Extracellular Β-glucosidase Research Articles

English

The production of an extracellular β-glucosidase by Aspergillus niger NRRL 599 was optimized using submerged fermentation technique. Effect of different media, different carbon sources, initial pH of the fermentation medium, temperature, incubation period and inoculum size on the production of β- glucosidase enzyme was investigated. A. niger NRRL 599 produced maximum extracellular β- glucosidase (4.48 U/mg) in Eggins and Pugh medium with 1% wheat bran (w/v) at pH 5.5 inoculated with 4% conidial suspension after 96 h of incubation at 30°C. Purified β-glucosidase gave K m and V max values of 3.11 mM and 20.83 U/mg respectively for pNPG hydrolysis. The enzyme was optimally active at pH 4.8 and at temperature of 60°C. Thermodynamic parameters, E a , ΔH and ΔS were found to be 52.17 KJ/mol, 49.90 J/mol.K and -71.69 KJ/mol, respectively. The pKa1 and pKa 2 of ionizable groups of active site residues involved in V max were calculated to be 4.1 and 6.0 respectively. Key words : β-Glucosidase, Aspergillus niger, kinetics, thermodynamics.

Enhanced growth of lactobacilli and bioconversion of isoflavones in biotin-supplemented soymilk upon ultrasound-treatment

This study aimed at utilizing ultrasound treatment to further enhance the growth of lactobacilli and their isoflavone bioconversion activities in biotin-supplemented soymilk. Strains of lactobacilli ( Lactobacillus acidophilus BT 1088, L. fermentum BT 8219, L. acidophilus FTDC 8633, L. gasseri FTDC 8131) were treated with ultrasound (30 kHz, 100 W) at different amplitudes (20%, 60% and 100%) for 60, 120 and 180 s prior to inoculation and fermentation in biotin-soymilk. The treatment affected the fatty acids chain of the cellular membrane lipid bilayer, as shown by an increased lipid peroxidation ( P < 0.05). This led to increased membrane fluidity and subsequently, membrane permeability ( P < 0.05). The permeabilized cellular membranes had facilitated nutrient internalization and subsequent growth enhancement ( P < 0.05). Higher amplitudes and longer durations of the treatment promoted growth of lactobacilli in soymilk, with viable counts exceeding 9 log CFU/mL. The intracellular and extracellular β-glucosidase specific activities of lactobacilli were also enhanced ( P < 0.05) upon ultrasound treatment, leading to increased bioconversion of isoflavones in soymilk, particularly genistin and malonyl genistin to genistein. Results from this study show that ultrasound treatment on lactobacilli cells promotes ( P < 0.05) the β-glucosidase activity of cells for the benefit of enhanced ( P < 0.05) isoflavone glucosides bioconversion to bioactive aglycones in soymilk.

Purification and Characterization of an Extracellular β-Glucosidase Produced by Phoma sp. KCTC11825BP Isolated from Rotten Mandarin Peel

A beta-glucosidase from Phoma sp. KCTC11825BP isolated from rotten mandarin peel was purified 8.5-fold with a specific activity of 84.5 U/mg protein. The purified enzyme had a molecular mass of 440 kDa with a subunit of 110 kDa. The partial amino acid sequence of the purified beta-glucosidase evidenced high homology with the fungal beta- glucosidases belonging to glycosyl hydrolase family 3. Its optimal activity was detected at pH 4.5 and 60 degrees C, and the enzyme had a half-life of 53 h at 60 degrees C. The Km values for p-nitrophenyl-beta-D-glucopyranoside and cellobiose were 0.3 mM and 3.2 mM, respectively. The enzyme was competitively inhibited by both glucose (Ki=1.7 mM) and glucono-delta-lactone (Ki=0.1 mM) when pNPG was used as the substrate. Its activity was inhibited by 41% by 10 mM Cu2+ and stimulated by 20% by 10 mM Mg2+.

