Characteristics of the binding of a bacterial expansin (BsEXLX1) to microcrystalline cellulose

Plant expansin proteins induce plant cell wall extension and have the ability to extend and disrupt cellulose. In addition, these proteins show synergistic activity with cellulases during cellulose hydrolysis. BsEXLX1 originating from Bacillus subtilis is a structural homolog of a β‐expansin produced by Zea mays (ZmEXPB1). The Langmuir isotherm for binding of BsEXLX1 to microcrystalline cellulose (i.e., Avicel) revealed that the equilibrium binding constant of BsEXLX1 to Avicel was similar to those of other Type A surface‐binding carbohydrate‐binding modules (CBMs) to microcrystalline cellulose, and the maximum number of binding sites on Avicel for BsEXLX1 was also comparable to those on microcrystalline cellulose for other Type A CBMs. BsEXLX1 did not bind to cellooligosaccharides, which is consistent with the typical binding behavior of Type A CBMs. The preferential binding pattern of a plant expansin, ZmEXPB1, to xylan, compared to cellulose was not exhibited by BsEXLX1. In addition, the binding capacities of cellulose and xylan for BsEXLX1 were much lower than those for CtCBD3. Biotechnol. Bioeng. 2013; 110: 401–407. © 2012 Wiley Periodicals, Inc.     

Binding characteristics of a bacterial expansin (BsEXLX1) for various types of pretreated lignocellulose

BsEXLX1 from Bacillus subtilis is the first discovered bacterial expansin as a structural homolog of a plant expansin, and it exhibited synergism with cellulase on the cellulose hydrolysis in a previous study. In this study, binding characteristics of BsEXLX1 were investigated using pretreated and untreated Miscanthus x giganteus in comparison with those of CtCBD3, a cellulose-binding domain from Clostridium thermocellum. The amounts of BsEXLX1 bound to cellulose-rich substrates were significantly lower than those of CtCBD3. However, the amounts of BsEXLX1 bound to lignin-rich substrates were much higher than those of CtCBD3. A binding competition assay between BsEXLX1 and CtCBD3 revealed that binding of BsEXLX1 to alkali lignin was not affected by the presence of CtCBD3. This preferential binding of BsEXLX1 to lignin could be related to root colonization in plants by bacteria, and the bacterial expansin could be used as a lignin blocker in the enzymatic hydrolysis of lignocellulose.     

Inhibition of cellulases by phenols

Enzyme hydrolysis of pretreated cellulosic materials slows as the concentration of solid biomass material increases, even though the ratio of enzyme to cellulose is kept constant. This form of inhibition is distinct from substrate and product inhibition, and has been noted for lignocellulosic materials including wood, corn stover, switch grass, and corn wet cake at solids concentrations greater than 10 g/L. Identification of enzyme inhibitors and moderation of their effects is of considerable practical importance since favorable ethanol production economics require that at least 200 g/L of cellulosic substrates be used to enable monosaccharide concentrations of 100 g/L, which result in ethanol titers of 50 g/L. Below about 45 g/L ethanol, distillation becomes energy inefficient. This work confirms that the phenols: vanillin, syringaldehyde, trans-cinnamic acid, and hydroxybenzoic acid, inhibit cellulose hydrolysis in wet cake by endo- and exo-cellulases, and cellobiose hydrolysis by β-glucosidase. A ratio of 4 mg of vanillin to 1 mg protein (0.5 FPU) reduces the rate of cellulose hydrolysis by 50%. β-Glucosidases from Trichoderma reesei and Aspergillus niger are less susceptible to inhibition and require about 10× and 100× higher concentrations of phenols for the same levels of inhibition. Phenols introduced with pretreated cellulose must be removed to maximize enzyme activity.     

Kinetics of cellulose hydrolysis by halostable cellulase from a marine Aspergillus niger at different salinities

Kinetics of cellulose hydrolysis with halostable cellulase from a marine Aspergillus niger was analyzed at different salinities. Cellulase activity in 8% NaCl solution was 1.43 folds higher than that in NaCl free solution. Half saturation constant, K_m (15.6260 g/L) and the rate constant of deactivation, K_de (0.3369 g/L h) in 8% NaCl solution was lower than that (18.6364 g/L), 0.3754 (g/L h) in NaCl free solution. The maximum initial hydrolysis velocity, V_max (25.5295 g/L h), in 8% NaCl solution was higher than that in NaCl free solution (25.0153 g/L h). High salinity increased affinity to the cellulase to the substrate and thermostability. Halostable cellulase from a marine Aspergillus niger was valuable for cellulose hydrolysis under high salinity conditions.     

