thioxanthen-9-one and DBT sulfone. The activity was completely repressed in a medium with 0.5 mM sodium sulfate or 0.1 mM methanesulfonic acid even in the presence of DBT. The enzyme activity in the cell-free extracts of this strain was inhibited by a degradation product, 2-hydroxybiphenyl (2-HBP), and its analog, 2,2′-dihydroxybiphenyl (DHBP), but not by sodium sulfate and biphenyl. 2-HBP and DHBP also significantly inhibited the growth of this strain when it was cultivated in the medium supplemented with DBT as a sole source of sulfur.

Dibenzothiophene (DBT) degrading enzyme responsible for the first step of DBT desulfurization by Rhodococcus erythropolis D-1: Purification and characterization

Abstract Rhodococcus erythropolis D-1 possesses an enzymatic pathway that can remove covalently bound sulfur from dibenzothiophene (DBT) to form 2-hydroxybiphenyl. Enzymes involved in DBT degradation, catalyzing the first step of DBT desulfurization, have been identified and purified and characterized. This enzymes convert DBT to DBT sulfone. Enzyme activity was detected when two DEAE-Sepharose column chromatography fractions were combined. One enzyme, designated as component B, was purified from R. erythropolis D-1. Component B was found to have a molecular mass of 250 kDa and consist of six subunits each with a mass of 45 kDa. Analysis of the 20-residue N-terminal amino acid sequence revealed that component B is the dszC product. Other enzyme(s), contained in fraction A, were found to exhibit NADH-linked reductase activity with artificial electron acceptors, dichlorophenolindophenol, cytochrome c and ferricyanide. The DBT degrading enzyme exhibits the narrow substrate specificity; it acts on some DBT derivatives but not on DBT analogues, carbazole and fluorene. The DBT degrading enzyme activity is inhibited by 1,10-phenanthroline, 2,2′-bipyridyl, p -chloromercuribenzoic acid, 5,5′-dithiobis(2-nitrobenzoic acid), 8-quinolinol, Mn 2+ , Cu 2+ , and Zn 2+ , suggesting that thiol group(s) and metal(s) might be essential for the enzyme activity. The optimal temperature and pH of this enzyme are 40°C, and about 8.0, respectively.

CLONING AND EXPRESSION OF THE GENE FOR A VANADIUM-DEPENDENT BROMOPEROXIDASE FROM A MARINE MACRO-ALGA, CORALLINA PILULIFERA

The cDNAs for a vanadium‐dependent bromoperoxidase were cloned from a marine macro‐alga, Corallina pilulifera. The open reading frame of one clone (bpo1) encoded a protein of 598 amino acids with a calculated molecular mass of 65 312 Da in good agreement with that of 64 kDa determined for the native enzyme. The deduced amino acid sequence coincided well with partial sequences of peptide fragments of the enzyme. From the same cDNA library we also isolated another cDNA clone (bpo2) encoding a protein of 597 amino acids with an identity of about 90% to BPO1, suggesting a genetic diversity of the bromoperoxidase gene of C. pilulifera growing in a relatively narrow area. The carboxy‐terminal 123 residues of the enzyme (BPO1) showed an identity of 45% to that of the marine macro‐alga Ascophillum nodosum. The homology search of the sequences of bromoperoxidases from C. pilulifera (this study) and A. nodosum, and chloroperoxidase from the fungus Curvularia inaequalis indicated highly conserved sequences PxYxSGHA and LxxxxAxxRxxxGxHxxxD. Furthermore, it was found that the histidine residue directly bound to vanadium, other residues building up the metal center and catalytic histidine residue forming the active site of the chloroperoxidase from C. inaequalis are conserved in the primary structure of the bromoperoxidase from C. pilulifera. The cloned bpo1 was introduced into Escherichia coli, and the expressed BPO1 was purified from the recombinant strain. The N‐terminal amino acid sequence of the purified BPO1 was identical to the deduced sequence from the cDNA except the N‐terminal methionine.

L-serine production by a methylotroph and its related enzymes.

The production process of l-serine from methanol and glycine has been developed using a methylotroph with the serine pathway. Consecutive reactions of two enzymes, methanol dehydrogenase (MDH) and serine hydroxymethyltransferase (SHMT) are involved in the production. We screened a high producer, Hyphomicrobium methylovorum, which is an obligate methylotroph. With resting cells of the bacterium, 24 mg/ml of l-serine was produced from 100 mg/ml of glycine and 48 mg/ml of methanol in 3 days under optimal conditions. Next, a glycine-resistant mutant GM2 showed improved serine production (32–34 mg/ml). The mutant GM2 was found to have elevated activities of MDH and SHMT. Since there has so far been little report on the systematic characterization of enzymes of the serine pathway in methylotrophs, not only the above two enzymes but also the other three enzymes in H. methylovorum were purified and characterized: MDH, SHMT and hydroxypyruvate reductase (HPR) were crystallized; serine-glyoxylate aminotransferase (SGAT) and glycerate kinase (GK) were purified to homogeneity. As a result, all these enzymes were found to be stable against preservation and to exist abundantly in the bacterium. The gene of SHMT was cloned and its deduced amino acid sequence had homology to those of Escherichia coli (55%) and rabbit liver (44%), whereas the enzyme of the bacterium was immunochemically distinguishable from those of microorganisms other than Hyphomicrobium strains and mammalian livers.

