Effect of ionic liquids,organic solvents and supercritical CO2 pretreatment on the conformation and catalytic properties of Candida rugosa lipase

The objective of this work is to assess the structure and activity of Candida rugosa lipase (CRL) pretreated with seventeen ionic liquids (ILs), five organic solvents and super-critical carbon dioxide (SC-CO₂). The results revealed that anion selection of ILs showed generally much greater effects on CRL esterification activity than cation choice, and CRL pretreated by ILs with strong water miscible properties showed very low esterification activity. The highest CRL activity treated with ILs [Hmim][PF6] was obtained with the value of 45078.0 U/g-protein. Furthermore, the CRL activities pretreated with five conventional organic solvents were also examined and the values increased with the log P decrease of organic solvents when log P was lower than 2.0. Finally, the CRL activities were respectively 1.2- and 1.3-fold higher over the untreated ones after pretreatment with sub- and super-critical CO₂ under the pressures of 6 MPa and 15 MPa at 40 °C for 20 min. Further analyses via FT-IR demonstrated that the high activity of CRL pretreated with ILs, organic solvents and SC-CO₂ was probably caused by the changes of CRL secondary structure. In conclusion, the results in this work will be helpful for us to choose the suitable reaction medium in CRL biocatalysis and biotransformation reactions.     

Carbon mineralization of Chinese fir (Cunninghamia lanceolata) soils under different temperature and humidity conditions

Burned and unburned mineral soils (0–10 cm) from a 40-year-old Chinese fir (Cunninghamia lanceolata) forest in Nanping, Fujian, China were incubated for 90 days at different temperatures (25 °C and 35 °C) and humidity [25%, 50%, and 75% of water holding capacity (WHC)] conditions. Carbon (C) mineralization of all soils was determined using CO₂ respiration method. The results showed that CO₂ evolution rates of the burned and control soils exhibited similar temporal patterns, and similar responses to temperature and moisture. CO₂ evolution rates for all soil samples decreased with incubation time. At different humidity conditions, average rate of C mineralization and cumulative mineralized C from burned and control soils were significantly higher at 35 °C than at 25 °C. This implied that C mineralization was less sensitive to soil moisture than to temperature. In both soils at 25 °C or 35 °C, the amount of soil evolved CO₂ over the 90 days incubation increased with increasing moisture content from 25% to 75% WHC. A temperature coefficient (Q₁₀) varied with soil moisture contents. The maximum values recorded for Q₁₀ were 1.7 in control soil and 1.6 in burned soil both at 25% WHC. However, there were no significant differences in Q₁₀ values between the control and burned soils over all moisture ranges (P > 0.05). The data of cumulative C–CO₂ released from control and burned soils were fitted to two different kinetic models. The two simultaneous reactions model described mineralization better than the first-order exponential model, which reflected the heterogeneity of substrate quality. Based on these results, it is possible to conclude that temperature and moisture are important in the controls of C mineralization, and the combined effects of these variables need to be considered to understand and predict the response of CO₂ release in subtropical ecosystems to climate change.     

Temperature response of C4 species big bluestem (Andropogon gerardii) is modified by growing carbon dioxide concentration

