Single point mutations enhance activity of <Emphasis Type="Italic">cis</Emphasis>-epoxysuccinate hydrolase

Objectives

To enhance activity of cis-epoxysuccinate hydrolase from Klebsiella sp. BK-58 for converting cis-epoxysuccinate to tartrate.

Results

By semi-saturation mutagenesis, all the mutants of the six important conserved residues almost completely lost activity. Then random mutation by error-prone PCR and high throughput screening were further performed to screen higher activity enzyme. We obtained a positive mutant F10D after screening 6000 mutations. Saturation mutagenesis on residues Phe10 showed that most of mutants exhibited higher activity than the wild-type, and the highest mutant was F10Q with activity of 812 U mg^?1 (k _cat /K _m , 9.8 ± 0.1 mM^?1 s^?1), which was 230 % higher than that of wild-type enzyme 355 U mg^?1 (k _cat /K _m , 5.3 ± 0.1 mM^?1 s^?1). However, the thermostability of the mutant F10Q slightly decreased.

Conclusions

The catalytic activity of a cis-epoxysuccinate hydrolase was efficient improved by a single mutation F10Q and Phe10 might play an important role in the catalysis.

     

Parasitism Rate of <Emphasis Type="Italic">Habrobracon hebetor</Emphasis> to <Emphasis Type="Italic">Ephestia kuehniella</Emphasis> Larvae: Residual Effect of Deltamethrin,Fenvalerate and Azadirachtin

Sublethal concentrations of chemical insecticides may cause changes in some behavioral characteristics of natural enemies such as functional responses. The residual effect of three synthetic insecticides including deltamethrin, fenvalerate and azadirachtin were studied on functional response of Habrobracon hebetor Say to Ephestia kuehniella Zeller larvae. Seven host densities (2, 4, 8, 16, 32, 64 and 96) were used during a 24 h period. The resulting data were appropriately fit to Type II functional response models in all treatments: (1) control (0.0916 h^?1; and T _h  = 0.2011 h); (2) deltamethrin (a = 0.0839 h^?1; and T _h  = 0.3560 h); (3) fenvalerate (a = 0.0808 h^?1 and T _h  = 0.3623 h); and (4) azadirachtin (a = 0.0900 h^?1 and T _h  = 0.2042 h). Maximum theoretical parasitism rate (T/T _h ) was 119.34 estimated for control wasps. There was no significant difference between the values of attack rates (a and a + D _a ) in all treatments while the handling time was statistically affected in female wasps treated with fenvalerate. Our findings will be useful in safe application of these insecticides in pest management programmes.     

Characterization of a new multifunctional beta-glucosidase from <Emphasis Type="Italic">Musca domestica</Emphasis>

Objective

To engineer Pichia pastoris for heterologous production of cellulase from Musca domestica and explore its potential for industrial applications.

Results

A new beta-glucosidase gene (bg), encoding 562 amino acids, was cloned from M. domestica by using rapid amplification of cDNA ends. The gene bg was linked to pPICZαA and expressed in P. pastoris with a yield of 500 mg l^?1. The enzyme has the maximum activity with 27.6 U mg^?1 towards cellulose. The beta-glucosidase has stable activity from 20 to 70 °C and can tolerate one-mole glucose. It has the maximum activities for salicin (25.9 ± 1.8 U mg^?1), cellobiose (40.1 ± 2.3 U mg^?1) and cellulose (27.6 ± 3.5 U mg^?1). The wide-range substrate activities of the beta-glucosidase were further verified by matrix-assisted laser desorption/ionization mass spectra. Structural analysis shows that the beta-glucosidase belongs to glycoside hydrolase family Ι and possesses O-glycosylation sites.

Conclusions

Thus, a multifunctional beta-glucosidase was expressed from M. domestica and provides a potential tool for industrial application of cellulose.

