Enzymatic synthesis of galactosyl lactic ethyl ester and its polymer for use as biomaterials

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Journal of Biotechnology 132 (2007) 314–317

Short communication

Enzymatic synthesis of galactosyl lactic ethyl ester and its polymer for use as biomaterials Hongfei Jia a,1 , Ping Wang b,∗ a

b

Department of Chemical Engineering, University of Akron, Akron, OH 44325-3906, United States Department of Bioproducts and Biosystems Engineering, University of Minnesota, St. Paul, MN 55108, United States Received 12 October 2006; received in revised form 18 April 2007; accepted 28 June 2007

Abstract Lactate-based chemicals and polymers including poly(lactic acid) (PLA) are highly valuable materials for biomedical, food and general-purpose applications. Chemical synthesis, albeit the high reaction velocities achieved with it, often leaves chemical residues that are subject to health and safety concerns. Alternative biosynthesis is preferred in order to overcome these problems. Herein we report a novel enzymatic synthesis for the preparation of ␤-d-galactosyl-l-lactic acid ethyl ester (GLAEE). Such a product, which may find applications in food and personal care products, is generally difficult to synthesize via traditional chemical routes because the reactions have to be highly selective due to the multiple hydroxyl groups of sugars. We further explore the enzymatic polymerization of GLAEE to form a unique biopolymer, poly(␤-d-galactoside-co-l-lactic acid) (PGLA). Novozyme 435® was found efficient in catalyzing the polymerization reaction in acetone with a conversion yield of 60% within 100 h. The molecular weight of the polymer product ranged from about 800–2000 as analyzed by using ESI-MS. It is expected that a variety of sugar-hydroxyl acids copolymers can be prepared through the same approach and a new class of biomaterials can thus be developed. © 2007 Elsevier B.V. All rights reserved. Keywords: Enzymatic polymerization; Biosynthesis; Biomaterails; Transglycosylation; Poly(lactic acid); Lipase; Galactosidase

Lactic acid is a versatile chemical used for the derivation of a broad range of chemicals and materials. For example, ethyl lactate ester have been used widely in electronic industry as a processing solvent, while poly(lactic acid) (PLA) is used for purposes ranging from biomedical materials to disposable plastics, mostly attributing to its good mechanical properties and biodegradability (Ikada and Tsuji, 2000). In exploring even broader applications, people often feel restricted by the properties of these materials. For example, PLA’s applications can be largely limited by its low hydrophilicity, high rigidity and crystallinity, as well as the difficulty in controlling its degradation rate. To meet the diversified application requirements, many copolymers of PLA have been developed, such as those with propylene and ethylene glycols (Gilding and Reed, 1979; Yamaoka and Kimura, 1996; Jeong et al., 1997), caprolactone (Ni and Yu, 1998), and lysine (Barrera et al., 1993). ∗

Corresponding author. Tel.: +1 612 624 4792; fax: +1 612 624 6286. E-mail address: [email protected] (P. Wang). 1 Present address: Materials Research Department, Toyota Technical Center, 2350 Green Road, Ann Arbor, MI 48105, United States. 0168-1656/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2007.06.018

Sugars represent another major class of biomaterials. In contrast to PLA, sugar-based materials are highly hydrophilic and are subject to fast biodegradation. The last two decades have witnessed a growing interest in using enzymatic or chemoenzymatic methods for the preparation of sugar-containing polymers (Patil et al., 1991; Blinkovsky and Dordick, 1993; Chen et al., 1995; Wang et al., 1995; Kitagawa and Tokiwa, 1998; Wang and Dordick, 1998; Liu and Dordick, 1999). Enzymes are attractive for such applications because of their unparalleled regio- and stereo-selectivities. With the specificity of enzymes, linear polymers with sugar units can be synthesized without complicated protection/deprotection steps as normally needed in chemical methodologies (Okada and Aoi, 1995; Wulff et al., 1997; Wang et al., 2002). Incorporation of sugar units into PLA may afford a new class of materials with several desired features, such as controllable degradation rates, attachment of bioactive and other reagents through the hydroxyl groups, and easy gelation by crosslinking. To the best of our knowledge, hitherto there is no report for the development of this type of copolymers. In the present work, we demonstrate the enzymatic synthesis of

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Scheme 1. Enzymatic synthesis of ␤-d-galactosyl-l-lactic acid ethyl ester (GLAEE) and poly(␤-d-galactoside-co-l-lactate) (PGLA).

