AOX Activity Research Articles

Multifactor transcriptional control of alternative oxidase induction integrates diverse environmental inputs to enable fungal virulence

Metabolic flexibility enables fungi to invade challenging host environments. In Candida albicans, a common cause of life-threatening infections in humans, an important contributor to flexibility is alternative oxidase (Aox) activity. Dramatic induction of this activity occurs under respiratory-stress conditions, which impair the classical electron transport chain (ETC). Here, we show that deletion of the inducible AOX2 gene cripples C. albicans virulence in mice by increasing immune recognition. To investigate further, we examined transcriptional regulation of AOX2 in molecular detail under host-relevant, ETC-inhibitory conditions. We found that multiple transcription factors, including Rtg1/Rtg3, Cwt1/Zcf11, and Zcf2, bind and regulate the AOX2 promoter, conferring thousand-fold levels of inducibility to AOX2 in response to distinct environmental stressors. Further dissection of this complex promoter revealed how integration of stimuli ranging from reactive species of oxygen, nitrogen, and sulfur to reduced copper availability is achieved at the transcriptional level to regulate AOX2 induction and enable pathogenesis.

Novel Combination of the Biophysical, Nutritional, and Nutraceutical Properties in Subtropical Pigmented Maize Hybrids.

Maize (Zea mays L.) represents the main caloric source for much of the world's population. Pigmented maize varieties are an excellent source of nutraceutical compounds: blue and yellow maize are rich in anthocyanins as well as carotenoids and phenolic acids, respectively. However, blue maize is usually grown in small quantities as a specialty crop because it lacks the qualities and adaptations of commercial white and yellow varieties. Here, a new high-yield variety of blue maize called Vitamaiz was developed from inbred lines of subtropical blue, white, and yellow maize. The aim of this study was to characterize the nutraceutical and physical properties of 30 Vitamaiz hybrids in two subtropical locations. Kernel physical traits, nutrient composition, and nutraceutical components (free phenolic acids, FPA; cell wall-bound phenolic acids, BPA; total monomeric anthocyanin content, TAC; antioxidant capacity, AOX by oxygen radical absorbance capacity assay, and total carotenoid content, TCC) were evaluated. The biophysical traits of the hybrids were suitable for nixtamalized and flour maize industries. High levels of FPA (228 mg GAE/100 g), BPA (635 mg GAE/100 g), and AOX (2.0 and 8.1 mM Trolox equivalent/100 g for FPA and BPA, respectively) were also detected with elevated TAC levels (274 mg C3G/kg dw) and AOX activity (3.1 mM Trolox equivalent/100 g). This is the first study to characterize Blue × Yellow maize hybrids that adapt to subtropical environments.

Gly Betaine Surpasses Melatonin to Improve Salt Tolerance in Dalbergia odorifera.

Salinity is one of the most serious factors limiting plant growth which can provoke significant losses in agricultural crop production, particularly in arid and semi-arid areas. This study aimed to investigate whether melatonin (MT; 0.05 and 0.1 mM), which has pleiotropic roles, has a better effect than glycine betaine (GB; 10 and 50 mM) on providing salt tolerance in a woody plant Dalbergia odorifera T. Chen. Also, the alternative oxidase activity (AOX) in plant subjected to MT or GB under salinity (150 and 250 mM) was evaluated given that the effect of exogenous MT or GB on AOX has not been reported yet. The results showed that the exogenous application of GB on the seedlings of D. odorifera increased the plant growth parameters, relative water content, total of chlorophyll content, and carotenoid content compared with well-watered and MT treatments. Under severe salinity, the seedlings subjected to GB showed, a significant enhancement in water use efficiency, transpiration, and net photosynthetic rate regardless to MT-treated seedlings. The levels of proline and soluble sugar in the seedlings treated with MT or GB decreased significantly under mild and severe salinity correlated with those in salt-stressed seedlings. Furthermore, GB-treated plants exhibited a significant inhibition of malondialdehyde content compared with MT-treated plants. The concentration of thiols and phenolic compounds were significantly enhanced in the leaves of seedlings treated with MT compared with those treated with GB. Under salt stress condition, GB scavenged significantly higher levels of hydrogen peroxide than MT; while under severe salinity, plants subjected to MT showed better scavenging ability for hydroxyl radicals compared with GB-treated seedlings. The results demonstrated also an enhancement of the levels of superoxide dismutase (SOD), guaiacol peroxidase, and AOX activities in seedlings treated with GB or MT compared with salt-stressed plants. The catalase activity (CAT) was increased by 0.05 mM MT and 0.1 mM GB under mild salinity. Meanwhile, the AOX activity under severe salinity was enhanced only by GB 50 mM. The findings of this study suggested that GB-treated seedlings possessed a better salt tolerance in comparison with MT-treated seedlings.

