Purification of β-Glucosidase from Saccharomyces lactis Strains Y-14 and Y-1057A

Two strains of Saccharomyces lactis (Y-14 and Y-1057A), medium (B(m)) and low (B(1)) constitutive producers of beta-glucosidase, were grown in enriched medium. beta-Glucosidase was extracted by autolysis and purified by ammonium sulfate precipitation, gel filtration, and calcium phosphate gel adsorption-elution. The kinetics of release, purification, and stability of beta-glucosidase from strains Y-14 and Y-1057A were compared with the enzyme from strain Y-123. The ability of glycerol, sorbitol, and mannitol to stabilize the beta-glucosidases is presented. A lower molecular weight, labile form of the Y-14 enzyme is demonstrated. Differences in the initial specific activites of beta-glucosidase among the three strains are discussed.     

Purification of β-Glucosidase from Saccharomyces lactis Strain Y-123

Saccharomyces lactis strain Y-123, a constitutive high producer of beta-glucosidase (B(h)), was grown in an enriched medium. beta-Glucosidase, extracted most easily by cell autolysis, was purified by successive ammonium sulfate precipitation, ethyl alcohol precipitation, gel filtration, calcium phosphate gel adsorption-elution, and hydroxyapatite column chromatography. The specific activity of the enzyme increased 200-fold during the purification. The electrophoretic and catalytic properties of the enzyme did not change during the procedure. Polyacrylamide gel disc electrophoresis of the partially purified enzyme revealed one major and several minor protein-staining bands. beta-Glucosidase activity in the polyacrylamide gel columns was measured directly by assaying sections of columns frozen and sliced immediately after electrophoresis. Most of the activity coincided with the major protein-staining band. Prolonged assay produced minor activity coinciding with the less intense protein bands. Properties of the enzyme suggest that it is a single, unconjugated, intracellular, high molecular weight protein. The purification procedure is applicable to strains of S. lactis which possess alleles of the B locus for beta-glucosidase synthesis.     

Purification and Characterization of Yeast β-Glucosidases

Constitutive beta-glucosidases from Saccharomyces fragilis (Y-18) and S. dobzhanskii (Y-19) precipitated at the same concentration of ammonium sulfate. The partially purified enzymes had similar activation energies, molecular weights, affinities for certain natural and synthetic beta-glucosides, and optimal pH values for substrate hydrolysis, and they were stable over approximately the same pH range. The enzymes, however, could be clearly distinguished by other criteria. Affinities of the synthetic, sulfur-containing beta-glucosides for Y-18 enzyme were many times greater than for Y-19 enzyme. The latter enzyme was more resistant to heat. The two enzymes eluted from diethylaminoethyl cellulose at different concentrations of sodium chloride. In precipitin tests, homologous enzyme-antisera systems were highly specific. The beta-glucosidase synthesized by a hybrid, S. fragilis x S. dobzhanskii (Y-42), was unique. Characterization of this enzyme produced values which were intermediate to those for the enzymes from the parental yeast strains. Heat-inactivation slopes and Lineweaver-Burk plots for the Y-42 enzyme were anomalous. It is suggested that hydrolytic activity in Y-42 preparations is due to a spectrum of hybrid enzyme molecules composed of varying amounts of two distinct polypeptides. It is further suggested that these polypeptides may be identical to those synthesized by the parental Y-18 and Y-19 yeast strains.     

Restriction site variation,length polymorphism and changes in gene order in the mitochondrial DNA of the yeast Kluyveromyces lactics

