Process development for biocatalytic reactions is a complex task due to the required interaction of several different scientific disciplines. Additionally, there is a lack of standardized procedures for guiding development and for identifying the major process limitations in these systems. This work seeks to address this problem by providing a methodology based on a simple, systematic series of experiments. Application of the methodology helps identify the major bottleneck for process implementation, whether it be enzyme activity, enzyme stability, or substrate mass transfer. In addition, the underlying mechanism behind these limitations can also be inferred. The methodology is illustrated using a simulated reaction system and is also applied to three experimental case studies. This methodology provides a set of simple experiments that may be performed at an early stage of biocatalytic process development to guide effective improvement strategies, whether they be via protein engineering or reaction engineering. Ultimately, this should afford faster and more efficient implementation of biocatalysts in industrial processes.

As the application of biocatalysis to complement conventional chemical and catalytic approaches continues to expand, an increasing number of reactions involve poorly water-soluble substrates. At required industrial concentrations necessary for industrial implementation, this frequently leads to heterogeneous reaction mixtures composed of multiple phases. Such systems are challenging to sample, and therefore, it is problematic to measure representative component concentrations. Herein, an online method for following the progress of oxygen-dependent reactions through accurate measurement of the oxygen mass balance in the gas phase of a reactor is demonstrated and validated. The method was successfully validated and demonstrated by using two model reactions: firstly, the oxidation of glucose by glucose oxidase and, secondly, the Baeyer-Villiger oxidation of macrocyclic ketones to lactones. Initial reaction rate constants and time-course progressions calculated from the oxygen mass balance were validated against conventional online methods of dissolved oxygen tension and pH titration measurements. A feasible operating window and the sensitivity to dynamic changes of reaction rates were established by controlling oxygen transfer through the operating parameters of the reactor. Such kinetic data forms the basis for reaction characterisation, from which bottlenecks may be made evident and directed improvement strategies can be identified and implemented.

The effect of initial water activity of MTBE (methyl tert-butyl ether) medium on CALB (Candida antarctica lipase B) catalyzed esterification reaction is investigated using experimental methods and classical molecular dynamics (MD) simulations. The experimental kinetic studies show that the initial reaction rate of CALB-catalyzed esterification reaction between butyric acid and ethanol decreases with increasing initial water activity of the medium. The highest rate of esterification is observed at the lowest water activity studied. MD simulations were performed to gain a molecular insight on the effect of initial water activity on the rate of CALB-catalyzed reaction. Our results show that hydration has an insignificant effect on the structure and flexibility of CALB. Rather, it appears that water molecules bind to certain regions ("hot spots") on the CALB surface and form clusters. The size of the water clusters at these hot spot regions gradually increase and expand with increasing water activity. Consequently, the surface area of CALB covered by the water molecules also increases. Specifically, our results indicate that a particular water cluster located close to the active site partially cover the binding pocket of substrate at high water activity. As a consequence, the effective concentration of substrate at the catalytic site decreases. Therefore, the reaction rate slows down with increasing water activity, which correlates well with the observed decrease in the experimentally determined initial reaction rate.

Biotechnology and BioengineeringVolume 113, Issue 4 p. 681-685 Issue Information - TOCFree Access Biotechnology and Bioengineering: Volume 113, Number 4, April 2016 First published: 25 February 2016 https://doi.org/10.1002/bit.25752AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Volume113, Issue4April 2016Pages 681-685 RelatedInformation

