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Biomedical subjects

J N Saddler

Publications and source records attributed to J N Saddler.

At least 19 recordsLinked to original sources
PubMed2004

An ethanologenic yeast exhibiting unusual metabolism in the fermentation of lignocellulosic hexose sugars.

Three lignocellulosic substrate mixtures [liquid fraction of acid-catalyzed steam-exploded softwood, softwood spent sulfite liquor (SSL) and hardwood SSL] were separately fermented by the industrially employed SSL-adapted strain Tembec T1 and a natural galactose-assimilating isolate (Y-1528) of Saccharomyces cerevisiae to compare fermentative efficacy. Both strains were confirmed as S. cerevisiae via molecular genotyping. The performance of strain Y-1528 exceeded that of Tembec T1 on all three substrate mixtures, with complete hexose sugar consumption ranging from 10 to 18 h for Y-1528, vs 24 to 28 h for T1. Furthermore, Y-1528 consumed galactose prior to glucose and mannose, in contrast to Tembec T1, which exhibited catabolite repression of galactose metabolism. Ethanol yields were comparable regardless of the substrate utilized. Strains T1 and Y-1528 were also combined in mixed culture to determine the effects of integrating their distinct metabolic capabilities during defined hexose sugar and SSL fermentations. Sugar consumption in the defined mixture was accelerated, with complete exhaustion of hexose sugars occurring in just over 6 h. Galactose was consumed first, followed by glucose and mannose. Ethanol yields were slightly reduced relative to pure cultures of Y-1528, but normal growth kinetics was not impeded. Sugar consumption in the SSLs was also accelerated, with complete utilization of softwood- and hardwood-derived hexose sugars occurring in 6 and 8 h, respectively. Catabolite repression was absent in both SSL fermentations.

Cellulose↗
PubMed2002

Degradation of trilinolein by laccase enzymes.

Laccase enzymes were investigated for their potential to catalyze the oxidation of trilinolein and methyl linoleate. This study demonstrates that laccase enzymes can oxidize unsaturated fatty acid esters and their associated lipids. The reaction products resulting from laccase-catalyzed reactions with trilinolein were analyzed using combined reversed-phase high-performance liquid chromatography and mass spectrometry via an atmospheric pressure chemical ionization source. The dominant oxidation products detected were monohydroperoxides, bishydroperoxides, and epoxides. This paper presents the first detailed investigation into the interaction between laccase enzymes and lipids containing unsaturated fatty acids.

Chromatography, Gas↗
PubMed2002

The influence of bark on the fermentation of Douglas-fir whitewood pre-hydrolysates.

Douglas-fir ( Pseudotsuga menziesii) whitewood was supplemented with increasing concentrations of bark (0-30%) and was pretreated using SO(2)-catalysed steam explosion. The presence of bark in the feedstock resulted in the decreased recovery of total sugars, furfural and 5-hydroxymethylfurfural in the resultant pre-hydrolysate. No detrimental impact on monomer sugar recovery was observed. The concentration of lipophilic extractives present in the pre-hydrolysate increased with increasing bark loading, to a maximum of 0.43 g x l(-1). The water-soluble pre-hydrolysates were fermented by Saccharomyces cerevisiae to determine the impact of bark on sugar consumption and ethanol production. Despite the inclusion of bark, fermentation of all pre-hydrolysates resulted in the complete consumption of hexose sugars within 48 h. Ethanol yields were greater than 0.43 g x g(-1) for all pre-hydrolysates regardless of bark content, indicating that, up to a content of 30%, bark had a negligible impact on the fermentation of the pre-hydrolysates to ethanol.

Biotechnology↗
PubMed2001

The effect of shaking regime on the rate and extent of enzymatic hydrolysis of cellulose.

In an attempt to elucidate the effect of mixing on the rate and extent of enzymatic hydrolysis of cellulosic substrates, alpha-cellulose was hydrolysed using a commercial cellulase preparation at varying levels of substrate concentration (2.5,5 and 7.5% (w/v)) and by using three shaking regimes: continuous at low-speed (25 rpm), continuous at high-speed (150 rpm) and an intermittent regime comprised of high and low-speed shaking intervals. The continuous, high-speed shaking produced the highest conversion yields, whereas the intermittent and low-speed shaking regimes resulted in lower conversions. After 72 h, at all shaking regimes (150 rpm,25 rpm and intermittent), using a low substrate concentration (2.5%) produced conversion yields (82,79 and 80%) higher than those obtained at high (7.5%) substrate concentration (68,63 and 68%). As the substrate concentration increased, the conversion yields at intermittent shaking gradually approached those resulting from high-speed shaking. Thus, it appears that intermittent shaking could be a beneficial process option as it can reduce the mixing energy requirements while producing reasonably high conversion yields.

