Influence of deep eutectic solvents on redox biocatalysis involving alcohol dehydrogenases.

Redox biocatalysis plays an increasingly important role in modern organic synthesis. The recent integration of novel media such as deep eutectic solvents (DESs) has significantly impacted this field of chemical biology. Alcohol dehydrogenases (ADHs) are important biocatalysts where their unique specificity is used for enantioselective synthesis. This review explores aspects of redox biocatalysis in the presence of DES both with whole cells and with isolated ADHs. In both cases, the presence of DES has a significant influence on the outcome of reactions albeit via different mechanisms. For whole cells, DES was shown to be a useful tool to direct product formation or configuration - a process of solvent engineering. Whole cells can tolerate DES as media components for the solubilization of hydrophobic substrates. In some cases, DES in the growth medium altered the enantioselectivity of whole cell transformations by solvent control. For isolated enzymes, on the other hand, the presence of DES promotes substrate solubility as well as enhancing enzyme stability and activity. DES can be employed as a smart solvent or smart cosubstrate particularly for cofactor regeneration purposes. From the literatures examined, it is suggested that DES based on choline chloride (ChCl) such as ChCl:Glycerol (Gly), ChCl:Glucose (Glu), and ChCl:1,4-butanediol (1,4-BD) are useful starting points for ADH-based redox biocatalysis. However, each specific reaction will require optimisation due to the influence of several factors on biocatalysis in DES. These include solvent composition, enzyme source, temperature, pH and ionic strength as well as the substrates and products under investigation.

Approaches to Avoid Proteolysis During Protein Expression and Purification.

All cells contain proteases, which hydrolyze the peptide bonds between amino acids of a protein backbone. Typically, proteases are prevented from nonspecific proteolysis by regulation and by their physical separation into different subcellular compartments; however, this segregation is not retained during cell lysis, which is the initial step in any protein isolation procedure. Prevention of proteolysis during protein purification often takes the form of a two-pronged approach: first, inhibition of proteolysis in situ, followed by the early separation of the protease from the protein of interest via chromatographic purification. Protease inhibitors are routinely used to limit the effect of the proteases before they are physically separated from the protein of interest via column chromatography. In this chapter, commonly used approaches to reducing or avoiding proteolysis during protein expression and purification are reviewed.

Protein Extraction and Purification by Differential Solubilization.

The preparation of purified soluble proteins for biochemical studies is essential and the solubility of a protein of interest in various media is central to this process. Selectively altering the solubility of a protein is a rapid and economical step in protein purification and is based on exploiting the inherent physicochemical properties of a polypeptide. Precipitation of proteins, released from cells upon lysis, is often used to concentrate a protein of interest before further purification steps (e.g., ion exchange chromatography, size exclusion chromatography etc).Recombinant proteins may be expressed in host cells as insoluble inclusion bodies due to various influences during overexpression. Such inclusion bodies can often be solubilized to be reconstituted as functional, correctly folded proteins.In this chapter, we examine strategies for extraction/precipitation/solubilization of proteins for protein purification. We also present bioinformatic tools to aid in understanding a protein's propensity to aggregate/solubilize that will be a useful starting point for the development of protein extraction, precipitation, and selective re-solubilization procedures.

Listeria monocytogenes is a solvent tolerant organism secreting a solvent stable lipase: potential biotechnological applications.

PURPOSE: The emerging biobased economy will require robust, adaptable, organisms for the production and processing of biomaterials as well as for bioremediation. Recently, the search for solvent tolerant organisms and solvent tolerant enzymes has intensified. Resilient organisms secreting solvent stable lipases are of particular interest for biotechnological applications. METHODS: Screening of soil samples for lipase-producing organisms was carried out on Rhodamine B plates. The most productive lipase-producing organisms were further screened for their resistance to solvents commonly used in biotechnological applications. RESULTS: In the course of screening, one of the isolated organisms that exhibited extracellular lipase activity, was identified as the human pathogen Listeria monocytogenes through 16S rRNA sequencing. Further exploration revealed that this organism was resistant to solvents ranging from log P - 0.81 to 4.0. Moreover, in the presence of these solvents, L. monocytogenes secreted an extracellular, solvent tolerant, lipase activity. This lipase retained approximately 80% activity when incubated in 30% (v/v) methanol for 24 h. CONCLUSION: These findings identify L. monocytogenes as a potentially useful organism for biotechnological applications. However, the fact that Listeria is a pathogen is problematic and it will require the use of non-pathogenic or attenuated Listeria strains for practical applications. Nonetheless, the ability to adapt to rapidly changing environmental conditions, to grow at low temperatures, to resist solvents and to secrete an extracellular solvent tolerant lipase are unique and highly useful characteristics. The potential application of L. monocytogenes in wastewater bioremediation and plastics degradation is discussed.

