Production of hexanoic acid from d-galactitol by a newly isolated Clostridium sp. BS-1

In a study screening anaerobic microbes utilizing d-galactitol as a fermentable carbon source, four bacterial strains were isolated from an enrichment culture producing H₂, ethanol, butanol, acetic acid, butyric acid, and hexanoic acid. Among these isolates, strain BS-1 produced hexanoic acid as a major metabolic product of anaerobic fermentation with d-galactitol. Strain BS-1 belonged to the genus Clostridium based on phylogenetic analysis using 16S rRNA gene sequences, and the most closely related strain was Clostridium sporosphaeroides DSM 1294^T, with 94.4% 16S rRNA gene similarity. In batch cultures, Clostridium sp. BS-1 produced 550 ± 31 mL L⁻¹ of H₂, 0.36 ± 0.01 g L⁻¹ of acetic acid, 0.44 ± 0.01 g L⁻¹ of butyric acid, and 0.98 ± 0.03 g L⁻¹ of hexanoic acid in a 4-day cultivation. The production of hexanoic acid increased to 1.22 and 1.73 g L⁻¹ with the addition of 1.5 g L⁻¹ of sodium acetate and 100 mM 2-(N-morpholino)ethanesulfonic acid (MES), respectively. Especially when 1.5 g L⁻¹ of sodium acetate and 100 mM MES were added simultaneously, the production of hexanoic acid increased up to 2.99 g L⁻¹. Without adding sodium acetate, 2.75 g L⁻¹ of hexanoic acid production from d-galactitol was achieved using a coculture of Clostridium sp. BS-1 and one of the isolates, Clostridium sp. BS-7, in the presence of 100 mM MES. In addition, volatile fatty acid (VFA) production by Clostridium sp. BS-1 from d-galactitol and d-glucose was enhanced when a more reduced culture redox potential (CRP) was applied via addition of Na₂S·9H₂O.     

Effect of various salts on the growth and development of Geotrichum candidum,the causal agent of carrot sour rot

This study evaluated the efficacy of ammonium, calcium, potassium and sodium salts as possible alternatives to synthetic fungicides in the control of Geotrichum candidum, the causal agent of sour rot on carrots. In vitro mycelial growth of G. candidum was completely halted by ammonium bicarbonate and carbonate; calcium oxide; potassium benzoate, carbonate and sorbate; sodium benzoate, carbonate and fluoride (2% w/v). Potassium and sodium bicarbonate also reduced mycelial growth by 77.78% and 90.60%, respectively, and the difference between the effects of sodium bicarbonate and the first group of salts was not statistically significant (p < 0.05). With the exception of potassium and sodium bicarbonate, the above‐mentioned salts also halted or strongly reduced arthrospore germination. Potassium bicarbonate, and sodium bicarbonate, acetate and propionate significantly increased conidiation (p < 0.05). Of all the salts tested in vitro, only ammonium bicarbonate and carbonate, calcium oxide and sodium fluoride were toxic to G. candidum. In in vivo studies, all the calcium salts tested (acetate, chloride, citrate, formate, lactate, oxide, propionate and silicate), several of the sodium salts (acetate, bicarbonate, chloride and fluoride) and potassium bicarbonate exhibited both protective and curative activity against G. candidum, significantly reducing the severity of sour rot in comparison to pathogen‐inoculated controls (p < 0.05). Although no curative was observed with ammonium bicarbonate, ammonium carbonate, potassium carbonate, potassium chloride, sodium carbonate or sodium citrate, these salts also demonstrated significant protective activity against sour rot when compared to controls (p < 0.05). In sum, the study findings show that all of the selected salts may be used to control carrot sour rot, except for sodium fluoride, which exhibited phytotoxicity to carrots.     

