Effect Of Substrate Concentration Research Articles

TEMPO-Functionalized Nanoreactors from Bottlebrush Copolymers for the Selective Oxidation of Alcohols in Water.

Polymeric nanoreactors in water fabricated by the self-assembly of amphiphilic copolymers have attracted much attention due to their good catalytic performance without using organic solvents. However, the disassembly and instability of relevant nanostructures often compromise their potential applicability. Herein, the preparation of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-containing nanoreactors by the self-assembly of amphiphilic bottlebrush copolymers has been demonstrated. First, a macromonomer having a norbornenyl polymerizable group was prepared by RAFT polymerization of hydrophobic and hydrophilic monomers. The macromonomer was further subjected to ring-opening metathesis polymerization to produce an amphiphilic bottlebrush copolymer. Further, TEMPO, as a catalyst, was introduced into the hydrophobic block through the activated ester strategy. Finally, TEMPO-functionalized polymeric nanoreactors were successfully obtained by self-assembly in water. The nanoreactors exhibited excellent catalytic activities in selective oxidation of alcohols in water. More importantly, the reaction kinetics showed that the turnover frequency is greatly increased compared to that of the similar nanoreactor prepared from liner copolymers under the same conditions. The outstanding catalytic activities of the nanoreactors from bottlebrush copolymers could be attributed to the more stable micellar structure using the substrate concentration effect. This work presents a new strategy to fabricate stable nanoreactors, paving the way for highly efficient organic reactions in aqueous solutions.

Production of bioprotein from oil palm fronds by Aspergillus terreus strain UniMAP AA-1

Bioprotein is an alternative source that can be used to substitute the conventional protein source. In order to produce bioprotein, agricultural wastes can be utilized by microorganisms as substrates. Therefore, in this study, the utilization of oil palm fronds (OPF) as a substrate for the production of bioprotein by Aspergillus terreus strain UniMAP AA-1 through solid-state fermentation was investigated. The objectives of this study are to evaluate the production of bioprotein developed from OPF as a substrate and to optimize the physical parameters affecting bioprotein production. Solid state fermentation was carried out in conical flasks with 20 g of working volume at 30°C for 7 days. The fermentation time which produced the highest bioprotein was recorded at day 5. After that, the effects of temperature, substrate concentration and inoculum size were screened through 2-Level factorial design. Substrate concentration and temperature for the fermentation process were further optimized using Response Surface Methodology through Central Composite Design. As a result, maximum bioprotein produced was 0.7348 mg/mL at the temperature of 34.69°C, 58.06% w/v of substrate concentration and 5% v/v inoculum size. The data obtained in this study is potentially applicable in the scale-up production of bioprotein from OPF by A. terreus strain UniMAP AA-1 in the future.

Decolorization of Malachite green dye by Stenotrophomonas maltophilia a compost bacterium

BackgroundThe release of triphenylmethane dyes, like malachite green (MG) and crystal violet (CV), into the environment is a cause of concern due to its toxicity, mutagenicity and carcinogenicity.ResultA bacterial strain that is capable of decolorizing both dyes was isolated from the composted neem oil-seed cake. The strain was characterized as Stenotrophomonas maltophilia based on the 16S rRNA gene sequence and designated as isolate TPMD-1. The kinetic study of the dye degradation revealed the efficiency of the above isolate to degrade MG. The effect of substrate concentration, pH, temperature, and agitation on the decolorization of MG by the isolate was also studied. The MG degradation rate was slightly more in neutral pH at 28–30 °C and 150 rpm. The UV-spectroscopy, HPLC, and FTIR analyses of the dye sample before and after bacterial treatment revealed the high ability of S. maltophilia TPDM-1 in the removal of MG from the media. The genetic snapshot of the isolate by PCR amplification and sequencing showed the presence of genes 'lac’ and ‘tmr’ that codes for laccase and triphenylmethane reductase.ConclusionThis study presented the first report of Stenotrophomonas maltophilia in the degradation and detoxification of MG dye by oxidoreduction, which could be used for the bioremediation of aquatic environments contaminated by MG.

