Occurrence of lipopolysaccharide alterations among Tn5 mutants of Rhizobium leguminosarum bv. trifolii strain 24.1 with altered colony morphology.

Transposon mutants of Rhizobium leguminosarum bv. trifolii 24.1 showing less glossy or smaller colonies were screened for properties usually associated with lipopolysaccharide (LPS) defects in R. leguminosarum, i.e. motility, growth rate, tendency to agglutination in liquid media and symbiotic efficiency. Neither any of the above mutants nor the earlier isolated 24.12 strain, defective in LPS, showed all these properties changed simultaneously. According to PAGE/sodium deoxycholate analysis the mutant 24.12 was the only one producing defective lipopolysaccharide. GC-MS analysis revealed in this mutant qualitative changes in composition of its LPS in comparison with LPS isolated from the parent strain. Other Tn5 mutants produced LPSs similar in composition, however the proportion between LPS I and LPS II differed from that in the parent strain.     

Comparative studies of lipopolysaccharide and exopolysaccharide from a virulent strain of Pseudomonas solanacearum and from three avirulent mutants.

The composition of the Pseudomonas solanacearum lipolysaccharide (LPS) was found to be similar to that described for the LPS of enterobacteria. The lipid A contained fatty acids and glucosamine in a molar ratio of 5:2. The LPS fraction contained 2-keto-3-deoxyoctulosonic acid, L-glycero-D-mannoheptose, hexoses (glucose, rhamnose, and glucosamine), and a pentose (xylose). The LPSs from the wild-type strain (GMI1000), from the spontaneous rough mutant (GMI2000), and from their respective acridine orange-resistant (Acrr) mutants (GMI1178 and GMI2179) contained the same component sugars in their polysaccharide moieties, but the relative amounts of each sugar varied greatly. Spontaneous mutation to the rough type was characterized by a decrease in the ratio of rhamnose to glucose, whereas a reverse effect was seen for the acridine orange resistance mutation from the parent strains (GMI1000 and GMI2000) to the respective mutant strains (GMI1178 and GMI2179). The exopolysaccharide (EPS) from GMI1000 was found to be composed of two fractions: a heteropolysaccharide (galactosamine, glucose, and rhamnose) excluded from Sephadex G-50 and an additional glucan with a lower molecular weight. Strains GMI1000 and GMI1178 produced comparable amounts of EPS, GMI2179 synthesized less EPS, and GMI2000 produced no detectable EPS. High-pressure liquid chromatography and 13C nuclear magnetic resonance analyses revealed some differences between these EPSs. The glucan fraction seemed to be the major component of the EPS from GMI2179, whereas GMI1000 and GMI1178 EPSs contained both fractions and appeared to differ in the structures of their heteropolysaccharide fractions. Viscosity measurements confirmed differences between whole EPSs produced by the three strains.     

Isolation and characterization of mutants of Rhizobium leguminosarum bv. viciae 248 with altered lipopolysaccharides: possible role of surface charge or hydrophobicity in bacterial release from the infection thread

Effects of alterations in lipopolysaccharide (LPS) structure of Rhizobium leguminosarum bv. viciae on effective symbiosis and on a number of cell surface characteristics were studied. Tn5 mutants with altered LPSs were screened for their inability to bind monoclonal antibody 3, one of three monoclonal antibodies to the tentative O-antigenic part of the wild-type LPS of strain 248. Ten class I LPS mutants completely lacked the O-antigen-containing LPS species. The class II LPS mutant had a severely diminished amount of an antigenically altered O-antigen-containing LPS. The class III LPS mutant had normal amounts of an altered, O-antigen-containing LPS. Class I and II mutants, but not the class III mutant, showed abnormal nodule development (i.e., blocked in the stage of bacterial release from the infection thread) resulting in nodules in which very few, at the most, plant cells contained bacteroids and which were unable to fix nitrogen. Class I and II mutants were nonmotile and were more sensitive to hydrophobic compounds than the parent strain. The most striking difference between the symbiotically defective class I and II LPS mutants on one hand and the wild-type strain and the class III mutant on the other hand was that the class I and II mutants have a more hydrophobic cell surface and a higher electrophoretic mobility. A role for an O-antigen-containing LPS in bacterial release from the infection thread, through its effects on general physicochemical cell surface characteristics, is proposed.     

