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Applied Microbiology and Biotechnology (v.90, #6)

Microbial enzymes for aromatic compound hydroxylation by Patrizia Di Gennaro; Anna Bargna; Guido Sello (pp. 1817-1827).

Redox enzymes are ubiquitous in all living organisms. In fact, oxidation and reduction reactions are fundamental for the transformation of cellular and external compounds both for cell reproduction and for energy production. Redox enzymes share a common characteristic that is the capacity of transferring electrons to and from molecules. In addition, microorganisms contain many oxidative enzymes, and because they are relatively easier to cultivate and study, they have been investigated in details, in particular for potential use in biotechnological field. One important reaction that oxidative enzymes perform is the introduction of one or two oxygen atoms on aromatic compounds. The most representative classes of enzymes that perform this reaction are oxygenases/hydroxylases, peroxidases, and laccases; they differ in many aspects: the metal present in the active site, the used reductive cofactor, the final oxidant, and the number of electrons transferred in each step. Their essential features and mechanisms of action have been the subject of several studies, together with some structural analyses. This review reports recent developments and summarizes some of the most interesting results concerning both structural requirements and mechanisms implicated in aromatic hydroxylation.

Keywords: Oxygenases/hydroxylases; Peroxidases; Laccases; Bacteria; Aromatic compounds; Hydroxylation reaction

Fungal endophytes: unique plant inhabitants with great promises by Amal Hassan Aly; Abdessamad Debbab; Peter Proksch (pp. 1829-1845).

Fungal endophytes residing in the internal tissues of living plants occur in almost every plant on earth from the arctic to the tropics. The endophyte–host relationship is described as a balanced symbiotic continuum ranging from mutualism through commensalism to parasitism. This overview will highlight selected aspects of endophyte diversity, host specificity, endophyte–host interaction and communication as well as regulation of secondary metabolite production with emphasis on advanced genomic methods and their role in improving our current knowledge of endophytic associations. Furthermore, the chemical potential of endophytic fungi for drug discovery will be discussed with focus on the detection of pharmaceutically valuable plant constituents as products of fungal biosynthesis. In addition, selected examples of bioactive metabolites reported in recent years (2008–2010) from fungal endophytes residing in terrestrial plants are presented grouped according to their reported biological activities.

Keywords: Endophytic fungi; Host plants; Diversity; Host specificity; Bioactive secondary metabolites

Photocatalytic disinfection using titanium dioxide: spectrum and mechanism of antimicrobial activity by Howard A. Foster; Iram B. Ditta; Sajnu Varghese; Alex Steele (pp. 1847-1868).

The photocatalytic properties of titanium dioxide are well known and have many applications including the removal of organic contaminants and production of self-cleaning glass. There is an increasing interest in the application of the photocatalytic properties of TiO₂ for disinfection of surfaces, air and water. Reviews of the applications of photocatalysis in disinfection (Gamage and Zhang 2010; Chong et al., Wat Res 44(10):2997–3027, 2010) and of modelling of TiO₂ action have recently been published (Dalrymple et al. , Appl Catal B 98(1–2):27–38, 2010). In this review, we give an overview of the effects of photoactivated TiO₂ on microorganisms. The activity has been shown to be capable of killing a wide range of Gram-negative and Gram-positive bacteria, filamentous and unicellular fungi, algae, protozoa, mammalian viruses and bacteriophage. Resting stages, particularly bacterial endospores, fungal spores and protozoan cysts, are generally more resistant than the vegetative forms, possibly due to the increased cell wall thickness. The killing mechanism involves degradation of the cell wall and cytoplasmic membrane due to the production of reactive oxygen species such as hydroxyl radicals and hydrogen peroxide. This initially leads to leakage of cellular contents then cell lysis and may be followed by complete mineralisation of the organism. Killing is most efficient when there is close contact between the organisms and the TiO₂ catalyst. The killing activity is enhanced by the presence of other antimicrobial agents such as Cu and Ag.

Keywords: Antimicrobial; Disinfection; Mechanism; Photocatalysis; ROS; TiO₂ ; Titania

“Biofilmology”: a multidisciplinary review of the study of microbial biofilms by Esther Karunakaran; Joy Mukherjee; Bharathi Ramalingam; Catherine A. Biggs (pp. 1869-1881).

