www.babonline.

org
Biotechnol. Appl. Biochem. (2009) 53, 3949 (Printed in Great Britain) doi:10.1042/BA20090033
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Tomotake Morita, Tokuma Fukuoka, Tomohiro Imura and Dai Kitamoto1

Research Institute for Innovations in Sustainable Chemistry, National Institute of Advanced Industrial Science and Technology (AIST), Tukuba Central 5-2, Higashi 1-1, Tsukuba, Ibaraki 305-8565, Japan
BSs (biosurfactants) produced by various microorganisms show unique properties (e.g. mild production conditions, lower toxicity, higher biodegradability and environmental compatibility) compared with chemically synthesized surfactants. The numerous advantages of BSs have prompted applications not only in the food, cosmetic and pharmaceutical industries but also in environmental protection and energysaving technology. Among BSs, glycolipid types are the most promising, owing to their high productivity from renewable resources and versatile biochemical properties. MELs (mannosylerythritol lipids), which are glycolipid BSs abundantly produced by basidiomycetous yeasts such as strains of Pseudozyma, exhibit not only excellent interfacial properties, but also remarkable differentiation-inducing activities against human leukaemia cells. MELs also show high binding afnity towards different immunoglobulins and lectins. Recently, a cationic liposome bearing MEL has been demonstrated to increase dramatically the efciency of gene transfection into mammalian cells. These features of BSs should broaden their application in new advanced technologies. In the present review the current status of research and development on glycolipid BSs, especially their production by Pseudozyma yeasts, is described.

acid type, (iii) lipopeptide type and (iv) polymer type. Of these, the glycolipid types have been most intensively studied because of their high productivity from renewable resources and their versatile biochemical properties [3]. For instance, the yeast Starmerella bombicola (former names Candida bombicola and Torulopsis bombicola) is able to produce sophorolipids, a class of glycolipid BSs, at a level of 300 400 g/litre. Another class of glycolipid BSs, namely MELs (mannosylerythritol lipids), is also produced from vegetable oils by the yeast Pseudozyma antarctica at a high yield of over 100 g/litre. The glycolipid type is thus the most promising for commercial production and utilization. In the present review we summarize the latest research and development in MELs, including their physicochemical properties and possible applications. We would like to refer the reader to reviews that provide more detail about the structures and properties of the other types of BSs [49].

MELs
MELs are glycolipid BSs that contain 4-O- -D-mannopyranosylerythritol or 1-O- -D-mannopyranosylerythritol as a hydrophilic head group and fatty acids as the hydrophobic chain (Figure 1). MELs are abundantly produced by yeast strains of the genus Pseudozyma [10,11]. These MELs exhibit not only excellent interfacial properties [12], but also remarkable differentiation-inducing activities against human leukaemia cells [13,14], rat pheochromocytoma [15] and mouse melanoma cells [16,17]. MELs also show high binding
Key words: basidiomycetous yeast, biosurfactant (BS), glycolipid, mannosylerythritol lipid (MEL), Pseudozyma. Abbreviations used: BS, biosurfactant; CAC, critical aggregation concentrations; CMC, critical micelle concentration; DDPC, L--didodecanoylphosphatidylcholine (L--dilauroylphosphatidylcholine); EST, expressed sequence tag; ITS, internal transcribed spacer; LUV, large unilamellar vesicles; MEL, mannosylerythritol lipid; MEL-A (m), MEL-A (o) and MEL-A (s), MEL-A prepared from methyl myristate, olive oil and soybean oil respectively; NBD, 7-nitrobenz-2-oxa-1,3-diazole; rDNA, ribosomal DNA; SAXS, small-angle X-ray scattering; W/O, water and oil. 1 To whom correspondence should be addressed (email dai-kitamoto@aist.go.jp).
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Introduction
BSs (biosurfactants) are extracellular amphiphilic compounds produced by micro-organisms and have attracted considerable interest in recent years on account of their biodegradability, mild production conditions and variety of functions. The numerous advantages of BSs have promoted their application in a variety of situations, ranging from the food, cosmetic and pharmaceutical industries to environmental protection and energy-saving technology [1,2]. BSs are classied into four main groups based on the structure of their hydrophilic part: (i) glycolipid type, (ii) fatty

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Figure 1 Chemical structure of MELs MEL-A: R1 =Ac, R2 =Ac; MEL-B: R1 =Ac, R2 =H; MEL-C: R1 =H, R2 =Ac; n = 412; m = 616.

afnity towards different immunoglobulins and lectins [18 23]. In addition, MEL-A, the major component of MELs, dramatically increases the efciency of gene transfection mediated by cationic liposomes [2427]. However, MELs have not been used in industry, despite the above advantages for practical applications. The breakthrough for practical use of MELs was thus undertaken by a microbial screening strategy aiming to further improve their production efciency and to expand their structural and functional variety.