Lack of UV-induced respiration shutoff in a recF strain of Escherichia coli: temperature conditional suppression at 30 degrees C by the sfrA mutation

A mutation in the recF gene of Escherichia coli results in a radiation-sensitive strain. The RecF pathway and the RecBC pathway account for nearly all of the conjugative recombination occuring in E. coli. recBC cells are radiation-sensitive and carry only out a small amount of recombination but these deficiencies are suppressed by an sbcB as recombination is shunted to the RecF pathway. A recBC sbcB recF strain is very radiation-sensitive and is devoid of recombination ability. These deficiencies are suppressed by the srfA mutation; srfA is a recA allele. UV-induced respiration shutoff is a recA⁺, lexA⁺ and recBC⁺ dependent. We report in this paper that respiration does not shutoff in a recF strain at 37 and 30°C. an srfA mutation suppresses this lack of respiration shutoff effect in a recF srfA mutant at 30°C but not at 37°C; no suppression by this mutation occurs at either temperature in a recF recBC sbcB strain. An srfA strain also does not shut off its respiration at 37°C and shows a temperature conditional UV-induced respiration shutoff response at 30°C. The srfA mutation is thought to cause an altered RecA protein to be produced and we suggest that at 37° This altered protein is temperature sensitive. We conclude from the results in this paper that the recF gene product is required for UV-induced respiration shutoff and that the RecA protein plays a special role in the induction process.     

Purification and characterization of thermo-labile alkaline phosphatase from an Antarctic psychrotolerant Bacillus sp. P9

A psychrotolerant Bacillus sp. from Antarctica produced an alkaline phosphatase in the culture supernatant. The strain showed 98.4% 16s rDNA sequence identity with Bacillus sphaericus. The 76 kDa protein was purified 11.1-fold showing alkaline phosphomonoesterase activity. Enzyme was optimally produced at 25 °C and pH 7.0. This cold active alkaline phosphatase is heat labile and gets completely inactivated at 60 °C in 50 min and is active in broad pH range.     

Characterization of LPS mutants of peanut specific Bradyrhizobium (GN17)

Two mutants of Bradyrhizobium sp. (Arachis) strain GN17 having altered lipopolysaccharide (LPS) composition were isolated upon random Tn5 mutagenesis to study their binding with peanut root lectin (PRA II). These mutant strains designated as GN17M1 and GN17M2 produced rough colonies and showed autoagglutination. Flow cytometric analyses indicated that strain GN17M1 bind to PRA II with highest efficiency. Both the mutants synthesized only high molecular weight lipopolysaccharides as observed by silver staining of polyacrylamide gel. The LPSs from both the mutants cross-reacted with anti-GN17 LPS, however, GN17M1 LPS showed 3 times higher cross-reactivity as detected by ELISA. Carbohydrate analysis by high performance anion exchange chromatography (HPAEC) showed that glucose was the major constituent of the purified LPS from the parent strain whereas mannose appeared as major component in the GN17M2 LPS. Equivalent amount of glucose and galactosamine with significant amount of mannose and galactose was the characteristics of the GN17M1 LPS. Purified LPS from GN17M1 and GN17M2 were respectively 17 and 10 times more potent inhibitors of PRA II activity than that of parent strain GN17. Similar binding efficiencies of the mutant LPS towards PRA II was also observed by ELISA. The results of this study indicate that the composition and the arrangement of the LPS are crucial for lectin binding.     

