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Applied Microbiology and Biotechnology (v.97, #2)

Influence of mass transfer on stable isotope fractionation by Martin Thullner; Anko Fischer; Hans-Hermann Richnow; Lukas Y. Wick (pp. 441-452).

Biodegradation of contaminants is a common remediation strategy for subsurface environments. To monitor the success of such remediation means a quantitative assessment of biodegradation at the field scale is required. Nevertheless, the reliable quantification of the in situ biodegradation process it is still a major challenge. Compound-specific stable isotope analysis has become an established method for the qualitative analysis of biodegradation in the field and this method is also proposed for a quantitative analysis. However, to use stable isotope data to obtain quantitative information on in situ biodegradation requires among others knowledge on the influence of mass transfer processes on the observed stable isotope fractionation. This paper reviews recent findings on the influence of mass transfer processes on stable isotope fractionation and on the quantitative interpretation of isotope data. Focus will be given on small-scale mass transfer processes controlling the bioavailability of contaminants. Such bioavailability limitations are known to affect the biodegradation rate and have recently been shown to affect stable isotope fractionation, too. Theoretical as well as experimental studies addressing the link between bioavailability and stable isotope fractionation are reviewed and the implications for assessing biodegradation in the field are discussed.

Keywords: Bioavailability; Compound-specific stable isotope analysis; CSIA; Biodegradation; Groundwater contamination

Preparation of biosilica structures from frustules of diatoms and their applications: current state and perspectives by Yu Wang; Jun Cai; Yonggang Jiang; Xinggang Jiang; Deyuan Zhang (pp. 453-460).

Frustules, the silica shells of diatoms, have unique porous architectures with good mechanical strength. In recent years, biologists have learned more about the mechanism of biosilica shells formation; meanwhile, physicists have revealed their optical and microfluidic properties, and chemists have identified ways to modify them into various materials while maintaining their hierarchical structures. These efforts have provided more opportunities to use biosilica structures in microsystems and other commercial products. This review focuses on the preparation of biosilica structures and their applications, especially in the development of microdevices. We discuss existing methods of extracting biosilica from diatomite and diatoms, introduce methods of separating biosilica structures by shape and sizes, and summarize recent studies on diatom-based devices used for biosensing, drug delivery, and energy applications. In addition, we introduce some new findings on diatoms, such as the elastic deformable characteristics of biosilica structures, and offer perspectives on planting diatom biosilica in microsystems.

Keywords: Frustules; Nanotechnology; Biomimetic; Microfabrication; Elasticity; Microsystems

Enzyme-catalyzed protein crosslinking by Tobias Heck; Greta Faccio; Michael Richter; Linda Thöny-Meyer (pp. 461-475).

The process of protein crosslinking comprises the chemical, enzymatic, or chemoenzymatic formation of new covalent bonds between polypeptides. This allows (1) the site-directed coupling of proteins with distinct properties and (2) the de novo assembly of polymeric protein networks. Transferases, hydrolases, and oxidoreductases can be employed as catalysts for the synthesis of crosslinked proteins, thereby complementing chemical crosslinking strategies. Here, we review enzymatic approaches that are used for protein crosslinking at the industrial level or have shown promising potential in investigations on the lab-scale. We illustrate the underlying mechanisms of crosslink formation and point out the roles of the enzymes in their natural environments. Additionally, we discuss advantages and drawbacks of the enzyme-based crosslinking strategies and their potential for different applications.

Keywords: Cross-linking; Conjugation; Ligation; Fusion proteins; Transglutaminase; Sortase A

Macro and trace mineral constituents and radionuclides in mushrooms: health benefits and risks by Jerzy Falandysz; Jan Borovička (pp. 477-501).

This article reviews and updates data on macro and trace elements and radionuclides in edible wild-grown and cultivated mushrooms. A huge biodiversity of mushrooms and spread of certain species over different continents makes the study on their multi-element constituents highly challenging. A few edible mushrooms are widely cultivated and efforts are on to employ them (largely Agaricus spp., Pleurotus spp., and Lentinula edodes) in the production of selenium-enriched food (mushrooms) or nutraceuticals (by using mycelia) and less on species used by traditional medicine, e.g., Ganoderma lucidum. There are also attempts to enrich mushrooms with other elements than Se and a good example is enrichment with lithium. Since minerals of nutritional value are common constituents of mushrooms collected from natural habitats, the problem is however their co-occurrence with some hazardous elements including Cd, Pb, Hg, Ag, As, and radionuclides. Discussed is also the problem of erroneous data on mineral compounds determined in mushrooms.

Keywords: Environment; Food; Fungi; Organic food; Se bioenrichment; Wild food

Biotechnology of non-Saccharomyces yeasts—the ascomycetes by Eric A. Johnson (pp. 503-517).

Saccharomyces cerevisiae and several other yeast species are among the most important groups of biotechnological organisms. S. cerevisiae and closely related ascomycetous yeasts are the major producer of biotechnology products worldwide, exceeding other groups of industrial microorganisms in productivity and economic revenues. Traditional industrial attributes of the S. cerevisiae group include their primary roles in food fermentations such as beers, cider, wines, sake, distilled spirits, bakery products, cheese, sausages, and other fermented foods. Other long-standing industrial processes involving S. cerevisae yeasts are production of fuel ethanol, single-cell protein (SCP), feeds and fodder, industrial enzymes, and small molecular weight metabolites. More recently, non-Saccharomyces yeasts (non-conventional yeasts) have been utilized as industrial organisms for a variety of biotechnological roles. Non-Saccharomyces yeasts are increasingly being used as hosts for expression of proteins, biocatalysts and multi-enzyme pathways for the synthesis of fine chemicals and small molecular weight compounds of medicinal and nutritional importance. Non-Saccharomyces yeasts also have important roles in agriculture as agents of biocontrol, bioremediation, and as indicators of environmental quality. Several of these products and processes have reached commercial utility, while others are in advanced development. The objective of this mini-review is to describe processes currently used by industry and those in developmental stages and close to commercialization primarily from non-Saccharomyces yeasts with an emphasis on new opportunities. The utility of S. cerevisiae in heterologous production of selected products is also described.

Keywords: Yeast; Biotechnology; Ascomycetes; Non-Saccharomyces

Genome-scale metabolic model in guiding metabolic engineering of microbial improvement by Chuan Xu; Lili Liu; Zhao Zhang; Danfeng Jin; Juanping Qiu; Ming Chen (pp. 519-539).

In the past few decades, despite all the significant achievements in industrial microbial improvement, the approaches of traditional random mutation and selection as well as the rational metabolic engineering based on the local knowledge cannot meet today’s needs. With rapid reconstructions and accurate in silico simulations, genome-scale metabolic model (GSMM) has become an indispensable tool to study the microbial metabolism and design strain improvements. In this review, we highlight the application of GSMM in guiding microbial improvements focusing on a systematic strategy and its achievements in different industrial fields. This strategy includes a repetitive process with four steps: essential data acquisition, GSMM reconstruction, constraints-based optimizing simulation, and experimental validation, in which the second and third steps are the centerpiece. The achievements presented here belong to different industrial application fields, including food and nutrients, biopharmaceuticals, biopolymers, microbial biofuel, and bioremediation. This strategy and its achievements demonstrate a momentous guidance of GSMM for metabolic engineering breeding of industrial microbes. More efforts are required to extend this kind of study in the meantime.

Keywords: Genome-scale metabolic model; Systems biology; Metabolic engineering; Microbial improvement; Industrial application

Metabolic characteristics of the species Variovorax paradoxus by Barbara Satola; Jan Hendrik Wübbeler; Alexander Steinbüchel (pp. 541-560).

