Improving AOX1 promoter efficiency by overexpression of Mit1 transcription factor.

BACKGROUND: Reprogramming in transcriptional regulation provides an effective tool for adjusting cellular metabolic activities. The strong methanol-inducible alcohol oxidase-1 promoter (pAOX1) is commonly used for heterologous gene expression in the yeast Pichia pastoris. Here, we present a novel Pichia pastoris strain engineered to co-express methanol-induced transcription factor 1 (Mit1) and the target protein. Mit1 upregulates pAOX1 in response to methanol. METHODS AND RESULTS: Two model proteins (VEGF and eGFP) have been used as the target proteins under the control of pAOX1. The sequence of Mit1 had obtained from the yeast genome and likewise cloned under the control of pAOX1. The results indicated a 1.9 and 2.2 fold increase in the detected VEGF and eGFP, respectively, when co-expressed with Mit1. Furthermore, the double-recombinant cells, containing Mit-1 and eGFP, produced 1.3 fold more eGFP when the methanol feeding concentration was doubled. The real-time PCR indicated a slight increase in the Mit1 expression, probably due to the negative regulatory feedback loop that exists for the intrinsic yeast Mit1. Overexpression of Mit1 also led to duplication of AOX1 enzyme activity, which may enhance the yeast cells' capacity for methanol detoxification. CONCLUSION: Overexpression of Mit1 could be considered a promising strategy for upregulation of target recombinant proteins in Pichia pastoris. Intracellular overexpression of Mit1 upregulates the heterologous target gene (eGFP) production, which is expressed under the control of pAOX1.

Hydroxyapatite as a biomaterial - a gift that keeps on giving.

The synthetic analogue to biogenic apatite, hydroxyapatite (HA) has a number of physicochemical properties that make it an attractive candidate for diagnosis, treatment of disease and augmentation of biological tissues. Here we describe some of the recent studies on HA, which may provide bases for a number of new medical applications. The content of this review is divided to different medical application modes utilizing HA, including tissue engineering, medical implants, controlled drug delivery, gene therapies, cancer therapies and bioimaging. A number of advantages of HA over other biomaterials emerge from this discourse, including (i) biocompatibility, (ii) bioactivity, (iii) relatively simple synthesis protocols for the fabrication of nanoparticles with specific sizes and shapes, (iv) smart response to environmental stimuli, (v) facile functionalization and surface modification through noncovalent interactions, and (vi) the capacity for being simultaneously loaded with a wide range of therapeutic agents and switched to bioimaging modalities for uses in theranostics. A special section is dedicated to analysis of the safety of particulate HA as a component of parenterally administrable medications. It is concluded that despite the fact that many benefits come with the usage of HA, its deficiencies and potential side effects must be addressed before the translation to the clinical domain is pursued. Although HA has been known in the biomaterials world as the exemplar of safety, this safety proves to be the function of size, morphology, surface ligands and other structural and compositional parameters defining the particles. For this reason, each HA, especially when it comes in a novel structural form, must be treated anew from the safety research angle before being allowed to enter the clinical stage.

Recombinant Acetylcholinesterase purification and its interaction with silver nanoparticle.

Although the use of silver nanoparticles (AgNPs) has substantial benefits, their entrance into the environment, food chain, and human body and their toxicity have come under serious scrutiny. Multiple noncovalent attractive forces between AgNPs and bio-macromolecules are responsible for immediate corona formation upon exposure to biological tissue. Here, the influence of AgNPs with neuro-enzyme Acetylcholinesterase (AChE) was investigated. AgNPs to enzyme ratio had an effect on the enzyme and features of the treated samples. It was also observed that time increments had a positive effect on the size of AgNPs and caused an increase in their initial size. In other words, smaller AgNPs resulted in size increments after interaction with enzymes, while the larger ones showed size decrements. The nano-crystalline AgNPs were identified in x-ray powder diffraction analyses before and after treatment with AChE. The (220) crystalline plane is related to the internal crystallinity of cubic Ag. The results show that the interaction between AChE and AgNPs could lead not only to a decrease in AChE activity, but also to a reduction in the crystallinity and stability of AgNPs. The circular dichroism demonstrates that the secondary structure of AChE also declined after 30 min of incubation with AgNPs at 37 °C.

