High Production of β-Glucosidase by Aspergillus niger on Corncob

Using low-cost raw material is an effective approach for reducing the cost of cellulolytic enzymes. The farmland waste corncob was found in this study to be the best carbon source for the production of β-glucosidase by Aspergillus niger. The maximum yield of β-glucosidase activity was 48.7 IU ml(-1) by using 50 g?l(-1) of corncob powder as the substrate. It was found that the water-soluble components of the corncob could increase β-glucosidase production significantly only when mixed with Avicel or wheat bran. The soluble components could not enhance the biomass and β-glucosidase production when used alone. On the other hand, the water-insoluble components of the corncob still produced high level of β-glucosidase (30 IU ml(-1)) although lower than that of using whole corncob. The results suggested that the water-insoluble components of corncob were beneficial for β-glucosidase production. It was further demonstrated that the xylan in the water-insoluble parts of corncob was the important factor in producing β-glucosidase by A. niger.     

High production of β-glucosidase from a marine Aspergillus niger immobilized on towel gourd vegetable sponges

To produce β-glucosidase by consecutive batch fermentation, a marine Aspergillus niger was immobilized on a natural carrier, towel gourd vegetable sponges. The immobilized mycelia were 0.15 g/g carrier with the immobilized biomass percentage of over 95%. The immobilized mycelia possessed the long durability(22.5 days). The maximum production occurred 1.5 day earlier by the immobilized mycelia than by the free mycelia. β-Glucosidase production of five consecutive batches was over 110 U/m L. At high salinity,the activity and stability of β-glucosidase from the marine A. niger increased remarkable. Immobilizing the marine A. niger on the novel natural carrier achieved the efficient production of β-glucosidase.     

Selection of the most effective kinetic model of lactase hydrolysis by immobilized Aspergillus niger and free β-galactosidase

The kinetic model of the hydrolysis of lactose by β-galactosidase from Aspergillus niger immobilized on a commercial ceramic monoliths was estimated in the attendance of lactose and its hydrolysis reaction products galactose and glucose. The aim of this work was to developing kinetic model of lactase hydrolysis by Aspergillus niger. The variables in this study are temperature, pH, enzyme concentration, substrate concentration and final product. The optimum temperature used to achieve the best hydrolysis performance in the kinetic model selection was 55 and 60 °C. The optimum pH used for enzyme activity was about 3.5 to 4. Five kinetic models were proposed to confirm experimental data the enzymatic reaction of the lactose hydrolysis by the β-galactosidase. The kinetics of lactose hydrolysis by both Immobilized and soluble lactases were scrutinized in a batch reactor system in the lack of any mass conduction restriction. In both instance the galactose inhibition kinetic models predicted the experimental data. The model is capable to fit the experimental data correctly in the extensive experimental span studied.     

Purification and characterization of an extracellular β-glucosidase with high transglucosylation activity and stability from Aspergillus niger No. 5.1

An extracellular beta-glucosidase was extracted from the culture filtrate of Aspergillus niger No. 5.1 and purified to homogeneity by using ammonium sulfate precipitation, Chitopearl-DEAE chromatography, and Sephadex G-100 chromatography. The specific activity of the enzyme was enriched 6.33-fold, with a recovery of 11.67%. The enzyme was a monomer and the molecular mass was 67.5 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 66.5 kDa by gel-filtration chromatography. The enzyme had optimum activity at pH 6.0 and 60 degrees C and was stable over the pH range of 3.0-9.0. It showed specificity of hydrolysis for p-nitrophenyl-beta-D-glucoside and cellobiose. The Km and Vmax values of the enzyme for cellobiose and salicin were 5.34 mM, 2.57 micromol/(mL.s), and 3.09 mM, 1.34 micromol/(mL.s), respectively. Both amino acid composition and N-terminal amino acid sequence of the enzyme were determined, which provides useful information for cloning of this enzyme.     

