Kohtaro Kirimura

BIODESULFURIZATION OF DIBENZOTHIOPHENE AND ITS DERIVATIVES THROUGH THE SELECTIVE CLEAVAGE OF CARBON-SULFUR BONDS BY A MODERATELY THERMOPHILIC BACTERIUM BACILLUS SUBTILIS WU-S2B

Heterocyclic organosulfur compounds such as dibenzothiophene (DBT) in petroleum cannot be completely removed by hydrodesulfurization using chemical catalysts. A moderately thermophilic bacterium Bacillus subtilis WU-S2B, which could desulfurize DBT at 50 degrees C through the selective cleavage of carbon-sulfur (CS) bonds, was newly isolated. At 50 degrees C, growing cells of WU-S2B could degrade 0.54 mM DBT within 120 h to produce 2-hydroxybiphenyl, and the resting cells could also degrade 0.81 mM DBT within 12 h. The DBT-desulfurizing ability of WU-S2B is high over a wide temperature range from 30 to 50 degrees C, and highest at 50 degrees C for both the growing and resting cells, and this is an extremely advantageous property for the practical biodesulfurization. In addition, WU-S2B could also desulfurize DBT derivatives such as 2,8-dimethylDBT, 4,6-dimethylDBT and 3,4-benzoDBT. Therefore, B. subtilis WU-S2B is considered to have more beneficial properties than other desulfurizing bacteria such as Rhodococcus strains previously reported, particularly from the viewpoint of its capacity for thermophilic desulfurization through the CS bond cleavage.

Biodesulfurization of Naphthothiophene and Benzothiophene through Selective Cleavage of Carbon-Sulfur Bonds by Rhodococcus sp. Strain WU-K2R

ABSTRACT Naphtho[2,1-b]thiophene (NTH) is an asymmetric structural isomer of dibenzothiophene (DBT), and in addition to DBT derivatives, NTH derivatives can also be detected in diesel oil following hydrodesulfurization treatment. Rhodococcus sp. strain WU-K2R was newly isolated from soil for its ability to grow in a medium with NTH as the sole source of sulfur, and growing cells of WU-K2R degraded 0.27 mM NTH within 7 days. WU-K2R could also grow in the medium with NTH sulfone, benzothiophene (BTH), 3-methyl-BTH, or 5-methyl-BTH as the sole source of sulfur but could not utilize DBT, DBT sulfone, or 4,6-dimethyl-DBT. On the other hand, WU-K2R did not utilize NTH or BTH as the sole source of carbon. By gas chromatography-mass spectrometry analysis, desulfurized NTH metabolites were identified as NTH sulfone, 2′-hydroxynaphthylethene, and naphtho[2,1-b]furan. Moreover, since desulfurized BTH metabolites were identified as BTH sulfone, benzo[c][1,2]oxathiin S-oxide, benzo[c][1,2]oxathiin S,S-dioxide, o-hydroxystyrene, 2-(2′-hydroxyphenyl)ethan-1-al, and benzofuran, it was concluded that WU-K2R desulfurized NTH and BTH through the sulfur-specific degradation pathways with the selective cleavage of carbon-sulfur bonds. Therefore, Rhodococcus sp. strain WU-K2R, which could preferentially desulfurize asymmetric heterocyclic sulfur compounds such as NTH and BTH through the sulfur-specific degradation pathways, is a unique desulfurizing biocatalyst showing properties different from those of DBT-desulfurizing bacteria.

Purification and characterization of a novel β-agarase from an alkalophilic bacterium, Alteromonas sp. E-1

A novel beta-agarase (EC 3.2.1.81) was purified from an agar-degrading alkalophilic bacterium, Alteromonas sp. E-1 isolated from the soil. This enzyme was obtained from a cell-free extract after sonication and purified 40.9-fold through treatment with streptomycin, ammonium sulfate fractionation and successive chromatography on anion-exchange and gel filtration columns. The molecular weight was estimated to be 82 kDa by SDS-polyacrylamide gel electrophoresis and 180 kDa by Superdex 200 gel filtration. The enzyme was inhibited by Mn2+, Cu2+, Fe2+, Zn2+ and Hg2+, and activated by K+, Na+ and EDTA, and its optimum pH and temperature for agarose degradation were 7.5 and 40 degrees C, respectively. This beta-agarase hydrolyzed agarose with rapid reduction of viscosity, and neoagarobiose [O-3,6-anhydro-alpha-L-galactopyranosyl(1-->3)-D-galactose] was detected from the early stage of the reaction. Neoagarobiose as the final product was selectively released from agarose, neoagarohexaose and neoagarotetraose by the reaction with this beta-agarase. This observation was different from that of other beta-agarases which produced mixtures of neoagarobiose and neoagarotetraose as the final hydrolysis products. The N-terminal amino acid sequence of this beta-agarase shows no homology to those of other beta-agarases that were so far reported.

