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.     

Comparative study of the production of extracellular β-glucosidase by four different strains of Aspergillus using submerged fermentation

Four strains of Aspergillus (Aspergillus niger CDBB-H-176, A. niger CDBB-H-175, A. niger ATCC 9642, and Aspergillus terreus CDBB-H-194) were used to produce extracellular β-glucosidase. Using an orthogonal experimental design (L₉), we optimized the parameters of culture medium to maximize the activity of β-glucosidase. The optimal conditions (same for the four strains) were as follows: temperature, 30°C; pH, 6.0; orbital agitation, 200?rpm; concentration of sucrose, 0.5% (w/v). The most productive strain was A. niger CDBB-H-175, with a yield of 701.2?U/mL. In a second stage, we optimized (L₁₈) the concentration of nutrients in the culture medium to determine whether this modification would increase the production of β-glucosidase. The optimal conditions for A. niger CDBB-H-175 were as follows (%, w/v): NaNO₃, 0.3; KCl, 0.3; KH₂PO₄, 0.15; NH₄NO₃, 0.1; NH₄H₂PO₄, 0.1; MgSO₄?·?7H₂O, 0.05; yeast extract, 0.1. The production of β-glucosidase under these conditions was 1207.9?U/mL. Enzymatic assays were used to characterize the enzyme; the optimum temperature and pH of β-glucosidase produced by the four selected micro-organisms were found to be 65°C and 5.0, respectively. We determined the Michaelis–Menten constants (K_m) only for A. niger CDBB-H-175 and CDBB-H-176; the values were 2.7 and 2.2?mM, respectively.     

Production and characterization of β-glucosidases from different Aspergillus strains

-D-Glucosidase enzymes (-D-glucoside glucohydrolase, EC 3.2.1.21) from different Aspergillus strains (Aspergillus phoenicis, A. niger and A. carbonarius) were examined with respect to the enzyme production of the different strains using different carbon sources and to the effect of the pH and temperature on the enzyme activity and stability. An efficient and rapid purification procedure was used for purifying the enzymes. Kinetic experiments were carried out using p-nitrophenyl -D-glucopyranoside (pNPG) and cellobiose as substrates. Two different fermentation methods were employed in which the carbon source was glucose or wheat bran. Aspergillus carbonarius proved to be the less effective strain in -glucosidase production. Aspergillus phoenicis produced the highest amount of -glucosidase on glucose as carbon source however on wheat bran A. niger was the best enzyme producer. Each Aspergillus strain produced one single acidic -glucosidase with pI values in the range of pH 3.52–4.2. There was no significant difference considering the effect of the pH and temperature on the activity and stability among the enzymes from different origins. The enzymes examined have only -glucosidase activity. The kinetic parameters showed that all enzymes hydrolysed pNPG with higher efficiency than cellobiose. This shows that hydrophobic interaction plays an important role in substrate binding. The kinetic parameters demonstrated that there was no significant difference among the enzymes from different origins in hydrolysing pNPG and cellobiose as the substrates.     

Production of cellulases by Thermomucor indicae-seudaticae: characterization of a thermophilic β-glucosidase

Abstract

The current study evaluated the production and characterization of β-glucosidase by the thermophilic fungus Thermomucor indicae-seudaticae in solid-state fermentation of wheat bran. Isolated fungi have significant amounts of β-glucosidase, an enzyme that may be applied to different industrial processes, such as the production of fuels, food, and other chemical compounds. Maximal enzyme activity occurred in pH 3.5–4.5 and at 70?°C. The enzyme exhibited high thermostability, for 1?h, up to 60?°C, and good tolerance to glucose (10?mM) and ethanol (10%). The optimization of fermentative parameters on the production of β-glucosidase was carried out by evaluating the best supplementary nutrient source, pH of nutrient solution, initial substrate moisture and fermentation temperature. The optimization of the above fermentation parameters increased enzyme activity by 120.0%. The highest enzymatic activity (164.0?U/g) occurred with wheat bran containing 70% initial moisture, supplemented with 1.0% (NH₄)₂SO₄ solution at pH 5.5–6.0 and fungus incubated at 40?°C. A more detailed study of β-glucosidase suggested that Sulfur is an important component of the main amino acid present in this enzyme. The enhancer of the enzyme activity occurred when the fungus was grown on wheat bran supplemented with a sulfur-containing solution. In fact, increasing the concentration of sulfur in the solution increased its activity.     