The purpose of this work was to compare mitochondrial DNA restriction endonuclease patterns in strains of the yeast Kluyveromyces lactis, from different sources, to see how conserved is the organization of this organellar genome. The mitochondrial DNA of five independently-isolated strains and one of unknown origin were compared. Strains NRRL Y-1205, NRRL Y-8279 and NRRL Y-1140 gave identical patterns. Strain NRRL Y-1564 showed an insertion, with respect to the other three, of approximately 1250 bp. Strain W600B had also an insertion with extra restriction sites for EcoRI, HpaI, HaeIII, HincII and XbaI. On the other hand, strain Y-123 showed a restriction pattern quite different from the others.Sequences putatively encoding apocytochrome b, ATPase subunit 9 and ribosomal RNA large subunit, were localized on the physical maps of three strains. Results demonstrated that the order of these three genes shows a common feature in strains W600B and WM37 (auxotroph of Y-1140) but a different distribution in WM27 (auxotroph derived from Y-123). All these facts explain the extensive intraspecific polymorphism observed in the mtDNA of this yeast.     

NAD-linked formate dehydrogenase from methanol-grown Pichia pastoris NRRL-Y-7556

An NAD-linked formate dehydrogenase (EC 1.2.1.2.) from methanol-grown Pichia pastoris NRRL Y-7556 has been purified. The purification procedure involved ammonium sulfate fractionation, hollow-fiber H1P10 filtration, ion-exchange chromatography, and gel filtration. Both dithiothreitol (10 mm) and glycerol (10%) were required for stability of the enzyme during purification. The final enzyme preparation was homogeneous as judged by polyacrylamide gel electrophoresis and by sedimentation pattern in an ultracentrifuge. The enzyme has a molecular weight of 94,000 and consists of two subunits of identical molecular weight. Formate dehydrogenase catalyzes specifically the oxidation of formate. No other compounds tested can replace NAD as the electron acceptor. The Michaelis constants were 0.14 mm for NAD and 16 mm for formate (pH 7.0, 25 °C). Optimum pH and temperature for formate dehydrogenase activity were around 6.5–7.5 and 20–25 °C, respectively. Amino acid composition of the enzyme was also studied. Antisera prepared against the purified enzyme from P. pastoris NRRL Y-7556 form precipitin bands with isofunctional enzymes from different strains of methanol-grown yeasts, but not bacteria, on immunodiffusion plates. Immunoglobulin fraction prepared against the enzyme from yeast strain Y-7556 inhibits the catalytic activity of the isofunctional enzymes from different strains of methanol-grown yeasts.     

Identification and purification of the main components of cellulases from a mutant strain of Trichoderma viride T 100-14

A new mutant strain of fungus Trichoderma viride T 100-14 was cultivated on 1% microcrystalline cellulose (Avicel) for 120h and the resulting culture filtrate was prepared for protein identification and purification. To identify the predominant catalytic components, cellulases were separated by an adapted two-dimensional electrophoresis technique. The apparent major spots were identified by high performance liquid chromatography electrospray ionization mass (HPLC-ESI-MS). Seven of the components were previously known, i.e., the endoglucanases Cel7B (EG I), Cel12A (EG III), Cel61A (EG IV), the cellobiohydrolases Cel7A (CBH I), Cel6A (CBH II), Cel6B (CBH IIb) and the beta-glucosidase. The seven major components in the fermentation broth of T. viride T 100-14 probably constitute the essential enzymes for crystalline cellulose hydrolysis and they were further purified to electrophoretic homogeneity by a series of chromatography column. Hydrolysis studies of the purified elements revealed that three of the cellulases were classified as cellobiohydrolases due to their main activities on p-nitrophenyl-beta-d-cellobioside (pNPC). Three of the cellulases, with the abilities of hydrolyzing both carboxymethyl-cellulose (CMC) and Avicel indicate their endoglucanase activities. It deserved noting that the beta-glucosidase from the T 100-14 displayed an extremely high activity on p-nitrophenyl-beta-D-glycopyranoside (pNPG), which suggested it was a good candidate for the conversion of cellobiose to glucose.     

Hydrolysis of dietary flavonoid glycosides by strains of intestinal Bacteroides from humans.

