Gene cloning and characterization of recombinant L-Asparaginase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strain R5

L-asparaginase gene from Bacillus subtilis strain R5 (Asn-R5), comprising 990 nucleotides corresponding to a polypeptide of 329 amino acids, was cloned and expressed in Escherichia coli. Recombinant Asn-R5 was produced in soluble and active form exhibiting a specific activity of 223 μmol min^?1 mg^?1. The optimal temperature and pH for L-asparaginase activity of Asn-R5 were 35 °C and 9.0, respectively. Asn-R5 displayed a 50% activity with D-asparagine and 2% with L-glutamine compared to 100% with L-asparagine. No activity could be detected when D-glutamine was used as substrate. Half-life of the enzyme was 180 min at 35 °C and 40 min at 50 °C. There was no effect of metal ions and EDTA on the activity indicating that Asn-R5 enzyme activity is not metal ion dependent. The K_m and V_max values were 2.4 mM and 265 μmol min^?1 mg^?1, respectively. Activation energy for reaction catalyzed by Asn-R5 was 28 kJ mol^?1. High L-asparaginase activity and thermostability of recombinant Asn-R5 may be beneficial for industrial production and application.     

Characterization of alcohol dehydrogenase from <Emphasis Type="Italic">Kangiella koreensis</Emphasis> and its application to production of all-<Emphasis Type="Italic">trans</Emphasis>-retinol

A recombinant alcohol dehydrogenase (ADH) from Kangiella koreensis was purified as a 40 kDa dimer with a specific activity of 21.3 nmol min^?1 mg^?1, a K _m of 1.8 μM, and a k _cat of 1.7 min^?1 for all-trans-retinal using NADH as cofactor. The enzyme showed activity for all-trans-retinol using NAD ⁺ as a cofactor. The reaction conditions for all-trans-retinol production were optimal at pH 6.5 and 60 °C, 2 g enzyme l^?1, and 2,200 mg all-trans-retinal l^?1 in the presence of 5 % (v/v) methanol, 1 % (w/v) hydroquinone, and 10 mM NADH. Under optimized conditions, the ADH produced 600 mg all-trans-retinol l^?1 after 3 h, with a conversion yield of 27.3 % (w/w) and a productivity of 200 mg l^?1 h^?1. This is the first report of the characterization of a bacterial ADH for all-trans-retinal and the biotechnological production of all-trans-retinol using ADH.     

Extraction of sterols in brown macroalgae from Antarctica and their identification by liquid chromatography coupled with tandem mass spectrometry

This study reports for the first time the extraction and quantification of sterols in six species of brown macroalgae from Antarctica: Adenocystis utricularis, Ascoseira mirabilis, Cystosphaera jacquinotii, Desmarestia anceps, Desmarestia antarctica, and Himantothallus grandifolius. Ultrasound irradiation was used as a promotor to extract sterols from algal biomass. The extracts were analyzed by high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for sterol quantization (ergosterol, brassicasterol, fucosterol, β-sitosterol, campesterol, cholesterol, and stigmasterol). In general, fucosterol was the most abundant (6.60 to 48.13 mg kg^?1), followed by β-sitosterol (5.29 to 16.49 mg kg^?1), stigmasterol (2.69 to 14.84 mg kg^?1) in five of the six examined algae. The sterol campesterol was found in lower concentrations (0.07 to 0.15 mg kg^?1) in all studied samples.     

Kinetics investigation of the hydrogen abstraction reaction between CH<Subscript>3</Subscript>SS and CN radicals

The reaction mechanisms and rates for the H abstraction reactions between CH₃SS and CN radicals in the gas phase were investigated with density functional theory (DFT) methods. The geometries, harmonic vibrational frequencies, and energies of all stationary points were obtained at B3PW91/6-311G(d,p) level of theory. Relationships between the reactants, intermediates, transition states and products were confirmed, with the frequency and the intrinsic reaction coordinate (IRC) analysis at the same theoretical level. High accurate energy information was provided by the G3(MP2) method combined with the standard statistical thermodynamics. Gibbs free energies at 298.15 K for all of the reaction steps were reported, and were used to describe the profile diagrams of the potential energy surface. The rate constants were evaluated with both the classical transition state theory and the canonical variational transition state theory, in which the small-curvature tunneling correction was included. A total number of 9 intermediates (IMs) and 17 transition states (TSs) were obtained. It is shown that IM1 is the most stable intermediate by the largest energy release, and the channel of CH₃SS?+?CN?→?IM3?→?TS10?→?P1(CH₂SS?+?HCN) is the dominant reaction with the lowest energy barrier of 144.7 kJ mol^?1. The fitted Arrhenius expressions of the calculated CVT/SCT rate constants for the rate-determining step of the favorable channel is k =7.73?×?10⁶? T ^1.40exp(?14,423.8/T) s^?1 in the temperature range of 200–2000 K. The apparent activation energy E _a(app.) for the main channel is ?102.5 kJ mol^?1, which is comparable with the G3(MP2) energy barrier of ?91.8 kJ mol^?1 of TS10 (relative to the reactants).     

