The use of biodegradable material such as simple carbohydrates and recyclable material such as thermo-sensitive polymers is in need to develop a sustainable aqueous two-phase system (ATPS) for the purification of biomolecules. Accurate determination of sucrose concentration is important in liquid-liquid equilibrium (LLE) study of carbohydrate-based ATPS. The well-established phenol-sulfuric acid method has been widely employed in the measurement of carbohydrate concentration. However, the presence of thermo-sensitive polymers, which has a lower critical solution temperature (LCST) below room temperature, in carbohydrate samples could hamper the precision of spectrophotometric analysis due to the formation of two phases or cloudiness in the sample. Thus, the following modifications were made in an attempt to eliminate the interference occurred during conventional phenol-sulfuric acid assay.•The modified assay for sucrose quantification was performed at an ice-cold temperature throughout the reaction in order to avoid the interference from thermo-sensitive polymers.•This method required a sample volume of 3 μL and hence the volume of other reagents employed was also considerably reduced.•The absorbance was measured at 520 nm which allowed a longer linearity range (0.05-7.5%, w/v).
The effect of different carbon and nitrogen sources on the production of mannan-degrading enzymes, focussing on β-mannanase, by Aspergillus niger was investigated using shake flask culture. The β-mannanase activity obtained during growth of A. niger on guar gum (GG, 1495 nkat mL(-1)) was much higher than those observed on other carbon substrates, locust bean gum (1148 nkat mL(-1)), α-cellulose (10.7 nkat mL(-1)), glucose (8.8 nkat mL(-1)) and carboxymethylcellulose (4.6 nkat mL(-1)). For fermentation using GG as a carbon source, bacteriological peptone gave the highest β-mannanase activity (1744 nkat mL(-1)) followed by peptone from meat (1168 nkat mL(-1)), yeast extract (817 nkat mL(-1)), ammonium sulphate (241 nkat mL(-1)), ammonium nitrate (113 nkat mL(-1)) and ammonium chloride (99 nkat mL(-1)) when used as a nitrogen source. The composition of bacteriological peptone and initial pH of the medium were further optimized using response surface methodology (RSM). Medium consisted of 21.3 g L(-1) GG and 57 g L(-1) peptone with initial culture pH of 5.5 was optimum for β-mannanase production (2063 nkat mL(-1)) by A. niger. The β-mannanase production obtained in this study using A. niger was significantly higher than those reported in the literature.
One-time ultrasonication pre-treatment of Rhodobacter sphaeroides was evaluated for improving biohydrogen production via photofermentation. Batch experiments were performed by varying ultrasonication amplitude (15, 30, and 45%) and duration (5, 10, and 15 min) using combined effluents from palm oil as well as pulp and paper mill as a single substrate. Experimental data showed that ultrasonication at amplitude 30% for 10 min (256.33 J/mL) achieved the highest biohydrogen yield of 9.982 mL H2/mLmedium with 5.125% of light efficiency. A maximum CODtotal removal of 44.7% was also obtained. However, when higher ultrasonication energy inputs (>256.33 J/mL) were transmitted to the cells, biohydrogen production did not improve further. In fact, 20.6% decrease of biohydrogen yield (as compared to the highest biohydrogen yield) was observed using the most intense ultrasonicated inoculum (472.59 J/mL). Field emission scanning electron microscope images revealed the occurrence of cell damages and biomass losses if ultrasonication at 472.59 J/mL was used. The present results suggested that moderate ultrasonication pre-treatment was an effective technique to improve biohydrogen production performances of R. sphaeroides.
Rapid industrial developments coupled with surging population growth have complicated issues dealing with water scarcity as the quest for clean and sanitized water intensifies globally. Existing fresh water supplies could be contaminated with organic, inorganic and biological matters that have potential harm to the society. Turbidity in general is a measure of water cloudiness induced by such colloidal and suspended matters and is also one of the major criteria in raw water monitoring to meet the stipulated water quality guidelines. Turbidity reduction is often accomplished using chemical coagulants such as alum. The use of alum is widely associated with potential development of health issues and generation of voluminous sludge. Natural coagulants that are available in abundance can certainly be considered in addressing the drawbacks associated with the use of chemical coagulants. Twenty one types of plant-based natural coagulants categorized as fruit waste and others are identified and presented collectively with their research summary in this review. The barriers and prospects of commercialization of natural coagulants in near future are also discussed.