Highly sensitive oxidation of MBTH/DMAB by MnFe2O4 nanoparticles as a promising method for nanozyme-based sensor development

Nanozymes are human-made nanomaterials with an intrinsic enzyme-like activity that offer a versatile alternative to natural enzymes for disease treatment, environmental remediation, and sensing. In this work, we proposed a novel spectrophotometric assay for determining the oxidase-like activity of manganese ferrite nanoparticles (MnFe2O4 NPs) based on the oxidative coupling of 3-methyl-2-benzothiazolinone-hydrazone (MBTH) and 3-(dimethylamino) benzoic acid (DMAB). The effects of pH (2−8), temperature (20–50 °C), and kinetics parameters as the effect of substrate and nanoparticle concentration on the oxidase-like activity were evaluated. The MnFe2O4 NPs (~3.19 ± 0.62 nm size) were synthesized by a co-precipitation method and characterized by TEM, SEM/EDS, VSM-SQUID, PXRD, and FTIR-ATR analyses. The optimum condition for MBTH/DMAB oxidation catalyzed by MnFe2O4 NPs was observed at pH = 3.9 and 30 °C. The MBTH/DMAB oxidation was adjusted to the Michaelis Menten kinetics model (R2 = 0.96) and showed a Km = 13.59 µM MBTH, kcat = 5.25 × 107 s−1 and kcat/Km = 3.86 × 1012 M−1 s−1. These high kcat and kcat/Km values indicate that the MBTH/DMAB couple is more sensitive than previously reported oxidase substrates by about three orders of magnitude. Hence, our findings open up a wide range of new potential applications on the development of more sensitive sensors based on the MBTH/DMAB reaction catalyzed by MnFe2O4 NPs for detecting several analytes (including alkaline phosphatase, heavy metals, dissolved oxygen in water, among others) in the medical and environmental field.

PRODUCTION AND PARTIAL CHARACTERIZATION OF A MILK-CLOTTING PROTEINASE PRODUCED BY BACILLUS SUBTILIS SMDFS-2B MN715837 IN SUBMERGED CULTURES

This study focused on the production and partial characterization of a milk-clotting protease produced by Bacillus subtilis SMDFS 2B in submerged cultures, under partially optimized conditions. The crude enzyme was recovered in the culture supernatant and concentrate was produced after cell removal and subsequent dialysis. Inhibition studies were conducted employing four distinct protease inhibitors: Pepstatin-A, Phenylmethane-sulphonyl-fluoride (PMSF), Ethylenediaminetetraacetic acid (EDTA), and iodoacetamide (IA). The effect of temperature, pH, metal ions and substrate concentration on milk-clotting activity were also evaluated. The thermal stability of the enzyme was determined by incubating the crude enzyme at a temperature value ranging from 35 oC to 60 oC. Similarly, pH stability was determined at pH values ranging between 4.5 and 8.0. The highest milk-clotting activity was observed at a temperature of 55 oC and pH 5.5. The crude enzyme preparation remained stable on incubation at 35 oC and 40 oC for 15 min and at pH 5.5. The enzyme also showed the lowest residual milk-clotting activity in the presence of EDTA (7.94%) and Pepstatin-A (26.71%). The addition of Mg2+ and Mn2+ significantly increased milk-clotting activity. The enzyme also showed an elevation in its apparent milk-clotting activity upon increasing the substrate (skim-milk) concentration. Thus, the milk-clotting protease produced by B. subtilis SMDFS 2B by submerged fermentation revealed some interesting milk-clotting characteristics. This may open the way for applications in the food and dairy industries.

Statistical optimization of methane production from brewery spent grain: Interaction effects of temperature and substrate concentration

