Differential Precipitation and Solubilisation of Proteins

Differential protein precipitation is a rapid and economical step in protein purification and is based on exploiting the inherent physicochemical properties of the polypeptide. Precipitation of recombinant proteins, lysed from the host cell, is commonly used to concentrate the protein of choice before further polishing steps with more selective purification columns (e.g., His-Tag, Size Exclusion, etc.). Recombinant proteins can also precipitate naturally as inclusion bodies due to various influences during overexpression in the host cell. Although this phenomenon permits easier initial separation from native proteins, these inclusion bodies must carefully be differentially solubilized so as to reform functional, correctly folded proteins. Here, appropriate bioinformatics tools to aid in understanding a protein’s propensity to aggregate and solubilize are explored as a backdrop for a typical protein extraction, precipitation, and selective resolubilization procedure, based on a recombinantly expressed protein

Eight steps to facilitating more equitable education in undergraduate sciences

Pedagogical practices can influence students’ confidence and ability beliefs and affect their ambition to persevere in science. Given the continuing need to diversify science and retain students in scientific programmes, science education must be tailored to cater to the needs of varied student groups. Since early experience in university programmes can be decisive in determining students’ further academic and professional choices, pedagogies employed in undergraduate science courses can be particularly influential in supporting science careers. Undergraduate science instructors are therefore encouraged to consider their approaches to teaching and learning from a variety of perspectives that could help empower students from under-represented groups

Expertise and Bias in Political Communication

Citizens minimize information costs by obtaining political information and guidance from other individuals who have assumed the costs of acquiring and processing political information. A problem occurs because ideal informants, characterized by the joint presence of political expertise and shared viewpoints, are often unavailable or rare within the groups where individuals are located. Hence, individuals must often look beyond their own group boundaries to find such individuals, but obtaining information from individuals located beyond their own groups produces additional information costs. Moreover, the availability of ideal informants varies across groups and settings, with the potential to produce (1) biases in favor of some groups at the expense of others, (2) varying levels of polarization among groups, and (3) context dependent patterns of informant centrality. The paper’s analysis is based on a series of small group experiments, each of which involves two groups of seven subjects who communicate with one another via networked computers in order to obtain information on candidates. The aggregate implications of the experimental results are analyzed using an agent-based model

The Goldilocks Approach: A Review of Employing Design of Experiments in Prokaryotic Recombinant Protein Production

The production of high yields of soluble recombinant protein is one of the main objectives of protein biotechnology. Several factors, such as expression system, vector, host, media composition and induction conditions can influence recombinant protein yield. Identifying the most important factors for optimum protein expression may involve significant investment of time and considerable cost. To address this problem, statistical models such as Design of Experiments (DoE) have been used to optimise recombinant protein production. This review examines the application of DoE in the production of recombinant proteins in prokaryotic expression systems with specific emphasis on media composition and culture conditions. The review examines the most commonly used DoE screening and optimisation designs. It provides examples of DoE applied to optimisation of media and culture conditions

Enzymatic in-situ transesterification of neutral lipids from simulated wastewater cultured Chlorella emersonii and Pseudokirchneriella subcapitata to sustainably produce fatty acid methyl esters

Alternative, more sustainable and environmentally positive, sources of energy are one of the current global challenges. One approach to achieving more sustainable sources of energy is to use waste from one system as a raw material for energy production, following the circular biosystem philosophy. This study successfully adopted this approach whereby microalgae strains Chlorella emersonii and Pseudokirchneriella subcapitata were bioprospected and metabolically engineered in simulated wastewater supplemented with glucose to produce neutral lipids. Using a two-step cultivation approach neutral lipid content was enhanced in Chlorella emersonii and Pseudokirchneriella subcapitata biomass. Via in-situ transesterification, these neutral lipids were subsequently bioconverted to biodiesel feedstock fatty acid methyl esters using novel solvent stable lipase(s) from Pseudomonas reinekei and Pseudomonas brenneri. The culturing of appropriate microalgae on wastewater, and bioconversion via organo-stable lipases may provide a commercially viable and sustainable biodiesel feedstock to help address the current global energy challenge

The Goldilocks Approach: A Review of Employing Design of Experiments in Prokaryotic Recombinant Protein Production

The production of high yields of soluble recombinant protein is one of the main objectives of protein biotechnology. Several factors, such as expression system, vector, host, media composition and induction conditions can influence recombinant protein yield. Identifying the most important factors for optimum protein expression may involve significant investment of time and considerable cost. To address this problem statistical models, such as Design of Experiments (DoE), have been used to optimise recombinant protein production. This review explores the application of DoE in the production of recombinant proteins, focusing on prokaryotic expression systems with a specific emphasis on media composition and culture conditions. The review examines the most commonly used DoE screening and optimisation methods, including factorial and screening designs. It provides examples of DoE informed media optimisation and culture condition optimisation. The review concludes with a consideration of the benefits of the application of DoE in recombinant protein production

Isolation, Purification and Characterization of a Novel Solvent Stable Lipase From Pseudomonas Reinekei

