S. M. Chae

Biotransformation of Ginsenoside Rb1 into Rd by the Bacterium Lysobacter panaciterrae

Triterpenoid saponins and ginsenosides, the majority of which contain the aglycons protopanaxadiol or protopanaxatriol, are important biologically active compounds of Panax ginseng C. A. Meyer. The principal ginsenoside, the content of which in ginseng root is >20% of the total saponins, is Rb1 (1), which includes -sophorose and -gentiobiose residues (6-O-Dglucopyranosyl-D-glucose) in the C-3 and C-20 positions, respectively, of 20(S)-protopanaxadiol. The minor ginsenoside Rd (2) is a close structural analog of Rb1 and has a glucose instead of -gentiobiose in the C-20 position [1]. Rd has a broader spectrum of biological activity than Rb1, for example, it increases the differentiation of nerve stem cells, protects neurons from neurotoxic substances, and inhibits proliferation of HepG2 liver cancer cells [2]. However, the content of Rd in ginseng root is 4–5 times less than that of Rb1. Therefore, biotransformation of ginsenoside Rb1 into Rd, in particular by using microorganisms, is of practical importance [3].

Biotransformation of ginsenoside Rb1 into F-2 and compound K by bacterium Sphingomonas sp. BG 25

Triterpenoid saponins and ginsenosides, the majority of which contain protopanaxadiol or protopanaxatriol as the aglycon, are important biologically active compounds of Panax ginseng C. A. Meyer. One of the principal ginsenosides, Rb1 (1), has -sophorose and -gentiobiose residues in the C-3 and C-20 positions, respectively, of 20(S)-protopanaxadiol. The minor ginsenoside F-2 (2) with glucose residues on C-3 and C-20 and compound K (3) with only one glucose on C-20 have structures close to that of Rb1 and are practically absent in ginseng roots [1]. Saponins 2 and 3 have broader spectra of biological activity than Rb1. For example, compound K inhibits growth of cultivated melanoma B16-B6, hepatocellular carcinoma Hep-G2, and lung carcinoma 95-D cells whereas F-2 induces apoptosis of breast cancer stem cells [1, 2].

Fatty-Acid Compositions of Three Strains of Blue-Green Algae Biomass, a Potential Feedstock for Producing Biodiesel Fuel

The most promising renewable feedstock for producing third-generation biofuel next to eukaryotic microalgae is prokaryotic blue-green algae, also known as cyanobacteria [1]. The compositions of their fatty acids (FA), which are converted to methyl or ethyl esters for use in the production of biodiesel fuel, are highly variable, like the algae themselves. The most important physicochemical properties of the fuel such as the cetane and iodine numbers and stability to oxidation and freezing are directly related to the fatty-acid composition of the starting biomass. The contents of several FA in biodiesel fuel are regulated by international standards such as EN 14214 [2]. Therefore, the search for cyanobacteria strains with FA compositions that satisfy existing standards is the most important research stage, to which much attention is paid [1]. Herein, we communicate information on the FA compositions of three cyanobacteria strains, the biomass of which is considered feedstock for producing biodiesel fuel. Blue-green alga Anabaena sp. BD47 was isolated from a natural aquifer near Daejeon (South Korea) and was identified taxonomically as strain BD47 based on morphological and physiological and biochemical characteristics. Cyanobacteria Anabaena sp. AG10059 and Synechococcus elongatus KMMCC-1063 were obtained from the Korean Collection for Type Cultures (KCTC) and the Korea Marine Microalgae Culture Center (KMMCC), respectively. All three cyanobacteria strains were cultivated in 18-cm cylindrical glass vessels containing modified BG-11 medium (8 L) for 7 d at 27–28°C with illumination by luminescent daylight lamps of intensity 140 E m–2 s–1 and continuous air bubbling. Microalgae cells were precipitated by centrifugation, rinsed with H2O, lyophilized, and extracted with CHCl3– MeOH (2:1 v/v). FA contents were analyzed by the published method [3]. Pigments were isolated and carotinoids were analyzed as before [4]. The total FA contents in Anabaena sp. BD47, Anabaena sp. AG10059, and S. elongatus KMMCC-1063 were 9.8, 11.7, and 14.6%, respectively, calculated for lyophilized biomass (LBM) of these microalgae. We found earlier that strain Anabaena sp. BD47 also produced poly(3-hydroxybutyrate) (PHB) (2.3%) [5]. We experimented simultaneously with the other two aforementioned strains but were unable to isolate PHB from them. This may be one reason for the lower FA content in Anabaena sp. BD47 because FA biosynthesis and PHB formation are competing metabolic pathways. However, PHB production is viewed as an undesirable process when selecting microalgae strains for producing biodiesel fuel from them [6]. Table 1 shows that the studied strains contained C12-C20 acids, the carbon chains of which satisfied the requirements for biodiesel fuel. The principal FA were 16:0 and 16:1(n-7), the main biodiesel components [2]. The qualitative characteristics of biodiesel fuel depend primarily on the ratio of saturated (SFA) to unsaturated (USFA) FA. The comparatively high SFA content observed in S. elongatus KMMCC-1063 had a positive effect on the cetane number and increased the stability of the fuel to oxidation but had a negative effect on its freezing stability [2]. Conversely, the relatively high USFA content observed in the Anabaena strains improved the last property but degraded the biodiesel fuel stability. The linolenic acid (18:3, n-3) contents in the three studied strains were within the upper limit of 12% imposed by European standard EN 14214 [2]. However, Anabaena sp. AG10059 and S. elongatus KMMCC-1063 were significantly more preferred according to this parameter than Anabaena sp. BD47. It is noteworthy that S. elongatus KMMCC-1063 lacked polyunsaturated FA that had a negative effect on the iodine number and had a high myristic acid (14:0) content that was rare for cyanobacteria and, together with lauric acid (12:0), was considered in some studies to be an ideal FA for producing biodiesel fuel [7].