Climate change factors interact to modify plant growth and development. The objective of this study was to evaluate the response to temperature of big bluestem (Andropogon gerardii Vitman) development, growth, reproduction and biomass partitioning under low and high carbon dioxide concentrations ([CO₂]) grown in controlled environmental conditions. Ten sunlit soil–plant–atmosphere-research (SPAR) chambers were used to study the effects of two [CO₂] of low (360 μL L⁻¹) and high (720 μL L⁻¹), and five different day/night temperatures of 20/12, 25/17, 30/22, 35/27 and 40/32 °C. Big bluestem cv. Bonelli seeds were sown in pure, fine sand, in 11 rows at equal spacing and after emergence were thinned to 10 plants per row. At maturity, individual plants were harvested and divided into leaves, stems, panicles and roots. Biomass decreased either above or below the optimum temperature of 30/22 °C. The effect of high [CO₂] on biomass accumulation (12–30% increase) was visible at less than optimum temperature (30/22 °C) and absent at two high temperatures. With increase in temperature, irrespective of the [CO₂], biomass partitioned to leaves increased (35%) where as that to stems decreased (33%). Panicle weight was 6–7% of biomass at 25/17 °C and fell to 1.6% at 40/32 °C. The biomass partitioned to roots, across the temperatures, was constant for plants grown at low [CO₂] but decreased by 7% for those grown at high [CO₂]_. The decrease in panicle/seed production at two high temperatures (>30/22 °C) might reduce this species population and dominance in tallgrass prairies. The temperature response functions at different [CO₂] will be useful to improve the predictive capabilities of dynamic global vegetation models in simulating dynamics of rangelands, where big bluestem is the dominant species.     

Decay and termite resistance,water absorption and swelling of thermally compressed wood panels

This study evaluated decay and termite resistance of thermally compressed pine wood panels under pressure at either 5 or 7 MPa and either 120 or 150 °C for 1 h. Wood specimens from the panels were exposed to laboratory decay resistance tests by using the wood degrading fungi, Gloeophyllum trabeum and Trametes versicolor. The thermal compression process caused increases in density and decreases in thickness of the panels; however, laboratory decay resistance tests revealed that thermally compressed wood was not resistant against the wood degrading fungi tested. More interesting results were found in laboratory termite resistance tests by using the Eastern subterranean termites, Reticulitermes flavipes. As pressure and temperature applied to the specimens increased to 7 MPa and 120 °C, mass losses in the specimens gradually decreased in comparison with control specimens. However, the specimens compressed at 7 MPa and 150 °C showed higher mass losses when compared to the specimens compressed at 7 MPa and 120 °C. The lowest water absorption and swelling rates were seen in the specimens exposed to a pressure of 7 MPa at 120 °C. The thermal compression process at 7 MPa and 150 °C resulted in the highest water absorption and swelling in the specimens.     

Stability and activity of Dictyoglomus thermophilum GH11 xylanase and its disulphide mutant at high pressure and temperature

The functional properties of extremophilic Dictyoglomus thermophilum xylanase (XYNB) and the N-terminal disulphide-bridge mutant (XYNB-DS) were studied at high pressure and temperature. The enzymes were quite stable even at the pressure of 500 MPa at 80 °C. The half-life of inactivation in these conditions was over 30 h. The inactivation at 80 °C in atmospheric pressure was only 3-times slower. The increase of pressure up to 500 MPa at 80 °C decreased only slightly the enzyme's stability, whereas in 500 MPa the increase of temperature from 22 to 80 °C decreased significantly more the enzyme's stability. While the high temperature (80–100 °C) decreased the enzyme reaction with short xylooligosaccharides (xylotetraose and xylotriose), the high pressure (100–300 MPa) had an opposite effect. The temperature of 100 °C strongly increased the K_m but did not affect the k_cat to the same extent, thus indicating that the interaction of the substrate with the active site suffers before the catalytic reaction begins to decrease as the temperature rises. Circular dichroism spectroscopy showed the high structural stability of XYNB and XYNB-DS at 93 °C.     

