Nanozymatic Biofuel Cell-Enabled Self-Powered Sensing System for a Sensitive Immunoassay.

Self-powered sensing system (SPSS) integrating the enzymatic biofuel cell and biosensing platform has attracted tremendous interest. However, natural enzymes suffer from the intrinsic drawbacks of enzymes and enzymatic proteins. Nanozymes with enzyme-like activities are the ideal alternatives to enzymes, and it is greatly challenging to explore high-performance nanozymatic biofuel cell for SPSS. Herein, the advanced nanozymatic biofuel cell-enabled SPSS is developed for the sensitive detection of the prostate-specific antigen (PSA), where Ir single atoms supported by nitrogen-doped carbon and Au nanozymes serve as the cathode and anode, respectively. Based on the excellent electrochemical activity and stability, the resultant nanozymatic biofuel cell exhibits a higher power output and open-circuit potential than the Pt/C-based counterpart, which is beneficial for the application of SPSS. As a proof of concept, the nanozymatic biofuel cell-enabled SPSS shows a wide detection range of 0.2-500 ng mL-1 with a detection limit of 62 pg mL-1 for PSA, which provides new insight into broadening the application scenarios of nanozymes.

Inhibition of MALT1 Alleviates Spinal Ischemia/Reperfusion Injury-Induced Neuroinflammation by Modulating Glial Endoplasmic Reticulum Stress in Rats.

PURPOSE: Glial activation and the disorders of cytokine secretion induced by endoplasmic reticulum stress (ERS) are crucial pathogenic processes in establishing ischemia/reperfusion (I/R) injury of the brain and spinal cord. This present study aimed to investigate the effects of mucous-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) on spinal cord ischemia/reperfusion (SCI/R) injury via regulating glial ERS. METHODS: SCI/R was induced by thoracic aorta occlusion-reperfusion in rats. The MALT1-specific inhibitor MI-2 or human recombinant MALT1 protein (hrMALT1) was administrated for three consecutive days after the surgery. Immunofluorescent staining was used to detect the localization of MALT1 and ERS profiles in activated astrocyte and microglia of spinal cord. The ultrastructure of endoplasmic reticulum (ER) was examined by transmission electron microscopy. Blood-spinal cord barrier (BSCB) disruption and noninflammatory status were assessed. The neuron loss and demyelination in the spinal cord were monitored, and the hindlimb motor function was evaluated in SCI/R rats. RESULTS: Intraperitoneally postoperative MI-2 treatment down-regulated phos-NF-κB (p65) and Bip (ERS marker protein) expression in the spinal cord after SCI/R in rats. Intraperitoneal injection MI-2 attenuated the swelling/dilation of ER of the glia in SCI/R rats. Furthermore, MI-2 attenuated I/R-induced Evans blue (EB) leakage and microglia M1 polarization in spinal cord, implying a role for MALT1 in the BSCB destruction and neuroinflammation after SCI/R in rats. Furthermore, intrathecal injection of hrMALT1 aggravated the fragmentation of neuron, loss of neurofibrils and demyelination caused by I/R, while 4-PBA, an ERS inhibitor, co-treatment with hrMALT1 reversed these effects in SCI/R rats. hrMALT1 administration aggravated the motor deficit index (MDI) scoring, while 4-PBA co-treatment improved SCI/R-induced motor deficits in rats. CONCLUSION: Inhibition of MALT1 alleviates SCI/R injury-induced neuroinflammation by modulating glial endoplasmic reticulum stress in rats.

Semi-industrial scale (30 m3) fed-batch fermentation for the production of D-lactate by Escherichia coli strain HBUT-D15.

D(-)-lactic acid is needed for manufacturing of stereo-complex poly-lactic acid polymer. Large scale D-lactic acid fermentation, however, has yet to be demonstrated. A genetically engineered Escherichia coli strain, HBUT-D, was adaptively evolved in a 15% calcium lactate medium for improved lactate tolerance. The resulting strain, HBUT-D15, was tested at a lab scale (7 L) by fed-batch fermentation with up to 200 g L-1 of glucose, producing 184-191 g L-1 of D-lactic acid, with a volumetric productivity of 4.38 g L-1 h-1, a yield of 92%, and an optical purity of 99.9%. The HBUT-D15 was then evaluated at a semi-industrial scale (30 m3) via fed-batch fermentation with up to 160 g L-1 of glucose, producing 146-150 g L-1 of D-lactic acid, with a volumetric productivity of 3.95-4.29 g L-1 h-1, a yield of 91-94%, and an optical purity of 99.8%. These results are comparable to that of current industrial scale L(+)-lactic acid fermentation.

