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Applied Microbiology and Biotechnology (v.79, #4)

Random genome deletion methods applicable to prokaryotes by Nobuaki Suzuki; Masayuki Inui; Hideaki Yukawa (pp. 519-526).

Through their enabling of simultaneous identification of multiple non-essential genes in a genome, large-segment genome deletion methods are an increasingly popular approach to minimize and tailor microbial genomes for specific functions. At present, difficulties in identifying target regions for deletion are a result of inadequate knowledge to define gene essentiality. Furthermore, with the majority of predicted open reading frames of completely sequenced genomes still annotated as putative genes, essential or important genes are found scattered throughout the genomes, limiting the size of non-essential segments that can be safely deleted in a single sweep. Recently described large-segment random genome deletion methods that utilize transposons enable the generation of random deletion strains, analysis of which makes identification of non-essential genes less tedious. Such and other efforts to determine the minimum genome content necessary for cell survival continue to accumulate important information that should help improve our understanding of genome function and evolution. This review presents an assessment of technological advancements of random genome deletion methods in prokaryotes to date.

Keywords: Large-segment deletion; Essential genes; Transposon; Minimum bacterial genome

Improvement of poly(γ-glutamic acid) biosynthesis and redistribution of metabolic flux with the presence of different additives in Bacillus subtilis CGMCC 0833 by Qun Wu; Hong Xu; Ningning Shi; Jun Yao; Sha Li; Pingkai Ouyang (pp. 527-535).

Tween-80, dimethyl sulfoxide (DMSO), and glycerol could be used as novel materials to regulate the central carbon metabolic pathway and improve γ-PGA biosynthesis by Bacillus subtilis CGMCC 0833. With glycerol in the medium, the activity of 2-oxoglutarate dehydrogenase complex at the key node of 2-oxoglutarate was depressed, more carbon flux distribution was directed to synthesize glutamate, the substrate of γ-PGA, which led to overproducing of γ-PGA, reached 31.7 g/l, compared to the original value of 26.7 g/l. When Tween-80 or DMSO was in the medium, the activity of isocitrate dehydrogenase was stimulated, the branch flux from 2-oxoglutarate to glutamate was also enhanced due to the increasing of total flux from iso-citrate to 2-oxoglutarate, then a large amount of glutamate was produced, and formation of γ-PGA was also improved, which was a different process compared with that of glycerol. Moreover, with the addition of Tween-80 or DMSO, cell membrane permeability was increased, which facilitated the uptake of extracellular substrates and the secretion of γ-PGA by this strain; therefore, γ-PGA production was further stimulated, and 34.4 and 32.7 g/l γ-PGA were obtained, respectively. This work firstly employed additives to improve the biosynthesis of γ-PGA and would be helpful in understanding the biosynthesis mechanism of γ-PGA by Bacillus species deeply.

Keywords: Bacillus subtilis CGMCC 0833; Cell membrane permeability; Flux redistribution; Metabolic engineering; Poly(γ-glutamic acid); Additive

Evaluation of complexed NO reduction mechanism in a chemical absorption–biological reduction integrated NO x removal system by Shi-Han Zhang; Xu-Hong Mi; Ling-Lin Cai; Jin-Lin Jiang; Wei Li (pp. 537-544).