Rutin and quercitrin are hydrolysed to quercetin, and robinin is hydrolysed to kaempferol, by faecal flora from healthy subjects. The enzymes required for these hydrolyses, namely alpha-rhamnosidase and beta-galactosidase, were produced by some strains of Bacteroides distasonis; other strains, however, synthesized beta-glucosidase. The last-named enzyme was also elaborated by Bacteroides uniformis and Bacteroides ovatus. All the enzymes were produced constitutively. A cell-free extract of B. distasonis containing beta-glucosidase displayed an enzymic activity of 1 mumol/10 min per 10 mg of protein.     

Stagonospora avenae secretes multiple enzymes that hydrolyze oat leaf saponins

The phytopathogenic fungus Stagonospora avenae is able to infect oat leaves despite the presence of avenacoside saponins in the leaf tissue. In response to pathogen attack, avenacosides are converted into 26-desglucoavenacosides (26-DGAs), which possess antifungal activity. These molecules are comprised of a steroidal backbone linked to a branched sugar chain consisting of one alpha-L-rhamnose and two (avenacoside A) or three (avenacoside B) beta-D-glucose residues. Isolates of the fungus that are pathogenic to oats are capable of sequential hydrolysis of the sugar residues from the 26-DGAs. Degradation is initiated by removal of the L-rhamnose, which abolishes antifungal activity. The D-glucose residues are then hydrolyzed by beta-glucosidase activity. A comprehensive analysis of saponin-hydrolyzing activities was undertaken, and it was established that S. avenae isolate WAC1293 secretes three enzymes, one alpha-rhamnosidase and two beta-glucosidases, that carry out this hydrolysis. The major beta-glucosidase was purified and the gene encoding the enzyme cloned. The protein is similar to saponin-hydrolyzing enzymes produced by three other phytopathogenic fungi, Gaeumannomyces graminis, Septoria lycopersici, and Botrytis cinerea, and is a family 3 beta-glucosidase. The gene encoding the beta-glucosidase is expressed during infection of oat leaves but is not essential for pathogenicity.     

Glycoprotein enzymes secreted by Aspergillus fumigatus: purification and properties of a second beta-glucosidase.

A second extracellular beta-glucosidase (betalarge) of Aspergillus fumigatus was purified to homogeneity and shown to be a glycoprotein, as determined by polyacrylamide gel electrophoresis followed by staining for protein and for carbohydrate. Its molecular weight was approximately 340,000 by gel filtration, while sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave an apparent molecular weight of 170,000, suggesting that the enzyme has two subunits. The glucosidase contained covalently bound sugars consisting of about 2 mol of glucosamine and 16 mol of mannose per mol of protein. The carbohydrate was found to be attached to the peptide via glucosaminyl leads to peptide linkage, possibly to asparagine residues. At pH 4.5 this enzyme readily hydrolyzed p-nitrophenyl-beta-D-glucopyranoside (Km = 0.88 mM) and cleaved two glucose disaccharides: gentiobiose (beta,1 leads to 6; Km = 0.75 mM) and cellobiose (beta,1 leads to 4; Km = 0.84 mM). Although its activity is similar to that of a previously purified beta-glucosidase (betasmall), the two enzymes differ with respect to their pH activity profiles, substrate specificities, and molecular weights. Also double diffusion tests with anti-betasmall antiserum and both purified beta-glucosidases revealed a nonidentical cross-reaction. Microcomplement fixation of native and periodate-oxidized betasmall suggested that the oligosaccharide chain(s) was not a major antigenic site.     

Purification and characterization of the extracellular beta-glucosidase produced by Candida wickerhamii

Candida wickerhamii NRRL Y-2563 produced a cell-bound beta-glucosidase when grown in complex media containing 50 g of cellobiose per liter. The majority of the enzyme was located on the cell surface and was released into the supernatant upon treatment of intact cells with Zymolyase 60,000. Only about 10% of the total activity was associated with the cytoplasm. The enzyme was purified to homogeneity, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme had an apparent native molecular mass of about 198,000 Da and appeared to be composed of two subunits with approximate molecular masses of 94,000 Da. The beta-glucosidase contained approximately 30.5% (w/w) carbohydrate. Mannose was the only detected neutral carbohydrate associated with the purified enzyme. The enzyme demonstrated optimal activity at a pH of 4.0 to 5.0. The Km of the purified beta-glucosidase was 6.74 X 10(-2) M for cellobiose and 4.17 X 10(-3) M for p-nitrophenyl-beta-D-glucopyranoside. Glucose did not appear to inhibit the enzyme.     

