Efficient Production of Di- and Tri-acylated Mannosylerythritol Lipids as Glycolipid Biosurfactants by Pseudozyma parantarctica JCM T

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1 Journal of Oleo Science Copyright 2008 by Japan Oil Chemists Society Efficient Production of Di- and Tri-acylated Mannosylerythritol Lipids as Glycolipid Biosurfactants by Pseudozyma parantarctica JCM T Tomotake Morita 1, Masaaki Konishi 1, Tokuma Fukuoka 1, Tomohiro Imura 1, Hideki Sakai 2 and Dai Kitamoto 1 1 Research Institute for Innovations in Sustainable Chemistry, National institute of Advanced Industrial Science and Technology (AIST) (5-2 Tukuba Central, 1-1 Higashi, Tsukuba, Ibaraki , JAPAN) 2 Faculty of Science and Technology, Tokyo University of Science (2641 Yamazaki, Noda, Chiba , JAPAN) Abstract: Mannosylerythritol lipids (MELs) are one of the most promising biosurfactants known, because of their multifunctionality and biocompatibility. In order to attain an efficient production of MELs, Pseudozyma parantarctica JCM T, which is a newly identified strain of the genus, was examined for the productivity of MELs at different culture conditions. The yeast strain showed significant cell growth and production of di-acylated MELs even at 36. In contrast, on conventional high-level MEL producers including P. rugulosa, the MEL yield considerably decreased with an increase of the cultivation temperature at over 30. On P. parantarctica, soybean oil and sodium nitrate were the best carbon and nitrogen sources, respectively. Under the optimal conditions on a shake-flask culture at 34, the amount of di-acylated MELs reached over 100 g/l by intermittent feeding of only soybean oil. Interestingly, the yeast strain produced tri-acylated MELs as well as di-acylated ones when grown on the medium containing higher soybean oil concentrations than 8% (vol/vol). The production of tri-acylated MELs was significantly accelerated at between 34 and 36. With 20 % (vol/vol) of soybean oil at 34, the yield of tri-acylated MELs reached 22.7 g/l. The extracellular lipase activity considerably depended on the culture temperature, and became the maximum at 34 ; this would bring the accelerated production of triacylated MELs. Accordingly, the present strain of P. parantarctica provided high efficiency in MEL production at elevated temperatures compared to conventional MEL producers, and would thus be highly advantageous for the commercial production of the promising biosurfactants. Key words: mannosylerythritol lipid, biosurfactant, glycolipid, Pseudozyma 1 INTRODUCTION Biosurfactants (BS) are extracellular amphiphilic compounds produced by a variety of microorganisms, and have attracted considerable interest in recent years due to their biodegradability, mild production conditions, and variety of functions 1). The numerous advantages of BS have promoted applications not only in the food, cosmetic and pharmaceutical industries, but also in environmental protection and energy-saving technology 2). Mannosylerythritol lipids (MELs) are glycolipid biosurfactants that contain 4-O-b-D-mannopyranosyl-meso-erythritol as a hydrophilic headgroup and two moles of fatty acids as a hydrophobic chain ( ). MELs are abundantly produced by the yeast strains of the genus Pseudozyma, and are one of the most promising BS known 2-4). These MELs exhibit not only excellent surface-active properties 5,6), but also versatile biochemical actions including celldifferentiation towards human leukemia 7,8), rat pheochromocytoma 9) and mouse melanoma cells 10) as well as high affinity binding towards different immunoglobulins 11,12) and lectins 13). In addition, MEL-A, the major component of Correspondence to: Dai Kitamoto, Bio-chemical Material Group, Research Institute for Innovations in Sustainable Chemistry, National institute of Advanced Industrial Science and Technology (AIST), 5-2 Tsukuba Central, 1-1 Higashi, Tsukuba, Ibaraki , JAPAN dai-kitamoto@aist.go.jp Accepted June 11, 2008 (recieved for review April 17, 2008) Journal of Oleo Science ISSN print / ISSN online 557

