High-yield expression,purification, characterization,and structure determination of tag-free <Emphasis Type="Italic">Candida utilis</Emphasis> uricase

We report the successful high-yield expression of Candida utilis uricase in Escherichia coli and the establishment of an efficient three-step protein purification protocol. The purity of the recombinant protein, which was confirmed to be C. utilis uricase by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometer analysis, was >98% and the specific activity was 38.4 IU/mg. Crystals of C. utilis uricase were grown at 18°C using 25% polyethylene glycol 3350 as precipitant. Diffraction by the crystals extends to 1.93 Å resolution, and the crystals belong to the space group P2₁2₁2₁ with unit cell parameters a?=?69.16 Å, b?=?139.31 Å, c?=?256.33 Å, and α?=?β?=?γ?=?90°. The crystal structure of C. utilis uricase shares a high similarity with other reported structures of the homologous uricases from other species in protein database, demonstrating that the three-dimensional structure of the protein defines critically to the catalytic activities.     

Cloning,expression, and characterization of a thermostable glucose-6-phosphate dehydrogenase from <Emphasis Type="Italic">Thermoanaerobacter tengcongensis</Emphasis>

Glucose-6-phosphate dehydrogenases (G6PDs) are important enzymes widely used in bioassay and biocatalysis. In this study, we reported the cloning, expression, and enzymatic characterization of G6PDs from the thermophilic bacterium Thermoanaerobacter tengcongensis MB4 (TtG6PD). SDS-PAGE showed that purified recombinant enzyme had an apparent subunit molecular weight of 60 kDa. Kinetics assay indicated that TtG6PD preferred NADP⁺ (k _cat/K _m = 2618 mM^?1 s^?1, k _cat = 249 s^?1, K _m = 0.10 ± 0.01 mM) as cofactor, although NAD⁺ (k _cat/K _m = 138 mM^?1 s^?1, k _cat = 604 s^?1, K _m = 4.37 ± 0.56 mM) could also be accepted. The K _m values of glucose-6-phosphate were 0.27 ± 0.07 mM and 5.08 ± 0.68 mM with NADP⁺ and NAD⁺ as cofactors, respectively. The enzyme displayed its optimum activity at pH 6.8–9.0 for NADP⁺ and at pH 7.0–8.6 for NAD⁺ while the optimal temperature was 80 °C for NADP⁺ and 70 °C for NAD⁺. This was the first observation that the NADP⁺-linked optimal temperature of a dual coenzyme-specific G6PD was higher than the NAD⁺-linked and growth (75 °C) optimal temperature, which suggested G6PD might contribute to the thermal resistance of a bacterium. The potential of TtG6PD to measure the activity of another thermophilic enzyme was demonstrated by the coupled assays for a thermophilic glucokinase.     

<Emphasis Type="Italic">Haloquadratum walsbyi</Emphasis> Yields a Versatile,NAD<Superscript>+</Superscript>/NADP<Superscript>+</Superscript> Dual Affinity,Thermostable, Alcohol Dehydrogenase (<Emphasis Type="Italic">Hw</Emphasis>ADH)

This study presents the first example of an alcohol dehydrogenase (ADH) from the halophilic archaeum Haloquadratum walsbyi (HwADH). A hexahistidine-tagged recombinant HwADH was heterologously overexpressed in Haloferax volcanii. HwADH was purified in one step and was found to be thermophilic with optimal activity at 65 °C. HwADH was active in the presence of 10% (v/v) organic solvent. The enzyme displayed dual cofactor specificity and a broad substrate scope, and maximum activity was detected with benzyl alcohol and 2-phenyl-1-propanol. HwADH accepted aromatic ketones, acetophenone and phenylacetone as substrates. The enzyme also accepted cyclohexanol and aromatic secondary alcohols, 1-phenylethanol and 4-phenyl-2-butanol. H. walsbyi may offer an excellent alternative to other archaeal sources to expand the toolbox of halophilic biocatalysts.     