Extracellular secretion of a cutinase with polyester-degrading potential by E. coli using a novel signal peptide from Amycolatopsis mediterranei.

Recent studies in this laboratory showed that an extracellular cutinase from A. mediterranei (AmCut) was able to degrade the plastics polycaprolactone and polybutylene succinate. Such plastics can be slow to degrade in soils due to a lack of efficient polyester degrading organisms. AmCut also showed potential for the biocatalytic synthesis of esters by reverse hydrolysis. The gene for AmCut has an upstream leader sequence whose transcript is not present in the purified enzyme. In this study, we show using predictive modelling, that this sequence codes for an N-terminal signal peptide that directs transmembrane expression via the Sec secretion pathway. E. coli is a useful host for recombinant enzymes used in biocatalysis due to the ease of genetic manipulation in this organism, which allows tuning of enzymes for specific applications, by mutagenesis. When a truncated GST-tagged AmCut gene (lacking its signal peptide) was expressed in E. coli, all cutinase activity was observed in the cytosolic fraction. However, when GST-tagged AmCut was expressed in E. coli along with its native signal peptide, cutinase activity was observed in both the periplasmic space and the culture medium. This finding revealed that the native signal peptide of a Gram-positive organism (AmCut) was being recognised by the Gram-negative (E. coli) Sec transmembrane transport system. AmCut was transported into E. coli's periplasmic space from where it was released into the culture medium. Surprisingly, the presence of a bulky GST tag at the N-terminus of the signal peptide did not hinder transmembrane targeting. Although the periplasmic targeting was unexpected, it is not unprecedented due to the conservation of the Sec pathway across species. It was more surprising that AmCut was secreted from the periplasmic space into the culture medium. This suggests that extracellular AmCut translocation across the E. coli outer membrane may involve non-classical secretion pathways. This tuneable recombinant E. coli expressing extracellular AmCut may be useful for degradation of polyester substrates in the environment; this and other applications are discussed.

Novel Facet of an Old Dietary Molecule? Direct Influence of Caffeine on Glucose and Biogenic Amine Handling by Human Adipocytes.

Caffeine is a plant alkaloid present in food and beverages consumed worldwide. It has high lipid solubility with recognized actions in the central nervous system and in peripheral tissues, notably the adipose depots. However, the literature is scant regarding caffeine's influence on adipocyte functions other than lipolysis, such as glucose incorporation into lipids (lipogenesis) and amine oxidation. The objective of this study was to explore the direct effects of caffeine and of isobutylmethylxanthine (IBMX) on these adipocyte functions. Glucose transport into fat cells freshly isolated from mice, rats, or humans was monitored by determining [3H]-2-deoxyglucose (2-DG) uptake, while the incorporation of radiolabeled glucose into cell lipids was used as an index of lipogenic activity. Oxidation of benzylamine by primary amine oxidase (PrAO) was inhibited by increasing doses of caffeine in human adipose tissue preparations with an inhibition constant (Ki) in the millimolar range. Caffeine inhibited basal and insulin-stimulated glucose transport as well as lipogenesis in rodent adipose cells. The antilipogenic action of caffeine was also observed in adipocytes from mice genetically invalidated for PrAO activity, indicating that PrAO activity was not required for lipogenesis inhibition. These caffeine inhibitory properties were extended to human adipocytes: relative to basal 2-DG uptake, set at 1.0 ± 0.2 for 6 individuals, 0.1 mM caffeine tended to reduce uptake to 0.83 ± 0.08. Insulin increased uptake by 3.86 ± 1.11 fold when tested alone at 100 nM, and by 3.21 ± 0.80 when combined with caffeine. Our results reinforce the recommendation of caffeine's potential in the treatment or prevention of obesity complications.

An extracellular lipase from Amycolatopsis mediterannei is a cutinase with plastic degrading activity.