Stabilization of antibody VH-domains by proteolytic selection

Single variable domains of antibodies represent the smallest antigen binding fragments but are less stable than when associated with their cognate variable domains. Here we have attempted to improve the thermodynamic stability of a model heavy chain variable domain (V_H) by “proteolytic selection” a method whereby the protease-resistance of the displayed protein is coupled to the infectivity of a filamentous bacteriophage. The gene encoding the heavy chain variable domain was taken from the anti-lysozyme antibody HyHEL-10, mutated at random by error-prone PCR, and displayed on filamentous bacteriophage by fusion between the domains of the phage p3 protein. As the entire p3 protein is required for phage infectivity, treatment of the phage library with trypsin at an elevated temperature (which leads to cleavage of p3 fusions with unfolded variable domains) selects for infectious phages bearing the more stable variable domains. After several rounds of selection, a mutant (S65G/T70S/D99N) was obtained with improved stability (T_m=58.5 °C and ΔG_25°C=6.3 kcal/mol compared to 51.6 °C and 4.2 kcal/mol for the parent domain). These mutations are conservative and the mutant domain retains the ability to pair with its cognate light chain variable domain in an Fv fragment and to mediate binding to lysozyme. Our results show that the thermodynamic stability of antibody single domains can be improved by “proteolytic selection” and this may represent a step towards making useful antibody single domains for biotechnological application.     

The isolation and characterization of lipopolysaccharide-defective mutants of Pseudomonas aeruginosa PAC1.

Mutants with defective lipopolysaccharides (LPSs) were isolated from Pseudomonas aeruginosa PACIR (Habs serogroup 3) by selection for resistance to aeruginocin from P. aeruginosa PI6 Carbenicillin-sensitive mutants were isolated from P. aeruginosa PACI but not all had defective LPSs. Rough colonial morphology and resistance to bacteriophage II9X appeared to be independent of LPS composition. The LPSs from five mutants were analysed and compared with that of the parent strain. Separation of partially-degraded polysaccharides from LPS from PACI on Sephadex G75 yielded two different high molecular weight fractions and a phosphorylated low molecular weight fraction (L). The mutant LPSs lacked most or all of the high molecular weight fractions but retained some low molecular weight material. That from PACI and two of the mutants was separated by elution from Biogel P6 into two fractions. One, L2, was the core polysaccharide while the other, LI, contained short antigenic side-chains attached to the core like the semi-rough (SR) LPSs of the Enterobacteriaceae. The two mutants which gave the LI fraction with Habs 3 and PACI antisera as did the parent strain. The other three mutants were unreactive and their LPSs contained core components only. One appeared to have a complete core while the other two lacked rhamnose and rhammose plus glucose respectively. Thus there may be four types of LPS in PACI: one contains unsubstituted core polysaccharide and yields L2 on acid hydrolysis, another has short antigenic side-chains of the SR type and yields the LI fraction, while the two high molecular weight fractions are derived from core polysaccharides with different side-chains.     

Improved high thermal stability of pullulanase from a newly isolated thermophilic Bacillus sp. AN-7

A thermophilic Bacillus sp. strain AN-7, isolated from a soil in India, produced an extracellular pullulanase upon growth on starch–peptone medium. The enzyme was purified to homogeneity by ammonium sulfate precipitation, anion exchange and gel filtration chromatography. The optimum temperature and pH for activity was 90 °C and 6.0. With half-life time longer than one day at 80 °C the enzyme proves to be thermostable in the pH range 4.5–7.0. The pullulanase from Bacillus strain lost activity rapidly when incubated at temperature higher than 105 °C or at pH lower than 4.5. Pullulanase was completely inhibited by the Hg²⁺ ions. Ca²⁺, dithiothreitol, and Mn²⁺ stimulated the pullulanase activity. Kinetic experiments at 80 °C and pH 6.0 gave V_max and K_m values of 154 U mg⁻¹ and 1.3 mg ml⁻¹. The products of pullulan were maltotriose and maltose. This proved that the purified pullulanase (pullulan-6-glucanohydrolase, EC 3.2.1.41) from Bacillus sp. AN-7 is classified under pullulanase type I. To our knowledge, this Bacillus pullulanase is the most highly thermostable type I pullulanase known to date.     

