BSA treatment to enhance enzymatic hydrolysis of cellulose in lignin containing substrates

Cellulase and bovine serum albumin (BSA) were added to Avicel cellulose and solids containing 56% cellulose and 28% lignin from dilute sulfuric acid pretreatment of corn stover. Little BSA was adsorbed on Avicel cellulose, while pretreated corn stover solids adsorbed considerable amounts of this protein. On the other hand, cellulase was highly adsorbed on both substrates. Adding a 1% concentration of BSA to dilute acid pretreated corn stover prior to enzyme addition at 15 FPU/g cellulose enhanced filter paper activity in solution by about a factor of 2 and beta-glucosidase activity in solution by about a factor of 14. Overall, these results suggested that BSA treatment reduced adsorption of cellulase and particularly beta-glucosidase on lignin. Of particular note, BSA treatment of pretreated corn stover solids prior to enzymatic hydrolysis increased 72 h glucose yields from about 82% to about 92% at a cellulase loading of 15 FPU/g cellulose or achieved about the same yield at a loading of 7.5 FPU/g cellulose. Similar improvements were also observed for enzymatic hydrolysis of ammonia fiber explosion (AFEX) pretreated corn stover and Douglas fir treated by SO(2) steam explosion and for simultaneous saccharification and fermentation (SSF) of BSA pretreated corn stover. In addition, BSA treatment prior to hydrolysis reduced the need for beta-glucosidase supplementation of SSF. The results are consistent with non-specific competitive, irreversible adsorption of BSA on lignin and identify promising strategies to reduce enzyme requirements for cellulose hydrolysis.     

Production and characterization of cellulolytic enzymes from the thermoacidophilic fungal Aspergillus terreus M11 under solid-state cultivation of corn stover

The production of extracellular cellulases by a newly isolated thermoacidophilic fungus, Aspergillus terreus M11, on the lignocellulosic materials was studied in solid-state fermentation (SSF). The results showed that the high-level cellulase activity was produced at 45 degrees C pH 3 and moisture 80% with corn stover and 0.8% yeast extract as carbon and nitrogen sources. 581 U endoglucanase activity, 243 U filter paper activity and 128 U beta-glucosidase activity per gram of carbon source were obtained in the optimal condition. Endoglucanase and beta-glucosidase exhibited their maximum activity at pH 2 and pH 3, respectively, and both of them showed remarkable stability in the range of pH 2-5. The activities of endoglucanase and beta-glucosidase were up to the maximum at 70 degrees C and maintained about 65% and 53% of their original activities after incubation at 70 degrees C for 6h. The enzyme preparations from this strain were used to hydrolyze Avicel. Higher hydrolysis yields of Avicel were up to 63% on 5% Avicel (w/v) for 72 h with 20 U FPase/g substrate.     