Unwrapping the hydrolytic system of the phytopathogenic fungus Phoma exigua by secretome analysis

The present work describes the secretome profiling of a phytopathogenic fungus, Phoma exigua by liquid chromatography coupled tandem mass spectrometry (LC–MS/MS) based proteomics approach to highlight the suites of enzymes responsible for biomass hydrolysis. Mass spectrometry identified 33 proteins in the Phoma secretome when grown on α-cellulose as the sole carbon source. The functional classification revealed a unique extracellular enzyme system mainly belonging to the family of glycosyl hydrolase proteins (52%). This hydrolytic system consisted of cellulases (endo-1,4-β-glucanase, cellobiohydrolase I, exoglucanase, and β-glucosidase), hemicellulases (1,4-β-xylosidase and endo-1,4-β-xylanase) and other hypothetical proteins including GH3, GH5, GH6, GH7, GH11, GH20, GH32 and GH54. The synergistic action of this enzyme cocktail was assessed by the saccharification of alkali treated wheat straw. Since the Phoma secretome has limited β-glucosidase activity, it was supplemented with commercial β-glucosidase. After supplementation, this enzyme complex resulted in high yields of glucose (177.2 ± 1.0 mg/gds), xylose (209.2 ± 1.5 mg/gds) and arabinose (25.2 ± 0.3 mg/gds). The secretome analysis and biomass hydrolysis by P. exigua revealed its unique potential as a source of hydrolytic enzymes for lignocellulosic biomass hydrolysis.     

Valorization of olive stones for xylitol and ethanol production from dilute acid pretreatment via enzymatic hydrolysis and fermentation by Pachysolen tannophilus

Olive stones are an agro-industrial by-product abundant in the Mediterranean area that is regarded as a potential lignocellulosic feedstock for sugar production. Statistical modeling of dilute-sulphuric acid hydrolysis of olive stones has been performed using a response surface methodology, with treatment temperature and process time as factors, to optimize the hydrolysis conditions aiming to attain maximum d-xylose extraction from hemicelluloses. Thus, solid yield and composition of solid and liquid phases were assessed by empirical modeling. The highest yield of d-xylose was found at a temperature of 195 °C for 5 min. Under these conditions, 89.7% of the total d-xylose was recovered from raw material. The resulting solids from optimal conditions were assayed as substrate for enzymatic hydrolysis, while fermentability of hemicellulosic hydrolysates was tested using the d-xylose-fermenting yeast Pachysolen tannophilus. Both bioprocesses were considerably influenced by enzyme loading and inoculum size. In the enzymatic hydrolysis step, about 56% of cellulose was converted into d-glucose by using an enzyme/solid ratio of 40 FPU g⁻¹, while in the fermentation carried out with a cell concentration of 2 g L⁻¹ a yield of 0.44 g xylitol/g d-xylose and a global volumetric productivity of 0.11 g L⁻¹ h⁻¹ were achieved.     

Enzymatic hydrolysis of corncob and ethanol production from cellulosic hydrolysate

 Enzymatic hydrolysis of corncob and ethanol fermentation from cellulosic hydrolysate were investigated. After corncob was pretreated by 1% H₂SO₄ at 108 °C for 3 h, the cellulosic residue was hydrolyzed by cellulase from Trichoderma reesei ZU-02 and the hydrolysis yield was 67.5%. Poor cellobiase activity in T. reesei cellulase restricted the conversion of cellobiose to glucose, and the accumulation of cellobiose caused severe feedback inhibition to the activities of β-1,4-endoglucanase and β-1,4-exoglucanase in cellulase system. Supplementing cellobiase from Aspergillus niger ZU-07 greatly reduced the inhibitory effect caused by cellobiose, and the hydrolysis yield was improved to 83.9% with enhanced cellobiase activity of 6.5 CBU g⁻¹ substrate. Fed-batch hydrolysis process was started with a batch hydrolysis containing 100 g l⁻¹ substrate, with cellulosic residue added at 6 and 12 h twice to get a final substrate concentration of 200 g l⁻¹. After 60 h of reaction, the reducing sugar concentration reached 116.3 g l⁻¹ with a hydrolysis yield of 79.5%. Further fermentation of cellulosic hydrolysate containing 95.3 g l⁻¹ glucose was performed using Saccharomyces cerevisiae 316, and 45.7 g l⁻¹ ethanol was obtained within 18 h. The research results are meaningful in fuel ethanol production from agricultural residue instead of grain starch.     