The Isolation and Partial Characterization of the Lipopolysaccharides from Several Rhizobium trifolii Mutants Affected in Root Hair Infection

The lipopolysaccharides (LPSs) from Rhizobium trifolii ANU843 and several transposon (Tn5) symbiotic mutants derived from ANU843 were isolated and partially characterized. The mutant strains are unable to induce normal root hair curling (Hac- phenotype) or nodulation (Nod-phenotype) in clover plants. The LPSs from the parent and mutants are very similar in composition. Analysis by PAGE shows that the LPSs consist of higher and lower molecular weight forms. The higher molecular weight form of the LPSs exists in several aggregation states when PAGE is done in 0.1% SDS but collapses into a single band when PAGE is done in 0.5% SDS. Mild acid hydrolysis of all the LPSs releases two polysaccharides, PS1 and PS2. Immunoblots of the PAGE gels and enzyme linked immunosorbant assay inhibition assays show that the PS1 fractions contain the immunodominant sites of the LPSs and that these sites are present in the higher molecular weight form of the LPSs. All the PS1 fractions contain methylated sugars, 2-amino-2,6-dideoxyhexose, heptose, glucuronic acid, and 2-keto-3-deoxyoctonic acid (KDO). All the PS2 fractions contain galacturonic acid, mannose, galactose, and KDO. The PS2 fractions have a molecular weight of about 700. The KDO is present at the reducing end of both the PS1 and the PS2 fractions. The PS1 and PS2 fractions from the mutants contain more glucose than these fractions from the parent. The LPS from a deletion mutant contains less acyl groups than the other LPSs. Immunoblots of the LPSs show that the parent and nod A mutant LPSs contain an additional antigenic band which is not observed in the other LPSs.     

Production, optimization growth conditions and properties of the xylanase from Aspergillus carneus M34

Growth conditions, including incubation times, temperature, agitation rate and initial pH of medium, that affect xylanase production by Aspergillus carneus M34 were studied sequentially use the classical “change-one-factor-at-a-time” method. Our results showed that there was a similar trend between cellular xylanase activity and extracellular xylanase activity. The optimal conditions for xylanase production, different from their cell growth, were on the third day, 30 °C, 100 rpm and pH 4, respectively, in this test. Response surface methodology (RSM) was further introduced to optimize the cultivation conditions and to evaluate the significance of these factors. The optimal cultivation conditions predicted from canonical analysis of this model were achieved by incubation at 35.08 °C with an agitation rate of 111.9 rpm and an initial pH of 5.16. In addition, temperature was the most critical factor for xylanase production by A. carneus M34. Xylanase activity of 22.2 U/mL was verified using the predicted optimal conditions and confirmed the fitness and applicability of the model. The optimal temperature and pH of the crude xylanase activity was observed at 60 °C and acidic pH, respectively. Sustained xylanase activity in the crude extract was also detected over a broad range of pH from 3 to 10. Considering its higher specificity toward agricultural wastes, especially corn cob and coba husk, this strain can be used to develop low-cost media for the mass-production of xylanase.     

Determination of the thermodynamics of carbonic anhydrase acid-unfolding by titration calorimetry

The enthalpy of unfolding (Δ_uH) of carbonic anhydrase II was determined by titrating the protein with acid and measuring the heat using isothermal titration calorimetry (ITC) in the temperature range of 5 to 59 °C. By combining the ITC results with our previous findings by differential scanning calorimetry (DSC) in the temperature range of 39 to 72 °C, the Δ_uH dependence over a wide temperature range was obtained. The temperature dependence of the enthalpy displays significant curvature indicating that the heat capacity of unfolding (Δ_uC_p) is dependent on temperature. The T-derivative of Δ_uC_p was equal to 100 ± 30 J/(mol × K²), with the result that the Δ_uC_p is equal to 15.8 kJ/(mol × K) at 5 °C, 19.0 kJ/(mol × K) at 37 °C and 21.8 kJ/(mol × K) at 64 °C. The enthalpy of unfolding is zero at 17 °C. At lower temperatures, the Δ_uH becomes exothermic.

This method of determining protein unfolding thermodynamics using acid-ITC, significantly widens the accessible T-range, provides direct estimate of the thermodynamic parameters at physiological temperature, and gives further insight into the third T-derivative of the Gibbs free energy of unfolding.     

Metabolic characteristics of sea cucumber Apostichopus japonicus (Selenka) during aestivation

Metabolic characteristics of the sea cucumber Apostichopus japonicus (Selenka) during aestivation were studied in the laboratory. The effects of water temperature on oxygen consumption rate (OCR) and ammonia-N excretion rate (AER) in A. japonicus were determined by the Winkler and Hypobromite methods, respectively. Mature (large, 148.5 ± 15.4 g, medium 69.3 ± 6.9 g) and immature (small, 21.2 ± 4.7 g) individuals aestivated at water temperatures of 20 and 25 °C, respectively. The metabolic characteristics of mature individuals were different from immature individuals during this period. The OCR of mature sea cucumbers peaked at 20 °C, and then dropped significantly at higher temperatures, whereas the OCR of the immature animals continued to increase slightly, even beyond the aestivation temperature. The AER of mature individuals peaked at 20 °C, while that of the immature animals peaked at 25 °C. The relationships between dry weight (DW) and absolute oxygen consumption (R) and absolute ammonia-N excretion (N) could be described by the regression equation R or N = aW^b. With the exception of 15 °C, the O / N ratios (calculated in atomic equivalents) of large size sea cucumbers was close to 20 across the temperatures used in this study, indicating that their energy source was a combination of lipid and protein. On the other hand, apart from small individuals maintained at 10 °C, the O / N ratios of the medium and small sea cucumbers were close to 10, indicating that protein was their major energy source. The O / N ratios in all size groups remained unchanged after aestivation was initiated.     

Temperature as a factor regulating growth and toxin content in the dinoflagellate Alexandrium catenella

Controlled laboratory culture of Alexandrium catenella was used to determine the effects of a range of temperatures between 10 and 16 °C on the growth and saxitoxin content of this dinoflagellate, using strain ACC02 isolated from seawater at Aysen, XI Region, Southern Chile. Cell cultures were made using L1 culture medium at 30‰ salinity, and a photon flux density of 59.53 μmol m² s⁻¹. The results showed that the duration of the exponential growth phase was determined by the experimental temperature, with maximum cell concentrations obtained at 12 °C; significantly lower cell concentrations and growth rates were obtained at 16 °C. Cell dry weight and chlorophyll a values followed cell growth trends. The toxicity of A. catenella was variable at all the experimental temperatures, with a tendency towards having an inverse relation to temperature, with the highest values occurring at 10 °C and the lowest at 16 °C. The optimal range of temperature for the growth of the Chilean strain of A. catenella differed from rates reported for this species isolated at other latitudes, and was correlated with natural temperature conditions predominant in the environment from which it was isolated. The inverse relation of toxicity with temperature in the laboratory was broadly reflected in observations on the toxicity of this dinoflagellate in the field, and coincided with results from the literature.     

Generation and analyses of the transgenic potatoes expressing heterologous thermostable β-amylase

β-Amylase hydrolyzes the -1,4-glycosidic linkages of starch resulting in the release of maltose. This reaction is of industrial importance for maltose production and for the preparation process of fermented foods and alcoholic beverages. A demand for an acceleration of the rate of enzymatic cleavage of the starch macro-molecule is a prerequisite for large-scale and highly efficient production. Increasing the temperature up to the optimum of approximately 60 °C can significantly speed up the reaction. However, at higher temperatures, the effect on protein denaturation becomes dominant, and the conversion rate decreases. The primary objective of this study was to generate transgenic plants of the “Kennebec” potato variety for production of thermostable β-amylase using Agrobacterium-mediated transformation. Four chimeric genes encoding the β-amylase with or without signal peptide sequences for targeting expression in cytoplasm, amyloplasts, or vacuoles were constructed and driven by high tuber expression promoter from Sucrose synthetase gene Sus4. Forty-two transgenic lines were selected for this study. Transgenic lines with various β-amylase constructs were verified for the existence and expression of the transgenes by PCR approaches. The expression level of the introduced β-amylase protein was estimated by immunoblot analyses using polyclonal antibodies. Recombinant β-amylase was successfully expressed in Escherichia coli B21 (DE3), and temperature ranges of these inducible recombinant proteins were found to be between 40 and 90 °C. This enzymatic complex produced in the in vitro cultured microtubers and field-grown tubers from transgenic potatoes were proved to be stable and active at 60 °C. The relative activities of β-amylase in tubers of field-grown potatoes were compared, and the maximum increase was found with transgenic line #6A of the pSUS4-AMY construct which has an 11-fold greater increase than the untransformed “Kennebec”. Variations of the chemical compositions were found in the selected transgenic lines. Results of this study suggest the feasibility of utilizing thermostable β-amylase in transgenic potatoes for the starch-processing industries.     

Characterization of heat resistant mutant strains of Rhizobium sp. [Cajanus] for growth, survival and symbiotic properties

Fourteen heat resistant mutant strains were isolated from a wild-type strain (PP201, Nod⁺ Fix⁺) of Rhizobium sp. (Cajanus) by giving it a heat shock of 43°C. These mutant strains showed a greater increase in optical density (O.D.) and a higher viable cell count in both rhizospheric and non-rhizospheric soil at high temperature. Symbiotic studies showed that pigeon pea plants inoculated with a few mutant strains had ineffective nodules (Nod⁺ Fix⁻) under controlled temperature (43°C) conditions, but under natural high temperature (40–45°C) conditions, the host plants infected with all the mutant strains showed higher total shoot nitrogen than the plants inoculated with the parent strain. Four mutant strains (HR-3, HR-6, HR-10 and HR-12) were found to be highly efficient for all the symbiotic parameters, and thus have the potential to be used as bioinoculants in the North-Western regions of India during the summer season.     

Effect of sucA or sucC gene knockout on the metabolism in Escherichia coli based on gene expressions, enzyme activities, intracellular metabolite concentrations and metabolic fluxes by C-labeling experiments

The effect of sucA or sucC gene knockout on the metabolism in Escherichia coli was investigated for the aerobic cell growth in batch and continuous cultivations based on gene expressions, enzyme activities, intracellular metabolite concentrations and metabolic flux analysis. In the batch cultivation, the cell growth rate and the glucose uptake rate were lower for sucA mutant as compared with the parent strain, while it was not the case for sucC mutant. A significantly higher amount of acetate was produced, and it was not utilized in sucC mutant, while a little less acetate was produced in sucA mutant as compared with the parent strain. Unlike the parent strain and sucC mutant, sucA mutant excreted a little amount of l-glutamate. Enzyme activity results show that some of the glycolytic enzymes such as Tpi and Pgk were up-regulated, while Pfk, Fba and Pyk activities were down-regulated for sucA mutant as compared with the parent strain. For sucC mutant, the activities of Pfk, Fba, Tpi, GAPDH, Pgk and Pyk activities were down-regulated. As for the TCA cycle enzymes, the activities of CS and ICDH were down-regulated, while those of Icl, MS, Fum and MDH were up-regulated for sucA mutant. The activities of the oxidative pentose phosphate (PP) pathway enzymes such as G6PDH and 6PGDH and the gluconeogenic pathway enzyme such as Mez were up-regulated in sucA mutant. The Ack activity was down-regulated for sucA mutant, but not for sucC mutant. In continuous cultivation, the gene expression results indicate that the global regulatory genes such as fadR and iclR were slightly down-regulated in sucA mutant, which enhanced the expression of aceA gene and caused the up-regulation of the isocitrate lyase activity in sucA mutant, while fadR and iclR of sucC mutant changed little and no isocitrate lyase activation was observed for sucC mutant. Some other global regulatory genes such as arcA and fnr genes were down-regulated in both mutants, which caused some of the TCA cycle genes to be up-regulated. The effect of the sucA gene knockout on the metabolic flux distributions was investigated based on ¹H–¹³C NMR spectra and GC–MS signals obtained from ¹³C-labeling experiments. Flux analysis results indicate that the knockout of sucA gene caused the activation of PP pathway and the glyoxylate shunt. The fluxes through glycolysis and the TCA cycle were down-regulated in the sucA mutant. On the other hand, the fluxes through PP pathway and the anaplerotic reactions of Ppc-Pck and Mez increased.     

