Properties of an intracellular beta-glucosidase purified from the cellobiose-fermenting yeast Candida wickerhamii.

An intracellular beta-glucosidase was isolated from the cellobiose-fermenting yeast, Candida wickerhamii. Production of the enzyme was stimulated under aerobic growth, with the highest level of production in a medium containing cellobiose as a carbohydrate source. The molecular mass of the purified protein was approximately 94 KDa. It appeared to exist as a dimeric structure with a native molecular mass of about 180 KDa. The optimal pH ranged from 6.0 to 6.5 with p-nitrophenyl beta-D-glucopyranoside (NpGlc) as a substrate. The optimal temperature for short-term (15-min) assays was 35 degrees C, while temperature-stability analysis revealed that the enzyme was labile at temperatures of 28 degrees C and above. Using NpGlc as a substrate, the enzyme was estimated to have a Km of 0.28 mM and a Vmax of 525 mumol product min-1 mg protein-1. Similar to the extracellular beta-glucosidase produced by C. wickerhamii, this enzyme resisted end-product inhibition by glucose, retaining 58% of its activity at 100 mM glucose. The activity of the enzyme was highest against aryl beta-1,4-glucosides. However, p-nitrophenyl xylopyranoside, lactose, cellobiose, and trehalose also served as substrates for the purified protein. Activity of the enzyme was stimulated by long-chain n-alkanols and inhibited by ethanol, 2-propanol, and 2-butanol. The amino acid sequence, obtained by Edman degradation analysis, suggests that this beta-glucosidase is related to the family-3 glycosyl hydrolases.

Expression and secretion of the Candida wickerhamii extracellular beta-glucosidase gene, bglB, in Saccharomyces cerevisiae.

The yeast Candida wickerhamii exports a cell-associated beta-glucosidase that is active against cellobiose and all soluble cellodextrins. Because of its unique ability to tolerate end-product inhibition by glucose, the bglB gene that encodes this enzyme was previously cloned and sequenced in this laboratory. Using several different promoters and constructs, bglB was expressed in the hosts Escherichia coli, Pichia pastoris, and Saccharomyces cerevisiae. Expression was initially performed in E. coli using either the lacZ or tac promoter. This resulted in intracellular expression of the BglB protein with the protein being rapidly fragmented. Secretion and glycosylation of active beta-glucosidase was achieved with several different S. cerevisiae constructs utilizing either the adh1 or the gal1 promoter on 2-micro replicating plasmids. When either the invertase (Suc2) or the BglB secretion signal was used, BglB protein remained associated with the cell wall and appeared to be hyperglycosylated. Expression in P. pastoris was also examined to determine if higher activity and expression could be achieved in a yeast host that usually does not hyperglycosylate. Using the alcohol oxidase promoter in conjunction with either the pho1 or the alpha-factor secretion signal, the recombinant enzyme was successfully secreted and glycosylated in P. pastoris. However, levels of protein expression from the chromosomally integrated vector were insufficient to detect activity.

Production of beta-glucosidase and diauxic usage of sugar mixtures by Candida molischiana.

The fermentation of cellobiose is a rare trait among yeasts. Of the 308 yeast species that utilize cellobiose aerobically, only 12 species ferment it, and only 2 species, Candida molischiana and Candida wickerhamii, also ferment cellodextrins. Candida molischiana produced beta-glucosidase activity on all carbon sources tested, except glucose, mannose, and fructose. When these sugars were added to cultures growing on cellobiose, the synthesis of beta-glucosidase ceased. However, the total amount of enzyme activity remained constant, indicating that the C. molischiana beta-glucosidase is catabolite repressed and not catabolite inactivated. When grown in medium initially containing glucose plus xylose, cellobiose, maltose, mannitol, or glucitol, C. molischiana preferentially utilized glucose and produced little beta-glucosidase activity until glucose was nearly depleted from the medium. When grown in medium containing cellobiose plus either fructose or mannose, the yeast preferentially utilized the monosaccharides and produced little beta-glucosidase activity. Candida molischiana produced beta-glucosidase and co-utilized cellobiose and xylose, maltose, or trehalose. Glucose and fructose, mannose, or trehalose were co-utilized; however, no beta-glucosidase activity was detected. Thus, the order of substrate preference groups appeared to be (glucose, trehalose, fructose, mannose) > (cellobiose, maltose, xylose) > (mannitol, glucitol).