     

Decolorization of textile dye RB19 using volcanic rock matrix immobilized <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> cells with surface displayed laccase

A triplicate volcanic rock matrix–Bacillus thuringiensis–laccase WlacD (VRMs–Bt–WlacD) dye decolorization system was developed. WlacD was displayed on the B. thuringiensis MB174 cell surface to prepare a whole-cell laccase biocatalyst by using two repeat N-terminal domains of autolysin Mbg (Mbgn)₂ as the anchoring motif. Immunofluorescence microscopic assays confirmed that the fusion protein (Mbgn)₂–WlacD was anchored on the surface of the recombinant B. thuringiensis MB174. After optimization by a single factor test, L ₉(3⁴)-orthogonal test, Plackett–Burman test, steepest ascent method, and Box–Behnken response surface methodology, the whole-cell specific laccase activity of B. thuringiensis MB174 was improved to 555.2 U L^?1, which was 2.25 times than that of the primary culture condition. Optimized B. thuringiensis MB174 cells were further adsorbed by VRMs to prepare VRMs–Bt–WlacD, an immobilized whole-cell laccase biocatalyst. Decolorization capacity of as-prepared VRMs–Bt–WlacD toward an initial concentration of 500 mg L^?1 of an textile dye reactive blue 19 (RB19) aqueous solution reached 72.36% at a solid-to-liquid ratio of 10 g–100 mL. Repeated decolorization-activation operations showed the high decolorization capacity of VRMs–Bt–WlacD and have the potential for large-scale or continuous operations.     

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.     

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.     

Age validation and seasonal growth patterns of a subtropical marsh fish: The Gulf Killifish, <Emphasis Type="Italic">Fundulus grandis</Emphasis>

Fundulus grandis (Baird and Girard), the Gulf Killifish, is an abundant species throughout the marshes of the northern Gulf of Mexico. Its wide distribution and high site fidelity makes it an ideal indicator species for brackish and salt marshes, which experience a variety of anthropogenic disturbances. Despite the ecological, commercial, and scientific importance of F. grandis, age determination methods have not been validated and little is known of its growth pattern. By combining a tag-recapture study with a chemical marker to stain otoliths, we validated an ageing method for F. grandis adults (49–128 mm TL) using whole sagittal otoliths and determined growth rates of recaptured individuals in winter (n = 58) and summer (n = 36) in Louisiana. Mean somatic growth in length was significantly greater during the winter (0.085 mm d^?1) than summer (0.054 mm d^?1). In contrast, mean otolith growth was significantly greater in summer (1.37 μm d^?1) than winter (0.826 μm d^?1). The uncoupling of somatic and otolith growth may be primarily attributed to warm summer temperatures, which led to enhanced otolith growth while simultaneously reducing somatic growth. Fundulus grandis was aged to a maximum of 2.25 years. The parameters of the von Bertalanffy growth model were estimated as: L _∞  = 87.27 mm, k = 2.43 year^?1, and t ₀ = ?0.022. These findings reveal essential age and growth information for F. grandis and provide a benchmark to evaluate responses to environmental disturbances.     

Quercetin and silver nitrate modulate organogenesis in <Emphasis Type="Italic">Carissa carandas</Emphasis> (L.)

An in vitro organogenesis protocol for Carissa carandas L. was developed using an auxin transport inhibitor (quercetin) and silver nitrate (AgNO₃), an inhibitor of ethylene action, in association with cytokinins in the culture medium. This protocol produced the maximum number of shoots from aseptic seedling-derived shoot apex explants of C. carandas. The highest rate of shoot multiplication was recorded on MS medium containing 2.0 mg L^?1 6-benzylaminopurine; 0.5 mg L^?1 kinetin, and 0.75 mg L^?1 quercetin at after 4 wk of culture. Similar results were obtained when MS medium fortified with 2.0 mg L^?1 BAP, 0.5 mg L^?1 kinetin, and 1.5 mg L^?1 AgNO₃ was used. However, successful rooting was achieved on quarter strength MS medium with 0.5 mg L^?1 indole-3-acetic acid. In this study, an inhibitor of auxin transport and ethylene action maximized shoot multiplication in medium fortified with cytokinins. The established rapid micropropagation method could be used to conserve elite genotypes of C. carandas.     