Comparison of batch,fed-batch and continuous well-mixed reactors for enzymatic hydrolysis of orange peel wastes

An alternative potential feedstock for bioethanol in the automotive sector is citrus peel waste (CPW), which can be processed through enzymatic hydrolysis and fermentation. The present work considers mathematical modeling of orange peel wastes (OPW) hydrolysis with the use of free enzymes and compares the performance of batch, fed-batch and continuous well-mixed reactors after introducing appropriate rate equations in dynamic mass balances. MATLAB^® was used for model implementation.Following the Michaelis–Menten approach, the authors used their own kinetic parameters for the pectin hydrolysis rate equation. The parameters were generated in an apposite experimental program for OPW hydrolysis to galacturonic acid with consideration of product inhibition; the corresponding values were obtained after Lineweaver–Burk linearization and are: r_max = 0.28 g/(L min), K_m = 19.80 g/L and K_IGA = 6.96 g/L, respectively. Vice-versa, the authors adopted the Kadam's group kinetic schemes and parameters for cellulose hydrolysis to cellobiose and glucose. The mathematical model of a well-mixed batch reactor was perfectly validated against the experimental results of OPW hydrolysis to galacturonic acid. In the case of a continuous well-mixed reactor, high dilution rates determine low conversion of OPW. The increased complication of fed-batch operation does not add advantages when compared to batch processing.     

Production of hemicellulosic sugars and glucose from residual corrugated cardboard

Corrugated cardboard samples were subjected to two-step saccharification. A first prehydrolysis stage was carried out to solubilise the hemicellulosic fraction as hemicellulosic sugars, and the solid phase from prehydrolysis was used as a substrate for the enzymic hydrolysis of cellulose. The prehydrolysis step was carried out for 0–180 min in media containing 1–3 wt.% of H₂SO₄ and the fraction of solid recovered after treatments and the compositions of solid and liquid phases from treatments were measured. The susceptibility of prehydrolysed solids towards the enzymic hydrolysis was assessed in further experiments. Under selected prehydrolysis conditions (3% H₂SO₄, 180 min), 78.2% of initial hemicelluloses was saccharified, leading to liquors containing up to 10 g hemicellulosic sugars/l and 9.2 g glucose/l. The corresponding solid phase, enriched in cellulose, showed good susceptibility towards enzymatic hydrolysis, leading to solutions containing up to 17.9 g glucose/l (conversion yield=63.6%) and a glucose/total sugar ratio of 0.93 g/g. Mathematical models assessing the effects of the operational conditions on both the prehydrolysis stage and the susceptibility of substrates towards enzymic hydrolysis have been developed.     

Enzymatic hydrolysis and fermentation of palm kernel press cake for production of bioethanol

Palm kernel press cake (PKC) is a residue of palm oil extraction, which was found to contain 48.5% of total carbohydrates of which 35.2% was mannan. The present study examines enzymatic hydrolysis of polysaccharides from the cell-wall material present in PKC to obtain monosaccharides that can be substrate in various fermentation processes such as ethanol production. The requirements for pretreatment were investigated and it was found that mannan in PKC was readily hydrolysed without any pretreatment. Several enzyme preparations were tested and Mannaway 25L was found as the best for releasing mannose, and Gammanase 1.0L worked well in degrading cellulose and mannose. Binary mixtures of enzymes were tested to increase the conversion, and 1:1 mixture of Mannaway 25L and Gammanase 1.0L showed good synergistic effect releasing 30% more mannose than the sum obtained using these enzymes individually. Using an enzyme loading of 2.3 mg protein/g PKC resulted in 63% of mannan in PKC being hydrolysed to mannose in 24 h, and in 96 h a total of 365 g mannose and glucose could be produced per kg PKC. Finally, PKC was hydrolysed and fermented using Saccharomyces cerevisiae with an ethanol yield of 125 g/kg PKC.     

