Yeast Supernatant Research Articles

Effects of recombinant anti-lipopolysaccharide factor expressed by Pichia pastoris on the growth performance, immune response and disease resistance of Litopenaeusvannamei

The codon-optimized anti-lipopolysaccharide factor (ALF) sequence was introduced into pPICZαA vector and transformed into Pichia pastoris GS115. The recombinant ALF yeast supernatant (rALF-mix) was freeze-dried and evaluated as a feed additive for Litopenaeus vannamei. It was found by antibacterial activity test in vitro that the rALF-mix had antibacterial activity under different pH and temperature conditions. The 0, 0.00375%, 0.0075%, 0.015%, 0.03% and 0.06% of rALF-mix were added respectively to make the six experimental diets. After a 10-week feeding trial with shrimps (2.36 ± 0.02 g), it was found that the weight gain rate (WGR) and protein efficiency ratio (PER) of shrimp in the groups with 0.0075%, 0.015% and 0.03% of dietary rALF-mix supplementation were significantly higher than those in the control group (P < 0.05). Dietary rALF-mix supplementation significantly increased the total haemocyte count, respiratory burst, phagocytic activity, total anti-oxidative capacity (T-AOC), phenol oxidase activity, nitric oxide synthase activity, lysozyme (LYZ) activity, serum antibacterial capacity in the hemolymph and the T-AOC, LYZ in the hepatopancreas of shrimps (P < 0.05). The malondialdehyde contents in hemolymph and hepatopancreas were significantly decreased (P < 0.05). Meanwhile, the expression levels of toll, immune deficiency, heat shock protein 70, crustin and lipopolysaccharide-β-glucan binding protein in the gill of shrimps were significantly increased (P < 0.05). After the challenge test, it was showed that dietary rALF-mix supplementation significantly improved the resistance of L. vannamei to Vibrio parahaemolyticus (P < 0.05). In conclusion, the rALF-mix can be used as a functional feed additive to improve the growth, immunity and disease resistance of shrimp. Based on the quadratic regression analysis for WGR, the optimal supplemental level of rALF-mix in diet for shrimp was estimated to be 0.02813%.

Comparative Evaluation of Secreted Plant Carotenoid Cleavage Dioxygenase 1 (CCD1) Enzymes in Saccharomyces cerevisiae

Enabling technologies in synthetic biology now present the opportunity to engineer wine yeast for enhanced novel aromas. In doing so, improved wine products will increase the desirability of wine for the consumer and add value to the winemaker. The action of the enzyme carotenoid cleavage dioxygenase 1 (CCD1) on β-carotene to produce β-ionone is of interest to improve the aroma and flavour of the wine. Engineering the yeast, Saccharomyces cerevisiae, to produce higher concentrations of CCD1 in grape-must presents an opportunity to increase the levels of this volatile organic compound, thus enhancing the organoleptic properties of wine. To this end, four phylogenetically diverse plant CCD1 genes were synthesised with a secretion signal peptide and transformed into S. cerevisiae. The relative ability of each enzyme secreted into the yeast supernatant to cleave the deep orange C40 β-carotene was determined by spectrophotometry; furthermore, the by-product of such cleavage, the highly aromatic C13 β-ionone, was assessed by head-space solid-phase micro-extraction, with analysis and detection by GCMS. Reduction in β-carotene levels and release of β-ionone from the supernatant were validated by LCMS detection of CCD1. These experiments demonstrated that expression in yeast of the CCD1s derived from Petunia hybrida and Vitis vinifera and their subsequent secretion into the medium provided superior efficacy in both β-carotene reduction and β-ionone liberation. We anticipate this knowledge being of benefit to future winemakers in producing a vinous product with enhanced organoleptic properties.