Evaluation of microbial globin promoters for oxygen-limited processes using <Emphasis Type="Italic">Escherichia coli</Emphasis>

Oxygen-responsive promoters can be useful for synthetic biology applications, however, information on their characteristics is still limited. Here, we characterized a group of heterologous microaerobic globin promoters in Escherichia coli. Globin promoters from Bacillus subtilis, Campylobacter jejuni, Deinococcus radiodurans, Streptomyces coelicolor, Salmonella typhi and Vitreoscilla stercoraria were used to express the FMN-binding fluorescent protein (FbFP), which is a non-oxygen dependent marker. FbFP fluorescence was monitored online in cultures at maximum oxygen transfer capacities (OTR_max) of 7 and 11 mmol L^?1 h^?1. Different FbFP fluorescence intensities were observed and the OTR_max affected the induction level and specific fluorescence emission rate (the product of the specific fluorescence intensity multiplied by the specific growth rate) of all promoters. The promoter from S. typhi displayed the highest fluorescence emission yields (the quotient of the fluorescence intensity divided by the scattered light intensity at every time-point) and rate, and together with the promoters from D. radiodurans and S. coelicolor, the highest induction ratios. These results show the potential of diverse heterologous globin promoters for oxygen-limited processes using E. coli.     

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.     

Carbon dioxide and poultry waste utilization for production of polyhydroxyalkanoate biopolymers by <Emphasis Type="Italic">Nostoc muscorum</Emphasis> Agardh: a sustainable approach

The new paradigm is to view wastes as resources for sustainable development. In this regard, the feasibility of poultry waste and CO₂ utilization for cultivation of a filamentous nitrogen-fixing cyanobacterium, Nostoc muscorum Agardh, was investigated for production polyhydroxyalkanoates, the biodegradable polymers. This cyanobacterium showed profound rise in biomass yield with up to 10 % CO₂ supply in airstream with an aeration rate of 0.1 vvm. Maximum biomass yield of 1.12 g L^?1 was recorded for 8 days incubation period, thus demonstrating a CO₂ biofixation rate of 0.263 g L^?1 day^?1 at 10 % (v/v) CO₂-enriched air. Poultry litter (PL) supplementation also had a positive impact on the biomass yield. The nutrient removal efficiency of N. muscorum was reflected in the significant reduction in nutrient load of PL over the experimental period. A maximum poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) [P(3HB-co-3HV)] copolymer yield of 774 mg L^?1 (65 % of dry cell wt.), the value almost 11-fold higher than the control, was recorded in 10 g L^?1 PL-supplemented cultures with 10 % CO₂ supply under the optimized condition, thus demonstrating that N. muscorum has good potential for CO₂ biomitigation and poultry waste remediation while simultaneously producing eco-friendly polymers.     

Occurrence and distribution of multiple antibiotic-resistant <Emphasis Type="Italic">Enterococcus</Emphasis> and <Emphasis Type="Italic">Lactobacillus</Emphasis> spp. from Indian poultry: in vivo transferability of their erythromycin,tetracycline and vancomycin resistance

The objective of this study was to determine the occurrence and distribution of antibiotic resistant (AR) lactic acid bacteria (LAB) in Indian poultry. LAB from poultry farm feces (n = 21) and samples from slaughter houses comprising chicken intestine (n = 46), raw meat (n = 23), and sanitary water (n = 4) were evaluated and compared with those from organic chicken (OC) collected from nearby villages. Screening studies showed 5–7 log units higher erythromycin (ER), tetracycline (TC) and vancomycin (VAN) resistant LAB from conventional poultry chicken (CC) compared to OC. Molecular characterization of isolated cultures (n = 32) with repetitive-PCR profiling and 16S rRNA gene sequencing revealed their taxonomical status as Enterococcus faecium (n = 16), Enterococcus durans (n = 2), Lactobacillus plantarum (n = 10), Lactobacillus pentosus (n = 1) and Lactobacillus salivarius (n = 3). The isolates were found to harbor erm(B), msr(C), msr(A/B), tet(M), tet(L) and tet(K) genes associated with Tn916 and Tn917 family transposons. Expression studies through real-time PCR revealed antibiotic-induced expression of the identified AR genes. In vitro and in vivo conjugational studies revealed transfer of ER and TC resistant (ER^R and TC^R) genes with transfer frequencies of 10^?7 and 10^?4 transconjugants recipient^?1, respectively. Although no known VAN resistance (VAN^R) genes were detected, high phenotypic resistance was observed and was transferable to the recipient. From a public health point of view, this study reports Indian poultry as a major source of high levels of AR bacteria contaminating the food chain and the environment. Thus, urgent and determined strategies are needed to control the spread of multiple AR bacteria.     