Biotechnology↗
PubMed2000

Steam pretreatment of Douglas-fir wood chips. Can conditions for optimum hemicellulose recovery still provide adequate access for efficient enzymatic hydrolysis?

Douglas-fir sapwood and heartwood were impregnated with SO2 and steam exploded at three severity levels, and the cellulose-rich, water-insoluble component was enzymatically hydrolyzed. The high-severity conditions resulted in near complete solubilization and some degradation of hemicelluloses and a significant improvement in the efficiency of enzymatic digestibility of the cellulose component. At lower severity, some of the hemicellulose remained unhydrolyzed, and the cellulose present in the pretreated solids was not readily hydrolyzed. The medium-severity pretreatment conditions proved to be a good compromise because they improved the enzymatic hydrolyzability of the solids and resulted in the recovery of the majority of hemicellulose in a monomeric form within the water-soluble stream. Sapwood-derived wood chips exhibited a higher susceptibility to both pretreatment and hydrolysis and, on steam explosion, formed smaller particles as compared to heartwood-derived wood chips.

Cellulase↗
PubMed1999

The nature of lignin from steam explosion/ enzymatic hydrolysis of softwood: structural features and possible uses: scientific note.

Effective utilization of the lignin by-product is a prerequisite to the commercial viability of ethanol production from softwood wastes using a steam explosion (SE)/enzymatic hydrolysis (EH)/fermentation process. Changes in the chemical composition of Douglas fir wood on SO2-catalyzed SE followed by EH were assessed using conventional analytical methods and new halogen-probe techniques. A significant solubilization of hemicelluloses was observed in the SE stage, the severity of which affected subsequent fermentation of cellulose and sorption of enzymes. SE of softwood resulted in dramatic changes in the chemical structure of lignin in the residual material involving chemical reactions via the benzyl cation. This leads to a more condensed lignin with partly blocked alpha-reaction centres. Possible uses for this lignin are discussed.

Journal Article↗
PubMed1998

Analysis of molecular size distributions of cellulose molecules during hydrolysis of cellulose by recombinant Cellulomonas fimi beta-1,4-glucanases.

Four beta-1,4-glucanases (cellulases) of the cellulolytic bacterium Cellulomonas fimi were purified from Escherichia coli cells transformed with recombinant plasmids. Previous analyses using soluble substrates had suggested that CenA and CenC were endoglucanases while CbhA and CbhB resembled the exo-acting cellobiohydrolases produced by cellulolytic fungi. Analysis of molecular size distributions during cellulose hydrolysis by the individual enzymes confirmed these preliminary findings and provided further evidence that endoglucanase CenC has a more processive hydrolytic activity than CenA. The significant differences between the size distributions obtained during hydrolysis of bacterial microcrystalline cellulose and acid-swollen cellulose can be explained in terms of the accessibility of beta-1,4-glucan chains to enzyme attack. Endoglucanases and cellobiohydrolases were much more easily distinguished when the acid-swollen substrate was used.

Actinomycetales↗
PubMed1997

Cellobiose dehydrogenase, an active agent in cellulose depolymerization.

The ability of cellobiose dehydrogenase purified from Phanerochaete chrysosporium to modify a Douglas fir kraft pulp was assessed. Although the addition of cellobiose dehydrogenase alone had little effect, supplementation with cellobiose and iron resulted in a substantial reduction in the degree of polymerization of the pulp cellulose. When the reaction was monitored over time, a progressive depolymerization of the cellulose was apparent with the concomitant production of cellobiono-1,5-lactone. Analysis of the reaction filtrates indicated that glucose and arabinose were the only neutral sugars generated. These sugars are derived from the degradation of the cellobiose rather than resulting from modifications of the pulp. These results suggest that the action of cellobiose dehydrogenase results in the generation of hydroxyl radicals via Fenton's chemistry which subsequently results in the depolymerization of cellulose. This appears to be the mechanism whereby a substantial reduction in the degree of polymerization of the cellulose can be achieved without a significant release of sugar.

Journal Article↗
PubMed1997

Microbiology and biodegradation of resin acids in pulp mill effluents: a minireview.

