Recombinant Protein Production and Purification Using Eukaryotic Cell Factories.

Cloning proteins enables their production and characterization for further studies. This requires inserting the gene of the studied protein to be inserted in a vector, which then will be transformed to the host cell used as "factory." Consequently, the "biomass" of host cells will be produced using bioreactors. Here we describe the production of Rhizomucor miehei lipase (RML) by cloning the corresponding genes in the yeast Pichia pastoris. This enzyme is used as a biocatalyst for biofuel production. The successfully produced recombinant proteins are then purified using ion exchange chromatography.

Comparative Analysis of Different Isolated Oleaginous Mucoromycota Fungi for Their γ-Linolenic Acid and Carotenoid Production.

γ-Linolenic acid (GLA) and carotenoids have attracted much interest due to their nutraceutical and pharmaceutical importance. Mucoromycota, typical oleaginous filamentous fungi, are known for their production of valuable essential fatty acids and carotenoids. In the present study, 81 fungal strains were isolated from different Egyptian localities, out of which 11 Mucoromycota were selected for further GLA and carotenoid investigation. Comparative analysis of total lipids by GC of selected isolates showed that GLA content was the highest in Rhizomucor pusillus AUMC 11616.A, Mucor circinelloides AUMC 6696.A, and M. hiemalis AUMC 6031 that represented 0.213, 0.211, and 0.20% of CDW, respectively. Carotenoid analysis of selected isolates by spectrophotometer demonstrated that the highest yield of total carotenoids (640 µg/g) was exhibited by M. hiemalis AUMC 6031 and M. hiemalis AUMC 6695, and these isolates were found to have a similar carotenoid profile with, ß-carotene (65%), zeaxanthin (34%), astaxanthin, and canthaxanthin (5%) of total carotenoids. The total fatty acids of all tested isolates showed moderate antimicrobial activity against Staphylococcus aureus and Salmonella Typhi, and Penicillium chrysogenum. To the best of our knowledge, this is the first report on the highest yield of total lipid accumulation (51.74% CDW) by a new oleaginous fungal isolate R. pusillus AUMC 11616.A. A new scope for a further study on this strain will be established to optimize and improve its total lipids with high GLA production. So, R. pusillus AUMC 11616.A might be a potential candidate for industrial application.

Kinetics and Optimization of Lipophilic Kojic Acid Derivative Synthesis in Polar Aprotic Solvent Using Lipozyme RMIM and Its Rheological Study.

The synthesis of kojic acid derivative (KAD) from kojic and palmitic acid (C16:0) in the presence of immobilized lipase from Rhizomucor miehei (commercially known as Lipozyme RMIM), was studied using a shake flask system. Kojic acid is a polyfunctional heterocycles that acts as a source of nucleophile in this reaction allowing the formation of a lipophilic KAD. In this study, the source of biocatalyst, Lipozyme RMIM, was derived from the lipase of Rhizomucor miehei immobilized on weak anion exchange macro-porous Duolite ES 562 by the adsorption technique. The effects of solvents, enzyme loading, reaction temperature, and substrate molar ratio on the reaction rate were investigated. In one-factor-at-a-time (OFAT) experiments, a high reaction rate (30.6 × 10-3 M·min-1) of KAD synthesis was recorded using acetone, enzyme loading of 1.25% (w/v), reaction time of 12 h, temperature of 50 °C and substrate molar ratio of 5:1. Thereafter, a yield of KAD synthesis was optimized via the response surface methodology (RSM) whereby the optimized molar ratio (fatty acid: kojic acid), enzyme loading, reaction temperature and reaction time were 6.74, 1.97% (w/v), 45.9 °C, and 20 h respectively, giving a high yield of KAD (64.47%). This condition was reevaluated in a 0.5 L stirred tank reactor (STR) where the agitation effects of two impellers; Rushton turbine (RT) and pitch-blade turbine (PBT), were investigated. In the STR, a very high yield of KAD synthesis (84.12%) was achieved using RT at 250 rpm, which was higher than the shake flask, thus indicating better mixing quality in STR. In a rheological study, a pseudoplastic behavior of KAD mixture was proposed for potential application in lotion formulation.

A novel aspartic protease from Rhizomucor miehei expressed in Pichia pastoris and its application on meat tenderization and preparation of turtle peptides.