Physiological and Biochemical Mechanisms Preventing Cd Toxicity in the New Hyperaccumulator <Emphasis Type="Italic">Abelmoschus manihot</Emphasis>

Abelmoschus manihot, an ornamental plant, was examined for phytoremediation purposes in accordance with the ability to accumulate cadmium and physiological mechanisms of cadmium tolerance. A net photosynthetic rate (A _N) glasshouse experiment for 60 days was conducted to investigate the influence of different cadmium amounts (0–100 mg kg^?1) on the growth, biomass, photosynthetic performance, reactive oxygen species (ROS) production, antioxidative enzyme activities, Cd uptake and accumulation of A. manihot. Exposure to cadmium enhanced plant growth even at 100 mg kg^?1, without showing symptoms of visible damage. The cadmium concentration of shoots (stems or leaves) and roots was more than the critical value of 100 mg kg^?1 and reached 126.17, 185.26 and 210.24 mg kg^?1, respectively. BCF values of A. manihot plants exceeded the reference value 1.0 for all the Cd treatments, and TF values were greater than 1 at 15–60 mg kg^?1 Cd treatment. The results also showed that cadmium concentrations of 60 mg kg^?1 or less induced a significant enhancement in plant net photosynthetic rate (A _N), stomatal conductance (G _s), transpiration rate (T _r), photosynthetic pigments and F _v/F _m. These parameters were slightly decreased at the higher concentration (100 mg kg^?1). The ROS production (O₂ ^?, H₂O₂) and antioxidative response including SOD, CAT and POD were significantly enhanced by increasing cadmium. These results suggest that A. manihot can be considered as a Cd-hyperaccumulator and the hormetic effects may be taken into consideration in remediation of Cd contamination soil.     

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.     

Toxicity of an Annonin-Based Commercial Bioinsecticide Against Three Primary Pest Species of Stored Products

The effects of a bioinsecticide formulation based on extract of Annona squamosa L. (Annonaceae) containing 10,000 mg L^?1 of acetogenin annonin as the main active ingredient were investigated against three primary pest species of stored grains in Brazil [maize weevil Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae), Mexican bean weevil Zabrotes subfasciatus (Boheman) (Coleoptera: Chrysomelidae: Bruchinae), and cowpea weevil Callosobruchus maculatus (Fabricius) (Coleoptera: Chrysomelidae: Bruchinae)] by means of residual contact bioassays. In a concentration-dependent manner, the annonin-based commercial bioinsecticide caused significant adult mortality of C. maculatus (LC₅₀ = 6890 μL kg^?1), S. zeamais (LC₅₀ = 2781 μL kg^?1), and Z. subfasciatus (LC₅₀ = 2120 μL kg^?1) after 120 h of residual contact exposure. In addition to acute toxicity, the tested bioinsecticide also promoted a significant reduction of the number of eggs laid by females of C. maculatus (EC₅₀ = 5949.7 μL kg^?1) and Z. subfasciatus (EC₅₀ = 552.7 μL kg^?1). Moreover, the bioinsecticide significantly reduced the number of emerged insects (F₁ generation) of C. maculatus (EC₅₀ = 2763.0 μL kg^?1), S. zeamais (EC₅₀ = 1380.8 μL kg^?1), and Z. subfasciatus (EC₅₀ = 561.5 μL kg^?1). The bioinsecticide also reduced the percentage of damaged grains for the three pest species studied, and its grain-protectant properties are comparable to or superior in efficacy in relation to a diatomaceous earth-based insecticide (Insecto® at 1000 mg kg^?1) used as a positive control. Thus, this standardized formulation has promising bioactivity against stored insect species and can be a useful component for IPM of stored grains in Brazil and elsewhere.     