Cloning and high-level expression of ß-xylosidase from Selenomonas ruminantium in Pichia pastoris by optimizing of pH, methanol concentration and temperature conditions.

ß-xylosidase and several other glycoside hydrolase family members, including xylanase, cooperate together to degrade hemicelluloses, a commonly found xylan polymer of plant-cell wall. ß-d-xylosidase/α-l-arabinofuranosidase from the ruminal anaerobic bacterium Selenomonas ruminantium (SXA) has potential utility in industrial processes such as production of fuel ethanol and other bioproducts. The optimized synthetic SXA gene was overexpressed in methylotrophic Pichia pastoris under the control of alcohol oxidase I (AOX1) promoter and secreted into the medium. Recombinant protein showed an optimum pH 4.8 and optimum temperature 50 °C. Furthermore, optimization of growth and induction conditions in shake flask was carried out. Using the optimum expression condition (pH 6, temperature 20 °C and 1% methanol induction), protein production was increased by about three times in comparison to the control. The recombinant SXA we have expressed here showed higher turnover frequency using ρ-nitrophenyl ß-xylopyranoside (PNPX) substrate, in contrast to most xylosidase experiments reported previously. This is the first report on the cloning and expression of a ß-xylosidase gene from glycoside hydrolase (GH) family 43 in Pichia pastoris. Our results confirm that P. pastoris is an appropriate host for high level expression and production of SXA for industrial applications.

Substrate affinity and catalytic efficiency are improved by decreasing glycosylation sites in Trichoderma reesei cellobiohydrolase I expressed in Pichia pastoris.

OBJECTIVES: To modify two main N-glycosylation residues of cellobiohydrolase I from Trichoderma reesei by site-directed mutagenesis for decreasing the extent of glycosylation and exploring possible effects on its properties. RESULTS: Asparagine 45 and 64 residues were mutated to alanine to make single/double mutants and expressed in P. pastoris. Decreasing N-glycosylation of the recombinant CBH I resulted in an increased affinity of the enzyme for carboxymethylcellulose and also improved the Kcat/Km while the specific activity was decreased. Also, the enzymes were stable up to 80 °C. There was no significant change of the optimum pH and temperature by decrease of glycosylation in the mutated enzymes in comparison to the wild-type at constant incubation time of assay. CONCLUSION: Post-translational glycan-modification of CBH I in P. pastoris has different impacts on the properties of the secreted enzymes. Substrate affinity and catalytic efficiency were improved significantly while the activity and high temperature stability were negatively affected.

Simulation and experimental study of a cold atmospheric pressure plasma and comparison of efficiency in boosting recombinant Endoglucanase II production in Pichia pastoris.

Recombinant proteins are essential in various industries, and scientists employ genetic engineering and synthetic biology to enhance the host cell's protein production capacity. Stress response pathways have been found effective in augmenting protein secretion. Cold atmospheric pressure plasma (CAP) can induce oxidative stress and enhance protein production. Previous studies have confirmed the applicability of CAP jets on Phytase and green fluorescent protein (GFP) production in Pichia pastoris hosts. This study investigates the effect of CAP treatment on another valuable recombinant protein, Endoglucanase II (EgII), integrated into the Pichia pastoris genome. The results demonstrated that plasma induction via two different ignition modes: sinusoidal alternating current (AC) and pulsed direct current (DC) for 120, 180, and 240 s has boosted protein secretion without affecting cell growth and viability. The AC-driven jet exhibited a higher percentage increase in secretion, up to 45%. Simulation of plasma function using COMSOL software provided a pattern of electron temperature (Te) and density distribution, which determine the plasma cocktail's chemistry and reactive species production. Furthermore, electron density (ne) and temperature were estimated from the recorded optical spectrum. The difference in electron properties may explain the moderately different impressions on expression capability. However, cell engineering to improve secretion often remains a trial-and-error approach, and improvements are, at least partially, specific to the protein produced.