A novel aspartic protease gene (RmproA) was cloned from the thermophilic fungus Rhizomucor miehei CAU432 and expressed in Pichia pastoris. The RmproA was successfully expressed in P. pastoris as an active extracellular protease. High protease activity of 3480.4 U/mL was obtained by high cell-density fermentation. The protease was purified by the two step protocols to homogeneity. The molecular mass of the RmproA was estimated to be 52.4 kDa by SDS-PAGE and 50.6 kDa by gel filtration. The purified enzyme was optimally active at pH 5.5 and 55 °C, respectively. The enzyme exhibited a broad range of substrate specificity. RmproA-treated pork muscle showed lower shear force than papain-treated sample at a relative low concentration, suggesting its effectiveness on meat tenderization. Moreover, turtle hydrolysis by RmproA resulted in a large amount of small peptides, which exhibited high ACE-inhibitory activity. Thus, RmproA may be a potential candidate for several industrial applications.

Novel FRET-substrates of Rhizomucor pusillus rennin: Activity and mechanistic studies.

The development of sensitive, easy and reliable methods for the determination of Rhizomucor pusillus rennin (MPR) activity, in free and in immobilized form, along with the elucidation of the mechanism of action, represent challenges for the widespread use of the enzyme in industrial cheese production. These could be accomplished by using highly specific and sensitive substrates, as well as direct assay methods. We designed and synthesized novel substrates based on Fluorescence Resonance Energy Transfer (FRET) for the MPR by employing computational simulation techniques and peptide synthesis in liquid phase. Three FRET-substrates (Abz-GFY-pNA, Abz-SFY-pNA and Abz-GFI-pNA) were found active, while the Abz-GFY-pNA showed the highest reliability, sensitivity and specificity among them. Subsequently, a novel mechanism of MPR action was elucidated, with the development of novel methods for assaying activity in free and immobilized form, which both may contribute in the wider use of rennin in cheese production and other biotechnological applications.

Rhizomucor miehei lipase immobilized on reinforced chitosan-chitin nanowhiskers support for synthesis of eugenyl benzoate.

An alternative environmentally benign support was prepared from chitosan-chitin nanowhiskers (CS/CNWs) for covalent immobilization of Rhizomucor miehei lipase (RML) to increase the operational stability and recyclability of RML in synthesizing eugenyl benzoate. The CS/CNWs support and RML-CS/CNWs were characterized using X-ray diffraction, fluorescent microscopy, and Fourier transform infrared spectroscopy. Efficiency of the RML-CS/CNWs was compared to the free RML to synthesize eugenyl benzoate for parameters: reaction temperature, stirring rate, reusability, and thermal stability. Under optimal experimental conditions (50°C, 250 rpm, catalyst loading 3 mg/mL), a twofold increase in yield of eugenyl benzoate was observed for RML-CS/CNWs as compared to free RML, with the former achieving maximum yield of the ester at 62.1% after 5 hr. Results demonstrated that the strategy adopted to prepare RML-CS/CNWs was useful, producing an improved and prospectively greener biocatalyst that supported a sustainable process to prepare eugenyl benzoate. Moreover, RML-CS/CNWs are biodegradable and perform esterification reactions under ambient conditions as compared to the less eco-friendly conventional acid catalyst. This research provides a facile and promising approach for improving activity of RML in which the resultant RML-CS/CNWs demonstrated good operational stability for up to eight successive esterification cycles to synthesize eugenyl benzoate.

Improved Performance of Magnetic Cross-Linked Lipase Aggregates by Interfacial Activation: A Robust and Magnetically Recyclable Biocatalyst for Transesterification of Jatropha Oil.

Lipases are the most widely employed enzymes in commercial industries. The catalytic mechanism of most lipases involves a step called "interfacial activation". As interfacial activation can lead to a significant increase in catalytic activity, it is of profound importance in developing lipase immobilization methods. To obtain a potential biocatalyst for industrial biodiesel production, an effective strategy for enhancement of catalytic activity and stability of immobilized lipase was developed. This was performed through the combination of interfacial activation with hybrid magnetic cross-linked lipase aggregates. This biocatalyst was investigated for the immobilization of lipase from Rhizomucor miehei (RML). Under the optimal conditions, the activity recovery of the surfactant-activated magnetic RML cross-linked enzyme aggregates (CLEAs) was as high as 2058%, with a 20-fold improvement over the free RML. Moreover, the immobilized RML showed excellent catalytic performance for the biodiesel reaction at a yield of 93%, and more importantly, could be easily separated from the reaction mixture by simple magnetic decantation, and retained more than 84% of its initial activities after five instances of reuse. This study provides a new and versatile approach for designing and fabricating immobilized lipase with high activation and stability.