<Emphasis Type="Italic">Talaromyces heiheensis</Emphasis> and <Emphasis Type="Italic">T. mangshanicus</Emphasis>, two new species from China

Two new species, Talaromyces heiheensis from rotten wood and T. mangshanicus isolated from soil, are illustrated and described as new to science in sections Trachyspermi and Talaromyces. The phylogenetic positions of the two new species inferred from the internal transcribed spacer, beta-tubulin, calmodulin and RNA polymerase II second largest subunit regions were carried out. Talaromyces heiheensis is phylogenetically closely related to T. albobiverticillius, T. rubrifaciens, T. solicola and T. erythromellis, and characterised by slow growth on Czapek yeast autolysate agar at 25 °C, orange conidia en masse on malt extract agar at 25 °C, biverticillate and terverticillate conidiophores, acerose phialides and subglobose to ellipsoidal, smooth-walled conidia. Talaromyces mangshanicus is related to T. kendrickii, T. qii and T. thailandensis, and characterised by slow-growing colonies with absent or sparse sporulation on CYA agar at 25 °C, conidia en masse greyish purple, purplish red soluble pigment on yeast extract agar (YES) at 25 °C, biverticillate conidiophores, ampulliform phialides and subglobose to ellipsoidal conidia with echinulate walls. They are distinguished from the known species in culture characteristics on four standard media, microscopic features and sequence data.     

Water-related environments: a multistep procedure to assess the diversity and enzymatic properties of cultivable bacteria

Studying the culturable portion of environmental bacterial populations is valuable for understanding the ecology, for discovering taxonomically interesting isolates and for exploiting their enzymatic abilities. In this study, diverse water-related samples, iced water (3 °C) from river, the sediment (29 °C) and water (55 °C) of a hot-spring, were investigated by two cultivation strategies, Dry and novel Wet approach. The isolates were clustered by fluorescent internal transcribed spacer PCR and identified by 16S rRNA sequencing. Several bacterial groups were also sub-typed through the application of Random Amplified Microsatellite Polymorphisms method. A broad enzymatic screening of all bacterial isolates was performed in order to assess the proteolytic, cellulolytic, lipolytic, esterolytic, amylolytic properties, as well as catalase and peroxidase activities. The Wet cultivation demonstrated to be suitable for the isolation of potential new species belonging to genera Massilia, Algoriphagus, Rheinheimera and Pandoraea. Valuable microbial resources with extensive enzymatic activities were recognized among the psychrophilic (Pantoea brenneri and Serratia sp.), mesophilic (Pandoraea, Massilia, Pseudomonas, Stenotrophomonas, Bacillus and Aeromonas) and thermophilic bacteria (Aeribacullus pallidus and Geobacillus kaustophilus). The experimental strategy developed in this study includes simple investigation tools able to reveal the genetic and enzymatic peculiarities of isolated microorganisms. It can be applied to different kinds of aquatic samples and extreme environments similar to those described in this study.     

Deletion of the <Emphasis Type="Italic">Clostridium thermocellum recA</Emphasis> gene reveals that it is required for thermophilic plasmid replication but not plasmid integration at homologous DNA sequences

A limitation to the engineering of cellulolytic thermophiles is the availability of functional, thermostable (≥?60 °C) replicating plasmid vectors for rapid expression and testing of genes that provide improved or novel fuel molecule production pathways. A series of plasmid vectors for genetic manipulation of the cellulolytic thermophile Caldicellulosiruptor bescii has recently been extended to Clostridium thermocellum, another cellulolytic thermophile that very efficiently solubilizes plant biomass and produces ethanol. While the C. bescii pBAS2 replicon on these plasmids is thermostable, the use of homologous promoters, signal sequences and genes led to undesired integration into the bacterial chromosome, a result also observed with less thermostable replicating vectors. In an attempt to overcome undesired plasmid integration in C. thermocellum, a deletion of recA was constructed. As expected, C. thermocellum ?recA showed impaired growth in chemically defined medium and an increased susceptibility to UV damage. Interestingly, we also found that recA is required for replication of the C. bescii thermophilic plasmid pBAS2 in C. thermocellum, but it is not required for replication of plasmid pNW33N. In addition, the C. thermocellum recA mutant retained the ability to integrate homologous DNA into the C. thermocellum chromosome. These data indicate that recA can be required for replication of certain plasmids, and that a recA-independent mechanism exists for the integration of homologous DNA into the C. thermocellum chromosome. Understanding thermophilic plasmid replication is not only important for engineering of these cellulolytic thermophiles, but also for developing genetic systems in similar new potentially useful non-model organisms.     