Effect of Ultrasound on the Growth of Probiotics and Bioconversion of Isoflavones in Prebiotic-Supplemented Soymilk

The objective of the present study was to evaluate the effects of ultrasound on the growth of probiotics and bioconversion of isoflavones in prebiotic-soymilk. Previous studies have shown that ultrasound elevated microbial enzymatic activity and growth by altering cellular membranes. The growth of probiotics was significantly decreased (P < 0.05) immediately after ultrasound treatment, attributed to membrane permeabilization, cell lysis, and membrane lipid peroxidation upon ultrasound treatment. The ultrasound treatment also caused alteration at the acyl chain, polar head, and interface region of the probiotic membrane phospholipid bilayers. The cells treated with ultrasound showed recovery from injury with subsequent increase in growth upon fermentation in soymilk (P < 0.05). Ultrasound treatment at 100 W for 2 and 3 min also enhanced (P < 0.05) the intracellular and extracellular β-glucosidase activity of probiotics, leading to increased (P < 0.05) bioconversion of glucosides to aglycones in the prebiotic-soymilk. Our present study illustrated that ultrasound treatment could produce bioactive synbiotic-soymilk with increased concentrations of bioactive aglycones.

Wine yeasts from Brazil: identification and screening for extracellular β-glucosidases

Wine yeasts from Brazil: identification and screening for extracellular β-glucosidases

A novel extracellular β-glucosidase from Issatchenkia terricola: Isolation, immobilization and application for aroma enhancement of white Muscat wine

Since most Saccharomyces strains show no β-glucosidase activity, the importance of non- Saccharomyces β-glucosidases in the development of wine aroma has been highlighted. However, only a few enzymes from yeasts isolated from grape-must and enological ecosystems are active in the stringent conditions of wine and the wine-making process (low pH, high concentrations of ethanol or glucose). A purified extract of extracellular β-glucosidase from Issatchenkia terricola, proved to be very active in the presence of glucose (100 g/L), ethanol (18%) and metabisulfite (60 mg/L). It is also active and relatively stable at acidic pH over 3.0. Immobilization onto Eupergit C greatly improved its stability, allowing the aromatization of white Muscat wine over a 16-day experiment. The released aroma compounds of control and treated wine were analyzed by GC–MS. The enzymatic treatment significantly increased the amount of monoterpenes and norisoprenoids, showing the potential of the immobilized enzyme for aroma development in wines.

Resistance and resilience of benthic biofilm communities from a temperate saltmarsh to desiccation and rewetting

Periods of desiccation and rewetting are regular, yet stressful events encountered by saltmarsh microbial communities. To examine the resistance and resilience of microbial biofilms to such stresses, sediments from saltmarsh creeks were allowed to desiccate for 23 days, followed by rewetting for 4 days, whereas control sediments were maintained under a natural tidal cycle. In the top 2 mm of the dry sediments, salinity increased steadily from 36 to 231 over 23 days, and returned to seawater salinity on rewetting. After 3 days, desiccated sediments had a lower chlorophyll a (Chl a) fluorescence signal as benthic diatoms ceased to migrate to the surface, with a recovery in cell migration and Chl a fluorescence on rewetting. Extracellular β-glucosidase and aminopeptidase activities decreased within the first week of drying, but increased sharply on rewetting. The bacterial community in the desiccating sediment changed significantly from the controls after 14 days of desiccation (salinity 144). Rewetting did not cause a return to the original community composition, but led to a further change. Pyrosequencing analysis of 16S rRNA genes amplified from the sediment revealed diverse microbial responses, for example desiccation enabled haloversatile Marinobacter species to increase their relative abundance, and thus take advantage of rewetting to grow rapidly and dominate the community. A temporal sequence of effects of desiccation and rewetting were thus observed, but the most notable feature was the overall resistance and resilience of the microbial community.