Assessing the potential of Chlorella sp. for treatment and resource utilization of brewery wastewater coupled with bioproduct production

Wastewater from various industries is the ideal substrate for the cultivation of microalgae, leading to efficient wastewater treatment and the production of a valuable microalgal biomass. However, further research is needed before wastewater can be used and treated on a large scale. The purpose of the paper was to explore the cultivation of the microalga Chlorella sp. in brewery wastewater (BWW) as well as under applied treatments of cultivating mode, dilution ratios, pretreatment method, and feeding strategies, and investigate the potential value of biomass obtained. Cultivation in BWW was initiated from 30% (v/v) loading while enrichment with 10% waste beer yeast supernatant (WYS) increased specific growth rate (μ) and biomass production, reaching 0.12 d−1 and 1.77 g L−1, respectively. Increasing BWW concentration up to 50% with 10% WYS can obtain the maximum biomass concentration of 2.03 g L−1 that is 0.14 g L−1 higher than 1.89 g L−1 of biomass in BG11. Even when the concentration of BWW reached 100%, biomass production can still reach 1.96 g L−1. In terms of BWW remediation, the maximum removal of ammonia nitrogen (NH4+-N), chemical oxygen demand, total nitrogen, and total phosphorus reached 94.38%, 88.52%, 96.71%, and 98.68%, respectively. Furthermore, the applied conditions determined the composition of Chlorella sp. biomass, which was mostly rich in lipid and carbohydrates. The maximum lipid, carbohydrate, and protein contents were 36.19%, 36.51%, and 19.77%, respectively, depending on culture conditions, and heterotrophic conditions were beneficial to protein accumulation in Chlorella sp. Photosynthetic fluorescence parameter electron transport rate was positively correlated with the biomass concentration of Chlorella sp.

Evaluation of the effect of Saccharomyces cerevisiae on the expression of enterotoxin genes in Escherichia coli O157: H7 (EHEC) and Escherichia coli H10407 (ETEC)

Enterotoxigenic (ETEC) and enterohemorrhagic Escherichia coli (EHEC) are the most important intestinal pathogens. Probiotics play an effective role in reducing the expression of virulence factor genes in intestinal pathogenic bacteria. The aim of the present study is to investigate the effect of probiotic Saccharomyces cerevisiae S3 on the expression of enterotoxin genes in both ETEC and EHEC. Supernatant and lysate of S. cerevisiae S3 are prepared. Subminimal inhibitory concentrations (sub-MIC) of supernatant and lysate are individually exerted to O157: H7 and H10407. The genes' expression of enterotoxins (elt, est, stx1, and stx2) are then determined using real-time PCR technique. The results showed, the yeast supernatant could decrease the expression of the elt gene in ETEC and that of stx1 in EHEC. Of note, in other cases, stx1 and est genes’ expression increased. The lysate had no inhibitory effect on the expressions of elt, est, and stx2 genes, but it increased the expression of genes in both ETEC and EHEC. Lysate extract only decreased the expression of stx1 in O157: H7. Our study shows some interesting results regarding the effectiveness of the compounds produced by S. cerevisiae S3 in the expressions of toxin genes in both ETEC and EHEC. We recommend more similar studies be performed in this regard.

Studies on the Catalytic Properties of Crude Freeze-Dried Preparations of Yarrowia lipolytica Extracellular Lipases for Geranyl Ester Derivative Synthesis.

The study aimed to evaluate the impact of selected factors of the freeze-drying process on the hydrolytic and synthetic activity of the extracellular lipases of Y. lipolytica KKP 379 and to attempt the use of the crude enzyme preparation as a biocatalyst in the synthesis of geranyl 4-hydroxyphenylpropanoate. Antioxidant and antibacterial properties of the geranyl ester derivative were also investigated in order to evaluate their usefulness as a novel food additive. The studies confirmed that freeze-drying was an effective method of dehydrating yeast supernatant and allowed for obtaining lyophilizates with low water activity from 0.055 to 0.160. The type and concentration of the additive (2–6% whey protein hydrolyzate, 0.5% and 1% ammonium sulphate) had a significant effect on the hydrolytic activity of enzyme preparations, while the selected variants of drying temperature during the freeze-drying process were not significant (10 °C and 50 °C). Low yield of geranyl 4-hydroxyphenylopropionate was shown when the lyophilized supernatant was used (5.3%), but the yield of ester synthesis increased when the freeze-dried Y. lipolytica yeast biomass was applied (47.9%). The study confirmed the antioxidant properties of the synthesized ester by the DPPH• and CUPRAC methods, as well as higher antibacterial activity against tested bacteria than its precursor with 0.125 mM MIC (minimal inhibitory concentration) against L. monocytogenes.

VHH-Photosensitizer Conjugates for Targeted Photodynamic Therapy of Met-Overexpressing Tumor Cells.

