Cellulose hydrolysis under extremely low sulfuric acid and high-temperature conditions.

The kinetics of cellulose hydrolysis under extremely low acid (ELA) conditions (0.07 wt%) and at temperatures >200 degrees C was investigated using batch reactors and bed-shrinking flow-through (BSFT) reactors. The maximum yield of glucose obtained from batch reactor experiments was about 60% for alpha-cellulose, which occurred at 205 and 220 degrees C. The maximum glucose yields from yellow poplar feedstocks were substantially lower, falling in the range of 26-50%. With yellow poplar feedstocks, a large amount of glucose was unaccounted for at the latter phase of the batch reactions. It appears that a substantial amount of released glucose condenses with nonglucosidic substances in liquid. The rate of glucan hydrolysis under ELA was relatively insensitive to temperature in batch experiments for all three substrates. This contradicts the traditional concept of cellulose hydrolysis and implies that additional factors influence the hydrolysis of glucan under ELA. In experiments using BSFT reactors, the glucose yields of 87.5, 90.3, and 90.8% were obtained for yellow poplar feedstocks at 205, 220, and 235 degrees C, respectively. The hydrolysis rate for glucan was about three times higher with the BSFT than with the batch reactors. The difference of observed kinetics and performance data between the BSFT and the batch reactors was far above that predicted by the reactor theory.

Effect of pretreatment on simultaneous saccharification and fermentation of hardwood into acetone/butanol.

The effectiveness of pretreatments on hardwood substrate was investigated in connection with its subsequent conversion by simultaneous saccharification and fermentation (SSF), using Clostridium acetobutylicum. The main objectives of the pretreatment were to achieve efficient separation of lignin from carbohydrates, and to obtain maximum sugar yield on enzymatic hydrolysis of pretreated wood. Two methods have given promising results: (1) supercritical CO2-SO2 treatment, and (2) monoethanolamine (MEA) treatment. The MEA pretreatment removed above 90% of hardwood lignin while retaining 83% of carbohydrates. With CO2-SO2 pretreatment, the degree of lignin separation was lower. Under the scheme of SSF, the pretreated hardwood was converted to acetone, butanol, and ethanol (ABE) via single stage processing by cellulase enzyme system and C. acetobutylicum cells. The product yield in the process was such that 15 g of ABE/100 g of dry aspen wood was produced. In the overall process of SSF, the enzymatic hydrolysis was found to be the rate-limiting step. The ability of C. acetobutylicum to metabolize various 6-carbon and 5-carbon sugars resulted in efficient utilization of all available sugars from hardwood.

Analysis of the coordinate expression of 3-hydroxy-3-methylglutaryl coenzyme A synthase and reductase activities in Chinese hamster ovary fibroblasts.

Decreased activities of both 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase and HMG CoA reductase are observed in the presence of sterol in the Chinese hamster ovary (CHO) fibroblast. In three different genotypes of CHO cell mutants resistant to 25-hydroxycholesterol both enzyme activities exhibit a decreased response to 25-hydroxycholesterol compared to wild-type cells. Permanently repressed levels of both HMG CoA synthase and HMG CoA reductase activities are observed in another CHO mutant, phenotypically a mevalonate auxotroph. Mevinolin, a competitive inhibitor of HMG CoA reductase, has no effect on HMG CoA synthase activity measured in vitro. Incubation of CHO cells with sublethal concentrations of mevinolin produces an inhibition of the conversion of [14C]acetate to cholesterol and results in elevated levels of both HMG CoA synthase and HMG CoA reductase activities. Studies of CHO cells in sterol-free medium supplemented with cycloheximide indicate that continuous protein synthesis is not required for the maximal expression of HMG CoA synthase activity and provide an explanation for the lack of temporal similarity between HMG CoA synthase and reductase activities after derepression. These results support the hypothesis of a common mode of regulation for HMG CoA synthase and HMG CoA reductase activities in CHO fibroblasts.

Complementary recessive 25-hydroxycholesterol-resistant somatic cell mutants--assay of 25-hydroxycholesterol binding activity.

Complementation analysis of recessive 25-hydroxycholesterol-resistant mutants of the CHO-Kl cell shows the existence of at least two complementation groups, one of which is missing a binding activity for 25-hydroxycholesterol. Both complementation groups are shown to be refractory to inhibition of cellular HMG-CoA reductase activity and in the inhibition of biosynthesis of this enzyme by 25-hydroxycholesterol.

Analysis of regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in a somatic cell mutant auxotrophic for mevalonate.

Regulation of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase activity and synthesis by 25-hydroxycholesterol is aberrant in a somatic cell mutant of the Chinese hamster ovary K1 cell auxotrophic for mevalonate by virtue of a defect in HMG-CoA synthase activity. Normal regulation of HMG-CoA reductase activity and synthesis in this mutant by 25-hydroxycholesterol can be restored by simultaneous incubation with a small (0.4 mM) mevalonate supplement. Normal regulation of HMG-CoA reductase is also observed in a revertant of the mutant cell with normal HMG-CoA synthase activity.

Isolation and characterization of a mammalian cell mutant defective in 3-hydroxy-3-methylglutaryl coenzyme A synthase.