This study evaluated the effects of thermal pretreatment of brewery spent grain (BSG) (by autoclave 121 °C, 1.45 atm for 30 min) on methane production (CH4). Operation temperature (31–59 °C) and substrate concentration (8.3–19.7 g BSG.L−1) factors were investigated by Response Surface Methodology (RSM) and Central Composite Design (CCD). Values ranging from 81.1 ± 2.0 to 290.1 ± 3.5 mL CH4.g−1 TVS were obtained according to operation temperature and substrate concentration variation. The most adverse condition for methanogenesis (81.1 ± 2.0 mL CH4.g−1 TVS) was at 59 °C and 14 g BSG.L−1, in which there was increase in the organic matter concentration from 173.6 ± 4.94 to 3036 ± 7.78 mg.L−1) result of a higher final concentration of volatile fatty acids (VFA, 2662.7 mg.L−1). On the other hand, the optimum condition predicted by the statistical model was at 35 °C and 18 g BSG.L−1 (289.1 mL CH4.g−1 TVS), which showed decrease in the organic matter concentration of 78.6% and a lower final concentration of VFA (533.2 mg.L−1). Hydrogenospora and Methanosaeta were identified in this optimum CH4 production condition, where acetoclastic methanogenic pathway prevailed. The CH4 production enhancement was concomitant to acetic acid concentration decrease (from 578.9 to 135.7 mg.L−1).

Converting waste molasses liquor into biohydrogen via dark fermentation using a continuous bioreactor

This study investigated the effects of substrate concentration, HRT (hydraulic retention time), and pre-treatment of the substrate molasses on biohydrogen production from waste molasses (condensed molasses fermentation solubles, CMS) with a CSTR (continuously-stirred tank reactor). First, the hydrogen production was performed with various CMS concentrations (40–90 g COD/L, total sugar 8.7–22.6 g/L) with 6 h HRT. The results show that the maximal hydrogen production rate (HPR) occurred at 80 g COD/L substrate (19.8 g ToSu/L, ToSu: Total Sugar), obtaining an HPR of 0.417 mol/L/d. However, maximum hydrogen yield (HY) of 1.44 mol H2/mol hexose and overall hydrogen production efficiency (HPE) of 25.6% were achieved with a CMS concentration of 70 g COD/L (17.3 g ToSu/L). The substrate inhibition occurred when CMS concentration was increased to 90 g COD/L (22.6 g ToSu/L). Furthermore, it was observed that the optimal HPR, HY, and HPE all occurred at HRT 6 h. Operating at a lower HRT of 4 h decreased the hydrogen production performance because of lower substrate utilization efficiency. The employment of pre-heating treatment (60 °C for 1 h) of the substrate could markedly enhance the fermentation performance. With 6 h HRT and substrate pre-heating treatment, the HPE raised to 29.9%, which is 18% higher than that obtained without thermal pretreatment.

Enantioselective resolution of (±)-1-phenylethyl acetate using the immobilized extracellular proteases from deep-sea Bacillus sp. DL-1

Bacillus sp. DL-1 was isolated from the deep sea of the Western Pacific Ocean and behaved very good resistance to NaCl. The extracellular proteases of Bacillus sp. DL-1 were found to exhibit excellent enantioselectivity for the kinetic resolution of (±)-1-phenylethyl acetate. To improve the stability of the enzyme, the immobilized extracellular proteases were preparated by using 60 g kieselguhr and 1-L crude fermentation broth containing extracellular proteases of Bacillus sp. DL-1, shaking at 25 °C, 200 r/min for 10 h. Every gram of kieselguhr adsorbed 25.7 mg extracellular protease and the enzymatic activity recovery was 79.86%. The immobilized proteases preserved about 35.8% of its activity after 6 repeated uses, which were also used as biocatalyst to asymmetrically hydrolyse (±)-1-phenylethyl acetate for the preparation of (R)-1-phenylethanol and (S)-1-phenylethyl acetate with high optical purities. The effects of pH, temperature, enzyme concentration, substrate concentration, reaction time and additives (metal ions/surfactants) on the resolution were investigated by single factor experiments. Under the optimal reaction conditions (10 mM (±)-1-phenylethyl acetate, 40 mg/mL immobilized extracellular proteases, pH 7.5 (Tris-HCl), 5% (v/v) methanol and 45 °C for 2 h), (R)-1-phenylethanol was generated with the e.e.p being > 97%, and the yield being 53%, respectively. Analogously, under the optimal reaction conditions (10-mM (±)-1-phenylethyl acetate, 360 mg/mL immobilized extracellular proteases, pH 6.0 (PB), 5% (v/v) DMSO and 35 °C for 1.5 h), (S)-1-phenylethyl acetate was generated with the e.e.s being over 99% and the yield being 79%, respectively. Compared with the extracellular proteases from Bacillus sp. DL-2, the immobilized extracellular proteases from Bacillus sp. DL-1 exhibited higher hydrolytic activity and could asymmetrically hydrolyse (±)-1-phenylethyl acetate by using higher substrate concentrations, shorter reaction times to obtain higher yields. Notably, the extracellular proteases of Bacillus sp. DL-1 were demonstrated to behave the same enantio-preference as those of most other reported esterases/lipases. Proteases from deep-sea Bacillus sp. DL-1 are promising biocatalysts for the synthesis of valuable chiral chemicals.