Photodynamic therapy (PDT) is an approach that kills (cancer) cells by the local production of toxic reactive oxygen species upon the local illumination of a photosensitizer (PS). The specificity of PDT has been further enhanced by the development of a new water-soluble PS and by the specific delivery of PS via conjugation to tumor-targeting antibodies. To improve tissue penetration and shorten photosensitivity, we have recently introduced nanobodies, also known as VHH (variable domains from the heavy chain of llama heavy chain antibodies), for targeted PDT of cancer cells overexpressing the epidermal growth factor receptor (EGFR). Overexpression and activation of another cancer-related receptor, the hepatocyte growth factor receptor (HGFR, c-Met or Met) is also involved in the progression and metastasis of a large variety of malignancies. In this study we evaluate whether anti-Met VHHs conjugated to PS can also serve as a biopharmaceutical for targeted PDT. VHHs targeting the SEMA (semaphorin-like) subdomain of Met were provided with a C-terminal tag that allowed both straightforward purification from yeast supernatant and directional conjugation to the PS IRDye700DX using maleimide chemistry. The generated anti-Met VHH-PS showed nanomolar binding affinity and, upon illumination, specifically killed MKN45 cells with nanomolar potency. This study shows that Met can also serve as a membrane target for targeted PDT.

Interactions among yeast and probiotic bacteria enhance probiotic properties and metabolism offering augmented protection to Artemia franciscana against Vibrio anguillarum

The interactions of the probiotics Bacillus subtilis, Lactococcus lactis and Lactobacillus plantarum with the yeast Saccharomyces cerevisiae were examined in terms of probiotic and biochemical characteristics. Yeast supernatant had a positive effect on the aggregation biofilm formation capacity and hydrophobicity of probiotics, and resulted in increased lactic acid levels, reduced pH values as well as lower RS and FAN levels of probiotics. The effect of probiotics supernatants on yeast was more complex but best results were obtained in the yeast: probiotic CFS ratio of 1:2 for B. subtilis and of 2:1 for the other probiotics. The observed effects depended on the volume ratio of the cell free supernatant to the culture it was applied on. Best results were obtained by the volume ratio probiotic: yeast of (2:1) for B. subtilis and of (1:2) probiotic: yeast for L. plantarum and L. lactis. These ratios were used for further evaluation in vitro against V. anguillarum, resulting in reduced survival and attachment properties of the pathogen. Moreover, the administration of the corresponding combination of bacteria and yeast to Artemia nauplii greatly improved their survival following a challenge with the pathogen. Our results demonstrate that yeast enhances the protective effect of probiotics in a strain specific manner.

Cell-Free Identification of S. cerevisiae Strains by Analysis of Supernatant Using LC-MS

Current literature shows a gap for methods which can identify yeast sub-species (strains or serovars) in samples where there are no viable cells remaining. Presented here is a technique for the analysis of yeast supernatant, including solid phase extraction, data-dependent acquisition liquid chromatography/mass spectrometry (LC-MS), and two chemometric methods to identify and classify yeast strains. Five strains of Saccharomyces cerevisiae were successfully identified in various stages of growth. In addition, peptide/protein identification was performed, without the need for additional data acquisition. Graphical Abstract ᅟ.

Introducing a Cell-Free Approach for the Identification of Brewing Yeast (Saccharomyces cerevisiae) Strains Using MALDI-TOF MS.

Matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry (TOF MS) is now accepted as a quick, easy-to-use, cost-effective, and accurate technique for the identification of microorganisms. However, the successful identification of microorganisms is dependent upon careful attention to factors such as growth conditions, extraction methods, mass spectral data collection, and data analysis procedures. Currently, most microorganism identification has been limited to the species level, and only a limited number of publications have been successful in achieving strain-level identification. In this work, a "cell-free" approach is introduced where peptide analytes secreted by several Saccharomyces cerevisiae strains during their growth period are analyzed. The analysis of the cell supernatant generates mass spectral patterns that are specific to each strain. The patterns generated in combination with a robust data analysis workflow using the open-source programs MALDIquant and Mass-Up allows for strain-level identification of S. cerevisiae. The cell-free approach using the yeast supernatant to accurately identify yeast strains is presented here as a proof of concept. Graphical Abstract.

The impact of epidermal growth factor supernatant on pig performance and ileal microbiota.