An extracellular lipase from Amycolatopsis mediteranei (AML) with potential applications in process biotechnology was recently cloned and examined in this laboratory. In the present study, the 3D structure of AML was elucidated by comparative modelling. AML lacked the 'lid' structure observed in most true lipases and shared similarities with plastic degrading enzymes. Modelling and substrate specificity studies showed that AML was a cutinase with a relatively exposed active site and specificity for medium chain fatty acyl moieties. AML rapidly hydrolysed the aliphatic plastics poly(ε-caprolactone) and poly(1,4-butylene succinate) extended with 1,6-diisocyanatohexane under mild conditions. These plastics are known to be slow to degrade in landfill. Poly(L-lactic acid) was not hydrolysed by AML, nor was the aromatic plastic Polyethylene Terephthalate (PET). The specificity of AML is partly explained by active site topology and analysis reveals that minor changes in the active site region can have large effects on substrate preference. These findings show that extracellular Amycolatopsis enzymes are capable of degrading a wider range of plastics than is generally recognised. The potential for application of AML in the bioremediation of plastics is discussed.

Methylxanthines Inhibit Primary Amine Oxidase and Monoamine Oxidase Activities of Human Adipose Tissue.

Background: Methylxanthines including caffeine and theobromine are widely consumed compounds and were recently shown to interact with bovine copper-containing amine oxidase. To the best of our knowledge, no direct demonstration of any interplay between these phytochemicals and human primary amine oxidase (PrAO) has been reported to date. We took advantage of the coexistence of PrAO and monoamine oxidase (MAO) activities in human subcutaneous adipose tissue (hScAT) to test the interaction between several methylxanthines and these enzymes, which are involved in many key pathophysiological processes. Methods: Benzylamine, methylamine, and tyramine were used as substrates for PrAO and MAO in homogenates of subcutaneous adipose depots obtained from overweight women undergoing plastic surgery. Methylxanthines were tested as substrates or inhibitors by fluorimetric determination of hydrogen peroxide, an end-product of amine oxidation. Results: Semicarbazide-sensitive PrAO activity was inhibited by theobromine, caffeine, and isobutylmethylxanthine (IBMX) while theophylline, paraxanthine, and 7-methylxanthine had little effect. Theobromine inhibited PrAO activity by 54% at 2.5 mM. Overall, the relationship between methylxanthine structure and the degree of inhibition was similar to that seen with bovine PrAO, although higher concentrations (mM) were required for inhibition. Theobromine also inhibited oxidation of tyramine by MAO, at the limits of its solubility in a DMSO vehicle. At doses higher than 12 % v/v, DMSO impaired MAO activity. MAO was also inhibited by millimolar doses of IBMX, caffeine and by other methylxanthines to a lesser extent. Conclusions: This preclinical study extrapolates previous findings with bovine PrAO to human tissues. Given that PrAO is a potential target for anti-inflammatory drugs, it indicates that alongside phosphodiesterase inhibition and adenosine receptor antagonism, PrAO and MAO inhibition could contribute to the health benefits of methylxanthines, especially their anti-inflammatory effects.

The Statistical Optimisation of Recombinant ß-glucosidase Production through a Two-Stage, Multi-Model, Design of Experiments Approach.

ß-glucosidases are a class of enzyme that are widely distributed in the living world, with examples noted in plants, fungi, animals and bacteria. They offer both hydrolysis and synthesis capacity for a wide range of biotechnological processes. However, the availability of native, or the production of recombinant ß-glucosidases, is currently a bottleneck in the widespread industrial application of this enzyme. In this present work, the production of recombinant ß-glucosidase from Streptomyces griseus was optimised using a Design of Experiments strategy, comprising a two-stage, multi-model design. Three screening models were comparatively employed: Fractional Factorial, Plackett-Burman and Definitive Screening Design. Four variables (temperature, incubation time, tryptone, and OD600 nm) were experimentally identified as having statistically significant effects on the production of S.griseus recombinant ß-glucosidase in E. coli BL21 (DE3). The four most influential variables were subsequently used to optimise recombinant ß-glucosidase production, employing Central Composite Design under Response Surface Methodology. Optimal levels were identified as: OD600 nm, 0.55; temperature, 26 °C; incubation time, 12 h; and tryptone, 15 g/L. This yielded a 2.62-fold increase in recombinant ß-glucosidase production, in comparison to the pre-optimised process. Affinity chromatography resulted in homogeneous, purified ß-glucosidase that was characterised in terms of pH stability, metal ion compatibility and kinetic rates for p-nitrophenyl-ß-D-glucopyranoside (pNPG) and cellobiose catalysis.