Lactate and acetate applied in dual‐chamber microbial fuel cells with domestic wastewater

For about 20 years, microbial fuel cells (MFCs) are an emerging technology that has gained attention for its new wastewater treatment and energy generation, especially its ability to convert chemical energy from a broad range of substances into electricity. However, MFC has not been widely commercialized due to low efficiency. Studies have shown that substrate loading is an important factor in scaling up. Therefore, this study investigates the effect of substrate type and concentration on honeycomb MFCs (HCMFCs). The effect of different concentrations ranging from 10 to 40 mM of lactate and acetate (1:1 ratio) substrates was investigated. Power efficiency was analyzed using polarization and power density curves. Results showed that the performance of MFCs and biofilm formation is affected by the substrates. Scanning electron microscopy showed some changes in biofilm formation. Mixing lactate and acetate at 30 mM gave the best performance with a power density of 956.75 mW m−2 and chemical oxygen demand removal of 87.8%. Furthermore, effective substrate degradation, having COD removal of 91.4%, was observed with acetate. Highlights Substrate type and concentration affect MFCs performance significantly Mixed substrate promotes robust mix bacterial culture Substrate concentration of 30 mM gave the best power generation Adverse effect of high substrate concentration on power generation was confirmed Effective substrate degradation with COD removal of 91.4% was observed with acetate

Lactic Acid Fermentation from Durian Seeds (Durio zibethinus Murr.) Using Lactobacillus plantarum

Durian is one of the abundant seasonal fruits at harvest time. Generally, the part consumed in durian is its salute fruit or its meat. Durian seeds contain 70-80% carbohydrates, but not yet utilized. Carbohydrates can be fermented into lactic acid using Lactobacillus plantarum bacteria. Lactic acid is a raw material for biodegradable plastic/poly lactic acid (PLA). The purposes of this study were to determine the effect of substrate concentration (7.5%, 10%, 12.5%, 15% and 17.5%), inoculum concentration (2%, 5%, 7%, 10% and 12%), and yeast extract concentration ( 0 g/L, 2.5 g/L, 5 g/L, 7.5 g/L and 10 g/L) on the yield and the concentration of lactic acid produced. The research was initialized by making durian seed flour, testing the proximate value of the materials, bacterial inoculation, fermentation of durian seed flour at 37°C, agitation speed of 100 rpm for 72 hours, then the fermentation results were analyzed for lactic acid concentration using UPLC (Ultra Performance Liquid Chromatography). The highest yield and lactic acid concentration at substrate concentration of 15% were 12.51% and 13.75g/L, at inoculum concentration of 2% were 19.44% and 21.3 8g/L, while the highest yield and lactic acid concentration at 10 g/L yeast extract concentration were 21.76% % and 23.92 g/L.

Anaerobic digestion of dissolved air floatation slurries: Effect of substrate concentration and pH