Cold atmospheric plasma treatment enhances recombinant model protein production in yeast Pichia pastoris.

Cold atmospheric pressure plasma (CAP) has been described as a novel technology with expanding applications in biomedicine and biotechnology. In the present study, we provide a mildly stressful condition using non-lethal doses of CAP (120, 180, and 240 s) and evaluate its potential benefits on the recombinant production of a model protein (enhanced green fluorescent protein (eGFP)) in yeast Pichia pastoris. The measured eGFP fluorescence augmented proportional to CAP exposure time. After 240 s treatment with CAP, the measured fluorescent intensity of culture supernatant (after 72 h) and results of real-time PCR (after 24 h) indicated an 84% and 76% increase in activity and related RNA concentration, respectively. Real-time analysis of a list of genes involved in oxidative stress response revealed a significant and durable improvement in their expression at five h and 24 h following CAP exposure. The improvement of the recombinant model protein production may be partly explained by the impact of the RONS on cellular constituents and altering the expression of specific stress genes. In conclusion, using CAP strategy may be considered a valuable strategy to improve recombinant protein production, and deciphering the molecular background mechanism could be inspiring in the reverse metabolic engineering of host cells.

Development of a carbon quantum dot-based sensor for the detection of acetylcholinesterase and the organophosphate pesticide.

In this study, a proper and reliable fluorometric method is introduced for screening acetylcholinesterase (AChE) and its inhibitors, using carbon quantum dots (CQDs) as the signal reporter. Pure, S-doped, and P-doped CQDs, were synthesized and their recoverable fluorescence quenching properties were observed, when exposed to Hg2+, Cu2+, and Fe3+ quenching ions, respectively. The study on the recovery of their emission showed that after the introduction of another guest substance with a stronger affinity to the quenching ions, their fluorescence is restored. The Design Expert software was employed to compare the performance of the three CQDs, as fluorescent probes, based on their quenching efficiency and the percentage of their emission recovery in the presence of AChE and acetylthiocholine (ATCh). Based on the statistical analysis, among the studied CQDs, S-doped CQD was the most suitable candidate for sensor designing. The detection mechanism for the proposed S-doped CQD-based sensor is as follows: The strong binding of Cu2+ ions to carboxyl groups of S-doped CQD quenches the fluorescence signal. Then, hydrolysis of ATCh into thiocholine (TCh) in the presence of AChE causes fluorescence recovery, due to the stronger affinity of Cu2+ to the TCh, rather than the CQD. Finally, in the presence of malathion and chlorpyrifos inhibitors, AChE loses its ability to hydrolyze ATCh to TCh, so the fluorescence emission remains quenched. Based on the proposed detection technique, the designed sensor showed detection limits of 1.70 ppb and 1.50 ppb for malathion and chlorpyrifos, respectively.

An overview of the vaccine platforms to combat COVID-19 with a focus on the subunit vaccines.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging virus that has caused the recent coronavirus disease (COVID-19) global pandemic. The current approved COVID-19 vaccines have shown considerable efficiency against hospitalization and death. However, the continuation of the pandemic for more than two years and the likelihood of new strain emergence despite the global rollout of vaccination highlight the immediate need for the development and improvement of vaccines. mRNA, viral vector, and inactivated virus vaccine platforms were the first members of the worldwide approved vaccine list. Subunit vaccines. which are vaccines based on synthetic peptides or recombinant proteins, have been used in lower numbers and limited countries. The unavoidable advantages of this platform, including safety and precise immune targeting, make it a promising vaccine with wider global use in the near future. This review article summarizes the current knowledge on different vaccine platforms, focusing on the subunit vaccines and their clinical trial advancements against COVID-19.

Expression of functional eGFP-fused antigen-binding fragment of ranibizumab in Pichia pastoris.