Regeneration of denatured polyphenol oxidase in Toona sinensis (A.Juss.) Roam

The regeneration of polyphenol oxidase (PPO) from leaves of Toona sinensis (A.Juss.) Roam (TS), denatured by boiling with 0.4% (w/v) sodium dodecyl sulfate (SDS) and 1% (v/v) β-mercaptoethanol, and was studied with SDS-polyacrylamide gel electrophoresis (PAGE) and the gel-activity-stained assay. The optimal regenerating conditions for the denatured TS-PPO were as follows: extracting with buffer of 150 mM Na-Pi, pH 7.2; regenerating with buffer of 50 mM Tris–HCl, pH 7.2, 2% Triton X-100 for 45 min. In addition, the PPO regeneration was promoted by the addition of Cu²⁺ in the recovery buffer. The regenerating activity of the denatured-PPO was reduced in samples extracted with buffers containing polyvinylpyrrolidone (PVP), polyvinylpolypyrrolidone (PVPP) or Triton X-100.     

High‐Throughput Chiral LC–MS/MS Method Using Overlapping Injection Mode for the Determination of Pantoprazole Enantiomers in Human Plasma with Application to Pharmacokinetic Study

A sensitive and high‐throughput chiral liquid chromatography–tandem mass spectrometry method was developed and validated for the quantification of R‐pantoprazole and S‐pantoprazole in human plasma. Sample extraction was carried out by using ethyl acetate liquid–liquid extraction in 96‐well plate format. The separation of pantoprazole enantiomers was performed on a CHIRALCEL OJ‐RH column and an overlapping injection mode was used to achieve a run time of 5.0 min/sample. The mobile phase consisted of 1) 10 mM ammonium acetate in methanol: acetonitrile (1:1, v/v) and 2) 20 mM ammonium acetate in water. Isocratic elution was used with flow rate at 500 μL/min. The enantiomers were quantified on a triple‐quadrupole mass spectrometer under multiple reaction monitoring (MRM) mode with m/z 382.1/230.0 for pantoprazole and m/z 388.4/230.1 for pantoprazole‐d7. Linearity from 20.0 to 5000 ng/mL was established for each enantiomer (r² > 0.99). Extraction recovery ranged from 91.7% to 96.4% for R‐pantoprazole and from 92.5% to 96.5% for S‐pantoprazole and the IS‐normalized matrix factor was 0.98 to 1.07 for R‐pantoprazole and S‐pantoprazole, respectively. The method was demonstrated with acceptable accuracy, precision, selectivity, and stability and the method was applied to support a pharmacokinetic study of a phase I clinical trial of racemic pantoprazole in healthy Chinese subjects. Chirality 28:569–575, 2016. © 2016 Wiley Periodicals, Inc.     

Nitrile-hydrolyzing enzyme from Meyerozyma guilliermondii and its potential in biosynthesis of 3-hydroxypropionic acid

3-Hydroxypropionic acid (3-HP) is an important platform chemical in organic synthesis. Traditionally, 3-HP was produced by chemical methods and fermentation process. In this work, a novel enzymatic method was developed for green synthesis of 3-HP. A yeast strain harboring nitrile-hydrolyzing enzyme was newly isolated from environmental samples using 3-hydroxypropionitrile (3-HPN) as the sole nitrogen source. It was identified to be Meyerozyma guilliermondii CGMCC12935 by sequencing of the 18S ribosomal DNA and internal transcribed spacer, together with analysis of the morphology characteristics. The catalytic properties of M. guilliermondii CGMCC12935 resting cells were determined, and the optimum activity was achieved at 55 °C and pH 7.5. The enzyme showed broad substrate specificity towards nitriles, especially 3-HPN, aminoacetonitrile and 3-cyanopyridine. The presence of Ag⁺, Pb²⁺ and excess substrate inhibited the enzyme activity, whereas 5% (v/v) ethyl acetate had a positive effect on the enzyme activity. M. guilliermondii CGMCC12935 resting cells by addition of 3% glucose could thoroughly hydrolyze 500 mM 3-HPN into 3-HP within 100 h and the maximal accumulative production of 3-HP reached 216.33 mM, which was over twofolds than the control group with no additional glucose. And this work would lay the foundation for biological production of 3-HP in industry.