The Pseudomonas sp. have been long recognized for their exogenous lipolytic activities yet the genus still contains a lot of unexplored strains. Due to the versatile metabolic machinery and their potential for adaptation to fluctuating environmental conditions Pseudomonas sp. are of great interest for biotechnological applications. In this study, a new extracellularly produced lipolytic enzyme from Pseudomonas sp. (P. reinekei) was purified and characterized. The production of lipase from P. reinekei (H1) was enhanced 10-fold by optimizing the nitrogen source. The 50 kDa H1 lipase was purified using negative and positive mode anion exchange chromatography. The purified lipase was active over a broad pH range (5.0-9.0) and was stable for 24h at 40°C. The lipase showed significant stability, and indeed activation, in the presence of organic solvents with log P≥ 2.0. These features render this lipase of interest as a biocatalyst for applications such as biodiesel production, detergent formulations and biodegradation of oil in the environment

The Effect of Calcium Alginate Entrapment on the Stability of Novel Lipases from P. Reinekei and P. brenneri

The high cost of soluble enzymes can limit their use for commercial and industrial purposes. Immobilization can enhance enzyme reusability, thereby reducing product isolation costs and overcoming this economic barrier. In the current study, two novel, purified lipases from Pseudomonas sp. (Pseudomonas reinekei and Pseudomonas brenneri) were entrapped in a calcium alginate matrix, with the aim of simultaneously enhancing enzyme reusability and stability. Following entrapment, the retained activity of the enzyme-alginate composite was verified by an enzymatic hydrolysis reaction of a p-nitrophenol palmitate substrate. The effect of the enzyme-alginate entrapment against various physiochemical parameters such as pH, temperature, metal ions, and solvents were subsequently examined. The entrapment was found to have minimal beneficial stability gains. However, enhanced enzyme reusability (up to 3 cycles) and stability (up to 18 days at 4°C) of the calcium alginate entrapped lipase, as indicated by residual hydrolysis of p-nitrophenol palmitate, was observed, suggesting potential roles for calcium alginate entrapped lipases in cost efficient enzyme catalysis. HIGHLIGHTS•Two novel lipases have been entrapped in calcium alginate for the first time.•A statistically enhanced stability in 1M EDTA was observed following entrapment.•The novel entrapped lipases display excellent storage stability and reusability

β-glucosidase from Streptomyces griseus: ester hydrolysis and alkyl glucoside synthesis in the presence of Deep Eutectic Solvents

Deep Eutectic Solvents (DES) are ionic liquid analogs that have attracted considerable attention as green solvents for biocatalytic transformations. The use of DES as part of a ‘solvent engineering’ approach to enhance enzyme stability holds great promise since they are biodegradeable, relatively inexpensive and environmentally safe media for enzyme reactions. However, the behaviour of specific enzymes in such solvents is complex; some enzymes are inhibited in DES, while others appear to be activated. Glucosidases are among the most widely used enzymes for commercial chemoenzymatic synthesis. In particular, their application in the synthesis of biodegradable alkyl glucosides by reverse hydrolysis is of great interest. Previous work in this laboratory identified Streptomyces griseus glucosidase (Sgβgl) as an interesting enzyme for biotechnological applications. In this study, we examined its behaviour in the presence of DES as a co-solvent using choline chloride as hydrogen bond donor and using glycerol, glucose and urea as hydrogen bond acceptors. We show that Sgβgl activity depends on both the nature of DES components, and their ratio in the eutectic mixture, as well as the water content of the reaction medium. A choline chloride/glycerol DES eutectic mixture at a level of 40% (v/v) caused activation of Sgβgl and increased its optimum temperature from 70 to 80oC: it also led to a striking increase in its thermostability, doubling its half-life at 60oC and almost tripling its half-life at 80oC. The synthesis of alkyl glucosides was explored using DES as a co-solvent. In the presence of DES, Sgβgl catalysed the formation of a range of alkyl glucosides. The presence of DES resulted in enhanced product yield, which was observed to increase with increasing temperature, up to 60oC. These studies show that the application of DES at relatively low % (v/v) levels can dramatically effect enzyme activity and stability. Specifically, enhanced thermostability can significantly increase the operating range for glucosidases for biocatalytic applications. Solvent engineering offers a simple and effective way to enhance glucosidase stability and will be useful as an alternative and/or adjunct to more complex methods such as immobilisation or protein engineering

Cutinase from Amycolatopsis mediterannei: marked activation and stabilisation in Deep Eutectic Solvents

Amycolatopsis mediterranei cutinase (AmCut) has potential biocatalytic applications in plastics degradation and ester synthesis. Deep Eutectic Solvents (DES) are next generation biodegradable solvents for biocatalysis. However, the behaviour of cutinase enzymes in DES is little studied. Herein, we examine the effect of selected DES, and their components, on AmCut activity and stability. Low amounts (10% v/v) of DES (choline chloride:glycerol; 1:1 mole ratio) caused striking activation of AmCut (over 2-fold). Further examination showed that the choline chloride component of DES caused the observed activation. This is the first report of activation of a cutinase by a small molecule. At higher concentrations (50% v/v), DES composed of a choline chloride with glycerol as hydrogen bond donor dramatically increased the thermostability of AmCut - the enzyme lost no activity after incubation at 50oC for 2 hours. The biotechnological utility and physiological relevance of choline chloride activation and stabilisation is discussed