2 T. Morita, M. Konishi, T. Fukuoka et al. Fig. 1 Chemical Structure of Mannosylerythritol Lipids. (a) di-acylated MELs, (b) tri-acylated MELs. MELs, dramatically increases the efficiency of gene transfection mediated by cationic liposomes 14-16). Recently, in addition to conventional di-acylated MELs ( ), novel types of MELs, namely mono-acylated MELs 17) and tri-acylated MELs 18) ( ) have also been reported. They show different surface-active and selfassembling properties depending on the hydrophilic and lipophilic balance (HLB), compared to conventional di-acylated MELs. The structural variety of MELs would thus facilitate their broad range of applications. Pseudozyma antarctica T-34 19), P. aphidis DSM ), and P. rugulosa NBRC (JCM T ) 4) were reported to be high-level MEL producers, which provide the yield of MELs over 100 g/l. Recently, three strains of the genus, P. parantarctica JCM T, P. fusiformata JCM 3931, P. tsukubaensis JCM T were also found to be MEL producers 21). Among the three stains, P. parantarctica, which is newly identified strain of the genus, gave the highest yield of 30 g/l from soybean oil ; the yield is comparable to that of the above high-level MEL producers. The development of new biosurfactant producers is of great interest, considering the commercial production of biosurfactants and their broad range of applications. We thus focused our attention on the feasible use of the newly identified strain for an efficient production of MELs. In this study, we investigated the culture conditions leading to the maximal production of di- and tri-acylated MELs at higher temperature compared to conventional MEL production. We also addressed the relationship between the MEL production and extracellular lipase activity. 2 EXPERIMENTAL 2 1 Pseudozyma parantarctica JCM T and P. rugulosa JCM T were obtained from the RIKEN BioResource Center. Stock cultures were cultivated for 3 days at 25 on an agar medium containing 4% glucose (wt/vol), 0.3% NaNO 3, 0.03% MgSO 4, 0.03% KH 2 PO 4, and 0.1% yeast extract. They were stored at 4 and renewed every 2 weeks. 2 2 Seed cultures were prepared by inoculating cells grown on slants into test tubes containing a growth medium [4% glucose (wt/vol), 0.3% NaNO 3, 0.03% MgSO 4, 0.03% KH 2 PO 4, 0.1% yeast extract (ph 6.0)] at 30 on a reciprocal shaker (150 strokes/min) for 2 d. To optimize the culture conditions for MEL production by P. parantarctica JCM T, seed cultures (0.1 ml) were transferred to test tubes containing 2 ml of an experimental medium [0.3% NaNO 3, 0.03% MgSO 4, 0.03% KH 2 PO 4, 0.1% yeast extract (ph 6.0) containing an adequate amount of carbon sources], and then incubated at different temperatures on a reciprocal shaker (150 strokes/min) for 8 d. MEL production by fed-batch culture was carried out using 1.0 ml of seed cultures with 200 ml Erlenmeyer flasks containing 20 ml of a fermentation medium [16% soybean oil (vol/vol), 0.3% NaNO 3, 0.03% MgSO 4, 0.03% KH 2 PO 4, 0.1% yeast extract (ph 6.0)] at 34 on a rotary shaker (250 rpm). After 7 d incubation, further 2.7 g of soybean oil (16%, vol/vol) was added directly to the flasks: this procedure was repeated for 4 weeks. 558

3 Production of mannosylerythritol lipids by Pseudozyma parantarctica 2 3 The produced MELs were extracted from the culture medium (initial 20 ml) with an equal amount of ethyl acetate. The extracts were analyzed by thin-layer chromatography (TLC) on silica plates (Silica gel 60F; Wako) with a solvent system consisting of chloroform-methanol- 7N ammonium hydroxide (65: 15: 2, by vol.). The compounds on the plates were located by charring at 110 for 5 min after spraying the anthrone reagent 22). Glycolipids and residual oils were detected blue and brown spots, respectively. The purified MEL-A, MEL-B and MEL-C were prepared from MELs produced by P. antarctica T-34 as reported previously 4), and then used as a standard on TLC and in the following experiments. Quantification of di-acylated MELs was carried out by high-performance liquid chromatography (HPLC) on a silica gel column (Inertsil SIL 100A 5 mm, mm; GL Science Inc, Japan) with a low temperature-evaporative light scattering detector (ELSD-LT; Shimadzu, Japan) using a gradient solvent program consisting of various proportions of chloroform and methanol (from 100:0 to 0: 100, vol/vol) at a flow rate of 1 ml/min 20). On HPLC, quantification of the MELs was performed based on the standard curves of purified MEL fractions 4). After cultivation, the volume of the culture medium was slightly decreased. All measurements reported here are calculated values from at least three independent experiments. Tri-acylated MELs was unable to quantify by the above HPLC method, because their peaks partially overlapped with those of residual oils and free fatty acids. The amount of tri-acylated MELs was thus measured after purification as follows. The ethyl acetate extracts were concentrated and subjected to the recycling preparative HPLC (Model LC-9104, Japan Analytical Industry Co., Ltd) equipped with refractive index (RI) and UV detectors as previously described 18). After separating tri-acylated MELs from diacylated MELs with the recycling HPLC, the tri-acylated fraction was concentrated and weighted directly. 