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Applied Microbiology and Biotechnology (v.67, #5)

Xylanases from fungi: properties and industrial applications by M. L. T. M. Polizeli; A. C. S. Rizzatti; R. Monti; H. F. Terenzi; J. A. Jorge; D. S. Amorim (pp. 577-591).

Xylan is the principal type of hemicellulose. It is a linear polymer of β-D-xylopyranosyl units linked by (1–4) glycosidic bonds. In nature, the polysaccharide backbone may be added to 4-O-methyl-α-D-glucuronopyranosyl units, acetyl groups, α-L-arabinofuranosyl, etc., in variable proportions. An enzymatic complex is responsible for the hydrolysis of xylan, but the main enzymes involved are endo-1,4-β-xylanase and β-xylosidase. These enzymes are produced by fungi, bacteria, yeast, marine algae, protozoans, snails, crustaceans, insect, seeds, etc., but the principal commercial source is filamentous fungi. Recently, there has been much industrial interest in xylan and its hydrolytic enzymatic complex, as a supplement in animal feed, for the manufacture of bread, food and drinks, textiles, bleaching of cellulose pulp, ethanol and xylitol production. This review describes some properties of xylan and its metabolism, as well as the biochemical properties of xylanases and their commercial applications.

Microbial production and applications of chiral hydroxyalkanoates by Guo-Qiang Chen; Qiong Wu (pp. 592-599).

Polyhydroxyalkanoates (PHA) are a family of polyesters consisting of over 150 chiral hydroxyalkanoic acids (HA). This paper reviews the physiological functions of (R)-3-hydroxybutyric acid (3HB) and (R)-4-hydroxybutyric acid and summarizes the technologies developed to produce various HA [3HB, (R)-3-hydroxyoctanoic acid, (R)-3-hydroxydecanoic acid, etc.] and the applications of chiral HA. Their outlooks and perspectives are discussed.

Biodegradation of xenobiotics by anaerobic bacteria by Chunlong Zhang; George N. Bennett (pp. 600-618).

Xenobiotic biodegradation under anaerobic conditions such as in groundwater, sediment, landfill, sludge digesters and bioreactors has gained increasing attention over the last two decades. This review gives a broad overview of our current understanding of and recent advances in anaerobic biodegradation of five selected groups of xenobiotic compounds (petroleum hydrocarbons and fuel additives, nitroaromatic compounds and explosives, chlorinated aliphatic and aromatic compounds, pesticides, and surfactants). Significant advances have been made toward the isolation of bacterial cultures, elucidation of biochemical mechanisms, and laboratory and field scale applications for xenobiotic removal. For certain highly chlorinated hydrocarbons (e.g., tetrachlorethylene), anaerobic processes cannot be easily substituted with current aerobic processes. For petroleum hydrocarbons, although aerobic processes are generally used, anaerobic biodegradation is significant under certain circumstances (e.g., O₂-depleted aquifers, oil spilled in marshes). For persistent compounds including polychlorinated biphenyls, dioxins, and DDT, anaerobic processes are slow for remedial application, but can be a significant long-term avenue for natural attenuation. In some cases, a sequential anaerobic-aerobic strategy is needed for total destruction of xenobiotic compounds. Several points for future research are also presented in this review.

Production of Lactobacillus kefir cells for asymmetric synthesis of a 3,5-dihydroxycarboxylate by Holger Pfruender; Maya Amidjojo; Florian Hang; Dirk Weuster-Botz (pp. 619-622).

An efficient fedbatch process for the production of Lactobacillus kefir DSM 20587 cells was developed. An improvement in space time yield of 270% (3.7 g_DCW l⁻¹ day⁻¹) and in final enzyme activity of 440% (9.1 U/ml) was achieved on a 150 l scale by controlling the oxygen transfer rate to 7–9 mmol l⁻¹ h⁻¹. The cells exhibited good and highly stereoselective reducing activities against tert-butyl 6-chloro-3,5-dioxohexanoate. tert-Butyl (3R,5S)-6-chloro-dihydroxyhexanoate, a chiral building block for HMG-CoA reductase inhibitor synthesis, was produced with 47.5% yield and >99% ee at C₃3 and C₅5 in a simple batch biotransformation process.