Introduction: Ranibizumab is a mouse monoclonal antibody fragment antigen-binding (Fab) against human vascular endothelial growth factor-A (VEGF-A), inhibiting angiogenesis. This antibody is commercially produced in Escherichia coli host and used to treat wet age-related macular degeneration (AMD). Methods: In this study, the heavy and light chains of ranibizumab were expressed in Pichia pastoris. The expressed chains were incubated overnight at 4°C for interaction. The formation of an active structure was evaluated based on the interaction with substrate VEGF-A using an indirect ELISA, and an electrochemical setup. Furthermore, reconstruction of split enhanced green fluorescent protein (eGFP) reporter, chimerized at the C-terminus of the heavy and light chains, was used to characterize chains' interaction. Results: P. pastoris efficiently expressed designed constructs and secreted them into the culture medium. The anti-Fab antibody detected the constructed Fab structure in western blot analysis. Reconstruction of the split reporter confirmed the interaction between heavy and light chains. The designed ELISA and electrochemical setup results verified the binding activity of the recombinant Fab structure against VEGF-A. Conclusion: In this work, we indicated that the heavy and light chains of ranibizumab Fab fragments (with or without linkage to split parts of eGFP protein) were produced in P. pastoris. The fluorescence of reconstructed eGFP was detected after incubating the equal ratio of chimeric-heavy and light chains. Immunoassay and electrochemical tests verified the bioactivity of constructed Fab. The data suggested that P. pastoris could be considered a potential efficient eukaryotic host for ranibizumab production.

Extraction of sugarcane bagasse arabinoxylan, integrated with enzymatic production of xylo-oligosaccharides and separation of cellulose.

Sugarcane processing roughly generates 54 million tonnes sugarcane bagasse (SCB)/year, making SCB an important material for upgrading to value-added molecules. In this study, an integrated scheme was developed for separating xylan, lignin and cellulose, followed by production of xylo-oligosaccharides (XOS) from SCB. Xylan extraction conditions were screened in: (1) single extractions in NaOH (0.25, 0.5, or 1 M), 121 °C (1 bar), 30 and 60 min; (2) 3 × repeated extraction cycles in NaOH (1 or 2 M), 121 °C (1 bar), 30 and 60 min or (3) pressurized liquid extractions (PLE), 100 bar, at low alkalinity (0-0.1 M NaOH) in the time and temperature range 10-30 min and 50-150 °C. Higher concentration of alkali (2 M NaOH) increased the xylan yield and resulted in higher apparent molecular weight of the xylan polymer (212 kDa using 1 and 2 M NaOH, vs 47 kDa using 0.5 M NaOH), but decreased the substituent sugar content. Repeated extraction at 2 M NaOH, 121 °C, 60 min solubilized both xylan (85.6% of the SCB xylan), and lignin (84.1% of the lignin), and left cellulose of high purity (95.8%) in the residuals. Solubilized xylan was separated from lignin by precipitation, and a polymer with ß-1,4-linked xylose backbone substituted by arabinose and glucuronic acids was confirmed by FT-IR and monosaccharide analysis. XOS yield in subsequent hydrolysis by endo-xylanases (from glycoside hydrolase family 10 or 11) was dependent on extraction conditions, and was highest using xylan extracted by 0.5 M NaOH, (42.3%, using Xyn10A from Bacillus halodurans), with xylobiose and xylotriose as main products. The present study shows successful separation of SCB xylan, lignin, and cellulose. High concentration of alkali, resulted in xylan with lower degree of substitution (especially reduced arabinosylation), while high pressure (using PLE), released more lignin than xylan. Enzymatic hydrolysis was more efficient using xylan extracted at lower alkaline strength and less efficient using xylan obtained by PLE and 2 M NaOH, which may be a consequence of polymer aggregation, via remaining lignin interactions.

A novel thermostable alkaline histamine oxidase from Glutamicibacter sp. N1A3101, induced by histamine and its analogue betahistine.