2 4 The tri-acylated MELs fraction was further purified by silica gel column chromatography as reported previously 18). The structure of the purified tri-acylated MEL was characterized by 1 H and 13 C nuclear magnetic resonance (NMR) with a Varian INOVA 400 (400 MHz) at 30 using the CD 3 OD solution. The fatty acid profile of the MEL was examined mainly by the method described previously 23). The methyl ester derivatives of the fatty acids were prepared by mixing the above purified glycolipids (10 mg) with 5% HCl-methanol reagent (1 ml). After the reaction was quenched with water (1 ml), the methyl ester derivatives were extracted with n-hexane and then analyzed by gas chromatography-mass spectrometry (GC-MS) (Hewlett Packard 6890 and 5973N) with a TC-WAX (GL-science, Tokyo) with the temperature programmed from 90 (held for 3 min) to 240 at 5 /min. 2 5 Lipase activity was measured with p-nitrophenyl ester (Sigma, USA) as the substrate 24). The reaction mixture was composed of 0.95 ml of substrate [5 mm p-nitrophenyl laurate (Sigma, USA) in 50 mm sodium acetate buffer (ph 5.6) containing 4% triton X-100] and 50 ml of culture solution. The reaction mixture was incubated at 37 for 15 min. The reaction was stopped by adding 1.0 ml of acetone and the absorbance was measured at 410 nm. The enzyme activity was expressed in terms of IU. One unit of the enzyme activity was defined as the amount of enzyme required to release 1 ml of p-nitrophenol per min. 3 RESULTS 3 1 P parantarctica a) Effect of carbon and nitrogen sources We previously reported that high-level MEL producers such as P. antarctica T-34 and P. rugulosa NBRC produce a large amount of di-acylated MELs ( ) from different vegetable oils. Here, the effect of the carbon source on the MEL production by P. parantarctica JCM T were studied using 8% (vol/vol) of various vegetable oils on the experimental medium ( ). The produced di-acylated MELs were quantified by HPLC as indicated above. Except for palm oil, all vegetable oils tested here gave good yields of the MELs. Since the highest yield was obtained from soybean oil, the oil was exclusively used as the sole carbon source in the following experiments. The type of nitrogen source considerably affected the MEL formation ( ). Sodium nitrate (0.3% wt/wt) provided a high yield of the MELs, while ammonium nitrate or sulfate (each 0.3% wt/wt) did no MELs. Sodium nitrate was thus exclusively used as the nitrogen source in the following experiments. To estimate the optimum concentration of the carbon source, the yeast strain was cultured in the experimental medium containing different concentrations of soybean oil ( ). The amount of di-acylated MELs significantly increased with an increase of soybean oil concentration up to 16% (vol/vol). When 16% (vol/vol) of soybean oil was used, the maximum yield and yield coefficient (on a weight basis to soybean oil supplied) were approximately 43.0 g/l and 0.32 g/g, respectively. b) Effect of temperature To estimate the effect of temperature on the MEL production, the yeast strain was cultured in the experimental 559

