Enhancing plastic biodegradation process: strategies and opportunities.

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.

Enhanced activity of Candida antarctica lipase B in cholinium aminoate ionic liquids: a combined experimental and computational analysis.

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.

Probing the molecular interactions between cholinium-based ionic liquids and insulin aspart: A combined computational and experimental study.

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.

PERISCOPE-Opt: Machine learning-based prediction of optimal fermentation conditions and yields of recombinant periplasmic protein expressed in Escherichia coli.

Optimization of the fermentation process for recombinant protein production (RPP) is often resource-intensive. Machine learning (ML) approaches are helpful in minimizing the experimentations and find vast applications in RPP. However, these ML-based tools primarily focus on features with respect to amino-acid-sequence, ruling out the influence of fermentation process conditions. The present study combines the features derived from fermentation process conditions with that from amino acid-sequence to construct an ML-based model that predicts the maximal protein yields and the corresponding fermentation conditions for the expression of target recombinant protein in the Escherichia coli periplasm. Two sets of XGBoost classifiers were employed in the first stage to classify the expression levels of the target protein as high (>50 mg/L), medium (between 0.5 and 50 mg/L), or low (<0.5 mg/L). The second-stage framework consisted of three regression models involving support vector machines and random forest to predict the expression yields corresponding to each expression-level-class. Independent tests showed that the predictor achieved an overall average accuracy of 75% and a Pearson coefficient correlation of 0.91 for the correctly classified instances. Therefore, our model offers a reliable substitution of numerous trial-and-error experiments to identify the optimal fermentation conditions and yield for RPP. It is also implemented as an open-access webserver, PERISCOPE-Opt (http://periscope-opt.erc.monash.edu).

In silico screening and heterologous expression of soluble dimethyl sulfide monooxygenases of microbial origin in Escherichia coli.

Sequence-based screening has been widely applied in the discovery of novel microbial enzymes. However, majority of the sequences in the genomic databases were annotated using computational approaches and lacks experimental characterization. Hence, the success in obtaining the functional biocatalysts with improved characteristics requires an efficient screening method that considers a wide array of factors. Recombinant expression of microbial enzymes is often hampered by the undesirable formation of inclusion body. Here, we present a systematic in silico screening method to identify the proteins expressible in soluble form and with the desired biological properties. The screening approach was adopted in the recombinant expression of dimethyl sulfide (DMS) monooxygenase in Escherichia coli. DMS monooxygenase, a two-component enzyme consisting of DmoA and DmoB subunits, was used as a model protein. The success rate of producing soluble and active DmoA is 71% (5 out of 7 genes). Interestingly, the soluble recombinant DmoA enzymes exhibited the NADH:FMN oxidoreductase activity in the absence of DmoB (second subunit), and the cofactor FMN, suggesting that DmoA is also an oxidoreductase. DmoA originated from Janthinobacterium sp. AD80 showed the maximum NADH oxidation activity (maximum reaction rate: 6.6 µM/min; specific activity: 133 µM/min/mg). This novel finding may allow DmoA to be used as an oxidoreductase biocatalyst for various industrial applications. The in silico gene screening methodology established from this study can increase the success rate of producing soluble and functional enzymes while avoiding the laborious trial and error involved in the screening of a large pool of genes available. KEY POINTS: • A systematic gene screening method was demonstrated. • DmoA is also an oxidoreductase capable of oxidizing NADH and reducing FMN. • DmoA oxidizes NADH in the absence of external FMN.

Enhanced structural stability of insulin aspart in cholinium aminoate ionic liquids.

Cholinium aminoates [Ch][AA] have gained tremendous interest as a promising ionic liquid medium for the synthesis and storage of proteins. However, high alkalinity of [Ch][AA] limits its usage with pH-sensitive proteins. Here, we probed the structure, stability, and interactions of a highly unstable therapeutic protein, insulin aspart (IA), in a range of buffered [Ch][AA] (b-[Ch][AA]) using a combination of biophysical tools and in silico pipeline including ultraviolet-visible, fluorescence, and circular dichroism spectroscopies, dynamic light scattering measurements and molecular docking. b-[Ch][AA] used in the study differed in concentrations and their anionic counterparts. We reveal information on ion and residue specific solvent-protein interactions, demonstrating that the structural stability of IA was enhanced by a buffered cholinium prolinate. In comparison to the glycinate and alaninate anions, the hydrophilic prolinate anions established more hydrogen bonds with the residues of IA and provided a less polar environment that favours the preservation of IA in its active monomeric form, opening new opportunities for utilizing [Ch][AA] as storage medium.

Valorisation of carrot peel waste by water-induced hydrocolloidal complexation for extraction of carotene and pectin.

Food processing waste is a potential resource of a variety of bioactive compounds. Carrot peel is a good example of phytonutrient-rich agroindustrial byproducts generated from the processing of carrots. The conventional methods for the extraction of phytonutrients typically involve large volume of organic solvents, complex procedures and expensive equipment. Hence, the development of green and simpler extraction method is advantageous to the valorisation of agroindustrial waste in terms of economic and sustainability. In this study, the applicability of carotene-pectin hydrocolloidal complexation to the co-extraction of carotenoids and pectin from carrot peel waste was evaluated. Carrot peel waste is a potential feedstock for this extraction method because it is rich in carotenoids and pectin, which could form the colloidal complex induced by water. The operating conditions of complexation process were optimized using response surface methodology. The maximum yield and purity of ß-carotenes extracted from carrot peel are 1.17 mg/100 g wet sample and 96%, respectively. In comparison to the conventional solvent extraction method, the number of operating steps in carotene-pectin hydrocolloidal complexation is significantly lower and the antioxidant activity of ß-carotenes was higher. The carotene-pectin hydrocolloidal complexation method is therefore a green extraction method that enables the valorisation of agricultural waste to recover carotenoids.

Stepwise optimization of recombinant protein production in Escherichia coli utilizing computational and experimental approaches.

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.

Kinetic modeling of bacteriocin-like inhibitory substance secretion by Pediococcus acidilactici Kp10 and its stability in food manufacturing conditions.

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.

Valorization of Dacryodes rostrata fruit through the characterization of its oil.

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.

Reusing colored industrial wastewaters in a photofermentation for enhancing biohydrogen production by using ultrasound stimulated Rhodobacter sphaeroides.

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.

Isolation, characterization and the potential use of starch from jackfruit seed wastes as a coagulant aid for treatment of turbid water.

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.

Periscope: quantitative prediction of soluble protein expression in the periplasm of Escherichia coli.

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/.

Towards more accurate prediction of protein folding rates: a review of the existing Web-based bioinformatics approaches.

The understanding of protein-folding mechanisms is often considered to be an important goal that will enable structural biologists to discover the mysterious relationship between the sequence, structure and function of proteins. The ability to predict protein-folding rates without the need for actual experimental work will assist the research work of structural biologists in many ways. Many bioinformatics tools have emerged in the past decade, and each has showcased different features. In this article, we review and compare eight web-based prediction tools that are currently available and that predominantly predict the protein-folding rate. The prediction performance, usability and utility, together with the prediction tool development and validation methodologies for these tools, are critically reviewed. This article is presented in a comprehensible manner to assist readers in the process of selecting the most appropriate bioinformatics tools to meet their needs.

Utilization of plant-based natural coagulants as future alternatives towards sustainable water clarification.

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.

Nutritional compositions and bioactivities of Dacryodes species: a review.

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.

Bioinformatics approaches for improved recombinant protein production in Escherichia coli: protein solubility prediction.

The solubility of recombinant protein expressed in Escherichia coli often represents the production yield. However, up-to-date, instances of successful production of soluble recombinant proteins in E. coli expression system with high yield remain scarce. This is mainly due to the difficulties in improving the overall production capacity, as most of the well-established strategies usually involve a series of trial and error steps with unguaranteed success. One way to concurrently improve the production yield and minimize the production cost would be incorporating the potency of bioinformatics tools to conduct in silico studies, which forecasts the outcome before actual experimental work. In this article, we review and compare seven prediction tools available, which predict the solubility of protein expressed in E. coli, using the following criteria: prediction performance, usability, utility, prediction tool development and validation methodologies. This comprehensive review will be a valuable resource for researchers with limited prior experience in bioinformatics tools. As such, this will facilitate their choice of appropriate tools for studies related to enhancement of intracellular recombinant protein production in E. coli.

Colorimetric quantification of sucrose in presence of thermo-sensitive polymers present in aqueous two-phase systems.

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).

Assessment of molecular recognition element for the quantification of human epidermal growth factor using surface plasmon resonance

Background: A method for the selection of suitable molecular recognition element (MRE) for the quantification of human epidermal growth factor (hEGF) using surface plasmon resonance (SPR) is presented. Two types of hEGF antibody, monoclonal and polyclonal, were immobilized on the surface of chip and validated for its characteristics and performance in the quantification of hEGF. Validation of this analytical procedure was to demonstrate the stability and suitability of antibody for the quantification of target protein. Results: Specificity, accuracy and precision for all samples were within acceptable limit for both antibodies. The affinity and kinetic constant of antibodies-hEGF binding were evaluated using a 1:1 Langmuir interaction model. The model fitted well to all binding responses simultaneously. Polyclonal antibody (pAb) has better affinity (K D = 7.39e-10 M) than monoclonal antibody (mAb) (K D = 9.54e-9 M). Further evaluation of kinetic constant demonstrated that pAb has faster reaction rate during sample injection, slower dissociation rate during buffer injection and higher level of saturation state than mAb. Besides, pAb has longer shelf life and greater number of cycle run. Conclusions: Thus, pAb was more suitable to be used as a stable MRE for further quantification works from the consideration of kinetic, binding rate and shelf life assessment.

Isolation of Pediococcus acidilactici Kp10 with ability to secrete bacteriocin-like inhibitory substance from milk products for applications in food industry.

BACKGROUND: 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. RESULTS: LAB isolated from a range of traditional fermented products were screened for the production of bacteriocin-like inhibitory substances. A total of 222 LAB strains were isolated from fermented milk products in the form of fresh curds, dried curds, and ghara (a traditional flavor enhancer prepared from whey), and fermented cocoa bean. Eleven LAB isolates that produced antimicrobial substances were identified as Lactococcus lactis, Lactobacillus plantarum, and Pediococcus acidilactici strains by biochemical methods and 16S rDNA gene sequencing. Of these, the cell-free supernatant of Kp10 (P. acidilactici) most strongly inhibited Listeria monocytogenes. Further analysis identified the antimicrobial substance produced by Kp10 as proteinaceous in nature and active over a wide pH range. Kp10 (P. acidilactici) was found to be catalase-negative, able to produce ß-galactosidase, resistant to bile salts (0.3%) and acidic conditions (pH 3), and susceptible to most antibiotics. CONCLUSION: Traditionally prepared fermented milk products are good sources of LAB with characteristics suitable for industrial applications. The isolate Kp10 (P. acidilactici) shows potential for the production of probiotic and functional foods.