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Applied Microbiology and Biotechnology (v.90, #1)

Engineering tubulin: microtubule functionalization approaches for nanoscale device applications by Jennelle L. Malcos; William O. Hancock (pp. 1-10).

With the emergences of engineered devices at microscale and nanoscale dimensions, there is a growing need for controlled actuation and transport at these length scales. The kinesin–microtubule system provides a highly evolved biological transport system well suited for these tasks. Accordingly, there is an ongoing effort to create hybrid nanodevices that integrate biological components with engineered materials for applications such as biological separations, nanoscale assembly, and sensing. Adopting microtubules for these applications generally requires covalent attachment of biotin, fluorophores, or other biomolecules to tubulin enable surface or cargo attachment, or visualization. This review summarizes different strategies for functionalizing microtubules for application-focused as well as basic biological research. These functionalization strategies must maintain the integrity of microtubule proteins so that they do not depolymerize and can be transported by kinesin motors, while adding utility such as the ability to reversibly bind cargo. The relevant biochemical and electrical properties of microtubules are discussed, as well as strategies for microtubule stabilization and long-term storage. Next, attachment strategies, such as antibodies and DNA hybridization that have proven useful to date, are discussed in the context of ongoing hybrid nanodevice research. The review concludes with a discussion of less explored opportunities, such as harnessing the utility of tubulin posttranslational modifications and the use of recombinant tubulin that may enable future progress in nanodevice development.

Keywords: Fluorescence; Biotinylation; Microfabrication; Nanotechnology; Kinesin; Cytoskeleton

Mupirocin: biosynthesis, special features and applications of an antibiotic from a Gram-negative bacterium by Rachel Gurney; Christopher M. Thomas (pp. 11-21).

Mupirocin is a polyketide antibiotic produced by Pseudomonas fluorescens. The biosynthetic cluster encodes 6 type I polyketide synthase multifunctional proteins and 29 single function proteins. The biosynthetic pathway belongs to the trans-AT group in which acyltransferase activity is provided by a separate polypeptide rather than in-cis as found in the original type I polyketide synthases. Special features of this group are in-cis methyltransferase domains and a trans-acting HMG-CoA synthase-cassette which insert α- and β- methyl groups respectively while enoyl reductase domains are absent from the condensing modules. In addition, for the mupirocin system, there is no obvious loading mechanism for initiation of the polyketide chain and many aspects of the pathway remain to be elucidated. Mupirocin inhibits isoleucyl-tRNA synthetase and has been used since 1985 to help prevent infection by methicillin-resistant Staphylococcus aureus, particularly within hospitals. Resistance to mupirocin was first detected in 1987 and high-level resistance in S. aureus is due to a plasmid-encoded second isoleucyl-tRNA synthetase, a more eukaryotic-like enzyme. Recent analysis of the biosynthetic pathway for thiomarinols from marine bacteria opens up possibilities to modify mupirocin so as to overcome this resistance.

Keywords: Mupirocin; Polyketide; Antibiotic; Trans-AT; Staphylococcus aureus ; Pseudomonas fluorescens

Lights on and action! Controlling microbial gene expression by light by Thomas Drepper; Ulrich Krauss; Sonja Meyer zu Berstenhorst; Jörg Pietruszka; Karl-Erich Jaeger (pp. 23-40).

Light-mediated control of gene expression and thus of any protein function and metabolic process in living microbes is a rapidly developing field of research in the areas of functional genomics, systems biology, and biotechnology. The unique physical properties of the environmental factor light allow for an independent photocontrol of various microbial processes in a noninvasive and spatiotemporal fashion. This mini review describes recently developed strategies to generate photo-sensitive expression systems in bacteria and yeast. Naturally occurring and artificial photoswitches consisting of light-sensitive input domains derived from different photoreceptors and regulatory output domains are presented and individual properties of light-controlled expression systems are discussed.

Keywords: Optogenetics; Photocaging; Photo-switching; Light–oxygen–voltage (LOV) domains; Phytochrome (Phy); Photoactive yellow protein (PYP); Yeast two hybrid (Y2H); Control of gene expression

Recent developments in the taxonomic affiliation and phylogenetic positioning of fungi: impact in applied microbiology and environmental biotechnology by Kerstin Voigt; Paul M. Kirk (pp. 41-57).

The goal of modern taxonomy is to understand the relationships of living organisms in terms of evolutionary descent. Thereby, the relationships between living organisms are understood in terms of nested clades—every time a speciation event takes place, two new clades are produced. Life comprises three domains of living organisms, these are the Bacteria, the Archaea and the Eukaryota. Within the eukaryotic domain, the fungi form a monophyletic group of the eukaryotic crown group and are thus high up in the evolutionary hierarchy of life. Fungus-like organisms possess certain morphological features of fungi, such as the hyphal organization of the Oomycota or the spores and reproductive structures inside a fructification of plasmodiophorids (Plasmodiophoromycota) and slime moulds (Mycetozoa). The first group are algae which secondarily lost their plastids during evolution and contain cellulose in their cell walls. Both osmotrophic phyla, the Oomycota and the Plasmidiophoromycota belong to the Chromista and Rhizaria, respectively, whereas the last group, the cellular and plasmodial slime moulds (Mycetozoa) are phagotrophic amoeboid protists belonging to the Amoebozoa. These fungus-like organisms are not considered further in this review. The Fungi sensu stricto comprise a heterogenous, often inconspicuous group of microorganisms which (1) are primarily heterotrophic with an (2) osmotrophic style of nutrition containing (3) chitin and its derivatives in the cell wall. This review discusses species concepts and current strategies in fungal taxonomy, phylogenetic affiliations of miscellaneous fungus-like groups like the microsporidia, perspectives of fungal nomenclature, and their impact on natural product research.

Keywords: Fungal nomenclature; Systematics; Phylogenomics; Evolution; Aquatic and terrestrial fungi; Fungal barcoding

Glyceollin, a soybean phytoalexin with medicinal properties by Tzi Bun Ng; Xiu Juan Ye; Jack Ho Wong; Evandro Fei Fang; Yau Sang Chan; Wenliang Pan; Xiu Yun Ye; Stephen Cho Wing Sze; Kalin Yanbo Zhang; Fang Liu; He Xiang Wang (pp. 59-68).

This review covers the biosynthesis of glyceollin and its biological activities including antiproliferative/antitumor action (toward B16 melanoma cells, LNCaP prostate cancer cells, and BG-1 ovarian cancer cells), anti-estrogenic action (through estrogen receptors α- and β-), antibacterial action (toward Erwinia carotovora, Escherichia coli, Bradyrhizobium japonicum, Sinorhizobium fredii ), antinematode activity, and antifungal activity (toward Fusarium solani, Phakospora pachyrhizi, Diaporthe phaseolorum, Macrophomina phaseolina, Sclerotina sclerotiorum, Phytophthora sojae, Cercospora sojina, Phialophora gregata, and Rhizoctonia solani). Other activities include insulinotropic action and attenuation of vascular contractions in rat aorta.

Keywords: Soybean; Phytoalexin; Glyceollin; Medisin

Growth and recombinant protein expression with Escherichia coli in different batch cultivation media by Ralf Hortsch; Dirk Weuster-Botz (pp. 69-76).