Identification and Characterization of Aldehyde Oxidase 5 in the Pheromone Gland of the Silkworm (Lepidoptera: Bombycidae).

Aldehyde oxidases (AOXs) are a subfamily of cytosolic molybdo-flavoenzymes that play critical roles in the detoxification and degradation of chemicals. Active AOXs, such as AOX1 and AOX2, have been identified and functionally analyzed in insect antennae but are rarely reported in other tissues. This is the first study to isolate and characterize the cDNA that encodes aldehyde oxidase 5 (BmAOX5) in the pheromone gland (PG) of the silkworm, Bombyx mori. The size of BmAOX5 cDNA is 3,741 nucleotides and includes an open reading frame, which encodes a protein of 1,246 amino acid residues. The theoretical molecular weight and isoelectric point of BmAOX5 are approximately 138 kDa and 5.58, respectively. BmAOX5 shares a similar primary structure with BmAOX1 and BmAOX2, containing two [2Fe-2S] redox centers, a FAD-binding domain, and a molybdenum cofactor (MoCo)-binding domain. RT–PCR revealed BmAOX5 to be particularly highly expressed in the PG (including ovipositor) of the female silkworm moth, and the expression was further confirmed by in situ hybridization, AOX activity staining, and anti-BmAOX5 western blotting. Further, BmAOX5 was shown to metabolize aromatic aldehydes, such as benzaldehyde, salicylaldehyde, and vanillic aldehyde, and fatty aldehydes, such as heptaldehyde and propionaldehyde. The maximum reaction rate (Vmax) of benzaldehyde as substrate was 21 mU and Km was 1.745 mmol/liter. These results suggested that BmAOX5 in the PG could metabolize aldehydes in the cytoplasm for detoxification or participate in the degradation of aldehyde pheromone substances and odorant compounds to identify mating partners and locate suitable spawning sites.

Joint inhibition of mitochondrial complex IV and alternative oxidase by genetic or chemical means represses chloroplast transcription in Arabidopsis.

Changes in the functional state of mitochondria have profound effects on other cellular compartments. Genome-wide expression analysis of Arabidopsisrps10 mutants with an RNAi-silenced expression of mitoribosomal S10 protein has revealed extensive transcriptional reprogramming. A meta-analysis comparing expression datasets of 25 mitochondrial perturbations showed a high similarity of the aox1a:rpoTmp mutant, which is defective in the alternative oxidase (AOX1a) and dual-targeted mitochondrial and plastid RNA polymerase (RPOTmp), to rps10. Both rps10 and aox1a:rpoTmp showed a significantly decreased electron flux through both the cytochrome and the alternative respiratory pathways, and a markedly decreased the expression of nuclear-encoded components of the chloroplast transcription machinery. In line with this, a decreased level of plastid transcripts was observed in rps10 and aox1a:rpoTmp, which was reflected in a reduced rate of chloroplast transcription. Chemical treatment of wild-type seedlings with respiratory inhibitors showed that only simultaneous and direct inhibition of complex IV and AOX activity decreased the level of plastid transcripts. Taken together, both chemical and genetic studies show that the limitation of the activity of two mitochondrial terminal oxidases, complex IV and AOX, negatively impacts chloroplast transcription. Salicylic acid and oxygen are discussed as putative mediators of the signalling pathway between mitochondria, nucleus and chloroplasts. This article is part of the theme issue 'Retrograde signalling from endosymbiotic organelles'.

Alternative Oxidase Activity Reduces Stress in Vibrio fischeri Cells Exposed to Nitric Oxide.