Periplasmic expression of soluble proteins in Escherichia coli not only offers a much-simplified downstream purification process, but also enhances the probability of obtaining correctly folded and biologically active proteins. Different combinations of signal peptides and target proteins lead to different soluble protein expression levels, ranging from negligible to several grams per litre. Accurate algorithms for rational selection of promising candidates can serve as a powerful tool to complement with current trial-and-error approaches. Accordingly, proteomics studies can be conducted with greater efficiency and cost-effectiveness. Here, we developed a predictor with a two-stage architecture, to predict the real-valued expression level of target protein in the periplasm. The output of the first-stage support vector machine (SVM) classifier determines which second-stage support vector regression (SVR) classifier to be used. When tested on an independent test dataset, the predictor achieved an overall prediction accuracy of 78% and a Pearson's correlation coefficient (PCC) of 0.77. We further illustrate the relative importance of various features with respect to different models. The results indicate that the occurrence of dipeptide glutamine and aspartic acid is the most important feature for the classification model. Finally, we provide access to the implemented predictor through the Periscope webserver, freely accessible at http://lightning.med.monash.edu/periscope/.
Response surface methodology (RSM) and artificial neural network (ANN) were used to optimize the effect of four independent variables, viz. glucose, sodium chloride (NaCl), temperature and induction time, on lipase production by a recombinant Escherichia coli BL21. The optimization and prediction capabilities of RSM and ANN were then compared. RSM predicted the dependent variable with a good coefficient of correlation determination (R² and adjusted R² values for the model. Although the R (2) value showed a good fit, absolute average deviation (AAD) and root mean square error (RMSE) values did not support the accuracy of the model and this was due to the inferiority in predicting the values towards the edges of the design points. On the other hand, ANN-predicted values were closer to the observed values with better R², adjusted R², AAD and RMSE values and this was due to the capability of predicting the values throughout the selected range of the design points. Similar to RSM, ANN could also be used to rank the effect of variables. However, ANN could not predict the interactive effect between the variables as performed by RSM. The optimum levels for glucose, NaCl, temperature and induction time predicted by RSM are 32 g/L, 5 g/L, 32°C and 2.12 h, and those by ANN are 25 g/L, 3 g/L, 30°C and 2 h, respectively. The ANN-predicted optimal levels gave higher lipase activity (55.8 IU/mL) as compared to RSM-predicted levels (50.2 IU/mL) and the predicted lipase activity was also closer to the observed data at these levels, suggesting that ANN is a better optimization method than RSM for lipase production by the recombinant strain.
Over the past few decades, Escherichia coli (E. coli) remains the most favorable host among the microbial cell factories for the production of soluble recombinant proteins. Recombinant protein production (RPP) via E. coli is optimized at the level of gene expression (expression level) and the process condition of fermentation (process level). Presently, the reported studies do not give a clear view on the selection of methods employed in the optimization of RPP. Here, we have reviewed various optimization methods and their preferences with respect to the factors at expression and process levels to achieve the optimal levels of soluble RPP. With a greater understanding of these optimization methods, we proposed a stepwise methodology linking the factors from both levels for optimizing the production of soluble recombinant protein in E. coli. The proposed methodology is further explained through five sets of examples demonstrating the optimization of RPP at both expression and process levels.Key Points• Stepwise methodology of optimizing recombinant protein production is proposed.• In silico tools can facilitate the optimization of gene- and protein-based factors.• Optimization of gene- and protein-based factors aids host-vector selection.• Statistical optimization is preferred for achieving optimal levels of process factors.