Parallel operated milliliter-scale stirred tank bioreactors were applied for recombinant protein expression studies in simple batch experiments without pH titration. An enzymatic glucose release system (EnBase), a complex medium, and the frequently used LB and TB media were compared with regard to growth of Escherichia coli and recombinant protein expression (alcohol dehydrogenase (ADH) from Lactobacillus brevis and formate dehydrogenase (FDH) from Candida boidinii). Dissolved oxygen and pH were recorded online, optical densities were measured at-line, and the activities of ADH and FDH were analyzed offline. Best growth was observed in a complex medium with maximum dry cell weight concentrations of 14 g L⁻¹. EnBase cultivations enabled final dry cell weight concentrations between 6 and 8 g L⁻¹. The pH remained nearly constant in EnBase cultivations due to the continuous glucose release, showing the usefulness of this glucose release system especially for pH-sensitive bioprocesses. Cell-specific enzyme activities varied considerably depending on the different media used. Maximum specific ADH activities were measured with the complex medium, 6 h after induction with IPTG, whereas the highest specific FDH activities were achieved with the EnBase medium at low glucose release profiles 24 h after induction. Hence, depending on the recombinant protein, different medium compositions, times for induction, and times for cell harvest have to be evaluated to achieve efficient expression of recombinant proteins in E. coli. A rapid experimental evaluation can easily be performed with parallel batch operated small-scale stirred tank bioreactors.

Keywords: Milliliter stirred-tank bioreactor; Protein expression; Optical sensors; Alcohol dehydrogenase; Formate dehydrogenase; Batch cultivation

Lactobacillus acidophilus as a live vehicle for oral immunization against chicken anemia virus by Hassan Moeini; Raha Abdul Rahim; Abdul Rahman Omar; Norazizah Shafee; Khatijah Yusoff (pp. 77-88).

The AcmA binding domains of Lactococcus lactis were used to display the VP1 protein of chicken anemia virus (CAV) on Lactobacillus acidophilus. One and two repeats of the cell wall binding domain of acmA gene were amplified from L. lactis MG1363 genome and then inserted into co-expression vector, pBudCE4.1. The VP1 gene of CAV was then fused to the acmA sequences and the VP2 gene was cloned into the second MCS of the same vector before transformation into Escherichia coli. The expressed recombinant proteins were purified using a His-tag affinity column and mixed with a culture of L. acidophilus. Whole cell ELISA and immunofluorescence assay showed the binding of the recombinant VP1 protein on the surface of the bacterial cells. The lactobacilli cells carrying the CAV VP1 protein were used to immunize specific pathogen-free chickens through the oral route. A moderate level of neutralizing antibody to CAV was detected in the serum of the immunized chickens. A VP1-specific proliferative response was observed in splenocytes of the chickens after oral immunization. The vaccinated groups also showed increased levels of Th1 cytokines interleukin (IL)-2, IL-12, and IFN-γ. These observations suggest that L. acidophilus can be used in the delivery of vaccines to chickens.

Keywords: Chicken anemia virus; Oral vaccine; AcmA anchor protein; Surface display; Lactobacillus acidophilus

Biomass production and nutrient uptake by Neochloris oleoabundans in an open trough system by Kyle E. Murray; Frank G. Healy; Rebecca S. McCord; Jeremy A. Shields (pp. 89-95).

The purpose of this paper is to present biomass and nutrient uptake data from Neochloris oleoabundans production in an open trough system. The growth medium used was BG11, temperature ranged from 16.7 °C to 25.3 °C, and pH ranged from 5.52 to 9.94 because the customary pH increase during algal biomass production was moderated by incoming CO₂ gas streams (atmospheric, 2%, 4%, and 6% CO₂). Peak concentrations of algal biomass ranged from 643 to 970 mg L⁻¹, specific growth rates ranged from 0.15 to 0.37 day⁻¹, and doubling times ranged from 4.8 to 1.9 days. Carbon, nitrogen, and phosphorus were incorporated into the biomass at 0.05%, 8.3%, and 54% of supplied amounts. Open growth systems supplemented with CO₂ should be designed to regulate medium pH within the range of 6.3 to 7.1. Future research should examine various media and alternative carbon sources to decrease doubling times, increase peak concentrations, and optimize nutrient uptake.

Keywords: Biofuel; Microalgae; Wastewater treatment; Carbon

Pycnoporus laccase-mediated bioconversion of rutin to oligomers suitable for biotechnology applications by Eva Uzan; Bénédicte Portet; Christian Lubrano; Sandrine Milesi; Anne Favel; Laurence Lesage-Meessen; Anne Lomascolo (pp. 97-105).

The Pycnoporus fungi are white-rot basidiomycetes listed as food- and cosmetic-grade microorganisms. Three high redox potential laccases from Pycnoporus coccineus and Pycnoporus sanguineus were tested and compared, with the commercial Suberase® as reference, for their ability to synthesise natural active oligomers from rutin (quercetin-3-rutinoside, one of the best-known naturally occurring flavonoid glycosides). The aim of this work was to develop a process with technical parameters (solvent, temperature, reaction time and raw materials) that were easy to scale up for industrial production and compatible with cosmetic and pharmaceutical formulation guidelines. The aqueous mixture of glycerol/ethanol/buffer described in this study met this requirement and allowed the solubilisation of rutin and its oxidative bioconversion into oligomers. The four flavonoid oligomer mixtures synthesised using laccases as catalysts were analysed by high performance liquid chromatography–diode array detection–negative electrospray ionisation–multistage mass spectrometry. Their chromatographic elution profiles were compared and 16 compounds were characterised and identified as dimers and trimers of rutin. The oligorutins were different in Suberase® and Pycnoporus laccase reaction mixtures. They were evaluated for their antioxidant, anti-inflammatory and anti-ageing activities on specific enzymatic targets such as cyclooxygenase (COX-2) and human matrix metalloproteinase 3 (MMP-3). Expressed in terms of IC₅₀, the flavonoid oligomers displayed a 2.5- to 3-fold higher superoxide scavenging activity than monomeric rutin. Pycnoporus laccase and Suberase® oligorutins led to an inhibition of COX-2 of about 35% and 70%, respectively, while monomeric rutin showed a near-negligible inhibition effect, less than about 10%. The best results on MMP-3 activity were obtained with rutin oligomers from P. sanguineus IMB W006-2 laccase and Suberase® with about 70–75% inhibition.

Keywords: Laccase; Sustainable chemistry; Pycnoporus ; Oligomerisation; Rutin

Metabolic production of a novel polymer feedstock, 3-carboxy muconate, from vanillin by Aaron Gosling; S. Jane Fowler; Michael S. O’Shea; Melissa Straffon; Geoff Dumsday; Michael Zachariou (pp. 107-116).