Alternative oxidase (Aox) is a non-energy-conserving respiratory oxidase found in certain eukaryotes and bacteria, whose role in physiology is not entirely clear. Using the genetically tractable bacterium Vibrio fischeri as a model organism, I have identified a role for Aox to reduce levels of stress in cells exposed to oxygen and nitric oxide (NO). In V. fischeri lacking the NO-detoxifying enzyme flavohemoglobin (Hmp), deletion of aox in cells grown in the presence of oxygen and NO results in alterations to the transcriptome that include increases in transcripts mapping to stress-related genes. Using fluorescence-based reporters, I identified corresponding increases in intracellular reactive oxygen species and decreases in membrane integrity in cells lacking aox Under these growth conditions, activity of Aox is linked to a decrease in NADH levels, indicating coupling of Aox activity with NADH dehydrogenase activity. Taken together, these results suggest that Aox functions to indirectly limit production of ferrous iron and damaging hydroxyl radicals, effectively reducing cellular stress during NO exposure.IMPORTANCE Unlike typical respiratory oxidases, alternative oxidase (Aox) does not directly contribute to energy conservation, and its activity would presumably reduce the efficiency of respiration and associated ATP production. Aox has been identified in certain bacteria, a majority of which are marine associated. The presence of Aox in these bacteria poses the interesting question of how Aox function benefits bacterial growth and survival in the ocean. Using the genetically tractable marine bacterium Vibrio fischeri, I have identified a role for Aox in reduction of stress under conditions where electron flux through the aerobic respiratory pathway is inhibited. These results suggest that Aox activity could positively impact longer-term bacterial fitness and survival under stressful environmental conditions.

Phosphorus concentration coordinates a respiratory bypass, synthesis and exudation of citrate, and the expression of high-affinity phosphorus transporters in Solanum lycopersicum.

Plants exhibit respiratory bypasses (e.g., the alternative oxidase [AOX]) and increase the synthesis of carboxylates in their organs (leaves and roots) in response to phosphorus (P) deficiency, which increases P uptake capacity. They also show differential expression of high-affinity inorganic phosphorus (Pi) transporters, thus avoiding P toxicity at a high P availability. The association between AOX and carboxylate synthesis was tested in Solanum lycopersicum plants grown at different soil P availability, by using plants grown under P-sufficient and P-limiting conditions and by applying a short-term (24hr) P-sufficient pulse to plants grown under P limitation. Tests were also performed with plants colonized with arbuscular mycorrhizal fungi, which increased plant P concentration under reduced P availability. The in vivo activities of AOX and cytochrome oxidase were measured together with the concentration of carboxylates and the P concentration in plant organs. Gene transcription of Pi transporters (LePT1 and LePT2) was also studied. A coordinated response between plant P concentration with these traits was observed, indicating that a sufficient P availability in soil led to a suppression of both AOX activity and synthesis of citrate and a downregulation of the transcription of genes encoding high-affinity Pi transporters, presumably to avoid P toxicity.

Estrés oxidativo, metabolismo lipídico y neurotransmisión en el caracol dulceacuícola (Pomacea patula) expuesto a la fracción hidrosoluble de petróleo crudo

Background. Crude oil is a super mixture of chemical compounds and is commonly found in aquatic environments. The tegogolo ( Pomacea patula Baker, 1922) is a Mexican freshwater snail endemic to Lake Catemaco in Veracruz; currently, however, its distribution has expanded to many freshwater ecosystems that suffer the impact of crude oil spills and oil byproducts like fuels. Goals . To assess a series of biomarkers involved in oxidative stress, neurotransmission, and fatty acid metabolism in tegogolos exposed to the water-accommodated fraction (WAF) of Maya crude oil (MCO). Methods . Tegogolo specimens were exposed to WAF of MCO obtained from loads of 0.1, 1, 10 and 100 mg/L. We evaluated ROS ( O2 -* and H2O2), oxidative stress (TBARS and RC=O), enzymes involved in antioxidant defense (SOD, CAT, and GPx), some enzymes involved in neurotransmission (AChE, GDA, and CbE activities), and biomarkers of fatty acids metabolism (fatty acids levels and AOX activity). Results. Clear biomarkers responses were observed only in some tissues. ROS were clearly higher than controls in the foot, head, and kidney; however, others biomarkers of oxidative stress remain statistically unchanged. SOD response was irregular with respect to controls and treatments. In contrast, CAT (foot) and GPx (foot and intestine) were the more active enzymes and their activities were higher than in controls. The responses of some enzymes involved in neurotransmission suggest that compensation mechanisms exist between AChE and GDA in the foot and head. Fatty acids metabolism increased with exposure to WAF; however, these types of biomarkers seem unsuitable for monitoring the toxic effects produced by WAF at low environmental concentrations. Conclusions. We can conclude that under the exposure conditions discussed herein, the tegogolos showed acclimation to WAF of Maya crude oil by complex mechanisms.