Weaning of pigs can lead to low-feed intake resulting in a lag in growth performance, reduced gut health, and diarrheal diseases. Epidermal growth factor (EGF), the most abundant growth factor in milk, increased weaned pig BW gain and feed efficiency in our previous work. It is believed that intestinal microbiota plays an important role in gut health and pig growth, but limited data are available on the impact of feed additives, such as EGF, on the microbial communities of the intestines. The objective of the study was to investigate if the positive influence of EGF supplementation on weight gain and gut health was related to differences in intestinal microbiota. To examine the efficacy of EGF, a 21-d animal trial was performed using 72 pigs (two equal blocks of 36 pigs with three barrows and three gilts/pen). Pigs were assigned to one of two dietary treatments at weaning (20 ± 2 d of age; n = 6 pens/treatment) balancing across treatment for litter, gender, and initial BW. Recombinant yeast supernatant containing EGF at 120 μg/kg BW/d and without EGF (control) was added to the feed for 21 d, followed by a common diet for 7 d. Pig performance was measured weekly and ileal digesta was collected at day 21 from six pigs/treatment for microbiome analysis. Pigs fed diets containing EGF fermentation supernatant had greater (P = 0.01) daily gain in week 3 and overall resulting in heavier (P = 0.029) BW at day 28, which was consistent to our previous finding. No difference in alpha-diversity (Chao1, Shanon, and Simpson indices) and beta-diversity (weighted and unweighted UniFrac distances) of ileal digesta microbiota between EGF supplemented and control pigs were observed. The relative abundances of bacterial taxa did not differ among treatment groups at the phylum level. The relative abundances of Corynebacterium (0.0 vs. 0.9%), Blautia (0.003 vs. 0.26%), and Coprococcus (0.0 vs. 0.05%) genera, and Rumminococcaceae family (0.001 vs. 0.08%) were decreased (P < 0.05) in EGF group compared to control and were negatively correlated (P < 0.05, r > 0.60) with growth performance. Pathways related to detoxification and carbohydrate metabolism were differentially represented in the luminal bacterial populations. The improved growth of pigs supplemented with EGF supernatant produced by Pichia pastoris may be related to changes in functional capacity of the gut microbial populations. However, the impact on mucosa-associated or large intestinal communities is still unknown.

Incubation of Aquilaria subintegra with Microbial Culture Supernatants Enhances Production of Volatile Compounds and Improves Quality of Agarwood Oil

Incubation with microbial culture supernatants improved essential oil yield from Aquilaria subintegra woodchips. The harvested woodchips were incubated with de man, rogosa and sharpe (MRS) agar, yeast mold (YM) agar medium and six different microbial culture supernatants obtained from Lactobacillus bulgaricus, L. acidophilus, Streptococcus thermophilus, Lactococcus lactis, Saccharomyces carlsbergensis and S. cerevisiae prior to hydrodistillation. Incubation with lactic acid bacteria supernatants provided higher yield of agarwood oil (0.45% w/w) than that obtained from yeast (0.25% w/w), agar media (0.23% w/w) and water (0.22% w/w). The composition of agarwood oil from all media and microbial supernatant incubations was investigated by using gas chromatography-mass spectrometry. Overall, three major volatile profiles were obtained, which corresponded to water soaking (control), as well as, both YM and MRS media, lactic acid bacteria, and yeast supernatant incubations. Sesquiterpenes and their oxygenated derivatives were key components of agarwood oil. Fifty-two volatile components were tentatively identified in all samples. Beta-agarofuran, α-eudesmol, karanone, α-agarofuran and agarospirol were major components present in most of the incubated samples, while S. cerevisiae-incubated A. subintegra provided higher amount of phenyl acetaldehyde. Microbial culture supernatant incubation numerically provided the highest yield of agarwood oil compared to water soaking traditional method, possibly resulting from activity of extracellular enzymes produced by the microbes. Incubation of agarwood with lactic acid bacteria supernatant significantly enhanced oil yields without changing volatile profile/composition of agarwood essential oil, thus this is a promising method for future use.

Evaluation of Food-Based Attractants for Drosophila suzukii (Diptera: Drosophilidae).