Simultaneous saccharification and fermentation of kraft pulp by recombinant Escherichia coli for phenyllactic acid production

Simultaneous saccharification and fermentation (SSF) of renewable cellulose for the production of 3-phenyllactic acid (PhLA) by recombinant Escherichia coli was investigated. Kraft pulp recovered from biomass fractionation processes was used as a model cellulosic feedstock and was hydrolyzed using 10–50 filter paper unit (FPU) g⁻¹ kraft pulp of a commercial cellulase mixture, which increased the glucose yield from 21% to 72% in an enzyme dose-dependent manner. PhLA fermentation of the hydrolyzed kraft pulp by a recombinant E. coli strain expressing phenylpyruvate reductase from Wickerhamia fluorescens TK1 produced 1.9 mM PhLA. The PhLA yield obtained using separate hydrolysis and fermentation was enhanced from 5.8% to 42% by process integration into SSF of kraft pulp (20 g L⁻¹) in a complex medium (pH 7.0) at 37 °C. The PhLA yield was negatively correlated with the initial glucose concentration, with a five-fold higher PhLA yield observed in culture medium containing 10 g L⁻¹ glucose compared to 100 g L⁻¹. Taken together, these results suggest that the PhLA yield from cellulose in kraft pulp can be improved by SSF under glucose-limited conditions.     

Immobilization of Candida rugosa lipase on electrospun cellulose nanofiber membrane

A biocatalyst with high activity retention of lipase was fabricated by the covalent immobilization of Candida rugosa lipase on a cellulose nanofiber membrane. This nanofiber membrane was composed of nonwoven fibers with 200 nm nominal fiber diameter. It was prepared by electrospinning of cellulose acetate (CA) and then modified with alkaline hydrolysis to convert the nanofiber surface into regenerated cellulose (RC). The nanofiber membrane was further oxidized by NaIO₄. Aldehyde groups were simultaneously generated on the nanofiber surface for coupling with lipase. Response surface methodology (RSM) was applied to model and optimize the modification conditions, namely NaIO₄ content (2–10 mg/mL), reaction time (2–10 h), reaction temperature (25–35 °C) and reaction pH (5.5–6.5). Well-correlating models were established for the residual activity of the immobilized enzyme (R² = 0.9228 and 0.8950). We found an enzymatic activity of 29.6 U/g of the biocatalyst was obtained with optimum operational conditions. The immobilized lipase exhibited significantly higher thermal stability and durability than equivalent free enzyme.     

Glucose and xylose stimulation of a β-glucosidase from the thermophilic fungus Humicola insolens: A kinetic and biophysical study

β-Glucosidases activated by glucose and xylose are uncommon yet intriguing enzymes that may enhance cellulose saccharification efficiency, and are of interest for application in bioethanol production processes. The molecular mechanisms of activation are completely unknown, and the aim of this study was the kinetic and biophysical characterization of the stimulation of a β-glucosidase from Humicola insolens by glucose and xylose. The effects of the monosaccharides were concentration dependent, where in a stimulatory range (0.1–50 mmol L⁻¹), the activity increased up to 2-fold; in a stimulatory-inhibitory range (50–450 mmol L⁻¹ glucose or 50–730 mmol L⁻¹ xylose), the enzyme continued to be stimulated, but the activity was lower than maximal. Above 450 mmol L⁻¹ glucose or 730 mmol L⁻¹ xylose, increasing inhibition occurred. Dynamic light scattering confirmed that the enzyme is monomeric (54 kDa) and kinetic, intrinsic tryptophan fluorescence emission and far ultraviolet circular dichroism analyses indicated that the enzyme possesses a catalytic site (CS) and a modulator binding site (MS). Glucose or xylose binding to the MS induces conformational changes that stimulate the catalytic activity at the CS. Glucose and xylose may compete with the substrate for the CS while the substrate competes with the monosaccharides for binding to the MS. The stimulation of the enzymatic activity by glucose and xylose, which compete for the same sites on the enzyme molecule, is not synergistic. These data reveal allosteric interactions between the MS and the CS in H. insolens β-glucosidase that result in fine modulation of the catalytic activity by the monosaccharides. A kinetic model was developed that accurately described the experimental data for enzyme stimulation by glucose and/or xylose. Understanding the regulatory mechanisms of the enzyme activity, with the aid of kinetic models, may be useful for the application of the enzyme in cellulose hydrolysis processes.     