Beta-glucosidase excretion in Trichoderma strains with different cell wall bound beta-1,3-glucanase activities

Two different strains of Trichoderma pseudokoningii (SE1 A8 and SE1 D81) and Trichoderma viride QM 9123 release into the medium different proportions of the total beta-glucosidase activity produced. This observation correlates with the degree of beta-1,3-glucanase binding to the cell wall found for each strain. DEAE-Sephadex ion-exchange chromatography revealed three peaks of beta-1,3-glucanase activity. These three enzymes (enzyme I, enzyme II, and enzyme III) differ in their extent of binding to the cell walls, their activity on isolated cell walls and Trichoderma beta-glucan, and their affinity for beta-glucan. Of these enzymes, enzyme II shows the largest variation in relative importance among the three strains and is located predominantly within the mural compartment. Enzyme II has the highest activity on and affinity for Trichoderma beta-glucan. Enzyme II is also the most active in releasing beta-glucosidase from cell walls of strain SE1 A8 (the strain excreting a high proportion of its beta-glucosidase into the culture fluid) as well as from strain SE1 D81 (little beta-glucosidase activity in the culture fluid). It is concluded that the action of beta-1,3-glucanase II on cell wall beta-glucan may be responsible for the in vivo release of cell wall bound beta-glucosidase into the culture fluid.     

Purification and characterization of two extracellular beta-glucosidases from Trichoderma reesei.

A major beta-glucosidase I and a minor beta-glucosidase II were purified from culture filtrates of the fungus Trichoderma reesei grown on wheat straw. The enzymes were purified using CM-Sepharose CL-6B cation-exchange and DEAE Bio-Gel A anion-exchange chromatography steps, followed by Sephadex G-75 gel filtration. The isolated enzymes were homogeneous in SDS-polyacrylamide gel electrophoresis and isoelectric focusing. beta-Glucosidase I (71 kDa) was isoelectric at pH 8.7 and contained 0.12% carbohydrate; beta-glucosidase II (114 kDa) was isoelectric at pH 4.8 and contained 9.0% carbohydrate. Both enzymes catalyzed the hydrolysis of cellobiose and p-nitrophenyl-beta-D-glucoside (pNPG). The Km and kcat/Km values for cellobiose were 2.10 mM, 2.45.10(4) s-1 M-1 (beta-glucosidase I) and 11.1 mM, 1.68.10(3) s-1 M-1 (beta-glucosidase II). With pNPG as substrate the Km and kcat/Km values were 182 microM, 7.93.10(5) s-1 M-1 (beta-glucosidase I) and 135 microM, 1.02.10(6) s-1 M-1 (beta-glucosidase II). The temperature optimum was 65-70 degrees C for beta-glucosidase I and 60 degrees C for beta-glucosidase II, the pH optimum was 4.6 and 4.0, respectively. Several inhibitors were tested for their action on both enzymes. beta-Glucosidase I and II were competitively inhibited by desoxynojirimycin, gluconolactone and glucose.     

Lactose-hydrolyzing beta-glycosidases of barley meal

A beta-glucosidase and a beta-galactosidase were purified to homogeneity from barley meal. The beta-glucosidase is a single basic polypeptide (pI greater than 8.5) with an Mr of 53,000 acting optimally at pH 4.5-5.0. The beta-galactosidase is composed of two subunits with an Mr of 42,000 and 33,000, respectively, and is acidic in nature (pI less than 5.7). Both enzymes are able to hydrolyze lactose with Michaelis constants lower than the concentration of this sugar in milk whey. Consequently, barley seems to be an inexpensive source of lactose-splitting enzymes.     

