Expression of scFv-anti-CHIKV-E2 in Escherichia coli with chaperones Co-expression, and its functional assay by electrochemical immunosensor.

Single Chain Variable Fragment (scFv), a small fragment of antibody can be used to substitute the monoclonal antibody for diagnostic purposes. Production of scFv in Escherichia coli host has been a challenge due to the potential miss-folding and formation of inclusion bodies. This study aimed to express anti-CHIKV E2 scFv which previously designed specifically for Asian strains by co-expression of three chaperones that play a role in increasing protein solubility; GroEL, GroES, and Trigger Factor. The scFv and chaperones were expressed in Origami B E. coli host under the control of the T7 promoter, and purified using a Ni-NTA column. Functional assay of anti-CHIKV-E2 scFv was examined by electrochemical immunosensor using gold modified Screen Printed Carbon Electrode (SPCE), and characterized by differential pulses voltammetry (DPV) using K3[Fe(CN)6] redox system and scanning microscope electron (SEM). The experimental condition was optimized using the Box-Behnken design. The results showed that co-expression of chaperone increased the soluble scFv yield from 54.405 µg/mL to 220.097 µg/mL (~5×). Furthermore, scFv can be used to detect CHIKV-E2 in immunosensor electrochemistry with a detection limit of 0.74048 ng/mL and a quantification limit of 2,24388 ng/mL. Thus, the scFv-anti-CHIKV-E2 can be applied as a bioreceptor in another immunoassay method.

Analyzing the Impact of Physicochemical Factors on Chlorella vulgaris Growth Through Design of Experiment (DoE) for Carbon Capture System.

The CO2 emission is increasing every year and threatening both humans and the ecosystem. Carbon capture technological innovations have emerged as a potential solution to mitigate this emissions. Due to its high capacity of photosynthetic activity, CO2 sequestration by microalgae, such as Chlorella vulgaris has attracted much attention as a carbon capture system. The growth of this microalgae is influenced by various physicochemical factors. By designing the Design of Experiment (DoE) with Response Surface Methodology (RSM), the effect of several independent factor can be evaluated to optimize Chlorella vulgaris growth condition and CO2 conversion. This study aims to identify the most impact factors affecting C. vulgaris growth through investigating the variations in physicochemical factors of aeration, initial pH, dark light regime, saline, and substrates concentration using DoE. In this study, C. vulgaris was cultivated in batch culture for 10 days with 8 experiments that were designed under various conditions as per experimental run. Biomass growth was observed using optical density and analyzed by first order regression. The result shows that aeration parameters was statistically significant affect microalgae growth, evidence by p-value below 0.05 at all observation points. Runs with aeration treatment showed a prolonged exponential growth phase and delayed onset of the deceleration phase. Additionally, this study also found that the initial pH level also significantly affects growth at the last day of cultivation. Cultures with a higher initial pH reached the stationary phase earlier than those with a lower pH. Thus, the growth of C. vulgaris can be optimized by adding aeration treatment into culture media and regulating initial pH around 8 to enhancing carbon fixation and biomass yield.

Effect of Lactic Acid and pH of Probiotic Yogurt on Peak Production of Laying Hens.

<b>Background and Objective:</b> Probiotic yogurt is beneficial for laying hens because it can improve the animal's hematological status which will improve livestock health, therefore it is hoped that probiotic yogurt can increase the production of laying hens. This research was conducted to determine the lactic acid levels and pH of probiotic yogurt, probiotic yogurt's effect on feed conversion ratio and total production of laying hens. <b>Materials and Methods:</b> The research was carried out using experimental methods using a Completely Randomized Design (CRD) with 5 treatments and 8 replications so the total sample was 40. The treatment consisted of P0: Basal ration; P1: Basal diet+2% probiotic powder B1 (<i>Bifidobacterium</i> spp. and <i>L. acidophilus</i>), P2: Basal ration+3% probiotic powder B1, P3: Basal diet+2% probiotic powder B2 (<i>L. bulgaricus</i>, <i>S. thermophilus</i>, <i>L. acidophilus</i> and <i>B. bifidum</i>) and P4: Basal ration+3% probiotic powder B2. The data were analyzed using Analysis of Variance (ANOVA) and followed by Duncan's Multiple Range Test. <b>Results:</b> Lactic acid content in probiotic yogurts B1 is 0.945% and B2 is 0.638%. Based on the results of statistical analysis using the variance test, show that giving probiotic powder to laying hens has a significant effect on the feed conversion ratio and has no significant effect on the production of laying hens. <b>Conclusion:</b> Based on the results of statistical analysis using the variance test, it shows that giving probiotic powder to laying hens has no significant effect on the production of laying hens during the peak period.

