Peroxygenase-Catalyzed Oxyfunctionalization Reactions Promoted by the Complete Oxidation of Methanol.

Peroxygenases catalyze a broad range of (stereo)selective oxyfunctionalization reactions. However, to access their full catalytic potential, peroxygenases need a balanced provision of hydrogen peroxide to achieve high catalytic activity while minimizing oxidative inactivation. Herein, we report an enzymatic cascade process that employs methanol as a sacrificial electron donor for the reductive activation of molecular oxygen. Full oxidation of methanol is achieved, generating three equivalents of hydrogen peroxide that can be used completely for the stereoselective hydroxylation of ethylbenzene as a model reaction. Overall we propose and demonstrate an atom-efficient and easily applicable alternative to established hydrogen peroxide generation methods, which enables the efficient use of peroxygenases for oxyfunctionalization reactions.

Direct ethanol production from cellulosic materials by Zymobacter palmae carrying Cellulomonas endoglucanase and Ruminococcus ß-glucosidase genes.

In order to reduce the cost of bioethanol production from lignocellulosic biomass, we conferred the ability to ferment cellulosic materials directly on Zymobacter palmae by co-expressing foreign endoglucanase and ß-glucosidase genes. Z. palmae is a novel ethanol-fermenting bacterium capable of utilizing a broad range of sugar substrates, but not cellulose. Therefore, the six genes encoding the cellulolytic enzymes (CenA, CenB, CenD, CbhA, CbhB, and Cex) from Cellulomonas fimi were introduced and expressed in Z. palmae. Of these cellulolytic enzyme genes cloned, CenA degraded carboxymethylcellulose and phosphoric acid-swollen cellulose (PASC) efficiently. The extracellular CenA catalyzed the hydrolysis of barley ß-glucan and PASC to liberate soluble cello-oligosaccharides, indicating that CenA is the most suitable enzyme for cellulose degradation among those cellulolytic enzymes expressed in Z. palmae. Furthermore, the cenA gene and ß-glucosidase gene (bgl) from Ruminococcus albus were co-expressed in Z. palmae. Of the total endoglucanase and ß-glucosidase activities, 57.1 and 18.1 % were localized in the culture medium of the strain. The genetically engineered strain completely saccharified and fermented 20 g/l barley ß-glucan to ethanol within 84 h, producing 79.5 % of the theoretical yield. Thus, the production and secretion of CenA and BGL enabled Z. palmae to efficiently ferment a water-soluble cellulosic polysaccharide to ethanol.

Expression and surface display of Cellulomonas endoglucanase in the ethanologenic bacterium Zymobacter palmae.

In order to reduce the cost of bioethanol production from lignocellulosic biomass, we developed a tool for cell surface display of cellulolytic enzymes on the ethanologenic bacterium Zymobacter palmae. Z. palmae is a novel ethanol-fermenting bacterium capable of utilizing a broad range of sugar substrates, but not cellulose. Therefore, to express and display heterologous cellulolytic enzymes on the Z. palmae cell surface, we utilized the cell-surface display motif of the Pseudomonas ice nucleation protein Ina. The gene encoding Ina from Pseudomonas syringae IFO3310 was cloned, and its product was comprised of three functional domains: an N-terminal domain, a central domain with repeated amino acid residues, and a C-terminal domain. The N-terminal domain of Ina was shown to function as the anchoring motif for a green fluorescence protein fusion protein in Escherichia coli. To express a heterologous cellulolytic enzyme extracellularly in Z. palmae, we fused the N-terminal coding sequence of Ina to the coding sequence of an N-terminal-truncated Cellulomonas endoglucanase. Z. palmae cells carrying the fusion endoglucanase gene were shown to degrade carboxymethyl cellulose. Although a portion of the expressed fusion endoglucanase was released from Z. palmae cells into the culture broth, we confirmed the display of the protein on the cell surface by immunofluorescence microscopy. The results indicate that the N-terminal anchoring motif of Ina from P. syringae enabled the translocation and display of the heterologous cellulase on the cell surface of Z. palmae.

Ethanol production from wood hydrolysate using genetically engineered Zymomonas mobilis.