Integrated analysis of hydrothermal flowthroughpretreatment

ABSTRACT: BACKGROUND: The impact of hydrothermal flowthrough (FT) pretreatment severity on pretreatment and solubilization performance metrics was evaluated for three milled feedstocks (corn stover, bagasse, and poplar) and two conversion systems (simultaneous saccharification and fermentation using yeast and fungal cellulase, and fermentation by Clostridium thermocellum). RESULTS: Compared to batch pretreatment, FT pretreatment consistently resulted in higher xylan recovery, higher removal of non-carbohydrate components and higher glucan solubilization by simultaneous saccharification and fermentation (SSF). Xylan recovery was above 90% for FT pretreatment below 4.1 severity but decreased at higher severities, particularly for bagasse. Removal of non-carbohydrate components during FT pretreatment increased from 65% at low severity to 80% at high severity for corn stover, and from 40% to 70% for bagasse and poplar. Solids obtained by FT pretreatment were amenable to high conversion for all of the feedstocks and conversion systems examined. The optimal time and temperature for FT pretreatment on poplar were found to be 16 minutes and 210 oC. At these conditions, SSF glucan conversion was about 85%, 94% of the xylan was removed, and 62% of the non carbohydrate mass was solubilized. Solubilization of FT-pretreated poplar was compared for C. thermocellum fermentation (10% inoculum), and for yeast-fungal cellulase SSF (5% inoculum, cellulase loading of 5 and 10 FPU/g glucan supplemented with beta-glucosidase at 15 and 30 U/g glucan). Under the conditions tested, which featured low solids concentration, C. thermocellum fermentation achieved faster rates and more complete conversion of FT-pretreated poplar than did SSF. Compared to SSF, solubilization by C. thermocellum was 30% higher after 4 days, and was over twice as fast on ball-milled FT-pretreated poplar. CONCLUSIONS: Xylan removal trends were similar between feedstocks whereas glucan conversion trends were significantly different, suggesting that factors in addition to xylan removal impact amenability of glucan to enzymatic attack. Corn stover exhibited higher hydrolysis yields than bagasse or poplar, which could be due to higher removal of non-carbohydrate components. Xylan in bagasse is more easily degraded than xylan in corn stover and poplar. Conversion of FT-pretreated substrates at low concentration was faster and more complete for C.thermocellum than for SSF.     

Simultaneous Saccharification and Ethanol Fermentation of Corn Stover at High Temperature and High Solids Loading by a Thermotolerant Strain Saccharomyces cerevisiae DQ1

The thermotolerant strain Saccharomyces cerevisiae DQ1 was applied to the simultaneous saccharification and fermentation (SSF) at high temperature and high solids loading of the dilute acid-pretreated corn stover in the present study. The SSF using S. cerevisiae DQ1 was operated at 30?% solids loading of the pretreated corn stover with three-step SSF mode and achieved up to ethanol titer of 48?g/L and yield of 65.6?%. S. cerevisiae DQ1 showed strong thermotolerance in both the regular one-step SSF and the three-step SSF with changing temperature in each step. The three-step SSF at 40°C using S. cerevisiae DQ1 tolerated the greater cellulase dosage and solids loading of the pretreated corn stover and resulted in increased ethanol production. The present study provided a practical potential for the future SSF of lignocellulose feedstock at high temperature to reach high ethanol titer.     

Purification and characterization of an intracellular beta-glucosidase from the protoplast fusant of Aspergillus oryzae and Aspergillus niger

Protoplasts of Aspergillus oryzae 3.481 and Aspergillus niger 3.316 were prepared using cellulose and snail enzyme with 0.6 M NaCl as osmotic stabilizer. Protoplast fusion has been performed using 35% polyethylene glycol 4.000 with 0.01 mM CaCl2. The fused protoplasts have been regenerated on regeneration medium and fusants were selected for further studies. An intracellular beta-glucosidase (EC 3.2.1.21) was purified from the protoplast fusant of Aspergillus oryzae 3.481 and Aspergillus niger 3.316 and characterized. The enzyme was purified 138.85-fold by ammonium sulphate precipitation, DE-22 ion exchange and Sephadex G-150 gel filtration chromatography with a specific activity of 297.14 U/mg of protein. The molecular mass of the purified enzyme was determined to be about 125 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The enzyme had an optimum pH of 5.4 and temperature of 65 degrees C, respectively. This enzyme showed relatively high stability against pH and temperature and was stable in the pH range of 3.0-6.6. Na+, K+, Ca2+, Mg2+ and EDTA completely inhibited the enzyme activity at a concentration of 10 mM. The enzyme activity was accelerated by Fe3+. The enzyme activity was strongly inhibited by glucose, the end product ofglucoside hydrolysis. The K(m) and V(max) values against salicin as substrate were 0.035 mM and 1.7215 micromol min(-1), respectively.     

