Effects of nitrogen sources on production and composition of sophorolipids by <Emphasis Type="Italic">Wickerhamiella domercqiae</Emphasis> var. <Emphasis Type="Italic">sophorolipid</Emphasis> CGMCC 1576

The effects of nitrogen sources on growth of sophorolipid-producing yeast, Wickerhamiella domercqiae var. sophorolipid CGMCC 1576 and on production and composition of sophorolipids were studied. Organic nitrogen sources are more favorable for accumulation of biomass than inorganic ones. Presence of ammonium ion from different inorganic nitrogen sources (except NH₄HCO₃) greatly inhibited the production of lactonic sophorolipids. However, when organic nitrogen sources were used, lactonic sophorolipid production was strongly increased. Production of crystalline lactonic sophorolipids from organic/inorganic nitrogen sources was enhanced with the increase of pH value adjusted by sodium hydroxide or sodium citrate solution. Fourier-transform infrared (FT-IR), gas chromatography mass spectrometry (GC-MS), high-performance liquid chromatography (HPLC), and mass spectra (MS) were employed to compare the composition of sophorolipid mixture obtained from different nitrogen sources. More than 15 acidic sophorolipid molecules and only 4 lactonic sophorolipid molecules were produced by using 1.27 g/l ammonium sulfate as nitrogen source; they were separated by preparative HPLC and their structures were elucidated by MS. These results suggest extraordinary regulatory effects of nitrogen source on growth and sophorolipid synthesis of W. domercqiae var. sophorolipid.     

Sophorolipid production from different lipid precursors observed with LC-MS

An HPLC-MSⁿ system was used to quantify and identify the structures of individual sophorolipid components produced in Torulopsis bombicola fermentation on glucose with or without hexadecane or soybean oil. With glucose alone, the SL production was minimal and the products were complex mixtures with mainly acidic SLs. The SLs produced with glucose plus soybean oil were also complex, containing both lactonic and acidic SLs with saturated and unsaturated C16 and C18 fatty acid moieties. The glucose plus hexadecane system gave the highest production rate and product selectivity, forming primarily two diacetylated lactonic isomers with palmitate as the fatty acid moiety. A close structure correspondence between the SL’s lipid moiety and the lipid precursor used was observed. The change of the composition of SL mixtures along batch fermentation was further examined. The concentrations of acidic SLs increased very gradually throughout the process. The production of lactonic SLs became appreciable following the addition of hexadecane or soybean oil at 24 h, and increased much more rapidly after the culture reached the stationary phase. The combined percentage of the main lactonic SLs leveled off at 80% for the hexadecane system and 50% for the soybean oil system. The yields of crude SLs were 0.84, 0.20, and 0.03 g per gram of hexadecane, soybean oil, and glucose consumed during the SL production phase. Hexadecane is thus a more efficient second C-source for sophorolipid production.     

Identification and quantification of sophorolipid analogs using ultra-fast liquid chromatography–mass spectrometry

An ultra-fast liquid chromatographic method combined with atmospheric pressure chemical ionization mass detection (UHPLC/APCI-MS) has been developed for the separation and quantification of sophorolipid analogs produced by the yeast Candida bombicola. The sophorolipid mixture was produced by growing the yeast in the presence of glucose and oleic acid under higher aeration. It was found that more than 95% of the analogs are lactonic sophorolipids and all the produced sophorolipids produced were either mono- or di-acetylated. Also observed was a sophorolipid analog with a tri-unsaturated fatty acid, which has not been reported previously.     