This review outlines information about the Gram-negative, aerobic bacterium Variovorax paradoxus. The genomes of these species have G+C contents of 66.5–69.4 mol%, and the cells form yellow colonies. Some strains of V. paradoxus are facultative lithoautotrophic, others are chemoorganotrophic. Many of them are associated with important catabolic processes including the degradation of toxic and/or complex chemical compounds. The degradation pathways or other skills related to the following compounds, respectively, are described in this review: sulfolane, 3-sulfolene, 2-mercaptosuccinic acid, 3,3′-thiodipropionic acid, aromatic sulfonates, alkanesulfonates, amino acids and other sulfur sources, polychlorinated biphenyls, dimethyl terephthalate, linuron, 2,4-dinitrotoluene, homovanillate, veratraldehyde, 2,4-dichlorophenoxyacetic acid, anthracene, poly(3-hydroxybutyrate), chitin, cellulose, humic acids, metal–EDTA complexes, yttrium, rare earth elements, As(III), trichloroethylene, capsaicin, 3-nitrotyrosine, acyl-homoserine lactones, 1-aminocyclopropane-1-carboxylate, methyl tert-butyl ether, geosmin, and 2-methylisoborneol. Strains of V. paradoxus are also engaged in mutually beneficial interactions with other plant and bacterial species in various ecosystems. This species comprises probably promising strains for bioremediation and other biotechnical applications. Lately, the complete genomes of strains S110 and EPS have been sequenced for further investigations.

Keywords: Variovorax paradoxus ; Comamonadaceae ; Biodegradation; Bioremediation; Metabolic diversity

Marine bacteria: potential candidates for enhanced bioremediation by Hirak R. Dash; Neelam Mangwani; Jaya Chakraborty; Supriya Kumari; Surajit Das (pp. 561-571).

Bacteria are widespread in nature as they can adapt to any extreme environmental conditions and perform various physiological activities. Marine environments are one of the most adverse environments owing to their varying nature of temperature, pH, salinity, sea surface temperature, currents, precipitation regimes and wind patterns. Due to the constant variation of environmental conditions, the microorganisms present in that environment are more suitably adapted to the adverse conditions, hence, possessing complex characteristic features of adaptation. Therefore, the bacteria isolated from the marine environments are supposed to be better utilized in bioremediation of heavy metals, hydrocarbon and many other recalcitrant compounds and xenobiotics through biofilm formation and production of extracellular polymeric substances. Many marine bacteria have been reported to have bioremediation potential. The advantage of using marine bacteria for bioremediation in situ is the direct use of organisms in any adverse conditions without any genetic manipulation. This review emphasizes the utilization of marine bacteria in the field of bioremediation and understanding the mechanism behind acquiring the characteristic feature of adaptive responses.

Keywords: Marine bacteria; Adaptation; Stress response; Diversity; Bioremediation

Development of a two-stage feeding strategy based on the kind and level of feeding nutrients for improving fed-batch production of l-threonine by Escherichia coli by Shuwen Liu; Yong Liang; Qian Liu; Tongtong Tao; Shujuan Lai; Ning Chen; Tingyi Wen (pp. 573-583).

Fed-batch fermentation is the predominant method for industrial production of amino acids. In this study, we comprehensively investigated the effects of four kinds of feeding nutrients and developed an accurate optimization strategy for fed-batch production of l-threonine. The production of l-threonine was severely inhibited when cell growth ceased in the bath culture. Similarly, l-threonine production was also associated with cell growth in the carbon-, phosphate-, and sulfate-limited fed-batch cultures, but the accumulation of l-threonine was markedly increased because of the extended production time in the growth stage. Interestingly, auxotrophic amino acid (l-isoleucine)-limited feeding promoted l-threonine production over the non-growth phase. Metabolite analysis indicates that substantial production of acetate and glutamate and the resulting accumulation of ammonium may lead to the inhibition of l-threonine production. During the growth phase, the levels of l-isoleucine were accurately optimized by balancing cell growth and production with Pontryagin’s maximum principle, basing on the relationship between the specific growth rate μ and specific production rate ρ. Furthermore, the depletion of l-isoleucine and phosphate at the end of the growth phase favored the synthesis of l-threonine in the subsequent non-growth phase. Combining the two-stage feeding profiles, the final l-threonine concentration and conversion rate were increased by 5.9- and 2.1-fold, respectively, compared to batch processes without feeding control. The identification of efficient feeding nutrient and the development of accurate feeding strategies provide potential guidelines for microbial production of amino acids.

Keywords: l-Threonine; Escherichia coli ; Fed-batch fermentation; Auxotrophic amino acid; Growth-limited feeding; Ammonium inhibition

Medium optimization and proteome analysis of (R,R)-2,3-butanediol production by Paenibacillus polymyxa ATCC 12321 by Jinshan Li; Wei Wang; Yanhe Ma; An-Ping Zeng (pp. 585-597).

Paenibacillus polymyxa can produce the (R,R)-stereoisomer of 2,3-butanediol (2,3-BDL) which is industrially very useful. Two important factors affecting (R,R)-BDL production by P. polymyxa ATCC 12321, medium composition, and addition of acetic acid to the culture were investigated in this study with accompanying comparative proteomic analysis. For this purpose, a simple control strategy of O₂ supply was applied on the basis of an optimized basal medium: after a short period of batch cultivation with relatively high O₂ supply, the culture is switched into strong O₂ limitation, thereby promoting BDL formation. Three parallel fed-batch cultures starting from the same batch culture in an early stationary phase were then comparatively studied: the first one was running as control with the only change of O₂ supply; the second was, in addition, supplemented with 0.5 g/L yeast extract; and the third one was further added with 6 g/L acetate. Proteomic analyses of the three fed-batch cultures identified more than 86 proteins involved primarily in the central carbon metabolism, amino acid biosynthesis, energy metabolism, and stress responses. The examination of expression patterns of selected proteins, especially combined with fermentation data, gave valuable insights into the metabolic regulation of P. polymyxa under the different given conditions. Based on the proteomic analysis and further medium optimization studies, methionine was identified as one major growth-limiting factor in the basal medium and explains well the effect of yeast extract. Acetic acid was found to trigger the so far less studied acetone biosynthesis pathway in this organism. The latter is suggested in turn to enhance the switch from acidogenesis to solventogenesis. Thus, these findings extended our knowledge about BDL formation in P. polymyxa and provided useful information for further strain and process optimization.

Keywords: Paenibacillus polymyxa ; (R,R)-2,3-BDL; Proteomic analysis; Yeast extract; Acetate; Acetone pathway

Advances in the bioconversion mechanism of lovastatin to wuxistatin by Amycolatopsis sp. CGMCC 1149 by Hong Zong; Bin Zhuge; Huiying Fang; Yanhui Cao; Lin Mu; Weilai Fu; Jian Song; Jian Zhuge (pp. 599-609).

Wuxistatin, a novel statin and more potent than lovastatin, was converted from lovastatin by Amycolatopsis sp. (CGMCC 1149). Product I, an intermediate product, was found in the fermentation broth, and the structure analysis showed that product I had an additional hydroxyl group at the methyl group attached to C3 compared to lovastatin, which indicates that product I is one isomer of wuxistatin. Isotope tracing experiment proved that hydroxyl group of wuxistatin was provided by product I and the reaction from product I to wuxistatin was an intramolecular transfer. Hydroxylation reaction established in a cell-free system could be inhibited by CO and enhanced by ATP, Fe²⁺, and ascorbic acid, which were consistent with the presumption that the hydroxylase was an induced cytochrome P450. Study on proteomics of Amycolatopsis sp. CGMCC 1149 suggested that three identified proteins, including integral membrane protein, Fe-S oxidoreductase, and GTP-binding protein YchF, were induced by lovastatin and required during hydroxylation reaction. In conclusion, bioconversion mechanism of wuxistatin by Amycolatopsis sp. CGMCC 1149 was proposed: lovastatin is firstly hydroxylated to product I by a hydroxylase, namely cytochrome P450, and then product I is rearranged to wuxistatin by isomerases.

Keywords: Lovastatin; Product I; Wuxistatin; Biconversion; Hydroxylation; Cytochrome P450

De novo creation of MG1655-derived E. coli strains specifically designed for plasmid DNA production by Geisa A. L. Gonçalves; Duarte M. F. Prazeres; Gabriel A. Monteiro; Kristala L. J. Prather (pp. 611-620).