Simultaneous saccharification and fermentation of ground corn stover for the production of fuel ethanol using Phanerochaete chrysosporium, Gloeophyllum trabeum, Saccharomyces cerevisiae, and Escherichia coli K011

Enzymatic saccharification of corn stover using Phanerochaete chrysosporium and Gloeophyllum trabeum and subsequent fermentation of the saccharification products to ethanol by Saccharomyces cerevisiae and Escherichia coli K011 were achieved. Prior to simultaneous saccharification and fermentation (SSF) for ethanol production, solid-state fermentation was performed for four days on ground corn stover using either P. chrysosporium or G. trabeum to induce in situ cellulase production. During SSF with S. cerevisiae or E. coli, ethanol production was the highest on day 4 for all samples. For corn stover treated with P. chrysosporium, the conversion to ethanol was 2.29 g/100 g corn stover with S. cerevisiae as the fermenting organism, whereas for the sample inoculated with E. coli K011, the ethanol production was 4.14 g/100 g corn stover. Corn stover treated with G. trabeum showed a conversion 1.90 and 4.79 g/100 g corn stover with S. cerevisiae and E. coli K011 as the fermenting organisms, respectively. Other fermentation co-products, such as acetic acid and lactic acid, were also monitored. Acetic acid production ranged between 0.45 and 0.78 g/100 g corn stover, while no lactic acid production was detected throughout the 5 days of SSF. The results of our experiment suggest that it is possible to perform SSF of corn stover using P. chrysosporium, G. trabeum, S. cerevisiae and E. coli K011 for the production of fuel ethanol.     

Enzymatic properties of two beta-glucosidases from Ceriporiopsis subvermispora produced in biopulping conditions

AIMS: Ceriporiopsis subvermispora produces endoglucanase and beta-glucosidase when cultivated on cellulose or wood, but biodegradation of cellulose during biopulping by C. subvermispora is low even after long periods. To resolve this discrepancy, we grew C. subvermispora on Pinus taeda wood chips and purified the major beta-glucosidases it produced. Kinetic parameters were determined to clear if this fungus produces enzymes capable of yielding assimilable glucose from wood. METHODS AND RESULTS: Ceriporiopsis subvermispora was grown on P. taeda wood chips under solid-state fermentation. After 30 days, the crude extract obtained from enzyme extraction with sodium acetate buffer 50 mmol l(-1), pH 5.4, was filtrated in membranes with a molecular mass exclusion limit of 100 kDa. Enzyme purification was carried out using successively Sephacryl S-300 gel filtration. The retained fraction attained 76% of beta-glucosidase activity with 3.7-fold purification. Two beta-glucosidases were detected with molecular mass of 110 and 53 kDa. We have performed a characterization of the enzymatic properties of the beta-glucosidase of 110 kDa. The optimum pH and temperature were 3.5 and 60 degrees C, respectively. The K(m) and V(max) values were respectively 3.29 mmol l(-1) and 0.113 micromol min(-1) for the hydrolysis of p-nitrophenyl-beta-glucopyranoside (pNPG) and 2.63 mmol l(-1) and 0.103 micromol min(-1), towards cellobiose. beta-Glucosidase activity was strongly increased by Mn(2+) and Fe(3+), while Cu(2+) severely inhibited it. CONCLUSIONS: Ceriporiopsis subvermispora produces small amounts of beta-glucosidase when grown on wood. The gel filtration and polyacrylamide gel electrophoresis data revealed the existence of two beta-glucosidases with 110 and 53 kDa. The 110 kDa beta-glucosidase from C. subvermispora can be efficiently purified in a single step by gel filtration chromatography. The enzyme has an acid pH optimum with similar activity on pNPG and cellobiose and is thus typical beta-glucosidase. SIGNIFICANCE AND IMPACT OF THE STUDY: Ceriporiopsis subvermispora produces beta-glucosidase with limited action during wood decay making able its use for the production of biomechanical and biochemical pulps. The results presented in this paper show the importance of studying the behaviour of beta-glucosidases during biopulping.     