The Drosophila suzukii (Matsumura) (Diptera: Drosophilidae) is a highly invasive species attacking a wide range of ripening soft-skinned fruits. A series of laboratory choice tests were conducted to determine whether different levels of rice vinegar affected attraction of Merlot wine to D. suzukii, as well as to compare attractiveness of two common fermentation food baits: wine-vinegar and yeast-sugar water mixtures. The relative attraction of various combinations was used to develop a bait whose effectiveness was tested in the field. In laboratory choice experiments, wine-vinegar (80:20, v:v, hereafter referred to as wine) was more preferred over a yeast-sugar water mixture (hereafter referred to as yeast) by D. suzukii. Combination baits, either a mixture of wine and yeast or a mixture of wine and a supernatant from the yeast (comboS), were significantly more attractive than each product alone. The two combination baits were equally attractive to D. suzukii, so were the yeast and its supernatant, suggesting that yeast supernatant could be used as a replacement for the yeast-sugar mixture currently used for trapping D. suzukii. The additive effect between wine and yeast supernatant in the field was not as profound as observed in the laboratory. In the field trial, numerically more male and female D. suzukii were captured in traps baited with comboS than those baited with the wine or yeast alone; however, significant differences were only found between the comboS and wine or between the comboS and yeast in some weeks over the period of the experiment.

Secretion metabolites of probiotic yeast, Pichia kudriavzevii AS-12, induces apoptosis pathways in human colorectal cancer cell lines

There is a common agreement on the important role of the gastrointestinal microbiota in the etiology of cancer. Benign probiotic yeast strains are able to ameliorate intestinal microbiota and regulate the host metabolism, physiology, and immune system through anti-inflammatory, antiproliferative, and anticancer effects. We hypothesized that Pichia kudriavzevii AS-12 secretion metabolites possess anticancer activity on human colorectal cancer cells (HT-29, Caco-2) via inhibiting growth and inducing apoptosis. This study aimed to assess the anticancer effect of P. kudriavzevii AS-12 secretion metabolites and the underlying mechanisms. The cytotoxicity evaluations were performed via 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide assay; 4′,6-diamidino-2-phenylindole staining; and FACS-flow cytometry tests. Also, the effects of P. kudriavzevii AS-12 secretion metabolites on the expression level of 6 important genes (BAD, Bcl-2, Caspase-3, Caspase-8, Caspase-9 and Fas-R) involved in the extrinsic and intrinsic apoptosis pathways were studied by real-time polymerase chain reaction method. P. kudriavzevii AS-12 secretion metabolites showed significant (P < .0001) cytotoxic effects on HT-29 cells (57.5%) and Caco-2 (32.5%) compared to KDR/293 normal cells (25%). Moreover, the cytotoxic effects of examined yeast supernatant on HT-29 cells were comparable with 5-fluorouracil, as a positive control (57.5% versus 62.2% respectively). Flow cytometric results showed that the induction of apoptosis is the main mechanism of the anticancer effects. Also, according to the reverse transcriptase polymerase chain reaction results, the expression level of proapoptotic genes (BAD, Caspase-3, Caspase-8, Caspase-9, and Fas-R) in treated HT-29 and Caco-2 cells was higher than untreated and normal cells, whereas the antiapoptotic gene (Bcl-2) was downregulated. P. kudriavzevii AS-12 secretion metabolites exert its anticancer effects by inhibiting cell proliferation and inducing intrinsic and extrinsic apoptosis in colon cancer cells.

Enhancement of bifidobacteria survival by Williopsis saturnus var. saturnus in milk.

The viability of three strains of probiotic Bifidobacterium lactis that were inoculated into UHT milk was examined with and without the presence of the yeast, Williopsis saturnus var. saturnus NCYC 22, in polypropylene tubes at 30 °C. The B. lactis viable cell count for strains HN019 and BB-12 remained above 6.0 Log cfu/ml, while strain B94 had 5.7 Log cfu/ml after six weeks of incubation in the presence of the co-inoculated yeast. Incubating the bifidus milk without added yeast under anaerobic condition did not improve the survival of B. lactis HN019, indicating that oxygen removal may not be responsible for W. saturnus NCYC 22's viability enhancing property. The addition of yeast supernatant or non-viable yeast also did not show any stabilising effects, suggesting that physical contact and/or interaction between viable W. saturnus and B. lactis plays an important role in sustaining the viability of the probiotic. W. saturnus NCYC 22 could increase the survival of B. lactis in bifidus milk under ambient temperature regardless of the initial concentration of yeast cells inoculated due to yeast growth. This study demonstrated the viability enhancing effect of viable W. saturnus NCYC 22 on B. lactis HN019, which could help towards extending the shelf-life of dairy beverages containing probiotic bifidobacteria.