Biochemical characterization and structural analysis of a new cold-active and salt-tolerant esterase from the marine bacterium <Emphasis Type="Italic">Thalassospira</Emphasis> sp.

A gene encoding an esterase, ThaEst2349, was identified in the marine psychrophilic bacterium Thalassospira sp. GB04J01. The gene was cloned and overexpressed in E. coli as a His-tagged fusion protein. The recombinant enzyme showed optimal activity at 45 °C and the thermal stability displayed a retention of 75 % relative activity at 40 °C after 2 h. The optimal pH was 8.5 but the enzyme kept more than 75 % of its maximal activity between pH 8.0 and 9.5. ThaEst2349 also showed remarkable tolerance towards high concentrations of salt and it was active against short-chain p-nitrophenyl esters, displaying optimal activity with the acetate. The enzyme was tested for tolerance of organic solvents and the results are suggesting that it could function as an interesting candidate for biotechnological applications. The crystal structure of ThaEst2349 was determined to 1.69 Å revealing an asymmetric unit containing two chains, which also is the biological unit. The structure has a characteristic cap domain and a catalytic triad comprising Ser158, His285 and Asp255. To explain the cold-active nature of the enzyme, we compared it against thermophilic counterparts. Our hypothesis is that a high methionine content, less hydrogen bonds and less ion pairs render the enzyme more flexible at low temperatures.     

Improved temperature characteristics of an <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> GHF11 xylanase,by <Emphasis Type="Italic">in silico</Emphasis> design and site-directed mutagenesis

To improve the temperature characteristics of a mesophilic glycoside hydrolase family (GHF) 11 xylanase AoXyn11A from Aspergillus oryzae, both introduction of a disulfide bridge and the substitution of a specific amino acid were carried out by in silico design and site-directed mutagenesis. Based on the analysis of a known crystal structure of thermophilic xylanase TlXynA from Thermomyces lanuginosus, and the alignment of primary structures between AoXyn11A and TlXynA, one mutant AoXyn11A^M with a disulfide bridge (Cys¹⁰⁸–Cys¹⁵²) was designed by replacing the Ser¹⁰⁸ and Asn¹⁵² of AoXyn11A with Cys residues, respectively. Additionally, based on the analysis of amino acid B-factor values, another mutant AoXyn11A^M-G22A was predicted by substituting Gly²² of AoXyn11A^M (having the maximum B-factor value of 69.25 Å, with the corresponding Ala²³ of TlXynA. Thereafter, two mutant xylanase-encoding genes, Aoxyn11A ^M and Aoxyn11A ^M-G22A, were constructed by site-directed mutagenesis. Aoxyn11A and two mutant genes were expressed in E. coli BL21(DE3) respectively, and three expressed recombinant xylanases, reAoXyn11A, reAoXyn11A^M and reAoXyn11A^M-G22A, were purified to homogeneity. The temperature optima of reAoXyn11A^M and reAoXyn11A^M-G22A were 60 and 65°C, respectively, being 5 and 10°C higher than that of reAoXyn11A. Their thermal inactivation half-lives at 50°C were 1.8- and 8.4-folds longer than that of reAoXyn11A. There were no obvious alterations after mutations in specific activity and enzymatic properties, except for the temperature characteristics.     

Kinetic characterization of a novel acid ectophosphatase from <Emphasis Type="Italic">Enterobacter asburiae</Emphasis>