Kinetin Regulates UV-B-Induced Damage to Growth,Photosystem II Photochemistry,and Nitrogen Metabolism in Tomato Seedlings

Cytokinins are a class of plant growth regulators that regulate several developmental processes in plants, and recently their role in counteracting the deleterious effects of abiotic stresses has been noted. The impacts of kinetin (10 µM, KN; an artificial cytokinin) on growth, photosystem II photochemistry, and nitrogen metabolism in tomato seedlings exposed to two levels (UV-B₁, ambient+?1.2 kJ m^?2 day^?1, and UV-B₂, ambient+?2.4 kJ m^?2 day^?1) of enhanced UV-B radiation were analyzed under open field condition. The growth, pigment contents, carbonic anhydrase activity, photosynthetic O₂ yield, and values of chlorophyll a fluorescence parameters: F _v/F ₀, F _v/F _m or φP₀, ψ ₀, φE ₀, and PI_ABS declined, whereas the values of energy flux parameters (ABS/RC, TR₀/RC, ET₀/RC, and DI₀/RC) of PS II, efficiency of water splitting complex (F ₀/F _v), and respiratory rate of O₂ uptake increased under UV-B stress. Likewise, UV-B exposure at both doses significantly inhibited the activity of enzymes involved in nitrogen metabolism: nitrate reductase, nitrite reductase, glutamine synthetase, and glutamate synthase. In contrast, an enhancing effect on glutamate dehydrogenase activity was observed under UV-B stress. Exogenous KN resulted in a significant attenuation in UV-B-induced negative effects on growth, pigments, photosynthesis, and nitrogen metabolism. The study concludes that exogenous KN improved the growth performance of tomato seedlings by attenuating the damaging effects of UV-B radiation on photochemistry of PS II and nitrogen metabolism, and the alleviating effect against the low dose (UV-B₁) of UV-B was more pronounced.     

Kinetic Study of the Acid Post-hydrolysis of Xylooligosaccharides from Hydrothermal Pretreatment

Hydrothermal pretreatment of sugarcane bagasse is a water-based and environment-friendly process that results in almost complete hemicellulose solubilization in oligomeric form as xylooligossacharides (XOs). However, the soluble XOs cannot be utilized by microorganisms such as yeasts, and therefore, a further break down is necessary to generate pentose (C5) monomers that can be then biotransformed into ethanol or other metabolites. The kinetics of XOs post-hydrolysis with sulfuric, maleic, and oxalic acids (the latter two being dicarboxylic acids) in a sugarcane bagasse hemicellulosic hydrolysate was assessed in a bench-scale reactor (2 L). By means of a 2² full factorial design with center point triplicate, acid mass loading and temperature were varied from 0.5 and 2.0% and from 120 to 150 °C, respectively. An irreversible first-order consecutive reaction model of the hydrolysis of XOs in liquid medium was employed. Based on an Arrhenius-type equation, a kinetic parameter estimation was performed with genetic algorithms and the Runge-Kutta methods. For the three acids, the calculated exponential factors, A _0n (n = 1, 2, and 3), ranged from 10¹² to 10¹⁵ min^?1; the dimensionless parameters, m _n (n = 1, 2, and 3), ranged from 0.86 to 1.97; and the activation energies ranged from 89 to 129.8 kJ·mol^?1. The model—developed at microscale—correctly described the observed XOs, C5, and furfural post-hydrolysis profiles in bench scale and proved the dicarboxylic acids were more selective towards post-hydrolysis by having slower kinetics than sulfuric acid.     

