A computational approach for the simulation and prediction of a linear three-step enzymatic cascade for the synthesis of -caprolactone (ECL) coupling an alcohol dehydrogenase (ADH), a cyclohexanone monooxygenase (CHMO), and a lipase for the subsequent hydrolysis of ECL to 6-hydroxyhexanoic acid (6-HHA). A kinetic model was developed with an accuracy of prediction for a fed-batch mode of 37% for substrate cyclohexanol (CHL), 90% for ECL, and >99% for the final product 6-HHA. Due to a severe inhibition of the CHMO by CHL, a batch synthesis was shown to be less efficient than a fed-batch approach. In the fed-batch synthesis, full conversion of 100 mM CHL was 28% faster with an analytical yield of 98% compared to 49% in case of the batch synthesis. The lipase-catalyzed hydrolysis of ECL to 6-HHA circumvents the inhibition of the CHMO by ECL enabling a 24% higher product concentration of 6-HHA compared to ECL in case of the fed-batch synthesis without lipase. Biotechnol. Bioeng. 2017;114: 1215-1221. 2017 Wiley Periodicals, Inc.
Poly-sigma-caprolactone (PCL) is chemically produced on an industrial scale in spite of the need for hazardous peracetic acid as an oxidation reagent. Although Baeyer-Villiger monooxygenases (BVMO) in principle enable the enzymatic synthesis of sigma-caprolactone (sigma-CL) directly from cyclohexanone with molecular oxygen, current systems suffer from low productivity and are subject to substrate and product inhibition. The major limitations for such a biocatalytic route to produce this bulk chemical were overcome by combining an alcohol dehydrogenase with a BVMO to enable the efficient oxidation of cyclohexanol to sigma-CL. Key to success was a subsequent direct ring-opening oligomerization of in situ formed sigma-CL in the aqueous phase by using lipase A from Candida antarctica, thus efficiently solving the product inhibition problem and leading to the formation of oligo-sigma-CL at more than 20g L(-1) when starting from 200mM cyclohexanol. This oligomer is easily chemically polymerized to PCL.
Title: A chemo-enzymatic route to synthesize (S)-gamma-valerolactone from levulinic acid Gotz K, Liese A, Ansorge-Schumacher M, Hilterhaus L Ref: Applied Microbiology & Biotechnology, 97:3865, 2013 : PubMed
Levulinic acid is a feasible platform chemical derived from acid-catalyzed hydrolysis of lignocellulose. The conversion of this substrate to (S)-gamma-valerolactone ((S)-GVL) was investigated in a chemo-enzymatic reaction sequence that benefits from mild reaction conditions and excellent enantiomeric excess of the desired (S)-GVL. For that purpose, levulinic acid was chemically esterified over the ion exchange resin Amberlyst 15 to yield ethyl levulinate (LaOEt). The keto ester was successfully reduced by (S)-specific carbonyl reductase from Candida parapsilosis (CPCR2) in a substrate-coupled cofactor regeneration system utilizing isopropanol as cosubstrate. In classical batch experiments, a maximum conversion of 95 % was achieved using a 20-fold excess of isopropanol. Continuous reduction of LaOEt was carried out for 24 h, and a productivity of more than 5 mg (S)-ethyl-4-hydroxypentanoate (4HPOEt) per mug CPCR2 was achieved. Afterwards (S)-4HPOEt (>99%ee) was substituted to lipase-catalyzed lactonization using immobilized lipase B from Candida antarctica to yield (S)-GVL in 90 % overall yield and >99%ee.
Triacylglycerol lipases (EC 3.1.1.3) catalyze both hydrolysis and synthesis reactions with a broad spectrum of substrates rendering them especially suitable for many biotechnological applications. Most lipases used today originate from mesophilic organisms and are susceptible to thermal denaturation whereas only few possess high thermotolerance. Here, we report on the identification and characterization of two novel thermostable bacterial lipases identified by functional metagenomic screenings. Metagenomic libraries were constructed from enrichment cultures maintained at 65 to 75 degrees C and screened resulting in the identification of initially 10 clones with lipolytic activities. Subsequently, two ORFs were identified encoding lipases, LipS and LipT. Comparative sequence analyses suggested that both enzymes are members of novel lipase families. LipS is a 30.2 kDa protein and revealed a half-life of 48 h at 70 degrees C. The lipT gene encoded for a multimeric enzyme with a half-life of 3 h at 70 degrees C. LipS had an optimum temperature at 70 degrees C and LipT at 75 degrees C. Both enzymes catalyzed hydrolysis of long-chain (C(12) and C(14)) fatty acid esters and additionally hydrolyzed a number of industry-relevant substrates. LipS was highly specific for (R)-ibuprofen-phenyl ester with an enantiomeric excess (ee) of 99%. Furthermore, LipS was able to synthesize 1-propyl laurate and 1-tetradecyl myristate at 70 degrees C with rates similar to those of the lipase CalB from Candida antarctica. LipS represents the first example of a thermostable metagenome-derived lipase with significant synthesis activities. Its X-ray structure was solved with a resolution of 1.99 A revealing an unusually compact lid structure.