日本根霉IFO5318胞外β—葡萄糖苷酶的纯化及部分特性

采用硫酸铵沉淀及柱层析等步骤纯化了日本根霉IFO5318的β—葡萄糖苷酶,回收率为22％。该酶分子量约为4.0×10~5,由四个相同大小的亚基组成;最适反应温度55℃,最适反应pH5.5;对热较敏感,但能在较大的pH范围内保持稳定。用对硝基苯基—β-D-吡喃葡糖苷为底物,测得的K_m和V_(max)值分别为0.825mg·ml~(-1)和135.4μmol·min~(-1)·mg~(-1)。该酶对纤维二糖的水解能力最强,SDS、Fe~(3 )、Hg~2 )等对酶活力有抑制作用。     

Xylanase from a newly isolated Fusarium verticillioides capable of utilizing corn fiber xylan

A fungus, Fusarium verticillioides (NRRL 26518), was isolated by screening soil samples using corn fiber xylan as carbon source. The extracellular xylanase from this fungal strain was purified to apparent homogeneity from the culture supernatant by ultrafiltration using a 30,000 cut-off membrane, octyl-Sepharose chromatography and Bio gel A-0.5 m gel filtration. The purified xylanase (specific activity 492 U/mg protein; MW 24,000; pI 8.6) displayed an optimum temperature at 50 degrees C and optimum pH at 5.5, a pH stability range from 4.0 to 9.5 and thermal stability up to 50 degrees C. It hydrolyzed a variety of xylan substrates mainly to xylobiose and higher short-chain xylooligosaccharides. No xylose was formed. The enzyme did not require metal ions for activity and stability.     

High solid simultaneous saccharification and fermentation of wet oxidized corn stover to ethanol

In this study ethanol was produced from corn stover pretreated by alkaline and acidic wet oxidation (WO) (195 degrees C, 15 min, 12 bar oxygen) followed by nonisothermal simultaneous saccharification and fermentation (SSF). In the first step of the SSF, small amounts of cellulases were added at 50 degrees C, the optimal temperature of enzymes, in order to obtain better mixing condition due to some liquefaction. In the second step more cellulases were added in combination with dried baker's yeast (Saccharomyces cerevisiae) at 30 degrees C. The phenols (0.4-0.5 g/L) and carboxylic acids (4.6-5.9 g/L) were present in the hemicellulose rich hydrolyzate at subinhibitory levels, thus no detoxification was needed prior to SSF of the whole slurry. Based on the cellulose available in the WO corn stover 83% of the theoretical ethanol yield was obtained under optimized SSF conditions. This was achieved with a substrate concentration of 12% dry matter (DM) acidic WO corn stover at 30 FPU/g DM (43.5 FPU/g cellulose) enzyme loading. Even with 20 and 15 FPU/g DM (corresponding to 29 and 22 FPU/g cellulose) enzyme loading, ethanol yields of 76 and 73%, respectively, were obtained. After 120 h of SSF the highest ethanol concentration of 52 g/L (6 vol.%) was achieved, which exceeds the technical and economical limit of the industrial-scale alcohol distillation. The SSF results showed that the cellulose in pretreated corn stover can be efficiently fermented to ethanol with up to 15% DM concentration. A further increase of substrate concentration reduced the ethanol yield significant as a result of insufficient mass transfer. It was also shown that the fermentation could be followed with an easy monitoring system based on the weight loss of the produced CO2.     

One-step purification and characterization of an intracellular β-glucosidase from Metschnikowia pulcherrima

A collection of 60 non-Saccharomyces yeasts isolated from grape musts in Uruguayan vineyards was screened for beta-glucosidase activity and Metschnikowia pulcherrima was the best source of this enzyme activity. Its major beta-glucosidase was successfully purified to homogeneity by ion-exchange chromatography on amino-agarose gel. The enzyme exhibited an optimum catalytic activity at 50 degrees C and pH 4.5 and was active against (1 --> 4)-beta and (1 --> 2)-beta glycosidic linkages. In spite of preserving 100% of its activity and stability in the presence of 12% (v/v) ethanol and 5 g glucose/l, the enzyme was unstable below pH 4. We characterized the beta-glucosidase from M. pulcherrima with a view to its potential applications in wine-making.     