The observation of biofilm formation is not a new phenomenon. The prevalence and significance of biofilm and aggregate formation in various processes have encouraged extensive research in this field for more than 40 years. In this review, we highlight techniques from different disciplines that have been used to successfully describe the extracellular, surface and intracellular elements that are predominant in understanding biofilm formation. To reduce the complexities involved in studying biofilms, researchers in the past have generally taken a parts-based, disciplinary specific approach to understand the different components of biofilms in isolation from one another. Recently, a few studies have looked into combining the different techniques to achieve a more holistic understanding of biofilms, yet this approach is still in its infancy. In order to attain a global understanding of the processes involved in the formation of biofilms and to formulate effective biofilm control strategies, researchers in the next decade should recognise that the study of biofilms, i.e. biofilmology, has evolved into a discipline in its own right and that mutual cooperation between the various disciplines towards a multidisciplinary research vision is vital in this field.

Keywords: Biofilms; Biofilmology; Extracellular; Intracellular; Multidisciplinary; Surface

Naringinases: occurrence, characteristics, and applications by Maria H. Ribeiro (pp. 1883-1895).

Naringinase, an enzyme complex, is commercially attractive due to its potential usefulness in pharmaceutical and food industries. It is of particular interest in the biotransformation of steroids, antibiotics, and mainly of glycosides hydrolysis. Moreover, it can be used in citrus juices debittering and wine industries. Naringinase expresses activity on α-l-rhamnosidase and β-d-glucosidase. Many natural glycosides, including naringin, rutin, quercitrin, hesperidin, diosgene, and ter-phenyl glycosides, containing terminal α-rhamnose and β-glucose can act as substrates of naringinase. The sources, production, activity, biochemical properties, and substrate specificity of naringinase are reviewed, along with a description of the enzymatic deglycosylation systems and applications, concluding with the identification of areas which need further extensive studies.

Keywords: Naringinase; α-l-Rhamnosidase; β-d-Glucosidase; Glycosides; Deglycosylation; Immobilization

Asymmetric biocatalytic reduction of 3,5-bis(trifluoromethyl) acetophenone to (1R)-[3,5-bis(trifluoromethyl)phenyl] ethanol using whole cells of newly isolated Leifsonia xyli HS0904 by Pu Wang; Jin-Bo Cai; Qi Ouyang; Jun-Yao He; Hui-Zhen Su (pp. 1897-1904).

A novel bacterial strain HS0904 was isolated from a soil sample using 3,5-bis(trifluoromethyl) acetophenone as the sole carbon source. This bacterial isolate can asymmetrically reduce 3,5-bis(trifluoromethyl) acetophenone to (1R)-[3,5-bis(trifluoromethyl)phenyl] ethanol with high enantiometric excess (ee) value. Based on its morphological, physiological characteristics, Biolog, 16S rDNA sequence and phylogenetic analysis, strain HS0904 was identified as Leifsonia xyli HS0904. To our knowledge, this is the first reported case on the species L. xyli exhibited R-stereospecific carbonyl reductase and used for the preparation of chiral (1R)-[3,5-bis(trifluoromethyl)phenyl] ethanol. The optimization of parameters for microbial transformation of 3,5-bis(trifluoromethyl) acetophenone to (1R)-[3,5-bis(trifluoromethyl)phenyl] ethanol catalyzed by whole cells of L. xyli HS0904 was carried out by examining some key factors including buffer pH, reaction temperature, shaking speed, substrate concentration, and reaction time. The obtained optimized conditions for the bioreduction are as follows: buffer pH 8.0, 70 mM of 3,5-bis(trifluoromethyl) acetophenone, 100 g l⁻¹ of glucose as co-substrate, 200 g l⁻¹ of resting cells as biocatalyst, reaction for 30 h at 30 °C and 200 rpm. Under above conditions, 99.4% of product ee and best yield of 62% were obtained, respectively. The results indicated that isolate L. xyli HS0904 is a novel potential biocatalyst for the production of (1R)-[3,5-bis(trifluoromethyl)phenyl] ethanol.

Keywords: Bioreduction; Leifsonia xyli HS0904; Isolation; Identification; (1R)-[3,5-bis(trifluoromethyl)phenyl] ethanol

β-Cyclodextrins enhance artemisinin production in Artemisia annua suspension cell cultures by Miriana Durante; Sofia Caretto; Angela Quarta; Angelo De Paolis; Rossella Nisi; Giovanni Mita (pp. 1905-1913).