Biological reduction of nitric oxide (NO) from Fe(II) ethylenediaminetetraacetic acid (EDTA)-NO to dinitrogen (N₂) is a core process for the continual nitrogen oxides (NO x) removal in the chemical absorption–biological reduction integrated approach. To explore the biological reduction of Fe(II)EDTA-NO, the stoichiometry and mechanism of Fe(II)EDTA-NO reduction with glucose or Fe(II)EDTA as electron donor were investigated. The experimental results indicate that the main product of complexed NO reduction is N₂, as there was no accumulation of nitrous oxide, ammonia, nitrite, or nitrate after the complete depletion of Fe(II)EDTA-NO. A transient accumulation of nitrous oxide (N₂O) suggests reduction of complexed NO proceeds with N₂O as an intermediate. Some quantitative data on the stoichiometry of the reaction are experimental support that reduction of complexed NO to N₂ actually works. In addition, glucose is the preferred and primary electron donor for complexed NO reduction. Fe(II)EDTA served as electron donor for the reduction of Fe(II)EDTA-NO even in the glucose excessive condition. A maximum reduction capacity as measured by NO (0.818 mM h⁻¹) is obtained at 4 mM of Fe(II)EDTA-NO using 5.6 mM of glucose as primary electron donor. These findings impact on the understanding of the mechanism of bacterial anaerobic Fe(II)EDTA-NO reduction and have implication for improving treatment methods of this integrated approach.

Keywords: Fe(II)EDTA-NO; Electron donor; Biological reduction; Mechanism; Stoichiometry

Uricase production by a recombinant Hansenula polymorpha strain harboring Candida utilis uricase gene by Zhiyu Chen; Zhaoyue Wang; Xiuping He; Xuena Guo; Weiwei Li; Borun Zhang (pp. 545-554).

Uricase is an important medical enzyme which can be used to determine urate in clinical analysis, to therapy gout, hyperuricemia, and tumor lysis syndrome. Uricase of Candida utilis was successfully expressed in Hansenula polymorpha under the control of methanol oxidase promoter using Saccharomyces cerevisiae α-factor signal peptide as the secretory sequence. Recombinant H. polymorpha MU200 with the highest extracellular uricase production was characterized with three copies of expression cassette and selected for process optimization for the production of recombinant enzyme. Among the parameters investigated in shaking flask cultures, the pH value of medium and inoculum size had great influence on the recombinant uricase production. The maximum extracellular uricase yield of 2.6 U/ml was obtained in shaking flask culture. The yield of recombinant uricase was significantly improved by the combined use of a high cell-density cultivation technique and a pH control strategy of switching culture pH from 5.5 to 6.5 in the induction phase. After induction for 58 h, the production of recombinant uricase reached 52.3 U/ml (about 2.1 g/l of protein) extracellularly and 60.3 U/ml (about 2.4 g/l) intracellularly in fed-batch fermentation, which are much higher than those expressed in other expression systems. To our knowledge, this is the first report about the heterologous expression of uricase in H. polymorpha.

Keywords: Candida utilis ; Recombinant uricase; pH control; Hansenula polymorpha

Cytochrome P450 (CYP105F2) from Streptomyces peucetius and its activity with oleandomycin by Pramod Shrestha; Tae-Jin Oh; Kwangkyong Liou; Jae Kyung Sohng (pp. 555-562).

The cytochrome P450 enzyme is one of the most versatile redox proteins and it is responsible for the oxidative metabolism of a wide variety of endogenous and exogenous compounds. The cytochrome P450 gene, CYP105F2, from Streptomyces peucetius was subcloned into the pET-32a(+) vector to overexpress the protein in E. coli BL21 (DE3) pLysS. The expressed enzyme was purified by fast protein liquid chromatography with a DEAE and UNO Q column. A 3D model was constructed based on the known crystallographic structures of cytochrome P450, and comparison with PikC and MoxA signified broad substrate specificity toward structurally diverse compounds. In addition, the in vitro hydroxylation of oleandomycin by purified CYP105F2 observed in liquid chromatography/mass spectrometry and mass/mass spectrometry indicated its flexibility towards alternative polyketides for the structural diversification of the macrolide by post-polyketide synthase hydroxylation.

Keywords: Cytochrome P450; Heterologous expression; Oleandomycin; Streptomyces peucetius ; Substrate flexibility

Efficient production of active form of recombinant cassava hydroxynitrile lyase using Escherichia coli in low-temperature culture by Hisashi Semba; Eita Ichige; Tadayuki Imanaka; Haruyuki Atomi; Hideki Aoyagi (pp. 563-569).

