SummaryAlthough nisin is a model lantibiotic, our knowledge of the specific interactions of prenisin with its modification enzymes remains fragmentary. Here, we demonstrate that the nisin modification enzymes NisB and NisC can be pulled down in vitro from Lactococcus lactis by an engineered His-tagged prenisin. This approach enables us to determine important intermolecular interactions of prenisin with its modification machinery within L. lactis. We demonstrate that (i) NisB has stronger interactions with precursor nisin than NisC has, (ii) deletion of the propeptide part keeping the nisin leader intact leads to a lack of binding, (iii) NisB point mutants of highly conserved residues W616, F342A, Y346F and P639A are still able to dehydrate prenisin, (iv) NisB D(77-79)Y80F mutant decreased the levels of NisB-prenisin interactions and resulted in unmodified prenisin, (v) substitution of an active site residue H331A in NisC leads to higher amounts of the co-purified complex, (vi) NisB is present in the form of a dimer, and (vii) the region FNLD (-18 to -15) of the leader is an important site for binding not only to NisB, but also to NisC.

Highlights• Butyl butyrate synthesis by Enzymatic Reactive Distillation is possible• Established rate-based model to describe Enzymatic Reactive Distillation • First time validation for an Enzymatic Reactive Distillation process model AbstractFor enzyme-catalyzed reactions, batch processes using stirred tank reactors are the state-ofthe-art production mode. The yield of the process may be limited by reaction equilibrium, product inhibition of the enzyme, low concentrations and possibly low reaction rates, while the recovery of the product may be limited due to thermodynamic constraints such as azeotropes. Using enzymes in an integrated reactive distillation process can overcome these limitations and provides a cost advantage over classic batch reactor processes.The aim of this paper is i) to report the successful pilot-scale experimental validation of an Enzymatic Reactive Distillation (ERD) process for the synthesis of butyl butyrate and ii) to Pilot-Scale Validation of Enzymatic Reactive Distillation for Butyl Butyrate ProductionWierschem et al.2 establish a rate-based model for conceptual process design which can be quickly adapted to other systems.The main novelty is the application of a continuous RD column with enzymes as a heterogeneous catalyst provided in two different types of catalytic packing: loosely filled immobilized enzyme beads in standard packings with catalyst pockets and gauze packings with catalytic coating.Experimental pilot-scale experiments show the feasibility of ERD and allow the comparison of the different packing types based on catalytic performance as well as stability.Furthermore, these experiments are used to validate a predictive rate-based model to describe ERD which can be used to check the sensitivity of process and design parameters as well as to provide a quick adaption to other systems for quick evaluation.

The integrated process setup of reaction and separation in reactive distillation can be favorable for enzyme-catalyzed reactions that are often equilibrium-limited and/or inhibited by the product of the reaction. However, until now, no appropriate way has described the implementation of the enzymes into distillation columns. In this work, a special type of biocatalytic coating for commercially available structured packings was developed to enable enzymatic reactions in reactive distillation columns. Lipase CALB was immobilized within the silica-gel based coating and showed high long-term stability, i.e., the coatings can be stored for at least 50 days at room temperature without any significant loss of enzyme activity. In addition, the silica-gel coatings were stable in organic media and at increased temperatures. Reactive distillation experiments were carried out in a batch reactive distillation setup comprising of structured A3-500 packings from Montz (Hilden, Germany) coated with the catalytic silica gel. Stepwise removal of the low boiling point product from the distillate shifted the equilibrium to the product side and increased the conversion of the reactants from 60% to 98%, in comparison with the corresponding setup in a standard batch reactor. The stability of the silica-gel coating and loss of the enzyme was investigated during the distillation runs. Overall, the integration of an enzymatic reaction into a reactive distillation column offers new possibilities for the application of biocatalysts in organic synthesis.

This work presents a feasibility study for an enzymatic reaction in a continuously operated reactive distillation column. As a model reaction, the transesterification of ethyl butyrate with n-butanol in the presence of lipase CALB was considered. For use in the distillation column, lipase CALB was immobilized by entrapment in a hydrophobic silica xerogel and introduced as granulate into the catalytic packing Katapak-SP-11. The reaction kinetics was experimentally determined for different concentration and temperature ranges and described by means of the Michaelis−Menten double-substrate kinetic model in combination with the Arrhenius model. With these kinetic data, process simulations were carried out with an Aspen Custom Modeler nonequilibrium-stage model validated for a DN50 pilot-scale column. The concentration of n-butanol in the reactive section was maintained low to decrease the inhibiting effects on the enzyme. For an optimized setup and operating conditions, conversion rates of more than 90% were achieved for n-butanol and 26% for ethyl butyrate. These results clearly demonstrate that lipase CALB can be applied in a continuously operated reactive distillation column.

An enantioselective biocatalytic reaction was carried out for the first time in a fully integrated batch reactive distillation setup. The investigated reaction was the lipase catalyzed kinetic resolution of a racemic mixture of (R/S)-2-pentanol with ethyl butyrate. The reaction is strongly limited by the reaction equilibrium so that the reactive distillation helped to shift the equilibrium toward the product side. The enantioselectivity of the applied lipase was high with ee values >99% for 2-pentanol at conversions of 69 ± 3%. As established in our previous work, the biocatalyst was again immobilized within a hydrophobic silica coating for structured packings. The production of the biocatalytic coating was further developed as a spray-coating method to allow reproducible coatings which also can be applied on larger surfaces. The influence of the coating on separation efficiency and pressure drop was studied as well as the stability of the coating under the required process conditions. Overall, this work demonstrates the first kinetic resolution in a reactive distillation setup with structured packings and presents the catalytic coating as an alternative structure for (bio)catalytic columns internals.