Saccharomyces cerevisiae EC-1118 enhances the survivability of probiotic Lactobacillus rhamnosus HN001 in an acidic environment.

The present study attempted to partially characterize and elucidate the viability-enhancing effect of a yeast strain Saccharomyces cerevisiae EC-1118 on a probiotic strain Lactobacillus rhamnosus HN001 under acidic conditions using a model system (non-growing cells). The yeast was found to significantly enhance (P < 0.05) the viability of the probiotic strain under acidic conditions (pH 2.5 to 4.0) by 2 to 4 log cycles, and the viability-enhancing effects were observed to be influenced by pH, and probiotic and yeast concentrations. Microscopic observation and co-aggregation assay revealed that the viability-enhancing effect of the yeast could be attributed to direct cell-cell contact co-aggregation mediated by yeast cell surface and/or cell wall components or metabolites. Furthermore, non-viable yeast cells killed by thermal means were observed to enhance the viability of the probiotic strain as well, suggesting that the surface and/or cell wall component(s) of the yeast contributing to co-aggregation was heat-stable. Cell-free yeast supernatant was also found to enhance the viability of the probiotic strain, indicating the presence of protective yeast metabolite(s) in the supernatant. These findings laid the foundation for further understanding of the mechanism(s) involved and for developing novel microbial starter cultures possibly without the use of live yeast for ambient-stable high-moisture probiotic foods.

Impact ofSaccharomyces Cerevisiaeon Viability of ProbioticLactobacillus Rhamnosusin Fermented Milk under Ambient Conditions

The viability of probiotic Lactobacillus rhamnosus HN001 was examined in fermented milk with and without Saccharomyces cerevisiae var. bayanus EC-1118. The yeast enhanced the viability of L. rhamnosus at 30C but not at 12C. High temperature (40C) was detrimental to yeast vitality and diminished its viability-enhancing effect. The yeast inoculated at 103 cfu/g multiplied to 106 cfu/g and enhanced the viability of L. rhamnosus. Viable yeast exerted maximum beneficial effects, although the cell-free yeast supernatant also enhanced the survival of L. rhamnosus. The addition of yeast after fermentation decreased its viability-enhancing effect. Removal of oxygen achieved the viability of L. rhamnosus that was comparable with that achieved in the presence of viable yeast. The results suggest that the improved viability of L. rhamnosus might involve yeast viability, oxygen removal and yeast metabolite(s). The findings would help develop novel microbial starter technology for ambient-stable fermented milks with live probiotics. Practical Applications Maintaining cell viability of probiotics is a scientific challenge, especially under ambient conditions. The findings of this study would lay the foundation for developing novel microbial starter technology for ambient-stable fermented foods with high cell counts of live probiotics, which entails stringent process and engineering control to ensure hygienic conditions and product safety. Benefits of such products would include: reducing distribution and storage costs by saving capital cost involved in chilled-chain distribution, storage and retailing; market penetration and growth where it was not possible due to a lack of or poor refrigeration; extending shelf life and increasing consumer convenience.

TOKSISITAS ASETAMINOFEN PADA KHAMIR Candida tropicalis

Use of the yeast for cell based method to do a screening substances having anti-oxidant property is considered to be simpler and cheaper. In order to develop yeast Candida tropicalis as a tool for evaluation of anti- or pro-oxidant property of substances in cell level, toxicity of an analgesic drug acetaminophen in the yeast had been preliminary studied. Incubation of yeast cell suspension in presence of 0.3% acetaminophen for 2 hours significantly decreased cell viability. Malon dialdehyde, a biochemical marker for cell oxidative damage, increased. Acetaminophen of 0.1% or 0.039% decreased when added in cell yeast suspension or supernatant respectively for 1 hour indicating drug metabolism by cellular and extracellular enzymes. The data indicated that toxicity of the drug in the yeast could be compared to that in mammalian cell where the drug was metabolized by cytochrome P-450 or peroxidase and followed with oxidative stress in cells caused by metabolite byproduct. Toxicity of the drug in the yeast may be in relation with formation of reactive oxygen species. These preliminary data used the yeast for screening antioxidant property of substances against acetaminophen toxicity.

