Microtiter Plate Immobilization Screening for Prototyping Heterogeneous Enzyme Cascades.

Immobilization is a key enabling technology in applied biocatalysis that facilitates the separation, recovery, and reuse of heterogeneous biocatalysts. However, finding a consensus immobilization protocol for several enzymes forming a multi-enzyme system is extremely difficult and relies on a combinatorial trial-and-error approach. Herein, we describe a protocol in which 17 different carriers functionalized with different reactive groups are tested in a 96-well microtiter plate to screen up to 21 immobilization protocols for up to 18 enzymes. This screening includes an activity and stability assay to select the optimal immobilization chemistry to achieve the most active and stable heterogeneous biocatalysts. The information retrieved from the screening can be rationalized using a Python-based application CapiPy. Finally, through scoring the screening results, we find the consensus immobilization protocol to assemble an immobilized four-enzyme system to transform vinyl acetate into (S)-3-hydroxybutyric acid. This methodology opens a path to speed up the prototyping of immobilized multi-enzyme pathways for chemical manufacturing.

CapiPy: python-based GUI-application to assist in protein immobilization.

SUMMARY: Protein immobilization, while widespread to unlock enzyme potential in biocatalysis, remains tied to a trial an error approach. Nonetheless, several databases and computational methods have been developed for protein characterization and their study. CapiPy is a user-friendly application for protein model creation and subsequent analysis with a special focus on the ease of use and interpretation of the results to help the users to make an informed decision on the immobilization approach which should be ideal for a protein of interest. The package has been tested with three separate random sets of 150 protein sequences from Uniprot with more than a 70% overall success rate (see Supplementary information and Supplementary Dataset). AVAILABILITY AND IMPLEMENTATION: The package is free to use under the GNU General Public License v3.0. All necessary files can be downloaded from https://github.com/drou0302/CapiPy or https://pypi.org/project/CapiPy/. All external requirements are also freely available, with some restrictions for non-academic users. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Construction of Highly Stable Cytotoxic Nuclear-Directed Ribonucleases.

Ribonucleases are proteins whose use is promising in anticancer therapy. We have previously constructed different human pancreatic ribonuclease variants that are selectively cytotoxic for tumor cells by introducing a nuclear localization signal into their sequence. However, these modifications produced an important decrease in their stability compromising their behavior in vivo. Here, we show that we can significantly increase the thermal stability of these cytotoxic proteins by introducing additional disulfide bonds by site-directed mutagenesis. One of these variants increases its thermal stability by around 17 °C, without affecting its catalytic activity while maintaining the cytotoxic activity against tumor cells. We also show that the most stable variant is significantly more resistant to proteolysis when incubated with proteinase K or with human sera, suggesting that its half-live could be increased in vivo once administered.

Combined chemoenzymatic strategy for sustainable continuous synthesis of the natural product hordenine.

To improve sustainability, safety and cost-efficiency of synthetic methodologies, biocatalysis can be a helpful ally. In this work, a novel chemoenzymatic stategy ensures the rapid synthesis of hordenine, a valuable phenolic phytochemical under mild working conditions. In a two-step cascade, the immobilized tyrosine decarboxylase from Lactobacillus brevis (LbTDC) is here coupled with the chemical reductive amination of tyramine. Starting from the abundant and cost-effective amino acid l-tyrosine, the complete conversion to hordenine is achieved in less than 5 minutes residence time in a fully-automated continuous flow system. Compared to the metal-catalyzed N,N-dimethylation of tyramine, this biocatalytic approach reduces the process environmental impact and improves its STY to 2.68 g L-1 h-1.

Multistep enzyme cascades as a route towards green and sustainable pharmaceutical syntheses.

Enzyme cascades are a powerful technology to develop environmentally friendly and cost-effective synthetic processes to manufacture drugs, as they couple different biotransformations in sequential reactions to synthesize the product. These biocatalytic tools can address two key parameters for the pharmaceutical industry: an improved selectivity of synthetic reactions and a reduction of potential hazards by using biocompatible catalysts, which can be produced from sustainable sources, which are biodegradable and, generally, non-toxic. Here we outline a broad variety of enzyme cascades used either in vivo (whole cells) or in vitro (purified enzymes) to specifically target pharmaceutically relevant molecules, from simple building blocks to complex drugs. We also discuss the advantages and requirements of multistep enzyme cascades and their combination with chemical catalysts through a series of reported examples. Finally, we examine the efficiency of enzyme cascades and how they can be further improved by enzyme engineering, process intensification in flow reactors and/or enzyme immobilization to meet all the industrial requirements.

A novel thymidine phosphorylase to synthesize (halogenated) anticancer and antiviral nucleoside drugs in continuous flow.

Four pharmaceutically relevant nucleoside analogues (5-fluoro-2'-deoxyuridine, 5-chloro-2'-deoxyuridine, 5-bromo-2'-deoxyuridine, and 5-iodo-2'-deoxyuridine) have been synthesized by using a novel thymidine phosphorylase from the halotolerant H. elongata (HeTP). Following enzyme immobilization on microbeads, the biocatalyst was implemented as a packed-bed reactor for the continuous production of halogenated nucleosides, achieving up to 90% conversion at the 10 mM scale with 30 min residence time. Taking the synthesis of floxuridine (5-fluoro-2'-deoxyuridine) as a study case, we obtained the highest space-time yield (5.5 g L-1 h-1) reported to date. In addition, bioinformatic tools such as MD analysis and CapiPy have contributed to shine light on the catalytic performance of HeTP as well as its immobilization, respectively.

Enhancing PLP-Binding Capacity of Class-III ω-Transaminase by Single Residue Substitution.

Transaminases are pyridoxal-5'-phosphate (PLP) binding enzymes, broadly studied for their potential industrial application. Their affinity for PLP has been related to their performance and operational stability and while significant differences in PLP requirements have been reported, the environment of the PLP-binding pocket is highly conserved. In this study, thorough analysis of the residue interaction network of three homologous transaminases Halomonas elongata (HeTA), Chromobacterium violaceum (CvTA), and Pseudomonas fluorescens (PfTA) revealed a single residue difference in their PLP binding pocket: an asparagine at position 120 in HeTA. N120 is suitably positioned to interact with an aspartic acid known to protonate the PLP pyridinium nitrogen, while the equivalent position is occupied by a valine in the other two enzymes. Three different mutants were constructed (HeTA-N120V, CvTA-V124N, and PfTA-V129N) and functionally analyzed. Notably, in HeTA and CvTA, the asparagine variants, consistently exhibited a higher thermal stability and a significant decrease in the dissociation constant (K d ) for PLP, confirming the important role of N120 in PLP binding. Moreover, the reaction intermediate pyridoxamine-5'-phosphate (PMP) was released more slowly into the bulk, indicating that the mutation also enhances their PMP binding capacity. The crystal structure of PfTA, elucidated in this work, revealed a tetrameric arrangement with the PLP binding sites near the subunit interface. In this case, the V129N mutation had a negligible effect on PLP-binding, but it reduced its temperature stability possibly destabilizing the quaternary structure.