Expression of acid ectophosphatase by Enterobacter asburiae, isolated from Cattleya walkeriana (Orchidaceae) roots and identified by the 16S rRNA gene sequencing analysis, was strictly regulated by phosphorus ions, with its optimal activity being observed at an inorganic phosphate concentration of 7 mM. At the optimum pH 3.5, intact cells released p-nitrophenol at a rate of 350.76 ± 13.53 nmol of p-nitrophenolate (pNP)/min/10⁸ cells. The membrane-bound enzyme was obtained by centrifugation at 100,000 × g for 1 h at 4°C. p-Nitrophenylphosphate (pNPP) hydrolysis by the enzyme follows “Michaelis-Menten” kinetics with V = 61.2 U/mg and K_0.5 = 60 μM, while ATP hydrolysis showed V = 19.7 U/mg, K_0.5 = 110 μM, and n_H = 1.6 and pyrophosphate hydrolysis showed V = 29.7 U/mg, K_0.5 = 84 μM, and n_H = 2.3. Arsenate and phosphate were competitive inhibitors with Ki = 0.6 mM and K_i = 1.8 mM, respectively. p-Nitrophenyl phosphatase (pNPPase) activity was inhibited by vanadate, while p-hydroxymercuribenzoate, EDTA, calcium, copper, and cobalt had no inhibitory effects. Magnesium ions were stimulatory (K_0.5 = 2.2 mM and n_H = 0.5). Production of an acid ectophosphatase can be a mechanism for the solubilization of mineral phosphates by microorganisms such as Enterobacter asburiae that are versatile in the solubilization of insoluble minerals, which, in turn, increases the availability of nutrients for plants, particularly in soils that are poor in phosphorus.     

Three New Species of <Emphasis Type="Italic">Eustomias</Emphasis> (Melanostomiidae) From the Southern Subtropical Waters with the Description of a New Subgenus

Eustomias diplomastiga (28° S, 84° W), E. mavka (36° S, 121° W), and E. kukuevi (23° S, 12° E) spp. n. are described from the southern subtropical waters of Atlantic and Pacific oceans. For the species E. diplomastiga sp. n. and E. crucis, a new subgenus Furcostomias subgen. n. is established. It is different from other subgenera based on the following features: stem of the chin barbel bifurcate at the middle, completely deprived of bulbs, with a pigmented shaft but without the external pigmentation of the stem and branches and without the spots associated with the photophores, the presence of a short ventral furrow, P 2, V 7. A review of the diagnostic features of the subgenera of Eustomias is given.     

Deciphering the mode of action,structural and biochemical analysis of heparinase II/III (<Emphasis Type="Italic">Ps</Emphasis>PL12a) a new member of family 12 polysaccharide lyase from <Emphasis Type="Italic">Pseudopedobacter saltans</Emphasis>

Heparinases are widely used for production of clinically and therapeutically important bioactive oligosaccharides and in analyzing the polydisperse, heterogeneous, and complex structures of heparin/heparan sulfate. In the present study, the gene (1911 bp) encoding heparinase II/III of family 12 polysaccharide lyase (PsPL12a) from Pseudopedobacter saltans was cloned, expressed, and biochemically and functionally characterized. The purified enzyme PsPL12a of molecular size approximately 76 kDa exhibited maximum activity in the temperature range 45–50 °C and at pH 6.0. PsPL12a gave maximum activity at 1% (w/v) heparin under optimum conditions. The kinetic parameters, K _m and V_max, for PsPL12a were 4.6?±?0.5 mg/ml and 70?±?2 U/mg, respectively. Ten millimolars of each Mg²⁺ and Mn²⁺ ions enhanced PsPL12a activity by 80%, whereas Ni²⁺ inhibited by 75% and Co²⁺ by 10%, and EDTA completely inactivated the enzyme. Protein melting curve of PsPL12a gave a single peak at 55 °C and 10 mM Mg²⁺ ions and shifted the peak to 60 °C. The secondary structure analysis of PsPL12a by CD showed 65.12% α-helix, 11.84% β-strand, and 23.04% random coil. The degradation products of heparin by PsPL12a analyzed by ESI-MS spectra displayed peaks corresponding to heparin di-, tetra-, penta-, and hexa-saccharides revealing the endolytic mode of enzyme action. Heparinase II/III (PsPL12a) from P. saltans can be used for production of low molecular weight heparin oligosaccharides for their utilization as anticoagulants. This is the first report on heparinase cloned from P. saltans.     