Enhancement of D-lactic acid production from a mixed glucose and xylose substrate by the Escherichia coli strain JH15 devoid of the glucose effect.

BACKGROUND: A thermal tolerant stereo-complex poly-lactic acid (SC-PLA) can be made by mixing Poly-D-lactic acid (PDLA) and poly-L-lactic acid (PLLA) at a defined ratio. This environmentally friendly biodegradable polymer could replace traditional recalcitrant petroleum-based plastics. To achieve this goal, however, it is imperative to produce optically pure lactic acid isomers using a cost-effective substrate such as cellulosic biomass. The roadblock of this process is that: 1) xylose derived from cellulosic biomass is un-fermentable by most lactic acid bacteria; 2) the glucose effect results in delayed and incomplete xylose fermentation. An alternative strain devoid of the glucose effect is needed to co-utilize both glucose and xylose for improved D-lactic acid production using a cellulosic biomass substrate. RESULTS: A previously engineered L-lactic acid Escherichia coli strain, WL204 (ΔfrdBC ΔldhA ΔackA ΔpflB ΔpdhR ::pflBp6-acEF-lpd ΔmgsA ΔadhE, ΔldhA::ldhL), was reengineered for production of D-lactic acid, by replacing the recombinant L-lactate dehydrogenase gene (ldhL) with a D-lactate dehydrogenase gene (ldhA). The glucose effect (catabolite repression) of the resulting strain, JH13, was eliminated by deletion of the ptsG gene which encodes for IIBC(glc) (a PTS enzyme for glucose transport). The derived strain, JH14, was metabolically evolved through serial transfers in screw-cap tubes containing glucose. The evolved strain, JH15, regained improved anaerobic cell growth using glucose. In fermentations using a mixture of glucose (50 g L(-1)) and xylose (50 g L(-1)), JH15 co-utilized both glucose and xylose, achieving an average sugar consumption rate of 1.04 g L(-1)h(-1), a D-lactic acid titer of 83 g L(-1), and a productivity of 0.86 g L(-1) h(-1). This result represents a 46 % improved sugar consumption rate, a 26 % increased D-lactic acid titer, and a 48 % enhanced productivity, compared to that achieved by JH13. CONCLUSIONS: These results demonstrated that JH15 has the potential for fermentative production of D-lactic acid using cellulosic biomass derived substrates, which contain a mixture of C6 and C5 sugars.

[Knockout of the ptsG gene in engineered Escherichia coli for homoethanol fermentation from sugar mixture].

To realize the simultaneous fermentation of xylose and glucose, ptsG (one of the glucose-PTS genes) was deleted from the engineered ethanologenic Escherichia coli SZ470 (deltapflB, deltafrdABCD, deltaackA, deltaldhA), resulting in loss of glucose effect in the mutant SZ470P (deltaptsG). When tested in 5% mixture of glucose (2.5%) and xylose (2.5%), SZ470P simultaneously used glucose (13 g/L) and xylose (20 g/L) whereas the parent strain SZ470 sequentially used glucose (25 g/L) then xylose (5 g/L). Upon completion of the fermentation, both strains achieved similar product yield of 89%. SZ470P produced 15.01 g/L of ethanol, which was 14.32% higher than that produced by SZ470 (12.86 g/L). Deleting ptsG gene enabled the mutant strain SZ470P to simultaneously use both glucose and xylose and achieve better ethanol production.

Engineering and adaptive evolution of Escherichia coli W for L-lactic acid fermentation from molasses and corn steep liquor without additional nutrients.

The D-lactic acid producing strain, Escherichia coli HBUT-D, was reengineered for L(+)-lactic acid fermentation by replacing the D-lactate dehydrogenase gene (ldhA) with an L(+)-lactate dehydrogenase gene (ldhL) from Pedicoccus acidilactici, followed by adaptive evolution in sucrose. The resulting strain, WYZ-L, has enhanced expression of the sucrose operon (cscA and cscKB). In 100 g L(-1) of sucrose fermentation using mineral salt medium, WYZ-L produced 97 g L(-1) of l(+)-lactic acid, with a yield of 90%, a maximum productivity of 3.17 g L(-1)h(-1) and an optical purity of greater than 99%. In fermentations using sugarcane molasses and corn steep liquor without additional nutrients, WYZ-L produced 75 g L(-1) of l(+)-lactic acid, with a yield of 85%, a maximum productivity of 1.18 g L(-1)h(-1), and greater than 99% optical purity. These results demonstrated that WYZ-L has the potential to use waste molasses and corn steep liquor as a resource for L(+)-lactic acid fermentation.

