Over-production of β-glucosidase by Aspergillus niger mutant from lignocellulosic biomass

The maximum yield of -glucosidase by A. niger KK2 mutant, grown on the basal medium for 7 days, was 514 I U g^–1 ground rice straw, and was about twice those obtained from wheat straw or bran by previous researchers. Optimal activity of -glucosidase was at 60–70 °C and pH 4.8.     

Optimization of β-glucosidase production from Aspergillus niger

本研究对Aspergillus niger Glu05生产β-葡萄糖苷酶的培养基组分及培养条件进行了优化.优化后的培养基组成和培养条件分别为:麸皮4％,tryptone 4％,1μmol MnSO4,1μmol NaCl,KH2PO40.2％,oH自然,摇床转速250 r/min,培养温度30℃,培养周期5d.优化后发酵液中酶活力达到44.11 IU/mL,与初始的产酶水平32.87 IU/mL相比,提高了36％.     

Kinetic properties of β-glucosidase from Aspergillus ornatus

Summary Kinetic properties of extracellular -glucosidase from Aspergillus ornatus were determined. The pH and temperature optima for the enzyme were found to be 4.6 and 60°C, respectively. Under these conditions, the enzyme exhibited a K _m (p-nitrophenyl--glucoside) value of 0.76±0.11 mM. The activation energy for the enzyme was 11.8 kcal/mol. Several divalent metal ions inhibited -glucosidase activity, some of which showed inhibition of enzyme activity only at higher concentrations. Ag²⁺ was the most potent inhibitor. A metal chelating agent, EDTA, also inhibited -glucosidase activity. Except for trehalose, glucose, glucono--lactone, cellobiose, gentiobiose, laminaribiose, maltose and isomaltose inhibited -glucosidase activity. Glucose was found to be a competitive inhibitor, whereas glucono--lactone and other -linked disaccharides were noncompetitive (mixed) inhibitors of the enzyme.     

Production and characterization of β-glucosidase from Aspergillus niger fermentation: Application for organic fraction of municipal solid waste hydrolysis and methane enhancement

The anaerobic digestion of the organic fraction of municipal solid waste (OFMSW) is currently an attractive treatment process with energy production in the form of biogas. Hydrolysis is the rate-limiting step for the anaerobic digestion of solid wastes. Thus, in the present study fungal enzymatic pretreatment of OFMSW was applied to enhance biogas production. Two enzyme cocktails rich on β-glucosidase were produced from submerged fermentation of Aspergillus niger on basal medium using OFMSW as carbon source and urea (Urea cocktail) and Ulva rigida as nitrogen source (Ulva cocktail). Ulva cocktail displayed an important effect on OFMSW solubilization. Therefore, an increase of reducing sugar concentration about 60% was obtained which was in correlation with chemical oxygen demand (COD) increase. The performance of enzymatic pretreatment on anaerobic digestion of OFMSW was studied by conducting biochemical methane potential tests. Results showed that the enzymatic pretreatment improved methane yield of OFMSW even at high solid concentration. High methane yield about 500 ml/g total volatile solid was obtained, which corresponds up to 68% enhancement over the control.     

N-phenylglucosylamine hydrolysis: a mechanistic probe of β-glucosidase

The spontaneous hydrolysis of glycosylamines, where the aglycone is either a primary amine or ammonia, is over a hundred million-times faster than that of O- or S-glycosides. The reason for this (as pointed out by Capon and Connett in 1965) is that, in contrast to the mechanism for O- or S-glycoside hydrolysis, hydrolysis of these N-glycosides (e.g., glc-NHR) involves an endocyclic C-O bond cleavage resulting in formation of an imine (iminium ion) which then reacts with water. Since ring-opening is kinetically favored with glycosylamines, compounds such as phenylglucosylamine can be a useful probes of enzymes that have been suggested to possibly follow this mechanism. With β-glucosidase from sweet almonds, the enzyme is highly efficient in catalyzing the hydrolysis of phenyl glucoside (k_cat/k_non ∼ 10¹⁴) and phenyl thioglucoside (k_cat/k_non ∼ 10¹⁰) while with either the almond or the Aspergillus niger enzyme or with yeast α-glucosidase, there is no detectable catalysis of phenylglucosylamine hydrolysis (k_cat/k_non < 20). These results are consistent with the generally accepted mechanism involving exocyclic bond cleavage by these enzymes.     