This paper deliberates the modelling and validation of bacteriocin-like inhibitory substance (BLIS) secretion by Pediococcus acidilactici Kp10 at different agitation speeds in a stirred tank bioreactor. A range of models namely the re-parameterised logistic, Luedeking-Piret and maintenance energy were assessed to predict the culture performance of the said bacterium. Growth of P. acidilactici Kp10 was enhanced with increased agitation speed up to 600 rpm while BLIS secretion was maximum at 400 rpm but decreased at higher agitation speed. Growth of P. acidilactici aptly subscribed to the re-parameterised logistic model while BLIS secretion and lactose consumption fitted well with the Luedeking-Piret model. The models revealed a relationship between growth of the bacterium and BLIS secretion. Bacterial growth and BLIS secretion were largely affected by the agitation speed of the stirred tank bioreactor which regulated the oxygen transfer to the culture. BLIS secretion by P. acidilactici Kp10 was however enhanced in oxygen-limited culture. The study also assessed BLIS from the perspective of its stability when subjected to factors such as temperature, pH and detergents. Results showed that BLIS produced by this strain was not affected by heat (at 25-100 °C for 20 min and at 121 °C for 15 min), surfactant (Tween 40, 60 and 80 and urea), detergents (up to 1% SDS), organic solvents (50% each of acetone, methanol and ethanol) and stable in a wide range of pH (2-10). The above information are pertinent with reference to commercial applications of this bacterial product in food manufacturing which invariably involve various sterilization processes and subjected to a wide pH range.
Fruit wastes constituting up to half of total fruit weight represent a large pool of untapped resources for isolation of starch with diverse applications. In this work, the possibility of isolating starch from tropical fruit wastes and its extended application as a natural coagulant was elucidated. Amongst the 12 various parts of fruit wastes selected, only jackfruit seeds contained more than 50% of total starch content. Using alkaline extraction procedures, starch has been successfully isolated from local jackfruit seeds with a yield of approximately 18%. Bell-shaped starch granules were observed under SEM with a granule size ranging from 1.1 to 41.6 μm. Detailed starch characteristics were performed to provide a comparison between the isolated seed starch and also conventional starches. Among them, chemical properties such as the content of starch, amylose, amylopectin and the corresponding molecular weights are some of the key characteristics which governed their performance as natural coagulants. The potential use of isolated seed starch as an aid was then demonstrated in both suspensions of kaolin (model synthetic system) and Chlorella sp. microalga (real-time application) with plausible outcomes. At optimized starch dosage of 60 mg/L, the overall turbidity removal in kaolin was enhanced by at least 25% at a fixed alum dosage of 2.1 mg/L. Positive turbidity and COD removals were also observed in the treatment of Chlorella suspensions. Starches which served as bridging agents aided in the linkage of neighbouring microflocs and subsequently, forming macroflocs through a secondary coagulation mechanism: adsorption and bridging.
As a class of ionic liquids with higher biocompatibility, cholinium aminoates ([Cho][AA]) hold potential as solvation media for enzymatic bioprocessing. Herein, solvation effect of [Cho][AA] on structural stability and enzymatic activity of Candida antarctica lipase B (CALB) was evaluated using experimental and computational approaches. Influence of [Cho][AA] on CALB stability was investigated using amino acid anions ([AA]-) with varying hydrophobicity levels. Choline phenylalaninate ([Cho][Phe]) resulted in 109.1% and 110.4% of relative CALB activity to buffer medium at 25 °C and 50 °C, respectively. Simulation results revealed the improvement of CALB's enzymatic activities by [AA]- with a strong hydrophobic character. Shielding of CALB from water molecules by [AA]- was observed. The level of CALB activity was governed by accumulation level of [AA]- at CALB's first hydration layer. The stronger interaction between His224 and Asp187 was postulated to be driven by [Cho][AA], resulting in the activity enhancement of CALB. The slight improvement of CALB activity in 0.05 M [Cho][Phe] at 50 °C could be due to the larger size of entrance to the catalytic site and the stronger interaction between the catalytic residues. The promising effect of [Cho][Phe] on CALB activation may stimulate research efforts in designing a 'fully green' bioreaction for various industrial applications.Communicated by Ramaswamy H. Sarma.
