Characterization of new glycolipid biosurfactants, tri-acylated mannosylerythritol lipids, produced by Pseudozyma yeasts

Transcription

1 Biotechnol Lett (2007) 29: DI /s RIGINAL RESEARCH PAPER Characterization of new glycolipid biosurfactants, tri-acylated mannosylerythritol lipids, produced by Pseudozyma yeasts Tokuma Fukuoka Æ Tomotake Morita Æ Masaaki Konishi Æ Tomohiro Imura Æ Dai Kitamoto Received: 21 December 2006 / Revised: 27 February 2007 / Accepted: 2 March 2007 / Published online: 7 April 2007 Ó Springer Science+Business Media B.V Abstract Mannosylerythritol lipids (MELs) are glycolipid biosurfactants produced by Pseudozyma yeasts. They show not only the excellent interfacial properties but also versatile biochemical actions. In the course of MEL production from soybean oil by P. antarctica and P. rugulosa, some new extracellular glycolipids (more hydrophobic than the previously reported di-acylated MELs) were found in the culture medium. The most hydrophobic one was identified as 1--alka(e)noyl-4--[(4,6 -di-acetyl-2,3 -di--alka(e)noyl)-b-d-mannopyranosyl]-d-erythritol, namely tri-acylated MEL. thers were tri-acylated MELs bearing only one acetyl group. The tri-acylated MEL could be prepared by the lipase-catalyzed esterification of a di-acylated MEL with oleic acid implying that the new glycolipids are synthesized from di-acylated MELs in the culture medium containing the residual fatty acids. Keywords Biosurfactant Enzymatic esterification Glycolipid Mannosylerythritol lipid Pseudozyma strains T. Fukuoka T. Morita M. Konishi T. Imura D. Kitamoto (&) Research Institute for Innovation in Sustainable Chemistry, National Institute of Advanced Industrial Science and Technology (AIST), AIST Tsukuba Central 5-2, Higashi, Tsukuba, Ibaraki , Japan dai-kitamoto@aist.go.jp Introduction Biosurfactants are surface-active compounds produced by a variety of microorganisms. They have been receiving increasing attention due to their unique properties, such as higher biodegradability and lower toxicity as well as versatile biological functions compared to chemically synthesized surfactants (Banat et al. 2000, Kitamoto et al. 2002). Many biosurfactants are produced from inexpensive natural resources, including n-alkanes, vegetable oils, carbohydrates, and even industrial waste products. They thus have additional advantages from the viewpoint of resource replacement and recycling (Lang 2002). Among the biosurfactants hitherto reported, mannosylerythritol lipids (MELs) are one of the most promising biosurfactants known (Kitamoto et al. 2002). They are produced from vegetable oils at over 100 g/l by Pseudozyma yeasts: P. antarctica (Kitamoto et al. 2001), P. aphidis (Rau et al. 2005) and P. rugulosa (Morita et al. 2006). In addition, MELs exhibit not only excellent interfacial properties (Kitamoto et al. 1993) but also versatile biochemical actions, including binding to immunoglobulin G (Im et al. 2003) and cell differentiation activities for human leukemia (Isoda et al. 1997), rat pheochromocytoma (Wakamatsu et al. 2001), and mouse melanoma cells (Zhao et al. 2001; Rodorigues et al. 2006).

