Harnessing the Phytase Production Potential of Soil-Borne Fungi from Wastewater Irrigated Fields Based on Eco-Cultural Optimization under Shake Flask Method

Indigenous fungi present in agricultural soils could have synchronized their inherent potentials to the local climatic conditions. Therefore, the fungi resident in the untreated wastewater irrigated agricultural field might develop their potential for producing various enzymes to handle the induced full organic load from domestic wastewater and toxic chemicals from the textile industry. Around 53 various fungal isolates were grown and separated from the soil samples from these sites through soil dilution, soil-culture plate, and soil-culture plate methods. All the purified fungi were subjected to a phosphatase production test, and only 13 fungal strains were selected as phosphatase producers. Among them, only five fungi identified as Aspergillus niger, Aspergillus flavus, Aspergillus fumigatus, Penicillium purourogenum, and Mucor rouxii based on morphological similarities, showing higher phosphate solubilizing indices, were utilized for eco-cultural fine-tuning to harness their full production potential under shake flask (SF) method. Among various media, orchestral tuning, 200 µM sodium phytate as substrate with 1.5 mL of inoculum size of the fungi, pH 7, temperature 30 °C, glucose, and ammonium nitrate as carbon and nitrogen additive with seven days of incubation were found to be the most appropriate cultural conditions to harness the phytase production potential of the selected fungi. Aspergillus niger and Aspergillus flavus showed initial phytase activity (5.2 Units/mL, 4.8 Units/mL) and phytase specific activity (2.85, 2.65 Units/mL per mg protein) during screening to be enhanced up to 17 ± 0.033 (Units/mL), 16 ± 0.033 (Units/mL) and (13 ± 0.012), 10 ± 0.066 (Units/mL per mg protein), respectively, with the above-mentioned conditions. The phytase enzyme produced from these fungi were found to be almost stable for a wide range of pH (4–8); temperature (20–60 °C); insensitive to Ca2+ and Mg2+ ions, and EDTA, Ni2+, and Ba2+ inhibitors but highly sensitive to Mn2+, Cu2+, and Zn2+ ions, and Co2+, Cr3+, Al3+, Fe2+ and Ag1+ inhibitors. It was suggested that both phytase-producing strains of A. niger and A. flavus or their crude phytase enzymes might be good candidates for application in soils to release phosphates from phytate and a possible valuable substitute of phosphate fertilizers.

METHOD VALIDATION OF SIMVASTATIN IN PCL-PEG-PCL TRIBLOCK COPOLYMER MICELLES USING UV-VIS SPECTROPHOTOMETRIC FOR SOLUBILITY ENHANCEMENT ASSAY

Objective: This study aims to increase the solubility of simvastatin (SIM), a hydrophobic drug, by incorporating it into PCL-PEG-PCL triblock copolymer micelles and validating the assay method used, namely Uv-Vis spectrophotometric. Methods: The shake flask method was used to determine the increase in solubility experienced by SIM after being incorporated into the micellar system. The values ​​of maximum wavelength (λmax), linearity, LOD, LOQ, accuracy, and precision were used as parameters measured to assess the validity of the assay method used. Results: The results showed that PCL-PEG-PCL triblock copolymer micelles could increase SIM solubility by 9.7 times (89.49±5.75 µg/ml) compared to SIM without modification (9.19±0.24 µg/ml). The validation results show the λmax value of 239 nm, a linear calibration curve with an R-value of 0.9994, LOD and LOQ of 0.33 µg/ml and 1.00 µg/ml, accurate measurement with recovery at concentrations of 80%, 100%, and 120% were 102.93±1.32%, 100.78±0.40%, and 104.58±0.79% and also had good precision ​​with RSD<2%. Conclusion: The PCL-PEG-PCL triblock copolymer micelles can increase SIM solubility and the Uv-Vis spectrophotometric method has been validated successfully for the quantitative analysis of SIM in PCL-PEG-PCL triblock copolymer micelles.