Biogenic amines (BAs) are low molecular weight organic bases formed by natural amino acids decarboxylation and trigger an array of toxicological effects in humans and animals. Bacterial amine oxidases enzymes are determined as practical tools to implement the rapid quantification of BAs in foods. Our study set out to obtain a new efficient, amine oxidase enzyme for developing new enzyme-based quantification of histamine. The soils from different sources were screened using histamine as sole carbon and nitrogen sources, and histamine oxidase producing bacteria were selected and identified using specific primers for histamine oxidase (HOD) gene. The HOD gene of six strains, out of 26 isolated histamine-utilizing bacteria, were amplified using our designed primers. The HOD enzyme from Glutamicibacter sp. N1A3101, isolated from nettle soil, was found to be thermostable and showed the highest substrate specificity toward the histamine and with no detected activity in the presence of putrescine, cadaverine, spermine, and spermidine. Its oxidation activity toward tyramine was lower than other HOD reported so far. The isolated enzyme was stable at 60 °C for 30 min and showed pH stability ranging from 6 to 9. Furthermore, we indicated the induction of identified HOD activity in the presence of betahistine as well, with nearly equal efficiency and without the consumption of the substrate.

Colloidal graphene oxide enhances the activity of a lipase and protects it from oxidative damage: Insights from physicochemical and molecular dynamics investigations.

Physical adsorption of lipase from Thermomyces lanuginosus onto single-layer sheets of graphene oxide (GO) was studied using the response surface methodology to evaluate the physicochemical factors - temperature, pH, ionic strength, and concentration - affecting the enzymatic activity and the immobilization efficiency. The immobilization efficiency and the activity of the enzyme were inversely proportional to each other. Specifically, higher pH values increased the immobilization efficacy, but produced changes in the aggregation state and secondary structure of the enzyme, thus decreasing its activity. Lower pH values, in turn, reduced the immobilization efficacy, but increased the activity of the adsorbed lipase. The adsorbed and the free lipase were followed during 600 ns and 3.5 µs, respectively, in molecular dynamics (MD) simulations. MD trajectories showed that irreversible adsorption freezes the enzyme in a state with a correctly opened catalytic cavity, while the active site remains without a direct interaction with the GO adsorbent. In contrast to the interfacial activation of lipases in a hydrophobic environment, where the catalytic pocket attaches to the hydrophobic surface, the adsorption onto GO made the active site of the lipase accessible by altering the tertiary structure of the enzyme, leading to a higher catalytic efficiency. Experimental investigations confirmed that the physical adsorption onto GO induces tertiary structure changes in the lipase and protects it from H2O2 by accepting the oxidative damage upon itself. In summary, the physical adsorption of the lipase onto GO is mainly affected by pH and could possibly provide a spreadable and robust catalytic interface for biotechnological applications.

Covalent immobilization of Drosophila acetylcholinesterase for biosensor applications.

The synthesized cDNA coding for AChE (acetylcholinesterase) was subcloned in pENTR/D-TOPO plasmid and expressed using baculovirus expression vector and Sf9 insect cells as host. Purified enzyme (specific activity 36374 micromol x min(-1) x mg(-1)) was immobilized on pre-activated perlite (a porous silica matrix) by silanization and glutaraldehyde treatment. The total enzyme immobilized was then measured, and total and specific activity of immobilized AChE was compared with that of soluble enzyme. Using this perlite support not only resulted in a great amount of maintained immobilized enzyme activity (more than 70%, specific activity 26238 micromolx min(-1) x mg(-1)), but also significantly improved stability against temperature (8.7- and 17.7-fold at 50 and 60 degrees C respectively), urea (2.7-fold) and acetonitrile (1.7-fold). Kinetic studies showed that the K(m) value for immobilized enzyme is very similar to the soluble one (0.088 and 0.081 mM respectively). In addition, immobilized enzymes retained 80% of their initial activity after 16 consecutive reactor batch cycles. A comparison of the inhibitory effect of paraoxon on soluble and immobilized AChE showed that immobilization increased the linearity of the inhibition plot particularly in the range 0.1 nM-0.1 microM.