Resin acids, a group of diterpenoid carboxylic acids present mainly in softwood species, are present in many pulp mill effluents and toxic to fish in recipient waters. They are considered to be readily biodegradable. However, their removal across biological treatment systems has been shown to vary. Recent studies indicate that natural resin acids and transformation products may accumulate in sediments and pose acute and chronic toxicity to fish. Several resin acid biotransformation compounds have also been shown to bioaccumulate and to be more resistant to biodegradation than the original material. Until recently, the microbiology of resin-acid degradation has received only scant attention. Although wood-inhabiting fungi have been shown to decrease the level of resin present in wood, there is no conclusive evidence that fungi can completely degrade these compounds. In contrast, a number of bacterial isolates have recently been described which are able to utilize dehydroabietic or isopimaric acids as their sole carbon source. There appears to be an unusually high degree of substrate specificity with respect of the utilization of abietane congeners and the presence of substituents. Pimaranes do not appear to be attacked to the same extent as the abietanes. This paper reviews the occurrence, chemistry, toxicity, and biodegradation of resin acids in relation to the biological treatment of pulp and paper mill effluents.

Biodegradation, Environmental↗
PubMed1995

Growth, induction, and substrate specificity of dehydroabietic acid-degrading bacteria isolated from a kraft mill effluent enrichment.

We investigated resin acid degradation in five bacteria isolated from a bleach kraft mill effluent enrichment. All of the bacteria grew on dehydroabietic acid (DHA), a resin acid routinely detected in pulping effluents, or glycerol as the sole carbon source. None of the strains grew on acetate or methanol. Glycerol-grown, high-density, resting-cell suspensions were found to undergo a lag for 2 to 4 h before DHA degradation commenced, suggesting that this activity was inducible. This was further investigated by spiking similar cultures with tetracycline, a protein synthesis inhibitor, at various times during the DHA disappearance curve. Cultures to which the antibiotic was added prior to the lag did not degrade DHA. Those that were spiked with the antibiotic after the lag phase (4 h) degraded DHA at the same rate as did controls with no added tetracycline. Therefore, de novo protein synthesis was required for DHA biodegradation, confirming that this activity is inducible. The five strains were also evaluated for their ability to degrade other resin acids. All strains behaved in a similar fashion. Unchlorinated abietane-type resin acids (abietic acid, DHA, and 7-oxo-DHA) were completely degraded within 7 days, whereas pimarane resin acids (sandaracopimaric acid, isopimaric acid, and pimaric acid) were poorly degraded (25% or less). Chlorination of DHA affected biodegradation, with both 12,14-dichloro-DHA and 14-chloro-DHA showing resistance to degradation. However, 50 to 60% of the 12-chloro-DHA was consumed within the same period.

Abietanes↗
PubMed1994

A streptavidin-cellulose-binding domain fusion protein that binds biotinylated proteins to cellulose.

A fusion protein, Sta-CBDCex, which comprises streptavidin with a cellulose-binding domain (CBDCex) fused to its C terminus, was produced in the cytoplasm of Escherichia coli, where it formed inclusion bodies. Renatured Sta-CBDCex, recovered from the inclusion bodies, adsorbed to Avicel, a microcrystalline cellulose. The cellulose-bound Sta-CBDCex in turn bound biotinylated alkaline phosphatase or biotinylated beta-glucosidase. The immobilized beta-glucosidase remained fully active during 2 weeks of continuous column operation at 50 degrees C.

Biotin↗
PubMed1993

A comparison of two xylanases from the thermophilic fungi Thielavia terrestris and Thermoascus crustaceus.

Two thermophilic xylanases (xylanase II from Thielavia terrestris 255B and the 32-kDa xylanase from Thermoascus crustaceus 235E) were studied to determine if they had different and complementary modes of action when they hydrolysed various types of xylans. Partial amino acid sequencing showed that these two enzymes belonged to different families of beta-1,4-glycanases. Xylanase II achieved faster solubilization of insoluble xylan whereas the 32-kDa xylanase was more effective in producing xylose and short xylo-oligomers. An assessment of the combined hydrolytic action of the two xylanases did not reveal any co-operative action. The sugars released when the two thermophilic xylanases were used together were almost identical to those released when the 32-kDa xylanase acted alone. The two xylanases were able to remove about 12% of the xylan remaining in an aspen kraft pulp. This indicated that either one of these thermophilic enzymes may be useful for enhancing the bleaching of kraft pulps.

Amino Acid Sequence↗
PubMed1992

Purification and characterization of a xylanase from the thermophilic ascomycete Thelavia terrestris 255B.