Production, purification, and characterization of a highly glucose-tolerant novel beta-glucosidase from Candida peltata.

Candida peltata (NRRL Y-6888) produced beta-glucosidase when grown in liquid culture on various substrates (glucose, xylose, L-arabinose, cellobiose, sucrose, and maltose). An extracellular beta-glucosidase was purified 1,800-fold to homogeneity from the culture supernatant of the yeast grown on glucose by salting out with ammonium sulfate, ion-exchange chromatography with DEAE Bio-Gel A agarose, Bio-Gel A-0.5m gel filtration, and cellobiose-Sepharose affinity chromatography. The enzyme was a monomeric protein with an apparent molecular weight of 43,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. It was optimally active at pH 5.0 and 50 degrees C and had a specific activity of 108 mumol.min-1.mg of protein-1 against p-nitrophenyl-beta-D-glucoside (pNP beta G). The purified beta-glucosidase readily hydrolyzed pNP beta G, cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose, with Km values of 2.3, 66, 39, 35, 21, and 18 mM, respectively. The enzyme was highly tolerant to glucose inhibition, with a Ki of 1.4 M (252 mg/ml). Substrate inhibition was not observed with 40 mM pNP beta G or 15% cellobiose. The enzyme did not require divalent cations for activity, and its activity was not affected by p-chloromercuribenzoate (0.2 mM), EDTA (10 mM), or dithiothreitol (10 mM). Ethanol at an optimal concentration (0.75%, vol/vol) stimulated the initial enzyme activity by only 11%. Cellobiose (10%, wt/vol) was almost completely hydrolyzed to glucose by the purified beta-glucosidase (1.5 U/ml) in both the absence and presence of glucose (6%). Glucose production was enhanced by 8.3% when microcrystalline cellulose (2%, wt/vol) was treated for 24 h with a commercial cellulase preparation (cellulase, 5 U/ml; beta-glucosidase, 0.45 U/ml) that was supplemented with purified beta-glucosidase (0.4 U/ml).     

Effect of pretreatment on the hydrolysis of cellulose by Penicillium funiculosum cellulase and recovery of enzyme

Penicillium funiculosum produces a complete cellulase which brings about 97% hydrolysis of cotton and has high beta-glucosidase, xylanase, laminarinase, and lichenase activities. This article deals with the effect of different pretreatments on the hydrolysis of sugarcane bagasse by P. funiculosum enzymes and the recovery of enzyme from the insoluble residues. Enzymic saccharification of bagasse pretreated with hot 1N NaOH followed by neutralization with HCI and steam treated under pressure (7 kg/cm(2)) gave 63 and 59% saccharification, respectively, in 48 h. Hemicellulose is not lost in these pretreatments. With a 30% slurry of steam-treated bagasse, a semisolid mass containing 14% sugar was obtained. A 90% recovery of CMCase, beta-glucosidase, and filter paper activity from the hydrolysates was obtained under the following conditions: (1) maintaining the ratio of enzyme to substrate high by stepwise addition of substrate, (2) brief grinding of the residual substrate with glass powder, and (3) addition of 0.4% Tween-80 to the eluting buffer. The high recovery of cellulolytic enzymes indicates that the adsorption of these enzymes on cellulose is not irreversible.     

In vitro water activity and pH dependence of mycelial growth and extracellular enzyme activities of Trichoderma strains with biocontrol potential