UVA, UVB and UVC Light Enhances the Biosynthesis of Phenolic Antioxidants in Fresh-Cut Carrot through a Synergistic Effect with Wounding.

Previously, we found that phenolic content and antioxidant capacity (AOX) in carrots increased with wounding intensity. It was also reported that UV radiation may trigger the phenylpropanoid metabolism in plant tissues. Here, we determined the combined effect of wounding intensity and UV radiation on phenolic compounds, AOX, and the phenylalanine ammonia-lyase (PAL) activity of carrots. Accordingly, phenolic content, AOX, and PAL activity increased in cut carrots with the duration of UVC radiation, whereas whole carrots showed no increase. Carrot pies showed a higher increase compared to slices and shreds. Phenolics, AOX, and PAL activity also increased in cut carrots exposed to UVA or UVB. The major phenolics were chlorogenic acid and its isomers, ferulic acid, and isocoumarin. The type of UV radiation affected phenolic profiles. Chlorogenic acid was induced by all UV radiations but mostly by UVB and UVC, ferulic acid was induced by all UV lights to comparable levels, while isocoumarin and 4,5-diCQA was induced mainly by UVB and UVC compared to UVA. In general, total phenolics correlated linearly with AOX for all treatments. A reactive oxygen species (ROS) mediated hypothetical mechanism explaining the synergistic effect of wounding and different UV radiation stresses on phenolics accumulation in plants is herein proposed.

Implications of evolutionary engineering for growth and recombinant protein production in methanol-based growth media in the yeast Pichia pastoris

BackgroundPichia pastoris is a widely used eukaryotic expression host for recombinant protein production. Adaptive laboratory evolution (ALE) has been applied in a wide range of studies in order to improve strains for biotechnological purposes. In this context, the impact of long-term carbon source adaptation in P. pastoris has not been addressed so far. Thus, we performed a pilot experiment in order to analyze the applicability and potential benefits of ALE towards improved growth and recombinant protein production in P. pastoris.ResultsAdaptation towards growth on methanol was performed in replicate cultures in rich and minimal growth medium for 250 generations. Increased growth rates on these growth media were observed at the population and single clone level. Evolved populations showed various degrees of growth advantages and trade-offs in non-evolutionary growth conditions. Genome resequencing revealed a wide variety of potential genetic targets associated with improved growth performance on methanol-based growth media. Alcohol oxidase represented a mutational hotspot since four out of seven evolved P. pastoris clones harbored mutations in this gene, resulting in decreased Aox activity, despite increased growth rates. Selected clones displayed strain-dependent variations for AOX-promoter based recombinant protein expression yield. One particularly interesting clone showed increased product titers ranging from a 2.5-fold increase in shake flask batch culture to a 1.8-fold increase during fed batch cultivation.ConclusionsOur data indicate a complex correlation of carbon source, growth context and recombinant protein production. While similar experiments have already shown their potential in other biotechnological areas where microbes were evolutionary engineered for improved stress resistance and growth, the current dataset encourages the analysis of the potential of ALE for improved protein production in P. pastoris on a broader scale.

High Osmolarity Environments Activate the Mitochondrial Alternative Oxidase in Debaryomyces Hansenii.

The oleaginous yeast Debaryomyces hansenii is a good model to understand molecular mechanisms involved in halotolerance because of its impressive ability to survive under a wide range of salt concentrations. Several cellular adaptations are implicated in this response, including the presence of a cyanide-insensitive ubiquinol oxidase (Aox). This protein, which is present in several taxonomical orders, has been related to different stress responses. However, little is known about its role in mitochondria during transitions from low to high saline environments. In this report, we analyze the effects of Aox in shifts from low to high salt concentrations in the culture media. At early stages of a salt insult, we observed that this protein prevents the overflow of electrons on the mitochondrial respiratory chain, thus, decreasing the production of reactive oxygen species. Interestingly, in the presence of high osmolite concentrations, Aox activity is able to sustain a stable membrane potential when coupled to complex I, despite a compromised cytochrome pathway. Taken together, our results suggest that under high osmolarity conditions Aox plays a critical role regulating mitochondrial physiology.