A xylanase from <Emphasis Type="Italic">Streptomyces sp.</Emphasis> FA1: heterologous expression,characterization, and its application in Chinese steamed bread

Xylanases (EC 3.2.1.8) are hydrolytic enzymes that have found widespread application in the food, feed, and paper-pulp industries. Streptomyces sp. FA1 xynA was expressed as a secreted protein in Pichia pastoris, and the xylanase was applied to the production of Chinese steamed bread for the first time. The optimal pH and the optimal temperature of XynA were 5.5 and 60 °C, respectively. Using beechwood as substrate, the K _m and V _max were 2.408 mg mL^?1 and 299.3 µmol min^?1 mg^?1, respectively. Under optimal conditions, a 3.6-L bioreactor produced 1374 U mL^?1 of XynA activity at a protein concentration of 6.3 g L^?1 after 132 h of fermentation. Use of recombinant XynA led to a greater increase in the specific volume of the CSB than could be achieved using commercial xylanase under optimal conditions. This study provides the basis for the application of the enzyme in the baking industry.     

Synthesis of natural variants and synthetic derivatives of the cyclic nonribosomal peptide luminmide in permeabilized <Emphasis Type="Italic">E. coli</Emphasis> Nissle and product formation kinetics

We used a recombinant, permeabilized E. coli Nissle strain harbouring the plu3263 gene cluster from Photorhabdus luminescens for the synthesis of luminmide type cyclic pentapeptides belonging to the class of nonribosomally biosynthesized peptides (NRP). Cells could be fully permeabilized using 1 % v/v toluene. Synthesis of luminmides was increased fivefold when 0.3 mM EDTA was added to the substrate mixture acting as an inhibitor of metal proteases. Luminmide formation was studied applying different amino acid concentrations. Apparent kinetic parameters for the synthesis of the main product luminmide A from leucine, phenylalanine and valine were calculated from the collected data. K _s ^app values ranged from 0.17 mM for leucine to 0.57 mM for phenylalanine, and r _max ^app was about 3 × 10^?8 mmol min^?1(g CDW)^?1). By removing phenylalanine from the substrate mixture, the formation of luminmide A was reduced tenfold while luminmide B was increased from 50 to 500 μg/l becoming the main product. Two new luminmides were synthesized in this study. Luminmide H incorporates tryptophan replacing phenylalanine in luminmide A. In luminmide I, leucine was replaced with 4,5-dehydro-leucine, a non-proteinogenic amino acid fed to the incubation mixture. Our study shows new opportunities for increasing the spectrum of luminmide variants produced, for improving production selectivity and for kinetic in vitro studies of the megasynthetases.     

The nitrite reductase activity of horse heart carboxymethylated-cytochrome <Emphasis Type="Italic">c</Emphasis> is modulated by cardiolipin

Horse heart carboxymethylated cytc (CM-cytc) displays myoglobin-like properties. Here, the effect of cardiolipin (CL) liposomes on the nitrite reductase activity of ferrous CM-cytc [CM-cytc-Fe(II)], in the presence of sodium dithionite, is reported between pH 5.5 and 7.6, at 20.0 °C. Cytc-Fe(II) displays a very low value of the apparent second-order rate constant for the NO₂ ^?-mediated conversion of cytc-Fe(II) to cytc-Fe(II)-NO [k _on = (7.3 ± 0.7) × 10^?2 M^?1 s^?1; at pH 7.4], whereas the value of k _on for NO₂ ^? reduction by CM-cytc-Fe(II) is 1.1 ± 0.2 M^?1 s^?1 (at pH 7.4). CL facilitates the NO₂ ^?-mediated nitrosylation of CM-cytc-Fe(II) in a dose-dependent manner, the value of k _on for the NO₂ ^?-mediated conversion of CL–CM-cytc-Fe(II) to CL–CM-cytc-Fe(II)-NO (5.6 ± 0.6 M^?1 s^?1; at pH 7.4) being slightly higher than that for the NO₂ ^?-mediated conversion of CL–cytc-Fe(II) to CL–cytc-Fe(II)-NO (2.6 ± 0.3 M^?1 s^?1; at pH 7.4). The apparent affinity of CL for CM-cytc-Fe(II) is essentially pH independent, the average value of B being (1.3 ± 0.3) × 10^?6 M. In the absence and presence of CL liposomes, the nitrite reductase activity of CM-cytc-Fe(II) increases linearly on lowering pH and the values of the slope of the linear fittings of Log k _on versus pH are ?1.05 ± 0.07 and ?1.03 ± 0.03, respectively, reflecting the involvement of one proton for the formation of the transient ferric form, NO, and OH^?. These results indicate that Met80 carboxymethylation and CL binding cooperate in the stabilization of the highly reactive heme-Fe atom of CL–CM-cytc.     