Ecology and evolution of Devonian trees in New York,USA

The first trees in New York were Middle Devonian (earliest Givetian) cladoxyls (?Duisbergia and Wattieza), with shallow-rooted manoxylic trunks. Cladoxyl trees in New York thus postdate their latest Emsian evolution in Spitzbergen. Progymnosperm trees (?Svalbardia and Callixylon–Archaeopteris) appeared in New York later (mid-Givetian) than progymnosperm trees from Spitzbergen (early Givetian). Associated paleosols are evidence that Wattieza formed intertidal to estuarine mangal and Callixylon formed dry riparian woodland. Also from paleosols comes evidence that Wattieza and Callixylon required about 350 mm more mean annual precipitation than plants of equivalent stature today, that Wattieza tolerated mean annual temperature 7 °C less than current limits of mangal (20 °C), and Callixylon could tolerate temperatures 14 °C less than modern mangal. Devonian mangal and riparian woodland spread into New York from wetter regions elsewhere during transient paleoclimatic spikes of very high CO₂ (3923 ± 238 ppmv), and subhumid (mean annual precipitation 730 ± 147 mm) conditions, which were more likely extrinsic atmospheric perturbations rather than consequences of tree evolution. For most of the Middle Devonian CO₂ was lower (2263 ± 238 ppmv), and paleoclimate in New York was semiarid (mean annual precipitation 484 ± 147 mm). Such transient perturbations and immigration events may explain the 40 million year gap between the late Emsian (400 Ma) evolution of trees and Famennian (360 Ma) CO₂ drawdown and expansion of ice caps.     

High hydrostatic pressure increased stability and activity of immobilized lipase in hexane

Lipases are important to high value product synthesis, modification, and enhancement. However, they are often unstable above 40 °C. While most current applications of high hydrostatic pressure (HHP) are for inactivating deleterious enzymes, there is evidence that HHP can stabilize and increase activity of some enzymes. This study examines the apparent kinetics of immobilized lipase-catalyzed synthesis of isoamyl acetate at HHP in hexane. HHP reduced thermal inactivation of lipase by up to 152% after 4 h at 80 °C and 400 MPa when compared to incubations at low pressure. No significant differences were found in activation energy (E_a) at different pressures, irrespectively of the pressurization and heating sequence, and were between 35.7 ± 3.5 and 47.8 ± 8.2 kJ mol^?1, depending on the method. In all methods utilized, activity at 63.5 and 80 °C at 400 MPa was greater (from about 20 to 96% increase) than at low pressure. Activity increased by 110% at low pressure versus a 239% increase at 350 MPa when the temperature was increased from 40 to 80 °C. Increasing pressure up to 350 MPa increased lipase activity while pressures greater than 350 MPa maintained or decreased lipase activity. Activation volume (ΔV^≠) appeared negative between ambient pressure and 200 MPa in contrast to a positive ΔV^≠ between 300 and 600 MPa. Apparent ΔV^≠ was 14.3 ± 1.7 or 15.2 ± 2.2 cm³ mol^?1 at 40 or 80 °C, respectively, between 300 and 500 MPa.     

High-pressure CO2 inactivation and induced damage on Saccharomyces cerevisiae evaluated by flow cytometry

The cultivability, integrity and permeabilisation of Saccharomyces cerevisiae in saline solution after high-pressure CO₂ treatment at 36 °C was assessed by using both conventional cultivation-based technique and flow cytometry. Conventional cultivation-based techniques do not allow the exact quantification of integer cells, which can be determined coupling the staining with propidium iodide and SYBR-Green I and the cell quantification by flow cytometry. A significant portion of cells injured by CO₂ treatment is incapable of forming colonies but is still integer and potentially metabolically active. The yeast cell damage was demonstrated to be dependent on the conditions applied. In particular the influence of different operative parameters on integrity and permeabilisation of yeast cells was evaluated: pressure (50–100 bar), treating time (10–20 min) and stirring rate (500–10,000 rpm). After a 20 min treatment at 100 bar, 36 °C and 10,000 rpm more than 95% of cells result with completely permeabilised membrane.     

Stability of phycocyanin extracted from Spirulina sp.: Influence of temperature,pH and preservatives

Temperature and pH play an important role in the stability of phycocyanin, a natural blue colorant. Systematic investigations showed the maximum stability of phycocyanin was in the pH range 5.5–6.0. Incubation at temperatures between 47 and 64 °C caused the concentration (C_R) and half-life of phycocyanin in solution to decrease rapidly. The C_R value remained at approximately 50% after incubating for 30 min at 59 °C. After heating at 60 °C for 15 min, the C_R value of phycocyanin at pH 7.0 was maintained at around 62–70% when 20–40% glucose or sucrose was added, and the half-life increased from 19 min to 30–44 min. 2.5% sodium chloride was found to be an effective preservative for phycocyanin at pH 7.0 as a C_R value of 76% was maintained and the half-life of 67 min was increased.     