An ethanologenic microorganism capable of fermenting all of the sugars released from lignocellulosic biomass through a saccharification process is essential for secondary bioethanol production. We therefore genetically engineered the ethanologenic bacterium Zymomonas mobilis such that it efficiently produced bioethanol from the hydrolysate of wood biomass containing glucose, mannose, and xylose as major sugar components. This was accomplished by introducing genes encoding mannose and xylose catabolic enzymes from Escherichia coli. Integration of E. coli manA into Z. mobilis chromosomal DNA conferred the ability to co-ferment mannose and glucose, producing 91 % of the theoretical yield of ethanol within 36 h. Then, by introducing a recombinant plasmid harboring the genes encoding E. coli xylA, xylB, tal, and tktA, we broadened the range of fermentable sugar substrates for Z. mobilis to include mannose and xylose as well as glucose. The resultant strain was able to ferment a mixture of 20 g/l glucose, 20 g/l mannose, and 20 g/l xylose as major sugar components of wood hydrolysate within 72 h, producing 89.8 % of the theoretical yield. The recombinant Z. mobilis also efficiently fermented actual acid hydrolysate prepared from cellulosic feedstock containing glucose, mannose, and xylose. Moreover, a reactor packed with the strain continuously produced ethanol from acid hydrolysate of wood biomass from coniferous trees for 10 days without accumulation of residual sugars. Ethanol productivity was at 10.27 g/l h at a dilution rate of 0.25 h(-1).

Production of ethanol by the white-rot basidiomycetes Peniophora cinerea and Trametes suaveolens.

The white-rot basidiomycetes, Peniophora cinerea and Trametes suaveolens, can produce ethanol from hexoses. Although T. suaveolens produced negligible amounts of ethanol under aerobic conditions, P. cinerea produced ethanol under both aerobic and semi-aerobic conditions and assimilated glucose, mannose, fructose, galactose, sucrose, maltose and cellobiose with yields of ethanol of 0.41, 0.45, 0.44, 0.19, 0.41, 0.44 and 0.45 g per g hexose, respectively. The corresponding results for T. suaveolens were 0.39, 0.3, 0.13, 0.2, 0.37, 0.35 and 0.31 g ethanol/g hexose. Furthermore, P. cinerea exhibited simultaneous saccharification and fermentation of amorphous cellulose.

A novel thermophilic lysozyme from bacteriophage phiIN93.

The lysozyme of bacteriophage phiIN93 was purified to apparent homogeneity with Carboxymethyl Sepharose and Hydroxyapatie columns from lysates of the phage grown on Thermus aquaticus TZ2. The enzyme is a single polypeptide chain with a molecular weight of 33,000. From the determined N-terminal amio acids of the enzyme, the locus of the gene was specified on a phiIN93 genome. The enzyme was not similar to egg white lysozyme, T4 phage lysozyme, or lambda phage lysozyme. The enzyme, phiIN93 lysozyme, was found to be a novel type of thermophilic lysozyme, which lyses specifically Thermus sp. cells, and exhibited conspicuous thermal stability at 95 degrees C for 1h in the presence of beta-mercaptoethanol.

Cyanide fishing and cyanide detection in coral reef fish using chemical tests and biosensors.

Sodium cyanide has been used in the Philippines to collect tropical marine fish for aquarium and food trades since the early 1960s. Cyanide fishing is a fast method to stun and collect fish. This practice is damaging the coral reefs irreversibly. In most countries cyanide fishing is illegal, but most of the exporting and importing countries do not have test and certificate systems. Many analytical methods are available for the detection of cyanide in environmental and biological samples. However, most of the techniques are time consuming, and some lack specificity or sensitivity. Besides, an ultra sensitive cyanide detection method is needed due to the rapid detoxification mechanisms in fish. The aim of this review is to give an overview of cyanide fishing problem in the south-east Asia and current strategies to combat this destructive practice, summarise some of the methods for cyanide detection in biological samples and their disadvantages. A novel approach to detect cyanide in marine fish tissues is briefly discussed.

Ethanol production from cellobiose by Zymobacter palmae carrying the Ruminocuccus albus beta-glucosidase gene.

Its metabolic characteristics suggest Zymobacter palmae gen. nov., sp. nov. could serve as a useful new ethanol-fermenting bacterium, but its biotechnological exploitation would require certain genetic improvements. We therefore established a method for transforming Z. palmae using the broad-host vector plasmids pRK290, pMFY31 and pMFY40 as a source of transforming DNA. Using electroporation, the frequency of transformation was 10(5) to 10(6) transformants/mug of DNA. To confer the ability to ferment cellobiose, which is a hydrolysis product from cellulosic materials treated enzymatically or with acid, the beta-glucosidase gene from Ruminococcus albus was introduced into Z. palmae, where its expression was driven by its endogenous promoter. About 56% of the enzyme expressed was localized on the cell-surface or in the periplasm. The recombinant Z. palmae could ferment 2% cellobiose to ethanol, producing 95% of the theoretical yield with no accumulation of organic acids as metabolic by-products. Thus, expression of beta-glucosidase in Z. palmae expanded the substrate spectrum of the strain, enabling ethanol production from cellulosic materials.