Oxygen transfer as a tool for fine-tuning recombinant protein production by <Emphasis Type="Italic">Pichia pastoris</Emphasis> under glyceraldehyde-3-phosphate dehydrogenase promoter

Effects of oxygen transfer on recombinant protein production by Pichia pastoris under glyceraldehyde-3-phosphate dehydrogenase promoter were investigated. Recombinant glucose isomerase was chosen as the model protein. Two groups of oxygen transfer strategies were applied, one of which was based on constant oxygen transfer rate where aeration rate was Q _O/V = 3 and 10 vvm, and agitation rate was N = 900 min^?1; while the other one was based on constant dissolved oxygen concentrations, C _DO = 5, 10, 15, 20 and 40 % in the fermentation broth, by using predetermined exponential glucose feeding with μ _o = 0.15 h^?1. The highest cell concentration was obtained as 44 g L^?1 at t = 9 h of the glucose fed-batch phase at C _DO = 20 % operation while the highest volumetric and specific enzyme activities were obtained as 4440 U L^?1 and 126 U g^?1 cell, respectively at C _DO = 15 % operation. Investigation of specific enzyme activities revealed that keeping C _DO at 15 % was more advantageous with an expense of relatively higher by-product formation and lower specific cell growth rate. For this strategy, the highest oxygen transfer coefficient and oxygen uptake rate were K _L a = 0.045 s^?1 and OUR = 8.91 mmol m^?3 s^?1, respectively.     

Bifunctional recombinant cellulase–xylanase (rBhcell-xyl) from the polyextremophilic bacterium <Emphasis Type="Italic">Bacillus halodurans</Emphasis> TSLV1 and its utility in valorization of renewable agro-residues

The thermostable bifunctional CMCase and xylanase encoding gene (rBhcell-xyl) from Bacillus halodurans TSLV1 has been expressed in Escherichia coli. The recombinant E. coli produced rBhcell-xyl (CMCase 2272 and 910 U L^?1 xylanase). The rBhcell-xyl is a ~62-kDa monomeric protein with temperature and pH optima of 60 °C and 6.0 with T_1/2 of 7.0 and 3.5 h at 80 °C for CMCase and xylanase, respectively. The apparent K _m values (CMC and Birchwood xylan) are 3.8 and 3.2 mg mL^?1. The catalytic efficiency (k _cat/K _m ) values of xylanase and CMCase are 657 and 171 mL mg^?1 min^?1, respectively. End-product analysis confirmed that rBhcell-xyl is a unique endo-acting enzyme with exoglucanase activity. The rBhcell-xyl is a GH5 family enzyme possessing single catalytic module and carbohydrate binding module. The action of rBhcell-xyl on corn cobs and wheat bran liberated reducing sugars, which can be fermented to bioethanol and fine biochemicals.     

Evaluation of growth and phycobiliprotein composition of cyanobacteria isolates cultivated in different nitrogen sources

Phycobiliproteins, light-harvesting pigments found in cyanobacteria and in some eukaryotic algae, have numerous commercial applications in food, cosmetic, and pharmaceutical industries. Colorant production from cyanobacteria offers advantages over their production from higher plants, as cyanobacteria have fast growth rate and high photosynthetic efficiency and require less space. In this study, three cyanobacteria strains were studied for phycobiliprotein production and the influence of sodium nitrate, potassium nitrate and ammonium chloride on the growth and phycobiliprotein composition of the strains were evaluated. In the batch culture period of 12 days, Phormidium sp. and Pseudoscillatoria sp. were able to utilize all tested nitrogen sources; however, ammonium chloride was the best nitrogen source for both strains to achieve maximum growth rate μ?=?0.284?±?0.03 and μ?=?0.274?±?0.13 day^?1, chlorophyll a 16.2?±? 0.5 and 12.2?±? 0.2 mg L^?1, and phycobiliprotein contents 19.38?±?0.09 and 19.99?±?0.14% of dry weight, whereas, for Arthrospira platensis, the highest growth rate of μ?=?0.304?±?0.0 day^?1, chlorophyll a 19.1?±?0.5 mg L^?1, and phycobiliprotein content of 22.27?±?0.21% of dry weight were achieved with sodium nitrate. The phycocyanin from the lyophilized cyanobacterial biomass was extracted using calcium chloride and food grade purity (A₆₂₀/A₂₈₀ ratio >?0.7) was achieved. Furthermore, phycocyanin was purified using two-step chromatographic method and the analytical grade purity (A₆₂₀/A₂₈₀ ratio >?4) was attained. SDS-PAGE demonstrated the purity and presence of two bands corresponding to α- and β-subunits of the C-phycocyanin. The results showed that Phormidium sp. and Pseudoscillatoria sp. could be good candidates for phycocyanin production.     