Overexpression and production of the high concentration of hydroxynitrile lyase from cassava (Manihot esculenta (MeHNL, EC 4.1.2.39)) were investigated. Hydroxynitrile lyase is a useful enzyme for the production of optically active cyanohydrin compounds. The production of MeHNL was increased by changing the rare codons of the original sequence of cassava MeHNL. However, most of the produced MeHNL was in the insoluble form. In order to increase the solubility of MeHNL, the effects of the cultivation temperature were investigated. When the cultivation temperature was reduced, the cell yield and the ratio of soluble MeHNL increased significantly. The enzyme activity and yield at low-temperature cultures (17 °C) were 850 times higher than those obtained at the optimum growth temperature of 37 °C. The rate of MeHNL production in the present study was calculated as 3,000 unit/h. Low-temperature cultivation was very effective in improving the productivity of the active form of MeHNL. Unlike the temperature-shift method, low-temperature cultivation has more potential for the large-scale production of MeHNL for the optically active cyanohydrin production.

Keywords: Hydroxynitrile lyase; Recombinant enzyme; High-density culture; Low temperature cultivation; Cyanohydrin

Characterization of a dITPase from the hyperthermophilic archaeon Thermococcus onnurineus NA1 and its application in PCR amplification by Yun-Jae Kim; Yong-Gu Ryu; Hyun Sook Lee; Yona Cho; Suk-Tae Kwon; Jung-Hyun Lee; Sung Gyun Kang (pp. 571-578).

In this study, we found that deoxyinosine triphosphate (dITP) could inhibit polymerase chain reaction (PCR) amplification of various family B-type DNA polymerases, and 0.93% dITP was spontaneously generated from deoxyadenosine triphosphate during PCR amplification. Thus, it was hypothesized that the generated dITP might have negative effect on PCR amplification of family B-type DNA polymerases. To overcome the inhibitory effect of dITP during PCR amplification, a dITP pyrophosphatase (dITPase) from Thermococcus onnurineus NA1 was applied to PCR amplification. Genomic analysis of the hyperthermophilic archaeon T. onnurineus NA1 revealed the presence of a 555-bp open reading frame with 48% similarity to HAM1-like dITPase from Methanocaldococcus jannaschii DSM2661 (NP_247195). The dITPase-encoding gene was cloned and expressed in Escherichia coli. The purified protein hydrolyzed dITP, not deoxyuridine triphosphate. Addition of the purified protein to PCR reactions using DNA polymerases from T. onnurineus NA1 and Pyrococcus furiosus significantly increased product yield, overcoming the inhibitory effect of dITP. This study shows the first representation that removing dITP using a dITPase enhances the PCR amplification yield of family B-type DNA polymerase.

Keywords: dITPase; dITP generation; Family B-type DNA polymerase; Hypoxanthine; Thermococcus

Bifunctional enhancement of a β-glucanase-xylanase fusion enzyme by optimization of peptide linkers by Ping Lu; Ming-Guang Feng (pp. 579-587).

The flexible peptides (GGGGS)n (n ≤ 3), the α-helical peptides (EAAAK)n (n ≤ 3) and two other peptides were used as linkers to construct bifunctional fusions of β-glucanase (Glu) and xylanase (Xyl) for improved catalytic efficiencies of both moieties. Eight Glu-Xyl fusion enzymes constructed with different linkers were all expressed as the proteins of ca. 46 kDa in Escherichia coli BL21 and displayed the activities of both β-glucanase and xylanase. Compared to all the characterized fusions with the parental enzymes, the catalytic efficiencies of the Glu and Xyl moieties were equivalent to 304–426% and 82–143% of the parental ones, respectively. The peptide linker (GGGGS)₂ resulted in the best fusion, whose catalytic efficiency had a net increase of 326% for the Glu and of 43% for the Xyl. The two moieties of a fusion with the linker (EAAAK)₃ also showed net increases of 262 and 31% in catalytic efficiency. Our results highlight, for the first time, the enhanced bifunctional activities of the Glu-Xyl fusion enzyme by optimizing the peptide linkers to separate the two moieties at a reasonable distance for beneficial interaction.