Interspecific variation in extracellular polysaccharide content and colony formation of <Emphasis Type="Italic">Microcystis</Emphasis> spp. cultured under different light intensities and temperatures

Single cells of five different Microcystis species (M. ichthyoblabe, M. viridis, M. flos-aquae, M. wesenbergii, and M. aeruginosa) were batch-cultured at different temperatures and light intensities: (a) 25 °C and 50 μmol photons m^?2 s^?1 (control culture); (b) 25 °C and 10 μmol photons m^?2 s^?1; and (c) 15 °C and 50 μmol photons m^?2 s^?1. The extracellular polysaccharide content was significantly higher in treatments b and c than in the control treatment. All Microcystis species existed as single cells under the control treatment but formed colonies in treatments b and c. All of the colonies were irregular with indistinct margins. M. ichthyoblabe, M. viridis, M. flos-aquae, and M. wesenbergii formed colonies with similar morphologies and their cells were loosely aggregated. In contrast, M. aeruginosa formed denser colonies with no distinct holes. The colony morphologies differed from the classic morphology of M. ichthyoblabe field-grown colonies but resembled that of small colonies found in Lake Taihu (Yangtze Delta Plain, China) during early spring. This indicates that field- and laboratory-grown colonies are governed by similar formation processes. We suggest that in laboratory and field environments, M. ichthyoblabe (or M. flos-aquae) colonies are representative of small colonies formed from single Microcystis cells, whereas the morphology of older colonies evolves to resemble M. wesenbergii and M. aeruginosa colonies.     

Characteristics and expression of genes encoding two small heat shock protein genes lacking introns from <Emphasis Type="Italic">Chilo suppressalis</Emphasis>

Small heat shock proteins (sHSPs) constitute a large, diverse, and functionally uncharacterized family of heat shock proteins. To gain insight regarding the function of sHSPs in insects, we identified genes encoding two sHSPs, Cshsp22.9b and Cshsp24.3, from the rice pest Chilo suppressalis. The cDNAs of Cshsp22.9b and Cshsp24.3 encoded proteins of 206 and 216 amino acids with isoelectric points of 5.79 and 9.28, respectively. Further characterization indicated that both Cshsp22.9b and Cshsp24.3 lacked introns. Real-time quantitative PCR indicated that Cshsp22.9b and Cshsp24.3 were expressed at higher levels within the fat body as compared to other tissues (head, epidermis, foregut, midgut, hindgut, Malpighian tubules, and hemocytes). Expression of Cshsp22.9b and Cshsp24.3 was lowest in the hindgut and Malpighian tubules, respectively. Cshsp22.9b and Cshsp24.3 showed identical patterns in response to thermal stress from ?11 to 43 °C, and both genes were up-regulated by hot and cold temperatures. The mRNA (messenger ribonucleic acid) expression levels of Cshsp22.9b (KY701308) and Cshsp24.3 (KY701309) were highest after a 2-h exposure at 39 °C and started to decline at 42 °C. In response to cold temperatures, both Cshsp22.9b and Cshsp24.3 showed maximal expression after a 2-h exposure to ?3 °C. The two Cshsps were more responsive to hot than cold temperature stress and were not induced by mildly cold or warm temperatures. In conclusion, Cshsp22.9b and Cshsp24.3 could play a very important role in the regulation of physiological activities in C. suppressalis that are impacted by environmental stimuli.     

Molecular characterization and homology modeling of spermidine synthase from <Emphasis Type="Italic">Synechococcus</Emphasis> sp. PCC 7942

Spermidine synthase (Spds) catalyzes the formation of spermidine by transferring the aminopropyl group from decarboxylated S-adenosylmethionine (dcSAM) to putrescine. The Synechococcus spds gene encoding Spds was expressed in Escherichia coli. The purified recombinant enzyme had a molecular mass of 33 kDa and showed optimal activity at pH 7.5, 37?°C. The enzyme had higher affinity for dcSAM (K _m, 20 µM) than for putrescine (K _m, 111 µM) and was highly specific towards the diamine putrescine with no activity observed towards longer chain diamines. The three-dimensional structural model for Synechococcus Spds revealed that most of the ligand binding residues in Spds from Synechococcus sp. PCC 7942 are identical to those of human and parasite Spds. Based on the model, the highly conserved acidic residues, Asp89, Asp159 and Asp162, are involved in the binding of substrates putrescine and dcSAM and Pro166 seems to confer substrate specificity towards putrescine.     