Kinetics of Bifidobacterium longum ATCC 15707 fermentations: effect of the dilution rate and carbon source by C. Shene; M. Mardones; P. Zamora; S. Bravo (pp. 623-630).

The effect of the dilution rate on biomass and product synthesis in fermentations of glucose, fructose and a commercial mixture of fructooligosaccharides (FOS) by Bifidobacterium longum ATCC 15707 was studied. Kinetic parameters (maximum specific growth rate, Monod constant, maintenance, and yield coefficients) in the mathematical model of the fermentation were estimated from experimental data. In the FOS mixture fermentations, approximately 12% of the total reducing sugars (mainly fructose) in the feed were not metabolized by the bacterium. In fermentations of fructose and the FOS mixture, biomass concentration increased as the dilution rate increased and, once maximum values were reached [3.90 (D=0.20 h⁻¹) and 2.54 g l⁻¹ (D=0.15 h⁻¹), respectively], decreased rapidly as the culture was washed out. Formic acid was detected at low dilution rates in glucose and fructose fermentations. The main products in fermentations of the three carbon sources were lactic and acetic acids. Average values of the molar ratio between acetic and lactic acids of 1.18, 1.21 and 0.83 mol mol⁻¹ were obtained in glucose, fructose and FOS mixture fermentations, respectively. In batch fermentations carried out without pH control this molar ratio was lower than 1.5 only when fructose was used as the carbon source.

Application of cyanide hydrolase from Klebsiella sp. in a biosensor system for the detection of low-level cyanide by Karen K. W. Mak; Alex W. C. Law; Shinsuke Tokuda; Hideshi Yanase; Reinhard Renneberg (pp. 631-636).

A partially purified preparation of cyanide hydrolase (cyanidase) from a bacterium, Klebsiella sp., was applied as a biocatalyst in a biosensor system for low-level cyanide detection. In the biosensor system cyanide hydrolase converts cyanide into formate and ammonia. The formate produced in the cyanide degradation was detected with a formate biosensor, in which formate dehydrogenase (FDH; E.C. 1.2.1.2) was co-immobilized with salicylate hydroxylase (SHL; E.C. 1.14.13.1) on a Clark electrode. The principle of the formate sensor is that FDH converts formate into carbon dioxide using β-nicotinamide adenine dinucleotide hydrate (NAD⁺). The corresponding NADH produced is then oxidized to NAD⁺ by SHL using salicylate and oxygen. The oxygen consumption is monitored with the Clark electrode. The optimum buffer pH and temperature for the enzymatic hydrolysis of potassium cyanide were studied. The preliminary experiments including the pretreatment of cyanide with cyanide hydrolase and then detection by the formate sensor gave a detection limit at 7.3 μmol l⁻¹ cyanide. The linear range of the calibration curve was between 30 μmol l⁻¹ and 300 μmol l⁻¹ cyanide.

Synthesis of ethyl phenylacetate by lyophilized mycelium of Aspergillus oryzae by A. Converti; R. Gandolfi; M. Zilli; F. Molinari; L. Binaghi; P. Perego; M. Del Borghi (pp. 637-640).

Lyophilized mycelia of Aspergillus oryzae CBS 102.07, Aspergillus oryzae MIM, Rhizopus oryzae CBS 112.07, Rhizopus oryzae CBS 391.34, Rhizopus oryzae CBS 260.28 and Rhizopus oryzae CBS 328.47 were tested in this study to select the best biocatalysts for ethanol acylation with phenylacetic acid. The mycelium-bound carboxylesterase activity of A. oryzae MIM, which exhibited the best performances, was initially investigated at 50°C, either in 0.1 M phosphate buffer or in n-heptane to catalyse the hydrolysis or the synthesis, respectively, of ethyl phenylacetate. The results in terms of product and substrate concentrations versus time were used to estimate the maximum molar conversions at equilibrium, the equilibrium constants, and the times needed to reach half maximum conversions, thus providing sufficient information about this biotransformation. The values of the apparent equilibrium constants, estimated at 20°C<T<50°C, were finally used to estimate the thermodynamic parameters of ethanol acylation by this biocatalyst.

Cloning and characterization of arabinoxylan arabinofuranohydrolase-D3 (AXHd3) from Bifidobacterium adolescentis DSM20083 by Lambertus A. M. van den Broek; Ruth M. Lloyd; Gerrit Beldman; Jan C. Verdoes; Barry V. McCleary; Alphons G. J. Voragen (pp. 641-647).