Accelerated molecular dynamics study to compare the thermostability of Bacillus licheniformis and Aspergillus niger α-amylase.

The thermostability of enzymes plays a significant role in the starch hydrolysis process in the industry. The structural difference between thermostable Bacillus licheniformis α-amylase (BLA) and thermolabile Aspergillus niger α-amylase (ANA) is interesting to be explored. This work aimed to study the thermostability-determining factor of BLA as compared to a non-thermostable enzyme, ANA, using molecular dynamics (MD) simulation at a high temperature. A 100 ns of classical MD, which was followed by 200 ns accelerated MD was conducted to explore the conformational changes of the enzyme. It is revealed that the intramolecular interactions through salt bridge interactions and the presence of calcium ions dominates the stability effect of BLA, despite the absence of a disulfide bond in the structure. These results should be useful in designing a thermostable enzyme that can be used in industrial processes.Communicated by Ramaswamy H. Sarma.

Theoretical and Experimental Studies on the Evidence of 1,3-ß-Glucan in Marennine of Haslea ostrearia.

Marennine, a blue pigment produced by the blue diatom Haslea ostrearia, is known to have some biological activities. This pigment is responsible for the greening of oysters on the West Coast of France. Other new species of blue diatom, H. karadagensis, H. silbo sp. inedit., H. provincialis sp. inedit, and H. nusantara, also produce marennine-like pigments with similar biological activities. Aside from being a potential source of natural blue pigments, H. ostrearia-like diatoms present a commercial potential for the aquaculture, food, cosmetics, and health industries. Unfortunately, for a hundred years, the exact molecular structure of this bioactive compound has remained a mystery. A lot of hypotheses regarding the chemical structure of marennine have been proposed. The recent discovery of this structure revealed that it is a macromolecule, mainly carbohydrates, with a complex composition. In this study, some glycoside hydrolases were used to digest marennine, and the products were further analyzed using nuclear magnetic resonance (NMR) and mass spectroscopy (MS). The reducing sugar assay showed that marennine was hydrolyzed only by endo-1,3-ß-glucanase. Further insight into the structure of marennine was provided by the spectrum of 1H NMR, MS, a colorimetric assay, and a computational study, which suggest that the chemical structure of marennine contains 1,3-ß-glucan.

Potential of Syzygnium polyanthum as Natural Food Preservative: A Review.

Food preservation is one of the strategies taken to maintain the level of public health. Oxidation activity and microbial contamination are the primary causes of food spoilage. For health reasons, people prefer natural preservatives over synthetic ones. Syzygnium polyanthum is widely spread throughout Asia and is utilized as a spice by the community. S. polyanthum has been found to be rich in phenols, hydroquinones, tannins, and flavonoids, which are potential antioxidants and antimicrobial agents. Consequently, S. polyanthum presents a tremendous opportunity as a natural preservative. This paper reviews recent articles about S. polyanthum dating back to the year 2000. This review summarizes the findings of natural compounds presented in S. polyanthum and their functional properties as antioxidants, antimicrobial agents, and natural preservatives in various types of food.

Enhancement Methods of Antioxidant Capacity in Rice Bran: A Review.

Rice (Oryza sativa L.) is a primary food that is widely consumed throughout the world, especially in Asian countries. The two main subspecies of rice are japonica and indica which are different in physical characteristics. In general, both indica and japonica rice consist of three types of grain colors, namely white, red, and black. Furthermore, rice and rice by-products contain secondary metabolites such as phenolic compounds, flavonoids, and tocopherols that have bioactivities such as antioxidants, antimicrobial, cancer chemopreventive, antidiabetic, and hypolipidemic agents. The existence of health benefits in rice bran, especially as antioxidants, gives rice bran the opportunity to be used as a functional food. Most of the bioactive compounds in plants are found in bound form with cell wall components such as cellulose and lignin. The process of releasing bonds between bioactive components and cell wall components in rice bran can increase the antioxidant capacity. Fermentation and treatment with enzymes were able to increase the total phenolic content, total flavonoids, tocotrienols, tocopherols, and γ-oryzanol in rice bran.

Molecular dynamics study to improve the substrate adsorption of Saccharomycopsis fibuligera R64 alpha-amylase by designing a new surface binding site.

Background: Carbohydrate binding module (CBM) and surface binding site (SBS) are two important parts of amylase which respond to the raw starch digestion. They are related to the enzyme ability to adsorb and to catalyze the starch hydrolysis. However, starch processing is still expensive due to the high temperature in the gelatinization step. Therefore, direct starch digestion is more favorable. One of the solutions is to use α-amylase with high starch adsorptivity, which is expected to be capable of digesting starch below the gelatinization temperature. In Indonesia, Saccharomycopsis fibuligera R64 α-amylase (Sfamy R64) is one of the enzymes with the highest activity on starch. However, its raw starch adsorptivity was low. The aim of this study was to propose an in-silico model of Sfamy R64 mutant by introducing a new SBS using molecular dynamics (MD) simulation. Methods: The structural behavior of Sfamy R64 and positive control were studied using MD simulation. Furthermore, the mutants of Sfamy R64 were designed to have a stable SBS which mimics the positive control. The substrate affinity in all systems was evaluated using the molecular mechanics generalized Born surface area (MM/GBSA) method. Results: The stability of a new SBS constructed by seven substitutions and a loop insertion was improved throughout MD simulation. The substrate was consistently bound to the SBS over 55 ns of simulation, as compared to 14 ns in wild-type. The structural behavior of SBS in mutant and positive control was similar. The interaction energies of the positive control, wild-type, and mutant were -17.6, -5.2, and -8.2 kcal/mol, respectively. Conclusion: The enhanced substrate binding in the mutant, due to the existence of a new SBS, suggests the potential of improving starch adsorptivity of Sfamy R64. This result should be useful in developing an enzyme with better substrate adsorption based on the rational computer-aided molecular design approach.

Computational Model of the Effect of a Surface-Binding Site on the Saccharomycopsis fibuligera R64 α-Amylase to the Substrate Adsorption.

α-Amylase is one of the important enzymes in the starch-processing industry. However, starch processing requires high temperature, thus resulting in high cost. The high adsorptivity of α-amylase to the substrate allows this enzyme to digest the starch at a lower temperature. α-Amylase from Saccharomycopsis fibuligera R64 (Sfamy R64), a locally sourced enzyme from Indonesia, has a high amylolytic activity but low starch adsorptivity. The objective of this study was to design a computational model of Sfamy R64 with increased starch adsorptivity using bioinformatics method. The model structure of Sfamy R64 was compared with the positive control, ie, Aspergillus niger α-amylase. The structural comparison showed that Sfamy R64 lacks the surface-binding site (SBS). An SBS was introduced to the structure of Sfamy R64 by S383Y/S386W mutations. The dynamics and binding affinity of the SBS of mutant to the substrate were also improved and comparable with that of the positive control.

Further investigation of relationships between membrane fluidity and ethanol tolerance in Saccharomyces cerevisiae.

Membrane lipid unsaturation index and membrane fluidity have been related to yeast ethanol stress tolerance in published studies, however findings have been inconsistent. In this study, viability reduction on exposure to 18% (v/v) ethanol was compared to membrane fluidity determined by laurdan generalized polarization. Furthermore, in the determination of viability reduction, we examined the effectiveness of two methods, namely total plate count and methylene violet staining. We found a strong negative correlation between ethanol tolerance and membrane fluidity, indicated by negative Pearson correlation coefficients of - 0.79, - 0.65 and - 0.69 for Saccharomyces cerevisiae strains A12, PDM and K7, respectively. We found that lower membrane fluidity leads to higher ethanol tolerance, as indicated by decreased viability reduction and higher laurdan generalized polarization in respiratory phase compared to respiro-fermentative phase cells. Total plate count better differentiated ethanol tolerance of yeast cells in different growth phases, while methylene violet staining was better to differentiate ethanol tolerance of the different yeast strains at a particular culture phase. Hence, both viability assessment methods have their own advantages and limitations, which should be considered when comparing stress tolerance in different situations.

The Importance of Surface-Binding Site towards Starch-Adsorptivity Level in α-Amylase: A Review on Structural Point of View.

Starch is a polymeric carbohydrate composed of glucose. As a source of energy, starch can be degraded by various amylolytic enzymes, including α-amylase. In a large-scale industry, starch processing cost is still expensive due to the requirement of high temperature during the gelatinization step. Therefore, α-amylase with raw starch digesting ability could decrease the energy cost by avoiding the high gelatinization temperature. It is known that the carbohydrate-binding module (CBM) and the surface-binding site (SBS) of α-amylase could facilitate the substrate binding to the enzyme's active site to enhance the starch digestion. These sites are a noncatalytic module, which could interact with a lengthy substrate such as insoluble starch. The major interaction between these sites and the substrate is the CH/pi-stacking interaction with the glucose ring. Several mutation studies on the Halothermothrix orenii, SusG Bacteroides thetaiotamicron, Barley, Aspergillus niger, and Saccharomycopsis fibuligera α-amylases have revealed that the stacking interaction through the aromatic residues at the SBS is essential to the starch adsorption. In this review, the SBS in various α-amylases is also presented. Therefore, based on the structural point of view, SBS is suggested as an essential site in α-amylase to increase its catalytic activity, especially towards the insoluble starch.