Structural variety of MELs and their producers

The MEL producers largely belong to the basidiomycetous yeasts of the genus Pseudozyma. Within this genus, P. antarctica, P. aphidis, P. rugulosa and P. parantarctica produce large amounts of conventional MELs mainly MEL-A, together with MEL-B and MEL-C from vegetable oil [2835]. The amount of MELs accumulated in the medium reaches over 100 g/litre in the resting cells when they are intermittently fed with substrates [2931,35]. P. fusiformata is also a MELA/MEL-B/MEL-C producer, although the production yield is lower than the other MEL producers [11]. P. tsukubaensis showed only MEL-B production [11]. The latest structural analysis revealed that the conguration of MEL-B from P. tsukubaensis was entirely opposite to that of conventional MEL [36]. The sugar moiety of MEL-B by P. tsukubaensis was identied to be 1-O- -D-mannopyranosylerythritol, which is stereochemically different from the 4-O- -Dmannopyranosylerythritol of conventional MEL. P. hubeiensis, P. graminicola, P. shanxiensis and P. siamensis were found to be MEL-C producers [3740]. Among these MEL-C producers, P. hubeiensis produced the highest amount of MEL-C when subjected to intermittent feeding culture; the maximum yield of MEL-C reached 72 g/litre [37]. Recently, P. crassa was also shown to produce MEL-A, MEL-B and MEL-C, and these products were the diastereomer of conventional MEL [41]. There is a structural diversity in the fatty acid moiety
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(Table 1) and their proportions vary according to the carbon source supplied [42]. Recently, the triacylated MEL, namely 1-O-alka(e)noyl4-O-{[4 ,6 -di-O-acetyl-2 ,3 -di-O-alka(e)noyl]- -D-mannopyranosyl}erythritol, was produced by P. antarctica, P. rugulosa and P. parantarctica with an excess amount of vegetable oil as the carbon source [31,43]. The highest production yield (22.7 g/litre) was obtained by P. parantarctica with soybean (Glycine max) oil at the concentration of over 20 % (w/v) [31]. Moreover, the mono-acylated MEL, 4-O[3 -O-alka(e)noyl- -D-mannopyranosyl]erythritol, was also discovered in the culture of P. antarctica and P. parantarctica grown with glucose as the sole carbon source [44]. The structural properties of the above MEL and their producers containing the others are listed in Table 1.

Hydrophilic structure of MEL: carbohydrate conguration

The latest breakthrough in the structural analysis of MEL is discovery of the diastereomer of the sugar moiety. Conventional MEL, such as MEL-A, MEL-B and MEL-C, consist of 4-O- -D-mannopyranosylerythritol as the hydrophilic part and two acyl groups as the hydrophobic part. Each homologue has one or two acetyl groups at C-4 and/or C-6 in the mannose moiety. Among the MEL producers, P. tsukubaensis was investigated in detail, owing to its unique production features; the strain produces only MEL-B without MEL-A and MEL-C. Interestingly, when subjected to structural analysis by 1 H- and 13 C-NMR, the glycolipid produced by P. tsukubaensis showed different peak patterns in part of the carbohydrate moiety (Figure 2) [36]. The two resonances arising from H-4a and H-4b in the erythritol ( 3.84.1 p.p.m.) were widely separated in the product from P. antarctica, whereas, in the product produced from P. tsukubaensis, these peaks overlapped. Furthermore, the resonance from the mannose anomeric hydrogen (H-1 ) of MEL-B by P. tsukubaensis was shifted to a lower eld, from 4.73 to 4.76 p.p.m.