4 T. Morita, M. Konishi, T. Fukuoka et al. Fig. 2 Production of Di-acylated MELs by P. parantarctica JCM T at Different Culture Conditions. The strain was cultivated at 30 for 8 d with the experimental medium containing different vegetable oils (8%, vol/vol) (a), nitrogen sources (0.3%, wt/vol) (b), and concentrations of soybean oil (c). Di-acylated MELs were quantified by HPLC with a silica gel column and a low temperature-evaporative light scattering detector. Vertical bars show the standard error of the mean based on three independent measurements. medium containing 8% (vol/vol) soybean oil at different cultivation temperatures. P. rugulosa JCM T, a highlevel MEL producer, was also used as a reference. Interestingly, between 30 and 38, P. parantarctica showed good growth even at 36, and provided the best yield of the MELs at 34 ; the maximum yield and yield coefficient reached at 40.3 g/l and 0.67 g/g ( and ). Thus, the yield coefficient increased more than 2-fold higher compared to the case of 30. In contrast, the growth and MEL production of P. rugulosa drastically decreased with an increase of the temperature ( ). The other high-level MEL producers, P. antarctica T ), P. antarctica JCM T, and P. aphidis JCM T, also showed low yields of the MELs at higher temperature over 30, compared to the case of P. parantarctica (data not shown). P. parantarctica JCM T is thus very likely to be more thermotolerable on the MEL production compared to other producers of the genus Pseudozyma, and highly advantageous for an efficient production of the MELs, which requires less energy for cooling in fermentation. 3 2 Under the optimal conditions obtained above, the production of di-acylated MELs using fed-batch culture was conducted to examine the feasibility of the continuous production. shows a typical time course of the MEL production with feeding of soybean oil at 34. Surprisingly, after incubation for 28 d using the fermentation medium, the total amount of MELs reached a concentration of approximately g/l; this value corresponded to approximately 0.20 g/g of yield coefficient. No cell growth was observed during the cultivation after 7 d, showing that the activity of the resting cells was maintained high enough for the MEL production during 4 weeks. The production rate may thus be improved by using a large-scale fermenter with larger numbers of resting cells. 3 3 P parantarctica Recently, P. antarctica and P. rugulosa were reported to produce a novel type of MEL, namely tri-acylated MELs ( ) as well as di-acylated MELs when soybean oil was used as the carbon source 18). On these strains, the amount of tri-acylated MELs in the culture medium increases with an increase of soybean oil concentration. However, there is still limited information on the production of tri-acylated MELs. As indicated above, P. parantarctica JCM T efficiently produced di-acylated MELs from high concentrations of soybean oil. We thus focused out attention on the formation of tri-acylated MELs by the present strain. The strain was cultivated at 34 for 8 d with the experimental medium containing different concentrations of soybean oil, considering the optimal culture conditions obtained above. With higher concentrations of soybean oil over 8% (vol/vol), ethyl acetate extracts of the culture medium gave spots of glycolipids showing higher Rf values than those of di-acylated MELs ( ). With 4% (vol/vol) of the oil, the extracts gave only di-acylated MELs. The strain was also cultivated for 8 days with the experimental 560

5 Production of mannosylerythritol lipids by Pseudozyma parantarctica Fig. 3 Cell Growth and Production of Di-acylated MELs by P. parantarctica JCM T and P. rugulosa JCM10323 T. P. parantarctica JCM T (black bar) and P. rugulosa JCM10323 T (white bar) were cultivated in the experimental medium containing 8% (vol/vol) of soybean oil for 8 d at different temperatures. Di-acylated MELs were quantified by HPLC. Vertical bars show the standard error of the mean based on three independent measurements. (a), weight of the dry cells (g/l); (b), the amounts of di-acylated MELs (g/l). medium containing 8% (vol/vol) of the oil at different temperatures ( ). At 34 and 36, the extracts also showed blue spots of glycolipids different from di-acylated MELs ( ), while those showed predominantly di-acylated MELs at 30 and 32. These new glycolipids showed less sensitivity toward anthrone reagent compared to diacylated MELs. These new glycolipids, which were identified as tri-acylated MELs as indicated below, were extracted and isolated by the recycling HPLC, and then subjected to weight measurement. Interestingly, the amount of tri-acylated MELs obtained from 8 and 20% (vol/vol) of soybean oil at 34 reached at 18.5 and 22.7 g/l; this corresponds to the yield coefficient of 0.28 and 0.14 g/g, respectively. Fig. 4 Time Course of MEL Production by P. parantarctica JCM T with Feeding of Soybean Oil. The strain JCM T was