Vanillin can be produced on a commercial scale by depolymerising renewable lignin. One product of microbial metabolism of vanillin by common soil microbes, such as Acinetobacter baylyi, is a tricarboxylic acid with a butadiene backbone known as 3-carboxy muconate (3CM). Three enzymes, 4-hydroxy benzaldehyde dehydrogenase, vanillate monooxygenase and protocatechuate 3,4-dioxygenase, catalyse the biotransformation of vanillin to 3CM. These three enzymes were metabolically engineered into an Escherichia coli host, giving a biocatalyst that converted vanillin into 3CM. The biocatalyst was found to give 100% yield of 3CM from 1 mM of vanillin after 39 h. The rate-limiting reaction was identified as the conversion of vanillate to 3,4-dihydroxybenzoate catalysed by vanillate monooxygenase. Low expression of the reductase subunit of this enzyme was identified as contributing to the reduced rate of this reaction. Proof of principle of a novel application for 3CM was demonstrated when it was converted into a trimethyl ester derivative and copolymerised with styrene.

Keywords: Biocatalysis; Metabolic engineering; Vanillin; 3-Carboxy muconate; Acinetobacter baylyi ; Unsaturated polyester

Identification and characterization of 4-hexylbenzoic acid and 4-nonyloxybenzoic acid as substrates of CYP102A1 by Rama Krishna Gudiminchi; Martha Sophia Smit (pp. 117-126).

CYP102A1 is an efficient medium- to long-chain fatty acid hydroxylase that is able to accept a wide range of non-natural substrates which bear no resemblance to the natural ones. 4-Hexylbenzoic acid (HBA) and 4-nonyloxybenzoic acid (NOBA) were identified as CYP102A1 substrates via screening studies using the BD Oxygen Biosensor System. Spectroscopic binding studies showed that these two substrates bind in the active site of CYP102A1 with K _d values of 2.6 ± 0.1 μM for HBA and 1.9 ± 0.2 μM for NOBA. NADPH consumption rates in the presence of HBA and NOBA were 45 ± 1 min⁻¹ and 61 ± 1 min⁻¹, respectively. The coupling efficiency for NADPH was 57% for NOBA, while it was 77% for HBA. During whole-cell biotransformations, HBA was converted into ω−1- and ω−2-hydroxyhexylbenzoic acid, whereas NOBA was oxidized to ω−2-hydroxynonyloxybenzoic acid and ω−2,ω−4-dihydroxynonyloxybenzoic acid. HBA was used as a fatty acid mimic to compare whole-cell biotransformations with cell-free extracts. Whole-cell biotransformations carried out in a biphasic system resulted in 86% conversion of 5 mM HBA, producing 3.8 mM ω−2- and 0.5 mM ω−1-hydroxyhexylbenzoic acid in 4 h with a turnover number of 4.1 min⁻¹, whereas 100% conversion of 5 mM HBA was obtained in 1 h with crude cell extracts and a cofactor regeneration system, giving a turnover number of 10.5 min⁻¹.

Keywords: CYP102A1; P450 BM-3; Substrate screening; Fluorescence-based oxygen consumption; 4-Hexylbenzoic acid; 4-Nonyloxybenzoic acid; Whole-cell biotransformation

Identification, cloning, heterologous expression, and characterization of a NADPH-dependent 7β-hydroxysteroid dehydrogenase from Collinsella aerofaciens by Luo Liu; Arno Aigner; Rolf D. Schmid (pp. 127-135).

A gene encoding an NADPH-dependent 7β-hydroxysteroid dehydrogenase (7β-HSDH) from Collinsella aerofaciens DSM 3979 (ATCC 25986, formerly Eubacterium aerofaciens) was identified and cloned in this study. Sequence comparison of the translated amino acid sequence suggests that the enzyme belongs to the short-chain dehydrogenase superfamily. This enzyme was expressed in Escherichia coli with a yield of 330 mg (5,828 U) per liter of culture. The enzyme catalyzes both the oxidation of ursodeoxycholic acid (UDA) forming 7-keto-lithocholic acid (KLA) and the reduction of KLA forming UDA acid in the presence of NADP⁺ or NADPH, respectively. In the presence of NADPH, 7β-HSDH can also reduce dehydrocholic acid. SDS-PAGE and gel filtration of the expressed and purified enzyme revealed a dimeric nature of 7β-HSDH with a size of 30 kDa for each subunit. If used for the oxidation of UDA, its pH optimum is between 9 and 10 whereas for the reduction of KLA and dehydrocholic acid it shows an optimum between pH 4 to 6. Usage of the enzyme for the biotransformation of KLA in a 0.5-g scale showed that this 7β-HSDH is a useful biocatalyst for producing UDA from suitable precursors in a preparative scale.

Keywords: Cholic acid; Hydroxysteroid dehydrogenase; Short-chain dehydrogenase; Ursodeoxycholic acid

Identification of a GH62 α-l-arabinofuranosidase specific for arabinoxylan produced by Penicillium chrysogenum by Tatsuji Sakamoto; Atsuhiro Ogura; Misako Inui; Sayaka Tokuda; Sachiko Hosokawa; Hideshi Ihara; Naoya Kasai (pp. 137-146).

An arabinoxylan arabinofuranohydrolase (AXS5) was purified from the culture filtrate of Penicillium chrysogenum 31B. A cDNA encoding AXS5 (axs5) was isolated by in vitro cloning using the N-terminal amino acid sequence of the native enzyme as a starting point. The deduced amino acid sequence of the axs5 gene has high similarities with those of arabinoxylan arabinofuranohydrolases of Aspergillus niger, Aspergillus tubingensis, and Aspergillus sojae. Module sequence analysis revealed that a “Glyco_hydro_62” was present at position 28–299 of AXS5. This is a family of α-l-arabinofuranosidases which are all members of glycoside hydrolase family 62. Recombinant AXS5 (rAXS5) expressed in Escherichia coli was highly active on arabinoxylan but not on branched sugar beet arabinan. ¹H-NMR analysis revealed that the rAXS5 cleaved arabinosyl side-chains linked to C-2 and C-3 of single-substituted xylose residues in arabinoxylan. Semi-quantitative RT-PCR analysis indicated that expression of the axs5 gene in P. chrysogenum 31B was strongly induced by adding d-xylose and arabinoxylan to the culture medium. Moreover, two binding sites of XlnR, a transcriptional activator that regulates the expression of the genes encoding xylanolytic enzymes, are present in the upstream region of the axs5 gene. These results suggest that AXS5 is involved in xylan degradation.

Keywords: Arabinoxylan arabinofuranohydrolase; α-l-Arabinofuranosidase; Glycoside hydrolase family 62; XlnR binding site; Penicillium chrysogenum

Bioconversion of substituted naphthalenes and β-eudesmol with the cytochrome P450 BM3 variant F87V by Norihiko Misawa; Miho Nodate; Toshihiko Otomatsu; Keiko Shimizu; Chie Kaido; Miho Kikuta; Akira Ideno; Hiroshi Ikenaga; Jun Ogawa; Sakayu Shimizu; Kazutoshi Shindo (pp. 147-157).