[Production of L-lactic acid from pentose by a genetically engineered Escherichia coli].

OBJECTIVE: In this study, we constructed a recombinant Escherichia coli strain for the production of high-purity L-lactic acid, using a homoethanol fermenting mutant E. coli SZ470 (deltafrdBC deltaldhA deltaackA deltafocA-pflB deltapdhR: :pflBp6-pflBrbs-aceEF-lpd) as the starting strain. METHODS: By using homologous recombination, we deleted the adhE gene from SZ470 to obtain a mutant Escherichia coli JH01, which could not grow under anaerobic conditions. Then we cloned the L-lactate dehydrogenase gene (ldhL) of Pediococcus acidilactici and inserted it into the chromosome of JH01 via electroporation to obtain a recombinant strain Escherichia coli JH12. We evaluated the L-lactic acid production of the recombinant strain in a 15 L fermenter. RESULTS: In 10 L LB medium supplemented with 6% glucose, JH12 maintained maximal cell growth and an efficient L-lactic acid production rate for 36 h. Glucose consumption rate achieved was 1.46 g/(L x h) and L-lactic acid production rate was 1.14 g/(L x h). The results also show that 41.13 g/L lactic acid was produced, achieving a purity of 95.69% (based on total fermentation products). Xylose consumption rate was 0.88 g/(L x h) and L-lactic acid production rate was 0.60 g/(L x h). The production of lactic acid was 34.73 g/L, achieving a purity of 98%. There were no succinic acid and formic acid detected and only little amount of acetic acid generated during the fermentation. CONCLUSION: We constructed a homolactic acid fermentation strain E. coli JH12, which could efficiently convert glucose and xylose into high-purity L-lactic acid. JH12 could have great potential in industrial fermentation for L-lactic acid production.

Homofermentative production of optically pure L-lactic acid from xylose by genetically engineered Escherichia coli B.

BACKGROUND: Polylactic acid (PLA), a biodegradable polymer, has the potential to replace (at least partially) traditional petroleum-based plastics, minimizing "white pollution". However, cost-effective production of optically pure L-lactic acid is needed to achieve the full potential of PLA. Currently, starch-based glucose is used for L-lactic acid fermentation by lactic acid bacteria. Due to its competition with food resources, an alternative non-food substrate such as cellulosic biomass is needed for L-lactic acid fermentation. Nevertheless, the substrate (sugar stream) derived from cellulosic biomass contains significant amounts of xylose, which is unfermentable by most lactic acid bacteria. However, the microorganisms that do ferment xylose usually carry out heterolactic acid fermentation. As a result, an alternative strain should be developed for homofermentative production of optically pure L-lactic acid using cellulosic biomass. RESULTS: In this study, an ethanologenic Escherichia coli strain, SZ470 (ΔfrdBC ΔldhA ΔackA ΔpflB ΔpdhR ::pflBp6-acEF-lpd ΔmgsA), was reengineered for homofermentative production of L-lactic acid from xylose (1.2 mole xylose = > 2 mole L-lactic acid), by deleting the alcohol dehydrogenase gene (adhE) and integrating the L-lactate dehydrogenase gene (ldhL) of Pediococcus acidilactici. The resulting strain, WL203, was metabolically evolved further through serial transfers in screw-cap tubes containing xylose, resulting in the strain WL204 with improved anaerobic cell growth. When tested in 70 g L-1 xylose fermentation (complex medium), WL204 produced 62 g L-1 L-lactic acid, with a maximum production rate of 1.631 g L-1 h-1 and a yield of 97% based on xylose metabolized. HPLC analysis using a chiral column showed that an L-lactic acid optical purity of 99.5% was achieved by WL204. CONCLUSIONS: These results demonstrated that WL204 has the potential for homofermentative production of L-lactic acid using cellulosic biomass derived substrates, which contain a significant amount of xylose.

Increasing reducing power output (NADH) of glucose catabolism for reduction of xylose to xylitol by genetically engineered Escherichia coli AI05.