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Table 1 Producers of MELs and the structures produced MEL producer P. antarctica Glycolipid MEL-A (as a main product) Mono-acylated MEL Triacylated MEL MEL-A (as a main product) Triacylated MEL MEL-A (as a main product) Mono-acylated MEL Triacylated MEL MEL-A (as a main product) MEL-A (as a main product) Diastereomer MEL-B MEL-C (as a main product) MEL-C (as a main product) MEL-C MEL-C Diastereomer MEL-A MEL-SY16 (homologous with MEL-B) MEL I-11 (homologous with MEL-B) MEL and cellobiose lipids MEL-C Fatty acids C8 , C10 C8 , C10 , C12 , C14 C8 , C10 , C18 C8 , C10 C8 , C10 , C18 C8 , C10 C8 , C10 , C12 , C14 C8 , C10 , C18 C8 , C10 C8 , C10 , C12 C8 , C12 , C14 C6 , C10 , C12 , C16 C6 , C8 , C12 , C14 C2 , C4 , C14 , C16 C2 , C4 , C14 , C16 C2 , C4 , C14 , C16 , C18 C8 , C12 , C14 C8 , C12 , C14 C6 , C14 , C16 C2 , C4 , C14 , C16 Yield (g/litre) 140 1.3 142 106.7 1.2 22.7 165 4 25 76.3 9.6 2.72 18.5 4.6 95 30a 1.4 Carbon source(s) n-Alkane Glucose Soybean oil Soybean oil, erythritol Soybean oil Soybean oil Glucose Soybean oil Soybean oil, glucose Soybean oil Soybean oil Soybean oil Soybean oil Soybean oil Safower oil Glucose, oleic acid Soybean oil, glucose Sunowerb oil Soybean oil Reference(s) [11,3235] [44] [43] [11,30] [43] [11,31] [44] [31] [11,28,29] [11] [11,36] [37] [38] [39] [40] [41] [54,68] [69] [51] [45]

P. rugulosa P. parantarctica

P. aphidis P. fujiformata P. tsukubaensis P. hubeiensis P. graminicola P. shanxiensis P. siamensis P. crassa Candida sp. SY16 Kurtzmanomyces sp. I-11 U. maydis U. cynodontis
a b

As a mixture. Helianthus annuus.

Figure 2 Partial 1 H-NMR spectra of mannosylerythritol derived from MEL-B produced by P. tsukubaensis (a) and P. antarctica (b)

Hydrophobic structure of MEL: fatty acid composition

As mentioned above, the existing varieties of MEL and its producers were found by microbiological study and detailed structural analysis. The fatty acid components of MEL were

also expanded as well as the acetylation of the mannose moiety (Table 1). The conventional MEL produced by P. antarctica, P. rugulosa, P. aphidis and P. parantarctica possess two fatty acids at C-2 and C-3 in the mannose moiety, and the fatty acid lengths were mainly C8 and C10 . The fatty acid length of the diastereomer MEL-B produced by
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Figure 3 Molecular phylogenetic tree constructed using ITS1, 5.8 S rRNA gene and ITS2 sequences of the genus Pseudozyma and Ustilago The DDBJ/GenBank /EMBL accession numbers are indicated in parentheses.

P. tsukubaensis was mainly C8 , C12 and C14 . The other strains producing mainly MEL-C showed different lengths of fatty acid in comparison with one another: P. hubeiensis was C6 , C10 , C12 and C16 , P. graminicola was C6 , C8 , C12 and C14 and P. shanxiensis and P. siamensis were C2 , C4 , C14 and C16 .