Keywords: Bifunctional fusion enzyme; β-glucanase; Xylanase; Peptide linker optimization

Purification and characterisation of a fructosyltransferase from Rhodotorula sp. by Saartje Hernalsteens; Francisco Maugeri (pp. 589-596).

The present work was devoted to investigations concerning the purification and characterisation of the fructooligosaccharide (FOS)-producing extracellular enzyme of Rhodotorula sp. LEB-V10. FOS are functional food ingredients showing prebiotic properties, meaning that it could stimulate selectively the growth and/or activity of probiotic bacteria in the gut. The purification of the enzyme was carried out according to the following sequential procedure: cell separation by centrifugation, recovering by ethanol precipitation and purification by anion exchange chromatography. The molecular weight was estimated to be 170 kDa by preparative gel filtration and 77 kDa by sodium dodecyl sulphate–polyacrylamide gel electrophoresis, signifying that the native enzyme exists as a dimer. With sucrose as substrate, the data failed to fit the Michaelis–Menten behaviour, rather showing a sigmoid shape similar to that of the allosteric enzymes (cooperative behaviour), requiring high sucrose concentrations to obtain high reaction rates. The enzyme showed both fructofuranosidase (FA) and fructosyl-transferase (FTA) activities. The optimum pH and temperature for FA activity were found to be around 4.0 and 72–75°C, respectively, while FTA showed optimum activity at pH 4.5 and 65–70°C. Both activities were very stable at temperatures below 66°C, while for FA, the enzyme was more stable at pH 4.0 and for FTA at pH 5.0.

Keywords: Rhodotorula sp.; Fructooligosaccharides; Enzyme characterisation; Enzyme kinetics

Homologous cloning, expression, and characterisation of a laccase from Streptomyces coelicolor and enzymatic decolourisation of an indigo dye by Etienne Dubé; François Shareck; Yves Hurtubise; Claude Daneault; Marc Beauregard (pp. 597-603).

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°C and was highly resistant toward conventional inhibitors. For instance, while the laccase of Trametes versicolor was completely inhibited by 1 mM NaN₃, 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.

Keywords: Laccase; Streptomyces coelicolor ; Homologous cloning; Expression; Characterisation; Dye decolourisation; Textile

A conserved Hpa2 protein has lytic activity against the bacterial cell wall in phytopathogenic Xanthomonas oryzae by Jiahuan Zhang; Xiaoyu Wang; Yan Zhang; Guiying Zhang; Jinsheng Wang (pp. 605-616).

The type III secretion system (TTSS) proteins form a needle-like structure injecting effector proteins into eukaryotic target cells. Although the TTSS forms an important pathway for bacterium–host interaction, its assembly process in vivo is poorly understood. The process is thought to include the opening of a pore before TTSS proteins are inserted into the bacterial cell wall. The proteins that break the bacterial cell wall have not yet been identified. We hypothesize that a hypersensitive response and pathogenicity (hrp) gene functions to digest the bacterial cell wall because it contains a conserved protein sequence similar to lytic transglycosylase. In this study, we cloned hrp-associated 2 (hpa2) genes from the bacteria Xanthomonas oryzae pathovars. We show in vitro that expressed Hpa2 protein has a lytic activity against bacterial cell walls. The analysis of a loss-of-function mutant of the hpa2 gene suggests that the hpa2 affects bacterial proliferation in host plants and a hypersensitive response in nonhost plants. As this is the first of such enzyme activity identified in the Hrp protein family, we speculate that the Hpa2 contributes to the assembly of the TTSS by enlarging gaps in the peptidoglycan meshwork of bacterial cell walls.