The production of β-glucosidase in shake-flasks by Aspergillus wentii

Studies in shake-flasks showed that Aspergillus wentii produces the maximum activity of β-glucosidase among the cultures tested. The activity against cellobiose was about 2–3 fold that against 4NPG. Aspergillus wentii produced a maximum activity of 16.5 U/ml in 14 days on malt extract. It also produced a comparable amount on other simple soluble sugars, which indicates that it is constitutive and does not require an inducer. Peptone was found to the best nitrogen source for β-glucosidase production. Optimum C/N ratio was found to be 7.3. Phosphate, magnesium and trace metals did not play significant roles in the production of β-glucosidase when they were used with malt extract as a carbon source. An inoculum of 6% (v/v) of 20-h-old culture grown on malt extract produced the maxium β-glucosidase activity.     

Hyper production of β-glucosidase by an Aspergillus sp.

Summary An Aspergillus sp. was isolated which secreted high levels of -glucosidase in growth medium. The maximum activity(10 IU/ml of -glucosidase and 22.6 IU/ml of cellobiase) was obtained in cellulose medium supplemented with wheat bran. The pH and temperature optima for this enzyme were 4.5 and 65°C respectively.NCL Communication No. 3616     

Purification and properties of a thermostable extracellular β-D-xylosidase produced by a thermotolerant Aspergillus phoenicis

A β-D-xylosidase was purified from cultures of a thermotolerant strain of Aspergillus phoenicis grown on xylan at 45°C. The enzyme was purified to homogeneity by chromatography on DEAE-cellulose and Sephadex G-100. The purified enzyme was a monomer of molecular mass 132 kDa by gel filtration and SDS-PAGE. Treatment with endoglycosidase H resulted in a protein with a molecular mass of 104 kDa. The enzyme was a glycoprotein with 43.5% carbohydrate content and exhibited a pI of 3.7. Optima of temperature and pH were 75°C and 4.0–4.5, respectively. The activity was stable at 60°C and had a K _m of 2.36 mM for p-nitrophenyl-β-D-xylopiranoside. The enzyme did not exhibit xylanase, cellulase, galactosidase or arabinosidase activities. The purified enzyme was active against natural substrates, such as xylobiose and xylotriose. Journal of Industrial Microbiology & Biotechnology (2001) 26, 156–160. Received 23 June 2000/ Accepted in revised form 29 September 2000     

Production of β-glucosidase byCladosporium resinae

Summary Cladosporium resinae QM 7998 produced high activities of extracellular and constitutive -glucosidase when grown on a variety of sugars or cellulose. Starch and ribose induced enzyme synthesis several fold.Cladosporium resinae could utilize agricultural waste residues for growth and -glucosidase production. The initial pH of the medium had a marked effect on enzyme prowduction and optimum pH was between 4.0 and 5.0 depending on the assay method. Mixed culturing ofC. resinae with yeasts, viz.Saccharomyces cerevisiae andCandida utilis, increased the -glucosidase production while that with other fungi decreased the enzyme yield. The- glucosidase preparation fromC. resinae significantly increased the saccharification of rice and wheat straw (untreated or delignified) withTrichoderma reesei QM 9414 cellulase preparation.

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Résumé Cladosporium resinae QM 7998 produit des concentrations élevées de -glucosidase tant extracellulaire que constitutive lorsqu'elle croît sur une variété de sucres ou sur la cellulose. On a trouvé que l'amidon et le ribose augmentent de plusieurs fois la quantité d'enzyme synthétisée.Cladosporium resinae peut utiliser des résidus agricoles pour sa croissance et pour la production de -glucosidase. Le pH initial du milieu exerce un effet marqué sur la production d'enzyme et le pH optimum est compris entre 4.0 et 5.0 selon les conditions de l'essai. La croissance mixte deCladosporium resinae avec diverses levures, notammentSaccharomyces cerevisiae etCandida utilis, augmente la production de -glucosidase tandis que celle avec d'autres moisissures diminue le rendement en enzyme. La -glucosidase deCladosporium resinae augmente de manière significative la saccharification des pailles de riz et de froment (non-traitées ou délignifiées) traités par la cellulase deTrichoderma reesei QM 9414.

     

Production of extracellular β-mannanases by yeasts and yeast-like microorganisms

A new screening method for simultaneous detection of endo-β-1,4-mannanase and endo-β-1,4-xylanase producing microorganisms is described. Two differently dyed substrate Ostazin Brilliant Red-galactomannan and Remazol Brilliant Blue-xylan were incorporated into the same agar media. Decolorizing of one or both substrates around the cell colonies indicates secretion of the corresponding enzyme(s). The method was used to screen 449 yeasts and yeast-like microorganisms belonging to 68 different genera. The secretion of endo-β-1,4-mannanases and/or endo-β-1,4-xylanases was found within 10 genera (42 positive strains out of 261 tested). A low frequency of occurrence of endo-β-1,4-mannanases was observed within the generaCryptococcus (1 positive strain out of 15 tested),Geotrichum (1 of 6) andPichia (1 of 35). The highest frequency of occurrence of endo-β-1,4-mannanases was found within the generaStephanoascus (2 of 2) andAureobasidium (14 of 14). Strains hydrolyzing Ostazin Brilliant Red-galactomannan were cultivated in liquid media containing 1 % locust bean gum. The best producers of extracellular endo-β-1,4-mannanases were found to be the strains ofAureobasidium pullulans.     