AIMS: Water activity (aw) and pH are probably the most important environmental parameters affecting the activities of mycoparasitic Trichoderma strains. Therefore it is important to collect information on the effects of these factors on mycelial growth and on the in vitro activities of extracellular enzymes involved in nutrient competition (e.g. beta-glucosidase, cellobiohydrolase and beta-xylosidase) and mycoparasitism (e.g. N-acetyl-beta-glucosaminidase, trypsin-like protease and chymotrypsin-like protease) of Trichoderma strains with biocontrol potential. METHODS AND RESULTS: Water activity and pH dependence of the linear mycelial growth of five examined Trichoderma strains belonging to three different species groups was examined on yeast extract and soil extract media. Maximal growth rates were observed at aw 0.997 and pH 4.0 in the case of all strains. The activities of the examined extracellular enzymes at different aw and pH values were determined spectrophotometrically after incubation with chromogenic p-nitrophenyl and p-nitroaniline substrates. Maximal enzyme activities were measured at aw 0.950 for beta-glucosidase, trypsin-like protease and chymotrypsin-like protease, at 0.910 for cellobiohydrolase and at 0.993 for beta-xylosidase and N-acetyl-beta-glucosaminidase enzymes. Optimal pH values are suggested to be at 5.0 for beta-glucosidase, cellobiohydrolase and N-acetyl-beta-glucosaminidase, at 3.0 for beta-xylosidase, at 6.0 for trypsin-like protease and between 6.0 and 7.0 for chymotrypsin-like protease activities, respectively. CONCLUSIONS: Extracellular enzymes of the examined mycoparasitic Trichoderma strains are able to display activities under a wider range of aw and pH values than those allowing mycelial growth. SIGNIFICANCE AND IMPACT OF THE STUDY: Data about the effects of aw and pH on mycelial growth and extracellular enzyme activities of Trichoderma reveal useful information about the applicability of biocontrol strains in agricultural soils with specific water and pH relations.     

Production, Purification, and Properties of a Thermostable beta-Glucosidase from a Color Variant Strain of Aureobasidium pullulans

A color variant strain of Aureobasidium pullulans (NRRL Y-12974) produced beta-glucosidase activity when grown in liquid culture on a variety of carbon sources, such as cellobiose, xylose, arabinose, lactose, sucrose, maltose, glucose, xylitol, xylan, cellulose, starch, and pullulan. An extracellular beta-glucosidase was purified 129-fold to homogeneity from the cell-free culture broth of the organism grown on corn bran. The purification protocol included ammonium sulfate treatment, CM Bio-Gel A agarose column chromatography, and gel filtrations on Bio-Gel A-0.5m and Sephacryl S-200. The beta-glucosidase was a glycoprotein with native molecular weight of 340,000 and was composed of two subunits with molecular weights of about 165,000. The enzyme displayed optimal activity at 75 degrees C and pH 4.5 and had a specific activity of 315 mumol . min . mg of protein under these conditions. The purified beta-glucosidase was active against p-nitrophenyl-beta-d-glucoside, cellobiose, cellotriose, cellotetraose, cellopentaose, cellohexaose, and celloheptaose, with K(m) values of 1.17, 1.00, 0.34, 0.36, 0.64, 0.68, and 1.65 mM, respectively. The enzyme activity was competitively inhibited by glucose (K(i) = 5.65 mM), while fructose, arabinose, galactose, mannose, and xylose (each at 56 mM) and sucrose and lactose (each at 29 mM) were not inhibitory. The enzyme did not require a metal ion for activity, and its activity was not affected by p-chloromercuribenzoate (0.2 mM), EDTA (10 mM), or dithiothreitol (10 mM). Ethanol (7.5%, vol/vol) stimulated the initial enzyme activity by 15%. Glucose production was enhanced by 7.9% when microcrystalline cellulose (2%, wt/vol) was treated for 48 h with a commercial cellulase preparation (5 U/ml) that was supplemented with the purified beta-glucosidase (0.21 U/ml) from A. pullulans.     