Media optimization for SHuffle T7 Escherichia coli expressing SUMO-Lispro proinsulin by response surface methodology

Background SHuffle is a suitable Escherichia coli (E. coli) strain for high yield cytoplasmic soluble expression of disulfide-bonded proteins such as Insulin due to its oxidative cytoplasmic condition and the ability to correct the arrangement of disulfide bonds. Lispro is an Insulin analog that is conventionally produced in E. coli as inclusion bodies (IBs) with prolonged production time and low recovery. Here in this study, we aimed to optimize cultivation media composition for high cell density fermentation of SHuffle T7 E. coli expressing soluble Lispro proinsulin fused to SUMO tag (SU-INS construct) to obtain high cell density fermentation. Results Factors including carbon and nitrogen sources, salts, metal ions, and pH were screened via Plackett–Burman design for their effectiveness on cell dry weight (CDW) as a measure of cell growth. The most significant variables of the screening experiment were Yeast extract and MgCl2 concentration, as well as pH. Succeedingly, The Central Composite Design was utilized to further evaluate and optimize the level of significant variables. The Optimized media (OM-I) enhanced biomass by 2.3 fold in the shake flask (2.5 g/L CDW) that reached 6.45 g/L (2.6 fold increase) when applied in batch culture fermentation. The efficacy of OM-I media for soluble expression was confirmed in both shake flask and fermentor. Conclusion The proposed media was suitable for high cell density fermentation of E. coli SHuffle T7 and was applicable for high yield soluble expression of Lispro proinsulin.

Solubility of Nifedipine by Shake Flask UV-Spectrometry; Review of Safety Concerns in Pregnancy

Nifedipine is chemically dimethyl 2,6-dimethyl-4-(2-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate, a dihydropyridine derivative used frequently as anti-hypertensive. It is a L- type calcium channel blocker (CCB). Few analogical discrepancies were found between Nifedipine’s clinical output report and chemical analysis of solubility. The ambition of this research is to conduct a re-check and proper quantification of partition co-efficient (logP) of Nifedipine and clarify the discrepancy and rectify if any mistake has been done in recent past. The method used is the “gold standard” shake-flask method followed by analysis through UV-scpectrophotmetry.

Synthesis and Characterization of a New Norfloxacin/Resorcinol Cocrystal with Enhanced Solubility and Dissolution Profile

A new cocrystal of Norfloxacin, a poorly soluble fluoroquinolone antibiotic, has been synthetized by a solvent-mediated transformation experiment in toluene, using resorcinol as a coformer. The new cocrystal exists in both anhydrous and monohydrate forms with the same (1:1) Norfloxacin/resorcinol stoichiometry. The solubility of Norfloxacin and the hydrated cocrystal were determined by the shake-flask method. While Norfloxacin has a solubility of 0.32 ± 0.02 mg/mL, the cocrystal has a solubility of 2.64 ± 0.39 mg/mL, approximately 10-fold higher. The dissolution rate was tested at four biorelevant pH levels of the gastrointestinal tract: 2.0, 4.0, 5.5, and 7.4. In a first set of comparative tests, the dissolution rate of Norfloxacin and the cocrystal was determined separately at each pH value. Both solid forms showed the highest dissolution rate at pH 2.0, where Norfloxacin is totally protonated. Then, the dissolution rate decreases as pH increases. In a second set of experiments, the dissolution of the cocrystal was evaluated by a unique dissolution test, in which the pH dynamically changed from 2.0 to 7.4, stepping 30 min at each of the four biorelevant pH values. Results were quite different in this case, since dissolution at pH 2 affects the behavior of Norfloxacin at the rest of the pH values.

Establishment of the Carrot-Made LTB-Syn Antigen Cell Line in Shake Flask and Airlift Bioreactor Cultures

Carrot (Daucus carota) cells have been used to effectively manufacture recombinant biopharmaceuticals such as cytokines, vaccines, and antibodies. We generated the carrot cell line Z4, genetically modified to produce the LTB-Syn antigen, which is a fusion protein proposed for immunotherapy against synucleinopathies. In this work, the Z4 cell suspension line was cultivated to produce the LTB-Syn protein in a 250 mL shake flask and 2 L airlift bioreactor cultures grown for 45 and 30 days, respectively. Maximum biomass was obtained on day 15 in both the airlift bioreactor (35.00 ± 0.04 g/L DW) and shake flasks (17.00 ± 0.04 g/L DW). In the bioreactor, the highest LTB-Syn protein yield (1.52 ± 0.03 µg/g FW) was obtained on day 15; while the same occurred on day 18 for shake flasks (0.92 ± 0.02 µg/g FW). LTB-Syn protein levels were analyzed by GM1-ELISA and western blot. PCR analysis confirmed the presence of the transgene in the Z4 line. The obtained data demonstrate that the carrot Z4 cell suspension line grown in airlift bioreactors shows promise for a scale-up cultivation producing an oral LTB-Syn antigen.