cultivated in 20 ml of the fermentation medium at 34, and then 2.7 g of soybean oil (16%, vol/vol) were directly added to the flask every 7 d as indicated by arrows. The produced MELs were extracted from culture medium with 20 ml of ethyl acetate. Filled circle, di-acylated MELs (g/l); and open circle, weight of dry cells (g/l). Di-acylated MELs were quantified by HPLC. 3 4 P parantarctica To confirm the structure of the glycolipids showing high Rf values compared to di-acylated MELs on TLC ( and ), the main blue spot was purified by the column chromatography and recycling HPLC, and then subjected to NMR studies. All the 1 H NMR spectra obtained from the main glycolipid corresponded well with those of tri-acylated MEL-A produced by P. rugulosa reported previously 18). The chemical shifts of the glycolipid were as follows; the mannose H-1 peak at 4.72 ppm (s), the H-2 peak at 5.51ppm (dd), the H-3 peak at 5.07 ppm (dd), the H-4 peak at 5.27 ppm (t), the H-5 peak at 3.72 ppm (dd), the H-6 peaks around 4.22 ppm (m), the erythritol H-1 peaks at 4.22 and 4.31 ppm (dd), the H-2 peak around 3.80 ppm (m), the H- 3 peaks around 3.68 ppm (m), and the H-4 peaks at 3.89 and 561

6 T. Morita, M. Konishi, T. Fukuoka et al. Fig. 5 Formation of Tri-acylated MELs by P. parantarctica JCM T at Different Culture Conditions. The strain was cultivated with the fermentation medium containing different concentrations of soybean oil at 34 for 8 d (a), or with the medium containing 8% (vol/vol) of soybean oil at different temperatures (b). The glycolipids produced in the medium were extracted with ethyl acetate, and the extracts were subjected to TLC plate. The glycolipids (blue spots) and soybean oil (brown spots) were expressed by anthrone reagent ppm (dd). The H-1 peak of the glycolipid shifted to a lower field by esterification of hydroxyl group at C-1 position of erythritol in comparison with di-acylated MEL-A. From these results, the main spot was identified as tri-acylated MEL-A. The result of 13 C NMR also confirmed the identification (data not shown). The fatty acids in the tri-acylated MEL-A produced by the strain were composed of medium-chain acids (C 8 -C 12, 30 % of the total) and long-chain acids (C 16 and C 18, 64 % of the total) ( ). On the other hand, the acids in the diacylated MEL-A produced by the same strain consisted of predominantly medium-chain acids (C 8 -C 12, 93.6 % of the total). This result was consistent with our previous results on P. antarctica and P. rugulosa, where tri-acylated MELs are synthesized from di-acylated MELs and free fatty acids in the culture medium by the mediation of extracellular lipase 18). 3 5 P parantarctica The activity of extracellular lipase is closely related to the productivity of di-acylated MELs 25,26). In addition, triacylated MELs should be synthesized in vitro from di-acylated MELs and free fatty acids by the lipase-catalyzed reaction 18). We thus checked the activity of extracellular lipase of P. parantarctica JCM T, aiming to address the function of the enzyme. The strain was then cultivated with the experimental medium containing 8% of soybean Table 1 Fatty acid C8:0 C8:1 C10:0 C10:1 C10:2 C12:0 C12:1 C14:2 C16:0 C18:0 C18:1 C18:2 Unknown a Morita et al., ) Fatty Acid Composition of MELs from Soybean Oil by P. parantarctica JCM T. Di-acylated MEL-A a Composition (%) Tri-acylated MEL-A oil at different temperatures for 8 d ( ). The lipase activity significantly changed depending on the cultivation temperature, and became the maximum at 34. This was in good agreement with the above results on the effect of temperature on the formation of di- and tri- 562

7 Production of mannosylerythritol lipids by Pseudozyma parantarctica Fig. 6 Extracellular Lipase Activity of P. parantarctica JCM T. The strain was cultured in the experimental medium containing 8% (vol/vol) of soybean oil for 8 d at different temperatures. The activities were measured by a spectrophotometric method using p-nitrophenyl laurate as substrate. Vertical bars show the standard error of the mean based on three independent measurements. acylated MELs ( and ). The enzyme activity is thus very likely to be crucial not only for the production of di-acylated MELs but also for the formation of tri-acylated MELs on P. parantarctica, although the details on the reaction and expression of the enzyme still remain unknown. 