Taxanomic analysis of the genus Pseudozyma

Aiming to expand the structural diversity of MELs and their producers, a search for novel MEL producers was done on the basis of the phylogenetic analysis of the genus Pseudozyma (Figure 3). Initially, P. rugulosa positioned near to the known MEL producers, P. antarctica and P. aphidis, was found to be a novel MEL producer [30]. The other Pseudozyma yeasts were then subjected to the taxonomic analysis, resulting in the three strains of yeasts (P. parantarctica, P. tsukubaensis and P. fusiformata) being newly categorized as MEL producers [11]. After that, P. graminicola, P. shanxiensis, P. siamensis and P. crassa were classied as additional MEL producers [3841]. By the same strategy, a strain of smut fungus, Ustilago cynodontis, was also found to produce mainly
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MEL-C from vegetable oil [45]. Accordingly, the taxonomic study was useful for screening of novel MEL producers, although the rDNA (ribosomal DNA) sequences, the ITS (internal transcribed spacer) region of rDNA, is the taxonomic index of micro-organisms. Furthermore, the environmental screening of MEL producers revealed that all the MEL producers obtained belong to the genus Pseudozyma [46]. Interestingly, the result of taxonomic distribution of the MEL producers obtained corresponded well with their properties of MEL production. The MEL-A producers such as P. antarctica, P. aphidis, P. rugulosa and P. parantarctica were closely positioned in the branch of the phylogenetic tree. P. crassa, producing the diastereomer MEL-A as the main product, was near the branch of the MEL-A producers. The diastereomer MEL-B producer, P. tsukubaensis, was independently positioned in the tree. The MEL-C producers were distantly categorized in the tree compared with the MEL-A producer, whereas P. shanxiensis, P. siamensis and U. cynodontis were closer than the other MEL-C producers. From the fatty-acid-composition point of view, the distal categorization for MEL-C producers seems to be appropriate (Table 1). Further study of the genetic index should enable us to efciently expand both

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Scheme 1

Presumptive biosynthetic pathway of MELs in P. antarctica

Grey hexagon/Man, mannose; black rhombus/Ery, erythritol; Man-Ery, MEL; white Ac in closed circle, acetyl group; zig-zag line in ellipse, fatty acid.

the structure of MELs and its producers, advancing their industrial applications.

Biosynthetic pathway of MEL

In the previous investigation of the biosynthetic pathway of fatty acids for MELs, it was suggested that the fatty acids are mostly -oxidation intermediates of the substrates supplied, and the pathway was clearly distinct from the pathways generally known in micro-organisms, that is, the chainshortening pathway (Scheme 1) [42]. Recently, an EST (expressed sequence tag) analysis was attempted with P. antarctica grown in a medium containing soybean oil, and the genes expressed during MEL production were tentatively categorized on the basis of putative function, although the details of each gene are almost unknown [47]. The tools for genetic engineering of the yeast the construction of the expression vector carrying efcient promoters and a simple transformation method by electroporation in Pseudozyma yeast were also developed in the past few years [4850]. On the other hand, the smut fungus U. maydis is able to produce MEL together with cellobiose lipids at a lower concentration compared with the genus Pseudozyma [51]. U. maydis is one of the most convenient micro-organisms

using advanced tools for genetic engineering. An identication of the genes for MEL biosynthesis has thereby been reported for U. maydis: emt1 encodes a mannosyltransferase involved in the formation of mannosylerythritol by mannosylation of erythritol, mat1 encodes an acetyltransferase catalysing the acetylation of mannosylerythritol at both the C-4 and C-6 hydroxy groups, and mac1 encodes an acyltransferase related to the acylation of mannosylerythritol [52,53]. Further investigation of the genomic analysis and development of the gene expression system for Pseudozyma yeasts should enable us to understand the biosynthetic pathway in detail and to improve the production of MEL by genetic modication.

Production of MEL
A large accumulation of MEL was for the rst time reported using P. antarctica T-34 (renamed from Candida antarctica T-34). The strain produced over 40 g of MEL/litre from soybean oil as the sole carbon source [32]. Moreover, the resting cells of the strain showed a production of 140 g/litre when fed intermittently with n-alkanes as the substrate (Figure 4) [35]. Recently, MEL production was reported using P. aphidis DSM 70725 (=CBS 6821, renamed from Candida
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cultivation, the subsequent downstream processing of the products is also critical to decrease the production cost. Therefore an easy-to-handle procedure required for economic isolation and purication of MEL has been investigated [55]. MELs were able to be transferred into a highly viscous phase by heating the culture supernatant without producing solvent waste. Recently, the advantage of using a water-soluble carbon source to design a simplied downstream process was also reported, as discussed below [56,57].

Carbon source for MEL production

From the point of view of MEL production yield, vegetable oil is the best substrate, since the production yield from vegetable oils reached over 100 g/litre [2931,35,54]. MEL producers are able to degrade vegetable oils by the reaction of extracellular lipases. However, when soybean oil is employed, the removal of the residual oil and its lipase degradation products (e.g. monoacylglycerols or diacylated glycerols and non-esteried fatty acids) are required. To improve the efciency of MEL production, the use of watersoluble carbon sources instead of vegetable oils would thus be highly desirable. Indeed, the purity of glucoseand glycerol-derived MELs was clearly higher than that of soybean oil-derived MELs; in the extract from the culture with glucose and glycerol, by-products such as the residual oil and its degradation products were not detected on TLC and HPLC analysis [56,57].