Characterization and gene cloning of l-xylulose reductase involved in l-arabinose catabolism from the pentose-fermenting fungus Rhizomucor pusillus.

l-Xylulose reductase (LXR) catalyzes the reduction of l-xylulose to xylitol in the fungal l-arabinose catabolic pathway. LXR (RpLXR) was purified from the pentose-fermenting zygomycetous fungus Rhizomucor pusillus NBRC 4578. The native RpLXR is a homotetramer composed of 29 kDa subunits and preferred NADPH as a coenzyme. The Km values were 8.71 mM for l-xylulose and 3.89 mM for dihydroxyacetone. The lxr3 (Rplxr3) gene encoding RpLXR consists of 792 bp and encodes a putative 263 amino acid protein (Mr = 28,341). The amino acid sequence of RpLXR showed high similarity to 3-oxoacyl-(acyl-carrier-protein) reductase. The Rplxr3 gene was expressed in Escherichia coli and the recombinant RpLXR exhibited properties similar to those of native RpLXR. Transcription of the Rplxr3 gene in R. pusillus NBRC 4578 was induced in the presence of l-arabinose and inhibited in the presence of d-glucose, d-xylose, and d-mannitol, indicating that RpLXR is involved in the l-arabinose catabolic pathway.

Naringin lauroyl ester inhibits lipopolysaccharide-induced activation of nuclear factor κB signaling in macrophages.

Naringin (Nar) has antioxidant and anti-inflammatory properties. It was recently reported that enzymatic modification of Nar enhanced its functions. Here, we acylated Nar with fatty acids of different sizes (C2-C18) using immobilized lipase from Rhizomucor miehei and investigated the anti-inflammatory effects of these molecules. Treatment of murine macrophage RAW264.7 cells with Nar alkyl esters inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) production, with Nar lauroyl ester (Nar-C12) showing the strongest effect. Furthermore, Nar-C12 suppressed the LPS-induced expression of inducible NO synthase by blocking the phosphorylation of inhibitor of nuclear factor (NF)-κB-α as well as the nuclear translocation of NF-κB subunit p65 in macrophage cells. Analysis of Nar-C12 uptake in macrophage cells revealed that Nar-C12 ester bond was partially degraded in the cell membrane and free Nar was translocated to the cytosol. These results indicate that Nar released from Nar-C12 exerts anti-inflammatory effects by suppressing NF-κB signaling pathway.

New alkaline lipase from Rhizomucor variabilis: Biochemical properties and stability in the presence of microbial EPS.

A new strain of Rhizomucor variabilis producing an active extracellular lipase was identified and characterized in the present studies. The culture conditions were optimized and the highest lipase production amounting to 136 U/mL was achieved after 4 days of cultivation. The optimum pH (5.5) and temperature (28 °C) were determined as the best conditions for R. variabilis lipase production. The isolated enzyme preparation exhibited maximum activity at 40 °C and pH 8.0. Lipase from R. variabilis was stable up to 50 °C during 2 H retaining 80% of its initial activity. The enzyme was highly stable in the pH range of 7.0-9.0. Moreover, the addition of naturally obtained exopolysaccharides (EPS) significantly enhanced lipase activity. The presence of EPS derived from Ganoderma applanatum and Rhizobium leguminosarum enhanced the lipase activity, which was 22% and 31%, respectively, higher than that in the control experiments. Simultaneously, the pH activity profiles remained unchanged. The Michaelis-Menten constant and the turnover number of the enzyme for p-nitrophenyl palmitate in the standard assay conditions were estimated at a level of 0.631 mM and 0.674 Sec(-1) . In conclusion, the results obtained in this work present a newly isolated lipase preparation stabilized with EPS or without modification as a very effective tool for industrial application.

Biochemical Characterization of a Novel Acidic Exochitinase from Rhizomucor miehei with Antifungal Activity.