Biotransformation of arsenite and bacterial aox activity in drinking water produced from surface water of floating houses: Arsenic contamination in Cambodia

The potential arsenite bioteansformation activity of arsenic was investigated by examining bacterial arsenic arsenite-oxidizing gene such as aoxS, aoxR, aoxA, aoxB, aoxC, and aoxD in high arsenic-contaminated drinking water produced from the surface water of floating houses. There is a biogeochemical cycle of activity involving arsenite oxidase aox system and the ars (arsenic resistance system) gene operon and aoxR leader gene activity in Alcaligenes faecalis SRR-11 and aoxS leader gene activity in Achromobacter xylosoxidans TSL-66. Batch experiments showed that SRR-11 and TSL-66 completely oxidized 1 mM of As (III) to As (V) within 35–40 h. The leaders of aoxS and aoxR are important for gene activity, and their effects in arsenic bioremediation and mobility in natural water has a significant ecological role because it allows arsenite oxidase in bacteria to control the biogeochemical cycle of arsenic-contaminated drinking water produced from surface water of floating houses.

Light regulation of mitochondrial alternative oxidase pathway during greening of etiolated wheat seedlings

This study deals with effects of de-etiolation (48h) of spring wheat (Triticum aestivum L., var. Irgina) seedlings on differential expression of AOX1 genes, levels of AOX protein and the alternative respiratory pathway (AP) capacity. As a result of exposure to continuous irradiation of dark-grown wheat seedlings, the respiratory activity and AP capacity in leaves significantly increased during the first 6h of studies. Expression of AOX1a was up-regulated by light and proved consistent with changes in the AP capacity. Effects on expression of AOX1c were less pronounced. Immunoblot analysis showed three distinct bands of AOX with molecular weights of 34, 36 and 38kDa, with no significant changes in the relative levels during de-etiolation. The lack of a clear correlation between AOX protein amount, AOX1a expression, and AP capacity suggests post-translational control of the enzyme activation. The AOX1a suppression and a decrease in the AP capacity correlated with the sugar pool depletion after 24h of the de-etiolation, which may mean a possible substrate dependence of the AOX activity in the green cells. More efficient malate oxidation by mitochondria as well as the higher AOX capacity during the first 6h of de-etiolation was detected, whereas respiration and AOX capacity with exogenous NADH and glycine increased after 6 and 24h, respectively. We conclude that AOX plays an important role during development of an actively photosynthesizing cell, and can rapidly adapt to changes in metabolism and photosynthesis.

Impact of NH4+ nitrogen source on the production of Rhizopus oryzae lipase in Pichia pastoris

BackgroundPichia pastoris is a highly successful system for heterologous expression. During the induction stage, the ammonium ion released into the fermentation broth has a deep impact on cell growth and protein expression. The impact of NH4+ concentration on the expression of the Rhizopus oryzae lipase proAROL in P. pastoris was investigated. ResultsThe lipase activity under the optimum NH4+ concentration of 440mmol/L reached 12,019U/mL. Increased concentrations of NH4+ in the broth prevented the protease production, resulting in higher specific lipase activity in the supernatant. Furthermore, analysis of carbon metabolism and energy regeneration pattern revealed that under the definite NH4+ concentrations more carbon source (methanol) was consumed with surged AOX activity and then the higher energy and amino acid precursors demand for recombinant protein synthesis is compensated for by the TCA cycle. ConclusionsIn this study, the R. oryzae lipase activity reaches the highest level ever reported under optimized NH4+ concentration and the analysis of the carbon metabolism provides useful information for future optimization of protein production by P. pastoris in a molecular level.