Biotransformation of Ginsenoside Rd into 20(S)-Rg3 by Bacterium Flavobacterium sp. BGS36

Triterpenoid glycosides, i.e., ginsenosides, are the main active principles of Panax ginseng C. A. Meyer and are responsible to the various pharmacological properties of this unique plant [1]. One of the most active minor constituents of P. ginseng is ginsenoside 20(S)-Rg3, which possesses a broad spectrum of biological activity, in particular, growth inhibition of malignant A549 lung cancer cells, U937 lymphoma, LNCaP prostate carcinoma, and SK-HEP-1 hepatoma. It is viewed as a potential anticancer drug among P. ginseng saponins [2]. 20(S)-Rg3 differs from the principal ginseng glycosides Rb1, Rb2, Rc, and Rd, which have protopanaxadiol as the aglycon, only by the lack of a carbohydrate group on C-20 [1]. This enables it to be prepared by selective incomplete deglycosylation of these saponins, including the use of various microorganisms [3]. We communicated earlier the isolation from soil samples taken from a ginseng field of several bacteria with -glucosidase activity [4] and the use of several of them to convert ginsenosides Rb1 into Rd [5] and Rd into the minor glycoside F-2 and compound K [6]. In continuation of these studies, we present data on the biotransformation of Rd (1), which has -sophorose and -D-glucose residues in the C-3 and C-20 positions, respectively, of 20(S)-protopanaxadiol, into ginsenoside 20(S)-Rg3 (2) using the bacterium Flavobacterium sp. BGS36.

Biotransformation of Ginsenosides Re and Rg1 by the Bacterium Microbacterium sp. GT35

The bacterium Microbacterium sp. GT35 was found to be capable of transforming Re and Rg1, principal ginsenosides of the 20(S)-protopanaxatriol series, into minor glycosides 20(S)-Rg2 and 20(S)-Rh1, respectively. The specificity of Microbacterium sp. GT35 differed from that of several other microorganisms by cleaving only the β-D-glucose on C-20 and not affecting the C-6 carbohydrates of 20(S)-protopanaxatriol.

Composition of Carotenoids from Cyanobacterium Anabaena sp. BD47 Biomass, Feedstock for Biodiesel Production