Earthworm Communities in the Bamboo Plantations of West Tripura (India)

Present study revealed the presence of 16 earthworm species belonging to 11 genera and four families viz. Megascolecidae (Amynthus alexandri, Metaphire houlleti, Lampito mauritii, Kanchuria sp1, Perionyx excavatus), Octochaetidae (Eutyphoeus gigas, Eutyphoeus comillahnus, Eutyphoeus orientalis, Octochaetona beatrix, Dichogaster bolaui, Lennogaster chittagongensis, Lennogaster yeicus), Moniligastridae (Drawida papillifer papillifer, Drawida assamensis, Drawida nepalensis) and Glossoscolecidae (Pontoscolex corethrurus) in the soils of five bamboo species [Bambusa balcooa (Sil Barak), Melocanna baccifera (Muli), Bambusa polumorpha (Bari), Bambus cacharensis (Bom) and Bambus bambus (Katabarak)] of West-Tripura. While four earthworm species viz. Metaphire houlleti, Drawida assamensis, Drawida papillifer papillifer and Pontoscolex corethrurus were common to all species of bamboo plantations, the rest showed restricted distribution. Among the earthworm species 4 were exotic (Amynthus alexandri, Metaphire houlleti, Dichogaster bolaui and Pontoscolex corethrurus) and the others were native to the Indian sub-continent. In general, earthworms under the bamboo plantations occurred within temperature range of 21.6 °C–28.0 °C, pH 4.0–7.0, organic matter 0.56–5.99 %, moisture 9.6–31.7 %, water holding capacity 14.6–43.9 % and bulk density 0.7–1.8 g cm^?3. The average density and biomass of the earthworms in the studied places were 108 ind m^?2 and 44 g m^?2 respectively. Earthworm diversity, dominance and evenness indices showed the values 1.00, 0.47 and 0.70 respectively. Earthworm density and biomass showed a negative correlation with temperature whereas those had a strong positive correlation with pH, moisture and organic matter of the soils.     

Cryopreservation of <Emphasis Type="Italic">Citrus</Emphasis> anthers in the National Crop Genebank of China

Genebank conservation of pollen is valuable because it makes genetic resources immediately available for use in breeding programs. In the case of Citrus species, conserved anthers or pollen can be easily transported and used to develop new varieties with pathogen resistance and desirable quality and yield traits. The aim of this study was to develop and improve air-desiccation cryopreservation protocols for Citrus cavaleriei and Citrus maxima anthers in genebanks. In the current study, warming, rehydration, and in vitro germination conditions were optimized to achieve high levels of in vitro germination in Citrus pollen for ten cultivars after liquid nitrogen (LN) exposure. The optimal warming, rehydration, and in vitro germination medium formulations affected the germination levels after pollen cryopreservation, with species- and cultivar-dependent effects. The Citrus anthers were dehydrated to the moisture content of 5–14% before LN exposure and warmed at 25 (cryopreserved Citrus anthers with a moisture content of lower than 10%) or 37°C (a moisture content of 10% or higher), then rehydrated, and cultured on medium with 150-g L^?1 sucrose, 0.1-g L^?1 boric acid, 1.0-g L^?1 calcium nitrate, 0.1-g L^?1 potassium nitrate, 0.3-g L^?1 magnesium sulfate, and 10-g L^?1 agar. After 2 yr of storage, in vitro germination levels of Citrus pollen after cryopreservation were significantly higher (> 22% for all ten cultivars) than those of samples that were stored at 4°C (0%). In vitro germination levels of pollen from six of ten cultivars after cryopreservation remained relatively high after 2 yr of storage (38–93%). The highest viability of 93% was obtained for C. cavaleriei ‘2–3’. The methods identified in the current study could be used to cryopreserve C. cavaleriei and C. maxima anthers.     