Artemisinin is a sesquiterpene antimalarial compound produced, though at low levels (0.1–1% dry weight), in Artemisia annua in which it accumulates in the glandular trichomes of the plant. Due to its antimalarial properties and short supply, efforts are being made to improve our understanding of artemisinin biosynthesis and its production. Native β-cyclodextrins, as well as the chemically modified heptakis(2,6-di-O-methyl)-β-cyclodextrin (DIMEB) and 2-hydroxypropyl-β-cyclodextrins, were added to the culture medium of A. annua suspension cultures, and their effects on artemisinin production were analysed. The effects of a joint cyclodextrin and methyl jasmonate treatment were also investigated. Fifty millimolar DIMEB, as well as a combination of 50 mM DIMEB and 100 μM methyl jasmonate, was highly effective in increasing the artemisinin levels in the culture medium. The observed artemisinin level (27 μmol g⁻¹ dry weight) was about 300-fold higher than that observed in untreated suspensions. The influence of β-cyclodextrins and methyl jasmonate on the expression of artemisinin biosynthetic genes was also investigated.

Keywords: Artemisia annua ; Artemisinin; Cyclodextrins; Methyl jasmonate; Plant cell cultures

Biosynthesis of isoprene in Escherichia coli via methylerythritol phosphate (MEP) pathway by Yaru Zhao; Jianming Yang; Bo Qin; Yonghao Li; Yuanzhang Sun; Sizheng Su; Mo Xian (pp. 1915-1922).

Isoprene is an aviation fuel of high quality and an important polymer building block in the synthetic chemistry industry. In light of high oil prices, sustained availability, and environmental concerns, isoprene from renewable materials is contemplated as a substitute for petroleum-based product. Escherichia coli with advantages over other wild microorganisms, is considered as a powerful host for biofuels and chemicals. Here, we constructed a synthetic pathway of isoprene in E. coli by introducing an isoprene synthase (ispS) gene from Populus nigra, which catalyzes the conversion of dimethylallyl diphosphate (DMAPP) to isoprene. To improve the isoprene production, we overexpressed the native 1-deoxy-d-xylulose-5-phosphate (DXP) synthase gene (dxs) and DXP reductoisomerase gene (dxr) in E. coli, which catalyzed the first step and the second step of MEP pathway, respectively. The fed-batch fermentation results showed that overexpression of DXS is helpful for the improvement of isoprene production. Surprisingly, heterologous expression of dxs and dxr from Bacillus subtilis in the E. coli expressing ispS resulted in a 2.3-fold enhancement of isoprene production (from 94 to 314 mg/L). The promising results showed that dxs and dxr from B. subtilis functioned more efficiently on the enhancement of isoprene production than native ones. This could be caused by the consequence of great difference in protein structures of the two original DXSs. It could be practical to produce isoprene in E. coli via MEP pathway through metabolic engineering. This work provides an alternative way for production of isoprene by engineered E. coli via MEP pathway through metabolic engineering.

Keywords: Isoprene; Biosynthesis; Escherichia coli ; MEP pathway

High-yield production of hydrophobins RodA and RodB from Aspergillus fumigatus in Pichia pastoris by Mona Højgaard Pedersen; Irina Borodina; Jacob Lange Moresco; Winnie Edith Svendsen; Jens Christian Frisvad; Ib Søndergaard (pp. 1923-1932).

Hydrophobins are small fungal proteins with amphipatic properties and the ability to self-assemble on a hydrophobic/hydrophilic interface; thus, many technical applications for hydrophobins have been suggested. The pathogenic fungus Aspergillus fumigatus expresses the hydrophobins RodA and RodB on the surface of its conidia. RodA is known to be of importance to the pathogenesis of the fungus, while the biological role of RodB is currently unknown. Here, we report the successful expression of both hydrophobins in Pichia pastoris and present fed-batch fermentation yields of 200–300 mg/l fermentation broth. Protein bands of expected sizes were detected by SDS-PAGE and western blotting, and the identity was further confirmed by tandem mass spectrometry. Both proteins were purified using his-affinity chromatography, and the high level of purity was verified by silver-stained SDS-PAGE. Recombinant RodA as well as rRodB were able to convert a glass surface from hydrophilic to hydrophobic similar to native RodA, but only rRodB was able to decrease the hydrophobicity of a Teflon-like surface to the same extent as native RodA, while rRodA showed this ability to a lesser extent. Recombinant RodA and native RodA showed a similar ability to emulsify air in water, while recombinant RodB could also emulsify oil in water better than the control protein bovine serum albumin (BSA). This is to our knowledge the first successful expression of hydrophobins from A. fumigatus in a eukaryote host, which makes it possible to further characterize both hydrophobins. Furthermore, the expression strategy and fed-batch production using P. pastoris may be transferred to hydrophobins from other species.