Pre-harvest fruit bagging influences fruit color and quality of apple cv. Delicious

An attempt was made to observe the effect of pre-harvest bagging with spun-bound fabric bags on color and quality of Delicious apple. Bagging was done about a month before harvesting and removed 3-day before harvesting. Bagged and non-bagged fruits were stored at 2℃ ± 1℃ and 90%-95% RH. Observations were recorded on color and fruit quality attributes such as total phenolics, AOX activity, fruit Ca contents, LOX activity, SSC and ascorbic acid contents at harvest and during storage. Our studies have revealed that bagged fruits have better color development (Hunter “a” = 52) than non-bagged fruits at harvest (Hunter “a” = 38), which declined slightly during storage. Similarly, at harvest, bagged fruits contained high amounts of Ca (5.38 mg/100g) and total phenolics (9.3 mg GAE/100gpulp) exhibited higher AOX activity (12.6 μmoles Trolox g-1), and had better SSC and ascorbic acid contents than non-bagged fruits, and there was a decline in all recorded parameters during storage. Bagged fruits exhibited lower LOX activity (1.38 μmoles min-1 g-1 FW) at harvest than non-bagged fruits (2.14 μmoles min-1 g-1 FW), indicating that non-bagged fruits were more senescent than bagged fruits. Further, LOX activity increased during storage both in bagged and non-bagged apples but increase in LOX activity was slower in bagged apples than in non-bagged apples.

Engineering the central pathways in Lactococcus lactis: Functional expression of the phosphofructokinase (pfk) and alternative oxidase (aox1) genes from Aspergillus niger in Lactococcus lactis facilitates improved carbon conversion rates under oxidizing conditions

The present work describes a novel central pathway engineering method that has been designed with the aim to increase the carbon conversion rates under oxidizing conditions in L. lactis fermentations. The nisin producer L. lactis ATCC11454 strain has been genetically engineered by cloning a truncated version of the phosphofructokinase gene (pfk13), along with the pkaC, encoding for the catalytic subunit of cAMP-dependent protein kinase, and the alternative oxidase (aox1) genes of A. niger. Functional expression of the above genes resulted in enhanced PFK activity and the introduction of AOX activity and alternative respiration in the presence of a source of heme in the substrate, under fully aerobic growth conditions. The constructed strain is capable of fermenting high concentrations of glucose as was demonstrated in a series of glucostat fed-batch fermentations with glucose levels maintained at 55, 138 and 277mM. The high maximum specific uptake rate of glucose of 1.8mMs−1gCDW−1 at 277mM glucose is characteristic of the improved ability of the microorganism to handle elevated glucose concentrations under conditions otherwise causing severe reduction of PFK activity. The increased carbon flow through glycolysis led to increased protein synthesis that was reflected in increased biomass and nisin levels. The pfk13–pkaC–aox1-transformant strain's fermentation at 277mM glucose gave a final biomass concentration of 7.5g/l and nisin activity of 14,000IU/ml which is, compared to the parental strain's production levels at its optimal 55mM glucose, increased by a factor of 2.34 for biomass and 4.37 for nisin.

Cloning and functional expression of the mitochondrial alternative oxidase gene ( aox1) of Aspergillus niger in Lactococcus lactis and its induction by oxidizing conditions

Lactococcus lactis is a widely used food bacterium mainly known for its fermentation metabolism. An important, and for long time overlooked, trait of this species is its ability to perform respiratory metabolism in the presence of heme and under aerobic conditions. There is no evidence however for the presence of an alternative respiration pathway and AOX activity. In this study, a cDNA fragment encoding the mitochondrial alternative oxidase, the enzyme responsible for alternative respiration, from a citric acid producing Aspergillus niger strain was cloned and expressed in L. lactis as a host strain. Expression of aox1 conferred on this organism cyanide-resistant and salicylhydroxamate-sensitive growth. Bioreactor cultures under fully aerobic conditions of the transformed L. lactis showed that the alternative respiratory pathway operates and improves significantly the microorganism's response to oxidizing stress conditions as it enhances biomass production, suppresses lactate formation, and leads to accumulation of large amounts of nisin.