     

Hydrolysis of cyclic poly(ethylene terephthalate) trimers by a carboxylesterase from Thermobifida fusca KW3

We have identified a carboxylesterase produced in liquid cultures of the thermophilic actinomycete Thermobifida fusca KW3 that were supplemented with poly(ethylene terephthalate) fibers. The enzyme hydrolyzed highly hydrophobic, synthetic cyclic poly(ethylene terephthalate) trimers with an optimal activity at 60°C and a pH of 6. V _max and K _m values for the hydrolysis were 9.3 μmol⁻¹ min⁻¹ mg⁻¹ and 0.5 mM, respectively. The esterase showed high specificity towards short and middle chain-length fatty acyl esters of p-nitrophenol. The enzyme retained 37% of its activity after 96 h of incubation at 50°C and a pH of 8. Enzyme inhibition studies and analysis of substitution mutants of the carboxylesterase revealed the typical catalytic mechanism of a serine hydrolase with a catalytic triad composed of serine, glutamic acid, and histidine.     

Purification and characterization of a thermostable intracellular β-xylosidase from the thermophilic fungus Sporotrichum thermophile

An intracellular β-xylosidase from the thermophilic fungus Sporotricum thermophile strain ATCC 34628 was purified to homogeneity by Q-Sepharose and Mono-Q column chromatographies. The protein properties correspond to molecular mass and pI values of 45 kDa and 4.2, respectively. The enzyme is optimally active at pH 7.0 and 50 °C. The purified β-xylosidase is fully stable at pH 6.0–8.0 and temperatures up to 50 °C and retained over 58% of its activity after 1 h at 60 °C. The enzyme hydrolyzes β-1,4-linked xylo-oligosaccharides with chain lengths from 2 to 6, releasing xylose from the non-reducing end, but is inactive against xylan substrates. The apparent K_m and V_max values from p-nitrophenyl β-d-xylopyranoside are 1.1 mM and 114 μmol p-nitrophenol min⁻¹ mg⁻¹, respectively. Alcohols inactivate the enzyme, ethanol at 10% (v/v) yields a 30% decrease of its activity. The enzyme is irreversibly inhibited by 2,3-epoxypropyl β-d-xylobioside while alkyl epoxides derived from d-xylose were not inhibitors of the enzyme. The enzyme catalyses the condensation reaction using high donor concentration, up to 60% (w/v) xylose.     

Changes in water relations,photosynthetic activity and proline accumulation in one-year-old olive trees (<Emphasis Type="Italic">Olea europaea</Emphasis> L. cv. Chemlali) in response to NaCl salinity

The comparative responses of young olive trees (Olea europaea L. cv “Chemlali”) to different NaCl salinity levels were investigated over 11 months. One-year-old own rooted plants were grown in 10-L pots containing sand and perlite mixture (1:3 v/v). Trees were subjected to three irrigation treatments: CP (control plants that were irrigated with fresh water); SS1 (salt stressed plants irrigated with water containing 100 mM NaCl) and SS2 plants (salt stressed plants irrigated with water containing 200 mM NaCl). Shoot elongation rate, relative water content, leaf water potential and net carbon dioxide exchange rates decreased significantly with increased NaCl salinity level. Under stressed conditions, the increase of Na⁺ and Cl⁻ ions in both leaves and roots was accompanied with that of proline and soluble sugars. The above results show that the accumulation of proline and sugars under stressed conditions could play a role in salt tolerance. The absence of toxicity symptoms under both stress treatments and the superior photosynthetic activity recorded in SS1-treated plants suggest that cv Chemlali is better able to acclimatize to 100 mM NaCl than at 200 mM NaCl. Our findings indicate that saline water containing 100 mM NaCl, the most available water in arid region in Tunisia, can be recommended for the irrigation of cv Chemlali in the arid south of Tunisia.     