Prokaryotic activities and abundance in pelagic areas of the Ionian Sea

The Ionian Sea represents a suitable basin for studying the biogeochemical processes mediated by microbial activities. Because of its characteristics as a crossing region between the western and eastern Mediterranean Sea, it is one of the sites most affected by changes in water mass composition and dynamics, caused by the Eastern Mediterranean Transient (EMT). To date, relatively few data exist on microbial activities in pelagic areas of the Ionian Sea. From 1998 to 2004, during different research cruises, prokaryotic parameters (abundance, extracellular enzyme activities leucine aminopeptidase, β-glucosidase, alkaline phosphatase, bacterial production and respiration) were measured together with culturable bacteria and the main physical, chemical and trophic parameters (temperature, salinity, nutrients, particulated organic matter). The aim of the study was to describe the spatial and temporal variability in microbial activities involved in the carbon and phosphorus cycles, in different layers. Results showed that organic matter transformation mediated by the microbial community displayed a significant increase in autumn, highlighting the occurrence of significant changes at meso- and bathypelagic depths. Unlike the dark ocean, bacterial growth efficiency in the Ionian Sea, which increased with depth, seemed to vary from being a source of carbon in the epipelagic layer to a sink in the meso- and bathypelagic layers. The mechanism of phosphatase regulation showed a weak inverse correlation between specific phosphatase and inorganic P in all seasons except autumn. It is worth mentioning that the reported results constitute, to the best of our knowledge, one of the available datasets giving information about microbial activities in the Ionian Sea.

Extracellular β-glucosidase production by the yeast Debaryomyces pseudopolymorphus UCLM-NS7A: optimization using response surface methodology

beta-Glucosidase production by Debaryomyces pseudopolymorphus UCLM-NS7A using a simple nutrient medium containing cellobiose was evaluated under several biochemical and physiological parameters in submerged fermentation. Enzyme induction was also examined using different carbon and nitrogen sources. Cellobiose and ammonium nitrate were the best C and N sources to enhance beta-glucosidase production. The addition of NaCl, MgSO(4), yeast extract, ethanol and Tween 80 to the nutrient medium before inoculation was also compared. A factorial design to optimize enzyme production was developed using four variables that most influenced beta-glucosidase production and data analyzed by the response surface method. Optimal conditions to produce beta-glucosidase in shake-flasks were 1.25% cellobiose, 0.05% Tween 80, 0.4% NH(4)NO(3) over 72 hours. In another factorial design to further increase enzyme production, a lab fermenter using prior-determined shake-flask optimized conditions resulted in higher beta-glucosidase titres at 72 hours, pH controlled at 6.25 and agitation of 200 rpm.

Purification and characterization of extracellular β-glucosidase from mycelia of the Tricholoma matsutake isolated from hardwood forest

Purification and characterization of extracellular β-glucosidase from mycelia of the Tricholoma matsutake isolated from hardwood forest

Vermicomposting of olive oil mill wastewaters

The disposal of olive oil mill wastewaters (OMW) represents a substantial environmental problem in Italy. A vermicompost process could be an alternative and valid method for the management of OMW. In a laboratory experiment, the OMW were absorbed onto a ligno-cellulosic solid matrix and 30 adult earthworms of Eisenia fetida specie were added. The experiment was carried out for 13 weeks. The number of earthworms increased throughout the experimental period and after 2 weeks about 90% of the earthworms had become sexually mature. The decrease in total organic carbon (about 35%), C : N ratio (from 31.2 to 12.3) and biochemical parameters (hydrolytic enzymes averagely 40% and dehydrogenase 23%), and the increase in humification rate (pyrophosphate extractable carbon (PEC) from 17.6 to 33.3 mg g(-1), and PEC : water-soluble carbon from 1.76 to 2.97) indicated the mineralization and the stabilization of organic matter at the end of the vermicomposting process. At the end of the experiment, the extracellular beta-glucosidase, phosphatase, urease and protease activities, measured in the pyrophosphate extract of the vermicompost, were found to be always higher or equal to that measured at the beginning of the vermicomposting process, suggesting that the enzymes bound to humic matter resisted biological attack and environmental stress. Moreover, the results obtained from the phyto-test showed that the OMW lose their toxicity and stimulate plant germination and growth.

Simultaneous saccharification and ethanol fermentation of oxalic acid pretreated corncob assessed with response surface methodology

Response surface methodology was used to evaluate optimal time, temperature and oxalic acid concentration for simultaneous saccharification and fermentation (SSF) of corncob particles by Pichia stipitis CBS 6054. Fifteen different conditions for pretreatment were examined in a 2 3 full factorial design with six axial points. Temperatures ranged from 132 to 180 °C, time from 10 to 90 min and oxalic acid loadings from 0.01 to 0.038 g/g solids. Separate maxima were found for enzymatic saccharification and hemicellulose fermentation, respectively, with the condition for maximum saccharification being significantly more severe. Ethanol production was affected by reaction temperature more than by oxalic acid and reaction time over the ranges examined. The effect of reaction temperature was significant at a 95% confidence level in its effect on ethanol production. Oxalic acid and reaction time were statistically significant at the 90% level. The highest ethanol concentration (20 g/l) was obtained after 48 h with an ethanol volumetric production rate of 0.42 g ethanol l −1 h −1. The ethanol yield after SSF with P. stipitis was significantly higher than predicted by sequential saccharification and fermentation of substrate pretreated under the same condition. This was attributed to the secretion of β-glucosidase by P. stipitis. During SSF, free extracellular β-glucosidase activity was 1.30 pNPG U/g with P. stipitis, while saccharification without the yeast was 0.66 pNPG U/g.