Bioconversion of various substituted naphthalenes that contain 1-methoxy- and 1-ethoxy-naphthalenes, methylnaphthalenes, dimethylnaphthalenes, and naphthalenecarboxylic acid methyl esters were performed using recombinant Escherichia coli cells, which expressed the gene coding for a cytochrome P450 BM3 variant F87V (P450 BM3 (F87V)) that was N-terminally fused to an archaeal peptidyl–prolyl cis-trans isomerase. In addition, bioconversion experiments with the same substrates were carried out using those that expressed the phnA1A2A3A4 genes for a polycyclic aromatic hydrocarbon (PAH)-dihydroxylating dioxygenase, which originated from a PAH-utilizing marine bacterium Cycloclasticus sp. strain A5. Consequently, a variety of mono-hydroxylated derivatives were generated from these substituted naphthalenes. Oxidative aryl coupling was found to produce a novel compound 4,4′-diethoxy-[2,2′]-binaphthalenyl-1,1′-diol from 1-ethoxynaphthalene with the E. coli cells expressing the P450 BM3 (F87V) gene. This recombinant E. coli was further shown to introduce the hydroxyl group regio- and stereo-specifically into a sesquiterpene β-eudesmol.

Keywords: P450 BM3; Cytochrome P450; Peptidyl–prolyl cis-trans isomerase; Dihydroxylating dioxygenase; Naphthalene; β-eudesmol

Discovery and characterization of d-phenylserine deaminase from Arthrobacter sp. TKS1 by Hisashi Muramatsu; Yuri Suzuki; Takeshi Imai; Sakuko Ueshima; Jun Ozaki; Yuji Matsui; Shin-ichiro Kato; Kouhei Ohnishi; Norihiro Kimoto; Hiroaki Yamamoto; Shinji Nagata (pp. 159-172).

We discovered a d-phenylserine deaminase that catalyzed the pyridoxal 5′-phosphate (PLP)-dependent deamination reaction from d-threo-phenylserine to phenylpyruvate in newly isolated Arthrobacter sp. TKS1. The enzyme was partially purified, and its N-terminal amino acid sequence was analyzed. Based on the sequence information, the gene encoding the enzyme was identified and expressed in Escherichia coli. The expressed protein was purified to homogeneity and characterized. The enzyme consisted of two identical 46-kDa subunits and showed maximum activity at pH 8.5 and 55°C. The enzyme was stable in the range of pH 7.5 to pH 8.5 and up to 50°C. The enzyme acted on the d-forms of β-hydroxy-α-amino acids, such as d-threo-phenylserine (K _m, 19 mM), d-serine (K _m, 5.8 mM), and d-threonine (K _m, 102 mM). As l-threonine, d-allo-threonine, l-allo-threonine, and dl-erythro-phenylserine were inert, the enzyme could distinguish d-threo-form from among the four stereoisomers of phenylserine or threonine. The enzyme was activated by ZnSO₄, CuSO₄, BaCl₂, and CoCl₂ and strongly inhibited by phenylhydrazine, sodium borohydride, hydroxylamine, and dl-penicillamine. The enzyme exhibited absorption maxima at 280 and around 415 nm. The enzyme has an N-terminal domain similar to that of alanine racemase, which belongs to the fold type III group of pyridoxal enzymes.

Keywords: d-Phenylserine deaminase; Arthrobacter sp. TKS1; Phenylserine metabolism; Pyridoxal 5′-phosphate

Mutational analysis of wheat (Triticum aestivum L.) nucleotide pyrophosphatase/phosphodiesterase shows the role of six amino acids in the catalytic mechanism by Iris J. Joye; Tim Beliën; Kristof Brijs; Wim Soetaert; Jan A. Delcour (pp. 173-180).

Nucleotide pyrophosphatases/phosphodiesterases (NPPs, PF01663) release nucleoside 5′-monophosphates from a wide range of nucleotide substrates. Only very recently, the first plant members of the NPP family were characterised (Joye et al. J Cereal Sci 51: 326–336, 2010), and little is known about their substrate-specifying residues. We elucidated the role of six amino acid residues of the recently identified and characterised Triticum aestivum L. NPP (Joye et al. J Cereal Sci 51: 326–336, 2010). Substitution of the highly conserved catalytic Thr132 into Ser or Ala completely abolished enzyme activity. Mutation of a highly conserved His255 residue into an apolar Ala suprisingly increased enzyme activity against most phosphodiester substrates. Four other residues moderately to highly conserved over NPPs of different organisms were studied as well. Mutation of the Asn153, Asn165 and Glu199 into an Arg, Ser and Asp residue, respectively, increased the relative enzyme activity against p-nitrophenyl phosphate. Furthermore, mutation of Phe194 into Ser increased the relative enzyme activity against adenosine 5′-monophosphate-containing substrates, although the overall enzyme activity of this mutant enzyme decreased. We conclude that the structural requirements and the conservation of the amino acids of the catalytic site of TaNPPr and, by extension, probably of all NPPs, are very stringent.

Keywords: Nucleotide pyrophosphatase/phosphodiesterase; Alkaline phosphatase; Mutagenesis

A rational design for hepatitis B virus X protein refolding and bioprocess development guided by second virial coefficient studies by Anindya Basu; Wei Ning Chen; Susanna Su Jan Leong (pp. 181-191).

The hepatitis B virus X (HBx) protein is well known for its role in hepatitis B virus infection that often leads to hepatocellular carcinoma. Despite the clinical importance of HBx, there is little progress in anti-HBx drug development strategies due to shortage of HBx from native sources. Consistent expression of HBx as insoluble inclusion bodies within various expression systems has largely hindered HBx manufacturing via economical biosynthesis routes. Confronted by this roadblock, this study aims to quantitatively understand HBx protein behaviour in solution that will guide the rational development of a refolding-based bioprocess for HBx production. Second virial coefficient (SVC) measurements were employed to study the effects of varying physicochemical parameters on HBx intermolecular protein interaction. The SVC results suggest that covalent HBx aggregates play a key role in protein destabilisation during refolding. The use of an SVC-optimised refolding environment yielded bioactive and soluble HBx proteins from the denatured–reduced inclusion body state. This study provides new knowledge on HBx solubility behaviour in vitro, which is important in structure–function elucidation behaviour of this hydrophobic protein. Importantly, a rational refolding-based Escherichia coli bioprocess that can deliver purified and soluble HBx at large scale is successfully developed, which opens the way for rapid preparation of soluble HBx for further clinical and characterisation studies.

Keywords: Hepatitis B virus X protein; Second virial coefficient; Solubility; Bioprocess; Refolding

Rapid evolution of arginine deiminase for improved anti-tumor activity by Ye Ni; Yongmei Liu; Ulrich Schwaneberg; Leilei Zhu; Na Li; Lifeng Li; Zhihao Sun (pp. 193-201).

Arginine deiminase (ADI), an arginine-degrading enzyme, has been studied as a potential anti-cancer agent for inhibiting arginine-auxotrophic tumors, such as melanomas and hepatocellular carcinomas. Based on our preliminary results, it was noticed that the optimum pH of ADI from Pseudomonas plecoglossicida (PpADI) was 6.0, and less than 10% of the activity was retained at pH 7.4 (pH of human plasma). Additionally, the K _m value for wild-type ADI (WT-ADI) was 2.88 mM (pH 6.0), which is over 20 times of the serum arginine level (100–120 μM). These are two major limitations for PpADI as a potential anti-cancer drug. A highly sensitive and efficient high-throughput screening strategy based on a modified diacetylmonoxime–thiosemicarbazide method was established to isolate ADI mutants with higher activity and lower K _m under physiological pH. Three improved mutants was selected from 650 variants after one round of ep-PCR, among which mutant 314 (M314: A128T, H404R, I410L) exhibiting the highest activity. Interestingly, sequence alignment shows that three amino acid substitutes in M314 are coincident with corresponding residues in ADI from Mycoplasma arginini. The specific activity of M314 (9.02 U/mg) is over 20-fold higher than that of WT-ADI (0.44 U/mg) at pH 7.4, and the K _m value was reduced to 0.65 mM (pH 7.4). Noticeably, the pH optimum was shifted from 6.0 to 6.5 in M314. Homology model of M314 was constructed to understand the molecular basis of the improved enzymatic properties. This work could provide promising drug candidate for curing arginine-auxotrophic cancers.