Enhanced γ-aminobutyric acid-forming activity of recombinant glutamate decarboxylase (gadA) from Escherichia coli

γ-aminobutyric acid (GABA) is an important bioactive regulator, and its biosynthesis is primarily through the α-decarboxylation of glutamate by glutamate decarboxylase (GAD). The procedures to obtain GABA by bioconvertion with high activity recombinant Escherichia coli GAD have been seldom understood. In this study, Escherichia coli GAD (gadA) was highly expressed (about 70–75% of total protein) as soluble protein in Escherichia coli BL21(DE3) containing pET28a-gadA, which was induced by 0.4 mM IPTG in LB medium, and maximal GABA-forming activity of the recombinant GAD was 40 U/mL at a concentration (0.15 mM) of pyridoxal phosphate (PLP) and a concentration (0.6 mM) of Ca²⁺ at optimal pH of 3.8. The optimal concentration (7.5 mM) of Mn²⁺ can also improve the activity of recombinant enzyme, but the co-effect of Ca²⁺ and Mn²⁺ exhibited antagonism effect when added simultaneously. LB and 0.1% (w/v) lactose were selected as culture medium and inducer, respectively. The relative activity was markedly higher activated by Ca²⁺ (174%), Mn²⁺ (164%) than that by other seven bivalent cations. Finally, the yield of GABA was high of 94 g/L detected by paper chromatography or HPLC in 1 L reaction system with 30 mL crude GAD (12 U/mL). By entrapping Escherichia coli glutamate decarboxylase into sodium alginate and carrageenan gel beads, the activity of immobilized GAD (IGAD) remained 85% during the initial five batches and the activity still remained 50% at the tenth batch, these results indicated that the recombinant Escherichia coli GAD was feasible for the future industrial production of GABA.     

Biomass and lipid productivities of <Emphasis Type="Italic">Chlorella vulgaris</Emphasis> under autotrophic,heterotrophic and mixotrophic growth conditions

Biomass and lipid productivities of Chlorella vulgaris under different growth conditions were investigated. While autotrophic growth did provide higher cellular lipid content (38%), the lipid productivity was much lower compared with those from heterotrophic growth with acetate, glucose, or glycerol. Optimal cell growth (2 g l⁻¹) and lipid productivity (54 mg l⁻¹ day⁻¹) were attained using glucose at 1% (w/v) whereas higher concentrations were inhibitory. Growth of C. vulgaris on glycerol had a similar dose effects as those from glucose. Overall, C. vulgaris is mixotrophic.     

A Shinella β-N-acetylglucosaminidase of glycoside hydrolase family 20 displays novel biochemical and molecular characteristics

β-N-Acetylglucosaminidases (GlcNAcases) are important for many biological functions and industrial applications. In this study, a glycoside hydrolase family 20 GlcNAcase from Shinella sp. JB10 was expressed in Escherichia coli BL21 (DE3). Compared to many GlcNAcases, the purified recombinant enzyme (rJB10Nag) exhibited a higher specificity activity (538.8 µmol min⁻¹ mg⁻¹) or V _max (1030.0 ± 82.1 µmol min⁻¹ mg⁻¹) toward p-nitrophenyl β-N-acetylglucosaminide and N,N′-diacetylchitobiose (specificity activity of 35.4 µmol min⁻¹ mg⁻¹) and a higher N-acetylglucosaminide tolerance (approximately 50% activity in 70.0 mM N-acetylglucosaminide). The degree of synergy on enzymatic degradation of chitin by a commercial chitinase and rJB10Nag was as high as 2.35. The enzyme was tolerant to most salts, especially 3.0–15.0% (w/v) NaCl and KCl. These biochemical characteristics make the JB10 GlcNAcase a candidate for use in many potential applications, including processing marine materials and the bioconversion of chitin waste. Furthermore, the enzyme has the highest proportions of alanine (16.5%), glycine (10.5%), and random coils (48.8%) with the lowest proportion of α-helices (24.9%) among experimentally characterized GH 20 GlcNAcases from other organisms.