Purification and characterization of extracellular 1,2-alpha-L-fucosidase from Bacillus cereus.

Bacillus cereus isolated from a soil sample, inductively produced alpha-L-fucosidase in culture medium containing porcine gastric mucin (PGM). The production of the enzyme was also weakly induced by L-fucose and D-arabinose, but not by other sugars including glucose. The enzyme was purified 61-fold with an overall recovery of 1.8% from the culture fluid supplemented with PGM by ammonium sulfate precipitation, acetone fractionation, and subsequent column chromatography. The purified enzyme was found homogeneous by SDS-PAGE and its molecular mass was estimated to be approximately 196,000 kDa. Its optimum pH was 7.0 and it was stable in the pH range of 5.0 to 9.0. The enzyme hydrolyzed the alpha-(1-->2)-L-fucosidic linkage in oligosaccharides such as Fucalpha1-2Galbeta1-4Glc (2'-fucosyllactose), Fucalpha1-2Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc (lacto-N-fucopentaose I), and the glycoprotein PGM. The enzyme was inactive on p-nitrophenyl alpha-L-fucoside, the alpha-(1-->3)-L-fucosidic linkages in Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4Glc (lacto-N-fucopentaose III) and orosomucoid, the alpha-(1-->4)-L-fucosidic linkage in Galbeta1-3(Fucalpha1-4)GlcNAcbeta1-3Galbeta1-4Glc (lacto-N-fucopentaose II), and the alpha-(1-->6)-L-fucosidic linkage in thyroglobulin.

Identification of functionally important amino acid residues in Zymomonas mobilis levansucrase.

The catalytic residues of levansucrase (sucrose:2,6-beta-D-fructan 6-beta-D-fructosyltransferase, EC 2.4.1.10) from Zymomonas mobilis were analyzed by random mutation and site-directed mutagenesis. We found that substitution of Glu278 with Asp and His reduced the k(cat) for sucrose hydrolysis 30- and 210-fold, respectively, strongly suggesting Glu278 plays a key role in catalyzing this reaction. Given the likelihood that another acidic amino residue was also involved, we constructed variants in which acidic amino acids located within homologous regions among bacterial levansucrases and fructosyltransferases were substituted, and found that substitution of Asp194, located in homologous region III, abolished sucrose hydrolysis. In addition, Glu278 was determined to be situated within the DXXER motif in homologous region IV conserved among bacterial levansucrases and fructosyltransferases, while Asp194 was within the triplet RDP motif conserved among bacterial levansucrases, fructosyltransferases and fructofuranosidases. Finally, comparison of our findings with published data on other site-directed mutated enzymes indicated His296, also located in homologous region IV, is crucial for catalysis of the transfructosylation reaction.

Metabolism of galactose in Zymomonas mobilis.

Zymomonas mobilis IFO 13 756 could take up galactose and metabolize it so that galactonic acid accumulated in the culture fluid. The oxidation of galactose was catalysed by a membrane-associated enzyme, which could be solubilized with a mixture of 1% Triton X-100 and 1 M: KC1. The strain also accumulated galactitol as a minor metabolite. To confer the ability to utilize galactose on Z. mobilis, a Gal(+) recombinant plasmid, pZG13, was constructed by the insertion of the galETK genes of Escherichia coli immediately downstream of the Z. mobilis promoter gene in pZA22, the plasmid introduced into a Zymomonas strain. Uridine diphospho-glucose 4-epimerase coded in the galE gene was expressed in Z. mobilis carrying pTG13. The recombinant strain could produce a small amount of ethanol from galactose.

Isolation and application of a styrene-degrading strain of Pseudomonas putida to biofiltration.

A bacterial strain ST201 capable of degrading styrene was isolated from soil and identified as Pseudomonas putida. This strain had high tolerance to styrene and could degrade it completely in 48 h at concentrations up to 600 mg/l. P. putida ST201 was also demonstrated to degrade a mixture of benzene, toluene, ethylbenzene and p-xylene. A packed tower biofilter inoculated with P. putida ST201 was constructed which removed styrene vapor with a styrene elimination capacity of 90 g/m3.h.

Characterization of membrane bioreactor for dry wine production.