Keywords: Xanthomonas oryzae ; hrp-conserved; Type III secretion system; Soluble lytic transglycosylase; hrp-associated; Plant-inducible promoter

Novel auto-inducing expression systems for the development of whole-cell biocatalysts by Patrizia Di Gennaro; Silvia Ferrara; Giuseppina Bestetti; Guido Sello; Dafne Solera; Enrica Galli; Francesco Renzi; Giovanni Bertoni (pp. 617-625).

Novel expression systems for the development of whole-cell biocatalysts were generated. Their novelty consists both in the host, Pseudomonas putida, and in the ability to auto-induce the expression of genes of interest at the exhaustion of the carbon source used for the biomass growth. The auto-induction relies on new expression vectors developed in this study and based on the activator TouR from Pseudomonas sp. OX1, which was shown to mediate the activation of target promoters in an effector-independent growth-phase-dependent manner when the carbon source is exhausted at the onset of the stationary phase. We validated the suitability of these expression systems through the production of (S)-styrene oxide by the styrene monooxygenase from Pseudomonas fluorescens ST. The yields of epoxides produced by these biocatalysts in flask experiments showed to be as efficient as those currently available based on inducible Escherichia coli systems. In addition, a larger scale of biomass production showed no reduction of biocatalysis efficiency. Therefore, the systems developed in this study constitute a valid alternative to current expression systems to use in bioconversion processes.

Keywords: Biocatalysis; Styrene monooxygenase; Stereoselective epoxidation; Whole-cell biocatalyst; Expression vector

Insertion sequence-based cassette PCR: cultivation-independent isolation of γ-hexachlorocyclohexane-degrading genes from soil DNA by Genki Fuchu; Yoshiyuki Ohtsubo; Michihiro Ito; Ryo Miyazaki; Akira Ono; Yuji Nagata; Masataka Tsuda (pp. 627-632).

γ-Hexachlorocyclohexane (γ-HCH) is a highly chlorinated pesticide that has caused serious environmental problems. Based on the frequently observed association of insertion sequence IS6100 with lin genes for γ-HCH degradation in several γ-HCH-degrading bacterial strains isolated to date, DNA fragments flanked by two copies of IS6100 were amplified by nested polymerase chain reaction (PCR) technique using a DNA sample extracted from soil contaminated with HCH. Four distinct DNA fragments with sizes of 6.6, 2.6, 1.6, and 1.3 kb were obtained, three of which carried lin genes: the 6.6-kb fragment carried linD and linE as well as linR; the 2.6-kb fragment showed a truncated form of linF; and the 1.6-kb fragment carried linB. Our approach, named as insertion sequence (IS)-based cassette PCR, was successful in the isolation of the lin genes from HCH-contaminated soil without cultivation of host cells and is applicable for the culture-independent isolation of other functional genes bordered by other IS elements.

Keywords: γ-Hexachlorocyclohexane; Insertion sequence; Soil DNA

Biosynthesis of enantiopure (S)-3-hydroxybutyric acid in metabolically engineered Escherichia coli by Sang-Hyun Lee; Si Jae Park; Sang Yup Lee; Soon Ho Hong (pp. 633-641).

A biosynthetic pathway for the production of (S)-3-hydroxybutyric acid (S3HB) from glucose was established in recombinant Escherichia coli by introducing the β-ketothiolase gene from Ralstonia eutropha H16, the (S)-3-hydroxybutyryl-CoA dehydrogenase gene from R. eutropha H16, or Clostridium acetobutylicum ATCC824, and the 3-hydroxyisobutyryl-CoA hydrolase gene from Bacillus cereus ATCC14579. Artificial operon consisting of these genes was constructed and was expressed in E. coli BL21 (DE3) codon plus under T7 promoter by isopropyl β-d-thiogalactoside (IPTG) induction. Recombinant E. coli BL21 (DE3) codon plus expressing the β-ketothiolase gene, the (S)-3-hydroxybutyryl-CoA dehydrogenase gene, and the 3-hydroxyisobutyryl-CoA hydrolase gene could synthesize enantiomerically pure S3HB to the concentration of 0.61 g l⁻¹ from 20 g l⁻¹ of glucose in Luria–Bertani medium. Fed-batch cultures of recombinant E. coli BL21 (DE3) codon plus were carried out to achieve higher titer of S3HB with varying induction time and glucose concentration during fermentation. Protein expression was induced by addition of 1 mM IPTG when cell concentration reached 10 and 20 g l⁻¹ (OD₆₀₀ = 30 and 60), respectively. When protein expression was induced at 60 of OD₆₀₀ and glucose was fed to the concentration of 15 g l⁻¹, 10.3 g l⁻¹ of S3HB was obtained in 38 h with the S3HB productivity of 0.21 g l⁻¹h⁻¹. Lowering glucose concentration to 5 g l⁻¹ and induction of protein expression at 30 of OD₆₀₀ significantly reduced final S3HB concentration to 3.7 g l⁻¹, which also resulted in the decrease of the S3HB productivity to 0.05 g l⁻¹h⁻¹.