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.     

Production of a thermostable extracellular lipase by Kluyveromyces marxianus

Kluyveromyces marxianus was grown in submerged culture in a complex medium with several potential inducers of lipolytic activity (triacylglycerols, fatty acids). The highest extracellular lipolytic enzyme production (about 80 U ml^–1 in 3 d) was obtained when the medium was supplemented with 2 g urea l^–1 plus 5 g tributyrin l^–1. Addition of surfactants (1 g l^–1) did not improve production. The lipase had a high thermal stability in aqueous solution (73% residual activity after 9 d at 50 °C, 16 min half-life time at 100 °C). It was also stable at acidic pH and showed good tolerance to organic solvents (70% residual activity after 2 d in n-hexane of cyclohexane).     

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.     

A highly glucose-tolerant GH1 β-glucosidase with greater conversion rate of soybean isoflavones in monogastric animals

In the feed industry, β-glucosidase has been widely used in the conversion of inactive and bounded soybean isoflavones into active aglycones. However, the conversion is frequently inhibited by the high concentration of intestinal glucose in monogastric animals. In this study, a GH1 β-glucosidase (AsBG1) with high specific activity, thermostability and glucose tolerance (IC₅₀ = 800 mM) was identified. It showed great glucose tolerance against substrates with hydrophobic aryl ligands (such as pNPG and soy isoflavones). Using soybean meal as the substrate, AsBG1 exhibited higher hydrolysis efficiency than the GH3 counterpart Bgl3A with or without the presence of glucose in the reaction system. Furthermore, it is the first time to find that the endogenous β-glucosidase of soybean meal, mostly belonging to GH3, plays a role in the hydrolysis of soybean isoflavones and is highly sensitive to glucose. These findings lead to a conclusion that the GH1 rather than GH3 β-glucosidase has prosperous application advantages in the conversion of soybean isoflavones in the feed industry.

     

Production of β-fructofuranosidase by Aspergillus japonicus in batch and fed-batch cultures

Summary A comparison of -fructofuranosidase (FFase, EC 3.2.l.26) production by Aspergillus japonicus TIT-90076 in batch and fed-batch cultures was investigated in shaken flasks. Results showed that fed-batch cultivation of A. japonicus using intermittent sucrose supply produced more FFase than batch culture, and the maximal enzyme production was 910 units ml^–1, which was about 20% higher than that in the batch cultures.     

Trans-glycosylation capacity of a highly glycosylated multi-specific β-glucosidase from Fusarium solani

An extracellular β-glucosidase from Fusaruim solani cultivated on wheat bran was purified by only two chromatographic steps. The purified enzyme exhibited optimal temperature and pH at 60 °C and pH 5, respectively. The purified β-glucosidase behaves as a very large protein due to its high degree of glycosylation. More interestingly, the endoglycosidase H (Endo H) treatment led to 97.55% loss of its initial activity after 24 h of treatment. Besides, the addition of Tunicamycin (nucleoside antibiotic blocking the N-glycosylation first step) during the culture of the fungus affected seriously the glycosylation of the enzyme. Both treatments (endo H and Tunicamycin) strengthened the idea that the hyperglycosylation is involved in the β-glucosidase activity and thermostability. This enzyme was also shown to belong to class III of β-glucosidases (multi-specific) since it was able to act on either cellobiose, gentiobiose or sophorose which are disaccharide composed of two units of d-glucose connected by β1–4, β1–6 and β1–2 linkage, respectively. The β-glucosidase activity was strongly enhanced by ferrous ion (Fe²⁺) and high ionic strength (1 M KCl). The purified enzyme exhibited an efficient transglycosylation capacity allowing the synthesis of cellotriose and cellotetraose using cellobiose as donor.

     

Production of β-carotene by a mutant of Rhodotorula glutinis

Wild strains of Rhodotorula glutinis and R. rubra were investigated concerning their carotenoid production, proportion of beta-carotene and cell mass yield. R. glutinis NCIM 3353 produced 2.2 mg carotenoid/l in 72 h; and the amount of beta-carotene was 14% (w/w) of the total carotenoid content (17 microg/g cell dry weight). It was subjected to mutagenesis using UV radiation for strain improvement. Out of 2,051 isolates screened, the yellow coloured mutant 32 produced 120-fold more beta-carotene (2,048 microg/g cell dry weight) than the parent culture in 36 h, which was 82% (w/w) of the total carotenoid content. Mutant 32 was grown on different carbon and nitrogen sources. The best yield of beta-carotene (33+/-3 mg/l) was obtained when glucose and yeast extract were supplied as carbon and nitrogen sources, respectively. Divalent cation salts further increased the total carotenoid content (66+/-2 mg/l) with beta-carotene as the major component (55+/-2%, w/w).     