Characterization of a novel thermophilic cyanobacterial strain from Taian hot springs in Taiwan for high CO2 mitigation and C-phycocyanin extraction

The photosynthetic thermophiles have advantage in sequestering CO₂ emitted from the energy sector due to their adaptation to high temperatures, growth at high concentrations of CO₂, and economically important metabolites. The characterization of such a microorganism, a cyanobacterium from Taian hot springs in Taiwan is described here. This thermophilic cyanobacterium is rod-shaped with a size of 1.2–2.5 μm × 6.0–9.0 μm. A comparison of the 16S RNA and cpcBA-IGS sequences revealed that it is closely related to Thermosynechococcus elongatus BP-1 and so named as Thermosynechococcus elongatus TA-1. This cyanobacterium has better growth at 10% and 20% CO₂, at 50 °C with 6000 lx light intensity, at a starting pH of 7–9 and in a medium with 20 mM NaCl. The preferred nitrogen source is NaNO₃ of which the minimal requirement is 10 mM. The purified phycocyanin (C-PC) from TA-1 is still kept native and active at a wide range of temperatures (4–60 °C) with a 65.65% activity even at 60 °C, as well as pH values from 4 to 9 and thus exhibiting a good thermal and acid–base stability. This thermophilic cyanobacterium could make integration of CO₂ mitigation from industrial flue gas and production of economically important product, like C-PC, more feasible.     

In vivo temperature limitations of photosynthesis in Phaseolus vulgaris L

The purpose of this study was to evaluate the temperature response of photosynthesis in two common bean genotypes differing in crop yield when grown under warm conditions. The cultivar Nobre is sensitive to high temperatures, whereas Diplomata shows better crop yield under high temperatures. Plants were grown in a greenhouse prior to transferring to a controlled environment cabinet for the temperature treatments. In a first experiment, 30 days-old plants were subjected to a short exposure (1 day) at temperatures that varied from 9 °C to 39 °C. Diplomata had lower net CO₂ assimilation rate (A) at 15 °C and 21 °C, but higher from 27 °C to 39 °C. Photosynthetic parameters calculated from modeling the response of A to the intercellular CO₂ concentration suggested that the different temperature responses of the two genotypes are caused by different rates of diffusion of CO₂ to the assimilation site, not by differences in biochemical limitations of photosynthesis. While stomatal conductance (g_s) did not differ between the genotypes, mesophyll conductance (g_m) was slightly greater for Nobre at 15 °C, but much higher in Diplomata from 21 °C to 39 °C. In a second experiment, no difference was observed in biomass accumulation between the two genotypes after growth for 24 days under a 35/20 °C (day/night) regime. Hence, the differences in photosynthesis did not cause variation in plant growth at the vegetative stage. The differential genotypic response of g_m to temperature suggests that g_m might be an important limitation to photosynthesis in Nobre, the common bean genotype sensitive to elevated temperature. However, more studies are needed employing other methods for g_m evaluation to validate these results.     

The role of nest surface temperatures and the brain in influencing ant metabolic rates