The interest in plasmid DNA (pDNA) as a biopharmaceutical has been increasing over the last several years, especially after the approval of the first DNA vaccines. New pDNA production strains have been created by rationally mutating genes selected on the basis of Escherichia coli central metabolism and plasmid properties. Nevertheless, the highly mutagenized genetic background of the strains used makes it difficult to ascertain the exact impact of those mutations. To explore the effect of strain genetic background, we investigated single and double knockouts of two genes, pykF and pykA, which were known to enhance pDNA synthesis in two different E. coli strains: MG1655 (wild-type genetic background) and DH5α (highly mutagenized genetic background). The knockouts were only effective in the wild-type strain MG1655, demonstrating the relevance of strain genetic background and the importance of designing new strains specifically for pDNA production. Based on the obtained results, we created a new pDNA production strain starting from MG1655 by knocking out the pgi gene in order to redirect carbon flux to the pentose phosphate pathway, enhance nucleotide synthesis, and, consequently, increase pDNA production. GALG20 (MG1655ΔendAΔrecAΔpgi) produced 25-fold more pDNA (19.1 mg/g dry cell weight, DCW) than its parental strain, MG1655ΔendAΔrecA (0.8 mg/g DCW), in glucose. For the first time, pgi was identified as an important target for constructing a high-yielding pDNA production strain.

Keywords: DNA vaccine; Plasmid biopharmaceuticals; Escherichia coli ; Strain engineering; Metabolic engineering

Purification of clinical-grade disulfide stabilized antibody fragment variable—Pseudomonas exotoxin conjugate (dsFv-PE38) expressed in Escherichia coli by Hua Jiang; Yueqing Xie; Andrew Burnette; John Roach; Steven L. Giardina; Toby T. Hecht; Stephen P. Creekmore; Gautam Mitra; Jianwei Zhu (pp. 621-632).

Immunotoxins are rationally designed cancer targeting and killing agents. Disulfide stabilized antibody Fv portion—toxin conjugates (dsFv-toxin) are third generation immunotoxins containing only the antibody fragment variable portions and a toxin fused to the V_H or V_L. Pseudomonas exotoxin fragment (PE-38) is a commonly used toxin in immunotoxin clinical trials. dsFv-toxin purification was previously published, but the recovery was not satisfactory. This report describes the development of a cGMP production process of the dsFv-toxin that incorporated a novel purification method. The method has been successfully applied to the clinical manufacturing of two dsFv-PE38 immunotoxins, MR1-1 targeting EGFRvIII and HA22 targeting CD22. The two subunits, V_L and V_H PE-38 were expressed separately in Escherichia coli using recombinant technology. Following cell lysis, inclusion bodies were isolated from the biomass harvested from fermentation in animal source component-free media. The dsFv-toxin was formed after denaturation and refolding, and subsequently purified to homogeneity through ammonium sulfate precipitation, hydrophobic interaction and ion-exchange chromatography steps. It was shown, in a direct comparison experiment using MR1-1 as model protein, that the recovery from the new purification method was improved three times over that from previously published method. The improved recovery was also demonstrated during the clinical production of two dsFv-PE38 immunotoxins—MR1-1 and HA22.

Keywords: Immunotoxin; Expression and purification; Refolding; cGMP production

One-step synthesis of 12-ketoursodeoxycholic acid from dehydrocholic acid using a multienzymatic system by Luo Liu; Michael Braun; Gabi Gebhardt; Dirk Weuster-Botz; Ralf Gross; Rolf D. Schmid (pp. 633-639).

12-ketoursodeoxycholic acid (12-keto-UDCA) is a key intermediate for the synthesis of ursodeoxycholic acid (UDCA), an important therapeutic agent for non-surgical treatment of human cholesterol gallstones and various liver diseases. The goal of this study is to develop a new enzymatic route for the synthesis 12-keto-UDCA based on a combination of NADPH-dependent 7β-hydroxysteroid dehydrogenase (7β-HSDH, EC 1.1.1.201) and NADH-dependent 3α-hydroxysteroid dehydrogenase (3α-HSDH, EC 1.1.1.50). In the presence of NADPH and NADH, the combination of these enzymes has the capacity to reduce the 3-carbonyl- and 7-carbonyl-groups of dehydrocholic acid (DHCA), forming 12-keto-UDCA in a single step. For cofactor regeneration, an engineered formate dehydrogenase, which is able to regenerate NADPH and NADH simultaneously, was used. All three enzymes were overexpressed in an engineered expression host Escherichia coli BL21(DE3)Δ7α-HSDH devoid of 7α-hydroxysteroid dehydrogenase, an enzyme indigenous to E. coli, in order to avoid formation of the undesired by-product 12-chenodeoxycholic acid in the reaction mixture. The stability of enzymes and reaction conditions such as pH value and substrate concentration were evaluated. No significant loss of activity was observed after 5 days under reaction condition. Under the optimal condition (10 mM of DHCA and pH 6), 99 % formation of 12-keto-UDCA with 91 % yield was observed.

Keywords: Cofactor regeneration; Dehydrocholic acid; Formate dehydrogenase-7β-hydroxysteroid dehydrogenase; 12-keto ursodeoxycholic acid; Ursodeoxycholic acid

Genome shuffling of Streptomyces viridochromogenes for improved production of avilamycin by Xing-An Lv; Ying-Yan Jin; Yu-Dong Li; Hong Zhang; Xin-Le Liang (pp. 641-648).

Avilamycin is one of EU-approved antimicrobial agents in feed industry to inhibit the growth of multidrug-resistant Gram-positive bacteria. Here, we applied a process of combining ribosome engineering and genome shuffling to achieve rapid improvement of avilamycin production in Streptomyces viridochromogenes AS 4.126. The starting mutant population was generated by ⁶⁰Co γ-irradiation treatments of the spores. After five rounds of protoplast fusion with streptomycin-resistance screening, an improved recombinant E-219 was obtained and its yield of avilamycin reached 1.4 g/L, which was increased by 4.85-fold and 36.8-fold in comparison with that of the shuffling starter Co γ-316 and the ancestor AS 4.126. Furthermore, the mechanism for the improvement of shuffled strains was investigated. Recombinants with enhanced streptomycin resistance exhibited significantly higher avilamycin production and product resistance, probably due to the mutations in the ribosome protein S12. The morphological difference between the parent mutant and shuffled recombinant was observed in conidiospore, and hyphae pellets. The presence of genetic diversity among shuffled populations with varied avilamycin productivity was confirmed by randomly amplified polymorphic DNA analysis. In summary, our results demonstrated that genome shuffling combined with ribosome engineering was a powerful approach for molecular breeding of high-yield industrial strains.

Keywords: Avilamycin; Genome shuffling; Ribosome engineering; RAPD; Streptomyces viridochromogenes

Characterization of the ginsenoside-transforming recombinant β-glucosidase from Actinosynnema mirum and bioconversion of major ginsenosides into minor ginsenosides by Chang-Hao Cui; Sun-Chang Kim; Wan-Taek Im (pp. 649-659).

This study focused on the cloning, expression, and characterization of ginsenoside-transforming recombinant β-glucosidase from Actinosynnema mirum KACC 20028^T in order to biotransform ginsenosides efficiently. The gene, termed as bglAm, encoding a β-glucosidase (BglAm) belonging to the glycoside hydrolase family 3 was cloned. bglAm consisted of 1,830 bp (609 amino acid residues) with a predicted molecular mass of 65,277 Da. This enzyme was overexpressed in Escherichia coli BL21(DE3) using a GST-fused pGEX 4T-1 vector system. The recombinant BglAm was purified with a GST·bind agarose resin and characterized. The optimum conditions of the recombinant BglAm were pH 7.0 and 37 °C. BglAm could hydrolyze the outer and inner glucose moieties at the C3 and C20 of the protopanaxadiol-type ginsenosides (i.e., Rb₁ and Rd, gypenoside XVII) to produce protopanaxadiol via gypenoside LXXV, F₂, and Rh₂(S) with various pathways. BglAm can effectively transform the ginsenoside Rb₁ to gypenoside XVII and Rd to F₂; the K _m values of Rb₁ and Rd were 0.69 ± 0.06 and 0.45 ± 0.02 mM, respectively, and the V _max values were 16.13 ± 0.29 and 51.56 ± 1.35 μmol min⁻¹ mg⁻¹ of protein, respectively. Furthermore, BglAm could convert the protopanaxatriol-type ginsenoside Re and Rg₁ into Rg₂(S) and Rh₁(S) hydrolyzing the attached glucose moiety at the C6 and C20 positions, respectively. These various ginsenoside-hydrolyzing pathways of BglAm may assist in producing the minor ginsenosides from abundant major ginsenosides.