Enhancing chimeric hydrophobin II-vascular endothelial growth factor A165 expression in Pichia pastoris and its efficient purification using hydrophobin counterpart.

We report cloning and expressing of recombinant human VEGF-A165, fused at the N-terminal with Hydrophobin II (HFBII) from Trichoderma reseei, in yeast Pichia pastoris. We validated the construct using SDS-PAGE and ELISA against VEGF-A165 and efficiently performed protein purification and enrichment based on HFBII counterpart and using an aqueous two-phase system (ATPS) with nonionic surfactant X-114. We studied the effects of various culture medium additives and interaction effects of positive factors to increase the recombinant HFBII-VEGF-A165 production. Supplementing the Pichia pastoris cell culture medium with Mg2+, Polysorbate 20 (PS 20), and 4-phenylbutyrate (PBA) improved the expression of the chimeric protein. Orthogonal experiments showed that the optimal condition to achieve maximal HFBII-VEGF-A165 production was with the addition of PBA, PS 20, and MgSO4. Under this condition, the production of the target protein was 4.5 times more than that in the medium without the additives. Overall, our approach to produce chimeric HFBII-VEGF-A165 and selectively capture it in ATPS is promising for large-scale protein production without laborious downstream processing.

Enzymatic hydrolysis of microalgae proteins using serine proteases: A study to characterize kinetic parameters.

Protein composition and molecular weight play an important role in the digestibility of microalgae proteins. In this study for the first time, proteinous materials of Dunaliella salina and Spirulina platensis were extracted and purified by fast protein liquid chromatography. Then, they are affected by trypsin and chymotrypsin as indicator intestinal enzymes. The results showed that the extracted protein from S. plantesis (ProS) was more rapidly hydrolysed than proteins from D. salina (ProD) because of their lower molecular weight and likely their greater flexibility and open structure. Also, the extent of hydrolysis by trypsin and chymotrypsin of ProS were higher and faster than ProD due to the more number of hydrolytic sites in ProS for both enzymes. The catalytic efficiency and kcat displayed that ProS were more suitable substrate than ProD for intestinal enzymes. The results exhibited that chymotrypsin can act better and faster than trypsin on peptide bonds of proteins.

The effect of non-thermal atmospheric plasma on the production and activity of recombinant phytase enzyme.

Atmospheric pressure cold plasma (ACP) is introduced as a useful tool in a variety of biological applications. Proteins are the most abundant macromolecules in living systems with a central role in all biological processes. These organic molecules are modified by ACP exposure that is responsible for many of ACP's biological effects. This study evaluated the effect of ACP on the production of recombinant phytase in yeast Pichia pastoris (P. pastoris) as well as the structure and function of the phytase enzyme. The results indicated that yeast cells treated with ACP, directly or indirectly, produced higher amounts of recombinant phytase, which was associated with the time of ACP treatment. The exposure of commercial phytase solution with ACP caused a significant increase in the enzyme activity (125%) after 4 hours. Evaluation of the phytase solution by far- and near-UV circular dichroism (CD) and fluorescence analysis indicated that this protein maintained its secondary structure when exposed to ACP while the tertiary structure was slightly unfolded. The effects of heat and H2O2 on the phytase structure and function were compared with the effect of ACP treatment. The modification of Cys, Tyr and Trp amino acids upon reactive oxygen/nitrogen spices was simulated using a molecular dynamics approach. RMSF and RMSD analysis suggested that this structural alteration occurs owing to changes made by reactive species in accessible amino acids.

Disulfide bonds elimination of endoglucanase II from Trichoderma reesei by site-directed mutagenesis to improve enzyme activity and thermal stability: An experimental and theoretical approach.