Arabinoxylan arabinofuranohydrolase-D3 (AXHd3) from Bifidobacterium adolescentis releases only C3-linked arabinose residues from double-substituted xylose residues. A genomic library of B. adolescentis DSM20083 was screened for the presence of the axhD3 gene. Two plasmids were identified containing part of the axhD3 gene. The nucleotide sequences were combined and three open reading frames (ORFs) were found. The first ORF showed high homology with xylanases belonging to family 8 of the glycoside hydrolases and this gene was designated xylA. The second ORF was the axhD3 gene belonging to glycoside hydrolase family 43. The third (partial) ORF coded for a putative carboxylesterase. The axhD3 gene was cloned and expressed in Escherichia coli. Several substrates were employed in the biochemical characterization of recombinant AXHd3. The enzyme showed the highest activity toward wheat arabinoxylan oligosaccharides. In addition, β-xylanase from Trichoderma sp. was able to degrade soluble wheat arabinoxylan polymer to a higher extent, after pretreatment with recombinant AXHd3. Arabinoxylan oligosaccharides incubated with a combination of recombinant AXHd3 and an α-l-arabinofuranosidase from Aspergillus niger did not result in a higher maximal release of arabinose than incubation with these enzymes separately.

Single-step purification of lipase from Burkholderia multivorans using polypropylene matrix by Namita Gupta; Pooja Rathi; Rajni Singh; Vineet Kumar Goswami; Rani Gupta (pp. 648-653).

Lipase from Burkholderia multivorans was purified with high yields directly from fermentation broth by a single-step purification protocol involving adsorption and desorption. The crude enzyme (lyophilized powder) from B. multivorans was loaded on Accurel (Membrana, Germany), a polypropylene matrix, using butanol as the solvent in a buffer at pH 9.0 and ambient temperature for a period of 12 h. The enzyme adsorbed onto the matrix with high specific activity (33 units mg⁻¹ protein). This was followed by desorption of the enzyme from the matrix using Triton X-100 as the eluent. The enzyme was finally recovered by precipitation with acetone (50%, v/v). Thus, an overall enzyme yield of 66% with a 3.0-fold purification was obtained. The purity of the enzyme was ascertained by SDS-PAGE. The phenomenon of adsorption and desorption on Accurel was studied for three more lipases, viz. Mucor meihei lipase (Sigma–Aldrich Co.), Lipolase (Novo Nordisk, Denmark) and Pseudomonas aeruginosa lipase (laboratory isolate).

Engineering of pyranose 2-oxidase from Peniophora gigantea towards improved thermostability and catalytic efficiency by Sabine Bastian; Matthias J. Rekowski; Klaus Witte; Dorothée M. Heckmann-Pohl; Friedrich Giffhorn (pp. 654-663).

To improve the stability and catalytic efficiency of pyranose 2-oxidase (P2Ox) by molecular enzyme evolution, we cloned P2Ox cDNA by RACE-PCR from a cDNA library derived from the basidiomycete Peniophora gigantea. The P2Ox gene was expressed in Escherichia coli BL21(DE3), yielding an intracellular and enzymatically active P2OxB with a volumetric yield of 500 units/l. Site-directed mutagenesis was employed to construct the P2Ox variant E540K (termed P2OxB1), which exhibited increased thermo- and pH-stability compared with the wild type, concomitantly with increased catalytic efficiencies (k_cat/K_m) for d-xylose and l-sorbose. P2OxB1 was provided with a C-terminal His₆-tag (termed P2OxB1H) and subjected to directed evolution using error-prone PCR. Screening based on a chromogenic assay yielded the new P2Ox variant K312E (termed P2OxB2H) that showed significant improvements with respect to k_cat/K_m for d-glucose (5.3-fold), methyl-β-d-glucoside (2.0-fold), d-galactose (4.8-fold), d-xylose (59.9-fold), and l-sorbose (69.0-fold), compared with wild-type P2Ox. The improved catalytic performance of P2OxB2H was demonstrated by bioconversions of l-sorbose that initially was a poor substrate for wild-type P2Ox. This is the first report on the improvement of a pyranose 2-oxidase by a dual approach of site-directed mutagenesis and directed evolution, and the application of the engineered P2Ox in bioconversions.