Synthesis of cello-oligosaccharides from cellobiose with β-glucosidase II from Aspergillus niger

An extracellular -glucosidase II of Aspergillus niger catalysed the synthesis of cello-oligosaccharides from cellobiose (15%, w/v). The enzyme was stable at and below 4°C for at least 230 days and also stable at 30°C with the presence of 2.0% (w/v) cellobiose. The maximum yield of cello-oligosaccharides was about 30% (mol/mol), based on cellobiose (130 mg/mL) consumed. © Rapid Science Ltd. 1998     

Production of β-glucosidase by Aspergillus terreus

The production of -glucosidase by Aspergillus terreus was investigated in liquid shake cultures. Enzyme production was maximum on the 7th day of growth (2.18 U/ml) with the initial pH of the medium in the range of 4.0–5.5. Cellulose (Sigmacell Type 100) at 1.0% (wt/vol) gave maximum -glucosidase activity among the various soluble and insoluble carbon sources tested. Potassium nitrate was a suitable nitrogen source for enzyme production. Triton X-100 at 0.15% (vol/vol) increased the enzyme levels of A. terreus. The test fungal strain showed an ability to ferment glucose to ethanol.     

Synthesis of alkyl β-glucosides from cellobiose with Aspergillus niger β-glucosidase II

An extracellular -glucosidase II of Aspergillus niger catalyzed the synthesis of methyl -glucoside and ethyl -glucoside with 5.0% (v/v) cellobiose as glucosyl donor in a biphasic media containing 20% (v/v) methanol and 30% (v/v) ethanol, respectively. The maximum yield of methyl -glucoside and ethyl -glucoside was 83% (mol/mol; 12 mg/ ml) and 53% (mol/mol; 5.5 mg/ml), based on cellobiose consumed. © Rapid Science Ltd. 1998     

Heterologous production and biochemical characterization of a new highly glucose tolerant GH1 β-glucosidase from Anoxybacillus thermarum

The enzymatic lignocellulosic biomass conversion into value-added products requires the use of enzyme-rich cocktails, including β-glucosidases that hydrolyze cellobiose and cellooligosaccharides to glucose. During hydrolysis occurs accumulation of monomers causing inhibition of some enzymes; thus, glucose/xylose tolerant β-glucosidases could overcome this drawback. The search of new tolerant enzymes showing additional properties, such as high activity, wide-pH range, and thermal stability is very relevant to improve the bioprocess. We describe a novel β-glucosidase GH1 from the thermophilic Anoxybacillus thermarum (BgAt), which stood out by the robustness combination of great glucose/xylose tolerance, thermal stability, and high Vmax. The recombinant his-tagged-BgAt was overexpressed in Escherichia coli, was purified in one step, showed a high glucose/xylose tolerance, and activity stimulation (presence of 0.4 M glucose/1.0 M xylose). The optimal activity was at 65 °C - pH 7.0. BgAt presented an extraordinary temperature stability (48 h – 50 °C), and pH stability (5.5–8.0). The novel enzyme showed outstanding Vmax values compared to other β-glucosidases. Using p-nitrophenyl-β-d-glucopyranoside as substrate the values were Vmax (7614 U/mg), and K_M (0.360 mM). These values suffer a displacement in Vmax to 14,026 U/mg (glucose), 14,886 U/mg (xylose), and K_M 0.877 mM (glucose), and 1.410 mM (xylose).     

Construction of a recombinant Trichoderma reesei strain expressing Aspergillus aculeatus β-glucosidase 1 for efficient biomass conversion

To develop a Trichoderma reesei strain appropriate for the saccharification of pretreated cellulosic biomass, a recombinant T. reesei strain, X3AB1, was constructed that expressed an Aspergillus aculeatus β-glucosidase 1 with high specific activity under the control of the xyn3 promoter. The culture supernatant from T. reesei X3AB1 grown on 1% Avicel as a carbon source had 63- and 25-fold higher β-glucosidase activity against cellobiose compared to that of the parent strain PC-3-7 and that of the T. reesei recombinant strain expressing an endogenous β-glucosidase I, respectively. Further, the xylanase activity was 30% lower than that of PC-3-7 due to the absence of xyn3. X3AB1 grown on 1% Avicel-0.5% xylan medium produced 2.3- and 3.3-fold more xylanase and β-xylosidase, respectively, than X3AB1 grown on 1% Avicel. The supernatant from X3AB1 grown on Avicel and xylan saccharified NaOH-pretreated rice straw efficiently at a low enzyme dose, indicating that the strain has good potential for use in cellulosic biomass conversion processes.     

Production of high activity Aspergillus niger BCC4525 β-mannanase in Pichia pastoris and its application for mannooligosaccharides production from biomass hydrolysis

A cDNA encoding β-mannanase was cloned from Aspergillus niger BCC4525 and expressed in Pichia pastoris KM71. The secreted enzyme hydrolyzed locust bean gum substrate with very high activity (1625 U/mL) and a relatively high k_cat/K_m (461 mg^?1 s^?1 mL). The enzyme is thermophilic and thermostable with an optimal temperature of 70 °C and 40% retention of endo-β-1,4-mannanase activity after preincubation at 70 °C. In addition, the enzyme exhibited broad pH stability with an optimal pH of 5.5. The recombinant enzyme hydrolyzes low-cost biomass, including palm kernel meal (PKM) and copra meal, to produce mannooligosaccharides, which is used as prebiotics to promote the growth of beneficial microflora in animals. An in vitro digestibility test simulating the gastrointestinal tract system of broilers suggested that the recombinant β-mannanase could effectively liberate reducing sugars from PKM-containing diet. These characteristics render this enzyme suitable for utilization as a feed additive to improve animal performance.     

