A Fed‐Batch Synthetic Strategy for a Three‐Step Enzymatic Synthesis of Poly‐ϵ‐caprolactone

Abstract

A three‐step enzymatic reaction sequence for the synthesis of poly‐ϵ‐caprolactone (PCL) was designed running in a fed‐batch operation. The first part of the cascade consisted of two oxidation steps starting with alcohol dehydrogenase catalyzed oxidation from cyclohexanol to cyclohexanone and further oxidation to ϵ‐caprolactone (ECL) by means of a Baeyer–Villiger monooxygenase. As a third step, lipase‐catalyzed hydrolysis of the lactone to 6‐hydroxyhexanoic acid (6‐HHA) was designed. With this biocatalytic multistep process reported herein, severe substrate surplus and product inhibition could be circumvented by the fed‐batch operation by adding the cyclohexanol substrate and by in situ product removal of ECL by hydrolysis, respectively. Up to 283 mm product concentration of 6‐HHA was reached in the fed‐batch operated process without loss in productivity within 20 h. After extraction and subsequent polymerization catalyzed by Candida antarctica lipase B, analysis of the unfractionated polymer revealed a bimodal distribution of the polymer population, which reached a mass average molar mass (Mw) value of approximately 63 000 g mol−1 and a dispersity (Mw/Mn) of 1.1 for the higher molecular weight population, which thus revealed an alternative route to the conventional synthesis of PCL.

Kinetic insights into ϵ‐caprolactone synthesis: Improvement of an enzymatic cascade reaction

“…Therefore, the data of one of our recently published results (Scherkus et al, 2016) shown in Figure 7 were taken into account. Therefore, the data of one of our recently published results (Scherkus et al, 2016) shown in Figure 7 were taken into account.…”

“…Nomenclature a max maximum enzyme activity, mmol min À1 mL À1 (representing v max [mmol min À1 mg À1 ] multiplied by the enzyme concentration [mg mL À1 ]) a tot total maximum enzyme activity considering initial reaction volume (only simulation), mmol/min K ADH eq equilibrium constant for the ADH catalyzing the oxidation of CHL to CHN K X M Michaelis-Menten constant of compound X, mM k base rate constant of supplied volume by titration, L mmol À1 min À1 (k ECL divided by the molar concentration of NaOH [5 mol/L]) k ECL rate constant for lipase-catalyzed hydrolysis of ECL, min À1 k feed rate constant of supplied volume of CHL, L/min K ECL loss rate constant of uncatalyzed hydrolysisof ECL, min À1 K CHL loss rate constant of depletion of CHL, min À1 V reaction volume, L v feed feed rate of CHL, mmol/min Figure 6. Comparison of experimental and calculated data from the enhanced fedbatch synthesis of ECL and subsequent hydrolysis to 6-HHA (Scherkus et al, 2016). Conditions for calculation: V ¼ 0.05 L, a CHMO tot ¼ 28.5 mmol/min, a ADH;ox tot ¼ 518.5 mmol/min, a ADH;red tot ¼ 4229.4 mmol/min, k CHL loss ¼ 0.002 min À1 , v feed ¼ 19/ 20.4 mmol/min, k ECL loss ¼ 9.3 Á 10 À5 min À1 , k 0 ECL ¼ 0.004 L g À1 min À1 , [CALB] ¼ 0.5 g/L, k feed ¼ 1.9 Á 10 À6 /1.95 Á 10 À6 L/min, k base ¼ 8 Á 10 À6 L mmol À1 min À1 .…”

“…As the biocatalytic approach necessitates nicotinamide cofactors, the use of whole-cells has been of high interest (Alphand and Wohlgemuth, 2010;Alphand et al, 2003;Baldwin et al, 2008;Fink et al, 2011;Law et al, 2006) with the supply of a carbon source to regenerate the cofactor. The self-sufficient linear cascade combining an ADH (Weckbecker and Hummel, 2006) and a cyclohexanone monooxygenase (CHMO) (Donoghue et al, 1976) for the synthesis of ECL makes the biocatalytic route very efficient since only a single substrate ends up to a single final product (Mallin et al, 2013;Scherkus et al, 2016;Schmidt et al, 2015a,b;Staudt et al, 2013). The self-sufficient linear cascade combining an ADH (Weckbecker and Hummel, 2006) and a cyclohexanone monooxygenase (CHMO) (Donoghue et al, 1976) for the synthesis of ECL makes the biocatalytic route very efficient since only a single substrate ends up to a single final product (Mallin et al, 2013;Scherkus et al, 2016;Schmidt et al, 2015a,b;Staudt et al, 2013).…”

“…The self-sufficient linear cascade combining an ADH (Weckbecker and Hummel, 2006) and a cyclohexanone monooxygenase (CHMO) (Donoghue et al, 1976) for the synthesis of ECL makes the biocatalytic route very efficient since only a single substrate ends up to a single final product (Mallin et al, 2013;Scherkus et al, 2016;Schmidt et al, 2015a,b;Staudt et al, 2013). 1) (Scherkus et al, 2016). Very recently, we reported on the above-mentioned ADH-CHMO linear cascade which was extended by a lipase catalyzed hydrolysis of ECL to 6-HHA in the aqueous medium ( Fig.…”

Biocatalytic conversion of cycloalkanes to lactones using an in‐vivo cascade in Pseudomonas taiwanensis VLB120

“…Realizing multi‐step reactions in one‐pot circumvents several purification steps and provides opportunities to reduce waste, time, and costs (Muschiol, Peters, Oberleitner, & Mihovilovic, ; Oberleitner et al, ; Schrittwieser, Velikogne, & Kroutil, ). In this context, several biocatalytic cascades have been designed to synthesize ϵ‐caprolactone starting from cyclohexanol, 2‐cyclohexen‐1‐ol, or hexane‐1,6‐diol (Bornadel, Hatti‐Kaul, Hollmann, & Kara, ; Mallin, Wulf, & Bornscheuer, ; Oberleitner, Peters, Rudroff, Bornscheuer, & Mihovilovic, ; Scherkus et al, ; Scherkus et al, ; Staudt, Bornscheuer, Menyes, Hummel, & Gröger, ). Recently, a biocatalytic reaction concept that mimicked chemical reaction route to synthesize lactones from cycloalkanes was designed by utilizing an in‐vitro cascade of four isolated enzymes (Pennec, Hollmann, Smit, & Opperman, ).…”