2 1112 Biotechnol Lett (2007) 29: All known MELs, namely MEL-A, -B and -C, consist of 4--b-D-mannopyranosyl-D-erythritol as the hydrophilic part and two fatty acyl groups as the hydrophobic part (Fig. 1). The hydrophilic part is acetylated at C-4 and/or C-6, and each derivative shows specific phase behavior and selfassembled structures in aqueous solutions (Imura et al. 2004, 2005, 2006). For instance, MEL-A spontaneously forms an L 3 (sponge) phase at low concentrations, while MEL-B forms an L a (lamellar) phase. These MELs have thus great potential as environmentally advanced materials that can be produced from renewable resources. However, the structural variety of MELs hitherto reported, especially in the hydrophobic part, still remains limited. Various types of MELs with different hydrophobic structures would show different interfacial and biological properties and lead to a broad range of applications for them. We thus focused our attention on the expansion and integration of MEL species, and investigated the extracellular products and MEL production conditions of the yeast strains. In the course of detailed characterization of products from soybean oil by P. antarctica, we found new glycolipids in the culture medium. Here, we describe for the first time the new types of MELs, which comprise three fattyacyl esters. We undertook a detailed characterization of the new MELs by NMR, MALDI-TF/MS and GC- MS analyses. We also performed the synthesis of the tri-acylated MEL from a di-acylated MEL by lipase-catalyzed esterification that provided clue to its biosynthetic mode in the yeast strain. H 3 C R 2 n 4' R 1 6' 3' 5' 2' CH 3 n 1' H 1 H 2 C 2 C H H H 3 C H 4 CH 2 Fig. 1 Chemical structure of mannosylerythritol lipids. MEL, mannosylerythritol lipid. MEL-A, R 1 =CH 3 C, R 2 =CH 3 C; MEL-B, R 1 =CH 3 C, R 2 = H; MEL-C, R 1 =H,R 2 =CH 3 C. n = 6 to 12 Materials and methods Materials Lipase, Novozyme 435 (10000 PLU/g) was kindly donated by Novozymes Japan Ltd. (Chiba, Japan). ther reagents and solvents were commercially available and were used as received. Methyl oleate (purity: 60%) was purchased from Wako Pure Chemicals and used without further purification. Microorganisms Pseudozyma antarctica (formerly Candida antarctica) T-34 used in this study was isolated as a MEL producer using soybean oil as the sole carbon source (Kitamoto et al. 1990). P. rugulosa NBRC was obtained from the National Institute of Technology and Evaluation of Japan (Morita et al. 2006). Culture conditions The following standard procedure for the production of MEL by fermentation was adopted. Seed cultures were prepared by growing cells in medium [40 g glucose/l, 3 g NaN 3 /l, 0.3 g MgS 4 /l, 0.3 g KH 2 P 4 /l, 1 g yeast extract/l (ph 6.0)] at 25 C on a reciprocal shaker (150 rpm) for 2 days. Seed cultures (0.1 ml) were transferred to test tubes containing 2 ml of a basal medium [80 g soybean oil/l, 3 g NaN 3 /l, 0.3 g MgS 4 /l, 0.3 g KH 2 P 4 /l, 1 g yeast extract/l (ph 6.0)], and then incubated as above for 8 days, unless otherwise indicated. Isolation of glycolipids produced from soybean oil by P. antarctica The glycolipids were extracted from the culture medium with an equal volume of ethyl acetate. The extracts were analyzed by TLC on silica plates with chloroform/methanol/7 M NH 4 H (65:15:2, by vol.). The compounds on the plates were located by charring at 110 C for 5 min after spraying an anthrone/sulfuric acid reagent as previously reported (Kitamoto et al. 1990). The purified MEL fraction including MEL-A, -B, and