Critical thermal maximum and body water loss in first instar larvae of three Cetoniidae species (Coleoptera)

Critical thermal maximum (CT_max) and body water losses were measured in first instar larvae of Gnorimus nobilis, Osmoderma eremita (Trichiinae) and Cetonischema aeruginosa (Cetoniinae) when air temperature was increased gradually (0.5 °C/min) from 20 °C to the critical thermal maximum (CT_max), in dry air (near 0% R.H.).

The CT_max was significantly lower in O. eremita (45.6±0.7 °C) than in G. nobilis (48.5±0.6) and C. aeruginosa (51.4±0.9 °C).

An increase of 10 °C (30–40 °C) induced a 2-fold increase of the water loss in C. aeruginosa and O. eremita (Q₁₀=2.10±0.12 and 2.13±0.20, respectively). In the range from 40 to 45 °C to CT_max a strong increase of the water loss was observed in O. eremita and C. aeruginosa, respectively. Body water losses were significantly lower in C. aeruginosa than in O. eremita and G. nobilis over the range 20 °C—CT_max; no significant difference occurred between G. nobilis and O. eremita.     

Improved thermostability and the optimum temperature of Rhizopus arrhizus lipase by directed evolution

To expand the functionality of lipase from Rhizopus arrhizus (RAL) we have used error-prone PCR and DNA shuffling methods to create RAL mutants with improved thermostability and the optimum temperature. One desirable mutant with three amino acids substitution was obtained. The mutated lipase was purified and characterized. The optimum temperature of the mutant lipase was higher by 10 °C than that of the wild-type RAL (WT-RAL). In addition, the thermostability characteristic of the mutant was also improved as the result of directed evolution. The half-life (T_1/2) at 50 °C of the mutant exceeded those of WT-RAL by 12-fold. To confirm which substitution contributed to enhance thermostability and the optimum temperature for lipase activity, three chimeric lipases: chimeric lipase 1(CL-1; A9T), chimeric lipase 2 (CL-2; E190V) and chimeric lipase 3 (CL-3; M225I) from the WT-RAL gene were constructed. Each of the chimeric enzymes was purified and characterized. Amino acid substitution at position 190 was determined to be critical for lipase thermostability and the optimum temperature, while the residue at position 9 and 225 had only marginal effect. The mutational effect is interpreted according to a simulated three-dimensional structure for the mutant lipase.     

Effect of nifedipine on core cooling in rats during tail cold water immersion

Male rats (450 g, n=11/group) were heated at an ambient temperature of 42°C until a rectal temperature of 42.8°C was attained. Rats, then received either saline (30°C)+tail ice water immersion (F+I) or saline (30°C)+tail ice water immersion+Nifedipine, a peripheral vasodilator, (F+I+N) to determine cooling rate effectiveness and survivability. The time to reach a rectal temperature of 42.8°C averaged 172 min in both groups resulting in similar heating rates (0.029°C/min). The cooling rates in group F+I and F+I+N were not significantly different from each other. We conclude that since Nifedipine did not improve cooling rates when combined with fluid+tail ice water immersion, its use as a cooling adjunct does not seem warranted.