A novel chitinase gene (RmChi44) from Rhizomucor miehei was cloned and expressed in Escherichia coli as an intracellular soluble and active protein. The recombinant chitinase (RmChi44) was purified to homogeneity and biochemically characterized. The molecular mass of RmChi44 was estimated to be 44.6 kDa on SDS-PAGE. RmChi44 displayed an acidic pH optimum of 4.5 and was stable within pH 4.5-9.0. The optimal temperature of RmChi44 was found to be 50 °C. The Km values of RmChi44 for colloidal chitin and glycol chitin were 4.02 and 1.55 mg/mL, respectively. RmChi44 hydrolyzed colloidal chitin to yield mainly N-acetyl chitobiose, exhibiting an exotype cleavage pattern. Moreover, the enzyme displayed ß-N-acetylglucosaminidase activity, splitting N-acetyl COSs with degree of polymerization (DP) 2-5 into their monomer. In addition, RmChi44 showed antifungal activity against some phytopathogenic fungi. This is the first report on an exochitinase showing ß-N-acetylglucosaminidase activity and antifungal activity from Rhizomucor species.

The first crystal structure of a glycoside hydrolase family 17 ß-1,3-glucanosyltransferase displays a unique catalytic cleft.

ß-1,3-Glucanosyltransferase (EC 2.4.1.-) plays an important role in the formation of branched glucans, as well as in cell-wall assembly and rearrangement in fungi and yeasts. The crystal structures of a novel glycoside hydrolase (GH) family 17 ß-1,3-glucanosyltransferase from Rhizomucor miehei (RmBgt17A) and the complexes of its active-site mutant (E189A) with two substrates were solved at resolutions of 1.30, 2.30 and 2.27 Å, respectively. The overall structure of RmBgt17A had the characteristic (ß/α)8 TIM-barrel fold. The structures of RmBgt17A and other GH family 17 members were compared: it was found that a conserved subdomain located in the region near helix α6 and part of the catalytic cleft in other GH family 17 members was absent in RmBgt17A. Instead, four amino-acid residues exposed to the surface of the enzyme (Tyr135, Tyr136, Glu158 and His172) were found in the reducing terminus of subsite +2 of RmBgt17A, hindering access to the catalytic cleft. This distinct region of RmBgt17A makes its catalytic cleft shorter than those of other reported GH family 17 enzymes. The complex structures also illustrated that RmBgt17A can only provide subsites -3 to +2. This structural evidence provides a clear explanation of the catalytic mode of RmBgt17A, in which laminaribiose is released from the reducing end of linear ß-1,3-glucan and the remaining glucan is transferred to the end of another ß-1,3-glucan acceptor. The first crystal structure of a GH family 17 ß-1,3-glucanosyltransferase may be useful in studies of the catalytic mechanism of GH family 17 proteins, and provides a basis for further enzymatic engineering or antifungal drug screening.

High-level expression of a novel α-galactosidase gene from Rhizomucor miehei in Pichia pastoris and characterization of the recombinant enyzme.

The second α-galactosidase gene (designated as RmgalB) was cloned from the thermophilic fungus Rhizomucor miehei and expressed in Pichia pastoris. The gene belonging to glycoside hydrolase (GH) family 36 has an open reading frame (ORF) of 2241bp encoding 746 amino acids with two introns. The recombinant α-galactosidase (RmgalB) was secreted at high levels of 1953.9Uml(-1) in high cell density fermentor, which is the highest yield obtained for a α-galactosidase. The purified enzyme as a tetramer gave a single band corresponding to a molecular mass of 83.1kDa in SDS-PAGE. The enzyme exhibited a very high specific activity of 505.5Umg(-1). The optimum temperature and pH of RmgalB were determined to be 55°C and pH 5.5, respectively. It was stable within pH 5.5-9.5 and up to 55°C. RmgalB displayed specificity toward raffinose and stachyose, and completely hydrolyzed the anti-nutritive raffinose family oligosaccharides (RFOs). These properties make RmgalB useful in the food and feed industries.

Structural insights into the substrate specificity and transglycosylation activity of a fungal glycoside hydrolase family 5 ß-mannosidase.