Semi-solid-state fermentation of Eicchornia crassipes biomass as lignocellulosic biopolymer for cellulase and β-glucosidase production by cocultivation of Aspergillus niger RK3 and Trichoderma reesei MTCC164

An aquatic weed biomass, Eicchornia crassipes, present in abundance and leading to a threatening level of water pollution was used as substrate for cellulase and β-glucosidase production using wild-type strain Aspergillus niger RK3 that was isolated from decomposing substrate. Alkali treatment of the biomass (10%) resulted in a 60–66% increase in endoglucanase, exoglucanase, and β-glucosidase production by the A. niger RK3 strain in semi-solid-state fermentation. Similarly, the alkali-treated biomass led to a 45–54% increase in endo- and exoglucanase and a higher (98%) increase in β-glucosidase production by Trichoderma reesei MTCC164 under similar conditions. However, the cocultivation of A. niger RK3 and T. reesei MTCC164 at a ratio of 3:1 showed a 20–24% increase in endo- and exoglucanase activities and about a 13% increase in the β-glucosidase activity over the maximum enzymatic activities observed under single culture conditions. Multistep physical (ultraviolet) and chemical (N-methyl-N′-nitrosoguanidine, sodium azide, colchicine) mutagenesis of the A. niger RK3 strain resulted in a highly cellulolytic mutant, UNSC-442, having an increase of 136, 138, and 96% in endoglucanase, exoglucanase, and β-glucosidase, activity, respectively. The cocultivation of mutant UNSC-442 along with T. reesei MTCC164 (at a ratio of 3:1) showed a further 10–11% increase in endo- and exoglucanase activities and a 29% increase in β-glucosidase activity in semi-solid-state fermentation.     

Heterologous Expression of Aspergillus niger β-d-Xylosidase (XlnD): Characterization on Lignocellulosic Substrates

The gene encoding a glycosyl hydrolase family 3 xylan 1,4-beta-xylosidase, xlnD, was successfully cloned from Aspergillus niger strain ATCC 10864. The recombinant product was expressed in Aspergillus awamori, purified by column chromatography, and verified by matrix-assisted laser desorption ionization, tandem time of flight (MALDI-TOF/TOF) mass spectroscopy of tryptic digests. The T _max was determined using differential scanning microcalorimetry (DSC) to be 78.2 °C; the K _m and k _cat were found to be 255 μM and 13.7 s⁻¹, respectively, using pNP-β-d-xylopyranoside as substrate. End-product inhibition by d-xylose was also verified and shown to be competitive; the K _i for this inhibition was estimated to be 3.3 mM. XlnD was shown to efficiently hydrolyze small xylo-oligomers to monomeric xylose, making it a critical hydrolytic activity in cases where xylose is to be recovered from biomass conversion processes. In addition, the presence of the XlnD was shown to synergistically enhance the ability of an endoxylanase, XynA from Thermomyces lanuginosus, to convert xylan present in selected pretreated lignocellulosic substrates. Furthermore, the addition of the XynA/XlnD complex was effective in enhancing the ability of a simplified cellulase complex to convert glucan present in the substrates.     

Dynamic Changes in Xylanases and β-1,4-Endoglucanases Secreted by Aspergillus niger An-76 in Response to Hydrolysates of Lignocellulose Polysaccharide

Aspergillus niger is an effective secretor of glycoside hydrolases that facilitate the saprophytic lifestyle of the fungus by degrading plant cell wall polysaccharides. In the present study, a series of dynamic zymography assays were applied to quantify the secreted glycoside hydrolases of A. niger cultured in media containing different carbon sources. Differences in the diversity and concentrations of polysaccharide hydrolysates dynamically regulated the secretion of glycoside hydrolases. The secretion of β-1,4-endoglucanase isozymes was observed to lag at least 24 h behind, rather than coincide with, the secretion of xylanase isozymes. Low concentrations of xylose could induce many endoxylanases (such as Xyn1/XynA, Xyn2, and Xyn3/XynB). High concentrations of xylose could sustain the induction of Xyn2 and Xyn3/XynB but repress Xyn1/XynA (GH10 endoxylanase), which has a broad substrate specificity, and also triggers the low-level secretion of Egl3/EglA, which also has a broad substrate specificity. Mixed polysaccharide hydrolysates sustained the induction of Egl1, whereas the other β-1,4-endoglucanases were sustainably induced by the specific polysaccharide hydrolysates released during the hydrolysis process (such as Egl2 and Egl4). These results indicate that the secretion of glycoside hydrolases may be specifically regulated by the production of polysaccharide hydrolysates released during the process of biomass degradation.     