Keywords: Ginsenoside; Biotransformation; Glycoside hydrolase; Actinosynnema mirum

Oxidation of phenolic compounds by the bifunctional catalase–phenol oxidase (CATPO) from Scytalidium thermophilum by Gulden Koclar Avci; Nursen Coruh; Ufuk Bolukbasi; Zumrut B. Ogel (pp. 661-672).

The thermophilic fungus Scytalidium thermophilum produces a novel bifunctional catalase with an additional phenol oxidase activity (CATPO); however, its phenol oxidation spectrum is not known. Here, 14 phenolic compounds were selected as substrates, among which (+)-catechin, catechol, caffeic acid, and chlorogenic acid yielded distinct oxidation products examined by reversed-phase HPLC chromatography method. Characterization of the products by LC-ESI/MS and UV–vis spectroscopy suggests the formation of dimers of dehydrocatechin type B (hydrophilic) and type A (hydrophobic), as well as oligomers, namely, a trimer and tetramer from (+)-catechin, the formation of a dimer and oligomer of catechol, a dimer from caffeic acid with a caffeicin-like structure, as well as trimeric and tetrameric derivatives, and a single major product from chlorogenic acid suggested to be a dimer. Based on the results, CATPO oxidizes phenolic compounds ranging from simple phenols to polyphenols but all having an ortho-diphenolic structure in common. The enzyme also appears to have stereoselectivity due to the oxidation of (+)-catechin, but not that of epicatechin. It is suggested that CATPO may contribute to the antioxidant mechanism of the fungus and may be of value for future food and biotechnology applications where such a bifunctional activity would be desirable.

Keywords: Scytalidium thermophilum ; Catalase; Phenol oxidase; Phenolic compounds; Oxidation; Antioxidant

Combinatorial approach of statistical optimization and mutagenesis for improved production of acidic phytase by Aspergillus niger NCIM 563 under submerged fermentation condition by K. Bhavsar; P. Gujar; P. Shah; V. Ravi Kumar; J. M. Khire (pp. 673-679).

Combination of statistical optimization and mutagenesis to isolate hypersecretory strains is studied to maximize phytase production from Aspergillus niger NCIM 563 under submerged fermentation. The overall results obtained show a remarkable 5.98-fold improvement in phytase production rates when compared to that using basal medium. Optimization of culture conditions from parent strain is studied first by the Plackett–Burman technique to evaluate the effects of 11 variables for phytase production. The results showed that glucose, MgSO₄, KCl, incubation period, and MnSO₄ are the most significant variables affecting enzyme production. Further optimization in these variables, using a central composite design technique, resulted in 3.74-fold increase in the yield of phytase production to 254,500 U/l when compared with the activity observed with basal media (68,000 U/l) in shake flask. Our experiments show that the phytase from A. niger NCIM 563 exhibits desirable activity in simulated gastric fluid conditions with low pH and also improved thermostability when compared to commercial phytase. The improved yield demonstrates the potential applicability of phytase enzyme as a source of phytase supplement for phosphorus nutrition and environmental protection in animal feed industry. Physical and chemical mutagenesis experiments were carried out in parallel to isolate hypersecretory mutants that could possibly further enhance the enzyme production. Using optimized media conditions of the parent strain, our results show that mutant strain A. niger NCIM 1359 increased the phytase activity by another 1.6-fold to 407,200 U/l.

Keywords: Phytase; Aspergillus niger ; Mutagenesis; Statistical optimization; Animal feed

Reversible immobilization of glucoamylase onto magnetic chitosan nanocarriers by Jianzhi Wang; Guanghui Zhao; Yanfeng Li; Xiao Liu; Pingping Hou (pp. 681-692).

A simple preparation process for the monodispersed pH-sensitive core-shell magnetic microspheres was carried out consisting of chitosan self-assembled on magnetic iron oxide nanoparticles. Meanwhile, glucoamylase was immobilized as a model enzyme on this carrier of Fe₃O₄/CS microspheres by ionic adsorption. The morphology, inner structure, and high magnetic sensitivity of the resulting magnetic chitosan microspheres were studied, respectively, with a field emission scanning electron microscope (SEM), transmission electron microscope (TEM), FT-IR spectroscopy, thermogravimetric analysis (TGA), and a vibrating sample magnetometer (VSM). Subsequently, the properties of glucoamylase immobilized on the regenerated supports were also investigated by determining storage stability, pH stability, reusability, magnetic response, and regeneration of supports. The results from characterization and determination remarkably indicated that the immobilized glucoamylase obtained presents excellent storage stability, pH stability, reusability, magnetic response, and regeneration of supports. Therefore, this kind of magnetic Fe₃O₄/CS microspheres with perfect monodispersity should be an ideal support for enzyme immobilization.

Keywords: Magnetic chitosan; Ionic adsorption; Immobilization; Reversible; Glucoamylase

Amino acid substitutions in naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4 result in regio- and stereo-specific hydroxylation of flavanone and isoflavanone by Jiyoung Seo; Ji-Young Ryu; Jaehong Han; Joong-Hoon Ahn; Michael J. Sadowsky; Hor-Gil Hur; Youhoon Chong (pp. 693-704).

Wild-type naphthalene dioxygenase (NDO) from Pseudomonas sp. strain NCIB 9816-4 transforms relatively planar flavone and isoflavone to cis-dihydrodiols. However, this enzyme cannot catalyze the transformation of flavanone and isoflavanone in which a phenyl group bonds to the stereogenic C2 or C3 of the C-ring. Protein modeling suggested that Phe224 in the substrate binding site of NDO may play a key role in substrate specificity toward flavanone and isoflavanone. Site-directed mutants of NDO with substitution of Phe224 with Tyr biotransformed only the (S)-stereoisomers of flavanone and isoflavanone, producing an 8-OH group on the A-ring. In contrast, the Phe224Cys and Phe224Gln substitutions, which used (2S)-flavanone as a substrate, and Phe224Lys, which transformed (2S)-flavanone and (3S)-isoflavanone, each showed lower activity than the Phe224Tyr substitution. The remainder of the tested mutants had no activity with flavanone and isoflavanone. Protein docking studies of flavanone and isoflavanone to the modeled mutant enzyme structures revealed that an expanded substrate binding site, due to mutation at 224, as well as appropriate hydrophobic interaction with the residue at 224, are critical for successful binding of the substrates. Results of this study also suggested that in addition to the previously known Phe352, the Phe224 site of NDO appears to be important site for expanding the substrate range of NDO and bringing regiospecific and stereospecific hydroxylation reactions to C8 of the flavanone and isoflavanone A-rings.

Keywords: Naphthalene dioxygenase; Site-directed mutagenesis; Flavanone; Flavone; Biotransformation

Transcriptional analysis of Pleurotus ostreatus laccase genes by Cinzia Pezzella; Vincenzo Lettera; Alessandra Piscitelli; Paola Giardina; Giovanni Sannia (pp. 705-717).

Fungal laccases (p-diphenol:oxygen oxidoreductase; EC 1.10.3.2) are multi-copper-containing oxidases that catalyse the oxidation of a great variety of phenolic compounds and aromatic amines through simultaneous reduction of molecular oxygen to water. Fungi generally produce several laccase isoenzymes encoded by complex multi-gene families. The Pleurotus ostreatus genome encodes 11 putative laccase coding genes, and only six different laccase isoenzymes have been isolated and characterised so far. Laccase expression was found to be regulated by culture conditions and developmental stages even if the redundancy of these genes still raises the question about their respective functions in vivo. In this context, laccase transcript profiling analysis has been used to unravel the physiological role played by the different isoforms produced by P. ostreatus. Even if reported results depict a complex picture of the transcriptional responses exhibited by the analysed laccase genes, they were allowed to speculate on the isoform role in vivo. Among the produced laccases, LACC10 (POXC) seems to play a major role during vegetative growth, since its transcription is downregulated when the fungus starts the fructification process. Furthermore, a new tessera has been added to the puzzling mosaic of the heterodimeric laccase LACC2 (POXA3). LACC2 small subunit seems to play an additional physiological role during fructification, beside that of LACC2 complex activation/stabilisation.

