Biomacromolecules 2010, 11, 259–268 259 Two-Step Biocatalytic Route to Biobased Functional Polyesters from ω-Carboxy Fatty Acids and Diols Yixin Yang, Wenhua Lu, Xiaoyan Zhang, Wenchun Xie, Minmin Cai, and Richard A. Gross* NSF I/UCRC for Biocatalysis and Bioprocessing of Macromolecules, Department of Chemical and Biological Sciences, Polytechnic Institute of NYU, Six Metrotech Center, Brooklyn, New York 11201 Received September 29, 2009; Revised Manuscript Received November 25, 2009 Biobased ω-carboxy fatty acid monomers 1,18-cis-9-octadecenedioic, 1,22-cis-9-docosenedioic, and 1,18-cis- 9,10-epoxy-octadecanedioic acids were synthesized in high conversion yields from oleic, erucic and epoxy stearic acids by whole-cell biotransformations catalyzed by C. tropicalis ATCC20962. Maximum volumetric yields in shake-flasks were 17.3, 14.2, and 19.1 g/L after 48 h conversion for oleic acid and 72 h conversions for erucic and epoxy stearic acids, respectively. Studies in fermentor with better control of pH and glucose feeding revealed that conversion of oleic acid to 1,18-cis-9-octadecenedioic acid by C. tropicalis ATCC20962 occurred with productivities up to 0.5 g/L/h. The conversion of ω-carboxy fatty acid monomers to polyesters was then studied using immobilized Candida antarctica Lipase B (N435) as catalyst. Polycondensations with diols were performed in bulk as well as in diphenyl ether. The retension of functionality from fatty acid, to ω-carboxy fatty acid monomer and to corresponding polyesters resulted in polymers with with unsaturated and epoxidized repeat units and Mw values ranging from 25000 to 57000 g/mol. These functional groups along chains disrupted crystallization giving materials that are low melting (23-40 °C). In contrast, saturated polyesters prepared from 1,18-octadecanedioic acid and 1,8-octanediol have correspondingly higher melting transitions (88 °C). TGA results indicated that all synthesized polyesters showed high thermal stabilities. Thus, the preparation of functional monomers from C. tropicalis ω-oxidation of fatty acids provides a wide range of new monomer building blocks to construct functional polymers. Introduction It is well-known that many microorganisms can convert n-alkanes and fatty acids to their corresponding R,ω-diacids, R R Aliphatic ,ω-dicarboxylic acids ( ,ω-diacids) are widely including Candida tropicalis,4,6,7 Candida cloaca,8 Cryptococ- used as raw materials for the manufacture of engineered plastics, 1 9 1 cus neoforman, and Corynebacterium sp. Candida tropicalis perfumes, fragrances, lubricants, and adhesives. The majority and similar yeasts produce R,ω-diacids through an ω-oxidation R of currently used ,ω-diacids are produced by chemical means pathway. The terminal methyl group is first hydroxylated by a from nonrenewable petrochemical feedstocks. For examples, cytochrome P450 monooxygenase that is further transformed adipic acid is manufactured by a two-step process involving via the action of fatty alcohol oxygenase and aldehyde dehy- stoichiometricnitricacidoxidationofacyclohexanol-cyclohenanone drogenase to form diacids.10 Of particular interest with regards mixture, which is aerobically generated from cyclohexane using to this paper is the report of C. tropicalis ATCC20962, which a homogeneous cobalt-based catalyst.2 Dodecanedioic acid is was engineered to block its -oxidation pathway.4 Yi et al.11 manufactured by a nickel-catalyzed cyclic trimerization of used a mutant of C. tropicalis to convert oleic acid to 1,18- butadiene, followed by hydrogenation to cyclododecane, air cis 4 oxidation to a mixture of cyclododecanone and cyclododecanol, -9-octadecenedioic acid. Picataggio et al. used engineered and, finally, nitric acid oxidation to dodecanedioic acid.3 Also, C. tropicalis ATCC20962 and its P450 monooxygenase chemical routes to R,ω-diacids can be tedious and result in (P450alkl) and NADPH-oxidoreductase (CPR) amplified strain unwanted byproducts. Furthermore, chemical routes are unavail- AR40 to convert methyl esters of saturated (C14 to C18) and R able to synthesize R,ω-diacids with carbon numbers greater than unsaturated (oleic, erucic) fatty acids to their corresponding ,ω- 13.4 diacids. Bioconversion of C12 to C22 n-alkanes and fatty acids to R,ω-diacids by strain AR40 was successfully performed Nature uses long-chain unsaturated and epoxidized dicar- without undesired modification of substrates or products via the boxylic acids (mainly 9,10-epoxy octadecanedioic, 1,18-cis-9- -oxidation pathway. Fabritius et al.12,13 reported that a mutant octadecenedioic, and 9,10-dihydroxy octadecanedioic acids), as of C. tropicalis M25 converts oleic acid12 and linoleic acid13 well as ω-hydroxyl carboxylic acids (mainly 9,10-epoxy-18- to their corresponding 3-hydroxydicarboxylic