Gas exchange,carbon balance and stomatal traits in wild and cultivated rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) genotypes

Carbon balancing within the plant species is an important feature for climatic adaptability. Photosynthesis and respiration traits are directly linked with carbon balance. These features were studied in 20 wild rice accessions Oryza spp., and cultivars. Wide variation was observed within the wild rice accessions for photosynthetic oxygen evolution or photosynthetic rate (A), dark (R _d), and light induced respiration (LIR) rates, as well as stomatal density and number. The mean rate of A varied from 10.49 μmol O₂ m^?2 s^?1 in cultivated species and 13.09 μmol O₂ m^?2 s^?1 in wild spp., The mean R _d is 2.09 μmol O₂ m^?2 s^?1 and 2.31 μmol O₂ m^?2 s^?1 in cultivated and wild spp., respectively. Light induced Respiration (LIR) was found to be almost twice in wild rice spp., (16.75 μmol O₂ m^?2 s^?1) compared to cultivated Oryza spp., Among the various parameters, this study reveals LIR and A as the key factors for positive carbon balance. Stomatal contribution towards carbon balance appears to be more dependent on abaxial surface where several number of stomata are situated. Correlation analysis indicates that R _d and LIR increase with the increase in A. In this study, O. nivara (CR 100100, CR 100097), O. rufipogon (IR 103404) and O. glumaepatula (IR104387) were identified as potential donors which could be used in rice breeding program. Co-ordination between gas exchange and patchiness in stomatal behaviour appears to be important for carbon balance and environmental adaptation of wild rice accessions, therefore, survival under harsh environment.     

Response of <Emphasis Type="Italic">Tisochrysis lutea</Emphasis> [Prymnesiophycidae] to aeration conditions in a bench-scale photobioreactor

The effects of superficial gas velocity (U_gr), gas entrance velocity (ν), and bubble size on the growth of Tisochrysis lutea was investigated in 600-mL photobioreactors operated with airlift pumps. Superficial gas velocities, calculated from measured air flow rates, ranging from 7 to 93 mm s^?1 were created using a 1.6-mm diameter syringe. We tested the effects of sparger velocity over a range of 2.48 to 73.4 m s^?1 and the effects of bubble size by using two styles of air stones and an open glass pipette, which created a bubble sizes in the range of 0.5 to 5 mm. We calculated oxygen mass transfer coefficient, k_La, values for all experimental conditions. Cell growth increased linearly with increased superficial gas velocity and decreased with increased sparger velocity. Results indicated that smaller bubble size leads to some initial cell damage, but after time, the increased gas transfer as reflected by the k_La value produced higher growth than larger bubbles. Two mechanisms were observed to correlate with cell damage in T. lutea: increasing velocity at the sparger tip and bubble bursting at the surface. These results demonstrate a method to test sensitivity of T. lutea to aeration, which is important for the design of airlift systems.     

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.     

Specific light uptake rates can enhance astaxanthin productivity in <Emphasis Type="Italic">Haematococcus lacustris</Emphasis>

Lumostatic operation was applied for efficient astaxanthin production in autotrophic Haematococcus lacustris cultures using 0.4-L bubble column photobioreactors. The lumostatic operation in this study was performed with three different specific light uptake rates (q _e) based on cell concentration, cell projection area, and fresh weight as one-, two- and three-dimensional characteristics values, respectively. The q _e value from the cell concentration (q _e1D) obtained was 13.5 × 10^?8 μE cell^?1 s^?1, and the maximum astaxanthin concentration was increased to 150 % compared to that of a control with constant light intensity. The other optimum q _e values by cell projection area (q _e2D) and fresh weight (q _e3D) were determined to be 195 μE m^?2 s^?1 and 10.5 μE g^?1 s^?1 for astaxanthin production, respectively. The maximum astaxanthin production from the lumostatic cultures using the parameters controlled by cell projection area (2D) and fresh weight (3D) also increased by 36 and 22 % over that of the controls, respectively. When comparing the optimal q _e values among the three different types, the lumostatic cultures using q _e based on fresh weight showed the highest astaxanthin productivity (22.8 mg L^?1 day^?1), which was a higher level than previously reported. The lumostatic operations reported here demonstrated that more efficient and effective astaxanthin production was obtained by H. lacustris than providing a constant light intensity, regardless of which parameter is used to calculate the specific light uptake rate.