     

A novel cold-adapted lipase from <Emphasis Type="Italic">Acinetobacter</Emphasis> sp. XMZ-26: gene cloning and characterisation

Acinetobacter sp. XMZ-26 (ACCC 05422) was isolated from soil samples obtained from glaciers in Xinjiang Province, China. The partial nucleotide sequence of a lipase gene was obtained by touchdown PCR using degenerate primers designed based on the conserved domains of cold-adapted lipases. Subsequently, a complete gene sequence encoding a 317 amino acid polypeptide was identified. Our novel lipase gene, lipA, was overexpressed in Escherichia coli. The recombinant protein (LipA) was purified by Ni-affinity chromatography, and then deeply characterised. The LipA resulted to hydrolyse pNP esters of fatty acids with acyl chain length from C2 to C16, and the preferred substrate was pNP octanoate showing a k _cat = 560.52 ± 28.32 s⁻¹, K _m = 0.075 ± 0.008 mM, and a k _cat/K _m = 7,377.29 ± 118.88 s⁻¹ mM⁻¹. Maximal LipA activity was observed at a temperature of 15°C and pH 10.0 using pNP decanoate as substrate. That LipA peaked at such a low temperature and remained most activity between 5°C and 35°C indicated that it was a cold-adapted enzyme. Remarkably, this lipase retained much of its activity in the presence of commercial detergents and organic solvents, including Ninol, Triton X-100, methanol, PEG-600, and DMSO. This cold-adapted lipase may find applications in the detergent industry and organic synthesis.     

Interactive effects of potassium and sodium on root growth and expression of K/Na transporter genes in rice

Sufficient supply of potassium (K) can alleviate the adverse effects of excess sodium (Na) on plant growth. However, it remains unclear if such a beneficial function is related to regulation of root growth and/or expression of K/Na transporters. Herein we report the responses of a rice cultivar, which was pretreated with normal nutrient solution for 1 month, to three levels of Na (0, 25, and 100 mM) without or with supply of K for 9 days. High Na (100 mM) significantly decreased plant growth, root activity, and total K uptake, and increased biomass ratio of roots to shoots. Short-term removal of K supply (9 days) did not affect root morphology and biomass ratio of roots to shoots, but decreased root activity of seedlings grown in high Na solution. K deficiency increased uptake of Na and transport of K from roots to shoots. Moreover, expression of OsHAK1, a putative K transporter gene, was upregulated by low Na (25 mM) and downregulated by high Na (100 mM) in roots. In leaves, its expression was suppressed by the Na treatments when K supply was maintained. Expression of OsHKT2;1, which encodes a protein that acts mainly as a Na transporter, was downregulated by high Na, but was enhanced by K deficiency both in roots and leaves. Expression of five other putative K/Na transporter or Na⁺/H⁺ genes, OsHKT1;1, OsHKT1;2, OsHKT2;3, OsNHX1, and OsSOS1, was not affected by the treatments. The results suggest that OsHAK1 and OsHKT2;1 were involved in the interactive effects of K and Na on their uptake and distribution in rice. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Reduction of Cr(VI) by Desulfovibrio vulgaris and Microbacterium sp.

Many industrial wastes contain Cr(VI), a carcinogen and mutagen, the toxicity of which can be ameliorated by reduction to Cr(III). Microbacterium sp. NCIMB 13776 andDesulfovibrio vulgaris NCIMB 8303 reduced Cr(VI) to Cr(III) anoxically using 25 mM sodium citrate buffer (pH 7), with 25 mM sodium acetate and 25 mM sodium formate as electron donors at 30 °C, under which conditions the rates of reduction of 500 M sodium chromate were 77 and 6 nmol h^–1 mg dry cell wt for D. vulgaris and Microbacterium sp., respectively, these being increased to 127 and 17 nmol h^–1 mg dry cell wt in the presence of 20 mM MOPS/NaOH buffer.     

Increase of ethanol tolerance of Saccharomyces cerevisiae by error-prone whole genome amplification

Saccharomyces cerevisiae was transformed for higher ethanol tolerance by error-prone whole genome amplification. The resulting PCR products were transformed back to the parental strain for homologous recombination to create a library of mutants with the perturbed genomic networks. A few rounds of transformation led to the isolation of mutants that grew in 9% (v/v) ethanol and 100 g glucose l⁻¹ compared to untransformed yeast which grew only at 6% (v/v) ethanol and 100 g glucose l⁻¹.     