Proteomics analysis of proteins in green alga Haematococcus lacustris (Chlorophyceae) expressed under combined stress of nitrogen starvation and high irradiance

This study is aimed at identifying the proteins that are up-regulated during astaxanthin accumulation in Haematococcus lacustris. For this H. lacustris cells were cultivated in photobioreactors under normal light irradiance of 40 μE m^?2 s^?1 for 6 days and then induced to accumulate astaxanthin for 3 days further by exposure to continuous high irradiance of 200 μE m^?2 s^?1 with fluorescent lamps as light source after the cells reached the stationary phase in a nitrogen-depleted condition. Under this condition, the average astaxanthin content per cell increased from 91 mg/l up to 406 mg/l after 3 days of induction. The proteomics data from a two-dimensional electrophoretic comparison demonstrated that a combination of nitrogen source depletion and 1 h high light have significantly changed the pattern of protein expression in H. lacustris. A total of 49 protein spots were picked after 1 h of stress induction. They consisted of 13 down-regulated proteins and 36 up-regulated proteins. Fifteen proteins which had highly up-regulated expression were further analyzed by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The results will point toward interesting proteins that can be pursued for further analysis of astaxanthin biosynthesis pathway.     

Novel Penicillium cellulases for total hydrolysis of lignocellulosics

The (hemi)cellulolytic systems of two novel lignocellulolytic Penicillium strains (Penicillium pulvillorum TUB F-2220 and P. cf. simplicissimum TUB F-2378) have been studied. The cultures of the Penicillium strains were characterized by high cellulase and β-glucosidase as well moderate xylanase activities compared to the Trichoderma reesei reference strains QM 6a and RUTC30 (volumetric or per secreted protein, respectively). Comparison of the novel Penicillium and T. reesei secreted enzyme mixtures in the hydrolysis of (ligno)cellulose substrates showed that the F-2220 enzyme mixture gave higher yields in the hydrolysis of crystalline cellulose (Avicel) and similar yields in hydrolysis of pre-treated spruce and wheat straw than enzyme mixture secreted by the T. reesei reference strain. The sensitivity of the Penicillium cellulase complexes to softwood (spruce) and grass (wheat straw) lignins was lignin and temperature dependent: inhibition of cellulose hydrolysis in the presence of wheat straw lignin was minor at 35 °C while at 45 °C by spruce lignin a clear inhibition was observed. The two main proteins in the F-2220 (hemi)cellulase complex were partially purified and identified by peptide sequence similarity as glycosyl hydrolases (cellobiohydrolases) of families 7 and 6. Adsorption of the GH7 enzyme PpCBH1 on cellulose and lignins was studied showing that the lignin adsorption of the enzyme is temperature and pH dependent. The ppcbh1 coding sequence was obtained using PCR cloning and the translated amino acid sequence of PpCBH1 showed up to 82% amino acid sequence identity to known Penicillium cellobiohydrolases.     

Effect of mechanical agitation on the production of cellulases by Trichoderma reesei RUT-C30 in a draft-tube airlift bioreactor

With the aim to produce cellulases and to study the effect of mechanical agitation, a 35 L draft-tube airlift bioreactor equipped with a mechanical impeller was developed and validated to grow Trichoderma reesei RUT-C30 in a cellulose culture medium with lactose and lactobionic acid as fed batch. Cultures carried out without mechanical agitation resulted in higher volumetric enzyme productivity (200 U L⁻¹ h⁻¹), filter paper activity (17 U mL⁻¹), carboxymethyl cellulase activity (11.8 U mL⁻¹) and soluble proteins (3.2 mg mL⁻¹) when compared to those with agitation. Stereo and polarized light microscopy analyses reveal that mechanical agitation resulted in shorter mycelial hyphae and larger numbers of tips.     