Keywords: Hydrophobin; RodA; RodB; Aspergillus fumigatus ; Pichia pastoris ; Protein expression; Fed-batch fermentation

Characterization of a bifunctional xylanase/endoglucanase from yak rumen microorganisms by Lei Chang; Mozhu Ding; Lei Bao; Yingzhi Chen; Jungang Zhou; Hong Lu (pp. 1933-1942).

A new gene, RuCelA, encoding a bifunctional xylanase/endoglucanase, was cloned from a metagenomic library of yak rumen microorganisms. RuCelA showed activity against xylan and carboxymethylcellulose (CMC), suggesting bifunctional xylanase/endoglucanase activity. The optimal conditions for xylanase and endoglucanase activities were 65°C, pH 7.0 and 50°C, pH 5.0, respectively. In addition, the presence of Co⁺ and Co²⁺ can greatly improve RuCelA's endoglucanase activity, while inhibits its xylanase activity. Further examination of substrate preference showed a higher activity against barley glucan and lichenin than against xylan and CMC. Using xylan and barley glucan as substrates, RuCelA displayed obvious synergistic effects with β-1,4-xylosidase and β-1,4-glucosidase. Generation of soluble oligosaccharides from lignocellulose is the key step in bioethanol production, and it is greatly notable that RuCelA can produce xylo-oligosaccharides and cello-oligosaccharides in the continuous saccharification of pretreated rice straw, which can be further degraded into fermentable sugars. Therefore, the bifunctional RuCelA distinguishes itself as an ideal candidate for industrial applications.

Keywords: Bifunctional; Metagenomic; Xylanase; Endoglucanase

Mutational analyses of Cry protein block7 polypeptides that facilitate the formation of protein inclusion in Escherichia coli by Tohru Hayakawa; Yoshitaka Shimizu; Tatsuhiko Ishida; Hiroshi Sakai (pp. 1943-1951).

4AaCter is the polypeptide from the C-terminal extension of mosquitocidal Cry4Aa toxin, and facilitates formation of protein inclusion in Escherichia coli. It has been demonstrated that the use of 4AaCter as a peptide tag results in the efficient production of heterologous protein in E. coli. It has also been demonstrated that proteins are integrated, without losing their biological activities, into the protein inclusions. Although the mechanism to form protein inclusions in E. coli is unclear, highly conserved block7 sequence in 4AaCter is thought to be one of the functional factors. In this study, to analyze the ability of block7 to form protein inclusion, synthetic genes encoding the block7 polypeptide from selected 15 Cry proteins were constructed and expressed to produce glutathione S-transferase fusions in E. coli. Unexpectedly, only three of them (Cry5Ba, Cry32Aa, and Cry48Aa) formed protein inclusion as efficiently as that of Cry4Aa (>90% efficiency). The efficiencies in forming the protein inclusion were ranging from 39% to 66% for most of the tested block7s, and almost no protein inclusion was observed in Cry47Aa block7. This suggested that the ability of block7 to form the protein inclusion may vary with the type of Cry protein or the amino acid sequences. Mutational analyses revealed that substitution of the hydrophobic amino acids in block7 significantly affected the formation of protein inclusion, suggesting some important roles of these hydrophobic amino acid residues. Present results will contribute to develop a compact peptide tag based on block7 which forms the protein inclusion efficiently.

Keywords: Bacillus thuringiensis ; Cry protein; Block7 polypeptide; Formation of protein inclusion; Escherichia coli ; Mutagenesis

A novel l-aspartate dehydrogenase from the mesophilic bacterium Pseudomonas aeruginosa PAO1: molecular characterization and application for l-aspartate production by Yinxia Li; Norika Kawakami; Henry Joseph Oduor Ogola; Hiroyuki Ashida; Takahiro Ishikawa; Hitoshi Shibata; Yoshihiro Sawa (pp. 1953-1962).