ß-Mannosidases are exo-acting glycoside hydrolases (GHs) that catalyse the removal of the nonreducing end ß-D-mannose from manno-oligosaccharides or mannoside-substituted molecules. They play important roles in fundamental biological processes and also have potential applications in various industries. In this study, the first fungal GH family 5 ß-mannosidase (RmMan5B) from Rhizomucor miehei was functionally and structurally characterized. RmMan5B exhibited a much higher activity against manno-oligosaccharides than against p-nitrophenyl ß-D-mannopyranoside (pNPM) and had a transglycosylation activity which transferred mannosyl residues to sugars such as fructose. To investigate its substrate specificity and transglycosylation activity, crystal structures of RmMan5B and of its inactive E202A mutant in complex with mannobiose, mannotriose and mannosyl-fructose were determined at resolutions of 1.3, 2.6, 2.0 and 2.4 Å, respectively. In addition, the crystal structure of R. miehei ß-mannanase (RmMan5A) was determined at a resolution of 2.3 Å. Both RmMan5A and RmMan5B adopt the (ß/α)8-barrel architecture, which is globally similar to the other members of GH family 5. However, RmMan5B shows several differences in the loop around the active site. The extended loop between strand ß8 and helix α8 (residues 354-392) forms a `double' steric barrier to `block' the substrate-binding cleft at the end of the -1 subsite. Trp119, Asn260 and Glu380 in the ß-mannosidase, which are involved in hydrogen-bond contacts with the -1 mannose, might be essential for exo catalytic activity. Moreover, the structure of RmMan5B in complex with mannosyl-fructose has provided evidence for the interactions between the ß-mannosidase and D-fructofuranose. Overall, the present study not only helps in understanding the catalytic mechanism of GH family 5 ß-mannosidases, but also provides a basis for further enzymatic engineering of ß-mannosidases and ß-mannanases.

Molecular analysis of NAD⁺-dependent xylitol dehydrogenase from the zygomycetous fungus Rhizomucor pusillus and reversal of the coenzyme preference.

The zygomycetous fungus Rhizomucor pusillus NBRC 4578 is able to ferment not only d-glucose but also d-xylose into ethanol. Xylitol dehydrogenase from R. pusillus NBRC 4578 (RpXDH), which catalyzes the second step of d-xylose metabolism, was purified, and its enzymatic properties were characterized. The purified RpXDH preferred NAD(+) as its coenzyme and showed substrate specificity for xylitol, d-sorbitol, and ribitol. cDNA cloning of xyl2 gene encoding RpXDH revealed that the gene included a coding sequence of 1,092 bp with a molecular mass of 39,185 kDa. Expression of the xyl2 in R. pusillus NBRC 4578 was induced by d-xylose, and the expression levels were increased with accumulation of xylitol. The xyl2 gene was expressed in Escherichia coli, and coenzyme preference of the recombinant RpXDH was reversed from NAD(+) to NADP(+) in the double mutant D205A/I206R by site-directed mutagenesis.

Biochemical characterization of the first fungal glycoside hydrolyase family 3 ß-N-acetylglucosaminidase from Rhizomucor miehei.

A novel ß-N-acetylglucosaminidase gene (RmNag) from Rhizomucor miehei was cloned and expressed in Escherichia coli. RmNag shares the highest identity of 37% with a putative ß-N-acetylglucosaminidase from Aspergillus clavatus. The recombinant enzyme was purified to homogeneity. The optimal pH and temperature of RmNag were pH 6.5 and 50 °C, respectively. It was stable in the pH range 6.0-8.0 and at temperatures below 45 °C. RmNag exhibited strict substrate specificity for p-nitrophenyl ß-N-acetylglucosaminide (pNP-GlcNAc) and N-acetyl chitooligosaccharides. The apparent Km of RmNag toward pNP-GlcNAc was 0.13 mM. The purified enzyme displayed an exo-type manner as it released the only end product of GlcNAc from all the tested N-acetyl chitooligosaccharides. Besides, RmNag exhibited relatively high N-acetyl-ß-D-glucosaminide tolerance with an inhibition constant Ki value of 9.68 mM. The excellent properties may give the enzyme great potential in industries. This is the first report on a glycoside hydrolyase family 3 ß-N-acetylglucosaminidase from a fungus.

Foot and Mouth Disease virus-loaded fungal chitosan nanoparticles for intranasal administration: impact of formulation on physicochemical and immunological characteristics.