Enhanced Pichia pastoris biomass under increased air pressure: batch and fed-batch strategies

The effects of pH on recombinant human erythropoietin (rHuEPO) production by Pichia pastoris was investigated at pH = 4.0, 4.5, 5.0, 5.5 and 6.0, where sorbitol and methanol were supplied to the bioreactor in batch and fed-batch mode, respectively, in the recombinant protein production phase (PP). For the first three phases, glycerol batch (GB), glycerol fed-batch (GFB) and methanol transition (MT), the growth profiles were almost the same for pH ≥ 5.0; however, at pH < 5.0 the growth rate was lower. The highest cell concentration was obtained as 81.4 g L−1 where GB and GFB were operated at pH = 5.0 and MT and PP were operated at pH = 4.5. Sorbitol added at the beginning of PP, was totally consumed within 15 h, at pH ≤ 5.0; however, at pH > 5.0 sorbitol consumption rate decreased. The highest rHuEPO concentration was achieved at pH = 4.5 as 0.16 g L−1, where the overall cell and rHuEPO yield on substrate, were 0.5 g g−1 and 1.5 mg g−1. pH did not have a significant effect on extracellular protease activity. The highest specific AOX activity (110 U g−1 CDW) was attained at pH = 4.5 parallel to the highest rHuEPO production. At pH = 4.5, higher oxygen demand of the cells resulted in higher oxygen KLa, 0.11 s−1, values.

Enhanced recombinant human erythropoietin production by Pichia pastoris in methanol fed-batch/sorbitol batch fermentation through pH optimization

The effects of pH on recombinant human erythropoietin (rHuEPO) production by Pichia pastoris was investigated at pH = 4.0, 4.5, 5.0, 5.5 and 6.0, where sorbitol and methanol were supplied to the bioreactor in batch and fed-batch mode, respectively, in the recombinant protein production phase (PP). For the first three phases, glycerol batch (GB), glycerol fed-batch (GFB) and methanol transition (MT), the growth profiles were almost the same for pH ≥ 5.0; however, at pH < 5.0 the growth rate was lower. The highest cell concentration was obtained as 81.4 g L −1 where GB and GFB were operated at pH = 5.0 and MT and PP were operated at pH = 4.5. Sorbitol added at the beginning of PP, was totally consumed within 15 h, at pH ≤ 5.0; however, at pH > 5.0 sorbitol consumption rate decreased. The highest rHuEPO concentration was achieved at pH = 4.5 as 0.16 g L −1, where the overall cell and rHuEPO yield on substrate, were 0.5 g g −1 and 1.5 mg g −1. pH did not have a significant effect on extracellular protease activity. The highest specific AOX activity (110 U g −1 CDW) was attained at pH = 4.5 parallel to the highest rHuEPO production. At pH = 4.5, higher oxygen demand of the cells resulted in higher oxygen K La, 0.11 s −1, values.

Arbuscular mycorrhizal fungi on growth, nutrient status, and total antioxidant activity of Melilotus albus during phytoremediation of a diesel-contaminated substrate

This research evaluated the effects of arbuscular mycorrhizal fungi (AMF) on growth, nutritional status, total antioxidant activity (AOX), total soluble phenolics content (TPC), and total nitrate reductase activity (NRA) of leaves and roots of Melilotus albus Medik planted in diesel-contaminated sand (7500 mg kg(-1)). Seedlings of Melilotus either Non inoculated (Non-AMF) or pre-inoculated plants (AMF) with the AMF-inoculum Glomus Zac-19 were transplanted to non-contaminated or contaminated sand. After 60 days, diesel significantly reduced plant growth. AMF- plants had no significant greater (64% and 89%, respectively) shoot and leaf dry weight than Non-AMF plants, but AMF plants had lower specific leaf area. AMF-plants had significantly greater content of microelements than non-AMF plants. Regardless diesel contamination, the total AOX and TPC were significantly higher in leaves when compared to roots; in contrast, NRA was higher in roots than leaves. Diesel increased total AOX of leaves, but AMF-plants had significantly lower AOX than non-AMF plants. In contrast, roots of AMF-plants had significantly higher AOX but lower NRA than non-AMF plants. AMF-colonization in roots detected via the fungal alkaline phosphatase activity was significantly reduced by the presence of diesel. AMF-inoculation alleviated diesel toxicity on M. albus by enhancing plant biomass, nutrient content, and AOX activity. In addition, AMF-plants significantly contributed in higher degradation of total petroleum hydrocarbons when compared to non-AMF-plants.