Enzyme structure and function have been studied for subtilisin BPN‘ solubilized in organic solvents by ion pairing with low concentrations of an anionic surfactant (Aerosol OT) in the absence of reversed micelles. Soluble subtilisin shows strikingly different behavior in octane and tetrahydrofuran (THF). In octane, the k cat/K m for the transesterification of N-acetyl-l-phenylalanine ethyl ester (APEE) is 370 M-1 s-1, within one order of magnitude of the enzyme's hydrolytic activity in water. Moreover, the observed half-life of the soluble enzyme in octane is nearly three orders of magnitude greater than in water, presumably because of the absence of autolysis in the organic solvent. In contrast, the catalytic efficiency of the enzyme dissolved in the polar solvent THF is 0.04 M-1 s-1, and the enzyme loses 99% of its activity within 10 min. Comparable enzyme inactivation could also be observed in octane, but only at elevated temperatures such as 70 °C. Therefore, the mechanisms of deactivation of the soluble enzyme were investigated in both octane and THF. Kinetic and spectroscopic (CD and EPR) studies support the existence of multiple inactive forms of the soluble enzyme in THF at 25 °C and in octane at 70 °C. Notably, in both cases a denatured form can be renatured in anhydrous octane at 25 °C, the first demonstration of enzyme renaturation in a bulk organic solvent. A model explaining the THF- and thermally-induced inactivation processes of soluble subtilisin BPN‘ is proposed, and the apparent reasons for the exceptionally high activity and stability of the soluble enzyme in octane are discussed.
It has been half a century since investigators first began experimenting with adding ion exchange resins during the fermentation of microbial natural products. With the development of nonionic polymeric adsorbents in the 1970s, the application of in situ product adsorption in bioprocessing has grown slowly, but steadily. To date, in situ product adsorption strategies have been used in biotransformations, plant cell culture, the production of biofuels, and selected bulk chemicals, such as butanol and lactic acid, as well as in more traditional natural product fermentation within the pharmaceutical industry. Apart from the operational gains in efficiency from the integration of fermentation and primary recovery, the addition of adsorbents during fermentation has repeatedly demonstrated the capacity to significantly increase titers by sequestering the product and preventing or mitigating degradation, feedback inhibition and/or cytotoxic effects. Adoption of in situ product adsorption has been particularly valuable in the early stages of natural product-based drug discovery programs, where quickly and cost-effectively generating multigram quantities of a lead compound can be challenging when using a wild-type strain and fermentation conditions that have not been optimized. While much of the literature involving in situ adsorption describes its application early in the drug development process, this does not imply that the potential for scale-up is limited. To date, commercial-scale processes utilizing in situ product adsorption have reached batch sizes of at least 30,000 l. Here we present examples where in situ product adsorption has been used to improve product titers or alter the ratios among biosynthetically related natural products, examine some of the relevant variables to consider, and discuss the mechanisms by which in situ adsorption may impact the biosynthesis of microbial natural products.
A significant number of marketed pharmaceuticals contain active pharmaceutical ingredients that are manufactured in part using biocatalysis as a key enabling technology. The utilization of biocatalysis is growing due to significant advances in technologies for enzyme discovery, supply, and improvement, as well as an increased focus on applications for chiral drugs and green chemistry. Nevertheless, there still remains a lack of clarity around quality and regulatory expectations when using biotransformations in research and manufacturing, and this lack of clarity can be a barrier to the uptake and adoption of biocatalysis. This commentary will explore and offer some rational, coherent, and achievable strategies for the use of biocatalysis in the manufacture of small molecule active pharmaceutical ingredients (APIs) based on a scientific, risk-based approach to drug quality and patient safety. We also seek to invite other interested parties to contact us with their views to add to the topics discussed here with the goal of expanding these thoughts into an industry-based white paper.
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