Keywords: Laccase transcriptional regulation; Laccase physiological role; Heterodimeric laccase; Fungal developmental stage; Quantitative RT-PCR; Laccase gene family

Characterization of glyceraldehyde-3-phosphate dehydrogenase gene RtGPD1 and development of genetic transformation method by dominant selection in oleaginous yeast Rhodosporidium toruloides by Yanbin Liu; Chong Mei John Koh; Longhua Sun; Mya Myintzu Hlaing; Minge Du; Ni Peng; Lianghui Ji (pp. 719-729).

The oleaginous yeast Rhodosporidium toruloides, which belongs to the Pucciniomycotina subphylum in the Basidiomycota, has attracted strong interest in the biofuel community recently due to its ability to accumulate more than 60% of dry biomass as lipid under high-density fermentation. A 3,543-nucleotide (nt) DNA fragment of the glyceraldehyde-3-phosphate dehydrogenase gene (GPD1) was isolated from R. toruloides ATCC 10657 and characterized in details. The 1,038-nt mRNA derived from seven exons encodes an open reading frame (ORF) of 345 amino acids that shows high identity (80%) to the Ustilago maydis homolog. Notably, the ORF is composed of codons strongly biased towards cytosine at the Wobble position. GPD1 is transcriptionally regulated by temperature shock, osmotic stress, and carbon source. Nested deletion analysis of the GPD1 promoter by GFP reporter assay revealed that two regions, −975 to −1,270 and −1,270 to −1,429, upstream from the translational start site of GPD1 were important for responses to various stress stimuli. Interestingly, a 176-bp short fragment maintained 42.2% promoter activity of the 795-bp version in U. maydis whereas it was reduced to 17.4% in R. toruloides. The GPD1 promoter drove strong expression of a codon-optimized enhanced green fluorescent protein gene (RtGFP) and a codon-optimized hygromycin phosphotransferase gene (hpt-3), which was critical for Agrobacterium tumefaciens-mediated transformation in R. toruloides.

Keywords: Glyceraldehyde-3-phosphate dehydrogenase; Oleaginous yeast; Rhodosporidium toruloides ; Agrobacterium tumefaciens-mediated transformation; Microbial oil

Selective breeding for desiccation tolerance in liquid culture provides genetically stable inbred lines of the entomopathogenic nematode Heterorhabditis bacteriophora by Samuel Anbesse; Nanette Hope Sumaya; Anna Verena Dörfler; Olaf Strauch; Ralf-Udo Ehlers (pp. 731-739).

The entomopathogenic nematode (EPN) Heterorhabditis bacteriophora is used in biological plant protection to control pest insects. In the past, several attempts targeted at an enhancement of the desiccation tolerance of EPN by genetic selection in order to improve their storage stability. The subsequent loss of improved beneficial traits after release of selection pressure has often been reported. In order to stabilize progress of selective breeding, selection during liquid culturing was tested against propagation in host insects. After release of the selection pressure, the tolerance was monitored over additional reproductive cycles in vivo and in vitro to compare the stability of the trait. Furthermore, it was tested whether the virulence of the selected strains would be impaired. Exposure to desiccation stress prior to propagation, in vivo or in vitro, both resulted in increasing desiccation tolerance. When selection pressure was released, the gained tolerance was lost again during in vivo production, whereas the tolerance was maintained at a high level when EPNs were cultured in liquid culture. In Heterorhabditis sp., liquid culture conditions produce highly homozygous, genetically stable inbred lines. The investigation provides easily applicable methods to improve and stabilize beneficial traits of heterorhabditid EPNs through selective breeding in liquid culture. Compared to nematodes from in vivo propagation, production in liquid media yielded EPN of higher virulence.

Keywords: Biological control; Genetic selection; Desiccation tolerance; Infectivity; Inbred lines

Cytoplasmic expression, antibody production, and characterization of the novel zinc finger protein 637 by Kai Li; Yuyan Wei; Jie Zhang; Jun Li; Bo Gao; Lugang Huang; Ping Lin; Yuquan Wei (pp. 741-749).

Zinc finger protein 637 (zfp637), belonging to the Kruppel-like protein family, comprises one atypical C₂H₂ and six consecutive typical zinc finger motifs. Based on the structural characterization of zfp637 and its location in the cell nucleus, we predict that zfp637 might function as a DNA-binding protein to regulate gene transcription. However, the absence of both a purified zfp637 protein and any commercial antibody for detecting it in cells and tissues has limited functional studies of zfp637 to date. Here, we developed and optimized an expression system by fusing zfp637 with glutathione S-transferase (GST) to achieve a maximal yield of soluble GST-zfp637 fusion protein in Escherichia coli BL21(DE3) cells. The yield was about 10 mg/l of the original bacterial culture. The recombinant GST-zfp637 fusion protein was purified and used for polyclonal antibody production in rabbits. In addition, we developed a method to remove the anti-GST antibody component and obtained a highly purified anti-zfp637 antibody, as demonstrated by an enzyme-linked immunosorbent assay. Western blotting showed that the anti-zfp637 antibody recognized not only the recombinant zfp637 protein but also endogenous zfp637 in several cell lines. The protein was localized mainly in the cell nucleus by immunofluorescence and immunohistochemistry. The expression levels of zfp637 mRNA and protein were significantly increased in NIH3T3 cells treated with 200 μM of H₂O₂ in a time-dependent manner. The recombinant GST-zfp637 fusion protein and our purified anti-zfp637 antibody will help in elucidating the function of zfp637.

Keywords: Zfp637; Recombinant protein; Cytoplasmic expression; Anti-zfp637-specific antibody; Oxidative stress

A Pseudomonas putida bioreporter for the detection of enzymes active on 2-alkyl-4(1H)-quinolone signalling molecules by Christine Müller; Susanne Fetzner (pp. 751-760).

The quorum sensing signalling molecules 2-heptyl-3-hydroxy-4(1H)-quinolone, termed the “Pseudomonas quinolone signal” (PQS), and 2-heptyl-4(1H)-quinolone (HHQ) play an important role in the control of virulence gene expression in Pseudomonas aeruginosa. To construct a bioreporter for the specific and sensitive detection of these compounds, a plasmid with the pqsR gene encoding the PQS- and HHQ-responsive transcriptional regulator PqsR, and with the PqsR-controlled pqsA promoter fused to the lacZ gene, was established in Pseudomonas putida KT2440. The bioreporter responds to HHQ and PQS at concentrations in the range of 0.1–10 and 0.01–5 μM, respectively, with EC₅₀ values of 1.50 ± 0.25 μM for HHQ and 0.15 ± 0.02 μM for PQS. 2,4-Dihydroxyquinoline, a metabolite produced abundantly by P. aeruginosa, did not elicit an increase in reporter enzyme activity. To test whether the bioreporter can be used for the detection of enzymes active on AQ signalling molecules, the hodC gene coding for 2-methyl-3-hydroxy-4(1H)-quinolone 2,4-dioxygenase was expressed in the reporter strain. This dioxygenase catalyses the cleavage of PQS, albeit with very low activity. The response of the bioreporter to PQS was significantly quenched by co-expression of the hodC gene, and HPLC analysis of culture extracts verified that the PQS levels decreased during cultivation. The bioreporter can be applied to screen for AQ-converting enzymes, which will be useful tools to interfere with quorum sensing and thus virulence in P. aeruginosa.

Keywords: Quorum sensing; Quorum quenching; Pseudomonas aeruginosa ; 2-Alkyl-4(1H)-quinolone; Pseudomonas quinolone signal; Bioreporter

High production of llama variable heavy-chain antibody fragment (VHH) fused to various reader proteins by Aspergillus oryzae by Hiromoto Hisada; Hiroko Tsutsumi; Hiroki Ishida; Yoji Hata (pp. 761-766).