Study of beta-glucosidase production by wine-related yeasts during alcoholic fermentation. A new rapid fluorimetric method to determine enzymatic activity

AIMS: The beta-glucosidase activity is involved in the hydrolysis of several important compounds for the development of varietal wine flavour. The aim of the present study was to investigate the production of beta-glucosidase in a number of wine-related yeast strains and to measure and identify this activity over the course of grape juice fermentation. METHODS AND RESULTS: beta-glucosidase activity was measured as the amount of 4-methylumbelliferone released from 4-methylumbelliferyl-beta-d-glucopyranoside substrate. Intact cells of some grape and wine-spoilage yeasts showed beta-glucosidase activity much higher than those observed in wine yeasts "sensu stricto". During fermentation, three Saccharomyces cerevisiae strains, one Hanseniaspora valbyensis strain and one Brettanomyces anomalus strain showed beta-glucosidase activity both intra- and extracellularly. CONCLUSIONS: In the studied strains, beta-glucosidase activity was at its maximum when the cells were in the active growth phase. However, a lowering of medium pH to values around 3 during fermentation led to total loss of activity. SIGNIFICANCE AND IMPACT OF THE STUDY: During the course of this study, a new, rapid and reproducible method to assay beta-glucosidase activity was developed. The fact that Saccharomyces and non-Saccharomyces yeast strains are able to express beta-glucosidase activity during the alcoholic fermentation sheds new light on the contribution of these yeasts in the aroma expression of wines.     

Optimization of cellulase mixture for efficient hydrolysis of steam-exploded corn stover by statistically designed experiments

To improve the enzymatic hydrolytic efficiency and reduce production cost, a statistically designed experimental approach was used to optimize the composition of cellulase mixture so as to maximize the amount of glucose produced from steam-exploded corn stover (SECS). Using seven purified enzymes (cellobiohydrolases, Cel7A, Cel6A, Cel6B; endoglucanases, Cel7B, Cel12A, Cel61A; and beta-glucosidase) from Trichoderma viride T 100-14 mutant strain, a multi-enzyme mixture was constituted after screening and optimization. The final optimal composition (mol%) of the multi-enzyme mixture was Cel7A (19.8%), Cel6A (37.5%), Cel6B (4.7%), Cel7B (17.7%), Cel12A (15.2%), Cel61A (2.3%) and beta-glucosidase (2.8%). The subsequent verification experiments followed by glucose assay together with scanning electron microscopy (SEM) observation confirmed the validity of the models. The multi-enzyme mixture displayed a high performance in converting the cellulosic substrate (SECS). The amount of glucose produced (15.5mg/ml) was 2.1 times as that of the crude cellulase preparation. The results indicated that the optimized cellulase mixture is an available and efficient paradigm for the hydrolysis of lignocellulosic substrate. The enhanced cellulolytic activity displayed by the constructed cellulase mixture could be used as an effective tool for producing bioethanol efficiently from cellulose.     

Subcellular Localization of Dhurrin beta-Glucosidase and Hydroxynitrile Lyase in the Mesophyll Cells of Sorghum Leaf Blades

Studies with purified mesophyll and epidermal protoplasts and bundle sheath strands have shown that the cyanogenic glucoside dhurrin (p-hydroxy-(S)-mandelonitrile-beta-d-glucoside) is localized in the epidermis of sorghum leaves whereas the enzymes involved in its degradation (dhurrin beta-glucosidase and hydroxynitrile lyase) are localized in the mesophyll tissue (Kojima M, JE Poulton, SS Thayer, EE Conn 1979 Plant Physiol 63: 1022-1028). The subcellular localization of these enzymes has now been examined using linear 30 to 55% (w/w) sucrose gradients by fractionation of mesophyll protoplast components. The hydroxynitrile lyase is found in the supernatant fractions suggesting a cytoplasmic (soluble cytoplasm, microsomal or vacuolar location). The dhurrin beta-glucosidase (dhurrinase) is particulate and mostly chloroplast-associated. The dhurrinase activity peak has a shoulder of activity more dense than that of the intact chloroplasts. This shoulder does not coincide with markers of any other cell fraction.In studies of chloroplasts isolated from ruptured mesophyll protoplasts by differential, low-speed centrifugation, the dhurrinase partitions in the same manner as the chloroplast marker triose phosphate dehydrogenase. Chloroplast localization of the beta-glucosidase has also been shown in histochemical studies using 6-bromo-2-naphthyl-beta-d-glucoside substrate coupled with fast Blue B.