Sophorolipids: improvement of the selective production by Starmerella bombicola through the design of nutritional requirements

Microtiter plates were used as minireactors to study Starmerella bombicola growth and sophorolipid (SL) production. Compositional analysis of SL mixtures by liquid chromatography with electrospray ionization tandem mass spectrometry showed similar results on SLs produced using the laboratory scale (shake flask) and the microscale (24-well microtiter plates (MTP)) approach. MTP suitability on SL production was proven, being this approach, especially advantageous on SL screening. Several hydrophilic carbon sources, hydrophobic co-substrates and nitrogen sources were supplied to culture media, and their influence on SL production was evaluated. The selection of specific hydrophobic co-substrate and nitrogen sources influenced the ratio acidic/lactonic SLs. In fact, it was observed that the production of acidic C18:1 diacetylated hydroxy fatty acid SLs was favoured when culture media was supplied with avocado, argan, sweet almond and jojoba oil or when NaNO₃ was supplied instead of urea. This last case was observed after 144 h of cultivation. A new SL, lactonic C18:3 hydroxy fatty acid diacetylated SL, was detected when borage and onagra oils were used individually as co-substrates. Overall results indicated the potential of the selective production of different and new sophorolipids by Starmerella bombicola based on the selection of carbon and nitrogen sources to culture media.     

Purification of lactonic sophorolipids by crystallization

Various experimental methods for purifying lactonic sophorolipids (SLs) via crystallization were studied. The commonly used solvent, ethanol, was found to have much higher solubility of the lactonic SLs than the acid SLs. Consequently, for purification of lactonic SLs, ethanol not only lacked the selectivity in removing acidic SLs but also resulted in significant loss of desired products. Aqueous buffers were subsequently studied as the solvent, based on the rationale that acidic SLs, having the free carboxylic acid group(s), are more hydrophilic than the lactonic SLs, especially at higher pH. Both phthalate and phosphate buffers were found more suitable for purifying lactonic SLs than ethanol. A practical and effective method for purifying lactonic SLs to about 99% purity using phosphate buffers was developed. The FTIR spectra also showed significantly less SL components with free acid groups in the purified SLs than in the crude SLs, confirming the removal of acidic SLs in the purification.     

Utilization of sweetwater as a cost-effective carbon source for sophorolipids production by Starmerella bombicola (ATCC 22214)

Biosurfactants are microbially synthesized surfactants that are environmental friendly due to low toxicity. Sophorolipid is one of the simplest biosurfactants with well-defined structure produced by Starmerella bombicola(ATCC 22214) on glucose and vegetable oil as the carbon source. The raw material cost accounts for 10-30% of the overall cost. Glycerol is readily available from a commercial fat-splitting process as sweetwater at a very low cost. Sophorolipids was synthesized using glycerol and sweetwater as a cost-effective carbon source. The glycerol was further replaced with sweetwater as a source of glycerol. Optimum glycerol concentration was 15% w/v with 10% w/v sunflower oil, giving 6.6 g/L of sophorolipids. The crude sophorolipid contains two major components; both of them were lactonic sophorolipids as analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC), liquid chromatography-mass spectroscopy (LC-MS), and nuclear magnetic resonance ((1)H-NMR).     

Production of sophorolipids with eicosapentaenoic acid and docosahexaenoic acid from Wickerhamiella domercqiae var. sophorolipid using fish oil as a hydrophobic carbon source

Sophorolipids (SLs) were synthesized by Wickerhamiella domercqiae var. sophorolipid CGMCC 1576 grown on fish oil and glucose. They were purified using preparative HPLC and their structures were identified by MS/MS. The yields of total and lactonic SLs were 47 and 19 g l^?1 in shake-flasks when fish oil 4 % (v/v) was used with glucose in the medium. The composition of SL mixture contained more than 20 SL molecules. Several unconventional SL molecules with eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) including zero-, mono- and di-acetylated acidic SLs with EPA and a di-acetylated acidic SL with DHA were obtained. Two unconventional lactonic SL molecules, non-acetylated lactonic SL with 22:3 and non-acetylated lactonic SL with 20:0, were also obtained.     