Anaerobic homofermentative production of reduced products requires additional reducing power (NADH and/or NADPH) output from glucose catabolism. Previously, with an anaerobically expressed pyruvate dehydrogenase operon (aceEF-lpd), we doubled the reducing power output to four NADH per glucose (or 1.2 xylose) catabolized anaerobically, which satisfied the NADH requirement to establish a non-transgenic homoethanol pathway (1 glucose or 1.2 xylose --> 2 acetyl-CoA + 4 NADH --> 2 ethanol) in the engineered strain, Escherichia coli SZ420 (∆frdBC ∆ldhA ∆ackA ∆focA-pflB ∆pdhR::pflBp6-pflBrbs-aceEF-lpd). In this study, E. coli SZ420 was further engineered for reduction of xylose to xylitol by (1) deleting the alcohol dehydrogenase gene (adhE) to divert NADH from the ethanol pathway; (2) deleting the glucose-specific PTS permease gene (ptsG) to eliminate catabolite repression and allow simultaneous uptake of glucose and xylose; (3) cloning the aldose reductase gene (xylI) of Candida boidinii to reduce xylose to xylitol. The resulting strain, E. coli AI05 (pAGI02), could in theory simultaneously uptake glucose and xylose, and utilize glucose as a source of reducing power for the reduction of xylose to xylitol, with an expected yield of four xylitol for each glucose consumed (YRPG = 4) under anaerobic conditions. In resting cell fermentation tests using glucose and xylose mixtures, E. coli AI05 (pAGI02) achieved an actual YRPG value of ~3.6, with xylitol as the major fermentation product and acetate as the by-product.

Homofermentative production of D-lactic acid from sucrose by a metabolically engineered Escherichia coli.

Escherichia coli W, a sucrose-positive strain, was engineered for the homofermentative production of D-lactic acid through chromosomal deletion of the competing fermentative pathway genes (adhE, frdABCD, pta, pflB, aldA) and the repressor gene (cscR) of the sucrose operon, and metabolic evolution for improved anaerobic cell growth. The resulting strain, HBUT-D, efficiently fermented 100 g sucrose l(-1) into 85 g D-lactic acid l(-1) in 72-84 h in mineral salts medium with a volumetric productivity of ~1 g l(-1) h(-1), a product yield of 85 % and D-lactic acid optical purity of 98.3 %, and with a minor by-product of 4 g acetate l(-1). HBUT-D thus has great potential for production of D-lactic acid using an inexpensive substrate, such as sugar cane and/or beet molasses, which are primarily composed of sucrose.

Engineering a homobutanol fermentation pathway in Escherichia coli EG03.

A homobutanol fermentation pathway was engineered in a derivative of Escherichia coli B (glucose [glycolysis] => 2 pyruvate + 2 NADH; pyruvate [pyruvate dehydrogenase] => acetyl-CoA + NADH; 2 acetyl-CoA [butanol pathway enzymes] + 4 NADH => butanol; summary stoichiometry: glucose => butanol). Initially, the native fermentation pathways were eliminated from E. coli B by deleting the genes encoding for lactate dehydrogenase (ldhA), acetate kinase (ackA), fumarate reductase (frdABCD), pyruvate formate lyase (pflB), and alcohol dehydrogenase (adhE), and the pyruvate dehydrogenase complex (aceEF-lpd) was anaerobically expressed through promoter replacement. The resulting strain, E. coli EG03 (ΔfrdABCD ΔldhA ΔackA ΔpflB Δ adhE ΔpdhR ::pflBp6-aceEF-lpd ΔmgsA), could generate 4 NADH for every glucose oxidized to two acetyl-CoA through glycolysis and the pyruvate dehydrogenase complex. However, EG03 lost its ability for anaerobic growth due to the lack of NADH oxidation pathways. When the butanol pathway genes that encode for acetyl-CoA acetyltransferase (thiL), 3-hydroxybutyryl-CoA dehydrogenase (hbd), crotonase (crt), butyryl-CoA dehydrogenase (bcd, etfA, etfB), and butyraldehyde dehydrogenase (adheII) were cloned from Clostridium acetobutylicum ATCC 824, and expressed in E. coli EG03, a balanced NADH oxidation pathway was established for homobutanol fermentation (glucose => 4 NADH + 2 acetyl-CoA => butanol). This strain was able to convert glucose to butanol (1,254 mg l(-1)) under anaerobic condition.

Partial deletion of rng (RNase G)-enhanced homoethanol fermentation of xylose by the non-transgenic Escherichia coli RM10.

