General aspects of immobilized biocatalysts and their applications in flow

Biocatalysis provides a unique opportunity to create complex and chiral molecules with unprecedented selectivity.[1-8] Enzymes are the preferred catalysts.[9-13] "White biotechnology" exploits biocatalysis on an industrial level, and the number of applications is increasing;[ 14-19] it is the application of "nature's toolset", for example microorganisms and enzymes, for the production of (fine) chemicals, pharmaceuticals, food ingredients, materials, and biofuels from renewable resources. It was estimated that the market share of biochemicals would increase to 12-20% of chemical production by 2015.[20] The degree of biopharmaceuticals increased from 7.8 to 15.6% from 2001 to 2011.[21] In Europe's biopharmaceutical sector, already some 20% of the current medicines are derived from biotechnology, and up to 50% of new medicines.[20] As with homogeneous chemocatalysis,[22] catalyst separation after completion of the reaction is the big issue to solve, for reasons of avoiding product contamination, cost of catalyst, and environmental friendliness. In a similar fashion to chemocatalysis, the immobilization of biocatalysts on a solid support is a prime approach,[23-26] as the "separation" is effectively done in one step ahead of the reaction and no further efforts downstream are needed. The catalyst can be used many times until its destabilization becomes significant. Enzyme fixation has been known for a long time,[7,27,28] and commercially fixed catalyst- carrier systems are available, such as Novozymes' immobilized lipase enzymes.[29] While fixed enzymes have been successfully used with conventional reactor technology, their more recent application in continuous small-scale reactors provides a seemingly ideal technology fit.[30,31] In 2015, the U.S. Food and Drug Administration (FDA) called on pharmaceutical manufacturers to change within one decade from batch to continuous reactions.[ 32] The essential tool in small-scale production is the microreactor or more commonly named flow reactor.[33-41] It can be microfabricated with inner dimensions of some hundred micrometers, but may be a microcapillary or a tube filled with material composed of "microspaces". There is a trend to move to somewhat larger milliscale reactors for reasons of throughput, reliability (sensitivity to clogging), and easiness of handling, while possibly compromising somewhat the high process intensification achieved on the microscale. Nonetheless, such milliscale systems generally still offer much better performance than conventional equipment. Continuous processing has been standard in petroleum and bulk chemistry from the beginning;[42] however, for a long time, the specialty and pharma industries preferred batch production due to its high flexibility, which fits well with the plethora of chemical products that are made. It also allows a fast change (shutdown and startup) in case new products are needed or production capacity is to be decreased or increased. With the increasing need for quality control and assurance and the steadily higher pressure for time-to-market, continuous systems have, in the last decade, become more important for small-scale fine-chemical production[43-46] and pharmaceuticals manufacture.[47-49] The term "flow chemistry" was coined for this[50-55] and has also shown cost and environmental advantages due to, among others, higher conversions and selectivity and lower solvent loads.[56,57] Due to the smallness of the processed volume, safety limitations are largely overcome, opening the door to forbidden and forgotten chemistries. In novel process windows, the needed decrease of reaction times to the short time scales of such processing can be achieved.[58-67] The unusual parameters even sometimes allow the exploration of completely new chemical pathways that are not possible with conventional equipment. Some processes have been taken to industrial scale.[39] Prime future research involves combining flow chemistry and artificial intelligence to a microprocessor control system for process optimization in a fully automated manner ("March of the Machines";[ 68,69] "The Robo-chemist"[70]). Rapid data exchange between machines operated at different locations and with different chemistries in foci will result in a chemical "Internet of Things".[71] Motivated by the considerations outlined above, pharmaceutical companies have aligned in a Pharmaceutical Roundtable and established the development of continuous production as their top priority.[72] Second-highest priority is given to bioprocesses. Consequently, both have been recently combined into "bio-flow chemistry"; enzymatic flow reactors are the most commonly used concept, and enzyme immobilization within these units is largely employed. Mini-packed-bed reactors, which are commonly found in small-scale continuous production, are ideal matrices for the immobilization of enzymes.[73-75] This is done on small beads, which are compressed within a tube to result in a packed bed. The range of bead materials spans from polymers to inorganic materials (typically oxides of metals and half metals) to modern artificial materials such as graphene oxide,[76] hierarchical porous monolithic rods,[77] polymer brushes,[78] and silica nanosprings.[79] Often these materials are porous, so that a much higher catalyst density and loading can be achieved in the small reactor volumes, which ensures higher productivity. Various ways of immobilizing the enzymes have been developed, as the surfaces of the above-mentioned support materials comprise different attachment groups, as do the enzymes.[73-79] Typically, a bifunctional chain provides the needed spatial flexibility so that the enzyme and its active center can behave as they would in a homogeneous environment.[ 73-75] The costs of enzyme immobilization are crucial for the overall capital costs of a bioflow- chemistry-based industrial system.[80] The quality of enzyme immobilization ensures sufficient operational time, i.e. a large number of process cycles before the enzyme is deactivated. This, together with the above-mentioned enzyme-density-triggered productivity, is essential to achieve industrial cost targets. This naturally also holds for biocatalytic batch processes. Thus, there is good reason to examine in more detail the diverse methods of enzyme immobilization; the following sections aim to provide such an overview and will also examine the role of these catalysts in bio-flow chemistry.