The ability of enzymes to operate in organic solvent is now widely accepted and is the basis for extensive research in enzymology. The challenge is to select the solvent media that allows the modulation of enzyme activity. For a rational selection of a solvent, it is necessary to understand the effect of organic solvent molecules on enzyme structure and the enzymatic reaction on a molecular level. To gain such insight, we combined experimental kinetics studies with full atomic molecular dynamics simulations and found a correlation between the activity of Candida antarctica lipase B (CALB) [for the esterification reaction between butyric acid and ethanol at a fixed water activity] and the binding of the solvent/substrate molecules in the active site region of CALB. We have investigated the influence of four organic solvents hexane (HEX), methyl tertiary butyl ether (MTBE), acetonitrile (ACN), and tertiary butanol (TBU)-on the catalytic activity of CALB for the esterification reaction. The solvents have been chosen on the basis of different polarity/functional groups. Our study shows that these organic solvents do not alter the overall conformation of CALB; rather, the solvent effects on the performance of the enzyme may be ascribed to binding of solvent molecules to the enzyme active site region and the solvation energy of substrate molecules in the different solvents. Polar solvent molecules interact strongly with CALB and compete with the substrate to bind to the active site region, resulting in an inhibitory effect which is also confirmed by the binding free energies for the solvent and substrate molecules estimated from the simulations. Consequently, the catalytic activity of CALB decreases in polar solvents. This effect is significant, and CALB is over 10 orders of magnitude more active in nonpolar solvents (HEX and MTBE) than in the polar solvents (ACN and TBU). TBU molecules show an exceptional behavior because the solvent molecule forms an extensive hydrogen bond network within the CALB active site region suggesting that solvent molecules rich on hydrogen bond acceptors and donors are poor solvents when used for lipase-catalyzed esterification reactions.

ABSTRACT In this work, we demonstrate the scale‐up from an 80 L fed‐batch scale to 40 m 3 along with the design of a 4 m 3 continuous process for enzymatic biodiesel production catalyzed by NS‐40116 (a liquid formulation of a modified Thermomyces lanuginosus lipase). Based on the analysis of actual pilot plant data for the transesterification of used cooking oil and brown grease, we propose a method applying first order integral analysis to fed‐batch data based on either the bound glycerol or free fatty acid content in the oil. This method greatly simplifies the modeling process and gives an indication of the effect of mixing at the various scales (80 L to 40 m 3 ) along with the prediction of the residence time needed to reach a desired conversion in a CSTR. Suitable process metrics reflecting commercial performance such as the reaction time, enzyme efficiency, and reactor productivity were evaluated for both the fed‐batch and CSTR cases. Given similar operating conditions, the CSTR operation on average, has a reaction time which is 1.3 times greater than the fed‐batch operation. We also showed how the process metrics can be used to quickly estimate the selling price of the enzyme. Assuming a biodiesel selling price of 0.6 USD/kg and a one‐time use of the enzyme (0.1% (w/w oil ) enzyme dosage); the enzyme can then be sold for 30 USD/kg which ensures that that the enzyme cost is not more than 5% of the biodiesel revenue. Biotechnol. Bioeng. 2016;113: 1719–1728. © 2016 Wiley Periodicals, Inc.

ABSTRACT An experimental platform based on scaled‐down unit operations combined in a plug‐and‐play manner enables easy and highly flexible testing of advanced biocatalytic process options such as in situ product removal (ISPR) process strategies. In such a platform, it is possible to compartmentalize different process steps while operating it as a combined system, giving the possibility to test and characterize the performance of novel process concepts and biocatalysts with minimal influence of inhibitory products. Here the capabilities of performing process development by applying scaled‐down unit operations are highlighted through a case study investigating the asymmetric synthesis of 1‐methyl‐3‐phenylpropylamine (MPPA) using ω‐transaminase, an enzyme in the sub‐family of amino transferases (ATAs). An on‐line HPLC system was applied to avoid manual sample handling and to semi‐automatically characterize ω‐transaminases in a scaled‐down packed‐bed reactor (PBR) module, showing MPPA as a strong inhibitor. To overcome the inhibition, a two‐step liquid–liquid extraction (LLE) ISPR concept was tested using scaled‐down unit operations combined in a plug‐and‐play manner. Through the tested ISPR concept, it was possible to continuously feed the main substrate benzylacetone (BA) and extract the main product MPPA throughout the reaction, thereby overcoming the challenges of low substrate solubility and product inhibition. The tested ISPR concept achieved a product concentration of 26.5 g MPPA · L −1 , a purity up to 70% g MPPA · g tot −1 and a recovery in the range of 80% mol · mol −1 of MPPA in 20 h, with the possibility to increase the concentration, purity, and recovery further. Biotechnol. Bioeng. 2017;114: 600–609. © 2016 Wiley Periodicals, Inc.