␤-d-galactosyl-l-lactate ethyl ester (GLAEE) and the subsequent polymerization to produce a linear sugar-lactate copolymer, poly(␤-d-galactoside-co-l-lactic acid) (PGLA) (Scheme 1). Ethyl-l-lactate is an environmentally benign solvent that has been widely used for processing of foods, pharmaceuticals and cosmetics, as well as in the manufacturing processes of electronic device (Rauckman, 1996; Clary et al., 1998). It can also be used as a monomer for the synthesis of PLA (Distel et al., 2005). In this work, GLAEE was prepared via ␤-galactosidasecatalyzed transglycosylation reaction between d-lactose and ethyl-l-lactate (Scheme 1, step 1). Since ethyl-l-lactate is miscible with water, the reaction was conducted in aqueous buffer at pH 4.5 (acetate, 0.05 M), which is an optimal value for the enzyme applied in this work (Tanaka et al., 1975). The formation of product was monitored by using HPLC. No product was detected in the control experiment without enzyme (Fig. 1). The reaction solution was quenched by adding acetonitrile to precipitate out sugars and ␤-galactosidase. The product GLAEE was then purified by the removal of solvents and unreacted ethyl-l-

Fig. 1. Time course of GLAEE concentration change in the transglycosylation reaction. (): reaction catalyzed by ␤-galactosidase; (* ): control reaction without enzyme. Concentration was determined by HPLC (Shimadzu VP) with a Symmetry C18 column and a refractive index detector. The mobile phase consisted of water, acetonitrile and methanol (3/1/1 by volume, respectively).

lactate from the supernatant on a rotary evaporator. About 95% of the product detected analytically was recovered through this purification process. In our initial typical preparations, 2 g of lactose and 25 mg of ␤-galactosidase was first dissolved in 5 ml of buffer, and then mixed with 6 ml of ethyl-l-lactate. A yield of 4.6 ± 0.4% (based on lactose) was achieved within 14 h at room temperature. One factor that limits the yield of this reaction may come from the hydrolysis reaction of lactose that competes with the transglycosylation reaction. Ethyl-l-lactate appeared to be stable enough in the aqueous solution during the reaction. According to our tests, less than 0.05% of the ethyl l-lactate was hydrolyzed in a period of 24 h. However, lactose was found hydrolyzed significantly, because ␤-galactosidase catalyzes the hydrolysis of lactose and generates glucose and galactose in aqueous environments (Whitaker, 1994). This side reaction might limit the yield of GLAEE because the hydrolytic activity of ␤-galactosidase is usually much higher than its transglycosylation activity in aqueous reaction media. We observed that the concentration of GLAEE reached a peak value at a reaction time of about 14 h (Fig. 1), and thereafter decreased slightly. We believe that it is also related to the instability of lactose, whose continuous and fast hydrolysis may shift the reaction equilibrium and eventually drive the transglycosylation reaction toward the reverse direction. By using excess amount of ethyl-l-lactate to counter balance that effect, we were able to improve the yield to 27 ± 2%. The structure of GLAEE was characterized by 13 C NMR analysis (Fig. 2). The formation of C1(sugar)-O-CH(lactate) link was confirmed by chemical shifts of: (1) C1(sugar): 104.65 ppm (GLAEE), an evident shift as compared with 105.72 and 105.70 ppm (d-lactose) and 97.90 ppm (␤-d-galactose); (2) CH(lactate, C4 ): 75.98 ppm (GLAEE), shifting from 69.55 ppm of ethyl-l-lactate. GLAEE was dissolved in a mixed-solvent of water and acetonitrile (1/1, v/v) and further studied by electron spray ionization mass spectrometry (ESI-MS). A peak of m/z = 303 was identified on the mass spectrum, corresponding to a GLAEE molecule (280 g/mol) associated with a Na+ (23.0 g/mol). GLAEE is highly soluble in water and polar solvents such as acetone, acetonitrile, chloroform, DMF and