Cloning and characterization of a gene encoding a cell-bound, extracellular beta-glucosidase in the yeast Candida wickerhamii.

The ability of yeasts to ferment cellodextrins is rare. Candida wickerhamii is able to use these sugars for alcohol production because of a cell-bound, extracellular, beta-glucosidase that is unusual by not being inhibited by glucose. A cDNA expression library in lambda phage was prepared with mRNA isolated from cellobiose-grown C. wickerhamii. Immunological screening of the library with polyclonal antibodies against purified C. wickerhamii cell-bound, extracellular beta-glucosidase yielded 12 positive clones. Restriction endonuclease analysis and sequence data revealed that the clones could be divided into two groups, bglA and bglB, which were shown to be genetically distinct by Southern hybridization analyses. Efforts were directed at the study of bglB since it appeared to code for the cell-bound beta-glucosidase. Sequence data from both cDNA and genomic clones showed the absence of introns in bglB. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting of cell lysates from Escherichia coli bglB clones confirmed the presence of an expressed protein with an apparent molecular mass of 72 kDa, which is consistent with that expected for an unglycosylated form of the enzyme. Amino acid comparisons of BglB with other beta-glucosidase sequences suggest that it is a member of family 1 glycosyl hydrolases but is unusual in that it contains an additional 100 to 130 amino acids at the N terminus. This sequence did not have homologies to other known protein sequences and may impart unique properties to this beta-glucosidase.

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.

Kinetic characterization of a beta-glucosidase from a yeast, Candida wickerhamii.

The extracytoplasmic, cell-bound beta-1,4-glucosidase of Candida wickerhamii was characterized kinetically. The enzyme was found to produce glucose from cellobiose and cellodextrins (degree of polymerization from three to six) by catalyzing the removal of the terminal glucose moiety from the nonreducing end of these beta-glucans. The Km values for the series, cellobiose through cellohexaose, were 210.7, 106.6, 106.3, 105.9, and 79.8 mM, respectively, whereas the kcat values were 14.79, 13.24, 13.78, 15.13, and 7.66 mumol of glucose.min-1.mg-1 of protein, respectively. A computer program was developed to estimate the integrated rate equation. When the above kinetic constants were used in the computer model, the predicted rates of glucose formation agreed well with the experimental data. Saccharomyces cerevisiae, which is unable to ferment cellobiose or cellodextrins, ferments glucose about twice as fast as C. wickerhamii. If S. cerevisiae is cultured on cellobiose or cellodextrins and the purified C. wickerhamii beta-glucosidase is added to the S. cerevisiae culture at levels that mimic the production of beta-glucosidase by a C. wickerhamii culture with time, the two cultures produce ethanol at equivalent rates. This suggests that the rate-limiting step in the fermentation of cellobiose/cellodextrins by C. wickerhamii is the production of beta-glucosidase.

Purification and characterization of the extracellular alpha-amylase from Streptococcus bovis JB1.

The extracellular alpha-amylase (1,4-alpha-D-glucanglucanohydrolase; EC 3.2.1.1) from maltose-grown Streptococcus bovis JB1 was purified to apparent homogeneity by ion-exchange chromatography (Mono Q). The enzyme had an isoelectric point of 4.50 and an apparent molecular mass of 77,000 Da, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was rich in acidic and hydrophobic amino acids. The 15-amino-acid NH2-terminal sequence was 40% homologous with the Bacillus subtilis saccharifying alpha-amylase and 27% homologous with the Clostridium acetobutylicum alpha-amylase. alpha-Amylase activity on soluble starch was optimal at pH 5.0 to 6.0. The enzyme was relatively stable between pH 5.5 and 8.5 and at temperatures below 50 degrees C. When soluble potato starch was used as the substrate, the enzyme had a Km of 0.88 mg.ml-1 and a kcat of 2,510 mumol of reducing sugar.min-1.mg of protein-1. The enzyme exhibited neither pullulanase nor dextranase activity and was 40 to 70% as active on amylopectin as on amylose. The major end products of amylose hydrolysis were maltose, maltotriose, and maltotetraose.