Characteristics of yeast growth and ethanol fermentation were examined in membrane bioreactor using a grape juice. After inoculation, batch fermentation was carried out for 24 h. When yeast growth reached the stationary phase, continuous fermentation was initiated. In continuous fermentation, a linear relationship was observed between cell concentration and dilution rate. In single-vessel membrane bioreactor, the cell concentrations of 18.7 g/l and 76.9 g/l (15 and 60 times higher than that of the batch fermentation, respectively) were observed at dilution rates of 0.1 h(-1) and 0.3 h(-1), respectively. The residual sugar concentration was higher than 10 g/l at the dilution rate of 0.1 h(-1), 0.2 h(-1) or 0.3 h(-1), therefore the single-vessel membrane bioreactor was not suitable for producing dry wine (sugar concentration: 4 g/l or less). In the double-vessel membrane bioreactor, it is most suitable to set the recycle ratio at 0.15 for keeping the sugar concentration below 4 g/l. The productivity of dry wine in the double-vessel membrane bioreactor was 28 times higher than that in the batch fermentation.

Biosynthesis of p-anisaldehyde by the white-rot basidiomycete Pleurotus ostreatus.

The white-rot basidiomycete Pleurotus ostreatus produced sweet flavor compounds on a liquid medium. The major and minor compounds identified by GC-MS analysis were p-anisaldehyde (4-methoxybenzaldehyde) and 3-chloro-p-anisaldehyde (3-chloro-4-methoxybenzaldehyde), respectively. p-Anisaldehyde was only produced under static culture conditions. Differences in the type and quantity of flavor compounds produced among wild strains of P. ostreatus were observed. Aryl alcohol oxidase and manganese peroxidase activities increased parallel to the production of p-anisaldehyde. These results indicated that the biosynthesis of p-anisaldehyde is concerned in generating H2O2-activated peroxidase in the lignin-degradation system. Addition of L-tyrosine to the culture led to higher production of p-anisaldehyde. The flavor extract, which contains p-anisaldehyde, exhibited antimicrobial activity against Bacillus subtilis, Pseudomonas aeruginosa, Aspergillus niger and Fusarium oxysporum.

Production of gamma-lactones by the brown-rot basidiomycete Piptoporus soloniensis.

A wild strain of brown-rot basidiomycete Piptoporus soloniensis produced a sweet flavor similar to tropical fruits in liquid cultures. The major and minor compounds were identified to be gamma-decalactone and gamma-octanolactone by gas chromatography-mass spectrometry analysis, respectively. The growth and production of gamma-decalactone by P. soloniensis in broth to which fatty acids had been added were investigated. The addition of 12-hydroxystearic acid and ricinoleic acid to the culture markedly enhanced the production of gamma-decalactone. On the other hand, addition of myristic acid, palmitic acid, stearic acid and oleic acid to the culture resulted in a higher production of gamma-octanolactone. The addition of hexanoic acid, octanoic acid, decanoic acid, lauric acid, linoleic acid and linolenic acid to the culture reduced the growth of P. soloniensis and production of gamma-decalactone and gamma-octanolactone. This strain accumulated oxalic acid in liquid culture and grew sufficiently under strongly acidic conditions.

Bioconversion of xylose, hexoses and biomass to ethanol by a new isolate of the white rot basidiomycete Trametes versicolor.

Second-generation bioethanol production requires the development of economically feasible and sustainable processes that use renewable lignocellulosic biomass as a starting material. However, the microbial fermentation of xylose, which is the principal pentose sugar in hemicellulose, is a limiting factor in developing such processes. Here, a strain of the white rot basidiomycete Trametes versicolor that was capable of efficiently fermenting xylose was newly isolated and characterized. This strain, designated KT9427, was capable of assimilating and converting xylose to ethanol under anaerobic conditions with a yield of 0.44 g ethanol per 1 g of sugar consumed. In culture medium containing low yeast extract concentrations, xylose consumption and ethanol productivity were enhanced. Adjusting the initial pH between 3.0 and 5.0 did not markedly influence xylose fermentation. T. versicolor KT9427 also produced ethanol from glucose, mannose, fructose, cellobiose and maltose at yields ranging from 0.45 to 0.49 g ethanol per 1 g of sugar consumed. In addition, strain KT9427 exhibited favourable conversion of non-pretreated starch, cellulose, xylan, wheat bran and rice straw into ethanol compared to common recombinant yeast strains. Taken together, the present findings suggest that T. versicolor KT9427 is a promising candidate for environmentally friendly ethanol production directly from lignocellulosic biomass.

Efficient xylose fermentation by the brown rot fungus Neolentinus lepideus.