Thermal limits of insects can be influenced by recent thermal history: here we used thermolimit respirometry to determine metabolic rate responses and thermal limits of the dominant meat ant, Iridomyrmex purpureus. Firstly, we tested the hypothesis that nest surface temperatures have a pervasive influence on thermal limits. Metabolic rates and activity of freshly field collected individuals were measured continuously while ramping temperatures from 44 °C to 62 °C at 0.25 °C/minute. At all the stages of thermolimit respirometry, metabolic rates were independent of nest surface temperatures, and CT_max did not differ between ants collected from nest with different surface temperatures. Secondly, we tested the effect of brain control on upper thermal limits of meat ants via ant decapitation experiments (‘headedness’). Decapitated ants exhibited similar upper critical temperature (CT_max) results to living ants (Decapitated 50.3±1.2 °C: Living 50.1±1.8 °C). Throughout the temperature ramping process, ‘headedness’ had a significant effect on metabolic rate in total (Decapitated V̇CO₂ 140±30 µl CO₂ mg⁻¹ min⁻¹: Living V̇CO₂ 250±50 CO₂ mg⁻¹ min⁻¹), as well as at temperatures below and above CT_max. At high temperatures (>44 °C) pre- CT_max the relationships between I. purpureus CT_max values and mass specific metabolic rates for living ants exhibited a negative slope whilst decapitated ants exhibited a positive slope. The decapitated ants also had a significantly higher Q₁₀:25–35 °C when compared to living ants (1.91±0.43 vs. 1.29±0.35). Our findings suggest that physiological responses of ants may be able to cope with increasing surface temperatures, as shown by metabolic rates across the thermolimit continuum, making them physiologically resilient to a rapidly changing climate. We also demonstrate that the brain plays a role in respiration, but critical thermal limits are independent of respiration levels.     

Impressive effect of immobilization conditions on the catalytic activity and enantioselectivity of Candida rugosa lipase toward S-Naproxen production

Candida rugosa lipase (CRL) was immobilized on Amberlite XAD 7 and the advantage of immobilization under the best reaction conditions in achieving high activity and enantioselectivity was shown for the hydrolysis of racemic Naproxen methyl ester. The performance of CRL was found to be better when the enzyme was immobilized at the temperature and pH values where higher conversion and enantioselectivity were obtained. The effects of immobilized lipase load, temperature, pH and substrate concentration on the conversion and enantioselectivity toward S-Naproxen production in aqueous phase/isooctane biphasic batch system were also evaluated. The increase in immobilized lipase load in 320–800 U/mL range increased the conversion of the substrate and enantioselectivity for S-Naproxen. The kinetic resolution of racemic Naproxen methyl ester conducted at the temperatures of 40, 45 and 50 °C and at the pH values of 4, 6, 7.5 and 9 resulted in the highest conversion and enantioselectivity at 45 °C and pH 6. Higher concentration of racemic Naproxen methyl ester than 10 mg/mL decreased both the conversion and enantioselectivity. CRL, which was immobilized at the temperature and pH values where the enzyme was more enantioselective, was successfully used in three successive batch runs each of 180 h. The highest enantiomeric ratio achieved in the S-Naproxen production was 174.2 with the conversion of 49%.     

A novel low-temperature-active exo-inulinase identified based on Molecular-Activity strategy from Sphingobacterium sp. GN25 isolated from feces of Grus nigricollis

A novel glycosyl hydrolase family 32 exo-inulinase (InuAGN25) gene was cloned from Sphingobacterium sp. GN25 isolated from feces of Grus nigricollis. InuAGN25 showed the highest identity of 54.3% with a putative levanase recorded in GenBank. Molecular-Activity strategy was proposed to predict InuAGN25 to be a low-temperature-active exo-inulinase before experiments performance. Molecular analyses included progressive sequential, phylogenetic and structural analyses. InuAGN25 was effectively expressed in Escherichia coli. The purified recombinant InuAGN25 showed characteristics of low-temperature-active enzymes: (1) the enzyme retained 55.8% of the maximum activity at 20 °C, 35.8% at 10 °C, and even 8.2% at 0 °C; (2) the enzyme exhibited 75.8, 30.5 and 10.8% of the initial activity after preincubation for 60 min at 45, 50 and 55 °C, respectively; (3) K_m values of the enzyme toward inulin were 2.8, 3.0, 3.2 and 5.8 mg ml⁻¹ at 0, 10, 20 and 40 °C, respectively. Fructose was the main product of inulin and Jerusalem artichoke tubers hydrolyzed by the purified recombinant InuAGN25 at room temperature, 10 °C and 0 °C. These results suggested the Molecular-Activity strategy worked efficiently and made InuAGN25 promising for the production of fructose at low temperatures.     