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Fig. 2. 13 C NMR spectra of GLAEE. C1–C6 (sugar part): 104.65, 73.57, 75.48, 71.32, 78.08 and 63.70 ppm; C1 –C5 (lactate part): 16.09, 65.26, 177.59, 75.98 and 20.94 ppm. NMR analysis was performed on a Gemini 300 system using D2 O as the solvent and trimethylsilyl propionic acid (sodium salt) as the external reference.

methanol. After purification, it appeared to be a viscous amber liquid, which may be directly used for cosmetic applications as other ␣-hydroxyl acid derivatives (Gilchrest, 1996). We further explored the enzymatic polymerization of GLAEE for production of a unique sugar-lactate alternative copolymer. The polymerization of GLAEE was achieved via a transesterification reaction catalyzed by an immobilized lipase, Novozyme 435® (Scheme 1, step 2). In lipase-catalyzed acylation reactions of sugars, C6 was the dominant reaction site when C1 was occupied (Therisod and Klibanov, 1986; Bjorkling et al., 1989; Adelhorst et al., 1990; Park et al., 2000). Novozyme 435® has been previously used for the transesterification reaction between ethyl-l-lactate and n-octyl glycoside (Bousquet et al., 1998; Torres et al., 2000), which was very close to the current system. To prepare PGLA, 160 mg of GLAEE monomer was dissolved in 2 ml of acetone, followed by the addition of ˚ molecular sieves. 80 mg of Novozyme 435® and 0.8 g of 3 A The molecular sieves were added as an attempt to absorb the ethanol produced during this reaction, as suggested by others (Holderich and Van Bekkum, 1991). HPLC analysis showed that over 60% of monomer was converted after 4 days of reaction (Fig. 3). In the control tests without the enzyme, the concentration of monomer only dropped about 10%, which is suspected to be a result of the adsorption of the monomer GLAEE onto

Fig. 3. Time course of GLAEE concentration change in the polymerization process. (): reaction catalyzed by Novozyme 435® ; (): control reaction without enzyme. GLAEE concentration was determined by HPLC analysis.

Fig. 4. Mass spectra of PGLA products. Mw of the repeating units was identified as 234, corresponding to C9 H14 O7 as shown in Scheme 1.

molecular sieves. GLAEE was very stable in acetone and showed no disappearance without the addition of molecular sieves. The resulting polymer was insoluble in acetone so that it had to be collected with deionized water after the reaction. The product was then dried via lyophilization. Based on weight, 53 ± 5% of the added monomer was recovered in form of water-soluble polymer product. ESI-MS tests with PGLA products observed a repeating m/z of 234, which matched the copolymer structure (Fig. 4). A maximum molecular weight (Mw) of 1942 was detected, corresponding to eight repeating units. Size exclusion chromatography measurements also detected the formation of a polymer product, but that does not provide much useful information regarding the size of the polymer product since we do not have a proper polymer standard to calibrate the chromatograms. Low Mw products have been observed in many enzyme-catalyzed polycondensation studies (Kobayashi, 1999; Mahapatro et al., 2004). For our reaction system, the solubility of PGLA in acetone may decrease quickly as its molecular size increases, and it can be expected that its subsequent precipitation terminates the polymerization. Nevertheless, the oligomeric product, as well as its monomer GLAEE, may still prove valuable for various applications such as food and cosmetic product additives. For applications that require higher Mw polymers, products may be developed by optimizing the reaction media with respect to enzyme activity and monomer/product solubility. Overall, we demonstrated that a new class of biomaterials with tunable size and properties could be prepared by taking advantages of the efficiency of enzymatic catalysis. In summary, GLAEE and PGLA were synthesized for the first time via enzymatic transglycosylation and transesterification reactions. More attractively, both of the starting materials, ethyl-l-lactate and d-lactose, can be obtained from renewable resources. It is expected that various sugar-carboxyl acids copolymers can be prepared through the same methodology and thus develop a wide array of biorenewable and biocompatible materials.

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