Analysis of the <Emphasis Type="Italic">xynB5</Emphasis> gene encoding a multifunctional GH3-BglX β-glucosidase-β-xylosidase-α-arabinosidase member in <Emphasis Type="Italic">Caulobacter crescentus</Emphasis>

The Caulobacter crescentus (NA1000) xynB5 gene (CCNA_03149) encodes a predicted β-glucosidase-β-xylosidase enzyme that was amplified by polymerase chain reaction; the product was cloned into the blunt ends of the pJet1.2 plasmid. Analysis of the protein sequence indicated the presence of conserved glycosyl hydrolase 3 (GH3), β-glucosidase-related glycosidase (BglX) and fibronectin type III-like domains. After verifying its identity by DNA sequencing, the xynB5 gene was linked to an amino-terminal His-tag using the pTrcHisA vector. A recombinant protein (95 kDa) was successfully overexpressed from the xynB5 gene in E. coli Top 10 and purified using pre-packed nickel-Sepharose columns. The purified protein (BglX-V-Ara) demonstrated multifunctional activities in the presence of different substrates for β-glucosidase (pNPG: p-nitrophenyl-β-D-glucoside) β-xylosidase (pNPX: p-nitrophenyl-β-D-xyloside) and α-arabinosidase (pNPA: p-nitrophenyl-α-L-arabinosidase). BglX-V-Ara presented an optimal pH of 6 for all substrates and optimal temperature of 50 °C for β-glucosidase and α-l-arabinosidase and 60 °C for β-xylosidase. BglX-V-Ara predominantly presented β-glucosidase activity, with the highest affinity for its substrate and catalytic efficiency (K_m 0.24 ± 0.0005 mM, V_max 0.041 ± 0.002 µmol min^?1 mg^?1 and K_cat/K_m 0.27 mM^?1 s^?1), followed by β-xylosidase (K_m 0.64 ± 0.032 mM, V_max 0.055 ± 0.002 µmol min^?1 mg^?1 and K_cat/K_m 0.14 mM^?1s^?1) and finally α-l-arabinosidase (K_m 1.45 ± 0.05 mM, V_max 0.091 ± 0.0004 µmol min^?1 mg^?1 and K_cat/K_m 0.1 mM^?1 s^?1). To date, this is the first report to demonstrate the characterization of a GH3-BglX family member in C. crescentus that may have applications in biotechnological processes (i.e., the simultaneous saccharification process) because the multifunctional enzyme could play an important role in bacterial hemicellulose degradation.     

An NADPH-dependent <Emphasis Type="Italic">Lactobacillus composti</Emphasis> short-chain dehydrogenase/reductase: characterization and application to (<Emphasis Type="Italic">R</Emphasis>)-1-phenylethanol synthesis

A new anti-Prelog short-chain dehydrogenase/reductase (SDR) encoding gene lcsdr was cloned from Lactobacillus composti DSM 18527, and heterologously expressed in Escherichia coli. LcSDR is nicotinamide adenine dinucleotide phosphate (NADPH)-dependent and has a molecular weight of approximately 30 kDa. The optimal pH and temperature were 6.5 and 30?°C, respectively. The maximal reaction rate V_max was 133.9 U mg^?1; the Michaelis–Menten constant K _m of LcSDR were 0.345 mM for acetophenone (1a), and 0.085 mM for NADPH. Through introducing an EsGDH-catalyzed NADPH regeneration system, a biocatalytic process for (R)-1-phenylethanol ((R)-1b) was developed with outstanding time–space yield. Under the optimized conditions, 50 g l^?1 1a was converted to (R)-1b in 2 h with a yield of 93.8%, enantiomeric excess of product (e.e._p) above 99% and space–time yield of 562.8 g l^?1 d^?1.     

Changes in Immunity,Expression of some Immune-Related Genes of Shabot Fish, <Emphasis Type="Italic">Tor grypus</Emphasis>, Following Experimental Infection with <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis>: Effects of Autochthonous Probiotics