Extracellular β-glucosidase production by a marine Aspergillus sydowii BTMFS 55 under solid state fermentation using statistical experimental design

A potential fungal strain producing extracellular β-glucosidase enzyme was isolated from sea water and identified as Aspergillus sydowii BTMFS 55 by a molecular approach based on 28S rDNA sequence homology which showed 93% identity with already reported sequences of Aspergillus sydowii in the GenBank. A sequential optimization strategy was used to enhance the production of β-glucosidase under solid state fermentation (SSF) with wheat bran (WB) as the growth medium. The two-level Plackett-Burman (PB) design was implemented to screen medium components that influence β-glucosidase production and among the 11 variables, moisture content, inoculums, and peptone were identified as the most significant factors for β-glucosidase production. The enzyme was purified by (NH4)2SO4 precipitation followed by ion exchange chromatography on DEAE sepharose. The enzyme was a monomeric protein with a molecular weight of ∼95 kDa as determined by SDS-PAGE. It was optimally active at pH 5.0 and 50°C. It showed high affinity towards pNPG and enzyme has a Km and Vmax of 0.67 mM and 83.3 U/mL, respectively. The enzyme was tolerant to glucose inhibition with a Ki of 17 mM. Low concentration of alcohols (10%), especially ethanol, could activate the enzyme. A considerable level of ethanol could produce from wheat bran and rice straw after 48 and 24 h, respectively, with the help of Saccharomyces cerevisiae in presence of cellulase and the purified β-glucosidase of Aspergilus sydowii BTMFS 55.

The characterisation of a novelPichia anomala β-glucosidase with potentially aroma-enhancing capabilities in wine

The production and characterisation of β-glucosidase from an isolated yeast strain classified as aPichia anomala MDD24 were studied. The result shows that cellobiose is a good inducer for extracellular β-glucosidase production and optimum concentration is 1.5 percent cellobiose in yeast peptone dextrose medium. The purified β-glucosidase fromPichia anomala MDD24 exhibited a specific activity of 614±14 U mg−1 of protein and a molecular mass of 42 kDa. This enzyme was slightly inhibited by fructose and sucrose in the range of 4 to 20% (w/v). An ethanol concentration between 4 and 20% (v/v) activated β-glucosidase activity, at presence 16% (v/v) ethanol, β-glucosidases obtained maximum relative activity around 150%. The optimum pH and optimum temperature for β-glucosidase activity were 4.5 and 40 °C, respectively. Although the activity under the pH and temperature of wine production (pH 3.5–4.0 and 15–20 °C) was quite low, the enzyme was stable and the relative activities were higher than commercial enzyme under those conditions. The extracellular β-glucosidase fromPichia anomala MDD24 makes it possible to release glucosidically-bound monoterpenes, which are the major contributors to floral and fruity aromas in wines fromMuscat-type varieties, at final stage of alcoholic fermentation.

Application ofTrichoderma reeseiCellulase and Xylanase Promoters through Homologous Recombination for Enhanced Production of Extracellular β-Glucosidase I

One of the limiting factors for the application of Trichoderma reesei to degrade cellulosic biomass is its low beta-glucosidase activity, required to convert cellobiose to glucose. The egl3 and the xyn3 promoters were used to express beta-glucosidase 1 gene bgl1 through homologous recombination to improve the cellulose degradation ability of T. reesei. The recombinant strains expressing beta-glucosidase 1 (BGLI) under the control of either the egl3 or the xyn3 promoter had 4.0 and 7.5 fold higher beta-glucosidase activity than the native strain, which compares well to the finding that in wild-type T. reesei PC-3-7, the levels of egl3 and xyn3 mRNA expression were 6.0 and 12 fold higher respectively than that of bgl1. Matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis of proteins secreted by the recombinant strains demonstrated that BGLI was overproduced. The increase in the transcription of bgl1 and the concomitant elevated level of BGLI in these recombinant strains were sufficient to degrade the cellobiose and cellotriose formed during the degradation of pretreated cedar powder.