Production,partial characterization,and immobilization in alginate beads of an alkaline protease from a new thermophilic fungus <Emphasis Type="Italic">Myceliophthora</Emphasis> sp.

Thermophilic fungi produce thermostable enzymes which have a number of applications, mainly in biotechnological processes. In this work, we describe the characterization of a protease produced in solidstate (SSF) and submerged (SmF) fermentations by a newly isolated thermophilic fungus identified as a putative new species in the genus Myceliophthora. Enzyme-production rate was evaluated for both fermentation processes, and in SSF, using a medium composed of a mixture of wheat bran and casein, the proteolytic output was 4.5-fold larger than that obtained in SmF. Additionally, the peak of proteolytic activity was obtained after 3 days for SSF whereas for SmF it was after 4 days. The crude enzyme obtained by both SSF and SmF displayed similar optimum temperature at 50°C, but the optimum pH shifted from 7 (SmF) to 9(SSF). The alkaline protease produced through solid-state fermentation (SSF), was immobilized on beads of calcium alginate, allowing comparative analyses of free and immobilized proteases to be carried out. It was observed that both optimum temperature and thermal stability of the immobilized enzyme were higher than for the free enzyme. Moreover, the immobilized enzyme showed considerable stability for up to 7 reuses.     

The cellulase complex of Neurospora crassa: activity, stability and release

The temperature and pH optima, and the temperature and pH stability, of crude and purified enzymes of the cellulase complex of the cellulolytic ascomycete fungus Neurospora crassa were investigated. The effects of some non-ionic surfactants and fatty acids on the production/release of enzymes of cellulase complex were also examined. For the different enzymes of the complex, activity maxima occurred between pH 4.0 and 7.0, with pH 5.0 being close to optimal for stability of all. Temperature optima for activity ranged between 45 and 65 degrees C, with the stability optimum between 45 and 50 degrees C. The presence of C18 fatty acids and surfactants resulted in increased production of both endoglucanase and exoglucanase in the medium. Oleic acid was the most effective fatty acid tested, and Tween 80 the most effective surfactant. Oleic acid had no detectable effect on production of beta-glucosidase, and Tween 80 actually reduced its production.     

Bioconversion of corn stover derived pentose and hexose to ethanol using cascade simultaneous saccharification and fermentation (CSSF)

A cascade type of fermentation, designated the cascade simultaneous saccharification and fermentation (CSSF), was studied to convert corn stover derived pentose and hexose to ethanol with reduced enzyme input. In detail, each step of CSSF utilizes two sequential SSF phases operating on pentose and hexose, i.e., pentose conversion using xylanase, endo-glucanase, and recombinant Escherichia coli (KO11) with minimal glucose conversion in the first phase SSF, and hexose conversion in the second phase SSF using cellulase, β-glucosidase, and Saccharomyces cerevisiae (D(5)A). In this cascade scheme, multiple stages of 1st and 2nd phase SSF were performed in series; enzymes are recycled from the fermentation broth of the last stage for the use of the next stage. This bioconversion process yielded up to 60% of the theoretical maximum ethanol yield based on the total sugars in untreated corn stover, while enzyme loadings were reduced by 50% (v/v) and the final ethanol concentration reached 27 g/l.     