The non‐specific lipid transfer protein, Ara h 9, is an important allergen in peanut

Plant food allergy in the Mediterranean area is mainly caused by non-specific lipid transfer proteins (nsLTP). The aim of this study was to characterize peanut nsLTP in comparison with peach nsLTP, Pru p 3, and assess its importance in peanut allergy. Peanut-allergic patients from Spain (n=32) were included on the basis of a positive case history and either a positive skin prick test or specific IgE to peanut. For comparison, sera of 41 peanut-allergic subjects from outside the Mediterranean area were used. Natural Ara h 9 and two isoforms of recombinant Ara h 9, expressed in Pichia pastoris, were purified using a two-step chromatographic procedure. Allergen characterization was carried out by N-terminal sequencing, circular dichroism (CD) spectroscopy, immunoblotting, IgE inhibition tests and basophil histamine release assays. Compared with natural peanut nsLTP, the recombinant proteins could be purified in high amounts from yeast supernatant (> or =45 mg/L). The identity of the proteins was verified by N-terminal amino acid sequencing and with rabbit nsLTP-specific antibodies. CD spectroscopy revealed similar secondary structures for all preparations and Pru p 3. The Ara h 9 isoforms showed 62-68% amino acid sequence identity with Pru p 3. IgE antibody reactivity to rAra h 9 was present in 29/32 Spanish and 6/41 non-Mediterranean subjects. Recombinant Ara h 9 showed strong cross-reactivity to nPru p 3 and similar IgE-binding capacity as nAra h 9. The two Ara h 9 isoforms displayed similar IgE reactivity. In peanut-allergic patients with concomitant peach allergy, Ara h 9 showed a weaker allergenic potency than Pru p 3 in histamine release assays. Ara h 9 is a major allergen in peanut-allergic patients from the Mediterranean area. Ara h 9 is capable of inducing histamine release from basophils, but to a lesser extent than Pru p 3.

High-level secretory production of recombinant human platelet-derived growth factor-BB by Saccharomyces cerevisiae under the non-selective conditions

Recombinant human platelet-derived growth factor-BB (rhPDGF-BB) was produced by Saccharomyces cerevisiae. A two-stage cultivation strategy with mixture of glucose and galactose was developed to enhance rhPDGF-BB production, with its concentration being 32 mg/l fermentation broth under optimal conditions: corn steep powder as nitrogen source, 2 g/1 of glucose concentration at the beginning of induction phase, a pH of 5.0 and 6.5 for cell growth and rhPDGF-BB accumulation. The purification process consisted of yeast supernatant ultrafiltration followed by ion exchange chromatography and molecular sieve, and the recovery of rhPDGF-BB was estimated to be 20.28%. Biological activity of the purified rhPDGF-BB was 3.05 ng/ml.

Swollenin, a Trichoderma reesei protein with sequence similarity to the plant expansins, exhibits disruption activity on cellulosic materials.

Plant cell wall proteins called expansins are thought to disrupt hydrogen bonding between cell wall polysaccharides without hydrolyzing them. We describe here a novel gene with sequence similarity to plant expansins, isolated from the cellulolytic fungus Trichoderma reesei. The protein named swollenin has an N-terminal fungal type cellulose binding domain connected by a linker region to the expansin-like domain. The protein also contains regions similar to mammalian fibronectin type III repeats, found for the first time in a fungal protein. The swollenin gene is regulated in a largely similar manner as the T. reesei cellulase genes. The biological role of SWOI was studied by disrupting the swo1 gene from T. reesei. The disruption had no apparent effect on the growth rate on glucose or on different cellulosic carbon sources. Non-stringent Southern hybridization of Trichoderma genomic DNA with swo1 showed the presence of other swollenin-like genes, which could substitute for the loss of SWOI in the disruptant. The swollenin gene was expressed in yeast and Aspergillus niger var. awamori. Activity assays on cotton fibers and filter paper were performed with concentrated SWOI-containing yeast supernatant that disrupted the structure of the cotton fibers without detectable formation of reducing sugars. It also weakened filter paper as assayed by an extensometer. The SWOI protein was purified from A. niger var. awamori culture supernatant and used in an activity assay with Valonia cell walls. It disrupted the structure of the cell walls without producing detectable amounts of reducing sugars.