Biotechnology and BioengineeringVolume 113, Issue 8 p. 1611-1615 Issue Information - TOCFree Access Biotechnology and Bioengineering: Volume 113, Number 8, August 2016 First published: 27 June 2016 https://doi.org/10.1002/bit.25780AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Volume113, Issue8August 2016Pages 1611-1615 RelatedInformation

The industrial implementation of biocatalysis for production of pharma and fine chemicals has grown substantially over recent years. An upcoming application is that of chiral synthesis of optically pure amines, a technology known for many years but that is now seeing a renewed and wider interest in industry. The technology has been demonstrated in a few selected cases, but widespread implementation and for a broader range of target molecules requires a deeper understanding of the underlying thermodynamic as well as economic constraints for the different choices that can be made in designing the process, in particular the choice of amine donor. This paper discusses these constraints and demonstrates, through simple thermodynamic and economic models, the process targets that need to be set and achieved for a process dependent on allowed process costs and quality targets.

The alkaline process for making biodiesel (fatty acid methyl esters, or FAME) is highly efficient at the transesterification of glycerides. However, its performance is poor when it comes to using oil that contain significant amounts of free fatty acids (FFA). The traditional approach to such feedstocks is to employ acid catalysis, which is slow and requires a large excess of methanol, or to evaporate FFA and convert that in a separate process. An attractive option would be to convert the FFA in oil feedstocks to FAME, before introducing it into the alkaline process. The high selectivity of enzyme catalysis makes it a suitable basis for such a pretreatment process. In this work, we present a characterization of the pretreatment of high-FFA rapeseed oil using immobilized Candida antarctica lipase B (Novozym 435), focused on the impact of initial FFA and methanol concentration. Based on experimental results, we have identified limitations for the process in terms of FFA concentration in the feedstock and make suggestions for process operation. It was found that, using 5% catalyst and 4% methanol at 35°C, the FFA concentration could be reduced to 0.5% within an hour for feedstock containing up to 15% FFA. Further, the reaction was observed to be under kinetic control, in that the biocatalyst converts FFA (and FAME) at a much higher rate than glyceride substrates. There is thus, both a minimum and a maximum reaction time for the process to achieve the desired concentration of FFA. Finally, an assessment of process stability in a continuous packed bed system indicates that as much as 15 m(3) oil could potentially be pretreated by 1 kg of biocatalyst at the given process conditions.

In this this paper, we use mechanistic modelling to guide the development of a continuous enzymatic process that is performed as a fed-batch operation. In this work we use the enzymatic biodiesel process as a case study. A mechanistic model developed in our previous work was used to determine the reactor operating conditions for a desired conversion. However, in using a detailed mechanistic model, given the large number of parameters and few experimental data points, the parameters were found not identifiable. The model is then only applicable within the limited operating range for which the model was validated. We hypothesize that fitting this model to fed-batch and continuous stirred tank reactor (CSTR) data together will enable us to use the model for determination of residence times to reach a specified conversion in a CSTR. With this approach, the model fits the experimental data for the five measured components (triglycerides, diglycerides, monoglycerides, free fatty acid and fatty acid methyl esters (biodiesel)) much better than using fed-batch data alone given the smaller residuals. We also observe a reduction in the correlation between the parameters. The model was then used to predict that 5 reactors are required (with a combined residence time of 30 hours) to reach a final biodiesel concentration within 2 % of the 95.6 mass % achieved in a fed-batch operation, for 24 hours.