Identification of glucose tolerant acid active β-glucosidases from thermophilic and thermotolerant fungi

Thirteen thermophilic and thermotolerant fungal cultures isolated from composting soils produced diverse β-glucosidases as indicated by zymograms of PAGE developed using 4-methylumbelliferyl-β-d-glucoside. IEF profiling revealed the presence of 28 β-glucosidases separated on the basis of their pI. Eleven of the β-glucosidases were active under acidic conditions. Two β-glucosidase isoforms, ASCβG-II of Aspergillus caespitosus and HIβG-I of Humicola insolens were resistant to inhibition by glucose and were active in the presence of 300 and 100 (mM) glucose, respectively.     

Site-saturation mutagenesis for β-glucosidase 1 from Aspergillus aculeatus to accelerate the saccharification of alkaline-pretreated bagasse

Applied Microbiology and Biotechnology - Aspergillus aculeatus β-glucosidase 1 (AaBGL1) is one of the best cellobiose hydrolytic enzymes without transglycosylation products, among...     

Immobilized β-glucosidase on magnetic chitosan microspheres for hydrolysis of straw cellulose

β-Glucosidase immobilized on magnetic chitosan microspheres for potential recycling usage in hydrolysis of cellulosic biomass was investigated. The immobilized enzyme had an activity of 6.4 U/g support under optimized condition when using cellobiose as substrate. Immobilization resulted in less increase of the apparent K_m, low drift of the optimal pH, as well as improved stability relative to the free enzyme. The immobilized β-glucosidase was applied to enzymatic hydrolysis of corn straw to produce 60.2 g/l reducing sugar with a conversion rate of 78.2% over the course of a 32-h reaction. This conversion rate was maintained above 76.5% after recycling the enzyme for use in eight batches (total 256 h), showing favorable operational stability of the immobilized enzyme.     

Effect of pH on transfructosylation and hydrolysis by β-fructofuranosidase from Aspergillus oryzae

 β-Fructofuranosidase was purified from commercial alkaline protease (Aspergillus oryzae origin). The optimal pH of its transfructosylating activity was more alkaline (pH 8) than that of its hydrolyzing activity (pH 5). In the case of a 24-h reaction with sucrose, the hydrolysis and transfructosylation reaction were optimal at pH 4–5 and pH 8, respectively. In the reaction at pH 8 1-kestose and nystose were the main fructooligosaccharides produced. The transfer ratio was hardly different between pH 5 and pH 8 early in the reaction, but the transfer products (1-kestose and nystose) were decreased at pH 5 as the reaction proceeded because of their hydrolysis. Received: 18 January 1995/Received last revision: 23 August 1995/Accepted: 13 September 1995     

Investigation of apigenin-7-O-β-glucoside hydrolysis by β-glucosidase from almonds

Summary The enzymatic transformation of apigenin-7-O--glucoside into apigenin is effected using -glucosidase from almonds. Kinetic studies gave the following values K_m=7.63×10^–4mol/dm³, v_max=10.37 mol mg^–1 min^–1 and E₁=65.21 kJ/mol when the substrate was pure apigenin-7-O--glucoside, while in case of apigenin-7-O--glucoside from dry extract of camomile ligulate flowers E₂ = 65.57 kJ/mol.Nomenclature S_o initial substrate concentration (g/100 cm³) - P product concentration (g/100 cm³) - k reaction rate constant (min^–1) - t reaction time (min)     

β-glucosidases from a new Aspergillus species can substitute commercial β-glucosidases for saccharification of lignocellulosic biomass