Characterization of Dibenzothiophene Desulfurization Reaction by Whole Cells of Rhodococcus erythropolis H-2 in the Presence of Hydrocarbon

Desulfurization of dibenzothiophene (DBT) by whole cells of Rhodococcus erythropolis H-2 proceeded efficiently in the presence of two model petroleums-kerosene or a mixture of hydrocarbons. The reaction rate was enhanced by the addition of these model petroleums at 40 and 20%, respectively. The optimal temperatures for the degradation of DBT and the formation of 2-hydroxybiphenyl (2-HBP), the DBT desulfurization product, were 45°C and 40°C, respectively, which were higher than the upper temperature limit (37°C) for growth with DBT as a sole source of sulfur. These results are evaluated from the viewpoint of practical microbial desulfurization.

Crystal Structure and Desulfurization Mechanism of 2′-Hydroxybiphenyl-2-sulfinic Acid Desulfinase

The desulfurization of dibenzothiophene in Rhodococcus erythropolis is catalyzed by two monooxygenases, DszA and DszC, and a desulfinase, DszB. In the last step of this pathway, DszB hydrolyzes 2′-hydroxybiphenyl-2-sulfinic acid into 2-hydroxybiphenyl and sulfite. We report on the crystal structures of DszB and an inactive mutant of DszB in complex with substrates at resolutions of 1.8Å or better. The overall fold of DszB is similar to those of periplasmic substrate-binding proteins. In the substrate complexes, biphenyl rings of substrates are recognized by extensive hydrophobic interactions with the active site residues. Binding of substrates accompanies structural changes of the active site loops and recruits His60 to the active site. The sulfinate group of bound substrates forms hydrogen bonds with side chains of Ser27, His60, and Arg70, each of which is shown by site-directed mutagenesis to be essential for the activity. In our proposed reaction mechanism, Cys27 functions as a nucleophile and seems to be activated by the sulfinate group of substrates, whereas His60 and Arg70 orient the syn orbital of sulfinate oxygen to the sulfhydryl hydrogen of Cys27 and stabilize the negatively charged reaction intermediate. Cys, His, and Arg residues are conserved in putative proteins homologous to DszB, which are presumed to constitute a new family of desulfinases.

Occurrence of bromoperoxidase in the marine green macro-alga, ulvella lens, and emission of volatile brominated methane by the enzyme

Abstract Bromoperoxidase activity was detected in the marine green macro-alga, Ulvella lens, which is used to induce the larval metamorphosis of sea urchin in aquaculture in Japan. The enzyme activity was enhanced 8.5- and 2.2-fold by the addition of cobalt and vanadium ions to the reaction mixture, respectively. The volatile halogenated compounds dibromomethane and tribromomethane were formed in the reaction mixture when the enzyme was incubated with oxaloacetate, hydrogen peroxide and potassium bromide. These results suggest that dibromomethane, which was reported to be released by U. lens and play an important role as the inducer of larval settlement and metamorphosis, is produced by bromoperoxidase in the alga.

Modification of halogen specificity of a vanadium‐dependent bromoperoxidase

The halide specificity of vanadium‐dependent bromoperoxidase (BPO) from the marine algae, Corallina pilulifera, has been changed by a single amino acid substitution. The residue R397 has been substituted by the other 19 amino acids. The mutant enzymes R397W and R397F showed significant chloroperoxidase (CPO) activity as well as BPO activity. These mutant enzymes were purified and their properties were investigated. The maximal velocities of CPO activities of the R397W and R397F enzymes were 31.2 and 39.2 units/mg, and the Km values for Cl− were 780 mM and 670 mM, respectively. Unlike the native enzyme, both mutant enzymes were inhibited by NaN3. In the case of the R397W enzyme, the incorporation rate of vanadate into the active site was low, compared with the R397F and the wild‐type enzyme. These results supported the existence of a specific halogen binding site within the catalytic cleft of vanadium haloperoxidases.

Enhancing effect of calcium and vanadium ions on thermal stability of bromoperoxidase from Corallina pilulifera.

Bromoperoxidase from the macro-alga Corallina pilulifera is an enzyme that possesses vanadate in the catalytic center, and shows a significant thermostability and stability toward organic solvents. The structural analysis of the recombinant enzyme overexpressed in yeast revealed that it contains one calcium atom per subunit. This has been confirmed by inductively coupled plasma emission spectrometry experiments. The study of the effect of metal ions on the apo-enzyme stability has shown that the calcium ion significantly increased the enzyme stability. In addition, vanadate also increased the thermostability and strontium and magnesium ions had similar effects as calcium. The holo-enzyme shows high stability in a range of organic solvents. The effect of the different ions and solvents on the structure of the enzyme has been studied by circular dichroism experiments. The high stability of the enzyme in the presence of organic solvents is useful for its application as a biocatalyst.