l-aspartate dehydrogenase (EC 1.4.1.21; l-AspDH) is a rare member of amino acid dehydrogenase superfamily and so far, two thermophilic enzymes have been reported. In our study, an ORF PA3505 encoding for a putative l-AspDH in the mesophilic bacterium Pseudomonas aeruginosa PAO1 was identified, cloned, and overexpressed in Escherichia coli. The homogeneously purified enzyme (PaeAspDH) was a dimeric protein with a molecular mass of about 28 kDa exhibiting a very high specific activity for l-aspartate (l-Asp) and oxaloacetate (OAA) of 127 and 147 U mg⁻¹, respectively. The enzyme was capable of utilizing both nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) as coenzyme. PaeAspDH showed a T _m value of 48°C for 20 min that was improved to approximately 60°C by the addition of 0.4 M NaCl or 30% glycerol. The apparent K _m values for OAA, NADH, and ammonia were 2.12, 0.045, and 10.1 mM, respectively; comparable results were observed with NADPH. The l-Asp production system B consisting of PaeAspDH, Bacillus subtilis malate dehydrogenase and E. coli fumarase, achieved a high level of l-Asp production (625 mM) from fumarate in fed-batch process with a molar conversion yield of 89.4%. Furthermore, the fermentative production system C released 33 mM of l-Asp after 50 h by using succinate as carbon source. This study represented an extensive characterization of the mesophilic AspDH and its potential applicability for efficient and attractive production of l-Asp. Our novel production systems are also hopeful for developing the new processes for other compounds production.

Keywords: l-Aspartate dehydrogenase; Mesophilic l-AspDH; Pseudomonas aeruginosa ; Thermostability; l-Aspartate production; Amino acid dehydrogenase

Characterization of a gene cluster and its putative promoter region for violacein biosynthesis in Pseudoalteromonas sp. 520P1 by Xi Zhang; Keiichi Enomoto (pp. 1963-1971).

Violacein, a purple pigment produced by some Gram-negative bacteria, has various physiological properties, such as antitrypanosomal and antitumoral activities. A gene cluster that encodes five enzymes, VioA–VioE, is responsible for synthesizing violacein. The expression of these enzymes is known to be regulated by a quorum sensing mechanism in Chromobacterium violaceum and Pseudoalteromonas sp. 520P1. To clarify the molecular mechanism of regulation of violacein synthesis, we cloned and characterized the gene cluster from Pseudoalteromonas sp. 520P1. A fosmid library of strain 520P1 was constructed and clones containing the gene cluster were isolated. The gene cluster was 7383 bp in length and encoded five enzyme genes, vioA–vioE. A putative promoter sequence was predicted in the upstream region of the cluster. In the promoter region, two contiguous palindromic sequences, a possible quorum sensing regulatory site, were found. However, the isolated Escherichia coli clones harboring the gene cluster and its upstream region were unable to produce violacein probably due to the lack of quorum sensing machinery for expression. To further examine the ability of vioA–vioE genes to synthesize violacein in vivo, the upstream promoter region was removed from the cluster and heterologous expression of the treated cluster was performed in E. coli using a recombinant pET vector with T7 promoter. Purple pigment was expressed, and the pigment was identified to be violacein using ultraviolet and visible light and HPLC analysis. These results will contribute to further studies regarding violacein biosynthesis and its mass production.

Keywords: Violacein; Pseudoalteromonas; Gene cluster; Fosmid library; Promoter prediction; Heterologous expression

Identification and characterization of a putative endolysin encoded by episomal phage phiSM101 of Clostridium perfringens by Hirofumi Nariya; Shigeru Miyata; Eiji Tamai; Hiroshi Sekiya; Jun Maki; Akinobu Okabe (pp. 1973-1979).

Clostridium perfringens produces potent toxins and histolytic enzymes, causing various diseases including life-threatening fulminant diseases in humans and other animals. Aiming at utilizing a phage endolysin as a therapeutic alternative to antibiotics, we surveyed the genome and bacteriophage sequences of C. perfringens. A phiSM101 muramidase gene (psm) revealed by this study can be assumed to encode an N-acetylmuramidase, since the N-terminal catalytic domain deduced from the gene shows high homology of those of N-acetylmuramidases. The psm gene is characteristic in that it is present in phiSM101, an episomal phage of enterotoxigenic C. perfringens type A strain, SM101, and also in that homologous genes are present in the genomes of all five C. perfringens toxin types. The psm gene was cloned and expressed in Escherichia coli as a protein histidine-tagged at the N-terminus (Psm-his). Psm-his was purified to homogeneity by nickel-charged immobilized metal affinity chromatography and anion-exchange chromatography. The purified enzyme lysed cells of all C. perfringens toxin types but not other clostridial species tested, as was shown by a turbidity reduction assay. These results indicate the Psm-his is useful as a cell-wall lytic enzyme and also suggest that it is potentially useful for biocontrol of this organism.