Xylem anatomy and calculated hydraulic conductance of four <Emphasis Type="Italic">Nothofagus</Emphasis> species with contrasting distribution in South-Central Chile

Nothofagus obliqua, N. dombeyi, N. alpina and N. antarctica are characteristic tree species of the temperate forests on the western slopes of the Andes with centres of distribution that differ in their temperature and moisture regimes. We tested branch wood from co-occurring specimens of these species for the inherent differences in xylem anatomy and theoretical hydraulic conductance to evaluate their resistance to drought or frost. The hydraulic conductivity of the xylem was calculated using a modified Hagen–Poiseuille equation and related to wood density. Conduit dimensions were used to predict the water potential that would cause 50 % loss of hydraulic conductivity (Ψ ₅₀). Nothofagus alpina, which mainly grows at sites with low frost frequency, exhibited the largest conduits and the highest mean values for conduit area, fraction of conduit area in the cross-section and hydraulic conductivity, but the lowest wood density. Opposite relationships were found in the plastic N. antarctica, whose xylem seems to be least vulnerable to freezing-induced, but also to drought-induced embolism. Calculated Ψ ₅₀ was highest (least negative) in N. alpina, indicating a relatively high susceptibility to cavitation. The xylem of the thermophilic N. obliqua and of N. dombeyi, which mainly occurs under oceanic climate, but can also survive at sporadically dry and warm sites, is not particularly adapted to periods of drought stress. Across all species, wood density was negatively correlated with the calculated hydraulic conductance. The xylem traits of N. alpina might contribute to its relatively high growth rate and facilitate its spread into forest gaps.     

Thermophilic and thermotolerant aerobic methanotrophs

The review generalizes the modern data on the taxonomic, structural, and functional diversity of aerobic methanotrophs growing at 25–50°C (Methylococcus capsulatus), 30–62°C (Methylocaldum szegediense, Methylocaldum gracile, and Methylocaldum tepidum), and 50–65°C (Methylothermus thermalis), which belong mainly to the Gammaproteobacteria. The specific features of adaptation of these methanotrophs to the temperature influences are considered on the metabolic and genetic levels. The recent sensational reports on the discovery and primary characterization of thermoacidophilic methanotrophs of the phylum Verrucomicrobia surviving at extreme pH (1–2) and temperature (65°C) values, corresponding to extremely low levels of CH₄ and O₂ solubility, are analyzed. The possibilities of implementation of the biotechnological potential of thermophilic and thermotolerant methanotrophs are discussed.     

HU histone-like DNA-binding protein from <Emphasis Type="Italic">Thermus thermophilus</Emphasis>: structural and evolutionary analyses

The histone-like DNA-binding proteins (HU) serve as model molecules for protein thermostability studies, as they function in different bacteria that grow in a wide range of temperatures and show sequence diversity under a common fold. In this work, we report the cloning of the hutth gene from Thermus thermophilus, the purification and crystallization of the recombinant HUTth protein, as well as its X-ray structure determination at 1.7 Å. Detailed structural and thermodynamic analyses were performed towards the understanding of the thermostability mechanism. The interaction of HUTth protein with plasmid DNA in solution has been determined for the first time with MST. Sequence conservation of an exclusively thermophilic order like Thermales, when compared to a predominantly mesophilic order (Deinococcales), should be subject, to some extent, to thermostability-related evolutionary pressure. This hypothesis was used to guide our bioinformatics and evolutionary studies. We discuss the impact of thermostability adaptation on the structure of HU proteins, based on the detailed evolutionary analysis of the Deinococcus–Thermus phylum, where HUTth belongs. Furthermore, we propose a novel method of engineering thermostable proteins, by combining consensus-based design with ancestral sequence reconstruction. Finally, through the structure of HUTth, we are able to examine the validity of these predictions. Our approach represents a significant advancement, as it explores for the first time the potential of ancestral sequence reconstruction in the divergence between a thermophilic and a mainly mesophilic taxon, combined with consensus-based engineering.     