Previously, a native homoethanol pathway was engineered in Escherichia coli B by deletions of competing pathway genes and anaerobic expression of pyruvate dehydrogenase (PDH encoded by aceEF-lpd). The resulting ethanol pathway involves glycolysis, PDH, and alcohol dehydrogenase (AdhE). The E. coli B-derived ethanologenic strain SZ420 was then further improved for ethanol tolerance (up to 40 g l(-1) ethanol) through adaptive evolution. However, the resulting ethanol tolerant mutant, SZ470, was still unable to complete fermentation of 75 g l(-1) xylose, even though the theoretical maximum ethanol titer would have been less than 40 g l(-1) should the fermentation have reached completion. In this study, the cra (encoding for a catabolite repressor activator) and the HSR2 region of rng (encoding for RNase G) were deleted from SZ470 in order to improve xylose fermentation. Deletion of the HSR2 domain resulted in significantly increased mRNA levels (47-fold to 409-fold) of multiple glycolytic genes (pgi, tpiA, gapA, eno), as well as the engineered ethanol pathway genes (aceEF-lpd, adhE) and the transcriptional regulator Fnr (fnr). The higher adhE mRNA level resulted in increased AdhE activity (>twofold). Although not measured, the increase of other mRNAs might also enhance expressions of their encoding proteins. The increased enzymes would then enable the resulting strain, RM10, to achieve increased cell growth and complete fermentation of 75 g l(-1) xylose with an 84% improved ethanol titer (35 g l(-1)), compared to that (19 g l(-1)) obtained by the parent, SZ470. However, deletion of cra resulted in a negative impact on cell growth and xylose fermentation, suggesting that Cra is important for long-term fermentative cell growth.

Structure elucidation and complete NMR spectral assignments of glucosylated saponins of cantalasaponin I.

Five new glucosylated steroidal glycosides, cantalasaponin I-B(1) (1), I-B(2) (2), I-B(3) (3), I-B(4) (4) and I-B(5) (5), were isolated and purified from the transformed product of the cantalasaponin I by using Toruzyme 3.0 l as biocatalyst. Their structures were elucidated on the basis of high-resolution electrospray ionization mass spectrometry, one-dimensional ((1) H and (13) C NMR) and two-dimensional [COSY, heteronuclear single-quantum correlation (HSQC), HMBC and HSQC-TOCSY] NMR spectral analyses and chemical evidence.

Adaptive evolution of nontransgenic Escherichia coli KC01 for improved ethanol tolerance and homoethanol fermentation from xylose.

Due to its excellent capability to ferment five-carbon sugars, Escherichia coli has been considered one of the platform organisms to be engineered for production of cellulosic ethanol. Nevertheless, genetically engineered ethanologenic E. coli lacks the essential trait of alcohol tolerance. Development of ethanol tolerance is required for cost-effective ethanol fermentation. In this study, we improved alcohol tolerance of a nontransgenic E. coli KC01 (ldhA pflB ackA frdBC pdhR::pflBp6-aceEF-lpd) through adaptive evolution. During ~350 generations of adaptive evolution, a gradually increased concentration of ethanol was used as a selection pressure to enrich ethanol-tolerant mutants. The evolved mutant, E. coli SZ470, was able to grow anaerobically at 40 g l(-1) ethanol, a twofold improvement over parent KC01. When compared with KC01 for small-scale (500 ml) xylose (50 g l(-1)) fermentation, SZ470 achieved 67% higher cell mass, 48% faster volumetric ethanol productivity, and 50% shorter time to complete fermentation with ethanol titer of 23.5 g l(-1) and yield of 94%. These results demonstrate that an industry-oriented nontransgenic E. coli strain could be developed through incremental improvements of desired traits by a combination of molecular biology and traditional microbiology techniques.

Enzymatic synthesis of alpha-glucosyl-timosaponin BII catalyzed by the extremely thermophilic enzyme: Toruzyme 3.0L.

Timosaponin BII (BII), a steroidal saponin showing potential anti-dementia activity, was converted into its glucosylation derivatives by Toruzyme 3.0L. Nine products with different degrees of glucosylation were purified and their structures were elucidated on the basis of (13)C NMR, HR-ESI-MS, and FAB-MS spectra data. The active enzyme in Toruzyme 3.0L was purified to electrophoretic homogeneity by tracking BII-glycosylase activity and was identified as Cyclodextrin-glycosyltransferase (CGTase, EC 2.4.1.19) by ESI-Q-TOF MS/MS. In this work, we found that the active enzyme catalyzed the synthesis of alpha-(1-->4)-linked glucosyl-BII when dextrin instead of an expensive activated sugar was used as the donor and showed a high thermal tolerance with the most favorable enzymatic activity at 100 degrees C. In addition, we also found that the alpha-amylases and CGTase, that is, GH13 family enzymes, all exhibited similar activities, which were able to catalyze glucosylation in steroidal saponins. But other kinds of amylases, such as gamma-amylase (GH15 family), had no such activity under the same reaction conditions.