Application of acetate buffer in pH adjustment of sorghum mash and its influence on fuel ethanol fermentation

A 2 M sodium acetate buffer at pH 4.2 was tried to simplify the step of pH adjustment in a laboratory dry-grind procedure. Ethanol yields or conversion efficiencies of 18 sorghum hybrids improved significantly with 2.0–5.9% (3.9% on average) of relative increases when the method of pH adjustment changed from traditional HCl to the acetate buffer. Ethanol yields obtained using the two methods were highly correlated (R ² = 0.96, P < 0.0001), indicating that the acetate buffer did not influence resolution of the procedure to differentiate sorghum hybrids varying in fermentation quality. Acetate retarded the growth of Saccharomyces cerevisiae, but did not affect the overall fermentation rate. With 41–47 mM of undissociated acetic acid in mash of a sorghum hybrid at pH 4.7, rates of glucose consumption and ethanol production were inhibited during exponential phase but promoted during stationary phase. The maximum growth rate constants (μ _max) were 0.42 and 0.32 h⁻¹ for cells grown in mashes with pH adjusted by HCl and the acetate buffer, respectively. Viable cell counts of yeast in mashes with pH adjusted by the acetate buffer were 36% lower than those in mashes adjusted by HCl during stationary phase. Coupled to a 5.3% relative increase in ethanol, a 43.6% relative decrease in glycerol was observed, when the acetate buffer was substituted for HCl. Acetate helped to transfer glucose to ethanol more efficiently. The strain tested did not use acetic acid as carbon source. It was suggested that decreased levels of ATP under acetate stress stimulate glycolysis to ethanol formation, increasing its yield at the expense of biomass and glycerol production. Names are necessary to report factually on available data; however, the U.S. Department of Agriculture neither guarantees nor warrants the standard of the product, and use of the name by the U.S. Department of Agriculture implies no approval of the product to the exclusion of others that may also be suitable.     

Kinetics of solvent-free geranyl acetate synthesis by Rhizopus oligosporus NRRL 5905 lipase immobilized on to cross-linked silica

The objective of the present work was to study the kinetics of the solvent-free synthesis of geranyl acetate by a novel lipase (activity 60 U g^?1) made by immobilization of lipase from Rhizopus oligosporous NRRL 5905 on to cross-linked silica gel. Transesterification was performed with vinyl acetate as the acyl donor. Vinyl acetate was used in large excess compared to geraniol, which made the reaction pseudo first order with respect to geraniol and the reaction rate followed Michaelis–Menten kinetics for a single substrate. To obtain the highest yield for geranyl acetate, various relevant physical parameters such as shaking speed, reaction time, enzyme concentration, initial water amount and reaction temperature that influence the activity of lipase were investigated. A maximum molar conversion of 67% was achieved after 48 h of reaction at 30°C, at an enzyme concentration of 25% w/v of reaction mixture. Substrate conversion remained constant for five successive cycles; thereafter the conversion dropped by only 11%. Using a pseudo first-order kinetic model for geranyl acetate synthesis in the absence of organic solvents, apparent K_m and V_max values were evaluated as 60 mM and 141 µmol g^?1 h^?1, respectively.     

Incorporation of [1-14C]acetate into the fatty acyl groups of soybean root plasma membrane phospholipids

The incorporation of [¹⁴C]acetate into fatty acids in a plasma membrane enriched fraction from mature soybean root (Glycine max) was studied by time-course experiments. Mature sections of 4-day-old dark-grown soybean roots were incubated with [1-¹⁴C]acetate, 1 mM sodium acetate and 50 μ/ml chloramphenicol. Plasma membrane vesicles were isolated at pH 7.8 and in the presence of 5 mM EDTA, 5 mM EGTA and 10 mM NaF. Lipid extracts analysed for phospholipid class and acyl chain composition revealed that relatively long incubation times did not alter the phospholipid composition of the plasma membrane enriched fraction. Radioactivity was incorporated into all the phospholipid classes proportional to their concentration in the membrane fraction. The distribution of ¹⁴C within the fatty acids of phosphatidylcholine and phosphatidylethanolamine differed from the respective fatty acid compositions and changed with time. Radioactivity also appeared more rapidly in the unsaturated acyl groups of phosphatidylcholine when compared with phosphatidylethanolamine. The rate and pattern of fatty acid incorporation into phosphatidylcholine differed from that for phosphatidylethanolamine.     

