Immobilization and Stabilization of an Engineered Acyltransferase for the Continuous Biosynthesis of Simvastatin in Packed-Bed Reactors

Abstract

Simvastatin is a top-selling cholesterol-lowering drug traditionally obtained through a semi-synthetic process starting from lovastatin. However, this process is cost-demanding and makes use of chemical reagents that can generate considerable waste. The sustainability concerns underlying the current semisynthetic process encouraged us to immobilize the engineered acyltransferase LovD−BuCh2 on different pore carriers to develop an innovative and sustainable process for the continuous biomanufacturing of simvastatin. We systematically assessed the effect of the immobilization on the functional and structural properties of the immobilized enzyme, the enzyme spatial distribution across the solid material, and the thermal stability of the immobilized biocatalysts. After screening several immobilization carriers, we selected LovD−BuCh2 immobilized on the controlled porous glass particles functionalized with Fe 3+ − catechol complexes as the most suitable heterogeneous biocatalyst to optimize the continuous synthesis of simvastatin. This immobilized enzyme was four times more thermally stable than its free counterpart and maintained >60% product yield during five operational cycles in the batch. Under the optimal flow conditions, we achieved 100% of simvastatin yield with a space-time yield of 4.61 g L −1 h −1 and a specific productivity of 17 mg product × mg enzyme −1 L −1 . This flow-biocatalysis system improves some green chemistry metrics such as the reaction mass efficiency and the atom economy when compared to previous studies of simvastatin manufacturing.

Accurate Prediction of Enzyme Thermostabilization with Rosetta Using AlphaFold Ensembles

“…We present this approach and compare its performance with Rosetta ddg for the Monacolin J acid acyltransferase LovD and Lipase A LipA. LovD, which has been the object of a number of studies from our group, has been evolved through directed evolution by Tang and co-workers in collaboration with Codexis, Inc. for maximizing the production of simvastatin, a blockbuster drug for controlling cholesterol levels in the blood. ,, The directed evolution campaign produced nine variants (LovD1-9), accumulating 29 mutations in total. We measured the T M of each variant, shown in Table along with the number of mutations introduced at each round (see Table S1 for further details).…”

Multienzyme Coimmobilization on Triheterofunctional Supports

“…The relative anisotropy of immobilized enzymes with respect to the anisotropy of their free counterpart reflects the changes in protein mobility promoted by the immobilization process. Normally, this relative anisotropy is greater as more stable the immobilized enzyme is, thus presenting a positive correlation with the half-life time of the immobilized biocatalysts . Finally, we determined the unfolding transition temperature ( T m ) of both free and immobilized enzymes by a thermal shift assay.…”

“…On one hand, we studied the intrinsic fluorescence spectrum of both immobilized and free enzymes (Figure S6) to acquire information about their microenvironment within the protein structure. 44 On the other hand, we determined the relative anisotropy of free and immobilized enzymes labeled with fluorescein B isocyanate. The fluorescent anisotropy of small fluorophores tethered to the enzyme structure informs us about the apparent mobility of the protein through its rotational tumbling.…”

Sustainable and Robust Closed-Loop Enzymatic Platform for Continuous/Semi-continuous Synthesis of Ursodeoxycholic Acid

“…The use of enzymes as catalysts for biosynthesis of chemical compounds is popular in the manufacturing of pharmaceuticals and active pharmaceutical ingredients. − Generally, biocatalysts are appealing for pharmaceutical manufacturing because their high enantio-, regio- and chemoselectivities reduce the generation of undesired side products. − Furthermore, biocatalysts are usually used under mild and environmentally friendly conditions, which means that enzymatic biotransformations are more sustainable compared with conventional chemical syntheses. , Nevertheless, the application of enzymatic systems can be limited by low reaction efficiencies, enzyme instability, and tedious optimization and scale-up processes. , …”

“…This integration can shorten the manufacturing cycle, reinforce bioprocesses, and improve productivity. − Additionally, in situ product removal in continuous flow systems may alleviate product inhibition of enzymes, which can maximize their catalytic function . Immobilized enzymes are usually used to realize long-term stable operation in continuous flow reactors. ,− Immobilization of enzymes on solid supports is an effective approach for improving the stability of biocatalysts either in organic solvents or under high temperatures and at extreme pH values. , Furthermore, the utilization of immobilized enzymes in a continuous flow reactor means the reaction operation and catalyst recovery steps can be performed simultaneously in a single unit, which greatly simplifies the downstream processes for product separation and purification . Therefore, the use of immobilized enzymes was hypothesized to be an effective strategy to enhance the bioprocess of ursodeoxycholic acid (UDCA) synthesis.…”