Keywords: Arginine deiminase; Directed evolution; pH optimum; Anti-tumor; Pseudomonas plecoglossicida ; Hepatocellular carcinomas

Processing and maturation of carboxypeptidase Y and alkaline phosphatase in Schizosaccharomyces pombe by Hiroyuki Mukaiyama; Tomoko Iwaki; Alimjan Idiris; Kaoru Takegawa (pp. 203-213).

Schizosaccharomyces pombe carboxypeptidase Y (CPY) is synthesized as a zymogen and transported into the vacuole where maturation and activation occurs. The 110-kDa S. pombe CPY precursor is processed twice and finally converted to a mature form consisting of polypeptides of approximately 19 and 32 kDa linked by a single disulfide bond. In Saccharomyces cerevisiae, maturation of CPY occurs mostly through the activity of vacuolar aspartyl protease Pep4p, whereas a Pep4p homolog has not been found in the S. pombe genome database. Based on analysis of protease-deficient mutants, we found that S. pombe CPY was not able to be processed or activated in isp6Δpsp3Δ double disruptants. Both Isp6p and Psp3p are subtilase-type serine proteases with related sequences. Moreover, alkaline phosphatase of S. pombe was found to be localized at the vacuolar membrane and was also unprocessed in isp6Δpsp3Δ double disruptants. Vacuolar localization of GFP-fused Isp6p and Psp3p was determined by fluorescence microscopy. These results suggest that the two serine proteases Isp6p and Psp3p are functional in the vacuole and are involved in proteolytic processing of vacuolar proteins.

Keywords: Schizosaccharomyces pombe ; CPY; Alkaline phosphatase; Isp6; Psp3

Involvement of a putative response regulator Brrg-1 in the regulation of sporulation, sensitivity to fungicides, and osmotic stress in Botrytis cinerea by Leiyan Yan; Qianqian Yang; Jinhua Jiang; Themis J. Michailides; Zhonghua Ma (pp. 215-226).

The response regulator protein is a core element of two-component signaling pathway. In this study, we investigated functions of BRRG-1 of Botrytis cinerea, a gene that encodes a putative response regulator protein, which is homologous to Rrg-1 in Neurospora crassa. The BRRG-1 gene deletion mutant ΔBrrg1-62 was unable to produce conidia. The mutant showed increased sensitivity to osmotic stress mediated by NaCl and KCl, and to oxidative stress generated by H₂O₂. Additionally, the mutant was more sensitive to the fungicides iprodione, fludioxonil, and triadimefon than the parental strain. Western-blot analysis showed that the Bos-2 protein, the putative downstream component of Brrg-1, was not phosphorylated in the ΔBrrg1-62. Real-time polymerase chain reaction assays showed that expression of BOS-2 also decreased significantly in the mutant. All of the defects were restored by genetic complementation of the ΔBrrg1-62 with the wild-type BRRG-1 gene. Plant inoculation tests showed that the mutant did not show changes in pathogenicity on rapeseed leaves. These results indicated that Brrg-1 is involved in the regulation of asexual development, sensitivity to iprodione, fludioxonil, and triadimefon fungicides, and adaptation to osmotic and oxidative stresses in B. cinerea.

Keywords: Botrytis cinerea ; Dicarboximide fungicide; Osmotic and oxidative stresses; Response regulator

C-terminal processing of tyrosinase is responsible for activation of Pholiota microspora proenzyme by Yasuko Kawamura-Konishi; Saya Maekawa; Mariko Tsuji; Hideyuki Goto (pp. 227-234).

Tyrosinase is expressed as a 67-kDa protein in Pholiota microspora (synonym Pholiota nameko), whereas the same enzyme purified from fruiting bodies of P. microspora is a 42-kDa protein that is cleaved with a C-terminal 25-kDa polypeptide from the 67-kDa protein. To confirm the role of C-terminal processing in enzyme activity, we expressed a recombinant 67-kDa tyrosinase in Escherichia coli cells. To obtain a soluble protein, the recombinant tyrosinase was expressed as a thioredoxin fusion protein with an enterokinase-cleavable site. Enterokinase digestion of the fusion protein produced a recombinant 67-kDa tyrosinase that did not have any catalytic activity. However, chymotrypsin digestion of the fusion protein produced a recombinant 44-kDa tyrosinase that was catalytically active and had a 25-kDa cleaved C-terminal. Kinetic parameters of the 44-kDa tyrosinase were similar to those of the 42-kDa tyrosinase purified from the fruiting bodies. These results suggest that tyrosinase is expressed in P. microspora as a latent 67-kDa proenzyme and is converted to the mature active 42-kDa enzyme by proteolytic processing of the C-terminal.

Keywords: Pholiota microspora ; Pholiota nameko ; Tyrosinase; Proenzyme; Activation

A proteomic analysis of the Pichia pastoris secretome in methanol-induced cultures by Chung-Jr Huang; Leonardo M. Damasceno; Kyle A. Anderson; Sheng Zhang; Lloyd J. Old; Carl A. Batt (pp. 235-247).

The secreted proteome of Pichia pastoris X-33 was investigated in methanol-induced cultures with a goal to enhance the secretion and purification of recombinant proteins. In a fed-batch fermentation at 30 °C, more host proteins were found in greater concentrations compared to cultures grown at 25 °C. Protein samples collected directly from the culture media at 25 °C, as well as separated by two-dimensional (2D) gel, were subjected to ESI-MS/MS analysis. A total of 75 proteins were identified in the media from different conditions including pre- and post-methanol induction and in a strain overexpressing a recombinant schistosomiasis vaccine, Sm14-C62V. The identified proteins include native secreted proteins and some intracellular proteins, most of which have low isoelectric points (pI < 6). 2D gel analyses further revealed important characteristics, such as abundance, degradation, and glycosylation of these identified proteins in this proteome. Cell wall-associated proteins involved in cell wall biogenesis, structure, and modification comprised the majority of the secreted proteins which have been identified. Intracellular proteins such as alcohol oxidase and superoxide dismutase were also found in the proteome, suggesting some degree of cell lysis. However, both protocols show that their concentrations are significantly lower than the native secreted proteins. This study identifies proteins secreted or released into the culture media in the methanol-induced fermentation cultures of P. pastoris X-33 and suggests potential biotechnology applications based on the discovery of this proteome.