One-step production of unacetylated sophorolipids by an acetyltransferase negative Candida bombicola

Sophorolipids from the non-pathogenic yeast Candida bombicola are applied commercially as biodegradable, eco-friendly surface active agents. These sophorolipids are produced by cultivation in presence of a hydrophobic carbon source and are always constituted of a mixture of structurally related molecules. For some applications however, certain structural variants perform better than others. Acetylation of the sophorolipid molecule is such a parameter that gains interest because of its influence on water solubility, foaming properties, and biological activity. Fully unacetylated sophorolipids therefore are interesting metabolites but cannot be produced in a pure way by conventional cultivation. Here we report the identification of the acetyltransferase gene AT, responsible for acetylation of de novo synthesized sophorolipids in Candida bombicola. By the creation of a Δat deletion mutant, we could create a yeast strain producing purely unacetylated sophorolipids with a yield of 5 ± 0.7 g/L using rapeseed oil as hydrophobic carbon source. In contrast to the chemical production of unacetylated sophorolipids used nowadays, the microbial production leads to mainly lactonic sophorolipids, in addition to minor amounts of acidic sophorolipids.     

Selective recovery of acidic and lactonic sophorolipids from culture broths towards the improvement of their therapeutic potential

Sophorolipids (SLs) were produced by Starmerella bombicola. The separation and purification of SLs are a complex process, since they are produced as a mixture of compounds with few structural differences. Solvent extraction is commonly used in downstream processing. In this work, an environmental friendly approach was developed for SLs recovery and purification, based on neutral polymeric sorbents, Amberlite XAD16N^TM, XAD18^TM, and XAD1600N^TM. In batch microassays, key parameters of sorption/desorption process (e.g., contact time, temperature, sorbents, and SLs concentrations) were optimized for separation of acidic and lactonic SLs. Sorption equilibrium was reached after 2–3 h, for all the sorbents tested. Among them XAD1600N^TM showed a higher sorption capacity (q _max 230 mg g^?1), a higher removal (≈100 %) of acidic and lactonic SLs [1 and 2.5 % (w/v)], and the best selectivity. Methanol, ethanol, and acetone were suitable for SLs elution. A selective desorption of SLs was attained with acetonitrile aqueous solutions (v/v): (1) 25 % led to 88.3 % of acidic SLs and (2) 55 % followed by methanol solution (100 %) led to 93.2 % of purified lactonic SLs. This achievement was particularly important regarding SLs potential therapeutic applications, since acidic and lactonic SLs show different biologic activities. In fact, acid SLs show higher virucidal and pro-inflammatory cytokine activity, while lactonic SLs show stronger spermicidal and anti-cancer activity.     

Characterization of a novel enzyme—<Emphasis Type="Italic">Starmerella bombicola</Emphasis> lactone esterase (SBLE)—responsible for sophorolipid lactonization

We recently discovered a novel enzyme in the exoproteome of Starmerella bombicola, which is structurally related to Candida antarctica lipase A. A knockout strain for this enzyme does no longer produce lactonic sophorolipids, prompting us to believe that this protein is the missing S. bombicola lactone esterase (SBLE). SBLE catalyzes a rather unusual reaction, i.e., an intramolecular esterification (lactonization) of acidic sophorolipids in an aqueous environment, which raised questions about its activity and mode of action. Here, we report the heterologous production of this enzyme in Pichia pastoris and its purification in a two-step strategy. Purified recombinant SBLE (rSBLE) was used to perform HPLC and liquid chromatography mass spectrometry (LCMS)-based assays with different sophorolipid mixtures. We experimentally confirmed that SBLE is able to perform ring closure of acetylated acidic sophorolipids. This substrate was selected for rSBLE kinetic studies to estimate the apparent values of K _m . We established that rSBLE displays optimal activity in the pH range of 3.5 to 6 and has an optimal temperature in the range of 20 to 50 °C. Additionally, we generated a rSBLE mutant through site-directed mutagenesis of Ser₁₉₄ in the predicted active site pocket and show that this mutant is lacking the ability to lactonize sophorolipids. We therefore propose that SBLE operates via the common serine hydrolase mechanism in which the catalytic serine residue is assisted by a His/Asp pair.     