Theobromine and related methylxanthines as inhibitors of Primary Amine Oxidase.

Methylxanthines are among the most widely consumed drugs in the world and evidence of their health benefits has been growing in recent years. Primary Amine Oxidase (PrAO) has been recognized as a therapeutic target for the amelioration of inflammatory, vascular, and neurodegenerative diseases. Previous work in our laboratories showed that caffeine inhibited Bovine PrAO with a Ki of 1.0 mM using benzylamine as substrate. This study aimed to extend our previous work and explore the possibility that related methylxanthines might influence PrAO activity. While paraxanthine, theophylline, and 7-methylxanthine had little effect on PrAO, theobromine was a noncompetitive inhibitor with a Ki of 276 ± 44 µM. The specific structural elements of methylxanthines that are required for inhibition allow us to suggest that their binding site on PrAO may be a target for therapeutics. The health benefits associated with dietary methylxanthine consumption could involve PrAO inhibition. PRACTICAL APPLICATIONS: Inhibition of PrAO by methylxanthines may be significant in conferring health benefits. The design of PrAO inhibitors based on the structural motifs identified in this study (N-methylation at specific locations) is indicated. Existing therapeutics based on a core xanthine structure can be evaluated for their effects on PrAO. PrAO inhibition must be considered as a potential mediator of the beneficial health effects of some methylxanthines. If inhibition in human tissues is comparable to, or greater than, that found in these studies it points to an important role for these compounds in human health.

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 24 h 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.

Solvent stable microbial lipases: current understanding and biotechnological applications.

OBJECTIVE: This review examines on our current understanding of microbial lipase solvent tolerance, with a specific focus on the molecular strategies employed to improve lipase stability in a non-aqueous environment. RESULTS: It provides an overview of known solvent tolerant lipases and of approaches to improving solvent stability such as; enhancing stabilising interactions, modification of residue flexibility and surface charge alteration. It shows that judicious selection of lipase source supplemented by appropriate enzyme stabilisation, can lead to a wide application spectrum for lipases. CONCLUSION: Organic solvent stable lipases are, and will continue to be, versatile and adaptable biocatalytic workhorses commonly employed for industrial applications in the food, pharmaceutical and green manufacturing industries.

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.

Avoiding Proteolysis During Protein Purification.

All cells contain proteases which hydrolyze the peptide bonds between amino acids in a protein backbone. Typically, proteases are prevented from nonspecific proteolysis by regulation and by their physical separation into different subcellular compartments; however, this segregation is not retained during cell lysis, which is the initial step in any protein isolation procedure. Prevention of proteolysis during protein purification often takes the form of a two-pronged approach; firstly inhibition of proteolysis in situ, followed by the early separation of the protease from the protein of interest via chromatographical purification. Protease inhibitors are routinely used to limit the effect of the proteases before they are physically separated from the protein of interest via column chromatography. Here, commonly used approaches to reducing or avoiding proteolysis during protein purification and subsequent chromatography are reviewed.

New inhibitors of the Kvß2 subunit from mammalian Kv1 potassium channels.

The role of the redox state of Kvß subunits in the modulation of Kv1 potassium channels has been well documented over the past few years. It has been suggested that a molecule that binds to or inhibits the aldo-keto reductase activity of Kvß might affect the modulation of channel properties. Previous studies of possible modulators of channel activity have shown that cortisone and some related compounds are able to physically dissociate the channel components by binding to a site at the interface between α and ß subunits. Herein, we describe some new inhibitors of rat brain Kvß2, identified using an assay based on multiple substrate turnover. This approach allows one to focus on molecules that specifically block NADPH oxidation. These studies showed that, at 0.5mM, 3,4-dihydroxphenylacetic acid (DOPAC) was an inhibitor of Kvß2 turnover yielding a ∼ 40-50% reduction in the aldehyde reductase activity of this subunit. Other significant inhibitors include the bioflavinoid, rutin and the polyphenol resveratrol; some of the known cardioprotective effects of these molecules may be attributable to Kv1 channel modulation. Cortisone or catechol caused moderate inhibition of Kvß2 turnover, and the aldo-keto reductases inhibitor valproate had an even smaller effect. Despite the importance of the Kv1 channels in a number of disease states, there have been few Kvß2 inhibitors reported. While the ones identified in this study are only effective at high concentrations, they could serve as tools to decipher the role of Kvß2 in vivo and, eventually, inform the development of novel therapeutics.

