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.

Design and Use of de novo Cascades for the Biosynthesis of New Benzylisoquinoline Alkaloids.

The benzylisoquinoline alkaloids (BIAs) are an important group of secondary metabolites from higher plants and have been reported to show significant biological activities. The production of BIAs through synthetic biology approaches provides a higher-yielding strategy than traditional synthetic methods or isolation from plant material. However, the reconstruction of BIA pathways in microorganisms by combining heterologous enzymes can also give access to BIAs through cascade reactions. Most importantly, non-natural BIAs can be generated through such artificial pathways. In the current study, we describe the use of tyrosinases and decarboxylases and combine these with a transaminase enzyme and norcoclaurine synthase for the efficient synthesis of several BIAs, including six non-natural alkaloids, in cascades from l-tyrosine and analogues.

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.

Data on a thermostable enzymatic one-pot reaction for the production of a high-value compound from l-arabinose.

The dataset presented in this article is related to the research article entitled "One-pot, two-step transaminase and transketolase synthesis of l-gluco-heptulose from l-arabinose" (Bawn et al., 2018 in press) [1]. This article presents data on initial experiments that were carried out to investigate new thermostable transketolase (TK) activities with l-arabinose. Transaminase (TAm) sequences from an in-house library of thermophilic strains were analyzed to compare homologies to characterized TAms with desired activity. DNA and amino acid sequences are presented for all the enzymes investigated. Calibration curves for products of the TK and TAm reactions are also presented along with chromatographic analysis of the various one-pot reactions.

One-pot, two-step transaminase and transketolase synthesis of l-gluco-heptulose from l-arabinose.

The use of biocatalysis for the synthesis of high value added chemical building blocks derived from biomass is becoming an increasingly important application for future sustainable technologies. The synthesis of a higher value chemical from l-arabinose, the predominant monosaccharide obtained from sugar beet pulp, is demonstrated here via a transketolase and transaminase coupled reaction. Thermostable transketolases derived from Deinococcus geothermalis and Deinococcus radiodurans catalysed the synthesis of l-gluco-heptulose from l-arabinose and ß-hydroxypyruvate at elevated temperatures with high conversions. ß-Hydroxypyruvate, a commercially expensive compound used in the transketolase reaction, was generated in situ from l-serine and α-ketoglutaric acid via a thermostable transaminase, also from Deinococcus geothermalis. The two steps were investigated and implemented in a one-pot system for the sustainable and efficient production of l-gluco-heptulose.

An integrated biorefinery concept for conversion of sugar beet pulp into value-added chemicals and pharmaceutical intermediates.

Over 8 million tonnes of sugar beet are grown annually in the UK. Sugar beet pulp (SBP) is the main by-product of sugar beet processing which is currently dried and sold as a low value animal feed. SBP is a rich source of carbohydrates, mainly in the form of cellulose and pectin, including d-glucose (Glu), l-arabinose (Ara) and d-galacturonic acid (GalAc). This work describes the technical feasibility of an integrated biorefinery concept for the fractionation of SBP and conversion of these monosaccharides into value-added products. SBP fractionation is initially carried out by steam explosion under mild conditions to yield soluble pectin and insoluble cellulose fractions. The cellulose is readily hydrolysed by cellulases to release Glu that can then be fermented by a commercial yeast strain to produce bioethanol at a high yield. The pectin fraction can be either fully hydrolysed, using physico-chemical methods, or selectively hydrolysed, using cloned arabinases and galacturonases, to yield Ara-rich and GalAc-rich streams. These monomers can be separated using either Centrifugal Partition Chromatography (CPC) or ultrafiltration into streams suitable for subsequent enzymatic upgrading. Building on our previous experience with transketolase (TK) and transaminase (TAm) enzymes, the conversion of Ara and GalAc into higher value products was explored. In particular the conversion of Ara into l-gluco-heptulose (GluHep), that has potential therapeutic applications in hypoglycaemia and cancer, using a mutant TK is described. Preliminary studies with TAm also suggest GluHep can be selectively aminated to the corresponding chiral aminopolyol. The current work is addressing the upgrading of the remaining SBP monomer, GalAc, and the modelling of the biorefinery concept to enable economic and Life Cycle Analysis (LCA).