LC/MS analysis and lipase modification of the sophorolipids produced by Rhodotorula bogoriensis **

The extracellular glycolipids produced by the yeast, Rhodotorula bogoriensis (formerly Candida bogoriensis), were analyzed using an LC/API-MS method. The analysis confirmed that the predominant form the sophorolipid structure contained a C22 hydroxy carboxylic acid. A minor amount (<10%) of a C24 hydroxy carboxylic acid in the sophorolipid was also found, which had not been reported previously. The sophorolipid product, which contained varying degrees of acetylation at the primary hydroxy groups of the sophorose sugar, was deacetylated with sodium methoxide. The des-acetylated sophorolipid was esterified using an immobilized lipase as catalyst in tetrahydrofuran and the product analyzed by mass spectrometric techniques. The product was screened for dimer or polymer formation but only a monomeric lactonized sophorolipid structure was detected.     

Integrated sophorolipid production and gravity separation

A novel method for the integrated gravity separation of sophorolipid from a fermentation broth has been developed, enabling removal of a sophorolipid phase of either higher or lower density than the bulk fermentation broth, while cells and other media components are recirculated and returned to the bioreactor. The capability of the separation system to recover an enriched sophorolipid product phase was demonstrated on three sophorolipid producing fed batch fermentations using Candida bombicola, giving an 11% reduction in fermenter volume required whilst maintaining sophorolipid production. Sophorolipid recoveries of up to 86% (280 g) of the total produced over a whole fermentation were achieved at an enrichment of up to 9. Furthermore, the broth viscosity reduction achieved by removal of the sophorolipid phase enabled a 34% reduction in mixing power to maintain the same dissolved oxygen level by the end of the fermentation, with a 9% average reduction over the course of the fermentation. Fermentation duration could be extended to 1023 h, allowing production of 623 g sophorolipid from 1 l initial batch volume. These benefits could lead to a substantial decrease in the cost of sophorolipid production, making high volume applications such as enhanced oil recovery economically feasible.     

Vegetable oil enhances sophorolipid production by <Emphasis Type="Italic">Rhodotorula bogoriensis</Emphasis>

The yeast Rhodotorula bogorensis produces sophorolipids of different structures to those produced by Candida bombicola. However, the yield is very low. To improve sophorolipid production by R. bogoriensis, vegetable oil was supplemented to the medium as a hydrophobic substrate: with rapeseed oil the sophorolipid yield was 1.26 g/l but without oil was 0.33 g/l. Cultures with meadowfoam oil produced 0.77 g sophorolipids/l. Lipase-treated meadowfoam oil, however, gave no significant increase in sophorolipid production. Possible explanations for the enhanced sophorolipid synthesis are discussed.     

代谢改造熊蜂生假丝酵母提高酸型槐糖脂产量

【目的】槐糖脂是一类生物表面活性剂,不仅具有常规表面活性剂所具有的增溶、乳化、润湿、发泡、分散、降低表面张力等通用性能,且对环境的耐受性极强。熊蜂生假丝酵母(Starmerella bombicola)能够发酵生产槐糖脂,但槐糖脂具有酸型、内酯型和乙酰化型等不同类型,结构多样,难以分离。本文拟通过代谢工程改造,构建高产酸型槐糖脂的熊蜂生假丝酵母工程菌株。【方法】利用潮霉素抗性基因构建了标记基因重复利用系统Rec-six基因编辑系统,在此基础上将合成内酯型槐糖脂的关键基因——内酯酶基因SBLE敲除获得一株只产酸型槐糖脂的工程菌株Δsble,进一步同源过量表达葡萄糖基转移酶基因UGTB并敲除过氧化物酶体膜转运蛋白编码基因PXA1,构建了高产酸型槐糖脂的酵母工程菌。【结果】与出发菌株相比,重组熊蜂生假丝酵母发酵油酸能够合成单一的酸型槐糖脂,而不再合成内酯型槐糖脂,同时酸型槐糖脂的产量由20 g/L提高到44 g/L,提高了2.1倍。【结论】通过敲除PXA1、SBLE和过表达UGTB来改造熊蜂生假丝酵母,能够有效提高重组菌的酸型槐糖脂产量,为发酵法生产酸型槐糖脂奠定了基础。     