4-Aminopyridine: Effects on electrical activity during ischemia and reperfusion in perfused rat hearts

We investigated the effects of 2 and 4 mM 4-aminopyridine (4-AP, – blocker of the transient outward current I_to) on the electrophysiological response to regional ischemia and reperfusion. Spontaneously beating rat hearts were subjected to coronary occlusion (10 min) followed by reperfusion. The surface electrogram and the membrane potential from subepicardial left ventricular cells were recorded throughout. The basal effect of 4-AP was a dose dependent increase in the action potential duration (APD₉₀) without changes in the resting potential or the heart rate. During early ischemia resting depolarization (from 87.4 ± 1.9–70.1 ± 2.5 mV in the controls) was enhanced by 4 mM, 4-AP (84.3 ± 1.4 mV vs. 61.7 ± 1.3 mV) whereas APD₉₀ increased by 73.5%. These effects resulted in a marked reduction in the duration of diastolic intervals that led to conduction failure and aborted responses. A partial recovery was found by the end of ischemia concomitant with APD₉₀ shortening in both, control and 4-AP treated hearts. On reperfusion, 4-AP did not influence the initial incidence of ventricular tachyarrhythmias but decreased their duration from 531.5 ± 56.3–260.7 ± 100 sec (2 mM) and to 75.6 ± 10.5 sec (4 mM). These data confirm others obtained by Henry et al. [11] in isolated cells indicating that ischemia induces sequential changes in several K⁺ conductances. In addition, they show that changes in action potential characteristics may exert beneficial effects on reperfusion arrhythmias by acting on the arrhythmic substrate without suppressing the trigger mechanism.     

Reactivation of immobilized penicillin G acylase: Influence of cosolvents and catalytic modulators

Reactivation of penicillin G acylase immobilized in glyoxyl-agarose after inactivation was studied with the purpose of increasing the lifespan of the biocatalyst by simple and reproducible strategies, considering unfolding–refolding and direct incubation in reactivation media. Reactivation yields were increased with respect to the control (fully aqueous medium) when cosolvents were added to the reactivation medium at concentrations below 50% (v/v). Best results were obtained with 30% (v/v) ethyleneglycol (EG) in both reactivation strategies. An increase in reactivation yield from 36.0 to 62.8% was obtained using the unfolding–refolding strategy, while an increase from 50.0 to 68.4% was obtained by direct incubation in aqueous media with respect to control. Catalytic modulators were also included in the reactivation medium: competitive inhibitors (phenylacetic acid and 2-thienylacetic acid) caused a reduction while non-competitive (7-ADCA and 6-APA) caused an increase in reactivation yield. Combining cosolvent and catalytic modulators, best results in both strategies were obtained with 30% (v/v) EG plus 100 mM 7-ADCA, where an increase in reactivation yield from 36.0 to 96.0% and from 50.0 to 98.0% was achieved with unfolding–refolding and direct incubation in reactivation media respectively. Apparent reactivation rate was higher in the case of direct incubation in reactivation media, best results being obtained when using 100 mM 7-ADCA and 30% (v/v) EG, with an increase with respect to the control (fully aqueous medium with no modulator) from 0.309 h^?1 to 1.129 h^?1, while for unfolding–refolding strategy increase was only from 0.124 h^?1 to 0.384 h^?1. Results indicate that direct incubation is a better strategy for penicillin G acylase reactivation and opens up the possibility of significantly increasing the operational lifespan of the biocatalyst by operating the reactor with repeated cycles of reaction and reactivation.