ABSTRACTCallera™ Trans L, a liquid formulation of Thermomyces lanuginosus lipase, has recently shown great promise as a cost‐efficient catalyst for methanolysis of triglyceride substrates, specifically in the BioFAME process. However, identifying the right combination of temperature and concentrations of catalyst, water and methanol to realize the full potential of the reaction system has remained a challenge. This study presents an investigation of the impact of temperature, enzyme and water concentration on the reaction, as well as the effect of methanol feed rate for the conversion of rapeseed oil in a fed‐batch reaction system. It was observed that the reaction can be divided into two distinct parts. The first part of the reaction, during which primarily tri‐ and diglycerides are converted, proceeded at a high rate and thus required a high rate of methanol supply. The second part of the reaction, where the remaining di‐ and monoglycerides are converted, proceeded at a much lower rate. Consequently, it is necessary to reduce the methanol feed rate during the latter part of the reaction to avoid inhibition or even inactivation of the enzyme. Since the second part of the reaction occupied most of the 24‐h reaction time, it was concluded that this is the part of the process where further development efforts should be targeted. This point was demonstrated by partially substituting the catalyst with a lipase with a different specificity, which enhanced the performance during the second phase of the reaction. Biotechnol. Bioeng. 2014;111: 2446–2453. © 2014 Wiley Periodicals, Inc.

In this article, a kinetic model for the enzymatic transesterification of rapeseed oil with methanol using Callera™ Trans L (a liquid formulation of a modified Thermomyces lanuginosus lipase) was developed from first principles. We base the model formulation on a Ping‐Pong Bi‐Bi mechanism. Methanol inhibition, along with the interfacial and bulk concentrations of the enzyme was also modeled. The model was developed to describe the effect of different oil compositions, as well as different water, enzyme, and methanol concentrations, which are relevant conditions needed for process evaluation, with respect to the industrial production of biodiesel. The developed kinetic model, coupled with a mass balance of the system, was fitted to and validated on experimental results for the fed‐batch transesterification of rapeseed oil. The confidence intervals of the parameter estimates, along with the identifiability of the model parameters were presented. The predictive capability of the model was tested for a case using 0.5% (wt. Enzyme/wt. Oil), 0.5% (wt. Water /wt. Oil) and feeding 1.5 times the stoichiometric amount of methanol in total over 24 h. For this case, an optimized methanol feeding profile that constrains the amount of methanol in the reactor was computed and the predictions experimentally validated. Monte‐Carlo simulations were then used to characterize the effect of the parameter uncertainty on the model outputs, giving a biodiesel yield, based on the mass of oil, of 90.8 ± 0.55 mass %. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1277–1290, 2014

In this work a kinetic model for the enzymatic transesterification of rapeseed oil using a solubilised lipase (Callera Trans L-Thermomyces lanuginosus) was developed from first principles. The model is based on a Ping-Pong Bi-Bi mechanism, with methanol inhibition, along with consideration of the differences in the interfacial and bulk concentrations of the enzyme. The model is then used to evaluate various feeding strategies to improve the enzymatic biodiesel production. The feeding strategies investigated, gave insight into how the methanol should be fed to potentially mitigate enzyme deactivation while improving the biodiesel yield. The best experimental results gave a yield of 703.76 g FAME L-1 and a reactor productivity of 28.12 g FAME L-1 h-1. In comparison, to reach the same yield, the optimised two step feeding strategy took 6.25 hours less, which equates to an increase the reactor productivity of 36.9 %.

In this contribution we extend our modelling work on the enzymatic production of biodiesel where we demonstrate the application of a Continuous‐Discrete Extended Kalman Filter (a state estimator). The state estimator is used to correct for mismatch between the process data and the process model for Fed‐batch production of biodiesel. For the three process runs investigated, using a single tuning parameter, q x = 2 × 10 −2 which represents the uncertainty in the process model, it was possible over the entire course of the reaction to reduce the overall mean and standard deviation of the error between the model and the process data for all of the five measured components (triglycerides, diglycerides, monoglycerides, fatty acid methyl esters, and free fatty acid). The most significant reduction for the three process runs, were for the monoglyceride and free fatty acid concentration. For those components, there was over a ten‐fold decrease in the overall mean error for the state estimator prediction compared with the predictions from the pure model simulations. It is also shown that the state estimator can be used as a tool for detection of outliers in the measurement data. For the enzymatic biodiesel process, given the infrequent and sometimes uncertain measurements obtained we see the use of the Continuous‐Discrete Extended Kalman Filter as a viable tool for real time process monitoring. © 2014 American Institute of Chemical Engineers Biotechnol. Prog. , 31:585–595, 2015