Engineering a Cytidine Aminotransferase for Biocatalytic Production of the Antiviral Molnupiravir

The COVID-19 pandemic highlights the urgent need for cost-effective processes to rapidly manufacture antiviral drugs at scale. Here we report a concise biocatalytic process for Molnupiravir, a nucleoside analogue currently in phase 3 clinical trials as an orally available treatment for SARS-CoV-2. Key to the success of this process was the development of a cytidine aminotransferase for the production of N-hydroxy-cytidine through evolutionary adaption of the hydrolytic enzyme cytidine deaminase. This engineered biocatalyst performs >100,000 turnovers in less than 30 minutes, operates at 180 g/L substrate loading and benefits from in situ crystallization of the N-hydroxy-cytidine product (>90% yield), which can be converted to Molnupiravir by a selective 5’-acylation using Novozym® 435.

Biocatalytic Production of Aldehydes: Exploring the Potential of Lathyrus cicera Amine Oxidase

Aldehydes are a class of carbonyl compounds widely used as intermediates in the pharmaceutical, cosmetic and food industries. To date, there are few fully enzymatic methods for synthesizing these highly reactive chemicals. In the present work, we explore the biocatalytic potential of an amino oxidase extracted from the etiolated shoots of Lathyrus cicera for the synthesis of value-added aldehydes, starting from the corresponding primary amines. In this frame, we have developed a completely chromatography-free purification protocol based on crossflow ultrafiltration, which makes the production of this enzyme easily scalable. Furthermore, we determined the kinetic parameters of the amine oxidase toward 20 differently substituted aliphatic and aromatic primary amines, and we developed a biocatalytic process for their conversion into the corresponding aldehydes. The reaction occurs in aqueous media at neutral pH in the presence of catalase, which removes the hydrogen peroxide produced during the reaction itself, contributing to the recycling of oxygen. A high conversion (>95%) was achieved within 3 h for all the tested compounds.

Magnetic Nanoparticle-Containing Supports as Carriers of Immobilized Enzymes: Key Factors Influencing the Biocatalyst Performance

In this short review (Perspective), we identify key features of the performance of biocatalysts developed by the immobilization of enzymes on the supports containing magnetic nanoparticles (NPs), analyzing the scientific literature for the last five years. A clear advantage of magnetic supports is their easy separation due to the magnetic attraction between magnetic NPs and an external magnetic field, facilitating the biocatalyst reuse. This allows for savings of materials and energy in the biocatalytic process. Commonly, magnetic NPs are isolated from enzymes either by polymers, silica, or some other protective layer. However, in those cases when iron oxide NPs are in close proximity to the enzyme, the biocatalyst may display a fascinating behavior, allowing for synergy of the performance due to the enzyme-like properties shown in iron oxides. Another important parameter which is discussed in this review is the magnetic support porosity, especially in hierarchical porous supports. In the case of comparatively large pores, which can freely accommodate enzyme molecules without jeopardizing their conformation, the enzyme surface ordering may create an optimal crowding on the support, enhancing the biocatalytic performance. Other factors such as surface-modifying agents or special enzyme reactor designs can be also influential in the performance of magnetic NP based immobilized enzymes.

Integrated Cleaner Biocatalytic Process for Biodiesel Production from Crude Palm Oil Comparing to Refined Palm Oil

An integrated cleaner biocatalyst process was performed for biodiesel production from crude palm oil (CPO) and refined palm oil (RPO). It was evaluated on process efficiency in terms of high purity of biodiesel as well as by-products without purification, less wastewater, less time consuming, and a simple downstream process. A first saponification step was carried out in both f CPO and RPO, a high purity of glycerol (86.25% and 87.5%) was achieved, respectively, while free fatty acids (FFASs) in soap were obtained after hexane extraction. High yields of FFASs were obtained from both CPO and RPO (98.83% and 90.94%). Subsequently, the FFAs were esterified to biodiesel by a biocatalyst of immobilized lipase. The highest biodiesel yields achieved were of 92.14% and 92.58% (CPO and RPO). Remarkably, biodiesel yields obtained from CPO and RPO achieved satisfactory values and the biocatalyst used could be reused for more than 16–17 cycles.

Application Fields, Positions, and Bioinformatic Mining of Non-active Sites: A Mini-Review

Active sites of enzymes play a vital role in catalysis, and researchhas been focused on the interactions between active sites and substrates to understand the biocatalytic process. However, the active sites distal to the catalytic cavity also participate in catalysis by maintaining the catalytic conformations. Therefore, some researchers have begun to investigate the roles of non-active sites in proteins, especially for enzyme families with different functions. In this mini-review, we focused on recent progress in research on non-active sites of enzymes. First, we outlined two major research methodswith non-active sites as direct targets, including understanding enzymatic mechanisms and enzyme engineering. Second, we classified the positions of reported non-active sites in enzyme structures and studied the molecular mechanisms underlying their functions, according to the literature on non-active sites. Finally, we summarized the results of bioinformatic analysisof mining non-active sites as targets for protein engineering.

New insights into the molecular mechanism behind mannitol and erythritol fructosylation by β-fructofuranosidase from Schwanniomyces occidentalis

The β-fructofuranosidase from Schwanniomyces occidentalis (Ffase) is a useful biotechnological tool for the fructosylation of different acceptors to produce fructooligosaccharides (FOS) and fructo-conjugates. In this work, the structural determinants of Ffase involved in the transfructosylating reaction of the alditols mannitol and erythritol have been studied in detail. Complexes with fructosyl-erythritol or sucrose were analyzed by crystallography and the effect of mutational changes in positions Gln-176, Gln-228, and Asn-254 studied to explore their role in modulating this biocatalytic process. Interestingly, N254T variant enhanced the wild-type protein production of fructosyl-erythritol and FOS by $$\sim$$ ∼ 30% and 48%, respectively. Moreover, it produced neokestose, which represented $$\sim$$ ∼ 27% of total FOS, and yielded 31.8 g l−1 blastose by using glucose as exclusive fructosyl-acceptor. Noteworthy, N254D and Q176E replacements turned the specificity of Ffase transferase activity towards the synthesis of the fructosylated polyols at the expense of FOS production, but without increasing the total reaction efficiency. The results presented here highlight the relevance of the pair Gln-228/Asn-254 for Ffase donor-sucrose binding and opens new windows of opportunity for optimizing the generation of fructosyl-derivatives by this enzyme enhancing its biotechnological applicability.