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

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

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

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

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

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

Isolation and Characterization of a Novel Thermo-solvent-stable Lipase from Pseudomonas Brenneri and its Application in Biodiesel Synthesis

Pseudomonads are one of the most studied species of bacteria as they display remarkable metabolic and physiological versatility. This enables them to colonize a wide variety of terrestrial and aquatic habitats, generating biotechnologically interesting enzymes. Here, the partial purification and characterization of a novel, extracellularly-produced, lipase from Pseudomonas brenneri is described. The partially purified lipase was active over a broad pH range (5.0–9.0) and was stable at 70 °C for 45 min. The lipase displayed significant stability, and in some cases activation, in the presence of organic solvents with log P≥2.0. Such stability characteristics indicated that this lipase could potentially be useful as a biocatalyst for biodiesel production. This was subsequently demonstrated through the facile production of Fatty Acid Methyl Esters in the presence of olive oil and methanol. Possible applications for this novel, stable lipase include the bioremediation of oil in the environment

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

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 AmCut gene (lacking its signal peptide) was expressed in E. coli, all cutinase activity was observed in the cytosolic fraction. However, when AmCut was expressed in E. coli along with its native signal peptide, cutinase activity was observed in the periplasmic space and in the culture medium. This finding revealed that the native signal peptide of a Gram-positive organism (A. mediterranei) 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. Although the periplasmic targeting was surprising, 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