Enzymatic synthesis of α-arbutin by α-anomer-selective glucosylation of hydroquinone using lyophilized cells of Xanthomonas campestris WU-9701

Abstract α-Arbutin, a useful cosmetic ingredient, was selectively synthesized by α-anomer-selective glucosylation of hydroquinone with maltose as a glucosyl donor using lyophilized cells of Xanthomonas campestris WU-9701 as a biocatalyst. When 45 mM hydroquinone and 120 mg of lyophilized cells showing 11 nkat of α-glucosyl transfer activity were shaken in 2 ml of 10 mM H 3 BO 3 NaOHKCl buffer (pH 7.5) containing 1.2 M maltose at 40°C, only one form of hydroquinone glucoside was selectively obtained as a product and identified as hydroquinone 1- O -α- d -glucopyranoside (α-arbutin) by 13 C-NMR, 1 H-NMR and two-dimensional HMBC analysis. Although hydroquinone has two phenolic -OH groups at the para position in its structure, only one -OH group, but not both -OHs, was glucosylated and no other glucosylated products such as maltotriose were detected in the reaction mixture. The reaction at 40°C for 36 h under optimum conditions yielded 42 mM α-arbutin, and the maximum molar conversion yield based on the amount of hydroquinone supplied reached 93%.

Cloning and expression of the cDNA encoding an alternative oxidase gene from Aspergillus niger WU-2223L.

Abstract A cDNA fragment encoding the mitochondrial alternative oxidase, the enzyme responsible for cyanide-insensitive and salicylhydroxamic acid (SHAM)-sensitive respiration, from the citric acid-producing fungus Aspergillus niger WU-2223L was cloned and expressed in Escherichia coli as a host strain. Synthetic primers were designed from the conserved nucleotide sequences of the alternative oxidase genes from higher plants and a yeast. The 210-bp DNA fragment was amplified by PCR with these primers using chromosomal DNA of WU-2223L as a template, and was employed to screen a cDNA library of A. niger. One full-length cDNA clone of 1.2 kb was obtained, and was sequenced to reveal that the clone contained an open reading frame (ORF-AOX1) encoding a polypeptide of 351 amino acids. The predicted amino-acid sequence exhibited 50%, 55%, and 52% homology to the alternative oxidases of Hansenula anomala, Neurospora crassa and Sauromatum guttatum, respectively. In the 5′-terminus region of the ORF-AOX1, a mitochondrial targeting motif was found. The whole open reading frame of ORF-AOX1 was ligated to plasmid pKK223-3 to construct the expression vector pKAOX1. The E. coli transformant harboring pKAOX1 showed cyanide-insensitive and SHAM-sensitive respiration, and expression was increased approximately two-fold by the addition of IPTG. These results indicated that the ORF-AOX1 encodes an alternative oxidase of A. niger.

Citric acid production by 2-deoxyglucose-resistant mutant strains of Aspergillus niger

SummaryMany mutant strains showing resistance to 2-deoxy-d-glucose (DG) on minimal medium containing glycerol as a carbon source were induced from Aspergillus niger WU-2223L, a citric acid-producing strain. The mutant strains were classifiable into two types according to their growth characteristics. On the agar plates containing glucose as a sole carbon source, mutant strains of the first type showed good growth irrespective of the presence or absence of DG. When cultivated in shake cultures, some strains of the first type, such as DGR1–2, showed faster glucose consumption and growth than strain WU-2223L. The period for citric acid production shortened from 9 days for strain WU-2223L to 6–7 days for these mutant strains. The levels and yields of citric acid production of the mutant strains were almost the same as those of strain WU-2223L. The mutant strains of the second type, however, showed very slow or no growth on both the agar plates containing glucose and fructose as sole carbon sources. In shake cultures, mutant strains such as DGR2-8 showed decreased glucose consumption rates, resulting in very low production of citric acid.

Identification and functional analysis of the genes encoding dibenzothiophene-desulfurizing enzymes from thermophilic bacteria

Thermophilic bacteria Bacillus subtilis WU-S2B and Mycobacterium phlei WU-F1 desulfurize dibenzothiophene (DBT) and alkylated DBTs through specific cleavage of the carbon-sulfur bonds over a temperature range up to 52°C. In order to identify and functionally analyze the DBT-desulfurization genes, the gene cluster containing bdsA, bdsB, and bdsC was cloned from B. subtilis WU-S2B. The nucleotide and amino acid sequences of bdsABC show homologies to those of the other known DBT-desulfurization genes and enzymes; e.g. a nucleotide sequence homology of 61.0% to dszABC of the mesophilic bacterium Rhodococcus sp. IGTS8 and 57.8% to tdsABC of the thermophilic bacterium Paenibacillus sp. A11-2. Deletion and subcloning analysis of bdsABC revealed that the gene products of bdsC, bdsA and bdsB oxidized DBT to DBT sulfone (DBTO2), converted DBTO2 to 2′-hydroxybiphenyl-2-sulfinate (HBPSi), and desulfurized HBPSi to 2-hydroxybiphenyl (2-HBP), respectively. Resting cells of a recombinant Escherichia coli JM109 harboring bdsABC converted DBT to 2-HBP over a temperature range of 30–52°C, indicating that the gene products of bdsABC were functional in the recombinant. The activities of DBT degradation at 50°C and DBT desulfurization (2-HBP production) at 40°C in resting cells of the recombinant were approximately five times and twice, respectively, as high as those in B. subtilis WU-S2B. The recombinant E. coli cells also degraded alkylated DBTs, such as 2,8-dimethylDBT and 4,6-dimethylDBT. The nucleotide sequences of B. subtilis WU-S2B bdsABC and the corresponding genes from M. phlei WU-F1 were found to be completely identical to each other although the strains are genetically different.