The influence of carbon and nitrogen nutrition on fusarin C biosynthesis by Fusarium moniliforme.

The influence of various carbon and nitrogen sources on fusarin C synthesis was examined in submerged cultures of Fusarium moniliforme NRRL 13616. Using a zinc-deficient, synthetic medium, highest levels of fusarin C were produced by cultures grown with urea or ammonium sulfate as the nitrogen source and fructose, sucrose, or glucose as the carbon source. In media supplemented with various concentrations of glucose and ammonium sulfate, glucose concentrations which provided excess carbohydrate significantly increased fusarin C synthesis, regardless of the ammonium sulfate concentration.

Fermentation and aerobic metabolism of cellodextrins by yeasts.

The fermentation and aerobic metabolism of cellodextrins by 14 yeast species or strains was monitored. When grown aerobically, Candida wickerhamii, C. guilliermondii, and C. molischiana metabolized cellodextrins of degree of polymerization 3 to 6. C. wickerhamii and C. molischiana also fermented these substrates, while C. guilliermondii fermented only cellodextrins of degree of polymerization less than or equal to 3. Debaryomyces polymorphus, Pichia guilliermondii, Clavispora lusitaniae, and one of two strains of Kluyveromyces lactis metabolized glucose, cellobiose, and cellotriose when grown aerobically. These yeasts also fermented these substrates, except for K. lactis, which fermented only glucose and cellobiose. The remaining species/strains tested, K. lactis, Brettano-myces claussenii, B. anomalus, K. dobzhanskii, Rhodotorula minuta, and Dekkera intermedia, both fermented and aerobically metabolized glucose and cellobiose. Crude enzyme preparations from all 14 yeast species or strains were tested for ability to hydrolyze cellotriose and cellotretose. Most of the yeasts produced an enzyme(s) capable of hydrolyzing cellotriose. However, with two exceptions, R. minuta and P. guilliermondii, only the yeasts that metabolized cellodextrins of degree of polymerization greater than 3 produced an enzyme(s) that hydrolyzed cellotretose.

Transport of glucose and cellobiose by Candida wickerhamii and Clavispora lusitaniae.

The cellular location of beta-1,4-glucosidase activity from, as well as the transport of glucose and cellobiose into, cells of Clavispora lusitaniae NRRL Y-5394 and Candida wickerhamii NRRL Y-2563 was investigated. The beta-glucosidase from Cl. lusitaniae appeared to be a soluble cytoplasmic enzyme. This yeast transported both glucose and cellobiose when grown in medium containing cellobiose as the sole carbon source. Glucose, but not cellobiose, uptake was observed for cells grown on glucose. The Ks and Vmax values for cellobiose transport were different when Cl. lusitaniae was cultured either aerobically (0.11 mM, 6.28 nmol.min-1.mg-1) or anaerobically (0.25 mM, 3.88 nmol-1.min-1.mg-1). The Ks and Vmax values for glucose transport (0.23-1.10 mM and 17.2-33.9 nmol.min-1.mg-1) also differed with the various growth conditions. The beta-glucosidase from C. wickerhamii was extracytoplasmically located. This yeast transported glucose, but not cellobiose, under all growth conditions tested. The Ks for glucose uptake was 0.13-0.28 mM when C. wickerhamii was cultured on cellobiose and 0.25-0.30 mM when cultured on glucose. The Vmax values for glucose uptake were greater for cells cultured on cellobiose (35.0-37.9 nmol.min-1.mg-1) than for cells cultured on glucose (15.6-21.4 nmol.min-1.mg-1). Cellobiose did not inhibit glucose uptake in either yeast. Glucose partially inhibited cellobiose transport in C. lusitaniae, but only if the yeast was grown aerobically. In both yeasts, sugar transport was sensitive to carbonyl cyanide p-trifluoromethoxyphenylhydrazone and 1799, but insensitive to valinomycin.