Copper,zinc superoxide dismutase of Curcuma aromatica is a kinetically stable protein

This study details on cloning and characterization of Cu,Zn superoxide dismutase (Ca–Cu,Zn SOD) from a medicinally important plant species Curcuma aromatica. Ca–Cu,Zn SOD was 692 bp with an open reading frame of 459 bp. Expression of the gene in Escherichia coli cells followed by purification yielded the enzyme with K_m of 0.047 ± 0.008 μM and V_max of 1250 ± 24 units/mg of protein. The enzyme functioned (i) across a temperature range of −10 to +80 °C with temperature optima at 20 °C; and (ii) at pH range of 6–9 with optimum activity at pH 7.8. Ca–Cu,Zn SOD retained 50% of the maximum activity after autoclaving, and was stable at a wide storage pH ranging from 3 to 10. The enzyme tolerated varying concentrations of denaturating agent, reductants, inhibitors, trypsin, was fairly resistant to inactivation at 80 °C for 180 min (k_d, 6.54 ± 0.17 × 10⁻³ min⁻¹; t_1/2, 106.07 ± 2.68 min), and had midpoint of thermal transition (T_m) of 70.45 °C. The results suggested Ca–Cu,Zn SOD to be a kinetically stable protein that could be used for various industrial applications.     

Novel regenerable potassium-based dry sorbents for CO2 capture at low temperatures

A novel potassium-based dry sorbent (KZrI) was developed for CO₂ capture at a low temperature range between 50 °C and 200 °C. The CO₂ absorption and regeneration properties of this novel regenerable potassium-based dry sorbent were measured in a fixed-bed reactor during multiple absorption/regeneration cycles at low temperature conditions (CO₂ absorption at 50–100 °C and regeneration at 130–200 °C). The total CO₂ capture capacity of the KZrI sorbent was maintained during the multiple CO₂ absorption/regeneration cycles. The XRD patterns and FTIR analyses of the sorbents after CO₂ absorption showed the KHCO₃ phase only except for the ZrO₂ phase used as support. Even after 10 cycles, any other new structures resulting from the by-product during CO₂ absorption were not observed. This phase could be easily converted into the original phase during regeneration, even at a low temperature (130 °C). The KZrI sorbent developed in this study showed excellent characteristics in CO₂ absorption and regeneration in that it satisfies the requirements of a large amount of CO₂ absorption (91.6 mg CO₂/g sorbent) and the complete regeneration at a low temperature condition (1 atm, 150 °C) without deactivation.     

A novel control of enzymatic enantioselectivity through the racemic temperature influenced by reaction media

The influence of reaction media on the racemic temperature (T_r) in the lipase-catalyzed resolution of ketoprofen vinyl ester was investigated. An effective approach to the control of the enzymatic enantioselectivity and the prediction of the increasing tendency was developed based on the T_r influenced by reaction media. The T_r for the resolution catalyzed by Candida rugosa lipase (CRL) was found at 29 °C in aqueous and S-ketoprofen was obtained predominantly at 40 °C. However, CRL showed R-selectivity at 40 °C in diisopropyl ether because the T_r was changed to 56 °C. CRL, lipase from AYS Amano^® and Mucor javanicus lipase were further applied for the investigation of the enzymatic enantioselectivity in dioxane, DIPE, isooctane and their mixed media with water. The effects of the reaction medium on T_r could be related to the solvent hydrophobicity, the lipase conformational flexibility and the interaction between the enantiomers and the lipase.     