Llama variable heavy-chain antibody fragment (VHH) fused to four different reader proteins was produced and secreted in culture medium by Aspergillus oryzae. These fusion proteins consisted of N-terminal reader proteins, VHH, and a C-terminal his-tag sequence which facilitated purification using one-step his-tag affinity chromatography. SDS-PAGE analysis of the deglycosylated purified fusion proteins confirmed that the molecular weight of each corresponded to the expected sum of VHH and the respective reader proteins. The apparent high molecular weight reader protein glucoamylase (GlaB) was found to be suitable for efficient VHH production. The GlaB-VHH-His protein bound its antigen, human chorionic gonadotropin, and was detectable by a new ELISA-based method using a coupled assay with glucoamylase, glucose oxidase, peroxidase, maltose, and 3,3′,5,5′-tetramethylbenzidine as substrates. Addition of potassium phosphate to the culture medium induced secretion of 0.61 mg GlaB-VHH-His protein/ml culture medium in 5 days.

Keywords: Llama variable heavy-chain antibody fragment (VHH); Reader protein; Overproduction; Aspergillus oryzae ; His-tagged protein

Mycoepoxydiene suppresses RANKL-induced osteoclast differentiation and reduces ovariectomy-induced bone loss in mice by Jingwei Zhu; Qiang Chen; Xiaochun Xia; Pingli Mo; Yuemao Shen; Chundong Yu (pp. 767-774).

Mycoepoxydiene (MED) is a compound isolated from the marine fungal Diaporthe sp. HLY-1 associated with mangroves. MED has various biological effects such as anti-microbial, anti-cancer, and anti-inflammatory activities. However, the effect of MED on the differentiation of osteoclasts, the multinucleated bone-resorbing cells which play a crucial role in bone remodeling, is still unknown. In this study, we showed that MED could inhibit receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation and the expression of three well-known osteoclast markers such as tartrate-resistant acid phosphatase, calcitonin receptor, and cathepsin K in bone marrow-derived macrophages. Furthermore, we found that MED inhibited the expression of nuclear factor of activated T cells c1, a key transcriptional factor in osteoclast differentiation, via inhibiting the phosphorylation of TAK1 and then blocking the activation of NF-κB and ERK1/2 pathways. Moreover, MED could prevent bone loss in ovariectomized mice. Taken together, we demonstrate for the first time that MED can suppress RANKL-induced osteoclast differentiation in vitro and ovariectomy-induced osteoporosis in vivo, suggesting that MED is a potential lead compound for the development of novel drugs for osteoporosis treatment.

Keywords: Mycoepoxydiene; Osteoclast differentiation; NF-кB; ERK1/2; NFATc1

Biogenic antimicrobial silver nanoparticles produced by fungi by Alexandre G. Rodrigues; Liu Yu Ping; Priscyla D. Marcato; Oswaldo L. Alves; Maria C. P. Silva; Rita C. Ruiz; Itamar S. Melo; Ljubica Tasic; Ana O. De Souza (pp. 775-782).

Aspergillus tubingensis and Bionectria ochroleuca showed excellent extracellular ability to synthesize silver nanoparticles (Ag NP), spherical in shape and 35 ± 10 nm in size. Ag NP were characterized by transmission electron microscopy, X-ray diffraction analysis, and photon correlation spectroscopy for particle size and zeta potential. Proteins present in the fungal filtrate and in Ag NP dispersion were analyzed by electrophoresis (sodium dodecyl sulfate polyacrylamide gel electrophoresis). Ag NP showed pronounced antifungal activity against Candida sp, frequently occurring in hospital infections, with minimal inhibitory concentration in the range of 0.11–1.75 μg/mL. Regarding antibacterial activity, nanoparticles produced by A. tubingensis were more effective compared to the other fungus, inhibiting 98.0 % of Pseudomonas. aeruginosa growth at 0.28 μg/mL. A. tubingensis synthesized Ag NP with surprisingly high and positive surface potential, differing greatly from all known fungi. These data open the possibility of obtaining biogenic Ag NP with positive surface potential and new applications.

Keywords: Silver nanoparticles; Antimicrobial activity; Aspergillus tubingensis ; Bionectria ochroleuca ; Mangrove

Symbiotic efficiency and phylogeny of the rhizobia isolated from Leucaena leucocephala in arid–hot river valley area in Panxi, Sichuan, China by Kai Wei Xu; Petri Penttinen; Yuan Xue Chen; Qiang Chen; Xiaoping Zhang (pp. 783-793).

In search of effective nitrogen-fixing strains for inoculating Leucaena leucocephala, we assessed the symbiotic efficiency of 41 rhizobial isolates from root nodules of L. leucocephala growing in the arid–hot river valley area in Panxi, China. The genetic diversity of the isolates was studied by analyzing the housekeeping genes 16S rRNA and recA, and the symbiotic genes nifH and nodC. In the nodulation and symbiotic efficiency assay, only 11 of the 41 isolates promoted the growth of L. leucocephala while the majority of the isolates were ineffective in symbiotic nitrogen fixation. Furthermore, one fourth of the isolates had a growth slowing effect on the host. According to the 16S rRNA and recA gene analyses, most of the isolates were Ensifer spp. The remaining isolates were assigned to Rhizobium, Mesorhizobium and Bradyrhizobium. The sequence analyses indicated that the L. leucocephala rhizobia had undergone gene recombination. In contrast to the promiscuity observed as a wide species distribution of the isolates, the results implied that L. leucocephala is preferentially nodulated by strains that share common symbiosis genes. The symbiotic efficiency was not connected to chromosomal background of the symbionts and isolates carrying a similar nifH or nodC showed totally different nitrogen fixation efficiency.

Keywords: Leucaena leucocephala ; Rhizobia; Housekeeping genes; Genetic diversity; Phylogeny; Symbiotic efficiency

A vacuolar membrane protein affects drastically the biosynthesis of the ACV tripeptide and the beta-lactam pathway of Penicillium chrysogenum by Marta Fernández-Aguado; Fernando Teijeira; Juan F. Martín; Ricardo V. Ullán (pp. 795-808).

The knowledge about enzymes’ compartmentalization and transport processes involved in the penicillin biosynthesis in Penicillium chrysogenum is very limited. The genome of this fungus contains multiple genes encoding transporter proteins, but very little is known about them. A bioinformatic search was made to find major facilitator supefamily (MFS) membrane proteins related to CefP transporter protein involved in the entry of isopenicillin N to the peroxisome in Acremonium chrysogenum. No strict homologue of CefP was observed in P. chrysogenum, but the penV gene was found to encode a membrane protein that contained 10 clear transmembrane spanners and two other motifs COG5594 and DUF221, typical of membrane proteins. RNAi-mediated silencing of penV gene provoked a drastic reduction of the production of the δ-(l-α-aminoadipyl-l-cysteinyl-d-valine) (ACV) and isopenicillin N intermediates and the final product of the pathway. RT-PCR and northern blot analyses confirmed a reduction in the expression levels of the pcbC and penDE biosynthetic genes, whereas that of the pcbAB gene increased. Localization studies by fluorescent laser scanning microscopy using Dsred and GFP fluorescent fusion proteins and the FM 4-64 fluorescent dye showed clearly that the protein was located in the vacuolar membrane. These results indicate that PenV participates in the first stage of the beta-lactam biosynthesis (i.e., the formation of the ACV tripeptide), probably taking part in the supply of amino acids from the vacuolar lumen to the vacuole-anchored ACV synthetase. This is in agreement with several reports on the localization of the ACV synthetase and provides increased evidence for a compartmentalized storage of precursor amino acids for non-ribosomal peptides. PenV is the first MFS transporter of P. chrysogenum linked to the beta-lactam biosynthesis that has been located in the vacuolar membrane.

Keywords: Penicillium chrysogenum ; Penicillin biosynthetic pathway; MFS transporter; Vacuolar membrane protein

Antioxidant properties of potentially probiotic bacteria: in vitro and in vivo activities by Alberto Amaretti; Mattia di Nunzio; Anna Pompei; Stefano Raimondi; Maddalena Rossi; Alessandra Bordoni (pp. 809-817).