Cyanobacteria (blue-green algae) in addition to eukaryotic microalgae are the most promising feedstocks for producing third-generation biofuel [1]. Therefore, biotechnological processes have been conceptualized mainly with advanced biomass processing in order to utilize as much as possible all its constituents [2]. We studied the blue-green alga Anabaena sp. BD47, the biomass of which is used as feedstock for producing biodiesel fuel and is simultaneously being studied as a source of other required substances such as poly(3-hydroxybutyrate) (PHB) that was isolated by us [3]. Herein we communicate data on the carotenoid content in Anabaena sp. BD47 biomass remaining after extraction of neutral lipids designated for processing into biodiesel fuel. Cyanobacterium Anabaena sp. BD47 was isolated from a natural aquifer in the vicinity of Daejeon (South Korea). Strain BD47 was identified taxonomically based on morphological, physiological, and biochemical characteristics. Standard carotenoids were acquired from DHI LAB (Denmark). NMR spectra were recorded on a Varian Unity Inova 6000 spectrometer; UV and Vis absorption spectra, on a Cary 300 spectrophotometer (Varian); IR spectra, on an FT-IR spectrometer (JASCO FTIR 4100), MALDI-TOF mass spectra, on an Autoflex III spectrometer (Bruker Daltonics). Cyanobacterium Anabaena sp. BD47 was cultivated in modified BG-11 medium for two weeks at 26–28°C with illumination by luminescent daylight lamps (150 E m–2 s–1) and constant air bubbling. Microalga cells were precipitated by centrifugation, lyophilized, and extracted with hexane to isolate neutral lipids that were used to produce biodiesel fuel. Remaining biomass was used for sequential isolation of carotenoids and PHB biopolymer using MeOH, in which the PHB characterized by us earlier [3] is insoluble, to extract pigments. Then, the MeOH extract was fractionated over a column of silica gel (Silica gel 60, Merck, Germany) as before [4]. Fractions containing pigments were analyzed for individual carotenoid contents by HPLC as described by us earlier [5]. The carotenoid content in Anabaena sp. BD47 II, which was obtained directly as a result of the cultivation, was also determined for comparison by using CH2Cl2–MeOH for total extraction of the pigments [5]. Table 1 presents the results. Table 1 shows that total carotenoids extracted from the processed biomass were ~60% of their total content in the starting biomass. As expected, the least polar echinenone and -carotene were most poorly extracted. Polar carotenoids remained practically completely in the processed biomass and could be extracted by MeOH. The most interesting of them was the most polar carotenoid, which had a retention time corresponding to specific carotenoid myxoxanthophyll glycosides, which are produced exclusively by cyanobacteria [4, 6]. This carotenoid was isolated from processed Anabaena sp. BD47 biomass by semi-preparative HPLC over a column of reversed-phase Supelco Discovery C18 (250 10 mm, 5 m) and identified by spectral methods. A comparison of its PMR and 13C NMR (Table 2), UV, IR, and mass spectra with those in the literature [4, 6, 7] indicated that the isolated carotenoid glycoside 1 was (3R,2 S)-myxol-2 -L-fucoside. The ability to form carotenoid glycosides such as myxol-2 -fucoside, ketomyxol-2 -fucoside, and oscillaxanthin is a distinguishing feature characteristic only of blue-green algae [4, 6]. The carotenoid glycoside composition of Anabaena sp. BD47 was similar to those of Anabaena variabilis IAM M-3 and several strains of the genus Nostoc [4], which form glycoside 1, but different from that of Anabaena variabilis ATCC 29413, which produces free myxol and 4-hydroxymyxol [8].

Production of Poly(3-Hydroxybutyrate) by Cyanobacterium Anabaena sp. BD47

Blue-green algae (cyanobacteria) are prokaryotes that together with eukaryotic microalgae are the most promising renewable raw material for manufacturing third-generation biofuels [1]. Recently developed biotechnological flow sheets are based on the concept of advanced processing of biomass that combines the processes and equipment for manufacturing biofuel, energy, and high value-added science-based products [2, 3]. The last includes high-quality edible proteins and polysaccharides, carotinoids, polyunsaturated fatty acids, phycobiliproteins, vitamins, and polyhydroxyalkanoates (biopolymers) [3]. We reported earlier on carotinoids as potential additional products from green microalga biomass used as starting material for biofuel [4]. Herein we communicate the isolation of poly(3-hydroxybutyrate) from biomass of the blue-green alga Anabaena sp. BD47 that is discarded after extracting neutral lipids designated for manufacturing of biodiesel fuel. Cyanobacterium Anabaena sp. BD47 was isolated from a natural aquifer near Daejeon (South Korea). Strain BD47 was identified taxonomically based on morphological, physiological, and biochemical characteristics. Standard poly(3-hydroxybutyrate) (PHB) and methyl-3-hydroxybutyrate were purchased from Sigma-Aldrich. PMR and 13C NMR spectra were recorded on a Bruker AM 500 spectrometer at operating frequencies 500 and 125 MHz, respectively. Gas chromatography–mass spectrometry (GC/MS) was performed on an HP-5890 GC connected to an HP 5972 mass-selective detector and equipped with a data monitoring and processing module (Hewlett-Packard, USA). We used a Supelco MDN-5 capillary column (30 m 0.25 mm 0.25 m). IR spectra were recorded on an FT-IR spectrometer (JASCO FT-IR 4100) using attenuated total internal reflection (ATR). The biopolymer molecular weight was determined by gel-permeation chromatography using a Styragel HT 5 column (7.8 300 mm) (Waters Corp., USA) and a set of polystyrene standards (Sigma). Cyanobacterium Anabaena sp. BD47 was cultivated in modified BG-11 medium for two weeks at 26–28°C with illumination by luminescent daylight lamps of intensity 150 E m–2 s–1 and continuous air bubbling. Microalga cells were precipitated by centrifugation, lyophilized, irradiated by ultrasound, and extracted with hexane to isolate neutral lipids and then MeOH to remove pigments. The remaining biomass was used for biopolymer isolation. For this, dry biomass (3 g) was extracted with refluxing CHCl3 (300 mL) for 6 h. The suspension was cooled and filtered through membrane filters. The filtrate was evaporated to 70 mL, treated with ten times the volume of hexane, left overnight at room temperature, and centrifuged. The supernatant was discarded. The precipitate was resuspended in hexane and again centrifuged. The resulting precipitate was dissolved in CHCl3 (70 mL), treated with ten times the volume of MeOH, and left overnight. The resulting precipitate was separated by centrifugation and dissolved in CHCl3. The solvent was distilled off to afford compound 1 as a translucent film with a gray tint (2.3% yield based on the weight of lyophilized cells). The structure of 1 was elucidated by spectral methods. PMR spectra showed characteristic resonances at 1.26, 2.49–2.62, and 5.25 ppm that corresponded to methyl, methylene, and methine protons of PHB [5]. The 13C NMR spectrum of 1 contained resonances for C atoms of the same groups at 19.7, 40.7, and 67.6 ppm, respectively, in addition to a resonance at 169.1 ppm that corresponded to carbonyl C atoms [5]. The IR spectrum of 1 exhibited absorption bands at 2976–2935, 1725, 1382, 1228, and 1050 cm–1 that corresponded to CH3, CH2, C=O, C–H, and C–O vibrations. This agreed with the literature data for PHB isolated from other blue-green algae [6] and with the IR spectrum of standard PHB [6]. The weightaverage molecular weight of 1 was 140 kDa. This was within the limits found for this biopolymer isolated from various cyanobacteria strains [7].