A somatic cell mutant of the Chinese hamster ovary (CHO)-K1 cell auxotrophic for mevalonic acid has been isolated by means of the bromodeoxyuridine-visible light technique. This mutant can incorporate labeled mevalonate but not labeled acetate into cholesterol and, thus, is apparently defective in mevalonate biosynthesis. The mutant is recessive with respect to the parental cell phenotype. Assessment of the in vitro activities of the enzymes responsible for mevalonate biosynthesis under varying growth conditions indicates that the mutant, Mev-1, is defective in 3-hydroxy-3-methylglutaryl coenzyme A synthase.

Radioimmune precipitation of 3-hydroxy-3-methylglutaryl coenzyme A reductase from Chinese hamster fibroblasts. Effect of 25-hydroxycholesterol.

Antibody prepared against 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase of rat liver can be shown to inhibit this enzyme in extracts prepared from cultured Chinese hamster ovary (CHO-K1) cells. The molecular weight (53,000) of the HMG-CoA reductase subunits of rat liver and Chinese hamster liver is identical with a [35S]methionine-labeled polypeptide that can be precipitated from CHO-K1 lysates by this antibody used in conjunction with protein A Sepharose. It is shown that 25-hydroxycholesterol which lowers HMG-CoA reductase activity in cultured fibroblasts blocks the incorporation of labeled methionine into this polypeptide. Furthermore, the antibody immune precipitates two other polypeptides with molecular weights of 127,000 and 60,000. The latter polypeptide responds to 25-hydroxycholesterol in the same fashion as the 53,000-dalton polypeptide. In a dominant 25-hydroxycholesterol-resistant mutant of the CHO-K1 cell, 25-hydroxycholesterol did not inhibit incorporation of labeled methionine into either the 53,000- or 60,000-dalton polypeptides.

Somatic cell genetic analysis of regulation of expression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

Three new types of regulatory somatic cell mutants defective in expression of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase are described. They include a recessive mutant with abnormally high enzyme activity, apparently defective in degradation of the enzyme, and one, phenotypically a mevalonate auxotroph, that maintains permanently repressed levels of enzyme activity and are recessive, constitutive mutants. These mutants offer means for analyses of the mechanism of regulation of expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase in cultured fibroblasts.

Differences between the anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferases of Salmonella typhimurium strains LT2 and LT7.

The anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferases (PRT), coded by the second structural gene (trpB) of the tryptophan (trp) operon in strains LT2 and LT7 of Salmonella typhimurium, differ from each other in a number of parameters. These include the apparent Km values for their substrates anthranilic acid and 5-phosphoribosylpyrophosphate, thermostability, sensitivity to substrate inhibition by anthranilic acid, as well as end-product inhibition by tryptophan and specific activity. The PRT of strain LT7 further differs from that of strain LT2 in that its apparent Km for 5-phosphoribosylpyrophosphate is three to seven times higher when associated with anthranilate synthase in the enzyme complex which catalyses the first two steps of tryptophan biosynthesis than in its free uncomplexed form, which the PRT of strain LT2 shows the same apparent Km for this substrate in both its free and complexed forms. These results confirm and extend the finding of Stuttard (1975) that strains LT2 and LT7 differ genetically form each other at a single site within region II of the trpB gene.

The binding of polyamines and of ethidium bromide to tRNA.

The binding of spermidine and ethidium bromide to mixed tRNA and phenylalanine tRNA has been studied under equilibrium conditions. The numbers and classes of binding sites obtained have been compared to those found in complexes isolated by gel filtration a low ionic strength. The latter complexes contain 10-11 moles of either spermidine or ethidium per mole of tRNA; either cation is completely displaceable by the other. In ethidium complexes, the first 2-3 moles are bound in fluorescent binding sites; the remaining 7-8 molecules bind in non-fluorescent form. At least one of the binding sites for spermidine appears similar to a binding site for fluorescent ethidium. Similar results are found with E. coli formylmethionine tRNA. Spermine, in excess of 18-20 moles per mole tRNA, causes precipitation of the complex. Putrescine does not form isolable complexes with yeast tRNA and displaces ethidium less readily from preformed ethidium-tRNA complexes. Under equilibrium conditions, in the absence of Mg++, there are 16-17 moles of spermidine bound per mole of tRNA as determined by equilibrium dialysis. Of these, 2-3 bind with a Ksence of 9 mM Mg++, the total number of binding sites is decreased slightly and there appears to be only one class of sites with a Ka = 600 M(-1). Quantitatively similar results are obtained for the binding of spermidine to yeast phenylalanine tRNA. When the interaction between ethidium bromide and mixed tRNA is studied by equilibrium dialysis or spectrophotometric titration, two classes of binding sites are obtained: 2-3 molecules bind with an average Ka = 6.6 x 10(5) M(-1) and 14-15 molecules bind with an average Ka = 4.1 x 10(4) M(-1). Spermidine, spermine, and Mg++ compete effectively for both classes of ethidium sites and have the effect of reducing the apparent binding constants for ethidium. When the binding of ethidium is studied by fluorometry, there are 3-4 highly fluorescent sites per tRNA. These sites are also affected by spermidine, spermine and Mg++. Putrescine has little effect on any of the classes of binding sites. These data are consistent with those found under non-equilibrium conditions. They suggest that polyamines bind to fairly specific regions of tRNA and may be involved in the maintenance of certain structural features of tRNA.