Figure 4 Time course of MEL production by P. rugulosa ()<grey square> [30], P. antarctica () [35] and P. hubeiensis ( ) [37]

antarctica CBS 6821). The fed-batch bioreactor production of MELs by the strain showed 165 g/litre as the maximum production yield with soybean oil and an excess amount of glucose as the carbon sources [28]. The production of MEL-SY16 was also achieved by fed-batch fermentation, and the maximum yield was reached at 95 g/litre with soybean oil as the substrate [54]. In P. rugulosa, supplementation with erythritol drastically enhanced the MEL production yield by 7090 %, and the maximum yield reached 142 g/litre using intermittent feeding of soybean oil and erythritol [30]. Furthermore, in fed-batch culture, the MEL-C production yield of P. hubeiensis KM-59 reached 76.3 g/litre [37]. P. siamensis produced 18.5 g of MEL-C [which possesses a shorter-chain fatty acid (C2 or C4 ) at the C-2 position and a longer-chain fatty acid (C16 ) at the C-3 position of the mannose moiety]/litre, from safower (Carthamus tinctorius) oil as the sole carbon source [40]. The efcient production of triacylated MEL was investigated using P. parantarctica, and the production yield reached at 22.7 g/litre with an excess amount of soybean oils [31]. The triacylated MELs were produced when the cells were grown in a medium with the excess amount of vegetable oils. Triacylated MEL seems to be synthesized by the esterication of diacylated MEL with fatty acids in the culture medium by extracellular lipase and/or esterase [43].

Physicochemical properties of MELs

MELs are surface-active compounds capable of reducing surface and interfacial tension at the interface between liquids, solids and gases, thereby allowing them to mix or disperse readily as emulsions in water or other liquids [12]. The all diacylated MELs (i.e. MEL-A, MEL-B and MELC) show excellent surface and interfacial-tension-lowering activities and low CMCs (critical micelle concentrations), as shown in Table 2. The mono-acylated MELs showed lower surface activity than those of diacylated MEL, as expected from their structural properties [CMC, 3.6 10 4 M; CMC (surface tension at the CMC), 33.8 mN/m]. The MELs thus show excellent surface properties regardless of the functional group and fatty acid length.

Downstream processing
To date, the practical use of MELs has not taken place, owing to high production costs, in spite of its potentially interesting applications. Besides high-yield production using bioreactor
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Self-assembling properties of MELs

The self-assembly of biologically based amphiphilic molecules has been the subject of intense study in recent years. Many types of amphiphilic molecules self-assemble to form a wide

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Table 2 Surface activities of MEL MEL species MEL-A Producer P. antarctica P. crassa P. antarctica P. graminicola P. hubeiensis P. siamensis P. antarctica Substrate Soybean oil Glucose and oleic acid Soybean oil Soybean oil Soybean oil Safower oil Glucose CMC (M) 2.7 10 6 5.2 10 6 4.5 10 6 4.0 10 6 6.0 10 6 4.5 10 6 3.6 10 4 CMC (mN/m) 28.4 26.5 28.2 24.2 25.1 30.7 33.8 Reference [12] [41] [12] [38] [37] [40] [44]