EndoglucanaseII (Cel5A) of Trichoderma reesei is widely used industrially with the high catalytic efficiency, but it is not stable high temperatures. Structural comparison with the closest thermophilic endoglucanase homolog, Cel5A from Thermoascus aurantiacus, demonstrates disulfide bond differences. Replacement of Cysteine99 with Valine and Cysteine323 with Histidine by site directed mutagenesis caused elimination of two disulfide bonds. Recombinant expression in Pichia pastoris showed the catalytic efficiency (kcat/Km) increment toward CMC for single mutant enzymes, C99V and C323H, about 1.87 and 1.3 folded respectively. This indicates that the elimination of disulfide bond in substrate binding cleft around the catalytic domain of mutant EndoglucanaseII may be increased the flexibility of protein, to form a suitable E-S complex. In direct contrast with previous studies suggesting the existence of disulfide bonds increase the protein stability, the results showed mutant endoglucanase enzymes with disulfide bond reduction have higher thermal stability. The thermal stability of C99V and C323H in 80 °C were increased 2.4 and 2.34 folded, respectively. In this project, theoretical data had a good agreement with the experimental results. Because of high enzyme activity and thermal stability, both of C99V and C323H mutant have high potential suitable for different industrial applications.

Investigation of the effects of starch on the physical and biological properties of polyacrylamide (PAAm)/starch nanofibers.

Here, we report the development of a new polyacrylamide (PAAm)/starch nanofibers' blend system and highlight its potential as substrate for efficient enzyme immobilization. PAAm was synthesized and blended with starch. The final blend was then electrospun into nanofibers. The response surface methodology was used to analyze the parameters that control nanofiber's diameter. Electrospun mat was then modified either by cross-linking or phytase immobilization using silane coupling agent and glutaraldehyde chemistry. Physico-chemical properties of blends were investigated using spectroscopic and thermal studies. The evaluation of immobilized enzyme kinetics on both pure and the starch blended PAAm nanofibers was performed using Michaelis-Menten kinetic curves. Fourier transform infrared spectroscopy results along with differential scanning and X-ray diffraction confirmed that blending was successfully accomplished. TGA analysis also demonstrated that the presence of starch enhances the thermal degradability of PAAm nanofibers. Finally, it was shown that addition of starch to PAAm increases the efficacies of enzyme loading and, therefore, significantly enhances the activity as well as kinetics of the immobilized enzyme on electrospun blend mats.

Enhancement of Alpha 1-antitrypsin Production in Pichia pastoris by Designing and Optimizing Medium Using Elemental Analysis.

Background: Human alpha 1-antitrypsin (AAT) is a monomeric glycosylated protein; it is the potent inhibitor of a whole range of serine proteases and protects tissues against their destructive effects. The human plasma-derived AAT, which is currently used to augment the AAT level in patients, is limited due to high cost and source limitation. Recombinant production of AAT can be considered as a potential alternative. Objectives: This study aims to develop and optimize a new chemically defi ned medium based on an elemental analysis of the yeast Pichia pastoris for an effi cient culture of the recombinant yeast-producing secretory AAT. Materials and Methods: An elemental analysis of Carbon (C), Hydrogen (H), Nitrogen (N), Sulfur (S); CHNS in its abbreviated form, and metallic elements was performed to determine the exact molecular constituent of the P. pastoris. The medium components were selected according to the obtained formula; they were optimized by the response surface methodology (RSM). The grown yeast cell was measured at the end of 18 h glycerol batch culture. The amounts of AAT production and elastase inhibitory capacity (EIC) were measured at the end of three days' methanol feeding. Results: The optimized medium compositions consist of glycerol (40 g.L-1), KH2PO4 (24.78 g.L-1), NaCl, (0.88 g.L-1), MgSO4 .7H2 O (1.95 g.L-1), (NH4 )2 SO4 (22.76 g.L-1), and trace elements (20 mL.L-1). The presented quadratic models show that KH2 PO4 and (NH4)2 SO4, are the most abundant ones in the P. pastoris biomass and have the greatest effect on the cell growth, EIC, and AAT protein production responses. Conclusions: According to the results of this study, it can be concluded that the characterizing cell composition formula could be considered as an appropriate method to design culture media in order to improve cell growth and productivity. Compared to the common P. pastoris chemically defi ned media, FM22 and BSM, production of AAT protein increased by 1.5 and 1.4 times, respectively, in this new medium.