Keywords: Clostridium perfringens ; Bacteriophage; Endolysin; N-acetylmuramidase; N-acetylmuramoyl-l-alanine amidase

Transcriptome analysis shows differential gene expression in the saprotrophic to parasitic transition of Pochonia chlamydosporia by Laura C. Rosso; Mariella M. Finetti-Sialer; Penny R. Hirsch; Aurelio Ciancio; Brian R. Kerry; Ian M. Clark (pp. 1981-1994).

Expression profiles were identified in the fungus Pochonia chlamydosporia, a biological control agent of plant parasitic nematodes, through a cDNA-amplified fragment length polymorphism approach. Two isolates with different host ranges, IMI 380407 and IMI 331547, were assayed in conditions of saprotrophic-to-parasitic transition, through in vitro assays. Gene expression profiles from three different nutritional conditions and four sampling times were generated, with eggs of host nematodes Globodera pallida and Meloidogyne incognita. Expression of transcripts changed in RNA fingerprints obtained under different nutritional stresses (starvation in presence/absence of eggs, or rich growth media). Transcript derived fragments (TDFs) obtained from the expression profiles corresponded to 6,800 products. A subset was sequenced and their expression profile confirmed through RT PCR. A total of 57 TDFs were selected for further analysis, based on similarities to translated or annotated sequences. Genes expressed during egg parasitism for both IMI 380407 and IMI 331547 were involved in metabolic functions, cellular signal regulation, cellular transport, regulation of gene expression, DNA repair, and other unknown functions. Multivariate analysis of TDF expression showed three groups for IMI 380407 and one for IMI 331547, each characterized by expression of genes related to eggs parasitism. Common amplification profiles among TDF clusters from both isolates also reflected a pool of constitutive genes, not affected by the nutritional conditions and nematode associations, related to general metabolic functions. The differential expression of parasitism related genes suggest a network of induced/repressed products, playing a role in fungal signaling and infection, with partial overlaps in host infection and parasitism traits.

Keywords: Transcriptomics; Nematode eggs; Parasitism; Differential gene expression; Saprotrophic; Pochonia chlamydosporia

Effects of hydroxypropyl-β-cyclodextrin on cell growth, activity, and integrity of steroid-transforming Arthrobacter simplex and Mycobacterium sp. by Yanbing Shen; Min Wang; Liting Zhang; Yinhu Ma; Bing Ma; Yu Zheng; Hao Liu; Jianmei Luo (pp. 1995-2003).

A comparative investigation was performed on the effects of hydroxypropyl-β-cyclodextrin (HP-β-CD) on the growth, biocatalytic activity, and cell integrity of Arthrobacter simplex TCCC 11037 (ASP) and Mycobacterium sp. NRRL B-3683 (MSP). The addition of HP-β-CD to ASP medium improved its cell wall permeability for lipophilic compounds but significantly inhibited its growth and biocatalytic activity. On the other hand, the addition of HP-β-CD to MSP broth had lesser effects. Atomic force microscopy scanning analysis revealed that HP-β-CD damaged the cell integrity in ASP, especially the outermost cell surface structure, but not in MSP, which remained intact, owing to the differences in their cell wall and cell membrane composition. Protein leaking and lipid content in ASP increased with increased HP-β-CD concentration, indicating possible alterations in ASP cell membrane features caused by HP-β-CD. These alterations may also explain the slow cell growth and decreased cell ΔΨm in ASP upon the addition of HP-β-CD.

Keywords: Steroid; Hydroxypropyl-β-cyclodextrin; Arthrobacter simplex ; Mycobacterium sp.; Biotransformation

The ncgl1108 (PheP _Cg) gene encodes a new l-Phe transporter in Corynebacterium glutamicum by Zhi Zhao; Jiu-Yuan Ding; Tang Li; Ning-Yi Zhou; Shuang-Jiang Liu (pp. 2005-2013).