Heterologous expression of codon optimized Trichoderma reesei Cel6A in Pichia pastoris

The Cel6A deficiency has become one of the limiting factors for cellulose saccharification in biochemical conversion of cellulosic biomass to fuels and chemicals. The work attempted to use codon optimization to enhance Trichoderma reesei Cel6A expression in Pichia pastoris. Two recombinants P. pastoris GS115 containing AOX1 and GAP promotors were successfully constructed, respectively. The optimal temperatures and pHs of the expressed Cel6A from two recombinants were consistent with each other, were also in the extremely similar range to that reported on the native Cel6A from T. reesei. Based on the shake flask fermentation, AOX1 promotor enabled the recombinant to produce 265 U/L and 300 mg/L of the Cel6A enzyme, and the GAP promotor resulted in 145 U/L and 200 mg/L. High cell density fed batch (HCDFB) fermentation significantly improved the enzyme titer (1100 U/L) and protein yield (2.0 g/L) for the recombinant with AOX1 promotor. Results have showed that the AOX1 promotor is more suitable than the GAP for the Cel6A expression in P. pastoris. And the HCDFB cultivation is a favorable way to express the Cel6A highly in the methanol inducible yeast.     

Sono-chemical preparation of cellulose nanocrystals from lignocellulose derived materials

This study examined the production of cellulose nanocrystals from microcrystalline wood cellulose, Avicel and recycled pulp of wood pulp using sono-chemical-assisted hydrolysis. Two hydrolysis systems: deionized water and maleic acid were evaluated. In deionized water, Avicel produced cellulose nanocrystals with average diameter of 21 ± 5 nm (minimum 15 nm and maximum 32 nm). Cellulose nanocrystals from recycled pulp were not distinctively spherical and had an average diameter of 23 ± 4 nm (minimum 14 nm and maximum 32 nm). Maleic acid (50 mM) sono-chemical assisted hydrolysis of Avicel at 15 °C and 90% power output for 9 min produced cellulose nanocrystals which were cylindrical in shape and were of dimensions, length 65 ± 19 nm and width 15 nm.     

Molecular cloning and high-level expression of a β-galactosidase gene from Paecilomyces aerugineus in Pichia pastoris

A β-galactosidase gene (designated PaGalA) was cloned for the first time from Paecilomyces aerugineus and expressed in Pichia pastoris under the control of the AOX1 promoter. The coding region of 3036 bp encoded a protein of 1011 amino acids with a deduced molecular mass of 108.7 kDa. The PaGalA without the signal peptide was cloned into a vector pPIC9K and was expressed successfully in P. pastoris as active extracellular β-galactosidase. The recombinant β-galactosidase (PaGalA) was secreted into the medium at an extremely high levels of 22 mg ml⁻¹ having an activity of 9500 U ml⁻¹ from high density fermentation culture, which is by far the highest yield obtained for a β-galactosidase. The purified enzyme with a high specific activity of 820 U mg⁻¹ had a molecular mass of 120 kDa on SDS-PAGE. PaGalA was optimally active at pH 4.5 and a temperature of 60 °C. The recombinant β-galactosidase was able to hydrolyze lactose efficiently at pH 5.0 and 50 °C. It also possessed transglycosylation activities at high concentrations of lactose. PaGalA exhibited better lactose hydrolysis efficiency in whey than two other widely used commercial lactases. The extremely high expression levels coupled with favorable biochemical properties make this enzyme highly suitable for commercial purposes in the hydrolysis of lactose in milk or whey.     

Pathways and kinetics analysis of biotransformation of Dioscorea zingiberensis by Aspergillus oryzae

In this paper, the pathways and kinetics for the production of diosgenin via biotransformation of Dioscorea zingiberensis C.H. Wright by Aspergillus oryzae CICC 2436 were analyzed. After 120 h of biotransformation at 30 °C, the concentration of diosgenin in the culture reached 36.87 ± 1.27 μmol/g raw herb, which was 21.2 times its initial concentration. A number of steroidal compounds were also isolated as minor products from the biotransformation, and one of these was identified as a novel compound named 3-O-β-d-glucopyranosyl (1 → 3) – β-d-glucopyranosyl (1 → 4) – β-d-glucopyranosyl-diosgenin (diosgenin-triglucoside). The biotransformation consisted of two stages: the release of steroids from the herb (accompanied by fungal growth) and hydrolysis of the steroids by glycosidases. Kinetic analysis and mathematical modelling showed that the process of biotransformation could be described by first-order kinetics under the condition of high K_m/[S] values. It consisted of a cascade of consecutive and parallel reactions involving three kinds of enzymes, five steroid saponins and their sapogenin. The main hydrolysis reactions that led to the production of diosgenin were also discussed.