Improving the Plasmid Stability by a Hok/Sok System for L-Homoserine Production in Escherichia Coli

Background: The production of bioactive compounds using microbial hosts is considered a safe, cost competitive and scalable approach. However, the efficient engineering of cell factories with well stability, such as for the production of L-aspartate family amino acids and derivatives, remains an outstanding challenge.Results: In the work, the toxin/antitoxin system and genome modification strategy were used to construct a stable Escherichia coli strain for L-homoserine production. The metabolic engineering strategies were focused on the enhancement of precursors for L-homoserine synthesis, reinforcement of the NADPH generation and efflux transporters using CRISPR-Cas9 system at the genome level. To improve the plasmid stability, two strategies were explored, including construction of the aspartate-auxotrophic and hok/sok systems. Constructing the auxotrophic complementation system to maintain plasmid stability was failed herein. The plasmid stability was improved by introducing the hok/sok system, resulting in 6.1 g/L (shake flask) and 44.4 g/L (5 L fermenter) L-homoserine production of the final engineered strain SHL19 without antibiotics addition. Moreover, the hok/sok system was also used to improve the plasmid stability for ectoine production, resulting in 36.7% and 46.5% higher titer of ectoine at shake flask and 5L fermenter without antibiotics addition, respectively. Conclusion: This work provides valuable strategies to improve plasmid stability for producing L-aspartate family amino acids and derivatives and eliminate environmental concerns associated with the application of antibiotics.

Genomics Characterization of an Engineered Corynebacterium glutamicum in Bioreactor Cultivation Under Ionic Liquid Stress

Corynebacterium glutamicum is an ideal microbial chassis for production of valuable bioproducts including amino acids and next generation biofuels. Here we resequence engineered isopentenol (IP) producing C. glutamicum BRC-JBEI 1.1.2 strain and assess differential transcriptional profiles using RNA sequencing under industrially relevant conditions including scale transition and compare the presence vs absence of an ionic liquid, cholinium lysinate ([Ch][Lys]). Analysis of the scale transition from shake flask to bioreactor with transcriptomics identified a distinct pattern of metabolic and regulatory responses needed for growth in this industrial format. These differential changes in gene expression corroborate altered accumulation of organic acids and bioproducts, including succinate, acetate, and acetoin that occur when cells are grown in the presence of 50 mM [Ch][Lys] in the stirred-tank reactor. This new genome assembly and differential expression analysis of cells grown in a stirred tank bioreactor clarify the cell response of an C. glutamicum strain engineered to produce IP.

Predictive Monitoring of Shake Flask Cultures with Online Estimated Growth Models

Simplicity renders shake flasks ideal for strain selection and substrate optimization in biotechnology. Uncertainty during initial experiments may, however, cause adverse growth conditions and mislead conclusions. Using growth models for online predictions of future biomass (BM) and the arrival of critical events like low dissolved oxygen (DO) levels or when to harvest is hence important to optimize protocols. Established knowledge that unfavorable metabolites of growing microorganisms interfere with the substrate suggests that growth dynamics and, as a consequence, the growth model parameters may vary in the course of an experiment. Predictive monitoring of shake flask cultures will therefore benefit from estimating growth model parameters in an online and adaptive manner. This paper evaluates a newly developed particle filter (PF) which is specifically tailored to the requirements of biotechnological shake flask experiments. By combining stationary accuracy with fast adaptation to change the proposed PF estimates time-varying growth model parameters from iteratively measured BM and DO sensor signals in an optimal manner. Such proposition of inferring time varying parameters of Gompertz and Logistic growth models is to our best knowledge novel and here for the first time assessed for predictive monitoring of Escherichia. coli (E. coli) shake flask experiments. Assessments that mimic real-time predictions of BM and DO levels under previously untested growth conditions demonstrate the efficacy of the approach. After allowing for an initialization phase where the PF learns appropriate model parameters, we obtain accurate predictions of future BM and DO levels and important temporal characteristics like when to harvest. Statically parameterized growth models that represent the dynamics of a specific setting will in general provide poor characterizations of the dynamics when we change strain or substrate. The proposed approach is thus an important innovation for scientists working on strain characterization and substrate optimization as providing accurate forecasts will improve reproducibility and efficiency in early-stage bioprocess development.