Thirty-four strains of lactic acid bacteria (seven Bifidobacterium, 11 Lactobacillus, six Lactococcus, and 10 Streptococcus thermophilus) were assayed in vitro for antioxidant activity against ascorbic and linolenic acid oxidation (TAA_AA and TAA_LA), trolox-equivalent antioxidant capacity (TEAC), intracellular glutathione (TGSH), and superoxide dismutase (SOD). Wide dispersion of each of TAA_AA, TAA_LA, TEAC, TGSH, and SOD occurred within bacterial groups, indicating that antioxidative properties are strain specific. The strains Bifidobacterium animalis subsp. lactis DSMZ 23032, Lactobacillus acidophilus DSMZ 23033, and Lactobacillus brevis DSMZ 23034 exhibited among the highest TAA_AA, TAA_LA, TEAC, and TGSH values within the lactobacilli and bifidobacteria. These strains were used to prepare a potentially antioxidative probiotic formulation, which was administered to rats at the dose of 10⁷, 10⁸, and 10⁹ cfu/day for 18 days. The probiotic strains colonized the colon of the rats during the trial and promoted intestinal saccharolytic metabolism. The analysis of plasma antioxidant activity, reactive oxygen molecules level, and glutathione concentration, revealed that, when administered at doses of at least 10⁸ cfu/day, the antioxidant mixture effectively reduced doxorubicin-induced oxidative stress. Probiotic strains which are capable to limit excessive amounts of reactive radicals in vivo may contribute to prevent and control several diseases associated with oxidative stress.

Keywords: Bifidobacterium ; Lactobacillus ; Probiotic; Antioxidant; In vivo; In vitro

Characterization of the chimeric seven-transmembrane protein containing conserved region of helix C–F of microbial rhodopsin from Ganges River by Ah Reum Choi; Se Jun Kim; Byung Hoon Jung; Kwang-Hwan Jung (pp. 819-828).

Proteorhodopsin (PR) is a light-driven proton pump that has been found in a variety of marine bacteria. Recently, many PR-like genes were found in non-marine environments. The goal of this study is to explore the function of rhodopsins that exist only as partial proteo-opsin genes using chimeras with marine green PR (GPR). We isolated nine partial genes of PR homologues using polymerase chain reaction (PCR) and chose three homologues of GPR from the surface of the Ganges River, which has earned them the name “CFR, Chimeric Freshwater Rhodopsin.” In order to characterize the proteins, we constructed the cassette based on GPR sequence without helices C to F and inserted the isolated conserved partial sequences. When expressed in E. coli, we could observe light-driven proton pumping activity similar to proteorhodopsin, however, photocycle kinetics of CFRs are much slower than proteorhodopsin. Half-time decay of O intermediates of CFRs ranged between 143 and 333 ms at pH 10; their absorption maxima were between 515 and 522 nm at pH 7. We can guess that the function of native rhodopsin, a retinal protein of fresh water bacteria, may be a light-driven proton transport based on the results from chimeric freshwater rhodopsins. This approach will enable many labs that keep reporting partial PCR-based opsin sequences to finally characterize their proteins.

Keywords: Opsin; Membrane protein; Proton pumping; Environmental genomics; Freshwater rhodopsin

A dual ELP-tagged split intein system for non-chromatographic recombinant protein purification by Changhua Shi; Qing Meng; David W. Wood (pp. 829-835).

Self-cleaving elastin-like protein (ELP) tags provide a very promising tool for recombinant protein purification. With this method, the target protein is purified by simple ELP-mediated precipitation steps, followed by self-cleavage and removal of the ELP tag. Unfortunately, however, inteins usually experience some level of pre-cleavage during protein expression, which can significantly decrease final yields. In this study, we solve this problem by splitting the intein into two ELP-tagged segments. Each segment is incapable of pre-cleavage alone, but the assembled segments release the target protein rapidly when assembled in vitro. The result is the very tight control of the tag cleaving reaction, combined with the simplicity of the ELP purification method. Using this system, we successfully purified four different sizes of target proteins with final yields comparable to or higher than our original contiguous intein–ELP system. Further, we demonstrate a streamlined split intein method, where cells expressing the tagged intein segments are combined prior to cell lysis, allowing the segments to be co-purified in a single reaction mixture.

Keywords: Elastin-like protein; Split intein; Protein purification; Self-cleaving tag

A simple and effective strategy for solving the problem of inclusion bodies in recombinant protein technology: His-tag deletions enhance soluble expression by Shaozhou Zhu; Cuiyu Gong; Lu Ren; Xingzhou Li; Dawei Song; Guojun Zheng (pp. 837-845).

The formation of inclusion bodies (IBs) in recombinant protein biotechnology has become one of the most frequent undesirable occurrences in both research and industrial applications. So far, the pET System is the most powerful system developed for the production of recombinant proteins when Escherichia coli is used as the microbial cell factory. Also, using fusion tags to facilitate detection and purification of the target protein is a commonly used tactic. However, there is still a large fraction of proteins that cannot be produced in E. coli in a soluble (and hence functional) form. Intensive research efforts have tried to address this issue, and numerous parameters have been modulated to avoid the formation of inclusion bodies. However, hardly anyone has noticed that adding fusion tags to the recombinant protein to facilitate purification is a key factor that affects the formation of inclusion bodies. To test this idea, the industrial biocatalysts uridine phosphorylase from Aeropyrum pernix K1 and (+)-γ-lactamase and (−)-γ-lactamase from Bradyrhizobium japonicum USDA 6 were expressed in E. coli by using the pET System and then examined. We found that using a histidine tag as a fusion partner for protein expression did affect the formation of inclusion bodies in these examples, suggesting that removing the fusion tag can promote the solubility of heterologous proteins. The production of soluble and highly active uridine phosphorylase, (+)-γ-lactamase, and (−)-γ-lactamase in our results shows that the traditional process needs to be reconsidered. Accordingly, a simple and efficient structure-based strategy for the production of valuable soluble recombinant proteins in E. coli is proposed.

Keywords: Inclusion bodies; His-tag; Uridine phosphorylase; (+)-γ-Lactamase; (−)-γ-Lactamase

Adaptation of nitrifying microbial biomass to nickel in batch incubations by Chok-Hang Yeung; Christopher A. Francis; Craig S. Criddle (pp. 847-857).

Nitrification—microbial oxidation of ammonia to nitrate—is sensitive to an array of inhibitors. Currently, little is known regarding the ecological processes that enable adaptation to inhibitors and recovery of nitrification. This study evaluated inhibition and recovery of nitrification in batch cultures of activated sludge incubated with different levels of nickel (Ni), a model inhibitor. Incubation with 1 mg/L of added Ni did not adversely affect nitrification, and little inhibition occurred at 5 and 10 mg/L Ni. Incubation with 50 mg/L Ni resulted in significant inhibition, decreased amoA transcript abundance, and delayed recovery of nitrification until amoA transcript abundance rebounded after 24 h. For this dosage, recovery of nitrification occurred without a significant change in ammonia-oxidizing bacteria (AOB) community structure. By contrast, incubation with 150 mg/L of added Ni strongly inhibited nitrification and delayed recovery until a shift in AOB community structure occurred after ∼6 weeks of incubation. The results indicate that inhibitor-resistant nitrifying cultures can be obtained from long-term batch incubations of decaying activated sludge incubated with high levels of added inhibitor.

Keywords: Adaptation; Nitrification; Nickel; Ammonia-oxidizing bacteria (AOB); Transcription; Microbial community structure

A biomimetic approach towards synthesis of zinc oxide nanoparticles by Navin Jain; Arpit Bhargava; Jagadish C. Tarafdar; Sunil K. Singh; Jitendra Panwar (pp. 859-869).

Using natural processes as inspiration, the present study demonstrates a positive correlation between zinc metal tolerance ability of a soil fungus and its potential for the synthesis of zinc oxide (ZnO) nanoparticles. A total of 19 fungal cultures were isolated from the rhizospheric soils of plants naturally growing at a zinc mine area in India and identified on the genus, respectively the species level. Aspergillus aeneus isolate NJP12 has been shown to have a high zinc metal tolerance ability and a potential for extracellular synthesis of ZnO nanoparticles under ambient conditions. UV–visible spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction analysis, transmission electron microscopy, and energy dispersive spectroscopy studies further confirmed the crystallinity, morphology, and composition of synthesized ZnO nanoparticles. The results revealed the synthesis of spherical nanoparticles coated with protein molecules which served as stabilizing agents. Investigations on the role of fungal extracellular proteins in the synthesis of nanoparticles indicated that the process is nonenzymatic but involves amino acids present in the protein chains.