β-Glucosidase activity plays an essential role for efficient and complete hydrolysis of lignocellulosic biomass. Direct use of fungal fermentation broths can be cost saving relative to using commercial enzymes for production of biofuels and bioproducts. Through a fungal screening program for β-glucosidase activity, strain AP (CBS 127449, Aspergillus saccharolyticus ) showed 10 times greater β-glucosidase activity than the average of all other fungi screened, with Aspergillus niger showing second greatest activity. The potential of a fermentation broth of strain AP was compared with the commercial β-glucosidase-containing enzyme preparations Novozym 188 and Cellic CTec. The fermentation broth was found to be a valid substitute for Novozym 188 in cellobiose hydrolysis. The Michaelis-Menten kinetics affinity constant as well as performance in cellobiose hydrolysis with regard to product inhibition were found to be the same for Novozym 188 and the broth of strain AP. Compared with Novozym 188, the fermentation broth had higher specific activity (11.3?U/mg total protein compared with 7.5 U/mg total protein) and also increased thermostability, identified by the thermal activity number of 66.8 vs. 63.4?°C for Novozym 188. The significant thermostability of strain AP β-glucosidases was further confirmed when compared with Cellic CTec. The β-glucosidases of strain AP were able to degrade cellodextrins with an exo-acting approach and could hydrolyse pretreated bagasse to monomeric sugars when combined with Celluclast 1.5L. The fungus therefore showed great potential as an onsite producer for β-glucosidase activity.     

Highly selective hydrolysis for the outer glucose at the C-20 position in ginsenosides by β-glucosidase from Thermus thermophilus and its application to the production of ginsenoside F2 from gypenoside XVII

β-Glucosidase from Thermus thermophilus has specific hydrolytic activity for the outer glucose at the C-20 position in protopanaxadiol-type ginsenosides without hydrolysis of the inner glucose. The hydrolytic activity of the enzyme for gypenoside XVII was optimal at pH 6.5 and 90 °C, with a half-life of 1 h with 3 g enzyme l^?1 and 4 g gypenoside XVII l^?1. Under the optimized conditions, the enzyme converted the substrate gypenoside XVII to ginsenoside F₂ with a molar yield of 100 % and a productivity of 4 g l^?1 h^?1. The conversion yield and productivity of ginsenoside F₂ are the highest reported thus far among enzymatic transformations.     

Characterization of the co-purified invertase and β-glucosidase of a multifunctional extract from Aspergillus terreus

The filamentous fungus Aspergillus terreus secretes both invertase and β-glucosidase when grown under submerged fermentation containing rye flour as the carbon source. The aim of this study was to characterize the co-purified fraction, especially the invertase activity. An invertase and a β-glucosidase were co-purified by two chromatographic steps, and the isolated enzymatic fraction was 139-fold enriched in invertase activity. SDS-PAGE analysis of the co-purified enzymes suggests that the protein fraction with invertase activity was heterodimeric, with subunits of 47 and 27 kDa. Maximal invertase activity, which was determined by response surface methodology, occurred in pH and temperature ranges of 4.0–6.0 and 55–65 °C, respectively. The invertase in co-purified enzymes was stable for 1 h at pH 3.0–10.0 and maintained full activity for up to 1 h at 55 °C when diluted in water. Invertase activity was stimulated by 1 mM concentrations of Mn²⁺ (161 %), Co²⁺ (68 %) and Mg²⁺ (61 %) and was inhibited by Al³⁺, Ag⁺, Fe²⁺ and Fe³⁺. In addition to sucrose, the co-purified enzymes hydrolyzed cellobiose, inulin and raffinose, and the apparent affinities for sucrose and cellobiose were quite similar (K_M = 22 mM). However, in the presence of Mn²⁺, the apparent affinity and V_max for sucrose hydrolysis increased approximately 2- and 2.9-fold, respectively, while for cellobiose, a 2.6-fold increase in V_max was observed, but the apparent affinity decreased 5.5-fold. Thus, it is possible to propose an application of this multifunctional extract containing both invertase and β-glucosidase to degrade plant biomass, thus increasing the concentration of monosaccharides obtained from sucrose and cellobiose.     

Properties of β-glucosidase purified from Aspergillus niger mutants USDB 0827 and USDB 0828

Summary Two extracellular -glucosidases (EC 3.2.1.21) were isolated from Aspergillus niger USDB 0827 and A. niger USDB 0828, and their physical and kinetic properties studied. Both enzymes were very similar in terms of molecular size (230000 Da), pH optimum (pH 4.6), temperature optimum (65° C), stability at high temperatures and substrate preferences. They were capable of hydrolysing -linked disaccharides, phenyl -d-glucoside, p-nitrophenyl -d-glucoside (PNPG), o-nitrophenyl -d-glucoside, salicin and methyl -d-glucoside but lacked activity towards -linked disaccharides, a range of p-nitrophenyl monoglycosides and p-nitrophenyl diglycosides. Both -glucosidases were better at hydrolysing cellobiose than cellotriose, cellotetraose or cellopentaose. For both enzymes, glucose showed competitive inhibition with PNPG as substrate but had no effect with cellobiose. However, the two -glucosidases differed in inhibition by glucono-1,5-lactone and affinity for cellobiose. -Glucosidase from A. niger USDB 0827 also gave lower specific activity, and was more susceptible to metal ions (Ag⁺, Fe²⁺ and Fe³⁺) inhibition than that of A. niger USDB 0828. Correspondence to: Y. K. Hoh