Thielavia terrestris 255B, a thermophilic ascomycete, produced two major forms of xylanase with pIs of 4.6 (xylanase I) and 6.1 (xylanase II). The latter enzyme could be purified to greater than 99% homogeneity using anion-exchange chromatography and gel filtration. Xylanase II had a mol wt of 25.7 kDa (SDS-PAGE) and a pH and a temperature optimum of 3.6-4.0 and 60-65 degrees C, respectively. The ratio of the enzyme's activity against xylan and carboxymethylcellulose was 500-1000 to 1, indicating a possible application of this enzyme in biobleaching processes. The amino acid sequence of this protein is being determined, and initial data suggest that the enzyme belongs to a group of low-mol wt xylanases that have been isolated from both bacteria and fungi.

Amino Acid Sequence↗
PubMed1987

Crystallization and preliminary X-ray diffraction study of a xylanase from Trichoderma harzianum.

A 20,000 Mr xylanase from Trichoderma harzianum has been purified and crystallized from 20% (w/v) saturated ammonium sulphate solutions. The unit cell is orthorhombic, space group P2(1)2(1)2(1), with unit cell lengths a = 44.2 A, b = 94.1 A, c = 51.6 A. Data from native crystals and several potential heavy-atom derivatives have been collected. An X-ray analysis to at least 2.8 A resolution appears to be feasible.

Crystallization↗
PubMed1987

The copurification of beta-glucosidase, beta-xylosidase, and 1,3-beta-glucanase in two separate enzyme complexes isolated from Trichoderma harzianum E58.

Two enzyme complexes, each with beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21), beta-xylosidase (beta-D-xylan xylohydrolase, EC 3.2.1.37), and 1,3-beta-glucanase (laminarinase, EC 3.2.1.39) activity, were purified to near homogeneity from the cellulolytic fungus Trichoderma harzianum E58. The two complexes had the same isoelectric point of pH 8.3 and identical subunit molecular masses of 75,400 daltons. The two complexes were also similar in that all activities were sensitive to inhibition by mercuric chloride (2 mM) and D-glucono-1,5-lactone (0.2% w/v). The activity ratios of the major and minor complexes were 1:1.7:4.3 and 1:1.6:3.1 for the beta-xylosidase, beta-glucosidase, and 1,3-beta-glucanase, respectively. Both complexes had approximately the same Km values for p-nitrophenyl beta-D-glucopyranoside and salicin. The pH optima of corresponding activities of the two complexes were also similar. The major and minor complexes differed in that the Km of the former for laminarin was almost threefold lower than that of the latter. Whereas all three activities of the minor complexes were inhibited by D-glucono-1,5-lactone with the same inhibition constant, the beta-glucosidase and 1,3-beta-glucanase of the major complex had inhibition constants which differed by more than 80,000 times. In addition, the inhibition on the 1,3-beta-glucanase in the major and minor complexes using D-glucono-1,5-lactone were noncompetitive and competitive, respectively. From the inhibition studies, the beta-glucosidase, beta-xylosidase, and 1,3-beta-glucanase activities in the minor complex were deduced to be more interdependent than the same activities in the major complex.

Chromatography, Ion Exchange↗
PubMed1986

Factors limiting the efficiency of cellulase enzymes.

The major reasons behind the low efficiency of enzymatic hydrolysis of cellulose are reviewed. The problem is a result of the heterogeneous nature of the hydrolysis reaction which involves a multicomponent soluble catalyst, an insoluble substrate and products which are both.

Bacteria↗
PubMed1985

Butanediol production from cellulose and hemicellulose by Klebsiella pneumoniae grown in sequential coculture with Trichoderma harzianum.

The bioconversion of cellulose and hemicellulose substrates to 2,3-butanediol by a sequential coculture approach was investigated with the cellulolytic fungus Trichoderma harzianum E58 and the fermentative bacterium Klebsiella pneumoniae. Vogel medium optimal for the production of the cellulolytic and xylanolytic enzymes of the fungus was found to be inhibitory to butanediol fermentation. This inhibition appeared to be due to a synergistic effect of various ingredients, particularly the salts, present in the fungal medium. The removal or replacement of such ingredients from Vogel medium led to the relief of fermentation inhibition, but the treatments also resulted in a significant decrease in fungal enzyme production. Resting cells of K. pneumoniae could be used for butanediol production in the fungal medium, indicating that the inhibitory effect on solvent production under such conditions was due to the indirect result of growth inhibition of the bacterial cells. The resting-cell approach could be combined with a fed-batch system for the direct conversion of 8 to 10% (wt/vol) of Solka-Floc or aspenwood xylan to butanediol at over 30% of the theoretical conversion efficiencies.

Butylene Glycols↗