Keywords: Pichia pastoris ; Secretome; Fermentation; Methanol-induced culture; ESI/MS/MS

Selection of a DGLA-producing mutant of the microalga Parietochloris incisa: I. Identification of mutation site and expression of VLC-PUFA biosynthesis genes by Umidjon Iskandarov; Inna Khozin-Goldberg; Zvi Cohen (pp. 249-256).

Chemical mutagenesis of the phototrophic green microalga Parietochloris incisa, producing high amounts of arachidonic acid (ARA), resulted in selection of a mutant deficient in ARA and rich in dihomo-γ-linolenic acid (DGLA) and thus ∆5 desaturase defective. The mutagenesis produced a nonsense mutation in the ∆5 desaturase gene, resulting in alteration of the 62nd codon TGG into a stop codon. Thus, the polypeptide encoded by the mutant ∆5 desaturase gene is severely truncated and biochemically inactive, as was confirmed by heterologous expression in Saccharomyces cerevisiae. The mutation did not affect the oleogenic nature of the strain, and the total fatty acid content in the mutant biomass reached 39%, in comparison to 34% in the wild type, after 14 days of nitrogen starvation; biomass yields amounted to 5.1 and 3.6 g/l, respectively. While in the wild type, DGLA and ARA comprised about 1% and 58% of total fatty acids, respectively; the mutation annulled ARA almost totally but increased the DGLA proportion to 32% only with a corresponding increase in the proportion of oleic acid. Consequently, DGLA comprised 12.3% of dry weight, in comparison to 19.4% ARA in the wild type. The expression profiles of the genes coding enzymes involved in VLC-PUFA biosynthesis, ∆12, ∆6, ∆5 desaturases and ∆6 PUFA elongase, during nitrogen starvation, were compared. The transcript levels of all four genes, which were coordinately up-regulated in the wild type, appeared to be drastically reduced in the mutant, indicating their co-regulated transcription.

Keywords: LC-PUFA; DGLA; ARA; Arachidonic acid; ∆5 Desaturase; Dihomo-γ-linolenic acid; Microalgae; MNNG mutagenesis; Parietochloris incisa ; VLC-PUFA

Production of arabitol from glycerol: strain screening and study of factors affecting production yield by Srujana Koganti; Tsung Min Kuo; Cletus P. Kurtzman; Nathan Smith; Lu-Kwang Ju (pp. 257-267).

Glycerol is a major by-product from biodiesel production, and developing new uses for glycerol is imperative to overall economics and sustainability of the biodiesel industry. With the aim of producing xylitol and/or arabitol as the value-added products from glycerol, 214 yeast strains, many osmotolerant, were first screened in this study. No strains were found to produce large amounts of xylitol as the dominant metabolite. Some produced polyol mixtures that might present difficulties to downstream separation and purification. Several Debaryomyces hansenii strains produced arabitol as the predominant metabolite with high yields, and D. hansenii strain SBP-1 (NRRL Y-7483) was chosen for further study on the effects of several growth conditions. The optimal temperature was found to be 30°C. Very low dissolved oxygen concentrations or anaerobic conditions inhibited polyol yields. Arabitol yield improved with increasing initial glycerol concentrations, reaching approximately 50% (w/w) with 150 g/L initial glycerol. However, the osmotic stress created by high salt concentrations (≥50 g/L) negatively affected arabitol production. Addition of glucose and xylose improved arabitol production while addition of sorbitol reduced production. Results from this work show that arabitol is a promising value-added product from glycerol using D. hansenii SBP-1 as the producing strain.

Keywords: Arabitol; Xylitol; Biodiesel; Glycerol; Osmotolerant yeast; Debaryomyces hansenii

Phototaxis in the magnetotactic bacterium Magnetospirillum magneticum strain AMB-1 is independent of magnetic fields by Chuanfang Chen; Qiufeng Ma; Wei Jiang; Tao Song (pp. 269-275).

Magnetotactic bacteria (MTB) can rapidly relocate to optimal habitats by magneto-aerotaxis. Little is known about MTB phototaxis, a response that might also aid navigation. In this study, we analyzed the relationship between phototaxis and magnetotaxis in Magnetospirillum magneticum strain AMB-1. Magnotactic AMB-1 cells migrated toward light, and migration increased with higher light intensity. This response was independent of wavelength, as AMB-1 cells migrated equally toward light from 400 to 750 nm. When AMB-1 cells were exposed to zero magnetic fields or to 0.2 mT magnetic fields that were opposite or orthogonal to the light beam, cells still migrated toward the light, indicating that phototaxis was independent of magnetotaxis. The R _mag value and coercive force (H _c) of AMB-1 increased when the bacteria were illuminated for 20 h, consistent with an increase in magnetosome synthesis or in magnetosome-containing cells. These results demonstrated that the M. magneticum AMB-1 responded to light as well as other environmental factors. To our knowledge, this is the first report of phototactic behavior in the bacteria of Magnetospirillum.

Keywords: Magnetotactic bacteria; Phototaxis; Magnetospirillum magneticum AMB-1

Toxicogenomic response of Mycobacterium bovis BCG to peracetic acid and a comparative analysis of the M. bovis BCG response to three oxidative disinfectants by Chantal W. Nde; Freshteh Toghrol; Hyeung-Jin Jang; William E. Bentley (pp. 277-304).

Tuberculosis is a leading cause of death worldwide and infects thousands of Americans annually. Mycobacterium bovis causes tuberculosis in humans and several animal species. Peracetic acid is an approved tuberculocide in hospital and domestic environments. This study presents for the first time the transcriptomic changes in M. bovis BCG after treatment with 0.1 mM peracetic acid for 10 and 20 min. This study also presents for the first time a comparison among the transcriptomic responses of M. bovis BCG to three oxidative disinfectants: peracetic acid, sodium hypochlorite, and hydrogen peroxide after 10 min of treatment. Results indicate that arginine biosynthesis, virulence, and oxidative stress response genes were upregulated after both peracetic acid treatment times. Three DNA repair genes were downregulated after 10 and 20 min and cell wall component genes were upregulated after 20 min. The devR–devS signal transduction system was upregulated after 10 min, suggesting a role in the protection against peracetic acid treatment. Results also suggest that peracetic acid and sodium hypochlorite both induce the expression of the ctpF gene which is upregulated in hypoxic environments. Further, this study reveals that in M. bovis BCG, hydrogen peroxide and peracetic acid both induce the expression of katG involved in oxidative stress response and the mbtD and mbtI genes involved in iron regulation/virulence.

Keywords: Microarrays; Mycobacterium bovis BCG; Peracetic acid; Sodium hypochlorite; Hydrogen peroxide; Transcriptomics

A halotolerant Alcanivorax sp. strain with potential application in saline soil remediation by Seyed Mohammad Mehdi Dastgheib; Mohamad Ali Amoozegar; Khosro Khajeh; Antonio Ventosa (pp. 305-312).