Adult plasticity of cold tolerance in a continental-temperate population of Drosophila suzukii

Drosophila suzukii (Matsumura) (Diptera: Drosophilidae) is a worldwide emerging pest of soft fruits, but its cold tolerance has not been thoroughly explored. We determined the cold tolerance strategy, low temperature thermal limits, and plasticity of cold tolerance in both male and female adult D. suzukii. We reared flies under common conditions (long days, 21 °C; control) and induced plasticity by rapid cold-hardening (RCH, 1 h at 0 °C followed by 1 h recovery), cold acclimation (CA, 5 days at 6 °C) or acclimation under fluctuating temperatures (FA). D. suzukii had supercooling points (SCPs) between −16 and −23 °C, and were chill-susceptible. 80% of control flies were killed after 1 h at −7.2 °C (males) or −7.5 °C (females); CA and FA improved survival of this temperature in both sexes, but RCH did not. 80% of control flies were killed after 70 h (male) or 92 h (female) at 0 °C, and FA shifted this to 112 h (males) and 165 h (females). FA flies entered chill coma (CT_min) at approximately −1.7 °C, which was ca. 0.5 °C colder than control flies; RCH and CA increased the CT_min compared to controls. Control and RCH flies exposed to 0 °C for 8 h took 30–40 min to recover movement, but this was reduced to <10 min in CA and FA. Flies placed outside in a field cage in London, Ontario, were all killed by a transient cold snap in December. We conclude that adult phenotypic plasticity is not sufficient to allow D. suzukii to overwinter in temperate habitats, and suggest that flies could overwinter in association with built structures, or that there may be additional cold tolerance imparted by developmental plasticity.     

Effects of constant temperature,exposure period,and age on diapause induction in Trichogramma dendrolimi

Trichogramma dendrolimi can be successfully reproduced in fresh eggs dissected from ovaries of the Chinese tussah silkworm (Antheraea pernyi) and is widely used in biological control of lepidopteran agricultural and forest pests in China. Diapause induction of T. dendrolimi in A. pernyi eggs was investigated through exposing the parasitoid to six constant temperatures (16, 13, 10, 7, 4, and 1 °C) for 19 exposure periods between 10 and 46 days. The sensitive age of T. dendrolimi for diapause induction was explored through a separate experiment to examine the parasitoids that had developed for 2, 3, 4, 5, 6, 7, and 8 days at 26 °C after parasitization, under the six constant temperatures, respectively. Diapause was induced at 10 or 7 °C, and the induction period was 4–6 weeks. The sensitive age of T. dendrolimi to react at the induction temperature was 2–3 days (at 26 °C). At 7 and 10 °C, the diapause rate increased with increasing exposure period and decreased with increased T. dendrolimi age at exposure. The optimum method to induce diapause in T. dendrolimi consisted of exposing hosts for parasitization at 26 °C for 8 h, and then keeping them at 26 °C for 40 h, finally, moving them into 10 °C for 4 weeks.     

Structural characterization of thermostable,solvent tolerant,cytosafe tannase from Bacillus subtilis PAB2

Tannase production by Bacillus subtilis PAB2, was investigated under solid state fermentation using tamarind seed as sole carbon source and it was found as the highest titer (73.44 U/gds). The enzyme was purified to homogeneity, which showed the molecular mass around 52 kDa (K_m = 0.445 mM, V_max = 125.8 mM/mg/min and K_cat = 2.88 min^–1). The enzyme was found stable in a range of pH (3.0–8.0) and temperature (30–70 °C) with an optimal activity at pH 5.0, pI of 4.4 and at 40 °C temperature. It exhibited half-life (t_1/2) of 4.5 h at 60 °C. The enzyme comprised a typical secondary structure containing α-helix (9.3%), β-pleated sheet (33.6%) and β-turn (17.2%). The native conformation of the enzyme was alike a 44 nm spherical nanoparticle upon aggregation. Thermodynamic parameters of tannase revealed that it was stable at 40 °C and showed Q₁₀, ΔG_d and ΔS_d values of 2.08, 99.37 KJ/mol and 252.38 J mol⁻¹ K⁻¹, respectively. Organic solvents were stimulatory with regard to enzyme activity. Moreover, the altered enzyme activity was determined to be correlated with the changes in structural conformation in presence of inducer and inhibitor. Tannase was explored to have no cytotoxicity on Vero cell line as well as rat model study.