In this study, the effects of orally administrated two native probiotics (Lactobacillus plantarum and Lactobacillus delbrueckii ssp. bulguricus), isolated from the intestine of Shabot fish, Tor grypus, on some immune response parameters and immune-related genes expression against Aeromonas hydrophila in T. grypus were evaluated. Four hundred and eighty juveniles weighing 45?±?10 g were randomly divided into four groups (with three replications) and fed with the experimental diet containing 5?×?10⁷ cfu g^?1 of L. plantarum (G1), Lactobacillus bulgaricus (G2), Lactobacillus casei (G3), and a control diet (without probiotics) for 60 continuous days. At the end of the dietary treatments, fish were challenged with a lethal concentration of A. hydrophila (5?×?10⁸ CFU ml^?1) via intra peritoneal (i.p) injection. Blood and head kidney samples were taken from six fish in each treatment before challenging and 6, 12, 24, and 48 h and also 7 days after injection. The results showed that lysozyme, complement, bactericidal, and NBT activity of probiotic-treated groups were significantly elevated (P?<?0.05). The IL-8, IL-1β, and TNF-α gene expressions were significantly higher in all probiotic-treated groups (P?<?0.05). Meanwhile, a high direct correlation was observed between serum immune parameters and expression of immune-related genes (P?<?0.0001); furthermore, the highest correlation (R ²?=?0.634, P?<?0.0001) was recorded between IL-1β expression and NBT activity. It can be concluded that not only two native probiotics strains stimulate serum immune responses parameters and immune-related gene expression in T. grypus, but also a high correlation was seen among these indices. The study suggests that gastrointestinal colonization is preferred for host specificity as the strain previously derived from shabot fish displayed better colonization than the non-indigenous bacteria strain such as L. casei. Therefore, these native probiotics bacteria can be accounted as suitable candidates to immune stimulation in fish.     

A chimeric α-amylase engineered from <Emphasis Type="Italic">Bacillus acidicola</Emphasis> and G<Emphasis Type="Italic">eobacillus thermoleovorans</Emphasis> with improved thermostability and catalytic efficiency

The α-amylase (Ba-amy) of Bacillus acidicola was fused with DNA fragments encoding partial N- and C-terminal region of thermostable α-amylase gene of Geobacillus thermoleovorans (Gt-amy). The chimeric enzyme (Ba-Gt-amy) expressed in Escherichia coli displays marked increase in catalytic efficiency [K _cat: 4 × 10⁴ s^?1 and K _cat/K _m: 5 × 10⁴ mL^?1 mg^?1 s^?1] and higher thermostability than Ba-amy. The melting temperature (T _m) of Ba-Gt-amy (73.8 °C) is also higher than Ba-amy (62 °C), and the CD spectrum analysis revealed the stability of the former, despite minor alteration in secondary structure. Langmuir–Hinshelwood kinetic analysis suggests that the adsorption of Ba-Gt-amy onto raw starch is more favourable than Ba-amy. Ba-Gt-amy is thus a suitable biocatalyst for raw starch saccharification at sub-gelatinization temperatures because of its acid stability, thermostability and Ca²⁺ independence, and better than the other known bacterial acidic α-amylases.     

Acetaldehyde kinetics of enological yeast during alcoholic fermentation in grape must

Acetaldehyde strongly binds to the wine preservative SO₂ and, on average, causes 50–70 mg l^?1 of bound SO₂ in red and white wines, respectively. Therefore, a reduction of bound and total SO₂ concentrations necessitates knowledge of the factors that affect final acetaldehyde concentrations in wines. This study provides a comprehensive analysis of the acetaldehyde production and degradation kinetics of 26 yeast strains of oenological relevance during alcoholic fermentation in must under controlled anaerobic conditions. Saccharomyces cerevisiae and non-Saccharomyces strains displayed similar metabolic kinetics where acetaldehyde reached an initial peak value at the beginning of fermentations followed by partial reutilization. Quantitatively, the range of values obtained for non-Saccharomyces strains greatly exceeded the variability among the S. cerevisiae strains tested. Non-Saccharomyces strains of the species C. vini, H. anomala, H. uvarum, and M. pulcherrima led to low acetaldehyde residues (<10 mg l^?1), while C. stellata, Z. bailii, and, especially, a S. pombe strain led to large residues (24–48 mg l^?1). Acetaldehyde residues in S. cerevisiae cultures were intermediate and less dispersed (14–34 mg l^?1). Addition of SO₂ to Chardonnay must triggered significant increases in acetaldehyde formation and residual acetaldehyde. On average, 0.33 mg of residual acetaldehyde remained per mg of SO₂ added to must, corresponding to an increase of 0.47 mg of bound SO₂ per mg of SO₂ added. This research demonstrates that certain non-Saccharomyces strains display acetaldehyde kinetics that would be suitable to reduce residual acetaldehyde, and hence, bound-SO₂ levels in grape wines. The acetaldehyde formation potential may be included as strain selection argument in view of reducing preservative SO₂ concentrations.