Keywords: (S)-3-hydroxybutyric acid; Escherichia coli ; Metabolic engineering; Fed-batch culture

Enhanced uptake of dissolved oxygen and glucose by Escherichia coli in a turbulent flow by Amer Al-Homoud; Miki Hondzo (pp. 643-655).

Laboratory experiments were conducted to study the effect of turbulence on Escherichia coli cells in an oscillating grid reactor under conditions of no oxygen transfer to the liquid phase. Fluid flow was quantified at a submillimeter resolution using a particle image velocimetry measuring technique. The root-mean-square estimates of the velocity gradient tensor components indicated the dominance of shear rate deformation in the fluid surrounding E. coli. The E. coli growth rate, dissolved oxygen (DO), and glucose uptake rates were facilitated by fluid-flow energy dissipation in the turbulent fluid. The Kolmogorov length scale (η K) and velocity (u K) underlined characteristic scales at which enhanced DO and glucose uptake by E. coli were determined in a turbulent flow in comparison to still-water controls. A first-order power–law relation between the mass transport to the cells and the moving fluid is developed. The combined effects of the enhanced rate of strain at η_K scale and uniform velocity at u K determined the facilitated DO and glucose fluxes to E. coli. The mass transport to the E. coli was modeled by the Sherwood (Sh)–Péclet (Pe) number relationship by $$Sh = 1 + { ext{1}}{ ext{.08 }}Pe_{u_K }^{0.62}$$ where $$Pe_{u_K } $$ is the Péclet number defined by the u K velocity scale. The proposed first-order model described experimental data fairly well.

Keywords: Fluid flow; E. coli ; Scale up; Turbulence; Mass transfer

Intergeneric coaggregation of strains isolated from phenol-degrading aerobic granules by Sunil S. Adav; Duu-Jong Lee; Juin-Yih Lai (pp. 657-661).

This work aims at exploring the intergeneric coaggregation of the pairs of strains, Acinetobacter calcoaceticus I6 and Bacillus thuringiensis I2 or Candida tropicalis I9 (with GenBank accession numbers EU250016, EU036759, and DQ515822) isolated from phenol-degrading aerobic granules. The I2 and I6 are functionally similar stains, while the I6 and I9 are functionally dissimilar strains. The lectin–saccharide interaction controlled the coaggregation of both the I2+I6 and I6+I9 pairs, with the protein adhesin being associated with the strain I6, and the complementary galactosamine-like or fucose-like sugar receptor with the strain I2 or I9, respectively. The rod-like I2 cells bridged the clusters of I2 or I6 cells to form aggregates, while the small I6 cells attached on and modified the surface of I9 to form aggregates.

Keywords: Coaggregation; Aerobic granules; Lectin–saccharide interaction

Identification of fungal metabolites of anticonvulsant drug carbamazepine by Su-Il Kang; Seo-Young Kang; Hor-Gil Hur (pp. 663-669).