3 Biotechnol Lett (2007) 29: C prepared as reported previously (Kitamoto et al. 2000) was used as a standard. The organic layer was separated and evaporated. The concentrated glycolipids were dissolved in chloroform and then purified by silica-gel (Wako-gel C-200) column chromatography using a gradient elution of chloroform/ acetone (10:0 3:7, v/v) mixtures as solvent systems (Kitamoto et al. 1990, 2000). Further purification of the hydrophobic glycolipids, which showed higher Rf values than that of MEL-A on TLC, was performed by the combination of the column chromatography and recycling preparative HPLC (Model LC-9104, Japan Analytical Industry Co., Ltd.) equipped with refractive index (RI) and UV detectors under the following conditions; JAI- GEL-1H-40 and JAIGEL-2H-40 size exclusion chromatography (SEC) columns, eluent: chloroform, flow rate: 14 ml/min, temperature: 25 C. Structure determination of the purified glycolipids Structure determination of the purified glycolipids was performed by 1 H-, 13 C-NMR analysis dissolved in chloroform-d using at 400 MHz. Detailed structural characterization was performed by two-dimensional NMR spectra, 1 H- 1 H correlation spectroscopy (CSY), heteronuclear multiple quantum correlation (HMQC), and heteronuclear multiple bond correlation (HMBC), using a JEL ECA-800 (800 MHz). In the NMR study, the purified MEL-A was used as a reference. The molecular weight of the purified glycolipids was measured by MALDI-TF/MS (Voyager-DE PR) with an a-cyano-4-hydroxycinnamic acid matrix. The fatty acid profiles of the glycolipids were examined by GC-MS (see Morita et al. 2006). Enzymatic preparation of tri-acylated MELs MEL-A comprising only saturated fatty acids was prepared from methyl myristate by P. antarctica T-34 as reported previously (Kitamoto et al. 2000). The obtained MEL-A (35 mg) and methyl oleate (150 mg) were mixed with 100 ll culture supernatant of P. rugulosa grown on the basal medium for 10 days as described above. The same reaction was also performed with Novozyme 435 (5 mg) in acetone (100 ll) instead of the supernatant. The reaction mixture was stirred for 48 h at 25 C, and it was then analyzed by TLC as described above. Results and discussion Isolation of glycolipids produced from soybean oil by P. antarctica Preliminary experiments on MEL production by P. antarctica when grown on a medium containing a high concentration of vegetable oil for over a week, revealed the production of unknown glycolipids showing higher Rf values compared to MEL-A on TLC. We investigated the extracellular products from soybean oil, using a two-step purification with silica gel column chromatography and recycling preparative HPLC. After cultivation in soybean oil (80 g/l) medium of for 8 days, the products from the culture medium were extracted and analyzed on TLC. We detected some unknown glycolipids that eluted together with residual oils in the chloroform/acetone (8:2, v/v) fraction on column chromatography. These were subjected to the recycling preparative HPLC using only chloroform, and the unknown glycolipids with high molecular weight were isolated. The isolated unknown glycolipids were again subjected to the column chromatography using a gradient elution of chloroform/acetone (85:15 75:25, v/v), and were purified into component A and component B (Fig. 4). Characterization of the purified glycolipids 1 H-NMR spectrum of component A (data not shown) showed a similar pattern to that of MEL- A (Morita et al. 2006), but it had different peak patterns at around ppm and ppm. The former peak was assigned as H-1 proton, which shifted to a lower field by esterification of hydroxyl group at the C-1 position of erythritol. In addition, on component A, the number of protons at ppm ( CH 3 ) increased (from 6 to 9) without an increase of protons at 2.0 ppm

4 1114 Biotechnol Lett (2007) 29: (derived from an acetyl group). These results indicated that component A is likely to have a structure consisting of MEL-A and a carboxylic acid ester. 13 C-NMR, 1 H- 1 H CSY, and HMQC analyses also supported this supposition, and revealed the presence of five esterified carbons in component A. The position of esterified carbons was determined by HMBC spectrometry (Fig. 2). n the HMBC spectrum, two carbonyl carbons (C = ) derived from two acetyl residues were assigned at and ppm, and correlated with H-4 and H-6 protons, respectively. Three carbonyl carbons derived from three acyl residues were assigned at 172.9, and ppm, and correlated with H-3, H-2, and H-1 protons, respectively. From these results, component A produced from soybean oil by P. antarctica was identified as 1--alka(e)noyl-4--[(4,6 -di--acetyl-2,3 di--alka(e)noyl)-b-d-mannopyranosyl]-d-erythritol (Fig. 3). The chemical shifts of component A are summarized in Table 1. The molecular weight of component A was determined by the main peak ([M + Na] + ) on MALDI-TF/MS analysis; this is well consistent with the above structure. The structure of component B was estimated in the same way. The NMR study showed that the component B is a mixture of glycolipids, which have three fatty esters but only one acetyl group. In MALDI-TF/MS analysis, the molecular weight of component B gave a main peak at 893.9, which corresponds to the structure of a deacetylated derivative of component A. Component B may thus be a mixture of acylated derivatives of MEL-B and MEL-C. Recently, Kida et al. reported (2006) that glycolipids bearing triple fatty acids show micrometer-sized supramolecular assemblies with unique morphologies. Sumida et al. reported (2000) that triple-chain amphiphiles highly stabilize bilayer membrane systems composed of phosphatidylcholines. The present tri-acylated Fig. 2 HMBC spectrum of component A. Detailed structural characterization of Component A was performed by HMBC analysis, using a JEL ECA-800 (800 MHz). HMBC, heteronuclear multiple bond correlation

5 Biotechnol Lett (2007) 29: H 3 C Ac n Ac CH 3 MELs are more hydrophobic than conventional di-acylated MELs, and would thus show great advantages for W/ emulsification and/or reverse micelle systems. We further investigated the fatty acid profiles of these tri-acylated MELs. The fatty acids in component A were composed of medium-chain acids (C 8 C 12, 41% of the total) and long-chain acids (C 16 and C 18, 56% of the total), while those in MEL-A were of only medium-chain acids (C 8 C 14 ) (Table 2). This means that the third fatty acid in the erythritol moiety of component A is mainly composed of the long-chain acids. Based on an earlier study (Kitamoto et al. 1998), most of the fatty acids in MEL-A are b-oxidation intermediates of the fatty acids in soybean oil supplied as the carbon source. The observed long-chain acids in component A are thus likely to be introduced into the sugar backbone in a different way from that of the medium-chain acids. Formation of tri-acylated MELs from soybean oil by P. rugulosa n ur previous study demonstrated that P. rugulosa gave a higher yield of MEL than P. antarctica T-34 (Morita et al. 2006). We thus investigated the formation of tri-acylated MELs from soybean oil by P. rugulosa with different culture conditions. As expected, the yeast also produced glycolipids showing higher Rf values compared to MEL-A on TLC. Based on the characterization as indicated above, these glycolipids were identified as component A and B. Interestingly, the H H H 2 C C C CH 2 C H H CH 3 Fig. 3 Chemical structure of component A. n = 6 10, m = 6 16 m yeast produced tri-acylated MELs at 80 g/l or more of soybean oil, but not at 40 g soybean oil/l (Fig. 4a). At 80 g soybean oil/l, the amount of triacylated MELs produced clearly increased along with the cultivation time (Fig. 4b). Similar results were obtained on P. antarctica T-34 (data not shown). Interestingly, in both cases, the formation of tri-acylated MELs took place when free fatty acids existed at some amount in the culture medium. As described above, the third fatty acid of component A would be long-chain acids. Taking these results into consideration, component A is very likely to be synthesized from di-acylated MELs and free fatty acids in the culture medium, where the fatty acids would be directly introduced into the primary hydroxyl group of erythritol by the mediation of extracellular lipase and/or esterase. In fact, P. antarctica as well as P. rugulosa secrete lipase and/or esterase when grown on soybean oil, and the activities significantly increase along with the cultivation time (Morita et al. 2007). We thus tried to prepare triacylated MELs by enzymatic methods, considering the efficient production of the new MEL species. Enzymatic preparation of tri-acylated MELs We examined the esterification of the purified MEL-A with methyl oleate