Thermophilic biodesulfurization of hydrodesulfurized light gas oils by Mycobacterium phlei WU-F1

Recalcitrant organosulfur compounds such as dibenzothiophene (DBT) derivatives in light gas oil (LGO) cannot be removed by conventional hydrodesulfurization (HDS) treatment using metallic catalysts. The thermophilic DBT-desulfurizing bacterium Mycobacterium phlei WU-F1 grew in a medium with hydrodesulfurized LGO as the sole source of sulfur, and exhibited high desulfurizing ability toward LGO between 30 and 50 degrees C. When WU-F1 was cultivated at 45 degrees C with B-LGO (390 ppm S), F-LGO (120 ppm S) or X-LGO (34 ppm S) as the sole source of sulfur, biodesulfurization resulted in around 60-70% reduction of sulfur content for all three types of hydrodesulfurized LGOs. In addition, when resting cells were incubated at 45 degrees C with hydrodesulfurized LGOs in the reaction mixtures containing 50% (v/v) oils, biodesulfurization reduced the sulfur content from 390 to 100 ppm S (B-LGO), from 120 to 42 ppm S (F-LGO) and from 34 to 15 ppm S (X-LGO). Gas chromatography analysis with an atomic emission detector revealed that the peaks of alkylated DBTs including 4-methyl-DBT, 4,6-dimethyl-DBT and 3,4,6-trimethyl-DBT significantly decreased after biodesulfurization. Therefore, thermophilic M. phlei WU-F1, which could effectively desulfurize HDS-treated LGOs over a wide temperature range up to 50 degrees C, may be a promising biocatalyst for practical biodesulfurization of diesel oil.

Purification and characterization of an extracellular β-glucosidase from the wood-grown fungus Xylaria regalis

Abstract.Xylaria regalis, a wood-grown ascomycete isolated in Taiwan, produces β-glucosidase (EC 3.2.1.21) extracellularly. The β-glucosidase was purified to homogeneity by ammonium sulfate precipitation, ion-exchange, and gel filtration chromatography. The molecular mass of the purified enzyme was estimated to be 85 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. With p-nitrophenyl β-D-glucopyranoside (PNPG) as the substrate at pH 5.0 and 50°C, the Km was 1.72 mM and Vmax was 326 μmol/min/mg. Optimal activity with PNPG as the substrate was at pH 5.0 and 50°C. The enzyme was stable at pH 5.0 at temperatures up to 50°C. The purified β-glucosidase was active against PNPG, cellobiose, sophorose, and gentiobiose, but did not hydrolyze lactose, sucrose, Avicel, and o-nitrophenyl β-D-galactopyranoside. The activity of β-glucosidase was stimulated by Ca2+, Mg2+, Mn2+, Cd2+ and β-mercaptoethanol, and inhibited by Ag+, Hg2+, SDS, and p-chloromercuribenzoate (PCMB).

Enzymatic Kolbe―Schmitt reaction to form salicylic acid from phenol: Enzymatic characterization and gene identification of a novel enzyme, Trichosporon moniliiforme salicylic acid decarboxylase

Salicylic acid decarboxylase (Sdc) can produce salicylic acid from phenol; it was found in the yeast Trichosporon moniliiforme WU-0401 and was for the first time enzymatically characterized, with the sdc gene heterologously expressed. Sdc catalyzed both reactions: decarboxylation of salicylic acid to phenol and the carboxylation of phenol to form salicylic acid without any byproducts. Both reactions were detected without the addition of any cofactors and occurred even in the presence of oxygen, suggesting that this Sdc is reversible, nonoxidative, and oxygen insensitive. Therefore, it is readily applicable in the selective production of salicylic acid from phenol, the enzymatic Kolbe-Schmitt reaction. The deduced amino acid sequence of the gene, sdc, encoding Sdc comprises 350 amino acid residues corresponding to a 40-kDa protein. The recombinant Escherichia coli BL21(DE3) expressing sdc converted phenol to salicylic acid with a 27% (mol/mol) yield at 30 degrees C for 9h.