Comparative enzymatic hydrolysis of pretreated spruce by supernatants, whole fermentation broths and washed mycelia of Trichoderma reesei and Trichoderma atroviride

Cellulase and β-glucosidase production on steam pretreated spruce (SPS) with Trichoderma reesei Rut C30, Trichoderma atroviride TUB F-1505 and TUB F-1663 was investigated. The enzymes were compared in term of activity, temperature optima and hydrolytic capacity. The T. atroviride cellulases proved to have lower temperature optima for filter paper activity (FPA) assay (50 °C) and for hydrolysis of SPS (40 °C) than the Rut C30 enzymes (60 °C for FPA and 50 °C for hydrolysis). Due to high levels of extracellular β-glucosidases, the T. atroviride enzyme supernatants hydrolyzed the washed SPS to glucose more efficiently than the enzymes produced by T. reesei. On the other hand, when the whole fermentation broths were used instead of the supernatants, thus the mycelium bound enzymes were also present, the hydrolytic capacity of T. reesei Rut C30 was enhanced by approximately 200%, while an improvement of only 15% was observed in case of the T. atroviride isolates.

Comparison of extraction methods for recovery of extracellular β-glucosidase in two different forest soils

A study was made of the efficiency of three different extractants, 0.1 M sodium pyrophosphate (pH 7), 67 mM phosphate buffer (pH 6) and 0.5 M potassium sulphate (pH 6.6), in recovering the protein quantity and the β-glucosidase enzyme activity from two natural forest soils: (1) an Inceptisoil located in Tuscany (Italy) in a mild Mediterranean climate, and (2) a Lithic Calcixeroll soil located in Murcia (south-east of Spain) in a dry-semiarid climate. The pyrophosphate was used to determine the activity of extracellular-humic-bound proteins, while the phosphate buffer and potassium sulphate were used to extract dissolved extracellular proteins. The latter extractant, after chloroform fumigation, was also used to measure total proteins in soil. A preliminary screening, using SDS-PAGE in one dimension, was also carried out in order to optimize the separation condition of soil proteins extracted with different buffers. To remove the interfering co-extracted substances (humic acid) a purification step using a column packed with insoluble polyvinylpyrrolidone was performed. The highest β-glucosidase activity was recovered in the pyrophosphate extract, thus confirming its capability of extracting humic-bound β-glucosidase enzyme in a stable and active form. The extractants performed differently with the two soil types and band patterns obtained with SDS-PAGE were extractant-specific, demonstrating that each was selective for a particular class of proteins. Surprisingly, protein bands were also obtained using pyrophosphate, in spite of the very dark extract colour due to the presence of humic substances.

Purification and Biochemical Characterization of Extracellular β-Glucosidases from the Hypercellulolytic Pol6 Mutant of Penicillium occitanis

The Pol6 mutant of Penicillium occitanis fungus is of great biotechnological interest since it possesses a high capacity of cellulases and beta-glucosidase production with high cellulose degradation efficiency (Jain et al., Enzyme Microb Technol, 12:691-696, 1990; Hadj-Taieb et al., Appl Microbiol Biotechnol, 37:197-201, 1992; Ellouz Chaabouni et al., Enzyme Microb Technol, 16:538-542, 1994; Ellouz Chaabouni et al., Appl Microbiol Biotechnol, 43:267-269, 1995). In this work, two forms of beta-glucosidase (beta-glu 1 and beta-glu 2) were purified from the culture supernatant of the Pol6 strain by gel filtration, ion exchange chromatography, and preparative anionic native electrophoresis. These enzymes were eluted as two distinct species from the diethylamino ethanol Sepharose CL6B and anionic native electrophoresis. However, both behaved identically on sodium dodecyl sulfate polyacrylamide gel electrophoresis (MW, 98 kDa), shared the same amino acid composition, carbohydrate content (8%), and kinetic properties. Moreover, they strongly cross-reacted immunologically. They were active on cellobiose and pNPG with Km values of 1.43 and 0.37 mM, respectively. beta-glu 1 and beta-glu 2 were competitively inhibited by 1 mM of glucose and 0.03 mM of delta-gluconolactone. They were also significantly inhibited by Hg(2+) and Cu(2) at 2 mM. The addition of purified enzymes to the poor beta-glucosidase crude extract of Trichoderma reesei increased its hydrolytic efficiency on H(3)P0(4) swollen cellulose but had no effect with P. occitanis crude extract. Besides their hydrolytic activities, beta-glu 1 and beta-glu 2 were endowed with trans-glycosidase activity at high concentration of glucose.