Derepressed 2-deoxyglucose-resistant mutants of Aspergillus niger with altered hexokinase and acid phosphatase activity in hyperproduction of β-fructofuranosidase

Aspergillus niger NRRL330 produces extracellular β-fructofuranosidase (Ffase), and its production is subject to repression by hexoses in the medium. After ultraviolet mutagenization and selection, seven derepressed mutants resistant to 2-deoxyglucose (2-DG) were isolated on Czapek’s minimal medium containing glycerol. One of the mutants, designated DGRA-1, produced higher levels of Ffase. A considerable difference occurred in the mutants with reference to hexokinase and intracellular acid phosphatase activities. The hexokinase activity of the mutant DGRA-1 (0.69 U/mg) was 1.8-fold higher than the wild type (0.38U/mg). Intracellular acid phosphatase activity of the mutant DGRA-1 (0.83 U/g of mycelia) was twofold higher than that of the wild type (0.42U/g of mycelia), suggesting that phosphorylation and dephosphorylation steps could attribute to the 2-DG resistance of A. niger. However, additional mutations could account for the increased production of Ffase in the mutant DGRA-1.     

Hydrolysis of cellobiose by immobilized β-glucosidase entrapped in maintenance-free gel spheres

A crude preparation of Aspergillus niger β-glucosidase (27.5 cello-biase U/mg protein at 40°C, pH 5.0) was immobilized on concanavalin A-Sepharose (CAS). The cellobiase activity of the immobilized enzyme was 1334 U/mg dried CAS or 108 U/mL CAS gel. The β-glucosidase-CAS complex was entrapped within crosslinked propylene glycol alginate/bone-geletin gel spheres that possessed between 0.67 and 2.35 cellobiase U/mL spheres, depending on their size. The effect of cellobiose concentration (10–300 mM) on the activity of native, immobilized, and gel-entrapped enzyme was determined. It was shown that concentrations of cellobiose between 10 and 180 mM were not inhibitory to the entrapped enzyme, although inhibition was found to occur with the native and immobilized enzyme. Exogenous ion addition was not necessary to maintain the structural integrity of the spheres, which were stable for 4 d at 40°C.     

The predicted unfolding order of the β-strands in the starch binding domain from Aspergillus niger glucoamylase

The unfolding of the β-strands in the starch binding domain from Aspergillus niger glucoamylase was predicted to follow the order of β3→β2→β6→β5→β4→β1→β7 by 600 ps molecular dynamics simulations at 300, 400, and 600 K. The interior region around β-strands 2 and 3 acts as the initiation site for unfolding. β-Strands 1 and 7 are probably stabilized by the disulfide bond formed between Cys509 and Cys604. β-Strand 4 is stabilized by forming an antiparallel β-sheet with β-strand 1. Hydrophobic and electrostatic interactions between side chains instead of the hydrogen bonds are important in stabilizing these β-strands, thus the entire starch binding domain.     

Use of hemicellulose hydrolysate for β-glucosidase fermentation

Hydrolysis of cellulose byTrichoderma cellulases often results in a mixture of glucose, cellobiose, and low-mol-wt cellodextrins. Cellobiose is nonfermentable for most yeasts, and therefore it has to be hydrolyzed to glucose by β-glucosidase prior to ethanol fermentation. In the present study, the β-glucosidase production of onePenicillium and threeAspergillus strains, which were previously selected out of 24 strains, was investigated on steam pretreated willow. Both steam-pretreated willow and hemicellulose hydrolysate, released during steam explosion of willow, were used as carbon sources. Reference cultivation runs were performed using prehydrolyzed Solka Floc and glucose: The four strains were compared withTrichoderma reesei regarding sugar consumption and β-glucosidase production.Aspergillus niger andAspergillus phoenicis proved to be the best enzyme producers on hemicellulose hydrolysate. The maximum β-glucosidase activity, 4.60 IU/mL, was obtained whenA. phoenicis was cultivated on the mixture of hemicellulose hydrolysate and steam-pretreated willow. The maximum yield of enzyme activity, 502 IU/g total carbohydrate, was obtained whenAspergillus foetidus was cultivated on the hemicellulose hydrolysate.     