Dissolved air floatation (DAF) slurries are an unconventional substrate for anaerobic digestion owing to their high COD concentrations, mainly consisting of lipids. Batch anaerobic digestion of DAF slurry was conducted to investigate the methane production through dilution of the slurry (with mineral salt medium, MSM) and by adjusting the initial pH (to 6.0, 5.5, 5.0 and 4.5) The performance of two different inocula, namely waste activated sludge (WAS) treating domestic wastewater and anaerobic granular sludge (AGS) treating dairy wastewater, was compared. The DAF slurry had a pH of 7.03 ( ± 0.07), conductivity of 6.69 ( ± 0.54) mS/cm, and a COD and ammonium concentration of 85.96 ( ± 8.32) g/L and 168.49 ( ± 1.79) mg N/L, respectively. DAF slurry without dilution produced a maximum methane yield of 58.60 ( ± 1.97) mL/g COD after 30 days of incubation. Dilution with MSM up to three folds, improved the methane yield by about 67%–105% and 67%–177% with WAS and AGS as the inoculum, respectively. Higher methane productivity was achieved at an inoculum to substrate ratio of at least 1.5 for WAS and 2.0 for AGS, respectively. AGS was more effective with a 20% increase in methane production from 3-fold diluted DAF slurry when compared with WAS. Adjustment of the initial pH of the DAF slurry to 6.0 resulted in a methane yield of 359.71 ( ± 32.72) mL/g COD, which was 23% higher than the control without pH adjustment. This study demonstrated that the methane yield from DAF slurries can be enhanced through substrate dilution and initial pH adjustment. • Methane yield enhanced by 23% upon adjustment of initial pH of the DAF slurry to 6.0. • 3-fold dilution increased methane production rate by 275% with granular sludge. • Anaerobic granular sludge is an efficient inoculum for methanogenesis of DAF slurry. • Lag phase duration was not shortened by dilution or pH adjustment of the DAF slurry. • Optimal inoculum to substrate ratio at least 1.5 and 2.0 for WAS and AGS, respectively.

Kinetic model for the enzymolysis of Paphia undulate by marine low-temperature alkaline proteinase

The kinetics of enzymatic hydrolysis of paphia sinensis was studied. The molecular weight distribution of the small peptide mixture was determined by liquid and mass spectrometry. The effects of initial substrate concentration on enzymatic hydrolysis reaction, the effects of initial enzyme concentration on enzymatic hydrolysis reaction, the effects of enzymatic hydrolysis time on enzymatic hydrolysis reaction and the mechanism of enzymatic hydrolysis reaction were investigated. In this experiment, the basic kinetic model of enzymatic hydrolysis of protein was derived, and the kinetic parameters and enzyme inactivation constant were determined.

Extraction of pectin from banana (Musa acuminata x balbisiana) peel waste flour using crude enzymes secreted by Aspergillus niger

Intensive cultivation has maintained annual banana production in Indonesia at about 7 million tons since 2015. Banana fruits bear about 35 - 40% weight of peel waste, which contains 20.28 g pectin/100 g the dry matter. The peel waste may trigger serious environmental problems. This study aimed to investigate the effect of substrate concentrations and time on the pectin yield extraction from banana peel waste flour using crude pectinase secreted by Aspergillus niger. Crude pectinases were prepared through batch fermentation of banana peel flour using A. niger. The enzymes were used to hydrolyze banana peel waste flour at various substrate concentrations and time at 323 K. The results indicate that the increase of both extraction parameters leads to increase the pectin yield to some extent and then level off. However, further increase of both extraction parameters cause inhibition by galacturonic acid. The highest pectin yield (10.8% weight) was achieved at 323 K and 0.067g/mL substrate concentration for 120 minutes. Applications of this result are expected to increase the economic value of banana peel waste. However, further study in the effect of other extraction parameters such as temperature, enzyme loading, pH, flour size and pectin purification are required.

Using microalgae for remediation of crude petroleum oil–water emulsions

Crude petroleum oil spills are among the most important organic contaminations. While the separated oils accumulated on the surface water are relatively easily removed, the emulsified portions are more difficult to remove and pose significant threats to the environment. Bioremediation using bacteria has proven to be an effective method, but the biomass produced in this case does not have any significant remunerative value. In this work, microalgae were proposed to combine emulsified oil remediation process with the potential of microalgae as a biofuel feedstock, thus enhancing the economic and environmental benefits of the process. A freshwater strain of Chlorella vulgaris was grown in water containing different concentrations of emulsified crude oil at different temperatures. The specific growth rate (μmax ) of the microalgae for each initial oil concentration was determined and was found to increase with the increase in initial oil concentration. For example, at 30°C, the specific growth rate, μ increased from 0.477 to 0.784 per day as the oil concentration increased from 57 to 222 mg/L. At 30°C, the effect of substrate concentration agreed with that of the microalgae growth, whereas at 40°C, the drop in oil concentration decreased with the increase in concentration. The results were fitted to a modified Monod kinetics model that used specific interfacial area as the influential substrate instead of the actual concentration. The results of this study clearly show the potential of using microalgae for emulsified oil remediation at relatively high concentrations.