Carbamazepine, which has been used in the treatments of epilepsy, is often found in the environment. Although metabolism of carbamazepine by humans and rats has been characterized, the environmental fate of carbamazepine has not been studied. In this study, two model fungi Cunninghamella elegans ATCC 9245 and Umbelopsis ramanniana R-56, which have previously shown diverse metabolic activities, were tested for metabolism of carbamazepine. Both fungi produced three metabolites each (C1–C3 and M1–M3). All six metabolites showed [M + H]⁺ at m/z 253, suggesting addition of one oxygen to the parent compound. High-performance liquid chromatography and liquid chromatography–mass spectrometric analysis detected 10, 11-dihydro-10, 11-epoxycarbamazepine as a major product (C3 (47%) and M3 (85%)) and 3-hydroxycarbamazepine (C2 (15%) and M2 (7%)) from carbamazepine through mixed mono-oxidation reactions in both fungal strains. C. elegans was confirmed to produce 2-hydroxycarbamazepine (C1 (38%)) while U. ramanniana produced a yet unidentified ring-hydroxylated metabolite (M1 (8%)). The current study suggests that carbamazepine is likely to be subjected to initially diverse mono-oxygenation reactions by fungal metabolisms, resulting in the formation of the corresponding metabolites, which were similarly found in mammalian metabolisms.

Keywords: Carbamazepine; Cunninghamella elegans ; Umbelopsis ramanniana ; Epoxidation; Mono-oxygenation

Adsorption of monorhamnolipid and dirhamnolipid on two Pseudomonas aeruginosa strains and the effect on cell surface hydrophobicity by Hua Zhong; Guang Ming Zeng; Jian Xiao Liu; Xiang Min Xu; Xing Zhong Yuan; Hai Yan Fu; Guo He Huang; Zhi Feng Liu; Ying Ding (pp. 671-677).

Previously, adsorption feature of a dirhamnolipid biosurfactant on diverse microbial cells was studied and the effect of the adsorption on cell surface hydrophobicity was compared. In this paper, the adsorption behavior of a monorhamnolipid and a dirhamnolipid on cells of two Pseudomonas aeruginosa strains was investigated in order to further reveal the influence of biosurfactant structure and cell property on the adsorption and the relation between the adsorption and cell surface hydrophobicity. Experimental results showed that the adsorption capacity of all the cells to monorhamnolipid was much stronger than to dirhamnolipid, and the rhamnolipid-sourced P. aeruginosa cells, no matter grown on glucose or hexadecane, released extra dirhamnolipid when aqueous concentration of dirhamnolipid was too high. Length of surfactant alkyl chain as well as the type of carbon source used to cultivate the cell adsorbents had only minor influence on the adsorption. The adsorption was assumed to be driven by polar interaction between the rhamnolipid molecules and the cell surface chemical groups. The directional orientation of the rhamnolipid molecules with hydrophobic moiety extending to the environment may account for the rapid increase of cell surface hydrophobicity at low aqueous concentrations of the surfactant, while the stable or decreased cell hydrophobicity was probably the consequence of multiple surfactant layer formation or hemimicelle accumulation.

Keywords: Biosurfactant; Monorhamnolipid; Dirhamnolipid; Adsorption; Cell surface hydrophobicity; Polar interaction

Organics and nitrogen removal and sludge stability in aerobic granular sludge membrane bioreactor by Jingfeng Wang; Xuan Wang; Zuguo Zhao; Junwen Li (pp. 679-685).