The efficient production of bioethanol on an industrial scale requires the use of renewable lignocellulosic biomass as a starting material. A limiting factor in developing efficient processes is identifying microorganisms that are able to effectively ferment xylose, the major pentose sugar found in hemicellulose, and break down carbohydrate polymers without pre-treatment steps. Here, a basidiomycete brown rot fungus was isolated as a new biocatalyst with unprecedented fermentability, as it was capable of converting not only the 6-carbon sugars constituting cellulose, but also the major 5-carbon sugar xylose in hemicelluloses, to ethanol. The fungus was identified as Neolentinus lepideus and was capable of assimilating and fermenting xylose to ethanol in yields of 0.30, 0.33, and 0.34 g of ethanol per g of xylose consumed under aerobic, oxygen-limited, and anaerobic conditions, respectively. A small amount of xylitol was detected as the major by-product of xylose metabolism. N. lepideus produced ethanol from glucose, mannose, galactose, cellobiose, maltose, and lactose with yields ranging from 0.34 to 0.38 g ethanol per g sugar consumed, and also exhibited relatively favorable conversion of non-pretreated starch, xylan, and wheat bran. These results suggest that N. lepideus is a promising candidate for cost-effective and environmentally friendly ethanol production from lignocellulosic biomass. To our knowledge, this is the first report on efficient ethanol fermentation from various carbohydrates, including xylose, by a naturally occurring brown rot fungus.

Direct ethanol production from starch, wheat bran and rice straw by the white rot fungus Trametes hirsuta.

The white rot fungus Trametes hirsuta produced ethanol from a variety of hexoses: glucose, mannose, cellobiose and maltose, with yields of 0.49, 0.48, 0.47 and 0.47 g/g of ethanol per sugar utilized, respectively. In addition, this fungus showed relatively favorable xylose consumption and ethanol production with a yield of 0.44 g/g. T. hirsuta was capable of directly fermenting starch, wheat bran and rice straw to ethanol without acid or enzymatic hydrolysis. Maximum ethanol concentrations of 9.1, 4.3 and 3.0 g/l, corresponding to 89.2%, 78.8% and 57.4% of the theoretical yield, were obtained when the fungus was grown in a medium containing 20 g/l starch, wheat bran or rice straw, respectively. The fermentation of rice straw pretreated with ball milling led to a small improvement in the ethanol yield: 3.4 g ethanol/20 g ball-milled rice straw. As T. hirsuta is an efficient microorganism capable of hydrolyzing biomass to fermentable sugars and directly converting them to ethanol, it may represent a suitable microorganism in consolidated bioprocessing applications.

Characterization of two acidic ß-glucosidases and ethanol fermentation in the brown rot fungus Fomitopsis palustris.

Two acidic ß-glucosidases (ßGI and ßGII) from the brown rot fungus Fomitopsis palustris were purified to homogeneity by several chromatographic steps. ßGI and ßGII had molecular weights of 130 and 213 kDa, respectively, and exhibited optimum activity at pH 2.5 and 55°C. The K(m) values of ßGI and ßGII for p-nitrophenyl-ß-d-glucopyranoside were 0.706 and 0.971 mM, respectively. Although the effect of metal ions and inhibitors differed between the two enzymes, both ß-glucosidases exhibited preferential glucose release during hydrolysis of cello-oligosaccharides, indicating that ßGI and ßGII possess effective exo-type activities. Notably, F. palustris was able to produce ethanol when cultured on medium containing 20 g/l of glucose, mannose, cellobiose, and maltose, in which the maximum ethanol concentrations measured were 9.2, 8.7, 9.0, and 8.9 g/l, corresponding to 90.2%, 85.3%, 88.2%, and 87.3% of the theoretical yield, respectively. These findings suggest that F. palustris has the ability not only to secrete ß-glucosidase enzymes effective at low pH, but also to function as a biocatalyst, which may be suitable for the conversion of lignocellulosic materials into ethanol.

The genomic structure of thermus bacteriophage {phi}IN93.

We have determined the complete nucleotide sequence of the phage IN93 is 19,604-bp long and contains 39 putative open reading frames. The functions for 20% of IN93 gene products are similar to those expressed by other known phages and bacteria, and include peptidase, lytic enzymes, integrase, repressor protein and replication protein. The structural proteins of the IN93 virion were identified through sodium dodecyl sulphate-polyacrylamide gel electrophoresis and found to have no similarity to those of other phages. We also determined the transcription initiation sites and classified four transcription units using the primer extension method. Three transcription units were transcribed in the same direction as part of the lytic cycle, while the remaining unit was transcribed in the opposite direction as part of the lysogenic cycle.