Transformation of Ginsenoside Rc into (20S)-Rg3 by the Bacterium Leuconostoc sp. BG78

Ginsenoside (20S)-Rg3 is one of the most active minor dammarane glycosides of Panax ginseng C. A. Meyer and exhibits a broad spectrum of biological activity including hepatoprotective, immunostimulating, neuroprotective, and antitumor. It inhibits cell growth of malignant A549 lung carcinoma, U937 lymphoma, LNCaP prostate carcinoma, and SK-HEP-1 hepatoma and is of interest in medicine as a potential antitumor agent [1]. One factor limiting the application of ginsenoside (20S)-Rg3 is its low content in the total saponins of Panax ginseng. (20S)-Rg3 differs from the principal dammarane glycosides Rb1, Rb2, Rc (1), and Rd (2) that have the aglycon protopanaxadiol only by the lack of a carbohydrate on C-20 [2]. This enables it to be produced by selective incomplete deglycosylation of these saponins, including by using various microorganisms [3, 4].

Biotransformation of Ginsenoside Rc into C-Mc1 by the Bacterium Sphingopyxis sp. BG97

The principal biologically active compounds of Panax ginseng C. A. Meyer are triterpenoid glycosides, ginsenosides [1], which exhibit immunostimulating, anti-inflammatory, anticarcinogenic, antitumor, antidiabetic, hepatoprotective, neuromodulating, and other properties [2]. Greater than 50 different ginsenosides have been isolated from P. ginseng. Six of these (Rb1, Rb2, Rc, Rd, Re, and Rg1) make up >80% of total saponins and have protopanaxadiol or protopanaxatriol as the aglycon [1]. Furthermore, it is well known that minor glycosides of P. ginseng in several instances have broader spectra of biological activity or are more active than them [2]. This stimulates great interest in producing the minor ginsenosides, including biotransformation of the principal ginsenosides using various microorganisms [3, 4]. We reported earlier the transformation of ginsenoside Rb1 into the minor glycoside F-2 and compound K [5] and glycoside Rd into minor ginsenoside (20S)-Rg3 [6] using bacteria isolated from soil samples taken from a ginseng field. Among these, strains belonging to the genus Sphingopyxis were identified [7]. Herein we communicate data on the transformation of principal ginsenoside Rc (1) into minor compound C-Mc1 (2) by the bacterium Sphingopyxis sp. BG97.

Biotransformation of the Principal Ginsenosides of Panax ginseng Into Minor Glycosides Through the Action of Bacterium Paenibacillus sp. BG134

The bacterium Paenibacillus sp. BG134 was capable of biotransforming the principal 20(S)-protopanaxadiol ginsenosides Rc, Rb2, Rd, and Rb1 into the corresponding minor glycosides C-Mc1, C-O, and F-2. The specificity of Paenibacillus sp. BG134 differed from that of several other microorganisms by cleaving only the terminal C-3 and C-20 β -D-glucose from their carbohydrate components.