Transformation of geniposide into genipin by immobilized β-glucosidase in a two-phase aqueous-organic system

Genipin is the bioactive compound of geniposide and a natural cross-linking agent. In order to improve the preparation process of genipin, the hydrolysis of geniposide to genipin by immobilized β-glucosidase in an aqueous-organic two-phase system was studied. β-glucosidase was immobilized by the crosslinking-embedding method using sodium alginate as the carrier. The optimum reaction temperature, pH value and time were 55 °C, 4.5 and 2.5 h, respectively. To reduce genipin hydrolysis and byproduct production the reaction was carried out in an aqueous-organic two-phase system comprising ethyl acetate and sodium acetate buffer. The product was analyzed by HPLC, UV, IR, and NMR. The yield of genipin was 47.81% and its purity was over 98% (HPLC).     

Glycosidase-catalysed synthesis of mannobioses by the reverse hydrolysis activity of α-mannosidase: partial purification of α-mannosidases from almond meal,limpets and Aspergillus niger

Two disaccharides, α-d-Manp-(1→2)-d-Manp 6 and α-d-Manp-(1→3)-d-Manp 7, were synthesised from mannose using the reverse hydrolysis activity of the partially purified α-mannosidases from almond (Prunus amygdalus) meal and limpets (Patella vulgata). Both disaccharides were isolated by carbon–Celite chromatography. Attempts were also made towards the synthesis of core pentasaccharide using the purified α-mannosidase from Aspergillus niger.     

Offline and online capillary electrophoresis enzyme assays of β-N-acetylhexosaminidase

Enzyme assays of β-N-acetylhexosaminidase from Aspergillus oryzae using capillary electrophoresis in the offline and online setup have been developed. The pH value and concentration of the borate-based background electrolyte were optimized in order to achieve baseline separation of N,N′,N″-triacetylchitotriose, N,N′-diacetylchitobiose, and N-acetyl-d-glucosamine. The optimized method using 25 mM tetraborate buffer, pH 10.0, was evaluated in terms of repeatability, limits of detection, quantification, and linearity. The method was successfully applied to the offline enzyme assay of β-N-acetylhexosaminidase, which was demonstrated by monitoring the hydrolysis of N,N′,N″-triacetylchitotriose. The presented method was also utilized to study the pH dependence of enzyme activity. An online assay with N,N′-diacetylchitobiose as a substrate was developed using the Transverse Diffusion of Laminar Flow Profiles model to optimize the injection sequence and in-capillary mixing of substrate and enzyme plugs. The experimental results were in good agreement with predictions of the model. The online assay was successfully used to observe the inhibition effect of N,N′-dimethylformamide on the activity of β-N-acetylhexosaminidase with nanoliter volumes of reagents used per run and a high degree of automation. After adjustment of background electrolyte pH, an online assay with N,N′,N″-triacetylchitotriose as a substrate was also performed.

Biotransformation of 14-Deacetoxy-13-oxo sinenxan A by Ginkgo Cell Cultures

Sinenxan A, 2a, 5a, 10b,14b-tetraacetoxy-4(20),11-taxadiene, is a taxoid isolated from the callus cultures of Taxus spp. in high yield (ca. 1~2% of dry weight)1,2. The rich resources and its taxane-skeleton vest it valuable potential for the semisynthesis of paclitaxel or other structurally related bioactive compounds, such as 搒econd -generation?taxoid anticancer agents and taxane-based multidrug resistant anticancer agents3-5. Many remarkable studies on its structural modification by chemi…     

Biosynthetic activity of recombinant Escherichia coli-expressed Pichia etchellsii β-glucosidase II

The hydrolysis and biosynthetic reactions of partially purified Pichia etchellsii β-glucosidase II from recombinant Escherichia coli pBG22:JM109 are described. With 167 mmol/L of initial glucose, the products of synthetic reactions, glucobiose and glucotriose, accumulated to 18 and 6 mmol/L, respectively. In transglycosylation reactions with 79 mmol/L of initial cellobiose, glucotriose and glucopentaose were obtained at 4.5 and 2 mmol/L, respectively. The effects of incubation time and substrate concentration were studied on the yield of synthesized oligosaccharides. In a reaction time of 24 h with 468 mmol/L of initial cellobiose, glucotriose and glucopentaose levels of 21.6 and 6.6 mmol/L, respectively, were obtained. The addition of dimethyl sulfoxide (DMSO) further increased the yields of the products by 10%. Detailed kinetic analysis indicated a significant (about twofold) increase in V _max/K _M of synthetic reactions in the presence of DMSO. A study of other disaccharides in transglycosylation reactions indicated biosynthetic activity in the order of sophorose > gentiobiose > cellobiose.     