by using the culture supernatant of P. rugulosa and Novozyme 435. Here, we used the purified MEL-A comprising of only saturated fatty acids as a substrate (Table 3). In both reactions, a glycolipid showing a higher Rf value compared to the MEL-A on TLC was obtained. The yield reached 40% using Novozyme 435. Based on the characterization as above, these glycolipids were identified to have the same structure as component A, except for the fatty acid profile. n the product obtained with Novozyme 435, the fatty acids were composed of medium-chain acids (C 8 C 12, 45 % of the total) and long-chain acids (C 16 and C 18, 55% of the total) (Table 3). The fatty acid profile was nearly the same as that of component A produced from soybean oil by P. rugulosa. This clearly indicated that fatty acids are directly introduced into the erythritol moiety of MEL-A to give a tri-acylated

6 1116 Biotechnol Lett (2007) 29: Table 1 NMR data for component A (chloroform-d, 400 MHz) 13 C-NMR d (ppm) 1 H-NMR d (ppm) D-Mannose C H d C H dd C H dd C H t C H m C H m meso-erythritol C H-1a 4.22 dd H-1b 4.31 dd C H m C H m C H-4a 3.89 dd H-4b 3.99 dd Acetyl groups C= (C-4 ) (C-6 ) CH 3 (C-4 ) s (C-6 ) s Acyl groups C= (C-2 ) (C-3 ) (C-1) C-CH 2 (C-2 ) m (C-3 ) m (C-1) m C-CH 2 CH b (CH 2 ) n b CH=CH b CH=CH-CH b CH b Table 2 The fatty acid profiles of MEL Fatty acid Composition (%) MEL-A 6: : : : : : : : : : : : : : unknown Component A MEL by the lipase. The observed C 16 and C 18 acids other than oleic acids in the tri-acylated MEL would originate from impurities coexisting with methyl oleate used, considering the low purity (60%) of the reagent. Accordingly, triacylated MELs were demonstrated to be synthesized from di-acylated MELs and fatty acids in the culture medium by the mediation of extracellular enzymes. Conclusion In this study, we have found a new type of MEL, namely tri-acylated MEL, in the culture medium of P. antarctica as well as P. rugulosa grown on high concentrations of soybean oil. The tri-acylated MEL was identified as 1--alka(e)noyl-4-- [(4,6 -di--acetyl-2,3 -di--alka(e)noyl)- b-d-mannopyranosyl]-d-erythritol. The fatty acids in the tri-acylated MELs comprised a large amount of long-chain acids, and were quite different from those in conventional di-acylated MELs.

7 Biotechnol Lett (2007) 29: Fig. 4 TLC patterns of the extracellular products from soybean oil by P. rugulosa under different culture conditions. The extracellular products extracted from the culture medium, which was prepared from soybean oil by P. rugulosa under different culture conditions, were analyzed by TLC. (a) The effect of soybean oil concentration on the glycolipids production, the standard of MELs and the extracellular products produced by P. rugulosa from 40, 80, and 120 g/l of soybean oil, incubated for 8 days (b) The time course of the glycolipids production, the standard of MELs and the extracellular products produced by P. rugulosa from 80 g/l of soybean oil, incubated for 2, 4, 6, 8, and 10 days. TLC, thin-layer chromatography Table 3 The fatty acid profiles of glycolipids Fatty acid Composition (%) Substrate a Product b 8: : : : : : : :2 4.0 a Substrate is MEL-A comprising of only saturated fatty acids prepared from methyl myristate b Product was obtained by lipase-catalyzed esterification of substrate (MEL-A) with methyl oleate Tri-acylated MELs are likely to be synthesized by the esterification of di-acylated MEL with fatty acids in the culture medium by extracellular lipase and/or esterase. Based on the study, tri-acylated MELs can be synthesized by lipase-catalyzed esterification from various substrates and provides a new strategy for creating different types of MELs having different interfacial properties. Further investigations on enzymatic syntheses of new MELs and the characterization are underway. Acknowledgements We would like to thank Dr. Tadashi Nemoto of our institute and Dr. Katsuo Asakura of JEL Ltd. Co., Japan, for their help on NMR measurement. We also wish to thank Ms. Akiko Sugimura, a fellow of the Japan Industrial Technology Association, for her