Measurement of protein biomass by Fourier transform infrared-photoacoustic spectroscopy.

A relatively new analytical technique, Fourier transform infrared-photoacoustic spectroscopy (FTIR-PAS), provides spectra of bacteria, fungi, and other microorganisms in solid states not suitable for conventional absorption spectroscopy. In this paper the feasibility of quantitative measurement of protein biomass on solid substrates by FTIR-PAS is examined and discussed. By measuring photoacoustic absorption bands from amide groups in the protein of microorganisms, the increase in biomass that occurs during growth was monitored directly and accurately. Incorporation of polyacrylonitrile into the sample as an internal standard was shown to be a convenient method for improving both the reliability and the range of detection by photoacoustic spectroscopy. Results of FTIR-PAS measurements of known quantities of microbial mass in simulated growth experiments suggest that the technique may be especially suitable for assays of microorganisms used in solid-state biosyntheses of drugs, hormones, and other biological agents.

Purification and characterization of an extracellular endoglucanase from the marine shipworm bacterium.

Bacterial cultures isolated from the gland of Deshayes of marine shipworm (Psiloteredo healdi) produced extracellular endoglucanase activity when cultured with 1% cellulose. An endoglucanase of subunit relative molecular mass 58,000, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was purified to homogeneity from cell-free culture medium. Similarly, the relative molecular mass of the native enzyme was 60,100 as determined by gel permeation chromatography. No carbohydrate appeared to be associated with the purified protein. The action of the purified enzyme on various cellodextrins was also studied. Only interior glucosyl linkages of cellodextrin chains larger than cellotriose were cleaved by the enzyme and the centermost bond of cellohexaose was preferentially cleaved. The Km values of the purified endoglucanase were 0.12 mM for cellotetraose, 0.05 mM for cellopentaose, and 0.11 mM for cellohexaose. Glucose, cellobiose, and cellotriose did not inhibit enzymatic activity.

Extracellular endoglucanase activity by a novel bacterium isolated from marine shipworm.

An extracellular enzyme preparation from shipworm bacterium cultures dramatically increased reducing sugar content of carboxymethylcellulose (CMC3), but did not solubilize sugar from particulate cellulose. The preparation degraded cellodextrins larger than cellotriose (G3). Only interior cellodextrin chain linkages were cleaved and the center-most bond of cellohexaose (G6) was preferentially cleaved. Activity maxima were observed at 60 degrees C and between pH 5.0 and 7.0. The activity was resistant to protease treatment and little loss of activity was observed after 14 d at 25 degrees C.

Growth characteristics of a novel nitrogen-fixing cellulolytic bacterium.

Growth characteristics of a cellulolytic nitrogen-fixing bacterium isolated from a marine shipworm by Waterbury et al. (J. B. Waterbury, C. B. Calloway, and R. D. Turner, Science 221:1401-1403, 1983) are described. When grown microaerobically, the bacterium exhibited doubling times of about 2 days in cellulose-supplemented synthetic medium devoid of combined nitrogen. Maximum growth was reached 12 to 16 days after inoculation. Growth optima for pH, temperature, and NaCl concentration were 8.5, 30 to 35 degrees C, and 0.3 M, respectively. During growth the bacterium produced succinic acid (0.026%) and acetic acid (0.010%). Formic acid (0.010%) was produced during the stationary growth phase. No growth was observed when glucose was the sole carbon source. Cellobiose supported weak growth, while longer-chain-length cellodextrins supported extensive growth. Analysis of residual carbohydrates in the medium during growth indicated that the bacterium catabolized a terminal glucose moiety from the cellodextrin chain.