Corynebacterium glutamicum played a central role in the establishment of fermentative production of amino acids, and it is a model for genetic and physiological studies. The general aromatic amino acid transporter, AroP Cg, was the sole functionally identified aromatic amino acid transporter from C. glutamicum. In this study, the ncgl1108 (named as pheP Cg), which is located upstream of the genetic cluster (ncgl1110 ∼ ncgl1113) for resorcinol catabolism, was identified as a new l-Phe specific transporter from C. glutamicum RES167. The disruption of pheP Cg resulted in RES167∆ncgl1108, and this mutant showed decreased growth on l-Phe (as nitrogen source) but not on l-Tyr or l-Trp. Uptake assays with unlabeled and ¹⁴C-labeled l-Phe and l-Tyr indicated that the mutants RES167∆ncgl1108 showed significant reduction in l-Phe uptake than RES167. Expression of pheP Cg in RES167∆ncgl1108/pGXKZ1 or RES167∆(ncgl1108-aroP Cg)/pGXKZ1 restored their ability to uptake for l-Phe and growth on l-Phe. The uptake of l-Phe was not inhibited by nine amino acids but by l-Tyr. The K _m and V _max values of RES167∆(ncgl1108-aroP Cg)/pGXKZ1 for l-Phe were determined to be 10.4 ± 1.5 μM and 1.2 ± 0.1 nmol min⁻¹ (mg DW)⁻¹, respectively, which are different from K _m and V _max values of RES167∆(ncgl1108-aroP Cg) for l-Phe [4.0 ± 0.4 μM and 0.6 ± 0.1 nmol min⁻¹ (mg DW)⁻¹]. In conclusion, this PheP Cg is a new l-Phe transporter in C. glutamicum.

Keywords: Aromatic amino acid transporter; Corynebacterium glutamicum ; PheP Cg ; AroP Cg ; l-Phenylalanine

Microbial community of sulfate-reducing up-flow sludge bed in the SANI^® process for saline sewage treatment by Jin Wang; Manyuan Shi; Hui Lu; Di Wu; Ming-Fei Shao; Tong Zhang; George A. Ekama; Mark C. M. van Loosdrecht; Guang-Hao Chen (pp. 2015-2025).

This study investigated the microbial community of the sulfate-reducing up-flow sludge bed (SRUSB) of a novel sulfate reduction, autotrophic denitrification, and nitrification integrated (SANI®) process for saline sewage treatment. The investigation involved a lab-scale SANI® system treating synthetic saline sewage and a pilot-scale SANI® plant treating 10 m³/day of screened saline sewage. Sulfate-reducing bacteria (SRB) were the dominant population, responsible for more than 80% of the chemical oxygen demand removal, and no methane-producing archaea were detected in both SRUSBs. Thermotogales-like bacteria were the dominant SRB in the pilot-scale SRUSB while Desulforhopalus-like bacteria were the major species in the lab-scale SRUSB.

Keywords: Microbial community analysis; SANI® process; Sulfate-reducing up-flow sludge bed (SRUSB); Saline sewage; Sulfate-reducing bacteria (SRB)

Enhancing Jatropha oil extraction yield from the kernels assisted by a xylan-degrading bacterium to preserve protein structure by Ahmad Marasabessy; Maelita R. Moeis; Johan P. M. Sanders; Ruud A. Weusthuis (pp. 2027-2036).

We investigated the use of bacterial cells isolated from paddy crab for the extraction of oil from Jatropha seed kernels in aqueous media while simultaneously preserving the protein structures of this protein-rich endosperm. A bacterial strain—which was marked as MB4 and identified by means of 16S rDNA sequencing and physiological characterization as either Bacillus pumilus or Bacillus altitudinis—enhanced the extraction yield of Jatropha oil. The incubation of an MB4 starter culture with preheated kernel slurry in aqueous media with the initial pH of 5.5 at 37 °C for 6 h liberated 73% w/w of the Jatropha oil. Since MB4 produces xylanases, it is suggested that strain MB4 facilitates oil liberation via degradation of hemicelluloses which form the oil-containing cell wall structure of the kernel. After MB4 assisted oil extraction, SDS-PAGE analysis showed that the majority of Jatropha proteins were preserved in the solid phase of the extraction residues. The advantages offered by this process are: protein in the residue can be further processed for other applications, no purified enzyme preparation is needed, and the resulting oil can be used for biodiesel production.

Keywords: Jatropha curcas ; Aqueous oil extraction; Bacillus pumilus ; Bacillus altitudinis ; Protease; Xylanase

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Applied Microbiology and Biotechnology (v.90, #6)