Expression and characterization of glutamate decarboxylase from <Emphasis Type="Italic">Lactobacillus brevis</Emphasis> HYE1 isolated from <Emphasis Type="Italic">kimchi</Emphasis>

A putative gene (gadlbhye1) encoding glutamate decarboxylase (GAD) was cloned from Lactobacillus brevis HYE1 isolated from kimchi, a traditional Korean fermented vegetable. The amino acid sequences of GADLbHYE1 showed 48% homology with the GadA family and 99% identity with the GadB family from L. brevis. The cloned GADLbHYE1 was functionally expressed in Escherichia coli using inducible expression vectors. The expressed recombinant GADLbHYE1 was successfully purified by Ni–NTA affinity chromatography, and had a molecular mass of 54 kDa with optimal hydrolysis activity at 55 °C and pH 4.0. Its thermal stability was determined to be higher than that of other GADs from L. brevis, based on its melting temperature (75.18 °C). Kinetic parameters including K_m and V_max values for GADLbHYE1 were 4.99 mmol/L and 0.224 mmol/L/min, respectively. In addition, the production of gamma-aminobutyric acid in E. coli BL21 harboring gadlbhye1/pET28a was increased by adding pyridoxine as a cheaper coenzyme.     

Cloning and expression of <Emphasis Type="Italic">Perilla frutescens FAD2</Emphasis> gene and polymorphism analysis among cultivars

?12 fatty acid desaturase (FAD2) is a key enzyme for linoleic acid and linolenic acid biosynthesis. Perilla frutescens is a special oil plant species with highest linolenic acid content. In this study, based on RACE, two alleles for one FAD2 gene were isolated from P. frutescens cultivar C2: the 3956 bp PfFAD2a and the 3959 bp PfFAD2b, both with a full-length cDNA of 1526 bp, and both encoding a 382aa basic protein. The alleles have identities of over 98%, and their encoded proteins differ only by substitution of a strongly similar residue. Saccharomyces cerevisiae heterologous expression suggested that PfFAD2a/b both encode a bio-functional FAD2 enzyme. Phylogenetic analyses indicated that PfFAD2 shows the highest homologies to FAD2 genes from dicots such as Boraginaceae and Burseraceae. PfFAD2a/b expressions are mainly restricted to developing seeds. PfFAD2a/b expression in the seedling leaf is upregulated by cold (4 °C) and repressed by heat (42 °C). Each of the eight cultivars contains two alleles for one PfFAD2 and 40 SNP sites are found. One allelic gene in cultivars C1 and P1 is pseudogene because of premature stop codon mutation in 5′ coding region. All other normal PfFAD2 genes/allelic genes encode identical or very similar proteins. PfFAD2a/b expression level in developing seeds also varies among the eight cultivars. This study provides systemic molecular and functional features of PfFAD2 and enables its application in the study of plant fatty acids traits.     

A new source of resistance to 2-furaldehyde from <Emphasis Type="Italic">Scheffersomyces</Emphasis> (<Emphasis Type="Italic">Pichia</Emphasis>) <Emphasis Type="Italic">stipitis</Emphasis> for sustainable lignocellulose-to-biofuel conversion

Aldehyde inhibitory compounds derived from lignocellulosic biomass pretreatment have been identified as a major class of toxic chemicals that interfere with microbial growth and subsequent fermentation for advanced biofuel production. Development of robust next-generation biocatalyst is a key for a low-cost biofuel production industry. Scheffersomyces (Pichia) stipitis is a naturally occurring C-5 sugar utilization yeast; however, little is known about the genetic background underlying its potential tolerance to biomass conversion inhibitors. We investigated and identified five uncharacterized putative aryl-alcohol dehydrogenase genes (SsAADs) from this yeast as a new source of resistance against biomass fermentation inhibitor 2-furaldehyde (furfural) by gene expression, gene cloning, and direct enzyme assay analysis using partially purified proteins. All five proteins from S. stipitis showed furfural reduction using cofactor NADH. An optimum active temperature was observed at 40 °C for SsAad1p; 30 °C for SsAad3p, SsAad4p, and SsAad5p; and 20 °C for SsAad2p. SsAad2p, SsAad3p, and SsAad4p showed tolerance to a wide range of pH from 4.5 to 8, but SsAad1p and SsAad5p were sensitive to pH changes beyond 7. Genes SsAAD2, SsAAD3, and SsAAD4 displayed significantly enhanced higher levels of expression in response to the challenge of furfural. Their encoding proteins also showed higher levels of specific activity toward furfural and were suggested as core functional enzymes contributing aldehyde resistance in S. stipitis.