Biodegradation of petroleum compounds in saline environments seems intricate and needs more attention. In this study, tetracosane was used to enrich alkane-degrading bacteria from oil-contaminated saline soils. Among the isolates, strain Qtet3, with the highest 16s rRNA gene sequence similarity to Alcanivorax dieselolei B-5^T, was able to grow at a wide range of NaCl concentrations and was shown by GC analysis to degrade more than 90% of tetracosane in 10 days. This strain has at least two alkB genes and could grow on crude oil and diesel fuel, and utilize various pure aliphatic hydrocarbon substrates (from C₁₂ to C₃₄). Highly hydrophobic cell surfaces and lack of significant surface tension reduction in the media suggest that the main mechanism of the cells for accessing substrate is to attach directly to hydrocarbon particles. Application of this strain for remediating crude oil-contaminated soils irrigated with defined saline water demonstrated that this halotolerant bacterium could survive and grow in saline soils irrigated with NaCl solutions up to 5% w/v, with the highest hydrocarbon degradation of 26.1% observed at 2.5% NaCl. This strain is promising for future industrial applications especially in bioremediation of saline soils and wastes.

Keywords: Alcanivorax ; Hydrocarbon; Tetracosane; Biodegradation; Halotolerant

Ceramic honeycomb as support for covalent immobilization of laccase from Trametes versicolor and transformation of nuclear fast red by Regina Plagemann; Ludwig Jonas; Udo Kragl (pp. 313-320).

The covalent immobilization of laccase on an inorganic ceramic support was investigated. The intention was to find a system of enzyme and reactor for a universal immobilization procedure. Laccase from Trametes versicolor as model enzyme was chosen. The special honeycomb structure of the monolith can be applied for intensive mixing of the reaction compounds. An appropriate reactor with ceramic material was constructed allowing different setup for enzyme immobilization and its application. To test the success of the immobilization, 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) was used. The immobilized laccase was found to be stable over a time period of over 3 months. As an example for possible application for treatment of wastewater containing dyes, the conversion of nuclear fast red as model substrate was tested.

Keywords: Biocatalysis; Ceramic monolith; Immobilization; Laccase; Nuclear fast red

A novel conico-cylindrical flask aids easy identification of critical process parameters for cultivation of marine bacteria by Sayani Mitra; Sreyashi Sarkar; Ratan Gachhui; Joydeep Mukherjee (pp. 321-330).

A polymethylmethacrylate (PMMA) conico-cylindrical flask (CCF) with an inner arrangement consisting of eight equidistantly spaced rectangular strips mounted radially on a circular disk to provide additional surface area for microbial attachment and ports to allow air supply was employed for melanin production by Shewanella colwelliana and antibiotic production by Pseudoalteromonas rubra. The design allowed comparison of production between (1) CCF with hydrophobic surface (PMMA-CCF), (2) CCF with hydrophilic glass surface (GS-CCF), and (3) standard unbaffled Erlenmeyer flask (EF). Melanin production in the PMMA-CCF was higher by at most 33.5% and growth of S. colwelliana by at most 309.2% compared to the other vessels. Melanin synthesis was positively correlated with reactor surface area and hydrophobicity, suspended cell growth, and biofilm formation. Antibiotic production in the EF was higher by at most 83.3%, but growth of P. rubra was higher in the PMMA-CCF by at most 54.5% compared to the other vessels. A hydrophilic vessel surface, abundant air supply, but low shear stress enhanced antibiotic production. The CCF together with the EF allowed identification of the crucial parameters (vessel surface characteristics, growth, biofilm formation, and aeration) influencing productivity, knowledge of which in the initial stages of process development will facilitate informed decisions at the later phases.

Keywords: Polymethylmethacrylate; Melanin; Pseudoalteromonas rubra ; Hydrophilic; Hydrophobic; Biofilm

An in vitro protocol for direct isolation of potential probiotic lactobacilli from raw bovine milk and traditional fermented milks by Federico Baruzzi; Palmiro Poltronieri; Grazia Marina Quero; Maria Morea; Lorenzo Morelli (pp. 331-342).

A method for isolating potential probiotic lactobacilli directly from traditional milk-based foods was developed. The novel digestion/enrichment protocol was set up taking care to minimize the protective effect of milk proteins and fats and was validated testing three commercial fermented milks containing well-known probiotic Lactobacillus strains. Only probiotic bacteria claimed in the label were isolated from two out of three commercial fermented milks. The application of the new protocol to 15 raw milk samples and 6 traditional fermented milk samples made it feasible to isolate 11 potential probiotic Lactobacillus strains belonging to Lactobacillus brevis, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus plantarum, Lactobacillus reuteri, and Lactobacillus vaginalis species. Even though further analyses need to ascertain functional properties of these lactobacilli, the novel protocol set-up makes it feasible to isolate quickly potential probiotic strains from traditional milk-based foods reducing the amount of time required by traditional procedures that, in addition, do not allow to isolate microorganisms occurring as sub-dominant populations.

Keywords: Probiotic bacteria; Complex microflora; Lactobacillus acidophilus species group; Bile tolerance

Cunninghamella elegans biomass optimisation for textile wastewater biosorption treatment: an analytical and ecotoxicological approach by Valeria Tigini; Valeria Prigione; Ilaria Donelli; Antonella Anastasi; Giuliano Freddi; Pietro Giansanti; Antonella Mangiavillano; Giovanna Cristina Varese (pp. 343-352).

The effect of pre-treatments on the composition of Cunninghamella elegans biomass and on its biosorption yields in the treatment of simulated textile wastewaters was investigated. The inactivated biomass was subjected to physical treatments, such as oven drying and lyophilisation, and chemical treatments using acid or alkali. The wastewater colour, COD and toxicity variations were evaluated. The lyophilisation sped up the biosorption process, whereas the chemical pre-treatment changed the affinity of biomass for different dyes. The alkali per-treated biomass achieved the highest COD reduction in the treatment of alkali wastewaters, probably because no release of alkali-soluble biomass components occurred under the alkaline pH conditions. Accordingly, only the acid pre-treated biomass decreased the COD of the acidic effluent. The ecotoxicity test showed significant toxicity reduction after biosorption treatments, indicating that decolourisation corresponds to an actual detoxification of the treated wastewaters. Fourier transform infrared spectroscopy, differential scanning calorimetry and thermogravimetric analyses of biomasses allowed highlighting their main chemical and physical properties and the changes induced by the different pre-treatments, as well as the effect of the chemical species adsorbed from wastewaters.

Keywords: Biosorption; Wastewaters; Remediation; Fungi; Biomass characterisation

Effects of organic nutrients and growth factors on biostimulation of tributyltin removal by sediment microorganisms and Enterobacter cloacae by Arthit Sakultantimetha; Helen E. Keenan; Tara K. Beattie; Sornnarin Bangkedphol; Olga Cavoura (pp. 353-360).

Natural attenuation can reduce contamination of tributyltin (TBT), but persistence of the xenobiotic can cause long-term issues in the environment. Biostimulation is used to accelerate biodegradation. This study investigated the ability of individual organic nutrients and growth factors to enhance TBT biodegradation by sediment microorganisms (SED) and Enterobacter cloacae strain TISTR1971 (B3). The supplements that produced high biomass yield were selected for degradation enhancement. For TBT degradation at initial concentration of 0.1 mg/l, negative or limited degradation was observed in some selected supplements indicating that increasing the biomass did not necessarily promote degradation. Consequently, the addition of nutrients was expected to increase both dioxygenase activity and the degrader population. At different concentrations of supplements, a mixture of succinate/glycerol showed the highest removal for SED which reduced TBT by 77%, 75%, and 68% for 0.1×, 1×, and 10× supplement concentration, respectively. For B3, the addition of succinate showed degradation of 49% (0.1×), 75% (1×), and 77% (10×). Most nutrients and amino acids had an inhibitory effect at 1× or 10× levels. Excess amount of the nutrients added can inhibit the initial degradation of TBT. Therefore, TBT biostimulation requires supplements that increase the capability of TBT degraders at an appropriate amount.