Over-expression in Escherichia coli of a thermally stable and regio-selective nitrile hydratase from Comamonas testosteroni 5-MGAM-4D by Kelly L. Petrillo; Shijun Wu; Eugenia C. Hann; Frederick B. Cooling; Arie Ben-Bassat; John E. Gavagan; Robert DiCosimo; Mark S. Payne (pp. 664-670).

The genes encoding a thermally stable and regio-selective nitrile hydratase (NHase) and an amidase from Comamonas testosteroni 5-MGAM-4D have been cloned and sequenced, and active NHase has been over-produced in Escherichia coli. Maximal activity requires co-expression of a small open reading frame immediately downstream from the NHase beta subunit gene. Compared to the native organism, the E. coli biocatalyst has nearly threefold more NHase activity on a dry cell weight basis, and this activity is significantly more thermally stable. In addition, this biocatalyst converts a wide spectrum of nitrile substrates to the corresponding amides. Such versatility and robustness are desirable attributes of a biocatalyst intended for use in commercial applications.

Mycobacterium sp. mutant strain producing 9α-hydroxyandrostenedione from sitosterol by M. V. Donova; S. A. Gulevskaya; D. V. Dovbnya; I. F. Puntus (pp. 671-678).

Mycobacterium sp. VKM Ac-1815D and its derivatives with altered resistance to antibacterial agents were able to produce androst-4-ene-3,17-dione (AD) as a major product from sitosterol. In this study, those strains were subjected to subsequent mutagenization by chemical agents and UV irradiation in combination with sitosterol selection pressure. The mutant Mycobacterium sp. 2-4 M was selected, being capable of producing 9α-hydroxyandrost-4-ene-3,17-dione (9-OH-AD) as a major product from sitosterol, with a 50% molar yield. Along with 9-OH-AD, both AD and 9α-hydroxylated metabolites with a partially degraded side-chain were formed from sitosterol by the mutant strain. The strain was unable to degrade 9-OH-AD, but degraded androsta-1,4-diene-3,17-dione (ADD), thus indicating a deficiency in steroid 1(2)-dehydrogenase and the presence of 9α-hydroxylase activity.

Bile acids are new products of a marine bacterium, Myroides sp. strain SM1 by Suppasil Maneerat; Teruhiko Nitoda; Hiroshi Kanzaki; Fusako Kawai (pp. 679-683).

Strain SM1 was isolated as a biosurfactant-producing microorganism from seawater and presumptively identified as Myroides sp., based on morphology, biochemical characteristics and 16S rDNA sequence. The strain produced surface-active compounds in marine broth, which were purified, using emulsification activity for n-hexadecane as an indicator. The purified compounds were identified by thin-layer chromatography, ¹H- and ¹³C-NMR spectra and fast atom bombardment mass spectrometry as cholic acid, deoxycholic acid and their glycine conjugates. Type strains of the genus Myroides, M. odoratus JCM7458 and M. odoramitimus JCM7460, also produced these compounds. Myroides sp. strain SM1 possessed a biosynthetic route to cholic acid from cholesterol. Thus, bile acids were found as new products of prokaryotic cells, genus Myroides.

Amino acid supplementation improves heterologous protein production by Saccharomyces cerevisiae in defined medium by Johann F. Görgens; Willem H. van Zyl; Johannes H. Knoetze; Bärbel Hahn-Hägerdal (pp. 684-691).

Supplementation of a chemically defined medium with amino acids or succinate to improve heterologous xylanase production by a prototrophic Saccharomyces cerevisiae transformant was investigated. The corresponding xylanase production during growth on ethanol in batch culture and in glucose-limited chemostat culture were quantified, as the native ADH2 promoter regulating xylanase expression was derepressed under these conditions. The addition of a balanced mixture of the preferred amino acids, Ala, Arg, Asn, Glu, Gln and Gly, improved both biomass and xylanase production, whereas several other individual amino acids inhibited biomass and/or xylanase production. Heterologous protein production by the recombinant yeast was also improved by supplementing the medium with succinate. The production of heterologous xylanase during growth on ethanol or glucose could thus be improved by supplementing metabolic precursors in the carbon- or nitrogen-metabolism.