Effects of Temperature and Substrate Concentration on N2O Release of ANAMMOX Process

The greenhouse gas N2O is released during the biological nitrogen removal process. ANAMMOX (anaerobic ammonium oxidation) is regarded as a promising nitrogen removal process for treating municipal wastewater, and the N2O emission patterns and mechanisms need further investigation. In this study, batch tests were performed to study the release of N2O at different temperatures and substrate concentrations, and the microbial mechanisms of N2O emission were discussed. The results showed that the increase of the influent substrate concentration of the ANAMMOX process promoted the release of N2O. At 35℃, when the influent nitrite increased from 40 mg·L-1 to 60 mg·L-1, 120 mg·L-1, the maximum accumulated concentration of N2O increased from 0.5 mg·L-1 to 1.5 mg·L-1 and 2.4 mg·L-1, accounting for 0.85%, 1.43%, and 1.11% of the total nitrogen removal, respectively. Lowering temperature had an obvious inhibitory effect on ANAMMOX activity. The specific ANAMMOX activity at 15℃ was only 6% of that at 30℃. Furthermore, the intracellular ATP concentration was reduced. At 15℃, the intracellular ATP concentration was 4% of that at 30℃. The decrease in temperature led to a decrease in the release of N2O in the ANAMMOX process. When the temperature decreased, the denitrification rate would decrease, leading to a lower N2O production rate and lower N2O accumulation. 16S rRNA amplicon sequencing showed that ANAMMOX bacteria Candidatus Brocadia and Ca. Jettenia were enriched, accounting for 6.9%-13.8% and 1.4%-2.6% of microbial community, respectively. Abundant heterotrophic bacteria were also found in the microbial community. The accumulation of N2O in the ANAMMOX process was mainly attributed to denitrifying bacteria producing and consuming N2O. This study provides support for controlling N2O emission during the ANAMMOX process for treating municipal wastewater.

Effect of initial pH and substrate concentration on the lactic acid production from cassava wastewater fermentation by an enriched culture of acidogenic microorganisms.

Recently, cassava processing wastewater has been considered an alternative substrate for lactic acid production due to its appreciable carbohydrate levels. The authors carried out different batch reactor trials aiming to favor the production of lactic acid through the fermentation of non-sterilized cassava wastewater by an enriched culture of acidogenic microorganisms. To this end, the impact of different initial pHs (4.5, 5.0, 5.7, 6.5, and 7.0) and different initial substrate concentrations (10, 15.8, 30, 44.2, and 50g/L) in terms of glucose on lactic acid production yield (Y) was evaluated by applying the design of experiment (DoE) known as central composite rotatable design (CCRD). The highest rate of lactic acid production (40g/L) occurred with an initial pH of 6.5 and an initial substrate concentration of 50g/L. The maximum yield was higher in trials T1, T2, T4, T5, and T8, reaching values of 0.80, 0.62, 0.60, 0.96, and 0.70g/g, respectively. The maximum lactic acid productivity (P), of 0.60 and 0.73gL-1 hr-1 , was observed in trials T5 and T8, respectively. The enriched culture of acidogenic microorganisms was shown to favor the production of lactic acid, since the production of other acids, such as acetic and propionic acid, did not exceed 3.5 and 4.5g/L, respectively. © 2020 Water Environment Federation PRACTITIONER POINTS: Cassava wastewater presented potential to lactic acid production. The CCRD showed that highest lactic acid concentrations (40g/L). The adoption of cassava wastewater or manipueira as a substrate resulted in important information on the tendency to obtain value-added products such as lactic acid.

The effect of substrate concentration on granular fermentation for caproic acid production

Contribution to the International Chain Elongation Conference 2020 | ICEC 2020. An abstract can be found in the right column.