technical assistance. This work was supported by the Industrial Technology Research Grant Program in 05A33008c from the New Energy and Industrial Technology Development rganization (NED) of Japan. References Banat IM, Makkar RS, Cameotra SS (2000) Potential commercial applications of microbial surfactants. Appl Microbiol Biotechnol 53: Im JH, Ikegami T, Yanagishita H, Takeyama Y, Idemoto Y, Koura N, Kitamoto D (2003) Mannosylerythritol lipids, yeast glycolipid biosurfactants, are potential affinity ligand materials for human immunoglobulin G. J Biomed Mater Res 65: Imura T, Yanagishita H, Kitamoto D (2004) Coacervate formation from natural glycolipid: one acetyl group on the headgroup triggers coacervate-to-vesicle transition. J Am Chem Soc 126: Imura T, Yanagishita H, hira J, Sakai H, Abe M, Kitamoto D (2005) Thermodynamically stable vesicle formation from glycolipid biosurfactant sponge phase. Colloids and Surfaces B: Biointerfaces 43: Imura T, hta N, Inoue K, Yagi H, Negishi H, Yanagishita H, Kitamoto D (2006) Naturally engineered glycolipid biosurfactants leading to distinctive self-assembled structures. Chem Eur J 12:

8 1118 Biotechnol Lett (2007) 29: Isoda H, Shinmoto H, Kitamoto D, Matsumura M, Nakahara T (1997) Differentiation of human promyelocytic leukemia cell line HL60 by microbial extracellular glycolipids. Lipids 32: Kida T, Tanaka T, Nakatsuji Y, Akashi M (2006) Formation of micrometer-sized supramolecular assemblies with unique morphologies from triple-chain lipids with two sugar head groups. Chem Lett 35: Kitamoto D, Akiba S, Hioki C, Tabuchi T (1990) Extracellular accumulation of mannosylerythritol lipids by a strain of Candida antarctica. Agric Biol Chem 54:31 36 Kitamoto D, Yanagishita H, Shinbo T, Nakane T, Kamisawa C, Nakahara T (1993) Surface active properties and antimicrobial activities of mannosylerythritol lipids as biosurfactants produced by Candida antarctica. J Biotech 29:91 96 Kitamoto D, Ghosh S, urisson G, Nakatani Y (2000) Formation of giant vesicles from diacylmannosyleryhtritols, and their binding to concanavalin A. Chem Commun 10: Kitamoto D, Ikegami T, Suzuki T, Sasaki A, Takeyama Y, Idemoto Y, Koura N, Yanagishita H (2001) Microbial conversion of n-alkanes into glycolipid biosurfactants, mannosylerythritol lipids, by Pseudozyma antarctica. Biotechnol Lett 23: Kitamoto D, Isoda H, Nakahara T (2002) Functions and potential applications of glycolipid biosurfactants from energy-saving materials to gene delivery carriers. J Biosci Bioeng 94: Lang S (2002) Biological amphiphiles (microbial biosurfactants). Curr pin Colloid Interface Sci 7:12 20 Morita T, Konishi M, Fukuoka T, Imura T, Kitamoto D (2006) Discovery of Pseudozyma rugulosa NBRC as a novel producer of the glycolipid biosurfactants, mannosylerythritol lipids, based on rdna sequence. Appl Microbiol Biotechnol 73: Morita T, Konishi M, Fukuoka T, Imura T, Kitamoto H, Kitamoto D (2007) Physiological differences in the formation of the glycolipid biosurfactants, mannosylerythritol lipids, between Pseudozyma antarctica and Pseudozyma aphidis. Appl Microbiol Biotechnol 74: Rau U, Nguyen LA, Schulz S, Wray V, Nimtz M, Roper H, Koch H, Lang S (2005) Formation and analysis of mannosylerythritol lipids secreted by Pseudozyma aphidis. Appl Microbial Biotechnol 66: Rodorigues L, Banat IM, Teixeria J, liveira R (2006) Biosurfactants: potential applications in medicine. J Antimicrob Chemothera 57: Sumida Y, Masuyama A, Takasu M, Kida T, Nakatsuji Y, Ikeda I, Nojima M (2000) Behavior of self-organized molecular assemblies composed of phosphatidylcholines and synthetic triple-chain amphiphiles in water. Langmuir 16: Wakamatsu Y, Zhao X, Jin C, Day N, Shibahara M, Nomura N, Nakahara T, Murata T, Yokoyama KK (2001) Mannosylerythritol lipid induces characteristics of neuronal differentiation in PC12 cells through an ERK-related signal cascade. Eur J Biochem 268: Zhao X, Murata T, hno S, Day N, Song J, Nomura N, Nakahara T, Yokoyama KK (2001) Protein kinase Ca plays a critical role in mannosylerythritol lipid-induced differentiation of melanoma B16 cells. J Biol Chem 276: