Characterisation of a hyperthermophilic transketolase from Thermotoga maritima DSM3109 as a biocatalyst for 7-keto-octuronic acid synthesis.

Transketolase (TK) is a fundamentally important enzyme in industrial biocatalysis which carries out a stereospecific carbon-carbon bond formation, and is widely used in the synthesis of prochiral ketones. This study describes the biochemical and molecular characterisation of a novel and unusual hyperthermophilic TK from Thermotoga maritima DSM3109 (TKtmar). TKtmar has a low protein sequence homology compared to the already described TKs, with key amino acid residues in the active site highly conserved. TKtmar has a very high optimum temperature (>90 °C) and shows pronounced stability at high temperature (e.g. t1/2 99 and 9.3 h at 50 and 80 °C, respectively) and in presence of organic solvents commonly used in industry (DMSO, acetonitrile and methanol). Substrate screening showed activity towards several monosaccharides and aliphatic aldehydes. In addition, for the first time, TK specificity towards uronic acids was achieved with TKtmar catalysing the efficient conversion of d-galacturonic acid and lithium hydroxypyruvate into 7-keto-octuronic acid, a very rare C8 uronic acid, in high yields (98%, 49 mM).

Enzymatic synthesis of chiral amino-alcohols by coupling transketolase and transaminase-catalyzed reactions in a cascading continuous-flow microreactor system.

Rapid biocatalytic process development and intensification continues to be challenging with currently available methods. Chiral amino-alcohols are of particular interest as they represent key industrial synthons for the production of complex molecules and optically pure pharmaceuticals. (2S,3R)-2-amino-1,3,4-butanetriol (ABT), a building block for the synthesis of protease inhibitors and detoxifying agents, can be synthesized from simple, non-chiral starting materials, by coupling a transketolase- and a transaminase-catalyzed reaction. However, until today, full conversion has not been shown and, typically, long reaction times are reported, making process modifications and improvement challenging. In this contribution, we present a novel microreactor-based approach based on free enzymes, and we report for the first time full conversion of ABT in a coupled enzyme cascade for both batch and continuous-flow systems. Using the compartmentalization of the reactions afforded by the microreactor cascade, we overcame inhibitory effects, increased the activity per unit volume, and optimized individual reaction conditions. The transketolase-catalyzed reaction was completed in under 10 min with a volumetric activity of 3.25 U ml-1 . Following optimization of the transaminase-catalyzed reaction, a volumetric activity of 10.8 U ml-1 was attained which led to full conversion of the coupled reaction in 2 hr. The presented approach illustrates how continuous-flow microreactors can be applied for the design and optimization of biocatalytic processes.

Novel Computational Protocols for Functionally Classifying and Characterising Serine Beta-Lactamases.

Beta-lactamases represent the main bacterial mechanism of resistance to beta-lactam antibiotics and are a significant challenge to modern medicine. We have developed an automated classification and analysis protocol that exploits structure- and sequence-based approaches and which allows us to propose a grouping of serine beta-lactamases that more consistently captures and rationalizes the existing three classification schemes: Classes, (A, C and D, which vary in their implementation of the mechanism of action); Types (that largely reflect evolutionary distance measured by sequence similarity); and Variant groups (which largely correspond with the Bush-Jacoby clinical groups). Our analysis platform exploits a suite of in-house and public tools to identify Functional Determinants (FDs), i.e. residue sites, responsible for conferring different phenotypes between different classes, different types and different variants. We focused on Class A beta-lactamases, the most highly populated and clinically relevant class, to identify FDs implicated in the distinct phenotypes associated with different Class A Types and Variants. We show that our FunFHMMer method can separate the known beta-lactamase classes and identify those positions likely to be responsible for the different implementations of the mechanism of action in these enzymes. Two novel algorithms, ASSP and SSPA, allow detection of FD sites likely to contribute to the broadening of the substrate profiles. Using our approaches, we recognise 151 Class A types in UniProt. Finally, we used our beta-lactamase FunFams and ASSP profiles to detect 4 novel Class A types in microbiome samples. Our platforms have been validated by literature studies, in silico analysis and some targeted experimental verification. Although developed for the serine beta-lactamases they could be used to classify and analyse any diverse protein superfamily where sub-families have diverged over both long and short evolutionary timescales.

Mechanoenzymatic reactions for the hydrolysis of PET.

Recent advances in the enzymatic degradation of poly(ethylene terphthalate) (PET) have led to a number of PET hydrolytic enzymes and mutants being developed. With the amount of PET building up in the natural world, there is a pressing need to develop scalable methods of breaking down the polymer into its monomers for recycling or other uses. Mechanoenzymatic reactions have gained traction recently as a green and efficient alternative to traditional biocatalytic reactions. For the first time we report increased yields of PET degradation by whole cell PETase enzymes by up to 27-fold by utilising ball milling cycles of reactive aging, when compared with typical solution-based reactions. This methodology leads to up to a 2600-fold decrease in the solvent required when compared with other leading degradation reactions in the field and a 30-fold decrease in comparison to reported industrial scale PET hydrolysis reactions.

The impact and effectiveness of the general public wearing masks to reduce the spread of pandemics in the UK: a multidisciplinary comparison of single-use masks versus reusable face masks.

During the coronavirus (COVID-19) pandemic, the UK government mandated the use of face masks in various public settings and recommended the use of reusable masks to combat shortages of medically graded single-use masks in healthcare. To assist decision-making on the choice of masks for future pandemics, where shortages may not be a contributing factor, the University College London (UCL) Plastic Waste Innovation Hub has carried out a multidisciplinary comparison between single-use and reusable masks based on their anatomy, standalone effectiveness, behavioural considerations, environmental impact and costs. Although current single-use masks have a higher standalone effectiveness against bacteria and viruses, studies show that reusable masks have adequate performance in slowing infection rates of respiratory viruses. Material flow analysis (MFA), life cycle assessment (LCA) and cost comparison show that reusable masks have a lower environmental and economic impact than single-use masks. If every person in the UK uses one single-use mask each day for a year, it will create a total of 124,000 tonnes of waste, 66,000 tonnes of which would be unrecyclable contaminated plastic waste (the masks), with the rest being the recyclable packaging typically used for transportation and distribution of masks. Using reusable masks creates >85% less waste, generates 3.5 times lower impact on climate change and incurs 3.7 times lower costs. Further behavioural research is necessary to understand the extent and current practices of mask use; and how these practices affect mask effectiveness in reducing infection rates. Wearing single-use masks may be preferred over reusable masks due to perceptions of increased hygiene and convenience. Understanding behaviour towards the regular machine-washing of reusable masks for their effective reuse is key to maximise their public health benefits and minimise environmental and economic costs.

Insights into proteolytic processing of the major peanut allergen Ara h 2 by endogenous peanut proteases.

BACKGROUND: The major peanut allergens are Ara h 1, Ara h 2 and Ara h 6. Proteolytic processing has been shown to be required for the maturation process of Ara h 6. The aim of this study was to examine whether Ara h 2 undergoes proteolytic processing and, if so, whether proteolytic processing influences its ability to bind human immunoglobulin E (IgE). RESULTS: Ara h 2 isolated from peanut extract under conditions of protease inhibition revealed a single additional peak for its two known isoforms (Ara h 2.01 and Ara h 2.02), corresponding to a C-terminally truncated form lacking a dipeptide (RY). Ara h 2 isolated in the absence of protease inhibition, however, yielded two additional peaks, identified as C-terminally truncated forms lacking either a dipeptide (RY) or a single tyrosine residue. The IgE-binding capacity of the Ara h 2 truncated forms was not altered. CONCLUSION: Ara h 2 undergoes proteolytic processing by peanut proteases that involves C-terminal removal of a dipeptide. Hence Ara h 2 isolated from peanut extract is a complex mixture of two isoforms expressed by different genes, Ara h 2.01 and Ara h 2.02, as well as truncated forms generated by the proteolytic processing of these isoforms.

Identification and catalytic properties of new epoxide hydrolases from the genomic data of soil bacteria.

Epoxide hydrolases (EHs) catalyse the conversion of epoxides into vicinal diols. These enzymes have extensive value in biocatalysis as they can generate enantiopure epoxides and diols which are important and versatile synthetic intermediates for the fine chemical and pharmaceutical industries. Despite these benefits, they have seen limited use in the bioindustry and novel EHs continue to be reported in the literature. We identified twenty-nine putative EHs within the genomes of soil bacteria. Eight of these EHs were explored in terms of their activity. Two limonene epoxide hydrolases (LEHs) and one ⍺/ß EH were active on a model compound styrene oxide and its ring-substituted derivatives, with low to good percentage conversions of 18-86%. Further exploration of the substrate scope with enantiopure (R)-styrene oxide and (S)-styrene oxide, showed different epoxide ring opening regioselectivities. Two enzymes, expressed from plasmids pQR1984 and pQR1990 de-symmetrised the meso-epoxide cyclohexene oxide, forming the (R,R)-diol with high enantioselectivity. Two LEHs, from plasmids pQR1980 and pQR1982 catalysed the hydrolysis of (+) and (-) limonene oxide, with diastereomeric preference for the (1S,2S,4R)- and (1R,2R,4S)-diol products, respectively. The enzyme from plasmid pQR1982 had a good substrate scope for a LEH, being active towards styrene oxide, its analogues, cyclohexene oxide and 1,2-epoxyhexane in addition to (±)-limonene oxide. The enzymes from plasmids pQR1982 and pQR1984 had good substrate scopes and their enzymatic properties were characterised with respect to styrene oxide. They had comparable temperature optima and pQR1984 had 70% activity in the presence of 40% of the green solvent MeOH, a useful property for bio-industrial applications. Overall, this study has provided novel EHs with potential value in industrial biocatalysis.

pET expression vector customized for efficient seamless cloning.

Here we present a modification of the widely used pET29 expression vector for use in rapid and straightforward parallel cloning via a gene replacement and Golden Gate strategy. The modification can be applied to other expression vectors for Gram-negative bacteria. We have used the modified vectors to clone large numbers of bacterial natural enzyme variants from genomic and metagenomic sources for applications in biocatalysis.

The identification and use of robust transaminases from a domestic drain metagenome.

Transaminases remain one of the most promising biocatalysts for use in chiral amine synthesis, however their industrial implementation has been hampered by their general instability towards, for example, high amine donor concentrations and organic solvent content. Herein we describe the identification, cloning and screening of 29 novel transaminases from a household drain metagenome. The most promising enzymes were fully characterised and the effects of pH, temperature, amine donor concentration and co-solvent determined. Several enzymes demonstrated good substrate tolerance as well as an unprecedented robustness for a wild-type transaminase. One enzyme in particular readily accepted IPA as an amine donor giving the same conversion with 2-50 equivalents, as well as being tolerant to a number of co-solvents, and operational in up to 50% DMSO - a characteristic as yet unobserved in a wild-type transaminase. This work highlights the value of using metagenomics for biocatalyst discovery from niche environments, and here has led to the identification of one of the most robust native transaminases described to date, with respect to IPA and DMSO tolerance.

Novel extremophilic proteases from Pseudomonas aeruginosa M211 and their application in the hydrolysis of dried distiller's grain with solubles.

Proteases are the most important group of industrial enzymes and they can be used in several fields including biorefineries for the valorization of industrial byproducts. In this study, we purified and characterized novel extremophilic proteases produced by a Pseudomonas aeruginosa strain isolated from Mauritia flexuosa palm swamps soil samples in Peruvian Amazon. In addition, we tested their ability to hydrolyze distillers dried grains with solubles (DDGS) protein. Three alkaline and thermophilic serine proteases named EI, EII, and EIII with molecular weight of 35, 40, and 55 kDa, respectively, were purified. EI and EIII were strongly inhibited by EDTA and Pefabloc being classified as serine-metalloproteases, while EII was completely inhibited only by Pefabloc being classified as a serine protease. In addition, EI and EII exhibited highest enzymatic activity at pH 8, while EIII at pH 11 maintaining almost 100% of it at pH 12. All the enzymes demonstrated optimum activity at 60°C. Enzymatic activity of EI was strongly stimulated in presence of Mn2+ (6.9-fold), EII was stimulated by Mn2+ (3.7-fold), while EIII was slightly stimulated by Zn2+ , Ca2+ , and Mg2+ . DDGS protein hydrolysis using purified Pseudomonas aeruginosa M211 proteases demonstrated that, based on glycine released, EIII presented the highest proteolytic activity toward DDGS. This enzyme enabled the release 63% of the total glycine content in wheat DDGS protein, 2.2-fold higher that when using the commercial Pronase®. Overall, our results indicate that this novel extremopreoteases have a great potential to be applied in DDGS hydrolysis. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2728, 2019.

Metagenomic ene-reductases for the bioreduction of sterically challenging enones.

Ene-reductases (ERs) of the Old Yellow Enzyme family catalyse asymmetric reduction of activated alkenes providing chiral products. They have become an important method in the synthetic chemists' toolbox offering a sustainable alternative to metal-catalysed asymmetric reduction. Development of new biocatalytic alkene reduction routes, however needs easy access to novel biocatalysts. A sequence-based functional metagenomic approach was used to identify novel ERs from a drain metagenome. From the ten putative ER enzymes initially identified, eight exhibited activities towards widely accepted mono-cyclic substrates with several of the ERs giving high reaction yields and stereoselectivities. Two highly performing enzymes that displayed excellent co-solvent tolerance were used for the stereoselective reduction of sterically challenging bicyclic enones where the reactions proceeded in high yields, which is unprecedented to date with wild-type ERs. On a preparative enzymatic scale, reductions of Hajos-Parish, Wieland-Miescher derivatives and a tricyclic ketone proceeded with good to excellent yields.

Functional Comparison of Bacteria from the Human Gut and Closely Related Non-Gut Bacteria Reveals the Importance of Conjugation and a Paucity of Motility and Chemotaxis Functions in the Gut Environment.

The human GI tract is a complex and still poorly understood environment, inhabited by one of the densest microbial communities on earth. The gut microbiota is shaped by millennia of evolution to co-exist with the host in commensal or symbiotic relationships. Members of the gut microbiota perform specific molecular functions important in the human gut environment. This can be illustrated by the presence of a highly expanded repertoire of proteins involved in carbohydrate metabolism, in phase with the large diversity of polysaccharides originating from the diet or from the host itself that can be encountered in this environment. In order to identify other bacterial functions that are important in the human gut environment, we investigated the distribution of functional groups of proteins in a group of human gut bacteria and their close non-gut relatives. Complementary to earlier global comparisons between different ecosystems, this approach should allow a closer focus on a group of functions directly related to the gut environment while avoiding functions related to taxonomically divergent microbiota composition, which may or may not be relevant for gut homeostasis. We identified several functions that are overrepresented in the human gut bacteria which had not been recognized in a global approach. The observed under-representation of certain other functions may be equally important for gut homeostasis. Together, these analyses provide us with new information about this environment so critical to our health and well-being.

Patented biotechnological applications of serpin: an update.

The Serine Protease Inhibitors (Serpins) have been a focus of research by biomedical industries due to their critical role in human health. The use of serpin in the treatment of many diseases was widely investigated through the identification of new genes encoding these proteins in all kingdoms of life. The characterization of these genes revealed that they encoded proteins having low sequence homologies. Future developments are focusing not only on the protease inhibition activity, but also on the other effects due to the interactions of serpins with other components such as hormone transport. Here we give a concise overview of the most recent patents that have been reported in this field of research.

High-throughput system for the presentation of secreted and surface-exposed proteins from Gram-positive bacteria in functional metagenomics studies.

Complex microbial ecosystems are increasingly studied through the use of metagenomics approaches. Overwhelming amounts of DNA sequence data are generated to describe the ecosystems, and allow to search for correlations between gene occurrence and clinical (e.g. in studies of the gut microbiota), physico-chemical (e.g. in studies of soil or water environments), or other parameters. Observed correlations can then be used to formulate hypotheses concerning microbial gene functions in relation to the ecosystem studied. In this context, functional metagenomics studies aim to validate these hypotheses and to explore the mechanisms involved. One possible approach is to PCR amplify or chemically synthesize genes of interest and to express them in a suitable host in order to study their function. For bacterial genes, Escherichia coli is often used as the expression host but, depending on the origin and nature of the genes of interest and the test system used to evaluate their putative function, other expression systems may be preferable. In this study, we developed a system to evaluate the role of secreted and surface-exposed proteins from Gram-positive bacteria in the human gut microbiota in immune modulation. We chose to use a Gram-positive host bacterium, Bacillus subtilis, and modified it to provide an expression background that behaves neutral in a cell-based immune modulation assay, in vitro. We also adapted an E. coli-B. subtilis shuttle expression vector for use with the Gateway high-throughput cloning system. Finally, we demonstrate the functionality of this host-vector system through the cloning and expression of a flagellin-coding sequence, and show that the expression-clone elicits an inflammatory response in a human intestinal epithelial cell line. The expression host can easily be adapted to assure neutrality in other assay systems, allowing the use of the presented presentation system in functional metagenomics of the gut and other ecosystems.