Glucosylation of steroidal saponins by cyclodextrin glucanotransferase.

It is known that the sugar chains of steroidal saponins play an important role in the biological and pharmacological activities. In order to synthesize steroidal saponins with novel sugar chains in one step for further studies on pharmacological activity, we here describe the glucosylation of steroidal saponins, and 5 compounds, timosaponin AIII (1), saponin Ta (2), saponin Tb (3), trillin (4) and cantalasaponin I (5), were converted into their glucosylated products by Toruzyme 3.0 L, a cyclodextrin glucanotransferase (CGTase). 12 glucosylated products were isolated and their structures elucidated on the basis of spectral data; they were all characterized as new compounds. The results showed that Toruzyme 3.0 L had the specific ability to add the α-D-glucopyranosyl group to the glucosyl group linked at the sugar chains of steroidal saponins, and the glucosyl group was the only acceptor. This is the first report of steroidal saponins with different degrees of glucosylation. The substrates and their glucosylated derivatives were evaluated for their cytotoxicity against HL-60 human promyelocytic leukemia cell by MTT assay. The substrates all exhibited high cytotoxicity (IC(50) < 10 µmol/L), excluding compound 5 (IC(50) > 150 µmol/L), and the cytotoxicity of most of the products showed no obvious changes compared with those of their substrates.

One new triterpenoid from biotransformation product of glycyrrhizic acid.

A new triterpenoid compound (1) and a known compound (2) were isolated from the product of biotransformation of glycyrrhizic acid by Aspergillus niger. On the basis of the 1D and 2D NMR ((1)H-(1)H COSY, HSQC, HMBC and NOESY) and MS spectrometry, their structures were established as 7beta, 15alpha-dihydroxy-3,11-dioxo-oleana-12-en-30-oic acid (1) and 15alpha-hydroxy-3,11-dione-oleana-12-en-30-oic acid (2), respectively.

[The clinical study on decompensatory cirrhotic patients treated by Bie Jia Jian].

OBJECTIVE: To observe the clinical curative effect on decompensatory cirrhotic patients treated by Bie Jia Jian. METHODS: 98 decompensatory cirrhotic patients were randomly divided into two groups: 49 patients in treatment group and 49 in control group. Both groups were treated with the same western medicine of protecting and supporting liver. Except that, treatment group were treated by Bie Jia Jian. RESULTS: The Contents of AST, ALT, total bilirubin (TB), direct bilirubin (DB), hyaluronic acid (HA), Laminin (LN) , procollagen III (pc III), and type IV collagen (IV.C) in both groups decreased after treatment, and prothrombin time activity (PTA) increased. Among them, the decrease of TB, DB, HA, LN, PC-III and IV-C, and the increase of PTA in treatment group were more obvious than those in control group (P < 0. 05). CONCLUSION: Bie Jia Jian is effective in treating decompesatory cirrhotic patients.

Purification, characterization, and substrate specificity of a glucoamylase with steroidal saponin-rhamnosidase activity from Curvularia lunata.

It has been previously reported that a glucoamylase from Curvularia lunata is able to hydrolyze the terminal 1,2-linked rhamnosyl residues of sugar chains at C-3 position of steroidal saponins. In this work, the enzyme was isolated and identified after isolation and purification by column chromatography including gel filtration and ion-exchange chromatography. Analysis of protein fragments by MALDI-TOF/TOF proteomics Analyzer indicated the enzyme to be 1,4-alpha-D-glucan glucohydrolase EC 3.2.1.3, GA and had considerable homology with the glucoamylase from Aspergillus oryzae. We first found that the glucoamylase was produced from C. lunata and was able to hydrolyze the terminal rhamnosyl of steroidal saponins. The enzyme had the general character of glucoamylase, which hydrolyze starch. It had a molecular mass of 66 kDa and was optimally active at 50 degrees C, pH 4, and specific activity of 12.34 U mg of total protein(-1) under the conditions, using diosgenin-3-O-alpha-L-rhamnopyranosyl(1-->4)-[alpha-L-rhamnopyranosyl (1-->2)]-beta-D-glucopyranoside (compound II) as the substrate. Furthermore, four kinds of commercial glucoamylases from Aspergillus niger were investigated in this work, and they had the similar activity in hydrolyzing terminal rhamnosyl residues of steroidal saponin.