Keywords: Biostimulation; Biodegradation; Enterobacter cloacae ; Sediment; Supplement; Tributyltin

Frequent concomitant presence of Desulfitobacterium spp. and “Dehalococcoides” spp. in chloroethene-dechlorinating microbial communities by Katia Rouzeau-Szynalski; Julien Maillard; Christof Holliger (pp. 361-368).

The presence of chloroethene dechlorination activity as well as several bacterial genera containing mainly organohalide-respiring members was investigated in 34 environmental samples from 18 different sites. Cultures inoculated with these environmental samples on tetrachloroethene and amended weekly with a seven organic electron donor mixture resulted in 11 enrichments with cis-DCE, ten with VC, and 11 with ethene as dechlorination end product, and only two where no dechlorination was observed. “Dehalococcoides” spp. and Desulfitobacterium spp. were detected in the majority of the environmental samples independently of the dechlorination end product formed. The concomitant presence of Dehalococcoides spp. and Desulfitobacterium spp. in the majority of the enrichments suggested that chloroethene dechlorination was probably the result of catalysis by at least two organohalide-respiring genera either in parallel or by stepwise catalysis. A more detailed study of one enrichment on cis-DCE suggested that in this culture Desulfitobacterium spp. as well as Dehalococcoides spp. dechlorinated cis-DCE whereas dechlorination of VC was only catalyzed by the latter.

Keywords: Organohalide respiration; Dehalorespiration; Halorespiration; Reductive dechlorination; Bioremediation

Structure and dynamics of nitrifier populations in a full-scale submerged membrane bioreactor during start-up by Tao Yu; Dong Li; Rong Qi; Sheng-tao Li; Shi-wei Xu; Min Yang (pp. 369-376).

Changes of microbial characteristics in a full-scale submerged membrane bioreactor system (capacity, 60,000 m³ day⁻¹) treating sewage were monitored over the start-up period (96 days). Fluorescence in situ hybridization analysis showed that the percentages of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (nitrobacter-related population) in total bacteria counted with DAPI staining increased significantly from 1.9% and 0.9% to 4.5% and 2.8%, corresponding to an increase of the specific ammonium oxidizing rate (from 0.06 to 0.12 kg N kg⁻¹ mixed liquor suspended solids (MLSS) per day) and the specific nitrate forming rate (from 0.05 to 0.10 kg N kg⁻¹ MLSS day⁻¹). Both the denaturing gradient gel electrophoresis of polymerase chain reaction and clone library results showed that the AOB was dominated by the genus Nitrosomonas, the diversity of which increased markedly with operational time. Most of the day 2 clones were closely related with the uncultured Nitrosomonas sp. clone Ninesprings-49S amoA gene (AY356450.1) originated from activated sludge, while the day 96 clone library showed a more diverse distribution characterized by the appearance of the oligotrophic nitrifiers like the Nitrosomonas oligotropha- and Nitrosomonas ureae-like bacteria, perhaps due to the interception by membrane and the low food-to-microorganisms ratio environment. The above results show that the membrane bioreactor system was characterized by the increased diversity and percentage of nitrifiers, which made it possible to achieve a stable and high efficient nitrification. Ammonia-oxidizing archaea with the changing population structures were also detected, but their roles for ammonia oxidation in the system need further studies.

Keywords: Membrane bioreactor; Nitrification; Ammonia-oxidizing bacteria; Ammonia-oxidizing archaea; Sludge characteristics

Direct ethanol production from cassava pulp using a surface-engineered yeast strain co-displaying two amylases, two cellulases, and β-glucosidase by Waraporn Apiwatanapiwat; Yoshinori Murata; Akihiko Kosugi; Ryosuke Yamada; Akihiko Kondo; Takamitsu Arai; Prapassorn Rugthaworn; Yutaka Mori (pp. 377-384).

In order to develop a method for producing fuel ethanol from cassava pulp using cell surface engineering (arming) technology, an arming yeast co-displaying α-amylase (α-AM), glucoamylase, endoglucanase, cellobiohydrase, and β-glucosidase on the surface of the yeast cells was constructed. The novel yeast strain, possessing the activities of all enzymes, was able to produce ethanol directly from soluble starch, barley β-glucan, and acid-treated Avicel. Cassava is a major crop in Southeast Asia and used mainly for starch production. In the starch manufacturing process, large amounts of solid wastes, called cassava pulp, are produced. The major components of cassava pulp are starch (approximately 60%) and cellulose fiber (approximately 30%). We attempted simultaneous saccharification and ethanol fermentation of cassava pulp with this arming yeast. During fermentation, ethanol concentration increased as the starch and cellulose fiber substrates contained in the cassava pulp decreased. The results clearly showed that the arming yeast was able to produce ethanol directly from cassava pulp without addition of any hydrolytic enzymes.

Keywords: Arming yeast; Cell surface display; Ethanol; Amylases; Cassava pulp; Cellulases

Optimization of process parameters for ethanol production from sugar cane molasses by Zymomonas mobilis using response surface methodology and genetic algorithm by Bodhisatta Maiti; Ankita Rathore; Saurav Srivastava; Mitali Shekhawat; Pradeep Srivastava (pp. 385-395).

Ethanol is a potential energy source and its production from renewable biomass has gained lot of popularity. There has been worldwide research to produce ethanol from regional inexpensive substrates. The present study deals with the optimization of process parameters (viz. temperature, pH, initial total reducing sugar (TRS) concentration in sugar cane molasses and fermentation time) for ethanol production from sugar cane molasses by Zymomonas mobilis using Box–Behnken experimental design and genetic algorithm (GA). An empirical model was developed through response surface methodology to analyze the effects of the process parameters on ethanol production. The data obtained after performing the experiments based on statistical design was utilized for regression analysis and analysis of variance studies. The regression equation obtained after regression analysis was used as a fitness function for the genetic algorithm. The GA optimization technique predicted a maximum ethanol yield of 59.59 g/L at temperature 31 °C, pH 5.13, initial TRS concentration 216 g/L and fermentation time 44 h. The maximum experimental ethanol yield obtained after applying GA was 58.4 g/L, which was in close agreement with the predicted value.

Keywords: Ethanol production; Zymomonas mobilis ; Sugar cane molasses; Genetic algorithm; Response surface methodology; Box–Behnken design

Erratum to: Discovery of a new tyrosinase-like enzyme family lacking a C-terminally processed domain: production and characterization of an Aspergillus oryzae catechol oxidase by Chiara Gasparetti; Greta Faccio; Mikko Arvas; Johanna Buchert; Markku Saloheimo; Kristiina Kruus (pp. 397-397).

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Applied Microbiology and Biotechnology (v.90, #1)

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Applied Microbiology and Biotechnology (v.90, #1)