4 DISCUSSION Here, we accomplished for the first time an effective production of di- and tri-acylated MELs using P. parantarctica JCM T at elevated temperatures compared to conventional MEL production systems. In our previous report, P. parantarctica JCM T, which is a newly identified strain of the genus, produced approximately 30 g/l of MELs from soybean oil at 30 ; the yield is comparable to that of high-level MEL producers such as P. antarctica, P. aphidis and P. rugulosa 21). Here we further investigated the productivity of MELs by the newly identified stain under different culture conditions. Surprisingly, P. parantarctica showed good cell growth and efficiently produced di- and tri-acylated MELs even at 36. In contrast, on other high-level MEL producers, the yields drastically decreased with an increase of the cultivation temperature at over 30. Therefore, the present strain would be very promising for commercial production of MELs, considering the thermotolerant property compared to conventional MEL producers. In the past years there has been an upsurge of interest in thermophilic microorganisms mainly due to the increase in most reaction rates, product yield and final product resistance to degeneration at higher temperatures. In microbial processes, advantages of the use of high temperature include energy savings achieved by reducing cooling costs or improvement of overheating problems usually encountered in areas/seasons of high ambient temperature where cooling is unavailable 27). The yeast strains that can grow at over 40 are referred to be thermophilic or thermotolelant yeasts 28,29). To the best of our knowledge, P. parantarctica JCM T is the most thermotolerable yeast among the MEL producers hitherto reported. Indeed, the strain grows can grow and significantly produce MELs at 38 ( and ), although it is not definitely thermotolerant yeast. Based on our previous study, P. rugulosa NBRC provides di-acylated MELs at a yield of 142 g/l 4). Rau et al. reported that P. aphidis DSM (=NBRC T ) gives the MELs at a yield of 165 g/l on bioreactor with the feeding of soybean oil and additional supplements (glucose, sodium nitrate and yeast extract) 30). However, both strains did not show any thermotolerant properties like P. parantarctica JCM T, and gave only a trace amount of MELs at 34 from soybean oil. In contrast, P. parantarctica gave the MEL yield of g/l at 34. Accordingly, the present stain may have a great potential as a novel MEL producer, which enables us to attain a large scale and energy saving MEL production system. Recently, tri-acylated MEL-A, which has two fatty acids on the mannose moiety and one fatty acid on the erythritol moiety, was newly found in the culture medium of P. antarctica and P. rugulosa 18). Tri-acylated MEL-A is assumed to show a smaller critical micelle concentration and hydrophilic-lipophilic balance (HLB) compared to diacylated MEL-A, and may thus broaden the functional variety of MELs. As expected, P. parantarctica JCM T successfully produced tri-acylated MELs as well as di-acylated MELs at higher temperatures over 30. When grown with 20% (vol/vol) of soybean oil at 34, the strain provided tri-acylated MELs at a yield of 22.7 g/l. The obtained yield is a surprisingly high level compared to hitherto reported yields of tri-acylated MELs. The gene expression mechanism for MEL biosynthesis still remains unclear on Pseudozyma yeasts, although Hewald et al. recently reported a gene cluster for biosynthesis of MEL in Ustilago maydis 31). P. parantarctica JCM T showed the highest productivity of di- and tri-acylated MELs when the cells were cultivated at around 34, considerably different from the case of other MEL producers. In addition, the activity of extracellular lipase was also 563