A short chain NAD(H)-dependent alcohol dehydrogenase (HpSCADH) from Helicobacter pylori: a role in growth under neutral and acidic conditions.

Toxic aldehydes produced by alcohol dehydrogenases have been implicated in the pathogenesis of Helicobacter pylori-related damage to the gastric mucosa. Despite this, the enzymes that might be responsible for producing such aldehydes have not been fully described. It was, therefore, of considerable interest to characterize the alcohol oxidizing enzymes in this pathogen. Previous work in this laboratory characterized two such H. pylori enzymes that had broad specificity for a range of aromatic alcohol substrates. However, an enzyme with specificity for aliphatic alcohols is likely to be required in order that H. pylori can metabolize the wide range of substrates encountered in the gastric mucosa. In this study we describe HpSCADH, an alcohol dehydrogenase from H. pylori 26695 with broad specificity for aliphatic alcohols. HpSCADH was classified in the cD1e subfamily of classical short chain alcohol dehydrogenases. The enzyme was a monomer of approximately 29kDa with a preference for NAD(+) as cofactor. Pyrazole was found to be a competitive inhibitor of HpSCADH. The physiological role of this enzyme was explored by construction of an HpSCADH isogenic mutant. At pH 7.0 the mutant showed reduced growth which became more pronounced when the pH was lowered to 5.0. When pyrazole was added to wild type H. pylori cells it caused growth profiles to be reduced to match those of the isogenic mutant suggesting that HpSCADH inhibition alone was responsible for growth impairment. Taken together, the data relating to the alcohol metabolizing enzymes of this pathogen indicate that they play an important role in H. pylori growth and adaptation to acidic environments. The therapeutic potential of targeting H. pylori alcohol dehydrogenases is discussed.

Overview of approaches to preventing and avoiding proteolysis during expression and purification of proteins.

Proteases are enzymes that cleave proteins. They occur widely in nature and play a fundamental role in cellular homeostasis; however, their presence can result in unwanted protein degradation during recombinant protein expression and purification. This unit introduces proteases, specifically focusing on the types commonly encountered during production of recombinant proteins. The strategies used to avoid and to prevent proteolysis are also described, with extensive consideration of the molecular, technical, and logistic methodologies involved.

Occupational noise exposure of nightclub bar employees in Ireland.

Due to the transposition of the EU Directive 2003/10/EC into Irish Law, the entertainment sector was obligated to comply with the requirements of the Safety, Health and Welfare at Work (General Application) Regulations 2007, Chapter 1 Part 5: Control of Noise at Work since February 2008. Compliance with the Noise Regulations was examined in 9 nightclubs in Ireland. The typical daily noise exposure of 19 bar employees was measured using 2 logging dosimeters and a Type 1 fixed position sound level meter. Physical site inspections identified nightclub noise control measures. Interviews and questionnaires were used to assess the managers and employees awareness of the noise legislation. The average bar employee daily noise exposure (L(EX, 8h)) was 92 dBA, almost 4 times more than the accepted legal limit. None of the venues examined were fully compliant with the requirements of the 2007 Noise Regulations, and awareness of this legislation was limited.

Purification and properties of Amycolatopsis mediterranei DSM 43304 lipase and its potential in flavour ester synthesis.

An extracellular thermostable lipase from Amycolatopsis mediterranei DSM 43304 has been purified to homogeneity using ammonium sulphate precipitation followed by anion exchange chromatography and hydrophobic interaction chromatography. This protocol resulted in a 398-fold purification with 36% final recovery. The purified A. mediterranei DSM 43304 lipase (AML) has an apparent molecular mass of 33 kDa. The N-terminal sequence, AANPYERGPDPTTASIEATR, showed highest similarity to a lipase from Streptomyces exfoliatus. The values of K(m)(app) and V(max)(app) for p-nitrophenyl palmitate (p-NPP) at the optimal temperature (60°C) and pH (8.0) were 0.099±0.010 mM and 2.53±0.06 mmol/min mg, respectively. The purified AML displayed significant activity towards a range of short and long chain triglyceride substrates and p-nitrophenyl esters. Hydrolysis of glycerol ester bonds occurred non-specifically. The purified AML displayed significant stability in the presence of organic solvents (40%, v/v) and catalyzed the synthesis of the flavour ester isoamyl acetate in free and immobilized states.