Novel aerobic granular sludge membrane bioreactor (GMBR) was established by combining aerobic granular sludge technology with membrane bioreactor (MBR). GMBR showed good organics removal and simultaneous nitrification and denitrification (SND) performances for synthesized wastewater. When influent total organic carbon (TOC) was 56.8–132.6 mg/L, the TOC removal of GMBR was 84.7–91.9%. When influent ammonia nitrogen was 28.1–38.4 mg/L, the ammonia nitrogen removal was 85.4–99.7%, and the total nitrogen removal was 41.7–78.4%. Moreover, batch experiments of sludge with different particle size demonstrated that: (1) flocculent sludge under aerobic condition almost have no denitrification capacity, (2) SND capacity was caused by the granular sludge, and (3) the denitrification rate and total nitrogen removal efficiency were enhanced with the increased particle size. In addition, study on the sludge morphology stability in GMBR showed that, although some granular sludge larger than 0.9 mm disaggregated at the beginning of operation, the granular sludge was able to maintain the stability of its granular morphology, and at the end of operation, the amount of granular sludge (larger than 0.18 mm) stabilized in GMBR was more than 56–62% of the total sludge concentration. The partial disaggregation of large granules is closely associated with the change of operating mode from sequencing batch reactor (SBR) system to MBR system.

Keywords: Aerobic granular sludge; Membrane bioreactor; Simultaneous nitrification and denitrification (SND); Sludge stability

Chitin purification from shrimp wastes by microbial deproteination and decalcification by Y. Xu; C. Gallert; J. Winter (pp. 687-697).

Chitin was purified from Penaeus monodon and Crangon crangon shells using a two-stage fermentation process with anaerobic deproteination followed by decalcification through homofermentative lactic acid fermentation. Deproteinating enrichment cultures from sewage sludge and ground meat (GM) were used with a proteolytic activity of 59 and 61 mg N l⁻¹ h⁻¹ with dried and 26 and 35 mg N l⁻¹ h⁻¹ with wet P. monodon shells. With 100 g wet cells of proteolytic bacteria per liter, protein removal was obtained in 42 h. An anaerobic spore-forming bacterium HP1 was isolated from enrichment GM. Its proteolytic activity was 76 U ml⁻¹ compared to 44 U ml⁻¹ of the consortium. Glucose was fermented with Lactobacillus casei MRS1 to lactic acid. At a pH of 3.6, calcium carbonate of the shells was solubilised. After deproteination and decalcification of P. monodon or C. crangon shells, the protein content was 5.8% or 6.7%, and the calcium content was 0.3% or 0.4%, respectively. The viscosity of the chitin from P. monodon and C. crangon was 45 and 135 mPa s, respectively, whereas purchased crab shell chitin (practical grade) had a viscosity of 21 mPa s, indicating a higher quality of biologically purified chitin.

Keywords: Shrimp waste; Chitin; Deproteination; Decalcification by lactic acid fermentation; Viscosity

Conditional chromosome splitting in Saccharomyces cerevisiae using the homing endonuclease PI-SceI by Kazuo Yamagishi; Minetaka Sugiyama; Yoshinobu Kaneko; Satoshi Harashima (pp. 699-706).

A novel chromosome engineering technology is described which enables conditional splitting of natural chromosomes in haploid cells of the yeast Saccharomyces cerevisiae. The technology consists of introduction of a recognition sequence for the homing endonuclease PI-SceI into the S. cerevisiae genome and conditional expression of the gene encoding the PI-SceI enzyme under the control of the MET3 promoter. To test the technology, we split chromosome V upstream of GLC7 by use of the autonomously replicating sequence (ARS)-added polymerase-chain-reaction-mediated chromosome-splitting (ARS-PCS) method that we recently developed. A recognition sequence for PI-SceI was subsequently introduced downstream of the GLC7 locus. Splitting was analyzed following induction of the PI-SceI-encoding gene. Approximately 50% of the clones tested had the expected minichromosome harboring only the GLC7 gene, suggesting that any desired chromosomal region may be converted into a new chromosome by use of this method. Because this technology allows initial construction of a strain harboring multiple constructs prior to subsequent induction of random chromosome loss events under specific selective conditions, we propose that this technology may be applicable to reconstructing the S. cerevisiae genome by means of combinatorial loss of minichromosomes.

Keywords: Single-gene chromosome; Conditional splitting; PI-SceI; Yeast; Genome reconstruction; PCS technology

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Applied Microbiology and Biotechnology (v.79, #4)