Directed Evolution of a Thermophilic β-glucosidase for Cellulosic Bioethanol Production

Characteristics that would make enzymes more desirable for industrial applications can be improved using directed evolution. We developed a directed evolution technique called random drift mutagenesis (RNDM). Mutant populations are screened and all functional mutants are collected and put forward into the next round of mutagenesis and screening. The goal of this technique is to evolve enzymes by rapidly accumulating mutations and exploring a greater sequence space by providing minimal selection pressure and high-throughput screening. The target enzyme was a β-glucosidase isolated from the thermophilic bacterium, Caldicellulosiruptor saccharolyticus that cleaves cellobiose resulting from endoglucanase hydrolysis of cellulose. Our screening method was fluorescence-activated cell sorting (FACS), an attractive method for assaying mutant enzyme libraries because individual cells can be screened, sorted into distinct populations and collected very rapidly. However, FACS screening poses several challenges, in particular, maintaining the link between genotype and phenotype because most enzyme substrates do not remain associated with the cells. We employed a technique where whole cells were encapsulated in cell-like structures along with the enzyme substrate. We used RNDM, in combination with whole cell encapsulation, to create and screen mutant β-glucosidase libraries. A mutant was isolated that, compared to the wild type, had higher specific and catalytic efficiencies (k _cat/K _M) with p-nitrophenol-glucopyranoside and -galactopyranoside, an increased catalytic turnover rate (k _cat) with cellobiose, an improvement in catalytic efficiency with lactose and reduced inhibition (K _i) with galactose and lactose. This mutant had three amino acid substitutions and one was located near the active site.     

Modification of β2 I by glutardialdehydeI by glutardialdehyde

Autoantibodies from patients with antiphospholipid syndrome (APS) recognize an epitope on β₂glycoprotein I (β₂GPI) only when native β₂GPI is adsorbed on surfaces composed of anionic phospholipids or oxidized polystyrene, β₂GPI was modified with the crosslinking agent, glutardialdehyde (GDA), which induced exposure of the anti-β₂GPI epitope at GDA: β₂GPI mol ratios in the range of 500–2000. A second crosslinking agent, dimethyl-suberimidate (DMS), did not expose the epitope, which may be a consequence of its having less tendency than GDA to form intermolecular links. SDS-PAGE experiments demonstrate that GDA does promote extensive intermolecular crosslinking of β₂GPI, and DMS does not. Formaldehyde also reacts with the lysine residues of β₂GPI, but does not expose the epitope. The circular dichroism spectra of native and modified β₂GPI confirm that GDA induces changes in conformation that are qualitatively different from those caused by formaldehyde. These data provide evidence that binding of lysine residues is not a sufficient condition for exposure of the autoepitope, and also support the likelihood that β₂GPI antibodies bind only to aggregates of the protein. Thus, by synthesizing an active holoantigen of β₂GPI, conditions were defined that are necessary for binding of human autoantibodies. The authors also suggest that treatment of phospholipid-binding proteins with chemical agents might provide a strategy to modify their structure and permit exposure of epitopes, resulting in synthetic antigens for therapeutic and diagnostic use.     

Preparation of trehalase inhibitor validoxylamine A by biocatalyzed hydrolysis of validemycin a with honeybee (Apis cerana fabr.) β-glucosidase

Validoxylamine A is structurally similar to trehalose and acts a potent competivive inhibitor of trehalase. It has recently been receiving increased attention as a potential material for the development of new insecticides or drugs. In this study, β-glucosidase extracted from honeybees (Apis cerana Fabr.) was used as a catalyst to produce validoxylamine A through enzymatic hydrolysis of validamycin A. β-Glucosidase was separated and purified from honeybees, and its characteristics were examined. The results showed that β-glucosidase was stable across a range of temperatures from 30 to 40°C and across a relatively wide range of pH values from 5.0 to 7.5. Investigation of the biocatalyzed hydrolysis process from validamycin A to validoxylamine A with β-glucosidase revealed that both the substrate (validamycin A) and the product (validoxylamine A) inhibited β-glucosidase activity. The inhibition constant of the substrate K value was 5.01 mM, and that of the product K _ip value was 1.32 mM. This product inhibition was competitive.