Simultaneous overproduction of extracellular endoglucanase and intracellular β-glucosidase by genetically engineered Bacillus subtilis

Summary Simultaneous overproduction of intracellular -glucosidase and extracellular endoglucanase was attempted by constructing two artificial operon systems comprising the -glucosidase-endoglucanase gene(E) or the endoglucanase--glucosidase gene(E) under the control of a strong engineered promoter, BJ27U88 and expressing them in Bacillus subtilis DB104. Two artificial operon systems contained 30 bp or 5 bp gap between the termination codon of the upstream gene and the SD sequence of the downstream gene, respectively. These operon systems were expressed well in B. subtilis and overproduced the -glucosidase cell extract as well as the endoglucanase supernatant. The level of expression in the operon system was almost the same as that in a single expression system.     

Formation of γ-glutamylglycyglycine by extracellular glutaminase of Aspergillus oryzae

γ-Glutamylglycylglycine (γ-GluGlyGly) was formed through the γ-glutamyltranspeptidase (GGT) reaction catalyzed by glutaminase in a water extract of wheat bran koji obtained with Aspergillus oryzae MA-27-IM. The yield of γ-GluGlyGly was about 18% from l-glutamine in a reaction mixture containing 50 mM l-glutamine, 50 mM glycylglycine, and the extract (0.1 unit ml as GGT activity) in a 100 mM Tris-HCl buffer solution (pH 7.2), which was incubated for 7 h at 30°C. The γ-GluGlyGly formed was purified by ion exchange chromatographies, and the identified by chemical and enzymatic methods as well as by infrared and PMR spectroscopic analyses.     

Purification and characterization of extracellular β-glucosidase from Myceliophthora thermophila

The extracellular -glucosidase has been purified from culture broth of Myceliophthora thermophila ATCC 48104 grown on crystalline cellulose. The enzyme was purified approximately 30-fold by (NH₄)₂SO₄ precipitation and column chromatography on DEAE-Sephadex A-50, Sephadex G-200 and DEAE-Sephadex A-50. The molecular mass of the enzyme was estimated to be about 120 kD by both sodium dodecyl sulphate gel electrophoresis and gel filtration chromatography. It displayed optimal activity at pH 4.8 and 60°C. The purified enzyme in the absence of substrate was stable up to 60°C and pH between 4.5 and 5.5. The enzyme hydrolysed p-nitrophenyl--d-glucoside, cellobiose and salicin but not carboxymethyl cellulose or crystalline cellulose. The K _m of the enzyme was 1.6mm for p-nitrophenyl--d-glucoside and 8.0mm for cellobiose. d-Glucose was a competitive inhibitor of the enzyme with a K of 22.5mm. Enzyme K activity was inhibited by HgCl₂, FeSO₄, CuSO₄, EDTA, sodium dodecyl sulphate, p-chloromercurobenzoate and iodoacetamide and was stimulated by 2-mercaptoethanol, dithiothreitol and glutathione. Ethanol up to 1.7 m had no effect on the enzyme activity.The authors are with the Department of Microbiology, Bose Institute, 93/1, A.P.C. Road, Calcutta 700 009, India. S.K. Raha is presently with the Department of Medicine, University of Saskatchewan, Saskatoon, Canada S7N OXO.     

Production and characterization of β-1,4-glucosidase from a strain of Penicillium pinophilum

A high β-glucosidase (BGL)-producing strain was isolated and identified as Penicillium pinophilum KMJ601 based on its morphology and internal transcribed spacer rDNA gene sequence. Under the optimal culture conditions, a maximum BGL specific activity of 3.2 U ml⁻¹ (83 U mg-protein⁻¹), one of the highest levels among BGL-producing microorganisms was obtained. An extracellular BGL was purified to homogeneity by sequential chromatography of P. pinophilum culture supernatants on a DEAE-Sepharose column, a gel filtration column, and then on a Mono Q column. The relative molecular weight of P. pinophilum BGL was determined to be 120 kDa by SDS-PAGE and size exclusion chromatography, indicating that the enzyme is a monomer. The hydrolytic activity of the BGL had a pH optimum of 3.5 and a temperature optimum of 32 °C. P. pinophilum BGL showed a higher activity (V_max = 1120 U mg-protein⁻¹) than most BGLs purified from other sources. The internal amino acid sequences of P. pinophilum BGL showed a significant homology with hydrolases from glycoside hydrolase family 3. Although BGLs have been purified and characterized from several other sources, P. pinophilum BGL is distinguished from other BGLs by its high activity.