Nasal vaccination is a promising, needle-free alternative route for parenteral vaccination. This study introduces a simple, scalable nasal vaccine delivery formulation for Foot and Mouth Disease virus (FMDv) using chitosan (CS) nanoparticles and assesses the potential of fungal CS for use as nanocarriers for mucosal vaccines. Fungal CS was extracted from fungal biomass and physiochemically characterized. FMDv-loaded CS nanoparticles, prepared using an ionic gelation technique, were characterized for particle size, zeta potential, morphology, loading efficiency and virus particle release. The immunogenicities of nasally applied FMDv-loaded fungal or commercial shrimp CS were compared with intraperitoneally administered fluid vaccine in guinea pigs. The nanoparticles had varied sizes (221.9-281.2 nm), positive electrical charge (+7 to +13 mV) and excellent antigen-loading capacity (93-97%). In vitro release studies revealed a biphasic virus particle release for all CS nanoparticles. Higher serum titers were developed with CS formulations than with free virus and were comparable with the titers for intraperitoneally administered fluid vaccine. Significantly higher IgA levels were found after the administration of nasal vaccine than after fluid vaccine or free virus. Overall, CS-FMDv nanoparticles stimulated humoral and mucosal immunity following intranasal administration. Fungal CS polymers were potent mucosal immunoadjuvants and showed promise as alternative sources of CS for mucosal vaccine formulations.

Biotransformation of oleanolic and maslinic acids by Rhizomucor miehei.

Microbial transformation of oleanolic acid by Rhizomucor miehei produced three metabolites. A known compound, a 30-hydroxyl derivative (queretaroic acid), and two 7ß,30- and 1ß,30-dihydroxylated metabolites, respectively. The action of the same fungus (R. miehei) on maslinic acid produced an olean-11-en-28,13ß-olide derivative, a metabolite hydroxylated at C-30, an 11-oxo derivative, and two metabolites with an 11α,12α-epoxy group, hydroxylated or not at C-30. Their structures were elucidated by extensive analyses of their spectroscopic data, and also by chemical correlations.

Pellet formation of zygomycetes and immobilization of yeast.

Pelleted growth provides many advantages for filamentous fungi, including decreased broth viscosity, improved aeration, stirring, and heat transfer. Thus, the factors influencing the probability of pellet formation of Rhizopus sp. in a defined medium was investigated using a multifactorial experimental design. Temperature, agitation intensity, Ca(2+)-concentration, pH, and solid cellulose particles, each had a significant effect on pelletization. Tween 80, spore concentration, and liquid volume were not found to have a significant effect. All of the effects were additive; no interactions were significant. The results were used to create a simple defined medium inducing pelletization, which was used for immobilization of a flocculating strain of Saccharomyces cerevisiae in the zygomycetes pellets. A flor-forming S. cerevisiae strain was also immobilized, while a non-flocculating strain colonized the pellets but was not immobilized. No adverse effects were detected as a result of the close proximity between the filamentous fungus and the yeast, which potentially allows for co-fermentation with S. cerevisiae immobilized in pellets of zygomycetes.

Characterization of two novel family 12 xyloglucanases from the thermophilic Rhizomucor miehei.

Two novel glycoside hydrolase (GH) family 12 xyloglucanase genes (designated RmXEG12A and RmXEG12B) were cloned from the thermophilic fungus Rhizomucor miehei. Both genes contained open reading frames of 729 bp encoding 242 amino acids. Their deduced amino acid sequences shared 68% identity with each other and less than 60% with other xyloglucanases. The two genes, without the sequences for the signal peptides, were cloned and successfully expressed in Escherichia coli as active xyloglucanases, designated RmXEG12A and RmXEG12B, with similar molecular masses--25.6 and 25.9 kDa, respectively. RmXEG12A showed optimal activity at pH 6.5 and 65 °C, RmXEG12B at pH 5.0 and 60 °C. Both recombinant xyloglucanases displayed very high specific activities, 6,681.4 and 3,092.2 U mg(-1), respectively, toward tamarind xyloglucan, but no activity toward carboxymethylcellulose, Avicel, or p-nitrophenyl derivatives. The main products of tamarind xyloglucan hydrolysis by the two xyloglucanases were XXXG, XXLG/XLXG, and XLLG (where G is an unsubstituted ß-D-Glc residue, X is a xylosylated ß-D-Glc residue, and L is a ß-D-Glc residue substituted by xylosyl-galactose).