Characterization of a Thermostable Extracellular β-Glucosidase with Activities of Exoglucanase and Transglycosylation from Paecilomyces thermophila

The purification and characterization of a novel extracellular beta-glucosidase from Paecilomyces thermophila J18 was studied. The beta-glucosidase was purified to 105-fold apparent homogeneity with a recovery yield of 21.7% by DEAE 52 and Sephacryl S-200 chromatographies. Its molecular masses were 116 and 197 kDa when detected by SDS-PAGE and gel filtration, respectively. It was a homodimeric glycoprotein with a carbohydrate content of 82.3%. The purified enzyme exhibited an optimal activity at 75 degrees C and pH 6.2. It was stable up to 65 degrees C and in the pH range of 5.0-8.5. The enzyme exhibited a broad substrate specificity and significantly hydrolyzed p-nitrophenyl-beta- d-glucopyranoside ( pNPG), cellobiose, gentiobiose, sophorose, amygdalin, salicin, daidzin, and genistin. Moreover, it displayed substantial activity on beta-glucans such as laminarin and lichenan, indicating that the enzyme has some exoglucanase activity. The rate of glucose released by the purified enzyme from cellooligosaccharides with a degree of polymerization (DP) ranging between 2 and 5 decreased with increasing chain length. Glucose and glucono-delta-lactone inhibited the beta-glucosidase competitively with Ki values of 73 and 0.49 mM, respectively. The beta-glucosidase hydrolyzed pNPG, cellobiose, gentiobiose, sophorose, salicin, and amygdalin, exhibiting apparent Km values of 0.26, 0.65, 0.77, 1.06, 1.39, and 1.45 mM, respectively. Besides, the enzyme showed transglycosylation activity, producing oligosaccharides with higher DP than the substrates when cellooligosaccharides were hydrolyzed. These properties make this beta-glucosidase useful for various biotechnological applications.

Purification and biochemical characterization of an extracellular β-glucosidase from the wood-decaying fungusDaldinia eschscholzii(Ehrenb.:Fr.) Rehm

An extracellular beta-glucosidase was purified from culture filtrates of the wood-decaying fungus Daldinia eschscholzii (Ehrenb.:Fr.) Rehm grown on 1.0% (w/v) carboxymethyl-cellulose using ammonium sulfate precipitation, ion-exchange, hydrophobic interaction and gel filtration chromatography. The enzyme is monomeric with a molecular weight of 64.2 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and has a pI of 8.55. The enzyme catalyzes the hydrolysis of p-nitrophenyl-beta-D-glucopyranoside (PNPG) as the substrate, with a K(m) of 1.52 mM, and V(max) of 3.21 U min mg(-1) protein. Glucose competitively inhibited beta-glucosidase with a K(i) value of 0.79 mM. Optimal activity with PNPG as the substrate was at pH 5.0 and 50 degrees C. The enzyme was stable at pH 5.0 at temperatures up to 50 degrees C. The purified beta-glucosidase was active against PNPG, cellobiose, sophorose, laminaribiose and gentiobiose, but did not hydrolyze lactose, sucrose, Avicel or o-nitrophenyl-beta-d-galactopyranoside. The activity of beta-glucosidase was stimulated by Ca(2+), Co(2+), Mg(2+), Mn(2+), glycerol, dimethyl sulfoxide (DMSO), dithiothreitol and EDTA, and strongly inhibited by Hg(2+). The internal amino acid sequences of D. eschscholziibeta-glucosidase have similarity to the sequences of the family 3 beta-glucosyl hydrolase.