MEL-B MEL-C

Mono-acylated MEL

variety of objects, including micelles, vesicles, tubes and coacervates [58]. In most cases the biological molecules have complicated structures, with one or more chiral centres and functional groups, which allow them to self-assemble into hierarchically ordered structures by using hydrogen bonding, and hydrophobic and van der Waals interactions [59]. The self-assembling properties of MEL-A and MEL-B were revealed by a uorescence-probe method, dynamic light-scattering analysis, freezefracture transmission microscopy and synchrotron SAXS (small-angle X-ray scattering)/wide-angle X-ray scattering [60]. Both MEL-A and MEL-B produced by P. antarctica exhibit excellent selfassembling properties at extremely low concentrations; they self-assemble into LUVs (large unilamellar vesicles) just above their CAC (critical aggregation concentrations). The self-assembled structure of MEL-A above a CAC value of 2.0 10 5 M was found to drastically change into a sponge structure (L3) composed of a network of randomly connected bilayers that are usually obtained from a complicated multi-component synthetic surfactant system. The average water-channel diameter of the sponge structure was 100 nm. The self-assembled structure of MEL-B seems to gradually move from LUVs to multilamellar vesicles. On a water-penetration scan, the lyotropic-liquid-crystal phase observation at high concentration tentatively demonstrated the formation of an inverted hexagonal phase (H2 ) for MELA, together with a lamellar phase (L ) for MEL-B, indicating a difference between MEL-A and MEL-B molecules in the spontaneous curvature of the assemblies. The structures of the liquid crystal of MEL-A were determined as L3 , bicontinuous cubic (V2 ) and L phases on the basis of SAXS measurement [61]. Consequently, the difference in the spontaneous curvature caused by the single acetyl group on the head group probably decides the direction of selfassembly of MEL (Figure 5). MEL-A produced by P. antarctica forms a thermodynamically stable vesicle (L1 ) from the L3 phase with DDPC (L- -didodecanoylphosphatidylcholine) [62]. The mixed vesicle solution kept its dispersion stability at 25 C for

more than 3 months, showing the formation of the thermodynamically stable vesicle. The addition of DDPC to a MEL-A L3 phase which has both negative and positive spontaneous curvature is likely to immediately determine the direction of the self-assembly because negatively curved MEL-A favours distributing in inner monolayers. Therefore this leads to the facial formation of thermodynamically stable vesicles. Microemulsions are thermodynamically stable isotropically homogeneous transparent systems containing substantial amounts of two immiscible liquids [i.e. W/O (water and oil)] stabilized by appropriate surfactants. The formation of a W/O microemulsion of MEL-A produced by P. antarctica was investigated using dynamic light scattering and freezefracture electron microscopy [63]. The W/O microemulsion by the addition of water to a MEL-A/ndecane mixture kept their single-phase transparent appearance. Recently, the aqueous-phase behaviour and vesicle formation of diastereomer MEL-B produced by P. tsukubaensis was investigated [64]. MEL-B was found to selfassemble into the L phase over wide concentration and temperature ranges. The obtained L phase was found to be stable up to 95 C below a MEL-B concentration of 85 % (w/w). Furthermore, MEL-B was able to form relatively large vesicles (15 m) at low MEL-B concentrations as judged by confocal-laser-scanning-microscopy studies. Consequently, MEL-B produced by P. tsukubaensis may prove to be useful in various elds of application as an L phase- and/or vesicleforming lipid. The formation of liquid crystal phases of the other MELs were tentatively estimated by water penetration scan (Figure 5). MEL-C produced by P. hubeiensis formed liquidcrystal phases similar to L phase and myelins, as well as MEL-B from P. antarctica [37]. MEL-C from P. graminicola appeared to show a liquid-crystal phase corresponding to the L phase of MEL-B by P. antarctica [38]. MEL-C from P. siamensis formed liquid-crystal phases similar to the hexagonal and L phase over a wide range of concentrations [40]. MEL-A produced by P. crassa, which is a diastereomer of the
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Figure 5 Water-penetration scans of MEL-A from P. antarctica (a), MEL-B from P. antarctica (b) [60], MEL-C from P. hubeiensis (c) [37] and MEL-C from P. siamensis (d) [40] L , lamella phase; L3 , sponge structure; V2 , bicontinuous cubic; W, water phase; S, neat surfactant phase.

conventional MEL-A produced by P. antarctica, seems to form liquid-crystal phases distinct from those of conventional MEL-A.