Enzymatic synthesis of a galactopyranose sophorolipid fatty acid-ester**

Sophorolipid lactones produced by Candida bombicola were deacetylated and ring opened with sodium methoxide to their corresponding methyl esters. The methyl esters, after re-acetylation with vinyl acetate using an immobilized lipase, were transesterified with 1,2-3,4-di-O-isopropylidene-d-galactopyranose in tetrahydrofuran using the same lipase catalyst. The di-O-isopropylidene sophorolipid sugar esters were hydrolyzed to give the galactopyranose sophorolipid esters as the final products.     

Effect of surfactants on cellulose hydrolysis

The effect of surfactants on the heterogeneous enzymatic hydrolysis of Sigmacell 100 cellulose and of steam-exploded wood was studied. Certain biosurfactants (sophorolipid, rhamnolipid, bacitracin) and Tween 80 increased the rate of hydrolysis of Sigmacell 100, as measured by the amount of reducing sugar produced, by as much as seven times. The hydrolysis of steam-exploded wood was increased by 67% in the presence of sophorolipid. At the same time, sophorolipid was found to decrease the amount of enzyme adsorbed onto the cellulose at equilibrium. Sophorolipid had the greatest effect on cellulose hydrolysis when it was present from the beginning of the experiment and when the enzyme/cellulose ratio was low. (c) 1993 John Wiley & Sons, Inc.     

Nouvelle Synthèse de l’α-Damascone

The gardenia absolute was separated into basic, phenolic, acidic, lactonic and neutral fractions and analysed by using GC, combined GC-MS, IR and NMR. A total of 130 components was identified. Among them, the characteristic constituents responsible for the sweet-green odor of this flower were jasmin lactone, cis-3-hexenol, esters of cis-3-hexenol, cis-3-hexenoic acid and tiglic acid.     

Importance of the cytochrome P450 monooxygenase CYP52 family for the sophorolipid-producing yeast Candida bombicola

Three cytochrome P450 monooxygenases belonging to the CYP52 family were isolated from the genome of the sophorolipid-producing yeast Candida bombicola using degenerate PCR and genomic walking. One gene displayed high identity with the CYP52E members and was classified into this group ( CYP52E3 ), whereas the other genes belonged to new groups: CYP52M and CYP52N. CYP52E3 and CYP52N1 turned out to be of no relevance for sophorolipid production, but show clear upregulation when the yeast cells are grown on alkanes as the sole carbon source. On the other hand, CYP52M1 is clearly upregulated during sophorolipid synthesis and very likely takes part in sophorolipid formation.     

Sophorolipid produced from the new yeast strain Wickerhamiella domercqiae induces apoptosis in H7402 human liver cancer cells

The effects of sophorolipid on the growth and apoptosis of H7402 human liver cancer cells were investigated. By treatment with sophorolipid, a dose- and time-dependent inhibition of cell proliferation was observed. The cells developed many features of apoptosis, including condensation of chromatin, nuclear fragmentation, and appearance of apoptotic bodies, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling positive cells were stained dark brown. Sophorolipid treatment induced apoptosis in H7402 cells by blocking cell cycle at G1 phase and partly at S phase, activating caspase-3, and increasing Ca2+ concentration in cytoplasm. These findings may suggest a potential use of sophorolipid for liver cancer treatment.