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

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

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

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 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

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

Development of novel low-mass module concepts based on MALTA monolithic pixel sensors

The MALTA CMOS monolithic silicon pixel sensors has been developed in the Tower 180 nm CMOS imaging process. It includes an asynchronous readout scheme and complies with the ATLAS inner tracker requirements for the HL-LHC. Several 4-chip MALTA modules have been built using Al wedge wire bonding to demonstrate the direct transfer of data from chip-to-chip and to read out the data of the entire module via one chip only. Novel technologies such as Anisotropic Conductive Films (ACF) and nanowires have been investigated to build a compact module. A lightweight flex with 17 {\mu}m trace spacing has been designed, allowing compact packaging with a direct attachment of the chip connection pads to the flex using these interconnection technologies. This contribution shows the current state of our work towards a flexible, low material, dense and reliable packaging and modularization of pixel detectors.Comment: 5 pages + 1 page references,8 figure

MALTA monolithic pixel sensors in TowerJazz 180 nm technology

Depleted Monolithic Active Pixel Sensors are of highest interest at the HL-LHC and beyond for the replacement of the Pixel trackers in the outermost layers of experiments where the requirement on total area and cost effectiveness is much bigger. They aim to provide high granularity and low material budget over large surfaces with ease of integration. Our research focuses on MALTA, a radiation hard DMAPS with small collection electrode designed in TowerJazz 180 nm CMOS imaging technology and asynchronous read-out. Latest prototypes are radiation hard up to 2 × 1015 1 MeV neq/cm2 with a time resolution better than 2 ns

Timing performance of radiation hard MALTA monolithic Pixel sensors

The MALTA family of Depleted Monolithic Active Pixel Sensor (DMAPS) produced in Tower 180 nm CMOS technology targets radiation hard applications for the HL-LHC and beyond. Several process modifications and front-end improvements have resulted in radiation hardness up to $2 \times 10^{15}~1~\text{MeV}~\text{n}_{eq}/\text{cm}^2$ and time resolution below 2 ns, with uniform charge collection efficiency across the Pixel of size $36.4 \times 36.4~\mu\text{m}^2$ with a $3~\mu\text{m}^2$ electrode size. The MALTA2 demonstrator produced in 2021 on high-resistivity epitaxial silicon and on Czochralski substrates implements a new cascoded front-end that reduces the RTS noise and has a higher gain. This contribution shows results from MALTA2 on timing resolution at the nanosecond level from the CERN SPS test-beam campaign of 2021.Comment: 8 pages, 8 figures. Submitted to Journal of Instrumentation (JINST). Proceedings of the 23rd International Workshop on Radiation Imaging Detectors IWORID 202

Depletion depth studies with the MALTA2 sensor, a depleted monolithic active pixel sensor

MALTA2 is a depleted monolithic active pixel sensor (DMAPS) developed in the Tower 180 nm CMOS imaging process. Monolithic CMOS sensors offer advantages over current hybrid imaging sensors both in terms of increased tracking performance due to lower material budget but also in terms of ease of integration and construction costs due to the monolithic design. Current research and development efforts are aimed towards radiation-hard designs up to 100 Mrad in Total Ionizing Dose and 3 × 1015 1 MeV neq / cm2 in Non-Ionizing Energy Loss. One important property of a sensor’s radiation hardness is the depletion depth at which efficient charge collection is achieved via drift movement. Grazing angle test-beam data was taken during the 2023 SPS CERN test beam with the MALTA telescope and Edge Transient Current Technique studies were performed at DESY in order to develop a quantitative study of the depletion depth for un-irradiated, epitaxial MALTA2 samples. The study is planned to be extended for irradiated and Czochralski MALTA2 samples