A comparison between simultaneous saccharification and fermentation and separate hydrolysis and fermentation using steam-pretreated corn stover

Two different process configurations, simultaneous saccharification and fermentation (SSF) and separate hydrolysis and fermentation (SHF), were compared, at 8% water-insoluble solids (WIS), regarding ethanol production from steam-pretreated corn stover. The enzymatic loading in these experiments was 10 FPU/g WIS and the yeast concentration in SSF was 1 g/L (dry weight) of a Saccharomyces cerevisiae strain. When the whole slurry from the pretreatment stage was used as it was, diluted to 8% WIS with water and pH adjusted, SSF gave a 13% higher overall ethanol yield than SHF (72.4% versus 59.1% of the theoretical). The impact of the inhibitory compounds in the liquid fraction of the pretreated slurry was shown to affect SSF and SHF in different ways. The overall ethanol yield (based on the untreated raw material) decreased when SSF was run in absence on inhibitors compared to SSF with inhibitors present. On the contrary, the presence of inhibitors decreased the overall ethanol yield in the case of SHF. However, the SHF yield achieves in the absence of inhibitors was still lower than the SSF yield achieves with inhibitors present.     

Screening of carbon sources for beta-glucosidase production by Aspergillus saccharolyticus

The fungus Aspergillus saccharolyticus was found to produce a culture broth rich in beta-glucosidase activity, an enzyme which plays an essential role for efficient and complete hydrolysis of lignocellulosic biomass. Direct application of fungal fermentation broths produced on-site in a biorefinery may be an integral part of a biorefinery for lowering the cost associated with the use of commercial enzymes for saccharification of biomass. Utilization of low value slip streams from the biorefinery as substrates for such an on-site enzyme production would be ideal to reduce costs. In order to understand which carbon sources that support growth and trigger A. saccharolyticus to produce beta-glucosidases, carbon sources, ranging from monomer sugars to complex lignocellulosic biomasses, including pretreated and hydrolyzed corn stover fractions, were investigated as substrates and inducers of enzyme production. A convenient micro titer plate experimental setup was developed that facilitated a fast screening for beta-glucosidase activity on the different carbon sources. The greatest beta-glucosidase activity was found when A. saccharolyticus was cultivated on media containing xylose, xylan, wheat bran, and pretreated corn stover. In a refinery, beta-glucosidase production by A. saccharolyticus could with success be based on the biomass hemicelluloses and their degradation products which cannot be converted by conventional yeast.     

Simultaneous saccharification and fermentation of cellulose for lactic acid production

Lactic acid production from α-cellulose by simultaneous saccharification and fermentation (SSF) was studied. The cellulose was converted in a batch SSF using cellulase enzyme Cytolase CL to produce glucose sugar andLactobacillus delbrueckii to ferment the glucose to lactic acid. The effects of temperature, pH, yeast extract loading, and lactic acid inhibition were studied to determine the optimum conditions for the batch processing. Cellulose was converted efficiently to lactic acid, and enzymatic hydrolysis was the rate controlling step in the SSF. The highest conversion rate was obtained at 46°C and pH 5.0. The observed yield of lactic acid from α-cellulose was 0.90 at 72 hours. The optimum pH of the SSF was coincident with that of enzymatic hydrolysis. The optimum temperature of the SSF was chosen as the highest temperature the microorganism could withstand. The optimum yeast extract loading was found to be 2.5 g/L. Lactic acid was observed to be inhibitory to the microorganisms’ activity.     

Purification and Characterization of an alpha-l-Arabinofuranosidase from Butyrivibrio fibrisolvens GS113