acids. Unfortu- hydroxy octadecanoic and 9,10,18-trihydroxy octadecanoic nately, bioconversions using of C. tropicalis described in Yi et acids) as building blocks to synthesize important plant polyesters 11 12,13 5 al. and Fabritius et al. resulted in low yields and product such as suberin and cutin. These and related monomers would R be useful to design and synthesize unique functional polyesters mixtures, including ,ω-diacids with different carbon-chain that would also biodegrade. However, they are difficult to lengths or having variable contents of hydroxylation along synthesize by chemical methods and are currently unavailable chains. Furthermore, comparison of C. tropicalis ATCC20962- commercially. catalyzed conversion rates of unsaturated acids differing in chain lengths to their corresponding R,ω-diacids has not been studied. * To whom correspondence should be addressed. Telephone: 718-260- Moreover, whereas the in vitro ω-hydroxylation of 9,10- 3024. Fax: 718-260-3075. E-mail: [email protected]. epoxystearic acid, catalyzed by cytochrome P450s in plants such 10.1021/bm901112m 2010 American Chemical Society Published on Web 12/10/2009 260 Biomacromolecules, Vol. 11, No. 1, 2010 Yang et al. as CYP86A1, CYP86A8, CYP94A1, and CYP94A5, has been monomers 1,18-cis-9-octadecenedioic acid (ω-carboxyl OA), studied,14 corresponding conversions of epoxy-containing fatty 1,22- cis-9-docosenedioic acid (ω-carboxyl EA), and 1,18-cis- acids to their corresponding epoxidized R,ω-diacids by C. 9,10-epoxy-octadecanedioic acid (ω-carboxyl epoxy SA). Ef- tropicalis has not been reported. Resulting R,ω-diacid monomers fects of fatty acid chain length and presence of unsaturation with an internal double bond, epoxy moiety, or other naturally versus epoxy groups at C18 carbons on the C. tropicalis occurring functionality in fatty acids can provide important ATCC20962-catalyzed conversion of these fatty acid substrates building blocks for polymer synthesis. Also, conversions of to diacids was studied. Diacid monomers were purified and unsaturated and epoxy functionalized R,ω-diacids to corre- characterized by gas chromatography (GC)-mass spectrometry sponding polyesters would be best performed by lipase catalysis (MS) and nuclear magnetic resonance (1H NMR and 13C NMR). for reasons discussed below. Conversion of unsaturated and epoxidized ω-carboxyl fatty acids Normally, polyesters are synthesized by ester interchange to polyesters was performed by condensation copolymerizations reactions or by direct esterification of diacid/diol or hydroxy- with a diol using an immobilized CALB (N435) as catalyst. acids.15 These reactions often require harsh reaction conditions Comparison of CALB-catalyzed copolymerizations in-bulk, and and metal catalysts, which are not compatible with retention of in diphenyl ether, was performed and chain growth was functional group structure during polymer synthesis. An alterna- monitored as a function of time. Effects of changes in ω-car- tive approach to traditional chemical polymerization methods boxyl fatty acid building blocks on thermal properties of is the use of cell-free, enzyme-catalyzed polycondensation resulting polyesters were also investigated. reactions.16 For example, lipase-catalyzed polymerizations of monomers containing a double bond or an epoxy group to Experimental Section synthesize polyesters have been reported by several authors.17 ∼ ∼ In one example described by Olsen and Sheares,17b trans-- Materials. Oleic acid (technically, 90% pure), erucic acid ( 99% hydromuconic acid (HMA) was copolymerized (90 °C, in-bulk, pure), 1,3-propanediol, 1,8-octanediol, 1,16-hexadecanediol, antifoam 48 h) with 1,8-octanediol using immobilized Candida antarctica 204, and Glucose (HK) Assay Kit were purchased from Sigma-Aldrich (St. Louis, U.S.A.). The diols were obtained in the highest available Lipase B (CALB) to give polyesters with M 10500 g/mol (M / n w purity and were used as received. 1,18-Octadecanedioic acid (∼95% M ) 2.0) whose double bonds remained intact.17b n pure) and thin layer chromatography (TLC) aluminum sheets were A natural bifunctional fatty acid of interest as a monomer purchased from TCI (Portland, U.S.A.) and Merck (Darmstadt, for polymer synthesis is ricinoleic acid [(Z,R)-12-hydroxy-9- Germany), respectively. Novozym 435 (abbreviated as N435, specified octadecenoic acid], derived from castor oil. However, esterifi- activity 10000 PLU/g) was a gift from Novozymes (Bagsvaerd, cation of secondary hydroxyl groups by lipase-catalysis is Denmark) and consists of CALB physically adsorbed within the generally slow.18,19 Furthermore, to use ricinoleic acid as a macroporous resin Lewatit VPOC 1600 (poly[methyl methacrylateco- bifunctional monomer to prepare high molecular weight prod-