Bacillus subtilis M4 decreases plant susceptibility towards fungal pathogens by increasing host resistance associated with differential gene expression by Marc Ongena; Francéline Duby; Emmanuel Jourdan; Thierry Beaudry; Victor Jadin; Jacques Dommes; Philippe Thonart (pp. 692-698).

Results presented in this paper describe the ability of Bacillus subtilis strain M4 to reduce disease incidence caused by Colletotrichum lagenarium and Pythium aphanidermatum on cucumber and tomato, respectively. Disease protection in both pathosystems was most probably due to induction of resistance in the host plant since experiments were designed in order to avoid any direct contact between the biocontrol agent and the pathogen. Pre-inoculation with strain M4 thus sensitised both plants to react more efficiently to subsequent pathogen infection. In cucumber, the use of endospores provided a disease control level similar to that obtained with vegetative cells. In contrast, a mixture of lipopeptides from the surfactin, iturin and fengycin families showed no resistance-inducing potential. Interestingly, treatment with strain M4 was also associated with significant changes in gene transcription in the host plant as revealed by cDNA-AFLP analyses. Several AFLP fragments corresponded to genes not expressed in control plants and specifically induced by the Bacillus treatment. In support to the macroscopic protective effect, this differential accumulation of mRNA also illustrates the plant reaction following perception of strain M4, and constitutes one of the very first examples of defence-associated modifications at the transcriptional level elicited by a non-pathogenic bacterium in a host plant.

Combined carbon and nitrogen removal from acetonitrile using algal–bacterial bioreactors by Raul Muñoz; Marco Jacinto; Benoit Guieysse; Bo Mattiasson (pp. 699-707).

When compared with Chlorella vulgaris, Scenedesmus obliquus and Selenastrum capricornutum, C. sorokiniana presented the highest tolerance to acetonitrile and the highest O₂ production capacity. It also supported the fastest acetonitrile biodegradation when mixed with a suitable acetonitrile-degrading bacterial consortium. Consequently, this microalga was tested in symbiosis with the bacterial culture for the continuous biodegradation of acetonitrile at 2 g l⁻¹ in a stirred tank photobioreactor and in a column photobioreactor under continuous illumination (250 μE m⁻² s⁻¹). Acetonitrile removal rates of up to 2.3 g l⁻¹ day⁻¹ and 1.9 g l⁻¹ day⁻¹ were achieved in the column photobioreactor and the stirred-tank photobioreactor, respectively, when operated at the shortest retention times tested (0.4 days, 0.6 days, respectively). In addition, when the stirred-tank photobioreactor was operated with a retention time of 3.5 days, the microbial culture was capable of assimilating up to 71% and nitrifying up to 12% of the NH₄⁺ theoretically released through the biodegradation of acetonitrile, thus reducing the need for subsequent nitrogen removal. This study suggests that complete removal of N-organics can be combined with a significant removal of nitrogen by using algal–bacterial systems and that further residual biomass digestion could pay-back part of the operation costs of the treatment plant.

Comparing activated sludge and aerobic granules as microbial inocula for phenol biodegradation by Stephen Tiong-Lee Tay; Benjamin Yan-Pui Moy; Abdul Majid Maszenan; Joo-Hwa Tay (pp. 708-713).

Activated sludge and acetate-fed granules were used as microbial inocula to start up two sequencing batch reactors (R1, R2) for phenol biodegradation. The reactors were operated in 4-h cycles at a phenol loading of 1.8 kg m⁻³ day⁻¹. The biomass in R1 failed to remove phenol and completely washed out after 4 days. R2 experienced initial difficulty in removing phenol, but the biomass acclimated quickly and effluent phenol concentrations declined to 0.3 mg l⁻¹ from day 3. The acetate-fed granules were covered with bacterial rods, but filamentous bacteria with sheaths, presumably to shield against toxicity, quickly emerged as the dominant morphotype upon phenol exposure. Bacterial adaptation to phenol also took the form of modifications in enzyme activity and increased production of extracellular polymers. 16S rRNA gene fingerprints revealed a slight decrease in bacterial diversity from day 0 to day 3 in R1, prior to process failure. In R2, a clear shift in community structure was observed as the seed evolved into phenol-degrading granules without losing species-richness. The results highlight the effectiveness of granules over activated sludge as seed for reactors treating toxic wastewaters.

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Applied Microbiology and Biotechnology (v.67, #5)