Gluconeogenic fructose-1,6-bisphosphatase from the mature sporocarps of common aquatic ferns: partial purification and basic characterization of this enzyme from Marsilea minuta (Polypodiopsida)

The present communication reports substantial activity of gluconeogenic fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) in three common heterosporous aquatic ferns (Marsilea minuta, Salvinia natans, and Azolla pinnata) and also describes a protocol for its partial purification from mature sporocarps of Marsilea minuta. The cytosolic FBPase, obtained from Marsilea minuta, Salvinia natans, and Azolla pinnata was recognized as gluconeogenic enzyme due to its drastic catabolic inactivation in presence of externally administered glucose and its insensitivity towards photosynthetic light illumination. Cytosolic gluconeogenic FBPase was partially purified from mature sporocarps of Marsilea minuta to about 22-fold over homogenate following low-speed centrifugation (11, 400 × g), 30–80% ammonium sulfate fractionation followed by subsequent chromatography using matrices like CM-Cellulose, Sephadex G-200, and Ultrogel AcA 34. The profile of partially purified FBPase in PAGE under non-denaturing condition was recorded. The enzyme activity increased linearly with respect to protein concentration to about 100 µg and with respect to time up to 75 minutes. Temperature optimum was found at 35 °C. The effect of substrate concentration and kinetic analyses for FBPase were carried out using D-fructose-1,6-bisphosphate (D-FBP, the substrate) in the range of 0.0 to 1.0 mM at an interval of 0.1 mM concentration. The Km value for D-FBP of FBPase was 0.06129 mM and Vmax was 4525 nmole Pi released (mg)-1 protein h-1 as determined by nonlinear regression kinetics using Prism 8 software (Graph Pad). The enzyme was functional in a constricted pH range of 7.0 to 8.0, giving maxima at pH 7.5. This cytosolic enzyme was significantly stimulated by Mg2+ and strongly inhibited by Hg2+, Cu2+ and Zn2+.

Mathematical modeling of a continuous-flow packed-bed reactor with immobilized lipase for kinetic resolution of (R,S)-2-pentanol.

In this study, the kinetic resolution of (R,S)-2-pentanol via transesterification to achieve S-2-pentanol, a key intermediate required in the synthesis of anti-Alzheimer drugs, was investigated in continuous-flow packed-bed reactors. The effects of residence time, substrate concentration, and operation time of the enzyme were investigated. Under steady state conditions, 50% conversion and enantiomeric excess of the substrate (ee S >99% were achieved at a residence time of 0.04 min. Productivity of the continuous-flow process (1.341 mmol/min/g)was about 4 times higher than that of the corresponding batch process (0.363 mmol/min/g). In addition, the mathematical modeling of the packed-bed reactor was conductedusing an axial dispersion model. Ping Pong Bi Bi kinetics was used in this model. Design parameters were determined and the developed equations were solved using an algorithm for solving boundary value problems for ordinary differential equations by collocation (bvp4c) using MATLAB. The results, obtained from the model, fitted the experimental data very well.).

Optimization of Culture Conditions for the Efficient Biosynthesis of Trilobatin from Phloretin by Engineered Escherichia coli Harboring the Apple Phloretin-4'-O-glycosyltransferase.

Trilobatin, a dihydrochalcone glucoside and natural sweetener, has diverse biological and therapeutic properties. In the present study, we developed a microbial system to produce trilobatin from phloretin using Escherichia coli (E. coli) overexpressing the phloretin-4'-O-glycosyltransferase from Malus x domestica Borkh. Various optimization strategies were employed for the efficient production of trilobatin using a one-factor-at-a-time method. The effect of UDP-glucose supplementation, substrate, and inducer concentrations, time of substrate feeding as well as protein induction, and different culture media combinations were evaluated and optimized to enhance the production of trilobatin. As a result, the highest trilobatin production, 246.83 μM (107.64 mg L-1), was obtained with an LB-TB medium combination, 22 h of induction with 0.1 mM IPTG followed by 4 h of feeding with 250 μM phloretin and without extracellular UDP-glucose supplementation. These results demonstrate the efficient production of trilobatin and constitute a promising foundation for large-scale production of the dihydrochalcone glycosides in engineered E. coli.