Keywords: Biomimetics; ZnO nanoparticles; Rhizosphere; Soil fungi; Metal tolerance; Aspergillus

Isolation and lipid degradation profile of Raoultella planticola strain 232-2 capable of efficiently catabolizing edible oils under acidic conditions by Daisuke Sugimori; Mika Watanabe; Tomohiro Utsue (pp. 871-880).

The lipids (fats and oils) degradation capabilities of soil microorganisms were investigated for possible application in treatment of lipids-contaminated wastewater. We isolated a strain of the bacterium Raoultella planticola strain 232-2 that is capable of efficiently catabolizing lipids under acidic conditions such as in grease traps in restaurants and food processing plants. The strain 232-2 efficiently catabolized a mixture (mixed lipids) of commercial vegetable oil, lard, and beef tallow (1:1:1, w/w/w) at 20–35 °C, pH 3–9, and 1,000–5,000 ppm lipid content. Highly effective degradation rate was observed at 35 °C and pH 4.0, and the 24-h degradation rate was 62.5 ± 10.5 % for 3,000 ppm mixed lipids. The 24-h degradation rate for 3,000 ppm commercial vegetable oil, lard, beef tallow, mixed lipids, and oleic acid was 71.8 %, 58.7 %, 56.1 %, 55.3 ± 8.5 %, and 91.9 % at pH 4 and 30 °C, respectively. R. planticola NBRC14939 (type strain) was also able to efficiently catabolize the lipids after repeated subculturing. The composition of the culture medium strongly influenced the degradation efficiency, with yeast extract supporting more complete dissimilation than BactoPeptone or beef extract. The acid tolerance of strain 232-2 is proposed to result from neutralization of the culture medium by urease-mediated decomposition of urea to NH₃. The rate of lipids degradation increased with the rates of neutralization and cell growth. Efficient lipids degradation using strain 232-2 has been achieved in the batch treatment of a restaurant wastewater.

Keywords: Lipids (fats and oils)-catabolyzing bacterium; Triacylglyceride degradation; Lipase; Wastewater treatment; Acid-tolerant bacterium; Raoultella planticola

Effective bioremoval and detoxification of textile dye mixture by Alishewanella sp. KMK6 by Yogesh M. Kolekar; Pallavi D. Konde; Vijay L. Markad; Sharwari V. Kulkarni; Ashvini U. Chaudhari; Kisan M. Kodam (pp. 881-889).

Alishewanella sp. strain KMK6 was able to degrade mixture of textile dyes (0.5–2.0 g l⁻¹) within 8 h. An initial 28 % reduction in COD was observed immediately after decolorization at static anoxic conditions which on further incubation at shaking conditions reduced by 90 %. Partially purified azoreductase was able to utilize different azo dyes as substrates. The HPLC profile of dye degradation showed formation of metabolic products. Further FTIR analysis showed significant changes in the peaks corresponding to functional groups present in dye mixture and its degradation products. The genotoxicity assessment showed that the dye degradation products were non-toxic compared to dye mixture.

Keywords: Alishewanella sp. KMK6; Azoreductase; Biodegradation; Azo dyes; COD

Isolation of cholesterol- and deoxycholate-degrading bacteria from soil samples: evidence of a common pathway by E. Merino; A. Barrientos; J. Rodríguez; G. Naharro; J. M. Luengo; E. R. Olivera (pp. 891-904).

Nineteen different steroid-degrading bacteria were isolated from soil samples by using selective media containing either cholesterol or deoxycholate as sole carbon source. Strains that assimilated cholesterol (17 COL strains) were gram-positive, belonging to the genera Gordonia, Tsukamurella, and Rhodococcus, and grew on media containing other steroids but were unable to use deoxycholate as sole carbon source. Surprisingly, some of the COL strains unable to grow using deoxycholate as sole carbon source were able to catabolize other bile salts (e.g., cholate). Conversely, strains able to grow using deoxycholate as the sole carbon source (two DOC isolates) were gram-negative, belonging to the genus Pseudomonas, and were unable to catabolize cholesterol and other sterols. COL and DOC were included into the corresponding taxonomic groups based on their morphology (cells and colonies), metabolic properties (kind of substrates that support bacterial growth), and genetic sequences (16S rDNA and rpoB). Additionally, different DOC21 Tn5 insertion mutants have been obtained. These mutants have been classified into two different groups: (1) those affected in the catabolism of bile salts but that, as wild type, can grow in other steroids and (2) those unable to grow in media containing any of the steroids tested. The identification of the insertion point of Tn5 in one of the mutants belonging to the second group (DOC21 Mut1) revealed that the gene knocked-out encodes an A-ring meta-cleavage dioxygenase needed for steroid catabolism.

Keywords: Sterols; Bile salts; Steroidic hormones; Catabolism; Catabolon; Peripheral pathways; Environmental isolates; Soil isolation

Comparison of solid-state and submerged-state fermentation for the bioprocessing of switchgrass to ethanol and acetate by Clostridium phytofermentans by Abhiney Jain; Charles K. Morlok; J. Michael Henson (pp. 905-917).

The conversion of sustainable energy crops using microbiological fermentation to biofuels and bioproducts typically uses submerged-state processes. Alternatively, solid-state fermentation processes have several advantages when compared to the typical submerged-state processes. This study compares the use of solid-state versus submerged-state fermentation using the mesophilic anaerobic bacterium Clostridium phytofermentans in the conversion of switchgrass to the end products of ethanol, acetate, and hydrogen. A shift in the ratio of metabolic products towards more acetate and hydrogen production than ethanol production was observed when C. phytofermentans was grown under solid-state conditions as compared to submerged-state conditions. Results indicated that the end product concentrations (in millimolar) obtained using solid-state fermentation were higher than using submerged-state fermentation. In contrast, the total fermentation products (in weight of product per weight of carbohydrates consumed) and switchgrass conversion were higher for submerged-state fermentation. The conversion of xylan was greater than glucan conversion under both fermentation conditions. An initial pH of 7 and moisture content of 80 % resulted in maximum end products formation. Scanning electron microscopy study showed the presence of biofilm formed by C. phytofermentans growing on switchgrass under submerged-state fermentation whereas bacterial cells attached to surface and no apparent biofilm was observed when grown under solid-state fermentation. To our knowledge, this is the first study reporting consolidated bioprocessing of a lignocellulosic substrate by a mesophilic anaerobic bacterium under solid-state fermentation conditions.

Keywords: Solid-state fermentation; Ethanol; Xylan; Lignocellulose; Clostridium phytofermentans

Characterization of an ammonium transporter in the oleaginous alga Chlorella protothecoides by Dong Yan; Junbiao Dai; Qingyu Wu (pp. 919-928).

A suppression subtractive hybridization cDNA library was used to screen the differently expressed (up-regulated) genes in the photosynthesis–fermentation approach (PFA) of Chlorella protothecoides cultivation. A total of 87 clones were obtained and sequenced, in which 78 clones were homologous to known genes in databases. Among them, the ammonium transporter gene (CpAMT1) was characterized in detail. Quantitative real-time PCR showed that the expression of CpAMT1 was significantly induced by PFA and correlated with lipid accumulation. The up-regulation of CpAMT1 was suppressed by glutamine, while the lipid biosynthesis was also inhibited. Further analysis showed that the expression of CpAMT1 was correlated with glutamine synthetase activity, suggesting that CpAMT1, along with glutamine synthetase/glutamate synthase, may be responsible for nitrogen sensing in C. protothecoides. Together, these results imply that the ammonium transporter CpAMT1 could be the initial sensor of nitrogen deficiency and channels the carbon excess toward lipid biosynthesis.

Keywords: Microalga Chlorella protothecoide ; Oil; Biodiesel; Ammonium transporter; C/N metabolism

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Applied Microbiology and Biotechnology (v.97, #2)

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Applied Microbiology and Biotechnology (v.97, #2)