Binding afnity towards immunoglobulins and lectins of MEL

IgG is widely used in immunodiagnostic and therapeutic applications. Protein A is useful for immunoafnity chromatography of IgG because of its high afnity. However, Protein A has two major drawbacks: the high cost of the protein and the highly acidic conditions needed to elute IgG. Previously Im et al. [21] reported that MEL-A shows high binding afnity towards human IgG. The binding behaviour was further investigated by using surface plasmon resonance and the measurement of surface pressure ( )area (A) isotherms [23]. Three kinds of MEL-A were prepared: MEL-A (m) from methyl myristate, MEL-A (o)
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from olive (Olea europea) oil and MEL-A (s) from soybean oil. The main fatty acids were C8 and C10 acids in all MELAs, and the content of unsaturated fatty acids was 0 % for MEL-A (m), 9.1 % for MEL-A (o), and 46.3 % for MEL-A (s). The acid contents signicantly inuenced their binding afnity, and the monolayer of MEL-A (o) gave a higher binding afnity than that of MEL-A (m) and MEL-A (s). On the basis of the measurement of the A isotherms involved in the monolayer packing density, the exhibited area of the monolayer was MEL-A (o) > MEL-A (m) > MEL-A (s), which is the same order as that of binding afnity. Accordingly, it was suggested that the binding afnity of MEL-A towards IgG is thus likely to be closely related to the monolayer packing density. MEL-A produced by P. antarctica also has binding afnity toward lectins on a self-assembled monolayer system [20]. The binding afnity of diastereomer MEL-A produced by P. crassa was studied on a self-assembled monolayer system as well as the above conventional MEL [41]. The MEL-A showed no binding afnity to human IgG.

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Additionally, MEL-B and MEL-C showed lower binding capacity to human IgG than did MEL-A [22].

Gene transfection
Gene transfection across cell membranes is a fundamental technology for molecular cell biology and also for clinical therapy. Although the most efcient methods for gene transfection involve the use of viral vectors, there are still arguments about risks regarding propagation and immunogenicity. However, a variety of non-viral gene-delivery systems have been investigated and cationic liposomes have proved to be useful tools for delivery of plasmid DNA and RNA into cells [66]. MEL-A produced by P. antarctica dramatically increased the efciency of gene transfection mediated by cationic liposomes with a cationic cholesterol derivative using NIH3T3 (mouse embryonic broblast) and COS-7 (African-green-monkey simian-virus-40-transformed kidney broblast) cells [24]. On the other hand, MEL-B and MEL-C from P. antarctica did not enhance the efciency of gene transfection [25]. Moreover, it was found that the FITCconjugated antisense oligonucleotides were temporarily present on the plasma membrane of the target cells and thereafter they were transferred into the nucleus of the target cells. These results suggested that MEL-A induces membrane fusion between target cells and cationic liposomes, accelerating the efciency of gene transfection drastically. Furthermore, the mechanism of the transfection mediated by cationic liposomes with NBD-conjugated MEL-A was investigated using Hala 229 human cervixcarcinoma cells [26]. MEL-A gradually distributed on the intracellular membranes through the plasma membranes of target cells, whereas the cationic liposomes with MEL-A fused to the plasma membranes in 2035 min. Thereafter, the oligonucleotide released from the vesicles was immediately transferred to the nucleus. Consequently, MEL-A is able to exhibit rapid promotion of transfection efciency by inducing membrane fusion between liposomes and the plasma membrane of target cells.

cells. MEL-A and MEL-B also signicantly induce the neurite outgrowth of rat pheochromocytoma PC-12 cells and partial cellular differentiation. MEL-A inhibits the growth of mouse melanoma B16 cells in a dose-dependent manner. Moreover, MEL-A is useful for environmentally friendly cold thermal storage since it suppresses the agglomeration of ice particles (an eco-friendly ice slurry system [67]). Details of other applicable properties of MEL have been described in previous reviews [19]. Recent ndings have permitted us to overcome problems in the commercial application of MEL by virtue of improved production and expanded structural diversity of MEL, in combination with microbiological, biochemical and physicochemical strategies. In fact, the commercial application of MEL in the cosmetic eld (an antiaging agent) has now been patented by a group of Japanese workers [70]. However, there is still limited available information on biosynthetic mechanisms, physicochemical properties and practical applications. Further breakthroughs from different elds are required to promote the research and development of MELs.

Funding
This work was supported by the New Energy and Industrial Technology Development Organization (NEDO) of Japan [Industrial Technology Research Grant Programme number 06A17501c].

References
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C

Further perspectives
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There are many potential practical uses of MEL besides the abovementioned functions. For instance, MELs show strong activity against Gram-positive bacteria, weak activity against Gram-negative bacteria and no activity against fungi. MELA and MEL-B have growth inhibition and differentiationinducing activities against human leukaemia cells, including K562 myelogenous-leukaemia cells, HL60 promyelocyticleukaemia cells and the human KU812 basophilic-leukaemia

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Received 23 January 2009/20 February 2009; accepted 2 March 2009 Published on the Internet 6 April 2009, doi:10.1042/BA20090033

2009 Portland Press Ltd