An alpha-l-arabinofuranosidase (EC 3.2.1.55) was purified from the cytoplasm of Butyrivibrio fibrisolvens GS113. The native enzyme had an apparent molecular mass of 240 kDa and was composed of eight polypeptide subunits of 31 kDa. The enzyme displayed an isoelectric point of 6.0, a pH optimum of 6.0 to 6.5, a pH stability of 4.0 to 8.0, and a temperature optimum of 45 degrees C and was stable to 55 degrees C. The K(m) and V(max) for p-nitrophenyl-alpha-l-arabinofuranoside were 0.7 mM and 109 mumol/min/mg of protein, respectively. The enzyme was specific for the furanoside configuration and also readily cleaved methylumbelliferyl-alpha-l-arabinofuranoside but had no activity on a variety of other nitrophenyl- or methylumbelliferyl glycosides. When the enzyme was incubated with cellulose, carboxymethyl cellulose, or arabinogalactan, no release of sugars was found. Arabinose was found as the hydrolysis product of oatspelt xylan, corn endosperm xylan, or beet arabinan. No activity was detected when either coumaric or ferulic acid ester linked to arabinoxylobiose was used as substrates, but arabinoxylobiose was degraded to arabinose and xylobiose. Since B. fibrisolvens GS113 possesses essentially no extracellular arabinofuranosidase activity, the major role of the purified enzyme is apparently in the assimilation of arabinose-containing xylooligosaccharides generated from xylosidase, phenolic esterase, xylanase, and other enzymatic activities on xylans.     

Purification and characterization of beta-glucosidase from honey bees (Apis mellifera)

beta-glucosidase has been purified from the ventriculus and honey sac of Apis mellifera using a combination of anion- and cation-exchange, hydroxyapatite and gel-permeation chromatography. In addition, beta-glucosidase from the hypopharyngeal glands has been partially purified using anion-exchange and gel-permeation chromatography. The purified beta-glucosidase gave a positive result by glycoprotein staining. This beta-glucosidase consists of only one subunit and has M(r) of 72 kDa as determined by SDS-PAGE. IEF-PAGE showed several bands with pIs ranging from 4.5 to 4.8. These multiform proteins have been proposed as having different degrees of glycosylation. The pH optimum of the purified beta-glucosidase from the ventriculus and honey sac are 5.0. These enzymes were stable at temperatures up to 50 degrees C and have a relatively wide pH stability range of 4.0 to 9.0. MALDI-TOF-MS peptide mass maps of purified beta-glucosidase from the ventriculus, honey sac and hypopharyngeal glands showed six matching masses. These results indicate that the beta-glucosidase isolated from the hypopharyngeal glands, honey sac and ventriculus is the same. It is proposed that beta-glucosidase is produced in the hypopharyngeal glands, secreted into the mouth during feeding and then passes to the honey sac. From the honey sac, this enzyme is transferred into honeycomb cells and the ventriculus.     

Purification and Characterization of an Intracellular β-Glucosidase from the Methylotrophic Yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Pichia pastoris beta-glucosidase was purified to apparent homogeneity by salting out with ammonium sulfate, gel filtration, and ion-exchange chromatography with Q-Sepharose and CM-Sepharose. The enzyme is a tetramer (275 kD) made up of four identical subunits (70 kD). The pH optimum is 7.3, and it is fairly stable in the pH range 5.5-9.5. The temperature optimum is 40 degrees C. The purified beta-glucosidase is effectively active on p-/o-nitrophenyl-beta-D-glucopyranosides (p-/o-NPG) and 4-methylumbelliferyl-beta-D-glucopyranoside (4-MUG) with Km values of 0.12, 0.22, and 0.096 mM and Vmax values of 10.0, 11.7, and 6.2 micromol/min per mg protein, respectively. It also exhibits different levels of activity against p-nitrophenyl-1-thio-beta-D-glucopyranoside, cellobiose, gentiobiose, amygdalin, prunasin, salicin, and linamarin. The enzyme is competitively inhibited by gluconolactone, p-/o-nitrophenyl-beta-D-fucopyranosides (p-/o-NPF), and glucose against p-NPG as substrate. o-NPF is the most effective inhibitor of the enzyme activity with Ki value of 0.41 mM. The enzyme is more tolerant to glucose inhibition with Ki value of 7.2 mM for p-NPG. Pichia pastoris has been employed as a host for the functional expression of heterologous beta-glucosidases and the risk of high background beta-glucosidase activity is discussed.