8 T. Morita, M. Konishi, T. Fukuoka et al. highest at 34. This fact supports that the enzyme catalyzes not only hydrolysis of soybean oil but also esterification of di-acylated MELs with free fatty acids in the culture medium. In conclusion, we accomplished for the first time an effective production of di- and tri-acylated MELs using P. parantarctica JCM T at elevated temperatures compared to conventional MEL production systems. Further study of the gene analysis for P. parantarctica should help us to understand the unique MEL biosynthetic manner depending on the cultivation temperature. ACKNOWLEDGEMENTS The authors thank to Ms. A. Sugimura, a fellow of the Japan Industrial Technology Association, for her technical assistance. This study was supported by the Industrial Technology Research Grant Program in 06A17501c from the New Energy and Industrial Technology Development Organization (NEDO) of Japan. 1. Banat, I.M.; Makkar, R.S.; Cameotra, S.S. Potential commercial applications of microbial surfactants. Appl. Microbiol. Biotechnol. 53, (2000). 2. Kitamoto, D.; Isoda, H.; Nakahara, T. Functional and potential application of glycolipid biosurfactants. J. Biosci. Bioeng. 94, (2002). 3. Lang, S. Biological amphiphiles (microbial biosurfactants). Curr. Opin. Coll. Int. Sci. 7, (2002). 4. Morita, T.; Konishi, M.; Fukuoka, T.; Imura, T.; Kitamoto, D. Discovery of Pseudozyma rugulosa NBRC as a novel producer of the glycolipid biosurfactants, mannosylerythritol lipids, based on rdna sequence. Appl. Microbiol. Biotechnol. 73, (2006). 5. Imura, T.; Yanagishita, H.; Kitamoto, D. Coacervate formation from natural glycolipid: one acetyl group on the headgroup triggers coacervate-to-vesicle transition. J. Am. Chem. Soc. 126, (2004). 6. Imura, T.; Yanagishita, H.; Kitamoto, D. Thermodynamically stable vesicle formation from glycolipid biosurfactant sponge phase. Colloids Surf. B Biointerfaces 43, (2005). 7. Isoda, H.; Kitamoto, D.; Shinomoto, H.; Matsumura, M.; Nakahara, T. Microbial extracellular glycolipid induction of differentiation and inhibition of the protein kinase C activity of human promyelocytic leukemia cell line activity of human promyelocytic leukemia cell line HL60. Biosci. Biotechnol. Biochem. 61, (1997). 8. Isoda, H.; Shinomoto, H.; Kitamoto, D.; Matsumura, M.; Nakahara, T. Differentiation of human promyelocytic leukemia cell line HL60 by microbial extracellular glycolipids. Lipids 32, (1997). 9. Wakamatsu, Y.; Zhao, X.; Jin, C.; Day, N.; Shibahara, M.; Nomura, N.; Nakahara, T.; Murata, T.; Yokoyama, K.K. Mannosylerythritol lipid induces characteristics of neuronal differentiation in PC12 cells through an ERKrelated signal cascade. Eur. J. Biochem. 268, (2001). 10. Zhao, X.; Wakamatsu, Y.; Shibahara, M.; Nomura, N.; Geltinger, C.; Nakahara, T.; Murata, T.; Yokoyama, K.K. Mannosylerythritol lipid is a potent inducer of apoptosis and differentiation of mouse melanoma cells in culture. Cancer Res. 59, (1999). 11. Imura, T.; Ito, S.; Azumi, R.; Yanagishita, H.; Sakai, H.; Abe, M.; Kitamoto, D. Monolayers assembled from a glycolipid biosurfactant from Pseudozyma (Candida) antarctica serve as a high-affinity ligand system for immunoglobulin G and M. Biotechnol. Lett. 29, (2007). 12. Ito, S.; Imura, T.; Fukuoka, T.; Morita, T.; Sakai, H.; Abe, M.; Kitamoto, D. Kinetic studies on the interactions between glycolipid biosurfactants assembled monolayers and various classes of immunoglobulins using surface plasmon resonance. Colloids Surf. B Biointerfaces 58, (2007). 13. Konishi, M.; Imura, T.; Morita, T.; Fukuoka, T.; Kitamoto, D. A yeast glycolipid biosurfactant, mannosyl-erythritol lipid, shows high binding affinity towards lectins on a self-assembled monolayer system. Biotechnol. Lett. 29, (2007). 14. Inoh, Y.; Kitamoto, D.; Hirashima, N.; Nakanishi, M. Biosurfactants of MEL-A increase gene transfection mediated by cationic liposomes. Biochem. Biophys. Res. Commun. 289, (2001). 15. Inoh, Y.; Kitamoto, D.; Hirashima, N.; Nakanishi, M. Biosurfactant MEL-A dramatically increases gene transfection via membrane fusion. J. Control Release 94, (2004). 16. Igarashi, S.; Hattori, Y.; Maitani, Y. Biosurfactant MEL-A enhances cellular association and gene transfection by cationic liposome. J. Control. Release 112, (2006). 17. Fukuoka, T.; Morita, T.; Konishi, M.; Imura, T.; Kitamoto, D. Structural characterization and surface-active properties of a new glycolipid biosurfactant, mono-acylated mannosylerythritol lipid, produced from glucose by Pseudozyma antarctica. Appl. Microbiol. Biotechnol. 76, (2007). 18. Fukuoka, T.; Morita, T.; Konishi, M.; Imura, T.; Kitamoto, D. Characterization of new glycolipid biosurfactants, tri-acylated mannosylerythritol lipids, produced by Pseudozyma yeasts. Biotechnol. Lett. 29, (2007). 19. Kitamoto, D.; Haneishi, K.; Nakahara, T.; Tabuchi, T. 564

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