Despite extensive studies revealing the potential of cholinium-based ionic liquids (ILs) in protein stabilization, the nature of interaction between ILs' constituents and protein residues is not well understood. In this work, we used a combined computational and experimental approach to investigate the structural stability of a peptide hormone, insulin aspart (IA), in ILs containing a choline cation [Ch]+ and either dihydrogen phosphate ([Dhp]-) or acetate ([Ace]-) as anions. Although IA remained stable in both 1 M [Ch][Dhp] and 1 M [Ch][Ace], [Dhp]- exhibited a much stronger stabilization effect than [Ace]-. Both the hydrophilic ILs intensely hydrated IA and increased the number of water molecules in IA's solvation shell. Undeterred by the increased number of water molecules, the native state of IA's hydrophobic core was maintained in the presence of ILs. Importantly, our results reveal the importance of IL concentration in the medium which was critical to maintain a steady population of ions in the microenvironment of IA and to counteract the denaturing effect of water molecules. Through molecular docking, we confirm that the anions exert the dominant effect on the structure of IA, while [Ch]+ have the secondary influence. The computational results were validated using spectroscopic analyses (ultra-violet, fluorescence, and circular dichroism) along with dynamic light scattering measurements. The extended stability of IA at 30 °C for 28 days in 1 M [Ch][Dhp] and [Ch][Ace] demonstrated in this study reveals the possibility of stabilizing IA using cholinium-based ILs.
Plastic biodegradation has emerged as a sustainable approach and green alternative in handling the ever-increasing accumulation of plastic wastes in the environment. The complete biodegradation of polyethylene terephthalate is one of the most recent breakthroughs in the field of plastic biodegradation. Despite the success, the effective and complete biodegradation of a wide variety of plastics is still far from the practical implementation, and an on-going effort has been mainly devoted to the exploration of novel microorganisms and enzymes for plastic biodegradation. However, alternative strategies which enhance the existing biodegradation process should not be neglected in the continuous advancement of this field. Thus, this review highlights various strategies which have shown to improve the biodegradation of plastics, which include the pretreatment of plastics using UV irradiation, thermal, or chemical treatments to increase the susceptibility of plastics toward microbial action. Alternative pretreatment strategies are also suggested and compared with the existing techniques. Besides, the effects of additives such as pro-oxidants, natural polymers, and surfactants on plastic biodegradation are discussed. In addition, considerations governing the biodegradation performance, such as the formulation of biodegradation medium, cell-free biocatalysis, and physico-chemical properties of plastics, are addressed. Lastly, the challenges and future prospects for the advancement of plastic biodegradation are also highlighted.
Lactic acid bacteria (LAB) can be isolated from traditional milk products. LAB that secrete substances that inhibit pathogenic bacteria and are resistant to acid, bile, and pepsin but not vancomycin may have potential in food applications.
Dacryodes species are evergreen, perennial trees with fleshy fruits and belong to the family Buseraseae. Many Dacryodes species are underutilized but are widely applied in traditional folk medicine to treat malaria, fever and skin diseases. The nutritional compositions, phytochemicals and biological activities of Dacryodes edulis, Dacryodes rostrata, Dacryodes buettneri, Dacryodes klaineana and Dacryodes hexandra are presented. The edible fruits of D. edulis are rich in lipids, proteins, vitamins, fatty acids and amino acids. Its extracts (leaf, fruit and resin) exhibit antioxidant, anti-microbial, anti-carcinogenic and other bioactivities. D. rostrata fruit has significant nutrient content, and is rich in proteins, lipids and minerals. These fruits are also highly rich in polyphenols, anthocyanins and antioxidant activities. This comprehensive review will assist the reader in understanding the nutritional benefits of Dacryodes species and in identifying current research needs.
Dacryodes rostrata (kembayau) is an important food and oil resource for local communities in Borneo, but it is not commonly known to wider community. The objective of this work is to valorize kembayau fruit by evaluating the characteristics of the oil from the fruit. In this study, the physicochemical characteristics and the lipophilic essential nutrient; the fatty acid composition, vitamin E and beta-carotene content of oils obtained from the peel, pulp and seeds of kembayau fruits were studied. The pulp of the kembayau fruit contained highest proportion of oil, followed by peel and seed. Kembayau fruit contained vitamin E and had trace amount of beta-carotene. Besides, kembayau fruit oils were not toxic to BRL3A cells, provided hepatoprotection and reversed lipid peroxidation in paracetamol-induced toxicity. Our results suggest that kembayau can be a potential source for cooking oil as the physicochemical characteristics are comparable with commercial source such as oil palm.