Affinity Purifications of Aldose Reductase and Xylitol Dehydrogenase from the Xylose-Fermenting Yeast Pachysolen tannophilus.

Although xylose is a major product of hydrolysis of lignocellulosic materials, few yeasts are able to convert it to ethanol. In Pachysolen tannophilus, one of the few xylose-fermenting yeasts found, aldose reductase and xylitol dehydrogenase were found to be key enzymes in the metabolic pathway for xylose fermentation. This paper presents a method for the rapid and simultaneous purification of both aldose reductase and xylitol dehydrogenase from P. tannophilus. Preliminary studies indicate that this method may be easily adapted to purify similar enzymes from other xylose-fermenting yeasts.

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.

Regulation of beta-1, 4-Glucosidase Expression by Candida wickerhamii.

Candida wickerhamii NRRL Y-2563 expressed beta-glucosidase activity (3 to 8 U/ml) constitutively when grown aerobically in complex medium containing either glycerol, succinate, xylose, galactose, or cellobiose as the carbon source. The addition of a high concentration of glucose (>75 g/liter) repressed beta-glucosidase expression (<0.3 U/ml); however, this yeast did produce beta-glucosidase when the initial glucose concentration was

Fermentation of cellodextrins to ethanol using mixed-culture fermentations.

The potential for enhancing ethanol production from cellodextrins by employing mixed-culture (Candida wickerhamii-Saccharomyces cerevisiae) fermentations was investigated. Initially, ethanol production was monitored in fermentation medium containing 50 g/L glucose plus 45 g/L cellobiose. Inoculum levels and times of inoculum addition were varied. Of the conditions tested, the most rapid rates of ethanol formation occurred in fermentations in which either C. wickerhamii and S. cerevisiae were coinoculated at a ratio of 57 : 1 cell/mL or in fermentations in which a 10-fold-greater S. cerevisiae inoculum was added to a pure culture C. wickerhamii fermentation after 1 day incubation. These conditions were used to attempt to enhance fermentations in which cellodextrins produced by trifluoroacetic acid hydrolysis of cellulose served as the sole carbon source. Cellodextrins that were not further purified after cellulose hydrolysis contained compounds that were slightly inhibitory to C. wickerhamii. In this case the mixed-culture fermentations produced 12-45% more ethanol than a pure culture C. wickerhamii fermentation. However, if the substrate was treated with Darco G-60 charcoal, the toxic materials were apparently removed and the pure culture C. wickerhamii fermentations performed as well as the mixed-culture fermentations.

Characterization of cellobiose fermentations to ethanol by yeasts.

Twenty-two different yeasts were screened for their ability to ferment both glucose and cellobiose. The fermentation characteristics of Candida lusitaniae (NRRL Y-5394) and C. wickerhamii (NRRL Y-2563) were selected for further study because their initial rate of ethanol production from cellobiose was faster than the other test cultures. C. lusitaniae produced 44 g/L ethanol from 90 g/L cellobiose after 5-7 days. When higher carbohydrate concentrations were employed, fermentation ceased when the ethanol concentration reached 45-60 g/L. C. lusitaniae exhibited barely detectable levels of beta-glucosidase, even though the culture actively fermented cellobiose. C. wickerhamii produced ethanol from cellobiose at a rate equivalent to C. lusitaniae; however, once the ethanol concentration reached 20 g/L, fermentation ceased. Using p-nitrophenyl-beta-D-glucopyranoside (pNPG) as substrate, beta-glucosidase (3-5 U/mL) was detected when C. wickerhamii was grown anaerobically on glucose or cellobiose. About 35% of the beta-glucosidase activity was excreted into the medium. The cell-associated activity was highest against pNPG and salicin. Approximately 100-fold less activity was detected with cellobiose as substrate. When empolying these organisms in a simultaneous saccharification-fermentation of avicel, using Trichoderma reesei cellulase as the saccharifying agent, 10-30% more ethanol was produced by the two yeasts capable of fermenting cellobiose than by the control, Saccharomyces cerevisiae.